role of geitlerinema sp. de2011 and scenedesmus sp. de2009 ... · researcharticle role of...

Research ArticleRole of Geitlerinema sp DE2011 andScenedesmus sp DE2009 as Bioindicators and Immobilizers ofChromium in a Contaminated Natural Environment

Laia Millach Antoni Soleacute and Isabel Esteve

Departament de Genetica i Microbiologia Facultat de Biociencies Universitat Autonoma de BarcelonaBellaterra Cerdanyola del Valles 08193 Barcelona Spain

Correspondence should be addressed to Isabel Esteve isabelesteveuabcat

Received 25 March 2015 Revised 25 May 2015 Accepted 31 May 2015

Academic Editor Qaisar Mahmood

Copyright copy 2015 Laia Millach et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this work was to study the potential of the two phototrophic microorganisms both isolated from Ebro Delta microbialmats to be used as bioindicators and immobilizers of chromium The results obtained indicated that (i) the Minimum MetalConcentration (MMC) significantly affecting Chlorophyll a intensity in Geitlerinema sp DE2011 and Scenedesmus sp DE2009 was025 120583M and 075 120583M respectively these values being lower than those established by current legislation and (ii) Scenedesmussp DE2009 was able to immobilize chromium externally in extracellular polymeric substances (EPS) and intracellularly inpolyphosphate (PP) inclusions Additionally this microorganism maintained high viability including at 500120583M Based on theseresults we postulate that Geitlerinema sp DE2011 and Scenedesmus sp DE2009 are good chromium-indicators of cytotoxicity andfurther that Scenedesmus sp DE2009 plays an important role in immobilizing this metal in a contaminated natural environment

1 Introduction

Metal contamination is a serious environmental problemthat affects life forms and changes the natural microbiotaof aquatic ecosystems Currently metals are released fromnatural and anthropogenic sources (eg industry transportfossil fuel combustion the mining industry and agriculture)into natural aquatic environments [1] These metals areaccumulated in waters sediments and biota generatingresistance inmicroorganisms that leads to environmental andpublic health problems To study and predict the effects andremoval of heavy metals on different ecosystems nematode[2] plants [3] and algae [4] among others have been usedCyanobacteria and algae are particularly very abundant inaquatic ecosystems playing an important role in primaryproduction in rivers and their deltas where metals very oftenaccumulate

The Ebro River is 928 km long flows from the north ofthe Iberian Peninsula to the Mediterranean Sea and drainsan area of 85000 km2 approximatelyThe Ebro Delta locatedat the outfall of the Ebro River is the second most important

wetland in Spain after Guadalquivir River marshes and thesecond one of the Mediterranean area after the Camargue(France) The Ebro Delta is also considered the third largestdelta in the Mediterranean with a 320 km2 triangular surfaceand it is located at the northeastern coastline of the IberianPeninsula (0∘351015840Endash0∘561015840E 40∘331015840Nndash40∘471015840N) [5] In 1983some of the most outstanding natural areas of the delta wereincluded in the Ebro Delta Natural Park (Parc Natural delDelta de lrsquoEbre) because of its ornithological importance aswell as for other geological biological economic and culturalaspects [6]

Microbial mats developed in water-sediment interfacesare formed by multilayered benthic microbial communitiesthat are distributed along vertical microgradients of differentphysical-chemical parameters These ecosystems are widelydistributed around the world in different extreme environ-ments such as lakes [7] marine waters [8] and cold waters[9] among others Ebro Delta microbial mats are formedby different microorganisms principally cyanobacteria andmicroalgae are the most abundant prokaryotic bacterialocated mainly in the upper layers of microbial mats [10]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 519769 11 pageshttpdxdoiorg1011552015519769

2 BioMed Research International

These microbial mats receive waters and contaminantsincluding heavy metals dragged by the River Ebro into itsestuary (delta) For this reason in the last few years ourwork group has isolated various microorganisms of thisecosystem and has developed several methods in particularConfocal Laser Scanning Microscopy (CLSM) to determinetheir capacity to tolerate or resist metals as well as evaluatethe effect of these in vivo at both cell and population levelsThese methods used for the in vivo study of phototrophicmicroorganisms have led to obtaining quantitative resultsmore quickly This is mainly due to the minimal necessarymanipulation of the specimens and since these emit naturalfluorescence they do not require staining protocols Fur-thermore the majority of works have evaluated the effectof lead and copper toxicity in isolated microorganisms [11]and the capacity of various microorganisms to uptake thesemetals extra- andor intracellularly using Scanning ElectronMicroscopy (SEM) and Transmission Electron Microscopy(TEM) both coupled to an Energy Dispersive X-Ray (SEM-EDX and TEM-EDX) [12]

However the role that microorganisms play in thissame habitat on chromium detoxification is still unknownChromium can exist in the environment as Cr(III) or Cr(VI)[13 14] and particularly in the Cr(VI) form is extremelytoxic mutagenic and carcinogenic In the environmentchromium is introduced as the by-product of industries[15] and phosphate fertilizers [16] In highly contaminatedhabitats [17 18] the reduction of Cr(VI) to Cr(III) is aneffective method of Cr(VI) detoxification Nevertheless theimmobilization efficiency of Cr(III) is still unclear and dif-ferent reports suggest that soluble organo-Cr(III) complexesare present in various chromate-reducing bacterial systems[19 20] The permanence of soluble forms of Cr(III) causesa serious problem since they can be reoxidized to Cr(VI) Itis for this reason that there is great interest in studying theimmobilization of Cr(III) in pilot-scale experiments [21]

Nowadays there is little information on this process inthe natural environment where the levels of contaminationby chromium are very low as in the River Ebro (lt2120583g Lminus1 Craccording to data from the Ebro Hydrographic Associationin the last 10 years) In these cases although the sameprobably occurs the Cr(VI) is biotransformed to Cr(III) andthis can remain in ecosystems immobilized or not and couldhave a toxic effect on life forms Likewise very little is knownabout the role of indigenous microorganisms in these naturalenvironments with low levels of chromium and also with aprolonged permanence of the metal in the ecosystem

The aim of this work is to determine the role of Geit-lerinema sp DE2011 and Scenedesmus sp DE2009 bothisolated fromEbroDeltamicrobial mats as bioindicators andimmobilizers of chromium and additionally to analyse theeffect of this metal on their biomass and cellular viability

2 Material and Methods

21 Microorganisms and Culture Conditions Geitlerinemasp DE2011 (cyanobacterium) and Scenedesmus sp DE2009(microalga) were isolated from Ebro Delta microbial mats(Tarragona) Spain Isolation and purification of the isolates

were performed by dilution and plating of microbial matssamples Isolated microorganisms were grown in liquidmineral Pfennig medium [22] in 100mL flasks Cultureswere exposed and maintained at 27∘C in a growth chamber(Climas Grow 180 ClimasLab Barcelona) under continuousillumination with a light intensity of 35 120583Emminus2 sminus1 for thecyanobacterium and 10 120583Emminus2 sminus1 for the microalga pro-vided by cold white fluorescence lights These cultures wereused as control in all the experiments performed

22 Preparation of Chromium Stock Solution The stocksolution of chromium was prepared by dissolving Cr(NO

3)3

(Sigma-Aldrich Bellefonte PA US) in deionized Milli-Qwater at the concentration of 1mM Cr(III) and sterilizedby filtration in Millex-GP 02 120583m filters (Millipore USA)Working concentrations of Cr(III) were obtained by serialdilution This solution was stored in the dark at 4∘C

23 Pigment Analysis of the Strains Using Confocal LaserScanning Microscopy The tolerance and the in vivo effectof chromium on cultures of Geitlerinema sp DE2011 andScenedesmus sp DE2009 were determined by 120582scan functionof CLSM (CLSM Leica TCS SP5 Leica Heidelberg Ger-many) Moreover in order to evaluate the effect of chromiumon the biomass and viability of Scenedesmus sp DE2009 amodification of the FLU-CLSM-IA (Fluorochrome-CLSM-Image Analysis) method described by Puyen et al [23] wasused

231 120582scan Function Cultures of Geitlerinema sp DE2011and Scenedesmus sp DE2009 were contaminated at differentCr(NO

3)3concentrations 0025 0050 01 025 050 075

1 and 5 120583M for the cyanobacterium DE2011 and 025 050075 1 5 10 15 and 25120583M for the microalga DE2009 Allexperiments were performed for 9 days under the sameconditions mentioned in Section 21

Pigment analysis was realized by the 120582scan function ofCLSM This technique provides information on the state ofthe photosynthetic pigments of phototrophic microorgan-isms on the basis of the emission wavelength region andthe fluorescence intensity emitted (autofluorescence) Eachimage sequence was obtained by scanning the same 119909119910optical section throughout the visible spectrum Images wereacquired at the 119911 position at which the fluorescence wasmaximal and acquisition settings were constant throughouteach experiment The sample excitation was carried out withan Argon Laser at 488 nm (120582exe 488) with a 120582 step size of3 nm for an emission wavelength between 550 and 748 nm

In order to measure the mean fluorescence intensity(MFI) of the 119909119910120582 data sets the Leica Confocal Software(Leica Microsystems CMS GmbH) was used In these con-focal images the pseudocolour palette 4 was selected wherewarm colours represented the maximum intensities and coldcolours represented the low intensities of fluorescence Theregions-of-interest (ROIs) function of the software was usedto measure the spectral signature For each sample 70 ROIsof 1 120583m2 taken from cells were analysed

BioMed Research International 3

This method allowed us to evaluate the physiologicalstate of the phototrophic microorganisms at single-cell levelconsidering changes in the spectrum of Chlorophyll a (Chl a)used as a marker For this purpose the state of pigments wasconsidered bymeans of theMaximum Intensity Fluorescence(MIF) signal detected at 661 nm (Chl a) for Geitlerinema spDE2011 and 685 nm for Scenedesmus sp DE2009 (Chl a)

232 FLU-CLSM-IA Modified Method To determine theeffect of chromium on biomass and cellular viability ofScenedesmus sp DE2009 cultures experiments at differentCr(NO

3)3concentrations 075 25 100 200 and 500 120583M

were performed for a period of 9 days under the sameconditions mentioned in Section 21 following a modificationof the FLU-CLSM-IA method [23] This method combinesthe use of specific fluorochrome the CLSM microscope andthe ImageJ v148s software

In this study Scenedesmus sp DE2009 autofluorescence(emission at 616ndash695 nm) and SYTOX Green Nucleic AcidStain fluorescence (emission at 520ndash580 nm Invitrogen LifeTechnologies) were used simultaneously as markers for liveand dead cells respectively in a simple dual-fluorescenceviability assay [24] Both the red and green fluorescencesignals were captured separately in a sequential scan pro-cess in two channels from each same 119909119910119911 optical section(Figures 1(a) and 1(c))

In order to differentiate between living and dead cells red(live cells) and green (dead cells) pseudocolors were used and20 red and green confocal images were acquired from everyculture of Scenedesmus sp DE2009 to determine the biomassand cellular viability at each Cr-concentration

The CLSM images were transformed to binary images(blackwhite) applying fluorescence threshold values of 30(red pixels) and 35 (green pixels) by means of the ImageJv148s software (Figures 1(b) and 1(d)) To minimize thebackground detected in every pair of images a smoothingfilter was used

To obtain biovolume values the Voxel Counter plug-in was applied to these filtered images [25] This specificapplication calculates the ratio between the thresholdedvoxels (red and green fluorescent voxel counts) to all voxelsfrom every binary image analysed The biovolume value(volume fraction) was finally multiplied by a conversionfactor of 310 fgC 120583m3 to convert it to biomass [26]

24 Ascertaining Chromium Immobilization through Elec-tronic Microscopy Techniques With the aim of determin-ing whether Geitlerinema sp DE2011 and Scenedesmus spDE2009 could immobilize metals extra- and intracellularlycells from cultures growing with andwithout chromiumwereanalysed by EDX coupled to SEM and TEM

241 Scanning Electron Microscopy and Energy Dispersive X-Ray Analysis Phototrophic microorganisms cultures werecontaminated at different Cr(NO

3)3concentrations 1 5 10

25 50 100 and 200120583M Cr(III) and incubated under thesame conditions as mentioned above for a period of 9 days

For SEM analysis cultures were filtrated in Nucleporepolycarbonate membranes (Whatman Ltd) and then werefixed in 25 glutaraldehyde diluted in Millonig phosphatebuffer (01M pH 4) at 4∘C for 2 hours and washed four timesin the same buffer dehydrated in increasing concentrationsof ethanol (30 50 70 90 and 100) and driedby critical-point (CPD 030 Critical Point Drier BAL-TECGmbH 58579 Schalksmuhle) Finally samples weremountedon aluminium metal stubs and coated with a 5 120583m gold layer(K550 Sputter Coater Emitech AshfordUK) for better imagecontrast A Zeiss EVOMA 10 scanning electron microscope(Carl Zeiss NTS GmbH Oberkochen Germany) was used toview the images

For EDX microanalysis cells were homogenously dis-tributed and filtered on polycarbonate membrane filtersThese filters were fixed dehydrated and dried by critical-point drying and then coated with gold An EDX spec-trophotometer Link Isis-200 (Oxford Instruments BucksEngland) coupled to the microscope operating at 20 kV wasused Finally EDX-SEM spectra from individual cells wereobtained

242 Transmission Electron Microscopy and Energy Disper-sive X-Ray Analysis TEM was used in order to observethe ultrastructure of the phototrophic microorganisms andTEM-EDX to assess whether Geitlerinema sp DE2011and Scenedesmus sp DE2009 were able to bioaccumulatechromium intracellularly So cyanobacterium DE2011 andthe microalga DE2009 were contaminated with 200120583MCr(NO

3)3for a period of 9 days Culture conditions were the

same as described for SEMFor TEM analysis samples were fixed in 25 glutaralde-

hyde diluted inMillonig phosphate buffer (01M pH 4) at 4∘Cfor 2 hours and washed four times (15min) in the same bufferat 4∘C Then samples were postfixed in 1 OsO

4at 4∘C for 2

hours washed in the same buffer and centrifuged in order toobtain a pellet They were then dehydrated in a graded seriesof acetone (50 70 90 95 and 100) and embeddedin Spurrrsquos resin Once the samples were included in the resinultrathin sections (70 nm) obtained with a Leica EM UC6Ultramicrotome (Leica Microsystems GmbH HeidelbergGermany) were mounted on carbon-coated titanium gridsand stained with uranyl acetate and lead citrate Sampleswere viewed in a Hitachi H-7000 transmission electronmicroscope (Hitachi Ltd Tokyo Japan)

For EDX microanalysis sections 200 nm thick were alsostained with uranyl acetate and mounted on carbon-coatedtitanium grids Samples were analysed with an EDX spec-trophotometer Link Isis-200 (Oxford Instruments BucksEngland) coupled to a Jeol Jem-2011 (Jeol Ltd Tokyo Japan)operating at 20 kV Finally EDX-TEM spectra from individ-ual cells were obtained

25 Statistical Analysis Statistical analyses were carried outby one-way analysis of variance (ANOVA) and Tukey andBonferronirsquos comparison post hoc tests Significant differenceswere accepted at119875 lt 005The analyseswere performedusingIBM SPSS Statistics software (version 200 for Windows 7)


ImageJ v148sCLSM Leica TCS SP5

Natural fluorescence-live cells

SYTOX green fluorescence-dead cells

(a)

(c) (d)

(b)

Figure 1 119909119910119911CLSM optical sections (a) and (b) and their corresponding binary images of live (b) and dead (d) Scenedesmus sp DE2009 cellsanalysed using the modified FLU-CLSM-IA method

3 Results and Discussion

31 Morphological Characteristics of Geitlerinema sp DE2011and Scenedesmus sp DE2009 Thephototrophicmicroorgan-isms isolated fromEbroDeltamicrobialmats were identifiedas Geitlerinema sp DE2011 [27] and Scenedesmus sp DE2009[12] by molecular biology methods Both microorganismsare very abundant in Ebro Delta microbial mats and play animportant role in the stabilization of deltaic sediments

Geitlerinema sp DE2011 is a cyanobacterium whichforms individual filaments sometimes densely packed andsurrounded by a sheath Cells from filaments vary in sizefrom 313 to 375 120583m On the other hand Scenedesmus spDE2009 is a microalga which like Geitlerinema sp DE2011forms a consortiumwith different heterotrophic bacteriaThemicroalga cells are spherical with a diameter of 7ndash9 120583m andtheir chloroplasts are distributed laterally in the cell

32 Chromium Tolerance in Phototrophic MicroorganismsIn order to calculate the Minimum Metal Concentration

(MMC) that significantly affects pigment intensity in Geit-lerinema sp DE2011 and Scenedesmus sp DE2009 twoexperiments were performed In the preliminary experimenta wide range of chromium concentrations was assayedDisplacement of the fluorescence peak was observed onlyin Geitlerinema sp DE2015 from 661 nm (MIF) towardsto 670 nm at maximum Cr-concentration assayed (5120583Mfluorescence spectrum) In both cases highly statisticallysignificant differences (119875 lt 005) were found between thecontrol and all the concentrations tested (Figures 2(a) and2(b))

For this reason a second experiment was carried out onGeitlerinema sp DE2011 with lower doses from 25 nM to075 120583M Cr(III) The 119909119910119911 optical sections of this microor-ganism corresponding to the autofluorescence detected incontrol and contaminated cultures were shown in Figures3(a) and 3(b) The results indicated that the MMC ofchromium (when compared with the control) that signifi-cantly (119875 lt 005) affected the intensity of the pigment (Chl a)


600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

050120583M075120583M

1120583M5120583M

(a)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

1120583M5120583M

10120583M15120583M25120583M

(b)

Figure 2 120582scan plots of Geitlerinema sp DE2011 (a) and Scenedesmus sp DE2009 (b) contaminated with a wide range of chromiumconcentrations

of Geitlerinema sp DE2011 was 025 120583M Cr An analogousexperiment to that mentioned above was performed withlower doses from 025 120583M to 5 120583M Cr(III) on cultures ofScenedesmus sp DE2009 The autofluorescence detected incontrol and contaminated cultures was shown in Figures 4(a)and 4(b) In this case the MMC that significantly (119875 lt005) affected the intensity of the pigment in Scenedesmus spDE2009 was 075120583M Cr and therefore this microorganismwasmore tolerant to chromium thanGeitlerinema sp DE2011(025 120583MCr)

On the other hand the 120582scan plots graphs of bothmicroorganisms indicated how the MIF peak (Chl a)decreased while the Cr-concentration increased followingmainly the same pattern as the control culture (Figures 3(c)and 4(c)) These results are in agreement with those obtainedby different authors which demonstrated in Scenedesmusobliquus andNostoc muscorum respectively that metal stressresults in direct inactivation of the photosystem II (PS II)reaction center and consequently a decrease of Chlorophylla fluorescence intensity (F

685) [28 29] Furthermore other

authors have demonstrated that in response to varyingphysical-chemical parameters photosynthetic microorgan-isms undergo changes in their physiological characteristicsmainly changing the quality and concentration of their light-harvesting pigments [30]

It is worth highlighting that the MMC values obtainedwere below the level permitted in continental surface waters(50120583g Lminus1 Cr) (in accordance with theDirective 2008105CEof the European Parliament and the Council on Envi-ronmental Quality Standards in the field of Water Policytransposed into Spanish law ldquoReal Decreto 602011 AnexoIIrdquo) which demonstrated that both microorganisms shouldbe considered as good indicators of cytotoxicity

33 Metal Immobilization in Phototrophic MicroorganismsCr-contaminated cultures of Geitlerinema sp DE2011 wereanalysed by SEM-EDX and chromium was not detected inthe extracellular polymeric substances (EPS) (Figures 3(d)3(e) and 3(f)) Nevertheless in the contaminated samplesof Scenedesmus sp DE2009 the results confirmed that themicroalga had the ability to sequester chromium in the EPS(Figures 4(d) 4(e) and 4(f)) Different parts of the filterwere also tested as a control in all samples to be sure thatchromium was retained only in cells

Both microorganisms have dense EPS envelopes whichexplain the external uptake of heavy metals Various authorshave suggested that the overall negative charge of EPS maybe essential for sequestering metal cations that are necessaryfor cell growth but present at low concentrations in theirsurroundings andor preventing the direct contact betweenthe cells and toxic heavymetals dispersed in the environment[31] The functions of EPS in metal uptake are knownbut other roles have been proposed for these polymerssuch as protection against dehydration or UV radiationbiomineralization phagocytosis and adhesion capacity to thesurrounding substrate [32]

Although Geitlerinema sp DE2011 gave a negative resultfor chromium uptake in previous studies it has beenshown that this cyanobacterium was able to capture leadand copper extracellularly [27] These differences in metalimmobilization were probably due to the fact that the samemicroorganism can capture distinct metals using differentfunctional groups in the EPS In accordance with studiescarried out by Ozturk et al [33] an increase in uronic acidglucuronic acid and galacturonic acid content was shown inthe EPS of Synechocystis sp BASO671 cultures contaminatedby chromium In addition Celekli et al [34] also confirmed


(a) (b)

600

610

620

630

640

650

660

670

680

690

700

710

720

730

740

750

020406080

100120140160180200

ControlWavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

652

655

658

661

664

667

670

673

676

165170175180185190195

Wavelength (nm)

MFI

075120583M025120583M100 nM

50 nM25 nM

(c)

(d) (e)

CaPd

AuAuNaO

Au

Ca

AuP

C

0 2 4 6 8 10(keV)

(f)

Figure 3 Continued


(g) (h)

Ca TiNa ClCu

ClTi

CuCuTi

PO

CaC

0 2 4 6 8 10(keV)

(i)

Figure 3 CLSM images of control (a) and chromium contaminated (b) cultures of Geitlerinema sp DE2011 (scale bars represent 10120583m) and120582scan plot (c) SEM images of control (d) and 200120583M chromium contaminated (e) cultures Scale bars represent 2120583m Contaminated EDXspectrum (f) TEM images of control (g) and 200120583M chromium contaminated (h) cultures Scale bars represent 1120583m Contaminated EDXspectrum (i)

that specific anionic groups played a significant role inthe biosorption of Cd2+ by Scenedesmus quadricauda varlongispina

On the other hand TEM micrographs of the ultrathinsections of Geitlerinema sp DE2011 and Scenedesmus spDE2009 growing at 200120583MCr showed abundant high elec-tron dense intracytoplasmic inclusions of different sizes intheir cytoplasm identified as polyphosphate inclusions (PP)(Figures 3(h) and 4(h)) In many cases similar inclusionshave been found when cells are grown in adverse cultureconditions [35] Chromium was not detected internally inGeitlerinema sp DE2011 or Scenedesmus sp DE2009 incontrol cultures (Figures 3(g) and 4(g))

The results obtained through EDX analysis of the inclu-sions demonstrated thatGeitlerinema sp DE2011 did not havethe capacity to accumulate chromium as no Cr peak wasdetected (Figure 3(i)) In contrast to this a significant Crpeakwas detected in Scenedesmus spDE2009 demonstratingthat this microorganism was able to immobilize this metalinternally in PP inclusions (Figure 4(i)) These results agree

with studies of Goldberg et al [36] which suggested that thiskind of inclusions has a detoxifying effect and a large affinityby sequestering heavy metals In general algae seem to bemore effective than cyanobacteria in capturing heavy metals[37 38] and as has been shown in this work Scenedesmussp DE2009 due to its ability to capture chromium bothextra- and intracellularly probably plays an important role inchromium detoxification in Ebro Delta microbial mats

34 Effect of Chromium on Biomass and Cellular Viability ofScenedesmus sp DE2009 For this objective previously thered and green fluorescent voxels counts were measured asmentioned in Section 232 The red voxels (live cells) rangedfrom 162097 plusmn 9220 (control experiment) to 143390 plusmn 6638(at 500120583M) and the green voxels (dead cells) varied from23450 plusmn 1822 (control experiment) to 32113 plusmn 2277 (at500120583M) The conversion of this data into biomass valuesmade it possible to observe that the live biomass slightlydecreased from 4792 plusmn 273mgC cmminus3 in the control cultureto 4239 plusmn 196mgC cmminus3 at 500120583MCr


(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)

Figure 4 CLSM images of control (a) and chromium contaminated (b) cultures of Scenedesmus sp DE2009 (scale bars represent 10 120583m) and120582scan plot (c) SEM images of control (d) and 200120583M chromium contaminated (e) cultures Scale bars represent 2120583m Contaminated EDXspectrum (f) Arrow indicates the main Cr peak at 54 keV TEM images of control (g) and 200 120583M chromium contaminated (h) culturesScale bars represent 1 120583m Contaminated EDX spectrum (i) Cr peaks are indicated by arrows

The changes in viability were shown in Figure 5 Theseresults were expressed as the percentages () of live cellsand dead cells for each contaminated culture These valuesshowed low significant differences (119875 lt 005) for all ofthem compared to the control culture which indicated aslight effect of the metal in the viability of Scenedesmus spDE2009 However there were no significant differences (119875 lt005) between the various concentrations tested with thepercentage of viable cells in all the Cr-concentrations testedremaining stable

Thus on comparing the growth of Scenedesmus spDE2009 in control culture and the maximum tested concen-tration (500120583M) it was observed that in the control experi-ment live cells represented 8719 and dead cells 1281 andin the contaminated culture live cells represented 8161 anddead cells 1839 (Figure 5) These results confirmed that ahigh level of viability of the microalga is maintained even atthe highest concentration of chromium tested

4 Conclusions

The results obtained in this paper lead to the conclusion thatScenedesmus sp DE2009 is more tolerant to chromium thanGeitlerinema sp DE2011 and that bothmicroorganisms couldbe considered as good indicators of chromium toxicity in lowcontaminated natural ecosystems

On the other hand Scenedesmus sp DE2009 maintainsan elevated biomass and viability at high Cr-concentrationsand also has the ability to capture chromium extracellularly inEPS and intracellularly in PP inclusions which demonstratesits capacity to immobilize this metal

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Con

trol

0

20

40

60

80

100

Dead cells ()Live cells ()

()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r

Cr(III) concentrations (120583M)

Figure 5 Percentages of live and dead Scenedesmus sp DE2009 cellsat different Cr(III) concentrations The bars indicate the StandardError of the Means (SEM)

Acknowledgments

This research was supported by the following grants DGI-CYT (CGL2008-01891BOS) and the UAB postgraduatescholarship to Laia Millach The authors express their grat-itude to the staff of Servei de Microscopia for technical assis-tance with the confocal and electron microscopes and Serveide Llengues both at Universitat Autonoma de BarcelonaTheyalso thank Eneko Mitxelena for his support on images digitalprocessing and Cristina Sosa for her help in this work

References

[1] B Nogales M P Lanfranconi J M Pina-Villalonga andR Bosch ldquoAnthropogenic perturbations in marine microbialcommunitiesrdquo FEMS Microbiology Reviews vol 35 no 2 pp275ndash298 2011

[2] P Salamun E Kucanova T Brazova D Miklisova M Rencoand V Hanzelova ldquoDiversity and food web structure of nema-tode communities under high soil salinity and alkaline pHrdquoEcotoxicology vol 23 no 8 pp 1367ndash1376 2014

[3] F C Chang C H Ko M J Tsai Y N Wang and C YChung ldquoPhytoremediation of heavy metal contaminated soilby Jatropha curcasrdquo Ecotoxicology vol 23 no 10 pp 1969ndash19782014

[4] AMagdaleno C G Velez M TWenzel andG Tell ldquoEffects ofcadmium copper and zinc on growth of four isolated algae froma highly polluted Argentina riverrdquo Bulletin of EnvironmentalContamination and Toxicology vol 92 no 2 pp 202ndash207 2014

[5] RGuerreroM Piqueras andMBerlanga ldquoMicrobialmats andthe search for minimal ecosystemsrdquo International Microbiologyvol 5 no 4 pp 177ndash188 2002

[6] S Manosa R Mateo and R Guitart ldquoA review of the effectsof agricultural and industrial contamination on the Ebro deltabiota and wildliferdquo Environmental Monitoring and Assessmentvol 71 no 2 pp 187ndash205 2001

[7] J K Cole J R Hutchison R S Renslow et al ldquoPhototrophicbiofilm assembly in microbial-mat-derived unicyanobacte-rial consortia model systems for the study of autotroph-heterotroph interactionsrdquo Frontiers in Microbiology vol 5article 109 2014

[8] D Hoffmann J Maldonado M F Wojciechowski and FGarcia-Pichel ldquoHydrogen export from intertidal cyanobacterialmats sources fluxes and the influence of community composi-tionrdquo Environmental Microbiology 2015

[9] D E Antibus L G Leff B L Hall J L Baeseman and C BBlackwood ldquoCultivable bacteria from ancient algal mats fromthe McMurdo Dry Valleys Antarcticardquo Extremophiles vol 16no 1 pp 105ndash114 2012

[10] I Esteve D Ceballos M Martınez-Alonso N Gaju andR Guerrero ldquoDevelopment of versicolored microbial matssuccession of microbial communitiesrdquo in Microbial Mats L JStal and P Caumette Eds vol 35 of NATO ASI Series pp 415ndash420 Springer Berlin Germany 1994

[11] M Seder-Colomina A Burgos J Maldonado A Sole andI Esteve ldquoThe effect of copper on different phototrophicmicroorganisms determined in vivo and at cellular level byconfocal laser microscopyrdquo Ecotoxicology vol 22 no 1 pp 199ndash205 2013

[12] J Maldonado A de los Rios I Esteve et al ldquoSequestrationand in vivo effect of lead on DE2009 microalga using high-resolutionmicroscopic techniquesrdquo Journal of HazardousMate-rials vol 183 no 1ndash3 pp 44ndash50 2010

[13] D Rai L E Eary and J M Zachara ldquoEnvironmental chemistryof chromiumrdquo Science of the Total Environment vol 86 no 1-2pp 15ndash23 1989

[14] V Gomez and M P Callao ldquoChromium determination andspeciation since 2000rdquo TrAC Trends in Analytical Chemistryvol 25 no 10 pp 1006ndash1015 2006

[15] S E Manahan Environmental Chemistry CRC Press Taylorand Francis Group Boca Raton Fla USA 9th edition 2009

[16] GNziguheba andE Smolders ldquoInputs of trace elements in agri-cultural soils via phosphate fertilizers in European countriesrdquoScience of the Total Environment vol 390 no 1 pp 53ndash57 2008

[17] M Faisal and S Hasnain ldquoComparative study of Cr(VI)uptake and reduction in industrial effluent by Ochrobactrumintermedium and Brevibacterium sprdquo Biotechnology Letters vol26 no 21 pp 1623ndash1628 2004

[18] S Sultan and S Hasnain ldquoReduction of toxic hexavalentchromium by Ochrobactrum intermedium strain SDCr-5 stim-ulated by heavy metalsrdquo Bioresource Technology vol 98 no 2pp 340ndash344 2007

[19] G J Puzon J N Petersen A G Roberts D M Kramer andL Xun ldquoA bacterial flavin reductase system reduces chromateto a soluble chromium (III)-NAD+ complexrdquo Biochemical andBiophysical Research Communications vol 294 no 1 pp 76ndash812002

[20] G J Puzon R K Tokala H Zhang D Yonge BM Peyton andL Xun ldquoMobility and recalcitrance of organo-chromium(III)complexesrdquo Chemosphere vol 70 no 11 pp 2054ndash2059 2008

[21] Y Cheng F Yan F Huang et al ldquoBioremediation of Cr(VI)and immobilization as Cr(III) by Ochrobactrum anthropirdquoEnvironmental Science and Technology vol 44 no 16 pp 6357ndash6363 2010


[22] N Pfennig and H G Trupper ldquoThe family Chromatiaceaerdquo inThe Prokaryotes A Balows H G Trupper M Dworkin andK H Schleifer Eds pp 3200ndash3221 Springer Berlin Germany2nd edition 1992

[23] Z M Puyen E Villagrasa J Maldonado I Esteve and ASole ldquoViability and biomass of Micrococcus luteus DE2008 atdifferent salinity concentrations determined by specific fluo-rochromes and CLSM-image analysisrdquo Current Microbiologyvol 64 no 1 pp 75ndash80 2012

[24] M Sato Y Murata M Mizusawa H Iwahashi and S Oka ldquoAsimple and rapid dual-fluorescence viability assay for microal-gaerdquo Microbiology and Culture Collections vol 20 pp 53ndash592004

[25] W S Rasband ImageJ US National Institutes of HealthBethesda Md USA 1997ndash2014 httpimagejnihgovij

[26] J C Fry ldquoDirect methods and biomass estimationrdquoMethods inMicrobiology vol 22 pp 41ndash85 1990

[27] A Burgos J Maldonado A de los Rios A Sole and I EsteveldquoEffect of copper and lead on two consortia of phototrophicmicroorganisms and their capacity to sequestermetalsrdquoAquaticToxicology vol 140-141 pp 324ndash336 2013

[28] N Mallick and F H Mohn ldquoUse of chlorophyll fluorescencein metal-stress research a case study with the green microalgaScenedesmusrdquo Ecotoxicology and Environmental Safety vol 55no 1 pp 64ndash69 2003

[29] S M Prasad J B Singh L C Rai and H D Kumar ldquoMetal-induced inhibition of photosynthetic electron transport chainof the cyanobacterium Nostoc muscorumrdquo FEMS MicrobiologyLetters vol 82 no 1 pp 95ndash100 1991

[30] M Roldan C Ascaso and J Wierzchos ldquoFluorescent finger-prints of endolithic phototrophic cyanobacteria living withinhalite rocks in the atacama desertrdquo Applied and EnvironmentalMicrobiology vol 80 no 10 pp 2998ndash3006 2014

[31] S Pereira E Micheletti A Zille et al ldquoUsing extracellularpolymeric substances (EPS)-producing cyanobacteria for thebioremediation of heavy metals do cations compete for theEPS functional groups and also accumulate inside the cellrdquoMicrobiology vol 157 no 2 pp 451ndash458 2011

[32] R de Philippis and M Vincenzini ldquoOutermost polysaccharidicinvestments of cyanobacteria nature significance and possibleapplicationsrdquo Recent Research Developments in Microbiologyvol 7 pp 13ndash22 2003

[33] S Ozturk B Aslim Z Suludere and S Tan ldquoMetal removal ofcyanobacterial exopolysaccharides by uronic acid content andmonosaccharide compositionrdquo Carbohydrate Polymers vol 101no 1 pp 265ndash271 2014

[34] A Celekli M Kapı and H Bozkurt ldquoEffect of cadmiumon biomass pigmentation malondialdehyde and proline ofScenedesmus quadricauda var longispinardquo Bulletin of Environ-mental Contamination andToxicology vol 91 no 5 pp 571ndash5762013

[35] T E Jensen and L M Sicko ldquoPhosphate metabolism in bluegreen algae I Fine structure of the lsquopolyphosphate overplusrsquophenomenon in Plectonema boryanumrdquo Canadian Journal ofMicrobiology vol 20 no 9 pp 1235ndash1239 1974

[36] J Goldberg H Gonzalez T E Jensen and W A CorpeldquoQuantitative analysis of the elemental composition and themass of bacterial polyphosphate bodies using STEM EDXrdquoMicrobios vol 106 no 415 pp 177ndash188 2001

[37] G-J Zhou F-Q Peng L-J Zhang and G-G Ying ldquoBiosorp-tion of zinc and copper from aqueous solutions by two fresh-water green microalgae Chlorella pyrenoidosa and Scenedesmus

obliquusrdquo Environmental Science and Pollution Research vol 19no 7 pp 2918ndash2929 2012

[38] J Kovacik P Babula J Hedbavny O Krystofova and IProvaznik ldquoPhysiology andmethodology of chromium toxicityusing alga Scenedesmus quadricauda as model objectrdquo Chemo-sphere vol 120 pp 23ndash30 2015


These microbial mats receive waters and contaminantsincluding heavy metals dragged by the River Ebro into itsestuary (delta) For this reason in the last few years ourwork group has isolated various microorganisms of thisecosystem and has developed several methods in particularConfocal Laser Scanning Microscopy (CLSM) to determinetheir capacity to tolerate or resist metals as well as evaluatethe effect of these in vivo at both cell and population levelsThese methods used for the in vivo study of phototrophicmicroorganisms have led to obtaining quantitative resultsmore quickly This is mainly due to the minimal necessarymanipulation of the specimens and since these emit naturalfluorescence they do not require staining protocols Fur-thermore the majority of works have evaluated the effectof lead and copper toxicity in isolated microorganisms [11]and the capacity of various microorganisms to uptake thesemetals extra- andor intracellularly using Scanning ElectronMicroscopy (SEM) and Transmission Electron Microscopy(TEM) both coupled to an Energy Dispersive X-Ray (SEM-EDX and TEM-EDX) [12]

However the role that microorganisms play in thissame habitat on chromium detoxification is still unknownChromium can exist in the environment as Cr(III) or Cr(VI)[13 14] and particularly in the Cr(VI) form is extremelytoxic mutagenic and carcinogenic In the environmentchromium is introduced as the by-product of industries[15] and phosphate fertilizers [16] In highly contaminatedhabitats [17 18] the reduction of Cr(VI) to Cr(III) is aneffective method of Cr(VI) detoxification Nevertheless theimmobilization efficiency of Cr(III) is still unclear and dif-ferent reports suggest that soluble organo-Cr(III) complexesare present in various chromate-reducing bacterial systems[19 20] The permanence of soluble forms of Cr(III) causesa serious problem since they can be reoxidized to Cr(VI) Itis for this reason that there is great interest in studying theimmobilization of Cr(III) in pilot-scale experiments [21]

Nowadays there is little information on this process inthe natural environment where the levels of contaminationby chromium are very low as in the River Ebro (lt2120583g Lminus1 Craccording to data from the Ebro Hydrographic Associationin the last 10 years) In these cases although the sameprobably occurs the Cr(VI) is biotransformed to Cr(III) andthis can remain in ecosystems immobilized or not and couldhave a toxic effect on life forms Likewise very little is knownabout the role of indigenous microorganisms in these naturalenvironments with low levels of chromium and also with aprolonged permanence of the metal in the ecosystem

The aim of this work is to determine the role of Geit-lerinema sp DE2011 and Scenedesmus sp DE2009 bothisolated fromEbroDeltamicrobial mats as bioindicators andimmobilizers of chromium and additionally to analyse theeffect of this metal on their biomass and cellular viability

2 Material and Methods

21 Microorganisms and Culture Conditions Geitlerinemasp DE2011 (cyanobacterium) and Scenedesmus sp DE2009(microalga) were isolated from Ebro Delta microbial mats(Tarragona) Spain Isolation and purification of the isolates

were performed by dilution and plating of microbial matssamples Isolated microorganisms were grown in liquidmineral Pfennig medium [22] in 100mL flasks Cultureswere exposed and maintained at 27∘C in a growth chamber(Climas Grow 180 ClimasLab Barcelona) under continuousillumination with a light intensity of 35 120583Emminus2 sminus1 for thecyanobacterium and 10 120583Emminus2 sminus1 for the microalga pro-vided by cold white fluorescence lights These cultures wereused as control in all the experiments performed

22 Preparation of Chromium Stock Solution The stocksolution of chromium was prepared by dissolving Cr(NO

3)3

(Sigma-Aldrich Bellefonte PA US) in deionized Milli-Qwater at the concentration of 1mM Cr(III) and sterilizedby filtration in Millex-GP 02 120583m filters (Millipore USA)Working concentrations of Cr(III) were obtained by serialdilution This solution was stored in the dark at 4∘C

23 Pigment Analysis of the Strains Using Confocal LaserScanning Microscopy The tolerance and the in vivo effectof chromium on cultures of Geitlerinema sp DE2011 andScenedesmus sp DE2009 were determined by 120582scan functionof CLSM (CLSM Leica TCS SP5 Leica Heidelberg Ger-many) Moreover in order to evaluate the effect of chromiumon the biomass and viability of Scenedesmus sp DE2009 amodification of the FLU-CLSM-IA (Fluorochrome-CLSM-Image Analysis) method described by Puyen et al [23] wasused

231 120582scan Function Cultures of Geitlerinema sp DE2011and Scenedesmus sp DE2009 were contaminated at differentCr(NO

3)3concentrations 0025 0050 01 025 050 075

1 and 5 120583M for the cyanobacterium DE2011 and 025 050075 1 5 10 15 and 25120583M for the microalga DE2009 Allexperiments were performed for 9 days under the sameconditions mentioned in Section 21

Pigment analysis was realized by the 120582scan function ofCLSM This technique provides information on the state ofthe photosynthetic pigments of phototrophic microorgan-isms on the basis of the emission wavelength region andthe fluorescence intensity emitted (autofluorescence) Eachimage sequence was obtained by scanning the same 119909119910optical section throughout the visible spectrum Images wereacquired at the 119911 position at which the fluorescence wasmaximal and acquisition settings were constant throughouteach experiment The sample excitation was carried out withan Argon Laser at 488 nm (120582exe 488) with a 120582 step size of3 nm for an emission wavelength between 550 and 748 nm

In order to measure the mean fluorescence intensity(MFI) of the 119909119910120582 data sets the Leica Confocal Software(Leica Microsystems CMS GmbH) was used In these con-focal images the pseudocolour palette 4 was selected wherewarm colours represented the maximum intensities and coldcolours represented the low intensities of fluorescence Theregions-of-interest (ROIs) function of the software was usedto measure the spectral signature For each sample 70 ROIsof 1 120583m2 taken from cells were analysed




















4at 4∘C for 2








(a)

(c) (d)

(b)









600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

050120583M075120583M

1120583M5120583M

(a)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

1120583M5120583M

10120583M15120583M25120583M

(b)











(a) (b)

600

610

620

630

640

650

660

670

680

690

700

710

720

730

740

750

020406080

100120140160180200


Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

652

655

658

661

664

667

670

673

676

165170175180185190195

Wavelength (nm)

MFI

075120583M025120583M100 nM

50 nM25 nM

(c)

(d) (e)

CaPd

AuAuNaO

Au

Ca

AuP

C

0 2 4 6 8 10(keV)

(f)

Figure 3 Continued


(g) (h)

Ca TiNa ClCu

ClTi

CuCuTi

PO

CaC

0 2 4 6 8 10(keV)

(i)








(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References




























































4at 4∘C for 2








(a)

(c) (d)

(b)









600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

050120583M075120583M

1120583M5120583M

(a)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

1120583M5120583M

10120583M15120583M25120583M

(b)











(a) (b)

600

610

620

630

640

650

660

670

680

690

700

710

720

730

740

750

020406080

100120140160180200


Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

652

655

658

661

664

667

670

673

676

165170175180185190195

Wavelength (nm)

MFI

075120583M025120583M100 nM

50 nM25 nM

(c)

(d) (e)

CaPd

AuAuNaO

Au

Ca

AuP

C

0 2 4 6 8 10(keV)

(f)

Figure 3 Continued


(g) (h)

Ca TiNa ClCu

ClTi

CuCuTi

PO

CaC

0 2 4 6 8 10(keV)

(i)








(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References













































(a)

(c) (d)

(b)









600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

050120583M075120583M

1120583M5120583M

(a)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

1120583M5120583M

10120583M15120583M25120583M

(b)











(a) (b)

600

610

620

630

640

650

660

670

680

690

700

710

720

730

740

750

020406080

100120140160180200


Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

652

655

658

661

664

667

670

673

676

165170175180185190195

Wavelength (nm)

MFI

075120583M025120583M100 nM

50 nM25 nM

(c)

(d) (e)

CaPd

AuAuNaO

Au

Ca

AuP

C

0 2 4 6 8 10(keV)

(f)

Figure 3 Continued


(g) (h)

Ca TiNa ClCu

ClTi

CuCuTi

PO

CaC

0 2 4 6 8 10(keV)

(i)








(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References










































600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

050120583M075120583M

1120583M5120583M

(a)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Control

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

1120583M5120583M

10120583M15120583M25120583M

(b)











(a) (b)

600

610

620

630

640

650

660

670

680

690

700

710

720

730

740

750

020406080

100120140160180200


Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

652

655

658

661

664

667

670

673

676

165170175180185190195

Wavelength (nm)

MFI

075120583M025120583M100 nM

50 nM25 nM

(c)

(d) (e)

CaPd

AuAuNaO

Au

Ca

AuP

C

0 2 4 6 8 10(keV)

(f)

Figure 3 Continued


(g) (h)

Ca TiNa ClCu

ClTi

CuCuTi

PO

CaC

0 2 4 6 8 10(keV)

(i)








(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References










































(a) (b)

600

610

620

630

640

650

660

670

680

690

700

710

720

730

740

750

020406080

100120140160180200


Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

652

655

658

661

664

667

670

673

676

165170175180185190195

Wavelength (nm)

MFI

075120583M025120583M100 nM

50 nM25 nM

(c)

(d) (e)

CaPd

AuAuNaO

Au

Ca

AuP

C

0 2 4 6 8 10(keV)

(f)

Figure 3 Continued


(g) (h)

Ca TiNa ClCu

ClTi

CuCuTi

PO

CaC

0 2 4 6 8 10(keV)

(i)








(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References










































(g) (h)

Ca TiNa ClCu

ClTi

CuCuTi

PO

CaC

0 2 4 6 8 10(keV)

(i)








(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References










































(a) (b)

630 640 650 660 670 680 690 700 710 720 730 740 7500

20406080

100120140160180200

Wavelength (nm)

Mea

n flu

ores

cenc

e int

ensit

y (M

FI)

679 682 685 688 691 694160165170175180185

Wavelength (nm)

MFI

Control 075120583M025120583M050120583M

1120583M5120583M

(c)

(d) (e)

AuCrAu

Pd Ca

AuMg

NaCr

AuP

CrO

CaC

0 2 4 6 8 10(keV)

(f)

Figure 4 Continued


(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References










































(g) (h)

OsCr OsS TiNa

Ti

OsCl Ca

Cr

Cl

TiP

Cr

OCCa

0 2 4 6 8 10(keV)

(i)




4 Conclusions






Con

trol

0

20

40

60

80

100


()

100120583

M C

r

200120583

M C

r

500120583

M C

r

075120583

M C

r

25120583

M C

r



Acknowledgments


References










































Con

trol

0

20

40

60

80

100


()

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Acknowledgments


References









































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