abstracts€¦ · keynote lectures (k1 – k8) ... keren kahi1, neta varsano1, andrea sorrentino2,...
TRANSCRIPT
Abstracts BIOMIN XV: 15th International Symposium on Biomineralization
9–13 September 2019 • Munich, Germany
1. Keynote lectures (K1 – K8) ................................................................................................................. 2
2. Talks (T1 – T89) ................................................................................................................................... 4
3. Posters (P1 – P107) ............................................................................................................................. 31
Design/Layout Layout: www.conventus.de
Editorial Deadline: 31 August 2019
1
K 1
On ion transport and concentration toward mineral formation
in sea urchin larvae Keren Kahi1, Neta Varsano1, Andrea Sorrentino2, Eva Pereiro2, Peter Rez3,
Steve Weiner1 and Lia Addadi*1
1Department of Structural Biology, Weizmann Institute of Science, Rehovot,
Israel 2ALBA Synchrotron Light Source, MISTRAL Beamline−Experiments Division, Barcelona, Spain 3Department of Physics, Arizona State University, Tempe, AZ, USA
During mineralized tissue formation, organisms are faced with a major
problem in ion transport and concentration. Suffice it to consider that marine
animals must concentrate calcium by >4 orders of magnitude relative to its concentration in sea water to deposit calcite or aragonite minerals. Sea urchin
larvae are good model organisms for studying ion processing during skeleton
formation, because of the extensive knowledge accumulated on the processes related to mineral deposition. Sea urchin larvae have endoskeletons
composed of two calcitic spicules, deposited by primary mesenchymal
spicule-forming cells (PMCs). PMCs take up seawater through endocytosis1 into a complex network of vacuoles. Within the PMCs, calcium
ions are translocated from the seawater vacuoles to various organelles and
vesicles where they accumulate, and subsequently precipitate as an amorphous calcium carbonate (ACC). The amorphous precipitates are finally
translocated to the spicule, where they crystallize.
We address the question of the form in which calcium ions are stored in
different locations in the cell, whether dissolved or solid and in which
structural phase. In order to locate and characterize calcium content in individual vesicles we performed cryo-soft X-ray microscopy (cryo-SXM)
on dispersed PMCs. The presence of concentrated calcium ions was detected
by imaging the cells in the energy range before and after the calcium L-absorption edges. We characterized the chemical environment of the calcium
ions using X-ray absorption spectroscopy. We observe hundreds of particles
containing Ca in each PMC. The particles are composed of different forms of highly disordered phases of calcium salts, presumably carbonate. We also
developed methods for quantitative evaluation of calcium ion concentrations.
We observed concentrations diluted relative to ACC (19M), but concentrated relative to sea water (10mM). The spectroscopic and analytical data thus
together indicate a transition through a series of amorphous calcium
carbonate phases in the low molar concentration range. These data shed light on the intracellular transport and concentration pathways of calcium ions in
PMCs. This may well be relevant to other organisms, and thus lead to a
deeper understanding of biogenic mineral formation. 1. N. Vidavsky, S. Addadi, A. Schertel, D. Ben-Ezra, M. Shpigel, L. Addadi,
S. Weiner, Calcium transport into the cells of the sea urchin larva in relation
to spicule formation. Proc. Natl. Acad. Sci. U.S.A, 113(45), 12637-12642,
2016. 2. E. Beniash, J. Aizenberg, L. Addadi, S. Weiner, Amorphous
calcium carbonate transforms into calcite during sea urchin larval spicule
growth. P. Roy. Soc. B-Biol. Sci., 264 (1380), 461-465, (1997).
K 2
Biomineralization in echinoderms: developmental mechanisms
and evolution. Charles Ettensohn*1 1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh,
PA USA
All adult echinoderms have a calcite-based endoskeleton. Embryonic and
larval patterns of skeletogenesis, however, vary greatly across the phylum,
revealing a rich history of evolutionary modifications to the developmental programs that underlie biomineral formation in this group. The formation of
the skeleton has been particularly well studied in embryos of euechinoid sea
urchins, which have served as a major experimental model for developmental biologists for more than a century. The cellular behaviors that underlie
skeleton formation in sea urchin embryos have been described in detail and
many gene products that play essential roles in biomineral formation have
been identified. In addition, a complex transcriptional network that underlies
skeletogenesis has recently been elucidated. This gene regulatory network
links the early specification of embryonic skeletogenic cells (primary mesenchyme cells, or PMCs) to their cellular behaviors and biomineral-
forming properties. The PMC gene network is proving to be a powerful tool
for understanding the genetic and molecular control of skeletogenesis in echinoderms and the evolution of biomineralization.
K 3
Getting to the roots of apatite-based biomineralization of dental
hard tissues: from Conodonts and Cichlids to related
bioinspired materials
Elena V. Sturm*1 (née Rosseeva) 1Physical Chemistry, Zukunftskolleg, University of Konstanz, Konstanz,
Germany
Chordates and especially vertebrates represent the most highly advanced and
complex group of organisms. The formation of their hierarchical apatite-organic based hard tissues is evolutionary optimized and exhibits high
structural complexity on various length scales and amazing mechanical performance. The major objective of our research is to explore the
biomineralization processes involved in the formation of dental (and dental-
like) apatite-based hard tissues of earliest and modern vertebrates. Specifically, in my presentation I will focus on the detailed characterization
of morphology-structure-composition-property relationships of hard tissues
of feeding apparatus of Conodonts and Cichlid Fishes. We elaborate and combine exciting evolutionary model systems with cutting-edge
spectroscopy, microscopy and diffraction techniques to analyze the
structural, chemical and morphogenetic basis of the dental hard tissue biomineralization process. Our new understanding of dentical and tooth
structure in Conodont and Cichlids could also advance strategies for
synthesizing bioinspired and biomimetic materials and deepen our knowledge of their morphogenesis process.
K 4
Coral Biomineralization: linking pieces of the puzzle
Sylvie Tambutté*1 1Department of Marine Biology, Centre Scientifique de Monaco, Monaco
Coral biomineralization is the process that leads to the formation of a calcium carbonate exoskeleton. As for other biominerals, two essential questions are:
What do we know about the control of coral biomineralization? How can we
link biological control with physico-chemical processes? I will show how studies performed at the Centre Scientifique de Monaco since the 90’s have
provided insight into the coral biomineralization process from the whole
organism to the gene by combining molecular and physiological approaches. I will present how data obtained from experiments conducted in different
coral compartments: the tissues, the extracellular calcifying medium and the
skeleton can be linked together and can help in deciphering where and how biological control occurs. More specifically, I will present 1) the
measurements of pH, carbonate and calcium in the extracellular medium that
we have obtained by developing in vivo approaches, and 2) the mechanisms involved in transepithelial ion transport that we have characterized by
molecular and physiological approaches. Finally, I will show how changes
in environmental parameters such as seawater pH influence biological control and can provide information on coral biomineralization.
K 5
Towards bone-on-a-chip: cell differentiation and extracellular
matrix organization Sana Ansari1,2,3, Esther Cramer1,2, Johanna Melke2,3, Keita Ito2,3, Sandra
Hofmann2,3, Nico Somemrdijk1,3, Anat Akiva*1,3 1Laboratory of Materials and Interface Chemistry and Center for Multiscale Electron Microscopy, Department of Chemical Engineering and Chemistry.
Eindhoven University of Technology, Eindhoven, The Netherlands. 2Institute for Complex Molecular Systems. Eindhoven University of Technology, Eindhoven, The Netherlands. 3Department of Biomedical Engineering. Eindhoven University of
Technology, Eindhoven, The Netherlands.
Introduction
The formation of bone involves a cascade of biological and chemical events that control the process of collagen mineralization in bone. In this process,
bone cells named osteoblasts form a 3D organized collagen matrix and
secrete several non-collagenous proteins (NCPs) that control the mineralization process. However, the precise mechanism by which these
NCPs control the mineralization of collagen fibrils is not yet clear. In an
attempt to get a direct observation on the time and place of the expression of these proteins, and to study their role in collagen mineralization, we use an
advanced in vitro model system.
Materials and methods. Here, human mesenchymal stromal cells (hMSCs) are seeded on 3D silk
scaffold and are exposed to osteogenic medium and to continuous
mechanical stimulation. Depending on the composition of the osteogenic medium and the specific mechanical load, we can control the differentiation
of the hMSCs to osteoblasts and sequentially into osteocytes – the last step
in osteoblast differentiation.
Results
Using a unique bioreactor, we monitor the cellular and extracellular
development of the system. The stem cells transformed from long and
2
elongated cells to round shaped cells, which are typical to osteoblasts. In this stage, the cells also expressed specific osteoblastic proteins. These proteins,
such as osteocalcin, osteopontin, and osteonectin where located intra and
extracellular, embedded in the collagen matrix. In a later time point, when the cells are embedded in the collagen matrix, another morphological change
occured, the transformation into osteocytes as judged by the formation of
long processes. The osteocyte formation was further supported by the expression of unique osteocyte marker proteins such as DMP-1 and
sclerostin.
Conclusion
In this study, we show for the first time that in vitro differentiation of hMSCs
into osteoblast and osteocytes is achievable. The system shows the main
characteristic features of bone, resembling the process of bone formation. This system will allow to obtain a better understanding of the role of specific
proteins on collagen mineralization, in a system that is close as possible to in
vivo bone.
K 6
Bioinspired magnetite nanoparticles as smart drug
nanocarriers and hyperthermia agents C. Jimenez-Lopez*1, A. Peigneux1, Y. Jabalera1, F. Oltolina2, G. Iglesias3, M. Prat2 1Universidad de Granada (España), Department of Microbiology,
Granada, Spain 2Università del Piemonte Orientale A. Avogadro, Dipartimento di Scienze
della Salute, Novara, Italy 3Universidad de Granada (España), Department of Applied Physics, Granada, Spain
Among the biologically controlled biomineralization process, few has raised so much expectations as the magnetosome formation by magnetotactic
bacteria. Magnetosomes are composed of a magnetite (or greigite) crystal
enveloped by a lipidic bilayer and they are crucial for those bacteria survival. Therefore, magnetotactic bacteria control magnetosome formation at the
genetic level, so the magnetite crystals have the necessary characteristics to
maximize their response to the magnetic field. Magnetosome research has raised interest in several areas, from Astrobiology to Nanotechnology, since
magnetosomes are the ideal magnetic nanoparticle that could be used in
many applications, mainly clinics, both in diagnosis and in therapeutics. However, the problem for their use is the extremelly low yields in which
magnetosomes are produced. In particular, the need for a directed targeted chemotherapy has become a
matter of growing interest in order to increase efficiency, to reduce the cost
of systemic treatments and, even more important, to reduce the undesirable secondary effects related to the systemic distribution of the chemotherapeutic
molecule and thus, its interaction with healthy cells. This is not only true and
crucial in tumor treatment, either by means of chemotherapy or immunotherapy, but also for local targetable diseases like local infections. In
this context, novel MamC-mediated biomimetic (magnetosome-like)
magnetic nanoparticles (BMNPs) are proposed as valuable carriers for targeted chemotherapy because of the size (36 ± 12 nm) and of surface
properties conferred by MamC coating. They are superparamagnetic at room
and body temperatures, have a large magnetic moment per particle, mediate hyperthermia, are cytocompatible, and, having a negative surface charge at
physiological pH, can be efficiently coupled with a variety of molecules and
antibodies directed against receptors overexpressed in target cells displaying coupling stability, while releasing DOXO at acidic pH. This release can be
enhanced by hyperthermia. As an example, the nanoassembly DOXO-mAb-
BMNPs has been characterized and demonstrated that it selectively recognizes the Met receptor (overexpressed in many cancers), binds
efficiently to Met+ tumor cells, and discharges DOXO within their nuclei
more efficiently than DOXO-BMNPs, exerting cytotoxicity. These data represent proof of concept for in vivo experiments in which the controlled
dual targeting (mAb-mediated and magnetic) approach and combined
(chemotherapy and hyperthermia) therapy are been studied. Peigneux, A., Oltolina, F., Colangelo, D., Iglesias, G. R., Delgado, A. V.,
Prat, M., Jimenez-Lopez, C. (2019) Functionalized Biomimetic Magnetic
Nanoparticles as Effective Nanocarriers for Targeted Chemotherapy. Particle and Particle Systems Characterization. DOI:10.1002/ppsc.201900057.
Nudelman, H,, Valverde-Tercedor, C., Kolusheva, S., Widdrat, M.,
Grimberg, N., Levi, H., Nelkenbaum, O., Davidov, G., Faivre, D., Jimenez-Lopez, C., Zarivach, R. (2016) Structure function studies of the magnetite-
biomineralizing magnetosome-associated protein MamC. Journal of
Structural Biology 194, 244-252.
K 7
The unique molecular physiologies of biomineralizing
phytoplankton: Animal, vegetable and mineral C. Brownlee*1 1Marine Biological Association, UK University of Southampton, UK
The marine eukaryotic phytoplankton account for around one quarter of
global productivity, roughly equivalent to the terrestrial rainforests. They also underpin much of global biomineralization in the form of biogenic
silica (diatoms) and calcite (coccolithophores). Despite the major
importance of these groups in the Earth’s largest ecosystems, their physiologies remain relatively poorly studied. Combined functional
genomics and single cell biophysics approaches are beginning to reveal
surprising novel features, including ion channels not previously found in eukaryotes. Their roles in excitability, signalling and cellular homeostasis
will be discussed. The discovery of novel silicon transporters is also
shedding new light on mechanisms of coccolithophore biomineralization. Together, these studies are providing new insights into the evolution of
major classes of eukaryotic ion channels and membrane transporters. The
rationale and approaches for studying single cell physiology in situ in the oceans will also be discussed.
K 8
Patterns in the evolutionary acquisitions of mineralized
skeletons
S. Porter*1, J. Moore1, L. A. Riedman1, R. Wood1, R. Rickaby1 1University of California at Santa Barbara, Earth Science, Santa Barbara, United States
Mineralized skeletons evolved many times within the eukaryotes, including
dozens of times within the Metazoa. These numerous independent acquisitions provide an opportunity to test hypotheses about the origin and
evolution of skeletons, including the factors controlling the initial choice of
mineralogy and the reasons why mineralized skeletons evolved in the first place. Earlier work in this area focused on the evolution of carbonate
skeletons by metazoans, showing that acquisitions of aragonitic skeletons
were clustered during times of aragonite seas (when the Mg/Ca ratio of seawater favored aragonite precipitation) and those of calcitic skeletons
during calcite seas. We have since expanded this dataset to include other
skeletal mineralogies and other eukaryotic clades, including both extinct and extant taxa, and have identified more than 80 independent acquisitions of
mineralized skeletons in eukaryotes, along with their time of first appearance,
their mineralogy, and, for the earliest animals, their microstructures and habitats at the time they first evolved.
Several interesting patterns have emerged from this preliminary dataset.
First, skeletal acquisitions among animals are clustered in time, with half appearing in the early Cambrian, and another 25% in the Ordovician through
Devonian periods. No acquisitions are recorded for the later Paleozoic. A
smaller cluster of acquisitions occursed in the mid-Triassic to Jurassic; nearly all of these later skeletons evolved within the cnidarians and annelids. In
contrast, skeletal acquisitions in non-metazoan eukaryotes are distributed throughout the Phanerozoic, with no obvious clustering in time. This
supports the view that ecological factors affecting only animals (e.g., the
appearance of carnivores), rather than physical factors affecting all marine organisms (e.g., increased Ca2+), were a primary driver of skeletal
biomineralization in animals. Second, acquisitions of phosphatic skeletons
are concentrated in the Neoproterozoic and Cambrian, perhaps reflecting higher levels of phosphate in the oceans at this time, though our count of
independent acquisitions may be inflated by post-mortem phosphatization of
organic skeletons., In contrast with calcareous and siliceous skeletons, which are distributed about equally among autotrophs and heterotrophs, phosphatic
skeletons are only known to occur in heterotrophs, perhaps reflecting the
higher cost for autotrophs to use a limiting nutrient to form a skeleton. Finally, the skeletal microstructures and habitats of the earliest carbonate
biomineralizers suggest phases of increasing biological control over
biomineralization, with the earliest carbonate skeletons forming disorganized microstructures and inhabiting only carbonate environments. These were
succeeded in the latest Ediacaran and early Cambrian by calcifying animals
that lived in both carbonate and siliciclastic environments and formed more organized, though still relatively simple, fibrous microstructures.
3
T 1 Nanostructure and growth of calcite crystals of the shell plates of the
barnacle Austromegabalanus psittacus (Crustacea, Cirripedia,
Balanidae)
A. Checa*1,2, E. Macías-Sánchez3, A. Sánchez-Navas4, A. Rodríguez-
Navarro4, N. A. Lagos5 1Universidad de Granada, Estratigrafía y Paleontología, Granada, Spain 2CSIC-Universidad de Granada, Instituto Andaluz de Ciencias de la Tierra,
Armilla, Spain 3Max Planck Institute of Colloids and Interfaces, Potsdam, Germany 4Universidad de Granada, Mineralogy and Petrology, Granada, Spain 5Universidad Santo Tomás, Centro de Investigación e Innovación para el Cambio Climático, Santiago, Chile
The calcareous plates enclosing the body of the barnacle Austromegabalanus
psittacus are mainly made by calcite crystals. The crystals are arranged into
bundles, within which all crystals are strictly co-oriented. The c-axes of crystals of different bundles are roughly perpendicular to the growth
surfaces. Observations of crystals appearing on the free growth surfaces
reveal changes in size (from 1 to 5 µm) and morphology (from irregular to euhedral rhombohedral). A remarkable feature of these crystals is their
coarse nanogranular ultrastructure. The {104} rhombohedral faces are
distinctly much smoother than other types of surfaces. In turn, the nanogranules making up the crystals are arranged into rod-like structures
(which we call lineations), which diverge at large angles from neat lines.
Examination of incomplete rhombohedra indicate that the lines from which the lineations diverge are at large angles to the {104} faces and clearly in
continuity with the rhombohedron edges. That is, they align in parallel to ˂-
441> directions of calcite. The complete sequence of growth from irregular granules to {104} rhombohedra can be reconstructed. The {104} faces begin
to appear at or close to their centers and spread towards the edges by the
progressive lateral addition of nanogranule lineations. The last rhombohedral elements completed are the edges. Rhombohedra are usually incomplete,
because the grains are quickly overgrown by other neighbors during plate
growth. TEM observations reveal that the nanostructure consists of crystalline domains, which are surrounded by amorphous areas. This is in
agreement with AFM observations, which show the existence of two phases
with different contrast. All the above observations give important insight into the growth mechanisms of these calcite biocrystals. We propose that during
the transformation of ACC into calcite, due to the force of crystallization,
organic molecules are expelled from the crystal lattice. Growth of the crystalline domains takes place preferentially along the directions, which are
the strongest periodic bond chains (PBCs) in calcite. Accordingly, organic
molecules are barely incorporated along these directions and become expelled sidewards. This implies that the formation of nanogranules and their
arrangement into lineations in barnacle calcite crystals is a
crystallographically-controlled phenomenon and cannot be explained by a particle-aggregation process, as previously shown for molluscan biogenic
aragonite.
T 2
Towards understanding larval bivalve calcification
mechanisms in a changing ocean F. Melzner*1, K. Ramesh2, M. Clark3, G. Nehrke4 1GEOMAR, Marine Ecology, Kiel, Germany 2University of Gothenburg, Gothenburg, Sweden 3British Antarctic Survey, Cambridge, United Kingdom 4AWI Bremerhaven, Bremerhaven, Germany
Marine larval bivalves (mussels, oysters) rapidly secrete first shells within
less than one day, thereby precipitating a calcium carbonate mass that corresponds to their own body mass. This remarkable synthesis effort
requires massive transfer of inorganic carbon and calcium, as well as proton
removal from the site of calcification. These transport mechanisms are unknown so far. Similarly, it is not clear, whether amorphous precursor
phases are formed intracellularly, or whether carbonate formation is located
in dedicated extracellular spaces at the organism-mineral interface. A better mechanistic characterization of shell formation processes is important, as it
allows us to understand the high vulnerability of larvae to ongoing ocean
acidification. We use mussel larvae (Mytilus) as model species and utilize a variety of
approaches, ranging from in vivo confocal microscopy, in vivo
confocal Raman microscopy, microelectrode recordings to transcriptomic approaches to learn more about early shell mineralization and how it is
impacted by ocean acidification.
Using in vivo calcein pulse-chase and confocal microscopy, we can show that (1) it is unlikely that biomineral precursors are formed in large vesicles in
cells. This finding is supported by in vivo confocal Raman microscopical analysis of first secreted shell components at the trochophore stage, which
(2) exclusively indicates presence of aragonite in the shell. While it is
possible that very small intracellular carbonate particles are produced intracellularly, it is equally possible that mineral formation is an extracellular
process. We therefore characterized the carbonate chemistry environment at the shell-tissue interface using minute microsensors for pH, calcium and
carbonate and find (3) that veliger larvae can elevate calcium carbonate
saturation state of the calcification space, which could be instrumental in increasing rates of carbonate precipitation. However, larvae (4) are not able
to maintain this regulatory effort when seawater pH decreases. This finding
could explain reduced calcification and dissolution rates when larvae are exposed to simulated ocean acidification. In order to identify ion transport
proteins that support calcification substrate transport and pH regulation, we
use substrate limitation assays in combination with transcriptomics and can (5) show that SLC4 and 26 family member candidate genes are probably vital
for bicarbonate transport and pH regulation.
References: Ramesh et al. 2017 Nature Communications 8:1709
Ramesh et al. 2018 Journal of the Royal Society Interface 15: 20170723
Ramesh et al. 2019 Ecology & Evolution (in press)
T 3
Terebratulide brachiopod shell biomineralization by mantle
epithelial cells M. Simonet Roda*1, A. Ziegler2, E. Griesshaber1, X. Yin1, U. Rupp2, D. Henkel3, V. Häussermann4,5, J. Laudien6, U. Brand7, A. Einsenhauer3, W.
W. Schmahl1 1Ludwig Maximilians Universität München, Department of Earth and Environmental Sciences, Munich, Germany 2University of Ulm, Central Facility for Electron Microscopy, Ulm,
Germany 3GEOMAR Helmhöoltz Centre for Ocean Research, Marine
Biogeochemistry/Marine Systems, Kiel, Germany 4Pontificia Universidad Católica de Valparaíso, Facultad de Recursos Naturales, Escuela de Ciencias del Mar, Valparaíso, Chile 5Huinay Scientific Field Station, Puerto Montt, Chile 6Alfred-Wegener-Institut Helmholtz-Zentrum fur Polar- und Meeresforschung, Bremerhaven, Germany 7Brock University, Department of Earth Sciences, Ontario, Canada
The shell of modern rhynchonellide and terebratulide brachiopods is a hybrid
composite where an extracellular biopolymer matrix is reinforced by calcite.
Both material components are secreted by outer mantle epithelium (OME) cells.
In order to understand mineral transport pathways for shell secretion and to assess differences in cellular activity during mineralization, we imaged with
TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium
(OME) cells of juvenile and live Magellania venosa shells. Imaging was carried out on embedded/etched, chemically fixed/decalcified and high-
pressure frozen/freeze-substituted shell pieces taken from the commissure,
central shell portions and from puncta. Imaging results are complemented with morphometric evaluations of volume fractions of membrane-bound
organelles (Simonet Roda et al. 2019a, Simonet Roda et al. 2019b).
At the commissure, the OME is multi-cell layered, while in central shell regions it is single-cell layered. OME cells form at the commissure oblique
extensions that, in cross-section, are round below the primary layer and flat
underneath the fibres. At central shell regions, OME cells are considerably thinner in comparison to cells at the commissure.
When actively secreting shell carbonate extrapallial space is lacking as OME
cells are in direct contact with the calcite of the forming fibres. Upon termination of secretion, OME cells attach via apical hemidesmosomes to
extracellular matrix membranes that line the proximal surface of fibres and
tonofilaments connect apical to basal hemidesmosomes. This stabilizes the contact of epithelium and the fibres and keeps the mantle in place. Individual
fibres are secreted by several cells at the same time. This requires
communication and tight cooperation of neighbouring OME cells for the coordinated secretion of organic membrane and mineral, explaining the high
abundance of gap junctions between cells.
There is not any observation in the cell ultrastructure in our study that can be taken as evidence for a vesicular transport based mineralization process. On
the contrary, the absent or very narrow (in the range of nanometers) space
between the epithelium and the growing fibres, together with the absence of significant differences in the volume fraction of vesicles between secreting
and non-secreting regions of the OME, as well as the extreme reduction in
cell thickness at sites of mineral secretion suggests, that in modern Magellania venosa (and likely in all Rhynchonellida and Terebratulida
forming the fibrous microstructure) mineral transport to the sites of
mineralization occurs via ion transport mechanisms through the cell membrane and not by transport of mineral by organelles such as vesicles.
Simonet Roda, M., Ziegler, A., Griesshaber, E., Yin, X., Rupp, U., Greiner,
M., Henkel, D., Häussermann, V., Eisenhauer, A., Laudien, J., Schmahl, W.W., Terebratulide brachiopod shell biomineralization by mantle epithelial
cells, Journal of Structural Biology,
https://doi.org/10.1016/j.jsb.2019.05.002 Simonet Roda, M., Griesshaber, E., Ziegler, A., Rupp, U., Yin, X., Henkel,
D., Häussermann, V., Laudien, J., Brand, U., Eisenhauer, A., Checa, A.G.,
4
Schmahl, W.W., 2019b. Calcite fibre formation in modern brachiopod shells. Sci. Rep. 9, 598.
T 4
How hepatopancreas cells of a terrestrial crustacean take up
and release calcium after ingestion of mineral from the old
shed cuticle U. Rupp*1, A. Ziegler1 1Ulm University, Central Facility For Electron Microscopy, Ulm, Germany
The hepatopancreas of isopods serves for food digestion, storage of lipids and carbohydrates, storage of essential metals as well as accumulation and
detoxification of xenobiotic metals. Metal is accumulated in lysosomal metal
containing granules in the two major cell types (S and B) of the hepatopancreas (Dallinger and Prosi, 1989). A recent µCT study on mineral
shifts in moulting Porcellio scaber has shown that mineral is present within
the hepatopancreas lumen, however, only when the animal has ingested the old shed cuticle (exuviae) after moult (Ziegler et al., 2017). This suggests
uptake of mineral from the ingested exuviae by the hepatopancreas and
recycling for mineralisation of the new cuticle. The aim of this study was i) to reveal if the cells of the hepatopancreas
contain mineral from the exuviae, ii) if the metal-containing granules
contribute to the uptake of calcium and its release for mineral recycling, and iii) to find pathways for mineral/calcium transport from the exuviae to the
hemolymph. We have therefore investigated the mineral distributions within
the cytosol and organelles of hepatopancreas S- and B-cells. We used P. scaber at the postmoult stage that have ingested their exuviae after
the moult, those that have not ingested their exuviae as a negative control, and animals at the intermoult stage, 9-10 days after exuviae ingestion that
have a fully mineralized cuticle.
We used STEM, TEM multi-image acquisition, EDX and EFTEM for the analysis of the ultrastructure and mineral distribution in high pressure frozen
and freeze substituted hepatopancreas tissue. Ultramicrotomy of resin
embedded samples was performed using a piezo-driven oscillating diamond knife and propane-1,3-diol as floatation medium for the sections to minimize
loss of mineral.
The cryofixed samples reveal numerous extracellular vesicles (exosomes) and many multivesicular bodies containing proexosomes in both cell
types. After high-pressure freezing and freeze-substitution, granules in
sections of S as well as B-cells contain well-preserved mineral deposits. In animals that have ingested the exuviae, we found a significantly higher
calcium concentration in the metal granules of S cells in comparison to
control animals. In B cells the calcium concentration in lysosomal granules was much lower and independent of moulting stage or exuviae ingestion. We
observe intracellular seams of mineral along the microvilli and the lateral
plasma membranes, consisting of phosphorus co-localizing with calcium and occasionally with calcium and zinc in S-cells and with iron or calcium in B-
cells. Small granules composed of calcium and phosphorous occur also
between cells and within the basal lamina. In intermoult animals, such mineral seams and granules are less abundant and absent in
control postmoult animals that have not ingested their cuticle.
The results indicate that calcium uptake from the ingested exuviae takes place across the apical plasma membrane of the cells and that
lysosomal metal granules in S-cells accumulate calcium for mineralization
of the new cuticle. Transport into the haemolymph occurs in the form of calcium phosphate via the basolateral membrane and the basal lamina. Co-
localization of zinc with calcium suggests similar pathways for the two
elements. Prosi F. and Dallinger R. 1988. Cell Biol. and Toxicol. 4: 81–96.
Ziegler A. Neues F. Janáček J. Beckmann F. Epple. M. 2017. Arthropod
Struct. Dev. 46: 63–76. Supported by the DFG ZI 368/11-1.
T 5
Identification of extracellular vesicles involved in the
biomineralization of the hen eggshell L. Stapane1, N. Le Roy1, J. Gautron*1 1French National Institute of Agricultural Research (INRA), Bird Biology and Poultry, Nouzilly, France
Question The eggshell is a critical barrier against mechanical stresses and microbial penetration. Its integrity is essential to maintain the hygienic quality of this
basic human food and to limit the number of downgraded eggs. In such a
context, we are looking for eggshell strength specific markers in order to optimize egg quality.
The eggshell is made of 95% mineral phase (calcium carbonate on calcite
form) and an organic matrix (3.5%) mostly containing proteins. Eggshell formation arises from an extra-cellular biomineralization process, which
takes place in a fluid that contains eggshell precursors and involves a
transient phase of amorphous calcium carbonate (ACC). This work aims at
exploring the presence and the role of extracellular vesicles to stabilize ACC and to address it to the mineralization site.
Results In a first approach, we used real time qRT-PCR to assess the expression of
vesicular target genes in several tissues. The results confirmed a high
expression of vesicular target genes (edil3, anxa1, anxa2, pdcd6ip) in oviduct portions where mineralization takes place. In this study, we have also
explored the role of EDIL3 and MFGE8 proteins in chicken shell at key
stages of shell mineralization, and confirmed they could bind Ca2+ and vesicles, thanks to an EGF-like calcium-binding domain and a F5/8C
phospholipid-binding domain. It was therefore suggested that both proteins
could be involved in the vesicular transport of calcium. In a second approach, electronic microscopy coupled with elementary analysis was used to observe
the uterine fluid collected during eggshell biomineralization. Data obtained
highlighted the presence of extracellular vesicles (~ 300 nm) containing calcium carbonate. Finally, Western Blot analysis confirmed the presence of
EDIL3, a key vesicular protein in the purified vesicle fraction.
Conclusions The results of this study showed for the first time the involvement of
extracellular vesicles in the transport of calcium carbonate for the
biomineralization of hen"s eggshell. We proposed a model of calcification using vesicles to stabilize ACC and explaining the fast deposition of the
crystalline calcite oriented layer in the shell. The proteins described in this
study will have to be explored as biological markers for a selection of chicken layers with improved mechanical properties.
T 6
STIM1 a calcium sensor promotes the assembly of an ECM
that contains extracellular vesicles and factors that modulate
mineralization A. George*1, Y. Chen1 1University of Illinois at Chicago, Oral Biology, Chicago, United States
Introduction
Biomineralization is a dynamic process in which living organisms deposit
mineral in the extracellular matrix. Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. Several
proteins work in coordination and contribute to this tightly regulated process.
STIM1 (Stromal interaction molecule 1) is one such protein that has been recently identified to function in bone and enamel mineralization. The
STIM1 protein is a calcium sensor localized to the membrane of the
endoplasmic cells and is well recognized for its physiological role in the endoplasmic reticulum. We have demonstrated earlier that DMP1
stimulation of preosteoblasts lead to calcium release from internal Ca2+
stores and this store depletion is sensed by the ER Ca 2+ sensor STIM1. Store-operated calcium entry is one of the major Ca 2+ influx mechanisms
following store depletion in the ER.
Objectives
To demonstrate a role for STIM1 in dentin matrix mineralization.
Materials and Methods
In order to understand the function of STIM1 during dentin mineralization, we overexpressed STIM1 in dental pulp stem cells (DPSCs) to generate p-
EF1α-STIM1 in which EF1a promoter drives the expression of STIM1. Cell
lines were characterized for the overexpression and knock down of STIM1. The transgenic cells along with the control were cultured for 7, 14 and 21
days in growth and differentiation media and the morphology of the cells and
topology of the matrix were examined by Field Emission Scanning Electron Microscopy. Further, we examined matrix mineralization by Alizarin red and
von Kossa staining. Gene expression analysis by RT-PCR was performed to
identify cell differentiation in the presence and absence of STIM1. Immunofluoresence was performed to confirm the localization of osteogenic
markers in the presence and absence of STIM1. Results: Transgenic cell
lines overexpressing and knockdown of STIM1 was successfully generated as assessed by fold changes in STIM1 mRNA expression. FESEM results
demonstrate that STIM1 overexpressing cells released large amount of
extracellular microvesicles and matrix mineralization. Interestingly, knockdown of STIM1 resulted in fewer microvesicles and less mineralized
matrix. Alizarin and von Kossa staining demonstrate the differentiation of
stem cells into odontogenic phenotype with STIM1 overexpression.
Conclusion
STIM1 is a crucial molecule in biomineralization as it influences release of
large amounts of extracellular vesicles and formation of mineralized matrix. This was impaired in the absence of STIM1. Downregulation of
differentiation markers suggest that STIM1 promotes differentiation
probably by mobilizing intracellular calcium ions. As Ca2+ functions as second messengers, therefore their role in cellular differentiation suggest that
STIM1 can promote intracellular Ca 2+ oscillations and thus provide a signal for activation of downstream and upstream effectors that promote
differentiation and matrix mineralization.
5
Acknowledgements
NIH-NIDCR DE 11657 and the Brodie Endowment Fund.
T 7
Intraperiostracal calcification in the Bivalvia- tuning in to the
aerials of Catillopecten E. Harper*1, G. Kamenev2, F. Varela- Feria3, J. Taylor4, E. Glover4, A.
Checa5
1University of Cambridge, Earth Sciences, Cambridge, United Kingdom 2Russian Academy of Science, 2. National Scientific Center of Marine
Biology, Vladivostok, Russian Federation 3University of Seville, Seville, Spain 4The Natural History Museum, Life Sciences, London, United Kingdom 5Uniiversity of Granada, 5. Department of Stratigraphy and Paleontology,
Granada, Spain
Most studies of molluscan biomineralization concentrate on deposition of
shell layers onto and below the periostracum. However, in the last decade there has been an increased appreciation of intraperiostracal calcification –
where biominerals are secreted within the periostracum - producing often
intricate and elaborate structures. Thus far most of this intraperiostracal calcification has been reported in the Bivalvia, where it is widespread in the
Anomalodesmata, Palaeoheterodonta, Pteriomorphia and Imparidentia. This
distribution hints that this is either a plesiomorphic character or that there is such a facility within the Bivalvia for such intraperiostracal calcification that
it has evolved polyphyletically in different situations.
In this study we present a detailed study of recently discovered intraperiostracal calcification in a clade in which it has not be observed
before. Members of the Propeamussiidae, the "glass scallops", are a denizens
of the deeper part of the oceans, where they produce fragile thin shells and pursue an actively carnivorous lifestyle. Amongst them, Catillopecten
natalyae from the abyssal plain (4,860-5,680 m depths) adjacent to the Kuril-
Kamchatka and Japan trenches (Pacific Ocean) produce an extraordinary ornament of intricate processes with multiple projections, looking like tiny
aerials and antennae. They are interesting because of their intricacy and the
fact that they are calcitic rather than the more typical aragonitic nature of intraperiostracal calcifications. Using a combination of SEM and EBSD we
show that that these intricate structures are produced entirely within the periostracum and must form in a way that allows growth in a progressively
outwards direction (i.e. entirely different from normal secretion whereby
growth occurs inwards from the surface of the shell). Each aerial is crystallographically continuous with the underlying prism. The stem of each
aerial is shown to be parallel to the c-axis of calcite and the typical three-fold
bifurcations of the antennae correspond with the three short diagonals of the calcite rhombohedra. There may be multiple aerials from a single prism in
the outer layer of the shell, and those of a single prisms show a remarkable
co-orientation, whilst those on adjacent prisms show a slightly different orientation. We will propose a model for the formation of these intricate
aerials and also to compare these with other forms of intraperiostracal
calcification in the Bivalvia. The function of these fascinating structures remains unknown!
We acknowledge Russian Foundation for Basic Research (grant no. 19-04-
00281-a) and CGL2017-85118-P (Spanish Ministry of Economy, Industry and Competitivity)
T 8
The dynamics of structural mesoscale dislocations in nacre M. Beliaev*1, I. Zlotnikov1 1Technische Universitaet Dresden, B CUBE - Center for Molecular Bioengineering, Dresden, Germany
Molluscan shells ultrastructures are a paradigm of complex hierarchical
biocomposite structures formed in the course of extracellular biomineralization. Morphogenesis of these ultrastructures is known to follow
thermodynamically driven self-assembly processes in accordance with the principles of classical materials science. Sheet nacre ultrastructure is a highly
regular and periodic architecture composed of thin organic membranes and
aragonitic layers, which are made of flat mesocrystalline platelets. Similar to classical crystal growth, nacre deposition is accompanied by incorporation of
two-dimensional defects, such as dislocations and twinning, that are integral
to its formation process. However, in contrast to a generic atomic-scale lattice, in sheet nacre, these defects occur on the mesoscale level.
Specifically, twinning in aragonite is known to be responsible for the shape
of nacre platelets and structural dislocations in the layered assembly are key to its morphogenesis. In the present work, we employ synchrotron-based
nanotomographic imaging combined with machine learning post-processing
techniques to visualize and understand the nature and the dynamics of mesoscale dislocations in the nacre of the bivalve Unio Pictorum in 3D. By
drawing an analogy to processes in classical materials science, we shed light
on the role of structural dislocations and their interaction in sheet nacre formation. This work is a step towards a deeper understanding of
fundamental thermodynamic and kinetic principles that drive the self-assembly of nacre.
T 9
Morphogenesis in the mollusc Atrina vexillum - an epitome for
ideal coarsening D. Zöllner*1, I. Zlotnikov1 1B CUBE - Center for Molecular Bioengineering, TU Dresden, Dresden, Germany
The microstructure of polycrystalline materials is closely linked to many
materials properties. This fact makes the phenomenon of the migration of individual grain boundaries between neighboring crystals and therewith the
coarsening of the whole grain structure a major focal point in classical
materials science. Out of the different growth modes observed in natural and synthetic materials—such as rocks, metals and alloys—the case of ideal
coarsening resp. grain growth is given special attention. Even though the
basic assumption for ideal coarsening—which is homogeneity of the physical properties of the boundaries—seems highly unrealistic, many analytical as
well as numerical investigations focus on this case due to the reduction in
problem complexity. As a result, the large variety of analytical models were rarely found to describe the microstructural evolution of experimentally
measured polycrystalline grain networks. In the present work, it is shown that
biomineralization of the prismatic architecture in the shell of the mollusc Atrina vexillum can be described qualitatively and quantitatively by
conventional thermodynamic, kinetic and topological considerations from
classical materials science. Hence, a biogenic polycrystalline material is presented as an epitome for ideal coarsening.
T 10
The role of residual stresses in biomineral morphogenesis
revealed by 3D dark-field x-ray microscopy V. Schoeppler*1, P. Cook2, I. Zlotnikov1 1TU Dresden, B CUBE - Center for Molecular Bioengineering, Dresden,
Germany 2University of Natural Resources and Life Sciences, Institute of Physics and Materials Science, Vienna, Austria
Residual internal stresses occur in numerous synthetic, geological and
biogenic crystals having desirable or undesirable effects on materials
performance. Specifically, a number of recent studies have demonstrated the significance of residual stresses in the mechanical functionality of a number
of biomineralized tissues. However, the role of these forces in biomineral
morphogenesis was never previously examined, mainly due to the lack of an appropriate characterization approach, which requires spatial and
crystallographic characterization of the mineral on several length scales.
Most of the state-of-the-art methods are either surface techniques yielding 2D information or allow only limited 3D analysis of very small sample
volumes.
In this work, we employed the recently developed technique—dark-field x-ray microscopy—to study the relationship between residual stresses and
crystallographic properties of biogenic calcite in the prismatic ultrastructure in the bivalves Pinna nobilis and Pinctada nigra in 3D. This method,
developed at the European Synchrotron Radiation Facility (ESRF), utilizes
magnifying refractive lenses to map the intensity profile of hard x-rays diffracted from crystalline materials with an angular resolution of 0.01° and
allows to analyze millimeter sized samples with a spatial resolution of 50 nm.
Whereas the prisms in P. nobilis have an almost perfect single crystalline character, the growing prisms in P. nigra gradually change their
crystallographic orientation and split into sub-prismatic domains. Due to the
high angular resolution of the method, we were able to obtain unprecedented detail on the mosaicity of prisms in the two organisms and to demonstrate a
correlation between internal lattice strains and local crystallographic
properties of biogenic calcite in 3D. By comparing the experimental data from the two species, we not only shed a new light on the relationship
between structure and texture during the formation of biomineralized tissues,
but also demonstrate the role of internal stresses in biomineral morphogenesis.
T 11
Stabilizing the shell as an inducible defense - morphological,
microstructural and chemical shell-modifications in the
freshwater snail Physella acuta in response to different crayfish
species H. Eck*1, A. Schenk1, V. Grün1, C. Laforsch1 1University of Bayreuth, Animal Ecology I, Bayreuth, Germany
Predator induced morphological defenses are a widespread phenomenon in
the phylum of mollusks. It has been previously demonstrated that mollusks respond to shell-crushing crustacean predators with changes in their shell,
6
including shell size, weight and thickness. These changes in shell morphology are often associated with an increased shell stability, i.e. shell
crush resistance. However, there is still very limited understanding of the
mechanism, by which such an adaptive increase in mechanical stability is achieved, e.g. microstructural or chemical modifications of the shells.
Moreover, it remains elusive whether there is a specificity of predator
kairomones (semiochemicals released by the predator that allows prey organisms to detect the predator), i.e. if mollusks can differentiate between
closely related crustacean predators and adjust the expression of inducible
morphological defenses accordingly. Therefore, we studied if freshwater snails express different inducible morphological defenses when exposed to
different crayfish species. Furthermore, the chemical and microstructural
modifications, leading to an increasing crush resistance in shells of predator-exposed snails were analyzed. In this study, we particularly focus on the
inducible morphological defenses in the acute bladder snail Physella acuta
in response to the marbled crayfish Procambarus virginalis and the signal crayfish Pacifastacus leniusculus. The snails were exposed to kairomones
from the two crayfish species until they reached maturity. Subsequently, the
morphology, weight and stability was compared across treatments. The shells were then analyzed for predator induced changes in microstructure and
chemical composition using a wide range of analytical methods including
scanning electron microscopy (SEM), Raman- and Fourier-transform infrared (FTIR) spectroscopy, powder x-ray diffraction (PXRD), small-angle
x-ray scattering (SAXS) and thermogravimetric analysis (TGA). Our results
demonstrate that P. acuta responds to both predators with the expression of inducible defenses. Compared to non-exposed individuals, crayfish exposed
snails characteristically show an increased shell size and weight, a narrower aperture of the shell and an increased crush resistance of the shell.
Interestingly, individuals from both predator treatments do not differ
significantly from each other. Regarding chemical modifications, shells of crayfish exposed snails exhibit higher volumes of carotenoids, while there
are no quantitative differences in the other components of the organic or
inorganic fraction. The microstructure of the shell did not show pronounced differences between non-induced and crayfish induced snails. Our study
demonstrates that P. acuta expresses inducible morphological defenses in
response to two different crayfish species. The observation that the expression of the defensive traits did not differ between the two crayfish
species provides first indications that there may exist a universal crayfish cue.
T 12
Mural mechanics and morphology of cephalopod shells -
adaptation or artifact? R. Lemanis*1, D. Stier1, I. Zlotnikov1 1Technische Universität Dresden, B CUBE – Center for Molecular Bioengineering, Dresden, Germany
Cephalopods have exploited their biomineralized skeleton over the course of hundreds of millions of years for the purposes of buoyancy, hydrodynamics,
and protection against predation. Over this time, they have explored a huge
disparity of ultrastructures and shell geometries that reflect constructional and ecological boundaries. Testing the biomechanics of these structures
allows us to begin to untangle the evolutionary history and ecology of these
animals. Here, we aim to test the potential function of mural modifications in coleoid cephalopods using comparative computational mechanics. The
spiral cephalopod phragmocone is a hollow tube divided into chambers by
mineralized walls; the attachment zone between these walls and the shell is the mural zone. The only extant coleoid with a spiral phragmocone, Spirula
spirula, is imaged using high resolution computed tomography and used as a
basis to construct a representative coleoid shell model. Mechanical data from nanoindentation from the Spirula shell are used to inform finite element
models to test how the presence of mural modifications affect stress and
strain distributions due to hydrostatic pressure. Indentation data reveals a significant difference between the moduli of the shell and septa, however not
one large enough to arrest cracks through the region. Furthermore, the septa
show a stiffness gradient whose origins are not currently understood. Shells with and without the mural flap show no differences in their response to the
applied pressure, while the rear, adapical flaps seem to redistribute peak
stress values away from the attachment site and onto the stiffer shell wall. We are able to show that mural modifications have only a minor effect on the
mechanics of the shell. The mural flap, a defining character of Decabrachia,
seems to have no apparent mechanical function. This result challenges prior hypotheses that proposed the mural flap as an adaptation to deeper water
depths that facilitated the ecological separation between Decabrachia and
belemnites.
T 13
In-Situ TEM of calcium carbonate mineralization in the presence
of L-aspartic acid M. Longuinho1, N. Peña2, D. Ihiawakrim2, M. Farina*3, A. Rossi4, O.
Ersen2 1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 2Institut de Physique et Chimie des Matériaux de Strasbourg, Strasbourg,
France 3Federal University of Rio de Janeiro, Rio de Janeiro, Brazil 4Brazilian Center for Physics Research, Rio de Janeiro, Brazil., Rio de Janeiro, Brazil
Introduction
Biomimetic syntheses are widely performed in the development of new
materials for pharmaceutical purposes and tissue regeneration, besides seeking to reproduce in vitro biomineralization processes. A better
understanding on how mineralization occurs in biological environment
allows the synthesis of materials with special properties, which ensure the reliability of the production. Among the biominerals, calcium carbonates
(CaCO3) are the most abundant, being present in primitive to complex
organism like the spicules in calcareos sponges and otoliths in the inner ear of vertebrates. Therefore, CaCO3 minerals are of great interest for
fundamental studies as a model for nucleation, growth and crystallization of
biogenic minerals. A great challenge in the biomineralization field is the understanding of the initial stages of the process, since the formation of a
critical nucleus, what occurs in a short period of time and at the molecular
level. This makes difficult the direct observation of this phenomenon with routine analytical imaging techniques. To overcome this restriction, we used
in situ techniques that allowed the observation of crystallization in the
aqueous medium in real time, like morphological changes, crystallinity and structural dynamic. Specifically, in situ Transmission Electron Microscopy
(in situ-TEM) enables acquisition of images and videos at Angstrom
resolution, as chemical and structural information during the development of mineral phases.
Objectives
The present work aims to understand how organic molecules, specifically aminoacids, influence CaCO3 biomineralization. As described in the
literature, there are specific proteins directly related to the biomineralization
which are capable of influencing the structure and kinetic of CaCO3 crystallization due to polarity, electrophilicity and presence of sulfate or
phosphate groups.
Material & methods
The experiment was carried out by mixing CaCl2 and Na2CO3 solutions,
with and without L-aspartic acid, and then the mixture was dropped in a chip of the in situ sample holder (Protochips) and analyzed in a JEOL-2100F
TEM.
Results & conclusion
Preliminary results show a region more electron dense, probably with
accumulation of ions where crystalline nuclei are formed. However, most of
them undergoes dissolution, before reaching a critical size (70 to 120 nm) in the samples without addition of the amino acid. The dissolution may have
happened because of the higher energy of the system leading to instability or
because of the energy of the electron beam during the analysis. In the experiment containing L-aspartic acid, it was also possible to observe
crystals with calcite-like morphology and organic vesicular-like structures
containing crystalline material. The existence of nanocrystals inside vesicular-like structures suggests that the amino acid can accumulate ions
inside these structures, inducing the precipitation of crystals. Such
phenomena are described in micron and millimeter scale using polymers and polypeptides as polymer induced liquid phase (PILP), however, never
described for small molecules like aminoacids forming nanoscale crystals.
Further studies will be carried out to better understand of this mechanism.
T 14
Affimer-directed control over calcium carbonate polymorphs I. Sandei*1, T. Gaule 2, F. Meldrum 1 1University of Leeds , Chemistry , Leeds, United Kingdom 2University of Leeds, Biological Sciences , Leeds, United Kingdom
A key factor in biomineralization is the use of organic molecules to direct the
formation of inorganic materials including silicates and magnetite. Being able to emulate this strategy synthetically and use organic molecules to
control the formation of inorganic materials is therefore a long-standing goal
in materials synthesis and crystallisation studies. Significant efforts have therefore been made to identify molecules that can produce inorganic crystals
with well-defined size, structure and morphology. One approach that has
received particular attention is the screening of libraries of polypeptides to identify individual molecules that are active in directing mineralisation.
There, polypeptides are typically displayed on phage, and active individuals
are selected based on multiple bio-panning rounds to identify strongly-binding individuals. These are anticipated to have a strong effect on
crystallisation.
7
Here, we use a phage-display approach to identify proteins that can direct calcium carbonate formation. In contrast to short polypeptides, these have
well-defined conformations that can be expected to be important in
controlling crystallisation. A 1.3∙1010 library of Affimers was employed, where these comprise a small protein scaffold of 81 amino acids. They are
extremely stable and displays significant beta sheet structure together with
one or two nine amino acid variable regions that display two variable loops. These were displayed on a modified form of the M13 phage major coat
protein (pVIII), and multiple bio-panning rounds were conducted to identify
individuals that bound strongly to calcite and aragonite at different pH conditions.
From the initial phage library, 14 different proteins were selected and
purified. The ones that strongly bound to calcite are particularly rich in basic and nonpolar amino acids in both loops. At pH 8.5 there is a prevalence of
negative charges (as the alpha carboxylic groups are mainly deprotonated)
with a great presence of histidine (H) and arginine (R) residues in Loop 1. For aragonite, we found a significant number of nonpolar amino acids, with
fewer basic residues than calcite and the appearance of aspartate (D) and
glutamate (E) residues in one of the two loops. Moreover, tryptophan (W) is one of the main residues present, together with polar amino acids carrying -
OH groups in the side chain.
Experiments are currently underway to explore the function of these proteins in directing calcium carbonate formation in bulk solution at
different pH values, and in the presence of magnesium ions. The proteins
will also be tethered to surfaces in the form of a monolayer, and their influence on calcium carbonate precipitation determined. This will enable
us to investigate whether the proteins behave differently in solution and when located on a solid substrate.
T 15
Synthetic, prismatic-type CaCO3 films via seeded
mineralization Y. Jiang*1
1Xiamen University, Xiamen University, China
Introduction
Biominerals, by taking advantage of their hierarchical architecture, reconcile
multiple functions which are otherwise contradictory in their synthetic counterparts. From the synthetic point of view, the delivery of hierarchical
architecture can be ascribed to the active roles of (multiple) soft matter,
which turns the mineralization proceeding in organism into self-organization processes. The control of a self-organization process for the delivery of life-
feathered synthetic minerals nonetheless remains technically challenging in
biomimetic mineralization.
Objectives
The primary focus of our work is to design specific microenvironment based
on the organization of multiple types of soft matter for the delivery of prismatic-type minerals with distinct architecture and remarkable mechanical
performance.
"Patients & methods" or "Materials & methods" We introduce seeded mineralization for the delivery of prismatic-type
CaCO3 films.
Results
The method starts with the deposition of granular CaCO3–polyacrylic acid
hybrid films on polymer substrates like chitosan, silk fibroin, or polyvinyl
alcohol. Next, an overgrowth procedure leads to prismatic-type CaCO3 overlayers. Our study shows that the presence of different additives can have
a tremendous impact on their morphological outcomes, while the selection
of polymer substrates determines the polymorphic form of the granular seed layer. In our very recent work, we study the impact of hydrogels on the
structural information of the prismatic overlayers, though hydrogels
themselves are not involved in the delivery of biogenic prismatic minerals. This prismatic CaCO3 films can be used for drug delivery applications.
Conclusion
To summarize, the synergistic effect of multiple types of soft matter and the proper use of biomimetic mineralization methods can create favored
microenvironments for the delivery of prismatic-type CaCO3 films in self-
organization processes. These CaCO3 films exhibit comparable mechanical performance to their biogenic counterparts. The rational design of
biomimetic mineralization systems helps for the mechanistic understanding
of biomineralization, and meanwhile, provides a practical tool to design proper materials for biomedical applications.
T 16
Synthesis of pyrite nanoparticles using the matrix protein from
the scaly-foot, Chrysomallon squamiferum T. Yamashita1, H. Matsuda1, Y. Suzuki2, N. Ahsan3, Y. Okada3, M.
Suzuki*1 1The University of Tokyo, Departiment of Applied Biological Chemistry, Bunkyo-ku, Tokyo, Japan 2The University of Tokyo, Deapartment of Earth and Planetary Science,
Bunkyo-ku, Tokyo, Japan 3The University of Tokyo, Research Center for Advanced Science and Technology, Meguro-ku, Tokyo, Japan
Introduction
Chrysomallon squamiferum (scaly-foot) is a deep-sea snail which was
discovered near the hydrothermal vent of the Central Indian Ocean Ridge "Kairei Field". This snail has scaly structures mineralized with iron sulfide
nanoparticles (pyrite, FeS2) on its foot, and the shell is covered with an iron
sulfide layer on the calcium carbonate layer. Pyrite nanoparticles have superior photovoltaic properties to be applied to photovoltaic power
generation in the solar panel. However, the methods of its inexpensive and
green chemical synthesis have not been established. Therefore, new industrial applications using the formation mechanism of pyrite nanoparticles
in the scaly-foot are required. In this research, in order to identify the
substances that have key factors to synthesize the sulfide nanoparticles in vitro, we tried to extract the organic molecules that bind to iron in the scaly-
foot, because the iron-binding organic molecules may have a function to
prevent the aggregation of particles.
Results and Discussion
In this study, we analyzed the organic molecules extracted from the scaly
structure and the shell in the scaly foot and searched for substances related to the production of iron sulfide nanoparticles by interaction with iron.
Pyrite in the scaly structure was dissolved by reducing with zinc and trivalent
chromium under anaerobic conditions. We used HPLC post column chelator method to identify the iron binding organic molecules. This result revealed
that large amount of low molecular weight organic matters having iron
binding ability existed in the scaly structure. This low molecular weight organic molecule was purified using cation exchange resin and reverse
phase-HPLC. We tried to determine its chemical structure using mass
spectrometry and NMR. On the other hand, 1 M acetic acid was used to dissolve the calcium carbonate
layer of the outer shell. Then, the insoluble iron sulfide layer in acetic acid was extracted using SDS-DTT. A specific protein band was detected from
the iron sulfide layer. LC-MS/MS analyses revealed that this band showed
an amino sequence of heme protein. Heme is consisting of tetrapyrrole ring that can make the complex with iron in vivo.
The heme protein may have some roles for the formation of nano pyrite in
vitro. We try to use the heme protein to synthesize an iron sulfide nanoparticles using various conditions.
T 17
Oriented crystallization of single crystalline strontium sulfate
in marine Acantharea V. Merk*1,2, P. Smeets2, J. Walker2, D. Joester2 1Florida Atlantic University, Department of Chemistry & Biochemistry;
Department of Ocean & Mechanical Engineering, Boca Raton, United States 2Northwestern University, Materials Science and Engineering, Evanston, United States
Introduction
Simple organisms, such as sea urchins, calcerous sponges, coccolithophores, or magnetotactic bacteria, pursue highly sophisticated strategies for growing
single-crystalline skeletal units. Acantharia radiolarians present yet another
prime example of single crystal engineering in nature. The marine protists build intricate star-shaped endoskeletons from smoothly curved strontium
sulfate (SrSO4) spicules, whose long axes coincide with the crystallographic
a-axis. Although Acantharia occur in the ocean's zooplankton from tropical to polar zones, the underlying crystallization mechanism remains virtually
unexplored.
Objectives
In this paper, we anticipate addressing the question how crystal texture is
controlled in a biological environment. The poor stability of Acantharia in
culture renders time-resolved observations of the spiculogenesis impossible. To infer the crystallization mechanism of biogenic celestite, we analyzed the
atomic- and nanostructure as well as the distribution of intercalated
biomolecules on various length scales.
Materials and Methods
We investigated the atomic and nanostructure of Acantharian skeletons from
the Solomon Islands using a combination of X-ray diffraction, Raman spectroscopy, electron-optical imaging and synchrotron-based techniques.
The sample preparation for transmission electron microscopy (TEM)
8
included targeted focused-ion beam (FIB) lift-outs from Acantharian spicules, mostly along primary crystallographic axes. Synchrotron X-ray
fluorescence mapping and Sulfur K-edge XANES spectra were obtained at
sector 13-ID-E at the Advanced Photon Source (APS), Argonne National Laboratory. Scanning wide-angle X-ray scattering data were acquired at the
P03 endstation, PETRA III at DESY.
Results
Acantharia grow highly ordered SrSO4 single crystals, whose crystal
structure is almost indistinguishable from geological and synthetic
counterparts. As markers for intracrystalline organic matter, sulfur-bearing amino acids and di-sulfides were detected across the protist"s skeleton by
microbeam X-ray absorption spectroscopy (XANES) at the sulfur K-edge.
Highly anisotropic small-angle X-ray scattering signals correspond to nanoscale electron density variations parallel to the crystallographic a-axis,
which are consistent with buried interphases in the SrSO4 biomineral.
Similarly, scanning TEM Z-contrast imaging showed nanodomains with a lower atomic number around the spicule center, which do not seem to
correspond to light metals (e.g. Na, K, Ca, etc.). In the diffraction contrast
image from the spicule cross-section, we observed concentric layers reflective of fluctuating growth.
Conclusions
Taken together, our findings suggest that biomolecules interact with specific planes and subsequently become introduced into the growing crystal. An
occlusion of specific biomolecules may guide the crystallographic
orientation of primary seed crystals, indicating genetic regulation of the crystal growth process. Furthermore, Acantharia present an excellent source
of bio-inspiration for alkali earth sulfates with tailored mechanical, electronic, and optical properties.
T 18
Influence of stress on biogenic calcite growth J. Colombani*1, B. Zareeipolgardani2, A. Piednoir1 1Université Claude Bernard Lyon 1, Institut Lumière Matière,
Villeurbanne, France 2Université Grenoble Alpes, ISTerre, Grenoble, France
Calcite, the most widespread crystalline form of calcium carbonate, is ubiquitous both in nature and in the industry. In particular, its precipitation
by living organisms under the form of shells is the major CO2 sink inside the
ocean, and it is used in multiple industrial sectors (ordinary cement manufacturing, paper bleaching, toothpastes, etc.). Therefore the knowledge
of its mechanisms of growth, both organic and inorganic, are of foremost
importance, in the modelling of the carbon cycle, in the simulation of oil reservoirs, or as a promising new route for biomimetic materials engineering.
One parameter, although almost always present during calcite growth, has
not been considered in most studies. When calcite grows from nuclei, they
eventually enter into contact, and stresses develop at their interfaces,
particularly if the material grows in a confined environment, for example
inside a living cell. So far, we have no idea on how this stress modifies the growth kinetics, or the morphology of the grown surface. To address this
issue, we have used an atomic force microscope (AFM), both to imply a local stress on a growing calcite surface, and to image it. We have found that the
stress has a double influence: it slows down significantly the growth, and
induces a change of the type of growing phase. Calcite biomineralization proceeds in presence of organic materials, among which amino acids are
widespread. Therefore we have investigated the influence of pentaglycine on
the growth kinetics. This amino acid shows the striking ability to cancel the phase transition induced by the applied force.
T 19
pH regulation in calcifying primary mesenchyme cells of the
sea urchin larva M. Hu*1 1University of Kiel, Institute of Physiology, Kiel, Germany
Question The sea urchin embryo develops an elaborate calcitic endoskeleton that has
been used by biologists to study the mechanisms of biomineralization. Amorphous calcium carbonate is formed in intracellular compartments and
exocytosed into the syncycial cable formed by primary mesenchyme cells
(PMCs). For intracellular precipitation of CaCO3, PMCs require HCO3-/CO32- concentrating as well as proton export mechanisms to promote
calcification. These processes are of fundamental importance in biological
calcification, but remain relatively unexplored even for mammalian systems.
Material & Methods Larval cultures: For our experiments we used the purple sea urchin
(Stongylocentrotus purpuratus) and larval cultures were maintained at 15°C in natural seawater for further experiments.
Recalcification assay: Skeletons of early pluteus larvae (3dpf) were
dissolved in MES buffered natural seawater adjusted to pH 6.0 for 12 h. After transfer to natural seawater (pH 8.1) larvae re-calcified their skeletons within
four days.
Life cell imaging: The pH sensitive dye BCECF_AM and BCECF-FA 10KD dextran were used to monitor intracellular as well as vesicular pH,
respectively. For cellular pH recordings the ectoderm was removed and
ratiometric pH recordings were performed. Immunocytochemistry and westernblot analyses: Localization and
quantification of PMC specific acid-base transporters was performed using
species-specific custom made antibodies. RT-qPCR and whole mount in situ hybridization: Expression levels of PMC
specific acid-base transporters were measured by qPCR and normalized to
the housekeeping gene SpZ12. Whole mount in-situ hybridization was performed to localize transcripts in the sea urchin larva.
Microinjection of GFP constructs and Morpholino knock-down: Micro-
injections were performed to insert morpholinos and BAC-GFP constructs into the fertilized egg (one-cell stage).
Results Here we demonstrate the SLC4 HCO3- transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic
endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary
mesenchyme cells with peak expression during de novo formation of the skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects
accompanied by decreased calcification rates and skeleton deformations. Re-
calcification experiments demonstrated an increase in PMC pHi and substantial elevations in intracellular [HCO3-] during skeleton rebuild driven
by SpSLc4a10. Despite overall reductions in proton export capacities of
PMCs in this phase, large filopodial cells containing acidic vesicles connect to the PMC syncytium and locally elevate pH regulatory capacities. These
cells do not express ALX1 a specific marker for skeletogenic mesenchyme cells. The process of skeleton rebuild and vesicular acidification in PMC
associated cells is sensitive to the V-Type-ATPase inhibitor bafilomycin.
Conclusion Intracellular accumulation of bicarbonate is a fundamental mechanism of
calcification in PMCs of the sea urchin embryo to develop and rebuild the
calcitic endoskeleton. We propose that during this phase PMCs shift from a NHE-based proton export mechanism to a vesicular storage of protons
generated through extensive precipitation of CaCO3. Non-PMC cells
associated to the skeletogenic syncytium potentially play a critical role in storage and removal of protons during extreme calcification efforts. These
results highlight the importance to better understand pH regulatory processes
in calcifying systems including cell-cell interactions and acid-base transport on the cellular and sub-cellular level.
T 20
The formation of fenestrations in Eucidaris tribuloides
embryonic spicules J. Walker*1, B. Moreno1, D. Joester1 1Northwestern University, Materials Science and Engineering, Evanston, United States
Skeletal patterning in sea urchin embryos is an important model system for
investigating biomineralization of calcium carbonate. Harnessing a tightly controlled growth mechanism, the organism is able to direct the formation of
single crystals that have complex curved and branched morphologies with
precise crystallographic directions. These single crystals of calcite, known as spicules, begin growing from a seed rhombohedral calcite crystal. The
deposition of these spicules and patterning of the skeletal structure is
controlled at the local level by the action of primary mesenchyme cells (PMCs). They fuse to create a syncytium, or privileged space, in which the
mineral is deposited and grows. The pattern of the skeleton is species
specific, and PMCs have been shown to be able to autonomously direct skeletal design including the formation of features such as fenestrations.
These are regularly spaced holes within the single-crystalline central rod that
is normal to the spicule direction, a property that is thought to increase the stiffness-to-weight ratios and fracture resistance of the structure. Replication
of this ability to produce consistent periodic features at multiple length scales
is vitally important for the creation of synthetic materials with specific and tunable properties.
In this work we investigated the formation of fenestrations in the embryonic
spicules of Eucidaris tribuloides, a tropical sea urchin, and the effect their presence has on crystallographic and morphological features of the spicule.
Spicule formation in both embryos of E. tribuloides and S. purpuratus were
investigated. Embryos from each species were separately grown in culture. Spicules were extracted from the embryonic cultures and examined by SEM
at Northwestern University Atomic and Nanoscale Experimental Centre
(NUANCE). The identified features were then further investigated by synchrotron white beam X-ray diffraction mapping using a 250 nm beam
size, which was obtained at beamline 34-ID-E at the Advanced Photon
Source, Argonne National Laboratory. SEM imaging of the extracted spicules at different time points allowed a
detailed visualization of the morphology of the fenestrations as the spicule
develops. Investigation of an individual spicule using white beam X-ray diffraction mapping enabled us to correlate the fenestrations to changes of
direction of the lattice planes within the crystal. Using a beam size of 250
9
nm it was possible to investigate these microscale features and observe changes to the (001) plane relating to the location in the spicule. Although
there was a high homogeneity in plane direction as would be expected for a
single crystal, small changes (>0.1 degrees) in rotation were identified in regions surrounding a fenestration.
White beam X-ray diffraction mapping can be used to correlatively with
electron microscopy to demonstrate local changes in lattice planes in and around micron-sized features. It can efficiently provide detailed
crystallographic information over a larger scale than other techniques such
as TEM, which can narrow down the area of interest for investigation using higher resolution but more labour intensive techniques. We have used it to
investigate lattice plane changes related to the formation of fenestration in
the growing spicule of a sea urchin embryo.
T 21
Effects of seawater Mg2+/Ca2+ ratio and diet on the
biomineralization and growth of sea urchins - applicability of
echinoderms in paleoenvironmental reconstructions D. Kołbuk*1, P. Dubois2, S. Di Giglio2, S. M’Zoudi2, J. Stolarski1, P.
Gorzelak1 1Polish Academy of Sciences, Institute of Paleobiology, Warsaw, Poland 2Université Libre de Bruxelles, Faculté des Sciences, Brussels, Belgium
It has been suggested that the skeletal Mg/Ca ratio of well-preserved fossil
calcifying marine invertebrates, including echinoderms, was predominantly
affected by the changes of Mg2+/Ca2+ ratio in the ancient seawater, which has varied between ~1.0 to 5.2 throughout the Phanerozoic. However, it has been
also demonstrated that the chemical composition of Recent and fossil echinoderms can be modified by a number of other factors, such as
temperature, salinity, metabolic processes, diet, and diagenesis. In this work,
we addressed the problem of diet vs. seawater chemistry impact on skeletal Mg/Ca ratio, in experimental conditions. Two phylogenetically distant
echinoid species, Psammechinus miliaris and Prionocidaris baculosa, were
cultured in low Mg2+/Ca2+ (~2.5 and 1.5) artificial seawater, prepared by lowering the Mg2+ and increasing the Ca2+ concentrations. Before the sea
urchins were exposed to low Mg2+/Ca2+ conditions, they were tagged with
manganese in order to identify newly-formed skeleton areas under cathodoluminescence (CL) and calibrate their growth rates. After tagging,
the echinoids were incubated for 21 days in seawater with different
Mg2+/Ca2+ ratios (~5.2, ~2.5, ~1.5 [mol/mol]) and fed with diets containing different amounts of magnesium (~0.3 wt% or ~20 wt%). Mg/Ca ratios in
the newly formed skeleton in different types of echinoid ossicles (test plates,
spines, teeth, and demipyramids) were determined using electron microprobe. Mg/Ca ratios in all ossicle types of both species decreased
proportionally with decreasing Mg2+/Ca2+ ratio of seawater, in which they
were growing. However, echinoids fed with magnesium-enriched diet typically displayed higher skeletal Mg/Ca ratios. Growth rates in some
ossicles (test plates and demipyramids) significantly decreased with the
reduction of ambient Mg2+/Ca2+ ratio. These results underscore the importance of diet in modulating Mg/Ca ratio of echinoderm skeleton and
suggest that caution should be exercised when using the skeletal Mg/Ca ratio
of fossil echinoderms as a direct paleoseawater Mg2+/Ca2+ proxy. [This research was funded by the National Science Centre (NCN) grant no.
2016/23/B/ST10/00990].
T 22
Genetic control over biomineralization in calcareous sponges O. Voigt*1, B. Fradusco1 1LMU Munich, Department of Earth- and Environmental Sciences, München, Germany
The ability to produce biominerals has been a key innovation in animal
evolution and allows animals to generate as essential structures as shells,
teeth and skeletons. The genetic mechanisms underlying the biomineralization process and their evolution are only poorly understood. It
seems that biomineralization evolved serval times in different animal linages,
but still utilize similar general principles and genetic pre-adaptations. We study biomineralization in the class of calcareous sponges, which belong
to the oldest animal phylum. These sponges gained the ability to produce
calcite spicules of different shapes in contrast to other sponge classes, which have siliceous spicules. They provide a simple system of
biomineralization: Calcareous sponges produce numerous spicules in a short
time (hours to days), and each is formed by only a few (e.g. two to seven) specialized cells.
Therefore, we aimed to identify the genetic mechanisms that control the
biomineralization system in calcareous sponges to understand general principles of biomineralization.
We applied a combination of genomics, transcriptomics, proteomics and
RNA in situ hybridization to identify key biomineralization genes of calcareous sponges and to obtain information about the spatial and temporal
expression during the spicule formation process.
Key components of the genetic biomineralization toolkit were identified, including carbonic anhydrases, bicarbonate transporters and secreted spicule
matrix proteins. We observed changes in the spatial and temporal expression
of these genes during the spicule formation process. Some matrix proteins are only produced by sclerocytes that form spicules of a specific shape.
Our results highlight the essential role of genetic control over the
biomineralization process even in as simple structures as the calcite spicules of calcareous sponges. Comparative analyses with multiple species will help
to reveal the genetic key-innovations obtained by the last common ancestor
of these sponge class.
T 23
Crystallographic misorientation between spicules in the
skeleton of the calcareous sponge sycettusa hastifera L. Souza Coelho1, M. Klautau2, M. Farina3, A. Rossi*1 1Brazilian Center for Research in Physics, Rio de Janeiro, Brazil 2Federal University of Rio de Janeiro, Biology, Rio de Janeiro, Brazil 3Federal University of Rio de Janeiro, Biomedical Science, Rio de Janeiro, Brazil
Introduction
The skeleton of calcareous sponges (phylum Porifera, class Calcarea) is
formed by Mg-calcite spicules ranging from approximately 10 to 2000 μm in
length and usually composed of two, three or four (rarely five) conics rays joined at the base [1,2]. One individual spicule behaves as a monocrystal
(trigonal system) with only few degrees of misorientation (<1°) [1,3]. The
species Sycettusa hastifera belong to the subclass Calcaronea. In this subclass the unpaired actine of tri and tetractine spicules is elongated
approximately in the [211] direction. The crystallographic direction of the
actines are conserved through the evolution of the classes. In the class Calcinea, the actines are elongated in the {210} directions. The spicules
normally have a specific position in the body of the sponge (cormus) and are
used as a taxonomic character. S. hastifera has triactine and tetractine spicules in the cortical, subcortical, subatrial and atrial regions of the
choanosome of the sponge.
Objectives
In this work, the crystallographic misorientations between spicules in the
cortex, subcortical, subatrial and atrial regions were identified in different
transversal sections of the tubular structure of the sponge (base, middle and top of the sponge tube) and in different area of each section of the tube. The
aim of this study is to relate the misorientation between spicules with the process of growth of the sponge skeleton and the resistance proprieties of the
aquiferous system tubes.
Materials and Methods
Samples from S. hastifera were collected in Arraial do Cabo city, Rio de
Janeiro, Brazil. The sponges were dehydrated with increasing concentrations
of ethanol and infiltrated with epoxy resin. The sponge embedded in resin was cut transversally to the tube direction using a diamond saw. Transversal
sections of the sponge were polished with alumina 1μm and then coated with
a thin carbon layer. Electron Backscatter Diffraction (EBSD) were performed in a Scanning Electron Microscope Jeol 7100FT equipped with an Oxford
EBSD detector. Kikuchi line patterns were recorded using Aztec and
Channel 5 softwares. Each transversal section was divided into four areas (1, 2, 3 and 4) from where cortical, subcortical, subatrial and atrial spicules were
studied. Stereographic projections were generated to study misorientation
between spicules. Spicule types were identified using an inverted optical microscope.
Results
The subcortical spicules were the less oriented spicule type from S. hastifera. The absence of a preferential orientation may be related with the existence of
pseudotriactine spicules which present similar morphology but different
crystallographic orientation compared to a conventional triactine. All spicule types in the middle section of the sponge tube were more well oriented than
in the base of the sponge. This result is related with the process of growth of
the sponge tube. The transversal section of the sponge tube had an oval symmetry. The areas of the section superposed to the larger diameter
presented spicules more well oriented compared to the areas in the smaller
diameter of the tube. The same spicule type may have different orientation in different area.
Conclusion
The spicule orientation in the cormus of S. hastifera present more or less misorientation depending on the transversal section of the sponge tube (base,
middle or top), the spicule type and the area of the transversal section.
10
T 24
Paradoxical mechanical properties of sponge spicules J. Werckmann*1, E. Bouzi2, E. Brodu2, M. Longuinho1, A. Porto Careiro3,
D. Ihiawakrim4, A. Rossi1, M. Klautau5, M. Farina5 1Brazilian Center of Physic Researsh, COMAN, Rio de Janeiro, Brazil 2University, Metz, France 3University, Marseille, France 4CNRS, Strasbourg, France 5University Fédérale, Rio de Janeiro, Brazil
Our study focuses on calcareous sponges. Their three-dimensional structure
is ensured by the entanglement of spicules within their soft cylindrical body,
composed mainly of collagen. These spicules have the distinction of being biogenic single crystals consisting of magnesium calcite. One of their
paradoxical properties is the conchoïdal fracture caused by mechanical stress,
whereas mineral calcite has fractures determined by {104} cleavage planes. The purpose of our work is to highlight the factors that are behind this
property. Thus we have implemented several characterization techniques
associated to transmission electron microscopy. Specimens of the species Paraleucilla magna were immersed in a 2.5% sodium hypochlorite solution
to completely remove the adhered organic tissue. The spicules were rinsed
with distilled water followed by 100% ethanol then air dried. Thin spicule cross section were obtained by Focused Ion Beam (FIB). Briefly, the
spicules were dispersed on a silicon substrate and metalized with a 200 nm
gold layer for surface protection and electrical conductivity. To improve the
protection, a 1 µ Pt layer was in situ deposited. To avoid ion damages,
progressive thinning process was implemented. To complete the process, a
current of 30 pA at an energy of 5 keV was used to clean and reduce the amorphous damaged layer produced during the former steps. Titan
transmission electron microscope operating at 300keV was used. The
electron diffraction obtained on sections extracted on the same spicule, whatever their orientation, are interpreted as originating a single crystal. In
addition, the high resolution images of the external spicule-membrane
interface show the presence of nanocluster attached or close to the surface of the spicule. Their FFT can be interpreted as coming from calcite
nanocrystals. The dark field image obtained when the microscope operates
in Scanning Transmission Electron Microscopy (STEM) mode reveals the existence of significant porosity. Transmission Kikuchi diffraction (TKD)
was implemented in a scanning microscope working at 30keV for mapping
the mosaicity. Before observation a thin layer of platinum was deposited to enhance the electrical conductivity of the sample and to protect them to the
beam damages. Spatial resolution was de of 10nm (pixel size) and the angular
resolution reached about 0.01°. Thanks to this angular precision, the maps of disorientation obtained show that the domains of the mosaic are smaller than
or equal to 10 nm. Based on our experimental results and bibliography (1, 2)
we conclude that the growth of spicules is a discontinuous process that leads
to the formation of a porous structure and that it is controlled by proteins that
catalyze the formation of nanocrystals that are deposited almost epitaxially
on the spicule in formation, resulting in the formation of a mosaic of very low disorientation (0.01°), undetectable by electron diffraction. During
growth a small amount of protein is included by chance in the spicules. These three factors: porosity, mosaicity and inclusion of proteins increase the
resistance of calcareous spicules to the mechanical stress entailing the
conchoidal fracture. This ensures a better resistance of the sponges to the mechanical stresses due to the ebb and flow of the sea and on the other hand
a better resistance to the action of predators.
1) Rossi et al. Acta Biomaterialia (2014)
2) L. Freeman et al. Angewandte Chemie (2010)
T 25
Rice plant biomineralization- Ultrastructure of biosilica, and
defense protein N. Ozaki*1,2, R. Abiko1, K. Tsuji1, T. Ishida1, M. Suzuki3, F. Nudelman2 1Akita Prefectural University, Department of Biotechnology, Faculty of
Bio-resource Sciences, Akita, Japan 2The University of Edinburgh, School of Chemistry, Edinburgh, United Kingdom 3The University of Tokyo, Graduate School of Agricultural and Life Sciences , Tokyo, Japan
Biologically formed amorphous silica (biosilica) are widely found in
bacteria, diatoms, marine sponges, terrestrial higher plants, some of which have been well characterized. Macromolecules in biominerals are known to
control mineralization. Biosilicas in diatom and marine sponge, are formed
under mild condition and neutral pH using unique macromolecules, such as silaffin, glassin, and long chain polyamines. The typical example of silicon
accumulating higher plants, gramineous plants (rice plants), produces a large
amount of biosilica in their leaf blades and rice husks. Although biosilicas deposited in rice plants play various important roles, such as enhancing
mechanical strength, improving disease resistance and photosynthetic activity, information on the molecular mechanisms involved in the biosilica
formation is very limited. In this study, we investigated the microstructure of biosilicas from rice plants (Oryza sativa) by using scanning electron
microscopy (SEM) and Cryo-FIB SEM. In addition, we extracted organic
matrices from biosilicas and characterized by LC-MS/MS analysis. SEM analyses revealed that the silicas of rice plants are composed of nanoparticles
that are several dozen nm in diameter, similar to diatom silica and to siliceous
spicules of sponge. We extracted a 11 kDa protein from silica from the leaf blades and a 140 kDa protein was identified as the only organic component
contained in rice husk"s silica. Protein Database searches revealed that the
11 kDa proteins belong to plant defense proteins known as LTPs, and the 140 kDa protein has an amino acid sequence similar to glucanase. Both proteins
are cationic proteins present in abundance in higher plants and are known to
play important roles in resistance to abiotic and biotic environment stress. Given their cationic nature, we hypothesize that they are involved in
regulating silica formation in the rice leaves and husks. Therefore, we are
currently investigating their function in controlling silica biomineralization.
T 26
The biology of silica deposition in leaf silica cell - a first full
description of plant bio-mineralization process R. Elbaum*1, S. Kumar2 1Hebrew University of Jerusalem, Institute of Plant Sciences, Rehovot,
Israel 2Weizmann Institute of Science, Plant & environmental sciences, Rehovot, Israel
Biomineralization in animals is a very common strategy creating skeletal elements. In plants, however, body design is flexible, and each organ is self-
supported by cell walls. Plant minerals are typically utilized in defense
mechanisms. Silica is a very abundant mineral in plants that may constitute 10% of the dry weight of certain tissues. Most astonishingly, very little is
known about the control over silica formation in plants. We discovered the
mechanism for silica deposition in epidermal silica cells of sorghum (Sorghum bicolor) leaves. Silica cell are almost completely filled with silica
when mature. We showed that silicification is confined to elongating leaves,
in a well-defined active silicification zone (ASZ). The mineralization initiates in live cells. Silica starts to form at the cell"s periphery, adjacent to
the primary cell wall. In contrast to other biomineralization processes, the
mineral is not formed inside vesicles. Instead, the silica cell protoplasts synthesize and secretes a unique protein that accelerates silica precipitation.
The protein is packed in vesicles and is secreted to the extracellular space, which is saturated with silicic acid. The silicic acid precipitates into silica,
creating a secondary wall inward to the primary cell wall, and restricting the
protoplast"s space. The thickening silica wall fills up the cell volume within a few hours. Alongside this fast process, the silica cell goes through
programmed cell death, the cell contents are possibly evacuated to
neighboring cells. This is a first description of silica deposition in plants.
T 27
Imaging and analysis of internal silicon pools in diatoms A. Gal*1 1Weizmann Institute of Science, Plant and Environmental Sciences, Rehovot, Israel
Diatoms are abundant unicellular algae that cover themselves with a cell wall
made of silica. The silicification process is under strict biological control, to
the extent that the delicate nanoscale architecture and fine ornaments are species-specific. The polymerization of silica from its soluble building
blocks is thought to take place inside a specialized organelle that is
responsible for precipitating the mineral phase and controlling its morphology. Our understanding of the inorganic precipitation mechanism of
diatom silica is still rudimentary, as conventional imaging and analytical
tools are inadequate to resolve the native-state structural information and chemistry related to the silicification process. For example, it is unclear what
are the mechanisms of silicon uptake and concentration from the
environment, and what are the conditions inside the silicifying organelle that
give rise to the precise morphology.
We use a suite of cryo electron microscopy techniques in order to extract intracellular structural and chemical information with nanoscale resolution.
3D serial imaging of whole Thalassiosira pseudonana cells, using cryo
focused-ion-beam scanning electron microscopy (cryoFIB-SEM), and energy dispersive spectroscopy (EDS), shows that the cells maintain high
intracellular concentration of Si throughout the cell cycle. In addition,
scanning transmission electron tomography (STEM tomography), enables us to visualize in 3D the ultrastructure of the silica deposition vesicle along the
silicification process. In the species Chaetoceros tenuissimus we followed
the formation of long silica needles with cryo electron tomography (cryoET) and discovered a new mechanism for the deposition of silica. Overall, our
direct approach to study the formation of diatom silica in situ by means of
advanced microscopy tools is yielding detailed understanding of the cellular controls that shape the silicification process in diatoms.
11
T 28
Control of biosilica morphology and mechanical performance
by the conserved diatom gene Silicanin-1 S. Görlich1, D. Pawolski1, I. Zlotnikov1, N. Kröger*1 1TU Dresden, B CUBE, Dresden, Germany
Diatoms represent a large group of unicellular, eukaryotic microalgae that
are most well known for their ability to produce cell walls made of
nanopatterned porous biosilica. The species-specifically patterned cell walls are paradigms for biological mineral morphogenesis and the evolution of
lightweight materials with exceptional mechanical performance. The
formation of biomineral building blocks often takes place within specialized intracellular vesicles, which in diatoms are called Silica Deposition Vesicles
(SDV). Recently two families of SDV membrane proteins have been
identified that are well conserved throughout all diatoms and may therefore play a fundamental role in diatom cell wall formation. One promising
candidate is Silicanin-1 (Sin1), which was shown to have moderate silica-
formation activity in vitro that is enhanced by the addition of long-chain polyamines. It was therefore speculated that Sin1 may influence the assembly
of biosilica-forming biomolecules within the SDV lumen. Here we describe
the CRISPR-Cas9 mediated gene knockout of Sin1 in Thalassiosira pseudonana. Although the mutants grew normally, the knockout cell lines
exhibit a reduced biosilica content and showed pleiotropic effects on silica
morphogenesis, which drastically compromised the strength and stiffness of their cell walls. These results identify Sin1 as a key player in the development
of specific structural features of the T. pseudonana cell wall and as essential
for the biogenesis of mechanically robust diatom cell walls, thus providing an explanation for the conservation of this gene throughout the diatom realm.
T 29
Investigation of the hybrid nanocomposites of the chiton tooth
stylus to find inspiration for new material development L. Stegbauer*1, E. E. Alp2, I. Moudrakovski3, P. Smeets1, R. Free1, S.
Wallace1, M. Hersam1, D. Joester1 1Northwestern University, Materials Science and Engineering, Evanston, United States 2Argonne National Laboratory, Advanced Photon Source, Sector 3, Lemont,
United States 3Max Planck Institute for Solid State Research , Stuttgart, Germany
Introduction
Organisms possess unparalleled control over the structure and properties of
mineralized tissues such as teeth and bones, creating curved single crystals
as well as tough and lightweight self-repairing skeletal structures. Organic matrices play an integral role in the selective formation of metastable mineral
precursors and their transformation into the final biomineral. The chiton is a
model system for the extracellular, matrix-mediated iron oxide.[1] Its radula is a ribbon-like rasping tongue with many rows of extremely hard, wear-
resistant, and self-sharpening teeth designed to withstand the stresses of
grazing algae on rocks.[2] So far, there have been numerous studies on the cuspid heads of the tooth, which consists of one of the hardest and most wear-
resistant biogenic materials, a magnetite/chitin composite. The underlying base, also called stylus, has not been investigated.
Objectives
In this study we aim to establish a better understanding of the biominerals in the radula tooth stylus of the chiton Cryptochiton stelleri. Modern material
fabrication requires large amount of energy to synthesize materials for
semiconductors, medicine, or construction, often employing extensive top-down fabrication steps at high temperatures and pressures. Biominerals can
serve as inspiration for finding innovative methods and new materials and
may also allow to decrease the carbon footprint of these processes. Such knowledge could inform the synthesis of new nanocomposite materials with
outstanding properties.
Materials and Methods
A comprehensive characterization of the stylus was performed with a suite
of techniques that provide insights into structure and compositions at
multiple length scales, including synchrotron Mößbauer spectroscopy (SMS), solid-state nuclear magnetic resonance (ss-NMR), X-ray
spectroscopy and electron imaging. Mechanical properties such as hardness
and reduced modulus were tested by nanoindentation. Results: We find evidence that the cusp-adjacent stylus is mineralized by
santabarbaraite, an amorphous iron hydroxy phosphate that was not known
to occur as a biomineral. The mineral occurs in very small nanoparticles adjacent to α-chitin nanofibers. The stylus possesses outstanding mechanical
properties that are comparable to steels. We propose that the organism
establishes complex spatial gradients in terms of the degree of mineralization (10-25 wt%) and mechanical properties across the stylus. Based on our
insights, we formulated bio-inspired inks for additive manufacturing,
demonstrating that the mechanical properties can be tuned over a wide range also in in vitro.
Conclusion
The stylus can bridge the gap of the vastly different mechanical properties of
the hard head and the radula membrane by varying the amount of deposition
of the biomineral santabarabaraite. The discovered mineralized component, the stylus of the chiton, is a biological nanocomposite material that exhibits
interweaved and co-aligned nanosized chitin fibers with nanosized
amorphous iron phosphate particles in a string-of-pearls manner. The combination of iron phosphate and chitin has impressive mechanical
properties over a wide tunable range which surpass many man-made
materials that require energy intensive manufacturing such as light-metal alloys. Deliberate selection of synthesis conditions inspired by the results of
the biomineral provide us with an artificial composite material that closely
resembles the stylus. The fabricated bulk AFP/chitosan is biodegradable, non-toxic, and processed at room temperature.
[1] L. R. Brooker, J. A. Shaw in Advanced topics in biomineralization,
IntechOpen, 2012, pp. 65–84. [2] a) J. C. Weaver, Q. Wang, A. Miserez, A. Tantuccio, R. Stromberg, K.
N. Bozhilov, P. Maxwell, R. Nay, S. T. Heier, E. DiMasi et al., Materials
Today 2010, 13, 42; b) H. A. Lowenstam, S. Weiner, On biomineralization, Oxford University Press, New York, 1989.
T 30
Reinforcement and adaptation of the mantis shrimp spike-
How crustacean cuticle became a perfect harpoon? Y. Delaunois*1, D. Ruffoni2, P. Compère1,3 1ULiege, Biologie, écologie évolution, Liege, Belgium 2Uliege, aerospace and mechanic, Liege, Belgium 3Uliege, Center for Applied Research and Education in Microscopy (CAREM), Liège, Belgium
Introduction
In the field of bioinspired materials, the crustacean cuticle is an example of
natural organo-mineral biomaterial able to endure a variety of strong stresses thanks to the combination of a complex fibre organization, adaptations and
controlled mineral deposition (Romano, Fabritius, & Raabe, 2007, Acta
Biomateriala 3, 301-309). Stomatopoda is a crustacean order including two groups: smashing and spearing mantis shrimps. Hence, the first group is
already well known for the mechanical abilities of its smashing limbs or
"hammer" (Patek & Caldwell, 2005, Journal of Experimental biology 208, 3655-3664), this study rather focuses on spearing mantis shrimps. Their
spearing appendages bear long spikes that impale fish in a fraction of second and are therefore designed to penetrate at high speed, to avoid escape of the
prey but also to resist to bending forces during the capture.
Objective
The aim of this study is to determine the structural and compositional
adaptations in the spike cuticle of spearing mantis shrimps to endure the
intense stress occurring during attacks.
Materials and methods
Spikes from Lysiosquillina maculata specimens were cut in segments and
either 2.5%-glutaraldehyde- or ethanol-fixed for TEM observation (after OsO4–staining and resin embedding) and for µCTscan and SEM-observation
respectively. Ethanol-fixed samples were also resin-embedded, then polished
for block-face imaging by BSE-SEM and EDAX under low vacuum conditions (0.4 Torr) in a ESEM-FEG XL-30 fitted with Bruker 129eV SDD.
Results
First, the µCTscan highlighted spike external features as curvature and serrations along both edges linked by grooves on the sides. When compared
to the decapod exoskeleton (classic model) and to the regular stomatopod
cuticle (cephalotoracic shield), the spike cuticle exhibits important modifications in its internal fibre architecture and mineralization. These
main changes are the lack of inner epicuticle and its replacement by a hyper-
mineralized exocuticle containing fluorapatite, and the alternating helicoidal (twisted plywood) and nematic (longitudinal orientation) arrangements of
chitin-protein fibres in the deeper procuticle layers. Also present in the
smashing limbs, theses procuticle layers were previously named the outer helicoidal layer, the striated layer and the inner helicoidal layer because no
clear subdivision between classical decapod layers (exo- and endocuticle)
was identified. The limit between exo- and endocuticle was identified in the thin outer helicoidal layer. In terms of composition, the endocuticle including
the 3 fibrous arrangements was seen to be mainly CaCO3-calcified with high
phosphate (and some fluor) proportion. The phosphate content gradually decreases toward the epidermis while inversely the substitution rates by Mg
increases. Nanoindentation tests also revealed significant variation in the
reduced modulus between the layers.
Conclusion
During evolution, the spearing limbs of mantis shrimps acquired a
specialized exoskeleton strongly modified in shape, in internal fibre architecture and mineral composition. These changes suit the intense
mechanical constraints, especially anisotropic stresses, resulting from the
spike shape and movement for rapid penetration in preys. They are also thought to cope with cracks propagations.
12
Acknowledgments: The first authors is a fellow of the FNRS-FRIA (Belgium)
T 31
Acid-induced demineralisation of enamel as a function of time
and pH R. Harper*1, R. Shelton1, J. James1, E. Salvati2, C. Besnard2, A.
Korsunsky2, G. Landini1 1University of Birmingham, School of Dentistry, Birmingham, United Kingdom 2University of Oxford, Department of Engineering Science, Oxford, United Kingdom
Acid-induced enamel demineralisation affects many people either by
exposure to acidic diets, acidic gases/ particulates from pollution (dental erosion) or to dental plaque acids (dental caries) with the latter currently
being the most prevalent disease in humans. This study aimed to develop in
situ micro-CT and light microscopy methods to determine progression of enamel demineralisation and the dynamic relationship between acid pH and
mineral density to aid in the development of improved prevention, diagnosis
and restorative treatments. Intact human third molars extracted for therapeutic reasons with full ethical
approval (National Research Ethics Committee; NHS-REC reference
09.H0405.33/ Consortium Reference BCHCDent332.1531.TB) were longitudinally sectioned into 500µm thick slices. A flat tipped 300µm
diameter needle was clamped perpendicularly on the external enamel smooth
surface before coating the entire surface with nail varnish to generate an ~300µm diameter circular non-varnished area on the slice. Varnished slices
for micro-CT in situ imaging were placed in a radiolucent Kapton® holder
(5 x 4.5cm pieces glued at the edges) containing lactic acid (10%, 0.5mL, pH 2.2) before sealing. Four samples were radiographically time-lapse imaged
hourly for 85h using a SkyScan 1172 micro CT scanner. Greyscale (related
to mineral density) loss was quantified for both the advance of the demineralisation front and extent of mineral density reduction. Varnished
slices for light microscopy in situ imaging were glued to the base of a petri
dish, to which 5ml of lactic acid (10%, pH 2.2) was added before sealing. Three samples were then time-lapsed imaged (every 100 seconds) using a
Zeiss Primotech D/A POL light microscope using a 5x objective. In addition,
18 varnished slices were incubated at 37°C in lactic acid (0.5%, 0.5mL) for three weeks at either pH 3.6, 4.0, 4.4, 4.8 and 5.2 whilst deionised water was
used as a control and were tomographically imaged using micro CT with lesion size and mineral loss quantified using an hydroxyapatite phantom.
Hourly micro-CT time-lapse sequences showed the depth of enamel
demineralisation progressed with time from the surface towards the dentine following a power-law function, which was 21% faster than the lateral
demineralisation progression after the 85h exposure to lactic acid (10%, pH
2.2). The minimum greyscale remaining (related to the remaining mineral density) within the induced enamel lesion followed an exponential decay,
while the total greyscale loss with time was linear, which showed a constant
anisotropic mineral release within the enamel architecture. Polarised light microscopy time-lapse sequences showed that once the demineralisation
front reached the Hunter-Schreger bands in enamel, there was preferential
demineralisation along those bands. Mineral density loss was linear with increasing pH acidity between pH 5.2 and pH 4.0 when incubated over a 3-
week period exposed to lactic acid (0.5%). At pH 4.0, there was complete
mineral loss at the centre of the demineralised area after the 3-week period and the linear function intercepted the x-axis at pH 5.6, near the critical HA
pH.
The time dependent linear total greyscale loss, exponential minimum greyscale remaining, power-law demineralisation front progression,
preferential lateral demineralisation and linear relationship between pH and
mineral density loss showed the intrinsic enamel structure affected the progression of demineralisation.
T 32
Remineralization potential of the enamel protein amelotin and
nano-hydroxyapatite M. Neshatian*1, J. Holcroft1, A. Kishen1, B. Ganss1 1University of toronto, Dentistry, TORONTO, Canada
Background Dental caries, which according to world health organization (WHO) is the
most prevalent chronic disease worldwide, is caused by acid-mediated demineralization of dental tissues. Resin-based composites are the most
commonly used restoration system for dental caries lesions. The integrity of
the interface between the tooth and the adhesive used to bond composite resin to the tooth tissue is crucial to the longevity of the restoration. Currently used
resin bonded composites have an average life span of only 5.7 years.
Therefore, remineralization of demineralized dentin, especially the tooth/restoration interface, is of considerable interest in restorative dentistry.
It may improve bond stability and delay or prevent restoration failures.
Amelotin (AMTN) is an enamel protein, which is expressed specifically during the enamel maturation stage and was shown to have a direct
promoting effect on mineral growth and formation both in vivo and in vitro.
Since AMTN is secreted on a pre-existing mineral layer during amelogenesis, we have used nano-hydroxyapatite (HA) to mimic the native
environment in which AMTN exerts its mineral promoting properties.
Objectives To investigate the ability of the combination of AMTN and Nano-HA to
improve mineralization in simulated body fluid (SBF) and remineralization
of demineralized dentin collagen. Hypothesis: combination of AMTN and nano-HA improves mineral formation both in SBF and dentin collagen.
Method
SBF was incubated with AMTN, nano-HA, or both at 37°C for up to 21 days. Light scattering was used to record the formation and growth of mineral
particles.
Coronal dentinal sections of human extracted molars were fully demineralized with 0.5M Ethylenediaminetetraacetic acid (EDTA).
Demineralized specimens were treated with AMTN, nano-HA or both and
incubated in SBF at 37°C. Specimens were fixed and imaged with scanning electron microscopy (SEM) to visualize mineral formation Results: The
combination of AMTN and nano–HA promoted mineral formation in SBF
significantly compared to controls (P<0.05) when tested by ANOVA. Minerals were visually identified on the surface of dentin disks as well as
within the dentinal tubules on the samples treated with both AMTN and
nano-HA in as little as 2 hours. Conclusion: combination of AMTN and nano-HA can accelerate the mineralization of demineralized dentin
Significance The accelerated mineral formation mediated by AMTN and nano-HA can be
further refined to engineer a complex that may enhance the durability of
dental restoration by creating a homogenous mineral interface between restorative material and native tooth tissue.
T 33
The hidden structure of mouse and human enamel P. Gilbert*1 1Univeristy of Wisconsin - Madison, Physics, Madison, WI, United States
Introduction Enamel is the hardest and most resilient tissue in any animal"s body. The
morphology of human and mouse enamel is well established: it consists of space-filling[1], aligned, parallel, ~50 nm wide, microns-long nanocrystals,
bundled into 5-micron-wide rods, previously known as prisms. The
orientation and arrangement of enamel crystals, however, are poorly understood.
Question 1. Are crystal orientations in human and mouse enamel as
previously assumed based on morphology alone, from SEM
images?
2. Do the observed crystal mis-orientations play a functional role in toughening enamel?
Methods We use polarization-dependent imaging contrast (PIC) mapping[2,3], a
synchrotron method that took a decade to refine and optimize[4,5], so it can
finally be used to measure and display the crystal c-axis orientation of carbonate or apatite biominerals. In a PIC map color quantitatively displays
the c-axis unit-vector orientation in polar coordinates, including the in-plane
and off-plane angles, displayed as hue and brightness, respectively, both referred to the polarization plane, not the image plane. The sample is
mounted vertically, and the beam illuminates it from the right with 30°
grazing incidence. The polarization plane is the plane in which the polarization vector rotates, which is perpendicular to the beam direction, thus
the polarization plane intersects the sample surface plane at 60°, and only a
vertical c-axis (displayed as cyan with full brightness), lies in both the polarization and the image plane[5]. Radiation damage to enamel is
minimal[6]. Before PIC mapping, enamel samples are embedded, polished,
coated[7].
Results PIC maps of mouse enamel show that, within a rod, crystals are co-oriented
with one another but not with the long axis of the rod[8], in human enamel they are not co-oriented with either: the c-axes of adjacent crystals are most
frequently mis-oriented by 1°-30°, and their orientation gradually changes up
to 30°-90° within each rod[9,5]. Molecular dynamics simulations demonstrate that mis-orientation of adjacent crystals induces crack
deflection[5], and thus toughens enamel.
Conclusions The newly observed mis-orientations contribute to make human enamel last
a lifetime.
References 1. L Yang, CE Killian, M Kunz, N Tamura, PUPA Gilbert, RSC-Nanoscale
(2011), DOI: 10.1039/C0NR00697A
2. PUPA Gilbert, A Young, SN Coppersmith, Proc Natl Acad Sci USA (2011), DOI: 10.1073/pnas.1107917108
13
3. CE Killian, RA Metzler, YUT Gong, TH Churchill, IC Olson, V Trubetskoy, MB Christensen, JH Fournelle, F De Carlo, S Cohen, J
Mahamid, FH Wilt, A Scholl, A Young, A Doran, SN Coppersmith,
PUPA Gilbert, Adv Funct Mater (2011), DOI: 10.1002/adfm.201001546 4. RA Metzler, M Abrecht, RM Olabisi, D Ariosa, CJ Johnson, BH Frazer,
SN Coppersmith, PUPA Gilbert, Phys. Rev. Lett. (2007), DOI:
10.1103/PhysRevLett.98.268102 5. E Beniash, CA Stifler, C-Y Sun, GS Jung, Z Qin, MJ Buehler, PUPA
Gilbert, submitted (2018),
6. T Parasassi, O Sapora, AM Giusti, G De Stasio, G Ravagnan, In J Rad Biol (1991), DOI: 10.1080/09553009114550061
7. G De Stasio, BH Frazer, B Gilbert, KL Richter, JW Valley,
Ultramicroscopy (2003), 10.1016/S0304-3991(03)00088-3 8. CA Stifler, N Kølln Wittig, M Sassi, C-Y Sun, MA Marcus, H Birkedal,
E Beniash, KM Rosso, PUPA Gilbert, J Am Chem Soc (2018), DOI:
10.1021/jacs.8b05547 9. RT DeVol, C-Y Sun, MA Marcus, SN Coppersmith, SCB Myneni,
PUPA Gilbert, J Am Chem Soc (2015), DOI: 10.1021/jacs.5b07931
Funding sources NSF: DMR-1603192; DOE: DE-FG02-07ER15899; DOE: DE-AC02-
05CH11231.
T 34
Unique three-dimensional structure of a fish mandible bone
subjected to unusually high mechanical loads E. Raguin*1, K. Rechav2, V. Brumfeld2, R. Shahar3, S. Weiner1 1Weizmann Institute of Science, Structural Biology, Rehovot, Israel 2Weizmann Institute of Science, Chemical Research Support, Rehovot,
Israel 3Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Koret School of Veterinary Medicine, Rehovot, Israel
Introduction
Much of our understanding of bone structure is based on two-dimensional
techniques. However, these methods provide limited insight into the
structural complexity found in these tissues, especially at the nanometer length scale. Here, we explore the structure-mechanics relations in 2D and
3D of a bone that is neither cortical nor trabecular at different hierarchical
levels of organization. This bone is found in the jaws of a fish that uses its tooth-jaw complex to crush hard-shelled bivalve mollusks. The bone is thus
subjected to unusually high compressive loads during mastication. The tooth-jaw complex is composed mainly of flat molar-like teeth characterized by the
absence of roots.
Objectives
To understand the structure-function relationships of the bone at the interface
of the tooth and the mandible.
Materials & methods
Jaws of adult black drum fish (Pogonias cromis) from Texas were studied.
Reflected light microscopy and back-scattered electron (BSE) microscopy of
polished sections, as well as micro-computed tomography (micro-CT) were used to assess at lower resolution the structural organization and density of
the bone near the contact area in 2D and 3D. We then used focused ion beam
with scanning electron microscopy (FIB-SEM) and the serial surface view (SSV) method to examine both demineralized (treated) bone and also dried
mineralized bone (untreated) to characterize the high resolution structure.
Both methods provide structural insights and the comparison shows that the untreated bone provides key structural information using FIB SEM, with less
risk of artifacts.
Results
Micro-CT and 2D polished sections of the mandibular bone revealed a highly
porous structure, with porosities ranging from tens to hundreds of microns in
diameter. MicroCT also showed that the teeth are supported by a circular narrow boney rim. The bone at the bone-tooth interphase is defined by a
small contact surface that is relatively more mineralized than most of the
jawbone. The interphase structure is characterized by the presence of fewer pores of approximately 5 to 50 μm in diameter compared to the other
mandibular bone. The nanoscale level presents the most unexpected
organization in both treated and untreated bones: rods of ordered collagen fibrils with their long axes parallel to the load direction are embedded inside
a disordered matrix. Untreated bones reveal the presence of nano-tubules
with diameters of 40 to 60 nm, which are aligned with the mineralized collagen fibrils.
Conclusions
We show an unexpected structure of bone at the tooth-bone interphase that is subjected to high mechanical loads: the collagen fibril nano-tubules fabric
is a novel structural motif not identified to date in bone. While the orientation
of the mineralized collagen fibrils in the rods provides the greatest resistance to compression, the function of the tubules remains enigmatic.
T 35
High resolution mechanical, elemental and ultrastructural
characterizations during zebrafish caudal fin regeneration P. Y. Chen*1, Y. R. Shih1, F. Bohns1, Y. J. Chuang2 1National Tsing Hua University, Materials Science and Engineering,
Hsinchu, Taiwan 2National Tsing Hua University, Life Science, Hsinchu, Taiwan
Introduction
Mineralization process during zebrafish fin regeneration has been studied,
mainly focused on the gene control mechanism, signal pathways and the roles
of collagen. Crystallization of mineral from amorphous to well-aligned crystal was evaluated by advanced X-ray analyses and cryo-electron
microscopy techniques. However, the micro/nano-scale mechanical
properties of zebrafish fin during regeneration have not been comprehensively investigated. In this study, micro-/nano-mechanical
properties at high spatial resolution of zebrafish caudal fin were investigated
by nanoindentation and PeakForce Quantitative Nanomechanical Mapping (PF-QNM). Mineralization and ultrastructure were evaluated by SEM,
Raman microscopy and electron probe X-ray microanalysis (EPMA) at
different locations and stages during the regeneration process. By combining these techniques, the relationship between the degree of mineralization and
mechanical properties during zebrafish caudal fin regeneration was
elucidated.
Materials & Methods
Wildtype AB strain zebrafish were used for the experiment. Fish caudal fins
were amputated and examined after 7 days post amputation (dpa), 14dpa, 21dpa and 28dpa. Nanoindentation (TI 980 TriboIndenter) was carried out to
probe localized mechanical properties (hardness and reduced elastic
modulus). AFM(PF-QNM) (Dimension Icon, Bruker) was used to analyze the nano-scale mechanical properties of the fin and characterize
nanostructure simultaneously. SEM (SU8010, Hitachi) and EPMA (JXA-
8500F, JEOL) were performed to characterize micro-structural features and high precision elemental analysis (Ca and P). In addition, by combing Raman
spectrometer (HORIBA, iHR550) and laser confocal microscope (Olympus,
IX71), Raman signal mappings were generated to correlate degree of mineralization and corresponding locations.
Results Nanoindentation results showed that the elastic modulus and hardness gradually decreased from proximal to distal regions of regenerated ray.
Nanoindentation mapping of individual proximal ray revealed that elastic modulus at the central region reached ~15GPa and dropped to ~3GPa toward
the edges and similar trends were observed in middle and distal rays. EPMA
analyses showed the decrease of Ca and P concentration from proximal to distal regions of the single ray and the mineral concentration declined from
the center to the edge within an individual ray, in agreement with
nanoindentation results. Additionally, PF-QNM showed low DMT modulus at distal region and higher modulus at the central region of individual ray.
SEM and AFM both showed non-mineralized collagen fibrils at the edge of
the ray while granular mineralized collagen fibrils were discovered at the center. Raman microscopy mapping showed a decrease of mineral signal
from proximal to distal region of regenerated fins after 7, 14, 21, 28 dpa.
Conclusion
We have applied multi-scale approaches, including elemental analysis,
ultrastructural characterization and mechanical testing to establish a
comprehensive analyses platform for the regeneration of zebrafish caudal fin. High precision EPMA elemental analysis and nanoindentation results both
confirmed that the regeneration and mineralization processes of zebrafish fin
start from the center and gradually spread to the edge of individual ray. This mechanical/materials science approach can not only explore bio-
mineralization process, but also have the potential to be used for in vivo
studies in the future.
T 36
Misorientation and enhanced hardness in tooth enamel C. Stifler*1, C. Y. Sun1, E. Beniash2, P. Gilbert1 1University of Wisconsin-Madison, Physics, Madison, United States 2University of Pittsburg, Oral Biology, Pittsburg, United States
Introduction
Teeth are subjected to extreme, repetitive forces and wear on a daily basis. Human enamel endures forces up to 770 Newtons, hundreds of times per day,
and must remain functional for decades [1]. By comparison, great white
shark enameloid exerts 7400 N of force when biting, but they shed their teeth regularly [2]. The mechanical stress that the teeth undergo suggests that there
are structural features in enamel that prevent catastrophic failure. While the
morphology of enamel is known to be space-filling [3], aligned groups of microns long, nanometers wide apatite crystals (called rods in humans and
bundles in sharks), the orientation of the crystals remains poorly understood.
Question
Is there a relationship between the degree of crystal misorientation in tooth
enamel of diverse animals and the hardness and elastic modulus?
14
Methods
We used a method called polarization-dependent imaging contrast(PIC)
mapping [4,5] to reveal the crystal orientations within rods in human or
mouse enamel and in bundles in sharkand parrotfish enameloid. PIC-mapping is a synchrotron method developed about a decade ago and has since
been refined, so that it can quantitatively measure c-axis orientations in
carbonate and apatite biominerals [6,7]. To create PIC maps, a series of photoelectron emission microscopy (PEEM) images ranging in x-ray
polarization from parallel to perpendicular is acquired the calcium L-edge
[8]. The intensity versus polarization curve that exists at every pixel in the stack is fit to a cosine squared curve according to Malus' law and orientation
information is extracted from the fit parameters.The colors in PIC maps
correspond to the c-axis orientation relative to the polarization of the x-rays: hue is the in-plane angle and brightness is the out-of-plane angle.The enamel
samples are embedded, polished and coated before analysis, and the x-rays
leave minimal damage to the sample [9,10].
Results
Analysis of PIC maps from mouse enamel [8],human enamel [7], great white
shark enameloid, and parrotfish enameloid [11] indicates that c-axis orientations of adjacent crystals are slightly misoriented by a few degrees,
the median misorientation ranging from 1.5-4°. In the case of human enamel,
crystals within a single rod can be misoriented up to 30-90°, but this change is gradual, similar to what was observed in nacre [7,12]. The observed
misorientation is positively correlated with the hardness and elastic modulus,
with R=0.86 and R=0.75, respectively.
Conclusions
Increased crystal misorientation in enamel is related to, and possibly causes enhanced hardness.
References
1.S Varga, S Spalj, ML Varga, SA Milosevic, S Mestrovic, M Slaj, European Journal of Orthodontics, (2011), DOI: 10.1093/ejo/cjq097
2.S Wroe, DR Huber, M Lowry, C McHenry, K Moreno, P Clausen, TL
Ferrara, E Cunningham, MN Dean, AP Summers, J. Zoology (2008), DOI: 10.1111/j.1469-7998.2008.00494.x
3.L Yang, CE Killian, M Kunz, N Tamura, PUPA Gilbert, RSC-Nanoscale
(2011), DOI: 10.1039/C0NR00697A 4.PUPA Gilbert, A Young, SN Coppersmith, Proc Natl Acad Sci USA
(2011), DOI: 10.1073/pnas.1107917108
5.CE Killian, RA Metzler, YUT Gong, TH Churchill, IC Olson, V Trubetskoy, MB Christensen, JH Fournelle, F De Carlo, S Cohen, J
Mahamid, FH Wilt, A Scholl, A Young, A Doran, SN Coppersmith, PUPA
Gilbert, Adv Funct Mater (2011), DOI: 10.1002/adfm.201001546 6.RA Metzler, M Abrecht, RM Olabisi, D Ariosa, CJ Johnson, BH Frazer,
SN Coppersmith, PUPA Gilbert, Phys. Rev. Lett. (2007), DOI:
10.1103/PhysRevLett.98.268102 7.E Beniash, CA Stifler, C-Y Sun, GS Jung, Z Qin, MJ Buehler, PUPA
Gilbert, submitted (2018),
8.CA Stifler, N Kølln Wittig, M Sassi, C-Y Sun, MA Marcus, H Birkedal, E Beniash, KM Rosso, PUPA Gilbert, J Am Chem Soc (2018),
DOI:10.1021/jacs.8b05547
9.T Parasassi, O Sapora, AM Giusti, G De Stasio, G Ravagnan, In J Rad Biol (1991), DOI:10.1080/09553009114550061
10.G De Stasio, BH Frazer, B Gilbert, KL Richter, JW Valley,
Ultramicroscopy (2003), DOI:10.1016/S0304-3991(03)00088-3 11.M Marcus, S Amini, CA Stifler, CY Sun, N Tamura, HA Bechtel, DY
Parkinson, HS Barnard, XXX Zhang, JQ Isaiah Chua, A Miserez, PUPA.
Gilbert, ACS Nano (2017), DOI:10.1021/acsnano.7b05044 12.RT DeVol, C-Y Sun, MA Marcus, SN Coppersmith, SCB Myneni, PUPA
Gilbert, J Am Chem Soc (2015), DOI:10.1021/jacs.5b07931
Funding Sources NSF: DMR-1603192
DOE: DE-FG02-07ER15899
DOE: DE-AC02-05CH11231
T 37
Investigation of Mg2+ incorporation into deciduous enamel and
its effect on the mechanical properties V. Kovacs Kis*1, A. Sulyok1, M. Hegedűs2, I. Kovács2,3, Z. Kovács2 1Centre for Energy Research Hungarian Academy of Sciences, Thin Film
Physics Laboratory, Budapest, Hungary 2Eötvös University, Department of Materials Physics, Budapest, Hungary 3Research Centre for Astronomy and Earth Sciences, HAS, Budapest, Hungary
The substitution of bivalent cations exhibiting biological activity, such as
Mg2+, in apatite, the main mineral reservoir of calcium and phosphorus in
vertebrates, is an intriguing research topic in biomaterial synthesis and fundamental biochemistry as well. Mg2+ is known to inhibit the growth of
apatite crystalline nuclei in biological environment [1]. Recently in has been
revealed that a Mg-rich amorphous calcium phosphate phase exists between the apatite nanocrystals in human dental enamel [2]. At the same time, it has
been demonstrated that Mg2+ incorporation into permanent tooth enamel
introduces changes in crystal structure, hardness and whiteness of the enamel [3]. Deciduous dental enamel has some specific microstructural features
which are different from permanent tooth enamel, e.g. the aprismatic outer
layer, having a decisive role in mechanical response and also in corrosion resistance and caries evolution.
In this contribution we investigate structural and mechanical properties of
sound deciduous molar enamel as function of Mg2+ incorporation via ion-exchange reactions. Furthermore, to elucidate the Mg2+ interaction with Ca-
phosphate nanoparticles, the same Mg-exchange experiments were
performed on synthetic hydroxylapatite (HAP) and amorphous calcium phosphate (ACP) nanopowders.
Structural investigation is carried out using micro-X-ray diffraction (µXRD)
scanning and transmission electron microscopy methods (SEM, TEM). Mechanical testing was done using depth sensing nanoindentation. The ion
exchange experiments were monitored by X-ray photoelectron spectroscopy
(XPS) and energy dispersive spectroscopy (EDS) in the TEM. Untreated sound enamel exhibits strong correlation between Mg2+
concentration, microstructure and nanohardness. The average (Ca+Mg)/P
value is 1.6, close to stoichiometric apatites. According to XPS, ion-exchange experiments on sound enmel resulted an increased Mg2+
concentration on the enamel surface up to 6 at% which stabilized at ca. 3 at%
with (Ca+Mg)/P =2.55 after 30 min sputtering time, corresponding to ca. 50 nm depth. Synthetic HAP sample exhibited less increase of Mg2+ content,
from 0.17 at% up to 1.95 at% and (Ca+Mg)/P from 1.64 up to 1.94. In case
of the ACP nanopowder, the formation of a new, probably amorphous phase was observed with characteristic morphology and Ca:Mg:P =1 : 1 : 2 ratio.
In case of HAP nanopowder, the Mg2+ incorporation occurs through surface hydration of the individual nanoparticles, as indicated by the increase of
(Ca+Mg)/P ratio. ACP nanopowder proved to be highly reactive in Mg-rich
environment. Enamel is a compact ceramic material, with crystallite size and morphology similar to HAP nanopowder but much less free crystalline
surfaces for hydration. The very high amount of incorporated Mg2+ even at
50 nm from the surface and the (Ca+Mg)/P ratio is supposed to be related to the low crystallinity material, probably with organic residue between the
enamel forming apatite nanowires. This intercrystalline material can serve as
either chemical or physical channel for Mg2+ ions, by faster dissolution rate or the presence of nanofractures, respectively. The effect of Mg2+ content on
the nanohardess is also discussed.
[1] Ding, Pan et al. (2014) Cryst. Growth Des. 14, 763−769 [2] La Fontaine, Zavgorodniy et al. (2016) Sci. Adv. 2, e1601145
[3] Abdallah, Eimar, Basset et al. (2016) Acta Biomaterialia 37, 174–183.
T 40
Osteoblast behavior and mineralization onto substrates with
controlled topographies R. Silva dos Santos1, P. Rougerie2, K. Anselme3, M. Farina*2 1Federal University of Rio de Janeiro, Institute of Biophysics Carlos Chagas Filho, Rio de Janeiro, Brazil 2Federal University of Rio de Janeiro, Institute of Biomedical Sciences, Rio
de Janeiro, Brazil 3Université de Haute-Alsace, Mulhouse, France
Introduction Topographical patterns can affect cell adhesion, proliferation, migration,
differentiation and gene expression. Osteoblasts in vivo encounter large,
curved topographical patterns such as Howship"s lacunae walls. Studying whether and how curved topography affect osteoblast biology and bone
matrix production thus can help us to understand bone formation in vivo and
enhance osteo-integrative processes in vivo. Interestingly, some studies report that artificial topographical elements like pillars or roughness can
improve matrix mineralization in vitro. However, these topographies are
usually irregular and/or contain edges. Therefore, they are of limited use to understand the reaction of osteoblast to curved surfaces more akin to what
they encounter in vivo. The aim of this work is thus to evaluate the influence
of curved substrate topography on osteoblast arrangement and mineralization, and the possibility to harness it to reproduce in vitro matrix
organization patterns observed in vivo, as in parallel fibered bone or osteons.
Methods Polydimethylsiloxane (PDMS) scaffolds were produced presenting three
types of cell-scale topographies: either edge-containing or smoothly curved
anisotropic substrates (grooves and ridges) or smoothly curved isotropic substrate (egg-box). The smoothly curved surfaces (isotropic or anisotropic)
are expected to better mimic in vivo environments compared to more classic
edge-containing surfaces. The scaffolds were subsequently coated with fibronectin, and further used for seeding primary rat calvaria preosteoblasts
(F-OST cells) that were then induced to mineralization. Cells attachment and
proliferation, quantity, composition and organization of the bone-like extracellular matrix were evaluated. Mineralized deposits were assessed with
Alizarin and Von Kossa stainings and cell distribution and orientation were
observed by staining for the actin cytoskeleton and laser scanning confocal microscopy.
15
Results Osteoblast cells aligned with the scaffold axes in all anisotropic substrates
although it was more evident in the topography with straight edges.
Mineralization occurred after two weeks without induction factors and after a few days upon induction, apparently also following the orientation of the
ridges and grooves, and it was more intense on the scaffold with straight
edges. The mineral deposits initiated in concave regions in all topographies, showing an alignment of the matrix with the substrates, which was not
observed in the control (flat PDMS). After approximately 10 days the
deposits tended to occupy the whole substrate. From confocal imaging we were able to generate topographical maps and 3D
reconstruction of our surfaces and determined the osteoblast positioning
relative to the local curvature at different stages of their proliferation and maturation.
Conclusions Osteoblasts cultivated over anisotropic surfaces orientate along the topography axis, indicating a topography-induced cell guidance and
suggesting a possible anisotropy of the produced mineralized matrix. We
found also that biomineralization initiated at concave regions. Putting together, these findings can help in the design of topography inducing
scaffolds for different purposes in biomineralization and bioengineering, as
the in vitro production of bone like tissue.
T 41
Influence of mineral properties and organic matrix
composition and structure on bone mineral dissolution A. Rodriguez-Navarro*1, N. Dominguez-Gasca1, C. Benavides-Reyes1, E. Sanchez-Rodriguez1, M. Greiner2, W. W. Schmahl2 1Universidad de Granada, Granada, Spain 2Ludwig-Maximilians-Universität, Department of Earth and Enviromental Sciences, Munich, Germany
Bone mineral dissolution is a highly complex process due to large chemical and structural heterogeneity of bone tissue. Bone mineral dissolution is
facilitated by the small crystal size of apatite crystals and large amount of
ionic substitutions. It is also dependent on the associated collagen matrix in which apatite crystals are integrated and on other proteins absorbed on crystal
surfaces. To better understand this process, we have studied in detail how
bone mineral chemistry and structure changes during demineralization using different analytical techniques such as electron microscopy, 2D X-ray
diffraction and infrared spectroscopy. We show that bone mineral and organic matrix characteristics of different types of bone tissues (i.e., bovine
enamel, bovine and avian cortical bone, avian medullary bone) strongly
influence bone mineral solubility and dissolution behavior. For instance, medullary bone mineral which is poorly crystalline and its organic matrix is
non-collagenous, dissolves much more rapidly than any other type of bone.
It is also shown than during demineralization, there is a selective dissolution of poorly crystalline disorganized bone mineral rich in labile carbonate.The
information gathered in this work provides a more complete picture of how
bone mineral dissolution occurs which is needed to understand basic but highly important processes such bone formation and resorption during the
bone remodeling.
T 42
Coral skeleton formation ? Concentrations and temporal
dynamics of pH, [CO32-] and [Ca2
+] in the extracellular
calcifying medium of a live tropical coral D. S. Sevilgen*1, A. Venn1, V. Planas-Bielsa1, E. Tarnbutté1, D. Zoccola1,
S. Tarnbutté1 1Centre Scientifique de Monaco, Monaco, Monaco
To build their skeleton, stony corals precipitate calcium carbonate (CaCO3 in
the form of aragonite) within an extracellular calcifying medium (ECM) which is located between the skeleton and the coral tissue. Increases in
important calcification parameters such as pH, calcium concentration [Ca2+]
and dissolved inorganic carbon species (DIC, e.g. [CO32-]) in the ECM can
facilitate the formation of CaCO3. Indeed, elevation of these parameters
increases the aragonite saturation state (Ωarag), a determinant that describes
the favorability of CaCO3 to form from solution. The objectives of our study were to access the ECM of live coral directly
with microsensors and conduct real time in vivo measurements in the ECM
of the growing edge of Stylophora pistillata microcolonies. The aim was to evaluate if and to which extend pH, [CO3
2-] and [Ca2+] are elevated.
Furthermore, we aimed at resolving temporal dynamics of the different ion
concentrations. Microsensor measurements were facilitated by inverted brightfield- and confocal microscopy and images of the studied coral region
were taken frequently throughout the experiments.
Our data showed that all parameters within the ECM, as well as derived DIC concentrations and Ωarag, were significantly elevated above seawater values.
Furthermore, by combining different approaches, we were able to follow
temporal dynamics of pH, [CO32-] and [Ca2
+] in the ECM and observe interrelations in the different ion concentrations.
Our study provides the most comprehensive in vivo characterization of
directly measured ECM carbonate chemistry parameters in a single live coral species to date, and highlights the role of active transport mechanisms for the
biological control of ECM carbonate chemistry. Moreover, the novel insights
into the temporal dynamics of the different ion concentrations improve understanding of carbonate chemistry dynamics during CaCO3 formation in
the ECM.
T 43
Modelling coral calcification - isotope and element fluxes in a
tightly constrained system F. Böhm*1, I. Taubner1, A. Eisenhauer1, M. Bleich2 1Geomar, Kiel, Germany 2University of Kiel, Institute of Physiology, Kiel, Germany
Isotopes are useful tools for studying biomineralization, where mineral formation mechanisms, material sources and transport pathways are reflected
in the isotopic composition of biominerals. On the other hand, applications
of isotopes as environmental proxies recorded in biominerals can profit siginificantly from a better understanding of the processing of elements and
isotopes during biomineralization.
We develop a numerical model of elemental and isotopic fluxes in scleractinian corals. Based on two recently published coral boxmodels (1-3)
our model quantifies ionic and molecular fluxes between seawater and coral
compartments: oral/aboral epithelia, coelenteron, calcifying medium, skeleton. It includes carbonic and boric acid chemistry and isotopes, Ca2+ and
its isotopes, and O2. Trans- and para-cellular fluxes are implemented.
The ion permeability of the oral epithelium was tuned to 45Ca tracer data and Ussing chamber results (4, 5). Diffusive ion permeabilities of the aboral
epithelium and the skeleton are constrained by electro-physiological data
from Ussing chamber experiments on Stylophora pistillata colonies (6). The para- and trans-cellular model fluxes were adjusted to yield calcification rates
equivalent to an extension on the order of 1 cm/year.
We find that the aboral epithelium is extremely tight. Para-cellular fluxes (2) and diffusion through the skeleton are of minor importance. Trans-cellular
transport is the only significant source of ions for calcification.
While small molecules like CO2 and B(OH)3 diffuse through cells, trans-membrane transport is necessary for ions like HCO3
-, Ca2+, Sr2+ or B(OH)4-.
Constraints from isotopes are used to design possible transport pathways in the model: While B/Ca ratios observed in coral skeletons can be achieved
with trans-cellular boric acid diffusion, the resulting boron isotope
composition disagrees with known coral values. However, correct isotope ratios are achieved assuming co-transport of borate ions with HCO3
- through
the cell membranes. Consequently, boron isotopes in corals are influenced
by pH and by bicarbonate transport rates. (1) Hohn S., Merico A. (2012) Biogeosci. 9, 4441-4454. (2) Hohn S., Merico
A. (2015) Front. Earth Sci. 2, 37. (3) Nakamura T. et al. (2013) Coral Reefs
32, 779-794. (4) Furla P.et al. (2000) J. exp. Biol. 203, 3445-3457. (5) Tambutté" E. et al. (2012) Proc. Royal Soc. B, 279, 19-27. (6) Taubner et al.
(2017) Limnol. Oceanogr. Meth., 15, 753-765.
T 44
Ion transporter gene expression is linked to the thermal
sensitivity of coral calcification C. Bernardet1, E. Tambutté1, N. Techer1, S. Tambutté1, A. Venn*1 1Centre Scientifique de Monaco, Marine Biology Department, Monaco, Monaco
Reef coral biomineralization is sensitive to temperature, which is one reason why coral reef ecosystems are threatened by climate change, but the
mechanisms underlying the thermal sensitivity of corals are poorly
understood. Furthermore, light is also a key factor in modulating biomineralization rates but a mechanistic understanding of how light
enhances coral calcification is lacking. We carried out a controlled laboratory
study that characterized the thermal performance curve (TPC) of calcification in light and darkness in the widely-used model coral species
Stylophora pistillata. Using the TPC to target low and high temperatures at
which calcification rates were depressed, we used gene expression analysis to investigate the role of ion transport mechanisms in the coral"s thermal
response. We focused the study on genes with functions in the transport and
regulation of dissolved inorganic carbon, calcium and H+. Our findings reveal a high degree of coherence between physiological responses (e.g.
calcification and respiration) with distinct gene expression patterns to the
different temperatures, and also to day and night conditions. At high and low temperatures, a core gene expression pattern emerged, pointing to thermal
effects on dissolved inorganic carbon transport mechanisms linked to
reductions in calcification rate. At high temperature but not low temperature, light stimulated calcification and the response of a more functionally diverse
group of genes. Overall, our results highlight biological control via ion
16
transport mechanisms linked to the thermal sensitivity of calcification, which is a subject of growing interest to emerging fields of study that seek to
improve the resilience of corals to climate change.
T 45
DNP-enhanced NMR of metamorphosing young corals shows
that skeleton construction entails modulation of organic
material S. Nasser1, M. Neder2, B. Uluca3, U. Akbey3, H. Heise3, T. Mass2, G.
Goobes*1 1Bar Ilan University, Chemistry, Ramat Gan, Israel 2University of Haifa, Marine Biology, Haifa, Israel 3Juelich Research Institute, Institute of Complex Systems, Juelich, Germany
The ability of corals to maintain homeostasis and mineralize has been
compromised by ocean acidification and temperature rise causing reefs to
recede and even vanish. Therefore, corals have been serving lately as important proxies of environmental impact on marine life. Early in their life,
corals undergo transformation from a swimming organism (planula) to an
benthic immobile one (polyp), that lives in colonies and forms exoskeleton for protection. During this metamorphosis process, they are vulnerable
leaving them exposed to detrimental external changes. It is therefore very
important to carefully characterize the two developmental states – planula and polyp- and to be able to assist corals withstand ongoing hazardous
variations in their surroundings.
Recently, we have shown using 13C MAS NMR on whole 13C-labeled young Stylophora pistillata corals that mineralization starts before the coral
settles. We also found using 2D 13C DARR that Glu-rich proteins bind soft amorphous mineral in the planula and Asp-rich proteins bind aragonite
crystals in the polyp. Regulation of the two mineral states by the disparate
proteins suited well the needs of the coral changing from motile to sessile. Here, we expand investigations of the metamorphosis using DNP-enhanced
MAS NMR measurements, obtaining favorable signal enhancements of
about 16- to 48-fold. Using 2D 13C DQ-SQ and PDSD measurements on the intact corals, labeled either via 13C carbonate/glycine or via 13C6
glucose/glycine, we follow the changes in organic level production related to
the transformation and the onset of aragonite precipitation. We monitor contribution of symbiotic dynoflagellates via carbonate metabolism by using
the latter as a food source.
The carbon fingerprint changes observed relate to increased carbohydrate production and certain proteins which were not detected without
enhancement. These changes are indicative of the transitions entailing onset
of colonization and expedited mineralization effort initiated after settling on the bottom of the ocean.
T 46
How inorganic spherulites grow- learning from corals C. Y. Sun*1,2, L. Gránásy3, C. Stifler1, T. Zaquin4, R. Chopdekar5, N.
Tamura5, J. Weaver6, J. Zhang1, S. Goffredo7, G. Falini8, M. Marcus5, T.
Pusztai3, T. Mass4, P. Gilbert1,2,9 1University of Wisconsin - Madison, Physics, Madison, United States 2University of Wisconsin - Madison, Materials Science, Madison, United States 3Wigner Research Centre for Physics, Institute for Solid State Physics and
Optics, Budapest, Hungary 4University of Haifa, Marine Biology, Haifa, Israel 5Lawrence Berkeley National Laboratory, Advanced Light Source,
Berkeley, United States 6Harvard University, Wyss Institute for Biologically Inspired Engineering,
Cambridge, United States 7University of Bologna, Biological, Geological and Environmental Sciences, Bologna, Italy 8University of Bologna, Chemistry, Bologna, Italy 9University of Wisconsin - Madison, Chemistry, Geoscience, Madison, United States
Introduction Modern coral skeletons are commonly assumed to consist of aragonite
crystals that grow spherulitically, that is, acicular crystals radiating from
common centers and fill space [1]. Although the name "spherulite" comes directly from its shape, morphology alone can be misleading. In an earlier
study, we determined quantitatively and found that spherulitic aragonite
crystals, both synthetic and biogenic from coral skeletons, exhibit a 0-35º misorientation of c-axes across grain boundaries [2]. Previously coral
skeletons were only been identified as spherulitic from morphological
observations, now we set out to characterize the crystal orientation distributions in coral skeletons from various genera and species to reveal
their structural differences.
Objectives The aim is to fully characterize the structure and crystal orientations in
diverse coral skeletons and to reveal the mechanism of spherulitic growth in
general.
Materials and Methods
Twelve different coral species analyzed here include different clades,
different growth morphologies, and different geographic origins. Polarization-dependent Imaging Contrast (PIC) mapping was used to
measure quantitative crystal orientations. PIC mapping was done using the
Photoelectron Emission spectroMicroscopy (PEEM) at ALS, LBNL. PIC mapping utilizes X-ray linear dichroism, which, for coral, is maximum at the
O K-edge π* peak (534 eV), and is done by rotating the linear polarization
of the illuminating X-rays from horizontal to vertical in 5º steps, acquiring a PEEM image at each polarization, and then fitting the intensity variation
versus polarization angle to a cosine-square curve, from which the c-axis for
each pixel can be deduced [3-5]. Electron backscattered diffraction and X-ray microdiffraction were done for further grain boundary analyses.
Phase-field simulation models with an orientation field [6] were used to
support the proposed formation mechanism.
Results Quantitative PIC maps confirmed that all coral skeletons form aragonite
spherulites. In addition, we discovered that 4 out of the 12 species show a different growth form of aragonite with tiny (0.2-2 µm), randomly oriented,
equant crystals, we termed "sprinkles". With quantitative PIC mapping, we
confirm that sprinkles are not spherulites due to their much larger c-axis misorientation angles across grain boundaries. We propose that the randomly
oriented sprinkles are the initially nucleated crystals, which later coarsen into spherulites: crystals with radially oriented c-axes have more space to grow
and thus become larger, at the expense of smaller, randomly oriented
sprinkles. Phase-field simulations support our proposed mechanism: in all spherulites the first nucleated crystals are randomly oriented sprinkles, which
then coarsen and become only radially oriented crystals.
Conclusions With quantitative crystal orientation analysis, we discovered randomly
oriented sprinkles in coral skeletons. We propose that sprinkles are the early-
stage nucleated crystals in all spherulites, whether biogenic, synthetic, or geologic, and then radially oriented crystals coarsen at the expense of non-
radially oriented ones. Phase-field theory confirms that this is indeed the
case. [1] Yang 2011, DOI: 10.1039/C0NR00697A
[2] Sun 2017, DOI: 10.1021/acsnano.7b00127
[3] Metzler 2007, DOI: 10.1103/PhysRevLett.98.268102 [4] Gilbert 2011, DOI: 10.1073/pnas.1107917108
[5] Killian 2011, DOI: 10.1002/adfm.201001546
[6] Gránásy 2005, DOI: 10.1103/PhysRevE.72.011605
T 47
Anisotropic lattice distortions caused by photosymbiosis in
scleractinian corals I. Coronado*1, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland
The symbiosis between scleractinian corals (host) and unicellular zooxanthellae (dinoflagellate algae, Symbiodinium spp), is the key to the
success of modern reefs. Symbionts deliver to coral host nutrients, resulting
from photosynthesis, and promote the light-enhanced skeletal calcification. Coral-algae symbiosis has had an important role in the reef evolution and
allowed them to thrive in different environments through the geological
record. Unfortunately, the identification of symbiosis in the fossil record is a paramount challenge. Several criteria have been applied with the purpose to
differentiate Recent and fossil symbiotic and asymbiotic corals on the basis
of their skeletons due to the lack of fossilization of zooxanthellae. Morphological, bio-geochemical, and lately microstructural differences have
been applied in order to distinguish the presence or absence of
photosymbionts. High-resolution X-ray diffraction (HR-XRD) studies have shown that crystal
lattice of bio-aragonite, in the molluscs and coral skeletons, is anisotropically
distorted as compared with that of geological counterparts. It has been suggested that these distortions result from the intercalation of organic
molecules into the crystallographic lattice during the biomineralization
process. It was assumed by our hypothesis that distinct biogeochemical differences found between symbiotic and asymbiotic corals should also be
reflected in changes of lattice parameters of bio-aragonite of these two
groups. In order to test this, we sampled a large suite of coral skeletons (30 symbiotic and 24 asymbiotic taxa) representing different phylogenetic lines.
High-resolution X-ray powder diffraction measurements were carried out at
MCX beamline of the Elettra Synchrotron Radiation Facility. The structural parameters were refined by Rietveld analysis and the lattice distortions (as
Δd/drf), crystallite size and microstrain analyses were performed. To
correlate the lattice distortions data with information about inorganic and organic molecules trapped into the crystals we collected also X-ray powder
diffraction data of isochronously annealed and bleached in block at different
17
time treatments of skeletons of selected taxa of both ecological groups. We also performed thermogravimetric analyses under an N2 atmosphere and
mass-spectroscopy measurements (using ICP-MS).
A linear variation between the lattice parameters of symbiotic and asymbiotic corals was observed. Orthorhombic constant ratios clustered both ecological
groups, confirming our original hypothesis. In addition, significant
differences in the biogeochemical composition were found, showing a complex correlation with anisotropic distortions. The observed
crystallographic vital effect seems to be related to physiological changes
resulting from the interaction between symbiont and coral host but also by the subsequent ecological variations of the studied samples.
Acknowledgements: This work was supported by the National Science
Centre (Poland) grant 2017/25/B/ST10/02221. The authors acknowledge the Elettra Synchrotron Radiation Facility (Trieste, Italy) for provision of MCX
beamline and synchrotron facilities (proposal: 20170388).
T 48
In-vivo and in-situ micro-Raman spectroscopy and imaging
study of mineral formation in the primary polyps of
pocilloporoidea corals I. Pinkas*1, M. Neder2, P. P. Laissue3, A. Akiva4, D. Akkaynak5, M.
Albéric6, O. Späker6, Y. Politi6, T. Mass2 1Weizmann Institute of Science, Chemical Research Support, Rehovot, Israel 2University of Haifa, Department of Marine Biology, Haifa, Israel 3University of Essex, School of Biological Sciences, Colchester, United
Kingdom 4Eindhoven University of Technology, Chemical Engineering and Chemistry, Eindhoven, Netherlands 5Princeton University, Department of Ecology & Evolutionary Biology ,
Princeton, United States 6Max Planck Institute of Colloids and Interfaces, Biomaterials, Potsdam, Germany
Introduction In reef-building corals, the earliest stages of mineral formation are critical to
the construction of reefs and their survival potential. The short time window of coral settlement, when massive, rapid calcification occurs, provides a
unique opportunity to study a range of bio-carbonate morphologies and to
understand the transformation of mineral phases into the mature coral exoskeleton.
Objectives To this end, we study two members of the pocilloporoid stony corals, Stylophora pistillata and Pocillopora acuta, during the first few days
after settlement. Following our discovery that mineralization starts even
before the polyp settlement and that the deposition of minerals is controlled by specific proteins1, we show that the initial mineral phase after settlement
is nascent magnesium calcite (Mg-Calcite), with rod-like structures in P.
acuta, and with dumbbell-like structures in S. pistillata.
Materials and methods The project utilizes several techniques to uncover the mechanism of skeleton
building in the coral larvae. Among them, SEM, EDS, Confocal fluorescence microscopy, XRD, XRF, molecular biology, bioinformatics, and in-
vivo and in-situ micro-Raman spectroscopy and imaging. The organisms
were collected from the Red Sea over several nights in February-May 2016 and 2017 by covering the corals with nets following peak releases of larvae.
Results Our measurements reveal that there are basic growth structures with similar characteristics for the two coral species that consist of Mg-Calcite (rods and
dumbbells). We verify this by both micro-Raman spectroscopy and
XRD/XRF. Confocal microscopy reveals vesicles filled with materials consisting of divalent ions (Ca2+, Mg2+, Sr2+) within the coral cell layers.
These rod and dumbbell-shaped structures, which, most likely, form the
centers of calcification (CoC), grow and merge to build the basic skeletal units of the polyp, the basal plate and the septa, in a spherulitic growth pattern
of aragonite needle shaped crystals. As these initial shapes (dumbbells) have
also been observed in calcium carbonate precipitation by bacteria, we have verified that these do not exist in the coral samples by fluorescence imaging
with fluorophores sensitive to bacteria, and by RNA sequencing of the
samples, showing that no relevant bacterial community is associated with the dumbbell structures.
Conclusions Based on our data, we suggest that mineralization in these corals occurs in three phases: first, ion rich vesicles of amorphous calcium carbonate (ACC)
are formed intracellularly. Then, the vesicles are transferred to the
calcification site, forming rod/dumbbell-shaped structures composed of nascent Mg-Calcite. During the third phase, the Mg-Calcite outer surface is
used for the growth of needle-shaped aragonite crystallites in a spherulitic growth pattern forming the septa and basal plate of the coral. These processes
take place at each center of calcification, starting at different times so that
one can observe all of them in the same polyp. We suggest a reason behind this complex process2.
References 1 Akiva A, Neder M, Kahil K, Gavriel R, Pinkas I, Goobes G, and Mass T,
Minerals in a Pre-Settled Coral Stylophora pistillata Crystallize via Protein
and Ion Changes, Nature Communications, 9, 1880; DOI: 10.1038/s41467-018-04285-7 (2018)
2 Neder M, Laissue P P, Akiva A, Akkaynak D, Albéric M, Spaeker O, Politi
Y, Pinkas I*, and Mass T*, Mineral formation in the primary polyps of pocilloporoid corals, Acta Biomaterialia, DOI:10.1016/j.actbio.2019.07.016
(2019)
T 49
Coral biomineralization toolkit differs in octocorallian vs
hexacorallian P. Ganot*1, N. Le Roy1,2, M. Fritz3, T. Rausch3, D. Aurelle4, A.
Haguenauer4, M. Arenda5, V. Benes3, D. Allemand1, S. Tambutté1 1Centre Scientifique de Monaco, Monaco, Monaco 2INRA, Nouzilly, France 3EMBL, Heidelberg, Germany 4CNRS, Marseille, France 5KAUST, Thuwal, Saudi Arabia
Introduction
Corals are calcifying organisms represented in diverse taxa of Cnidaria,
including Hexacorallia (e.g. reef-building corals), and Octocorallia (e.g. precious red corals). Corallium rubrum, the Mediterranean precious red
coral, produces two types of biomineralized structures, the sclerites and the
axial skeleton. Both are made of high Mg calcite precipitates and an organic matrix (OM) which is secreted by the scleroblast cells and the calcifying
epithelium, respectively. It is noteworthy that the red coral axial skeleton is
the material for one of the oldest forms of jewelry known to man.
Objectives
Hexacorallia and Octocorallia diverged around 550-700 Mya, before the first
mineralized cnidarian fossil dating. How evolutionary conserved are the processes controlling biomineralization between the reef-building corals and
the precious red corals? Although calcification in hexacorallia has been the
subject of several investigations, little is known on octocorallian calcification.
Mat&met
We combined microdissection technics with omics approaches to shed light on the synthesis and composition of the red coral biominerals. Proteomic
analysis of the decalcified biominerals allowed us to identify over 100 different proteins composing the sclerites and axial skeleton OMs.
Transcriptomic analysis of the calcifying tissues enabled us to ascertain
whether the OM proteins were specific to the calcification process or partaken with other regular extracellular matrix processes.
Results
Our results show that 1/ many (but not all) OM proteins are shared between the sclerites and the axial skeleton; 2/ in comparison to the known reef-
building coral OM proteins, octocorals seem to have evolved different
strategies: a marked utilization of collagen fibers but also, many novel proteins; 3/ only half of the red coral OM proteins are specifically expressed
by the calcifying cells; 4/ many of calcifying specific proteins appear to be
co-opted from already existing extracellular matrix gene families.
Conclusion
Coral is a vernacular name: calcification in different cnidarian taxa, although
using an evolutionary conserved "tool kit" (e.g. Carbonic Anhydrases), appears to result from independent recruitments of extracellular matrix genes
involved in other processes.
T 50
Biomineralization in cephalopods- first proteomic data on
Spirula spirula and Argonauta hians M. OUDOT*1, E. FARA1, P. NEIGE1, F. MARIN1 1CNRS / Université de Bourgogne - Franche-Comté, DIJON, France
Cephalopods constitute a major class of molluscs, from which only a part of
the living representatives possesses a calcified shell. The macro-evolutionary history of this clade shows a tendency to shell reduction, internalization and
finally, complete loss, from "basal" forms (nautilus) to the most derived ones
(octopus). Although phylogenetic relationships between living shell-bearing cephalopods are rather well established, molecular mechanisms of shell
formation are still poorly understood. In particular, skeleton-associated
proteins, which are supposed to constitute the 'molecular toolbox' for shell formation, have been partly characterized, and only in two cephalopod
genera, the nautilus and the cuttlefish.
The objective of the present study is to sketch the outlines of the skeletal matrix in cephalopods, to check whether representatives of this class use
similar or totally different 'molecular toolboxes'. In addition, our study aims
at describing precisely the microstructural characteristics observed in the models. Our ongoing study is primarily delimited to two species, the Ram's
horn squid Spirula spirula and the paper nautilus Argonauta hians. Note that
18
in this latter case, the shell is a calcitic eggcase secreted only by females and constitutes a remarkable example of a derived character, unique to argonautid
clade.
To this end, we have extracted - after two cleaning procedures - the shell calcifying matrices of the two studied species and performed proteomic
characterization followed by in silico investigations. The saccharidic
signature of soluble fractions has also been investigated by ELLA tests using a large set of lectins. In the case of S. spirula, all our investigations rest upon
two geographically different batches: one from Canary Islands, one from
Brazil. In parallel, we have studied the shell microstructural features of S. spirula and of A. hians by conventional SEM observations on polished
sections.
The shell of S. spirula has a complex microstructural organization. In particular, SEM observations evidence a peculiar feature, also described
among Spirula"s ancestors and defined as "mural flap": at the junction of the
septa with the shell wall, the lamello-fibrillar structure of the septa is inserted between two sub-layers of the internal microstructure of the wall, in the form
of a bevel. From a biochemical viewpoint, the organic matrix of S. spirula is
essentially proteinaceous and saccharidic with macromolecules of discrete and non-discrete molecular weight. The soluble fraction reacts with WGA,
LEL, jacalin and concanavalin A lectins. Our proteomic investigations
identified several protein hits. However, most of them occur with one peptide only and do not match with already known mollusc skeletal proteins. This
strongly suggests that the shell matrix of the ram"s horn squid is quite
different from that of other shell-bearing molluscs studied so far. We come to a similar preliminary conclusion by analyzing the proteomic data obtained
from the shell matrix of Argonauta hians, also characterized by a majority of 'one-peptide' hits. Our study emphasizes the diversity of shell matrix
components in cephalopods, which may put into question a single molecular
model for describing shell formation in this mollusc class.
T 51
Tools for understanding proteins of unknown function in
biomineralization A. Skeffington*1, A. Ohara2, A. Milentyev3, C. Heintze2, S. Görlich2, N.
Poulsen2, A. Fischer1, M. Brzezinka1, M. Gorka1, A. Graf1, N. Kröger2, A. Scheffel1 1MPIMP, Organelle Biology and Biotechnology, Potsdam-Golm, Germany 2B CUBE Center for Molecular Bioengineering, Dresden, Germany 3MPI of Molecular Cell Biology and Genetics, Dresden, Germany
Proteins have been found to be intimately associated with biominerals from
a wide range of organisms. Many roles for these proteins have been
postulated, including the stabilisation of amorphous phases, generation of hydrogels and supramolecular templates, control of crystal nucleation and
the control of crystal morphology. When examining the results of a
proteomics experiment designed to identify proteins associated with a given biomineral, studies often focus on those proteins with similarity to proteins
or domains of known function, which have normally been characterised in
organisms that do not biomineralize. However a number of the proteins identified in biomineralization proteomic experiments are normally
annotated as proteins of unknown function and indeed we would expect
proteins important for a particular mode of biomineralization to be largely restricted to those taxa that engage in this process. Thus, it is clear that to
understand taxon specific biomineralization processes we need to understand
the proteins of as yet unknown function associated with biominerals. Molecular genetic tools for many biomineralizing organisms are in the early
stages of development, meaning that experimental investigations into the
roles of proteins of unknown function are highly laborious. Thus, computational tools are required to investigate the properties of the proteins
and thereby guide and focus experimental work. In this presentation I will
describe a range of user-friendly computational tools for the analysis of proteins of unknown function. This includes tools to examine the statistical
properties of sequences, including local and global biases in amino acid
composition, the degree of intrinsic disorder and sequence complexity. In addition, the tools can be applied to find overrepresented motifs and can be
used for the visualisation and exploration of these data. I will also describe a
new method for the identification of novel domains independent of multiple sequence alignment methods. To illustrate the utility of these tools, I will use
our own proteomic data, focussing on the silica associated proteins of
diatoms and the calcite associated proteins of coccolithophores. I will also demonstrate that new information can be found in previously published data
sets using these methods, focussing on data from molluscs and echinoderms.
T 52
Osseointegrative one implant coatings H. Cölfen*1, J. Knaus1, M. Gießl1, D. Schaffarzcyck2 1Universität Konstanz, Physikalische Chemie, Konstanz, Germany 2stimOS GmbH, Konstanz, Germany
Implants are important materials to replace bone. The today commonly used
materials are titanium and polyetheretherketone (PEEK). While titanium is
very hard, PEEK has mechanical properties very similar to bone which is an advantage. However, while titanium is at least partly accepted by the bone
building osteoblast cells, PEEK is completely bioinert. This leads to a bad or
missing connection of the implant to the surrounding bone tissue and revision surgeries are often necessary with a chance of 15 – 40 % depending on the
implant. This is painful for the patient and causes enormous costs.
We have therefore developed a strategy to covalently coat the implant with a bone mimetic layer. We use a coupling chemistry to covalently bind gelatin
or collagen to a PEEK surface or alternatively to titanium. Afterwards, this
layer is mineralized with calcium phosphate resulting in an only 50 – 100 nm thick bone mimetic surface layer. Comparative cell tests show that
Osteoblasts like the bone mimetic layer and secrete collagen as a first step to
building new bone already within the first 12 h. Animal tests with a sheep model show the superior performance of the osseointegrative bone implant
surface in comparison to the common implant materials.
We have extended this work to covalently bound polysaccharide-based coatings using the same approach to bind and mineralize Hyaluronic acid.
Also, fully synthetic bone implant surface modifications are presented.
T 53
Multiscale characterization of bone matrix changes in a pre-
metastatic mouse model of breast cancer C. Liu*1, A. E. Chiou2, I. Moreno1, T. Tang1, W. Wagermaier1, M. Dean1,
P. Fratzl1, C. Fischbach2 1Max Planck Institute of Colloids and Interfaces, Department of
Biomaterials, Potsdam, Germany 2Cornell University, Meinig School of Biomedical Engineering, Ithaca, United States
Introduction Metastatic breast cancer often spreads to bone, resulting in incurable
osteolytic lesions and poor clinical outcome. While bone degradation is a
hallmark of bone metastasis, recent studies indicate that early-stage bone metastasis may depend on tumor cell adhesion to osteogenic regions.
Increasing evidence suggests that primary tumors can prepare metastatic
niches via circulating tumor-derived factors, but it remains unclear whether this also occurs in breast cancer bone metastasis, and if changes in the bone
matrix play a role in this process.
Objectives To test the hypothesis that primary breast cancer can alter bone matrix across
various length scales prior to metastasis, we have applied multiscale analysis techniques to characterize the bones of a mouse model of pre-metastatic
breast cancer.
Methods To model the effects of circulating tumor-derived factors, 3-week female
nude mice (n=5) received daily intraperitoneal injections of either tumor cell
conditioned media (TCM), collected from the human breast cancer cell line MDA-MB-231, or blank media (control), for a period of 3 weeks. Because
the proximal tibia and distal femur are common sites of bone metastasis in
mice, the hindlimbs were harvested for analyses. For each mouse, one tibia was fixed in 70% ethanol and subjected to micro-computed tomography
(micro-CT), backscattered electron (BSE) imaging, confocal laser scanning
microscopy (CLSM), Raman microspectroscopy and laboratory-based small-angle X-ray scattering (SAXS). The contralateral tibia was fixed, decalcified,
and processed for histological analysis. Distal femurs were used to harvest
RNA from trabecular bone for sequencing (RNAseq).
Results Micro-CT analysis of trabecular bone in the proximal tibia of TCM-injected
mice revealed that bone volume fraction, trabecular thickness, and trabecular separation increased significantly relative to control mice, while trabecular
number decreased. BSE agreed with these changes, and further indicated that
the increase in trabecular bone area for TCM-injected mice was most prominent within 200 µm to the metaphyseal growth plate. SAXS analysis
within this region demonstrated that the thickness of mineral crystals does
not change, but their degree of orientation was slightly decreased in TCM-injected mice compared to control mice. Bone dynamic histomorphometry
showed the cortical bone in TCM-injected mice has higher mineral
apposition rate compared to the control. Raman data showed increased mineral-matrix and carbonate-mineral ratios in the endocortical bone of the
TCM group. Ongoing experiments examine differences in bone remodeling
cells via immunostaining, collagen organization via second harmonic generation imaging and gene expression profiles via RNAseq.
Conclusion Together, these results suggest that circulating tumor-derived factors modulate both trabecular and cortical bone structures from the micro- to
nano-scales in sites prone to breast cancer bone metastasis. Changes in the
bone microstructure, degree of mineral crystal orientation, mineral apposition rate and carbonate-mineral ratio suggest that bone remodeling and
osteogenic activity may increase due to tumor-derived factors, which may
provide a favorable metastatic niche in bone, even before their arrival at bone tissues.
19
T 54
Effect of mother-of-pearl feeding on bone loss due to
ovariectomy in the rat K. D. Nguyen1, N. Laroche1, A. Vanden Bossche 1, Y. Bertache1, M. T.
Linossier 1, M. Thomas 1, S. Peyroche 1, M. Normand 1, L. Vico 1, M.
Rousseau*1,2 1U1059 Inserm - Sainbiose (Santé INgéniérie Biologie St-Etienne) Campus
Santé Innovation, Université Jean Monnet, Saint-Priest-en-Jarez, France 2UMR5510 Mateis, CNRS/Lyon University/INSA-Lyon, Lyon, France
Introduction
Previous in vivo and in vitro studies showed that mother-of-pearl or nacre, a natural material of marine origin, influence bone health. Nevertheless,
limited studies investigated the effects of oral nacre supplementation in bone
loss model. In addition, the model of bilaterally ovariectomized (OVX) rats, which mimics the acceleration of bone loss observed in postmenopausal
women due to estrogen deficiency, is well established in investigations of
osteoporotic therapies.
Objective
This study was aimed to investigate the pharmacological effect of oral
administration with powdered nacre on ovariectomy-induced postmenopausal osteoporosis in female rats.
Materials & Methods: Eight-week-old 58 femal Wistar rats were purchased
in the study. At 16 weeks of age, the Baseline group (n = 10) was sacrificed; 48 rats were either sham- operated (Sham group, n=12) or ovariectomized
(n=36). These 36 ovariectomized rats were randomly divided into three
subgroups (n = 12 each): OVX group (the standard diet), OVX CaCO₃ group (250 mg CaCO₃ / kg body weight / day in the standard diet) and OVXB Nacre
group (250 mg Nacre / kg body weight / day in the standard diet). Their
corresponding treatments were administrated orally for 4 weeks. This study was reviewed and approved by the Institutional Animal Care and Use
Committee (IACUC) of St-Etienne Univ (Approval No.: #13401-
2018020615412128 v6). Changes in body weight as well as those in the uterus were measured. PIXImus measured BMD and % fat mass in vivo. The
bone microstructure of the proximal tibia was assessed by μCT imagery in
vivo and ex vivo. The level of mRNA expression of genes involved in bone formation and resorption was analyzed by qRT-PCR. The Kruskal-Wallis
and Mann-Whitney U-tests with the FDR adjustment were used to analyze
the different data.
Results
Changes in body and uterine weights confirmed the OVX model inducing estrogen deficiency (p <0.001). The increase in body weight of the OVX
Nacre group was significantly limited after 2 weeks and 4 weeks compared
with OVX group (p <0.01). The presence of extensive trabecular deterioration (μCT in vivo) of the three OVX groups confirmed the onset of
bone loss. The ex vivo study showed that mother-of-pearl treatment improves
the trabecular bone volume. Representative genes for bone formation and resorption increased significantly in OVX groups compared to the Sham
group (p <0.01). Some genes (e.g. OPN, RANK) are overexpressed in OVX
Nacre group compared with OVX and OVX CaCO₃ group (p <0.05).
Conclusion
The results obtained provide evidence of the protective effect of mother-of-
pearl on weight gain related to estrogen deficiency. We also found an improvement in the trabecular parameters which determined bone
microstructure. In conclusion, our study is the first step to advance towards
the development of a new osteoporosis therapy based on mother-of-pearl.
T 55
Trabecular bone growth and architecture in tibia defects
implanted with carbonated hydroxyapatite microspheres A. Malta Rossi*1, V. Martinez-Zelaya2, N. Lopes Archilha2, M. Calasans-Maia3, M. Farina4, A. Linhares Rossi1
1Brazilian Center for Research in Physics, Condensed Matter, Applied
Physics and Nanoscience, Rio de Janeiro, Brazil 2Brazilian Synchrotron Light Laboratory, Campinas, Brazil, Campinas,
Brazil 3Federal Fluminense University,Niteroi, Brazil 4Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
The trabecular bone architecture and its dependence on the mechanical load have been intensively reported in the literature. In recent work, Nathaly
Reznikov et al. (2016) introduced the concept of inter-trabecular angle to
describe the architecture of the trabecular network in adult human femora. The authors concluded that the trabecular topology present geometrical
motifs with tensegrity.
In the present work, we used Synchrotron Radiation-based X-ray microtomography (SR-μCT), SEM-FIB (Slice and View) and transmission
electron microscopy (TEM) to investigate the trabecular architecture induced
in rat tibia diaphysis defects in the early times of bone growth, in the absence (group 1) and presence of a nanostructured biomaterial (group 2).
The defects were induced in the rat tibia diaphysis for 07, 14 and 21 days. Microspheres (400-600 μm in diameter) composed of nanostructured
carbonated hydroxyapatite (CHA) and sodium alginate were implanted in
group 2 for the same experimental periods. SR-μCT analyses of the tibia defects were conducted in the IMX beamline
of the Brazilian Synchrotron Light Laboratory (LNLS) in Campinas/Brazil.
The reconstructed 3D images were segmented and skeletonized with Avizo (FEI, Oregon, USA), Fiji/ImageJ (http://fiji.sc/Skeletonize3D), PNExtract (
Imperial College and ITA App (Weizmann Institute of Science) software.
The 3D reconstruction of the trabecular network and its skeletonization
were used to determine and quantify morphological parameters such as the
number of edges and nodes, length and thickness distribution, planarity, intra-trabecular angles and the amount biomaterial surface covered with
new bone. After seven days surgery, a trabecular network consisting of
small trabeculae (≈ 5 μm) was already formed in group 1 and progressed to the center of the gap, which was covered with new trabecular bone in 21
days. The trabecular architecture was based on geometrical motives
consisting of 3, 4 and 5 trabecular edges interconnected to a node (3N, 4N and 5N, Nathaly et. al, 2016). The distribution of inter-planar angles (ITA)
had a maximum at 118 °, 106 ° and 96°, for 3N, 4N and 5N motifs,
respectively. After 21 of the CHA implantation, new trabecular bone was adhered to 70% of the spheres fragments surface and occupies the majority
of the gap and the tibia medullary cavity. The presence of the biomaterial
inhibited the trabecular growth (length and thickness) but did not modify
the inter-trabecular angle distribution. The trabecular architecture is lost
when new bone is very close (50-150 μm) and in contact with the biomaterial surface. SEM-FIB and TEM analyses showed that
bone/biomaterial interface was composed of disordered mineral tissue
grown close to the surface, and into the surface pores smaller than 1 μm. The results demonstrated that bone repair induced in rat tibia defects
initiated from interconnected edges and nodes structures, with triangular
planar, tetrahedral and pentahedral geometries. Since the tibia diaphysis was not submitted to extensive mechanical load during the 07 days of the
animal life, we may conclude that trabecular topology was an intrinsic
characteristic of the trabecular bone formation in rats. This inherent feature of the trabecular bone was not altered with the implantation of the
biomaterial, and the presence of numerous nanostructured fragments that
occupied the defect volume and interacted with the newly formed bone. Mineral tissue may be grown in sub-micron porosity of the biomaterial
surface.
T 56
In-situ hydrostatic compression and diffraction reveal
increased stiffness of ashed dentine apatite mineral J. B. Forien*1, C. Fleck2, C. Krywka3, A. C. Deymier4, P. Zaslansky5
1Lawrence Livermore National Laboratory, Livermore, United States 2Technical Universit Berlin, Berlin, Germany 3Helmholtz-Zentrum Geesthacht, Geesthacht, Germany 4University of Connecticut, Farmington, Germany 5Department for Operative and Preventive Dentistry, Charité - Universitätsmedizin Berlin, Berlin, Germany
Tooth dentine is a bone-like material containing carbonated hydroxyapatite
nanoparticles (cHAp) within and surrounding a network of collagen fibrils
with submicrometer diameters. The role of the mineral particles is to stiffen the nanocomposite and to enhance both stiffness and toughness of dentine so
that it can sustain decades of intense daily, cyclic mechanical stress. The
nanometer size of the mineral particles makes the measurement of their elastic properties technically challenging. Consequently, it is widely assumed
that the elastic properties of biogenic hydroxyapatites are identical to those
of geological apatite. However, in a previous study we found that pristine dentine apatite particles are about 20% softer than geological and synthetic
apatites and that the mineral has an average bulk modulus K=82.7 GPa.
In a new series of measurements, we investigate the effect of dentine heat
treatment on the response of the apatite phase of the composite to
compression. X-ray diffraction combined with in situ hydrostatic water-
mediated pressurization were used to track changes in the c and a crystal lattice parameters. Dentine samples were measured wet under pristine
condition, after annealing for 1 hour at 250 deg, and after ashing for 10 hours
at 550 deg. Results show a decrease of ~3% in Young"s modulus from pristine to
partially annealed dentine (250 deg, 1 hr), and an increase of ~7% from
pristine to fully annealed dentine (550 deg, 10 hrs). Similarly, bulk modulus decrease by ~3% and increase by about 7% from pristine to partially and fully
annealed dentine, respectively. The change of properties in cHAp upon heat
treatment is attributed to an initial partial degradation of the organic phase followed by ashing leading to loss of carbonate impurities.
20
T 57
Heat-induced changes in bone mineral structure and
chemistry: transformation from carbonate-apatite to
hydroxyapatite M. Greiner*1, A. B. Rodríguez-Navarro2, M. F. Heinig1, K. Mayer3, B.
Kocsis1, A. Göhring3, A. Toncala3, G. Grupe3, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department of Earth- and
Environmental Science, Munich, Germany 2Universidad de Granada, Departamento Mineralogía y Petrología,
Granada, Spain 3Ludwig-Maximilians-Universität München, Fakultät für Biologie, Anthropologie und Humangenomik, Planegg-Martinsried, Germany
Bone is a complex hierarchically structured composite material constituted of carbonated apatite (bioapatite) nanocrystals mineralizing collagen (type I)
microfibrils. Biological apatite chemical composition is crucially different
from stoichiometric hydroxyapatite being particularly susceptible to marked changes in ionic substitutions on the cationic, phosphate and channel anionic
sites during heating.
Experimental incineration experiments were conducted in order to better understand changes in the complex biomineral during heating. The
experiments were motivated by archeological and forensic research
questions, that can also shed important light on our understanding of the bone mineral structure and properties.
Bovine cortical bone pieces were heated at 100, 200, 300…1000 °C for 150
minutes. In addition, short-time incinerations (10, 20, 30… 60 minutes) at 650 °C and 700 °C were performed to understand the time-dependence of the
reaction in a critical temperature range. Original and heated bone samples were investigated using complementary analytical methods such as X-ray
powder diffraction (XRPD) with advanced profile deconvolution by Rietveld
refinement, Fourier-transform infrared spectroscopy (FTIR), and infrared-coupled thermogravimetric analysis (TGA-FTIR).
No hydroxyl ions are discernible in FTIR spectra of original bone mineral
while CO32- and H2O bands are prominent. Original bone mineral has a XRD
crystallite size with an average of 65 Å in the ab plane and 174 Å along the
c-axis. A pronounced increase of crystallite size at 700 °C after 30 minutes
of heat treatment can be observed, from 205 Å along [001] and an average of 134 Å in the (001) plane (700 °C/30 min) to 392 Å along [001] and an
average of 266 Å in the (001) plane (700 °C/40 min).
Rather than a simple recrystallization, this process can be better described as a reaction with a decrease of CO3
2- and H2O and an increase of hydroxyl
groups in the apatite lattice, as detected by FTIR. Thus, the bovine bone
mineral should definitely be referred to as "carbonate-hydro-apatite" rather than hydroxyapatite.
The carbonate content in bone decreases further with higher treatment
temperatures and only weak carbonate signals are observed in bone heated at 1000 °C. Above 800 °C, buchwaldite (CaNaPO4) is formed from the Na
component of the bone mineral. Signals of combusted organic compounds in
the TGA-FTIR spectra clearly diminish at temperatures higher than 650 °C. The time-dependence of the recrystallization-reaction at 650°-700°C is much
more complex than a simple Boltzmann/Arrhenius exponential relation.
From our data we can conclude that the recrystallization reaction from bioapatite to hydroxyapatite accelerates dramatically after the complete
combustion of all organic compounds of the bone, i.e., when the apatite
grains come into direct contact to each other (Greiner et al. 2019). Greiner, M., Rodríguez-Navarro, A., Heinig, M.F., Mayer, K., Kocsis, B.,
Göhring, A., Toncala, A., Grupe, G., Schmahl, W.W. (2019) Bone
incineration: An experimental study on mineral structure, colour and crystalline state. Journal of Archaeological Science: Reports, 25, 507- 518.
T 58
Mineral deposition and structure in the avian leg tendon E. Macias-Sanchez*1, Z. Zou2, T. Tang1, L. Bertinetti1, N. Tarakina3, W. J.
Landis4, P. Fratzl1 1Max Planck Institute of Colloids and Interfaces, Biomaterials, Potsdam,
Germany 2Wuhan University of Technology, State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan, China 3Max Planck Institute of Colloids and Interfaces, Colloid Chemistry,
Potsdam, Germany 4The University of California, San Francisco, United States
Introduction Mineral deposition in bone and mineralized tendon has been well described
in electron microscopy of longitudinal tissue sections, based on the banding
pattern of collagen and mineral platelets observed mainly parallel to the collagen fiber long axis1. Deposition pattern in transverse sections2-5 is
more uncertain and remains controversial. In particular, the question to which
extent mineral particles are predominantly intra- or extrafibrillar is still debated3-5. The cross-section deposition pattern has been described in terms
of a "lacy pattern" that consists of groups of concentrically arranged, curved
agglomerates of thin mineral particles surrounding unmineralized spaces 30 to 50 nm in diameter3-5. Both the filamentous (longitudinal) and lacy pattern
(transverse) are thought to be different projections of the same ultrastructure,
but the underlying 3D mineral arrangement is yet unclear.
Objectives
In order to understand the formation of these mineral deposition patterns, we
studied early stages of mineralization using a simplified but equivalent model system to bone: the avian leg tendon. Its collagen fibrils, arranged in a
parallel fashion, mineralize as the animal ages and permit the mineralization
process to be monitored precisely. Study aims were to increase understanding of collagen fibril mineralization and to compare resulting data with recently
described bone mineral patterns2-5.
Materials and methods The present work combines Transmission Electron Microscopy (TEM),
Scanning Transmission Electron Microscopy (STEM), and Selected Area
Electron Diffraction (SAED) to elucidate crystal distribution and orientation through the mineralization process. Elemental analysis by means of Energy
Dispersive X-ray Spectroscopy (EDS) was used to characterize the organo-
mineral interface of structures occurring during mineral formation and to asses Ca/P molar ratio.
Results Early mineral aggregates were found in extrafibrillar collagen spaces, following the contour of the fibrils. Crystallites then propagated within fibrils
(at sites where mineral was also outside the same sites) following curved
trajectories. As these foci of mineralization grew, they appeared as radially-oriented clusters of relatively large crystal aggregates. Mineral foci expanded
until they coalesced, encompassing several fibrils and forming a continuous mineral network. The resulting pattern strikingly resembles the lacy pattern
described for bone ultrastructure2-5.
Conclusion These analytical observations support the concept that early stages of
mineralization in avian leg tendon lead to formation of deposition patterns
that are similar to those documented in bone. This result would suggest a possibly common mineralization mechanism for type I collagen-based
materials.
1. Landis, Song, Leith, McEwen, McEwen. 1993. J. Struct. Biol. 110, 39-54
2. Rubin, Jasiuk, Taylor, Rubin, Ganey, Apkarian. 2003. Bone 33, 270-282
3. McNally, Schwarcz, Botton, Arsenault. 2012 PLosOne 7(1): e29258 4. Grandfield, Vuong, Schwarcz. 2018. Calcif. Tiss. Int. 103, 606-616
5. Reznikov, Bilton, Lari, Stevens, Kröger. 2018. Science 360, eaao2189
T 59
Infrared imaging analysis of collagen and hydroxyapatite in
sclerotic aortic valve tissue A. Mieting*1, C. Dittfeld1, A. Jannasch1, K. Plötze1, S. M. Tugtekin1, K.
Matschke1, G. Steiner2 1Dresden University of Technology, Faculty of Medicine Carl Gustav
Carus, Department of Cardiac Surgery, Dresden, Germany 2Dresden University of Technology, Faculty of Medicine Carl Gustav Carus, Clinical Sensoring and Monitoring, Dresden, Germany
Introduction
Aortic valve (AV) fibrosis is accompanied by collagen remodelling which
promotes accumulation of hydroxyapatite, resulting in a pathological
biocomposite. Biomineralization is the main characteristic of calcified aortic valve disease (CAVD) and in glutaraldehyde cross-linked AV bioprostheses
but pathophysiology is still not fully understood. Infrared (IR) spectroscopic
imaging yields spatial information e.g. of the collagen and mineralic composition due to unsupervised chemical information of the spectroscopic
data set by multivariate chemometrics. An IR-spectrum of biomineralization
shows vibrational band assignments for phosphate of the apatite structure and for the carbohydrate moieties of collagen at 950-1200 cm-1 and for the
secondary structure of collagen by amid bands at 1500-1700 cm-1.
Objective
Aim of the study is the investigation and association of different chemical
properties of hydroxyapatite and amid I and II alterations in biomineralized
collagen of calcification nodules in CAVD tissues by IR-spectroscopic imaging followed by multivariate chemometrics.
Materials & methods: Tissue samples of calcified human AV and
bioprostheses were cryosectioned and transferred on CaF2 slides. IR-microscopy: FT-IR spectrometer Vertex 70; infrared microscope Hyperion
3000; MCT focal plane array detector. Before multivariate analysis, the
spectra were evaluated for outliers, corrected for baseline and area normalized (Matlab). Representative parts of the sections were defined from
visible light image. K-means clustering analysis was used to group the
preprocessed infrared spectra. Principle component analysis (PCA) was calculated on correlation matrix of the imaging data set in the spectral ranges
of 950-1200 cm-1 and 1480-1800 cm-1.
Results
Spectral information of initial sample set were grouped by cluster analysis in
spatially distinct mineralic and organic regions with characteristic spectral
21
band features for each tissue group. PC1-PC4 of amid I and II bands of mineralized tissue areas reveal an increased content of β-sheet collagen
conformation demonstrated by signals at 1623, 1673 and 1690 cm-1. In
addition a signal at 1515 cm-1 is detected referring to an increased content of tyrosine residues in mineralized tissue areas of the human AV. The non-
mineralized organic AV tissue is characterized by α-helical collagen
conformation assigned to 1558 and 1662 cm-1. In comparison, glutaraldehyde cross-linked pericardial tissue of bioprosthesis exhibits additionally signals
at 1532 cm-1 for anti-parallel β-sheets and signals at 1684 cm-1 for β-sheets
of collagen. In contrast, changes in the range of 950-1200 cm-1 are similar in AV and bioprosthesis: non-mineralized tissue is characterized by
carbohydrate groups of collagen at 973, 1030 and 1075 cm-1 and mineralized
tissue is characterized by PO43- of apatitic structures at 996 cm-1 and an
assignment for poorly crystalline apatites at 1110 cm-1.
Conclusion
Collagen conformation is modified in calcified AV tissue and in bioprosthesis due to chemical pretreatment or due to the origin of the
pericardial tissue. An increased β-sheet collagen content and a higher
tyrosine content in collagen of mineralized human AV tissue is detected by IR-spectroscopy in an initial dataset. Also in pericardial bioprosthetic tissue
an increased β-sheet content was monitored that may promote degeneration
by calcification.
T 60
In situ nanoscale biomineralization of calcium oxalate kidney
stone in presence of molecular modifiers T. Shokuhfar*1, R. Shahbazian 1 1University of Illinois at Chicago, Bioengineering, Chicago, United States
Calcium oxalate (CaOx) is the primary constituent of kidney stones, calcium storage medium in plants, and may play a role in plant-mediated capture of
atmospheric carbon. Here, CaOx nucleation was studied in real-time with
nanoscale resolution via graphene liquid cell transmission electron microscopy. We observed the co-existence of both classical nucleation and
non-classical nucleation pathways by which the structure and morphology of
CaOx crystals show distinct characteristics. Interestingly, we observed that the addition of citrate to the CaOx solution significantly reduced the
thermodynamic stability of CaOx nuclei, interfering with CaOx nucleation,
and promoted the formation of CaOx dihydrate (COD). This study shows that manipulation of nanoscale formation pathways may influence macroscale
properties dependent on the mineral morphology, crystal structure, and hydration state, such as mechanical properties, solubility, and biological cell
attachment of CaOx.
In Situ GLC-TEM Imaging In situ TEM imaging was achieved via encapsulation of 0.1M CaCl2 and
0.1M NaOx in control samples and 0.1M CaCl2, 0.1M NaOx, and 0.1M
citrate in the citrate treated samples. The samples were added to microcentrifuge tubes and mixed. 0.5µl of the solution was added to a
graphene coated grid. A secondary graphene coated grid was placed
graphene side down on the liquid sample. The grid was then placed into a TEM sample holder. The TEM holder was placed into a vacuum pump to
remove any encapsulated liquid from the TEM holder and the sample. The
sample was then imaged in TEM at 80kV. The increase in the contrast of the particle throughout the video was measured as a function of the grey scale of
the image, where C is the measured greyscale:
∆C=C_particle/C_bacground -1. The electron dose rate was calculated by:
Ψ=(10^5 SI)/(πa^2 )
Where S is the stopping power ((MeVcm^2)/g), I the current, a the radius of the electron beam (m), and 105 converts units SI units to Grays/s.
The STEM-EELS data was acquired using a Hitachi HD2300 STEM
microscope operated at 200 kV. The energy range of 270-577.2 eV was examined with 0.30 eV energy dispersion and EELS aperture of 3mm.
Energy windows of 50 eV were used to compare the ratio of O:Ca using the
O K edge and Ca L edge. Electron exposure was set to 15 seconds. The EELS data collection electron dose rate was between of 0.08 to 38.20e-/Å2s while
the total dose was between 1.16 to 573 e-/Å2.
CONCLUSIONS Here, real-time nanoscale studies show the nanoscale nucleation of CaOx via
two pathways. Classical nucleation causes formation of rhombohedral COM,
the most thermodynamically favorable crystal structure of CaOx, whereas rectangular COM forms via non-classical nucleation. Further, formation of
both ex situ and in situ nanoscale COM via classical nucleation indicates the
lower ∆G and 〖∆G〗^‡ of COM as compared to COD. Citrate increases the
〖∆G〗_COM^‡ over the relatively high 〖∆G〗_COD^‡ to induce the
formation of COD via cyclic aggregation of CaOx rather than via classical
nucleation. The inhibition of CaOx formation by citrate occurs at the
nanoscale, which prevents formation of COM, but with high mixing this energy barrier is overcome to form COD in bulk solutions.
. Since COD does not bind to cell walls as well as COM, citrate may prevent
kidney stone formation by preferentially forming COD to promote CaOx
excretion. Here, the results suggest that this inhibition of COM does not only occur at the microscale but upon the very nanoscale nucleation of CaOx.
T 61
Precipitation mechanisms in renal calcium phosphate plaques
and their relevance for the growth of calcium oxalate kidney
stones I. Sethmann*1, G. Wendt-Nordahl2, T. Knoll2, H. J. Kleebe1 1Technische Universität Darmstadt, Institut für Angewandte
Geowissenschaften, Darmstadt, Germany 2Klinikum Sindelfingen-Böblingen, Urologische Klinik Sindelfingen, Sindelfingen, Germany
Introduction
Calcium oxalate monohydrate (COM) kidney stones often occur attached to
calcium phosphate (CaP) plaques that form in the interstitium of the renal
inner medulla and can come in contact with urine at lesions of the epithelium of the renal pelvis. These CaP precipitates, called Randall"s plaque (RP),
seem to be a precondition for the formation of these COM stones. RP, as a
form of pathological biomineralization, forms in the course of ion re-absorption in the nephrons at high levels of ion concentration in the
interstitial fluid and possibly significantly increased levels of CaP
supersaturation.[1] Comprehensive knowledge of the conditions and mechanisms of the formation of RP may be the key to inhibiting the
precipitation of CaP and preventing the growth of COM renal stones.
Objective
The aim of this study was a microstructural investigation of RP attached to
COM stones in order to conclude on conditions and processes that lead to RP precipitation and initial COM stone growth. Since direct observation of
plaque formation and analyses of the local conditions at the formation sites
is hardly possible, model experiments mimicking the precipitation process have to be designed to enable monitoring the mechanisms involved. The
synthesis of structural analysis and experimental simulation is expected to
lead to a better understanding of RP formation.
Materials and Methods
RP attached to surgically removed COM kidney stones was investigated
using scanning electron microscopy (SEM) and integrated energy-dispersive X-ray (EDX) spectroscopy. Model experiments were performed using
hydrogels with simulated body fluid in a double diffusion set-up for CaP
precipitation mimicking the formation of RP. The precipitation process and its products are currently being characterized using vibrational spectroscopy,
X-ray diffraction and SEM techniques.
Results
In RP, the mesh size of the interstitial tissue appeared to be positively
correlated to the local size of the CaP particles. Larger particles showed
spherical morphologies and laminated internal structures suggesting an initial precipitation as metastable amorphous calcium phosphate (ACP) which may
have crystallized at a later stage.[2] In COM stones, crystals in contact with
RP showed morphologies different from the bulk of the stones and in many places they were encrusted with a thin layer of CaP.[2] Model experiments
show that the density of the gel network is negatively positively correlated
with the waiting time for precipitation and negatively correlated with the spreading rate of the cloud of CaP particles. Further characterization of the
precipitates is in progress.
Conclusions
We conclude that locally differing spatial confinements in the tissue and the
related restrictions of ion diffusion have an impact on particle sizes and
arrangements in RP. Furthermore, we propose that CaP-(super)saturated interstitial fluid may diffuse through RP and into the urine, where it could
trigger COM and CaP precipitation at the RP–urine interface and, hence,
initiate stone growth.
References [1] A.P. Evan, F.L. Coe, J. Lingeman, S. Bledsoe, E.M. Worcester, Am. J.
Physiol. Renal Physiol. 315 (2018) F1236–F1242. [2] I. Sethmann, G.Wendt-Nordahl, T. Knoll, F. Enzmann, L. Simon, H.-J.
Kleebe, Urolithiasis 45 (2017) 235–248.
T 62
Molecular structure of osteopontin fragment at the interface
with calcium ion interaction and within calcium oxalate
monohydrate composite H. Lu*1, S. Alamdari2, S. Roeters3, J. Pfaendtner 2, T. Weidner3, M. Bonn1 1Max Planck Institute for Polymer Research, Molecular Spectroscopy,
Mainz, Germany 2University of Washington, Department of Chemical Engineering, Seattle,
Washington, United States 3Aarhus University, Department of Chemistry, Aarhus, Denmark
Introduction
Calcium oxalate monohydrate (COM) is the major inorganic mineral
component of kidney stone – the chronic human disease threatening peoples's
22
lives for years. Understanding molecular mechanisms of biological control over COM crystallization will promote development of effective stone
disease therapies and stimulate novel strategies for synthesizing biologically
inspired materials. The most potent kidney stone inhibitor is osteopontin (OPN). OPN protein and its peptide fragments are considered to be
intrinsically disordered and they have significant flexibility to bind calcium
ions. Despite previous extensive literatures all attest the great importance of direct interaction between OPN (or OPN peptides) and COM mineral surface
– due to the general difficulty in probing protein (peptide) surface structure
– the exact interaction mechanism and molecular picture of OPN at its mineralization interface are all the time missing.
Objectives
Here we focus on the functional OPN peptide domain (i.e. OPN62-85 H2NSNESHDHMDDMDDEDDDDHVDSQDCOOH). We combine
surface specific sum frequency generation (SFG) spectroscopy and
molecular dynamics simulations to probe peptide structure at the interface with calcium ion interaction and within its inherent COM composite.
Materials & methods
OPN peptides firstly absorbed at air-water interface, by injecting calcium cations and subsequent oxalate anions, we were capable to use SFG to in situ
probe the peptide at the interface with calcium ion interaction and within
mineral composite. SFG spectroscopy is a second order non linear optical spectroscopy by overlapping visible and infrared laser pulse. Its selection
rules dictate that SFG spectroscopy only probes the "interface" without
interference from bulk solutions. Molecular dynamics simulations were performed to complement SFG results. Calculated SFG spectra from
the simulated structure were compared with experimental spectra, thereby further verifying the simulated peptide structure.
Results
OPN peptides restructure strongly with Ca ions at their interface prior to calcium oxalate nucleation. In contrary to one disordered motif, we disclose
that – induced by the bidentate binding Ca ions – OPN peptides adopt highly
ordered beta turn motif. The ordered peptide structure is retained after COM mineralization.
Conclusion
Our results, on the one hand, provide direct molecular picture of OPN key fragment at their inherent relevant interfaces, and on the other hand,
demonstrate the strength of combining SFG spectroscopy and molecular
simulation in probing weakly folded peptides at their functional interfaces.
T 63
Improved biomineralization of lead in aqueous media by
microbial carbonate precipitation assisted with cationic
polypeptide J. He1, W. Li1, V. Achal*1 1Guangdong Technion Israel Institute of Technology, Environmental Engineering, Shantou, China
Introduction
Ever increasing urban expansion, industrial development and anthropogenic
activities are major sources of polluting environment with heavy metals.
Posing with serious environmental problems, heavy metals are the environmental priority pollutants and require ultimate remediation to protect
both health and environment. In recent years, biomineralization involving
ureolytic bacteria producing enzyme urease that leads to carbonate precipitation, chiefly calcite, was found effective in combating heavy metal
toxicity. This biocalcification process, widely known as "microbially
induced carbonate precipitation (MICP)" has the ability to immobilize heavy metals efficiently by precipitating them during urea hydrolysis.
Objectives
Lead (Pb) is widely recognized by its highly toxic and mobile nature, also brings severe negative effect on human health and the environment. Thus,
the process of MICP was adopted in presented research to immobilize Pb.
Further, considering the role of cationic polypeptide in CaCO3 crystallization, study aimed to improve the MICP efficiency to immobilize
Pb significantly.
Materials & methods
To get Pb tolerant bacterial strains, bacteria were isolated from metals
contaminated industrial sludge samples. Urease selection pressure media
were further used to screen urease producing bacteria. The efficient urease producing Pb tolerant bacterial strain was identified based on 16S rRNA gene
sequencing. The bacterial strain was grown in optimized MICP nutrient
media containing urea, CaCl2, and poly-Lys, supplemented with 50 mg/L and 100 mg/L of Pb, for a week. The experiments were also conducted in the
absence of poly-Lys. Urease activity, pH, Pb concentration were measured
at regular interval. The concentration of Pb was analyzed by ICP-MS. The FTIR spectra were recorded in order to know the functional groups involved
in Pb immobilization. The morphological and elemental analyses were carried out with SEM-EDX. The biominerals formed in the process of
remediation of Pb was identified by XRD.
Results
One of the most efficient urease producing and Pb tolerant bacterial strain,
identified as Lysinibacillus sp. was selected in this research. During seven
days of experiment, urease production was significantly more in media with poly-Lys with maximum activity on the fifth day and pH was also on the rise.
The concentration of Pb decreased significantly on the fifth day with removal
efficiency of 81% and 74% without poly-Lys, when the initial Pb concentration was 50 and 100 mg/L, respectively. The addition of poly-Lys
led to further decrease in Pb concentration, with removal efficiency of 87%
and 83%, respectively. The FTIR spectra revealed number of peaks indicative of CO3 bonding and formation of carbonate compounds in MICP
samples; however, such peaks were more intense in the presence of poly-Lys.
XRD identified peaks of calcite, in addition to confirmation on immobilization of Pb in the form of PbCO3.
Conclusion
The presented study improved biomineralization efficiency of carbonate precipitation in metal immobilization with poly-Lys addition.
T 64
Biosynthesis of copper nanoparticles from mine waste J. Ordóñez*1, L. S. Wong-Pinto1 1Universidad de Antofagasta, Department of chemical and mineral process engineering, Antofagasta, Chile
Introduction Chile is one of the most important worldwide producers of copper, which
results in the generation of a large amount of waste, such as ore tailings.
Latest reports indicate that eight billion tons of tailings have been generated from the copper mining industry. In tailings remain a broad spectrum of
species among them copper, that. On the other hand, nanosized materials
have unique properties compared to the macromaterial counterparts, due to their quantum confinement. For this, the application of nanoparticles (NPs)
in strategical, medical and renewable energy devices are in full development.
Cheaper and more eco-friendly techniques for nanoparticles synthesis is the current challenge to increase the application of these materials.
Objective This work aims to synthesise copper nanoparticles (CuNPs) from copper ore tailings, using bacterial biomass of Pseudomonas stutzeri.
Material and methods Samples of ore tailings were collected from porphyry copper deposits located in active mining operations in northern Chile. Mineralogical and chemical
characterisation was carried out by X-ray diffraction (XRD),
microfluorescence short-wave infrared reflectance spectrometry (SWIR), and by atomic absorption spectroscopy (AAS).
Biosynthesis of CuNPs was performed using 10 g/L biomass of P. stutzeri in
two different solutions: i) CuSO4 6 mM, and ii) leached tailings (copper
concentration of 1.4 mM), stirred at 150 rpm for 24 h. After biosynthesis,
biomass was discarded by centrifugation, and the supernatant containing
CuNPs was then analysed by microfluorescence, Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM), X-ray
photoelectron spectroscopy (XPS) and field-emission scanning electron microscopy coupled with energy dispersity X-ray spectroscopy
(FESEM/EDX).
Results Tailings have high levels of copper remaining about 1000 ppm, mainly as
chalcopyrite (CuFeS2). The absence of copper oxides and the predominance
of kaolinite in the clays was verified, which was useful to define the leaching conditions. CuNPs were obtained both synthetic CuSO4 solution and leached
tailing. Preliminary results suggest that the use of fresh biomass show best
biogenic activity than lyophilised one. Analysis of structural variations in the cell wall indicates biosorption and eventually, bioreduction, activity in both
treatments, related to hydroxyl, methylene and carboxyl groups. Various
characterisation techniques confirmed bioreduction; microfluorescence analysis revealed that after biosynthesis, on the surface of biomass, Cu, Fe
and S were detected. AFM images showed the formation of agglomerated
CuNPs. Finer visualisation by FESEM/EDX shows that CuNPs are spherical
with sizes between 20-250 nm. XPS spectra confirmed that CuNPs are not
only Cu0 but also as Cu1+ and Cu2+, being possible the coexistence of copper
oxides and sulphides with the metallic form.
Conclusions Biomass of P. stutzeri can biosynthesise CuNPs from tailings deposits, which
is a novel biotreatment of diluted industrial effluents that allow their valorisation in an eco-friendly way and extracting value from mining waste.
One of the challenges that must be faced is the separation of the nanoparticles
and the reduction of the agglomerated mass.
23
T 65
Bacterial effect on the crystallization of mineral phases in the
human urinary system (based on biomimetic syntheses) A. Izatulina*1, A. Nikolaev1,2, M. Kuz’mina1, O. Frank-Kamenetskaya1, V.
Malyshev3 1St. Petersburg State University, Crystallography, St. Petersburg, Russian Federation 2Grebenshchikov Institute of Silicate Chemistry of the Russian Academy of
Sciences, St. Petersburg, Russian Federation 3S.M. Kirov Military Medical Academy, Microbiology, St. Petersburg, Russian Federation
Urolithiasis is an example of pathogenic mineral formation in the human
body. It is well known that the presence of a variety of bacteria in the urine
is very likely and bacterial inflammation often accompanies stone formation. Under the conditions of model experiments, the effect of bacteria that are
present in human urine (Escherichia coli, Pseudomonas aeruginosa,
Klebsiella pneumoniae and Staphylococcus aureus) on the formation of the renal stone mineral phases, such as brushite, struvite, vitlocite, octacalcium
phosphate, apatite, whewellite, and weddellite, was studied in systems
simulating the composition of human urine and using two types of nutrient media (Muller–Hinton Broth and Meat–Peptone Broth). Multidirectional
changes in the pH values of the solutions were analyzed, which are the result
of all system components" interactions with the crystallization process. It was shown that the presence of bacteria has a different effect on the phosphate
and oxalate phases" formation. The presence of pathogens and nutrient media
significantly affect the precipitant phase composition and the position of the resulting phosphate phase"s crystallization boundaries, which can shift both
to more acidic (struvite, apatite) and more alkaline (brushite) areas. Under
conditions of oxalate mineralization, bacteria accelerate the nucleation of calcium oxalates by almost two times and also increase the amount of oxalate
precipitates along with phosphates and stabilize the calcium oxalate
dihydrate to weddellite. As it can be seen from the reported results and the available literature data, the bacterial effect on oxalate and phosphate phase
formation is different. Thus, in the case of oxalate mineralization, primarily
(most likely), the inflammatory process will contribute to the decrease of oxalate supersaturation in urine due to calcium oxalate crystallization. In the
case of phosphate mineralization, the change in urine pH and the products of
bacterial metabolism will be of major importance. Studies aimed at identifying the specific action of certain microorganisms on the
crystallization of certain mineral phases should serve to develop individual methods of treatment and prevention of urolithiasis. The obtained results
could be regarded as the scientific basis for understanding the mechanisms
of bacterial participation stone formation in the human urinary system and the creation of biotechnological methods for the prevention of this disease.
This work was supported by the Russian Science Foundation (no. 18-77-
00026). The XRD studies have been performed at the X-ray Diffraction Centre of St.Petersburg State University.
T 66
Bio-dynamics of copper sulfide minerals and K+-jarosite
probed by Raman and FTIR microspectroscopy C. Varotsis*1 1Cyprus University of Technology, Environmental Science and Technology, Lemesos, Cyprus
Introduction
Environmental friendly approaches for the proper management of copper production including processing of low grade ore and tails have been
developed. Heap bioleaching is the appropriate technology to handle low
grade copper sulfide ores bearing chalcopyrite, idaite, bornite, chalcocite and covellite. A variety of chemical and biological processes have been applied
towards establishing the most efficient environmental friendly bio-
hydrometallurgy treatment technique of low-grade copper mixed ores. High copper extractions are achieved in environmental acceptable conditions and
in low cost, under optimum redox potentials with isolated and mixed cultures
of A. ferroxidans and A. thiooxidans, The understanding of synergistic effects which have resulted from the presence of mixed ores in the bioleaching
procedure is important for understanding the bioleaching behavior between
single and mixed ores and the origin of the existing differences.
Objectives
Raman and FTIR microspectroscopies are structure sensitive techniques and
have been applied towards our understanding of the characterization of the structure as well the structure-function relationship in minerals. The
combined application of the techniques for the bioleaching behavior of
bornite, chalcocite and covellite and the comparison with other bioleached Cu/Fe/S systems such as chalcopyrite provides valuable information on the
whole bio-hydrometallurgy system and overall visualizations of the
dynamics of the bioleached mineral ore.
Materials and Methods
Unleached samples of bornite, chalcocite and covellite were collected from
the mines of HCM in Cyprus, and placed in different test tubes containing 30
ml of the corresponding growth medium and a total of 10% inoculation of cell suspensions from stock cultures collected from the mine at pH 1.8. FTIR
and Raman microspectroscopy.
Results
We report the μm-FTIR and Raman microspectroscopic detection of bornite
[Cu5FeS4] -, chalcocite [Cu2S] -, and covelitte [CuS] -bacterial interactions
by a consortium of microorganisms. The absorption signals of amide I, K+-jarosite and of the produced extracellular polymeric substances (EPS) from
the mixed culture as a function of position on the surfaces of the bioleached
bornite, chalcocite and covellite demonstrated their heterogeneity within the surface of the minerals. To our knowledge this is the first combined
application of μm-FTIR and Raman microspectroscopy for the bioleaching
behaviour of bornite, chalcocite and covellite and the comparison with other bioleached systems such as chalcopyrite [CuFeS2] provides valuable
information on the whole bio-hydrometallurgy Cu/Fe/S system.
Conclusions
We report for the first time the ν(Cu-S) of bornite and chalcocite which are
intermediates in the bioleaching of chalcopyrite and the bioleaching behavior
of bornite, chalcocite and covellite by a consortium of microorganisms present in HCM by Raman and FTIR microspectroscopies and compare it
with that previously reported for chalcopyrite. Based on our results we
propose a mechanism for the sequential steps for chalcopyrite bioleaching in which chalcopyrite is converted initially to Cu2S, and subsequently to CuS.
T 67
Influence of bacterial EPS on mineral organization in EPS-
hydrogel-calcite composite aggregates - the chelating effect of
Bacillus subtilis, Mycobacterium phley, Mycobacterium
smegmatis, and Pseudomonas putida EPS X. Yin*1, F. Weitzel1, E. Griesshaber1, C. Jiménez-López2, L. Fernández-Díaz3,4, A. Ziegler5, A. Rodríguez-Navarro6, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department für Geo- und
Umweltwissenschaften, München, Germany 2Universidad de Granada, Departamento de Microbiología, Granada,
Spain 3Universidad Complutense de Madrid, Departamento de Mineralogía y Petrología, Madrid, Spain 4Universidad Complutense de Madrid, Instituto de Geociencias (ICMM,
CSIC), Madrid, Spain 5Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm,
Germany 6Universidad de Granada, Departamento de Mineralogía y Petrología, Granada, Spain
Mineralized structures generated under biological control are hierarchical composites that consist of two distinct materials: biopolymer matrix that is
reinforced by mineral(s). The biopolymer matrix is developed within the
biological hard tissue as membranes or network of fibrils. The biopolymers affect mineral organization as well as material properties of the biological
composite material. Hydrogel systems can be regarded to some extent as
model systems for understanding the influence of biopolymer matrices in biologic structural materials on nucleation, crystal growth, mineral
orientation, and hard tissue organization. The fibrous fabric of agarose and
gelatin gels forms compartments with specific diffusion rates, local concentrations, and supersaturation of solutes. Hence, an environment is
formed in an artificial system that shows some common features to those that
are present at sites of mineralization in biological hard tissues. However, despite many similarities, major differences still remain.
Microbial cells surround themselves by a self-produced matrix of hydrated
extracellular polymeric substances (EPS), which protects the cells and enhances their physiological activities. Bacterial EPS is not a denaturalized
product if compared to agarose or gelatin hydrogels. To understand the directing influence of biopolymers on mineral organization and composite
material formation, we synthesized with Bacillus subtilis, Mycobacterium
phley, Mycobacterium smegmatis, and Pseudomonas putida EPS hydrogel-calcite composite aggregates and investigated the influence of EPS matrices
on aggregate formation, growth and calcite organization. EPS composition
was investigated with FTIR, aggregate morphologies and hierarchical mineral organization were characterized with scanning electron microscopy
(FE-SEM) and electron backscatter diffraction (EBSD). Bacterial EPS and
agarose hydrogel distribution were visualized with selective etching procedures, micro-Raman and kernel misorientation analysis derived from
EBSD data.
Relative to reference aggregates devoid of bacterial EPS, aggregates that contain bacterial EPS are reduced in size and, for the EPS of a specific
bacterium, have distinctive crystal morphologies. Polymer (bacterial
EPS/agarose hydrogel) distribution is highly inhomogeneous in aggregates that contain EPS. In the case of P. putida and M. phley, the occluded polymer
(EPS/agarose hydrogel) is mainly present as membranes, while for M.
24
smegmatis and B. subtilis the EPS/hydrogel mixture is occluded within the aggregate as membranes and as network of fibrils. Relative to reference
aggregates devoid of EPS, where subunit formation is either absent or
negligible, subunit formation in aggregates containing bacterial EPS is extensive. Subunits vary significantly in shape, size, and mode of
organization within the aggregate. For M. smegmatis and B. subtilis subunit
organization is radial to spherulitic, while for P. putida subunit organization within the aggregate is almost random, while for M. phley subunit
organization is highly co-oriented.
In conclusion, bacterial EPS changes the microstructure and texture of the mineral in a specific manner. This is a specific characteristic for a given
bacterium and is a feature that can be used as a tool for the recognition and
identification of bacterially mediated calcification in present environments as well as in the geological record.
T 68
The red algae mineralized tissue- a low weight high strength
material N. Bianco Stein*1, B. Pokroy1, P. Zaslansky2 1Technion, Materials Science and Engineering, Haifa, Israel 2Charité - Universitätsmedizin Berlin, Department for Operative and Preventive Dentistry, Berlin, Germany
Biomineralization, the formation of minerals by living organisms, has long attracted researchers' attention due to the enhanced properties of biominerals
compared to their synthetic and geological counterparts. For example, the
toughness of the lenses on the arm plates of the brittle star O. wendtii was found to be twice as high compared to geological calcite.
Calcite, the most thermodynamically stable polymorph of calcium carbonate,
is an abundant structural component in the skeleton of many marine organisms. In many cases Mg is incorporated into the calcite lattice with
levels reaching as high as 45 mol%.
In this study we investigate the structure of the coralline red algae at various length-scales. Their mineralized tissue is composed of high-Mg calcite. In
addition, an organic phase comprised of polysaccharides serves as a template
for crystallization. The structure of these algae has not been widely studied before and information on their nanostructure is highly missing.
Structural characterization was performed utilizing various high-end
techniques such as synchrotron radiation macro- tomography and nano- tomography. In addition, we used high resolution SEM and high resolution
TEM for the study of the morphology and the nanostructure. Coralline red algae exhibit a remarkable macrostructure. We have discovered
that the structure of these algae is in fact a highly porous structure, with
porosity reaching as high as 70vol%. In addition, we have shown that their structure is hierarchical with several orders from the nano to the macro scale,
formed by crystals with diameter in the nanometric size. Moreover, we have
discovered unique structural elements that have not been previously reported. We have shown that these unique structural elements provide the algae with
greatly enhanced mechanical properties that allow them to endure stresses
applied by the sea waves present in shallow waters. The combination of high porosity along with a unique macrostructure allows
these algae to obtain a low weight structure with enhanced mechanical
properties. This study can lead toward a better understanding of the structure function relation of biomineralized structures and toward the synthesis of
novel materials with improved mechanical properties.
T 69
Deep-water scleractinian corals and their skeletal organic
matrices: A source of phylogenetic data? J. Stolarski1, K. Janiszewska1, I. Coronado1, T. Takeuchi2, L. Ravet3, J.
Thomas3, F. Marin*3 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2Okinawa Institute of Science and Technology Graduate University, Marine
Genomics Unit, Onna, Japan 3University of Burgundy-Franche-Comté, Dijon, France
The reef-building scleractinian corals contribute massively to the global formation of biogenic calcium carbonate. Although the physiology of
calcification in corals has been well studied, the molecular mechanisms
underlying this process are still poorly understood. Similarly to other mineralizing metazoans, scleractinian corals build their exoskeleton by
secreting inorganic precursors of calcification together with an organic
matrix, which consists of proteins and polysaccharides. This matrix - the main regulator of the deposition of calcium carbonate - remains occluded in
the growing biocrystals. In recent years, high-throughput screening
techniques have allowed the identification of a large number of skeletal proteins. To date, however, these data were retrieved only from shallow-
water, photosymbiotic taxa such as Acropora, Stylophora or Porites.
The main objective of this study was to supplement the skeletal database with data from deep-water, asymbiotic scleractinians. The selected samples
represent 3 major clades i.e., Desmophyllum (the Robusta clade)
Anthemiphyllia (the basalmost position in Complexa clade), and Letepsammia and Gardineria, both representing basal scleractinians. Large
phylogenetic distances on molecular tree topology and unique
microstructural skeletal organization of each taxon suggest that the composition of skeletal matrices may differ accordingly to the phylogenetic
position.
Following thorough cleaning of the skeleton, the organic content was extracted, divided into soluble and insoluble fractions and submitted to
proteomic analysis. Since no genomic/transcriptomic data are available on
the 4 species, the in silico analysis was performed against a large data set comprising all "non vertebrate proteins" available and the transcriptome of
the coral Porites australiensis. Aliquots of the soluble fractions were
analyzed on SDS-PAGE and screened against a set of 21 lectins in order to obtain their saccharidic signature.
For each sample, several peptides were identified, matching with a large
number of protein hits (32 to 109). Many of them occured with P. australiensis proteins; hits with "other metazoans" proteins (including
Acropora millepora) were significantly reduced. Only 8 to 29% of the hits
corresponded to 2 or more peptide matches. In spite of potential bias, our data suggest that the protein matrices of Desmophyllum and Letepsammia are
more similar between them than from the matrices of Gardineria and
Anthemiphyllia. The saccharide analysis indicates that only 5 (Desmophyllum, Anthemiphyllia), 7 (Gardineria) and 8 (Letepsammia)
lectins reacted. The lectin profiles of all samples were rather similar and
almost superimposable for Gardineria and Anthemiphyllia. In all cases, the highest reactivity was obtained with LEL (an N-acetylglucosamine-binding
lectin); STL lectin gave high reactivity with Desmophyllum and Letepsammia and moderate with Gardineria and Anthemiphyllia.
The composition of organic matrices may provide some background
information about the evolution of biomineralization in scleractinian corals. The protein similarity between Desmophyllum and Letepsammia that seem
only distantly phylogenetically related or similar lectin profiles of
Gardineria and Anthemiphyllia provide a hint to seek deep homology/convergence of their biomineralization paths. Lack of close
similarity between two members of basal clade (Gardineria and
Letepsammia) may point to their early evolutionary divergence. Acknowledgments: This work was supported by the National Science Center
(Poland) grants 2011/03/N/ST10/06471 (KJ) and 2017/25/B/ST10/02221
(JS).
T 70
Molecular and skeletal fingerprints of scleractinian coral
biomineralization along the depth gradient T. Mass*1, A. Malik1, S. Einbinder1, P. Zaslansky2, B. Pokroy3, I. Polishchuk3, J. Stolarski4 1University of Haifa, Marine Biology, Haifa, Israel 2Charité - Universitätsmedizin, Department for Operative and Preventive Dentistry, Berlin, Germany 3Technion - Israel Institute of Technology, Department of Materials Science
& Engineering, Haifa, Israel 4Polish Academy of Sciences, Institute of Paleobiology,, Warszawa, Poland
Reef building corals, the major producers of biogenic calcium carbonate, form skeletons in plethora of morphological forms. The skeleton shapes and
sizes are distinct for individual species but show also phenotypic plasticity
along environmental gradients. A fundamental question remains how these phenotypic skeletal modifications are reflected in molecular
biomineralization program of the coral organisms? This study provides the
first comprehensive macro- and microstructural skeletal analysis of scleractinian coral Stylophora pistillata (clade 4) collected across the depth
gradient from the sea surface to mesophotic depths of 60m and reciprocally
transplanted from 5->60 and 60->5 m in the Gulf of Eilat (Red Sea, Israel). Morphological study was combined with expression pattern of distinct gene
ontology (GO) and of biomineralization "tool kit" genes. Macro- and
microstructure of S. pistillata colonies significantly change over a depth gradient from spherical (surface depths) to flat and branching morphotypes
(mesophotic depths) and from intense (surface depths) and weak (mesophotic
depths) development of fine-scale regions of enhanced skeleton growth ("calcification centers"). In parallel, the trascriptome composition shifts from
overrepresentation of genes associated with oxygen stress response and DNA
repair (shallow-water), to coral-biomeniralization, cilia, extracellular matrix, and immune response (mesophotic depths). Interestingly, at both the
phenotypic and the gene level, transplanted corals partly adapt the typical
depth-specific properties. Accordingly, we show that organic matrix fraction is enriched in the skeleton at deep-water, in parallel with overrepresentation
of biomineralization "tool-kit" structural extracellular genes in deep water
colonies. Our results provide insights into molecular mechanisms of coral calcification in changing environment which are also encoded in the
skeleton. As such they provide exciting perspective of new paleogenomic
interpretations of fossil corals that preserve fine-scale skeletal features.
25
T 71
Modern corals vs. ancient oceans N. Conci*1, S. Vargas1, E. Griesshaber1, W. W. Schmahl1, G. Wörheide1,2,3 1Ludwig Maximilian University, Munchen, Germany 2SNSB — Bayerische Staatssammlung für Paläontologie und Geologie,
München, Germany 3GeoBio-Center, Ludwig-Maximilians-Universität München, München, Germany
Biomineralization is a taxonomically ubiquitous process by which organisms form minerals that they use for support, protection, or nutrient storage, such
as shells, skeletons, and bones. Among corals (Class Anthozoa, Phylum
Cnidaria) biomineralization is widespread and both aragonite and calcite structures can be observed. Coral biomineralization has been extensively
studied, primarily due to the ecological role these organisms play in the
formation and accretion of coral reefs. Nevertheless, how corals acquired the ability to form skeletons characterized by different calcium carbonate
(CaCO3) polymorphs, i.e. calcite and aragonite, remains elusive. One
standing question revolves around whether corals can biologically control the deposition of either polymorph. This has important evolutionary implications
as changes in seawater chemistry through the Earth"s history, especially the
Mg/Ca ratio, appear to favor the deposition of either one polymorph or the other.
To address this question, we have set-up calcite-inducing, Cretaceous-like
marine aquaria experiments to grow coral species from both clades of
biomineralizing anthozoans, namely the subclass Octocorallia (Heliopora
coerulea) and the order Scleractinia (Montipora digitata). We employed a
diverse array of mineralogical analyses, including electron backscatter diffraction (EBSD) and energy dispersive spectroscopy (EDS), to determine
and examine the presence of environmentally-induced modifications within
the coral skeleton. We then used RNA sequencing to investigate the transcriptional response of the corals to different polymorph favoring
environments. In conjunction, these data provide insights on how the
diversity of skeletal structures in corals evolved and may inform our predictions about the response of these ecologically important organisms to
future changes in ocean chemistry .
T 72
Biomineralization pathways in calcifying dinoflagellates-
uptake, storage in MgCaP-rich bodies and formation of the
shell A. Jantschke*1, I. Pinkas2, A. Hirsch2, A. Schertel3, L. Addadi2, S. Weiner2 1Technical University Dresden, Bioanalytical Chemistry, Dresden,
Germany 2Weizmann Institute of Science, Rehovot, Israel 3Carl Zeiss Microscopy GmbH, Oberkochen, Germany
Introduction Dinoflagellates are one of the most important contributors to both marine and
freshwater productivity. They have two main life stages, the thecate/motile stage and the cyst/resting stage. One family of dinoflagellates, the
Thoracosphaeraceae, produce calcitic shells in their cyst stage.
Objectives Although dinoflagellates are the second most important calcifying
phytoplankton group after coccolithophores, their biomineralization mechanism is still not fully understood and considered as one of the most
serious gaps in knowledge. For this reason, the main objectives are to gain
insight in the mineral architecture and stabilization mechanism, subcellular structures that may play a role in biomineralization, as well as into cyst
formation mechanism.
Materials & methods We investigate calcitic cyst formation in two representative members
(L. granifera and C. operosum aff.) from different clades of calcareous
dinoflagellates using cryo-electron microscopy (cryo SEM and cryo FIB SEM) in combination with various spectroscopic techniques (FT-IR, Raman,
Fluorescence, EDS). These imaging techniques allow investigation of cells
as close as possible to the natural state.
Results Only calcein AM and not calcein enters these cells, indicating active uptake
of calcium and other divalent cations. In both species, we observed vacuoles containing crystalline inclusions using cryo-SEM. So far, crystalline deposits
in the family Thoracosphaeraceae were assigned to calcite and it was
assumed that they are involved in the calcitic shell formation. Surprisingly, using in situ Raman spectroscopic imaging we could identify these
crystalline inclusions as anhydrous guanine in the biogenic β-form using their
low-wavenumber Raman signature. Live-cell imaging of cells stained with Calcein-AM, the use of cryo-
sectioning and cryo-EDS shows the presence of small MgCaP-rich mineral
bodies within the same vacuolar enclosures. 3D cryo-FIB-SEM serial block face imaging of a calcifying cell of C. operosum aff. shows a remarkably
large number (353) of these bodies distributed in the cell volume. Out of
these bodies, 52 (about 15%) are located between the two inner organic layers. We suggest that these MgCaP-rich bodies are secreted into the outer
matrix and are part of a Ca-concentrating or transport mechanism.
Calcite formation occurs inside the outer matrix via multiple independent nucleation events. Based on 2D and 3D cryo-electron microscopic datasets
we show that individual calcite crystals grow with preferred orientation into
a dense reticular network that forms the calcitic cyst. In the final calcification stage the main structural motif are calcite plates with evenly arranged pores
that form the highly regular, porous calcite shell.
Conclusion In summary, our results show the presence of a multifunctional vacuole. In
calcifying cells of L. granifera and C. operosum aff. vacuolar MgCaP-rich
bodies seem to be part of a Ca-concentrating or transport mechanism, indicating there is a common biomineralization pathway in the family
Thoracosphaeraceae: (1) Uptake of Ca and other cations through the
membranes. (2) Deposition of Mg and Ca ions inside a disordered MgCaP-body. (3) Secretion of these bodies to the extracellular space between the
outer membranes. (4) Formation and growth of calcite bodies into a dense
reticulate network that forms the mature calcitic shell.
T 73
Marine temperature recorded in bivalve shell ultrastructures -
proxy potential and current limitations N. Höche*1, E. O. Walliser1, M. Peharda2, B. R. Schöne1 1Johannes Gutenberg University Mainz, Dept. of Applied and Analytical
Paleontology, Mainz, Germany 2Institute of Oceanography and Fisheries, Split, Croatia
Introduction Bivalve shells are increasingly exploited as high-resolution paleoclimate archives. These mollusks are globally distributed in aquatic ecosystems,
occur in large numbers and individuals of some species are very long-lived.
Periodic growth patterns can be used to precisely date each shell portion. Furthermore, the shells form in equilibrium with the oxygen isotope
signature of the ambient water, so that δ18Oshell provides information on
temperature during growth. However, oxygen isotope-based temperature estimates can be challenging if the isotope value of the water is unknown or
if the isotope signal is diagenetically altered. Evidently, there is a strong need
for independent temperature proxies. One potential candidate is the shell ultrastructure, specifically the size and shape of individual biomineral units
(BMUs). So far, BMU-based temperature reconstructions have only been tested in a few species from a limited number of localities. In addition, the
recognition and morphometric analysis of BMUs (in SEM images) needs to
automated through suitable image-processing algorithms.
Objective In this study, we assessed the effect of temperature on BMUs of
ultrastructures, which have hitherto not been studied (i.e. crossed-lamellar, homogenous and crossed-acicular). To advance objective and quantitative
temperature reconstructions, we developed new image-processing
algorithms for automated BMU recognition.
Material and methods We analyzed the marine bivalves Glycymeris bimaculata and G. nummaria
from the Adriatic Sea, and Arctica islandica from Iceland and the Baltic Sea. The two glycymerids were used to study potential species-specific
differences, and A. islandica was used to evaluate potential locality-specific
differences in recording environmental variables.
Results All studied species showed the same relationship between temperature and
ultrastructure as reported in previously studied bivalves, i.e., larger and more elongated BMUs were formed in warmer waters. We calculated transfer
functions for crossed-lamellar ultrastructures of Glycymeris spp. enabling
quantitative temperature estimates. The homogenous outer shell layer of A. islandica is challenging to interpret. Specimens from the Baltic Sea showed
significantly larger variation in BMU size than specimens from Iceland.
Conclusions Studied bivalve species recorded temperature changes in their shell
ultrastructure: BMUs were larger and more elongated when formed in
warmer waters. Transfer functions are species-specific. SEM-based quantification of BMU morphometry is challenging in case of the
homogenous ultrastructure of A. islandica.
26
T 74
Ocean acidification impacts European abalone (Haliotis
tuberculata) shell microstructure and mechanical properties S. Auzoux-Bordenave*1,2, S. Avignon1, P. Dubois3, N. Richard1, M.
Coheleach4, A. Badou1, S. Di Giglio3, L. Malet5, S. Martin2,6, S. Roussel4,
S. Huchette7 1Museum national d'histoire naturelle, Station de Biologie Marine,
Concarneau, France 2Sorbonne Université, Paris, France 3Université Libre de Bruxelles, Laboratoire de Biologie Marine, Brussels,
Belgium 4Université de Brest, CNRS, IRD, Ifremer, LEMAR, Plouzané, France 5Université Libre de Bruxelles, Service 4Mat, Brussels, Belgium 6Station Biologique de Roscoff, CNRS/CU, Roscoff, France 7Ecloserie France-Haliotis, Plouguerneau, France
Ocean acidification (OA) is of major concern for marine organisms,
especially for calcifying species such as mollusks, with their shell made of calcium carbonate. Indeed, OA has been shown to reduce survival and
growth, alter morphology and/or impair shell formation and mineralization
in marine molluscs. The European abalone Haliotis tuberculata is a commercially and ecologically important gastropod species with a calcified
shell mostly composed of CaCO3 under aragonite form. Since aragonite is
more susceptible to dissolution compared to calcite, the abalone shell provides a relevant model to study the impact of OA on
biomineralization. This study investigated the effects of pCO2 induced OA
on shell microstructure, calcification and resistance in adult abalone. According to the most pessimistic scenario (-0.3 pH unit by 2100), adult 3.5
year-old abalone were exposed to two pH conditions (pHT = 7.71 ± 0.06,
pCO2 = 988 ± 143 µatm, vs pHT = 8.01 ± 0.05, pCO2 = 449 ± 63 µatm) during four months by increasing pCO2 into five experimental tanks per
condition. Shell surfaces as well as cross sections were examined by
Scanning Electron Microscopy (SEM) to assess whether lowering the pH had an influence on shell microstructure. The thickness of the shell layers (i.e.
periostracum, spherulitic and nacre) was determined on cross-section images
using Image J software. Biomechanical tests (such as compression tests and nanoindentation measurements) were performed to compare the shell
properties (strength, hardness and elasticity) between the two pH treatments.
After four month of exposure to low pH condition, the periostracum of abalone shell exposed to low pH appeared lighter and exhibited a corroded
surface compared to those exposed to control condition. This corrosion was confirmed by the decreased thickness of the periostracum observed at pH 7.7,
while no difference in spherulitic and nacre layers thickness were observed.
SEM observation of nacre microstructure revealed irregularities with a heterogeneous aragonite platelets and a corroded texture surface in abalone
shells exposed to low pH. The shell resistance of abalone exposed to pH 7.7
was significantly reduced by 29 % compared to those exposed to pH 8.0. In addition, nanoindentation assays revealed significant differences in shell
hardness and elasticity for abalone exposed to pH 7.7. Shell corrosion and
microstructure changes would likely result from indirect effects of OA either on the carbonate chemistry of the calcification site or on physiological
processes driving CaCO3 deposition.
Our results highlighted that OA negatively impacted the shell microstructure and resistance in adult abalone, leading to more fragile shell. In their natural
environment, abalone may be at greater risk under future pH condition as
their shells may not offer sufficient protection from predators and other environmental stressors. As a consequence for the abalone industry, OA
might represent two challenges (i) by reducing abalone protection and
threatening wild population stocks, and (ii) by enhancing the cost of its aquaculture due to an increased time to reach a marketable size.
T 75
Oyster biomineralisation is maintained under ocean
acidification K. Chandra Rajan*1, T. Vengatesan1 1The University of Hong Kong, Swire Institute of Marine Science, School of
Biological Sciences, Hong Kong Island, Hong Kong
Introduction Ocean acidification (OA), the on-going reduction of oceanic pH and the carbonate saturation state (Ω), is known to affect biomineralisation in several
marine invertebrates. Ion transport is an important aspect of maintaining
biomineralisation under OA. There are two concepts, related to ion homeostasis, that explain the negative effect of OA on marine calcifiers:
1) Substrate limitation theory, where the reduction in the carbonate saturation
state is considered as the main negative stressor. 2) Proton flux limitation model, where more energy will be required for
active transport of H+ ions from the site of biomineralisation when the H+ in
the ocean increases. However, little is known about the epigenetic ability of the marine calcifiers
to adapt to these two challenges. Hence, in this study we have investigated
the epigenetic response of an ecologically and commercially important aquaculture oyster species - Crassostrea hongkongensis to OA.
Objectives Mantle tissue specialises in biomineralisation and is conserved throughout molluscs. Thus, in our study we focus over the epigenetic response of the
mantle tissue under OA. The objectives are:
1) Understanding the epigenetic response of mantle to OA (gene expression and DNA methylation) with a focus over ion transporters and shell matrix
proteins.
2) Characterising the changes in the shell quality under OA via shell microstructure, density, Mg/Ca ratio and hardness.
Materials & methods Oyster spats (one day post settlement) were grown under ambient pH (8.1) and OA conditions (pH 7.4 and 7.7) for five months, a significantly long
duration for an OA study. mRNA-seq and Methyl-RAD techniques were
used for gene expression and DNA methylation analysis. Shell properties were analysed using Scanning Electron Microscope (SEM), SEM-EDS,
Micro-CT and Vicker's Hardness test.
Results The shell growth under OA was surprisingly maintained, like the ambient
conditions, throughout the experimental duration. The Mg/Ca ratio and
hardness also remains unchanged. However, there was complete dissolution of the prismatic layer under pH 7.4. In-spite of severe dissolution, the overall
shell growth was maintained.
Preliminary analysis of the RNA-seq data shows that the expression of well-known biomineralisation related genes such as alkaline phosphatase,
carbonic anhydrase and tyrosinase were maintained. Among the differentially expressed genes, above 80% of the genes where
downregulated. The top downregulated genes under OA are related to
mitosis, ribosomes and various energy metabolism such as amino acid, carbohydrates and glycan. Among the few upregulated genes, the top
upregulated genes are related to plasma membrane and calcium binding. The
notable genes among the upregulated were voltage dependent calcium channels. Preliminary analysis of the DNA methylation data shows that top
down-regulated methylation sites are also related to voltage gated
calcium/sodium channels, indicating a strong epigenetic base for adaptation in oysters.
Conclusion Crassostrea hongkongensis maintains biomineralisation even under extreme ocean acidification scenarios. The adaptation strategy involves: 1)
upregulating the ion transporter system, for supplying required substrate and
proton homeostasis. 2) The energy spent on upregulating ion transporters is compensated by undergoing metabolic depression.
T 76
Effect of polycarboxylated eggshell membrane on in-vitro
mineralization J. L. ARIAS*1, K. SILVA1, A. NEIRA-CARRILLO1, L. ORTIZ1, N.
BUTTO1, M. S. FERNANDEZ1 1University of Chile, SANTIAGO, Chile
Biomineralization concerns not only inorganic ions formation, but also the
regulatory role of an organic matrix mainly related to the action of anionic groups such as sulfate in proteoglycans or aspartate and glutamate in
proteins. The eggshell membrane (ESM) is a network of a fibrillar
biopolymer organized as two layers of protein fibers. Main protein of the ESM is type X collagen. Mineralization of chicken eggshell started on
specialized negative charged sites of the ESM referred to as mammillae,
associated to the occurrence of mammillan, a keratan sulfate-rich proteoglycan. Growth of calcite crystals occurs from the mammillae upwards
in association with the sequential secretion of ovoglycan, a dermatan sulfate-
rich proteoglycan. Although 22% of the ESM aminoacids are glutamic plus aspartic, there is no mineralization on the surface of ESM fibers, but only on
the afore mentioned sulfate-rich mammillae. Therefore, as a possible
explanation we hypothesize that the occurrence of a competitive effect between sulfated and carboxylic groups on the surface of the ESM could be
involved in driving calcium carbonate crystal nucleation to privileged sites.
In order to answer this question, an experimental enrichment of ESM with polycarboxylic groups was tested on its ability to crystallize calcium
carbonate in vitro.
Polycarboxylated ESM was obtained by coupling polyaspartic or polyglutamic acids to ESM fibrils crosslinked with water-soluble
carbodiimide. Selective protection and cleavage of carboxylic groups during
synthesis were followed by FTIR spectroscopy. Crystallization assays were based on a gas-diffusion chamber method consisting of a chamber built with
a plastic Petri dish having a central hole in its bottom and glued to a plastic
cylindrical vessel. Polystyrene microbridges were settled on the bottom of the petri dish. Microbridges were filled with 200 mM dihydrate calcium
chloride solution in 200 mM Tris buffer, pH 9.0. The cylindrical vessel
contained 25 mM ammonium carbonate solution. Intact or polycarboxylated ESM strips were deposited on the top of each microbridge with the
mammillary side facing down. Five replicates of each experiment were
27
carried out inside the chamber at 20 °C for 24 h. After the experiments, eggshell strips were taken out of the microbridges, air-dried at room
temperature, mounted on aluminum stubs with scotch double-sided tape, and
coated with gold. Crystal morphology was observed and number and size estimated in a Jeol JSM-IT300 EDS-Oxford Instrument scanning electron
microscope.
When functionalized ESM were located upside down on the calcification solution on the top of the microbridges for 24 hours, higher amount of small
2-3 µm rhombohedral calcite crystals adhered to the fibers were observed
when compared with the intact ESM where 7-10 µm calcite crystals were located only on the mammillae.
It is safe to conclude that the occurrence of polycarboxylic groups
immobilized on ESM is a strong attractor for calcium carbonate crystallization on functionalized ESM used as a template scaffold. Funded
by FONDECYT 1180734
T 77
Fungi-induced mineralization in vitro- controlling the crystal
morphology and polymorph I. Polishchuk*1, A. Livne1, B. Pokroy1 1Technion - Israel Institute of Technology, Materials Science and Engineering, Haifa, Israel
Fungi live within diverse environments and survive well under extreme conditions, which are usually beyond the tolerance of many other organisms.
In various environments fungi are known to induce precipitation of a wide
range of minerals. Specifically, it has been shown that various species of fungi facilitate calcium carbonate mineralization. Inspired by examples of
needle-fiber calcite formed via fungal biomineralization typically observed
in soils and sediments, herein we utilized active and inactivated fungus to induce mineralization synthetically. For the first time we report the ability of
fungi mycelium to serve as a template for growth of chlorhydroxy-apatite
tubes and hopeite cylinders. We also demonstrate the feasibility of growing aragonite needles from a fungal source in vitro. The obtained needles are
curved, display hexagonal facets and demonstrate high aspect ratios close to
60. The size and the shape of the synthetic needles match that of the natural fungus" mycelium. The morphology, micro- and nanostructure of the grown
crystals were studied utilizing high-resolution characterization techniques.
The presented findings demonstrate that fungus present in the crystallization environment can induce the formation of high aspect ratio fibers and stabilize
metastable polymorphs.
T 78
Post-mortem recrystallization of biogenic amorphous calcium
carbonate and crystal tailoring by the inherited
macromolecular framework J. Stolarski*1, I. Coronado1, G. Luquet2, M. Potocka3, M. Mazur4, A.
Baronnet5, O. Grauby5, A. Meibom6,7 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2Muséum National d'Histoire Naturelle, UMR BOREA, Paris, France 3Institute of Biochemistry and Biophysics , Department of Antarctic Biology, Warsaw, Poland 4University of Warsaw, Department of Chemistry, Warsaw, Poland 5CNRS - Aix Marseille Université, CINaM - UMR 7325, Marseille, France 6Ecole Polytechnique Fédérale de Lausanne , Lausanne, Switzerland 7Université de Lausanne, Center for Advanced Surface Analysis, Lausanne, Switzerland
Assembly of mineral deposits from transient, disordered and nanometric-size
precursor particles is a widespread skeleton-formation strategy of many groups of organisms (e.g., corals, mollusks, echinoderms). It enables
efficient cellular transport of mineral components to the skeleton formation
site and their aggregation within biologically confined space. The disordered particles enclosed by the organic matrix are then transformed through local
dissolution–co-precipitation processes into stable and crystalline phases.
Consequently, biologically-controlled minerals, in contrast to abiotic (synthetic/geological) crystals, are organo-mineral nanocomposites with
uniform particle sizes, high levels of spatial organization, complex
morphologies, controlled aggregation and texture, preferential crystallographic orientation and form higher-order hierarchical structures.
Such criteria are often used to distinguish biogenic and abiogenic minerals in
the fossil record. In our experiment, we induced the formation of crystalline mineral phase
from lobster gastroliths, originally formed mainly by biogenic amorphous
calcium carbonate (ACC). In natural conditions (inside the lobster body) the amorphous gastrolith mineral does not crystallize and is assimilated by the
organism immediately after molting. Inducing crystallization of the gastrolith
amorphous phase, which acted as a crystallization "precursor", we found that the resulting mineral is calcite, which shows all aforementioned properties of
biominerals, including highly elaborated hierarchical organization. These
newly formed structures were characterized by FESEM, AFM, EBSD,
Raman spectroscopy techniques. Such "biogenic" organization of the post-mortem formed calcite is explained by the inheritance of the original
macromolecular framework, which acts as an organic template in precisely
orchestrated dissolution-co-precipitation process. The finding has several implications for traditional, structural criteria in interpreting the pristine
structure of biominerals in paleontology and astrobiology.
Acknowledgments: Acknowledgments: This work was supported by the National Science Center (Poland) grant 2017/25/B/ST10/02221.
T 79
Spinodal decomposition in the formation of biogenic single
crystals of magnesium calcite E. Seknazi1, S. Kozachkevich1, I. Polishchuk1, N. Bianco Stein1, P.
Zaslansky2, A. Katsman1, B. Pokroy*1 1Technion Israel Institute if Technology, Materials Science and Engineering, Hiafa, Israel 2Charite, Berlin, Germany
As organisms can form crystals only under ambient conditions, they
demonstrate fascinating strategies to overcome this limitation. Recently, we
reported a previously unknown biostrategy for toughening brittle calcite crystals by means of pre-compression of the material, using coherently
incorporated Mg-rich nanoprecipitates arranged in a layered manner in the
lenses of a brittlestar, Ophiocoma wendtii. Here we propose the mechanisms of formation of this functional hierarchical structure under conditions of
ambient temperature and limited solid diffusion. For the first time, we
propose that formation proceeds via a spinodal decomposition of a liquid or gel-like magnesium amorphous calcium carbonate (Mg-ACC) precursor into
Mg-rich nanoparticles and a Mg-depleted amorphous matrix. In a second
step, crystallization of the decomposed amorphous precursor leads to the formation of high-Mg particles-rich layers. The model was supported by our
experimental results in synthetic Mg-calcite, which reinforce the concept of
a spinodal decomposition in the amorphous precursor. We also show that organics suppress the spinodal decomposition and that most probably the
paucity of organics present in the brittlestar"s mineralized tissue allows it to
occur in this system. These new insights have significant implications for fundamental understanding of the role of Mg-ACC material transformation
during crystallization and its subsequent stability.
T 80
Bionic synthesis of a magnetic calcite skeletal through living
foraminifera G. Magnabosco1, H. Hauzer2, C. Albonetti3, V. Morandi4, J. Erez2, G.
Falini*1 1Alma Mater Studiorum - Università di Bologna, Chemistry "Giacomo
Ciamician", BOLOGNA, Italy 2The Hebrew University of Jerusalem, Institute of Earth Sciences, Jerusalem, Israel 3National Research Council, Institute for Nanostructured Materials,
Bologna, Germany 4National Research Council, Institute for Microelectronics and Microsystems, Bologna, Italy
The peculiar functional properties of calcium carbonate biominerals, such as
shells, echinoderm spines and brittle stars, have stimulated both fundamental research in biomineralization and in vitro bio-inspired synthetic processes in
material science. As a consequence of the latter, bio-materials having
different/additional properties with respect to the natural ones have been produced by cell-free laboratory activities. Despite this effort, the production
of materials having functional properties even similar to the natural ones
remains elusive. Here we demonstrate that new nano-composite materials can be prepared in vivo exploiting the special mineralization pathway of the
foraminifer Amphistrigina lessoni, a calcifying organism producing a calcitic
skeleton by vacuolization of seawater. Accordingly, a bionic skeleton possessing a chamber entrapping magnetic nanoparticles has been obtained
growing the organism in seawater containing such nano-particles. Such a
bionic synthetic approach differs and goes beyond the biologically inspired synthetic processes and the biosynthesis of nano-particles by bacteria. It
represents the first research in which a bionic calcified tissue has been
prepared in vivo. This represents a new powerful tool for the preparation of nano-materials exploiting the capability of organisms to control the
calcification pathway with an accuracy that is unparalleled in laboratory cell
free synthetic processes.
28
T 81
Biomimetic precipitation of carbonates in tubular materials
formed in flow conditions C. I. Sainz-Diaz*1, E. Esacmilla-Roa2, J. Cartwright1 1Instituto Andaluz de Ciencias de la Tierra, CSIC-UGR, Armilla-Granada,
Spain 2Lulea University of Technology, Lulea, Sweden
Biomineral tubes are often secreted around an organism, or part of it, that forms a template about which the tube assembles. Another method of
forming biomineral tubes can be templating mineral tube formation around a
fluid jet. Such self-assembled fluid-flow-templated tubes are found in the physical sciences, in the so-called chemical gardens[1], and also in such
geophysical cases as volcanoes, soda straws in caves, and brinicles under sea
ice. Chemical gardens of carbonates have been prepared at laboratory forming different nano-morphology and crystallographic transformations by
nanoscale precipitation of carbonates and other metal oxides grown in the
surfaces of tubular materials generated in flow conditions out of equilibrium. The formed materials were characterized observing nanocrystals with
different morphology and chemistry depending on the growth conditions.
The interaction of several organic polymers has been explored during the precipitation of carbonates.
Molecular modeling calculations have been performed related with the
interaction of organic molecules on mineral solid surfaces. The adsorption of organic compounds showed to be an exothermic process. This indicates that
these nanomaterials can be good absorbants for organic compounds. These
results open interesting applications of these materials to clean polluted soils and as nanocarriers of bioactive compounds for therapeutic and
environmental applications.
[1] Barge, L. M.; Cardoso, S. S. S.; Cartwright, J. H. E.; Cooper, G. J. T.;
Cronin, L.; De Wit, A.; Dolobo↵, I. J.; Escribano, B.; Goldstein, R. E.; Haudin, F. et al. From chemical gardens to chemobrionics. Chem. Rev. 2015,
115, 8652–8703.
T 82
Curving crystals in biominerals and biomimetic minerals H. Imai*1, Y. Yukimasa1, M. Takasaki1, M. Suzuki1, Y. Oaki1, T. Sasaki2 1Keio University, Applied Chemistry, Yokohama, Japan 2The University of Tokyo, The University Museum, Tokyo, Japan
Introduction
A wide variety of biominerals are comprised of micrometric and nanometric
building blocks. We frequently observed unusual curving crystalline units as a building block in the biogenic hierarchical architectures. However, details
of the curving morphologies including their crystallographic structures and
their formation mechanism have not been clarified sufficiently. The clarification of the detailed structures would be important for understanding
of biogenic mineralization and development of biomimetic material
processing.
Objective
We characterized several kinds of curving crystals in biominerals to clarify
their crystallographic structure. Here we focused on aragonite helix of a pteropod shell and hydroxyapatite around the enamel-dentin junction of a
bovine incisor as typical examples of curving morphologies. Moreover, we
tried to produce biomimetic curving morphologies with calcium carbonate and calcium phosphate in artificial systems to discuss the formation
mechanism of the specifically designed crystals.
Materials and method
A petropod shell of C. globulosa was used for structural analysis of aragonitic
helical structures. Bovine incisors of cattle were used for characterization of
hydroxyapatite in tooth enamel. We fractured the teeth with a hammer and prepared small pieces of the specimen that exposed their cross section for
electron microscopies. Several pieces were treated with solutions of sodium
hypochlorite or disodium ethylenediaminetetraacetate to reveal their microstructures. The morphology of the shell surfaces and grown crystals
was observed using scanning microscopes. The crystallographic structures
were revealed by transmission electron microscope with selected area electron diffraction using cross-sectional samples prepared using focused ion
beam milling.
Rod-like crystals of calcium carbonate and calcium phosphate were prepared on several kinds of organic and inorganic substrates in supersaturated
aqueous solutions containing specific organic molecules. Curving
morphologies consisting of rod-like crystals were successfully produced with change in the direction of an ion flow.
Results and discussion
We observed the parallel assembly of curving aragonite fibers ~200−300 nm in width on the mildly etched shells of C. globulosa. The rod-like building
blocks 1−2 mm in length were found in the curving fibers as parts of the
helical architecture. The curving fibers are deduced to be composed of several rod-like building blocks whose a and b axes of the orthorhombic
crystal are intermittently deviated. We also characterized the crystallographic
structures in the enamel-dentin junction region of bovine incisors. The
enamel prisms of hydroxyapatite are regarded as a bundle of nanometer-scale fibers that are elongated in the c direction. Curving parts were observed in
the radial arrangements of the nanofibers around the root of the prisms. A
gradual stepwise change of the c direction is found to be essential for the formation of the curving parts.
We produced highly ordered arrays of c-axis-elongated aragonite nanorods
~100 nm diameter similar to the microstructure in lamellae of gastropods in a supersaturated solution containing specific organic molecules. We also
fabricated enamel-like fluorapatite nanorod arrays on an organic substrate in
an aqueous solution system that was based on the simulated body fluid. A gradual change in the growth direction along the c axis was induced through
a stepwise growth behavior. The direction of an ion flow is deduced to affect
a gradual change in the growth orientation of nanometer-scale crystalline rods.
Conclusion
We characterized aragonite helix of a pteropod shell and hydroxyapatite around the enamel-dentin junction of a bovine incisor. We produced
biomimetic curving morphologies with calcium carbonate and calcium
phosphate in artificial systems. The crystallographic orientation in the curving crystals in biominerals and biomimetic minerals was changed in a
stepwise fashion along nanometer-scale rods and fibers.
T 83
Minerobiolization - biomineralization at the origin of life J. Cartwright*1 1CSIC, Granada, Spain
If, as seems likely, life developed in hydrothermal vents it first used mineral membranes within the vents. The first photo-cells must have learnt to
manipulate the mineral membranes that formed their compartments in order
to control their metabolism. There must have occurred a biological takeover of the self-assembled mineral structures in the first proto-cells, with the
incorporation of proto-biological molecules within the mineral membranes
to alter their properties for life"s purposes, so that, for example, passive osmosis in a mineral membrane gradually became active chemiosmosis in a
proto-cell membrane. This biological takeover of a mineral system is what
we term minerobiolization, in contradistinction to the usual biomineralization in which the biology controls and assembles the mineral.
T 84
Insights into the evolution of biomineral (calcium and silicon)
transporters in viridiplantae M. A. Nawaz*1, I. Zemchenko1, X. Lin2, A. Zakharenko1, R. Muhammad
Atif3, T. F. Chan2, K. S. Golokhvast1 1Far Eastern Federal University, Education and Scientific Center of Nanotechnology, Vladivostok, Russian Federation 2The Chinese University of Hong Kong, Shatin, Hong Kong 3University of Agriculture, US-Pakistan Centre for Advanced Studies in Agriculture and Food Security, Faisalabad, Pakistan
Introduction
Biomineralization is a ubiquitous adaptive strategy in living organisms for
sustenance and structural integrity. Members of viridiplantae and especially
the vascular plants have mastered the art of transporting and accumulating silicon and calcium biominerals. To this respect silicon transporters (SITs),
NOD26-like major intrinsic proteins (NIPs) are responsible for silicon
transport and Ca2+ ATPases (pumps) and Ca2+ exchangers (CAXs) are primarily responsible for calcium transport.
Question
To resolve the sequestration mechanisms behind the deposition of these minerals i.e. the formation of phytoliths and cystoliths, it is essential to
understand the transport mechanism and especially how the transporters
evolved in plants and diverged functionally.
Methods
We performed comparative genomic studies supported by phylogenetic
reconstruction, gene structure analysis, duplication analysis, divergence
analysis, codon bias analysis, molecular evolution analysis, co-expression
networking and phylostratigraphic analysis to provide evolutionary insights.
Results and Conclusions
Here, we identified SITs, NIPs, Ca2+ pumps and CAXs in viridiplantae. Our
evidence suggests that segmental duplication was a prevalent evolutionary force for the expansion of viridiplantae biomineral transporters. Non-
terrestrial plants lost many members of biomineral transporters suggesting
lower biomineralization need/capacity. We identified that angiosperms experienced speciation followed by species/lineage-specific gene
duplications and similar gene modules existed across monocots and dicots.
The duplicated gene pairs have resulted in substantial neo-functionalization. Silicon transport-related genes expanded after embryophyte split. Dicots had
higher biomineral transporter genes owing to lineage-specific and species
specific whole genome duplications.
29
T 85
Evolution of planktonic gastropod calcification over short and
long timescales P. Ramos-Silva*1, D. Wall-Palmer1, L. Mekkes1,2, F. Marin3, F. Marlètaz4,
K. Peijnenburg1,2 1Naturalis Biodiversity Center, Marine Biodiversity, Leiden, Netherlands 2University of Amsterdam, Institute for Biodiversity and Ecosystem
Dynamics (IBED), Amsterdam, Netherlands 3University of Burgundy-Franche-Comté, Biogéosciences UMR CNRS 6282, Dijon, France 4Okinawa Institute for Science and Technology, Molecular Genetics Unit, Okinawa, Japan
Calcification by marine organisms can be drastically affected by ocean
acidification (OA) due to a reduced availability of calcium carbonate in the seawater. Pteropods and heteropods are planktonic gastropods believed to be
among the most vulnerable organisms to the effects of OA because they live
at the ocean surface and build thin shells of aragonite. Shelled pteropods have received considerable attention and are reported to decrease calcification
rates and experience shell dissolution under high CO2 conditions. Shelled
heteropods have received much less attention, but are expected to be equally vulnerable. Both groups are proposed as bioindicators to monitor the impacts
of global change on open ocean ecosystems. However, their vulnerability is
based on short-term exposures to extreme OA conditions. Similar to other mollusks, planktonic gastropods build shells through a biomineralization
process, which is biologically controlled at the molecular level. Still, little is
known about their evolutionary potential in the long-term. Using shell proteomics and transcriptomics we are currently identifying the
"biomineralization toolkit" in pteropod and heteropod species. Extensins,
collagens and whey acidic domain proteins are part of the repertoire of shell matrix proteins (SMPs) that is being identified in planktonic gastropods for
the first time. Next, SMPs are used to understand how biomineralization
evolved in the two independent plankton groups over short and long timescales. Over long timescales includes the evolutionary analysis of SMPs
using a fossil-calibrated phylogenomics tree. Over short timescales involves
studies at the population-level by measuring gene expression under past, present and future concentrations of CO2. Combining macro- with micro-
evolutionary approaches will shed light on the processes that drive diversity
and evolution of calcification in planktonic gastropods and will allow more realistic predictions of the consequences of global change on marine
calcifiers.
T 86
Biomineral growth kinetics and thermodynamics as an
architectural constraint on the evolution of molluscan shells I. Zlotnikov*1, V. Schoeppler1, R. Lemanis1 1B CUBE - Center for Molecular Bioengineering, Dresden, Germany
Molluscan shells are a classical model system to study formation-structure-
function relationships in biological materials and the process of biomineralized tissue morphogenesis. Typically, each shell consists of a
number of highly mineralized ultrastructures, each characterized by a specific three-dimensional mineral-organic architecture. Surprisingly, in
some cases, despite the lack of a mutual biochemical toolkit for
biomineralization or evidence of homology, shells from different independently evolved species contain similar shell ultrastructures. In the
current study, using a recently developed physical framework, which is based
on an analogy to the well known process of directional solidification, we compare the process of ultrastructural morphogenesis of shells from three
major molluscan classes: a bivalve Unio pictorum, a cephalopod Nautilus
pompilius and a gastropod Haliotis asinina. First, we demonstrate that the fabrication of these highly biomineralized tissues is guided by the organisms
by regulating the chemical and physical boundary conditions that control the
growth kinetics of the mineral phase. Second, we expand our understanding of the theoretical macroscopic morphospace of possible molluscan shell
shapes (Raup's concept of morphospace) to the level of possible
ultrastructures that comprise them. Finally, we shed a new light on the evolutionary aspect of molluscan shell ultrastructural fabrication within the
framework of Seilacher's Constructional Morphology and Morphodynamics.
We suggest that the repeated "discovery" of some mineral morphologies reflects a series of architectural constraints provided specifically by
biomineral growth kinetics.
T 87
The shell microstructure of the species of Gigantoproductus
(carboniferous), the giants of the phylum brachiopoda L. Angiolini*1, G. Crippa1, K. Azmy2, G. Capitani3, G. Confalonieri4, G.
Della Porta1, E. Griesshaber5, D. Harper6, M. Leng7, L. Nolan8, M. Orlandi3,
R. Posenato9, W. W. Schmahl5, V. Banks7, M. Stephenson7 1Università degli Studi di Milano, Scienze della Terra A. Desio, Milano,
Italy 2Memorial University of Newfoundland, St. John's, Canada 3Università degli Studi Milano Bicocca, Milano, Italy 4Università degli Studi di Torino, Torino, Italy 5Ludwig-Maximilians Universität München, Munchen, Germany 6Durham University, Durham, United Kingdom 7British Geological Survey, Keyworth, United Kingdom 8University of Leicester, Leicester, United Kingdom 9Università di Ferrara, Ferrara, Italy
The species of Gigantoproductusreach over 30 cm in width and are considered giants within the Palaeozoic sedentary marine benthos. Their
shell thickness is remarkable, reaching over 1 cm and consisting of a thin
pseudopunctate laminar secondary layer and a very thick columnar tertiary layer, made of a few hundreds of micrometres long, substructured columnar
units. The microstructure of several specimens has been investigated by
multiple analyses [petrography, cathodoluminescence (CL), Scanning Electron Microscopy (SEM), Electron Backscatter Diffraction (EBSD),
Transmission Electron Microscopy (TEM)]. The analysed shells are
generally well preserved, but locally altered by authigenic silica replacement of brownish, fibrous chalcedony and anhedral to subhedral microquartz
forming concentrically laminated spherulites, and euhedral megaquartz
crystals with hexagonal basal section, which embed calcite crystals and growth lines of the brachiopod shells. Petrographic and isotope analyses
show that silica replacement of the gigantoproductid outer shell occurred
during early diagenetic phase. Despite this, EBSD analyses show that the shells retain the presence of large pristine columnar units with crystallites
being highly co-oriented. MUD values are 41 to 71, except for occasionally
overprinted shell margins with low MUD values. TEM analyses also show that the columnar layer is formed by nanoscopic, biocomposite mesocrystal
calcite, that is built up by grains approximately co-oriented and that it
contains nanometres inclusions, with a dark contrast, forming trails between calcite grain borders similar to those observed in Recent brachiopod shells.
These inclusions were analysed by Nuclear Magnetic Resonance (NMR), and Gas Chromatography Mass Spectrometry (GC-MS) analyses, which showed
that an occluded organic fraction is preserved in these ancient fossil shells,
and its preserved amino acid composition is comparable with that observed in Recent brachiopod taxa.
Finally, the analyses of the carbon- and nitrogen-isotopic compositions of the
occluded organic matrix within their calcite shells allowed to explain the gigantic size and thick carbonate skeleton of these Palaeozoic benthic
brachiopods, as the result of a mixotroph lifestyle, by which they could rely
on the energy and nutrients derived both from photosymbiotic microbes and from suspension feeding.
T 88
How did the carrier shell Xenophora crispa build its shell?
Evidence from the recent and fossil record G. Crippa*1, G. Pasinetti1, M. Dapiaggi1 1University of Milan, Milano, Italy
The animal kingdom offers several examples of organisms forming their
exoskeletons selecting and agglutinating objects from the surrounding
environment. The most famous and spectacular among these agglutinating organisms is probably represented by the carrier shell Xenophora Fischer von
Waldheim, 1807. The genus Xenophora comprises species of marine
gastropods, known from the Cretaceous to the Recent, which are able to agglutinate fragments of different origins to form their shells; they show
different agglutination potentials, from species lacking attachments to
species completely covered by agglutinated materials, as the Mediterranean Xenophora crispa. Here, we analyse Recent and fossil specimens of
Xenophora crispa from the Mediterranean area at the Scanning Electron
Microscope and X-Ray Powder Diffraction, to better understand its biomineralization pattern and the mechanisms leading to the agglutination of
shells and bio/lithoclasts. Also, we provide new data on the poorly described
gastropod shell microstructures which knowledge is generally limited to a relatively small number of specific taxonomic groups.
We conclude that: a) most of the Xenophora crispa shell is composed by an
aragonitic crossed lamellar fabric, but a fibrous to spherulitic prismatic fabric seemingly of calcite has been found in the columella and in the peripheral
edge, i.e. the thickest parts of the shell; b) the attachment of objects is
mediated by a prismatic microstructure, indicating that this is the most functional fabric in attachment areas in molluscs; c) the functional meaning
of the agglutination in Xenophora crispa is related to a snowshoe strategy to
30
successfully colonize muddy substrates. Indeed, this species secretes in the columella and in the peripheral edge a less dense, less hard and more organic
rich calcitic fabric, to lighten the thickest parts of the shells in order not to
sink in soft sediments.
T 89
Molecular evolution of the matrix proteins of shells and darts
in terrestrial snail Euhadra quaesita K. Shimizu*1,2, K. Kimura3,4, Y. Isowa5, K. Oshima2, M. Ishikawa2,6, H. Kagi2, K. Kito5, M. Hattori2,7, S. Chiba3, K. Endo2 1University of Exeter, College of Life and Environmental Sciences, Exeter,
United Kingdom 2The University of Tokyo, Tokyo, Japan 3Tohoku University, Sendai, Japan 4Kyungpook National University, Bukgu, South Korea 5Meiji University, Kawasaki, Japan 6Yamazaki University of Animal Health Technology, Hachioji, Japan 7Waseda University, Tokyo, Japan
Mollusks are among the most diverse calcifying animals and are well studied
for various aspects of their biomineralization. Although many shell matrix proteins (SMPs) have already been reported in mollusks, most reports have
focused on marine mollusks, and the SMPs of terrestrial snails remain
unclear. In addition, some terrestrial stylommatophoran snails have evolved a novel calcified apparatus, known as "love dart", used for mating behavior.
We identified 54 SMPs in the terrestrial snail Euhadra quaesita. The SMP of
the highest abundance index value in E. quaesitais similar to the most abundant SMP in the other hitherto studied pulmonate Cepaea nemoralis.
These proteins contain Pro- and Gly-rich regions, but have no known specific
domains and have no homologous proteins in the GenBank non-redundant protein database except for the proteins reported from pulmonates. These
novel proteins may have evolved in the last common ancestor of pulmonates
with a key role in their shell mineralization under terrestrial or freshwater environments. We then identified four dart matrix proteins (DMPs) and
found that two of them are the same proteins as those identified as SMPs in
this species. Our results suggest that some DMPs possibly have evolved by independent gene co-option from SMPs during dart evolution events. These
results provide a new perspective on the evolution of SMPs in the terrestrial
environment and the novel calcified feature of "love darts" in land snails.
P 01
Self-assembling block copolymers in the nucleation of
hydroxyapaptite Y. Jhons*1, N. Judge1, C. Fowler2, F. Nudelman1 1University of Edinburgh, Chemistry, Edinburgh, United Kingdom 2GlaxoSmithKline, GSK Consumer Healthcare, Weybridge, United Kingdom
Dental erosion is a worldwide pandemic with over 3.6 billion people
suffering from tooth decay, with 486 million children with caries on their permanent teeth.1 Enamel is the outer layer of the teeth, is highly mineralised
with around 95 % (by volume) of the structure being formed of carbonated hydroxyapatite (HAP), a form of mineralised calcium phosphate (CAP). The
exposure of enamel to acids from bacterial fermentation or from our diets
results in dissolution of the mineral and the formation of irreversible lesions that can result in tooth loss if left untreated. To address these issues, polymers
that are able to bind to enamel and prevent demineralization have been
investigated, however the effect of the polymer structure on the growth of HAP have not yet been studied.2
The aim of this project is to design and synthesise self-assembling
phosphorous-containing polymers that either prevent demineralisation or promote controlled nucleation and growth of HAP in enamel. We
hypothesize that controlling the self-assembling morphology can result in
controlled HAP growth, which could be key to remineralizing enamel. Here reversible addition fragmentation chain transfer (RAFT) polymerisation has
been used to synthesize phosphorous-based triblock copolymers. Using
dynamic light-scattering (DLS) and scanning electron microscopy (SEM), we observed that the triblock copolymer forms hollow spheres 300 – 700 nm
in size at concentrations above 6.2 x 10-3 mg cm-3. When incubated in tris
buffer pH 7.4 containing 2 mM of CaCl2 and 1 mM of K2HPO4 at 37 ºC, DLS and SEM demonstrate that the polymer at concentrations above 7.5 x 10-3 mg
cm-3 promotes the precipitation of calcium phosphate. Control experiments
without additives or using similar polymers that are either devoid of the phosphate group or that do not self-assemble did not result in calcium
phosphate precipitation. In conclusion, we synthesized a triblock copolymer
that promotes the precipitation of calcium phosphate. Both the self-assembly into micelles and the presence of phosphate groups in the polymer are needed
to promote calcium phosphate precipitation. Next steps will be to use
transmission electron microscopy to study their 3D morphology and characterize the form of calcium phosphate precipitated.
References
1 T. Vos, A. A. Abajobir et. al., Lancet, 2017, 390, 1211–1259.
2 Y. Lei, T. Wang, J. W. Mitchell, L. Zaidel, J. Qiu and L. Kilpatrick-
Liverman, RSC Adv., 2014, 4, 49053–49060.
P 02
The effects of protein incorporation and crosslinking on the
mechanical properties of mineralized chitin matrices J. Elias*1, L. Gower1 1University of Florida, Materials Science and Engineering, Gainesville, United States
Many natural protective systems, such as the exoskeletons of various
arthropods, use specialized organic templates and additives to guide the
mineralization of calcium phosphate, calcium carbonate or other mineral species. These natural mechanisms create mineral-organic composite
structures with attractive mechanical properties tailored to specific
environments, making them a subject of interest for many materials researchers. Even though there have been many attempts to mimic the
structure and properties of the crustacean cuticle in synthetic materials, there
are still interactions that are not fully understood because of the limitations of studying natural organisms. In vitro model systems, however, can provide
for the modification and isolation of system variables, making them valuable
for the understanding of various natural systems. The goals of this proposal are to: 1) Synthesize ordered chitin templates, then incorporate silk fibroin
or methacrylated silk fibroin into the templates to create chitin-fibroin
structures with varying degrees of crosslinking, 2) mineralize templates with calcium carbonate using a polymer-induced liquid precursor (PILP) process
and evaluate the effects of fibroin incorporation and crosslinking on the
mineralized structures, and 3) determine the effect of fibroin incorporation on the hardness, modulus, and fracture toughness of mineralized templates
using nanoindentation. The liquid crystalline ordering and protein
stabilization is expected to produce structures that mimic the helicoidal ordering of the chitin-protein phase in the arthropod exoskeleton, and the
biomimetic mineralization of these templates can create structures that mimic
the crustacean cuticle composite structure. This research can provide insight into the possible mechanisms used by biological organisms to create unique
hierarchical structures as well as investigating unique mechanisms that may
be used as inspiration for the synthesis of new composite structures.
P 03
Influence of bioaragonite microstructure in the kinetics of its
pseudomorphic replacement by apatite L. Fernández-Díaz*1,2, M. Greiner3, E. Griesshaber3, D. Reinares4, M. Zenkert3, X. Yin3, A. Ziegler5, S. Veintemillas-Verdaguer4, W. W.
Schmahl3 1Universidad Complutense de Madrid, Mineralogy and Petrology, Madrid, Spain 2Institute of Geosciences (IGEO) , Geomaterials, Madrid, Spain 3Ludwig-Maximilians-Universität, Geo-und Umweltwissenschaften, Munich, Germany 4Institute of Material Sience of Madrid (ICMM, CSIC), Madrid, Spain 5University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany
Numerous features of carbonate biological hard tissues are inspirational for
the development of new functional materials. Their characteristic internal
hierarchically arranged porosity and their microstructure are among these features. Both can partially or totally be preserved during the transformation
of carbonate biominerals into apatite after interaction with boiling phosphate-
bearing aqueous solutions. This carbonate-apatite transformation involves the development of interface coupled dissolution-crystallization reactions
(ICDR) and commonly takes place with the preservation of the biomineral
external shape as well. In this work we study the kinetics of the pseudomorphic replacement by apatite of the bioaragonite of the cuttlebone
of the cephalopod Sepia officinalis and the different portions of the shells of
the bivalves Arctica islandica and Hyriopsis cumingii. All these biominerals have distinct microstructures and distribution of occluded organic
biopolymers. The kinetics of the transformation is fastest for Sepia
officinalis cuttlebone, which after only 10 hours of interaction with a (NH4)2HPO4 boiling aqueous solution is converted in a 90 wt % apatite
scaffold [1, 2]. This conversion is accompanied by a significant increase in
the biomineral surface area, which is attributed to the generation of new porosity that facilitates the progress of the ICDR. All other biominerals
transform into apatite at a much slower rate [2]. Both, the inner and outer
layers of the shell of Arctica islandica transform into apatite at similar rates, but after 14 days of interaction with the phosphate-bearing solution only
reach transformation percentages as small as 10 wt% and 15 wt%,
respectively. The prismatic portion of the shell of Hyriopsis cumingii is more reactive than the shell of Arctica islandica, with transformation percentages
above 25 after 14 days interaction with the phosphate bearing solution. In
31
contrast, the aragonite of the nacreous portion of this shell is extremely unreactive and remains virtually pristine during the whole duration of the
interaction experiment (14 days). We interpret that the distinctly different
kinetics of the into apatite transformation reflect the different accessibility of the phosphate-bearing aqueous solution to the mineral component of the hard
tissues studied. Differences in fluid accessibility arise from the
characteristics of each hard tissue regarding their porosity, their content of biopolymers and their ultra- and microstructure as well as the specific
evolution of each of these features as the ICDR progresses.
References
[1] Reinares-Fisac, D.; Veintemillas-Verdaguer, S.; Fernández-Díaz, L.
Conversion of biogenic aragonite into hydroxyapatite scaffolds in boiling
solutions. CrystEngComm, 2017, 19, 110-116. [2] Greiner, M.; Fernández-Díaz, L.; Griesshaber, E.; Zenkert, M.; Yin, X.;
Ziegler, A.; Veintemillas-Verdaguer, S.; Schmahl, W. W. Biomineral
reactivity: The kinetics of the replacement reaction of biological aragonite to apatite. Minerals, 2018, 8(8), 315.
P 04
Mineral replacement of bioaragonite by apatite – differences
between symbiotic and asymbiotic corals P. Forjanes*1, M. Greiner2, E. Griesshaber2, L. Fernández-Díaz1,3, I.
Coronado4, J. Stolarski4, M. Zenkert2, D. Joester5, L. Stegbauer5, U.
Rameshbabu5, S. Veintemillas-Verdaguer6, W. W. Schmahl2 1Universidad Complutense de Madrid , Mineralogía y Petrología, Madrid,
Spain 2LMU Munich, Department of Environment and Earth Sciences, Munich, Germany 3Institute of Geosciences (CSIC, UCM), Madrid, Spain 4Institute of Palaeobiology,, Warsaw, Poland 5Northwestern University, Materials Science and Engineering, Evanston,
United States 6Institute of Materials Science (CSIC), Madrid, Spain
Scaffolds for bone tissue engineering can be obtained through the
pseudomorphic mineral replacement of carbonate skeletons of invertebrates by apatite upon interaction with boiling phosphate-bearing aqueous solutions
[1, 2]. These skeletons exhibit porosity at different length-scales, which is
preserved during the mineral transformation process. Their characteristic pore sizes and pore distribution are closely matching those ideals for bone
grafting. In this work, we assessed the kinetics of the aragonite-apatite transformation of skeletons of four coral taxa: Acropora, Porites, Lophelia,
and Madrepora. Coral skeletons are organic-inorganic composites that show
distinct function-related characteristics, regarding their microstructure, skeletal density, original porosity compositional and distributional
differences in organic biopolymers and geochemistry. These distinct
features, which are significantly different in some symbiotic (Acropora and Porites) and asymbiotic (Lophelia and Madrepora) corals, have a complex
influence on the skeleton reactivity. Skeletons of Acropora and Porites
contain larger volumes of macro- and mesoscale porosities and have rougher surfaces. In contrast, the skeletons of Lophelia and Madrepora are compact
and show smooth surfaces. Moreover, the bioaragonite of symbiotic corals is
Mg-richer and Sr-poorer than that of asymbiotic corals. Further differences regard the characteristics of their organic matrix. The highest content of
organics is found in the skeleton of Madrepora oculata, with 6.05%,
followed by Lophelia pertusa, with 4.93%. The two symbiotic corals, Acropora sp. and Porites sp., show lower contents of organics, with 2.64%
and 2.10%, respectively.
The skeletons of Madrepora and Lophelia transform at a slower rate in contrast to Porites and Acropora into apatite during the first 4 days of
interaction with the phosphate-bearing solution. This is followed by a
subsequent latent period, without apatite replacement, that lasts 7 to 9 days and afterwards, the transformation rate rapidly increases. The fastest
transformation kinetics corresponds to the skeletons of the two symbiotic
corals Porites and Acropora, whose apatite content after 14 days of interaction reaches 39 wt% and 60 wt%, respectively. After this period the
skeleton of Lophelia contains only 15.3 wt% of apatite. Interestingly, the
skeleton of Madrepora is the least reactive, reaching only a 2.2 wt% of apatite at the end of the experiment. We conclude that the transformation of
coral bioaragonite into apatite is facilitated by the higher porosity and
rougher surfaces of the skeletons of symbiotic corals. The higher content of organics of asymbiotic coral skeletons seems to prevent aragonite
transformation. Further differences in aragonite into apatite transformation
kinetics might be modulated by both, biopolymer decomposition rate and composition-related differences in aragonite solubility.
References [1] D. Reinares-Fisac, S. Veintemillas-Verdaguer, L. Fernández-Díaz. Conversion of biogenic aragonite into hydroxyapatite scaffolds in boiling
solutions. CrystEngComm 2017, 19, 110-116.
[2] M. Greiner, L. Fernandez-Diaz, E. Griesshaber, M.N. Zenkert, X. Yin, A. Ziegler, S. Veintemillas-Verdaguer, W.W. Schmahl, Biomineral Reactivity:
The Kinetics of the Replacement Reaction of Biological Aragonite to Apatite. Minerals 2018, 8, 315.
P 05
Synergetic organic-inorganic interactions regulate
mineralization Y. C. Huang*1, B. Wu2, M. Drechsler3, S. J. Huang4, A. Rao5, J. C. C.
Chan4, D. Gebauer1,6 1University of Konstanz, Chemistry, Konstanz, Germany 2Forschungszentrum Jülich, Jülich Centre for Neutron Science, Garching,
Germany 3University of Bayreuth, Bayreuth Institute for Macromolecular Chemistry, Bayreuth, Germany 4National Taiwan University, Chemistry, Taipei, Taiwan 5University of Twente, Faculty of Science and Technology, Enschede, Netherlands 6Leibniz University of Hannover, Institute of Inorganic Chemistry, Hannover, Germany
Biomineralization is regulated by biomolecular and ionic species in complex
physiological environments. However, the underlying mechanism of how organisms precisely exert control over mineralization still remains elusive.
In the present study, we address the fundamental aspects of nucleation and
crystallization of calcium carbonate (CaCO3), a vital biological and geological mineral. The bidirectional relations between specific protein
moieties (CTL domains) of the sea urchin spicule proteome and distinct
inorganic entities in the course of mineralization are elucidated. Given the pH-dependent speciation of (bi)carbonate ions, a potentiometric titration
methodology was implemented for quantitatively investigating CaCO3
mineralization at near-neutral pH levels (pH 7.5–9.0). Notably, our investigation brings forth the mechanistic contributions of HCO3
- ions in
mineral nucleation as active soluble species interacting with ionic
components and structural constituents of the emergent solid amorphous phases. Moreover, exploring the influences of recombinant proteins
associated with sea urchin skeletons (CTL proteins), biophysical properties
involving ion-complexation and self-association are found to impact mineralization. In particular, in the presence of Mg2+ ions at lower pH levels,
the investigated CTL proteins exert enhanced control over mineralization.
This implies that in physiological scenarios, with the synergy of "spectator" ion species (e.g. Mg2+ and HCO3
- ions), minute quantity of biomolecules can
profoundly govern mineral nucleation. Collectively, our findings suggest that biomineralization, emerging as a biologically programmed multistep
crystallization reaction, is a bidirectional process which encompasses (i)
ionic and biomolecular additives that regulate the nucleation and crystallization of inorganics and (ii) distinct mineral entities that tune the
self-association of biomolecules.
P 06
Green synthesis of silver nanoparticles using Crocus sativus
corms aqueous extract and evaluation of their antibacterial
activity and cytotoxic effect on human ovarian cancer cell line
(A2780cp) A. Taghva*1, M. Entezari2, S. Ghafoori3 1Islamic Azad University Farahan Branch, Microbiology, Tehran, Iran 2Islamic Azad University Tehran Medical Sciences branch, Biology,
Tehran, Iran 3Payame Noor University, Biotechnology, Tehran, Iran
Introduction Recently, the application of nanoparticles is grown in various fields such as
biotechnology, nanotechnology, physics, chemistry, materials science, as
well as other new commercial applications. Noble metal nanoparticles such as gold, silver, and platinum present unique physicochemical properties,
which are not observed in larger metal nanoparticles, therefore, the metal
nanoparticles are used in different fields such as optical devices, catalysis,
biological labelling, drug delivery system, and cancer therapy.
Objectives In the present investigation, biosynthesizing silver nanoparticles (AgNPs) via Crocus sativus corm aqueous was explored. Then, antibacterial and
mutagenicity potential of the silver nanoparticles was investigated by Agar
Well Diffusion and Ames methods, respectively. Finally, the cytotoxic effect of AgNPs against human ovarian cancer cells was examined by the MTT
method.
Materials and methods
Biosynthesis of silver nanoparticles has done by subjecting Crocus sativus
corm aqueous extract and 0.001 Mm silver nitrate solution. After color
changing, nanoparticle production has investigated via spectrophotometry, XRD, and TEM. Then, antibacterial properties of produced silver
nanoparticles have investigated by Agar Well Diffusion and MIC determining Methods. Also, mutagenicity potential of produced silver
nanoparticles has determined by Ames test. Finally, cytotoxic effects of the
32
created the nanoparticles on A2780cp cell line has checked by cell culture and MTT method.
Results
The results indicated that nanoparticles were produced in 45 minutes by reducing silver nitrate ions and color changing occurred during this time.
Spectrophotometry assay showed an absorbance peak at 425 nm wavelength.
Also, XRD test was confirmed nanoparticles production. TEM microscopy indicated that the size of produced AgNPs was ranging from 5 to 25 nm with
a spherical shape. Antibacterial properties of silver nanoparticles were
approved by both Agar Well Diffusion and MIC test methods. Moreover, No mutagenic activity was seen in produced AgNPs. MTT assay after treatment
of human ovarian cancer cell line (A2780cp) with AgNPs in 24 h showed
that there is a dose-dependent cytotoxic activity in produced AgNPs against cancer cell line.
Conclusion It is clear that silver nanoparticles have a high potential to be used in industry and medicine. However, they should be examined from various aspects such
as safety, toxicity, and so on.
P 07
Enhancement in the photobiological hydrogen production of
chlorella-material hybrids by dimethyl sulfoxide Y. Zhao*1, L. Shu1, C. Shao1, W. Xiong2, R. Tang1 1Zhejiang University, Hangzhou, China 2Nanjing University, Nanjing, China
Photobiological production of hydrogen is low energy consumption and environmental friendliness, ensuring the generation of clean and renewable
energies. It has been reported that Chlorella aggregates, which is induced by
silica-based materials, can continuously produce hydrogen photobiologically under normal aerobic conditions. Even so, the yield is relatively low, which
equals only 0.42 % of the light‐to‐H2 energy‐conversion efficiency.
However, we find that a simple addition of dimethyl sulfoxide (DMSO) into an aqueous environment (to 0.5 vol %) can significantly promote the H2 yield
of Chlorella aggregates, reaching 0.69 % of the light‐to‐H2 energy‐
conversion efficiency. The improvement is explained by a DMSO-induced increase in the cellular respiration rate. This will lead to a decrease of the
oxygen content within the aggregates, and subsequently resulting in the
activation of more hydrogenases. In general, this strategy exhibits a functional enhancement by a combination of small molecules and organism-
material hybrids.
P 08
Non-classical crystallization of calcium carbonate towards
single crystal formation Z. Liu*1, H. Pan1, Z. Zhang2, Z. Wang3, B. Jin1, R. Tang1, J. J. De Yoreo3 1Zhejiang University, Chemistry, Hangzhou, China 2Xiamen University, Xiamen, China 3Pacific Northwest National Laboratory, Richland, WA, United States
Crystallization by particle attachment or two step nucleation has been widely
observed in both natural and synthetic environments. However, this non-classical crystallization towards single crystal formation cannot be explained
by classical nucleation theory, and many mysteries are still remained to
understand this process. We herein use calcium carbonate (CaCO3), which is a typical geological and biological mineral, as a model to investigate its
crystallization. It is revealed that a random particle attachment followed by a
self-orientation could occur on CaCO3 particles, leading to single crystal formation. And this is attributed by its surface stress induced grain-boundary
migration. Besides, we find the crystallization pathway of amorphous CaCO3
can be controlled by magnesium ion (Mg), which is a widely observed impurity in biominerals. In most cases, the crystallization of amorphous
CaCO3 is dominated by dissolution/re-precipitation process, and the resulting
crystal exhibit expected morphologies. Only at high Mg concentration, the crystallization occurs in the absence of a morphological change to give
spheroidal single crystalline Mg-calcite. It is explained by the high water
content in Mg doped amorphous CaCO3, in which the reorganization of molecules in solid-phase becomes available. Our findings promote the
understanding of crystallization in nature, implying some potential
mechanisms in biomineralization. Most importantly, these results can shed light on the construction of functional crystals with controllable crystalline
orientation and morphology, by design.
P 09
Biosynthesis and evaluation of the characteristics of silver
nanoparticles using Cassia fistula fruit aqueous extract and its
antibacterial activity S. Ghafoori*1, A. Taghva2, Z. Tayebi3, M. Hashemi4 1Payame Noor University, Biotechnology, Tehran, Iran 2Islamic Azad University, Farahan Branch, Microbiology,, Farmahin, Iran 3Islamic Azad University, Tehran Medical Sciences, Faculty of Medicine, Microbiology, Tehran, Iran 4Islamic Azad University Tehran Medical Sciences branch, Genetics, Tehran, Iran
Introduction
There are several ways of nanoparticles production, but the biological method of nanoparticles production is under the attention of researchers due
to its eco-friendly and energy saving properties.
Objectives In the present study biosynthesis of silver nanoparticle by Cassia fistula fruit
extracts were examined and mutagenesis potential of nanoparticles was
investigated. Furthermore, the antibacterial effect of the produced nanoparticles was investigated on In vitro and In vivo.
Materials and methods
In order to nanoparticles production, the fruit extract was subjected to the silver nitrate aqueous solution at the final concentration of 1 mMolar. After
nanoparticles production, the color changed reaction mixture was used for
characterization with spectrophotometry, X-ray diffraction analysis (XRD), Transition electron microscope (TEM) microscopy and DLS. Then, the
antibacterial effect of the produced nanoparticles was investigated by agar well diffusion method against three bacterial pathogenic strains. Mutagenesis
effect of silver NP was investigated by the Ames test. At the final, wound
healing repair of silver Nano NP in vivo was examined.
Results After nanoparticles production, the color of the plant extract was converted
to dark green attributed to the surface plasmon resonance band (SPR) of the silver nanoparticles. Visible spectra of the color changed extract had
maximum absorption peaks around 418 nm. Furthermore, the presence of the
silver nanoparticles was confirmed by the XRD. TEM analysis revealed that the obtained silver nanoparticles were triangle, hexahedron and spherical in
their shapes. DLS test has shown that average sizes of nanoparticles were
around 3/6-4/5nm. Antibacterial assays revealed that the produced nanoparticles had suitable effects against all of the three bacterial strains.
Mutagenic effects of nanoparticles were not observed. The silver
nanoparticles have a suitable effect on preventing wound infection in mice were tested.
Conclusion
It seems that the biological production of nanoparticles with the usage of these plant extracts is able to enhance their medicinal effects. In this study, it
is indicated that the antibacterial property of the extracts containing
nanoparticles was promoted considerably. Promoting other effects of these nanoparticles can be prospective for future studies.
P 10
SEM/EDS analysis of microbial induced calcium carbonate
crystal formation in agarose hydrogels B. Christgen*1, H. Mitrani1, M. Zhang2 1Newcastle University, School of Engineering, Newcastle upon Tyne,
United Kingdom 2Northumbria University, Health and Life Sciences, Newcastle upon Tyne, United Kingdom
Introduction
Microorganism mediated processes are present in every environment on the
Earth. Understanding how bacteria interact with and use their environment to survive is essential to translate to and use this knowledge in sustainable
biotechnologies. Microbial induced calcite precipitation (MICP) as a
potential low cost and environmental sustainable process has gathered extensive interest in the geotechnical and construction community for soil
improvement and as a sustainable construction material in recent years.
Underground carbon storage, sustainable biobricks, and healing and corrosion protection of cracks in cement based building materials are
examples of the multitude of applications investigated. Hydrogels have long
been used to investigate chemical crystal growth and morphology for calcium carbonate. Additionally hydrogels can be used as soil analogues to study
bacterial growth and MICP in situ in a more confined and easily accessible
environment.
Objectives
In this study we investigated the influence of variations in the cementation
media on micro-scale calcite growth and distribution in agarose hydrogels over time using S. pasteurii as a model organism.
33
Materials and Methods
S. pasteurii was grown over night in liquid culture before suspension in
agarose hydrogels which contained a cementation media of nutrient broth,
urea, CaCl2 (at concentrations between 30 mM and 480 mM), NH4Cl and NaHCO3. The hydrogels were grown between two to nine days at 30 °C and
were then analyzed by SEM/EDS and the calcium carbonate content was
measured. SEM specimen were prepared by flash freezing in liquid nitrogen to avoid structural deformations during the freeze drying process and then
freeze dried for 24 hours at -80 °C under vacuum. Before SEM analysis, the
specimen were sputter coated with a 5 nm layer of platinum using a high resolution sputter coater. The specimen were visualized at low voltage (1.8
kV) to avoid damaging the hydrogel structure while the EDS X-ray spectrum
for elemental analysis was obtained using 20 kV afterwards.
Results
SEM analysis shows spherical crystal growth after three days days incubation
of the hydrogels at 30 °C. Crystal size varied between 10µm and 300µ in different specimen depending on incubation time and CaCl2 concentration.
Higher calcium chloride concentrations in the cementation media led to more
and larger crystal formation in the hydrogel. EDS X-ray spectra of the crystals confirmed the presence of carbon, oxygen and calcium, the elements
that make up CaCO3 which verifies that the formed crystals are calcite.
Control hydrogel samples incubated without bacteria and/or without calcium chloride which were treated the same as the main specimen showed no crystal
formation.
Conclusion
SEM and EDS anlysis have been used to investigate calcite formation in
hydrogels from MICP induced by S. pasteurii and cementation media over time. EDS confirmed that the spherical crystals observed in the hydrogels
were calcite and growth depended on incubation length as well as the
concentration of calcium chloride in the cementation media used.
P 11
Use of biomineralized microsilica and highly nanostructured
CaCO3 particles for biosensors and bioseparation S. Kim1, M. B. Gu*1 1Korea University, Department of Biotechnology, College of Life Sciences and Biotechnology, Seoul, South Korea
Introduction
The calcium carbonate (CaCO3), which is most abundant biomineral found
in nature, is commonly used as exoskeletons of many life forms such as algae, clam, and sea urchins. Recently, there has been a growing interest in using
CaCO3 for diverse applications due to their versatility and unique properties.
Especially, CaCO3 can be promising materials in biotechnological areas. Furthermore, the various hierarchical structures of biominerals make CaCO3
based materials more attractive.
Objectives
We report the application of CaCO3 based biomineral in the biosensing and
bioseparation using aptamer as a bioreceptor. For biosensing application,
CaCO3 structure derived from unicellular algae was utilized to enhance the analytical performance of the biosensor. For bioseparation application,
aptamer conjugated magnetically separable silica coated CaCO3
microparticles were developed for antibiotics removal.
Materials and Methods
For biosensing application, CaCO3 microparticle derived from algae was
applied for surface modification of electrode. Briefly, 1 μl of algae-derived CaCO3 structure (45 mg/ml) was applied to the substrate, and the gold
working electrode was formed by sputtering. Sandwich-type binding
aptamers were applied for sensing type-2-diabetes. For bioseparation application, aptamer conjugated magnetically separable silica coated calcium
carbonate microparticles were synthesized by simple procedures. Briefly, 20
mM of CaCl2 in Tris-HCl (1 M, pH 8.3) solution with Poly-acrylic acid (0.1 mg/ml) and 100 μL of magnetic nanoparticles was stirred in a CO2 chamber
and coated with silica by the modified sol-gel. Finally, oxytetracycline
binding aptamers were immobilized on to the particle for specific removal oxytetracycline.
Results
The morphological, electrochemical characteristics and the performances of the newly developed biosensor were investigated by SEM, EDAX, cyclic
voltammetry, and chronoamperometry. The electroactive surface area was
increased about 3.8-fold compared to a commercial screen-printed electrode (SPGE) which is likely due to the roughened surface provided by the
nanostructured CaCO3 microparticles. The analytical performance of the
newly developed biosensor was enhanced, when compared to the SPGE, by 1.16-fold and 3-fold in buffer and serum condition, respectively. The
improved analytical performance could be explained by the surface
properties of algae-derived CaCO3 microparticles which increased electroactive surface area affecting the diffusion of electrolytes and analytes.
Aptamer-conjugated magnetically separable silica coated calcium carbonate
microparticles for antibiotics removal were successfully synthesized and characterized by SEM, FT-IR, EDAX, BET, and CLSM. About 6 μm sized
aptamer modified CaCO3 microparticles showed low non-specific adsorption
to oxytetracycline (OTC) which was less than 15%. The selective capturing efficiency towards OTC in both buffer and tap water was about 85% and
73%, respectively. Moreover, these bio-hybrid mineral microparticles were
found to be stable, even after 5 repeated usages, maintaining the initial capturing efficiency of 72%.
Conclusion
Using the biomineralized and highly nanostructured CaCO3 microparticles for biosensing and bioseparation, we could successfully detect and capture
the target molecules with improved performances. With the advantages of
biominerals, these new studies could open up new possibilities for biotechnological applications of biominerals.
P 12
Calcium carbonate crystals formation under the influence of
otolithic and otoconial matrix collagen-like protein otolin-1 K. Bielak*1, A. Zoglowek1, J. Stolarski2, A. Ożyhar1, P. Dobryszycki1 1Politechnika Wrocławska, Department of Biochemistry, Wrocław, Poland 2Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland
The creation of mineralized structures in invertebrates and vertebrates
requires cellular regulations, where the action of distinctive proteins control the deposition of biominerals as they have an influence on the nucleation,
growth, localization and morphology of the growing structure. Many of them
belong to intrinsically disordered proteins (IDPs) considered as the major regulators of the process. However, significant role of matrix proteins in the
process of biomineralization cannot be neglected. Matrix proteins provide an
organic scaffold and a surface for mineral deposition as in an epitaxial nucleation. The possible interactions between matrix proteins and inorganic
elements suggest that also crystals supporting proteins may have an influence
on the polymorphic form as well as biomineral morphology. In the formation of fish otoliths and higher vertebrates otoconia, a protein
named otolin-1 is involved as the scaffolding and possible tethering element
of these calcium carbonate structures of the inner ear. Otolin-1 is one of a few non-IDPs proteins involved directly in the process of ear stones and ear
dust biomineralization.
In this study, we would like to test the influence of recombinant homologues of Danio rerio and Homo sapiens otolin-1 on the formation of calcium
carbonate crystals in slow diffusion method.
Recombinant zebrafish and human otolin-1 was expressed in the bacterial system composed of Escherichia coli Arctic Express cells transformed with
pQE80L plasmid containing cDNA of zOtol1 and hOtol1 separately. Protein purification and sample preparation involved affinity chromatography to
cobalt or nickel cations followed by size exclusion chromatography. Calcium
carbonate crystals were grown in the range of protein concentrations in 96-wells plate-based biomineralization chambers with the use of a slow
diffusion method.
The proposed method of production and purification of recombinant zOtol1 and hOtol1 was sufficient to obtain highly pure samples in the yields ranging
from 0.86 mg up to 1 mg of the protein from one liter of bacterial culture.
zOtol1 and hOtol1 affected the size and morphology of calcium carbonate crystals obtained by slow diffusion method. Micro-Raman microscopy
determined the polymorphic form of these biominerals.
Concluding, the presence of otolith and otoconia matrix recombinant collagen-like otolin-1 has an impact on the formation of calcium carbonate
crystals in the slow diffusion system for Danio rerio and Homo sapiens
homologues. Acknowledgments: This work was supported by the National Science Center
(Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity
subsidy from the Polish Ministry of Science and High Education for the Faculty of Chemistry of Wroclaw University of Science and Technology.
P 13
Cryo-TEM study of the growth and crystallization processes of
calcium phosphate G. Dalmonico1, M. Farina2, A. Rossi*1 1Brazilian Center for Research in Physics, Rio de Janeiro, Brazil 2Federal University of Rio de Janeiro, Biomedical Science, Rio de Janeiro, Brazil
Introduction
Calcium orthophosphates are a class of materials that attracts strong interest
in many research areas in the fields of chemistry, physics, biology and
medicine. They are one of the main inorganic constituents of the calcified tissues of vertebrates (bones and teeth) and can be used in bone implants due
to their remarkable biocompatibility and bioactivity. The synthesis of HA in
a controlled manner is an important requirement for different applications and can also be used for elucidating the mechanism of mineralization in
biological systems (biomineralization).
Objective
In this work, the morphology and structural characteristics of calcium
phosphate (CaP) nanoparticles were investigated at different steps of the
34
synthesis process by analytical cryo-transmission electron microscopy (Cryo-TEM) in combination with other analytical techniques that need large
amounts of material.
Materials and Methods
The synthesis of HA was performed by dropwise addition of phosphoric acid
to calcium hydroxide solution in order to obtain a final solution with a Ca/P
ratio of 1.67. Calcium and phosphate ions react to form CaP and water. The whole synthesis (dropwise addition and aging) occurred over a period of 24
h. The reaction was performed at room temperature (~25°C), and the pH was
not kept constant. Samples were collected after different periods of time (5 min, 10 min, 30 min, 1 h, 2 h, 7 h, 11 h, 15 h, 19 h and 24 h) for the study of
the process of HA growth and crystallization. The samples were ultrafast-
frozen to be analysed by transmission electron microscopy or freeze-dried to be analysed by XPS, XRD and FTIR.
Results and Discussion
We showed that the process of drying the sample, instead of analyzing the sample in vitreous ice, did not significantly alter the morphology of the
nanoparticles but did induce significant changes in their crystallinity. As
shown by Cryo-TEM, crystallization of the nanoparticles in the frozen hydrated state was detected only after a long period of synthesis. In the first
5 min of reaction, which corresponds to a low amount of phosphate ions in
solution, we observed nanoparticles characterized by a high Ca/P ratio that transformed after 30-60 min into needle-like amorphous nanoparticles with
a comparatively lower Ca/P ratio. The mechanism of HA formation using
these chemical routes will also be discussed in the presentation regarding new paradigms of (bio)mineralization on the basis of a nonclassic mechanism
of nucleation induced by nanocluster aggregation, amorphous calcium phosphate and multiple stages of crystallization.
P 14
Stabilization of amorphous calcium oxalate (ACO) particles
with poly(acrylic) acid in pre-nucleation essays F. Diaz-Soler*1, M. Weber1, A. Neira-Carrillo1 1Universidad de Chile, Santiago, Chile
Introduction
Calcium Oxalate (CaOx) is one of the most widely distributed biominerals in
nature and recently a non-classical crystallization route (CNC) via ionic
aggregates (clusters) as amorphous precursors particles has been reported. It is well known that acidic biomolecules present a key role in classical
crystallization (Biomineralization) and pathological mineralization of CaOx, present in plants and animals. Thus, poly(acrylic) acid (PAA) is an acidic
synthetic polymer, described as an inhibitor, widely used to understand how
biomolecules modulate nucleation and the stages of aggregation and crystalline growth of biogenic materials. However, the effect of PAA on the
kinetics of the CaOx CNC is currently unknown.
General Objective
To investigate the kinetics and PAA concentration effect on the CNC of
CaOx through in vitro pre-nucleation assays.
Materials and methods
The CaOx pre-nucleation assays were performed using a computationally
controlled automatic titration system (Titrando 907, Metrohm) and
commercial computational software (Tiamo ™, current version: 2.3). CaOx pre-nucleation assays were performed by using an automated titration of
5mM sodium oxalate solutions with 20mM calcium chloride solutions at a
rate of 0.06 ml per minute in the absence and presence of PAA at concentrations of 10, 50 and 100 mg/L. During the whole pre-nucleation test,
the monitoring of free calcium ion concentration was carried out with a
selective ion electrode kept constant at pH 6.7. The determination and characterization of amorphous precursors (cluster) was studied by using
dynamic light scattering system (Nanosizer), transmission electron
microscopy (TEM) with selected area electron diffraction and Raman and FTIR spectroscopic techniques.
Results
Preliminary trials showed that the concentration of free calcium detected in the absence or presence of PAA differs from the amount of calcium added to
the solution. When PAA was used as an additive at concentration of 100
mg/L, there is a decrease in the slope of the kinetic curve of the CNC prior to nucleation point respect to the kinetic curves obtained in the absence of
additives. PAA acted as an inhibitor of CaOx crystallization, stabilizing the
pre-nucleation cluster, delaying the nucleation stage at longer times, which was proportional to the additive concentration. In addition, PAA allowed to
tolerate a higher concentration of free calcium, increasing its super-
saturation. Finally, PAA induces the formation of more soluble solid phases after nucleation. Ultra-structural characterization by HR-TEM is under
progress.
Conclusion
The current experimental results regarding to the kinetics of CNC of CaOx
suggest that PAA has the ability to stabilize in vitro amorphous precursors
formed during the pre-nucleation assays. We believe that the electrostatic interaction of the acid groups of PAA with the ionic CaOx clusters would be
stabilized by steric repulsion, preventing their aggregation.
Acknowledgment
The authors are grateful for the funding provided by Fondecyt, Project N°
1171520.
P 15
Biomimetic supertough and strong hybrid macrofibers Y. Yu*1, R. Tang1 1Zhejiang University, Department of Chemistry, hangzhou, China
Inspired by the special organic-inorganic hierarchical nanostructure of bone, the block copolymer with crystalline and amorphous protein regions as well
as special structural orientation of natural spider silk and their excellent
mechanical strength and toughness, here we show a simple down-up self-assembly method to prepare organic-inorganic hybrid macrofibers with
ordered mineralized polymer chain alignment. Owing to the coexistence of
crystalline and amorphous domains and the special rivet structure in the hybrid nanostructured macrofibers, the resultant hybrid macrofibers exhibits
superhigh tensile strength to ~950 MPa which is close to natural spider silk
(1150 ± 200 MPa), special toughness to 221 J g-1 which is proved to be the world"s highest special toughness among the PVA based materials and
surpasses natural spider silk (195 J g-1) and large stretchability (80.6%) as
well as excellent knitting properties and dyeability. This supertough and strong biomimetic hybrid macrofibers hence has promising applications in
textile fields such as flexible ballistic fabric. This down-top biomimetic
preparation approach of nanostructured macrofibers with excellent
mechanical properties is simple, scalable, and cost-effective, representing a
promising direction for the development of fiber industry.
P 16
The synergic effect of Sr2+ and Mg2+ on the stabilization of
amorphous calcium phosphate W. Jin*1, R. Tang1 1Zhejiang Unniversity, Hangzhou, China
Amorphous calcium phosphate (ACP) is a meta-stable precursor phase for
bone formation and it can transform into the thermodynamic stable mineral phase, hydroxyapatite (HAP, the main inorganic phase in bone), under
physiological conditions. In biological system, it has been documented that
Mg2+ can effectively stabilize ACP to ensure a regulation of biomineralization kinetics. In this study, we investigate the effect of another
alkaline earth metal ion, Sr2+, on ACP stabilization. In the human body,
99.9% Sr2+ is located in bones, and administered Sr2+ is almost exclusively precipitated in bone. Sr2+ is vital to young bone tissues formation. The role
of Sr2+ on ACP formation and its transformation needs to be clarified. The
precipitation of ACP and the crystallization of HAP are accompanied by an
abrupt drop in pH. We find that Sr2+ itself have less stabilization effect on
ACP in comparison with Mg2+ pH curves. However, the presence of Sr2+ can
significantly enhance the stabilization of Mg2+ on ACP due to a synergic effect. The chemical analysis reveals more Mg2+ should be excluded from
ACP to initiate the crystallization of HAP when Sr2+ ions are co-existed in
the amorphous phase. This change results in additional energy barriers for the solid phase transformation to provide a better stabilization effect on ACP,
which benefit bone formation. The revealing of multiple ions effects on the
phase transformation of amorphous minerals might shed lights on the understanding of biomineralization process as well as the fabrication of
stable amorphous phase, which has wide applications in drug delivery,
vaccine reservations, environment treatments, and lithium ion battery.This finding highlights the process of dopant ion exclusion in ACP and its control,
which enriches our understanding on the bioinspired regulation of
crystallization by using the cooperation of multiple ions.
P 17
Understanding the phase transformation from amorphous to
crystalline calcium phosphate by in situ transmission electron
microscopy B. Jin*1, C. Shao1, Z. Liu1, Z. Mu1, R. Tang1 1Zhejiang University, Hangzhou, China
In nature, the growth front of delicate mineral structures is covered with an amorphous continuous layer which is regarded as a precursor phase of
crystallized phase. For example, amorphous calcium phosphate (ACP)
widely exists in the new bone where ACP can transform into hydroxyapatite (HAP). Although the local rearrangement mediated direct phase
transformation mechanism from ACP to HAP has been reported, the detailed
phase transformation process still remains to be a great challenge due to the lack of direct experimental evidences. Here, we use in situ transmission
electron microscopy (TEM) to investigate the phase transformation
mechanism by skillfully designing a system in where spherical ACP particle closely contacts with HAP. It is found that the phase transformation is
achieved via epitaxial growth. Furthermore, in situ high resolution TEM
results show that the epitaxial growth induced crystallization advances via
35
the formation of kinks and steps. It is proposed that these steps and kinks are formed by the local rearrangement of posner"s clusters within ACP. This
finding highlights the key role of physicochemical effects in phase
transformation, contradicting the prior assumed epitaxial match between the structural organic matrix and the new produced mineral, which provides an
important implication in biomimetic mineralization and materials science.
P 18
Influence of Equisetum arvense extract on calcium oxalate
crystallization S. POLAT*1, P. SAYAN1 1Marmara University, Chemical Engineering, Istanbul, Turkey
Introduction Calcium oxalate crystallization is of great interest in medicine because it is the main constituent in the majority kidney stones. Calcium oxalate exists in
nature as calcium oxalate monohydrate (COM, whewellite), calcium oxalate
dihydrate (COD, weddellite) and calcium oxalate trihydrate (COT, caoxite). Calcium oxalate monohydrate and dihydrate forms are the major constituents
in most kidney stones. In this study, the effects on Equisetum arvense extract
on calcium oxalate crystallization were investigated detailed.
Objectives The main aim of this study was to investigate the potential inhibition effect
on Equisetum arvense extract on calcium oxalate crystallization to prevent kidney stone formation.
Materials & methods The calcium oxalate crystallization as a result of the reaction between calcium chloride and sodium oxalate was carried out in a cylindrical glass
crystallizer. The experiments were performed in batch crystallization mode
and the conditions were set at 37°C and pH 7.4 at a constant stirring rate. The crystallizer was kept at a constant temperature using a thermostat. During the
experiment, a pH meter was used to monitor the solution pH.
To investigate the effects Equisetum arvense, the extract was fed into crystallizer via a syringe pump. In this study, the concentration of the
aqueous Equisetum arvense extract used was 10 wt%. The end-products were
characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning
electron microscopy (SEM) were used.
Results XRD results showed that the crystals prepared in pure media include only the
calcium oxalate monohydrate form. According to the SEM analysis, the crystals prepared in pure medium had a hexagonal prismatic morphology.
The calcium oxalate monohydrate crystals had very fine structures and their
surfaces were slightly rough. The surface morphology of the crystals prepared in the presence of the Equisetum arvense was changed. In addition,
the functional groups and thermal properties of the crystals were investigated
in Equisetum arvense extract medium.
Conclusion Equisetum arvense has been showed to affect the crystal structure and
morphology of calcium oxalate using in vitro experiments. The structural and morphological transformations were confirmed using XRD, SEM, and FTIR
spectroscopy. Our results are useful and will contribute to the research efforts
on kidney stone formation, a significant clinical disease and important area of research in the field of biomineralization.
P 19
Synthesis, characterization and photocatalytic properties of Ti-
bearing hydroxyapatites A. Korneev*1, O. Frank-Kamanetskaya1, M. Kuz'mina1, V. Ryabchuk2, E.
Sturm3 1Saint Petersburg State University, Department of Crystallography, Saint Petesburg, Russian Federation 2Saint Petersburg State University, Department of Photonics, Saint
Petersburg, Russian Federation 3University of Kontanz, Department of Chemistry, Konstanz, Germany
According to [Wakamura et al., 2003; Tsukada et al., 2011] Ti-containing hydroxyapatites have photocatalytic activity comparable to activity of
anatase. However, there are doubts if this activity related to incorporation of
Ti-ions into hydroxyapatite or to small concentrations of TiO2 in precipitate, which can be not detected by X-ray diffraction.
Two series of hydroxyapatites were synthesized from solutions containing Ti
ions. The 1st series was precipitated from CaNO3, (NH4)2HPO4 and TiCl3 solutions, the 2nd series was precipitated from Ca(OH)2, H3PO4 and
C12H28O4Ti. Synthesized precipitates were studied with a wide set of
methods (X-ray powder diffraction, Raman spectroscopy, SEM, EDX, TEM, diffuse reflection spectroscopy).
According to the study results, hydroxyapatite has been formed in all
synthesis. Also precipitates of 1st series contain anatase impurity. Formation of anatase starts at atomic ratio Ti/Ca=0.56 in solution by XRD data, and at
Ti/Ca=0.01 by Raman spectroscopy.
Precipitates of 2nd series do not contain anatase by XRD data. However, after annealing samples at 700 °C for 6 hours peaks of anatase appear at diffraction
patterns. It evidences of formation of amorphous TiO2 during precipitation,
also confirmed by EDX data. After annealing amorphous TiO2 transits into anatase. Samples obtained from solutions with Ti/Ca=0.01 – 0.03 do not
contain any titanium dioxide phases by all used methods.
The increase of Ti concentration is assotiated with cell parameters variations. For 1st series, in the interval of Ti/Ca=0 – 0.13 titanium enters at Ca-sites
and in the interval of Ti/Ca =0.13 – 0.15 titanium enters at P-sites. For 2nd
series, titanium enters at both Ca and P-sites, but substitution of P is prevalent. Maximum concentration of Ti in 2nd series (9.5 wt%) is
significantly higher than in 1st series (5.4 wt%).
Diffuse reflection spectra of precipitates become more similar to anatase spectra (KRONOClean 7050) with increase of Ti concentration. Band gap
energy estimated by Tauc method using Kubelka-Munk transformation of
experimental diffuse reflectance spectra R(λ) ranges from 4,34 to 3.25 eV and is less than band gap energy of pure hydroapatite (Eg = 6 eV).
The obtained data confirm that Ti-containing hydroxyapatites can show
photocatalytic activity which depends on the entrance of titanium into apatite. The next step of our work is estimating their photocatalytic properties
through decomposition of organics.
The research was supported by RFBR grant №19-55-45019 IND_a and following resource centers of SPBU: Geo-Environmental Research and
Modelling (GEOMODEL), X-ray Diffraction Studies, Nanophotonics,
Optical and Laser Matetials Research, Microscopy and Microanalysis References
Wakamura, M., Hashimoto, K., Watanabe, T. Photocatalysis by Calcium Hydroxyapatite Modified with Ti(IV): Albumin Decomposition and
Bactericidal Effect. Langmuir, 2003, 19, 3428-3431.
Tsukada, M., Wakamura M., Yoshida, N., and Watanabe, T. Band gap and photocatalytic properties of Ti-substituted hydroxyapatite: Comparison with
anatase-TiO2, J. Mol. Catal. A: Chem., 2011, 338, 18–23.
P 20
Immobilization of Amino Acid Dehydrogenases by GO-PEI-IL
Hybrid Material induced Biomineralization K. Liu1, S. Wang*1 1Xiamen University, Department of Chemical and Biochemical Engineering, Hamburg, Germany
Introduction
L-Homophenylalanine (L-HPA) is a chiral unnatural amino acid, which is a
key intermediate of several pharmaceuticals. Biosynthesis of L-HPA
catalyzed by phenylalanine dehydrogenase (PheDH) is an ideal route. There are problems with limited substrate solubility in aqueous system and enzyme
denaturation in organic solvents.
Objectives
Immobilization of halophilic phenylalanine dehydrogenase with hybrid
materials based on biomineralization in order to create biocompatible
microenvironment and improve its stability and catalytic efficiency.
Materials & methods
Halophilic phenylalanine dehydrogenase (PheDH) from Bacillus
nanhaiensis is expressed in E.coil and purified. PheDH was absorbed to GO-PEI-IL and formed GO-PEI-IL-PheDH-TiO2 with the addition of titanium
bis(ammonium lactato)-dihydroxide (Ti-BALDH). Enzyme activity were
studied with 2-oxo-4-phenylbutyrate as substrate and NADH as cofactor.
Results
The effect of ionic liquid on [EMIM]BF4, [BMIM]BF4, [BMIM]Cl enhanced
enzyme activity by 2.1, 1.5 and 4.6 folds, respectively, while enzyme entrapment efficiency was 92.3%, 82.9% and 99.0%. ILs not only increased
the substrate solubility and also activated the enzyme activity, which was
accord to the salt active property of PheDH from Bacillus nanhaiensis. Compared with free PheDH, GO-PEI-PheDH, GO-PEI-IL-
PheDH, thermostability were greatly enhanced by induced biomineralization
of Ti-BALDH. Titania provided a rigid cage pocket for the protection from structure unfolding. A mechanistic illustration of the formation of hybrid
nanoparticles were proposed based on the multi-level interactions of enzyme,
PEI and ILs.
Conclusion
Polyethylenimine (PEI) and ionic liquids (ILs) functioned graphene oxide
(GO) were applied for PheDH immobilization by induced biomineralization of TiO2 with improved stability and activity for biosynthesis of L-
homophenylalanine. Hybrid materials GO-PEI-IL-TiO2 with synergistic
effect is are highly potential for enzyme immobilization, especially for bioelectrocatalysis, due to its biocompatibility and conductivity.
36
P 21
Biomimetic synthesis of bovine serum albumin and cysteine
modified ubiquitin-templated fluorescent inorganic
nanocomposites O. Akyüz*1, H. Cölfen1 1Konstanz University, Chemical Biology, Konstanz, Germany
Introduction
Biomineralization which is one of the interesting strategy found in nature for
the fabrication of advanced inorganic materials, is a source of inspiration for
many scientists searching methods for the synthesis of functional and bio-friendly nanomaterials. Proteins which possess well defined three-
dimensional structures and self-assembly properties based on their specific
binding sequences have been used as templates for biomineralization of various noble metals to functional nanostructures. By combination of the
functionalities of nanostructures with the biomineralization strategies;
fabrication of convenient functional and bio-friendly protein-templated fluorescent inorganic nanocomposites (FINCs) has been possible to use them
in various biomedical applications[1].
To sum up, proteins are promising biomolecules serve as scaffolds for the fabrication of FINCs with marvelous functions via the biomineralization
process. Therefore, developing new strategies for the synthesis of FINCs are
crucial to extend their properties and potential usages in bio nanotechnology.
Objectives
Synthesis of FINCs has been getting attention by many scientists due to their
attractive functions and features. However, the traditional chemical synthesis routes of such FINCs require harsh reaction conditions which are the
drawbacks for their activities in biological applications. Therefore, developing biomimetic synthesis strategies for the fabrication of FINCs
which are templated with natural and recombinant proteins under mild
reaction conditions, is pivotal to improve their functionalities. The purpose of this study is biomimetic synthesis of FINCs (gold
nanoclusters (AuNCs), cadmium selenide and lead sulfide quantum dots
(CdSeQD and PbSQD)) by using genetically engineered and natural proteins as biotemplates and investigating their properties via spectroscopic and
microscopic methods.
Materials & Methods
Bovine serum albumin (BSA), Ubiquitin (Ub) and Cysteine Modified
Ubiquitin (CMUb) were used as natural and recombinant proteins for the
synthesis of AuNCs, CdSeQD, and PbSQD in aqueous solutions at physiological temperature under alkaline conditions. The proposed
stabilizing/reducing mechanism behind the protein-templated biomimetic
mineralization of FINCs is as follows: (1) proteins containing abundant metal binding sites, such as cysteine (Cys), histidine residues can covalently
interact with the metal ions, (2) when the pH of protein environment is
adjusted to alkaline conditions by adding NaOH, i) the conformation of the protein changes and forms such a cage-like structure which is suitable for the
accumulation of metal ions (nucleation), ii) the phenolic groups on tyrosine
residue can be activated to a phenoxide ion at alkaline pH and then reduces metal ions to metal nanoclusters (reduction).
Results
The biomineralization experiments with the natural Ub and recombinant CMUb have clarified the pivotal role of the presence of Cys in protein
sequence by resulting in unstable and stable FINCs, respectively. As a result,
BSA and CMUb stabilized, functional, and stable FINCs possess photoluminescence and crystal size between 570-700 nm and 3-4 nm,
respectively, have been synthesized and characterized.
Conclusion
Green biomimetic synthesis strategies have been used to direct the formation
of FINCs having sub nanometer size and stable photoluminescence.
[1] J.Huang, L. Lin, D. Sun, H. Chen, D. Yang, Q. Li, Chem Soc Rev 2015,44,6330-6374
P 22
The coupled influence of Mg content and humidity on the
transition from amorphous calcium carbonate to calcite R. Thuemmler*1, B. Purgstaller2, E. Griesshaber1, M. Zenkert1, M. Dietzl2,
W. W. Schmahl1 1Ludwig-Maximillian Universitaet, Muenchen, Germany 2University of Technology, Institute of Applied Geosciences , Graz, Austria
Amorphous calcium carbonate (ACC) shows a remarkable stability, which is initiated by incorporation of impurities, e.g. ions or/and organic ligands.
Some impurities, e.g. Mg, stabilizes amorphous calcium carbonate to a high
degree and prevents effectively its transformation to crystallized calcite or aragonite.
In this study we discuss the coupled influence of Mg and humidity on the
transformation of ACC to crystalline carbonate. We describe for ACC stabilized with 5 and 50 wt% Mg contents the newly-formed carbonate phase
and the mode and speed from the amorphous to crystalline transformation.
Experiments were carried out in three humidity environments: low (about
24%), high (about 90%) and a wet state, where destilled water was sprayed onto the ACC tablet. Transformation experiments lasted for one, two, six and
nine weeks. We characterize our reference and newly-formed carbonate
material with XRD, FE-SEM, and electron backscatter diffraction (EBSD). We find that in the low humidity environment, even with low Mg contents,
ACC is highly stabilized and, for time periods up to six weeks, does not
transform to calcite or aragonite. The most significant transformation takes place when ACC contains 5 wt% Mg but is exposed for a long time to a high
humidity environment. Spraying the surface of the ACC tablet with water
induces transformation and crystalline carbonate formation, however, to a lesser extent as it is the case when the ACC tablet is exposed high humidity
for a long time period.
P 23
Size matters- nucleation kinetics of amorphous calcium
carbonate in confinement D. Joester*1, N. Metoki1, J. Cavanaugh1, M. Whittaker1, J. S. Evans1, K.
Alvares1 1Northwestern University, Materials Science and Engineering, Evanston, Illinois, United States
Introduction
Biomineralizing organisms routinely assemble materials with sophisticated
design and advanced functional properties, often using amorphous precursors to access compositional and structural states far from equilibrium. Organic
macromolecules and inorganic additives are thought to play an integral role
in controlling phase transformations in these systems. However, it has proven extremely challenging to accurately describe pathways and determine
mechanisms, even for extensively studied system such as amorphous calcium
carbonate (ACC).[1] A particular problem is the determination of accurate nucleation rates for the transformation of metastable precursors such as ACC
into the final, crystalline minerals.[2]
Objectives
Inspired by work in protein crystallization,[3] and guided by theoretical
considerations,[4] we set out to expand our earlier work on nucleation and
growth in liposomes[5] by developing a platform for the determination of nucleation rates in confinement based on droplet microfluidics.[6]
Materials and Methods
We designed and fabricated microfluidic devices that enable in situ observation of the crystallization of amorphous precursors confined in
emulsion droplets. Using time lapse polarized light microscopy, XRD, and Raman spectro-microscopy, we estimated the timepoint of individual
nucleation events in hundreds of droplets. Finally, statistical analysis of
nucleation events allowed us to determine steady state nucleation rates for the conversion of ACC to vaterite and calcite, and the impact of
biomacromolecules and inorganic ions on these rates.
Results In the absence of additives, statistical analysis revealed kinetics consistent
with classical nucleation theory. A steady-state nucleation rate of 1.2 cm-3s-
1 for the crystallization from ACC was determined. This low rate has important implications for phase transformation in biological systems. We
find that protein extracts from the sea urchin tooth (S. purpuratus) greatly
accelerate the nucleation rate. Recombinant spicule matrix protein (rSM30B/C) shows complex, concentration-dependent impact on
crystallization rates. To our surprise, this includes a polymorph switch at
elevated concentrations. Finally, we find that the presence of barium cations in droplets has a dramatic impact on nucleation mechanisms and rates.
Conclusion
Droplet microfluidic devices are powerful means to dissect nucleation and growth of amorphous precursors, and the impact of additives relevant in
biology and bio-inspired materials chemistry. I will discuss implications of
the absolute rates we determined for biological systems as well as mechanistic insights into the acceleration by rSM30B/C and barium cations.
References [1] J. J. De Yoreo, P. U. P. A. Gilbert, N. A. J. M. Sommerdijk, R. L. Penn, S. Whitelam, D. Joester, H. Zhang, Jeffrey D. Rimer, A. Navrotsky, J. F.
Banfield, A. F. Wallace, F. M. Michel, F. C. Meldrum, H. Cölfen, P. M.
Dove, Science 2015, 349. [2] L. M. Hamm, A. J. Giuffre, N. Han, J. Tao, D. Wang, J. J. De Yoreo, P. M. Dove, PNAS 2014, 111, 1304-1309. [3] S. V.
Akella, A. Mowitz, M. Heymann, S. Fraden, Crystal Growth and Design
2014, 14, 4487-4509. [4] R. P. Sear, CrystEngComm 2014, 16, 6506-6522. [5] C. C. Tester, M. L. Whittaker, D. Joester, Chem Commun 2014, 50
5619 - 5622. [6] J. Cavanaugh, M. L. Whittaker, D. Joester, RSC Chemical
Science 2019, available online.
37
P 24
Polymorph control of anhydrous guanine crystals Y. Ma*1, F. Chen1 1Beijing Institute of Technology, School of Chemistry and Chemical Engineering, Beijing, China
Amorphous guanine was proposed to be the mineralization precursor of biological anhydrous guanine β phase in vesica of the Koi fish scales1.
Guanine is one of the most widespread organic crystal existing in organisms
to produce structural colors. Guanine monohydrate and two anhydrous
guanine phases (AG), aand b , are three well known crystalline phases of
guanine. The excellent optical performances of guanine crystals are mainly
attributed to the crystalline AG β micro-platelets exposed (100) plane and high refractive index (1.83) 2. Herein, a novel crystal phase of anhydrous
guanine consisting of N9-G purine ring was obtained by dehydration of
guanine monohydrate (GM) at 111 °C, termed as dehydrated-GM, which is the first reported tautomeric polymorph of AG (N7-G purine ring). The
Dehydrated-GM has very good water harvest ability (8 wt%) with a fast rate
( in 30 min) even at relatively low room humidity (below 20 %) and low temperature (40 °C). We realized the synthesis of three polymorphs of
anhydrous guanine, AG a, AG b and dehydrated-GM in this work. A pure
phase of the anhydrous guanine (AG) β form was obtained via transformation of a hydrated amorphous guanine phase (HAmG), demonstrating short-range
order in solvents such as formamide, DMSO and DMF. Solid-state NMR
(ssNMR) characterization indicates that the HAmG precursor has similar
short-range order as AG β, which might be the reason for the formation of
AG β instead of the thermodynamically more stable AG α. The AG β nano-
platelets obtained in DMSO expose (100) plane when polyvinylpyrrolidone (PVP) was applied as additive. The sizes of AG β nano-platelets were about
100 nm in length, 40 nm in width, and 10 - 20 nm in thickness. The delicate
control on the polymorph and morphology of guanine crystals via an amorphous phase strategy may inspire the formation of highly ordered
hierarchical structures of guanine crystals with unique optical properties.
References [1] D. Gur, Y. Politi, B. Sivan, P. Fratzl, S. Weiner, L. Addadi, Angew. Chem.
Int. Ed. 2013, 52, 388.
[2] A. Levy-Lior, B. Pokroy, B. Levavi-Sivan, L. Leiserowitz, S. Weiner, L. Addadi, Cryst. Growth Des., 2008, 8, 507.
[3] F. H. Chen, Y. R. Ma*, Y. X. Wang, and L. M. Qi*, Cryst. Growth Des.,
2018, 18, 6497. [4] F. H. Chen, B.B. Wu, N. Elad, A. Gal, Y.N. Liu, Y. R. Ma * and L. M.
Qi, CrystEngComm, 2019, DOI: 10.1039/C9CE00245F.
P 25
The incorporation of organic and inorganic impurities into the
lattice of metastable vaterite E. Seknazi*1, B. Pokroy1 1Technion - Israel Institute of Technology, Material Science and Engineering, Haifa, Israel
Most of calcitic marine biominerals were shown to incorporate intracrystalline organic and inorganic inclusions. Inspired by these previous
findings, biomimetic calcite, containing organic or inorganic intracrystalline
inclusions was successfully synthesized in various routes. In the present study, we consider this feature in another calcium carbonate polymorph,
namely vaterite. Vaterite is the least thermodynamically stable and the least
abundant anhydrous crystalline phase of calcium carbonate. It is also the least well understood, as its crystal structure is still a matter of debate.
In this study, we show that vaterite is able to sustain high lattice strains and
incorporate Mg, Ba, and aspartic acid in its lattice. We report, for the first time, the formation of substituted Mg- and Ba-vaterite. We characterized and
quantified the structural distortions caused by incorporation of aspartic acid,
Mg or Ba into the vaterite hexagonal pseudo-cell by means of high-resolution synchrotron X-ray diffraction and micro-Raman spectroscopy. We show that
Mg substituted for up to 12% of Ca, and Ba for up to 2.5% of Ca, to form
crystals of Mg-vaterite and Ba-vaterite with lattice distortions of up to 0.9% and 0.14%, respectively.
Interestingly, this study proves that the presence of Mg and organics in biogenic vaterite can be parameters which affect its reported structural
variability. Therefore, this study, if accompanied by analyses of the Mg and
organics present in biogenic vaterite, could help clarify the true structure of vaterite.
P 26
Novel nanoscopic pathways in crystallisation and selfassembly
of barium carbonate nanoparticles in microemulsions W. Sager*1 1Forschungszentrum Jülich, Peter Grünberg Institute - Microstructure reserch (PGI-5), Jülich, Germany
Different nanoscopic paths to witherite nanoparticle formation via the birth
and deterioration and/or transformation of meta-stable amorphous and, so far unreported, crystalline barium carbonate nanoparticles using nonionic water-
in-oil microemulsions as precipitation media have been investigated in detail
by transmission electron microscopy and selected area diffraction. Compared to precipitation from homogeneous media, the employment of
microemulsions allows for a more precise control of the ongoing nucleation
and growth processes, not only via adjusting the concentration of the reacting species involved and temperature, but also by tuning the size and the
exchange kinetics of the isolated and compartmentalised nanometre-sized
water domains. Here we present a comprehensive study on how tuning the properties of the parental microemulsion can be implemented in tailoring the
crystal structure, morphology and self-assembling properties of nucleating
and growing nanoparticles. At low water content a variety of different meta-stable nanoparticles forms that ranges from in the electron beam amorphous
filaments to monoclinic (m) cubes and hexagonally shaped, probably trigonal
(t), thin platelets, which assemble into micrometre-long stacks. Slowing down of the exchange kinetics permits us to study the early stages of particle
formation and to gain full insight into unrevealed crystal transformation and
re-crystallisation processes. The genesis of the, at ambient conditions thermodynamically stable, orthorhombic (o) polymorph (witherite) evolves
either via direct transformation (e.g. a→o transition from filaments into rods)
or via dissolution/re-crystallisation processes. In the latter case the crystalline pre-structure particles dissolve, thereby freeing material for the re-nucleation
of the witherite phase (m/t→o transition), emphasising the double role of the
microemulsion domains as nucleation site and host and thus transport medium for water-soluble species. Depending on the different crystallisation
paths taken and the growth conditions encountered, a rich variety in final
nanoparticle morphologies is obtained. The ubiquitous microemulsion droplets induce attractive depletion forces between the forming nanoparticles
leading to different types of particle selfassembly, such as columnar stack
formation of nanoplatelets that is additionally characterised by small angle x-ray scattering and electron tomography.
P 28
Organization of prismatic layers in mollusk shells - Pinctada vs.
Pinna Y. Dauphin*1, E. Zolotoyabko2, A. Berner2, E. Lakin2, C. Rollion-Bard3,
J. P. Cuif4, P. Fratzl5 1Museum national d'histoire naturelle, ISYEB UMR 7205, Paris, France 2Technion-Israel Institute of Technology, Department of Materials Science
and Engineering, HAIFA, Israel 3Université Sorbonne Paris Cité, IPGP UMR 7154, Paris, France 4Museum national d'histoire naturelle, CR2P UMR 7207, Paris, France 5Max Planck Institute of Colloids and Interfaces, Department of Biomaterials, Potsdam, Germany
Introduction
Cross-sections of calcitic prismatic layers in mollusk shells, cut
perpendicular to growth direction, reveal well-defined polygonal shapes
clearly resolved by light and electron microscopy. These observations are typical for the prisms of Pinna, Pinctada, Atrina, and Ostrea. Taking into
account the rhombohedral symmetry of calcite, often presented in hexagonal
axes, this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In other words, the c-axis of calcite is thought to be
perpendicular to the surface of the shell. This paradigm is mostly based on
the long-term studies (since 1840) of Pinna nobilis shells, revealing the largest prism size; however other shells are much less investigated.
In this research, we address the question whether or not the crystallography
of calcitic prisms in Pinctada shells is similar to that well established in Pinna. As working example, we focused on the structure of Pinctada
margaritifera shells.
Materials & methods
The shells of Pinctada margaritifera were collected in French Polynesia,
while those of Pinna nobilis came from the Mediterranean Sea. The shell
morphologies and finer structural features were imaged by optical microscopies, SEM and AFM; crystallographic dissimilarities were analyzed
by X-ray diffraction and EBSD; in situ composition was studied using
Raman and FTIR spectroscopies, EPMA, XANES and X-ray fluorescence. Results: There are two kinds of prisms in Pinctada margaritifera: small
simple prisms and large prisms comprising several sectors. Only simple
prisms are known to exist in Pinna and Atrina shells. All prisms are built of nano-granular calcite (still being nearly single-crystalline) and organic
matrix. In contrast to Pinna nobilis, we find that in both large and small
prisms of Pinctada margaritifera the c-axis is mostly perpendicular to their
38
long morphological axis, i.e., parallel to the surface of the shell. Furthermore, the degree of this preferred orientation is higher in large prisms.
We relate these growth dissimilarities to the striking differences in the
organic contents. In fact, the soluble organic matrix of Pinna nobilis is rich in acidic sulphated polysaccharides, whereas that of Pinctada margaritifera
is rich in proteins. Lipid contents of Pinna and Pinctada also differ, as well
as Mg and S concentrations. In addition, small prisms have higher organic and organics-related sulfur contents than the large ones. In particular, atomic
percentage of sulfur in Pinna nobilis is three and twenty times higher than in
small and large prisms, respectively, of Pinctada margaritifera.
Conclusion
We conclude that, despite their morphological similarities and common
mineralogy, the prisms of Pinctada and Pinna, both Pteriomorpha, are far from being identical. The comparison of the organic contents reveals
significant differences in their nature and quantities. Based on these findings,
we suggest that in mollusk shells, the morphology of calcitic prisms is not primarily determined by the crystallographic structure of calcite, but largely
by organic components, the presence or absence of which drastically change
the growth mode.
P 29
Biomimetic cross-lamellar structures fabricated by ordered
self-assembly of CaCO3 nanorods M. Takasaki*1, M. Tago1, T. S. Suzuki2, Y. Oaki1, H. Imai1 1Keio University, Yokohama, Japan 2National Institute for Materials Science, Tsukuba, Japan
Introduction Biominerals have excellent mechanical and optical properties as compared
with geologic balky crystals. Especially, cross-lamellar structures of seashells consisting of CaCO3-based nanorods have received attention as an
excellent model for lightweight and tough structural materials because of
their superior mechanical properties derived from oriented architectures. Ordered self-assembly of nanocrystals is a non-classical way to fabricate
analogues of biogenic oriented architectures. However, artificial cross-
lamellar structures have not been produced by the self-assembly technique.
Objectives The aim of our study is fabrication of biomimetic cross-lamellar structure by
an evaporation-driven assembly method. We utilized calcite nanorods as a building block and an intense magnetic field to enhance their oriented
attachment.
Materials & methods Calcite nanorods ~50 nm in diameter that were elongated in the c direction
were obtained by oriented attachment of calcite nanograins synthesized by carbonation of Ca(OH)2 aqueous dispersion. We demonstrated a millimeter-
scale ordered assembly of calcite nanorods dispersed in ethanol by
combining the arrangement with an evaporation-driven capillary force and alignment under an intense magnetic force. Layer-by-layer accumulation of
the ordered assembly of the calcite nanorods was performed with a rotation
of the substrate at a 90-degree angle.
Results Calcite films ~10 μm in thickness were formed by oriented assembly with
lateral stacking of the nanorods on a substrate. The orientation of the nanorods in the films was improved by application of an intense magnetic
field at 12 T. The single-crystalline feature of the films similar to biogenic
cross-lamellae was characterized by X-ray and electron diffraction. Artificial cross-lamellar structures with crystal direction switching at 90-degree were
obtained by layer-by-layer accumulation of the ordered calcite films.
Conclusions Biomimetic cross-lamellar structures were fabricated through oriented
attachment of calcite nanorods by evaporation-driven self-assembly. Our
results suggest that the self-assembly techniques are applicable for fabrication of excellent analogues of biogenic oriented architectures.
P 30
The phenotypic plasticity in shell microstructures of vent and
seep pectinodontid limpets K. Sato*1, C. Chen2, R. G. Jenkins3, H. K. Watanabe2 1Waseda University, Department of Earth Sciences, School of Education,
Tokyo, Japan 2Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan 3Kanazawa university, Kanazawa, Japan
Gastropods often show intraspecific variation in phenotypes among different
habitats. Recently, the authors carried out molecular phylogenetic analyses
of pectinodontid limpets in two genera, Bathyacmaea and Serradonta, commonly associated with vent and seep environments in the west Pacific
(Nakano & Sasaki, 2011). Instead of revealing distinct lineages
corresponding to morphological identification, the analyses showed that all individuals from both genera were mixed in a single nested monophyletic
clade, except for one undescribed species from the South Chamorro
Seamount. This result strongly implies that most previously recognized "species" in fact belong to one species that is highly morphologically plastic
in shell form. The shell microstructure is the micro-scale morphological trait
of molluscan shells (Carter, 1990) and can represent a strong tool to estimate phyletic relationships and physiology. Therefore, the purpose of this study is
to assemble how genetic constraints and environmental factors affect the
expression of shell microstructure of pectinodontid limpets. Shell microstructures of pectinodontids were observed by SEM and their mineral
composition was detected by Raman spectrophotometry. As a result, we were
able to estimate specimens to genus level classification based on their shell microstructural compositions. Furthermore, the proportion of shell
microstructures with two different mineral compositions (aragonite or
calcite) clearly corresponds to differences in chemosynthetic habitat. Our study reports that mollusks regulate the mineral polymorphism in response
to their chemosynthetic habitat. This phenotypic plasticity in shell
mineralogy is significant not only in the context of the adaptive radiation of pectinodontid linages to chemosynthetic environments, but also to shed light
on mineralogical aspects regarding the biomineralization mechanism of the
molluscan shells.
P 31
Immunological detection and LC-MS/MS analysis of the shell
matrix protein ICP-1 in brachiopods Y. Isowa*1, K. Kito2, H. Sawada1, K. Endo3 1Nagoya university, Sugashima Marine Biological Laboratory, Toba, Japan 2Meiji University, Department of Life Sciences, Kawasaki, Japan 3University of Tokyo, Department of Earth and Planetary Science, Tokyo, Japan
Introduction
Brachiopods are sessile marine invertebrates belonging to Lophotrochozoa,
and have two valves composed of calcium carbonate or calcium phosphate.
Recently, proteomic analyses of shell matrix proteins have been performed on several brachiopod species to understand the molecular mechanisms and
evolution of brachiopod shell formation. In a previous shell proteomic
study of the rhynchonelliform brachiopod Laqueus rubellus, we identified a complete sequence of ICP-1, which was originally characterised as a
pigment-carrying intracrystalline protein from the three rhynchonelliform brachiopods Neothyris lenticularis, Calloria inconspicua, and Terebratella
sanguinea using Edman degradation. We showed that it is the most
abundant protein in the shell of L. rubellus by LC-MS/MS analysis. However, further details of ICP-1 remained to be clarified.
Objectives Antibodies are useful for localization of proteins within tissues. As a first
step to localize shell matrix proteins in the shells, we prepared an antibody
against ICP-1 in this study, and verified the binding specificity of the antibody. In addition, we carried out LC-MS/MS analysis to characterize
post-translational modifications of ICP-1.
Materials & methods The shell extracts from L. rubellus were subjected to SDS-PAGE, and
Western blotting was performed using the antibody against a synthetic
peptide designed from the ICP-1 sequence of L. rubellus. LC-MS/MS analysis was also performed against three major bands in the SDS-PAGE.
Results Western blotting showed a single band of approximately 60 kDa in size. This is substantially higher than the molecular mass expected from the theoretical
amino acid sequence of ICP-1 (17.5 kDa). We inferred that LrICP-1
undergoes post-translational modifications. Indeed, searches using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc) indicated
that LrICP-1 has one potential glycosylation site. Meanwhile, LC-MS/MS
analysis showed that a peptide of ICP-1 was identified from a band under 6.5 kDa, which is smaller than the molecular mass expected from the theoretical
amino acid sequence of ICP-1.
Conclusion The synthetic peptide used for antibody generation is located in the N-
terminal region, while the peptide identified by LC-MS/MS is located in the
C-terminal region. Therefore, one possibility is that ICP-1 undergoes post-translational cleavage, and the N-terminal fragment undergoes other post-
translational modifications. To confirm this hypothesis, we plan to perform
additional LC-MS/MS analysis to identify peptides in the N-terminal region of ICP-1.
39
P 32
Exploring biomineral formation at the nanoscale by electron
energy loss spectroscopy (EELS) M. de Frutos*1, O. Stéphan1, W. Ajili2, T. Azaïs2, N. Nassif2, S. Auzoux-
Bordenave3, D. Bazin4, M. Daudon5, E. Letavernier5 1CNRS Université de Paris Sud, Laboratoire de Physique des Solides, Orsay, France 2CNRS Sorbonne Université, LCMCP, Paris, France 3CNRS Sorbonne Université, BOREA, Paris, France 4CNRS Université de Paris Sud, Laboratoire de Chimie Physique, Orsay,
France 5Hopital Tenon AP-HP, Service des Explorations Fonctionnelles, Paris, France
Understanding of the hierarchical organization of biominerals and the mechanisms involved in their formation implies to unveil their chemical
composition and structure at different scales. Of particular relevance is the
characterization of the organo-mineral interface to understand how the organic fraction regulates the initiation of the calcification (nucleation) and
direct the crystal growth. Conventional TEM (transmission electron
microscopy) is limited, since it cannot easily distinguish the nature of the different components. Compared to other spectroscopic approaches, EELS
in a STEM (scanning transmission electron microscope) offers the advantage
of an outstanding spatial resolution (at the nanometer scale) in both chemical analysis and imaging.
To illustrate the interest of EELS for biomineral studies, we will present
selected results related to two different systems: kidney calcifications and abalone nacre. EELS was used to analyze the specimens with a resolution
defined by the diameter of the electron probe (typically better than a
nanometer). Kidney samples were prepared by ultramicrotomy from small pieces of
human kidney chemically fixed and embedded in epoxy resin. Nacre thin
section samples were obtained by Focused Ion Beam (FIB) on a selected area of the nacre layer of one-year-old Haliotis tuberculata shell. EELS data were
recorded on a VG HB01 STEM operated at 100 keV. The measurements were
obtained using a nitrogen-cooled sample stage and with the minimum electron dose to limit the radiation damage induced by the electron beam.
During an acquisition, the electron beam was scanned over the specimen and
an EELS spectrum was acquired for each position, giving access to an elemental mapping.
EELS data were compared with different reference samples, including mineral and organic compounds, in order to identify the chemical species
present in calcified tissues. The relative distributions of mineral and organic
fractions were obtained from the signal of the calcium and phosphorus edges and by the gaussian fitting of the peaks associated to carbonate and organic
compounds on the carbon K-edge. Two main minerals were found: calcium
carbonate in nacre and calcium phosphate, sometimes associated to carbonate, in kidney calcifications. The biological fraction were clearly
distinguished from the embedding resin.
Our study shows that STEM-EELS allows a nanoscale analysis of the chemical composition of biominerals in different calcified tissues giving
access to the mineral/organic interface. These data provides new insights to
elaborate possible mechanisms involved in biominerals formation. Acknowledgements: This work was partially funded via the CNRS-CEA
"METSA" French network (FR CNRS 3507) and the EU grant ESTEEM.
P 33
Sub-structures in bivalve simple prisms - a record of species-
specific control over crystallization during shell growth J. P. Cuif*1, Y. Dauphin2 1Museum Histoire Naturelle, CR2P-Paleontology, Paris, France 2Museum National Histoire Naturelle, umr ISYEB, Paris, France
Introduction
Since their very first observation in the shell of the Pteriomorphid Bivalve
Pinna nobilis, (Bowerbank 1844) "simple prism" as a microstructural
category has been used for shell description in many biological taxa, with taxonomic and evolutionary purposes. Most of the comparative studies are
based on static description focusing on morphological patterns and three
dimensional arrangements of the crystal-like units observed in adult shells. Here attention is drawn to diversity of mineral phase organizations and
growth mode variations in two simple prisms: P. nobilis and Pinctada
margaritifera.
Materials and Methods
Series of Pinna shells were kindly provided by Pr. N. Vicente, head of the
Les Embiez laboratory at Toulon-Six-Fours (France). Shells of Pinctada margaritifera were collected in Polynesia during a five years research project
involving the research department of the Direction des Ressources Marines
at Tahiti. Structural patterns were made visible through polished surfaces prepared on variously located areas of the prismatic layers followed when
necessary by appropriate etchings. From these specifically prepared surfaces,
information was obtained by using optical observation (mostly epi-polarisation), SEM on both secondary and back-scattered modes,
synchrotron based XANES method (ESRF Grenoble France) and
fluorescence (SOLEIL Saint-Aubin France).
Results
In Pinna nobilis etching shows that prisms are built by closely associated 6
to 8 µm thick sub-units (about 8 to 15 for every prism depending on specimens) whose overall orientations is slightly oblique to longitudinal
growth axis. Close correspondence of these mineral sub-structures between
adjacent prisms shows that their formation at the growing edge of the mantle is related to the pulsed stepping extension of the shell.
Prisms of the Pinctada margaritifera exhibit important morphological and
microstructural changes during growth. After an initial monocrystalline status increase in prism diameter is correlated to passage to a polycrystalline
status. Additionally, orientation of the crystallographic c axes becomes
perpendicular to growth direction, a surprising contrast to common views. Shift from single crystal to polycrystalline structure of the prisms, then
regression of the diameter and biochemical change in the final stages suggest
a continuous evolution of the mineralizing phase related to shell overall elongation and aging of the mineralizing cell layer.
Conclusion
These two prisms exhibit very distinct and contrasting internal sub-structures, revealing the biological control exerted over crystallization by the
mineralizing secretions of the epithelial cells during shell growth. From a
methodological view point, note can be made that such biological control of the structural and crystallographic patterns rises question regarding models
based on simple physical process (e.g. crystalline competition as well as predictive capability of thermodynamic grain growth theory and self-similar
crystallization).
P 34
Correlative 3D Raman imaging- a powerful method for
comprehensive studies of biomineralized samples U. Schmidt1, J. Englert1, A. Richter1, M. Böhmler1, T. Olschewski1, K.
Hollricher1, H. Fischer*1 1WITec GmbH, 89081, Germany
Introduction Raman microscopy is a useful tool for analying biominerals, as it can reveal the chemical composition of composite materials. Even more information
can be obtained by correlating the acquired images with data from other methods, such as Atomic Force Microscopy (AFM) or Scanning Electron
Microscopy (SEM). Such approaches can yield information that would not
be available with only one technique. Thus, Raman Imaging in combination with AFM or SEM can provide new insights into the fundamental processes
by which organisms produce biocomposites.
Objectives In this contribution we will introduce the principles of state-of-the-art
confocal Raman Imaging as a tool for analyzing the chemical and molecular
characteristics of a sample. We will show how this technique can be used in combination with AFM and SEM to correlate chemical information with
structural properties. The aim of this contribution is to describe and highlight
the unique features of such combined scientific analysis instruments, based on examples from various nano-biomaterials.
Materials & Methods For this study we use a Raman Imaging system which combines a confocal microscope with a highly sensitive Raman spectrometer. It delivers an
excellent depth resolution that enables the acquisition of 3D Raman images.
Raman microscopy allows the analysis of the distribution of the chemical components in a sample without requiring specialized sample preparation or
risking damage to the sample. It can easily be combined with other
techniques, such as AFM or SEM for correlative measurements.
Results SEM provides information on morphology, elemental composition and
crystalline structure of a sample. Confocal Raman Imaging of the same sample area reveals the chemical composition as well as polymorphism,
stress states and anisotropies. The combination of SEM and confocal Raman
microscopy in a single instrument allows the nondestructive characterization of biominerals with the highest resolution. Measurements of pearls and nacre
illustrate the benefits of combining these methods for investigating
biominerals. For example, a biomineralization defect in pearls was studied, which leads to loss of its pearlescence ("milky pearl").
Conclusion This submission highlights the power of correlative Raman techniques, such as Raman-AFM and Raman Imaging and Scanning Electron (RISE)
Microscopy, for the analysis of composite materials.
40
P 35
Asymmetric distribution of the crystal particle implies the
potential mechanism of aragonite tablets formation in the
nacreous layer of Pinctada fucata T. Jiang*1, J. Huang1, L. Li1, L. Xie1, R. Zhang1,2 1Tsinghua University, MOE Key Laboratory of Protein Sciences, School of
Life Sciences, Beijing, China 2Yangtze Delta Region Institute of Tsinghua University, Department of Biotechnology and Biomedicine, Jiaxing, China
The structure of the pearl oyster shell is intriguing because it implies that there are two biomineralization strategies co-existed in one system. The
thermodynamic aspect of the prismatic layer formation has already been
discussed but that of the nacreous layer seems lost in the whole picture. The mechanism governing the formation of the nacreous layer is convoluted.
However, the discussion about the pivotal component in the process is still
enlightenment. Here, with the help of the scanning electron microscope (SEM) and the transmission electron microscope (TEM), the crystal particle
distribution on the shell surface and the arrangement of aragonite tablets in
Pinctada fucata have been carefully examined. Strikingly, contrary to intuition, particles do not uniformly accumulate outward from the center
which might imply the dominant role played by the Marangoni effect. The
thickness of the aragonite tablets is not identical. The organic frame has evenly shifted among different layers. Taken together, these results imply the
existence of the Marangoni effect in the process of the nacreous layer
formation and the asymmetric distribution of the particle may lead to the even shift of the organic frame.
P 36
Microstrured biomineral particles from Emiliania huxleyi as a
raw material M. Chairopoulou*1, M. Herrmann2, U. Teipel1 1TH Nürnberg Georg Simon Ohm, Mechanical Process Engineering,
Nürnberg, Germany 2Fraunhofer-Institut für Chemische Technologie, Pfinztal, Germany
Microstructured particles have a number of interesting assets that can be beneficial for certain applications. To that counts for example the specific
surface area, which tends to increase with decreasing particle size, the pore structure, promising for material loading, and also the wetting behavior of
fine powder materials. When such structures are pursuit in natural systems
the algae family of Coccolithophorida pose a good representative. The sophisticated structures, formed according to strict biomineralization
mechanisms, are called coccoliths and are composed mainly of calcium
carbonate (CaCO3). Since CaCO3 is broadly used in numerous applications the potential of coccoliths is anticipated to be promising. However, in order
to use those uniquely formed particles in any future applications basic
questions and challenges still need to be improved. In an effort to address challenges associated with their separation process this study focuses on
recovering coccoliths from freshly cultivated media. The biogenic broth for
the experimental part was taken from laboratory cultivations of an Emiliania huxleyi strain in a 5 L fed-batch cultivation system. To that system, strategies
to clean and isolate the particles were followed according to literature
references and were tested for the first time for the same system. Through characterization of the treated particles, the applied methods could be
compared and two separation concepts could be distinguished. For the two
concepts further optimization steps were followed concluding in still open challenges that need to be phased in the coccolith separation chapter.
P 37
Proteins involved in Cyprinus carpio otoliths biomineralization M. Kalka*1, A. Ożyhar1, J. Stalarski2, P. Dobryszycki1 1Wroclaw University of Science and Technology, Department of
Biochemistry, Wrocław, Poland 2Institute of Paleobiology, Polish Academy of Science, Department of
Environmental Paleobiology, Warsaw, Poland
Otoliths, the ear stones of teleost fish, are biomineralized structures whose
major role is sound transduction and sensation of linear acceleration. They are mainly composed of calcium carbonate, which is deposited onto an
organic matrix. The organic matrix, which consists of proteins,
polysaccharides and lipids, accounts for only a few mass percent of an otolith. Nevertheless, the organic fraction, especially proteins, have been
shown to be essential for proper biomineralization of otoliths. Our recent
proteomics studies have revealed that otoliths contain more proteins than previously thought.
The aim of our work was the isolation and characterization of
macromolecules found in common carp otoliths. We paid particular attention to structural properties and biomineralization activity of isolated proteins.
In order to isolate proteins, asteriscus otoliths were decalcified with EDTA.
Then, obtained macromolecules were separated by various chromatographic
technics and examined with electrophoresis. Proteins compositions were analyzed with LC-MS/MS. Their structural properties were investigated by
CD. Influence of isolated proteins on calcium carbonate crystallization was
studied by a slow diffusion method. Crystals morphology and polymorph were investigated by SEM and micro-Raman microscopy. Moreover, post-
translational modifications, such as phosphorylation and glycosylation, were
also studied. Developed methods allowed the initial separation of otolith's proteins.
Besides previously studied otolith proteins, proteomics identified several
new ones that could be connected to biomineralization. Isolated macromolecules strongly affect the morphology of calcium carbonate
crystals. Appropriate staining of separated fractions indicates a possible high
percentage of proteoglycans and glycosaminoglycans in the ear stones. In addition, one protein that has a particularly strong affinity to the
hydroxyapatite column was isolated. In vitro studies of this protein indicate,
that it occurs in phosphorylated form in otoliths. The protein changes calcium carbonate crystals polymorph from calcite to vaterite. Our results pointed out
that the protein is present in high-molecular-weight aggregates and peptides
identified with MS/MS demonstrated that it might be Starmaker-like protein. In conclusion, obtained results indicate that otolith biomineralization process
might be far more complex than previously claimed. Our research allowed
isolation, initial separation and characterization of individual components of the organic fraction. Most probably it was the first time when the Starmaker-
like protein was successfully isolated from otolith matrix. Our results
revealed that proteins from common carp could be especially prone to phosphorylation and glycosylation.
Acknowledgments: This work was supported by the National Science Center (Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity
subsidy from the Polish Ministry of Science and High Education for the
Faculty of Chemistry of Wroclaw University of Science and Technology.
P 38
Proteomic investigation of the blue mussel larval shell organic
matrix A. Carini*1, T. Koudelka2, A. Tholey2, E. Appel3, S. Gorb3, F. Melzner4,
K. Ramesh5 1The University of Hong Kong, School of Biological Sciences, Hong Kong,
Hong Kong 2University of Kiel, Institute for Experimental Medicine - Systematic
Proteomics and Bioanalytics, Kiel, Germany 3University of Kiel, Department of Functional Morphology and Biomechanics, Institute of Zoology, Kiel, Germany 4GEOMAR, Marine Ecology, Kiel, Germany 5University of Gothenburg, Sven Lovén Centre for Marine Infrastructure, Fiskebäckskil, Sweden
Introduction Shell matrix proteins (SMPs) are occluded within molluskan shells and are
fundamental to the biological control over mineralization. While many
studies have been performed on adult SMPs, those of larval stages remain largely undescribed.
Objectives Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell
proteomes.
Methods Following development of a method for cleaning minute larval shells of
tissue contaminants, the whole blue mussel larval shell proteome was
extracted and subsequently sequenced using shotgun proteomics.
Results Forty-nine SMPs were identified in total. Twenty-one proteins were common
to all samples including: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP
domain-containing sequence and a ferric chelate reductase 1-like sequence.
Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. This study demonstrates
that the mussel larval shell proteome is unique: while key predictable
mollusk shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized. Further, comparison to adult mussels reveals
that only four domains are conserved among species and developmental
stages and nine domains are specific to mussel larvae.
Conclusion The bivalve larval shell proteome is not a subset of the adult one: blue mussel
larvae assemble a unique and novel shell proteome that serves specific physiological needs for their development. Further, the observed differences
in shell proteomes between species and life stages support the idea that strong
species-specific and ontogenetic variation exists in molluskan shell proteomes.
41
P 39
Deciphering the intricate 3D Structure of growing calcite
crystals during coccolith formation E. M. Avrahami*1, L. Aram1, A. Gal1 1Weizmann Institute of Science, Plant and Environmental Sciences, Rehovot, Israel
Coccolithophores are a widespread and ecologically important group of
unicellular calcifying phytoplankton. They are distinguished by a complex exoskeleton composed of multiple interlocking calcite plates, known as
coccoliths. Coccoliths are produced intracellularly within a specialized
membrane-bound organelle, the coccolith vesicle, and are subsequently exocytosed to the cell surface. Although extensive research in the past
century has contributed greatly to our understanding of coccolithophore
physiology and ecological significance, not much is known about the mechanisms by which they precipitate and control coccolith assembly.
Nearly thirty years ago, the V-R model for crystal nucleation, orientation,
and growth was suggested based on light- and electron microscopy, and to this day it is the ruling paradigm. However, the path by which crystals grow
into their final morphology remains unclear. In this work, we chose
Cacidiscus leptoporus as a model organism for exploring the growth of intracellular coccoliths, given that this species forms relatively large
coccoliths in comparison to the commonly studied species Emiliania huxleyi
and Pleurochrysis carterae, simplifying their analysis. A simple, yet effective, protocol was established for extracting developing coccoliths at
initial formation stages while preserving the inorganic phases and their
ultrastructure. These coccoliths were then imaged ex vivo using several electron microscopy techniques, including high-resolution STEM. By
recording a tilt-series of the crystals, a tomogram was created, and a 3D
model of the interlocking crystals was constructed. This model has allowed us to visualize the native interlocked R and V units with clear
crystallographic planes. Applying this methodology for multiple samples
will enable us to reconstruct the entire sequence of crystal growth, thus linking shape control to the crystallographic driving forces, and yielding a
mechanistic understanding about the control organisms have on biomineral
formation.
P 40
Diagenetic alterations reshape the hierarchical organization of
Cretaceous brachiopod shells - from nanoscale to higher scales D. Gaspard*1, J. Nouet2, C. Rountree3 1Muséum National d'Histoire Naturelle - Sorbonne Université,
Département Origines & Evolution, Centre de Recherche en Paléontologie
de Paris, UMR 7207, CR2P, Paris, France 2Université Paris-Saclay, Earth Sciences, Orsay, France 3SPEC, CEA, Gif-sur-Yvette, France
Introduction/objectives
After the attempt to reveal and understand the hierarchical organization of
the inner layers of the calcite shell of modern rhynchonelliform brachiopods (Gaspard, 2006, Gaspard & Nouet, 2016), the purpose here is to highlight the
modifications introduced in Cretaceous shells since the death till the state of fossil. Conservation, alterations in different palaeo-environmental situations
for these witnesses of old times are to be uncovered from the nanoscale to
continuum scale.
Materials and Methods Two and three-layered shells [cf. Sellithyris cenomanensisGaspard,
Moutonithyris dutempleana (d"Orbigny) among others], from different Cretaceous Western Europe locations, were observed with comparable
modern ones (Liothyrella neozelanica, Aerothyris kerguelenensis, Tichosina
cubensis, Notosaria nigricans…) using complementary throughput techniques (scanning electron microscopy (SEM), mapping elements,
including in the foremost rank the Bruker atomic force microscopy (AFM).
Results The observations reveal that from the death of the organisms, the exoskeleton
(biomaterial) composed of low-Mg calcite and organic matrices is subject of
chemical and physical aggressions leading to light or heavy alterations. More often the organic matrices are partly or entirely destroyed. First, after the
disappearance of the organic sheathes, several fibrous elements merge. As
well, after partial disappearance of the intracrystalline organic matrix in intimate relation with the individual homogeneous nanoparticles (in the
ordinary course of shell construction) in extant shells, these later become
more angular and amalgamate to shape blocky calcite and heterogeneous structures in fossil shells. AFM Peak Force tapping modes provide
topographical and nanomechanical information. Nanomechanical
information includes adhesion, deformation…measurements highlighting the most viscoelastic components (organic compounds) with respect to the
nanoparticles.
Conclusion As a matter of fact, the hierarchical organization observed at sub-micrometric
levels in the modern shells can be more or less heavily rubbed out by a
recrystallization (secondary calcite or silicic nodules) that contributes to destroying or partly strengthening the fossil shells. This aspect can be due to
bacterial activity using the organic matrices as food supply, presence of
microboring organisms that induce dissolution and recrystallization, and weathering.
P 41
High resolution study of nacre formation and organo-mineral
interface in the shell of the European abalone Haliotis
tuberculata S. Auzoux-Bordenave*1,2, W. Ajili1, N. Menguy1, I. Estève1, M. De Frutos3, K. Benzerara1, N. Nassif1, T. Azaïs1 1Sorbonne University, Paris, France 2Muséum national d’Histoire naturelle, Concarneau, France 3University Paris-Sud, Physics, Orsay, France
The European abalone Haliotis tuberculata is a commercially and ecologically important gastropod species and represents a key-model to study
the basic mechanisms of shell formation. The abalone shell is composed of
two calcified layers underlying the periostracum, i.e. an outer spherulitic layer and an inner nacreous layer. The nacreous layer is exclusively
composed of aragonite platelets, 6–8 μm in diameter, horizontally aligned in
layers and vertically in columns, arranged with a small amount of organic material. This typical architecture provides to nacre exceptional mechanical
properties with potential uses in jewellery and biomimetics. The organic
phase, which represents less than 5 wt.% of nacre, is proposed to guide nucleation and control the growth and orientation of the mineral phase.
Although the nacre structure is studied since many decades, the formation mechanisms and the interaction between organic and mineral components
still need to be clarified.
In this study, we used juvenile shell of one-year-old abalone H. tuberculata to investigate nacre formation and organo-mineral interface through high-
resolution (HR) microscopies, at the nanometer scale. A growing nacre area
was selected in juvenile shell for further investigation of nacre surface using scanning electronic microscopy (SEM) and Field Emission Gun (SEM-
FEG). HR imaging of the juvenile abalone shell resulted in an in-depth
characterization of the growing nacre, from the spherulitic edge to the mature zone of the nacre surface, providing a detailed sequence of nacre formation.
At a higher magnification, the tablet surface exhibits typical nanograins and
early stacks of aragonite platelets appeared separated by a porous layer of organic matrix. Additional focused ion beam (FIB) coupled with SEM
allowed the observation of the 3D organization of the aragonite platelets
stacks at early stages of their formation. Additional techniques such as high-resolution transmission electronic
microscopy (HR-TEM), scanning transmission X-ray microscopy (STXM)
and electron energy loss spectroscopy (EELS) were performed onto thin FIB section of aragonite platelets columns to gain local information on the nature
and atomic composition of the organo-mineral interface at the nanometer
scale. In growing nacre, large disordered organic inclusions were evidenced in the center of each aragonite platelet. These organic inclusions are oriented
along the c-axis direction, extending from one tablet to another through
organic bridges. It is suggested that these organic inclusions act as a vertical "backbone" controlling the longitudinal growth of the stacked tablets column
along the c-axis. In the case of lateral growth, a 20 nm thick disordered layer
was evidenced by HR-TEM at the surface of growing aragonite platelet. This disordered layer appeared mainly composed of an organic fraction where
carbonates ions appear to be absent. However, the presence of both calcium
and phosphate suggests the presence of calcium-binding acidic and phosphorylated proteins involved in the control of lateral growth of nacre.
Together, these results bring new insights to the fundamental mechanisms of
nacre formation and on the interaction between organic and mineral components involved in the control of aragonite platelets growth.
P 42
Growth dynamics and fine scale characterization of calcareous
granules of the annelid Lumbricus terrestris S. Mandera*1, I. Coronado1, M. Mazur2, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2University of Warsaw, Department of Chemistry, Warsaw, Poland
Calciferous glands of some species of earthworms (Annelida, Oligochaeta)
secrete amorphous calcium carbonate (ACC) particles that are transported toward a pair of oesophageal pouches where they coalesce and crystallize.
The final product is a solid calcareous granule that is excreted by the
organism to the soil. Spherical or sub-elliptical CaCO3 granules from 0.5 to 2 mm in diameter are composed mostly of calcite, although vaterite,
aragonite and ACC have also been found. The purpose of formation of such
mineralized structures still remains uncertain and possible hypotheses include: pH buffering of the internal body, adjustment of respiratory CO2,
and elimination of excess of Ca derived from diet and even spurious
mineralization. Little is known on the processes that control the final mineral
42
product secreted by earthworms, even though metals uptake by earthworms granules as a potential use for bioremediation have been studied in-depth and
the ACC formation by the calciferous glands have been characterized. The
main purpose of the current study is to elucidate the mechanisms of biocrystallization and the growth dynamics during granules formation.
Earthworm"s granules were obtained from laboratory culturing of fully
clitellate specimens of Lumbricus terrestris. Specimens were kept in artificial soil at a constant temperature, neutral pH, and controlled moisture. In order
to characterize the growth dynamics of the granules, an Mn-labelling
experiment in an amended artificial soil was carried out using two Mn-sources in separate lines of trials: 1) Mn-bearing CaCO3 (at 50 and 100 ppm,
respectively), and 2) a solution of MnSO4 (100 ppm of Mn). 15 specimens
were cultured during 28 days, 3 control specimens in an Mn-free soil and 12 specimens in amended soil (4 for each composition). Specimens were
exposed to amended soils and Mn-free soils every 2 to 7 days. An ulterior
structural, biogeochemical and crystallographic characterization of the granules was carried out, using petrographic microscopy,
cathodoluminescence (CL), SEM, AFM, EMPA, EBSD, and FTIR
spectroscopy. Growth dynamics experiment reveals that control specimens produce
between 6-9.8 mg of CaCO3 in 2-5 granules per week. Granules show a radial
texture formed mostly by calcite crystals with variable habits and some evidence of original concentric growth. The crystal habits are prismatic, lath,
and foliated–lamellar crystals, which occasionally are arranged forming
cross-lamellar-like microstructures. Moreover, some opaque, in transmitted light, and microgranular patches have been found. Organic matrix is
arranged, mostly, parallel to the growth axis of crystals and some calcite crystals surfaces exhibit nanogranular textures. CL data show a hidden
concentric sub-daily banding, with a thrombolite-like texture and kinked
zonation inside the calcite crystals. These features reveal a complex mechanism of formation that depends on physicochemical conditions
generated at microscale during the granule formation.
Acknowledgments: This work was supported by the National Science Centre (Poland) grant 2017/25/B/ST10/02221.
P 43
Towards identification of the protein machinery of coccolith
formation in the coccolithophore Pleurochrysis carterae E. Zschieschang*1, A. Skeffington1, M. Gorka1, A. Scheffel1 1Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
Coccolithophores are marine unicellular algae that produce mineralized
scales called coccoliths, which are 3D arrays of complex-shaped calcite crystals. How the arrangement of the crystals is guided and how crystal
morphogenesis is controlled is of interest for bioinspired materials synthesis.
Coccolith formation involves an insoluble organic structure called the base plate that templates calcite nucleation, as well as soluble acidic
polysaccharides. Recent work on the coccolithophore Pleurochrysis carterae
(recently reassigned to Chrysotila carterae) suggests that the base plate provides an attractive model system for the identification of macromolecules
controlling the crystal nucleation pattern. These base plates are composed of
polysaccharides and proteins. The exact molecular composition and identities of these components remain unknown. Here we tested different
extraction approaches to isolate individual components of the base plates of
P. carterae coccoliths. One extract was subjected to proteomic analysis. As the proteomic analysis of the coccolith base plates is at its infancy, we tested
different proteases to generate peptides. For protein identification we used
the publicly available protein database of P. carterae, which has been generated from short Illumina reads and is likely to contain incorrect protein
models due to misassembly of the reads. To improve our confidence in the
identifications we also used a database which we generated from long-read cDNA PacBio data. A comparison of the different data sets will be presented.
The workflow we have established will greatly aid attempts to elucidate the
protein machinery involved in coccolith formation.
P 44
Biological patterning in calcareous biomineralization of
terrestrial gastropod eggs J. Stolarski*1, I. Coronado1, M. Mazur2, A. Sulikowska-Drozd3 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2University of Warsaw, Department of Chemistry, Warsaw, Poland 3University of Lodz, Department of Invertebrate Zoology and Hydrobiology, Lodz, Poland
Gastropod mollusks are known from the formation of elaborated calcium carbonate shell patterns. Calcified protoconchs (larval shells) and complex
hierarchical structures of adult shells (teloconch) have been described among
various gastropod clades that include marine, fresh-water and terrestrial taxa. Much less is known about the earliest developmental phase of calcification
associated with the formation of eggs, especially among the pulmonate land
snails. Such calcified egg shells may supply the snail embryo with calcium ions to enable the formation of the earliest shell before hatching, provide
mechanical support, and potentially (not confirmed) reduce the water
evaporation from the egg. It was shown, that in contrast to almost invariably aragonite mineralogy of adult gastropod shells, the egg membranes are
mineralized with calcite crystals, often showing tetrahedral morphology.
This suggests a lack of strong biological control on this type of mineralization. Nonetheless, some authors noted a surprising variation of
ultrastructural features among more heavily calcified eggs.
Here, we have examined with SEM, micro-Computed Tomography, Raman and FT-IR spectroscopy), various structural and compositional features of
egg membrane mineralization in representatives of a highly diversified
family of door snails (Clausiliidae). The eggs were taken from oviparous (Caspiophaedusa, Oospira), viviparous (Idyla), and taxa with egg-retention
reproductive strategy (Pontophaedusa, Zaptyx, and Formosana). The
mineralized egg membranes show different patterns of crystal distribution, which invariably are composed of calcite. The calcite crystals are typically
embedded in the organic multilayered membrane (mixture of proteins and
polysaccharides), which can sandwich the crystal stacks. No evidence of amorphous precursors was found (common in systems of biologically
controlled mineralization), however distinct patterns of distribution of
crystals point to biological coordination of this process. A unique, spiral arrangement of crystal clusters is typical to eggs of Caspiophaedusa, more
homogenous distribution of tetrahedral crystals is characteristic of eggs of
Idyla and Formosana, whereas in eggs of Zaptyx and Oospira, the calcite crystals form pyramidal aggregates. In Pontophaedusa homogenously
distributed calcite crystals form a dense cover. Biological patterning that governs the calcite crystal distribution in door
snails egg membranes points to the potential use of this character in a
phylogenetic context. In fact, well-differentiated molecular clades (such as Caspiophaedusa, Pontophaedusa) show distinct crystal distribution patterns,
irrespective of reproductive strategy. Further proteomic and organic content
analyses are required to elucidate the precise biological mechanism of crystal growth coordination.
Acknowledgments: This work was supported by the National Science Center
(Poland) grant 2017/25/B/ST10/02221 (to JS) and 2016/21/B/NZ8/03086 (to ASD).
P 45
Crystallographic texture and microstructure of modern
Glycymeris shells G. Crippa*1, E. Griesshaber2, A. Checa3, E. Harper4, W. W. Schmahl2 1University of Milan, Milano, Italy 2Ludwig-Maximilians Universität München, Munich, Germany 3Universidad de Granada, Granada, Spain 4University of Cambridge, Cambridge, United Kingdom
Mollusk shells are biocomposites made of calcium carbonate crystals
(calcite, aragonite or both) and intra- and intercrystalline organic matrix,
resulting in a lightweight product of highly elaborate architectures, endowed with unique structural properties (stiffness, fracture toughness, tensile
strength). Calcite and/or aragonite crystals show different crystallographic
arrangements, which are hierarchically organized at the nano-, micro- and meso-scale, forming different microstructures.
The most common fabric among mollusk shells is represented by the
aragonitic crossed lamellar one, which forms the shells of both marine and continental taxa, thus showing a great adaptive potential and evolutionary
success; this is one of the hardest microstructures which has the ability to
dissipate and stop cracks through the shell. Species belonging to the genus Glycymeris Da Costa, 1778 are excellent taxa
to investigate for this type of microstructure, as they have an aragonitic shell
with an outer and an inner crossed lamellar layers. Also, they are widespread and common in fossil faunas and their applicability in the fossil record spans
from paleoecology, paleoclimatology, sclerochronology to
archaeomalacology. Although Glycymeris shells and the crossed lamellar fabric have been widely
study and documented, up to now a detailed characterization of the
crystallography of both this taxon and this microstructure with Electron Backscatter Diffraction (EBSD) is lacking. Here, we analyzed modern
Glycymeris shells combining the use of the traditional and easily available
Scanning Electron Microscopy with the more advanced EBSD technique, to improve the knowledge of the crossed lamellar microstructure and of its
relationship with other shell layers (i.e., myostraca). With the present
contribution we will be able to provide: i) a modern unaltered reference sample for detecting diagenetic alteration in fossil shells, which, due to the
widely use of Glycymeris fossil shells in paleoclimatic and
paleoenvironmental reconstructions, is of crucial importance, and ii) new data to better understand mollusk biomineralization processes, as well as the
formation of the crossed lamellar microstructure and its correlation with
myostracal layers, here characterized in detail for the first time.
43
P 46
Formation of biomimetic agar gel carbonate composites –
Influence of Mg and Sr S. Rudin1, M. Greiner*1, E. Griesshaber1, L. Fernández-Díaz2,3, W. W.
Schmahl1 1Ludwig-Maximilians-Universität München, Department of Earth- and Environmental Science, Munich, Germany 2Universidad Complutense de Madrid, Departamento de Cristalografía y
Mineralogía , Madrid, Germany 3Ciudad Universitaria, Instituto de Geociencias (UCM, CSIS), Madrid, Spain
Hydrogels are excellent models for understanding extracellular matrix
microenvironments and are therefore highly suitable for mimicking
biomineralization processes. Recent studies (Greiner et al. 2018) showed the combined influence of reagent solution concentration and agar solid content
on the formation of calcite aggregates grown in double-diffusion systems.
Biologic calcite hard tissues e.g. sea urchin teeth can incorporate up to 45 mol% Mg (Ma & Qi 2010). Coral skeletons, both aragonitic or calcitic,
incorporate small amounts of Sr (Smith et al. 1979).
We present results on the effect of additives (Mg, Sr) in agar gel on the formation of carbonate agar composites in double-diffusion systems. We
vary the additive concentration, the agar solid content and the concentration
of reagent solutions and compare our results to previous findings where additives were not used.
The aggregates are characterized by X-ray powder diffraction (XRPD),
scanning electron microscopy (SEM), electron backscatter diffraction (EBSD) and energy dispersive X-ray spectroscopy (EDX).
The presence of magnesium results in the formation of dumbbell shaped and
spherical calcite aggregates with a vast variation of surface morphologies containing a spherulitic inner structure, whose subunits are crystals with a
slight mosaic structure. The incorporated magnesium is (almost) evenly
distributed within the composites; occasionally it is slightly enriched at the rim of the aggregates. Crystallization under high Mg to Ca ratios in the
aqueous solution (0.1M Mg: 0.5M Ca or higher) appears to promote the
stabilization of amorphous calcium carbonate (ACC) and the formation of aragonite.
The presence of strontium in the growth medium leads to the formation of
calcite aggregates with predominantly spherical morphologies. These aggregates are polycrystals. Individual subunits of the aggregates show a low
mosaic-spread (high co-orientation). Incorporated Sr shows a zoned pattern
within the calcite aggregates, regardless of the Sr concentration in the agar hydrogel. For Sr to Ca ratios of 0.01M Sr:0.1M Ca and 0.1M Sr:0.5M Ca we
observe calcite composites overgrown by strontianite. A high agar solid content (2 wt%) promotes this overgrowth compared to a low agar solid
content (0.5 wt%).
Mg or Sr in the growth medium promotes misorientation in calcite-agar gel composites. This misorientation most likely arises from split growth
phenomena as dislocations generate to release Mg or Sr incorporation-related
lattice strain. Greiner, M., Yin, X., Fernández-Díaz, L., Griesshaber, E., Weitzel, F.,
Ziegler, A., Veintemillas-Verdaguer, S., Schmahl, W.W. (2018) Crystal
Growth and Design, 18, 1401-1414. Ma, Y. & Qi, L. (2010) Biomineralization of sea urchin teeth. Frontiers of
Chemistry in China, 5, 299-308.
Smith, S.V., Buddemeier, R.W., Redalje, R.C., Houck, J.E. (1979) Strontium-calcium thermometry in coral skeletons. Science, 204, 404-407.
P 47
Observation and simulation of 3D pigmentation patterns of
molluscan shells H. Sato*1, T. Sasaki2 1The University of Tokyo, Earth and Planetary Science, Tokyo, Japan 2The University of Tokyo, The University Museum, Tokyo, Japan
Molluscan shells show highly diverse, variable and complex pigmentation
patterns. Morphogenesis of the patterns can be understood by a mathematical model which represents dynamics of pattern formation. Several models have
been proposed so far. The previous models assume that secretory organs of
pigment are distributed in line along mantle edge, and the pattern on shell are regarded as a track of a temporally changing pattern of pigment secretion
along mantle edge. However, an observation of mantle edge suggests that the
secretory organs are two-dimensionally (2D) distributed; hence, the patterns in shells should be three-dimensional (3D). Here, we confirmed if 2D-
distributed secretory organs generated 3D pigmentation patterns in shells, by
observation and simulation. First, we observed pigmentation patterns on cross sections of Conidae shells
to describe 3D patterns. We found that some species had 3D pigmentation
patterns and different surface patterns had different patterns on cross sections, which cannot be explained by simple dynamics such as simple
diffusion in the direction of inner shell surface. This suggests that pigment
secretion may interacts mutually not only in the direction of mantle edge but also in the perpendicular direction.
Second, we proposed a new model assuming 2D-distributed secretory organs
and simulated the model numerically. Then, the results were compared with actual shell patterns. The new model was based on a reaction-diffusion
system. A simulation of the model generated triangles and stripes on surface,
which appear different patterns on cross sections respectively. The generated patterns corresponded with observed patterns of C. textile shells.
The correspondence between the observation and the simulation suggests
that Conidae species have mutually interacting secretory organs of pigment which are 2D-distributed, and form 3D pigmentation patterns. The new
model for 3D patterns provides a new interpretation of morphogenesis of
pigmentation patterns.
P 48
When predators become prey- shell repair patterns in Nucella
lapillus (Linnaeus, 1758) D. Mayk*1,2, L. Peck2, E. Griesshaber3, W. W. Schmahl3, E. Harper1 1University of Cambridge, Earth Sciences, Cambridge, United Kingdom 2British Antarctic Survey, Cambridge, United Kingdom 3Ludwig-Maximillian Universitaet, Muenchen, Germany
Introduction
The common intertidal gastropod Nucella lapillus (also referred to as dog whelk) is a predatory species feeding on e.g., blue mussels and barnacles. It
builds a massive calcium carbonate shell that shelters it from predators and
other environmental hazards. Young dog whelks which have not been able to grow thick shells yet may be subject to attacks from crabs, birds and even
conspecifics. If an attack turns out to be non-lethal it is of great importance
for the individual to repair the damaged shell as fast as possible to be fit for potential future attacks. It is very likely that the need for rapid repair goes
jointly with an increase in the energy requirement per unit calcium carbonate
formed due to [1] an elevation of the saturation state of the calcifying fluid or [2] an increase in the amount of organics used. Previous studies have
shown that increased calcification rates at damaged sites often result in a
disturbed microstructure and crystal alignment which possibly affects the shell integrity.
Objectives
We investigate the ability of N. lapillus specimens from a broad shell size range (i.e., juvenile to adult specimens), to repair artificially damaged shells
and compare the microstructure of the repaired regions with those of naturally occurred damage in conspecifics. We investigate the rate and mode
of shell repair in an attempt to link the capability of shell repair and mortality
rate of the specimens to energy availability among shell size/age groups.
Materials & methods
120 specimens of N. lapillus were collected from the rocky shore in Whitby,
UK in spring 2019 and reared in a seawater aquarium setup at the British Antarctic Survey [BAS] in Cambridge. Specimens were starved throughout
the experiment. After an acclimation phase of two weeks, long notches were
cut perpendicular to the aperture rim into the shells of ~60 specimens using a milling bit. This was done with great care to prevent damages of the
specimen's tissue. Notch repair increments were monitored weekly.
Subsequent analysis of the repaired notch regions of sections under the scanning-electron microscope [SEM] and microstructure orientation
mapping by means of electron-backscatter diffraction [EBSD] was carried
out.
Preliminary results
Our results suggest that smaller individuals (shell length < 1.5 cm) are readily
capable of repairing shell damage and start the repair earlier than larger individuals. Absolute shell repair measured as length, however, is greater in
larger/older individuals. The mortality rate during the experiment was > 10
times higher in < 1.5 cm long specimens than in > 1.5 cm long specimens suggesting that energy reserves in the younger dog whelks are smaller and
faster depleted by the repair process than in larger animals. The shells of N.
lapillus consist of three layers, namely cross-lamellar aragonite, homogeneous calcite, and the periostracum. The microstructure analysis
showed that N. lapillus produces a more regulated shell structure in the
repaired regions than is normally secreted in the undisturbed shell. We distinguish four steps observed in the repair process: [1] the damaged shell
surface is lined with a thin layer of cross-lamellar aragonite, [2] a mound-
shaped calcite structure is built on to the aragonite lining exhibiting a specific upward grading of crystal sizes from small to large and with a highly co-
orientated crystal orientation, [3] homogeneous and disorientated calcite is
built on top in a rather elongated way with a strongly frayed surface to the outside. This step happens rather quickly and covers up most of the damaged
area. [4] A lining of cross lamellar aragonite is laid over the inside of the
entire notch covering the inner surface.
Outlook
Thermogravimetry measurements of the notch material will be carried out to
investigate the organic content of the repaired shell regions. Results will be compared to the organic content of the shell layers in undisturbed shells.
44
Further SEM and high-resolution EBSD measurements will be carried out to further characterize N. lapillus' microstructure of the repair margins.
P 49
Shell-muscle attachment in the bivalves Ostrea stentina
(Payraudeau, 1826) and Anomia ephippium (Linnaeus, 1758) J. D. Castro-Claros1, C. Salas*1, C. Lucena2, A. Checa3 1Universidad de Malaga, Biologia Animal, Malaga, Spain 2Universidad de Malaga, SCAI, Malaga, Spain 3Universidad de Granada, Estratigrafia y Paleontologia, Granada, Spain
Introduction The muscles of the mantle in bivalves attach to the shell valves and retract
the mantle edges. Among them, the adductors are particularly important for
the survival of the animal. Most studies have addressed the type and morphology of the muscular fibers and filaments (Paniagua et al 1996), and
a few have focused on the attachment of these muscles to the shell (Nakahara
& Bevelander 1970). However, the mechanism of transport through the muscle to the myostracum was never addressed. The goal of this research is
to describe ultrastructure and the transport of material across the adductor
muscle-myostracum attachment in Ostrea stentina and Anomia ephippium.
Material and methods We examined 10 specimens of O. stentina and 10 specimens of A. ephippium,
collected in the littoral of Málaga (south Spain). Specimens were fixed in 2.5% glutaraldehyde (4 °C), decalcified in 2% EDTA, post-fixed in OsO4
(2%) and embedded in epoxy resin Epon 812 (EMS). Samples for calcium
detection were post-fixed in a mixture of OsO4 (2%) and potassium hexahydroxoantimonate (2%) in PBS. Ultra-thin sections (50 nm) were
stained with uranyl acetate (2%). They were observed in a TEM JEOL-
JEM1400 and a TEM-STEM FEI Talos 200X, with EDX analyzer. Collagenase was used to test the presence of collagen. One specimen of each
species was embedded in methacrylate (Technovit 7200 VLC). These
sections (50 μm) and the semi-thin sections (~0.5 μm) were stained with toluidine blue (1%)
Results The adductor muscles in both species are composed of "smooth" and "striated" muscles. The outer epithelium of the mantle continues across the
adductor muscle area and their cells contain many vesicles. Bundles of
filaments connect the muscular cells with the extrapallial space through hemidesmosomes. The extrapallial space (ca.100-150 nm thick) is filled with
organic secretions from the vesicles of the mantle cells. Additional bundles of microfilaments of collagen fibers cross the extrapallial sheet, from the
hemidesmosomes to inside the myostracum, where they form a network.
EDX analysis shows the presence of calcium inside vesicles from the mantle cells, the extrapallial sheet and the myostracum network.
Conclusion The presence of a layer of cells between the muscular cells and the shell was indicated by Nakahara & Bevelander (1970), who called them "adhesive
cells". Subsequently, Bubel (1984) called them "tendon cells". Our
observations confirm the existence of this cell layer, which is the continuation of the outer mantle epithelium across the adductor muscle. The
extrapallial space is replenished with secretions of the mantle cells and
collagen fibers, seemingly originated in the hemidesmosomes. The presence of numerous vesicles extruding from the mantle cells were probably mistaken
as a microvillous border by Nakahara & Bevelander (1970). However, no
microvilli have been observed in the basal mantle cell membranes. The presence of calcium inside some vesicles suggests that part of the calcium is
transported thereby. It would be interesting to study whether calcium is
transported as amorphous calcium carbonate or as a crystalline phase. Bubel A. 1984. Epidermal cells. In: Bereiter-Hahn et al. (eds.) Biology of the
Integument 1, Springer: 400-447.
Nakahara H., Bevelander G. 1970. Texas Rep. Biol. Med. 28(3): 279-286. Paniagua R., Royuela M., García-Anchuelo R.M., Fraile B. 1996. Histol.
Histopathol. 11(1): 181-201.
P 50
Structure - material property relationships in marine bivalve
and gastropod calcite and aragonite Z. Tang*1, E. Griesshaber1, E. Harper2, S. Zaefferer3, M. Zenkert1, N.
Lagos4, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department of Geoscience and
Environmental Sciences, Munich, Germany 2University of Cambridge , Department of Earth Sciences, Cambridge, United Kingdom 3Max-Planck-Institut für Eisenforschung, Department of Microstructure
Physics and Alloy Design, Düsseldorf, Germany 4Universidad Santo Tomás, Centro de Investigación e Innovación para el Cambio Climático, Santiago, Chile
Carbonate biological hard tissues are composite materials consisting of
biopolymer and biomineral components. Both material classes are present in
the composite hard tissue with complex hierarchical structures and architectures.
In this study we investgate the interlinkage between mineral organization
(microstructure, texture), biopolymer matrix distribution and biomaterial properties for some marine gastropods (Haliotis glabra, Patella vulgata,
Tegula sp.) and some marine bivalves (Mytilus edulis, Chama arcana). The
hard tissues of all these organisms consist of calcite and aragonite, however in characteristic organizational patterns. Haliotis laevigata and Mytilus
edulis contain two shell layers: an outer calcitic and an inner aragonitic shell
region. The aragonite in both organisms is nacreous aragonite, in Haliotis laevigata the calcite is prismatic, while in Mytilus edulis it is fibrous. In
Patella vulgata and Chama arcana the aragonitic shell portion is sandwiched
between two calcitic shell layers. These two calcitic shell portions have distinct microstructures; are neither prismatic (as it is the case in Haliotis
laevigata) nor fibrous (as it is the case in Mytilus edulis). Tegula sp. also has
an outer calcitic and an inner aragonitic shell portion. However, the aragonite in Tegula sp. is not developed in a nacreous microstructure; it is an assembly
of aragonitic prisms that increase in size towards the soft tissue of the animal.
Microstructure and texture characterization was done with SEM imaging and electron backscatter diffraction (EBSD), biopolymer content was analyzed
with Thermo-Gravimetric- Analysis (TGA), biopolymer distribution within
the biological hard tissue was visualized with various etching protocols and SEM imaging, biomaterial properties were measured for two hierarchical
levels with instrumented nanoindentation and microindentation.
We find that biopolymer contents is fairly similar for the hard tissues of the investigated species, however, mineral organization differs significantly
between the species, for both, the calcite and the aragonite. Accordingly, for the selected hard tissues, mineral organization exerts the strongest effect on
the observed variation of nano- and micro-hardness as well as elastic
modulus. With the wide range of selected microstructures we aim to highlight in this contribution the relation between specific microstructural construction
principles, biomaterial properties and biomaterial functions.
P 51
Pattern formation of interlocking calcite fibre hybrid
composite in brachiopod shells W. W. Schmahl*1, E. Griesshaber1, A. Ziegler2, M. Simonet Roda1, D.
Henkel3 1LMU Munich, Earth and Environmental Sciences, Munich, Germany 2University of Ulm, Central Facility of Electron Microscopy, Ulm,
Germany 3GEOMAR Helmholtz Centre for Ocean Research , Marine Biogeochemistry/Marine Systems, Kiel, Germany
Fibrous biological hybrid composites are an important class of materials.
Aragonite or calcite fibers are embedded in a pliant biopolymer matrix, the
latter being always cross-linked within the hard tissue. Most biological carbonate hard tissues are subject to compressive, bending and shearing
forces. As fibers within a matrix cannot be reorganized once they endure
these forces, they must be properly packed and oriented within the hard tissue from the onset of their formation. This is accomplished by the formation of
stacks of parallel fibers with interlocking concave/convex cross-sectional
shapes. On the next hierarchical level, the stacks are twisted in a plywood-like arrangement. This ensures that all components of the composite are
interleaved in three dimension and on all length scales.
The fibrous shell layer of modern brachiopod shells is such a hybrid composite material. The interlocked concave-convex packing morphology of
the calcite fibres is more sophisticated than the simple "brick and mortar"
pattern of mollusc nacre or the random cellular polygonal convex cross-sectional of columnar calcite of many invertebrates. In the fibrous calcite
composite, the convex surface of each fibre is lined by an organic membrane,
which is intricately connected to the fibre calcite. The four concave surfaces are adjacent to the linings of the convex sides of neighboring fibers.
When brachiopod fibre and nacreous tablet formation are compared,
significant differences emerge in biopolymer/mineral deposition and, hence, microstructure generation. In the case of modern brachiopod fibers, during
secretion, mantle epithelium cells are always in direct contact with the
mineral, whereas in molluscs the nacreous tablets are never in direct contact with epithelial cells. There is always an interlamellar (in bivalves) or surface
(in gastropods) membrane between secreting mantle cells and the growing
aragonite platelets. When brachiopod fibers form, secretion of the biopolymer membrane covering the convex surface of the fibre is the last and
terminal step in fibre growth. In contrast, when nacre forms, aragonite tablet
formation starts with the consecutive self-assembly by liquid crystallization of interlamellar membranes. This leads to the formation of compartments that
become successively infiltrated by aragonite. Accordingly, we find for
modern brachiopod shell and molluscan nacre development two divergent microstructure generation processes. One is biologically controlled through
direct cellular contact and activity with the mineral as it is the case for
brachiopods, the other is physically controlled through the self-organization of extracellular matrix membranes as it is the case for molluscan nacre.
45
P 52
Formation and mosaicity of coccolith segment calcite of the
marine algae X. Yin*1, A. Ziegler2, K. Kelm3, R. Hoffmann1, P. Watermeyer3, P. Alexa1,
C. Read2,4, L. Schlüter5, T. B. H. Reusch5, E. Griesshaber1, P. Walther2, W.
W. Schmahl1 1Ludwig-Maximilians-Universität München, Department für Geo- und
Umweltwissenschaften, München, Germany 2Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm, Germany 3Deutsches Zentrum für Luft- und Raumfahrt, Institut für Werkstoff-
Forschung, Köln, Germany 4Universitätsklinikums Ulm, Institut für Virologie, Ulm, Germany 5GEOMAR Helmholtz-Zentrum für Ozeanforschung Kiel, Marine Evolutionary Ecology, Kiel, Germany
Coccolithophores belong to the most abundant CaCO3 mineralizing
organisms.Coccolithophore calcification is a complex and highly regulated process, resulting in a biogenic product that differs in both, morphology and
chemical composition from the abiogenically produced equivalent. Unlike
extracellularly formed biological carbonate hard tissues, coccolithophore calcite is neither a hybrid composite, nor is it distinguished by a hierarchical
microstructure. This is remarkable, as the key to optimize crystalline
biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the specific microstructure.
To get insight into the pathway of biomineralization of E. huxleyi coccoliths
we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of
the calcite in the coccolith vesicle within the cell. With TEM diffraction and
annular dark field (ADF) imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic-block-boundaries.
As at these only minor misorientations occur, we attribute them to dislocation
networks creating small-angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E. huxleyi calcite mosaicity is not
caused by occluded biopolymers, as it is the case in extracellularly formed
hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical atom-by-atom growth
mechanisms. Using cryo-preparation techniques for SEM and TEM we found
that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well-controlled constant gap
of about 4 nm between them. We describe this conspicuous connection as a not yet described inter-organelle junction, the "nuclear envelope junction".
The narrow gap of this junction likely facilitates transport of Ca2+ ions from
the nuclear envelope to the coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear
envelope – endoplasmic reticulum Ca2+-store of the cell for the transport of
Ca2+ ions from the external medium to the coccolith vesicle and that E. huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle
accretion mechanism.
P 53
Skeletal repair of modern brachiopod shells E. Griesshaber*1, J. H. Robinson2, E. M. Harper3, M. Lamare4, M.
Williams4, M. Zenkert5, W. W. Schmahl5 1Ludwig-Maximilians University Munich, Department of Earth and
Environmental Sciences , Munich, Germany 2University of Otago, Department of Geology, Dunedin, New Zealand 3University of Cambridge, Department of Earth Sciences, Cambridge,
United Kingdom 4University of Otago, The Department of Marine Sciences, Dunedin, New Zealand 5LMU Munich, Department of Earth and Environmental Sciences, Munich, Germany
Invertebrates, brachiopods, molluscs, echinoderms, are known to repair
damage to their shells. This is either inflicted by predators or by physical
environmental impact, e.g. ice blocks or, in high surf regions, e.g. rocks.
Shells often show various and distinct repair traces. This indicates that a specific organism is often attacked by a range of animals as well as different
environmental impacts.
In this contribution we describe new shell formation at damaged shell portions for the modern terebratulid brachiopod. We investigate how
Calloria inconspicua copes with an injured shell, how quickly it repairs its
shell and how much control the animal exerts on the organization of the newly-formed mineral, the mineral that is secreted for repair. Optical and
electron microscopical techniques were used for imaging the shells, electron
backscatter diffraction analysis (EBSD) was applied for the determination of calcite organization and measurement of calcite crystal co-orientation
strength. Live Calloria inconspicua specimens were collected at Otago
harbor, New Zealand and were placed into aquaria. Animals were damaged mechanically either by drilling 1 to 2 mm sized holes into their shells or by
cutting slices off the shell. Subsequent to damage formation, brachiopods
were placed back into the tanks for repairing their shell. Most specimens survived and, thus, coped well with injured shells. They repaired damaged
shell parts quickly, within three to five months.
Calloria inconspicua secrets two-layered shells, a primary, outer, and a fibrous, inner, shell portion with, in the latter, well-aligned calcite fibres (Ye
et al. 2018, Journal of Structural Biology). As brachiopod fibres occlude very
minor amounts of biopolymers, calcite crystallites within the fibres are highly co-aligned (e.g. Casella et al. 2018, Biogeosciences). At repair of the
hole drilled into the shell, the mantle tissue secrets first a thin layer of calcite
that is added onto inner shell surfaces at still undisturbed shell portions. Relative to shell calcite secreted under normal living conditions, the calcite
that forms first at repair has an almost random microstructure and texture. It
is a thin layer of calcite that appears to serve as stabilization for the subsequently secreted calcite, the calcite that covers the hole drilled into the
shell. The microstructure and texture of the first-formed shell calcite is
significantly different to that secreted under normal conditions. In the former (the first-formed calcite) typical morphologies of mineral units forming the
primary and the fibrous shell layers are entirely absent as well as the high co-
orientation strength of calcite crystallites within a particular mineral unit. MUD values (indication for the strength of crystallite co-orientation, Casella
et al. 2018, Biogeosciences) of undisturbed shell portions of modern
Callorica inconspicua range between 60 to 70, while for the first-formed calcite and the calcite that covers the hole drilled into the shell MUD values
are between 18 and 20.
Despite low MUD values microstructure recovery starts soon when the shell is repaired. For the shell portion that covers the hole drilled into the shell, we
can clearly distinguish between primary and fibrous layer mineral units. With ongoing repair these gain more and more their normal morphologies. Recent
studies of cell ultrastructure secreting the calcite of the modern terebratulid
brachiopod Magellania venosa (Simonet Roda 2019a, Nature Scientific Reports; Simonet Roda 2019b: Journal of Structural Biology) showed that at
active secretion outer mantle epithelium cells are in direct contact to the
forming fibre ant the primary layer calcite. Taking this into account it can be assumed that the mantle portion beneath the drill-hole only secretes the
calcite that is needed for repair of that shell portion.
P 54
The role of innate immunity of aortic valve cells in human
aortic valve calcification X. Meng*1, L. Ao1, D. Fullerton1 1University of Colorado Denver, Surgery, Aurora, United States
Chronic inflammation and progressive calcification of the aortic valve
leaflets lead to calcific valvular heart disease (CVHD) that is common in the elderly. Currently, pharmacological prevention of CVHD progression is
unavailable although there is a wide window for intervention. In addition, the
interaction between the pro-inflammatory and pro-osteogenic mechanisms in aortic valve calcification is not well understood. Aortic valve interstitial cells
(AVICs) are actively involved in valvular calcification. Initial studies by our
group demonstrate that human AVICs express pro-osteogenic proteins (including BMP-2 and TGF-β1) in response to stimulation of Toll-like
receptor (TLR) 2 or 4. Further, the TLR-mediated innate immune response
in human AVICs leads to pro-osteogenic reprogramming characterized by the expression of Runx2 and alkaline phosphatase, and formation of calcium
deposits. These studies uncovered a novel mechanistic role of the AVIC
innate immunity in aortic valve calcification. Our recent work identified several endogenous factors that can elicit the osteogenic response in human
AVICs through TLR2/4, including oxidized low-density lipoprotein,
biglycan and matrilin 2. These endogenous factors (damage-associated molecular patterns, DAMPs) induce the osteogenic response in human
AVICs mainly through the NF-κB and ERK1/2 pathways. Together, our
findings demonstrate that DAMPs are capable of inducing osteogenic response in human AVICs and that the innate immune receptors have novel
functions in modulating the osteogenic response in human aortic valve cells.
These findings suggest that AVIC TLRs may play an important role in the pathogenesis of CVHD and that modulation of the signaling pathways
utilized by TLRs may have therapeutic potential for suppression of CVHD
progression.
46
P 55
Structural characterization of acellular components in
Ariolimax californicus (Gastropoda; Stylommatophora)
D. Montroni*1,2, X. Zhang1, J. Leonard3, M. Kaya4, C. Amemiya5, G.
Falini2, M. Rolandi1 1University of California Santa Cruz, Department of Electrical Engineering, Santa Cruz, California, United States 2Alma Mater Studiorum Università di Bologna, Department of Chemistry
“Giacomo Ciamician”, Bologna, Italy 3University of California Santa Cruz, Joseph M. Long Marine Laboratory,
Institute of Marine Science, Santa Cruz, California, United States 4Aksaray University, Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray, Turkey 5University of California, School of Natural Science, Merced, CA, United States
Biological materials such as the chiton"s tooth, the squid"s beak, and the
byssal threads of bivalves have inspired the development of new materials. In this regard, we have characterized the acellular components of the
terrestrial slug Ariolimax californicus (banana slug), which are three matrices
in the buccal mass and the internal shell. The buccal mass is an apparatus, very similar in function to the human mouth,
whose role is processing food. The components observed in the buccal mass
were the radula, the jaw, and the odontophore. In many marine mollusks the radula is a well studied organ, but no study has ever been done on terrestrial
soft food eating mollusks. Moreover, the jaw and the odontophore have never
been described before under a structural point of view. The other matrix studied was the internal shell. Along with the evolution,
slugs have lost their external shell to adapt to calcium poor environments, but
some of them maintained a vestigial internal shell. Differently from mollusks shells, which role, composition and mechanisms of formation have been
widely investigated, the internal shell of slugs has been poorly studied and
many aspects are still unknown. It has only been observed that mineralizes and demineralizes depending on the life cycle of the animal.
The research involved the sectioning of the matrices and the study of their
morphological structures using electron scanning and optical microscopy. Spectroscopy, X-ray diffraction, and chemical analysis were also used to
investigate the composition and the structure of the material in its different
regions. In the radula, calcium-rich denticles were tightly interlocked one to the other
on top of a nanofibrous chitin membrane. The jaw was observed having a nanostructured morphology made of chitin to achieve compression resistance
and is directly linked to the foregut cuticle, which has a protective
nanofibrous structure. Finally, in the odontophore, we observed a structurally elastic microstructure that interfaces soft tissues with a highly stressed radula
membrane. Based on those observations, we discussed the interaction
between these components and highlighted how the materials in these components have evolved together to perform their tasks.
The study of the internal shell showed two different calcite crystal
morphologies on the two faces of the shell. Those crystal phases grew at the interface of three chitin-protein based layers: a thick internal layer and two
external membranes on each side of it. In the ventral face spherulite crystals
were observed, while lamellar crystals were observed in the dorsal face. This polymorphic asymmetry was observed also in the organic matrix hosting the
crystals. Those observations led to the hypothesis that this matrix act as
calcium storage with a face working as short-term calcium reserve, while the other as a long-term one.
In conclusion, considering the structural characterization of the A.
californicus" matrices in relation to their functions, this study could give a new perspective on fabricating bioinspired materials.
P 56
Structure-function relationships in carbonate endo- and
exoskeletons E. Griesshaber*1, A. Checa2, A. Ziegler3, M. Zenkert1, W. W. Schmahl1 1Ludwig-Maximilians University Munich, Department of Earth and
Environmental Sciences , Munich, Germany 2Departamento de Estratigrafía y Paleontología Facultad de Ciencias
Universidad de Granada Avenida Fuentenueva s/n Granada, Granada,
Spain 3University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany
In this contribution we highlight and discuss differences in mineral
organisation of biocarbonate hard tissues used by organisms for diverse
purposes: as a buoyancy device, for protection of the soft tissue and as enforcement of the crustacean cuticle. We observe a huge diversity of
microstructure and texture patterns that range from almost unaligned over
graded to co-aligned crystal assemblies. Hence, a high order as well as a high disorder in mineral organization is advantageous and is fabricated by the
organism. Carbonate organization patterns were measured with conventional
and high-resolution EBSD (electron backscattered diffraction) and transmission Kikuchi diffraction (TKD); differences in structural
organization of the mineral were imaged with FE-SEM and STEM. We
characterize carbonate assembly in the buoyancy device of the cephalopod Spirula spirula, the shell of the molluscs Mytilus edulis and Haliotis glabra,
and the calcite occlusions within the cuticle of the isopods Tylus europaeus
and Porcellio scaber. The cephalopod Spirula spirula possesses a light-weight internal shell that is
divided into discrete chambers. These are separated from each other by
partitions, the septa. A thin organic siphuncle runs through all chambers and facilitates that liquid and gas diffuse into and out of the chambers, hereby
allowing for buoyancy adjustments. Spirula sp. covers daily almost 1 km
difference in altitude as the animal spends the night in deep waters, while for the day it traverses the water column up to about 200 to 300 km depth below
the water surface. The shell is coiled in a single plane, resembling a
logarithmic spiral, the coils do not touch one another. Both the shell wall and the septum are mineralized with the walls being almost devoid and the septa
consisting of a large proportion of organic matter. The septa allow the shell
to function as a buoyancy device. They are curved, with the concave side pointing towards the first chamber and consist of organic laminae enforced
by intercalated nanometric granular aragonite. In contrast, aragonite crystals
that form the shell walls are micrometer sized and are irregular in size and morphology. Their arrangement is structured, such that large crystals line the
outer sides, while an assembly of small aragonite crystallites form the inner
regions of the shell wall. Crystal axes co-orientation strengths is high for both the aragonite of the walls as well as for the granular aragonite within the septa
(MUD values scatter between 150 and 200). Aragonite c*-axes are perpendicular to the shell vault and rotate with its curvature.
The shell of the molluscs Mytilus edulis and Haliotis glabra consist of three
distinct layers separated from each other by either organic membranes or, for a given genera, by characteristic transition zones. The mineralized shell
portions consist of dense assemblages of carbonate mineral units. For both
organisms calcite forms the seaward pointing layer, while nacreous aragonite constitutes the shell part that is next to the soft tissue of the animal. In Mytilus
edulis the calcitic shell portion comprises calcite fibres, each of them
sheathed in organic substance. In Haliotis glabra the calcitic shell section is formed by irregularly sized and shaped calcite units that decrease
consistently in size towards the periostracum, the outermost layer of the shell.
For a given age, shell thicknesses are comparable for the two animals. Mytilus edulis lives attached to the substrate, in very shallow waters and in
high surf regions, while the benthic Haliotis glabra lives in water depths
between 50 to 80 m. Mytilus edulis developed a thin, flexible and tough shell, while Haliotis glabra protects its soft tissue with a hard and stiff shell. The
calcitic shell portion is rigid in Haliotis glabra, while it is ductile in Mytilus
edulis. For adult specimens and within the central portion of a cross-section through the shell in Haliotis glabra we find an about 60 to 70 mm thick layer
of prismatic calcite and of an about 280 to 300 mm thick layer of nacreous
aragonite. In a comparable section of the shell the calcitic shell region of Mytilus edulis comprises about 150 to 160 mm of fibrous calcite and an about
80 mm thick layer of nacreous aragonite. Thus, in Haliotis glabra nacreous
aragonite prevails, while in Mytilus edulis fibrous calcite forms the major part of the hard tissue. The outer shell portion of Haliotis glabra with its
irregularly shaped and sized mineral units (arranged with a low degree of co-
orientation; MUD below 20) provides the necessary stiffness, while in Mytilus edulis the (highly co-oriented; MUD above 300) calcite fibre
arrangement, with each fibre being sheathed by biopolymers, provides the
tensile strength and ductility. The common rough woodlouse Porcellio scaber and the sand burrowing
species Tylos europaeus reinforce the exo- and endocuticle layers with
mineral, amorphous calcium carbonate, calcite and phosphate. The amount of incorporated calcite as well as the pattern of calcite organization differs
significantly between the species. While the calcite layer within the
exocuticle of Tylos europaeus is thick and consists of a multitude of highly misoriented calcite domains, the calcite in the exocuticle of Porcellio scaber
is significantly thinner and is composed of a few large domains containing
highly co-oriented calcite crystallites. Differences in mineral incorporation and patterns of crystal assembly are relatable to functional and habitat
adaptations: The tergite of Porcellio scaber is thin and flexible. Upon
predation the animal either runs away or clings still and firmly to the substrate, hence it needs a lightweight and highly flexible cuticle. In contrast,
the beach dwelling isopod Tylos europaeus rolls into a sphere upon threat
and the animal relies on its thick cuticle for protection of its soft body tissue.
47
P 57
Nanoscale structuring of modern brachiopod calcite fibres E. Griesshaber*1, R. Schmidt2, T. Schmidt2, R. Steffen2, H. Gnägi3, M.
Simonet Roda1, A. Ziegler1, W. W. Schmahl1 1Ludwig-Maximilians University Munich, Department of Earth and
Environmental Sciences , Munich, Germany 2Hitachi High-Technologies Europe GmbH, Krefeld, Germany 3Diatome Ltd, Biel-Bienne, Switzerland
Organization of nanosized entities across many length scales poses a major challenge in the development and production of man-made materials with
advanced functions. In contrast, in biologically formed hard tissues, this
design feature and its formation principle is intrinsic. It began already with the emergence of first skeletal hard parts in late Precambrian times and, since
then, it was diversified by evolutionary adaptation.
Modern terebratulid and rhynchonellid brachiopod shells consist of up to three mineralized shell layers: the outermost primary, the inner fibrous, and,
where developed, an innermost columnar layer. In two-layered shells the
fibrous layer forms an extensive part of the shell. The fibers are hundreds of micrometers long and are almost single-crystalline mineral units. The shape
of brachiopod fibers is unique and was already developed in Lower Cambrian
times. In recent brachiopods, the morphology and dimension of fibers is characteristic for a given brachiopod species and is evolutionary adapted to
the animal"s habitat.
The aim of our study was to detect and visualize a possible nanostructuring
of modern brachiopod fibres. We investigated the fibrous layer of the modern
brachiopod Magellania venosa from micrometer to nanometer scale levels
with FE-SEM and EBSD, STEM imaging and TKD. Conventional EBSD measurements prove a strong co-orientation strength of calcite crystallites
within the fibres and document on micrometer scales their clossness to being
single crystals. However, with AFM and STEM imaging and transmission Kikuchi diffraction (TKD) measurements we are able to demonstrate the
internal nanoscale structuring of the fibres. Co-oriented nanosized calcite
crystals are stringently assembled to individual rows, a multitude of the latter comprise individual fibres.
Hence, the almost single crystalline calcite brachiopod fibres are
substructured internally. They comprise highly co-oriented calcite crystallites that are placed in a thin network of biopolymer fibrils, a finding
that has not yet been shown for modern brachiopod calcite fibres. Individual
fibres in modern brachiopod shells are not single crystals.
P 58
Diversity and function of biominerals in Ciliates M. L. Lemloh*1 1University of Stuttgart, Materials Testing Institute, Stuttgart, Germany
Eukaryotic single-celled organisms like Ciliates (Protista) represent an
excellent model system to discover biomineralization principles with respect
to intracellular mechanisms involved in ion enrichment, vesicular mineral transport and biomineral formation. There are over 8,000 species of the
Phylum Ciliophora and since Ciliates naturally occur in freshwater, brackish,
and marine habitats as well as in extreme environments, they can be used to study the impact of different environmental conditions, e.g. available mineral
sources. Although it is known that Ciliates form diverse intracellular mineral
structures, little is understood about their biomineralization processes. Therefore, we combine dynamic in vivo studies of mineral forming Ciliates
together with high-resolution methods like analytical electron microscopy, to
identify common principles and to distinguish diverse mechanisms with specific adaptations involved in mineral formation. The presentation will
outline selected examples of biomineralizing Ciliates with respect to the
characteristics and various functions of the formed minerals.
P 59
Effect of eggshell on polymorphic transformation of calcium
carbonate S. Polat*1, P. Sayan1 1Marmara University, Chemical Engineering, Istanbul, Turkey
Introduction Calcium carbonate precipitation has drawn a lot of research attention over
the last few decades due to its widespread industrial applications and
scientific value in the field of biomineralization. Calcium carbonate crystals can exist as three different forms, namely, calcite, vaterite, and aragonite.
Different polymorphs tend to have different chemical and physical
properties, which can strongly influence the properties of the products; thus, it is important to have control of the crystal form. This study investigated
precipitation of calcium carbonate in the presence of eggshell.
Objectives The purposes of this study were to determine the influence of eggshell on the
morphology of calcium carbonate and to describe the effects of eggshell on
the polymorphic transformation process and the thermal decomposition behavior of calcium carbonate.
Materials & methods Calcium chloride dihydrate and sodium carbonate were used as the reactants in the calcium carbonate precipitation process. The experiment was
conducted in a 1-L double-jacketed crystallizer. The temperature was
controlled using a thermostat and the experiments performed at 30 °C. During the experiments, the pH of the solution was monitored consistently
and fixed at pH 8.5. The solution was mixed at 500 rpm using a three-blade
propeller and mechanical stirrer. The calcium carbonate suspensions obtained as a result of the experiments were removed from the crystallizer,
filtered and then dried. The as-prepared products were characterized by X-
ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy
(SEM) were used.
Results To investigate the structure of the calcium carbonate formed in the absence
and the presence of additive, calcium carbonate crystal forms were
investigated using XRD and FTIR spectroscopy. All the diffraction peaks for the crystals obtained in pure media were attributed to the existence of calcite
crystals with a rhombohedral structure. The calcite form was fully converted
into the vaterite form in eggshell media, which was in agreement with the FTIR results.
SEM and particle size analysis were performed to investigate the effect of
the eggshell on the morphology and the size of the calcium carbonate. The crystals formed in pure media mainly consisted of uniform crystals with a
cubic-like shape and smooth surface. When using eggshell in the crystallization media, nearly all of the crystals occurred in vaterite form and
their appearance was spherical and the specific surface area of calcium
carbonate increased significantly. The filtration characteristics of the calcium carbonate crystals were
determined and the mean specific cake resistance and the mean cake porosity
of the crystals were calculated based on Darcy"s Law. In addition, the thermal decomposition of the calcium carbonate samples was analyzed using
thermogravimetric analysis.
Conclusion The present study investigated the effect of eggshell on the precipitation of
calcium carbonate. In the eggshell medium, the diffraction peaks of the
calcite crystals disappeared and only vaterite crystals were observed. SEM images illustrated that eggshell significantly improved the rounded and
spherical crystals. This study demonstrated the modification of the structure
and morphology of calcium carbonate as well as the formation of its vaterite form were possible using eggshell.
P 60
Crustacean cuticular matrix in transition- spatial and temporal
diversity N. Žnidaršič*1, P. Mrak1, J. Štrus1, K. Žagar Soderžnik2, M. Čeh2 1University of Ljubljana, Department of Biology, Biotechnical faculty,
Ljubljana, Slovenia 2Jožef Stefan Institute, Department for Nanostructured Materials, Ljubljana, Slovenia
Biological mineralized matrices display inhomogenity in structure and
composition. Coexistance of different mineral forms in well defined
functional layers was characterized in detail in several calcified and other biomineralized structures. Complex assemblages of hierarchically organized
organic macromolecules in biomineralized matrices also show structural
alterations in distinct regions. Additional complexity of biomineralized matrices refers to the time scale alterations in structure and composition due
to tissue differentiation during animal development and during replacement.
To address the questions in biomineralized matrix elaboration it is necessary to determine the ultrastructure and composition of the sample at the specific
site(s) and at the specific time. In addition, for microscopical characterization
it is beneficial to preserve both, mineral and organic components. We have applied a correlative microscopic approach that combines structure
determination with elemental and molecular localization data to characterize
the crustacean cuticle differentiation during development and to characterize the interface between calcified and non-calcified layers in adult animals.
Localizations with specific ligands, transmission electron microscopy and
block face imaging in BSE mode by scanning electron microscopy were supplemented by EDXS analyses. Different specimen preparation
procedures were implemented, including methanol fixed and resin embedded
specimens. This enables to obtain information on elemental composition in selected regions of the sample that was not exposed to water solutions and to
correlate this chemical information with ultrastructure as revealed by SEM
imaging of the block surface and by TEM imaging of the corresponding ultrathin section. We have focused to characterize the interface between the
two horizontal regions of the cuticle in the adult specimens, i.e. between the
calcified endocuticle and non-mineralized membranous layer that faces epidermal cells. This is a complex transition zone from the architectural and
compositional point of view. Next, the cuticle of different developmental
48
stages was analysed to determine the modifications of exoskeletal cuticle during differentiation. The earliest stage with an epidermal matrix displaying
typical structure of adult exoskeletal cuticle is the prehatching embryo. In
early postembryonic stages a new cuticle is formed and calcium sequestration in the matrix is evident. Major alterations of epidermal matrix correspond to
the transit from embryonic to postembryonic period that is accompanied by
embryo hatching from the vitelline membrane.
P 61
Crystallographic and biomineral organization of the cuticle of
Devonian trilobites- tailored armours of the past I. Coronado*1, J. Esteve2, J. A. Cruz3, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2Los Andes University, Geosciences Department, Bogotá, Colombia 3Complutense University, GEODESPAL, Madrid, Poland
Systematic characterization of well-preserved fossils can help to trace back
evolutionary changes in biomineralization processes that occurred along the Earth history, but also to provide inspiration to create biomaterials of unique
properties. These tasks can be reached insofar as the original crystallo-
chemical properties of biominerals have not been obliterated by diagenesis. Biominerals formed in biologically controlled process by marine and
terrestrial organisms are hierarchical organo-mineral composite
nanomaterials, with distinctive microstructures and well constrained crystallographic arrangements.
Trilobites are marine fossil arthropods that inhabited seas from Cambrian to
Permian, which have a characteristic articulated carapace. Trilobite cuticle is heavily mineralized assembled by layered structures that form a dense
framework, providing them tailored armours for protection. Cuticles moult
during ontogeny (creating exuviae) and they represent majority of trilobite finds, whereas only exceptionally the full skeletons are preserved. Although
microstructure of trilobite cuticles have been vastly described in the
literature, still, little is known about the mechanisms that ruled their formation and their characteristic biomineral properties. The purpose of this
work was assessed for the first time, the structure of Devonian trilobite
cuticles at micro- and nano-scale levels, as well as the crystallographic arrangement of selected parts of carapace.
Two complete trilobite carapaces belonging to the genera Phacops sp. and
Scabriscutellum sp. from Middle Devonian of Hamar Laghdad Formation (Erfoud, Morocco) were studied. The material was observed under
cathodoluminescence (CL) with a special emphasis to the preservation state. Micro- and nanostructure was characterized by SEM and AFM and
subsequently crystallographic arrangement of four skeletal parts was studied
(EBSD): cranidia, pygidia, articulated thoracic segments and hypostome. The calcitic cuticle is a hierarchical self-assembled structure composed by a
taxa-specific microstructure, dense packaging, forming curved and
interwoven textures. Although, some evidence of diagenesis have been observed in Scabriscutellum sp. cuticle (CL and EBSD data), most of
microcrystals are well preserved in both taxa, and they are composed of
nanogranules coated by dark envelopes, at nanoscale. Cuticles exhibit the c-axis of crystals oriented perpendicular to skeletal surface and although the
analysis shows a common crystallographic arrangement between both taxa,
small differences have been observed in the supra-specialized structures. External structures of carapace exhibit a dense and well-constrained
crystallographic organization in comparison with internal ones. Such
structural organization of trilobite cuticle may explain its mechanical resistance and contribute to a better understanding of the success of this
group during almost 350 million years.
Acknowledgments: This study was supported by the Spanish "Ministerio de Economía y Competitividad" (research projects CGL2016‐78738‐P).
P 62
Solid-state NMR studies of 13C, 15N,29Si-enriched biosilica of
the diatom Cyclotella cryptica F. Kolbe*1, H. Ehren2, M. Baldus2, E. Brunner1 1TU Dresden, Faculty of Chemistry and Food Chemistry, Dresden,
Germany 2Utrecht University, Bijvoet Center for Biomolecular Research, Utrecht, Netherlands
Introduction
Diatoms are unicellular algae producing micro- and nano-structured cell
walls consisting of amorphous silica. The shape and pattern of these cell walls is species-specific. Although some compounds which are involved in
the process of biomineralization like polycationic peptides called silaffins or
long-chain polyamines (LCPAs) are known, the process of biomineralization is not fully understood until today.
Objectives
Solid-state NMR spectroscopy is used to gain a deeper insight into the interactions at the organic/inorganic interface of the diatom cell wall. Special
solid-state NMR methods, i.e. the REDOR experiments, can be used for
distance determination between hetero nuclei. (REDOR: Rotational Echo DOuble Resonance)
Materilas & methods
Within the present contribution, the biosilica of Cyclotella cryptica, a centric maritime diatom, is studied. The silica phase of the purified cell walls can be
characterized using 29Si solid-state NMR spectroscopy whereas the organic
compounds are investigated by 13C, 15N, and 31P solid-state NMR spectroscopy. The sensitivity of the experiments can be greatly enhanced
through isotope-labeling of the diatoms during cultivation in isotope-
enriched culture medium. Moreover, DNP-supported solid-state NMR experiments (DNP: Dynamic Nuclear Polarization) provide further signal
enhancement, which is especially interesting for 2-dimensional NMR
experiments like PDSD (Proton-Driven Spin Diffusion) or DQSQ (Double Quantum Single Quantum).
Results
One important feature of C. cryptica is a massive insoluble organic matrix. Solid-state NMR spectroscopy is used to investigate the insoluble organic
matrix in C. cryptica as well as the silica/organic interface. The different
organic compounds like sugars as well as proteins and LCPAs are studied by the observation of hetero nuclei like 13C, 15N and 31P. In addition, the distance
between different organic compounds and the silica phase is determined
using a 1H-13C-29Si REDOR experiment. It can be shown, that the shortest distances between silica and organic compounds appear for different signals
in the 13C-chemical shift range of 40 – 60 ppm, which is typical for LCPAs.
This indicates a very close contact between the LCPAs and the biosilica. Moreover, DNP-supported NMR measurements confirm the presence of
different sugars. Especially, the presence of chitin is verified using a PDSD experiment. The calculated enhancement factors for different signals and
thus, for different organic compounds can help to get an idea for the
supramolecular architecture of the biosilica. Furthermore, a comparison to the biosilica of Thalassiosira pseudonana reveals both, similarities as well
as differences between the organic matrices of these diatom species.
Conclusion
Our studies show that solid-state NMR is one powerful technique to learn
more about biomolecules and their arrangement in diatom biosilica. NMR
experiments help to identify different organic compounds. Moreover, we get a deeper insight into the supramolecular arrangement of the different
biomolecules in the biosilica.
P 63
Biosilicification- phytolith formation in plants M. A. Nawaz1, I. Zemchenko*1, A. Zakharenko1, K. S. Golokhvast1 1Far Eastern Federal University, School of Engineering, Vladivostok, Russian Federation
Silica is deposited extra and intracellularly in plants in solid form as
phytoliths. Phytoliths have emerged as accepted proxies for reconstructing
ancient flora, agricultural economies, environment, climate, and taxonomic
tools. The discovery of silicon transporter genes has aided in understanding the mechanism of silicon transport within the plant body and reconstruct
plant phylogeny based on the ability of plants to accumulate silica. However, a clear understanding of silica deposition and the formation of phytoliths is
still an enigma and the information on the proteins involved in plant
biosilicification is still scarce. With the observation of various shapes and morphologies of phytoliths, it is essential to understand which factors control
this mechanism. During the last two decades, much research has been done
in this regard and silicon research has expanded as an earth-life science superdiscipline. We integrated the recent knowledge and new concepts on
the evolution of biosilicification in plants; uptake, transport and deposition,
shape, size, and chemistry. We also consider the questions such as how phytoliths of fixed shape are biogenically produced and discuss their
implications in taxonomy, palaeoenvironment, and palaeoflora. Finally, the
applications of phytoliths in the fields of agricultural and biomedical nanotechnology are discussed.
P 64
Regioselective immobilization of an enzyme cascade on diatom
biosilica E. kumari*1, N. Kröger1 1Technische Universitaet Dresden (BCUBE), Dresden, Germany
Introduction
Diatom biosilica is a favorable support material for enzyme immobilization,
as its hierarchical mesoporous structure provides a large surface area for the attachment and allows the efficient reactant diffusion. The immobilization of
multi-enzyme systems is an emerging technology that enables the rapid
multi-step conversion of a substrate into a desired product. Previously it was shown that the activity of immobilized glucose oxidase is strongly influenced
by the species-specific diatom biosilica structures. However, it has remained
unknown, whether the activity of an enzyme cascade is influenced by its positioning within structurally different regions of the biosilica from the
same diatom species. Thalassiosira pseudonana biosilica, which served as a
49
model diatom for this project, is composed of two morphologically distinct building blocks: girdle bands and valves. Girdle bands biosilica rings which
a smooth surface and perforated by uniformly sized mesopores (20 nm
diameter). In contrast, valves have very rough surfaces with a hierarchical pore patterns.
Objective
The aim of the present work is to test the hypothesis that silica structure is a key parameter for the activity of an immobilized enzyme cascade.
Furthermore, we investigated whether the activity of the cascade is
influenced by the relative positioning of the two enzymes on the biosilica surface.
Materials and methods
We have used an in vivo method (LiDSI) to immobilize on biosilica the enzymes glucose oxidase and horseradish peroxidase. Together they
constitute a minimal enzyme cascade. LiDSi is based on introducing into the
diatom T. pseudonana synthetic genes that encode the desired enzymes fused to a fluorescent proteins and a regioselective silica targeting peptide. The
enzymes were localized on the biosilica surface of transformant diatoms
using confocal fluorescence microscopy. After biosilica isolation the catalytic activity of the immobilized cascade was determined through a
colorimetric assay.
Result
We generated four T. pseudonana strains each carrying the same enzyme
cascade, but with different locations of the two enzymes inside the biosilica.
The four enzyme cascade configurations exhibited striking differences in specific catalytic activities (i.e. catalytic activity per immobilized enzyme
molecule). Transformants with both enzymes immobilized in the valve exhibited the lowest specific activity, whereas the specific activity of
transformants containing both enzymes in the girdle band region was 3- to 6-
fold higher. Further investigations into the cause for the observed differences in the cascade activity ruled out influences of quantity, surface exposure and
stability of the enzymes.
Conclusion
Our results confirm the remarkable, yet little understood, influence of
biosilica architecture on enzyme activity.
P 65
In vivo incorporation of iron oxide into diatom biosilica S. Machill*1, J. Kaden1, S. I. Brückner1, L. Köhler1, R. Reichenbächer1, M.
Schumacher1, E. Brunner1 1TU Dresden, Chair of Bioanalytical Chemistry, Faculty of Chemistry and Food Chemistry, Dresden, Germany
Introduction
Diatom biosilica is a species-specific, micro- and nanostructured material
which consists of silicon dioxide and tightly attached organic compounds.
Diatoms can accumulate "foreign" elements like germanium, aluminum or
gold and incorporate or attach them to the silica. These in vivo synthesized
nanocomposite materials can be used as catalysts, adsorbents or biosensors. The role of iron in the biosilification of diatoms is not fully
understood. Iron is an essential nutrient in the metabolism of diatoms and plays a central role for photosynthesis. Genomic analyses indicate a close
connection between the metabolism of silicon and iron in diatoms. Therefore,
the role of iron in biosilification should be further elucidated.
Objectives
The aim of this work is to examine weather or not iron can be accumulated
by diatoms and to determine the chemical state it is associated with the siliceous diatom cell walls. In order to quantify the maximum amount of
incorporated iron, we offered excessive iron concentrations up to the
tolerance limit in the culture medium during diatom growth. The morphology and the structure of the obtained materials were characterized by microscopic
and spectroscopic techniques to answer the question if iron is dispersed in
the biosilica or preferentially clustered. As a defined reference material, silica precipitates with different amounts of iron were also studied.
Materials & methods
The diatom species Stephanopyxis turris and Thalassiosira pseudonana were
cultured in an artificial seawater medium with increased iron concentrations.
To remove the physically bound organic matter and ions, the cell walls were
extracted by a lysis buffer. The obtained biosilica as well as samples of the reference material were analyzed by ICP-OES in order to determine total
Fe:Si ratios. For morphological studies, scanning electron microscopy
(SEM) was used. The chemical state of iron was characterized by Fourier transform infrared (FTIR), Raman, 29Si magic angle spinning (MAS) nuclear
magnetic resonance (NMR), as well as electron paramagnetic resonance
(EPR) spectroscopy.
Results
The amount of iron incorporated in the biosilica of Stephanopyxis turris does
not linearly increase with the amount of iron offered in the growth medium. It is concluded that iron deposition in biosilica is obviously regulated and
limited. The spectroscopic investigations revealed that iron within the silica
framework of the synthetic silicagels is preferentially dispersed leading to Si-O-Fe bond formation. In contrast, biosilica-associated iron in Stephanopyxis
turris exists almost exclusively as Fe2O3 clusters/nanoparticles which are tightly associated to the cell wall. T1 measurements in 29Si MAS NMR
experiments revealed that only a minor amount (below 5 %) is dispersed over
the biosilica. That means that iron does not replace silicon atoms at tetrahedral sites as it is known for aluminum incorporation.
Conclusion Iron is not mainly coprecipitated in dispersed form within the biosilica during cell wall synthesis of Stephanopyxis turris. It rather exists as Fe2O3 clusters.
The obtained material is thus not suitable for technical applications due to
the low amount of iron incorporation. A suitable material may therefore be an iron-silica-nanocomposite.
P 66
New data on the structure of the giant basal spicule of
monorhaphis sponge A. Pisera*1, M. Łukowiak1, K. Tabachnick2 1Institute of Paleobiology, PAS., Warszawa, Poland 2P.P. Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Russian Federation
The skeletal elements of sponges are called spicules and are made of silica or calcium carbonate. They are usually small (micrometers to millimetres)
and situated inside the sponge body. But the basal (siliceous) spicule of deep
water hexactinellid sponge Monorhaphis can reach almost 1 cm in diameter and over 2 meters of length being probably the largest single biosilica-based
structure on Earth. The spicule protrude from the sponge body and anchors
it in the sediment. This giant spicule became a model of hexactinellid spicules structure and
formation. According to previous studies (Wang et al. 2011 with references)
the majority of the spicule is composed of regular smooth siliceous laminae, but two most external layers differ in morphology and structure. The most
external layer called "banded ribbon layer" was first described as made of a
solid fibrous collagen but later its siliceous composition has been proven. The sculpture of the second layer, occurring directly below, was described as
depressions with elevated rims that housed sclerocytes.
The aim of the study was to better understand the structure of this spicule, and to interpret the observed features.
We have examined several spicules of Monorhaphis along the whole length
under a light microscope. Then the fragments which were different in appearance were studied under SEM. Mineral composition of the studied
samples was determined with EDAX. Our studies have shown that the two most external layers are not occurring
along the whole spicule length. All three structurally different units
superimposed occur only in the lower part of the spicules, the uppermost part display only central core of smooth solid silica. We have confirmed that the
banded ribbon layer is siliceous, not organic, but, composed of granular silica
and porous, in opposition to a solid glassy structure of all more internal lamellae. The structure of the penultimate layer is exactly the same as the
core of the spicule. i.e. glassy and without visible porosity. We also
demonstrated that this layer is covered with regularly developed and distributed elevations, not with depressions as previously thought. For this
reason they cannot house sclerocytes; additionally in late ontogenetic stages
in hexactinellids occurs only sclerosyncytium. The elevations in penultimate layer are developed by progressively stronger folding of the surface of
superimposed thinner layers. They fit tightly into depressions of the banded
ribbon layer, developed its lower surface. It is not clear why and how the banded ribbon and tuberculated layers
of Monorhaphis basal spicule have developed, but we speculate that the
banded ribbon surface may be an adaptation to help the sponge to stay fixed on the spicule, not to glide down on very smooth silica surface. The
elevations of the penultimate layer that fit into depressions of the lower
surface of banded ribbon layer help to stabilize (fix together) the two most external layers.
References 1. X. H. Wang, M. Wiens, H. C. Schröder, K. P. Jochum, U. Schloßmacher, H. Götz, H. Duschner, W. E. G. Müller, J. Experimental Biol. 214 (2011)
2047-2056.
Financial support by the Polish National Science Center Grant 2016/21/B/ST10/02332 to A. Pisera.
50
P 67
The accumulation of aluminum in diatom frustules and
modifications of the resulting alumosilicate L. Köhler*1, S. Machill1, A. Werner2, C. Selzer2, S. Kaskel2, E. Brunner1 1Chair of Bioanalytical Chemistry, Faculty of Chemistry and Food
Chemistry, Technische Universität Dresden, Dresden, Germany 2Chair of Inorganic Chemistry I, Faculty of Chemistry and Food Chemistry, Technische Universität Dresden, Dresden, Germany
Introduction
Diatoms stand out from other unicellular algae through their uniquely
patterned, siliceous cell walls. These so-called frustules contain mainly amorphous SiO2, but also foreign elements such as metalloid and metal
atoms. Aluminum is a particularly interesting constituent because it directly
replaces silicon in the silica framework, forming an alumosilicate.1,2 This aluminum insertion leads to a negative charge, which is probably
counterbalanced by cations like calcium.1
Objectives
In the present study, several ways of aluminum addition to the living diatom
cultures were evaluated. The structure model from literature1 was reviewed
with respect to the coordination of aluminum and the type of counter ions. Varying growth parameters were assessed to tailor materials properties of the
frustules, i.e., the molar ratio of aluminum to silicon. This characteristic
property affects possible future applications in sorption and catalysis. Besides the changes in vivo, frustules were further modified in vitro to
enhance porosity and acidic strength.
Materials & methods
The fast growing and resistant diatom species Thalassiosira pseudonana was
cultivated in aluminum-enriched media. After harvest, frustules were cleaned
with lysis buffer and calcined. This biosilica was characterized by various methods including infrared, nuclear magnetic resonance and optical emission
spectroscopy as well as nitrogen and ammonia sorption. To exchange the
counter ions created by aluminum incorporation, the biosilica material was stirred in an ammonia solution and subsequently heated. Furthermore,
frustules were etched with a mild base to increase the specific surface area.
Catalytic activity was investigated by an acid-catalyzed alkylation of aromatic compounds, which was monitored by gas chromatography coupled
with mass spectrometry.
Results
Diatom incorporated aluminum into their frustules, resulting in n(Al):n(Si)
ratios up to the scale of 1:10. With further in vitro modification steps, acidity was more than doubled to 320 µmol NH3 g
-1 and the specific surface slightly
increased to 70 m2g-1. Ion-exchanged biosilica was shown to be catalytically
active in contrast to non-modified biosilica.3
Conclusion
The current study shows that the siliceous cell walls of Thalassiosira
pseudonana can be aluminum enriched in vivo while preserving their structure. Through in vitro modification, the acidity and specific surface area
of the biosilica can be enhanced. As successful catalytic tests show, diatoms
are a possible source for "green" catalysts. (1) Gehlen, M.; Beck, L.; Calas, G.; Flank, A.-M.; Bennekom, A. J. Van;
Beusekom, J. E. E. Van. Geochim. Cosmochim. Acta 2002, 66 (9), 1601–
1609. (2) Machill, S.; Köhler, L.; Ueberlein, S.; Hedrich, R.; Kunaschk, M.;
Paasch, S.; Schulze, R.; Brunner, E. BioMetals 2013, 26 (1).
(3) Köhler, L.; Machill, S.; Werner, A.; Selzer, C.; Kaskel, S.; Brunner, E. Molecules 2017, 22 (12).
P 68
The role of aluminum in diatom biosilicification M. Soleimani*1, S. Maddala 1, A. Akiva1, L. Rotten1, I. Zlotnikov2, R.
A.T.M. van Benthem1, H. Friedrich1, N. A. J. M. Sommerdijk1 1Eindhoven University of Technology , chemical engineering and chemistry , Eindhoven, Netherlands 2 CUBE Center for Molecular Bioengineering, Dresden, Germany
Introduction Diatoms are unicellular photosynthetic algae which live in nearly every aquatic territory. Their unique morphological characteristic is the silica cell
wall (frustule), which is formed from two halves, like a petri dish, joined
together by girdle bands. Besides silicon, diatoms incorporate aluminum into their silica cell wall, impacting the solubility and dissolution rate of the
frustule. Due to the complexity of the structural arrangement of diatoms, the
chemical connectivity between aluminum and silicon inside the frustule has not yet been discovered. Here, we use various analytical techniques to get
insights into how aluminum plays a role in frustule formation.
Materials and methods Pinnularia sp (P.sp) and Thalassiosira pseudonana (T.p) were grown in
filtered, autoclaved seawater amended with nutrients according to Guillard"s
f/2-recipe. To synchronize cells at cytokinesis arrest, silicon starvation was applied with an incubation time of 48 h in Si free medium. Starvation was
terminated by adding Guillard"s (F/2) marine water enrichment solution and
varying amounts of aluminum to have Si and Al precursors simultaneously in the culture. After 12 days of cultivation, cells were harvested and washed
by centrifugation and kept at -20 for future characterizations. In order
to monitor the frustules formation inside the silica deposition vesicle (SDV) with fluorescence microscopy, synchronized growth was
initiated by repleting the culture with a final concentration of 100 µM silicic
acid and 1 µM PDMPO as a staining agent.
Results Fluorescence microscopy images show valve formation starts from the
central nodule and proceeding toward the outer valve edges. Following one hour the full two-dimensional size of the new valve has been reached. After
6 hours the fluorescence intensity of the SDV reaches the maximum,
implying at this point the two cells are relatively separated and cell division is taking place. EDX measurements of P.sp and T.p unveil the presence of
aluminum in the frustules. SEM images of P.sp show that the pore diameters
seem to decrease by increasing the concentration of aluminum in the cultures. The Solid-state NMR spectra show that the sample contained both
four and six coordinated Al (III) as indicated by peaks at 7.99 and 55.03 ppm,
respectively. Six coordinated Al is the most predominant Al (III) species in our sample which could be from our precursor bonded Al to organic
compounds inside and outside of the cells or adsorbed Al (III) on the surface
of frustules. However, the peak at 55.03 ppm shows the presence of four-fold coordinated Al suggesting it is incorporated inside the silica networks of the
frustules.
Conclusion EDX measurements show the presence of aluminum in both species. SEM
images of P.sp show that the pore size decreased by increasing the AlCl3 in the growth medium. Cell division in P.sp as shown in fluorescence
microscopy images takes six hours. Solid State 27Al NMR spectra show four
and six coordinated aluminum in the structure of T.p. In the future, we aim to perform 29Si and 27Al solid-state NMR of the frustule to not only
evaluate the coordination environment of aluminum but also to pinpoint the
effect of aluminum on cross-linking of silica. In addition, some mechanical properties of intact cells and frustule will be measured to investigate the
effect of aluminum on the mechanical characteristic of diatoms.
P 69
Highly efficient encapsulation of biocatalyst in diatom-inspired
silica nanoparticleforenzymatic CO2 capture and utilization B. H. Jo*1
1Gyeongsang National University, Division of Life Science, Jinju, South Korea
This study reports on the development and characterization of a carbonic anhydrase (CA)-based biocatalyst encapsulated in a biosilica matrix for use
in CO2 capture an utilization. Encapsulation occurred simultaneously with
autonomous silica synthesis by diatom-derived silica-condensing R5 peptide, which was genetically fused to recombinant CA. The encapsulation
efficiency was greater than 95%, and the encapsulated CA was not leached
from the silica matrix, demonstrating the highly efficient R5-mediated auto-encapsulation process. The catalytic efficiency for CO2 hydration was pH
dependent, suggesting that proton transfer from silica to water is a rate
limiting step. In addition to good reusability, the encapsulated CA exhibited outstanding thermostability, retaining 80% activity after 5 days at 50˚C. The
thermoactivity was also remarkable, showing ~10-fold higher activity at
60˚C compared to that at 25˚C. The physical structure was observed to be highly compact with a low surface area, stressing the importance of the
outermost surface for catalytic performance. We also demonstrated the
applicability of the silica nanoparticle to the sequestration of CO2 in carbonate minerals. The rate of CaCO3 precipitation was remarkably
accelerated by the encapsulated biocatalyst. Thus, this silica-CA
nanocomposite, efficiently synthesized via a biomimetic green route, can be successfully used as a robust biocatalyst for biomimetic sequestration of the
greenhouse gas CO2.
P 70
Design of biosilica-enveloped R5 ferritin cage for dual drug
delivery system development M. R. Ki1, M. A. A. Mohamed1, K. B. Yeo1, S. H. Kim1, K. H. Min1, S. P.
Pack*1 1Korea University, Biotechnology and Bioinformatics, Sejong, South Korea
Silica has unique properties such as good biocompatibility, excellent pH and thermal stability, as well as high porosity, low toxicity, and superior
mechanical stability. Consequently, silica-based nanomaterials have gained
considerable interest for their potential biotechnological applications such as in drug delivery, enzyme immobilization, and biocatalysis. However, the
classic approaches to silica synthesis require harsh reaction conditions such
as extreme pH, high temperature, and toxic chemicals. One approach to alleviating these limitations is to mimic the silica mineralization seen in
biological systems. In one of well-known examples, silaffin polypeptides
derived from diatom biosilica are a class of massively post-translationally
51
modified (PTM) proteins that are responsible for initiating silica formation at ambient temperature and pressure. Interstingly, an unmodified silaffin R5
peptide is capable of mediating silica precipitation in vitro under specific
conditions. Using recombinant DNA technology, R5 allows for in situ formation of biosilica matrices containing the R5-fused recomabinant
proteins. Here, we designed R5 peptide-fused ferritin (R5FT) for the
developement of an advanced dual drug delivery system (dDDS). Since the fused R5 can mediate biosilica deposition on the ferritin surface
(SiO2/R5FT), we could load two types of molecules into the core inside
(ferritin cage) and the shell outside (biosilica matrix), respectively. One model (D#1) was loaded into the cage by the reassembly of R5FT to obtain
R5FT(D#1), and then the other model drug (D#2) was captured in situ by
biosilica matrix formation to prepare SiO2(D#2)/R5FT(D#1). The captured D#2 in the shell exhibited a short-term release, while D#1 in the
core showed a long-term sustained release. This dDDS system with the
additional release of D#2 decreased the IC50 value of D#1 by two-fold compared to the use of only D#1 in dDDS. We further optimized the size and
performance of biosilica-enveloped ferritin cage in terms of drug delivery
system. The developed dDDS using biosilica-enveloped ferritin cage can provide more efficient combinational drug therapies.
P 71
Biological silica formation in diatoms - mineralization outside
the box? B. Mayzel*1, S. Wolf2, A. Gal1 1Weizmann Institute of Science, Department of Plant & Environmental
Sciences, Rehovot, Israel 2Weizmann Institute of Science, Department of Chemical Research Support, Rehovot, Israel
Introduction
Diatoms are unicellular algae, abundant in all aquatic environments. The
hallmark of diatoms is their mineralized cell wall, made of amorphous silica. The morphology of the cell wall is species-specific, forming distinct 3D
micro- and nano-metric architectural features. The prevailing paradigm to
explain the biological control over the formation of the inorganic phase is that each silica element is formed intracellularly inside a silica deposition
vesicle (SDV), and once completed it is exocytosed. The complex cellular
processes within the SDVs are thought to regulate the mineralization process. Chaetoceros tenuissimus is a diatom species with a cell size of 5µm in
diameter, which is characterized by four long extensions, called the setae, radiating from the main cell body. The setae are 15-30µm long and 250-
300nm in diameter and are covered by a silica shell. Seta growth commences
after cell division and continues at the seta tip until its full length is reached. Therefore, the growing tip of the C. tenuissimus seta is a unique system to
study the cellular process of silica formation.
Objective
Identifying the cellular organelles and structural elements responsible for
silica mineralization of C. tenuissimus setae.
Methods
C. tenuissimus cells were grown in culture and their cell cycle synchronized
to increase the abundance of growth-stage seta in cells. CryoTEM
tomography was used to study the 3D structure of C. tenuissimus setae at various growth stages. Samples were plunge-frozen in liquid ethane to
preserve the native structure of the setae. Tomograms of seta tips were
acquired using the 200kV Tecnai F20 and the 300kV Titan Krios G3i microscopes. IMOD software was used to reconstruct the tomograms from
the data.
Results
24 tomograms of setae were generated, 11 are assigned to growth-stage setae.
These tomograms clearly show the complex helical architecture of the thin
silica fibers enveloping the seta. A thin extra-cellular polysaccharide layer covering the mineralized silica cell wall was observed. The cytoplasmic
extension within the growing setae contained a single microtubule filament
inside the cell membrane. In some cases, small vesicles were noted in the cytoplasm. Importantly, none of the tomograms showed an SDV or other
associated cellular structures at the tip of the setae.
Conclusions
These results suggest a silicification mechanism in diatoms that is not SDV
(silica deposition vesicle) dependent, but rather a continuous process of
extracellular silicification. This mechanism may explain other silica elements in diatoms and solve fundamental unknowns about the biological process of
silicification.
P 72
Interactions between gold nanoparticles and the diatom
Stephanopyxis turris N. Pytlik*1, S. Machill1, B. Klemmed2, A. Eychmüller2, E. Brunner1 1Technische Universität Dresden, Bioanalytical Chemistry, Dresden,
Germany 2Technische Universität Dresden, Physical Chemistry, Dresden, Germany
Introduction
Diatoms are unicellular algae, which contribute very significantly to the
global carbon fixation by photosynthesis. They are especially famous for
their cell wall made from amorphous biosilica which presents a spectacular example for the beauty of biomineralization. The cell walls are highly
structured and exhibit regular pore patterns at micro- and nanoscale. Each
diatom species is characterized by a unique cell shape and pore pattern. However, the understanding of the complex process of three-dimensional cell
wall formation is still limited.1
Objectives
Diatoms, furthermore, find increasing research interest in the field of
catalysis, solar cells or as templates. For several potential applications, the
cells are combined with gold nanoparticles either in vivo or in vitro. However, only few information is available about the interactions and effects
of gold nanoparticles on the diatom cells.
Materials & methods
We have recently exploited the potential of the gold nanoparticles to generate
a SERS (surface enhanced Raman spectroscopy) effect for an in vivo
monitoring. Using a 3D setup, it was shown that gold nanoparticles, which were biosynthesized by the diatom Stephanopyxis turris, can even occur
inside the diatom cells.2 Consequently, these intracellular gold nanoparticles
must either a) be taken up by the cell after extracellular gold ion reduction or b) be produced inside the cell after the uptake of gold ions.2 To answer the
question whether or not gold nanoparticles can generally be taken up by
diatom cells, chemically synthesized gold nanoparticles were added to living diatom cells and their interactions were observed by 3D SERS.
Results
Gold nanoparticles with diameters ≥ 50 nm could clearly be localized inside the cells, whereas smaller gold nanoparticles were never detected in the cell
interior. This indicates a size-dependent uptake mechanism that comes along
with different toxicities.3
Conclusion
The consequences of gold nanoparticle contact with diatom cells have not yet been fully understood and long-term effects for the organism itself as well
as for the environment are hardly predictable. Having in mind the raising
gold nanoparticle implementation in industry, our findings point out the responsibility to intensify research concerning nano-bio interactions –
especially focusing on diatoms, as an essential biomineralizing organism for
our ecosystem. 1 N. Pytlik and E. Brunner. MRS Commun. 8, 2018, 322-331. 2 N. Pytlik et al. Algal Res. 28, 2017, 9-15. 3 N. Pytlik et al. Algal Res. 39, 2019, 101447.
P 73
Macrobiomineralogy- principles of biomineralization in giant
whale bones and perspectives for bioinspired materials science M. Wysokowski*1 1Poznan University of Technology, Faculty of Chemical Technology, Poznan, Poland
Introduction Nowadays, biomineralization is defined as ancient fundamental biological
process with high dynamics by which living organisms produce minerals with multifunctional properties including to harden existing subcellular
organic matrices and tissues, to produce protective armors and shells against
external damages of diverse origins (predators, UV irradiation, toxic metals, etc.), as well as to carry out magnetic navigation. Diverse biominerals and
biomineral-based skeletal structures are occurring in nature on nano-(virus
and bacteria), micro- (diatoms, coccolothophores) and macro- (up to 7-meter-long whale bones) levels. The mechanisms of biomineralization
remain hotly debated. The best way to understand the basic principles of
biomineralization on both molecular and macro-scale levels is by a coherent synergetic collaboration using explicit reasoning and well-tested explanatory
principles of multidisciplinary experience, knowledge and new technologies.
Giant bones of whales (Cetacea) represent the largest biomineral-based constructs known. The existence of such macrobiominerals with still
unknown mechanisms of their origin and development is an example of a
ground-breaking phenomenon unexplored in nature. In this study, the concept of macrobiomineralization is proposed for the first time.
Objectives The main objective is to discover the fundamental mechanisms of biomineralization in selected giant whale bones. This includes the analysis
of the structure-function relationship between organic (collagen, lipids) and
52
inorganic mineral phases in selected lipid-rich whale bones (mandibles, ribs and vertebrae).
Materials & methods Selected whale bones from registered museum collections have been gentle demineralized using EDTA-based solution at pH 7.2 at 37°C to extract
corresponding organic phases and to carry out their identification using
modern bioanalytical tools including GC, HPLC, ESI-MS, FTIR, Raman, CARS, 13C solid state NMR, XPS, XRD, HR-TEM and NEXAFS. Modern
3D stereo microscopy together with fluorescence microscopy and BET
measurements are used for additional characterization of porosity, inner structural organization and special surface area of the biomaterials studied.
Results Biomineralizers like whales, which are able to produce hydroxyapatite (HAP)-based skeletal structures in lipid-rich (hydrophobic) environment, are
of crucial scientific interest. For the first time we show that millimeter-sized
fibers of collagen remain to play significant role in formation of highly carboxylated HAP, especially in vertebrae. Strong difference with respect to
organic phases has been shown between the whale vertebrae and ribs using
NEXAFS spectroscopy. Phospholipids in ribs fraction have been found tightly bound to collagen fibres. It is suggested that in contrast to human
bones,calcium oleate can be the precursor of HAP in the lipid-rich bones of
whales.
Conclusion The fact that giant whale bones represent examples of large-scale
biocomposites which has been synthesized in situ at 36.6 °C is very motivating to resolve the outstanding questions. Consequently, based on
obtained results the objective with regard to "large-scale biomimetics" includes the goal to develop the key way for industrial production and design
at ambient conditions of "3D Ca-phosphate-lipid-collagen composites" using
naturally occurring sources of each component.
P 74
The unstructured proteins in biological structures- the case of
human teeth from a protein chemist's perspective V. Sharma*1, A. Srinivasan2, A. Roychoudhary3, F. Nikolajeff4, S. Kumar1 1All India Institute of Medical Sciences, Biophysics, New Delhi, India 2Jamia Hamdard University, Biochemistry, New Delhi, India 3All India Institute of Medical Sciences, Oral and Maxillofacial Surgery, New Delhi, India 4Uppsala University, Engineering Sciences, Uppsala, Sweden
Introduction
Proteins are the biomacromolecules that work downstream of genes in every
living system. It is these protein molecules that are responsible for a plethora of functions in our body, ranging from building skeleton tissues to
transmitting signal from one place to others. There was a period when
biologists have the view that for a protein to function, the structure is of supreme importance. Nevertheless, this structure-function paradigm is fairly
obeyed by most of the proteins, if not all of them. Last two decades have
witnessed the increased reports of proteins that do not have a stable three-dimensional structure but are very much indispensable for the cellular and
biological activities. These unstructured proteins are now known as
"Intrinsically disordered proteins" (IDPs). Interestingly, these IDPs function not only in isolation (e.g. transcription factors) but helps in the formation of
hard tissues like teeth, bone, and mollusk shell, by governing a process called
biomineralization. It is the process by which living organisms produce minerals, often to harden or stiffen existing tissues. Such tissues are called
mineralized tissues. The process of biomineralization is still considered as a
scientific puzzle.
Aim
To investigate the structural differences of protein extracts from four types
of human teeth (i.e. Molar, Premolar, Canine, and Incisor) and their implication in regulating the calcium phosphate mineralization in vitro.
Methods
1) Isolation and purification of proteins from different types of human teeth. 2) Characterization of tooth proteins by (a) Fourier transform Infrared
Spectroscopy(FTIR); (b) Circular Dichroism (CD)
3) In vitro calcium phosphate biomineralization assay followed by size measurement experiments by Nanoparticle tracking analysis and analysis by
scanning/transmission electron microscopy.
Results and Discussion
Protein extract was successfully purified from different types of human teeth
(i.e. Molar, Premolar, Incisor, and Canine) which were quantified and run on an SDS PAGE and subsequently stained by silver staining. FTIR was done
to understand the differences present among the different teeth extract and
there secondary structure characteristics. CD was done to validate the protein secondary structure details obtained by FTIR. We have also investigated the
effects of these protein extracts on mineralization of calcium phosphate
through in vitro biomineralization assay followed by size-based measurement analysis via Nanoparticle tracking analysis (NTA).
Conclusions
1) Secondary structural characteristics of different protein extracts were
studied using various biophysical techniques. The presence of IDPs in the
majority was confirmed within these protein extracts. 2) We have also investigated the effects of these protein extracts on
mineralization of calcium phosphate through in vitro biomineralization assay
followed by size-based measurement analysis via Nanoparticle tracking analysis (NTA).
This is the first time the effect of the whole protein extract was taken into
account which in our view more closely imitates the in vivo process of tooth biomineralization.
P 75
Brachiopod shells can be folded when wet F. Nudelman*1, J. Ihli2, A. Schenk3, M. Holler2, K. Wakonig2, M. Duer4, M.
Cusack5 1University of Edinburgh, School of Chemistry, Edinburgh, United
Kingdom 2Paul Scherrer institut, Villigen, Switzerland 3University of Bayreuth, Faculty of Biology, Chemistry and Earth Sciences,
Bayreuth, Germany 4University of Cambridge, Chemistry, Cambridge, United Kingdom 5University of Stirling, Biological and Environmental Sciences, Stirling, United Kingdom
Question
For hundreds of millions of years, nature has evolved a large assortment of
organic-inorganic hybrid materials that are optimally adapted for a wide range of functions, including navigation, protection, mechanical support and
protection. These materials not only exhibit exceptional material properties
but also display multifunctionality, including features such as adapting, sensing and self-healing. Among the most remarkable biominerals found in
nature are the shells of the phosphatic brachiopod Discinisca tenuis. We
empirically observed, for the first time, that the mechanical properties of these shells vary according to their water content. While they are hard and
stiff when dry, they become flexible when hydrated, to the point that the shell
can be folded in 2 without fracturing. Such capability of an organic-inorganic composite to switch reversibly between stiff and flexible and in real time,
adapting to changes in the environment that demand a different set of
mechanical properties, is truly unique among both biological and synthetic materials. The aim of this research was to characterise, from the molecular
to the micron-scale, how the structure the brachiopod shells changes as a
function of hydration, leading to changes in mechanical properties.
Methods
We used cryo-ptychographic X-ray computed tomography (PXCT) on dry,
partially hydrated (exposed to 70 % relative humidity) and fully hydrated
shells (exposed to 100 % relative humidity) to characterise how the structure
of the shell at the sub-micron and micron levels changed as a function of
hydration. Cryo-scanning electron microscopy (cryoSEM) measurements were performed on dry and wet shells to analyse changes at the shell nano-
structure as a function of hydration. Solid-state NMR (SSNMR) was used to determine the effect of hydration on the molecular conformation of the
organic components.
Results
PXCT measurements demonstrated the organic-rich layers in the shell
expanded in thickness, increasing from ~160 nm in the dry sample to ~180
nm in the partially hydrated and ~340 nm in the fully hydrated sample. CryoSEM suggests that the organic components of the shell swell upon
hydration. Using SSNMR, we identified that the macromolecular chains of
proteins, and in particular the methyl groups in proximity to the mineral, become more mobile with hydration.
Conclusions Our results show that the changes in the mechanical properties of the shell as it absorbs water arise from modifications in the mobility of the organic
components and structural changes, at the sub-micron and micrometer
levels, caused by the swelling of the organic layers.
P 76
Ion substitutions in apatites of human urinary stones A. Korneev*1, A. Izatulina1, O. Frank-Kamanetskaya1
1Saint Petersburg State University, Department of Crystallography, Saint
Petesburg, Russian Federation
Hydroxyapatite is the main mineral component of phosphate stones in human urinary system according to the powder XRD data: it also occurs as an
impurity in oxalate and urate stones. According to our collection of renal stones of St. Petersburg and the Leningrad region residents, apatite is present
in 64,7% stones. Using SEM and EDX it was revealed that individual apatite
grains are present almost in all oxalate urinary stones, which confirms the hypothesis of initializing calcium oxalates crystallization by hydroxyapatite.
Due to the low degree of crystallinity of bioapatites the conclusion of their
composition can be made according to the detailed EDX analysis and values
53
of lattice parameters (Frank-Kamenetskaya et al. 2011). According to our data values of the a unit cell parameter of urinary stones apatites vary from
9.395(2) to 9.457(4) Å, i.e. can be higher or lower than that of stoichiometric
hydroxyapatite (a = 9.418, JCPDS № 9-432). Values of the c unit cell parameter (6.849– 6.885(4) Å) can be significantly lower than in
stoichiometric hydroxyapatite (c = 6.884 Å, JCPDS № 9-432). The increase
of a parameter of urinary stone apatites, are associated with substitutions of OH- ions by H2O molecules in channels of crystal structure, the decrease - by
F- ions. EDX data show that fluorine is present in trace amounts in apatite of
phosphate kidney stones, and in apatite of oxalate stones its concentration significantly higher (1.6 – 4.2 wt.%). Reduction of c parameter relative to
stoichiometric hydroxyapatite is due to the large portion of vacancies in Ca
sites. Their appearance can be associated with substitutions of OH- ions by H2O molecules and PO4
3- ions by CO32- and HPO4
2- ions.
The range of urinary stones apatite lattice parameters variations is
significantly higher than in other pathogenic apatites that are formed in human organism, which indicates the essential variation of their forming
conditions.
This work was supported by the Russian Science Foundation (no. 18-77-00026). The XRD studies have been performed at the X-ray Diffraction
Centre of St. Petersburg State University. Scanning electron microscopy and
EDX analysis have been performed at «Center for Geo-Environmental Research and Modeling (GEOMODEL)» of St. Petersburg State University.
References
Frank-Kamenetskaya O., Kol'tsov A., Kuz'mina M., Zorina M., Poritskaya L. Ion substitutions and non-stoichiometry of carbonated apatite-(CaOH)
synthesised by precipitation and hydrothermal methods // Journal of Molecular Structure. 2011. Vol. 992. P. 9-18.
P 77
TEM study of compositional and structural changes of bone
crystals by heating M. Yosikawa*1, M. Kakei2 1Meikai University School of Health Sciences, Department of Oral Health
Sciences, Urayasu, Japan 2Medical English Institute[NPO], Saitama, Japan
Introduction
Octacalcium phosphate (OCP) is considered to be a candidate for the central dark line (CDL) which is observed in tooth enamel, dentin and bone crystals.
On the other hands, it has been reported that the formation of huntite minerals occurred prior to the nucleation of apatite crystal (Casciani et. al., 1979).
Regarding the physical property of OCP, it has been reported that OCP was
decomposed completely up to 150 ℃ (Bigi et. al., 1990), while the thermal decomposition of huntite minerals has been reported to begin from around
550 ℃ (Földvárl, M. 2011). From the ultrastructural viewpoint, transmission
electron microscopic (TEM) study has shown that after heating at about 600 ℃, a small number of crystals still preserved CDLs in their structures as
reported previously (Kakei et. al., 2005). This finding suggested that CDL
known as the nucleation site of biological apatite crystals might consist from huntite minerals and a subsequent induced initial lattice line of apatite.
Furthermore, the lattice line of CDL did not create two lattice lines of apatite,
meaning that CDL is not identical to OCP. Based on the previous findings, the present study was aimed to further clarify the involvement of huntite
minerals to the CDL formation. Also, we conducted to examine the structural
change of crystals by heating at over 700 ℃.
Objective
The present study is conducted to distinguish between CDL in apatite crystal
and OCP, and examined the structural changes of apatite crystals by heating.
Material and Methods
To clarify the composition of CDL, the results of both thermal analysis of
huntite minerals and TEM observation of bone crystals treated by heating at different temperatures were compared. After removing the surrounding soft
tissues, rat calvaria were treated with a plasma reactor to remove organic
substances. Thermogravimetry/Differential thermal analysis (TG/DTA) analysis of huntite minerals was conducted in air atmosphere with heating at
20 °C/min. For TEM study, samples were heated at 600, 700 and 1,000 ℃,
respectively, for one hour in the muffle furnace.
Results
Fig.1 showed the TG and DTG derivative curves of huntite minerals. The
decomposition of huntite minerals began from around 500 ℃ (Fig. 1). TEM study of the control of calvaria showed numerous tiny crystals showing the
presence of CDL (inset) (Fig. 2). After heating at 600 ℃, each crystal seemed
to be obscure and some crystals amalgamated with each other, though some crystals still preserved CDL (Fig. 3). After heating at 700 and 1,000 ℃,
respectively, TEM observations revealed that large crystals were formed
(Figs 4a and b). CDLs disappeared completely.
Conclusion
The preservation of CDLs by heating at around 600 ℃ clearly reveals that
CDL is not identical to octacalcium phosphate, meaning that two different mechanisms for apatite formation exist. Creating larger crystals by heating
at above 700 ℃ may contribute to the increase of crystallinity.
P 78
Correlation of bone morphology and quality with melatonin
intake in pregnant and lactating rats N. Ishikawa*1, H. Mishima2, A. Hattori3, Y. Maruyama3, N. Suzuki4, Y.
Matsumoto5
1Kagawa University, graduate school of Agriculture, Division of applied
biological and rare sugar sciences, MIki, Kita gun, Kagawa prefecture,
Japan 2Tsurumi University School of Dental Medicine, Dental Engineering,
Yokohama, Japan 3Tokyo Medical and Dental University, Biology, Ichikawa, Japan 4Kanazawa University, Nature and Environmental technology, Housu-gun,
Japan 5Kagawa University faculty of agriculture, Life Science Course, MIki, Japan
Introduction
Melatonin (MEL) is a hormone that plays a role in the circadian rhythm.
MEL increases bone density and inhibits the differentiation of osteoclasts
(Koyama, 2002; Hattori, 2017). In 6-day-old rats, MEL participates in the formation of incremental lines and calcification of dentin (Mishima et al.,
2018). However, the effects of MEL intake during osteogenesis on bone
formation in rats have not been clarified. Therefore, we analyzed the bone morphology and quality based on MEL intake in lactating rats.
Objectives
The present study evaluated the effects of MEL intake in lactating rats on the bone morphology and quality during lactation.
Materials and methods
Six SD pregnant rats were divided into 3 groups: (1) control group (Con; 0.5% alcohol-containing water), (2) low-concentration group (Low; 0.5%
alcohol + 20 µg/mL MEL-containing water), and (3) high-concentration
group (High; 0.5% alcohol + 100 µg/mL MEL-containing water). MEL was administered for 9 days, the last 3 days of pregnancy and the 6 days after
birth. Slaughter was carried out at midday and midnight on 6-day-old. The
concentrations of plasma melatonin metabolites (serotonin: 5HT, N-acetyl serotonin: NAS, melatonin: MEL, and 6-hydroxymelatonin: HaMT) were
measured by LC-MS/MS. The trabecular area ratio was observed and analyzed using a fluorescence microscopy. The calcification, crystallinity
and collagen maturity of bone were analyzed by FTIR. The bone composition
was analyzed by SEM-EDS.
Results
5HT was significantly higher in the MEL-treated group at midday and
midnight (p < 0.05). MEL was significantly higher in the High group than that of the Con group at midnight (p < 0.05). HaMT value increased in the
proportion with the MEL dose. The trabecular area ratio was significantly
higher in the Low group than that of the Con group at midday (p < 0.05). Calcification was significantly higher in the High group than that of the Con
group at midday (p < 0.05). The crystallinity and collagen maturity were
significantly lower in the MEL-treated group at midnight (p < 0.05). The mass ratios of Ca and P were significantly higher in the MEL-treated group
at midday (p < 0.05). At midnight, the mass ratio of P was significantly lower
in the High group than that of the Con group (p < 0.05).
Conclusion
In blood analysis, 5HT is secreted in the breast milk during lactation, thus we
think that 5HT was transferred from breast milk to rat pups and NAS was confirmed in rat pups plasma. Further, we think that MEL metabolism was
activated by MEL intake and 5HT in breast milk increased. The value of
MEL and HaMT in plasma suggested the transfer of MEL to rat pups through breast milk. In the MEL-treated group, the trabecular area increased, and
calcification was promoted, however bone was immature. Thus, bone
maturation probably did not catch up with bone formation in the MEL-treated group. The SEM-EDS analysis suggests that MEL may have promoted bone
formation and affected bone composition. The present results suggest that
MEL intake in the pregnant rats was transferred to rat pups through breast milk to promote bone formation and calcification. From Mishima's (2018)
finding that MEL changes the structure of dentin apatite crystals and collagen
fibers, the results of this study suggest that the mechanism of MEL effects on the bone calcification may differ from the dentin calcification.
54
P 79
Crystallographic and chemical vital effects in Dendrophyllia
skeleton - a by-product of the biocrystallization I. Coronado*1, A. Pérez-Huerta2, J. A. Cruz3, J. O. Cáceres4, S. Moncayo5,
V. Motto-Ros5, F. Trichard5, G. Panczer5, F. Pelascini6, J. Stolarski1 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 2The University of Alabama, Department of Geological Sciences,
Tuscaloosa, United States 3Complutense University, GEODESPAL, Madrid, Spain 4Complutense University, Departamento de Química Analítica, Madrid,
Spain 5Institut Lumière Matière, UMR5306 – UCBL- CNRS, Lyon University, Villeurbanne, Germany 6CRITT Matériaux Alsace, Schiltigheim, France
Coral skeletons are natural archives of the climate variability and a potential
proxy for paleoenvironmental reconstructions. Paleoclimate reconstructions
are based on changes in isotopic ratios and trace element uptake by bio-aragonite skeletons, as a response to environmental changes such as
temperature and ocean circulation . Unfortunately, corals are not longer
considered as passive recorders of environmental changes because the geochemical composition of their skeletons does not precipitate in
thermodynamic equilibrium with seawater. This desequilibrium has been
attributed to vital effects. Two classes of vital effects have been recognized: those derived from
physiological processes and those related to biocrystallization, from crystal
nucleation to stabilization of the mineral phases. Those vital effects linked to biocrystallization have not been studied in-depth in corals.Their
interpretation requires the incorporation of groundbreaking knowledge from
recent discoveries in coral biomineralization i.e., identification of amorphous precursor phases that fulfills a new paradigm of crystallization by particle
attachment (CPA) and biocomposite nature of coral skeleton. Therefore, in
order to retrieve a "purely" environmental signalfrom coral skeletons, chemical variations linked to precursor phases and the organic templates
involved in bio-aragonite crystallization have to be understood.
The purpose of this study is to identify and characterize the vital effect derived from biocrystallization processes. We performed a detailed study of
a cold-water scleractinian coral species Dendrophyllia ramea skeletons,
analyzing the micro- and nanostructures (using SEM and AFM), the crystallographic arrangement and lattice parameters (using EBSD and XRD),
and the biogeochemical composition (using FTIR of isolated organic matrices, Raman mapping, TG-analysis and a multi-elemental imaging using
LIBS).
The Dendrophyllia skeleton has a hierarchical mesocrystalline organization formed by aragonite fibers in the Thickened Deposits (TD) and
microgranular aggregates of amorphous nature in the Rapid Accretion
Deposits (RAD). We recognize several structural and biogeochemical heterogeneities in the different skeletal parts derived from biocrystallization
processes: a variable amount and composition of organic matrix and water in
septa and coenosteum, which may cause marked distortions in the orthorhombic constant ratios. Also, the edge layers of coenosteum (in
contrast to those formed earlier) are poorly crystallized, and show
enrichments of Mg, Sr and H. Acknowledgments: This work was supported by the National Science Centre
(Poland) grant 2017/25/B/ST10/02221.
P 80
The role of lacuna and canalicular network morphology in
osteocyte mechanosensation A. H. van Heteren*1, Y. Nakajima2, T. Näreoja3 1Ludwig-Maximilians-Universität München, Department Biologie II, München, Germany 2Tokyo City University, Department of Natural Science, Tokyo, Japan 3Karolinska Institutet, Department of Laboratory Science, Huddinge, Sweden
Introduction
There are differences in bone structure between anatomical sites and species
and these are reflected in canalicular networks and the lacunar shape of
osteocytes. For example, bone microanatomy is different in terrestrial and aquatic vertebrates. In bone, osteocytes are intertwined between type-1
collagen lamella and hydroxyapatite crystals that reform countless times as
the bone adapts to loading applied on it. Differently shaped or oriented osteocyte lacunae likely have a different volumetric deformation under a
specific load, which will change the load-induced fluid flow that osteocytes
feel (e.g., round osteocytes are more mechanosensitive than flat ones). The precise influence of the canalicular network has not yet been assessed either.
Objectives
Our primary goal is to establish how lacunar and osteocyte cell shape impact mechanosensation, using long bones of extant vertebrates, as well as
palaeontological specimens for a deep-time perspective. We aim to test the
mechanosensory response in 3D in vitro osteocyte cultures. Variations in osteocyte shape between bone regions with different loading modes raise the
question whether osteocyte shape represents an adaptive response.
Materials & methods
Osteocyte morphology will be modified by 3D bioprinting of polymers into
patterns that control lacunar shape. After we have differentiated the osteocyte cultures in these networks, we apply mechanical compression load
(nanoindentation) and fluid-flow shear stress to measure their ability to
produce a mechanosensory response. Lacunar shape will be digitized using high-resolution X-ray tomography in a Synchrotron. The shape of the
lacunae will be quantified in 3D using geometric morphometrics. The
canalicular network will be analysed in terms of connectivity, canalicular thickness, canalicular spacing, etc. Multivariate statistics will be used to
assess the correlation between osteocytic mechanosensation and morphology
in extant vertebrates. Extreme changes in mechanical loading at the evolutionary transition from land to water will be used to test whether
osteocyte shape is an adaptive response.
Results
We hypothesise that osteocyte morphology is predictive of mechanosensory
ability of the bone. We expect to find that osteocyte markers (e.g., sclerostin,
RANKL, osteoprotegerin, and extracellular vesicles) will vary with lacunar morphology and canalicular network topology in extant vertebrates. We also
expect to find a correlation between the morphological parameters and the
marker variations, as well as differences between secondary aquatic vertebrates and their land inhabiting counterparts.
Conclusion
If our hypotheses prove to be correct, we will have established that osteocyte
shape is an adaptive response to mechanical loading. We also expect to
establish that the osteocyte and the canalicular network change across extreme changes in loading modes, such as during the land to water transition
of secondary aquatic vertebrates.This information could be used to interpret
the loading regimes of fossils.
P 81
First finding of mineralized primary layer in Lingula anatina
(Lamarck, 1801) Brachiopoda, Lingulida A. Madison*1, T. Kuzmina2 1Paleontological Institute, Moscow, Russian Federation 2Lomonosov Moscow State University, Moscow, Russian Federation
The shell secretion of recent brachiopods remains poorly known, especially
if compared with the studied in detail biomineralization processes in recent
mollusks. The lingulids are best known of all brachiopods in microstructural sense as they possess the unique shell composed of alternating
biomineralized and organic layers that remained structurally stable
throughout the Phanerozoic. Three parts are distinguished in the lingulide shell: a periostracum and primary and secondary layers. The mineralized part
of the linguloid shell consists of up to 10 nm in diameter nanogranules of
apatite, specifically the fluorapatite francolite, aggregated into up to several microns in size spheroids or variously long rods. Though the superfamily
Linguloidea appeared in the Early Cambrian, only two genera, Lingula
Bruguière, 1797 and Glottidia Dall, 1870, survived up to recently. Studies on the linguloid primary layer provided quite controversial data on its presence
and structure. Watabe and Pan (1984) described 40–50 µm thick primary
layer composed of aggregates of spherulites for Glottidia pyramidata (Stimpson, 1860). However, Iwata (1981) did not find any mineralized
primary layer in Lingula unguis (L.) (this species was considered as a
synonym of Lingula anatina Lamarck, 1801 by Emig (1982). Williams et al. (1994) reported a 40 µm thick organic primary layer in L. anatina composed
mainly of glycoaminoglycans (GAGs).
We studied ten specimens of L. anatina from Vietnam and the Philippines. The Vietnam specimens were preserved in a 4% formaldehyde solution in
filtered sea water and the Philippine specimens were dried without any
chemical treatment. All specimens were studied with SEM first with periostracum and then after bleaching with collagenase/proteinase mixture.
The shell substance was studied externally and along the fractures. In many
places on the shell outer surface after bleaching preserved a well-ordered sheets of about 0.5 µm high and 0.2 mm thick cylindroids directed
perpendicularly to the valve surface. Such dolioform crystals were previously
unknown for linguliform brachiopods. The sheets look as a thin light film in SEM images in contrast with the underlying compact lamina and cover the
whole shell surface including the protegulum and brephic shell. On the
brephic and adult shells, the cylindroids are neatly packed and their outer and inner ends are obtuse while on the protegulum they are more random and
fusiform and somewhat resemble the acicular primary layer of
rhynchonelliform brachiopods with carbonate shell. The external surface of the sheets of cylindroids bear reflections of the radial striation of
periostracum and thus this is the first shell layer underlying the periostracum,
i.e. the primary layer. As it preserved after the bleaching, it is mineralized. Thus three types of the primary layer are known for recent linguloids: 40–50
µm thick primary layer composed of aggregates of spherulites in G.
55
pyramidata (Watabe and Pan 1984), about 40 µm thick primary layer composed of GAGs in L. anatina from Japan (Williams et al. 1994) and about
1 µm thick layer of cylindroids in L. anatina from Vietnam and the
Philippines. One of the possible explanations for strongly differing primary layers in one species is that in fact they are different taxa but additional data
on the molecular phylogenetics of these brachiopods are needed in order to
resolves this problem.
P 82
Guided mineral growth on amelogenin scaffolds promoted by
amelotin B. Ganss1, A. Danesi1, A. Mansouri1, A. Phen1, J. Holcroft*1, J. Bonde1, K. Carneiro1 1University of Toronto, Faculty of Dentistry, Toronto, Canada
Introduction
Enamel, the outermost layer of the tooth, is composed of intertwined
hydroxyapatite crystals; it is the hardest, most highly mineralized tissue in the body and incapable of regeneration. Amelogenesis is the matrix-guided
process of enamel formation, but its detailed mechanism is not well
elucidated. The most abundant protein present in the developing enamel matrix is amelogenin (AMEL). AMEL acts as a scaffolding protein that
templates hydroxyapatite (HA) crystal organization. In vitro, recombinant
AMEL self-assembles into nanoribbons in the presence of calcium and phosphate ions. Amelotin (AMTN), a recently discovered enamel matrix
protein essential for proper enamel mineralization and has been shown to
promote mineral formation in vitro and in vivo. The functional relationship between AMTN and AMEL as the major enamel matrix protein has yet to be
studied. We hypothesize that AMTN can promote mineralization on AMEL
structures.
Objectives
This study aims to 1) determine the self-assembly behavior of AMTN and 2)
determine the effect of self-assembled AMTN and AMEL, alone and in combination, on calcium-phosphate mineral formation and growth.
Materials & Methods
Human recombinant AMEL and AMTN proteins were expressed in Escherichia coli. Proteins were self-assembled in a calcium phosphate
solution for up to 28 days before being co-incubated for 20 minutes. Self-
assembled AMEL and AMTN, as well as AMEL-AMTN were characterized by Atomic Force Microscopy (AFM) and Transmission Electron Microscopy
(TEM).
Results
AMTN self-assembled into ribbon-like nanostructures over a period of up to
3 weeks. . Mineral deposits formed on top of these ribbons 14 days after assembly. Co-incubation of self-assembled AMTN with AMEL resulted in
the formation of needle-like crystals along AMEL nanoribbons. No
mineralization was observed on AMEL alone.
Conclusion
The co-incubation of self-assembled AMTN to self-assembled AMEL
nanoribbons promotes the formation of guided mineral growth along the AMEL template. This combined activity provides new insights on
mechanistic details of enamel biomineralization. Understanding this
combined effect will open opportunities for developing biological and/or synthetic regeneration strategies for dental enamel.
P 83
Calcium phosphate granules in hypodermal cells participate in
the mineralization of crustacean mandible incisors A. Ziegler*1, B. Nutz1, U. Rupp1 1University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany
The incisive regions of the mandibles, the pars incisivae (PI), of the terrestrial
crustacean Porcellio scaber can be subdivided into three regions, according to differences in mineral content. The distal tip of the PI is not mineralized,
the middle region is mineralized with amorphous calcium phosphate (ACP)
and the base, that connects the incisive region with the corpus of the mandible, with calcium carbonate (Huber et al., 2014). The presence of both,
ACP in the middle region and CaCO3 in the adjacent base and corpus is an
ideal situation to study mineral phase specific differences in epithelial calcium transport pathways.
Which intracellular compartments are involved in calcium transport is
currently the most important question to understand the contribution of cells in biological mineralization processes. Knowing the pathway of how
intracellular calcium is directed through the cell is indispensable for
understanding, which molecular transport mechanisms are possibly involved in biomineralization. How mineralization with calcium phosphate instead of
calcium carbonate affects the structural differentiation of the mandible
epithelial cells and transport of mineral through it, is completely unknown. Therefore, we investigated the hypodermis cells of the PI with the aim to
describe calcium compartmentation and cell structure differentiations for
epithelial calcium phosphate transport in comparison to those in CaCO3 transport.
We used TEM and STEM to investigate the ultrastructure of epithelial cells,
and X-ray spectral mapping to detect mineral containing organelles within cells. For elemental mapping, we used high pressure frozen material, freeze-
substituted in acetone containing uranyl acetate and glutaraldehyde. Thin
sections of resin embedded material were floated on propane-1,2 diol instead of water to minimize loss of diffusive elements within the sample.
The results show that the hypodermis of the PI consists of two different cell
types. The P-cells and the C-cells that secrete and mineralize the calcium phosphate and calcium carbonate containing part of the PI cuticle,
respectively. The P-cells have up to 400 µm long extensions that originate
from the cell somata that are situated in the base region and corpus and extending to the forming cuticle of the middle region. The extensions, of P-
cells contain numerous clathrin-coated vesicles and non-coated vesicle along
microtubules. Near the cuticle, the extensions contain calcium phosphate granules up to 1µm in diameter, likely of endosomal origin, that are filled
with calcium phosphate. The element composition of these granules
resembles that of the ACP in the PI cuticle. The C-cells have no large extensions and contain no mineral granules. They resemble the hypodermis
of the sternal integument for which the cellular mechanism for epithelial
calcium transport are well studied. The results suggest that within the PI the cellular mechanism for
mineralization of the cuticle follows two different pathways. In P-cells
mineral formation takes place within organelles of the endosomal pathway that contribute to cuticle mineralization, while in C-cells mineral is
transported across the epithelial cells by ion channels and carriers, and intracellular compartmentation of calcium ions within the endoplasmic
reticulum.
Huber J, Fabritius HO, Griesshaber E, Ziegler A (2014) Journal of Structural Biology 188, 1-15
Supported by DFG ZI 368/11-1.
P 84
Physical and anatomical variation of mammalian bone
bioapatite structure and composition B. Foley*1, M. Greiner1, G. McGlynn2, W. W. Schmahl1 1Ludwig-Maximilians-Universität München, Department of Earth- and Environmental Science, München, Germany 2Staatssammlung für Anthropologie und Paläoanatomie München, München, Germany
Bone is a hierarchical composite material primarily consisting of an
inorganic calcium phosphate phase—a widely substituted variant of apatite commonly referred to as bioapatite—dispersed in an organic collagen matrix.
Structural variation of the bioapatite lattice occurs with substitution of PO43-
and OH- by carbonate ions, which results in changes of lattice parameters (Handschin et al. 1995). Changes in structure and chemical composition of
bioapatite have been linked to aging and nutrition (Boskey et al. 2010).
Lattice parameters, crystallinity and ionic substitution of bioapatite also vary with anatomical location, pathological conditions, and physical stress in vivo
and with the environment during fossilization and cremation post mortem
(Greiner et al. 2018). As such, detailed investigation of bone bioapatite structure and chemical composition is essential to uncovering the stories of
forensic and archaeological bone samples and understanding bone resorption
processes in contexts such as disease pathology and tissue engineering. Human bone samples were analyzed by X-ray powder diffraction and
Fourier-transform infrared spectroscopy (FTIR) to investigate bone
bioapatite structure and composition. Specifically, Rietveld refinement of X-ray diffraction data was used to determine lattice parameters and crystallite
size. Samples of the femur, clavicle, sternum, talus, calcaneus, and parietal
bone were analyzed from the skeletal remains of three middle-aged human subjects: an average-build male, a robust-build male, and a petite-build
female. Lattice parameters and crystallite size were shown to vary with
respect to anatomical position, gender, and build. Bone samples from the robust-build male showed consistently larger a (=b) lattice parameters than
did samples from the average-build male and petite-build female for all
anatomical positions. FTIR spectra of analyzed samples showed consistent ratios of organic to inorganic content.
Preliminary results show consistent proportion of organic content but
structural variation with respect to physical build.
Boskey, A., & Coleman, R. (2010). Aging and Bone. Journal of Dental
Research,89(12), 1333-1348. doi:10.1177/0022034510377791 Greiner, M., Kocsis, B., Heinig, M. F., Mayer, K., Toncala, A., Grupe, G., &
Schmahl, W. W. (2018). Experimental Cremation of Bone: Crystallite Size
and Lattice Parameter Evolution. Biomineralization,21-29. doi:10.1007/978-981-13-1002-7_3
Handschin, R., & Stern, W. (1995). X-ray diffraction studies on the lattice
perfection of human bone apatite (Crista Iliaca). Bone,16(4). doi:10.1016/s8756-3282(95)80385-8
56
P 85
Infrared nanoscopy of biomaterials A. Cernescu*1, S. Amarie1, A. Huber1, F. Keilmann2 1neaspec GmbH, Haar, Munich, Germany 2Ludwig-Maximilians-University, Munich, Germany
Introduction
Scattering-type scanning near-field optical microscopy (s-SNOM) has
become a key technology to study the chemical composition of inorganic and
organic materials at the nanoscale.
Objectives
The investigated samples are nanocomposite biomaterials, namely human
bone sections [1], human tooth specimens [2] and mollusk shell which contain mineral nanocrystals in organic matrices [3]. The mineral parts are
highlighted by their resonantly enhanced contrast due to phonons.
Materials & methods
Our method is surface-sensitive, probing to a depth of about 20 nm.
Spectroscopic near-field imaging is enabled by combining 20nm-resolving
tip-scattering near-field microscopy (s-SNOM) with an infrared continuum source. Specific contrasting of biomineral components is enabled by simply
choosing the appropriate "fingerprint" infrared region that as in traditional
FTIR (Fourier-transform infrared spectroscopy) identifies virtually any chemical compound. Hence nano-FTIR stands for the successful realization
of combining s-SNOM and FTIR [3].
Results
Mineral distribution across dentine/enamel interface is resolved with 20nm
spatial resolution showing a gradient in the hydroxyapatite concentration.
Relatively large Ca2PO4 crystals (size >100 nm) are observed in both dentin and enamel regions, as well on the human bone samples (Fig. 1).
Fig. 1: Minerals in human bone. High resolution IR nanoscopy image
obtained simultaneously with the AFM topography shows the nanoscale distribution of Calcium phosphate and collagen within the cross section of a
human bone lamella extracted from a hip joint. Imaging at different
wavelengths maps different minerals within the tissue, which reveals important information about the bone structural integrity and could provide
details about the process of demineralization due to diseases, such as
osteoporosis. Interestingly, phosphate nano-crystals are also found in mollusk shell
specimens, close to the interface between calcite and aragonite biominerals.
Conclusion
The benefits of using the s-SNOM technology are the nanoscale resolution
combined with the chemical spectroscopic identification of molecules.
Straightforwardly applicable in many fields of biomaterials, general mineralogy and solid state research, this technology is becoming one of the
most powerful tools in nanoscale analytics.
References
[1] T. Geith et. al, Visualisation of methacrylate-embedded human bone
sections by infrared nanoscopy, J. of Biophotonics (2012);
[2] T. Sui et. al, Structure-Function Correlative Microscopy of Peritubular and Intertubular Dentine, Materials (2018);
[3] S. Amarie et al, Nano-FTIR chemical mapping of minerals in biological materials, Beilstein J. Nanotechnol. (2012).
P 86
Discovery of long-chain polyamines and their biosynthetic
enzyme in the biosilicifying bacterium Bacillus cereus T. Ikeda*1, K. Yamamoto1, R. Hirota1, A. Kuroda1
1Hiroshima University, Hiroshima, Japan
Introduction
Although silica biomineralization (biosilicification) has been intensively
investigated in several eukaryotes, such as diatoms, sponges, and higher
plants, prokaryotic biosilicification was not studied until recently. In 2010, we reported that biosilicification occurs in the gram-positive, spore-forming
bacteria, Bacillus cereus and its close relatives, and that silica is deposited in and around a spore coat layer as a protective coating against acids (Hirota et
al. 2010 J. Bacteriol. 192, 111). In a recent study, we demonstrated that the
spore coat protein, CotB1, which carries a characteristic C-terminal zwitterionic sequence, plays an essential role in biosilicification; however,
the underlying mechanism was not elucidated (Motomura et al. 2016 J.
Bacteriol. 198, 276).
Objectives
This study aimed to further investigate bacterial biosilicification
mechanisms. Since most eukaryotic silica-precipitating peptides and proteins were discovered by dissolving biosilica in hydrofluoric acid (HF) or
ammonium fluoride (NH4F), we hypothesized that the organic compounds
embedded in the B. cereus biosilica also play an important role in silica formation.
Materials & methods
A suspension of B. cereus spores was mixed with nitric and sulfuric acid and then boiled to degrade organic matter. Insoluble biosilica were harvested by
ultracentrifugation and repeatedly washed with distilled water until the pH of
the supernatant became neutral. The biosilica pellet was suspended in 6 M NH4F solution to dissolve the silica. After incubating at room temperature
for 30 min, the solution was repeatedly dialyzed against 200 mM ammonium
acetate to remove NH4F. The solution was then freeze-dried to remove the solvents and the remaining substances were dissolved in a small amount of
distilled water. Finally, the extract was analyzed by SDS-PAGE and mass
spectrometry.
Results
The extract from the biosilica showed a low-molecular-weight band on SDS-
PAGE gels. Mass spectrometric analysis revealed that this band contained long-chain polyamines (LCPAs) with long repeats of the -CH2CH2CH2NH-
unit. Notably, LCPAs were also identified in silica-accumulating eukaryotes
such as diatoms and siliceous sponges, although their chemical structures are slightly different from those of B. cereus LCPAs. These findings strongly
suggested that LCPAs play a common and important role in silica formation
in these organisms. Moreover, the chemical structure of B. cereus LCPAs strongly suggested that they were synthesized via repetitive aminopropyl-
transfer reactions. We also identified a plausible candidate for LCPA
synthase via homology searches using bacterial aminoproplytransferases as query sequences. Furthermore, gene disruption experiments showed that the
candidate gene is indeed essential for LCPA synthesis. Unexpectedly, the
disruptant lacking LCPAs formed biosilica around the spore coat, similar to the wild type, when cultivated in the presence of silicic acid, thereby
suggesting that LCPAs are not essential for biosilicification in B. cereus.
Conclusion
In this study, we demonstrated the presence of LCPAs in the B. cereus
biosilica and found a putative gene encoding LCPA synthase. Despite the common presence of LCPAs in different evolutionary lineages of silica-
accumulating organisms, gene disruption analysis unexpectedly showed that
LCPAs are not essential for biosilicification in this bacterium.
P 87
Crystallization of calcium oxalates inherent in mineralized
biofilms A. Rusakov*1, M. Kuz’mina1, O. Frank-Kamenetskaya1 1Saint Petersburg State University, Crystallography, Saint Petersburg, Russian Federation
Biofilms containing colonies of microscopic fungi (micromicetes) can often be found on the surface of carbonate rocks (marble, limestone etc.) in urban
environment. Micromycetes excrete substantial amounts of organic acids (oxalic, citric, malic, succinic, fumaric, lactic etc.) in the course of their
metabolism. These acids solubilize carbonate rocks and induce calcium
oxalate (calcium oxalate monohydrate - whewellite, calcium oxalate dihydrate - weddellite) crystallization. These minerals as well as micromicete
metabolites and several other chemical additives which come from the
environment form the so called oxalate patina. The aim of this work was to study the influence of different organic and inorganic biofilm components on
phase composition and the morphology of the formed calcium oxalates in
vitro. Calcium oxalate crystallization was performed by decantation of a mixture
of sodium oxalate and calcium chloride aqueous solutions with pH variation
(4.0-7.0) at room temperature (22-25С). Na+, K+, Mg2+, Fe3+, PO43-,SO42-, CO32- ions were added to the solution as the impurity components
as well as the organic acids, excreted by micromicetes. The compound
concentrations were close to the ones observed in the biofilms. It was found that whewellite crystallizes in the presence of all inorganic and
organic components. Weddellite formed in the presence of citric ions of
varying concentrations with no dependance on the presence of inorganic components, as well as in the presence of three acids (malic, succinic and
fumaric) with an equal ratio of their concentrations in the absence of
inorganic components. Weddellite also solely formed when Ca2+ ions to the oxalate ions concentration was set close to stoichiometry (1.5:1) at pH≥6,
and at an increased ratio of more than 7.5:1 at pH≥5. The addition of iron
cations to the system significantly increases the amount of weddellite in the sediment, while the admixture of carbonate ions or phosphate ions, on the
contrary, decreases the amount of weddellite in the sediment. The presence
of phosphate and carbonate ions in the crystallization medium can lead, in appropriate conditions, to the formation of very small amounts of additional
mineral phases, such as brushite and calcite, for example.
Whewellite crystallized in the following forms: small elongated lamellar crystals with side faces not very pronounced (in syntheses without any
additional impurities); large aggregates consisting of oval petals (in systems
with additional inorganic impurities); small spherical spherulites consist of small curved plates (in systems citrate ions); small spherical spherulites (in
systems with citrate ions and inorganic impurities); elongated cylindrical or
"kidney-shaped" aggregates which consist of thin plates (with the addition of additional organic acids).
Weddellite crystals had tetragonal dipyramid facetes (in systems with citrate
ions and inorganic impurities), and with the increase of Ca2+ concentration (while C2O42- concentration was set to 3 mmol/l) a transition from
dipyramidal to skeletal crystals was observed. In systems with Fe3+, Mg2+,
57
PO43- ions weddellite precipitated as real crystals with small tetrahonal dipyramid in the center of a large tetrahonal plate with an uneven edge. The
formation of prism facets of weddellite crystals was observed only in the
presence of citrate ions and MgSO4 in the solution. The results contribute to the research of the oxalate patina formation and give
evidence on the specific patina components which influence the mineral
composition and the crystal morphology. The obtained results allow to recreate the original pattern of biofilm
mineralization based on the morphology of calcium oxalate crystals found in
biofilms. Acknowledgements: This work was supported by the Russian Science
Foundation (no. 19-17-00141). The laboratory researches were carried out in
the Research Resource Centers of Saint Petersburg State University: SEM investigations - in the "Resource Center Microscopy and Microanalysis
(RСMM)" and "Interdisciplinary Resource Center for Nanotechnology"
"XRD measurements – in the «X-ray Diffraction Centre".
P 88
Modifying the marble surfaceinfluenced bybacteria Bacillus
spp. and bacterial-fungal associations K. Sazanova*1, O. Frank-Kamenetskaya2, D. Vlasov1,2, A. Vlasov2, M. Zelenskaya2, A. Rusakov2 1Komarov Botanical research institute RAS, laboratory of analytical
phytochemistry, Saint-Patersburg, Russian Federation 2Saint-Petersburg State University, Saint-Petersburg, Russian Federation
Metabolism products secreted by the microbial lithobiont community are a powerful factor in modern mineral formation. Most of the time rock surfaces
in natural systems are inhabited by the multi-species communities (mainly
fungi and bacteria). The biochemical activity of microorganisms in communities differs significantly from monocultures. The patterns of
microbial crystallizationunder influence of several species of
microorganisms are practically unexplored. The aim of this study was to receive a mophogenetic patterns of calcium
carbonates and oxalates crystallization on marble surface induced by
metabolism of bacteria and bacterial-fungal associationsin experimental conditions
Two series of experiments were performed with microorganisms: 1-
monoculture of bacteria Bacillus spp., 2 - co-culture of Bacillus subtilis and fungus Aspergillus niger.
Cultivation of microorganisms was carried out in a liquid medium and in wet chamber.Czapek-Dox medium with different glucose concentration (1, 5, 10,
30 g/l) was used as a nutrient medium. In the experiments in liquid medium
marble blocks were put on the bottom of Petri dishes and 15 ml liquid Czapek-Dox medium were added. The cultivation time: 14, 21, 30, 60
days. In experiments in wet chamber microorganism cell suspension was
applied on the marble block surface.The cultivation period for different marble blocks was 21 and 90 days.
Scanning electron microscopy (SEM) and energy-dispersive X-ray
spectroscopy (EDXS) were used for the identification of phase composition of crystallization products and examination of crystal morphology. The
determination of the metabolite composition was carried out in a liquid
culture of microorganisms using gas chromatography-mass spectrometry (GC-MS) by use of an Agilent mass spectrometer (MSD5975 mass selective
detector), column HP-5MS, 30 m 9 0.25 mm. The analysis of the EPS content
in the liquid monoculture of Bacillus subtilis and in co-culture of Bacillus subtilis and Aspergillus niger was performed by precipitation in cold ethanol,
centrifugation and weighing.
In a liquid medium with slightly alkaline pH values, the release of EPS by bacteria leads to crystallization of calcite, the intensity of which increases
with increasing sugar content in the crystallization medium. In oligotrophic
conditions of a moist chamber the acidifying activity of Bacillus subtilis prevales which leads to crystallization of calcium oxalate dehydrate
(weddellite). The metabolic activity of B. subtilis and A. niger association is
vastly different from the activity of the separate monocultures. The metabolic activity of micromycetes can suppress the formation of bacterial EPS and
prevent the formation of calcite crust. In a liquid medium, as the sugar
content increases, the acidifying activity of the fungus increases which leads to a shift of pH of the medium to the acidic region, carbonate crystallization
attenuation, and oxalate crystallization activation - the formation of calcium
oxalate monohydrate and dihydrate (whewellite and weddellite). The phase composition of microbial crystallization products is vastly
different in association of microorganisms from the activity of monocultures.
In the case of Bacillus subtilis and co-culture of Bacillus subtilis-Aspergillus niger it determined by the ratio of the concentrations of EPS and oxalic acid
in the crystallization medium.
This work was supported by the Russian Science Foundation (project No. 19-17-00141)
P 89
Cyanobacteria-related carbonate sedimentation in modern
rivers, Leningrad region, Russia O. Rodina*1, O. Vereshchagin2, D. Vlasov1, M. Nikitin3, M. Zelenskaya1 1Saint Petersburg State University, Biological, Saint Petersburg, Russian
Federation 2Saint Petersburg State University, Institute of Earth Sciences, Saint
Petersburg, Russian Federation 3The Bonch-Bruevich Saint-Petersburg State University of Telecommunications, Institute of Military Education, Saint Petersburg, Russian Federation
Introduction
Today, cyanobacteria (CB) continue to play a central role in the carbon and
nitrogen cycle. They are considered to be the pioneers of the colonization of mineral substrata. CB are not only biodegradation agents, but also they take
important place in biomineralization of carbonate in fresh water. CB are
common habitants of modern rivers riched in carbonates of the south of the Leningrad region, Russia. However, cyanobacteria-carbonates relation is still
in focus of scientific debates.
Objectives
Minerals composition of carbonate sediments from modern rivers of the
south of the Leningrad region was studied. The CB diversity was described.
An experiment of the community growing in cumulative culture for studying cyanobacteria-carbonates relation was performed.
Materials & methods
Thirty one samples of layers (biofilms) from the stone surfaces were selected in Leningrad region. Samples were dried for 3 month. X-ray diffraction
patterns (XRD) were recorded on a Rigaku Miniflex II diffractometer. The
chemical composition and micromorphology was studied by means of an Hitachi S-3400 N scanning electron microscope equipped with an Oxford
Instruments AzTec Energy X-Max 20. For the identification of CB, direct
microscopy was carried out after their settling in distilled water for a month. The cumulative culture of the microorganism community was grown in
distilled water for a year. Identification of species was carried out using light
microscopy (Leica DM 1000).
Results
The mineral composition of carbonate deposits is represented by calcite and
aragonite. The appearance of aragonite in the sediment is associated with an increased content of magnesium in calcite (MgO> 1 wt.%). CB recovered
and started to grow in solution only from samples, which contain aragonite. Twenty-nine taxa were identified in these samples. CB taxa are belonging to
5 orders, 10 families, 17 genera. The most widely distributed species were
from the genus Phormidium. Only Calothrix (C.) sp. from one sample has mucous cover with mineral crystals on it. This sample was used for further
community growing in cumulative culture. The sample of the community
with C. sp. started to be green after one week settling in distilled water and after that, it grew at room temperature in a 100 ml glass beaker under a glass
cover (ventilated) for a year. After a year of cultivation, the C. sp. began to
dominate in the community, and it was almost completely covered with mineral particles. Result of XRD showed that Mg-calcite is main phase of
this cover. However, no aragonite was found.
Conclusion
The communities of CB associated with the deposition of carbonates in the
modern rivers of Leningrad region was described. The fact of the association
of mineral particles with mucous covers of C. sp. is confirmed by means of optical and SEM microscopy, as well as by XRD. The natural samples of
carbonate sediments with CB contain calcite and aragonite. However, only
the mineral case of cyanobacteria grown in distilled water is composed of calcite. It is necessary to conduct further experiments for establishing the
relationship between cyanobacteria and carbonates. The authors thank the
Resource Center for X-ray diffraction studies, Geomodel Resource Centre, Resource Center for Microscopy and Microanalysis (RСMM) of Saint
Petersburg State University for providing instrumental and computational
resources. This work was supported by the Russian Science Foundation (project No. 19-17-00141).
P 90
Biodegradation of 2 - methoxyethanol by a new bacterium
isolate Pseudomonas sp. strain VB under aerobic conditions F. O. ekhaise*1 1University of Benin, Microbiology, Benin, Nigeria
Microbial biodegradation of 2-methoxyethanol also known as Methyl glycol
(MG) under anaerobic conditions has received much attention during the past
decade. However, not much is known about the aerobic degradation of 2-methoxyethanol. Samples from various environmental niches were enriched
to isolate and determine bacterial isolates capable of utilizing 2-
methoxyethanol as a sole source of carbon and energy under aerobic conditions. A 2-methoxyethanol degrading bacterium was isolated from
anaerobic sludge of a municipal sewage from a treatment plant in Bayreuth,
58
Germany by selective enrichment techniques. The isolate was designated strain VB after it was shown by the 16S rRNA phylogenetic sequence
analysis as belonging to the genus Pseudomonas. Under aerobic conditions
Pseudomonas sp. strain VB was capable of mineralizing 2-methoxyethanol and its intermediary metabolites. Stoichiometrically, the strain utilized one
mole of oxygen per one mole of 2-methoxyethanol instead of four mole
oxygen per one mole of 2-methoxythanol for the total oxidative metabolism
P 91
Formation of humboldtine-group oxalates Me2+(C2O4) 2H2O
(Me = Fe, Mn, Mg) under the influence of fungus Aspergillus
niger M. Zelenskaya*1, O. Frank-Kamenetskaya2, A. Izatulina2, A. Rusakov2, D.
Vlasov1 1St. Petersburg State University, Department of Botany, Saint Petersburg,
Russian Federation 2St. Petersburg State University, Crystallography , Saint Petersburg, Russian Federation
The interaction between the products of lichen metabolism and the rocks underlying the lichens leads to processes of modern mineral formation. The
present work describes the results of the laboratory experiments on the
formation of humboldtine-group oxalates under the influence of the fungus Aspergillus niger on the surface of todorokite (Ca,Sr)0.3-
0.5(Mn4+,Mn3+,Mg)6O12•3-4.5H2O, kutnohorite Ca (Mn,Mg,Fe)(CO3)2,
siderite (Fe,Mg,Mn)CO3, as well as ankerite Ca(Fe,Mg)CO3. The experiments were held at room temperature in liquid Czapek-Dox
medium. The synthetic products were studied under scanning electron microscope, as well as by EDX analysis and X-Ray powder diffraction
methods.
Magnesium oxalate dihydrate (glushinskite) formed under the influence of Aspergillus niger in the initial stages of the experiment with todorokite and
kutnohorite. The sequence of transformations of todorokite and kutnohorite
by fungus Aspergillus niger, leading to the formation of insoluble Mn2+ oxalates of different water content (mycogenic analogues of the minerals
falottaite and lindbergite), is different and depends on the valence of
manganese ions in the underlying mineral substrate. Due to the dissolution of todorokite by organic acids excreted by fungi, manganese ions (mostly
Mn3+ and Mn4+) get into the crystallization medium, where are then reduced
to Mn2+ along with a gradual pH increase, which all leads to oxalate crystallization. Kutnohorite on the other hand initially contains only Mn2+
manganese ions. The formation of manganese oxalate here is preceded by the
oxidation of Mn2+ ions to Mn3+,4+ during the initial stage of the experiment and the following crystallization of micogenic Mn-oxide (a todorokite
analog). Under the action of fungi micogenic todorokite immediately begins
to dissolve, and only after the reduction of manganese back to Mn2+ the oxalate crystallization begins. During todorokite transformation, manganese
oxalate trihydrate (falottaite) appears in the medium first followed by
manganese oxalate dihydrate (lindbergite), which we can explain by falottaite dehydration which feeds lindbergite crystallization. In the case of
the mycogenic transformation of kutnohorite, falottaite was not formed and
the crystallization of lindbergite occurs without the participation of falottaite. We can assume that the formation of manganese oxalates of different water
content is regulated by chemical compounds of their crystallization media,
compositions of which are substantially different for todorokite and kutnohorite.
Iron oxalate dihydrate (humboldtine) was first obtained in the experiments
with fungus Aspergillus niger on the surface of iron carbonates: siderite and ankerite. It formed plate-like crystals, close in habit to glushinskite: on
siderite surface it had an admixture of magnesium and manganese, on
ankerite surface - an admixture of magnesium. Acknowledgements: This work was supported by the Russian Science
Foundation (no. 19-17-00141). The laboratory researches were carried out in
the Research Resource Centers of Saint Petersburg State University: SEM investigations - in the "Resource Center Microscopy and Microanalysis
(RСMM)" and XRD measurements – in the «X-ray Diffraction Centre».
P 92
Improved CaCO3 biomineralization under extreme alkalinity
conditions of two native microbial consortia from extreme
Chilean ecosystem S. Marín*1, V. Zetola2, S. Olivares1, C. Demergasso*1 1Universidad Católica del Norte, Biotechnology Center, Antofagasta, Chile 2Universidad Católica del Norte, Facultad de Ciencias de Ingeniería y Construcción, Antofagasta, Chile
Introduction Biologically induced, controlled and influenced mineralization are three
avowed microbe-mineral interactions with noted implication in ecology, astrobiology, biogeochemistry and paleontology sciences. In the last decade,
the benefits of microbe-mineral interactions for the biocementation and
building materials industry are under extensive investigation and gradually being disclosed. One of the known challenges for reaching an effective
application of this kind of biotechnology in the concrete industry has been
the dramatical loss of cell growth and ureolytic activity under the extreme alkaline environment compatible with concrete sustainability. Obtaining
microorganisms naturally adapted to conditions of extreme alkalinity could
facilitate the adaptation process and improve the in situ mineralization performance on the concrete matrix. The aim of this study was to obtain and
adapt novel microbial consortia, native from extreme ambient of Chile, with
improved capacities to produce CaCO3 by biologically induced and controlled mineralization under extreme alkalinity conditions (pH > 12) and
useful for in situ concrete repairing and improving.
Materials and methods Ten microbial consortia with proven ability of ureolytic activity at laboratory
scale were obtained from different extreme alkaline lagoons (pH > 8.5), salt
flats and soils from the Altiplano, Chile, and immediately characterized by massive sequencing of 16S-rRNA gene. Consortia were enriched in both
Tryptic Soy Broth (TSB) and specific ureolytic culture media. Enriched
consortia were characterized again by DGGE. A gradual adaptation to extreme alkalinity conditions from pH 8.0 to 12.5 was performed for seven
months. Urease activity, pH and cell growth were constantly monitored. One
of the best representative species of CaCO3 biomineralizing microbes, Sporosarcina pasteurii (DSM 33), was used as a positive control. Mixes of
the native consortia were performed in an attempt to increase the adaptation
ability. Sand conglomeration and concrete repair ability for the best alkaline adapted and most efficient mineralizer consortium were evaluated. Obtained
CaCO3 mineral was characterized by X-ray Diffraction and Scanning Electron Microscopy.
Results and Conclusions
Natural consortia obtained from extreme alkalinity zones presented an interesting microbial diversity and confirm the microbial adaptation to these
extreme environments. Microbial consortia enriched with TSB medium was
more diverse than those enriched with ureolytic medium. Species that grow in ureolytic specific medium are not necessarily present in TSB cultures or
natural samples meaning that some ureolytic identified species are present in
natural environments but with low abundance. New native strains of Sporosarcina, Bacillus and Lysinibacillus species were obtained from
ureolytic cultures. All ureolytic consortia were active until pH 11.5 but only
two of them were able to actively growing and do hydrolyze urea at pH > 12. Moreover, the mix of these two native microbial consortia achieved a higher
ureolytic and mineralizing performance under all the evaluated conditions
showing an even better adaptive ability respect to each separate consortia and respect to control, S. pasteurii. According to the authors, this is the first work
performing successfully biologically induced CaCO3 mineralization at the
extreme alkaline conditions analyzed, with a great perspective in the concrete repair application.
P 93
Bacterial choreography- designing interactions through
biological induced mineralisation T. H. Arnardottir*1 1Newcastle University, Architecture, Newcastle, United Kingdom
This research explores the potential design role of bacterial-induced
biomineralisation. It sits within the speculations regarding our changing
relationship with nature through engineered biological systems and new material processes. In the convergence of design and biological fabrication,
the role of the designer is shifting from assuming the role of the sculptor to
adopting the perspective of the cultivator in the study and production of new living material assemblages. In this intersection, we are moving away from
the mass production of fast, cheap and repetitive elements by enabling the
production of biologically made materials. Much like properties of natural materials such as bones, which are not designed but instead and are shaped
by a set of natural constraints, biomineralisation has opened up the possibility
of utilising nature"s intelligence for the built environment. Such process has precise applications in the fabrication of materials as there has been extensive
research on utilising bacteria to induce biological cementation through
engineering solutions, such as crack repair in concrete, soils" improvement or forming energy-efficient bricks. By harnessing the biomineralisation
process, bacterial-induced fabrication can be used in the creation of new
materials with functionally graded and variable properties. This research aims to explore material- and fabrication methods that
incorporate living cells as an inherent part of the process and to outline
parameters that facilitate the synthesis of this biomaterial. It focuses on the assemblages of biologically fabricated matter through the interaction of
bacterial agents in a complex system, by challenging the designer"s thinking
from the application of an ideal form on an inert matter, to the shaping of a bottom-up emerging form. The goal is to set out material and fabrication
processes to enable designers to engage with these living systems as
resources. This research concentrates on structuring a biofabrication process whose
purpose is to partly control the physical geometry of a microbial induced
59
mineralised structure. The experimental approach entailed an exploration of established procedures and the testing of novel physical apparatuses that
were part bioreactors and part moulding vessels. These enabled the
biomaterial synthesis through the alteration and control of chemical, spatial and structural compositions of the environment. In this setting, the bacteria
Sporosarcina pasteurii was mixed in with sand in nutrient solution and
grown overnight. This allowed the culture to situate itself within the grain before being flushed at intervals with the cementation media of nutrient
broth, urea and CaCI2 over a few days.
The change in compositions led to a framework to reach biomineralisation at given points and, by optimising the biofabrication process, obtaining fully
cemented pieces through the hybrid moulding/bioreactor technique.
Based on these findings, this design framework can enable designers to visualise and generate interruptions and interactions with the mineralised
material. Designers can think of this process as an interactive, alive network
that can be used to choreograph parameters to produce form alongside microbes. By utilising this induced material, the framework can demonstrate
different ways the design paradigm can potentially shape living matter and
inherently evolve our relationship with biological design in new ways.
P 94
The directing effect of bacterial EPS on calcite organization
and EPS-carbonate composite aggregate formation X. Yin*1, F. Weitzel1, E. Griesshaber1, C. Jiménez-López2, L. Fernández-Díaz3, A. Rodríguez-Navarro4, A. Ziegler5, W. W. Schmahl1 1LMU Munich, Department of Earth and Environmental Sciences, Munich,
Germany 2Universidad de Granada, Departamento de Microbiología, Granada,
Spain 3Universidad Complutense de Madrid, Departamento de Mineralogía y Petrología, Madrid, Spain 4Universidad de Granada, Departamento de Mineralogía y Petrología,
Granada, Spain 5Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm, Germany
Mineralized structures generated under biological control are hierarchical
composites that consist of two distinct materials: a compliant biopolymer
matrix that is reinforced by stiff and hard minerals. The biopolymer matrix in the biological structural material is developed as a membrane or as a
network of fibrils and has structural as well as functional roles for the architecture and material properties of the composite hard tissue.
Microbial cells surround themselves with a fibrous biopolymer matrix (EPS)
for protection, orientation of cells and enhancement of physiological activities. For understanding the influence of biopolymer matrices on mineral
organization and composite material formation we conducted growth
experiments with the extracellular polymeric substance (EPS) of the gram negative bacteria Pseudomonas putida. We synthesized EPS-agarose
hydrogel-calcite composite aggregates and characterized aggregate
morphologies, EPS/hydrogel distribution, mineral organization and co-orientation strength.
We find that P. putida EPS exerts a tremendous influence on aggregate
morphology, pattern of polymer distribution and mode of mineral organization. Contrasting to reference aggregates that are devoid of bacterial
EPS, in aggregates that occlude EPS the pattern of polymer distribution is
highly inhomogenous and is developed mainly as membranes. Accordingly, subunit formation in these is extensive. Subunits are irregular in shape, size
and distribution and are highly misoriented to each other. Aggregates that
contain P. putida EPS are radial mosaic polycrystals, while the reference aggregates, devoid of EPS, are branched dendrites with the branches being
highly stepped and the calcite well co-oriented.
Incorporation of P. putida EPS into calcite changes the microstructure and texture of the mineral in a specific manner. This is a characteristic that can
be developed and used as a tool for the recognition and identification of
bacterially mediated calcification.
P 95
Development of CaCO3 materials through bacteria embedded
hydrogels R. Boons*1, G. Freitas1, G. Nyström1, A. Studart2, T. Zimmermann1 1EMPA, Dübendorf, Switzerland 2ETH Zürich, Zürich, Switzerland
Numerous types of mineral-based structures are used nowadays in a variety
of applications, ranging from construction to medicine. One possible way to
create a mineral formed structure is through a biomineralization process, which is utilised in this research to create mineral-based products.
Biomineralisation is a widespread phenomenon in several phylogenetic
groups including bacteria that are able to control, influence and induce mineralisation. Examples of the latter are certain Bacillus strains which are
known to induce calcium carbonate formation. This process is called
microbiologically induced calcium carbonate precipitation (MICP). Here the carbonate formed in presence of the bacteria will react with calcium in the
surrounding, resulting in precipitation of CaCO3. In order to obtain a specific
CaCO3–based structure using these strains, the bacteria have to be placed in a shape of interest. In this study, Sporosarcina pasteurii bacteria, formerly
known as Bacillus pasteurii, are embedded in bio-based polymer hydrogel
matrices (e.g. sodium alginate) together with the appropriate nutrients. The hydrogel should possess certain properties, such as biocompatibility,
porosity and high water content, optimised for the survival of the bacteria
and production of mineral crystals. The bacteria have to be provided with molecules specific to the bacterial strain, as well as supplementary products,
such as calcium sources, that are necessary for the mineralisation process.
The mineral formation is distinctively affected by different components added to the matrix and by the residing bacteria. Examining liquid media
cultures, this is observed as variations in S. pasteurii induced CaCO3
polymorphisms upon addition of different calcium sources. Additionally the ratio and transformation from one polymorph to the other are investigated
over time using several analysis methods, e.g. XRD and SEM. Finally, the
bacteria are embedded in the hydrogel and are shown to also induce CaCO3 formation in this condition.
P 96
Mineral deposition by lichens F. Bachmair*1, G. Lehrberger2, A. Beck3, M. Zenkert1, E. Griesshaber1,
W. W. Schmahl1 1Ludwig-Maximilians University Munich, Department of Earth and
Environmental Sciences , Munich, Germany 2Ingineurgeologie TU Munich, Munich, Germany 3Botanical State Collection Munich, Munich, Germany
Litchens deposit a variety of minerals, in particular, calcium oxalates in two hydration states: the monohydrate whewellite and the dehydrate weddelite.
Litchens release oxalic acid that reacts with the substrate mineral. Hence,
litchens etch and weather the surfaces of the rocks on which they grow. Etching rock minerals results in the extracellular formation of new mineral
deposits that most probably have no organic matrix associated with the
crystals. The purpose of our study is to identify and characterize those minerals that
are formed with the activity of litchens. In particular we aim to distinguish
between grains that constitute the substrate and the crystals that are deposited through litchen activity. We investigated Caloplaca falvescens litchens that
colonize limestone of the Jurassic Kehlheimer Kalkstein formation present
in the surroundings of Regensburg, Germany. Mineral characterization was performed with FE-SEM imaging, EBSD, EDX, XRD and FTIR. With the
applied combination of methods we detected both, chemical and
morphological differences between minerals and grains forming the substrate
and the minerals that were secreted by the litchens. While the limestone
substrate is composed of large, irregularly oriented carbonate grains,
substrate portions where the litchens settled are infused with a multiude of minute holes containing organic substance. In the latter regions we find
circular structures consisting of layered calcite, with the layers being assembled of minute calcite crystallites.
In conclusion, the distinctness of grain morphologies and crystallite sizes
coupled with some chemical characteristics enabled the clear distinction between original sedimentary carbonate deposits and carbonate minerals
formed as a mineralization product of litchens when colonizing and etching
the substrate mineral components.
P 97
Examination of the periodicity of incremental lines observed in
the otolith fossils of fishes from Nobori formation, Japan H. Mishima*1, Y. Kondo2, F. Ohe3, Y. Miake4 1Tsurumi University School of Dental Medicine, Department of Dental Engineering, Yokohama, Japan 2Kochi University, Science and Technology Unit, Natural Sciences Cluster, Kochi, Japan 3Nara National Research Institute of Cultural Properties, Nara, Japan 4Tsurumi University School of Dental Medicine, Oral anatomy, Yokohama, Japan
Introduction
The main component of the otolith in fishes is composed of a crystal of
calcium carbonate (CaCO3). As compared with the teeth and bone composed
of apatite crystal, the otolith is a different hard tissue. However, the incremental lines in the tissue of the otolith are formed in the same manner
as the teeth and bones. It has reported that the periodicity of the incremental
lines of otolith has a daily (circadian rhythm), a tidal, a lunar, a seasonal, and an annual periodicity (circannual) (Pannella, 1980). The ages of the fish were
estimated through the annual incremental lines. Little information is
available regarding the ultrastructure, chemical composition and the periodicity of incremental lines of otolith fossils of fish.
60
Objectives
The purpose of the present study is to examine the structure and composition
of incremental lines in the otolith fossils of fishes through the histological
and analytics studies.
Materials and methods
In this research, the otolith fossils of fishes (Nobori formation, Pliocene,
Muroto, Kochi Prefecture, about 3 million years ago) were used. The ten otolith fossils samples were the different fish genera habitats and used the
following samples (different fish genera).
(1) 1) Macrouridae spp. indet., 2) Cetonurus noboriensis (Aoki and Baba), 3) Caelorinchus anatirostris Jordan et Gilbert, 4) Ventrifossa sp. A, 5)
Coryhaenoides cinereus Gilbert. The habitat was continental shelf and slope
at a depth of 200m or deeper. (2) 1) Myctophidae spp. Indet., 2) Lobianchia gemellarii (Cocco), 3) Diaphus
gigas Gilbertt. The main habitats were the diurnal vertical movement.
(3) Sebates scythropus (Jordan and Starks). The habitat was continental shelf to upper slope (water depth 300m).
(4) Congriscus megastomus (Günther). The habitat was continental shelf -
upper slope habitat. Polyester resin-embedded samples and non-embedded samples were used in
this study. One-sided polish specimens and ground specimen were observed
and analyzed using light microscopy, digital microscopy, polarizing
microscopy, scanning electron microscopy(SEM), SEM-EDS analysis,
electron-probe microanalyzer (EPMA), laser Raman microprobe spectrometry, and X-ray diffraction method.
Results
The otolith crystals were aragonite by the X-ray diffraction method and the laser Raman microprobe spectrometry. The inside of the otolith was found to
consist of needle-like crystals crossed by incremental lines. The circadian
incremental lines (2-5 μm), and several long periods of incremental lines (tidal rhythm: about 14 days interval, monthly rhythm: about 28 days
interval, circannual rhythm) were observed in the otolith. By SEM images,
the incremental lines were observed as the dark bands. The incremental lines had content of C, O, and Ca, and additionally contained Si as a trace element.
Both Lobianchia gemellarii and Diaphus gigas were unclear in the circadian
incremental lines. Their habits were diurnal vertical movement. By comparison, Ventrifossa sp., Cetonurus nobonesis, Sebates scythropus, and
Congriscus megastomus were evident in the circadian incremental lines.
Their habitats were the slope from the continental shelf.
Conclusion
It is possible that the periodicity of the incremental lines of otolith changed
with the ecosystems.
P 98
Misorientation driven morphological evolution of the prismatic
ultrastructure in mollusc shells D. Stier*1, D. Zöllner1, I. Zlotnikov1 1TU Dresden, B CUBE - Center for Molecular Bioengineering, Dresden, Germany
Molluscs are a well-established model system to study biomineralization and
the process of biomineral morphogenesis. In addition to our growing
understanding of the different biochemical mechanisms that are responsible for mineral formation, a number of recent studies suggest that the deposition
of the various shell ultrastructures is a thermodynamically driven self-
assembly process. Specifically, it was shown that the formation of the prismatic ultrastructure, consisting of elongated mineral columns glued
together by an interprismatic organic membrane, can be quantified by
classical models of grain growth and coarsening. In this model, morphological evolution of the entire prismatic ultrastructure, which
proceeds parallel to the long axis of the prisms, is driven by the reduction of the amount of interfaces that separate the individual prisms. However, so far,
the energy of those interfaces was considered to be uniform. Therefore, the
influence of the crystallographic misorientation between adjacent prisms on the interface energy and thus, the morphogenesis of the corresponding
prismatic units, was never previously considered. In the current study, we
examine the prismatic ultrastructure in three mollusc species from the genus Pinctada. In these bivalves, the prismatic assemblies not only demonstrate a
coarsening behavior, which is predicted by the recently developed self-
assembly models, but also exhibit gradual changes in the crystallographic orientation of the individual prisms. Using electron backscatter diffraction
analysis (EBSD) and synchrotron-based X-Ray tomography to follow the
crystallographic properties and the shape of the prisms at different stages of growth, respectively, we show a correlation between the textural and
morphological evolution of the prismatic ultrastructures. In fact, we
demonstrate the key role of misorientation between neighboring mineral building blocks in the growth kinetics of biocomposite architectures.
P 99
The evolution of Theicideid brachiopods shell microstructure M. Simonet Roda*1, S. Milner2, E. Griesshaber1, H. Jurikova3, C. Rollion-
Bard2, L. Angiolini4, F. Ye4, A. Ziegler5, A. Bitner6, D. Henkel3, A. Einsenhauer3, W. W. Schmahl1 1Ludwig Maximilians Universität München, Department of Earth and
Environmental Sciences, Munich, Germany 2Institut de Physique de Globe de Paris, Department of Geochemistry and
Cosmochemistry, Paris, France 3GEOMAR Helmholtz Centre for Ocean Research, Marine Biogeochemistry/Marine Systems, Kiel, Germany 4University of Milan, Department of Earth Sciences "A. Desio", Milan, Italy 5University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany 6Polish Academy of Sciences, Institute of Palaeobiology, Warsaw, Poland
The interpretation of geochemical proxies allows the reconstruction of past
and present seawater chemical conditions and evolution with time. As proxy
data are obtained from geological archives, e.g. carbonate shells of marine invertebrates, a substantial understanding of shell microstructures is of
immense importance. With that knowledge a better understanding and
interpretation of geochemical proxy data is possible. Their abundance in the geological record together with the chemical and
structural stability of their low-magnesium calcite shells render brachiopods
to be an important group within the invertebrates. Further, within the phylum
Brachiopoda, the investigation of thecideidine shell morphology and
structure, histological characteristics of the mantle and metabolism in general
has been the subject of research for several decades and revealed the distinctiveness of this group of brachiopods relative to species of other
extinct and modern brachiopod genera (e.g. Pajaud, 1970, Grant, 1972,
Williams, 1972). Previous work suggests a progressive loss of the fibrous shell layer and the development of a "granular" primary layer, a process that
started in the Jurassic and carries on until now. Comparing FE-SEM, EBSD
and AFM results, on shell microstructure and texture, between modern rhynchonellide, terebratulide and thecideide brachiopod shells show that
mineral unit organization in modern thecideide brachiopod shells is entirely
different than the one present in the other groups. The study was completed with stable isotope and element analyses.
The objective of this study is to discuss in detail patterns of shell
microstructure of fossil and modern representatives of theicideid brachiopods and trace the evolution of shell organization and microstructure development
from the Triassic to the present time. Instead of the clear distinction between
an outer primary and an inward fibrous shell layer and the presence of stacks of fibers, as it is the case in modern rhynchonellide and terebratulide
brachiopod shells, we do not find any obvious mineral unit organization in
the shell of modern thecideides. The presence of fibers is clearly visible for
the Triassic and Jurassic specimens. It disappears subsequently, and from the
Cretaceous, to modern times granular microstructures prevail. The latter
consist of highly irregularly shaped mineral units with calcite being assembled with a low degree of co-orientation. Larger calcite granules,
acicular crystallites, and pseudo-polygonal mineral units are embedded in a matrix of small crystallites.
In modern species as Pajaudina atlantica and Kakanuiella chathamensis we
observe within the shell an extracellular organic matrix; however, this biopolymer matrix does not have a regular structure such as that of organic
sheaths encasing the fibers. We consider the granular microstructure of
thecideide shells as a recently evolved feature, followed its development with time, and discuss its interlinkage with the fibrous fabric from a
microstructural point of view.
Grant R. E. 1972. The lophophore and feeding mechanism of the Productidina (Brachiopoda). Journal of Paleontology, 46, 213- 249.
Pajaud D. 1970. Monographies des Thecidees (Brachiopodes). Memoire
Societe Geologique Francaise, 49, 112, 1-349. Williams A. 1972. The secretion and structural evolution of the shell of
Thecideidine brachiopods. Phil. Trans. Roy. Soc. B, 439-478.
P 100
Pineal gland calcification under hypoxic conditions M. Kopani*1 Bronislava Vraníková1, Daniel Kosnáč1, Michal Zeman2,
Vladimír Šišovský3
*1Comenius University, Institute of Pathological Anatomy, Faculty of
Medicine, Bratislava, Slovakia 1 Institute of Medical Physics, Biophysics, Informatics and Telemedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia 2 Department of Animal Physiology and Ethology, Faculty of Natural
Science, Comenius University, Bratislava, Slovakia 3 Institute of Pathological Anatomy, Faculty of Medicine, Comenius
University, Bratislava, Slovakia
Introduction The pineal gland (glandula pinealis) is neuroendocrine gland secreting melatonin. This hormone is involved in many physiological processes
61
including regulations/controls the circadian rhythm, body temperature, immunity and antioxidant activity.
Objectives The aim of this study is investigated the effect of hypoxia on the occurrence of pineal gland calcification. Distribution of calcerous material by light (LM)
and transmission electron microscopy (TEM) was investigated. Chemical
composition of foreign material by scanning electron microscopy with energy-dispersive microanalysis (SEM-EDX) was done. Melatonin
concentrations in blood plasma by direct radioimmunoassay were measured.
Materials & methods The experiments were performed on 24 adult male Wistar rats. First group
was exposed to prenatal hypoxia for 12 h at 20th day of development (n = 9),
second group to prenatal hypoxia for 2x8 h at 19th and 20th days of development (n = 5) and third group was control group (n = 10). All
procedures were conducted in accordance with the Declaration of Helsinki.
Results Scattered vacuoles and large focal calcium-rich particles in the pineal gland
and at the surface of pineal gland by LM were observed. The size of particles
is around 3 µm, sporadically up to 30 µm. SEM-EDX reveals that they consist of Ca, P, Na, S and Fe. TEM reveals vacuoles in the cytoplasm of
pinealocytes type I filled with both flocculent and fibrous material in 12h
hypoxia group. No significant differences in melatonin concentration among groups were found.
Conclusion Pineal gland consists of pinealocytes type 1 and type 2. Their function is secretion of melatonin. It was observed that the cytoplasm of pinealocytes
contains vacuoles filled with flocculent and fibrous material. Welsh (1984) showed that vacuoles play important role in calcification process. Our TEM
results confirmed the presence of flocculent and fibrous material in the
vacuoles. Our previous study revealed that calcerous material was mainly amorphous with the presence of calcite, aragonite and vaterite (nano)crystals
in the samples (Tofail et al. 2019). It is suggested that mineralization process
of pineal gland is similar like bone mineralization and hypoxia favors calcification under hypoxic conditions (Tan et al. 2018). Physical properties
(pyro-, piezo- and ferroelectricity) of crystals may play the important role in
the function of pineal gland.
References TAN DX, CHEN LD, POEGGELER BEA. Melatonin: a potent, endogenous
hydroxyl radical scavenger. Endocr J 1: 57– 60, 1993. TOFAIL SAM, MOURAS R, McNAMARA K, PATYK-KAZMIERCZAK
E, GEANEY H, ZAWOROTKO M, RYAN KM, SOULIMANE T, SILIEN
C, KOPÁNI M. Multimodal surface analyses of chemistry and structure of biominerals in rodent pineal gland concretions. Appl Surf Sci. 469: 378-386,
2019
WELSH MG. Cytochemical analysis of calcium distribution in the superficial pineal gland of the Mongolian gerbil. J Pineal Res. 1: 305-316,
1984.
P 101
Influence of organic additives on the crystallisation of gypsum
and portlandite C. Pritzel*1,2 1Building and Materials Chemistry University of Siegen, Chemistry, Siegen, Germany 2University of Siegen, Chemistry, Siegen, Germany
Gypsum (CaSO4*2H2O) and portlandite (Ca(OH)2) are used in inorganic
binding materials, in those materials organic additives are used to influence
technical properties. Most of the used additives are influencing the morphology of the formed crystals. This influence of organic additives on
the morphology of gypsum or portlandite was tested in crystal growth
experiments. Gypsum was prepared from Na2SO4 and CaCl2 or from calcium sulphate hemihydrate with and without different additives like betain
(used as foaming agent), fruit acids (used as retarder), poly carboxylate ethers
(used as superplasticizers) for example. The different additives influence the crystals morphology, because they are occupying different crystals surfaces
preferred. In case of citric acid the crystals morphology is changed from long
needle like crystals with a large aspect ratio and having more ramifications with the lack of acid to shorter, narrower crystals and less branches. The
smaller diameter of the crystals can be explained because the c-axis is firstly
covered by citric acid and as the consequence the fastest growing axis grows much slower. Owing to the collection the Ca2+ ions from the crystallization
solution leads to the lower ratio Ca/SO4 and the fewer branches can be
observed. These phenomena caused the first forming SO4-backbone which results in the fewer branches in the crystals afterward. The citric acid
adsorbed on the c-axis was shown by Raman-microscopy and AFM. The
calcium sorption was proved by Ca-ion selective electrode. The changed morphology of the created gypsum crystals is decreasing the strength of the
formed gypsum stone and increasing the forming accuracy. It was found that
the Polycarboxylate ethers are influencing the crystal morphology in the same way. The crystallisation of gypsum with and without additives from
hemihydrate was investigated with in-situ optical microscopy, ESEM, in-situ
XRD, particle size distribution measurement, ion chromatography, calcium sensitive electrode, ultra-sonic wave measurement and heat flow calorimetry.
The different techniques are needed to understand the process in detail.
The influence of betain on the crystallisation of portlandite was checked in a crystallisation experiment. Portlandite was produced from CaCl2 and NaOH
during a diffusion experiment with and without betain. The formed crystals
without betain are needle-like with a hexagonal shaped base. With betain thinner layers of those crystals grew together in different steps turned to each
other. The crystals morphology was investigated by ESEM and the sorption
of betain by RAMAN spectroscopy. The used techniques can also be applied to understand the crystal growth of different crystals in the presence of
organic additives. The influence of different organic substances which can
be found in bio-minerals should be investigated in future.
P 102
Reading between the lines in biomineralizations reaching out
for clues of environmental impact - case reports from studies
based on analytic spectroscopy, isotope geochemistry and
proteomics methods S. Berland*1, J. Arivalagan1, E. Feunteun1, A. Bartolini1, C. Thaler1,
A. Marie1 1MNHN, Adaptation du Vivant, Paris, France
Living organisms and mineral collectively speak in a rich and ancient story. Along evolution, complex biomineralized structures have appeared in a range
of species. These structures develop during ontogenesis under the control of
metabolism. The morphology and chemical composition of these structures are known to be shaped by environmental (external) factors under the limits
of functional outcomes. Here we present cases that illustrate calcium
carbonate biomineralisation responses to environmental factors, with special emphasis on technological investigations range, limits and stumbling blocks.
Calcareous tests of foraminifera were investigated. Among biomineralizing
organisms, foraminifera (unicellular eukaryotes) have been producing carbonates since geological time. Their tests (or shells) of calcium carbonate
trace environmental signals, such as ocean temperature, oxygen level,
contamination, salinity, etc. Our approach was to investigate the molecular and cellular machinery of biocalcification based on a benthic calcitic
formainifera, protozoan unicellular model. Coupling geochemical and
biological perspectives on foraminifera will enhance interpretation of the proxies used for environmental and climatic reconstructions.
The second case report arise from the Sub-Antarctic islands, in which severe
environmental conditions drive discrete ecosystems at meso-scale level. In the native land snail Notodiscus hookeri specific populations have evolved
into two ecophenotypes suited with shell either mineral-rich or organic-rich
flexible shells. The soil environment and especially the availability of
exchangeable calcium was the driving factor for ecotypes. Several
methodologies were applied: scanning electron microscopy of the shell, X-
Ray diffraction analysis of the soil, Solid-state Nuclear Magnetic Resonance and shell proteomics. This model enables to follow up the time over which
such adaptive response may have occurred towards phenotypic radiation.
In the context of global change, Baltic Sea was considered as a workshop area to study how native bivalve molluscs (Mytilus sp) can form shells under
lower calcium carbonate saturation states. A proteomic approach was
performed on the shells to learn about the composition of the nested matrix proteins, the local conductor of biomineralization. Result have shown
patterns of modulation in shell proteins which could be correlated to different
shell phenotypes in relationship with shell capacity to offer protection to the individuals.
Finally stable isotopes geochemistry (oxygen and carbon isotopes) in otolith
of glass eels (post larvae of European eels) was used with nanoscale mass spectrometry techniques (NanoSIMs) to track changes of the environment
along the early life history of this diadromous teleost fishes. The otolith
contains visible morphological checks that indicate life cycle key event, e.g hatching, first feeding, and metamorphosis. Minute differences in oxygen
isotopic ratios of oxygen (d18O) in otolith allow addressing the changes in temperature encountered by the larvae along their early life history.
Therefore, these analyses provide environmental information to trace
spawning places and dissemination routes of the eel larvae from the spawning places to the estuaries of Europe.
At the scale of the individual, community or species, biominerals stand as a
repository of environmental information alongside with evolution arrangements from lineage or acquisition. Enclosed time-frame range for the
process to apply needs to be looked for now.
References
Sabbatini A., Bédouet L., Marie A., Bartolini A., Landemarre L. , Weber M.
X., Gusti Ngurah Mahardika I., Berland S., Zito F., Vénec-Peyré M.T. (2014)
Biomineralization Of Schlumbergerella Floresiana, A Significant Carbonate-Producing Benthic Foraminifer. Geobiology: 12, 289-307
Charrier M, Marie A, Guillaume D, Bédouet L, Le Lannic J, Roiland C.,
Berland S., Pierre J.S. , Le Floch M. , Frenot Y., Lebouvier M. (2013) Soil Calcium Availability Influences Shell Ecophenotype Formation in the Sub-
Antarctic Land Snail, Notodiscus hookeri. PLoS ONE 8(12): e84527.
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Arivalagan, J., Yarra, T., Marie, B., Sleight, V. A., Duvernois-Berthet, E., Clark, M., Berland S. (2016). Insights from the shell proteome:
biomineralization to adaptation. Molecular Biology and Evolution, vol.34,
pp. 66-77
P 103
Role of biogenic carbonates in the sediment balance of
accumulative beaches of the Anapa Bay Bar (the Black Sea) A. Kosyan*1 1A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of
Sciences, Laboratory of morphology and ecology of marine invertebrates, Moscow, Russian Federation
Question
Sandy beaches of the Anapa Bay Bar are unique nature landscapes, possessing great esthetic and recreational value, as well as essential
significance for the economy of the Krasnodar region (Russia). The stability
of the beaches is determined by balance and dynamics of sediments. Biogenic sediments (carbonates) are provided by populations of coastal shelly
mollusks, mainly bivalves. In some sites, the share of biogenic carbonates
reaches 90%. Within the framework of the long-scale monitoring of the Anapa Bay Bar,
having been conducted by the researchers from IO RAS and IPEE RAS and
aimed to complex studying of lytho- and hydrodynamic processes, we assessed the quantitative role of mollusks in the sediment balance of the Bar.
Methods
We studied samples of benthic mollusks at five sections with stations at 2, 6 and 10 m depths (deeper mollusks cannot be transferred to the shore even by
stormy waves), taken in 2016-2018. Selected mollusks were counted and
measured. The age was determined by the lines of shell external growth. Determination of carbonate and mineral components of the sand samples was
carried out using 30% hydrochloric acid solution.
Results
The productivity of the two most widespread and abundant in the area species
of bivalves Chamelea gallina and Donax trunculus is not the same in
different years at different sections and depths. The contribution of C. gallina shells to the total carbonate sediments at depths of its mass
development 6-10 m was 327.2 g/m2 in 2016, 127.1 g/m2 in 2017, and 179.3
g/m2 in 2018; the contribution of D. trunculus shells at depths 2-10 m was 123.7, 35.9 and 31.3 g/m2, respectively. Gastropods Rapana venosa had an
order less number and biomass than bivalves, the biomass of other mollusk species was negligibly low. Carbonates content in the bottom sediments
varied from 3 to 30 % (average 11%). An increase in carbonates in bottom
sediments was not directly related to an increase in biomass of living mollusks on the same depths (r = -0.22). Thus, the distribution of carbonates
is rather explained by the movement of sediments, than the distribution of
live mollusks at the bottom. The data on the size-age structure of the mollusk population and earlier observations (Kosyan, 2016) show that the vast
majority of C. gallina within the Anapa Bay-bar (60%) die as a result of
rapana predation at an age of 2-3 years; most D. trunculus die at an age of one year (94% of the population). The width of the zone of mass development
of C. gallina at depths of 6–10 m is approximately 400 m, the width of such
zone of D. trunculus (2–10 m) is 900 m. Taking all above mentioned into account, we may calculate the annual income of the carbonates of biogenic
origin on the studied 45 km long site of the Anapa Bay Bar: 7834.5, 2227.5,
and 4257 tons in 2016, 2017 and 2018 respectively, or on average 4773 tons per year. There are about 1.0 to 4.0 million tons of sediments in constant
motion over the underwater coastal slope, thus restoring of the beaches by
means of shelly material amounts from 0.1 to 0.5%.
Conclusions
The annual contribution of shelly material to the total sediment balance of
the Anapa Bay Bar is on average 4773 tons per year which comprises from 0.1 to 0.5% of total amount of moving sediments. This indicates the
importance of taking measures to protect and restore the population of
mollusks living on the underwater coastal slope. The work was supported by RFBR grant No. 19-45-230001.
P 104
Biominerals in aquatic geosystems under extreme conditions L. Reykhard*1, N. Shulga1, N. Kozina1, Y. Novichkova1, O. Dara1, A.
Boev1, P. Sapozhnikov1, A. Izhitskiy1, N. Belyaev1, V. Gordeev1, O. Kalinina1, A. Reikhard2 1Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow,
Russian Federation 2State Secondary General School № 2086, Moscow, Russian Federation
Authigenic biominerals and their associations with minerals of other origin are used as indicators of the geological, hydrological, and ecological
characteristics of ancient and modern aquatic geosystems. Extreme
environments of biomineralization lead to the appearance of specific structural and chemical features of minerals that must be considered when
using the biomineral indicators. This work aimed to identify the properties
and the origin of biominerals under extreme conditions (high pressure and salinity, low temperatures, high content of hydrogen sulfide and methane,
etc.) in the aquatic geosystems.
Biominerals were studied during scientific surveys in the Arctic and the Antarctic, as well as the Caspian, the Black and the Aral seas. Biominerals
in ferromanganese nodules of the Clarion-Clipperton Fracture Zone (Pacific
Ocean) were also studied [1]. The sampling of biominerals was carried out from the ice cover, water column, bottom and coastal sediments; temperature
and salinity of the water column were measured in situ. Laboratory research
included light and scanning electron microscopy, electron probe microanalysis, X-ray diffraction, geochemical analyses,
micropaleontological and microbiological studies.
It was found out that specific associations of authigenic biominerals are formed as a result of biogenic and abiogenic interactions in each studied
geosystem. However, some biominerals, such as opal, framboidal pyrite (FP)
and some Fe-Mn oxyhydroxides behave like «cosmopolitans». Opal is formed under different extreme conditions and in different mineral
associations, including cryogenic and chemogenic minerals. Thus, opal
frustules of diatoms are formed in the ice cover and under-ice water of the Central Arctic Ocean [2]. In the hypersaline residual basins of the Aral Sea
(the Lake Tshchebas and the Chernyshev Bay) with a high content of
hydrogen sulfide and methane [3], opal is actively generated in the frustules of euryhaline diatoms in the water mass and involved in the structure of
coastal microcrystalline cortical deposits composed of chemogenic crystals
of konyaite, thenardite, blödite, eugsterite, halite, and gypsum. FP was found in bottom sediments of the areas under the variety of extreme
factors, such as low temperatures, high concentrations of hydrogen sulfide and methane [2, 4-6]. Influence of the hydrological and biogeochemical
conditions on the depth of formation, size, morphology, and structure of FP,
as well as the type of mineral/organic substrate, was determined for each studied area.
«Endemical» biominerals, such as discovered vivianite and polycrystalline
Mg-calcite are formed in very specific conditions during the diagenetic transformation of organic and mineral matter. Thus, in association with
aragonite and FP, Mg-calcite forms microcrystalline cement of the carbonate
concretions at a cold methane seep site in the Laptev Sea [6]. The results of the study can be used in the interpretation of the oceanographic
data, paleogeographic reconstructions, environmental monitoring, mining of
the oceanic ore deposits and development of new biocomposite materials. Acknowledgments. The work has been realized in the framework of the state
assignment (theme № 0149-2019-0004).
References [1] Reykhard, L.Ye., Shulga, N.А. (2019). Ore Geology Reviews. Vol. 110.
102933
[2] Reykhard, L.Ye. et al. (2018) Acta Cryst. A74, e250 [3] Izhitskaya, E.S. et al. (2019). Environmental Research Letters. ERL-
105929.R2
[4] Novichkova, Ye.A. et al. (2017). Doklady Akademii Nauk, Vol. 474, No. 3, pp. 365–369
[5] Kozina, N.V. et al. (2018). Russ. J. Earth. Sci., 18, ES6003
[6] Kravchishina, M.D. et al. (2017). Oceanology. V. 57. No. 1. P. 174–191
P 105
Biomineralization plasticity can maintain mechanical stability
of scallop shells exposed to ocean acidification and warming N. Lagos*1, A. Rodriguez-Navarro2, J. Vivanco3, C. Garcia4, C. Duarte5, M. Lardies6 1Universidad Santo Tomas, Centro de Investigación e Innovación para el
Cambio Climático, Santiago, Chile 2Universidad de Granada, Petrology and Mineralogy, Granada, Spain 3Universidad Adolfo Ibañez, Bioingenieria, Viña del Mar, Chile 4Universidad de Santiago de Chile, Ingenieria Mecanica, Santiago, Chile 5Universidad Andres Bello, Dept. Ecología y Biodiversidad, Santiago,
Chile 6Universidad Adolfo Ibañez, Fac. Ingeniería y Ciencias, Santiago, Chile
Ocean acidification (OA) is projected to impact the physiology and shell
carbonate precipitation in mollusks. However, warming may confer resistance to these impacts, and mollusks may trade-off growth and
calcification for maintain shell functionality under the influence of climate
stressors. In this study, we test this hypothesis by assessing the organic composition, crystallography and mechanical properties in shells of
Argopecten purpuratus juvenile scallops exposed to increased temperature
and pCO2-driven ocean acidification. Shell organic matter (%, TGA) increased at cold (14°C) and acidified (7.7) seawater condition. Discriminant
function analysis indicates that organic composition of the shells shown
systematic differences among treatments, with high levels of success (93%) in reclassifiyng the individuals to the combination of
temperature/acidification treatment at which were exposed based on their
organic composition data (ATR-FTIR). Amide and carbonate groups showed significant increments under warming (18ºC) and acidification conditions,
while sulphates and polysaccharides decreased under the same conditions.
63
Crystallographic orientation (XRD) of mineral phase was also variable, showing a significant reduction in the angular spread at the
warming/acidification treatment. In spite these changes in the biopolymer
and mineral phases of the shells of A. purpuratus, the structural resistance (elastic module) do no was affected, but a significant reduction in
microhardness was recorded in individuals shell exposed to
cold/acidification conditions. SEM and micro-CT observations indicate that exposure to these conditions promotes the erosion of the shell periostracum
and dissolution of microstructures associated to shell ribs of the outer surface.
Our results suggest that plasticity in both organic composition as mineral phases of the A. purpuratus shells could be a general compensatory
mechanism to confront climate stressors in order to maintain the shell macro-
structure functionality, but micro-scale dissolution and lesser hardness may compromise further ecological functions and the overall adaptive response.
P 106
Impedance-spectroscopy in biology and health-science I. Tobehn-Steinhäuser*1, A. Winzer1, T. Frank1, S. Herbst1, S. Görland1, T.
Ortlepp1 1CiS Forschungsinstitut für Mikrosensorik, Design, Erfurt, Germany
Introduction
We are partners for industrial research and development of silicon-based
sensors. The main areas of focus are MEMS and MOEMS with highest
stability and reliability and solutions for special photonic detectors and
detector arrays, as well as radiation and particle detectors. You will receive
customized solutions for your sensor systems as well as production in small
series and the qualification of reliability and service life. The contributions show the possibilities of impedance spectroscopy (IS) and
fluorescence in the field of microsystems technology with regard to
biological and medical applications.
Objectives
With the help of IS three projects were worked on. The first involved the
online determination of the diclofenac concentration in wastewater without major laboratory work (BioSam). Another project was the online detection
of mastitis pathogens in cow's milk directly at the milking process (Mastitis).
In addition, a project was conducted to enable the testing of new products for biocompatibility using IS and mouse fibroblasts (3DCellSens).
Materials & methods
To detect toxins, simple biological systems such as yeast cells are suitable (BioSam). These can be genetically modified so that they emit fluorescent
light when certain substances appear. To properly quantify this detectable
light, the number of yeast cells must be known. For this purpose one uses in this case the IS. In order to be able to combine both sensor principles,
interdigital structures made of ITO were realized on glass substrate, so that
the light can be detected simultaneously throw the optically transparent
structure.
In the project Mastitis the pathogens should be detected directly at the
milking process. without too much laboratory infrastructure. The structure consists of different variants of a switchable electrode array. It is possible to
interconnect random patterns to adapt the electric field to the measuring task (cell geometry). The switchable electrodes enable a fast changeover of the
electrode geometry. This makes it possible on the one hand to hide the
properties of the cells from the measurement signal (small electrode distances) and, on the other hand, to measure the polarization of the cells
directly (interconnection to electrode blocks).
From the behavior of certain biological cell cultures on different environmental influences and substances, conclusions can be drawn and on
the interactions in other applications. An important task here is the
determination of the biocompatibility of materials. The given new method allows the evaluation of biocompatibility already during the experiment. The
measuring device consists of a sensor with evaluation electronics and records
the data by means of IS. The basis for this is formed by adhesion coupling between the cells and the sensor. Vital cells behave like an insulator in the
nutrient medium, which binds to the cell carrier through adhesion. Upon the
onset of cell death, the cell membrane breaks and, as a result, the adhesive
contacts disapear.
The cell no longer acts as an insulator. As a result the impedance changes. 4.
Results Fully functional samples or prototypes could be produced in all three
projects. The results will be shown during the conference.
Conclusion
IS, also in combination with other measuring methods, is one powerful
method to caracterise biological and medical systems. With our contribution,
we may be able to show a way to solve an existing measurement task.
P 107
Fluorescence lifetime- sensor components I. Tobehn-Steinhäuser*1, A. Winzer1, C. Möller1, C. Heinze1, H. G.
Ortlepp1, T. Ortlepp1 1CiS Forschungsinstitut für Mikrosensorik, Design, Erfurt, Germany
Introduction
Determination of fluorescence lifetime is a key tool in the investigation of
cell metabolism and cell growth. The time decay of the fluorescence of
functionalized dyes facilitates the understanding for instance of intracellular temperature fluctuations originating from chemical processes within the
cells.
Objectives
For the development of our sensor components we establish the following
key requirements:
The excitation of the fluorescent dyes must be done with light
pulses as short as 1 nanosecond. And the excitation wavelength
must be about 370 nm, i.e. suitable functionality confirmation with a dye.
Our design of the optics module makes it possible to measure an
array of probe chambers in titer plates in parallel. Therefore the
width must not exceed 8 mm.
As detectors silicon photomultipliers (SiPM) are used which also
enable for miniaturization and thus for parallel measurements.
An application-specific integrated circuit (ASIC) allows for a fast
and accurate collection of the fluorescence decay statistics.
Materials & methods
The optical behavior of the lighting module was simulated by a ray tracing software tool (ZEMAX). For this purpose, a model of the UV 370nm LED
was established and adjusted to the measured LED radiance. For beam
shaping, UV antireflection-coated quartz lenses were used. The mounting of the optical elements (lenses, filters and aperture) was done with 3D printed
brass housing. As excitation filter a commercial bandpass filter is used.
A UV enhanced photodiode was integrated on the pulse excitation component for monitoring the radiated power of the LED.
For the fast determination of the fluorescence lifetime, we designed an ASIC
for the SiPM detectors. The functionality of the excitation module as well as the detection module is
demonstrated with the fluorescence dye ATTO 390.
Results
The possibility of generating nanosecond pulses with a LED is known in the
literature. However, the existing concepts need 200 to 400 V as supply
voltage for the avalanche transistors used in the circuitry. Our solution only needs 9 V of supply voltage, which is of benefit for assembling a compact
array of such sensors. The design allows a pitch of the PCB of 8 mm.
The generated light pulse of the LED was experimentally determined via a time-correlated single photon counting (TCSPC) measuring. The FWHM of
the LED pulse determined from the measurement is around 1 ns.
The functional test of the excitation component yielded a time constant of τ = 4.6 ± 0.3 ns for the used fluorescent dye ATTO 390. This value agrees
very well with τ = 5.0 ns given by the manufacturer.
The ASIC performance was determined and improved by simulations. Which suggest a high suitability for fluorescent lifetime measurements. Key
parameter is a time measurement accuracy of 80 ps and a measurement time
of up to 20 ns.
Conclusion
An illumination unit for fluorescence lifetime determination operates at a
wavelength of 370 nm and has a pulse width of around 1 ns. The width (8 mm) of the entire module is suitable for parallel measurements on titer
plates. As a confirmation example, the fluorescence lifetime of the dye
ATTO 390 was determined. Further, an ASIC for the TCSPC measurement with a SiPM was developed. The ASIC specifications as well as the first real
test results are discussed.
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P 108
Extracellular vesicles involved in the guinea fowl eggshell
quantitative proteomics yield new findings related to its unique
structure N. Le Roy1, L. Combes-Soia2, V. Labas2, A. Rodriguez-Navarro3, M.
Hincke3,4, Y. Nys1, J. Gautron*1
1French National Institute of Agricultural Research (INRA), Bird Biology
and Poultry, Nouzilly, France 2French National Institute of Agricultural Research, Physiology of
Reproduction and behaviour, Nouzilly, France 3Universitad of Granada, Departemento de mineralogia y petrologica, Granada, Spain 4University of Ottawa, Department of cellular and molecular medicine,
Ottawa, Canada Question The Guinea fowl (Numida meleagris) eggshell is a bioceramic material with
the remarkable mechanical property of being twice as strong as the chicken eggshell. In both species, the eggshell is composed of 95% mineral in the
form of calcite polymorph and 3.5% organic matrix including shell
membranes. However, the eggshell ultrastructure and microstructure are significantly different between these species. In the chicken, the eggshell is
made of columnar calcite crystal units arranged vertically. In the Guinea
fowl, the same crystal architecture is observed in its inner half, followed by a dramatic change in crystal size and orientation. The unique ultrastructural
characteristics of Guinea fowl eggshell confer a superior resistance to
breakage compared to eggshells from other bird species. In order to understand the underlying mechanisms controlling the formation and
structural organization of this highly resistant material at five key stages of
the mineralization process (4 h, 10 h, 11 h, 12 h and 18 h post-ovulation).
Methods FormingGuinea fowl Eggs were sampled at five stages: 4 h (n=5), 10 h (n=6),
11 h (n=6), 12 h (n=6) and 18 h (n=6) post-ovulation (p.o., i.e. time after the previous egg) when the nucleation sites appear and early mineralization starts
(4 h p.o.), or just before (10 h p.o.), during (11-12 h p.o.) and after the shift
in crystal orientation (18 h p.o.). The organic matrix was extracted from each eggshell and analyzed by a bottom-up quantitative proteomics approach. We
the used statistical and bioinformatics tools to determine the protein related
to the main structural shift and their functiuons.
Results The present work is the first Guinea fowl eggshell proteomic study, which
allowed the identification of 149 proteins. Comparison of the Guinea fowl eggshell proteome with that of other bird species leads to the identification
of 9 proteins that are only present in Guinea fowl. Among them Protein S100-
A6 and GDF6 are notable as they exhibit potential functions related to shell mineralization and especially at the point of the modification in crystal size
and orientation that confers remarkable strength on the Guinea fowl eggshell.
In addition to these two proteins, we also report that 61 proteins are more abundant during the secondary nucleation events associated with the change
in crystal orientation and the formation of the new layer. Additionally, we
identify the most abundant proteins involved in the different phases of Guinea fowl shell formation, from the first events of biomineralization until
the deposition of the new layer. Our study showed that proteins associated
with early mineralization are similar in chicken and Guinea fowl, but also revealed candidate proteins, which may be involved in a dramatic shift in
eggshell microstructure that is unique to Guinea fowl. The proteins more
abundant during the shift were ANXA2, S100A6, CALB1, TSKU, FAM20C, GPC-4, DCA-1-like and GDF6. Amongst them are calcium binding proteins,
protein cores of proteoglycans, proteins involved in the regulation of proteins
driving the mineralization.
Conclusion This is the first proteome survey in Guinea fowl eggshell which exhibits
exceptional mechanical proeprties. These data enriched the huge number of matrix proteins identified in various bird"s eggshell proteomics studies and
will allow genomic improvements and will give insights for material sciences. Genes coding matrix proteins will be used as biological markers for
genomic selection to reinforce eggshell breaking strength. The corresponding
transcripts will be associated with published and private SNPs and mapped in QTLs related to shell quality. They will constitute candidate genes to gain
precision for genomic selection to reinforce shell mechanical properties.
Industrial ceramics are made in high temperature and pressure. Material science explores the biomineralization to investigate how living organisms
build their shell in physiological conditions. Amongst various biominerals,
the bird"s eggshell are the most widely documented. Information on shell matrix proteins and how they contribute to the mechanical properties, gives
a chance to establish a list of natural organic compounds of benefit usable in
the fabrication of calcium carbonate materials/ceramics.
65