4th international symposium on bacterial … · joaquin caro-astorga, imperial college london, uk...

4th INTERNATIONAL SYMPOSIUM ON BACTERIAL NANOCELLULOSE

Book of abstracts

3-4th October, 2019

Almeida Garrett Library Auditorium, Porto-Portugal

WELCOME MESSAGE

Dear all,

On behalf of the Organizing Committee, we would like to warmly welcome you to the 4th International

Symposium on Bacterial Nano-Cellulose (4ISBNC)!

This event, which has been held biennially since 2013, mobilizes experts from the field, to discuss all

aspects of research related to Bacterial Nano-Cellulose Biotechnology, including the biology and genetics

of producing microorganisms, production biotechnologies and different areas of application, namely

biomedicine, food and cosmetic industries, composites, pulp and paper. Previous editions took place in

New Orleans (2013), Gdansk (2015) and Fukuoka (2017).

The 4th ISBNC will be held in Porto - Portugal, at the Almeida Garrett Library Auditorium, between 3 and

4, October, 2019.

As with previous events, this Symposium aims to attract international researchers to communicate and

share the latest developments in this fast-moving and continually expanding field of Bacterial Nano-

Cellulose.

Reasons to attend:

Present your latest research

Learn and share your knowledge with and from internationally renowned researchers

Understand the current state of research and the challenges to future discovery

Meet and socialize with fellow scientists from around the world

Take some time to explore the vibrant city of Porto!

We believe the next few years will witness the translation of BNC from Academia to the Bioeconomy. Be

a part of it and contribute to this exciting challenge!

Miguel Gama, PhD

Chair for 4th International Symposium of Bacterial Nano Cellulose

(4ISBNC 2019, Porto, Portugal)

ORGANIZING COMMITTE

SCIENTIFIC COMMITTEE

Chair: Miguel Gama (University of Minho, Portugal)

Fernando Dourado (University of Minho, Portugal)

Carmen Freire (University of Aveiro, Portugal)

Armando Silvestre (University of Aveiro, Portugal)

Feng Hong (Donghua University, China)

Yizao Wan (East China Jiaotong University, China)

Dieter Klemm (Friedrich Schiller University of Jena, Germany)

Stanislaw Bielecki (Lodz University of Technology, Poland)

Morsyleide Freitas Rosa (Embrapa Agroindústria Tropical, Brazil)

José Domingos Fontana (Federal University of Technology - Paraná, Brazil)

Inder Saxena (University of Texas, USA)

LOCAL ORGANIZING COMMITTEE

Miguel Gama (University of Minho, Portugal)

Fernando Dourado (University of Minho, Portugal)

Carmen Freire (University of Aveiro, Portugal)

Armando Silvestre (University of Aveiro, Portugal)

Ana Cristina Rodrigues (University of Minho, Portugal)

SPONSORS

ACKNOWLEDGMENTS

DETAILED PROGRAM

Day 1 (October 2nd)

18h Opening ceremony (Almeida Garret Library)

Day 2 (October 3rd)

9h Opening ceremony

SESSION 1 MOLECULAR TOOLS FOR IMPROVED PRODUCTION & PROPERTIES

chair person: Inder Saxena

9h15 Keynote lecture: Upstream processing leading to improvements in bacterial nanocellulose properties and productivity

Stanislaw Bielecki, Lodz University of Technology, Poland

9h45 In vitro cellulose synthesis using a recombinant cellulose synthase

Kenji Tajima, Hokkaido University, Japan

10h00 Bringing synthetic biology to bacterial nanocellulose

Tom Ellis, Imperial College London, UK

10h15 Genetics of cellulose biosynthesis in Gluconacetobacter xylinus

Inder Saxena, The University of Texas at Austin, USA

10h30 Genetically engineering bacteria to produce diverse bacterial nanocellulose material properties

Goosens VJ, Imperial College London, UK

10h45 Genomics-aided deciphering of BNC synthesis and regulation

Małgorzata Ryngajłło, Lodz University of Technology, Poland

11h00 COFFEE BREAK

SESSION 2 COMPOSITES & APPLICATIONS: PART 1

chair person: Yizao Wan

11h30 Keynote lecture: Bacterial Nano Cellulose: Exploring and developing the potential of an intriguing, low-cost and fully biobased product towards the demands of our contemporary society

Falk Liebner, BOKU University, Austria

12h00 Sustainable and recyclable thermoelectric paper by bacteria farming

Anna Roig, Institute of Materials Science of Barcelona, Spain

12h15 Bacterial nanocellulose a sustainable material: applications ranging from electronics to biosensors

Elvira Fortunato, CENIMAT/I3N and CEMOP-UNINOVA, Portugal

12h30 Optically transparent bacterial nanocellulose-based composites as substrate for flexible organic light emitting diode

Hernane Barud, University of São Paulo, Brazil

12h45 Cellulose patches in plants

Anna Laromaine, Institute of Materials Science of Barcelona, Spain

13h00 Bacterial Nanocellulose: A brief history and future prospects at “Embrapa”

Morsyleide de Freitas Rosa, EMBRAPA, Brazil

13h15 LUNCH

SESSION 3 COMPOSITES & APPLICATIONS: PART 2

chair person: Falk Liebner

14h45 Keynote lecture: Making fluffy Bacterial Cellulose Nanopapers: Useful filters with tailored properties

Koon-Yang Lee, University of Vienna, Austria

15h15 Bacterial NanoCellulose as a Natural Component of Next-Generation Bio-Composites

Karolina Ludwicka, Lodz University of Technology, Poland

15h30 Development and application of bacterial nanocellulose/graphene nanocomposites

Yizao Wan, East China Jiaotong University, China

15h45 BNC composites for the leather industry

Fernando Dourado, University of Minho & Satisfibre, Portugal

16h00 Transparent poly(methyl methacrylate) composites based on bacterial cellulose nanofibre networks with improved fracture resistance and impact strength

Koon-Yang Lee, Imperial College London, UK

16h15 Bacterial nanocellulose within the context of pulp-and-paper emerging biorefineries

Carlos Pascoal Neto, The Navigator Company, Portugal

16h30 Bacterial cellulose for edible films and coatings

Henriette M.C. Azeredo, EMBRAPA, Brazil

16h45 COFFEE-BREAK

SESSION 4 SHARP PRESENTATIONS: PART 1

chair person: Anna Roig

17h15 Functionalization, Regeneration and Modification of Bacterial NanoCellulose

Joaquin Caro-Astorga, Imperial College London, UK

17h22 Confined spatial nanoparticle distribution in bacterial nanocellulose millefeuille

Soledad Roig-Sánchez, Institute of Materials Science of Barcelona

17h29 Role of motA and motB genes in bacterialnanocellulose biosynthesis in Komagataeibacter genus Paulina Jacek, Lodz University of Technology, Poland

17h36 Bacterial nanocellulose as an ocular bandage material

Irene Anton-Sales, Institute of Materials Science of Barcelona, Spain

17h43 END OF THE SESSION

19h30 Dinner

Day 3 (October 4th)

SESSION 5 PRODUCTION AND FORMULATION

chair person: Stanislaw Bielecki

9h00 Keynote lecture: 3D-structured Cellulose Biofilms and Applications

Orlando J. Rojas, Aalto University, Finland

9h30 A Dry Bacterial Cellulose-Carboxymethyl Cellulose Formulation as Stabilizer for Pickering Oil-in-Water Emulsions

Daniela Martins, Universidade do Minho, Portugal

9h45 On to the impact of low cost substrates for BNC production

Miguel Gama, University of Minho, Portugal

10h00 Cider waste as a resource for bacterial nanocellulose production and new approaches for advanced applications

Leire Urbina Moreno, University of the Basque Country, Spain

10h15 Bacterial nanocellulose-based films with antibacterial and conductive properties for active and intelligent food packaging

Vilela, C., Universidade do Aveiro, Portugal

10h30 COFFEE-BREAK AND GROUP PHOTO

SESSION 6 BIOMEDICAL APPLICATIONS, PART 1

chair person: Tom Ellis

11h15 Keynote lecture: Pharmaceutical Insights: Overcoming the Challenges of Bacterial Nanocellulose for Controlled Drug Delivery

Dagmar Fischer, Friedrich Schiller University Jena, Germany

11h45 Adjusting BNC for 3D tissue engineering by modifications at different stages of its production Katarzyna Kubiak, Lodz University of Technology, Poland

12h00 Evaluation of Three Types of Bacterial Nanocellulose Tubes for Application as Vascular Graft: Bioreactors Determine Tubes Structure and Properties

Feng Hong, Donghua University, China

12h15 Preparation and properties of type I collagen/ quaternized chitosan/ Bacterial nanocellulose composite films for multifunctional wound dressings

Haiyong Ao, East China Jiaotong University, China

12h30 Bacterial NanoCellulose-Polyhydroxyalkanoate hydrogels with antimicrobial capacity against Staphylococcus aureus

Auxiliadora Prieto Jiménez, Biological Research Center (CIB-CSIC), Spain

12h45 LUNCH

SESSION 7 BIOMEDICAL APPLICATIONS, PART 2

chair person: Dana Kralisch

14h30 Study on preparation and properties of micro-nanofibers composite vascular scaffold

Shanshan Yang, East China Jiaotong University, China

14h45 Bacterial nanocellulose-hyaluronic acid microneedles for skincare applications

Daniela Filipa da Silva Fonseca, Universidade do Aveiro, Portugal

15h00 The Prospect of Bacterial NanoCellulose Impregnated with Antimicrobials in Treatment of Biofilm-Related Bone Infections

Adam Junka, Medical University of Wroclaw, Poland

15h15 Modified BNC as a vascular prosthesis and cardiac valve prosthesis - preclinical studies

Piotr Siondalski, Medical University of Gdansk, Bowil Biotech, Poland

15h30 Delivery of antiseptic solutions by a bacterial cellulose wound dressing: PHMB, octenidine and povidone-iodine

Ives Bernardelli de Mattos, Fraunhofer Institute for Silicate Research ISC, Germany

15h45 COFFEE-BREAK

SESSION 8 SHARP PRESENTATIONS: PART 2

chair person: Miguel Gama

16h15 Development and characterization of native and citric acid-modified cellulose's drug-delivery system for bacterial biofilm eradication

Karol Fijalkowski, West Pomeranian University of Technology, Poland

16h22 A biodegradable antibacterial nanocomposite based on oxidized bacterial nanocellulose for fast hemostasis and wound healing

Haibin Yuan, Donghua University, China

16h29 Peptide Functionalization of bacterial cellulose for antimicrobial activity

Ana M. Hernandez Arriaga, Biological Research Center (CIB-CSIC), Spain

16h36 Manufacturing of modified bacterial nanocellulose as tailor-made anti-inflammatory wound dressing

Uwe Beekmann, Friedrich Schiller University Jena, Germany

16h43 Modification of bacterial nanocellulose membranes for tailored drug carrier

Marcia Margarete Meier, Santa Catarina State University, Brazil

SESSION 9 BC COMPANIES AND MARKET PULL

chair person: Koon-Yang Lee

16h50 Keynote lecture: Bacterial nanocellulose product design towards biomedical and diagnostic applications

Dana Kralisch, JeNaCell, Germany

17h20 Microengineered biosynthesized cellulose as antifibrotic protection for implantable medical devices

Simone Bottan, Hylomorph, Switzerland

17h35 NullarborTM Tree-Free Fibre and other Commercial Applications for Bacterial Nanocellulose

Gary Cass, Nanollose, Australia

17h50 From lab to market

Kamil Palmikowsky, Bowil Biotech, Poland

18h05 END OF SYMPOSIUM/NEXT SYMPOSIUM

Table of Contents

ORAL COMMUNICATIONS: October 3rd ........................................................................................................ 1

SESSION 1 – Molecular tools for improved production & properties (chair person: Inder Saxena) ............ 1

KEYNOTE LECTURE: Upstream processing leading to improvements in bacterial nanocellulose

properties and productivity ...................................................................................................................... 2

In vitro cellulose synthesis using a recombinant cellulose synthase ........................................................ 4

Bringing synthetic biology to bacterial nanocellulose .............................................................................. 5

Genetics of cellulose biosynthesis in Gluconacetobacter xylinus ............................................................. 6

Genetically engineering bacteria to produce diverse bacterial cellulose material properties ................. 7

Genomics-aided deciphering of BNC synthesis and regulation ................................................................ 8

SESSION 2 – Composites & applications: part 1 (chair person: Yizao Wan) ................................................. 9

KEYNOTE LECTURE: Bacterial Nano Cellulose: Exploring and developing the potential of an intriguing,

low-cost and fully biobased product towards the demands of our contemporary society ................... 10

Sustainable and recyclable thermoelectric paper by bacteria farming .................................................. 12

Bacterial cellulose a sustainable material: applications ranging from electronics to biosensors .......... 13

Optically transparent bacterial cellulose-based composites as substrate for flexible organic light

emitting diode. ........................................................................................................................................ 14

Cellulose patches in plants ...................................................................................................................... 16

Bacterial Nanocellulose: A brief history and future prospects at “Embrapa” ........................................ 18

SESSION 3 – Composites & applications: part 2 (chair person: Falk Liebner)............................................. 19

KEYNOTE LECTURE: Making fluffy Bacterial Cellulose Nanopapers: Useful filters with tailored

properties ................................................................................................................................................ 20

Bacterial NanoCellulose as a Natural Component of Next-Generation Bio-Composites ....................... 21

Development and application of bacterial cellulose/graphene nanocomposites .................................. 23

Bacterial Nanocellulose composites for the textile and leather industries ............................................ 25

Transparent poly(methyl methacrylate) composites based on bacterial cellulose nanofibre networks

with improved fracture resistance and impact strength ........................................................................ 27

Bacterial cellulose within the context of pulp-and-paper emerging biorefineries ................................. 28

Bacterial cellulose for edible films and coatings ..................................................................................... 29

SESSION 4 - Sharp presentations: part 1 (chair person: Anna Roig) ........................................................... 30

Functionalization, Regeneration and Modification of Bacterial Cellulose.............................................. 31

Confined spatial nanoparticle distribution in bacterial nanocellulose millefeuille ................................ 32

Role of motA and motB genes in bionanocellulose biosynthesis in Komagataeibacter genus .............. 34

Bacterial nanocellulose as an ocular bandage material ......................................................................... 35

ORAL COMMUNICATIONS: October 4th ..................................................................................................... 36

SESSION 5 – Production and formulation (chair person: Stanislaw Bielecki) ............................................. 37

KEYNOTE LECTURE: 3D-structured Cellulose Biofilms and Applications ................................................ 38

A Dry Bacterial Cellulose-Carboxymethyl Cellulose Formulation as Stabilizer for Pickering Oil-in-Water

Emulsions ................................................................................................................................................ 39

On to the impact of low cost substrates for BNC production ................................................................. 40

Cider waste as a resource for bacterial cellulose production and new approaches for advanced

applications ............................................................................................................................................. 42

Bacterial nanocellulose-based films with antibacterial and conductive properties for active and

intelligent food packaging ....................................................................................................................... 44

SESSION 6 - Biomedical applications, part 1 (chair person: Dieter Klemm) ............................................... 46

KEYNOTE LECTURE: Pharmaceutical Insights: Overcoming the Challenges of Bacterial Nanocellulose

for Controlled Drug Delivery ................................................................................................................... 47

Adjusting BNC for 3D tissue engineering by modifications at different stages of its production .......... 48

Evaluation of Three Types of Bacterial Nanocellulose Tubes for Application as Vascular Graft:

Bioreactors Determine Tubes Structure and Properties ........................................................................ 49

Preparation and properties of type I collagen/ quaternized chitosan/ Bacterial cellulose composite

films for multifunctional wound dressings ............................................................................................. 50

Bacterial Cellulose-Polyhydroxyalkanoate hydrogels with antimicrobial capacity against

Staphylococcus aureus ............................................................................................................................ 51

SESSION 7 - Biomedical applications, part 2 (chair person: Dana Kralisch) ................................................ 53

Study on Preparation and properties of micro-nanofibers composite vascular scaffold ....................... 54

Bacterial nanocellulose-hyaluronic acid microneedles for skincare applications .................................. 56

The Prospect of Bacterial Cellulose Impregnated with Antimicrobials in Treatment of Biofilm-Related

Bone Infections ....................................................................................................................................... 58

Modified BNC as a vascular prosthesis and cardiac valve prosthesis - preclinical studies. .................... 60

Delivery of antiseptic solutions by a bacterial cellulose wound dressing: PHMB, octenidine and

povidone-iodine ...................................................................................................................................... 61

SESSION 8 - Sharp presentations: part 2 (chair person: Anna Roig) ........................................................... 63

Development and characterization of native and citric acid-modified cellulose's drug-delivery system

for bacterial biofilm eradication ............................................................................................................. 64

A biodegradable antibacterial nanocomposite based on oxidized bacterial cellulose for fast hemostasis

and wound healing.................................................................................................................................. 66

Peptide Functionalization of bacterial cellulose for antimicrobial activity ............................................ 67

Manufacturing of modified bacterial cellulose as tailor-made anti-inflammatory wound dressing ...... 69

Modification of bacterial nanocellulose membranes for tailored drug carrier ...................................... 71

SESSION 9 - BC companies and market pull (chair person: Koon-Yang Lee) .............................................. 73

KEYNOTE LECTURE: BioTech Cellulose: Process-controlled design of multilayered materials, coatings

and composites ....................................................................................................................................... 74

Microengineered biosynthesized cellulose as antifibrotic protection for implantable medical devices75

NullarborTM Tree-Free Fibre and other Commercial Applications for Bacterial Nanocellulose.............. 77

Bacterial cellulose product design towards biomedical and diagnostic applications ............................ 78

BNC products: From Lab to Market ........................................................................................................ 80

Posters ........................................................................................................................................................ 81

P1: Bacterial Cellulose as a Material for Innovative Cardiovascular Implants ........................................ 82

P2: Probiotic edible films from bacterial cellulose/cashew tree gum .................................................... 84

P3: Scaffolds of bacterial cellulose functionalized with bioactive metal phosphates ............................ 85

P4: Evaluation of bacterial cellulose degradation after exposure in different abiotic and biotic

components ............................................................................................................................................ 87

P5: Antimicrobial Wound Dressing Based on Bacterial Cellulose Containing Silver Nanoparticles ....... 88

P6: Nano-fibrillated bacterial cellulose with hydrophobic surface characteristics by heterogeneous

silylation .................................................................................................................................................. 90

P7: Construction and performance study of integrated bacterial cellulose hernia mesh ...................... 91

P8: Preparation and properties of graphene oxide/bacterial cellulose nanocomposites with gradient

structure .................................................................................................................................................. 93

P9: Enhancement of mechanical and biological properties of calcium phosphate bone cement by

incorporating bacterial cellulose ............................................................................................................ 95

P10: Step-by-step self-assembly of 2D few-layer reduced graphene oxide into 3D architecture of

bacterial cellulose for a robust, ultralight, and recyclable all-carbon absorbent ................................... 97

P11: Study on Microwave Absorbing Properties of Fe3O4/Bacterial Cellulose Composites ................... 99

P12: Silver nanowire-doped bacterial cellulose as potential antimicrobial wound dressing ............... 101

P13: Effect of Nanofibrillated bacterial cellulose (NFBC) inclusion on the physical properties of pulp

fiber sheets and polyvinyl alcohol films ................................................................................................ 103

P14: A Toolbox of Pharmaceutical Strategies for the Controllable Skin Delivery of Boswellia Extracts

using Bacterial Nanocellulose ............................................................................................................... 104

P15: Obtaining and characterization of new composite of bacterial nanocelullose containing

bioliquefied brown coal ........................................................................................................................ 106

P16: Biosynthesis of BNC and BNC-based nitrocellulose ...................................................................... 108

P17: Characterization of the proteins encoded by the second cellulose synthase operon ................. 109

P18: Novel chitosan-coated bacterial cellulose hydrogels for controlled delivery systems ................ 111

P19: Evaluation of in vitro cytotoxicity of Bacterial Cellulose obtained from agroindustrial byproducts

.............................................................................................................................................................. 113

P20: Effect of the combination of bacterial nanocellulose dressing with secondary dressings: an in

vitro and in vivo approach .................................................................................................................... 115

P21: Synthesis of bacterial cellulose by Komagataeibacter strains in agitated and static process

conditions.............................................................................................................................................. 116

P22: Evaluation of the synthesis of bacterial cellulose by acetic acid bacteria isolated from vinegar

industry using soybean molasses as fermentative substrate ............................................................... 117

P23: Surface modification of bacterial cellulose membranes for microfluidic applications ................ 119

P24: Identification and Engineering of Native and Non-native Secretion Systems in K. rhaeticus to

engineer biomaterials with unique properties ..................................................................................... 121

P25: Challenges on specific surface area analysis of cellulosic materials ............................................. 122

P26: Influence of different types of bacterial nanocellulose on development of oil-in-water Pickering

emulsions .............................................................................................................................................. 123

P27: Nanopapers based on bacterial cellulose with conductive, magnetic or photochromic properties

.............................................................................................................................................................. 125

P28: Synthesis of hydrophobic bacterial cellulose nanocrystals for the retention of oils .................... 127

P29: Life cycle assessment of bacterial cellulose production in soybean molasses culture medium .. 128

P30: Influence of fermentation time and bacterial strains on properties of bacterial cellulose produced

by soybean molasses fermentation ...................................................................................................... 130

P31: Microwave-assisted periodate oxidation and characterization of bacterial cellulose ................. 132

P32: Bioactivity and biocompatibility of hybrid systems based on bacterial cellulose/strontium apatite

.............................................................................................................................................................. 133

P33: BNC-based scaffolds for tissue engineering: preparation and characterization .......................... 135

P34: Bioactive dressing based on bacterial cellulose and papain for wound debridement ................. 137

P35: Towards Texture Control of Bacterial Cellulose ........................................................................... 138

P36: Spray-dried redispersible bacterial nanocellulose microspheres ................................................. 139

P37: Optimization of bacterial nanocellulose fermentation using lignocellulosic residues and

development of novel BNC-starch composites .................................................................................... 141

P38: Synthesis and characterization of oxidized bacterial cellulose through electrochemical methods:

its biodegradability and potential as hemostatic material ................................................................... 143

AUTHORS LIST ........................................................................................................................................... 145

ORAL COMMUNICATIONS: October 3rd


3-4th October, 2019


SESSION 1 – Molecular tools for improved production & properties (chair person: Inder Saxena)


3-4th October, 2019


KEYNOTE LECTURE: Upstream processing leading to improvements in bacterial nanocellulose

properties and productivity

Stanislaw Bielecki

Institute of Technical Biochemistry, Lodz University of Technology, Poland [email protected]

Abstract

Recently, the interest in efficient use of diverse natural substrates and processes becomes more and more

significant. An example of a rational usage of natural resources is the fabrication of bacterial nanocellulose

(BNC), which may be produced from various renewable materials. BNC is a natural polymer, synthesized

by a number of microorganisms, among which the strains from the Komagataeibacter genus are the most

recognized and efficient producers. These bacteria produce an extracellular, chemically purest cellulose

which is ten times stronger than the plant-based equivalent. Moreover, BNC is characterized by numerous

unique features, such as excellent biocompatibility, high crystallinity and water holding capacity,

mechanical durability. Because of these properties this natural biomaterial have found numerous

potential applications in fabrication of bioproducts and is already considered as a ‘bio-base’ for the

development of novel materials in various fields, among others food processing, electronics, defence

industry, paper making, chemical and textile industries, as well as in medicine, especially for tissue

engineering. However, the precise control of bacterial nanocellulose biosynthesis is a major challenge for

biotechnology, in particular when the properties of BNC should be specifically tailored depending on its

specific application. Biotechnological successes and experience gained for model bacterial and yeast

strains should be quickly adjusted and applied in Komagateibacter genus, along with the knowledge being

acquired at molecular level.

During the presentation current data about the molecular basis of BNC biosynthesis and its regulation, as

well as perspectives for genetic modifications of BNC producers, attainable owing to the newly developed

molecular tools, will be discussed. The requirements for upstream processing parameters like physico-

chemical culture conditions, supplementation of culture media and changes in bioreactor design will be

discussed, taking into consideration novel methods of BNC and BNC composites development. Molecular

tools and ‘omics’ technologies, as well as technological approaches enabling improvement of BNC

production efficiency and costs reduction will be also analysed.

References

(1) Jacek P, Dourado F, Gama M, Bielecki S, (2019) Molecular aspects of bacterial nanocellulose biosynthesis”. Microbial Biotechnology, 0(0).1-17

(2) J Wang, J Tavakoli.Y.Tang (2019) Bacterial cellulose production,properties and application with different culture metods – A review . Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2019.05.008

(3) Michael Florea et al ., (2016) Engineering control of bacterial cellulose production using a genetic

toolkit and a new cellulose-producing strain. PNAS Plus, E3431-E3440

mailto:[email protected]

https://doi.org/10.1016/j.carbpol.2019.05.008


3-4th October, 2019


(4) Jacek P, Szustak M, Kubiak K, Gendaszewska-Darmach E, Ludwicka K, Bielecki S, „Scaffolds for

chondrogenic cells cultivation prepared from bacterial cellulose with relaxed fibers structure induced

genetically”. Nanomaterials, 2018

(5) U.Romeling, M.Galperin (2015) Bacterial cellulose biosynthesis: Diversity of operons, subunits,

products and functions. Trends in Microbiology 23(9):545-557

(6) Cielecka I., Szustak M., Gendaszewska-Darmach E., Kalinowska H., Ryngajłło M., Maniukiewicz W.,

Bielecki S. (2018). Novel bionanocellulose/к-carrageenan composites for tissue engineering. Applied

sciences 8(8): 1352

(6) M. Kolodziejczyk, K.Ludwicka, T. Pankiewicz, S. Bielecki (2017) EP2787072


3-4th October, 2019


In vitro cellulose synthesis using a recombinant cellulose synthase

Kenji Tajima1),*, Bono Aoshima2), Hiroko Ninoyu2), Tomoya Imai3), Takuya Isono1), Takuya Yamamoto1), Toshifumi Satoh1), and Yao Min4)

1)Faculty of Engineering, Hokkaido University, N13W8, Kita-ku, Sapporo 060-8628, Japan 2)Graduate School of Chemical Sciences and Engineering, Hokkaido University, N13W8, Kita-ku, Sapporo 060-8628, Japan 3)RISH, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan 4)Faculty of Advanced Life Science, Hokkaido University, N10W8, Kita-ku, Sapporo, 060-0810, Japan

Abstract

Cellulose produced by bacteria, such as Gluconacetobacter species, is called bacterial cellulose (BC). BC

has exceptional physicochemical properties which are different from those of cellulose produced by

plants; therefore, BC has attracted much attention as a new renewable material. BC is synthesized by

cellulose synthase complexes [Terminal Complex (TC)] existing in cell membranes. TC is thought to be

consist of at least four subunits (CeSABCD) in Gluconacetobacter hansenii (G. hansenii), but overall

structure of TC has not been clarified so far. Since BC productivities for Gluconacetobacter species are

relatively low; therefore, their enhancement is strongly required. For achieving this object, our final goal

is to elucidate the synthetic mechanism of BC by analyzing the overall structure of TC. In this study, we

constructed plasmids including some of cellulose synthesis-related genes (Figure), and then introduced

into Escherichia coli and G. hansenii. Membrane fractions were prepared from these recombinants, and

their cellulose synthase activities were evaluated by in vitro assay system including bis-(3',5')-cyclic-di-

guanosine monophosphate (c-di-GMP). Furthermore, the structures of the obtained products were

analyzed by FT-IR, XRD, SPM, and cellulase treatment.

Figure A cellulose synthesis-related gene cluster from Gluconacetobacter hansenii ATCC23769.


3-4th October, 2019


Bringing synthetic biology to bacterial nanocellulose

Tom Ellis Imperial College London

Abstract

Synthetic biology offers a route to engineering cells and organisms by rewriting their DNA and

reprogramming their behaviour. This foundational technology is revolutionizing biotechnology while also

providing new ways to understand how natural living systems have evolved and operate. One of the most

fascinating and valuable properties of natural living systems is their ability to make diverse and

multifunctional materials. The plant kingdom in particular provides us with a myriad of different materials,

all produced by cells secreting cellulose and other polymers in different compositions and arrangements.

However, understanding how the DNA of plant cells encodes the production of these different material

properties and learning how this can be reprogrammed is sadly close-to-impossible because plants are

inherently complex and reliant on their native material production to survive.

Cellulose-producing bacteria offer the perfect ‘blank slate’ for understanding how DNA code can lead to

desired material properties. As an easy-to-work-with, transformable bacteria, they reliably produce large

amounts of pure nanocellulose that has minimal modification or macrostructure. Using synthetic biology

tools for engineering DNA and introducing this into Komagataeibacter rhaeticus cells, our group is

exploring how genetic coding can be used to change the grown material via the three main ways seen in

nature - via (i) self-patterning of structures, (ii) enzymatic modification and (iii) co-production of other

polymers to give composites. In this talk, I’ll summarise our efforts to better understand and genetically

engineer K. rhaeticus, and how our synthetic biology tools have been used to program the basics of self-

patterning in growing pellicles. Our progress on protein-cellulose composites and enzymatically self-

modified materials from this bacteria will be presented. Finally, the use of Kombucha-inspired bacteria-

yeast co-cultures will be described, and these offer exciting new opportunities for growing smart and

functional cellulose-based materials for diverse applications.


3-4th October, 2019


Genetics of cellulose biosynthesis in Gluconacetobacter xylinus

Inder Saxena

Department of Molecular Biosciences, The University of Texas at Austin, Austin, Tx 78712, USA [email protected]

Abstract

Bacterial cellulose production utilizes various strains of Gluconacetobacter xylinus, given that they are

some of the most prolific producers of cellulose. In fact, most studies of bacterial cellulose biosynthesis

are performed using this bacterium, and it is in this bacterium that the genes for cellulose biosynthesis

were first identified. In addition to identification of genes that have a role in cellulose biosynthesis, in this

bacterium a few other genes and DNA sequences were also identified using conventional genetic analysis.

Even as genome sequences of a number of G. xylinus strains are available, attempts are also being made

to make sense of the information obtained from genetic analysis of a number of cellulose synthesis

mutants. Analysis of a cellulose synthesis mutant of G. xylinus ATCC 23769 led to the identification of an

insertion element IS1238 in this bacterium. The significance of this discovery is interesting as this insertion

element has not been identified in any published genome sequence of various strains of this bacterium.

For developing a better understanding of bacterial cellulose synthesis, it will be important to integrate the

genetic, biochemical and structural studies with the genomic information. It is this strategy that will make

it possible to determine the similarities and differences amongst the various strains of G. xylinus, and

identify regions of the genome that contribute to the high level of cellulose production in this bacterium.



3-4th October, 2019


Genetically engineering bacteria to produce diverse bacterial cellulose material properties

Goosens VJ, Dekker L, Walker K, Matthews S, Lee KY, Ellis T* Imperial College London

Abstract

While plant-derived cellulose is one of the most abundant and widely used organic molecules on earth,

the high level of impurities in limits some of its applications. The bacterial cellulose (BC) produced by some

bacteria as a component their extracellular matrix is ultrapure. Our group isolated a proficient BC-

producing strain Komagataeibacter rhaeticus that is genetically tractable. Importantly we have

sequenced, examined the transcriptome and are developing an expanding Komagataeibacter tool kit

(KTK). Armed with this, our we aim to express and secrete new functional molecules in K. rhaeticus,

thereby altering the chemical nature of cellulose and programming diverse material properties into the

BC. The first system we are focusing on is Curli pili, Curli are amyloid proteins fibres that protrude off

bacterial surfaces. Importantly, amyloid proteins are exceptionally stable, resistant to heat, temperature

and chemicals – all attractive chemical properties to add to a cellulose matrix. We have developed a

Golden Gate-based tool kit that has assisted the cloning of a selection of inducible K. rhaeticus plasmids

expressing the Curli system. We have created a panel of Curli producing operons, resulting in varied

expression of the system as well as functionalized Amyloid fibres. We are currently examining mechanical

properties of the resultant cellulose-amyloid composite. These experiments will confirm whether or not

Curli pili alter the strength, flexibility and nature of the BC thereby enhancing its downstream industrial

applications.


3-4th October, 2019


Genomics-aided deciphering of BNC synthesis and regulation

Małgorzata Ryngajłło, Paulina Jacek, Izabela Cielecka, Katarzyna Kubiak, Marzena Jędrzejczak-Krzepkowska, Stanisław Bielecki

Lodz University of Technology, Institute of Technical Biochemistry, Łódź, Poland

Abstract

In the last decade, a great progress in understanding of the molecular background of bionanocellulose

(BNC) synthesis in Komagataeibacter was made through the research involving genome sequencing,

transcription profiling and genetic engineering (1). Despite these efforts, function of a large part of the

genome remains uncharacterized. This knowledge is necessary for generation of genome-scale metabolic

models and directed strain engineering towards synthesis of cellulose with high yields and of desired

properties.

The growing number of genome sequences of the Komagataeibacter strains opened an opportunity to

find common features of the genus. On the other hand, what remains the unique genetic characteristics

of the species may be the source of their phenotypic distinctiveness. We will discuss the results of our

comparative genomics study which highlighted the diversity of the genus in terms of carbon source

preference, synthesis of extracellular polysaccharides (EPS), c-di-GMP-based regulatory network and

genome stability (2).

We further harnessed the gathered knowledge to explain one of the common features of many of the

Komagataeibacter species which is the significant improvement of cellulose production through the

supplementation of culture media with ethanol. Why ethanol addition contributes to higher BNC yields is

not yet fully understood. It is postulated to provide an alternative energy source, enabling effective use

of glucose for cellulose biosynthesis rather than for energy acquisition. We investigated the effect of

ethanol supplementation on the global gene expression profile of Komagataeibacter xylinus E25 using

RNA sequencing technology (RNA-seq)(4). We will provide genetic evidence explaining the positive effect

of ethanol on cellulose biosynthesis by discussing the expression profiles of genes related to the central

and auxiliary metabolic pathways as well as regulatory circuits.

References

(1) Jacek, P., Dourado, F., Gama, M., Bielecki, S. (2019). Molecular aspects of bacterial nanocellulose biosynthesis. Microbial Biotechnology. 0:1–17

(2) Ryngajłło, M., Kubiak, K., Jędrzejczak-Krzepkowska, M., Jacek, P., Bielecki, S. (2018). Comparative genomics of the Komagataeibacter strains-efficient bionanocellulose producers. MicrobiologyOpen.

(3) Ryngajłło, M., Jacek, P., Cielecka, I., Bielecki, S. (2019). Effect of ethanol supplementation on the transcriptional landscape of bionanocellulose producer Komagataeibacter xylinus E25. Applied Microbiology and Biotechnology.


3-4th October, 2019


SESSION 2 – Composites & applications: part 1 (chair person: Yizao Wan)


3-4th October, 2019


KEYNOTE LECTURE: Bacterial Nano Cellulose: Exploring and developing the potential of an

intriguing, low-cost and fully biobased product towards the demands of our contemporary

society

Hubert Hettegger, Irina Sulaeva, Nicole Doyle nee Pircher, Emmerich Haimer, Huiqing Wang, Sakeena Quraishi, Antje Potthast, Thomas Rosenau, Falk Liebner

Institute for Chemistry of Renewable Resources, University of Natural Resources and Life Sciences Vienna, Konrad-Lorenz-Strasse 24, A-3430 Tulln an der Donau, AUSTRIA

Abstract

Cellulose material research and development is currently driven by vivid activities exploring the

opportunities of pulp-derived cellulose nanoparticles and their particular self-assembling behavior in

dilute aqueous media. However, largely unnoticed, tuning and modification of bacterial cellulose as the

purest form of cellulose has been further advanced paving way towards new applications, large-scale

production and commercialization.

This lecture intends to give an overview of the state of the art in bacterial nanocellulose (BNC) material

research. Owing to its unparalleled purity, hydrophilicity and physiological harmlessness, BNC is attractive

for a broad range of applications in food, cosmetics and biomedicine. While the use of BNC in food

technology relies on its properties as dietary fibers or thermal insulation materials, traditional cosmetic

and wound dressing applications employ primarily the excellent moistening capabilities of BNC. The latter

is employed in cosmetic facial masks to prevent human skin from ageing and wrinkle formation, while it

helps in wound dressings to reduce scarring (1).

The open-porous nanomorphology of BNC pellicles, sheets or spherical particles, their particular three-

dimensional networks composed of entangled cellulose ribbons, huge specific surface, high cellulose

crystallinity and abundance of hydroxyl groups literally invite for further modification, tuning and

adaptation to create smart materials satisfying the needs of our today’s contemporary society (1).

Complementing highlights of BNC materials research from the recent literature, this talk presents also

some contributions of our group exploring mainly the potential of BNC for selected biomedical

applications. This includes improvement of moisture holding capability by alginates, reducing the

stickiness to wounds through direct polymerization of ethyl 2-cyanoacrylate, hydrophobization with alkyl

ketene dimer (2), loading of BNC alcogels with bioactive compounds, such as D-panthenol and L-ascorbic

acid using single-batch scCO2 antisolvent precipitation and scCO2 drying to afford respective aerogels (3),

modeling of their release behavior into aqueous environment (3), bactericidal modification using

polyhexamethylene biguanide, covalent decoration of bacterial cellulose 3D networks with

semiconductor quantum dots and carbon dots for sensing applications (4), or reinforcement of bacterial

cellulose aerogels with interpenetrating networks of biocompatible polymers, such as PMMA (5). Finally,

the development of photoactive BNC matrices for inactivation of gram-positive bacteria S. aureus and B.

subtilis and destruction of odorous compounds emitted from chronic wounds will be reported.


3-4th October, 2019


References

(1) Liebner, F., Pircher, N., Rosenau, T. (2016). Bacterial cellulose aerogels. In: M. Gama, F. Dourado, S. Bielecki (eds.), Bacterial Nanocellulose: From Biotechnology to Bio-Economy. Elsevier 2016, ISBN 978-0-444-63458-0, chapter 5, pp. 73-108. (2) Russler, A., Wieland, M., Bacher, M., Henniges, U., Liebner, F., Miethe, P., Potthast, A., Rosenau, T. (2012). AKD-modification of bacterial cellulose aerogels in scCO2. Cellulose 19:1337-1349. (3) Haimer, E., Wendland, M., Rosenau, T., Liebner, F. (2010). Loading of bacterial cellulose aerogels with pharmaceutically active compounds by anti-solvent precipitation with supercritical carbon dioxide. Macromolecular Symposium. 294:64–74. (4) Quraishi, S., Plappert, S., Ungerer, B., Taupe, P., Altmutter, W., Liebner, F. (2019). Bacterial cellulose - carbon dot hybrid nanopaper for sensing applications. Applied Sciences 9/1:107-121. (5) Pircher, N., Veigel, S., Aigner, N., Nedelec, J.-M., Rosenau, T., Liebner, F. (2014). Reinforcement of bacterial cellulose aerogels with biocompatible polymers. Carbohydrate Polymers 111:505-513.


3-4th October, 2019


Sustainable and recyclable thermoelectric paper by bacteria farming

Deyaa Abol-Fotouh,a,b Bernhard Dörling,a Osnat Zapata-Arteaga, a Xabier Rodríguez-Martínez, a Andrés Gómez, a J. Sebastian Reparaz, a Anna Laromaine, a Mariano Campoy-Quiles, a Anna Roig a

a Institute of Materials Science of Barcelona (ICMAB-CSIC), Bellaterra, 08193, Spain. b City of Scientific Research & Technological Applications (SRTA-City), New Borg Al-Arab, 21934, Egypt

Abstract

Converting waste heat into electricity through sustainable, inexpensive and non-toxic thermoelectric

devices is an unmet challenge in the field of green power generation.

I will explain the growth of bacterial cellulose (BC) films with an embedded finely dispersed carbon

nanotubes network. The paper-like films are mechanically resistant, flexible and stable up to 250 °C. Films

exhibit low thermal conductivity and, despite a modest carbon nanotube content of just 10 % weight

percent, their electric conductivity is comparable to that of buckypaper. The films are fully bendable and

can be conformally wrapped around heat sources of any shape. The high porosity of the material

facilitates effective n-type doping, enabling the easy fabrication of thermoelectric modules. These devices

could be used to generate electricity from residual heat to feed low power sensors. Importantly, BC can

be enzymatically biodegraded and the carbon nanotubes reclaimed.

Because bacterial cellulose can be home made, perhaps we may be facing first baby steps towards a new

energy paradigm, where users will be able to farm bacteria to manufacture their own electric generators.

References

Farming thermoelectric paper. Deyaa Abol-Fotouh, Bernhard Dörling, Osnat Zapata-Arteaga, Xabier Rodríguez-Martínez, Andrés Gómez, J. Sebastian Reparaz, Anna Laromaine, Anna Roig and Mariano Campoy-Quiles. Energy Environ. Sci., 12 (2019) 716 – 726 DOI: 10.1039/C8EE03112F.


3-4th October, 2019


Bacterial cellulose a sustainable material: applications ranging from electronics to biosensors

Elvira Fortunato

CENIMAT/I3N and CEMOP-UNINOVA, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal

Abstract

In this talk we will discuss the state of the art and potential future directions in paper-based electronics

with special emphasis to the work developed at CENIMAT|i3N, covering electronic devices, smart displays,

printed electronics, sensors and diagnostic tests.

We have been observing a rapid and growing interest concerning the utilization of biological materials for

a wide range of applications. One of the most representative example is cellulose, not only in the form of

raw material mainly for pulp and paper production, but also in the development of advanced

materials/products with tailor-made properties, especially the ones based on nanostructures.

5 years ago paper electronics was pure science fiction, but today we have already several paper-based

electronics like integrated circuits, supercapacitors, batteries, fuel cells, solar cells, transistors, microwave

electronics, digital logic/computation, displays, force-sensing MEMS, user interfaces, transparent

substrates, substrates with high strength, wearable devices, and new rapid diagnostic test sensors. These

devices with their associated physics and processing will play an important and relevant to our society

ongoing efforts to in environmental sustainability, safety, communication, health, and performance.


3-4th October, 2019


Optically transparent bacterial cellulose-based composites as substrate for flexible organic

light emitting diode.

Hernane S. Barud (1), Cristiano Legnani (2), José M. A. Caiut (3), Robson Rosa da Silva (4), Sidney J. L. Ribeiro (5), Marco Cremona (6)

1- Laboratório de Biopolímeros e Biomateriais – BioPolMatUniversidade de Araraquara (Uniara)AraraquaraBrazil 2-.Laboratório de Eletrônica Orgânica, Departamento de FísicaUniversidade Federal de Juiz de Fora, UFJFJuiz de ForaBrazil 3-Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão PretoUniversidade de São PauloRibeirãoPretoBrazil 4- Chalmers University, Gootemburg, Sweeden. 5.Instituto de QuímicaUniversidade Estadual Paulista, UNESPAraraquaraBrazil 6.Laboratório de Optoeletrônica Molecular, Departamento de FísicaPontifícia Universidade Católica do Rio de Janeiro, PUC-Rio de JaneiroBrazil

Abstract

Optically transparent bacterial cellulose biocompatible composites membranes have been prepared in

the last year in our labs. Wet and dry BC membranes have been surface modified by casting association

with anothers polymers (polyurethane, polycaprolactone, polyetheremide) andboehmite-siloxane

organic-inorganic hybrids. These multifunctional composite membranes have been covered with silicon

dioxide (SiO2) and indium tin oxide (ITO) thin films deposited at room temperature using radio

frequency (RF) magnetron sputtering. Visible light transmission improves to up 88%, instead of 40%

previously achieved by pristine BC membranes. The electrical properties for BC/boehmite-

siloxane/SiO2/ITO substrate shows that the ITO deposited films are n-type doped semiconductors with

resistivity of 2.7 × 10−4 Ω cm, carrier concentration of − 1.48 × 1021 cm−3, and mobility of

15.2 cm2 V−1 s−1. ALL BC composites have been uses ad as a substrate for the fabrication of a small

molecule organic light-emitting diode OLED. The maximum efficiencies obtained were 1.95 cd/A and

1.68 cd/A for the glass reference OLED and the BC/boehmite-siloxane based-OLED, just to show one

composite. TheFOLED efficiency is then around 86% of the standard ITO-based OLED. These new

optically transparent bacterial cellulose biocompatible composites open opportunities to application

on optoeletronic devices and also photodynamic therapy (PDT).

Acknowledegments

I would like to thank to FINEP process 407822/2018-6.

References

C. Legnani, C. Vilani, V.L. Calil, H.S. Barud, W.G. Quirino, C.A. Achete, S.J.L. Ribeiro, M. Cremona, Thin Solid Films 517, 1016 (2008) E.R.P. Pinto, H.S. Barud, R.R. Silva, M. Palmieri, W.L. Polito, V.L. Calil, M. Cremona, S.J.L. Ribeiro, Y. Messaddeq, J. Mater. Chem. C 3, 11581 (2015)


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3-4th October, 2019


Cellulose patches in plants

Anna Laromaine1, Agnese Rabissi1-2, Alejandro Alonso-Díaz2, Jordi Floriach-Clark1, Montserrat Capellades2, Núria S. Coll2

1. Institute of Material Science of Barcelona (ICMAB), CSIC, Campus UAB, Bellaterra, Barcelona, 08193, Spain. 2. Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, Barcelona, 08193, Spain.

Abstract

Food security and the need to increase sustainably crop yields for a rapidly growing world population are

among the greatest social and economic challenges of our century.

The most extended agricultural practice to enhance crop yield is to increase host plant density, which in

turn tends to increase the severity of plant diseases.1 Furthermore, globalization has contributed to the

spreading of new pests into regions in which plants were not adapted, causing unaffordable agronomic

losses. Most plant diseases occurring in agriculture are caused by fungal pathogens.2 Plant diseases caused

by pathogenic bacteria are less prevalent; but their effects on agriculture are also devastating.3

In this work, we present an anti-bacterial and anti-fungal patch, which exploits the potential of smart-

based nanomaterials as pesticides and silver nanoparticles (AgNPs) as anti-bacterial active component,

improving the efficiency and environmental sustainability of pesticides application. In order to prevent

the release of the NPs to the environment and their runoff loss during application, we attached the active

silver nanoparticles to a bacterial cellulose (BC) matrix to obtain BC-AgNPs hybrid films. The bacterial

cellulose matrix has similar chemical composition to the plant cellulose present in leaves but shows higher

purity, crystallinity and water absorbance.4-5 We take advantage of the gel-like nature of the bacterial

cellulose matrix to in situ synthesize and embed AgNPs.

We evaluated the release and attachment of silver nanoparticles to a bacterial cellulose matrix and their

in vitro anti-pathogenic properties against an array of plant pathogens with high agro-economical impact,

such as the bacterium Pseudomonas syringae and the fungus Botrytis cinerea.

These anti-bacterial and anti-fungal capacities were also assessed in vivo using the plant species Nicotiana

benthamiana a close relative of tobacco and tomato; both widely used as a model organisms in plant

biology.6

References

(1) Sundström, J. F. et al. Future threats to agricultural food production posed by environmental degradation, climate change, and animal and plant diseases - a risk analysis in three economic and climate settings. Food Secur. 6, 201–215 (2014). (2) Agrios, G. N. Plant Pathology. Quinta edición. Academic Press. Nueva York. (2005). (3) V. Rajesh Kannan, K. K. B. Sustainable Approaches to controlling plant pathogenic bacteria. (2015). (4) Klemm, D. et al. Nanocelluloses: A new family of nature-based materials. Angew. Chemie - Int. Ed. 50, 5438–5466 (2011).


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(5) 5 Zeng, M., Laromaine, A. & Roig, A. Bacterial cellulose films: influence of bacterial strain and drying route on film properties. Cellulose 21, 4455–4469 (2014). (6)Enhancing Localized Pesticide Action through Plant Foliage by Silver-Cellulose Hybrid Patches A. Alonso-Díaz, J.i Floriach-Clark, J. Fuentes, M. Capellades, N. S. Coll, A. Laromaine; ACS Biomater. Sci. Eng., (2019), 5 (2), pp 413–419.


3-4th October, 2019


Bacterial Nanocellulose: A brief history and future prospects at “Embrapa”

Morsyleide de Freitas Rosa

Embrapa Agroindústria Tropical, Brazil

Abstract

The Brazilian Agricultural Corporation (Embrapa) has been involved in bacterial nanocellulose (BNC)

research since 2012. Our aim is to show our brief history and to share our main ongoings, as well as the

future prospects related to the BNC.

Many studies have been mainly focused in the use of alternative (low-cost) carbon sources, instead of the

usual synthetic medium. Furthermore, the effects of cultivation types and the bacterial strains have been

evaluated. The results suggested that we have promising substrates (e.g. soybean molasses) and they

could be an interesting option to produce BNC for various applications, which do not require such a high

purity degree as those required for the biomedical applications.

We have performed top-down approaches (e.g.: homogenization, hydrolysis and combined chemical-

mechanical processes) with the purpose to resize the bacterial cellulose fibers in small particles (BNC

nanocrystals and BNC nanofibrils) suspensions, adding functionality to the material and new versatility, in

terms of transport, storage and handling, as well as for processing purposes. This disassembly of the BNC

membranes produced a promising starting material for different applications.

Our first applications based on the BNC were focused in the biomedical usages (wound dressing and drug

delivery system). Currently, we have focused in the usage of the BNC as a promising material for food

packaging purposes (edible films and coatings). As it is hydrophilic, it allows fruits and vegetables to

maintain gas exchanges and respiration, nevertheless, at lower rates, while at the same time, being

sufficiently water resistant. Recently, we have been designing Pickering emulsions (surfactant-free

emulsions), based on the structural characteristics of the BNC colloidal nanoparticles. These nanoparticles

can be used to prepare a monodispersed emulsion without any further modification and stabilize it

irreversibly.

Presently, large efforts have been placed on identifying the critical environmental points in the processes

related to the BNC in a laboratory scale, applying life cycle assessment (LCA). The LCA is essential to

guarantee the quality of the processes and products to be developed, as well as to optimize the critical

stages of the previously mentioned processes, aiming at lowering environmental impacts. These stated

results do open opportunities for the development of environmentally friendly new materials based on

BNC.

References

(1) Azeredo, H. M. C., Barud, H., Farinas, C.S., Vasconcellos, V.M., Claro, A.M. (2019). Bacterial cellulose

as a raw material for food and food packaging applications. Frontiers in Sustainable Food Systems, 3: 7.


3-4th October, 2019


SESSION 3 – Composites & applications: part 2 (chair person: Falk Liebner)


3-4th October, 2019


KEYNOTE LECTURE: Making fluffy Bacterial Cellulose Nanopapers: Useful filters with tailored

properties

Andreas Mautner1, Koon Yang Lee2, Alexander Bismarck1,3 1 Polymer & Composite Engineering (PaCE) Group, Institute of Materials Chemistry,

Faculty of Chemistry, University of Vienna 2 Department of Aeronautics, Imperial College London, South Kensington Campus, London

3 Department of Chemical Engineering, Imperial College London, South Kensington Campus, London

Abstract

Papers have been used as filters since ancient times. However, conventional filter papers, produced from

cellulose pulp fibres or microfibrils cannot be used to remove nanoscale pollutants. Using a conventional

paper making process utilizing cellulose microfibrils fails to produce papers capable of UF or even NF

operations. Recently, we demonstrated the utility of bacterial cellulose papers, also often referred to as

nanopapers as tight aqueous UF membranes. These nanopapers were produced using a simple

papermaking process. Unfortunately, the permeance of these membranes is rather, which is due to the

low porosity of these papers.

To address this drawback we produced nanopapers from dispersions of BC in organic solvents rather than

water, which gives results in BC papers with a higher porosity. The higher paper porosity should result in

an increased permeance of BC papers but without affecting the pore size and thus the separation

efficiency of the membrane.

We here present nanopapers made from bacterial cellulose from a set of different organic solvents

produced using a simple paper making process including a solvent exchange step.


3-4th October, 2019


Bacterial NanoCellulose as a Natural Component of Next-Generation Bio-Composites

Karolina Ludwicka, Marzena Jedrzejczak-Krzepkowska, Edyta Gendaszewska-Darmach, Teresa Pankiewicz, Stanislaw Bielecki Institute of Technical Biochemistry, Lodz University of Technology, Poland

[email protected]

Abstract

High-performance, low-cost and biocompatible composites are today dream materials for biomedical

applications. With the development of a nanoscale synthesis and engineering technologies, also as

inspired by the structures of natural biological materials, biocomposites featuring superior stiffness,

strength and biocompatibility are reported as the next-generation multifunctional super-materials.

Bacterial NanoCellulose (BNC), as a polymer naturally produced by acetic acid bacteria, characterized by

extraordinary properties already widely exploited in biomedical applications, is an excellent substrate for

the development of such composites. Here, for the first time, BNC was stably connected, with very good

results, with such materials as polypropylene (PP) porous mesh, High Strength Metallurgical Graphene

(HSMG) or biosolubilized brown coal.

The components of BNC-PP composites, obtained in vertical stationary cultures of Komagataeibacter

xylinus, are stably integrated after the process of cultivation and purification. Bionanocellulose does not

negatively influence mechanical properties of the polypropylene mesh, preserving its tensile strength,

elasticity and load. Moreover application of bacterial cellulose makes the composites less immunogenic

by decreasing mast cell adhesion and degranulation as compared to polypropylene itself. Therefore, the

composites have the great potential of application in medicine, and depending on the applied porous

material, might be used either in hernioplasty (if porous hernia mesh is used), cranioplasty (if perforated

metal or polymeric cranial implant is applied) or as a protective barrier in any application that requires

biocompatibility or antiadhesive properties improvement.

The highly preliminary results of cellulose connected to the HSMG indicate changes of BNC chemical

properties. As a direct consequence of BNC and graphene differential chemical features, by connecting to

HSMG cellulose hydrophilic properties were changed to more hydrophobic. Graphene derivatives are

known for their capacity to uptake different compounds from water solutions, what gives perspectives

for BNC composites applications not only in strictly medical areas. Nevertheless, it is highly probable, that

the combination of such two high-performance materials of unique properties will offer new perspectives

for BNC applications.

The composites with biosolubilized brown coal are prepared by in situ and ex situ methods. Liquefied coal

has excellent sorption properties, e.g. in biosorption of numerous pollutants, and is obtained by a

chemical pre-treatment followed by a biosolubilization process conducted by specific consortia of

microorganisms. Since the product of biosolubilization is a mixture of humic, fulvic acids and other organic

compounds, BNC cultivated with solubilized coal may gain a new range of properties not reported before.



3-4th October, 2019


References

(1) Ludwicka, K. Kolodziejczyk, M., Gendaszewska-Darmach, E., Chrzanowski, M., Jedrzejczak-Krzepkowska, M., Rytczak, P., Bielecki, S. (2019). Stable composite of bacterial nanocellulose and perforated polypropylene mesh for biomedical applications. J Biomed Mater Res B Appl Biomater. 107(4):978-987 (2) Kula, P. J. et al. (2015). High Strength Metallurgical Graphene - Mechanisms of Growth and Properties. Archives of Metallurgy and Materials. 60(4):2535-41 (3) Romanowska I., Strzelecki B., Bielecki S. (2015). Biosolubilization of Polish brown coal by Gordonia alkanivorans S7 and Bacillus mycoides NS1020. Fuel Processing Technology. 131: 430–436


3-4th October, 2019


Development and application of bacterial cellulose/graphene nanocomposites

Yizao Wan1, Quanchao Zhang1, Haiyong Ao1, Zhiwei Yang1, Jian Hu1, Honglin Luo1

1Institute of Advanced Materials, East China Jiaotong University, Nanchang, 330013, China [email protected]

Abstract

Two-dimensional carbon-based nanomaterials, including graphene and its derivatives, have been

considered as candidates for biomedical applications such as artificial scaffolds, sensors, cell labelling,

bacterial inhibition, and drug delivery. On the other hand, bacterial cellulose (BC) has received growing

interest in recent decades due to its impressive inherent characteristics with potential applications in

diverse sectors. Accordingly, fabricating graphene-reinforced BC nanocomposites can be of particular

importance in many fields.

It has been well documented that the critical issue on the fabrication of composites is how to

homogeneously incorporate and distribute nanofillers in the matrices. In the graphene-BC system,

mechanical mixing and in situ biosynthesis [1] are two most common methods of making graphene/BC

nanocomposite. However, mechanical mixing breaks the integrated nanofibrous structure of BC, which is

not favorable since it reduces the mechanical robustness and damages the three-dimensional (3D)

network structure of pristine BC. In order to maintain the intrinsic 3D interconnected porous structure of

BC while allowing the dispersion of graphene nanosheets, our group developed a novel membrane-liquid

interface culture (MLIC) method, with its main steps illustrated in Fig. 1. In the first step, a graphene-

dispersed medium was sprayed onto the BC substrate to allow for BC growth. After the sprayed medium

was completely consumed by bacteria, another layer of medium was sprayed. Such process was repeated

until the designed thickness was achieved. In contrast to the conventional static culture method, which is

limited by the difficulty of oxygen and nutrient transport, the distinct feature of the MLIC method allows

us to obtain very thick membranes with homogenous structure and the thickness of the products can be

accurately controlled by the volume of sprayed medium. The resultant graphene/BC nanocomposites

demonstrate uniformly distributed graphene nanosheets that are closely bundled with BC nanofibers,

forming a unique nanostructured composite. This sophisticated nanostructure endows the graphene/BC

composite with extremely high mechanical properties and electrical conductivity [2, 3]. Therefore, the

graphene/BC composites hold great promise in various applications (such as electrodes, adsorbents, and

scaffolds for tissue engineering) which require excellent mechanical properties and electrical conductivity.

Furthermore, the MLIC method is scalable, versatile, and eco-friendly. More importantly, this method can

be extended to the production of other BC-based nanocomposites.



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Fig. 1. Fabrication processes of graphene/BC composites.

References

[1] H. Luo, P. Xiong, J. Xie, Z. Yang, Y. Huang, J. Hu, Y. Wan, Y. Xu, Uniformly dispersed freestanding carbon nanofiber/graphene electrodes made by a scalable biological method for high-performance flexible supercapacitors, Adv Funct Mater 28 (2018) 1803075. [2] H. Luo, J. Xie, J. Wang, F. Yao, Z. Yang, Y. Wan, Step-by-step self-assembly of 2D few-layer reduced graphene oxide into 3D architecture of bacterial cellulose for a robust, ultralight, and recyclable all-carbon absorbent, Carbon 139 (2018) 824-832. [3] H. Luo, J. Dong, X. Xu, J. Wang, Z. Yang, Y. Wan, Exploring excellent dispersion of graphene nanosheets in three-dimensional bacterial cellulose for ultra-strong nanocomposite hydrogels, Composites Part A 109 (2018) 290-297.


3-4th October, 2019


Bacterial Nanocellulose composites for the textile and leather industries

Fernando Dourado1,2, Marta Fernandes3, António Pedro Souto3, Miguel Gama2

1Satisfibre, S.A., Portugal 2CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar,

4710-057 Braga, Portugal 3 Centre for Textile Science and Technology, University of Minho, Campus de Azurém,

4800-058 Guimarães, Portugal

Abstract

The tannery industry faces several challenges associated with high environmental impact, scarcity of raw

materials and increasing consumer demand for environmentally friendly products. The worldwide

production of leather is approximately 20 billion square feet per year (1). To produce one ton of leather

6.7 tons of raw skin are processed (2) an 57,000 liters of water (3) and 3.35 tons of chemicals (4) are

needed. Worldwide, for bovine skin, 370 billion litres of water are consumed annually, generating 6.5

million tons of solid waste. The development of leather analogues has thus long been pursued, leading to

the appearance of various materials, some synthetic, other natural. Despite the increasing interest and

market pull, the market penetration of these alternative products has been relatively modest, due to high

production costs, low breathability, high stiffness, accelerated discoloration, among other limitations.

Also, recent market trends towards the identification of natural non-cotton derived textiles are emerging.

This research intends to contribute to the reduction of the animal hide dependency by the development

of composites from bacterial nanocellulose (BNC) as structural material and activated vegetable oils and

other hydrophobic polymers, as a flexibilizing, mechanical reinforcing and hydrophobizing agents. The

newly developed strategy here presented, based on BNC, aims at meeting the market pull from both the

shoe and textiles industries regarding the need for new high-performance natural materials. A novel

approach was tested for the bulk and surface modification of BC, combining simplicity, potential for

application at large scale and low cost, based on the use of an exhaustion process. Through this process,

hydrophobic polymers could be incorporated into the nanofibrillar matrix of BNC, aiming at obtaining a

malleable, breathable and water impermeable nanocomposites. This presentation will summarize the

main results on the preparation of BC-based composites featuring promising properties for application in

the textile and shoe industries (5).

Acknowledgments

This study was supported by FEDER funding on the Programa Operacional Regional do Norte (NORTE2020)

within the scope of the project NORTE-01-0247-FEDER-003435 (‘BUILD–Bacterial cellulose Leather’) and

Bio-TecNorte operation NORTE-01-0145-FEDER-000004, FEDER funding on the Programa Operacional

Factores de Competitividade–COMPETE within the scope of the project POCI-01-0145-FEDER-007136 and

POCI-01- 0145-FEDER-006684, national funds through FCT–Foundation for Science and Technology within

the scope of the project UID/CTM/00264/2019 and UID/BIO/ 04469/2013.


3-4th October, 2019


References

(1) FAO (2015) World statistical compendium for raw hides and skins, leather and leather footwear 1999-2015.

(2) Kanagaraj, J., Senthilvelan, T., Panda, R. C. and Kavitha, S. (2015) “Eco-friendly waste management strategies for greener environment towards sustainable development in leather industry: a comprehensive review,” Journal of Cleaner Production. Elsevier Ltd, 89, pp. 1–17. doi: 10.1016/j.jclepro.2014.11.013.

(3) Wool, R. (2013) “Composites having leather-like characteristics.” United States. doi: US 20100322867A1.

(4) Black, M., Casanova, M., Rydin, S., Scalet, B. M., Roudier, S. and Sancho, L. D. (2013) Best Available Techniques (BAT) Reference Document for the Tanning of Hides and Skins. doi: 10.2788/13548.

(5) Marta Fernandes, Miguel Gama, Fernando Dourado and António Pedro Souto. 2019. Development of novel bacterial cellulose composites for the textile and shoe industry. Microbial Biotechnology (https://doi.org/10.1111/1751-7915.13387)


3-4th October, 2019


Transparent poly(methyl methacrylate) composites based on bacterial cellulose nanofibre

networks with improved fracture resistance and impact strength

Alba Santmartí, Jiawei Teh, Koon-Yang Lee Department of Aeronautics, Imperial College London, SW7 2AZ London, United Kingdom

Abstract

Bacterial cellulose (BC) is an ultrapure form of cellulose nanofibres with mechanical performance

exceeding that of E-glass fibres. The tensile stiffness and strength of a single BC nanofibre have been

estimated to be up to 160 GPa and 6 GPa, respectively. BC is produced as a continuous nanofibre network

in the form of tough wet pellicle with high specific surface area. It is hypothesised that this continuous BC

nanofibre network will serve as excellent nano-reinforcement for polymers with similar refractive indices,

such as acrylic resin, for the production lightweight polymeric transparent armour with performance

beyond the state-of-the-art. The introduction of such continuous nanofibre network into acrylic resin will

introduce additional energy-absorbing mechanisms, including fibre de-bonding (due to an increase in

fibre-polymer matrix interface), fibre re-orientation and fracture, enhancing the impact resistant of the

resulting BC-reinforced acrylic resin without sacrificing optical transparency.

In this study, we report the tensile, fracture and impact properties of poly(methyl methacrylate) (PMMA)

composites reinforced with bacterial cellulose (BC) pellicle. It was found that BC pellicle-reinforced PMMA

composites with high optical clarity could be produced. By reinforcing PMMA with up to 5.0 wt.-% BC

pellicle, a significant increase in tensile modulus compared to neat PMMA was observed. However, this

improvement was accompanied by a significant decrease in tensile strength, strain-at-failure and impact

strength due to matrix embrittlement. To circumvent this effect, 3 novel composite designs were

developed: PMMA composites with uniformly dispersed BC throughout PMMA; sandwich structured

construction consisting of BC pellicle-reinforced PMMA sandwiched between two neat PMMA sheets;

hornified BC pellicle-reinforced PMMA composites. The tensile moduli of these composites were found

to increase compared to neat PMMA. Furthermore, no significant detrimental effect in the tensile

strength of the resulting composites was observed. This was attributed to the reinforcement architecture,

which was designed to reduce BC nanofibre- PMMA interface such that the effect of matrix embrittlement

was minimised. The best performing BC pellicle reinforced PMMA composites was reinforced with

hornified BC pellicle, whereby the impact strength was 25% higher than neat PMMA, without sacrificing

optical clarity.


3-4th October, 2019


Bacterial cellulose within the context of pulp-and-paper emerging biorefineries

Carlos Pascoal Neto

RAIZ - Forest and Paper Research Institute / The Navigator Company Quinta de São Francisco, 3801-501 Eixo – Aveiro, Portugal

Abstract

Pulp-and-paper mills are evolving to modern forest-based biorefineries, where wood and forest biomass

is comprehensively converted into pulp and paper products, bioenergy, biofuels and bioproducts. Within

this context, part of the biomass resulting from forest exploitation, as well as from wood processing in the

mill, may be submitted to saccharification processes, yielding sugars that constitute good a good substrate

for bacterial cellulose production. This bacterial nanocellulose, potentially produced on site, may find

interesting applications on pulp and paper production, namely on the developing of mechanical

properties, barrier properties as well as on the enhancing of printability of fine papers.

This presentation aims at giving an overview of the concept of pulp-and-paper biorefineries and of the

possibility to integrate the production of bacterial cellulose in pulp-and-paper mills. Potential applications

of bacterial cellulose on paper products are reviewed and illustrated with some R&D results.


3-4th October, 2019


Bacterial cellulose for edible films and coatings

Henriette M.C. Azeredo1,2 1Embrapa Agroindústria Tropical, 2Embrapa Instrumentação

Abstract

Bacterial cellulose (BC) may be combined to other components by impregnation or by formulation from

disassembled BC sheets. Both approaches have advantages and limitations; the second one being the one

of choice when several components are to be combined in accurate proportions1. Our group has been

working on some development on edible films from nanofibrillated bacterial cellulose (NFBC). Those NFBC

films have been reported to present much better tensile properties and water resistance than most other

materials used for edible films, giving NFBC an advantage for many potential applications. NFBC has been

also used in combination with cashew tree gum (CTG), improving the physical performance (barrier and

tensile properties, resistance to water) of CTG films. NFBC films containing sensory or health-appealing

components (e.g. fruit purees2, probiotics) have been also produced. Those and other examples will be

presented as potential applications of NFBC as a distinguished edible material for films and coatings.

References (1) Azeredo, H. M. C., Barud, H., Farinas, C.S., Vasconcellos, V.M., Claro, A.M. (2019). Bacterial cellulose as a raw material for food and food packaging applications. Frontiers in Sustainable Food Systems, 3: 7. (2) Viana, R. M., Sá, N. M. S. M., Barros, M. O., Borges, M. F., Azeredo, H. M. C. (2018). Nanofibrillated bacterial cellulose and pectin edible films added with fruit purees. Carbohydrate Polymers, 196: 27–32.


3-4th October, 2019


SESSION 4 - Sharp presentations: part 1 (chair person: Anna Roig)


3-4th October, 2019


Functionalization, Regeneration and Modification of Bacterial Cellulose

Caro-Astorga J., Gilbert C., Lee K., Ellis T. Department of Bioengineering, Imperial College London, United Kingdom

Abstract

Engineered living materials exhibit outstanding traits in terms of scalability and ease of production, and

offer new physical and mechanical properties enabled by morphology control and functionalization (1).

Nanocellulose is emerging as a promising biological material for the future, with a variety of applications

and a global market expected to increase 50 to 100 fold over the next decade (2). Komagataeibacter

rhaeticus offers an excellent strain to produce this material in industrial quantities, giving a high purity

product grown from cheap raw materials (3). Inspired by kombucha fermentation, we have reproduced

co-culturing of K. rhaeticus with yeasts including S. cerevisiae. Both organisms can be engineered enabling

the co-culture to produce complex enzymes and chemical products and degrade compounds, offering a

route to functionalizing nanocellulose so that the material can sense the external environment and

respond. As an example case, here we probe the functionalization of nanocellulose by engineering S.

cerevisiae to secrete an engineered β-lactamase with a cellulose binding domain as the nanocellulose

material grows. We also explore bioengineering methods to regenerate damaged nanocellulose, a step

that will increase the versatility of this material, and we test treatments of the grown nanocellulose and

identify compounds that can change physical and mechanical properties as desired, yielding material that

is tens-fold stronger under compression test.

References

(1) Nguyen PQ, Courchesne ND, Duraj-Thatte A, Praveschotinunt P, Joshi NS. (2018). Engineered Living Materials: Prospects and Challenges for Using Biological Systems to Direct the Assembly of Smart Materials. Advanced Materials, 30 (19): e1704847. (2) Globarl Market of Cellulose Nanofibres to 2030. (3) Michael Florea, Henrik Hagemann, Gabriella Santosa, James Abbott, Chris N. Micklem, Xenia Spencer-Milnes, Laura de Arroyo Garcia, Despoina Paschou, Christopher Lazenbatt, Deze Kong, Haroon Chughtai, Kirsten Jensen, Paul S. Freemont, Richard Kitney, Benjamin Reeve, and Tom Ellis. (2016). Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. PNAS, 113 (24) E3431-E3440.


3-4th October, 2019


Confined spatial nanoparticle distribution in bacterial nanocellulose millefeuille

Soledad Roig-Sánchez1, Erik Jungstedt2, Irene Anton-Sales1, David C. Malaspina1, Jordi Faraudo1, Lars A. Berglund2, Anna Laromaine1, Anna Roig1

1 Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08193 Bellaterra, Spain 2 Department of Fiber and Polymer Technology, Wallenberg Wood Science Center, Royal Institute of Technology (KTH), SE-100 44 Stockholm, Sweden [email protected] , www.icmab.es/nn , @NNgroup_ICMAB

Abstract

Bacterial nanocellulose (BC) exhibit properties such as high crystallinity, transparency, high water holding

capacity and hierarchical porosity. In addition, it is chemical and physical tunable. These properties

endorse BC as a sustainable resource to achieve advanced functional nanocomposites, which are in

increasing need for products and devices.1,2 Nevertheless, developing a high strength and flexible

nanocellulose-based biocomposite is still a challenge and a control on some parameters as the

nanoparticles loading fraction, their morphology and its disposition within the porous scaffold is needed.

A multifunctional laminated material with topographic confinement of nanoparticles using the bacterial

cellulose as a platform will be presented. A microwave-assisted synthesis route has been employed to in-

situ nucleate and grow metal and metal oxides nanoparticles on the cellulosic fibers (Au, Ag, TiO2, Fe2O3)

and to confer different functional properties (electrical conductivity, magnetism and photocatalysis) to

the BC films.3 The drying process has allowed us to attach on demand the different nanocomposite layers,

creating a millefeuille construction. Structural, functional and mechanical integrity of the millefeuille will

be discussed.4

References

(1) Torres, F. G., Arroyo, J. J., Troncoso, O. P. (2019). Bacterial Cellulose Nanocomposites: An All-Nano Type of Material. Mater. Sci. Eng. C. 98 (January), 1277–1293.

(2) Zeng, M., Laromaine, A., Roig, A. (2014). Bacterial Cellulose Films: Influence of Bacterial Strain and


http://www.icmab.es/nn


3-4th October, 2019


Drying Route on Film Properties. Cellulose. 21 (6), 4455–4469. (3) Zeng, M., Laromaine, A., Feng, W., Levkin, P. A., Roig, A. (2014). Origami Magnetic Cellulose:

Controlled Magnetic Fraction and Patterning of Flexible Bacterial Cellulose. J. Mater. Chem. C. 2, 6312–6318.

(4) Roig-Sanchez, S., Jungstedt, E., Anton-Sales, I., Malaspina, D. C., Faraudo, J., Berglund, L. A., Laromaine, A., Roig, A. (2019). Nanocellulose Films with Multiple Functional Nanoparticles in Confined Spatial Distribution. Nanoscale Horizons. 4, 634–341.


3-4th October, 2019


Role of motA and motB genes in bionanocellulose biosynthesis in Komagataeibacter genus

Paulina Jacek, Małgorzata Ryngajłło, Katarzyna Kubiak, Stanisław Bielecki

Institute of Technical Biochemistry, Lodz University of Technology, B. Stefanowskiego 4/10, 90-924 Lodz, Poland

[email protected]

Abstract

Bacterial nanocellulose (BNC) is a highly abundant natural polymer synthesized by some microorganisms

among which the Gram-negative bacteria Komagataeibacter hansenii and K. xylinus have been widely

used as a model organisms for studying BNC biosynthesis (1). In Gram-negative bacteria, cellular motility,

cell morphogenesis, metabolism, and material transport are described as one of the crucial factors for

biofilm formation. Many reports suggest that proteins belonging to the MotA/TolQ/ExbB and

MotB/TolR/ExbD families, which form transmembrane proton channels, are important in biofilm

formation.

Recently, we identified the motA and motB genes in Komagataeibacter genus. In order to elucidate the

role of motA and motB in Komagataeibacter, we constructed disruption and overexpression mutants (2-

4). Then we examined the influence of motA and motAB genes disruption on global gene expression using

RNA-seq technique.

The phenotype analysis of disruption and overexpression mutants has shown changes in cell morphology,

efficiency of cellulose production and structural changes in cellulose membranes (2-4). The results of RNA

sequencing of Komagataeibacter xylinus E25 disruption mutants suggest that tested genes have similar

function like their homologs.

References

1) Jacek P, Dourado F, Gama M, Bielecki S, „Molecular aspects of bacterial nanocellulose biosynthesis”. Microbial Biotechnology, 2019,

2) Jacek P, Ryngajłło M, Bielecki S “Structural changes of bacterial nanocellulose pellicles induced by genetic modification of Komagataeibacter hansenii ATCC 23769”. Applied Microbiology and Biotechnology, 2019,

3) Jacek P, Kubiak K, Ryngajłło M, Rytczak P, Paluch P, Bielecki S, „Modification of bacterial nanocellulose properties through mutation of motility related genes in Komagataeibacter hansenii ATCC 53582”. New Biotechnology, 2019,

4) Jacek P, Szustak M, Kubiak K, Gendaszewska-Darmach E, Ludwicka K, Bielecki S, „Scaffolds for chondrogenic cells cultivation prepared from bacterial cellulose with relaxed fibers structure induced genetically”. Nanomaterials, 2018,



3-4th October, 2019


Bacterial nanocellulose as an ocular bandage material

Irene Anton-Sales1, Justin D’Antin2, Ralph Michael2, Anna Laromaine1 and Anna Roig1

1Insitute of Materials Science of Barcelona (ICMAB-CSIC) 2Centro de Oftalmología Barraquer

Abstract

Bacterial nanocellulose (BNC) has already been proposed for diverse biomedical applications, mainly for

skin regeneration. Nevertheless, the potential of BNC to treat other damaged tissues, such as the cornea,

remains unexploited. Corneal trauma and ulcerations are responsible for more than 1.5 million new cases

of vision reduction every year worldwide. Management of these patients remains limited by the shortage

of high-quality donor corneas for transplantation. To overcome this issue, much hope is put on

regenerative medicine and biomaterial-based approaches. In line with this, we suggest using BNC patches

to treat ocular surface disorders expecting that they will provide both protection to the corneal wounds

and a vehicle for cell transplantation to the ocular surface.

First, structural characterization of our BNC films produced by G.xylinus will be presented. It will be

followed by detailed cytocompatibility tests of BNC performed with human dermal fibroblasts. This

includes studies about cell attachment, viability, proliferation and morphology of fibroblasts cultured on

top of different BNC films. The tested supports for cell culture include BNC films with different water

content, shape, topography and also BNC-TiO2 nanocomposites. Moreover, we will also show that BNC

films can be employed to cryopreserve adherent cells. These in vitro results highlight the versatility of BNC

as a substrate to maintain, expand and possibly transplant human cells to diverse body surfaces.

The second part of the talk will show our first attempts, performed in close collaboration with clinicians,

towards the applicability of BNC in regenerative ophthalmology. Ex vivo experiments to investigate the

interactions between BNC films and excised porcine corneas will be presented. These studies demonstrate

that BNC meets the basic requirements of mechanical resistance to suture, conformability to the dome

shape of the eye, ex vivo stability and ease of manipulation to be used as a new ocular bandage material

and/or as a vehicle for cell transplantation to the cornea.

References

1- Anton-Sales I, Beekmann U, Kralisch D, Laromaine A, Roig A. Opportunities of bacterial cellulose to treat epithelial tissues. Curr Drug Targets. (2018) In press

2- Tronser T, Laromaine A, Roig A, Levkin PA. Bacterial Cellulose Promotes Long-Term Stemness of mESC. ACS Appl Mater Interfaces. 2018 May 16;10(19):16260–9.

3- Roig-Sanchez S, Jungstedt E, Anton-Sales I, Malaspina DC, Faraudo J, Berglund LA, et al. Nanocellulose films with multiple functional nanoparticles in confined spatial distribution. Nanoscale Horizons. (2019) In press


3-4th October, 2019


ORAL COMMUNICATIONS: October 4th


3-4th October, 2019


SESSION 5 – Production and formulation (chair person: Stanislaw Bielecki)


3-4th October, 2019


KEYNOTE LECTURE: 3D-structured Cellulose Biofilms and Applications

Luiz G. Greca, Janika Lehtonen, Blaise L. Tardy, Orlando J. Rojas

Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Finland

Abstract

We discuss a platform to biosynthetically produce 3D cellulose biofilms with microbial cellulose using

Komagataeibacter medellinensis ID13488 (1). Moreover, a simple yet customizable biofabrication process

is proposed that uses hydrophobic particles to finely direct the morphology of bacterial cellulose biofilms

in three dimensions resulting in hollow, seamless, cellulose-based objects of tailored shape and size (2).

The robustness and integrity of the structured nanocelluloses were demonstrated against intense

mechanical and chemical stresses. The synergetic combination of the 3D shaping techniques introduced

in this work with cross disciplinary knowledge, brings a new perspective on the existing applications of

nanocellulose, and also extend the scope of functionalities that can be achieved for the benefit of different

fields. For instance, by keeping the biofilm aspect, plasmid transfection in 3D morphologies can be further

exploited. On the other hand, the same morphological feature of the cellulose biofilms can be used

interactively with functional moieties. This has implications across several research fields and particularly

for scaffolding, tissue engineering and food applications. We demonstrate here the combination of the

cellulosic biofilms with heat-resistant MOF-HRP and in reaction media (3), suggesting the possibility of

forming complex pathways usable in thermally and chemically harsh environments.

References

(1) Molina-Ramírez C., Enciso C., Torres-Taborda M., Zuluaga R., Gañán P., Rojas, O. J., Castro C. Effects of alternative energy sources on bacterial cellulose characteristics produced by Komagataeibacter medellinensis, International Journal of Biological Macromolecules, 117, 735-741 (2018). DOI: 10.1016/j.ijbiomac.2018.05.195. (2) Greca L.G., Lehtonen J., Tardy B.L., Guo J., Rojas O.J., Biofabrication of multifunctional nanocellulosic 3D structures: a facile and customizable route, Materials Horizons, 5, 408-415 (2018). DOI: 10.1039/C7MH01139C7. (3) Jeremic S., Djokic L., Ajdačić V., Božinović N., Pavlovic V., Manojlović D.D., Babu R., Senthamaraikannan R., Rojas O.J., Opsenica I., Nikodinovic-Runic J.,Production of bacterial nanocellulose (BNC) and its application as a solid support in transition metal catalysed cross-coupling reactions, International Journal of Biological Macromolecules, 129, 351-360 (2019). DOI: 10.1016/j.ijbiomac.2019.01.154.


3-4th October, 2019


A Dry Bacterial Cellulose-Carboxymethyl Cellulose Formulation as Stabilizer for Pickering Oil-

in-Water Emulsions

D. Martins, F. Dourado, F.M. Gama CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal

Abstract

Hydrocolloidal microcrystalline cellulose (MCC) from plant sources, is already widely used in industry to

regulate the stability, texture, rheology and organoleptic properties of many food and cosmetic

formulations. Bacterial cellulose (BC) is produced biotechnologically by different microorganisms, but

most efficiently by acetic acid bacteria from the genera Komagataeibacter. This biomaterial is a prominent

alternative to the already marketed celluloses, being more pure, crystalline, and having nanoscale fibres

with high aspect ratio which account for excellent mechanical properties. BC has already been used in its

hydrated form for the stabilization of oil-in-water (o/w) Pickering emulsions (particle-stabilized systems,

as an alternative for the conventional surfactant-stabilized). For the sake of storage, economy and

practicality, additives for industries are preferentially provided in a dry or powder form. Co-drying

cellulose fibres or crystals with water soluble polysaccharides helps maintaining the rheologic and

structuring properties after rehydration. Dry powdered, rehydratable bacterial cellulose (BC) formulations

are reported, being produced by different grinding, drying and dispersing methods which were studied in

terms of the impact in the final product’s properties.

The main objective of this study was to assess the stabilizing properties of BC in Pickering o/w emulsions.

For this, an equimassic formulation of BC and 90 kDa carboxymethyl cellulose (BC:CMC) was prepared and

spray dried. Isohexadecane-in-water emulsions (10:90) were prepared in the presence of 0.10%, 0.25%

and 0.50% of the BC:CMC formulation. Visual and microscopic aspect of the emulsions was registered over

time. Samples were also visualized in Cryo-SEM. Rheological tests were performed to assess the

emulsion’s viscosity profile, storage and loss moduli. Interfacial tension between the immiscible phases

was measured with the Pendant Drop and Du Noüy Ring methods. For benchmarking purposes, the same

emulsion preparation and analysis protocol was made with several different commercial cellulosic

products and xanthan gum. In short, BC:CMC showed formation of a three-dimensional network and

viscosity increasing (thickening) properties, crucial characteristics for emulsion stabilizing formulations.

BC has technically superior properties that will allow it to compete with, or even replace, plant celluloses

in industry.


3-4th October, 2019


On to the impact of low cost substrates for BNC production

Rodrigues, A. C.(1), Soares da Silva, F. A.G.(1), Oliveira, J. V.(1), Forte, A.(1), Dourado, F.(1,2), Alves, M. M.(1) and Gama, M.*(1)

(1)CEB - Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal (2)Satisfibre, S.A., Portugal * [email protected]

Abstract

Bacterial nanocellulose (BNC) is an exopolysaccharide produced by certain acetic acid bacteria. It has high

crystallinity, high mechanical strength, high purity and high water-holding capacity. These properties

make it useful in making artificial skin (1), electronic paper, composite reinforcement, development of

food and cosmetic applications (2). The cost of fermentation media is believed to contribute significantly

to the operational costs, especially if synthetic commercial media are used. Hence, much research on BNC

production using low-cost substrates has been done focusing on lowering the production costs (3). Also,

to meet the requirement for industrial applications, effective large-scale BNC production systems need to

be developed, which involves improving the fermentation conditions and identifying high yield BNC-

producing strains (4). However, as with many fermentation systems, while promoting the recycling of low

value-added products, the use of complex substrates may in fact represent a bottleneck in the BNC

fermentation processes. Some of these substrates present, comparatively to synthetic nutrients, high

chemical oxygen demand (COD), total and volatile solids (TS and VS), total nitrogen (TN), antimicrobial

components (such as phenols) Consequently, these alternative substrates may place an economic

problem either downhill, due to the need for wastewaters treatments and/or, uphill, due to the need of

substrates pre-treatment.

In this work, the optimization of alternative BNC culture medium (Molasses-Corn Steep Liquor, MOL-CSL),

using Response surface methodology – central composite design was used to evaluate the effect of

inexpensive and widely available nutrients sources, namely MOL, ethanol (EtOH), CSL and ammonium

sulphate on BNC production yield under static culture by komagataeibacter xylinus BPR 2001. The

optimized parameters for maximum BNC production were: % (m/v): MOL 5.38, CSL 1.91, ammonium

sulphate 0.63, disodium phosphate 0.270, citric acid 0.115 and ethanol 1.38 % (v/v). The maximum BNC

production yield were 7.5 ± 0.54 g/L versus 1.79 ± 0.04 g/L for MOL-CSL and synthetic medium (HS-EtOH)

culture medium, respectively. The resulting wastewater from each culture medium was characterized

regarding COD, TN, TS and VS, leading to the conclusion that the wastewaters generated using MOL-CSL

are more heavily charged with organic matter, increasing the final costs of BNC production due to the

higher costs associated to wastewater treatment. Anaerobic digestion (AD) was studied for wastewater

treatment and biogas production from the wastewaters of the BNC fermentation and purification process.

Finally, a preliminary Life Cycle Assessment of BNC production was performed and will be presented.

Acknowledgements: This study was supported by the Portuguese Foundation for Science and Technology

(FCT) (PhD grant SFRH/BD/89547/2012 attributed to Ana Cristina Rodrigues and under the scope of the

strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and



3-4th October, 2019


Multibiorefinery PAC (SAICTPAC/0040/2015) and BioTecNorte operation (NORTE-01-0145-FEDER-

000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa

Operacional Regional do Norte and project nº 003435: “BUILD – Bacterial cellulose Leather”, funded by

Fundo Europeu de Desenvolvimento Regional (FEDER) through the Programa Operacional do Regional do

Norte (NORTE 2020) The authors also acknowledge the financial support of the FCT (ESF) through the

grant given to J.V. Oliveira (SFRH/BD/111911/2015).

References

(1) Fontana, J.D., De Sousa, A.M., Fontana, C.K., Torriani, I.L., Moreschi, J.C., Gallotti, B.J., De Souza, S.J., Narcisco, G.P., Bichara, J.A. Farah, L.F.X. (1990). Acetobacter cellulose pellicle as a temporary skin substitute. Applied Microbiology and Biotechnology. 24-25: 253-264. (2) Jonas, R. & Farah, L.H. (1998). Production and Application of Microbial Cellulose. Polymer Degradation and Stability. 59:101–106. (3) Keshk, S. & Sameshima, K. (2006). The utilization of sugar cane molasses with/without the presence of lignosulfonate for the production of bacterial cellulose. Applied Microbiology and Biotechnology, 72(2): 291-296. (4) Vandamme, E. J., De Baets, S., Vanbaelen, A., Joris, K., De Wulf, P. (1998). Improved production of bacterial cellulose and its application potential. Polymer Degradation and Stability, 59:(1–3), 93-99.


3-4th October, 2019


Cider waste as a resource for bacterial cellulose production and new approaches for

advanced applications

Leire Urbina, María Ángeles Corcuera, Nagore Gabilondo, Arantxa Eceiza, Aloña Retegi ‘Materials + Technologies’ Group, Engineering School of Gipuzkoa. Department of Chemical and Environmental

Engineering, University of the Basque Country (UPV/EHU), Pza. Europa 1, 20018 Donostia-San Sebastian.

([email protected])

Abstract

In recent years, the investigation and utilization of bacterial cellulose in functional materials has been the

focus of numerous research studies, as this material has some remarkable properties such as excellent

mechanical properties due to the 3D network-like structure formed by cellulose nanofibers during its

biosynthesis, high water holding capacity, high crystallinity and purity(1). One of the major challenges to

address in bacterial derived polymer technology is to find suitable carbon sources as substrates that are

cheap for achieving large scale industrial applications(2). In this context, apple pomace is the main by-

product of apple juice and cider production. It contains minerals and also a large amount of carbohydrates.

Taking these features into account, cider by-products could be considered a good substrate for BC. In this

work, the viability of cider by-products from the Basque Country as a cheap and sustainable carbon

substrate for BC production was demonstrated. In addition, due to the interesting physicochemical

properties of the obtained product, different BC-based nanocomposites were developed for a wide range

of applications. Firstly, environmentally friendly membranes were prepared by in situ and ex situ routes

based on BC as template and chitosan as functional entity, for the elimination of copper in wastewaters.

Secondly, water-activated shape memory nanocomposites were prepared by the infiltration of a

waterborne polyurethane dispersion into BC membranes for possible biomedical applications. In addition,

mailto:([email protected]


3-4th October, 2019


BC-based films for potential food related applications with improved mechanical properties and

hydrophobicity and antioxidant properties were developed. Finally, BC/graphene oxide hybrid spheres

were developed in dynamic cultures for biomedical applications. As demonstrated in this work, bacterial

cellulose can be considered an advanced material and these new approaches open new possibilities for

the application of this biopolymer in different fields.

Figure 1. Schematic representation of the work.

References

(1) Badshah, M., Ullah, H., Khan, A.R., Khan, S., Park, J.K., Khan, T. (2018). Surface modification and evaluation of bacterial cellulose for drug delivery. International Journal of Biological Macromolecules. 113: 526-533.

(2) Araújo, D.J.C., Machado, A.V. Vilarinho, M.C.L.G. (2018). Availability and suitability of agroindustrial residues as feedstock for cellulose-based materials: brazil case study. Waste and Biomass Valorization. 1-16.

CIDER BY-PRODUCTS

Fermentation

Watercleaning

Shapememory

Foodpackaging

Drugdelivery

https://www.sciencedirect.com/science/article/pii/S0141813017336553#!





https://www.sciencedirect.com/science/journal/01418130


3-4th October, 2019


Bacterial nanocellulose-based films with antibacterial and conductive properties for active

and intelligent food packaging

Vilela, C.,1* Moreirinha, C.,1 Domingues, E.,2 Figueiredo, F. M. L.,2 Almeida, A.,3 Freire, C. S. R.

1Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, 3810-193 Aveiro, Portugal, [email protected];

2Department of Materials and Ceramic Engineering, CICECO – Aveiro Institute of Materials, University of Aveiro, 3810-193 Aveiro, Portugal; 3Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal

Abstract

Bacterial nanocellulose (BNC) is becoming an important substrate to design multifunctional nanomaterials

with singular and custom-made properties for application in several areas, including in active and

intelligent food packaging (1-3).

In the present study, antibacterial conductive nanocomposite films composed of poly(sulfobetaine

methacrylate) (PSBMA) and BNC were developed and investigated for application in food packaging. The

films were fabricated via the in situ polymerization of the zwitterionic sulfobetaine methacrylate (SBMA)

monomer inside the BNC nanofibrous network and in the presence of poly(ethylene glycol) diacrylate as

cross-linker. The ensuing nanocomposites are macroscopically homogeneous, more transparent than

pristine BNC, and present thermal stability up to 200 °C in N2 atmosphere. Additionally, the films have

good mechanical performance with Young’s modulus ≥ 3.1 GPa and viscoelastic properties with storage

modulus ≥ 181 MPa, UV-blocking properties and high water-uptake capacity (450%–559%). The zwitterion

film with 62 wt.% of cross-linked PSBMA showed bactericidal effect against Staphylococcus aureus (4.3–

log CFU reduction) and bacteriostatic activity towards Escherichia coli (1.1–log CFU reduction), together

with a maximum proton conductivity of ca. 1.5 mS cm−1 at 94 °C and 98% relative humidity.

In view of this set of assets, the zwitterion PSBMA/BNC nanocomposites show potential as films for

application as active and intelligent food packaging materials, because they can shield the food from the

effects of UV-radiation, inhibit the growth of pathogenic microorganisms responsible for food spoilage

and foodborne illness, absorb moisture and water, and act as conductimetric humidity sensors to control

humidity levels in foodstuff.

Funding

This work was developed within the scope of the projects CICECO – Aveiro Institute of Materials

(UID/CTM/50011/2019) and CESAM (UID/AMB/50017/2019), financed by national funds through the

FCT/MEC. The research contract of C.V. is funded by national funds (OE), through FCT – Fundação para a

Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of

the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. FCT is also

acknowledge for the research contract under Stimulus of Scientific Employment 2017 to C.S.R.F.

(CEECIND/00464/2017).



3-4th October, 2019


References

(1) Vilela, C., Kurek, M., Hayouka, Z., Röcker, B., Yildirim, S., Antunes, M. D. C., Nilsen-Nygaard, J., Pettersen, M. K., Freire, C. S. R. (2018). A concise guide to active agents for active food packaging. Trends Food Sci. Technol. 80:212–222. (2) Poyatos-Racionero, E., Ros-Lis, J. V., Vivancos, J. L., Martínez-Máñez, R. (2018) Recent advances on intelligent packaging as tools to reduce food waste. J. Clean. Prod. 172:3398–3409. (3) Azeredo, H. M. C., Barud, H., Farinas, C. S., Vasconcellos, V. M., Claro, A. M. (2019) Bacterial cellulose as a raw material for food and food packaging applications. Front. Sustain. Food Syst. 3:7.


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SESSION 6 - Biomedical applications, part 1 (chair person: Dieter Klemm)


3-4th October, 2019


KEYNOTE LECTURE: Pharmaceutical Insights: Overcoming the Challenges of Bacterial

Nanocellulose for Controlled Drug Delivery

D. Fischer*

Friedrich Schiller University Jena, Pharmaceutical Technology and Biopharmacy, Lessingstraße 8, 07743 Jena, Germany

*[email protected]

Abstract

Bacterial nanocellulose (BNC) is an innovative biofabricated material, generated in a biotechnological

process by Komagataeibacter bacteria strains from glucose and consisting of about 1% cellulose and 99%

water. The interest in BNC as drug delivery system dramatically increased during the last years. The

material offers unique characteristics such as high purity, excellent biocompatibility and extraordinary

tensile strength. Due to its nanosized 3D network resulting in an enormous surface area-to-volume ratio,

it is expected to hold a large amount of active ingredients. However, due to the hydrophilic character,

prolongation of the drug release over weeks and months or incorporation and transport of water-

insoluble drugs especially of BCS class III and IV as a major trend of pharmaceutical industry, require the

application of more sophisticated loading and release strategies.

We developed different loading techniques to accomplish a (i) fast and immediate or vice versa (ii)

sustained and extended drug release from several hours to weeks. Native and freeze-dried BNC was

equipped with additives like thermo-responsive Poloxamer micelles and gels as well as nanoemulsions

and liposomes to deliver drugs. One of the longest release profiles over up to one week could be realized

for the antiseptic octenidine in the presence of different types of Poloxamers in various concentrations in

BNC with improved mechanical and antimicrobial properties and unchanged biocompatibility. Lipophilic

drugs like coenzyme Q10 demonstrated a controllable release depending on the type of BNC (native vs.

freeze dried), the loading technique and the utilization of nanoemulsions. By varying these parameters,

differences in penetration depth and distribution of lipophilic drugs could be shown in porcine skin

experiments (Saarbrücken model and tape stripping). Beside small molecules, high molar mass drugs like

plasmid DNA were protected against enzymatic degradation by maintaining the high biocompatibility of

BNC and transfection efficacy of the plasmids. The release was found to be dependent on the type of BNC,

the plasmid and the loading technique and lasted over 2-50 days.

In conclusion, using sophisticated techniques, BNC can be tailored as immediate and sustained drug

release system for ready-to-use and patient-designed bedside applications e.g. for dermal wound

treatment, cosmetics or the use as implant.

Acknowledgments The authors gratefully acknowledge the Free State of Thuringia and the European Social Fund (2016 FGR 0045) for funding. K. xylinus culture was kindly provided by JeNaCell GmbH.

mailto:*[email protected]


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Adjusting BNC for 3D tissue engineering by modifications at different stages of its production

K. Kubiak, I. Cielecka, P. Jacek, M. Szustak, E. Gendaszewska-Darmach, S. Bielecki

Institute of Technical Biochemistry, Lodz University of Technology, 4/10 Stefanowskiego Str, 90-924 Lodz, Poland

Abstract

Development of three-dimensional scaffolds mimicking natural surroundings of cultivated in vitro cells is

an ongoing challenge for tissue engineering. Bacterial nano-cellulose (BNC) as a biomaterial well-known

from its biocompatibility have been applied in this field widely. However, research show that tight spatial

organization of cellulose fibers limits cell ingrowth and restricts practical use of BNC-based scaffolds.

Important advantage of BNC as scaffold is its water-holding capacity and therefore possibility to soak it

with substances promoting cells welfare and/or differentiation.

Recently, both issues have been addressed by independent interventions in chondrocytes-supporting

scaffolds production process: in situ BNC- carrageenan porous composite preparation (1) and by usage of

genetically modified Komagataeibacter hansenii ATCC 23769 strain to produce BNC membranes with

relaxed fibers structure (2). Composites and membranes were evaluated as scaffolds in in vitro assays to

verify chondrogenic ATDC5 cells line viability, glycosaminoglycan production and specific genes expression

as well as RBL-2H3 mast cells degranulation. Both studies showed improvement of pro-chondrogenic

properties of newly obtained materials when compared to unchanged BNC.

In this presentation we deliver some hypothesis how structure and composition of the BNC-based

scaffolds influenced the chondrogenic cells behavior. Interestingly, similar cell-protective properties of

BNC have been revealed in in vitro tests performed with BNC-glycerol composites on keratinocytes (3).

Furthermore, we summarize pros and cons of introducing improvements on different stages of BNC

production process including methods complexity, costs and ecology.

References

(1) Cielecka, I., Szustak, M., Gendaszewska-Darmach, E., Kalinowska, H., Ryngajłło, M., Maniukiewicz, W., Bielecki, S. (2018) .Novel Bionanocellulose/κ-Carrageenan Composites for Tissue Engineering, Applied Sciences. 8:1352.

(2) Jacek P., Szustak M., Kubiak K., Gendaszewska-Darmach E., Ludwicka K., Bielecki S. (2018) Scaffolds for Chondrogenic Cells Cultivation Prepared from Bacterial Cellulose with Relaxed Fibers Structure Induced Genetically. Nanomaterials (Basel). 8:1066.

(3) Cielecka, I., Szustak, M., Kalinowska, H., Gendaszewska-Darmach, E., Ryngajłło, M., Maniukiewicz, W., Bielecki, S. (2019) Glycerol-plasticized bacterial nanocellulose-based composites with enhanced flexibility and liquid sorption capacity, Cellulose. doi:10.1007/s10570-019-02501-1


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Evaluation of Three Types of Bacterial Nanocellulose Tubes for Application as Vascular Graft:

Bioreactors Determine Tubes Structure and Properties

Feng Hong*, Luhan Bao, Jingyu Tang, Lin Chen

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, North Renmin Road 2999, Shanghai 201620, China

* [email protected]

Abstract

Bacterial nanocellulose (BNC) has a promising prospect in application of vascular graft[1-3]. Several

bioreactors have been utilized for synthesis of the BNC tubes[1,4], however little knowledge is known about

differences in tube structures and properties. While the potential of BNC for artificial blood vessels has

been widely studied, a systematical comparison of BNC tubes prepared in different kinds of bioreactors

has never been performed. Herein, a newly designed device composed of a glass rod in the axis of a

silicone tube (G-BNC bioreactor) was compared with two reported bioreactors[4] in fabricating three types

of BNC tubes (G-BNC, S-BNC, and D-BNC tubes). The differences in physicochemical properties and

performances as vascular grafts, and morphologies especially the surface roughness were compared in

detail.

References

(1) Hong, F., Wei, B., Chen, L. (2015). Preliminary study on biosynthesis of bacterial nanocellulose tubes in a novel double-silicone-tube bioreactor for potential vascular prosthesis. BioMed Research International, 2015: 560365.

(2) Tang, J., Bao, L., Li, X., Chen, L., Hong, F. F. (2015). Potential of PVA-doped bacterial nano-cellulose tubular composites for artificial blood vessels. Journal of Materials Chemistry B, 3(43): 8537-8547.

(3) Li, X., Tang, J., Bao, L., Chen, L., Hong, F. F. (2017). Performance improvements of the BNC tubes from unique double-silicone-tube bioreactors by introducing chitosan and heparin for application as small-diameter artificial blood vessels. Carbohydrate Polymers, 178: 394-405.

(4) Tang, J., Li, X., Bao, L., Chen, L., Hong, F. F. (2017). Comparison of two types of bioreactors for synthesis of bacterial nanocellulose tubes as potential medical prostheses including artificial blood vessels. Journal of Chemical Technology and Biotechnology, 92 (6): 1218-1228.



3-4th October, 2019


Preparation and properties of type I collagen/ quaternized chitosan/ Bacterial cellulose

composite films for multifunctional wound dressings

Haiyong Ao, Chen Zhou, Yanjiao Nie, Wenwen Jiang, Yizao Wan

Institute of advanced materials, East China Jiaotong University, Nanchang, Jiangxi, CHina

Abstract

As a natural polymer produced by several strains of bacteria, BC is made up of nanofiber and has nano-

scaled three-dimensional network structure, and has several unique physical and chemical performances,

such as high purity, great water imbibition and permeability, high tensile strength, favorable

biocompatibility and non-toxic[1]. Due to these outstanding characteristics BC has wide application for

wound dressing. However, Pure BC has neither favorable bactericidal properties nor bioactive groups to

promote cute and severe skin injury repair.

In this study, Hydroxypropyltrimethyl ammonium chloride chitosan (HACC) with considerable

antimicrobial properties and type I collagen (ColI) with favorable cytocompatibility were introduced into

bacterial cellulose (BC) and ColI/HACC/BC composite films were fabricated by in situ static culture. The

results of SEM and FTIR effectively proved that HACC and ColI were incorporated with cellulose

successfully. As following HACC and ColI introduced, the crystallinity, porosity and water vapor

transmission of BC based composite films were reduced, while the water absorbency were improved. The

obtained ColI/HACC/BC composite films exhibited considerable antibacterial properties, which inhibited

bacterial colonization and biofilm formation of Staphylococcus aureus (S. aureus, ATCC 25923), compared

with BC and ColI/BC. Furthermore, NIH-3T3 fibroblast cells on ColI/HACC/BC composite films showed

further proliferation than cells on BC and HACC/BC films. The obtained ColI/HACC/BC composite films with

favorable antibacterial properties and cytocompatibility may be regarded as potential candidates for

wound dressings.

References

(1) Luo, H., Xiong, G., Wan, Y. (2014). In situ phosphorus K-edge X-ray absorption spectroscopy studies of calcium–phosphate formation and transformation on the surface of bacterial cellulose nanofibers. Cellulose. 21:3303–3309


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Bacterial Cellulose-Polyhydroxyalkanoate hydrogels with antimicrobial capacity against

Staphylococcus aureus

Virginia Rivero-Buceta1, María Rosa Aguilar2,3,4, Ana M. Hernández-Arriaga1, Francisco Blanco1, Julio San Román2,3,4, Auxiliadora Prieto1,4.

1Polymer Biotechnology Group, Biological Research Center (CIB-CSIC), CSIC, 28040, Madrid, Spain. 2Biomaterials Group, Institute of Polymer Science and Technology (ICTP-CSIC), Spain. 3Networking Biomedical Research Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. 4Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy‐Spanish National Research Council (SusPlast‐CSIC), Madrid, Spain

[email protected]

Abstract

Bacterial cellulose (BC) is an excellent wound dressing device due to its characteristics, such as its

biocompatibility, non-toxicity, mechanical stability and high moisture content. However, it lacks

antimicrobial activity, which is a critical function of the skin-barrier that is compromised during wound

healing.

One of the leading candidates with potential applications in the medical field is the group of naturally

produced bacterial polyesters or poly([R]-3-hydroxyalkanoates) (PHAs) that constitute a large class of

biodegradable biopolymers that show minimal tissue toxicity. The physical and mechanical properties of

PHAs are significantly influenced by their monomer composition and chemical structure, which can be

tailored by metabolic engineering approaches. In this work, antimicrobial BC membranes were obtained

by chemical blending of BC with PHACOS, a second generation polymer containing thioester groups in the

side chain with demonstrated antibacterial properties against Methicillin-resistant Staphylococcus aureus

(MRSA) (1). MRSA are especially troublesome in hospitals, prisons, schools, and nursing homes where

patients with open wounds, invasive devices, and/or weakened immune systems are at high risk of

infection.

BC was produced in the strain Komagataeibacter medellinenesis ID13488 (2). PHACOS was produced in

the model bacterium Pseudomonas putida KT2440 by metabolic engineering using decanoic acid as

inducer of the growth and polymer synthesis, and 6-acetylthiohexanoic acid as PHA precursor in a two-

stage strategy (1). Homogeneous mixtures of BC and PHA or PHACOS were obtained using the ionic liquid

(IL) BMIMCl as solvent. Hydroxyl groups of BC and carbonyl groups of PHA form new hydrogen bonds as

demonstrated by the FTIR experiments. TGA analysis showed that not only the blends show Td

(decomposition temperatures) values in between the individual components, but also the Td increases

with the cellulose content in the mixture. The SEM images show the morphology of the blends as a phase

separated structure in relation to the proportion of the polyhydroxyalkanoate in the blend. The

antimicrobial activities of the BC, BC/PHA-control and BC/PHACOS hydrogels were tested after the

controlled enzymatic hydrolysis of the blends with a medium-chain lenght PHA depolymerase of

Streptomyces exfoliatus K10 DSMZ 41693 showing a 1-log reduction in bacterial viability for S. aureus only

in hydrogels that contain PHACOS.



3-4th October, 2019


This study demonstrates the viability of developing new biodegradable blends based on BC and PHACOS

with antimicrobial activity against S. aureus and their potential uses as antimicrobial devices.

ACKNOWLEDGMENTS: The authors thank the financial support from grants BIO2017-83448-R,

P2018/NMT-4389 and MAT2017-84277-R.

REFERENCES

(1) Dinjaski, N., Fernández-Gutiérrez, M., Selvam, S., Parra-Ruiz, F., Lehman, SM., San Román, J., García, E., García, JL., García, AJ., Prieto, MA. (2014). Biomaterials. 35: 14-24 (2) Hernandez-Arriaga AM., del Cerro, C., Urbina, L., Eceiza, A., Corcuera, MA., Retegi, A., Prieto, MA. (2019). Genome sequence and characterization of the bcs clusters for the production of nanocellulose from the low pH resistant strain Komagataeibacter medellinensis ID13488. Microb Biotechnol. doi: 10.1111/1751-7915.13376


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SESSION 7 - Biomedical applications, part 2 (chair person: Dana Kralisch)


3-4th October, 2019


Study on Preparation and properties of micro-nanofibers composite vascular scaffold

Yang Shanshan1,2, Cui Teng1,2, Peng Mengxia1,2, Luo Honglin1,2, Yang Zhiwei1,2, Wan Yizao1,2 1 Institute of Advanced Materials, East China Jiaotong University, Jiangxi Province, Nanchang City, 330013 2 College of Materials Science and Engineering, East China Jiaotong University, Jiangxi Province, Nanchang City, 330013

[email protected]

Abstract

With the aging of the population and the change of living habits, cardiovascular patients are showing a

growing trend, and the clinical application of vascular stents is gradually increasing. In this study, a micro-

nano composite artificial blood vessel with the size of less than 6 mm based on bacterial cellulose (BC)

and cellulose acetate (CA) was constructed (Fig. 1a). Scanning electron microscopy characterization

showed that vessel scaffold was composed by the CA microfibers with the diameter of 0.82 μm and the BC

nanofibers with the diameter of 48.88 nm (Fig. 1b). Increase of BC content, the mechanical properties

and thermal stability of the stent materials were gradually improved. The mechanical properties of BC/CA

scaffolds increased from 0.3 MPa to 1.8 MPa after 10 hours of interfacial culture. Compared with pure BC

and CA, all BC/CA scaffolds had better proliferation promoting effect. It was worth noting that with the

increase of BC content, the promotion of cell proliferation behaviour first increased and then decreased,

likely due to the obvious pore structure change of BC/CA scaffolds caused by exceeded BC content, which

resulted into a change of cell proliferation behaviour. In addition, all BC/CA micro-nano composite stents

exhibited good blood compatibility.In conclusion, BC/CA micro-nano composite vascular stent has good

biocompatibility and blood compatibility, and has good application prospects. It is expected to be a good

substitute for natural blood vessels.

Figure 1 The macrophotographs of CA (a), SEM images of the BC/CA membranes(b)



3-4th October, 2019


References

(1) Kuang, H. Z., Wang, Y., Hu, J. F., Wang, C. S., Lu, S. Y., Mo, X. M. (2018). A Method for Preparation of an Internal Layer of Artificial Vascular Graft Co-Modified with Salvianolic Acid B and Heparin. Appl. Mater. Interfaces. 8:19365-19372

(2) Guo, F. Y., Wang, N., Wang, L., Hou, L. L., Ma, L., Liu, J., Chen, Y., Fan, B. B., Zhao, Y. (2015). An electrospun strong PCL/PU composite vascular graft with mechanical anisotropy and cyclic stability. J. Mater. Chem. A 3:4782-4787


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Bacterial nanocellulose-hyaluronic acid microneedles for skincare applications

Fonseca, D. F. S., Vilela, C., Silvestre, A. J. D., Freire, C. S. R. *

CICECO – Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal, *[email protected]

Abstract

Bacterial nanocellulose (BNC), a versatile biopolymer with excellent mechanical properties, porosity and

water holding ability, has gained a great deal of attention during the past few decades due to its peculiar

ultrafine network of micro- and nanofibrils. These auspicious features prompt the use of BNC as a

macromolecular support for the incorporation of multiple functional ingredients, and their controlled

release [1]. Within this context, the incorporation of cosmeceuticals in BNC provides the basis for the

development of exceptional materials with great potential in skincare applications.

This work describes the development of microneedles (MNs), viz. micron-sized needles designed to bypass

the stratum corneum [2], for skincare applications using hyaluronate and BNC. Specifically, hyaluronate-

based MNs will improve the overall skin appearance, due to its plumping, smoothing and filling effect [3].

And, BNC, which is employed as a back layer of this patch, enables a controlled release of active molecules

(e.g. antioxidants) through the microchannels created by the hyaluronate needles. Therefore, this

approach focuses on the quick release of hyaluronate coupled with a controlled delivery of an antioxidant

impregnated inside the BNC membrane. Rutin (a flavonoid glycoside), shows anti-aging effects on human

dermal fibroblasts and human skin, attracting increased attention in the cosmeceutical area, and was

selected as model compound due to its low skin permeability [4].

Notably, the as-prepared BNC-rutin loaded hyaluronate MNs displayed homogeneous and uniform arrays,

with sufficient mechanical force to withstand skin insertion (failure force > 0.15 N/needle). This work

unveiled the potential of using BNC as a versatile matrix for the incorporation of active molecules, as part

of MNs systems, and making them promising devices for cosmetic applications.

Funding

This work was developed within the scope of the projects CICECO – Aveiro Institute of Materials

(UID/CTM/50011/2019) and CESAM (UID/AMB/50017/2019), financed by national funds through the

FCT/MEC. The research contract of C.V. is funded by national funds (OE), through FCT – Fundação para a

Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of

the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. FCT is also

acknowledge for the doctoral grant to D.F.S.F. (PD/BD/115621/2016) and research contract under

Stimulus of Scientific Employment 2017 to C.S.R.F. (CEECIND/00464/2017).

References

1. Silvestre, A. J. D.; Freire, C. S. R.; Neto, C. P. (2014) Do bacterial cellulose membranes have potential in drug-delivery systems? Expert opinion on drug delivery. 11, 1113–1124.



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2. Larrañeta, E.; Lutton, R. E. M.; Woolfson, A. D.; Donnelly, R. F. (2016) Microneedle arrays as transdermal and intradermal drug delivery systems: Materials science, manufacture and commercial development. Materials Science and Engineering R: Reports. 104, 1–32. 3. Kang, G.; Tu, T. N. T.; Kim, S.; Yang, H.; Jang, M.; Jo, D.; Ryu, J.; Baek, J.; Jung, H. (2018) Adenosine-loaded dissolving microneedle patches to improve skin wrinkles, dermal density, elasticity and hydration. International Journal of Cosmetic Science. 40, 199–206. 4. Choi, S. J.; Lee, S. N.; Kim, K.; Joo, D. H.; Shin, S.; Lee, J.; Lee, H. K.; Kim, J.; Kwon, S. Bin; Kim, M. J.; Ahn, K. J.; An, I. S.; An, S.; Cha, H. J. (2016) Biological effects of rutin on skin aging. International Journal of Molecular Medicine. 38, 357–363.


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The Prospect of Bacterial Cellulose Impregnated with Antimicrobials in Treatment of Biofilm-

Related Bone Infections

Adam Junka1,2*, Bartłomiej Dudek2, Joanna Czajkowska2, Agnieszka Zegan2, Paweł Migdal2, Iwona Bil-Lula3, Karol Fijalkowski4

1 Pharmaceutical Microbiology and Parasitology Department, Medical University of Wroclaw, Borowska 211a, 50-556 Wroclaw 2 Laboratory of Microbiology of Łukasiewicz Research Network – PORT Polish Center for Technology Development, Wrocław, Poland, Stabłowicka 147 street, 54-066 Wroclaw 3 Department of Clinical Chemistry, Medical University of Wroclaw, Borowska 211a, 50-556 Wrocław, Poland 4 Department of Immunology, Microbiology and Physiological Chemistry, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology in Szczecin, Piastów 45, 70-311 Szczecin, Poland

Abstract

Introduction: Bone infections caused by microbial biofilms pose a significant threat not only to

homeostasis of patients but also to their life. As we already proven, biofilms are able to re-shape structure

of bone and to hide within [1]. It causes not only problems with biofilm eradication, but it also allows

microbes to stay dormant deep inside of bone and then to spread, causing recurrent infections [2].

Therefore, eradication of biofilm and prevention of microbes from spreading is an urgent clinical need.

The bacterial cellulose [BC], thanks to its excellent material and water-related properties [3] may be

applied to provide appropriate antimicrobial agents to bone and to meet clinical demand above-

mentioned.

Aim: To investigate in vitro applicability of bacterial cellulose, impregnated with gentamycin antibiotic or

bacteriophage, to eradicate biofilms formed on hydroxyapatite/bone and to sequestrate pathogens.

Materials and Methods: BC discs were produced using ATCC23679 Komagataeibacter xylinus strain and

subjected to standard purification. Next, discs were saturated with various concentrations of gentamycin

and Enterobacteria phage T4 units. The series of spectrometric and quantitative analyses aiming to

confirm presence of antibiotic or phage within cellulose were performed, respectively. Subsequently, BC

discs were wrapped around hydroxyapatite discs or rat femur bones with preformed Staphylococcus

aureus ATCC6538 or T4-sensitive Escherichia coli biofilm. After a contact time, number of bacterial cells

on HA/bone, BC and in medium was counted using quantitative culturing. Moreover, advanced Scanning

Electron Microscopy Imaging (SEM) was performed to visualize processes taking place within

experimental setting. In a separate line of investigation, solutions containing high concentrations of

analyzed microbes were placed into BC discs and left for 24 – 72h. Subsequently, the estimation of cell

number and penetrability through BC was assessed.

Results: BC eagerly absorbed gentamycin and T4 phage in applied range of concentrations. Gentamycin

released from BC was able to reduce bacterial biofilm from HA/bone in dose-dependent manner. In turn,

decrease of bacterial cell number in result of application of T4-containing BC was relatively low in this

experimental setting. Both gentamycin and phage-containing BC discs were able to efficiently impede


3-4th October, 2019


spreading and penetrability of microbes. Interestingly, also control BC discs (without antimicrobial agent)

were able to keep pathogens inside its structure, however within more limited period of time.

Conclusions: Obtained pilot in vitro results indicate promising potential of bacterial cellulose containing

antimicrobial agents in treatment of biofilm-related bone infections and in preventing pathogens from

spreading.

This work was funded from 2017/27/B/NZ6/02103 National Science Centre grant: Analysis of mechanisms

of increased efectivness of antimicrobial substances against biofilms in the presence of a rotating

magnetic field

References

[1] Junka, A., Szymczyk, P., Ziółkowski, G., Karuga-Kuźniewska, E., Smutnicka D., Bil-Lula I., Bartoszewicz, M., Mahabady, S., Sedghizadeh, P. (2017). Bad to the bone: on in vitro and ex vivo microbial biofilm ability to directly destroy colonized bone surfaces without participation of host immunity or osteoclastogenesis. PLoS One 12:1; e0169565 [2] Uçkay,I., Assal, M., Legout, L., Rohner,R., Stern, R., Lew, D., Hoffmeyer, P., Bernard, L. (2006). Recurrent Osteomyelitis Caused by Infection with Different Bacterial Strains without Obvious Source of Reinfection. J Clin Microbiol 44:3; 1194–1196 [3] Fijałkowski, K., Żywicka, A., Drozd, R., Junka A., Peitler, D., Kordas, M., Konopacki M., Szymczyk, P., Rakoczy, R. (2017) Increased water content in bacterial cellulose synthesized under rotating magnetic fields. Electromagn Biol Med 36:2; 192-201


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Modified BNC as a vascular prosthesis and cardiac valve prosthesis - preclinical studies.

Piotr Siondalski, M.D. Ph.D. Gdansk, Poland Medical University of Gdansk, Cardiac and Vascular Surgery Department, Gdansk, Poland

Abstract

Introduction:

Over the last years, a series of laboratory tests and BNC modifications were performed.

BNC material approval for in vivo testing was obtained. Final results were published in "Materials Science

& Engineering C" Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter

xylinus E25 as a new biological implant.

Purpose of the study:

Assessment of the suitability of BNC as a bioimplant in vascular surgery and cardiac surgery in an animal

model.

Material and method:

Sixteen operations performed on pigs descending aortic reconstruction. Final results were evaluated after

6 months of observation.

Three operations were performed on sheep with a three-lobe heart valve implant as a pulmonary conduit.

Results were evaluated after 6 months of observation.

Outcome:

General condition of animals observed during the study was assessed as good.

Function of the BNC bioimplant as aortic wall and heart valve remained normal (echocardiography).

Tests after performing planned euthanasia: macroscopic, histopathological, physical: X-ray, MikroCT, X-

ray diffractometry (XRD), showed no significant features of degeneration and biodegradation of the

implanted BNC.

Conclusions:

Bionanocellulose can be considered a very promising biological material used as aortic vascular prosthesis

and heart valve.


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Delivery of antiseptic solutions by a bacterial cellulose wound dressing: PHMB, octenidine

and povidone-iodine Bernardelli de Mattos, I.*, Funk, M., Tuca, A. C., Holzer, J. C. J., Popp, D.,

Mautner. S., Birngruber, T., Kamolz, L. P., Nischwitz, S. P., Groeber-Becker, F. Fraunhofer Institute for Silicate Research ISC, Translational Center Regenerative Therapies, Würzburg, Germany

Abstract

Bacterial nanocellulose (BNC) is considered a promising delivery system for various substances. Novel

approaches using BNC-based wound dressings in combination with commercially available antiseptics

could be used in the treatment of burn patients. This unique delivery capacity of the material associated

with the wound coverage characteristic of the dressing itself could be a key factor for the treatment of

extensive burn wounds which are often and prone to infections. The aim was to test the uptake and

release capacity of BNC for several antiseptics, e.g. polyhexanide (PHMB), octenidine and povidone-

iodine-based solutions, and the efficacy of the released antiseptics against infectious microorganisms.

Uptake and release for four different antiseptics, containing PHMB (Prontosan® and LAVANID® 2),

octenidine (Octenisept®) or povidone-iodine (Betaisodona®), was performed using commercially available

BNC-based wound dressings. UV spectrophotometry for the solutions was used to measure the

concentration of the molecules and the pace of the uptake and release was compared to dextran

molecules with different molecular weight coupled with fluorescent markers. Furthermore, the antiseptic

efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested. Dextran

molecules showed a size-dependent behaviour regarding to both uptake and release from the BNC. The

same behaviour was observed considering the molecular size of the active compounds contained in the

tested antiseptics. After 30min incubation most of the antiseptics showed at least a half maximal uptake

rate. At that time point the BNC was carrying, for all the solutions tested, a concentration of antiseptics

higher than needed to achieve the 24h MBC for MRSA [1, 2]. One of the PHMB-based (LAVANID® 2) and

the octenidine solution showed a prolonged release profile, whereas the other PHMB antiseptic

(Prontosan®) and the povidone-iodine-based solution achieved a more pronounced delivery within the

first 24h. All tested BNC combinations showed a dose-dependent efficacy against S. aureus using an

adapted disk diffusion assay in vitro. The efficacy of the BNC dressing-antiseptic combination against the

bacteria was higher, considering the comprised dose, when compared to commercially available wound

dressings, including a BNC-based dressing product releasing PHMB. Combination of tested antiseptics with

BNC showed to be an efficient approach to control bacterial infections. Considering the easy-handling

uptake process, and the releasing profile, which can be adapted, depending on the severity of the

infection, by simply changing the antiseptic used, the utilization of this procedure in clinical emergency

settings raise as a novel tool to aid the treatment of burn patients.


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References [1] Koburger T, Hubner NO, Braun M, Siebert J, Kramer A. (2010). Standardized comparison of antiseptic efficacy of triclosan, PVP-iodine, octenidine dihydrochloride, polyhexanide and chlorhexidine digluconate. J Antimicrob Chemother. 65:1712-9. [2] Hardy K, Sunnucks K, Gil H, Shabir S, Trampari E, Hawkey P, et al. (2018). Increased Usage of Antiseptics Is Associated with Reduced Susceptibility in Clinical Isolates of Staphylococcus aureus. MBio. 9.


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SESSION 8 - Sharp presentations: part 2 (chair person: Anna Roig)


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Development and characterization of native and citric acid-modified cellulose's drug-delivery

system for bacterial biofilm eradication

Karol Fijalkowski1*, Daria Ciecholewska1, Adam Junka2,3

1 Department of Immunology, Microbiology and Physiological Chemistry, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology in Szczecin, Piastów 45, 70-311 Szczecin, Poland

2 Pharmaceutical Microbiology and Parasitology Department, Medical University of Wroclaw, Borowska 211a, 50-556 Wrocław, Poland

3 Laboratory of Microbiology of Łukasiewicz Research Network – PORT Polish Center for Technology Development, 54-066 Wrocław, Poland

Abstract

Despite many advantages, bacterial cellulose (BC) itself does not have antibacterial properties. In order

to equip a BC material with antimicrobial activity, the polymer can be impregnated with drugs, e.g.

antibiotics or antiseptics. However, in the case of bacteria occurring in biofilms, not only the working

concentration of the antimicrobials, but also their release time is of paramount significance considering

practical applications of the active biomaterial [1]. A lasting antimicrobial effect requires a continued

release of the antimicrobial agent from the carrier. Therefore, to obtain a material of strictly defined

properties required for the medical applications, the BC is subjected to various specific modifications, e.g.

by using carbohydrate cross-linkers to maintain three dimensional structure of the material [2].

The aim of the current study was to develop and evaluate applicability of native and citric acid-modified

BC membranes as the antibiotics or antiseptics delivery systems with potential to eradicate biofilms

formed by pathogenic strains of bacteria.

BC membranes were produced using Komagataeibacter xylinus strain (ATCC 53524), purified by alkaline

treatment and then subjected to crosslinking reactions by immersion in the solution of citric acid and

various catalysts including disodium phosphate, sodium bicarbonate and ammonia. The obtained

materials were characterized in terms of its physical and biological properties using SEM and ATR-FTIR

and by the determination of its density, porosity, water-related properties and cytotoxicity. In a separate

line of investigation, BC membranes were saturated with various concentrations of antibiotics and

antiseptics and the series of spectrometric and quantitative analyses aiming to confirm the presence and

release behavior of antimicrobials from the BC samples were performed. The final analyzes concerned the

activity of modified BC impregnated with antimicrobial substances against bacterial biofilms and

evaluation of their cytotoxicity.

The obtained citric acid modified BC showed an altered macro- and micro-structure, degree of porosity,

were characterized by increased swelling ability (up to six times compared to the control) and reduced

rate of water release (up to seven times compared to the control). Furthermore, except for ammonia,

other catalysts were not cytotoxic. The antimicrobials-impregnated modified BC exhibited strong and

long-term antimicrobial activity against biofilm producing clinical bacterial isolates. Thus, it can be

concluded that a combination of antimicrobial agents and citric acid modified BC could be used for

production of a new class drug-delivery systems with both antimicrobial activity and biocompatibility.


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This study was supported by the National Centre for Research and Development in Poland (Grant No.

LIDER/011/221/L-5/13/NCBR/2014) and the National Science Center (Grant No. 2017/27/B/NZ6/02103).

References

[1] Żywicka, A., Fijałkowski, K., Junka, A., Grzesiak, J., El Fray, M. (2018). Modification of bacterial cellulose with quaternary ammonium compounds based on fatty acids and amino acids and the effect on antimicrobial activity. Biomacromolecules, 19(5): 1528-1538. [2] Meftahi, A., Khajavi, R., Rashidi, A., Rahimi, M. K., Bahador, A. (2018). Preventing the collapse of 3D bacterial cellulose network via citric acid. Journal of Nanostructure in Chemistry, 8(3): 311-320.


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A biodegradable antibacterial nanocomposite based on oxidized bacterial cellulose for fast

hemostasis and wound healing

Haibin Yuan1,2, Lin Chen2, Feng F. Hong1,2*

1 State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, China

2 College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, North Renmin Road 2999, Shanghai 201620, China

* [email protected]

Abstract

Developing biodegradable and antibacterial hemostatic materials with high blood absorption for internal

noncompressible hemorrhage remains a challenge1. Here we report a hemostatic composite (OBC/COL/CS)

by coupling oxidized bacterial cellulose (OBC) and chitosan (CS) with addition of collagen (COL) via

electrostatic attraction. The composite exhibited proper mechanical strength, broad spectrum

antimicrobial property and remarkable degradation in vivo within 30 days. Moreover, the notable

hemostatic efficacy of the OBC/COL/CS composite was confirmed on mouse livers used as models. The

results showed that the OBC/COL/CS exhibited better blood-clotting ability, higher blood cell adhesion,

lower blood loss, ultrafast bleeding cessation, which is superior to a commercial wound dressing

SurgicelTM film. Our results suggest that the OBC/COL/CS is a potential quick procoagulant agent to serve

as absorbable hemostats for internal bleeding control.

References

(1) Zhao, X., Guo, B. L., Wu, H., Liang, Y. P., Ma, P. X. (2018). Injectable antibacterial conductive nanocomposite cryogels with rapid shape recovery for noncompressible hemorrhage and wound healing. Nature Communications. 9: 2784.



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Peptide Functionalization of bacterial cellulose for antimicrobial activity

Ana M. Hernández-Arriaga1, Francisco Blanco Parte1, M. Auxiliadora Prieto1,2 1Microbial and Plant Biotechnology. Polymer Biotechnology. Biological Research Center (CIB-CSIC), Madrid, Spain; 2Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy‐Spanish National Research Council (SusPlast‐CSIC), Madrid, Spain

[email protected]

Abstract

Bacterial biopolymers are very attractive materials due to their biodegradability and non-toxicity. In a

Circular Economy context, they can be produced using wastes as feedstocks in sustainable bioprocesses.

Our group is interested in the sustainable production of bacterial cellulose (BC), and their application in

biomedicine by their functionalization with antimicrobial proteins such as enzybiotics (1). BC was

produced in Komagataeibacter medellinenesis ID13488 (2,3,4) which is able to produce crystalline

bacterial cellulose (BC) under high acidic growth conditions making this strain desirable for industrial BC

production from acidic residues (3) (e.g. wastes generated from cider production). To explore the

molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced. Genome

comparison analyses of K. medellinensis ID13488 with other cellulose‐producing related strains resulted

in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and

composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) and their organization in four operons

transcribed as four independent polycistronic mRNAs was determined. The BC produced has been applied

to developed functionalized BC hydrogels by a Cellulose Binding Domain from Clostridium cellulovorans

(CBDclos tag) for the immobilization of enzybiotics on BC-based devices. The coding gene sequence of the

synthetic gene EZB1-CBD was cloned in the expression vector pET29 and cloned in Escherichia coli BL21

(DE3) cells. Expression conditions were optimized by varying the temperature, inducer concentration and

culture time. Purification and immobilization of EZB1-CBD was performed by binding affinity of the CBD

domain of the fusion protein to BC membranes. The antimicrobial activity against Staphylococcus aureus

of EZB1-CBD was demonstrated by a particular antimicrobial assay that was specifically designed for these

materials, using cellulase for degrading the hydrogel to release the viable bacteria.

ACKNOWLEDGMENTS: The authors thank the financial support from grants BIO2017-83448-R and

P2018/NMT-4389.

REFERENCES

(1) Pastagia, M., Schuch R., Fischetti, VA., Huang, DB. (2013). Lysins: the arrival of pathogen-directed anti-infectives. Journal of Medical Microbiology. 62: 1506- 1516



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(2) Castro, C., Zuluaga, R., Álvarez, C., Putaux, J., Caro, G., Rojas, OJ., Mondragon, I., Gañán, P., (2012). Bacterial cellulose produced by a new acid-resistant strain of Gluconacetobacter Genus. Carbohydrate Polymers. 89: 1033– 1037 (3) Urbina, L., Hernández-Arriaga, AM., Eceiza, A., Gabilondo, N., Corcuera, MA., Prieto, MA., Retegi, A. (2017). By-products of the cider production: an alternative source of nutrients to produce bacterial cellulose. 24: 2071–2082 (4) Hernandez-Arriaga AM., del Cerro, C., Urbina, L., Eceiza, A., Corcuera, MA., Retegi, A., Prieto, MA. (2019). Genome sequence and characterization of the bcs clusters for the production of nanocellulose from the low pH resistant strain Komagataeibacter medellinensis ID13488. Microb Biotechnol. doi: 10.1111/1751-7915.13376


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Manufacturing of modified bacterial cellulose as tailor-made anti-inflammatory wound

dressing

U. Beekmann1,4,*, B. Karl1, P. Zahel1, D. Käfer1, L. Schmölz2, S. Lorkowski2, O. Werz3, D. Fischer1, D. Kralisch1,4

1 Friedrich Schiller University Jena, Pharmaceutical Technology and Biopharmacy, Lessingstraße 8, 07743 Jena, Germany 2 Friedrich Schiller University Jena, Nutritional Biochemistry and Physiology, Dornburger Straße 25, 07743 Jena, Germany 3 Friedrich Schiller University Jena, Pharmaceutical and Medicinal Chemistry, Philosophenweg 14, 07743 Jena, Germany 4 JeNaCell GmbH, Winzerlaer Straße 2, 07745 Jena * [email protected]

Abstract

Bacterial cellulose (BC) is a fascinating and sustainable hydropolymer with high potential in value added

applications such as modern wound management or drug delivery systems (1, 2). Although medical

products based on BC are already manufactured by several companies worldwide, wound dressings with

the additional benefit of active pharmaceutical ingredient (API) release are still rare. In the InflammAging

project, anti-inflammatory natural substances, such as triterpene acids from frankincense as well as

Vitamin E metabolites, are investigated as innovative APIs. The most promising candidates are

incorporated in BC in order to develop new, tailor-made active wound dressings for the treatment of local

inflammatory wounds. Since the loading of BC with lipophilic substances is still challenging due to the

hydrophilicity of the material (3), post- as well as in situ-modifications of BC during the bioprocess have

been investigated. At this, post-modifications selected should lead to a more lipophilic fiber network while

in situ-modifications should provide increased pore sizes. Both modifications where tested regarding its

effect on lipophilic model API uptake and release behaviour, before the results were transferred to the

natural substances of interest.

BC was synthesized by the bacteria strain Komagataeibacter xylinus (DSM 14666) at 28 °C for seven days

in 24-well-plates (2.27 cm²) and trays (400 cm²), respectively. For in situ-modification, different additives

(e.g. poly(ethylene glycol) (4)) were tested. As a main result, the pore sizes of the cellulose network could

be varied in the range of 2-10 µm having an influence on the transparency of the BC as well. In a next step,

the in situ-modification could be successfully transferred into pilot-plant-scale (1 m2). In case of post-

modification, acetylation or oxidation with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) and

subsequent conjugation with more hydrophobic compounds such as phenylalanine was investigated. In

case of both, in situ as well as post-modification, in vitro toxicity test MTT assay proved preserved

biocompatibility of the modified biomaterial. Clear differences in loading capacities for anti-inflammatory

model substances of varying lipophilicity (e.g. diclofenac & indomethacin) and release profiles could be

observed, underlining the potential of modified BC as controlled drug delivery system. These findings are

now used in the InflammAging project for the incorporation of lipophilic frankincense extract, quantified



3-4th October, 2019


by the lead substances 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and 11-keto-β-boswellic acid (KBA)

into BC for innovative and sustainable product design based on highly active natural compounds.

Acknowledgement

We gratefully acknowledge the Free State of Thuringia and the European Social Fund (2016 FGR 0045) for

funding.

References

(1) Pötzinger Y., Kralisch D., Fischer D. (2017). Bacterial nanocellulose: the future of controlled drug delivery? Ther Deliv. 8 (9): 753-761. (2) Anton-Sales I., Beekmann U., Laromaine A., Roig A., Kralisch D. (2018). Opportunities of bacterial cellulose to treat epithelial tissues. Curr. Drug Targets. 20. (3) Alkhatib Y., Dewaldt M., Moritz S., Nitzsche R., Kralisch D., Fischer D. (2017). Controlled extended octenidine release from a bacterial nanocellulose/Poloxamer hybrid system. Eur. J. Pharm. Biopharm. 112: 164-176. (4) Hessler N., Klemm D. (2009). Alteration of bacterial nanocellulose structure by in situ modification using polyethylene glycol and carbohydrate additives. Cellulose. 16(5): 899–910.


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Modification of bacterial nanocellulose membranes for tailored drug carrier

Streit, S.1, Inoue, B. S.1, Fonseca, V.2, Schneider, A.L.S.2, Meier, M. M*1 1Departament of Chemistry, Santa Catarina State University (UDESC), Joinville (SC), Brazil, [email protected]

2Engineering of Process Program, University of Joinville Region (UNIVILLE), Joinville (SC), Brazil,

Abstract

When using biomaterials as drug carriers it is often necessary to modify the polymer phase to enable

adequate loading and subsequent drug release in a modulated manner. Hydrophobic drugs may present

low loading levels in hydrophilic polymer matrices, such as bacterial cellulose (BC). On the other hand,

hydrophilic drugs exhibit rapid release in hydrophilic matrices, and / or strong physical adsorption on the

surface, as nanocellulose fibers, especially cationic drugs.

The high hydrophilic character of bacterial cellulose membranes, their high surface area and

biocompatibility are important requirements for controlled release systems, however, it presents

challenges for the incorporation of some drugs. In turn, the presence of hydroxyl groups in their chemical

structure makes BC a potential platform for chemical modification and consequently tailoring its polarity

and interaction with drugs.

In order to modulate the release of cationic drugs and prevent its adsorption on BC, in this study, β-

cyclodextrin (βCD) was combined with chlorhexidine digluconate (CHX). Cyclodextrins (CDs) constitute a

family of cyclic oligosaccharides with approximately 6 Å of diameter in its internal cavity, making possible

the accommodation of aromatic groups that are found in the structure of most drugs1, leaving its polar

structure to the outer side of the cavity, thus, forming inclusion complexes2.

In this way, this study presents chemical and physicochemical strategies to modify bacterial cellulose

membranes using cyclodextrin and studied its relationship with the in vitro release of a cationic model

drug, chlorhexidine digluconate (CHX).

CB membranes were synthesized using Komagataeibacter hansenii ATCC 23769 cultivated and transferred

to a 20% inoculum rate into an erlenmeyer flask containing 40 mL of the culture medium. The culture was

kept static for 12 days at 30°C. Membranes were modified ex-situ and in-situ using βCD. CHX loading

capacity and the in vitro release profile from the synthesized membranes were determined using UV-vis

spectroscopy assay.

The results demonstrate a significant effect of βCD on the CHX release profile from bacterial cellulose

membranes when compared to the control group. Suggesting the formation of inclusion complexes

between CHX:βCD, whose chemical equilibrium in aqueous media modulates the release of CHX.

Acknowledgements

This study was funded in part by FAPESC, CNPq, UDESC and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.



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References

(1) Balte, A. S., Goyal, P. K., & Gejji, S. P. (2012). Theoretical studies on the encapsulation of paracetamol in the α, β and γ cyclodextrins. Journal of Chemical and Pharmaceutical Research. 4(5):2391–2399

(2) Denadai, A. M. L., Teixeira, K. I., Santoro, M. M. ., Pimenta, A. M. C., Cortes, M. E., & Sinisterra, R. D. (2007). Supramolecular self-assembly of β-cyclodextrin: an effective carrier of the antimicrobial agent chlorhexidine. Carbohydrate Research. 342(05): 2286–2296.


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SESSION 9 - BC companies and market pull (chair person: Koon-Yang Lee)


3-4th October, 2019


KEYNOTE LECTURE: BioTech Cellulose: Process-controlled design of multilayered materials,

coatings and composites

Dieter Klemm*, Friederike Kramer, Katrin Petzold-Welcke, Thomas Richter, Wolfgang Fried

KKF Company – polymers for life [email protected]

Abstract

The only type of cellulose produced by synthesis is bacterial nanocellulose (BNC, BioTech cellulose). In

most cases, BNC is formed by cultivation of acetic acid bacteria in an aqueous culture medium under static

conditions. Our novel dynamic Mobile Matrix Reservoir Technology (MMR Tech) allows for the first time

the design of not only the shape and dimension of the hydrogel bodies, but also the most important BNC

properties such as structure of the surfaces and the internal nanofiber network architecture. These

product parameters can be process-controlled using a bioreactor constructed for this purpose.

Cellulose formation takes place layer by layer on a shaping template (e.g. flat or cylindrical). This template

is located - apart from the aqueous culture medium - in the air space of a synthesis robot. For loading with

bacteria and nutrient components the template is dipped vertically into the culture medium. This dipping

process causes turbulence in the culture medium, and the BNC formation at the surface area of the liquid

culture cannot take place. With increasing length of staying in the reactor's airspace, increasingly denser

BNC layers are formed. Repeated loading of culture medium results in delamination-stable multi-layer gel

materials with variable surface structure and nanofiber network architecture.

The applicability of the developed BNC materials as medical implants for the healing of damaged bile ducts

is demonstrated. Initial statements on efficacy in membrane technologies are under discussion - including

the development of multi-layer composites and coated supports.

References

(1) Klemm, D., Cranston, E. D., Fischer, D., Gama, M., Kedzior, S. A., Kralisch, D., Kramer, F., Kondo, T., Lindström, T., Nietzsche, S., Petzold-Welcke, K., Rauchfuß, F. (2018). Nanocellulose as a natural source for groundbreaking applications in materials science: Today’s state. Materials Today. 21:720-748



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Microengineered biosynthesized cellulose as antifibrotic protection for implantable medical

devices

Simone Bottan a, f, Francesco Robotti a, e, f , Giovanni Pellegrini b, Josep Monnè Rodriguezb, Christian Witzel c, Tanja Schmidtc, Volkmar Falkd, Dimos Poulikakos e, Christoph Starck d and Aldo Ferrarie a. Hylomorph AG, Zurich, Switzerland b. Laboratory for Animal Model Pathology, Institute of Veterinary Pathology, Vetsuisse

Faculty, University of Zurich, Zurich, Switzerland c. Forschungseinrichtungen für Experimentelle Medizin (FEM), Charité-Universitätsmedizin

Berlin, Berlin, Germany d. Department of Cardiothoracic and Vascular Surgery, German Heart Institute Berlin, Berlin,

Germany e. Laboratory of Thermodynamics in Emerging Technologies, Department of Mechanical and

Process Engineering, ETH Zurich, Sonneggstrasse 3, CH-8092 Zurich, Switzerland f. Wyss Zurich, ETH and University of Zurich, Switzerland

Abstract

Background

The surgical implantation of all medical devices coincides with the onset of foreign body reaction. This

innate immune system response aims at removing or isolating what the body perceives as a threat. It

ultimately leads to the deposition of a firm, thick and poorly vascularized fibrotic tissue around the target

object, i.e. the device. In this scenario, the recipients of cardiac implantable electronic devices (CIEDs) and

breast implants are exposed to a considerable danger of adverse events whenever the implant shall be

exchanged, upgraded, or revised. Risks include pocket infection and/or hematoma. Their likelihood and

severity are significantly higher in presence of fibrotic tissue. Owing the increasing number of implant-

based surgical procedures, the incidence of related adverse events is expected to rise, with significant

health-economics burden.

Objective

The present study establishes the feasibility, safety, and performance of an antifibrotic protection for CIED

and breast implants comprising a surface micro-engineered, non-resorbable biosynthesized cellulose

(SME-BNC) membrane.

Methods

SME-BNC membranes were generated by Guided Assembly-based Biolithography (1), to implement

selected microscale geometries on the cellulose membrane surface. The performance of the SME-BNC

protection was chronically evaluated in minipigs to report on their effect on fibrotic tissue formation.

Sixteen (n = 16) animals received each one SME-BNC covered pacemaker (PM) and breast implants (BI)

and one identical, respectively naked counterpart, at equivalent anatomical sites.

Results

SME-BNC protective layers were juxtaposed around PMs and BIs through a repeatable protocol which did

not significantly prolongate the surgical procedure.


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Explants were performed at 3 and 12 months after implantation. Endpoint histopathological analysis

showed that the SME-BNC protective layers were perfectly integer, with no sign of chemical or mechanical

degradation. They appeared as a thin layer of white-tan material, adherent to the underlying thin fibrous

capsule, from which it could be peeled off by gently pulling with forceps.

The protective effect of micro-engineered SME-BNC yielded an average reduction of 68% and 54% of the

fibrotic tissue thickness generated around PM and BI, respectively, as compared to the bare counterparts.

When protected by SME-BNC, PM generators and the proximal parts of the leads were completely free of

fibrotic tissue, yielding simplified and quicker removal.

Conclusions

The innovative application of BNC as medical device is presented and demonstrated through a long-term

chronic study in a large animal model. The insertion of an anti-fibrotic, non-resorbable and well-tolerated

SME-BNC layer at the interface between the target implant and the surrounding tissue in the surgical

pocket abated the formation of fibrotic tissue, ensuring an easy access to the device pocket, and thus

creating the conditions for de-risked CIED and breast implants revision surgeries.

References

(1) Robotti F. et al (2015). Surface-Structured Bacterial Cellulose with Guided Assembly-Based

Biolithography (GAB). ACSnano 9:206-219


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NullarborTM Tree-Free Fibre and other Commercial Applications for Bacterial Nanocellulose

Gary Cass Nanollose Ltd, Australia

Abstract

From a super lightweight porous matrix to an incredibly strong solid material; bacterial nanocellulose

(BNC) has quickly moved from its traditional Nata-de-coco food application to wide-ranging applications

for a variety of markets. Nanollose is an Australian based biotechnology company advancing innovative

eco-friendly technologies relating to the production, processing and applications of BNC. This seminar will

describe several of Nanollose’s research projects including;

• Rayon is traditionally produced from wood pulp in a process responsible for the felling of 150 million

trees each year. Nanollose has developed a world first method that transforms BNC into NullarborTM

Tree-Free rayon fibres using technology which is compatible with existing industry processing and

manufacturing equipment.

• Static or bioreactor? Nanollose is working with several partners to assess the best methods, bacterial

strains and culture media for growing BNC to meet future demand and price points. This includes static

cultures currently being used for the production of Nata de coco and various stirred-tank bioreactors.

• The method of post-harvest processing of BNC can be modified depending on its intended application.

Nanollose has developed several methods which are able to produce a range of purified BNC materials

with different properties from a super lightweight porous matrix to a soft fluffy fibre to an incredibly

strong and dense material.

These projects along with many other cellulose alternatives will be the driving forces that are rapidly

shaping our world to a more sustainable bio-based and circular society. Inspiration, Imagination and

Innovation will be key to overcome the challenges for the future.


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Bacterial cellulose product design towards biomedical and diagnostic applications

Dana Kralisch1,2,*, Uwe Beekmann1,2 1 JeNaCell GmbH, Winzerlaer Straße 2, 07745 Jena 2 Friedrich Schiller University Jena, Pharmaceutical Technology and Biopharmacy,

Lessingstraße 8, 07743 Jena, Germany * [email protected] [email protected]

Abstract

Bacterial cellulose (BC) has versatile application potentials in novel biomedical (1) and diagnostic (2)

products. Among them, combinations of BC with living cells or biological agents seem to be particularly

promising. An up-to-date overview will be given about research and product development activities in

both fields, ranging from application as cell carrier in in vitro models for drug discovery or transport studies

mimicking epithelial barrier and specific transport function (3, 4) to tailor-made delivery systems. In the

latter case, specific modifications of the BC network (5) were investigated up to demonstration scale

allowing an improved incorporation of biological components such as natural anti-inflammatory

substances for skin treatment or antibodies for immunotherapy (6). In all developments, the young BC

producing company JeNaCell benefits from its successful cooperation with a strong network of scientific

partners supporting the transfer from fundamental BC research to novel, innovative BC based products.

Acknowledgement

D.K. thankfully acknowledges the financial support by the German Federal Ministry for Economic Affairs

and Energy (funding ID: 16KN044035, ZIM-Gewebe), by the Free State of Thuringia and the European

Social Fund (funding ID: 2016 FGR 0045, InflammAging) as well as by the European Commission under a

MSCA-ITN award, grant number 675743 (ISPIC), and under a MSCA-RISE award, grant number 777682

(CANCER).

References

(1) Anton-Sales, I.; Beekmann, U.; Laromaine, A.; Roig, A.; Kralisch, D. (2019) Opportunities of bacterial cellulose to treat epithelial tissues. Current Drug Targets 20:1-15.

(2) Picheth, G. F.; Pirich, C. L.; Sierakowski, M. R.; Woehl, M. A.; Sakakibara, C. N.; de Souza, C. F.; Martin, A. A.; da Silva, R.; de Freitas, R. A. (2017) Bacterial cellulose in biomedical applications: A review. International Journal of Biological Macromolecules, 104:97-106.

(3) Fey, Ch.; Betz, J.; Rosenbaum, C., Kralisch, D.; Vielreicher, M.; Friedrich, O.; Zdzieblo, D; Metzger, M.; Bacterial nanocellulose as novel carrier for intestinal epithelial cells in drug delivery studies, submitted

(4) Vielreicher, M.; Kralisch, D.; Völkl, S.; Sternal, F.; Arkudas, A.; Friedrich, O. (2018) Bacterial nanocellulose stimulates mesenchymal stem cell expansion and formation of stable collagen-I networks as a novel biomaterial in tissue engineering. Scientific Reports 8 (1):9401.




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(5) Beekmann, U.; Zahel, P., Karl, B.; Schmölz, L.; Lorkowski, S.; Werz, O.; Fischer, D.; Kralisch, D.,

Modification of bacterial cellulose to enhance anti-inflammatory wound dressing properties, 5th Euro BioMAT, 08.-09.05.2019, Weimar, Germany.

(6) Chung, C. K.; Da Silva, C. G.; Kralisch, D.; Chan, A.; Ossendorp, F.; Cruz, L. J. (2018) Combinatory therapy adopting nanoparticle-based cancer vaccination with immune checkpoint blockade for treatment of post-surgical tumor recurrences. Journal of Controlled Release 285:56-66.


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BNC products: From Lab to Market

Kamil Palmowski, GM, BOWIL Biotech

BOWIL Biotech Sp. z o.o. (Ltd.) 7, Skandynawska Street 84 120 Wladyslawowo, Poland www.bowil.pl

Abstract

BOWIL Biotech, specializes in bionanocellulose production, according to the highest standards. Since 2011

BOWIL Biotech is focusing not only on R&D, but as well on final product development, including product

launches. Intelligent materials, developed with patented technologies, registered in EU and US, are finally

starting to be applied in clinical practices. During this short presentation we will debate on road blocks,

challenges and final success in introducing BNC products to the worldwide market.

http://www.bowil.pl/


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Posters


3-4th October, 2019


P1: Bacterial Cellulose as a Material for Innovative Cardiovascular Implants

Andree V., Pieger T., Rzany A., Hensel B.

Max Schaldach-Stiftungsprofessur für Biomedizinische Technik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

Abstract

Bacterial Cellulose (BC) shows excellent properties as a biomaterial for innovative medical implants. Based

on animal studies it is sensible to expect that no degradation will occur in the human body and that the

material is almost perfectly biocompatible. [1,2] The use in cardiovascular implants, however, is not yet

established. It is for this purpose that we cultivate and analyse BC with respect to the key properties that

are crucial for its use as material for the tissue components in transcatheter aortic valve prostheses. In

this context, BC will replace the conventionally used porcine or bovine pericardium. Furthermore, it might

be used as a material for vascular grafts or to fill cavities, e.g. aneurysms.

For the cellulose formation, the bacterial strain Gluconacetobacter hansenii of the company American

Type Culture Collection is used. A culture medium according to Hestrin and Schramm is inoculated with

the bacteria. [3] The culture medium and the bacterial suspension are mixed in a ratio of 12:1. The

cultivation takes place in an incubator at 28° C for seven days. [4]

The resulting fleece has a thickness of 5-7mm. By rinsing with endotoxin-free water for three days, the

remnants of the culture medium as well as bacterial cell debris are washed out. A purification process in

a 0.1 mol solution of sodium hydroxide is employed to remove endotoxins. Autoclaving at 121° C for 20

minutes ensures sterility. An air-drying process (72 hours) results in a reduction of thickness. After

rehydration in endotoxin-free water at 37° C, the final state of the BC is obtained. [4]

The thus prepared tissue is analysed with respect to its mechanical properties by using a uniaxial tensile

test setup. The water content, as well as the water retention capacity are also measured. A force at break

of 44 N (cross section 32 mm x 6.5 mm) and a strain at break of about 50 % are obtained. The water

content in this rehydrated state is 81 %, while the water retention capacity is 145 %. [4]

By scanning electron microscopy, the diameter of the individual fibres is determined to be in the range

30-60 nm independent from the cultivation time. The topology exhibits a compact fibre network and

clusters of bacteria. The combination of the properties makes bacterial cellulose a promising new

biomaterial for various medical applications.

References

(1) C. Xu, X. Ma u. a. Bacterial Cellulose Membranes Used as Artificial Substitutes for Dural Defection in Rabbits. In: Int. J. Mol. Sci 15 (2014), S. 10855-10867

(2) J. Wippermann, D. Schumann u. a. Preliminary Results of Small Arterial Substitute Performed with a New Cylindrical Biomaterial Composed of Bacterial Cellulose. In: Eur J Vasc Endovasc Surg 37 (2009), S. 592-596


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(3) S. Hestrin und M. Schramm. Synthesis of Cellulose by Acetobacter xylinum: 2. Preparation of freeze-dried Cells capable of Polymerizing Glucose to Cellulose. In: Biochemical Journal 58.2 (1954), S. 345-352.

(4) T. Pieger. Bakterielle Cellulose – ein vielseitiges Biomaterial für kardiovaskuläre Implantate. Dissertation. Friedrich-Alexander-Universität Erlangen-Nürnberg, 2019.


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P2: Probiotic edible films from bacterial cellulose/cashew tree gum

Ana Vitória Oliveira-Alcântara1, Ana Angel S. Abreu1, Catarina Gonçalves2, Pablo Fuciños2, Miguel A. Cerqueira2, Francisco M. P. da Gama3, Sueli Rodrigues1, Henriette M. C. Azeredo4,5,*

1Federal University of Ceara, Brazil; 2International Iberian Nanotechnology Laboratory, Portugal; 3University of Minho, Portugal; 4Embrapa Agroindústria Tropical, Brazil; 5Embrapa Instrumentação, Brazil.

Abstract

Edible films are thin layers of biopolymer-based materials, which are expected to help the packaging

system in protecting food against environmental factors. Besides passive protection, edible films may also

be carriers of active/bioactive components. Probiotic films are expected not only to bring health benefits

to the consumers, but also to extend food microbial shelf life due to competitive effects of probiotics1.

Bacterial cellulose (BC) has been presented as a promising matrix for immobilization of probiotics,

protecting them against adverse factors e.g. stomach pH2. In this study, BC was combined to cashew tree

gum (CG) to produce an edible film carrying a probiotic bacteria (Bacillus coagulans). CG was used to

decrease the viscosity of film forming dispersions. Four films were produced: BC/CG/Pro (containing the

probiotic B. coagulans), BC/CG/Pre (containing the prebiotic fructooligosaccharides – FOS), BC/CG/Syn

(containing both probiotic and prebiotic, making it synbiotic), and BC/CG (a control film). The presence of

the probiotic and/or prebiotic affected the tensile properties of the films, especially the tensile strength.

The survival rate of the probiotic on film drying and storage was increased by the presence of FOS. An in

vitro digestibility test was also carried out on films, demonstrating that the bacteria in BC/CG/Pro films

exhibited an enhanced survival rate on gastric environment when compared to the free probiotic.

References

(1) Espitia, P.J.P., Batista, R.A., Azeredo, H.M.C., Otoni, C.G. (2016). Probiotics and their potential applications in active edible films and coatings. Food Research International, 90: 42. (2) Fijałkowski, K., Peitler, D., Rakoczy, R., Żywicka, A. (2016). Survival of probiotic lactic acid bacteria immobilized in different forms of bacterial cellulose in simulated gastric juices and bile salt solution. LWT - Food Science and Technology, 68: 322.


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P3: Scaffolds of bacterial cellulose functionalized with bioactive metal phosphates

Pezzin, A.P.T.*1, Paterno, F. L.1, Gaulke, E.1, da Silva, D.F.2, Garcia, M.C.F.1, Meier, M.M.2, Apati, G.P.1, Schneider, A.L.S.1, Porto, L.M.3

1Paulo Malschitzki Street, 10. University of the Region of Joinville - UNIVILLE Joinville. [email protected]*, 2 University of the state of Santa Catarina (UDESC), 3 Federal University of Santa Catarina (UFSC)

Abstract

The use of biopolymers for guided bone regeneration (GBR) has attracted great interest due to the

possibility of processing in three-dimensional structures. Bacterial cellulose (BC), for example, is one of

the most promising biopolymers due to its high crystallinity, water retention capacity, three-dimensional

interconnected porous nanostructure, and excellent biocompatibility1 allowing it behave as a good

matrix/support for cell growth. On the other hand, hydroxyapatite (HAp) is the main constituent of

inorganic components of bone tissue, has excellent biocompatibility, bioactivity and osteoconductivity2.

In addition, incorporation of some elements such as Mg+2 or Sr+2 in the trace element form plays a vital

role in bone growth and repair, which can control degradation, increase the mechanical strength of

materials and positively regulate their bioactive properties3. The aim of this work was to develop BC

biocomposites functionalized by immersion cycles4 with hydroxyapatite (BC/HAp) and hybrids of copper

(BC/CuHAp), magnesium (BC/MgHAp), zinc (BC/ZnHAp) and strontium (BC/SrHAp) aiming to induce bone

growth for application in guided tissue regeneration (GTR). The freeze-dried samples were characterized

by Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR/ATR),

thermogravimetric (TGA), field emission scanning electron microscopy (SEM-FEG), X-ray diffraction (XRD),

and inductively coupled plasma optic emission (ICP-OES). In addition, the degree of swelling, porosity,

bioactivity, and release of ions after immersion in simulated body fluid (FBS), antimicrobial properties and

cytotoxicity were determined. TGA analysis demonstrated the presence of about 50 to 72% by mass of

mineral phase in BC, the diffraction patterns obtained by XRD confirmed the formation of apatite in

functionalized membranes. The hybrids showed adequate swelling and porosity, the release assay

confirmed the presence of all metal ions in PBS and the SEM micrographs showed the deposition of

different crystals of the metal phosphates in BC. Regarding the antimicrobial potential, it was observed

that the functionalization with Cu improved the antimicrobial properties of the biocomposite in relation

to the biomaterials incorporated with Mg. On the other hand, the biomaterials incorporated with Mg had

a lower negative effect on the cellular viability, being more indicated for the use as implantable materials.

These results indicate the formation of apatites with metallic elements in the BC membranes, suggesting

that these are promising biomaterials for use in biomedical applications due to their high bioactivity.

References

(1)Recouvreux, D.O.S., Rambo, C.R., Berti, F.V., Carminatti, C.A., Antônio, R.V., Porto, L.M. (2011). Novel three-dimensional cocoon-like hydrogels for soft tissue regeneration. Materials Science and Engineering: C, 31 (2): 151–157.

mailto:[email protected]*


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(2)Ehret, C., Aid-Launais, R., Sagardoy, T., Siadous, R., Bareille, R., Rey, S., Pechev, S., Etienne, L., Kalisky, J., Mones, E., Letourneur, D., Vilamitjana, J. A. (2017). Strontium-doped hydroxyapatite polysaccharide materials effect on ectopic bone formation. Plos One, 12 (9):1-21. (3)Pina, S., Canadas, R.F., Jiménez, G., Perán, M., Marchal, J.A., Reis, R.L., Oliveira, J.M. (2017). Biofunctional Ionic-Doped Calcium Phosphates: Silk Fibroin Composites for Bone Tissue Engineering Scaffolding. Cells Tissues Organs, 204: 150–163. (4)Hutchens, S. A., Benson, R. S., Evans, B. R., O’neill, H. M., Rawn, C. J. (2006). Biomimetic synthesis of calcium-deficient hydroxyapatite in a natural hydrogel. Biomaterials, 27: 4661-4670.


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P4: Evaluation of bacterial cellulose degradation after exposure in different abiotic and biotic

components

Pezzin, A.P.T.*, Camargo, M.S.A., Cercal, A.P., Fonseca, V., Garcia, M.C.F., Apati, G.P., Schneider, A.L.S. Paulo Malschitzki Street, 10. Universidade da Região de Joinville - UNIVILLE [email protected] *

Abstract

Bacterial cellulose (BC) is a polymer that has a range of applications in different industrial segments (health

area, food packaging, electronics among others) because it is biocompatible, non-toxic, has high liquid

absorption capacity and biodegradability. Within this context, the present work aimed to the study of the

degradation of BC in different environmental conditions, since they can be discarded in the environment.

The membranes of BC produced were analyzed dry and wet, being monitored as a function of degradation

time. The methodology employed involved the biosynthesis of BC membranes by Komagataeibacter and

purification with a solution of 0.1 M sodium hydroxide in a water bath at 80 ° C for 1 h. The degradation

of the BC membranes was evaluated at different degradation times in the following media: soil (SO),

estuarine environment (EE), natural weathering (NW) and accelerated aging (AA), using greenhouse dried

BC membranes air circulation and humid conditions. The membranes were removed at predetermined

periods and were monitored by visual analysis, Fourier transform infrared spectroscopy (FTIR) and

thermogravimetric analysis (TGA). In the visual analysis, it was possible to observe the physical alterations,

such as roughness, cracks, color change. The results of FTIR showed chemical changes during the time of

exposure to the environments, in relation to the structures of the control BC. The TGA analyzes indicated

the thermal and mass loss changes that occurred in the membranes after degradation. The results showed

that, although the tests took place under different conditions, in general the intensity of the membrane

degradation occurred in the following order: SO > EE > NW > AA. In the SO and EE the degradation was

more intense facilitated by the greater presence of microorganisms in these media, which promote the

microbial attack. The wet membranes suffered more rapid degradation than the dry ones. This fact can

be explained because the membrane of BC is extremely porous, but with drying in greenhouse, these

pores are collapsed, while in the wet membrane the pores remain intact, the water being a vehicle that

helps to transport the microorganisms to within the membrane and consequently accelerate degradation.

References

SCHRÖPFER, S.B. et al. Biodegradation evaluation of bacterial cellulose. vegetable cellulose and poly(3-hydroxybutyrate) in soil. Polímeros. v.25, n.2, p.154–160, 2015. SHI, Z. et al. Utilization of bacterial cellulose in food. Food Hydrocolloids. v.35. p. 539–545, 2014. WANG, B. et al. In vitro biodegradability of bacterial cellulose by cellulase in simulated body fluid and compatibility in vivo. Cellulose. v 23, n.5, p.3187–3198. 2016.



3-4th October, 2019


P5: Antimicrobial Wound Dressing Based on Bacterial Cellulose Containing Silver Nanoparticles

Rafael M. Sábio1, Karyn F. Manieri1, Andréia B. Meneguin1, Vanderlei S. Bagnato1, Hernane S. Barud2. 1São Carlos Institute of Physics, University of São Paulo, São Carlos, SP, Brazil.

2Biopolymers and Biomaterials Laboratory, Araraquara University – UNIARA, Araraquara, SP, Brazil.

Abstract

Bacterial cellulose (BC) consists of a natural biopolymer synthesized by bacteria composed largely of water

showing unique properties such as high chemical purity, biocompatibility, biodegradability, high

mechanical resistance and others [1]. BC has been extensively used in biomedical applications as dressing

for wounds and burns, providing a humid environment to the affected region leading to healing and

reducing pain in patients [2]. However, this kind of wound dressing do not present antimicrobial activity,

which consists of one of the critical functions of the cutaneous barrier in effective wound healing [1,2]. To

overcome this drawback, this work proposes to ally antimicrobial properties of silver nanoparticles (AgNP)

with wound healing properties of BC dressings to fabricate a new biocomposites (BC@AgNP) aiming to

avoid wounds and burns infections. BC membranes were prepared similarly as described by Pacheco et

al. [3]. The fabrication of BC@AgNP comprises the immersion of BC dressing in AgNP aqueous suspension

during 24 h under stirring and room temperature. AgNP, BC and BC@AgNP physicochemical

characterizations were performed using UV-Visible and FE-SEM techniques. Minimum inhibitory

concentration (MIC) of the AgNP was determined for gram-negative (Escherichia coli, 108 CFU mL-1) and

gram-positive (Staphylococcus aureus, 108 CFU mL-1) bacteria. Citotoxicity were evaluated by MTT assays

using GM07492 cell lines (normal human fibroblast). Antimicrobial activity were performed using E. coli

(108 CFU mL-1) and gram-positive S. aureus (108 CFU mL-1) bacteria during 24 h at 37 °C. UV-Visible data

shows a large size distribution of AgNP in aqueous solution. FE-SEM results confirm distinct average size

distribution of AgNP besides exhibited pristine CB composed by random 3D nanofibers network.

BC@AgNP displayed nanofibers structure intact relating to pristine BC with homogeneously distribution

of AgNP onto BC nanofibers surface. MIC from AgNP for E. coli and S. aureus bacteria were 62.5 and 125

ppm, respectively. Despites low concentration of free AgNP to present cell viability from MTT assays, after

incorporation onto BC surface (BC@AgNP) higher concentration of AgNP exhibited viability values above

70 %. In antimicrobial tests, the activities against gram-positive and negative bacteria were detected only

by contact method suggesting that the AgNP was not released from BC nanofibers. These results suggest

that the new BC@AgNP biocomposites are suitable for application as antimicrobial and healing wound

dressings. In vivo assays are been performed to confirmed the potential strategy to fabricate new

BC@AgNP biocomposites for clinical evaluation.

Acknowledegments I would like to thank to EMBRAPII (grant number: 18020005), CEPOF/Fapesp 407822/2018-6.

References [1] Wu, J., Zheng, Y., Song, W., Luan, J., Wen, X., Wu, Z., Chen, X., Wang, Q., Guo, S. (2014). Carbohydrate Polymers. 102:762-771.


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[2] Foresti, M. L., Vázquez, A., Boury, B. (2017). Carbohydrate Polymers. 157:447-467. [3] Pacheco, G., Nogueira, C. R., Meneguin, A. B., Trovatti, E., Silva, M. C. C., Machado, R. T. A., Ribeiro, S. J. L., Da Silva Filho, E. C., Barud, H. S. (2017). Industrial Crops & Products. 107:13-19.


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P6: Nano-fibrillated bacterial cellulose with hydrophobic surface characteristics by

heterogeneous silylation

Hiroyuki Kono*†, Taiki Uno†, Ryota Kishimoto†, Tokuo Matushima‡, Kenji Tajima§ † National Institute of Technology, Tomakomai College, Hokkaido, 059-1275, Japan

‡ Kusano Sakko Inc., Hokkaido, 067-0063, Japan § Faculty of Engineering, Hokkaido University, Hokkaido, 060-8628, Japan

Abstract

Hydrophobic nano-fibrillated bacterial cellulose (NFBC) was prepared by use of an efficient silylation

process in water. The starting material of NFBC was obtained by aerobic agitating cultivation of cellulose

producing bacterium (Gluconacetobacter intermedius NEDO-01) in a medium supplemented with sodium

carboxymethyl cellulose 1,2). The NFBC was heterogeneously silylated by methyltri-methoxysilane (MTMS)

sols in the acidic water (pH4) 3), and a series of the silylated NFBC was prepared by changing feed amount

of MTMS sols. Structural characterization of these samples using FTIR, solid-state NMR, and WAXS

revealed that the optimal condition for the selective silylation of the surface layer of NFBC. The optimally

silylated NFBC was well dispersed in chloroform, and the nano-fibril structure was retained in the solvent.

The silylated NFBC could be expected to be as a filler of the fiber-reinforced resins.

References

1) Kose, R., Sunagawa, N., Yoshida, M., Tajima, K., Cellulose, 20, 2971–2979 (2013). 2) Tajima, K., Kusumoto, R., Kose, R., et, al., Biomacromolecules, 18, 3432–3438 (2017). 3) Zhang, A., Sébe, G., Rentsch, D., et al., Chem. Mater, 26, 2659–2668 (2014).


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P7: Construction and performance study of integrated bacterial cellulose hernia mesh

Xie Jing1, 2, Cui Teng1, 2, Luo Honglin1, 2, Yang Zhiwei1, 2, Wan Yizao1, 2

1 Institute of Advanced Materials, East China Jiaotong University, Jiangxi Province, Nanchang City, 330013

2 College of Materials Science and Engineering, East China Jiaotong University, Jiangxi Province, Nanchang City, 330013

[email protected]

Abstract

Utilization of hernia mesh for the hernia repair assisting is a widely used clinical approach. An ideal

mesh should be beneficial for cell growth and anti-adhesion. Unfortunately, various degrees complication

caused by many available hernia repair mesh (HRM) limits their application, which is highly desired for

developing of new HRM. In this study, by using a membrane-liquid interface culture method and scarified

gelatin microspheres template (200-300 μm), an integrated bacterial cellulose mesh composed by

nanopore side and micropore side was constructed, which was demonstrated by scanning electron

microscopy characterization (Fig. 1). Fourier transform infrared spectroscopy clearly verified the complete

elimination of gelatin. Tensile test results showed that the stress at break and strain at break of hernia

mesh were 0.25 MPa and 50.0%, respectively. Additionally, in vitro study revealed that the mesh was non-

cytotoxic, the nanopores side of mesh showed the good anti-adhesion properties, while cells clustered

within the micropores could be observed in the micropores side. The results suggested that the integrated

mesh was fully biocompatible and could integrate into surrounding tissues, which is highly potential for

the application in the hernia repair.

Figure 1 SEM images of the integrated bacterial cellulose mesh. The surface morphology (left) and the

cross-sectional morphology (right).

References

(1) Lai, C., Hu, K. S., Wang, Q. L., Sheng, L. Y., Zhang, S. J., Zhang, Y. (2018). Anti-adhesion mesh for hernia repair based on modified bacterial cellulose. Starch – Stark. 1700319.



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(2) Zaborowska, M., Bodin, A., Bäckdahl, H., Popp, J., Goldstein, A., Gatenholm, P. (2010). Microporous bacterial cellulose as a potential scaffold for bone regeneration. Acta Biomaterialia, 7(6):2540-2547. (3) Khan, S., Ul-Islam, M., Ikram, M., Islam, S. U., Ullah, M. W., Israr, M., Park, J. K. (2018). Preparation and structural characterization of surface modified microporous bacterial cellulose scaffolds: A potential material for skin regeneration applications in vitro and in vivo. International journal of biological macromolecules, 117:1200-1210. (4) Liu, S., Chu, M., Zhu, Y., Li, L., Wang, L., Gao, H., & Ren, L. (2017). A novel antibacterial cellulose based biomaterial for hernia mesh applications. RSC Advances, 7(19):11601-11607.


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P8: Preparation and properties of graphene oxide/bacterial cellulose nanocomposites with

gradient structure

Liu JinZhi1,2, Yang ZhiWei1,2, Wan YiZao1,2 1 Institute of Advanced Materials, East China Jiaotong University, Jiangxi Province, Nanchang City, 330013

2 College of Materials Science and Engineering, East China Jiaotong University, Jiangxi Province, Nanchang City, 330013

Abstract

Natural osteochondral tissues exhibit a multivariate gradient structure, including chemical composition,

porosity, and mechanical properties. Developing a scaffolds to mimic the gradient structure of

osteochondral tissue for regeneration and repair of osteochondral tissue is a promising approach. In the

present work, a bacterial cellulose/graphene oxide hydrogel with gradient structures to mimic

osteochondral tissue was fabricated via a membrane liquid interface culture method. The hydrogel with

a gradient change in the ratio of graphene oxide to bacterial cellulose in the direction of thickness was

prepared. The gradient structure were formed by controlling the graphene oxide content (low, medium

and high content) in the hydrogels, which could be clearly observed by scanning electron microscopy

characterization (Fig. 1). Moreover, the mechanical properties of the hydrogel also change in a gradient

in the direction of thickness. The tensile test were carried out to assess the mechanical performance of

the hydrogels. The calculated tensile strength of the low, medium and high regions in the hydrogels were

1.1 MPa, 1.5 MPa, 2.0 MPa, respectively. The gradient bacterial cellulose/graphene oxide hydrogel

promoted and regulated osteogenic differentiation of human mesenchymal stem cells in an

osteoinductive environment in vitro. This gradient bacterial cellulose/graphene oxide hydrogel provides

materials for the preparation of biomimetic biological gradient scaffolds to facilitate regeneration and

repair of osteochondral tissue.

Figure 1 SEM images of bacterial cellulose/graphene oxide composite materials with low, medium and

high content of graphene oxide


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References

(1) Liu, Z., Meyers, M. A., Zhang, Z., & Ritchie, R. O. (2017). Functional gradients and heterogeneities in biological materials: Design principles, functions, and bioinspired applications. Progress in Materials Science, 88, 467-498.

(2) Luo, H., Dong, J., Yao, F., Yang, Z., Li, W., Wang, J. & Wan, Y. (2018). Layer-by-Layer Assembled Bacterial Cellulose/Graphene Oxide Hydrogels with Extremely Enhanced Mechanical Properties. Nano-micro letters, 10(3), 42.

(3) Guo, J., Li, C., Ling, S., Huang, W., Chen, Y., & Kaplan, D. L. (2017). Multiscale design and synthesis of biomimetic gradient protein/biosilica composites for interfacial tissue engineering. Biomaterials, 145, 44-55.


3-4th October, 2019


P9: Enhancement of mechanical and biological properties of calcium phosphate bone cement

by incorporating bacterial cellulose

Quanchao Zhang1, Deqiang Gan1, Yizao Wan1, Honglin Luo1 1Institute of Advanced Materials, East China Jiaotong University, Nanchang 330013, China

[email protected]

Abstract

Calcium phosphate cement (CPC) has been used as a clinical bone replacement material due to its close

resemblance in chemical composition to the mineral component of bone extracellular matrix as well as

its proven good biocompatibility, bioactivity, osteoconductivity, and injectable[1]. However, low

mechanical strength of CPC restricts its use in load-bearing defects. As a natural nanofibrous material,

bacterial cellulose (BC) shows various striking advantages and has been widely used to construct

composites. Herein, for the first time, we selected bacterial cellulose (BC) fibers to reinforce CPC (Fig. 1a).

The BC-reinforced CPC (BC/CPC) composites with varying BC content were prepared and compared in

terms of microstructure, compressive strength and cellular responses. Scanning electron microscope

(SEM) images revealed the uniform dispersion of BC in CPC matrix. Fourier-transform infrared

spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) results confirmed the interfacial

coordination interaction between BC and CPC. It was found that the introduction of BC enhanced the

compressive strength of CPC and the optimal BC content was 2 wt.% (Fig. 1b). Moreover, cell studies

showed that BC/CPC composites exhibited better cell performance than bare CPC. The results have

demonstrated that a new CPC composite with enhanced mechanical performance and cellular responses

may be developed by using BC as reinforcement.

Fig. 1. (a) The preparation procedure and the proposed mechanism of interface reaction between BC and CPC, (b)compressive strength for different CPC and BC/CPC composites. ** means significant difference at p < 0.01 and * means significant difference at p < 0.05. CPC composites with 1, 2, 3, and 4 wt.% of BC were prepared and named as BC/CPC-1, BC/CPC-2, BC/CPC-3, and BC/CPC-4, respectively.



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References

(1) Zhang, J., Liu, W.; Schnitzler, V.; et al. (2014). Calcium phosphate cements for bone substitution: chemistry, handling and mechanical properties. Acta Biomater. 10:1035-1049.


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P10: Step-by-step self-assembly of 2D few-layer reduced graphene oxide into 3D architecture

of bacterial cellulose for a robust, ultralight, and recyclable all-carbon absorbent

Jie Wang1, Honglin Luo1,2, Jing Xie1, Zhiwei Yang1 , Yizao Wan1,2

1Institute of Advanced Materials, East China Jiaotong University, Nanchang 330013, China 2School of Materials Science and Engineering, Tianjin University, Tianjin 300072, China

[email protected]

Abstract

Development of high performance absorbents is of particular importance in environmental protection.

Herein, we reported a mechanically robust, ultralight, and recyclable absorbent with highly dispersed

twodimensional (2D) few-layer reduced graphene oxide (FrGO) in a three-dimensional (3D) carbonized

bacterial cellulose (CBC) monolith via a novel step-by-step in situ biosynthesis (SBSB) method followed by

carbonization. The step-by-step self-assembly produced a mechanically entangled nanostructure

between FrGO and CBC nanofibers, which possessed the ideal properties as an absorbent for oils and

organics: low density, high porosity, and high hydrophobicity. As shown in Fig. a-c when approaching

gasoline (dyed with Sudan III) floating on water, the CBC/FrGO could completely absorb it within a few

seconds. Similarly, as shown in Fig. d-f the CBC/FrGO could completely absorb phenoxin, which sank to

the bottom of the beaker, in a few seconds. When used as absorbents, the monolithic CBC/FrGO aerogel

exhibited excellent recyclability and high absorption capacities toward numerous oils and organic

solvents.The absorption capacities of CBC/ FrGO was using various oils and solvents. As control, the

absorption capacities of bare CBC were also tested, and the obtained values were presented in Fig. g. As

expected, CBC/FrGO showed significantly higher absorption capacities than CBC for all oils and organic

liquids. These properties, together with the green, cost-effective and scalable production process, make

it very promising as a superior all-carbon absorbent for environmental protection.



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Fig. (a-c) Chronological images of a CBC/FrGO absorbent picking up gasoline (dyed with Sudan III) from

gasoline. (d-f) Chronological images of a CBC/FrGO absorbent picking up phenoxin (dyed with Sudan III)

at the bottom of the beaker. (g) Absorption efficiency of CBC/FrGO and CBC for various organic liquids.

References

(1) W. Wan, Y. Lin, A. Prakash, Y. Zhou. (2016). Three-dimensional carbon-based architectures for oil remediation: from synthesis and modification to functionalization. J. Mater. Chem. 4: 18687-18705 (2) W. Zhen-Yu, L. Chao, L. Hai-Wei, C. Jia-Fu, Y. Shu-Hong. (2013). Ultralight, flexible, andfire-resistant carbon nanofiber aerogels from bacterial cellulose. Angew.Chem. 125: 2997-3001


3-4th October, 2019


P11: Study on Microwave Absorbing Properties of Fe3O4/Bacterial Cellulose Composites

Zhihuan Huang1,2, Quanchao Zhang1, Yizao Wan1* 1 Institute of Advanced Materials, East China Jiaotong University (Nanchang 330013, China)

2 School of Materials Science and Engineering, East China Jiaotong University (Nanchang 330013, China) [email protected]

Abstract

With the rapid development of wireless communication technology and electronic equipment

around us, military stealth technology has become a research hotspot all over the world. The new three-

dimensional porous carbon-based absorbing material not only solves the problems of a complicated

process, high cost and low yield in traditional materials, but also exhibits the advantage of thin, light, and

mechanical strong [1]. In this work, it was proposed to pre-blend bacterial cellulose (BC) and Fe3O4, and

then adopt flexible vacuum filtration self-assembly [2] and hot pressing technology to prepare flexible and

magnetic Fe3O4/BC composite membrane. The composite membrane exhibited excellent flexibility

through macroscopic mechanical testing (Fig. 1). The material had a distinct layered structure of large

voids between different layers, which had a plurality of nanopores with interconnected, and the Fe3O4

nanoparticles could easily penetrate into the fiber layer and remained close to the nanofibers interaction.

Magnetic analysis showed that the saturation magnetization of Fe3O4/BC composites was much lower

than pure Fe3O4. This was primarily due to the addition of non-magnetic medium BC, which hindered the

magnetic coupling of Fe3O4 particles and formed a magnetization loss layer [3]. As expected, the

combination of Fe3O4 and BC effectively reduced the dielectric constant and improved the impedance

matching. Interestingly, the unique three-dimensional porous structure enhanced the energy loss of

electromagnetic waves by multiple reflection and scatteration of incident waves. Simultaneously, the

uniform dispersion of Fe3O4 in the matrix increased the interfacial polarization and hysteresis polarization,

so that part of the electromagnetic waves were transformed into heat energy, until electromagnetic

waves were absorbed more than 99.9%. These results suggested that the Fe3O4/BC composite membrane

has excellent mechanical properties, magnetic properties and absorption properties, which is expected to

be used in the field of electromagnetic shielding materials.



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References

(1) Zeng, Z. H., Jin, H., Chen, M. j., et al. (2016). Adv Funct Mater. 26(2):303–310. (2) Chen, K., Shi, B., Yue, Y., et al. (2015). ACS Nano. 9(8):8165-8175. (3) He, F., Fan, J., Ma, D., et al. (2010). Carbon. 48(11):3139-3144.

Fig. 1 Macro photo of Fe3O4/BC composite membrane (left), top view (a), sucked up by magnet (b), folded into different shapes (d-f) Fig. 2 SEM images of Fe3O4/BC composite membrane (right), cross section (a), surface (b), Fe element mapping of composite (c) and content (d)


3-4th October, 2019


P12: Silver nanowire-doped bacterial cellulose as potential antimicrobial wound dressing

Mengxia Peng1, Lingling Xiong1, Quanchao Zhang1, Yizao Wan1* 1 Institute of Advanced Materials, East China Jiaotong University, Jiangxi Province, Nanchang City, 330013

[email protected]

Key words：Bacterial cellulose; silver nanowire; antimicrobial; Wound dressing

Abstract

Bacterial infection is the main cause of chronic wound healing, therefore, development of wound

dressings with antimicrobial activity to accelerating the healing of chronic wounds is highly desired. In this

paper, silver nanowires(AgNWs) were used as broad-spectrum antimicrobial materials, while bacterial

cellulose(BC) was used as substrate for the preparation of BC-AgNWs by a membrane liquid culture

method. It could be seen from the XRD pattern of BC-AgNW1 that only the characteristic peaks of BC and

AgNWs could be observed (Fig. 1), which indicated that BC and AgNWs did not form new phase.

Subsequently, SEM characterization (Fig. 2) showed that silver nanowires were uniform distributed in BC.

More AgNWs could be found in BC with the raise content of silver nanowire in BC culture medium.

Antibacterial experiments (Fig. 3) revealed that BC did not show any antimicrobial activity, whereas BC-

AgNWs exhibited the inhibition rate of above 99%. In summary, BC-AgNWs has good potential in

antimicrobial wound dressing.

Fig. 1 XRD pattern of BC-AgNW1 Fig. 2 SEM photograph of (a) BC and (b-f) BC-AgNW1-BC-AgNW5

10 20 30 40 50 60 70 80 90

AgNW

(002)

(311)(222)

(220)(200)

Inte

nsity (

a.u

.)

2 Theta (degree)

(111)

(101)

BC



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Fig. 3 (a) Colony of dilute solution in co-culture of material with S. aureus (b) Fluorescent images of S.aureus stained with DAPI after incubation with BC and BC-AgNWs (c) Growth inhibition zone of the BC and BC-AgNWs against S.aureus.

References

(1) Déborah, S., Sónia, P. M., Ribeiro, M. P. (2018). Recent advances on antimicrobial wound dressing a review. European Journal of Pharmaceutics & Biopharmaceutics. 127:130-141.

(2) Thomas, S. (2008). A review of the physical, biological and clinical properties of a bacterial cellulose wound dressing. Journal of Wound Care. 17:349-352.

(3) Jones, R. S., Draheim, R. R., Roldo, M. (2018). Silver Nanowires: Synthesis, Antibacterial Activity and Biomedical Applications. Applied Sciences. 8:673.

(4) Wu, C. N., Fuh, S. C., Lin, S. P. (2018). TEMPO-oxidized bacterial cellulose pellicle with silver nanoparticles for wound dressing. Biomacromolecules. 19:544-554.


3-4th October, 2019


P13: Effect of Nanofibrillated bacterial cellulose (NFBC) inclusion on the physical properties of

pulp fiber sheets and polyvinyl alcohol films

Ryoko Maruyama1), Ryota Kose1), Kenji Tajima 2), Tokuo Matsushima3) 1) Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan

2) Faculty of Engineering, Hokkaido University, Sapporo, 060-8628, Japan 3) Kusano Sakko Inc., Ebetsu, 067-0063, Japan

Abstract

In recent years, we have succeeded in the mass production of a 20-nm-wide nanofibrillated bacterial

cellulose (NFBC)(1) as a new form of bacterial cellulose (BC). The NFBC, which exhibits high water

dispersibility, was produced by culturing a cellulose-producing bacterium in a medium supplemented with

carboxymethyl cellulose (CMC) as dispersing agent. For NFBC, CMC exists on the surface of microfibrils. In

this study, pulp fiber sheets and polyvinyl alcohol (PVA) film with NFBC were prepared to demonstrate

the expansion of the applications of NFBC as a filler, and their basic physical properties investigated.

Pulp fiber suspensions with NFBC were prepared, with membrane being filtrated under vacuum to form

a wet pulp fiber sheet. The chemically prepared CNF was used as a reference. Following water removal by

pressing, the sheet was dried using a hot press machine. We measured the drainage time of NFBC and

chemically prepared CNF suspension. The time is a very important factor in the production of paper

productivity. Compared to chemically prepared CNF suspension, NFBC suspension time was shorter.

Moreover, aggregations of pulp fibers were observed in the pulp fiber sheet without NFBC, while no

aggregations were observed in the sheet when NFBC was added. Therefore, the addition of NFBC could

allow production of the pulp fiber sheet with better visually uniformity. When the content rate of NFBC

was added at 1% or more, the tensile strength improved with increasing amount of added NFBC. The

chemically prepared CNF was more effective in improving the tensile index of pulp fiber sheets, while the

variation coefficient of the tensile index of pulp fiber sheets with NFBC was lower than that of the pulp

fiber sheet with chemically prepared CNF. Therefore, the NFBC addition also enhanced the mechanical

homogeneity for pulp fiber sheets.

The NFBC suspension was mixed with PVA aqueous solution in various proportions. PVA film with NFBC

was prepared by film casting method, after which the basic physical properties of the film were examined.

We will report the observed effects of the NFBC inclusion on the properties of the PVA film in the poster

session.

References

(1) Kose, R., Sunagawa, N., Yoshida, M., Tajima, K. (2013). One-step production of nanofibrillated bacterial cellulose (NFBC) from waste glycerol using Gluconacetobacter intermedius NEDO-01. Cellulose. 20: 2971–2979.


3-4th October, 2019


P14: A Toolbox of Pharmaceutical Strategies for the Controllable Skin Delivery of Boswellia

Extracts using Bacterial Nanocellulose

B. Karl1, Y. Alkhatib1, U. Beekmann1, G. Blume2, S. Lorkowski3, D. Kralisch1, O. Werz4, D. Fischer1*

1Friedrich Schiller University Jena, Pharmaceutical Technology and Biopharmacy, Lessingstraße 8, 07743 Jena, Germany 2Sopharcos, Im Schloss 7, 36396 Steinau an der Straße, Germany 3 Friedrich Schiller University Jena, Nutritional Biochemistry and Physiology, Dornburger Straße 25, 07743 Jena, Germany 4Friedrich Schiller University Jena, Pharmaceutical and Medicinal Chemistry, Philosophenweg 14, 07743 Jena, Germany * [email protected]

Abstract

Due to their anti-inflammatory properties, lipophilic extracts of the resin from Boswellia species are

proposed as an effective treatment of a wide variety of inflammation-related conditions and are discussed

as natural substitutes for non-steroidal anti-inflammatory drugs (NSAID) such as ibuprofen or naproxen.

Dermal application of Boswellia extracts with effective penetration into the skin is of interest for the

treatment of e.g. dermatitis, psoriasis and chronic wounds as well as in skin care for anti-aging strategies.

The biopolymer bacterial nanocellulose (BNC) was used as delivery system since it already proved to

support the wound healing process [2] due to its unique three-dimensional network of nanosized fibers

with excellent biocompatibility. However, the highly lipophilic character of the Boswellia extract required

new technological strategies to overcome the hydrophilicity of the BNC.

In the present study, BNC was synthesized by the bacterial strain Komagataeibacter xylinus DSM 14666

and effectively formulated with Boswellia extract using different techniques (adsorption, vortex and

reswelling technique) in combination with several additives (hydrophilic polymers, dimethyl isosorbide,

poly(ethylene glycol), nanoemulsions) as a toolbox of strategies that accomplished the homogenous and

stable incorporation of the lipophilic extract into the hydrophilic BNC. Generating and following Ishikawa

diagrams optimized the process of formulation according to the concept of Quality by Design (QbD).

Storage experiments showed constant values without loss of drug stability over more than 90 days. The

loading procedure did not change the preferential characteristics of the BNC like high water absorption

and retention, softness and pressure stability. Depending on the type of additive, fast release systems for

acute treatments (e.g. cosmetic masks or acute wounds) over up to 2 h as well as delivery systems for a

prolonged Boswellia extract release for chronic wounds over several days have been acquired. To

investigate the amount, depth and distribution of penetrated Boswellia extracts in the skin, tape-stripping

experiments were performed on porcine skin. Different application modes, application times and the

utilization of additives and nanoemulsions were examined and showed correlations, e.g. between

selected additive and depth of Boswellia extract penetration.

The obtained results provide the basis for the further development using in vivo systems to achieve a

tailor-made, enhanced, non-irritant, anti-inflammatory formulation that would serve as a better

alternative for the dermal treatment thus providing better patient comfort and compliance.



3-4th October, 2019


Acknowledgments

The authors gratefully acknowledge the Free State of Thuringia and the European Social Fund (2016 FGR

0045) for funding. K. xylinus culture was kindly provided by JeNaCell GmbH.

References

[1] Pötzinger, Y. et al.: Ther. Deliv. 2017, 8 (9): 753-761. [2] Sulaeva, I. et al.: Biotechnol Adv. 2015, 33 (8): 1547–71.


3-4th October, 2019


P15: Obtaining and characterization of new composite of bacterial nanocelullose containing

bioliquefied brown coal

Marzena Jędrzejczak-Krzepkowska, Karolina Ludwicka, Bartosz Strzelecki, Teresa Pankiewicz, Stanisław Bielecki

Institute of Technical Biochemistry, Lodz University of Technology, Poland [email protected]

Abstract (poster)

Bacterial nanocellulose (BNC) has unique properties, such as water holding capacity, high degree of

polymerization, high thermal properties and crystalinity, good mechanical properties with fiber network

structure, neutrally charged, moldability. With these features BNC can be applied as natural renewable

polymer. Furthermore, BNC can be modified and used to obtain biomaterials with new properties and new

potential applications in many industrial and medical fields, e.g. scaffolds for tissue engineering, packaging

industry, electronics, environmental protection [1]

Bioliquefied brown coal is obtained as a product in clean coal technologies, by means of biological

processes, e.g. when lignite (after pre-treatment with an oxidant, e.g. nitric acid, hydrogen peroxide) is

utilized as a carbon source by microbial cultures. The products of biosolubilization are a mixture of humic,

fulvic acids and other organic compounds. Liquefied lignite can be used in cosmetics, as well as excellent

sorbent, for example in biosorption of various pollutants or as fertilizer. What's more, it may also be used

as a raw material for production of various chemicals like alcohols, aromatic compounds, fatty acids,

methane etc. [2,3,4]

The aim of the research is the production and characterization of a new composite biomaterial, which is

composed of BNC and biosolubilized brown coal. Liquid coal was obtained by preliminary treatment of

brown coal (using HNO3 or O3) and biosolubilization process under culture conditions by a group of

microorganisms Gordonia alkanivorans S7 and Fusarium oxysporum 1101. New BNC composite with

biosolubilized brown coal was obtained in situ and ex situ. This composite was characterized by physico-

chemical methods. Their morphology, crystallinity, chemical composition and water retention capacity

were analysed. The mechanical properties were investigated by tensile testing. On the basis of the

obtained results, no negative influence of liquefied brown coal on the growth of K. xylinus E25 strain and

BNC biosynthesis was found. The new composite with the increase in the content of liquefied carbon in

BNC is characterized by an increase in water retention and a decrease in water absorption.

References

(1) Ludwicka K., Kolodziejczyk M., Gendaszewska-Darmach E., Chrzanowski M., Jedrzejczak-Krzepkowska M., Rytczak P., Bielecki S. (2019) Stable composite of bacterial nanocellulose and perforated polypropylene mesh for biomedical applications. Journal of Biomedical Materials Research Part B Applied Biomaterials 107(4):978-987

(2) Romanowska I., Strzelecki B., Bielecki S. (2015). Biosolubilization of Polish brown coal by Gordonia alkanivorans S7 and Bacillus mycoides NS1020. Fuel Processing Technology. 131: 430–436



3-4th October, 2019


(3) B. Strzelecki B., Romanowska I., Bielecki S., Marchut‐Mikołajczyk O. (2017) Method of biosolubilization of brown coal in order to obtain organic fertilizer with high content of humic acids. Polish Patent Application No P.423511

(4) Kwiatos N., Jędrzejczak‐Krzepkowska M., Strzelecki B., Bielecki S. (2018). Improvement of efficiency of brown coal biosolubilization by novel recombinant Fusarium oxysporum laccase. AMB Express 8:133


3-4th October, 2019


P16: Biosynthesis of BNC and BNC-based nitrocellulose

Vera V. Budaeva,* Galina F. Mironova, Yulia A. Gismatulina, Ekaterina A. Skiba, Evgenia K. Gladysheva, Ekaterina I. Kashcheyeva, Olga V. Baibakova, Anna A. Korchagina, Dmitry S. Golubev, Nikolay V. Bychin,

Gennady V. Sakovich

Bioconversion Laboratory, Institute for Problems of Chemical and Energetic Technologies, Siberian Branch of the Russian Academy of Sciences (IPCET SB RAS), Biysk 659322, Siberia, Russia

[email protected] (V.V. Budaeva, Head of Laboratory)

Abstract

Bacterial nanocellulose (BNC) possesses unique properties such as high purity, nanofibrous network

structure, 99% water content, and represents a mechanically and thermally stable hydrogel [1]. Because

BNC is highly demanded, the scale-up of its biosynthetic technology is a topical challenge, as is the

wasteless production of BNC due to its high cost. We have scaled up the biosynthesis of BNC in a 104-L

vessel with the Medusomyces gisevii Sa-12 symbiont. We carried out eleven biosynthetic cycles of BNC

using the preceding growth medium as inoculum for the next runs. Then, we freeze-dried the BNC and

nitrated it with mixed nitric-sulfuric acids under conditions optimal for plant cellulose [2]. Afterwards, we

characterized the BNC-based nitrocellulose, and the SEM analysis showed that the BNC-derived

nitrocellulose remained the 3D structure. The nitration study results we obtained with unique BNC, we

believe, are of fundamental importance and they differ from those obtained with plant cellulose nitrates

[3].

The research was supported by the Russian Science Foundation (project No. 17-19-01054).

References

(1) Klemm, D., Cranston, E. D., Fischer, D., Gama, M., Kedzior, S. A., Kralisch, D., Kramer, F. Kondo, T., Lindström, T., Nietzsche, S., Katrin Petzold-Welcke, K., Rauchfuß, F. (2018). Nanocellulose as a natural source for groundbreaking applications in materials science: Today’s state. Mater. Today. 21 (7):720-748. (2) Gismatulina, Y. A., Budaeva, V. V., Sakovich, G. V. (2018) Nitrocellulose synthesis from Miscanthus cellulose. Propellants Explos. Pyrotech. 43:96-100. (3) Liu, Y. H., Shao, Z., Wang, W. J., Li, L., Lu, Y. Y., Sun, J. (2018) System and method for simultaneous measurement of nitrogen content and uniformity of nitration of nitrocellulose. Cent. Eur. J. Energ. Mater. 15(4): 554-571.



3-4th October, 2019


P17: Characterization of the proteins encoded by the second cellulose synthase operon

Szymczak* I., Pietrzyk-Brzezińska A., Ryngajłło M., Duszyński K., Bielecki S.

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland

Abstract

Cellulose, the most abundant natural polymer in the world, can be obtained from plant material using

chemical methods such as alkali or acid hydrolysis in a process that threatens the environment . Therefore,

environmentally friendly bacterial cellulose (BC) produced extracellularly in the form of a biofilm by

various strains of acetic acid bacteria, is considered as a promising alternative to plant cellulose and

increases its importance [1,2]. The most effective cellulose producers are bacteria of the Komagateibacter

genus [3]. Bacterial cellulose (BC) produced by these microorganisms is characterized by unique

properties, therefore this polymer is widely used in various industries as well as in medicine. Cellulose

production on an industrial scale is still unprofitable. Great potential to improve the efficiency of cellulose

synthesis is the application of genetic engineering techniques of the producer's cell. However, there is still

a lack of knowledge about the molecular mechanisms of BC biosynthesis and its regulation. Although the

function of genes comprising the first cellulose synthase operon (bcsI) is understood to a large extent, the

second operon (bcsII) encoding three proteins: BcsX, BcsY and BcsZ, is not well characterized. Gene

expression analysis revealed that bcsX is expressed at almost three times higher level than the bcsAI

subunit of the bcsI operon (the main catalytic subunit). It can be supposed that bcsX may play an important

role in modulating the BC properties. While BcsX and BcsZ are supposed to belong to the SGNH/GDSL

hydrolase family protein, BcsY protein may be involved in acetylation of the BC [4]. The aim of the study

was to prepare constructs allowing expression of proteins coded by genes bcsX, bcsY and bcsZ present in

the Komagataeibacter xylinus E25 genome in the bacterial system. The genes were isolated from K. xylinus

E25 genomic DNA, amplified in the PCR and an attempt to prepare constructs using the bcsX, bcsY and

bcsZ genes and pETM-11 vector suitable for expression in E. coli BL21-Gold cells was made. Three

constructs allowing cloning and expression of proteins BcsX and BcsZ present in the K. xylinus E25 genome

were successfully prepared. The expression of BcsX led to obtain soluble protein, purified by affinity

chromatography. Initial crystallization trials, performed using commercial screens led to first crystal hits.

Unfortunately, very small crystals and low resolution (4 Å) of diffraction data did not allow for the solution

of the 3D crystal structure of BcsX. In the future, the further optimization of crystallization conditions is

necessary in order to obtain well-diffracting crystals.

References

(1) Gullo, M., La China, S., Falcone, P. M., Giudici, P. (2018). Biotechnological production of cellulose by acetic acid bacteria: current state and perspectives. Appl. Microbiol. Biotechnol., 102 (16): 6885–6898. (2) Jozala, A. F., de Lencastre Novaes, L. C., Lopes, A. M., Ebumina, V. (2016). Bacterial nanocellulose production and application: a 10-year overview. Appl. Microbiol. Biotechnol., 100 (5):2063–72.


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(3) Ryngajłło, M., Kubiak, K., Jędrzejczak-Krzepkowska, M., Jacek, P., Bielecki S. (2018). Comparative genomics of the Komagataeibacter strains-Efficient bionanocellulose producers. Microbiologyopen, p. e00731. (4) Umeda, Y., Hirano, A., Ishibashi, M., Akiyama, H., Onizuka, T., Ikeuchi, M., Inoue, Y. (1999). Cloning of cellulose synthase genes from Acetobacter xylinum JCM 7664: Implication of a novel set of cellulose synthase genes. DNA Research. 6:109-115


3-4th October, 2019


P18: Novel chitosan-coated bacterial cellulose hydrogels for controlled delivery systems

Gabriel Arner, Olatz Guaresti, Leire Urbina, Julen Diez, Arantxa Eceiza, Aloña Retegi, Nagore Gabilondo

‘Materials + Technologies’ Group, Engineering School of Gipuzkoa. Department of Chemical and Environmental Engineering, University of the Basque Country (UPV/EHU), Pza. Europa 1, 20018 Donostia-San Sebastian.

[email protected]

Abstract

The research on different technologies for the development of new systems for the controlled release of

drugs is of great interest for the pharmaceutical industry. To this end, research into the synthesis and

design of new polymer systems is booming and, particularly, that focused on natural polymers and

green/sustainable synthesis processes. Two fundamental factors drive the research in this field: greater

concern for sustainable development and interest in biomimetic/bioinspired approaches [1]. Natural

polymers generate a limited environmental impact, can be obtained in large quantities, at a low cost and

are biocompatible and chemically versatile. Moreover, living tissues are a model to follow for the design

of new functional materials with superior structures and properties. All above has led to a growing interest

in the design of new biomaterials based on bacterial cellulose (BC) for being applied in biomedicine with

several purposes [2,3]. In this work, dynamic BC cultures were performed in order to obtain chitosan and

alginate hydrogel particles with encapsulated BC nanofribrils. Bacterial cellulose/chitosan/alginate hybrid

particles were in situ biosynthesized in agitated cultures obtaining ionically crosslinked particles. The

obtained particles were characterized by FTIR and their morphology, water holding capacity and stability

was studied. This work provides a new route for designing chitosan/alginate/bacterial cellulose hydrogels

for drug delivery applications.

Figure 1. Schematic representation of the work.

References

(3) Badshah, M., Ullah, H., Khan, A.R., Khan, S., Park, J.K., Khan, T. (2018). Surface modification and evaluation of bacterial cellulose for drug delivery. International Journal of Biological Macromolecules. 113: 526-533.







https://www.sciencedirect.com/science/journal/01418130


3-4th October, 2019


(4) Algar, I., Fernandes, S.C.M., Mondragon, G., Castro, C., García-Astrain, C., Gabilondo, N., Retegi, A., Eceiza, A. (2015). Pineapple agroindustrial residues for the production of high value bacterial cellulose with different morphologies Journal of Applied Polymer Science. 132: 41237.


3-4th October, 2019


P19: Evaluation of in vitro cytotoxicity of Bacterial Cellulose obtained from agroindustrial

byproducts Fábia Karine Andrade1*, Catarina Gonçalves2, Lorenzo Miguel Pastrana2,

Maria de Fátima Borges3, Rodrigo Silveira Vieira1, Morsyleide de Freitas Rosa3

1 Federal University of Ceará (UFC), Department of Chemical Engineering, Bloco 709, CEP 60455-760, Fortaleza, Ceará, Brazil 2 Food Processing Group, International Iberian Nanotechnology Laboratory, Braga, Portugal 3 Embrapa Agroindústria Tropical, Rua Dra Sara Mesquita 2270, Planalto do Pici, CEP 60511-110, Fortaleza, CE, Brazil [email protected]

Abstract

Bacterial cellulose (BC) has a long history of its use in various food and biomedical applications. However,

the potential for its industrialization and commercialization at large scale is still a challenge due to high

fermentation cost, low productivity and expensive culture media. To overcome this problem, several

studies have proposed the use of low-cost substrates, such as biomass byproducts from different

industries, to cheapen the BC production1. However, safety assessment to disclose any potential health

risk of BC produced using alternative media remains necessary to ensure that it is safe for use in food and

medical products2. The present study was undertaken to evaluate the cytotoxicity effect on four different

cell lines (HUVEC, CCD-18Co, L-929 and SIRC) of BC produced in three byproducts from Brazilian

agroindustry (cashew juice, cashew permeate and soy molasse).

In order to produce BC pellicles, the Komagataeibacter xylinus microorganism was inoculated in the tested

media and incubated statically at 30 °C for 10 days. After fermentation, the pellicles were washed twice

with water (1 h at 100 °C) and transferred to a 2% NaOH solution at 80 °C for 1 h. Purification in NaOH

was performed five times for cashew permeate and juice and two times for soy molasse. After purification,

the BC pellicles were rinsed in tap water until a neutral pH, followed by washing in distilled water. The

pellicles were dried and then sterilized by autoclaving. To evaluate the cytotoxicity, a direct MTT assay

was performed. BC samples were placed vertically against the wall of the well of a 24-well polystyrene

plate. Then, the cells were seeded at 2.5 x 104cell/well and the plates were incubated in a 37 °C and 5%

CO2 incubator for 24h and finally the MTT assay was conducted.

The results showed that for CCD, HUVEC and SIRC cell lines, the values of cell viability after incubation

with BCs produced in agroindustrial byproducts in relation to BC produced in traditional Hestrin-Schramm

medium (assumed as 100% of cell viability) were above 82%, being the higher values for permeate (99-

115%) and the lower values to soy molasse (82-85%). For the L-929 cell line the viability obtained after

contact with BC produced in cashew permeate and juice were 99%, while for soy molasse was 76%. These

preliminary results showed that there are variations in cell viability of BC obtained in different culture

media, however in general the cell viability remained above 80%.



3-4th October, 2019


References

(1) Hussain, Z., Sajjad, W., Khan, T. et al. (2019). Production of bacterial cellulose from industrial wastes: a review. Cellulose. 26: 2895-2911.

(2) Pinto, F., De-Oliveira, A. C. A. X., De-Carvalho, R. R. et al. (2016). Acute toxicity, cytotoxicity, genotoxicity and antigenotoxic effects of a cellulosic exopolysaccharide obtained from sugarcane molasses. Carbohydrate Polymers. 137:556-560.


3-4th October, 2019


P20: Effect of the combination of bacterial nanocellulose dressing with secondary dressings:

an in vitro and in vivo approach Bernardelli de Mattos, I.*, Funk, M., Tuca, A. C., Palackic, A.,

Holzer, J. C. J., Birngruber, T., Kamolz, L. P., Groeber-Becker, F. Fraunhofer Institute for Silicate Research ISC, Translational Center Regenerative Therapies, Würzburg, Germany

Abstract

A controlled moisture balance on the wound surface can enhance the effect of various cytokines,

chemokines and growth factors, favoring cell growth and thereafter the healing process. Burn patients,

for instance, would benefit of this approach, since a higher efficiency on the healing process cloud help to

decrease the hospitalization time. To analyze the effect of the moisture balance on the healing process, a

bacterial nanocellulose (BNC) dressing comprising around 98% water as a sodium chloride solution was

tested in combination with different commercially available secondary wound dressings (Aquacel®

Extra™, cotton gauze, Jelonet and Opsite Flexflix). Evaporation rates from the BNC were observed in vitro

and the corresponding wound healing was studied in vivo using a porcine donor site model. In vitro

experiments showed highest evaporation rates of the water content from BNC when combined with

cotton gauze or Aquacel® Extra™. When Jelonet was used as secondary dressing, intermediate

evaporation rates were achieved. A strong water retention was found for the combination of BNC with

Opsite Flexflix. Histological results from the animal study showed that BNC dressing in combination with

Aquacel® Extra™ or cotton gauze achieved high rates of reepithelialization. Reduced reepithelization was

observed using Jelonet as a secondary dressing and the usage of Opsite Flexifix resulted in high water

retention leading to very bad to non-healing wounds and a high maceration rate. The results showed that

a controlled moisture environment on the wound is achievable by using BNC dressing in combination with

Aquacel® Extra™, cotton gauze, Jelonet, and Opsite Flexifix. The higher reepithelialization achieved by

combination of BNC with selected secondary dressings open a great opportunity to improve the healing

process in clinical wound management.


3-4th October, 2019


P21: Synthesis of bacterial cellulose by Komagataeibacter strains in agitated and static

process conditions

Catarino, R. P. F., Rosa, M. F., Gomes, R. J., Hata, N. N. Y., Marestoni, L. D., Brígida, A. I. S., Spinosa, W. A.*

* Department of Food Science and Technology, State University of Londrina, Londrina-PR, Brazil. [email protected]

Abstract

Some strains of acetic acid bacteria have been identified as producing BC and acetic acid. The economic

viability of these products is strongly linked to the cost of the raw material and the conditions employed

in the process, so the use of coproducts of industrial processes characterizes a promising source for this

purpose (1). Soybean molasses present potential for use as a substrate in fermentative processes and this

is due to the high amount of carbohydrates present in this co-product originated in the processing of soy

protein concentrate. The aim of this work was to evaluate the effect of methods (agitated and static),

strains (Komagataeibacter hansenii ATCC 23769, Komagataeibacter xylinus ATCC 700178 e

Komagataeibacter sp. V-05 MH036354) and culture media (Hestrin–Schramm and molasses with soy

ethanol) on the production of bacterial cellulose. A completely randomized design was used in a triple

factorial scheme (2 x 3 x 2), in two production methods, three strains and in two different culture media.

Cultivation was carried out for 10 days at 30 °C. BC production was determined by the dry mass per volume

of culture medium (g.L-1) obtained at the end of the process. The chemical structure, morphology and

thermal stability of BC were characterized by FTIR, SEM and TGA/DTG techniques. BC production resulted

from triple interaction between factors. The V-05 strain was more efficient than the other strains in HS

with production of 0.8203 g.L-1 in shaken medium and 3.3113 g.L-1 in static. The BC obtained in the static

process had the form of a uniform film in the shape of the vial (vessel) in which it was produced. Already

in the agitated process this one presented in the form of regular spheres with different diameters and

irregular and fragmented. All samples had high water retention capacity (77.62 to 247.76 g) and good

thermal stability. The membranes obtained by strain V-05 lower residual mass and higher thermal stability

compared to the others when produced in HS medium. The strain V-05 proved to be suitable to produce

cellulose. The soybean molasses medium is an alternative for the simultaneous production of acid and BC.

References

(1) Saichana, N., Matsushita, K., Adachi, O., Frébort, I., Frebortova J. (2015). Acetic acid bacteria: A group of bacteria with versatile biotechnological applications. Biotechnology Advances. 33: 1260–1271.



3-4th October, 2019


P22: Evaluation of the synthesis of bacterial cellulose by acetic acid bacteria isolated from

vinegar industry using soybean molasses as fermentative substrate

Gomes, R. J., Rosa, M. F., Catarino, R. P. F., Hata, N. N. Y., Marestoni, L. D., Brígida, A. I. S., Spinosa, W. A.*

* Department of Food Science and Technology, State University of Londrina, Londrina-PR, Brazil. [email protected]

Abstract

Soybean molasses is an industrial by-product, generated after the soy protein concentrate production

from defatted soybean meal. It has more than 50% of carbohydrates, mainly sucrose, stachyose and

raffinose (1). Due to its high concentration of sugars, soybean molasses can be used as a fermentative

substrate by microorganisms, aiming to obtain microbial metabolites, such as acetic acid and bacterial

cellulose, both synthesized by acetic acid bacteria. These bacteria comprise a variety of microorganisms

with important industrial applications, mainly in the vinegar production. Some strains are also able to

synthesize cellulose, a biopolymer recognized for its specific properties, such as high purity, crystallinity

and polymerization degree, great water holding and retention capacity, high tensile strength, elasticity

and excellent biocompatibility and biodegradability (2). Based on this, the present work aimed to isolate

acetic acid bacteria from a vinegar industry and evaluate their potential of synthesizing bacterial cellulose

in a medium containing soybean molasses as carbon and nitrogen source. Five strains of acid and cellulose

producing bacteria, identified after biochemical tests, were obtained. For these selected strains, cellulose

production and acidity yield were determined in the fermentation of soybean molasses. For this, 10% of

the inoculum was transferred to Erlenmeyers flasks containing soybean molasses (20º Brix) added of 2%

ethanol to improve the productivity or containing HS broth. The flasks were incubated at 30 ºC for 14 days

under static conditions. After the alkaline treatment (1M NaOH / 80 °C), the cellulose produced in soybean

molasses and standard medium were quantified and characterized in relation to crystallinity index by X-

Ray diffraction (XRD), chemical structure by infrared spectroscopy (FT-IR), thermogravimetric analyses

(TGA), water holding capacity and rehydration ratio. The V-05 strain exhibited the highest cellulose

production in both standard HS and soybean molasses-based medium. In this substrate, an increase in the

synthesis of the biopolymer was obtained, reaching a maximum production of 10 g L-1. There was

difference in production rate and hydrophilic properties for both producing microorganisms and

substrates used, possibly due to structural differences in the cellulose produced in each medium and for

each microorganism. Results of XRD, TGA and FT-IR demonstrated that the samples produced in soybean

molasses maintained the structural characteristics similar to those obtained from HS medium. These

results demonstrate that soybean molasses may represent an interesting and more economic culture

media for BC production that can provide higher fermentation yields than those obtained in the standard

HS medium.



3-4th October, 2019


References

(1) Long, C. C., Gibbons, W. R. (2013). Conversion of soy molasses, soy solubles, and dried soybean carbohydrates into ethanol. International Journal of Agricultural and Biological Engineering. 6:62-68

(2) Mohammadkazemi, F., Azin, M., Ashori, A. (2015). Production of bacterial cellulose using different carbon sources and culture media. Carbohydrate Polymers. 117:518-523


3-4th October, 2019


P23: Surface modification of bacterial cellulose membranes for microfluidic applications

S. L. Silvestre1, A. C. Marques1, C. Cristelo 2, M. F. Gama 2, R. Martins1, E. Fortunato1 1Departamento de Ciência dos Materiais, CENIMAT|I3N and CEMOP/UNINOVA, Faculdade de Ciências e Tecnologia

– Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. 2CEB- Centre of Biological Engineering, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal

Abstract

Although the great advances in medicine, there are still many limitations related to materials that can

interact with the human body and its cells. One of the major challenges is developing biocompatible

materials that can be used to skin repair treatments, tissue regeneration, inflammation, and wound

treatments, among other problems. The most critical step to explore new materials development still

remains in the interactive surface cell-contact to different biomaterials1. Bacterial cellulose (BC) has

attracted the attention of many in this field due to its advantageous characteristics being biodegradable,

non-toxic, non-carcinogenic, and biocompatible with the human body. This polymer could be the future

of regenerative medicine and for that, it is necessary to continue exploring their properties and possible

treatments that can improve them. The work reports the functionalization of bacterial cellulose (BC)

membranes with a view to their application in microfluidic devices for the growth of epidermal cells. Here,

is proposed a surface modification with Parylene C deposition by chemical vapor deposition (CVD), and

consecutively optimized oxygen (O2) plasma pre-treatment and sulfurhexafluoride (SF6) plasma treatment

in a reactive ion etching (RIE) system. Parylene C is a transparent polymer to the naked eye with an

excellent permeation barrier for liquid and gaseous types, required for this kind of application. This

chemically inert, biocompatible, biostable, flexible, hydrophobic and resistant polymer, has been

extensively used for several applications, including for medical devices and implants2. This proposed

technique3 produce a surface roughness and enhances the intrinsic hydrophobicity of Parylene-C, by

giving not only hydrophobic but superhydrophobic behavior on surface membranes, without altering the

material bulk (bacterial cellulose) properties. The best results were obtained by deposition of 10 g of

parylene-C, pre-treated with O2 plasma for 10 min, and then SF6 plasma for 1 min. After all the

optimization processes a superhydrophobic contact angle was obtained, approximately 155 degrees and

remained hydrophobic during 15 days of wettability tests. This work present exceptional results and

investigates the ability of the modified bacterial cellulose by integrating them into a chip to support the

growth of epidermal cells.

Acknowledgement

The authors thank funding through the project “SkinShip - Dispositivo de microfluídica inovador baseado

em celulose capaz de suportar a modelação 3D de pele”, with reference PTDC/BBB-BIO/1889/2014

supported by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Programa Operacional

Competitividade e Internacionalização - COMPETE 2020, do Programa Operacional Regional de Lisboa e

por Fundos Nacionais através da FCT - Fundação para a Ciência.


3-4th October, 2019


References

1. Picheth, G. F. et al. Bacterial cellulose in biomedical applications: A review. Int. J. Biol. Macromol. 104, 97–106 (2017).

2. Bi, X., Crum, B. P. & Li, W. Super hydrophobic Parylene-C produced by consecutive O2 and SF6 plasma treatment. J. Microelectromechanical Syst. 23, 628–635 (2014).

3. Brancato, L., Keulemans, G., Gijsenbergh, P. & Puers, R. Plasma enhanced hydrophobicity of parylene-C surfaces for a blood contacting pressure sensor. Procedia Eng. 87, 336–339 (2014).


3-4th October, 2019


P24: Identification and Engineering of Native and Non-native Secretion Systems in K.

rhaeticus to engineer biomaterials with unique properties

Amritpal Singh1, Dr Rodrigo Ledemsa Amaro1, Dr Tom Ellis1

1Bessemer Building, South Kensington Campus, Department of Bioengineering, Faculty of Engineering, Imperial College London

Abstract

Bacterial cellulose (BC) is an ultra-pure carbohydrate polymer which has a myriad of useful properties.

These properties are due to its purity from contaminating polysaccharides. It has a highly crystalline

structure, a high tensile strength and a high water holding potential. Therefore, it is of great interest to

the biotechnology industry as it provides a unique material that can be used in a range of applications

from filtration membranes to medical devices. Komagataeibacter rhaeticus (K. rhaeticus) is one of the

species in the Acetobacter family which produces BC as a product of cell growth. We aim to functionalise

the cellulose membrane by secreting proteins of interest into the bacterial cellulose membrane. Two

approaches will be used, 1) Identifying native secretion systems by investigating the secretome of K.

rhaeticus. This will indicate what secretion systems may be present within the organism and suggest

possible native secretion tags which will allow us to secrete proteins into the membrane. 2) Engineering

a Type V secretion system (SS), AIDA-I from Escherichia coli into K. rhaeticus. This autotransporter systems

will allow secretion of proteins of interest into the BC pellicle and functionalise it with desired properties.

Therefore, using tools developed in our lab we aim to genetically engineering the amenable strain from

our lab (K. rhaeticus iGEM). To express and secrete functional proteins into the BC matrix using identified

native secretion sytems or engineering in non-native secretion systems.


3-4th October, 2019


P25: Challenges on specific surface area analysis of cellulosic materials

Anett Kondor, Andreas Mautner, Koon-Yang Lee, Daryl Williams, Alexander Bismarck

Surface Measurement Systems Ltd., 5 Wharfside Rosemont Road, London, HA0 4PE, United Kingdom

Abstract

The interaction of a solid with its surroundings is through the available surface area for adsorption of gas

or vapour molecules. This also allows probing of materials surface including irregularities and pores. One

of the most successful methods is based on the BET method for gas adsorption onto a solid surface. The

adsorption method of Brunauer, Emmett and Teller (BET) is based on the physical adsorption of a vapour

or gas onto the surface of a solid. Traditionally, sorption studies were carried out at low temperatures to

obtain nitrogen isotherms at 77 K, which were then used to calculate BET surface areas. Considering that

material behavior varies with temperature, measurements at ambient temperatures may be more

relevant and also allow the use of various gases and vapours.

Presented is an alternative method to determine the BET specific surface area of cellulose materials

(freeze dried bacterial cellulose, BC nanopaper and Avicel) and specifically low surface area [particularly

less than 5.00 m2/g] materials at ambient temperature under the presence of relative humidity, and in

addition discussing the existence of the absolute surface area.


3-4th October, 2019


P26: Influence of different types of bacterial nanocellulose on development of oil-in-water

Pickering emulsions

Náyra Pinto1,2,5, Ana Isabel Bourbon2, Miguel Cerqueira2, Lorenzo Pastrana2, Miguel Gama3, Henriette Azeredo1,4, Morsyleide Rosa1,5, Catarina Gonçalves2

1Biomass Technology, Embrapa Tropical Agroindustry, Ceará, Brazil. 2Food Processing Group, International Iberian Nanotechnology Laboratory, Braga Portugal.

3Centre of Biological Engineering, University of Minho, Braga, Portugal.

4Department of Chemical Engineering, Federal University of Ceará, Ceará, Brazil.

Abstract

Bacterial nanocelluloses have been studied to stabilize oil-in-water emulsions due to its ability to adsorb

on the oil-water interface, promoting highly stable and surfactant-free systems. However, several features

of the bacterial nanocellulose may influence the resultant emulsion, such as the cellulose nature, size,

surface charge, shape and chemical surface. Thus, this work aims to produce sunflower oil-in-water

emulsions using bacterial nanocelluloses produced by fermentation of Komagataeibacter xylinus in

Hestrin e Schramm (HS) medium and processed by two different treatments: 2,2,6,6-tetramethyl-1-

piperidinoxyl (TEMPO) mediated oxidation for the production of cellulose nanofibrils (CNF) and acid

hydrolysis with sulfuric acid for the production of cellulose nanocrystals (CNC). The oxidized bacterial

cellulose suspension was further nanofibrillated by high-speed homogenizer that produced the CNF (82

nm diameter and -46.5 mV surface charge). Nanocrystals had an average length of 491 nm, mean diameter

of 70 nm and -50.3 mV of surface charge. For each nanocellulose, different o/w ratios were tested, in

order to produce stable emulsions. The emulsions were formulated with an oil phase of 10-1% and an

aqueous phase of 90-99%, with a nanocellulose concentration 0.5-1%. Different salt concentrations (NaCl)

were tested in the aqueous phase (0-50 mM). The emulsion stability was evaluated by visual inspection

considering that the absence of oil in the emulsion surface represents emulsion stability. Emulsions

stabilized by CNC exhibited a mean droplet diameter varying between 1.2 and 2.0 µm, with a white color

and fluid texture. On the other hand, the emulsions stabilized by CNF formed droplets above 2.0 µm with

a less fluid texture, which confirmed the influence of the bacterial nanocellulose features on the

characteristics of the emulsions formed. The microscopy analysis performed with polarized light

demonstrated the presence of fibrils and crystals on the oil-water interface of the droplets. The oil

droplets were also analysed by fluorescence microscopy after nile red staining. These results indicated

that pickering emulsions composed by CNC and CNF, can be used as a carrier to encapsulate active

compounds. Beta-carotene will be incorporated in the oil-in-water emulsions, as an hydrophobic model

molecule, and digestibility studies will be performed to assess the beta-carotene bioaccessibility in

different formulations.

Acknowledgments for the financial support by CAPES development agency.


3-4th October, 2019


References

(1) Nascimento, E. S., Pereira, A. L. S., Barros, M. O., Barroso, M. K. A, Lima, H. S., Borges, M. F., Feitosa, J. P. A., Azeredo, H. M. C., Rosa, M. F. (2019). TEMPO oxidation and high-speed blending as a combined approach to disassemble bacterial cellulose. Cellulose. 26:2291-2302.


3-4th October, 2019


P27: Nanopapers based on bacterial cellulose with conductive, magnetic or photochromic

properties

Agnieszka Tercjak1, Joseba Gomez-Hermoso-de-Mendoza1, Hernane S. Barud2,3, Sydney J. L. Ribeiro2 Junkal Gutierrez1

1Group `Materials + Technologies´ (GMT), Department of Chemical and Environmental Engineering, Faculty of Engineering Gipuzkoa, University of the Basque Country (UPV/EHU), Plaza Europa 1, 20018 Donostia-San Sebastián, 2Laboratory of Photonic Materials, Institute of Chemistry, São Paulo State University-UNESP, Araraquara, SP, Brazil, 3Laboratório de Química Medicinal e Medicina Regenerativa (QUIMMERA), Centro Universitário de Araraquara, Araraquara, SP, Brazil

Abstract

Bacterial cellulose (BC) is biosynthesized polymer with interesting properties such as very good

mechanical properties (high strength and stiffness), porosity, high crystallinity, high water absorption

capability as well as biodegradability and remarkable biocompatibility. Moreover, BC has capability to

form film with tridimensional nanofiber network structure. These remarkable properties of BC make it

interesting matrix to design nanopapers with conductive, magnetic, photochromic properties and others,

which can find wide range of application in Materials Science and Nanotechnology area fields (tissue

engineering, liquid filtration and purification, solar cells, electronic devices and others).

According to the application requirements different nanoentities can be added in situ (biosynthesized)

and ex situ (at post-production level) to the BC tridimensional nanofiber network film in order to design

nanopapers with tailored properties.

In this work, different strategies for the fabrication of multifunctional nanopapers were employed via ex

situ method, by inmersion of BC tridimensional nanofiber network papers into different nanoparticle

solutions. On the one hand, sol-gel synthesized nanoparticles (titanium, vanadium and a mixture of both

oxides) and on the other hand superparamagnetic iron oxide nanoparticles (SPION), Fe2O3-PEO solution

were used. The morphology of designed nanopapers was investigated by atomic force microscopy (AFM)

and scanning electron microscopy (SEM). In order to characterized obtained BC based nanopapers from

the point of view of future applications different techniques were employed. The conductive properties

were studied at nanoscale using electric force microscopy (EFM), tunneling atomic force microscopy

(TUNA) and at macroscale using Keithley semiconductor analyser. Magnetic properties were measured by

means of magnetic force microscopy (MFM).

References

(1) Barud, H. B., Tercjak, A., Gutierrez, J., Viali, W. R., Nunes, E. S., Ribeiro, S. J. L., Jafellici, M., Nalin, M., Marques, R. F. C. (2015). Biocellulose-based flexible magnetic paper. J. Appl. Phys. 117:17B734/1-17B734/4 (2) Gutierrez, J., Tercjak, A., Argal, I., Retegi, A., Mondragon I. (2012). Conductive properties of TiO2/bacterial cellulose hybrid fibres. J. Colloid Interf. Sci. 377:88-93


3-4th October, 2019


(3) Gutierrez, J., Fernandes, S. M. C., Mondragon, I., Tercjak, A. (2013). Multifunctional hybrid nanopapers based on bacterial cellulose and sol-gel synthesized titanium/vanadium oxide nanoparticles. Cellulose 20:1301-1311. (4) Tercjak, A., Gutierrez, J., Barud, H. S., Domeneguetti, R. R., Ribeiro, S. J. L. (2015). Nano- and macroscale structural and mechanical properties of in situ synthesized bacterial cellulose/PEO‑b‑PPO‑b‑PEO biocomposites ACS Appl. Mater. Inter. 7, 4142-4150. (5) Gutierrez, J., Fernandes, S. M. C., Mondragon, I., Tercjak, A. (2012). Conductive photoswitchable vanadium oxide nanopaper based on bacterial cellulose. ChemSusChem 5:2323-2326. (6) Tercjak, A., Gutierrez, J., Barud, H. S., Ribeiro, S. J. L. (2016) Switchable photoluminescence liquid crystal coated bacterial cellulose films with conductive response. Carbohydrate Polymers 143:188-197


3-4th October, 2019


P28: Synthesis of hydrophobic bacterial cellulose nanocrystals for the retention of oils

Pezzin, A.P.T.*, Giacomassi, W.N.B., Garcia, M.C.F., Meier, M.M., Apati, G.P., Schneider, A.L.S. Paulo Malschitzki Street, 10. Universidade da Região de Joinville - UNIVILLE Joinville. [email protected] *

Abstract

Because of the growth of offshore oil production and transportation, the probability of oil spill accidents

has dramatically increased over the past several decades. The removal of oil by sorbent materials is

efficient, economical, and ecologically friendly, as the pollutant can be properly discarded, and no

secondary pollution is created. Much effort has been dedicated to the development of sustainable and

inexpensive sorbent materials based on natural fibers, which combine attractive properties such as

renewability, biodegradability, and environmental friendliness [1]. In comparison with vegetable

cellulose, bacterial cellulose (BC) possesses outstanding merits, such as high crystallinity and purity with

the absence of lignin or hemicelluloses, low density, high modulus and tensile strength, excellent water

holding capacity and biodegradabibilty [2]. The main drawback stems from the strong hydrophilic

character of the BC chain, which restricts the oil / water selectivity of the adsorbent [1]. In this context,

the present work reports the synthesis of hydrophobic nanocrystals of bacterial cellulose by an efficient

process of silanization in water, which can be used as components of filters for the retention of oils,

presenting an alternative of prevention/treatment of contaminated effluents. Bacterial cellulose (BC) was

synthesized by the Komagataeibacter hansenii, which is a species capable of producing BC on an industrial

scale. The nanocrystals of bacterial cellulose (NCBC) were isolated from their matrices by acid hydrolysis

with 64% sulfuric acid and functionalized in an aqueous medium in the presence of methyltriethoxysilane

(MTES). The suspensions were freeze dried to preserve the web-like structure of the BC cellulose in the

dry state. X-ray diffraction (XRD) analyzes evidenced the increase in the crystallinity of the materials after

the acid hydrolysis, confirming the decrease of the amorphous regions of the cellulose chains. Fourier

transform infrared spectroscopy (FTIR) analysis showed the presence of silicon in the functionalized

samples, due to the presence of silicon bands and O-Si-CH3 type bonds. Thermogravimetric analysis (TGA)

showed that the thermal stability of the silanized nanocrystals was close. Functionalized nanocrystals

combined hydrophobic and oleophilic properties by retaining a drop of xylene and repulsing a drop of

water made simultaneously on the surface of the samples, indicating that both materials are an

alternative for the removal of oils from water surfaces.

References

(1) Zhang, Z., Sebe, C., Rentsch, D., Zimmermann, T., Tingaut, P. (2014). Ultralightweight and flexible silylated nanocellulose sponges for the selective removal of oil from water. Chemistry of Materials, 26:2659-2668. (2) Yan, H., Chen, X., Song, H., Li, J., Feng, Y., Shi, Z., Wang, X., Li, Q. (2017). Synthesis of bacterial cellulose and bacterial cellulose nanocrystals for their applications in the stabilization of olive oil pickering emulsion. Food Hydrocolloids, 72:127-135.



3-4th October, 2019


P29: Life cycle assessment of bacterial cellulose production in soybean molasses culture

medium

Renata de Araújo e Silva1, Ednaldo Benício de Sá Filho2, Ana Iraidy Santa Brigida3, Morsyleide de Freitas Rosa3, Maria Cléa Brito de Figueirêdo*3

1 State University of Ceará - Postgraduate Program in Natural Sciences 2 Cearense Foundation for Scientific and Technological Development Support - FUNCAP

3 Embrapa Agroindústria Tropical [email protected]

Abstract

Bacterial cellulose (BC) is a promising biopolymer for applications in several areas. Still, BC production cost

is very high, mainly because of the used culture medium Hestrin and Schramm (HS). In recent years,

alternative culture mediums, such as soybean molasses, have been proposed to produce BC, but without

evaluating the resulting environmental impacts (1). This work assessed the potential environmental

impacts of producing 1g of BC by Komagataeibacter xylinus ATCC 53582 and a strain isolated from a

vinegar industry, applying three substrates in a static culture, at the laboratory scale: i) HS; ii) acid-

hydrolyzed soybean molasses (HSM) and iii) diluted soybean molasses (DSM). Life Cycle Assessment (LCA)

was applied, according to ISO 14044 standards (2). The scope was "cradle-to-gate" type, and considered

the following impact categories, by the ILCD 2011 method (3): climate change, acidification, water

resource depletion, marine eutrophication, freshwater eutrophication, freshwater eco-toxicity, human

toxicity, cancer, and non-cancer effects. The production stages evaluated in these three processes were:

microorganism maintenance, pre-activation, activation, fermentative substrate preparation, static

cultivation, purification, neutralization, and drying of BC pellicles. Results showed that DSM presented

better performance than HSM and HS for most of the impact categories. HS presented worse

environmental impacts than HSM and DSM, for most categories. In DSM, the fermentative substrate

preparation stage led to major impacts because of emissions from soybean cultivation for soybean

molasses production.

The applications of organic and inorganic fertilizers as well as the transformation of land into arable areas

are the main aspects that contribute to the environmental impacts of soybean cultivation. These impacts

could be reduced by the use of better management practices such as No-till farming, Integrated Pest

Management, Nutrient management and soybean nodulation with rhizobia bacteria. In conclusion, BC

production in DSM can cause less environmental impacts than the other substrates, being indicated

efforts in process optimization, aiming technical-economic feasibility and scale up.

References

(1) Jozala, A. F., Pértile, R. A. N., Santos, C. A., Santos-Ebinuma, V. C., Seckler, M. M., Gama, F. M, Pessoa, A. (2015). Bacterial Cellulose Production by Gluconacetobacter xylinus by Employing Alternative Culture Media. Applied Microbiology and Biotechnology. 99(3):1181–1190



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(2) International Organization for Standardization (2009). ISO 14044: environmental management - Life cycle assessment - Requirements and guidelines. Genebra: ISO: 2009b.

(3) European Commission, Joint Research Centre, Institute for Environment and Sustainability. Characterisation factors of the ILCD Recommended Life Cycle Impact Assessment methods. Database and Supporting Information. First edition. February 2012. EUR 25167. Luxembourg. Publications Office of the European Union; 2012.


3-4th October, 2019


P30: Influence of fermentation time and bacterial strains on properties of bacterial cellulose

produced by soybean molasses fermentation

Matheus de Oliveira Barros1, Natália Tavares de Almeida1, Jessica Silva de Almeida1, João Pedro Bessa de Souza1, Maria de Fátima Borges2, Morsyleide de Freitas Rosa2, Ana Iraidy Santa Brígida2*

1. Universidade Federal do Ceará 2. Embrapa Agroindústria Tropical [email protected]

Abstract

Bacterial cellulose (BC) is a biopolymer secreted by various strains of microorganisms and composed by

naturally occurring nanoscale fibers with high crystallinity and purity. Due to its characteristics, BC has

been used in several different applications, such as food packaging, wound dressing, artificial blood

vessels among others[1]. Those applications require BC with different properties that can be manipulated

through the bacterial strain, fermentation time and culture medium used. However, its large-scale

production is limited by, mainly, the high cost of the fermentation process due to the price of the synthetic

culture medium. Many agroindustrial sources rich in sugars have been studied as culture media for BC

production aiming for cost reduction[2]. In this context, this study aimed to evaluate the bacterial

cellulose produced by soybean molasses fermentation analysing the effect of culture time and strains on

BC properties. A soybean molasses based culture medium (SMH75) was obtained by a hydrolysis process

of a 75 g.L-1 soybean molasses solution using H2SO4 0,1M at 90°C for 10 minutes followed by neutralization

with NaOH 30% (w/v) solution, vacuum filtration and supplementation with commercial grade ethanol

(2%, v/v). The BC was produced by using Komagateibacter (K.) xylinus ATCC 53582 and K. hansenii ATCC

23769 in static culture at 30ºC with fermentation time varying from 3 to 10 days and using two different

media: SMH75 and Hestrin & Schramm (HS, [2]). The BC pellicles were purified using water at 100°C for

1h twice followed by immersion in NaOH 0,5M at 80°C repeatedly until all the culture medium to be

removed, and neutralization by successive washes with tap water. Some of the BC produced were dried

at 50°C and cut for Dynamic Mechanical Analysis (DMA) to evaluate its mechanical properties and the rest

of the BC was freeze-dried after which it was characterized by Thermogravimetric Analysis (TGA), X-Ray

Diffraction (XRD) and Scanning Electron Microscopy (SEM). In all treatments BC production increased

alongside with the increase of fermentation time. TGA showed that all BC pellicles produced were

thermally stable showing degradation events typical of BC. The crystallinity indexes obtained by XRD

analysis showed that it was influenced by the culture medium, strain and fermentation time. In closing,

the SEM images showed an increase in the fibrils width as the fermentation time increased. Those

different properties in the BC produced can be the defining factor for those BC specific application.

References

[1] A. Margarita, A. Gallegos, H. Carrera, R. Parra, Bacterial Cellulose : A Sustainable Source to Develop Value-Added Products – A Review, 11 (2016) 5641–5655.

[2] H.L.S. Lima, E.S. Nascimento, F.K. Andrade, A.I.S. Brígida, M.F. Borges, A.R. Cassales, C.R. Muniz, M.D.S.M. Souza Filho, J.P.S. Morais, M.D.F. Rosa, Bacterial cellulose production by



3-4th October, 2019


Komagataeibacter hansenii ATCC 23769 using sisal juice - An agroindustry waste, Brazilian J. Chem. Eng. 34 (2017) 671–680. doi:10.1590/0104-6632.20170343s20150514.


3-4th October, 2019


P31: Microwave-assisted periodate oxidation and characterization of bacterial cellulose

Luisa Macedo Vasconcelos1*, Niédja Fittipaldi Vasconcelos2, Fábia Karine Andrade1, Rodrigo Silveira Vieira1, Diego Lomonaco Vasconcelos de Oliveira², Maria de Fátima Borges3, Morsyleide de Freitas Rosa3

1 Department of Chemical Engineering, Federal University of Ceará, Fortaleza, Ceará, Brazil 2 Department of Chemistry, Federal University of Ceará, Fortaleza, Ceará, Brazil

3 Embrapa Agroindustria Tropical, Fortaleza, Ceará, Brazil * [email protected]

Abstract

Bacterial cellulose (BC) is a bio‐compatible material suitable for various biomedical applications from

bandages for cutaneous wound‐healing to scaffolds for tissue growth. BC is not, however, bioactive or

degraded in vivo, features that limit its practical applications. Studies on BC are mainly focused on

conferring such new functionalities to the material. Among these studies, periodate oxidation of BC

introduces to its structure aldehyde groups capable of reacting with amine groups present in biomolecules,

thus promoting their immobilization along with augmenting general reactivity. Moreover, oxidized BC is

considerably more biodegradable in simulated physiological conditions1 than native BC. In this context,

microwave‐assisted heating presents advantages over conventional heating in the field of organic

synthesis by reducing reaction times and increasing general productivity and has been successfully applied

to the chemical modification of cellulose2, though has yet to be established towards oxidation. In this work,

BC membrane was obtained by static fermentation of K. hansenii (ATCC 53582) in Hestrin‐Schramm

medium. Then, the BC was purified with K2CO3 solution at 80 oC for 1h and oxidized with NaIO4 under

controlled conditions at 90oC for 30 minutes using microwave irradiation at a maximum of 300W. The same

reaction conditions were reproduced with conventional heating in a water bath. The products were

characterized by a variety of techniques including SEM, FTIR, TGA, DSC, XRD and by quantifying aldehyde

content. The main focus of this work was to study and compare the structural and physical‐chemical

properties of BC from both processes. Preliminary results indicate that microwave‐assisted oxidation of

BC produces similar oxidation profiles in a shorter reaction time, as well as similar chemical and

morphological modifications in cellulose structure when compared to conventional heating methods.

Therefore, the microwave‐assisted periodate oxidation is an interesting alternative method for BC

chemical modifications.

References

(1) J. Li, Y. Wan, L. Li, H. Liang and J. Wang, Materials Science and Engineering C 29, 1635–42 (2009) (2) D.M. dos Santos, A.L. Bukzem, D.P.R. Ascheri, R. Signini, G.L.B. de Aquino, Carbohydrate Polymers 131, 125–133, (2015).



3-4th October, 2019


P32: Bioactivity and biocompatibility of hybrid systems based on bacterial

cellulose/strontium apatite

Luz, E. P. C. G.1,Chaves, P. H. S.3, Vieira, L. A. P.2, Andrade, F. K.1,3, Borges, M. F.3, Vieira, R. S.1, Silva, I. I. C.2, Rosa, M. F.3*

1Department of Chemical Engineering/ Federal University of Ceará (UFC), Brazil 2Department of Biotechnology/ Federal University of Ceará (UFC/Sobral), Brazil

3Embrapa Agroindústria Tropical – CNPAT, Brazil *[email protected]

Abstract

Biomaterials are devices that come into contact with biological systems to repair or replace a damage

tissue or organ. To perform the proposed function, they must be biocompatible and present some

properties according to their application. The present work aimed to evaluate the in vitro bioactivity and

in vivo biocompatibility of hybrid systems based on bacterial cellulose/strontium apatite for future

applications as guided bone regeneration (GBR). BC is a polymer produced by fermentation and presents

several interesting properties, as biocompatibility and high mechanical strength. In turn, Sr has chemical

similarity with calcium stimulating bone formation and inhibiting bone resorption. The differential of this

release system is in the form of Sr delivery, presenting a slow and continuous desorption profile due to

the strong interaction that strontium apatite does with bacterial cellulose (BC). BC membranes were

produced after cultivation of Komagataeibacter xylinus (ATCC 53582) in Hestrin-Schramm medium at 30

°C for 5 days, and then purified for total removal of culture medium and bacterial microorganism. To

obtain the hybrid materials, the non-oxidized or periodate oxidized BC membranes were mineralized by

alternate immersion cycles in strontium chloride and sodium bibasic phosphate solutions. The produced

hybrid materials were characterized by the swelling degree, FTIR, TGA, SEM and XRD analysis. The in vitro

bioactivity of the hybrid systems was evaluated after immersion in simulated body fluid (SBF) during 90

days by scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) analysis. The in vivo

biocompatibility was analysed by histological evaluation of the subcutaneous hybrid grafts after 1, 3 and

9 weeks of implantation in mice. The results showed that all materials were bioactive, since after

immersion in SBF the precipitation of calcium, magnesium and potassium minerals occurred, which are

important elements in bone repair. As for biocompatibility study, all samples showed to be biocompatible

with low inflammatory response, especially in the oxidized samples, where the degradation of the

biomaterial was more intense, facilitating the action of both granulation and loose tissues and

accelerating the tissue regeneration.



3-4th October, 2019


References

Foresti, M. L., Vázquez, A., Boury, B. (2017). Applications of bacterial cellulose as precursor of carbon and composites with metal oxide, metal sulphide and metal nanoparticles: A review of recent advances. Carbohydrate Polymers. 157:447-467. Yang, M. et al. (2016) Biomimetic Design of Oxidized bacterial cellulose-gelatin-hydroxyapatite nanocomposites. Journal of Bionic Engineering. 13:631-640.


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P33: BNC-based scaffolds for tissue engineering: preparation and characterization

Izabela Cielecka1, Marcin Szustak1, Edyta Gendaszewska-Darmach1, Halina Kalinowska1, Małgorzata Ryngałło1, Waldemar Maniukiewicz2, Stanisław Bielecki1

1Institute of Technical Biochemistry, Lodz University of Technology, Poland 2Institute of General and Ecological Chemistry, Lodz University of Technology, Poland

Abstract

BNC exhibits many desirable and unique properties such as biocompatibility, high mechanical strength

and water holding capacity up to 100 times its dry mass as well as high crystallinity and porosity.

Biocompatibility of BNC-based products makes them suitable for biomedical applications, such as wound

dressings and scaffolds for tissue engineering. The interactions of the biomaterial with surrounding

mammalian cells depend on the proper distribution of functional groups in the three-dimensional space.

The native BNC-based scaffolds promote neither the excellent cell adhesion nor growth because cellulose

is biologically inactive, mainly due to its neutral charge (1). Therefore, there is a need to modulate the

intrinsic properties that may be particularly beneficial in the biomedical application.

In the presented research, the properties of bionanocellulose have been improved by introducing other

polysaccharides, such as κ-carrageenan (2), carboxymethyl cellulose and hydroxyethylcellulose into three

dimensional structure of bacterial cellulose. Additionally, BNC-based composites with CMC and HEC were

modified ex situ with glycerol to increase the absorption ability of materials (3). The presented

combination of in situ and ex situ modifications of bacterial cellulose synthesized by K. xylinus E25 under

stationary conditions, enabled production of nanocomposites that may replace traditional BNC dressings.

The glycerol-plasticized BNC, BNC-CMC and BNC-HEC membranes were flexible after drying, and absorbed

large amounts of artificial exudate and water after rehydration. On the other hand, bionanocellulose/κ-

carrageenan composites, meet the specific tissue engineering requirements, including the high tensile

and compression strength, water holding capacity, and water retention ratio as well as suitable swelling

properties. As the key properties of these composites may be easily modified during their fabrication, the

established procedure may lead to production of customized scaffolds.

This work presents the methods for in situ preparation of stable composites based on bionanocelulose

and discusses the most important parameters of these biomaterials in the context of medical

applications. Moreover, properties such as structure of fibres, crystallinity, mechanical strength, water

holding capacity and free swell absorption capacity were assessed.

References

(1) Barud, H.G.O., Silva, R.R., Barud, H.S., Tercjak, A., Gutierrez, J., Lustri, W.R., Ribeiro, S.J.L. (2016). A multipurpose natural and renewable polymer in medical applications: Bacterial cellulose. Carbohydrates Polymers. 153:406-420 (2) Cielecka I., Szustak M., Gendaszewska-Darmach E., Kalinowska H., Ryngajłło M., Maniukiewicz W., Bielecki S. (2018). Novel bionanocellulose/к-carrageenan composites for tissue engineering. Applied sciences 8(8): 1352


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(3) 3. Glycerol-plasticized bacterial nanocellulose-based composites with enhanced flexibility and liquid sorption capacity, I. Cielecka, M. Szustak, H. Kalinowska, E. Gendaszewska-Darmach, M. Ryngajłło, W. Maniukiewicz, S. Bielecki, in press in Cellulose; doi:10.1007/s10570-019-02501-1


3-4th October, 2019


P34: Bioactive dressing based on bacterial cellulose and papain for wound debridement

Vasconcelos, N. F. 1*; Andrade, F. K. 2; Vieira, R. S. 2; Mantovani, D. 3; Vieira, L. de A. P. 4; Borges, M. de F. 4; Rosa, M. F.4

1 Department of Chemistry, Federal University of Ceará (UFC), Fortaleza, Ceará, Brazil

2 Department of Chemical Engineering, Federal University of Ceará (UFC), Fortaleza, Ceará, Brazil 3 Department of Min-Met-Materials Engineering, Laval University; Quebec City, Canada

4 Embrapa Agroindústria Tropical, Fortaleza, Ceará, Brazil * [email protected]

Abstract

Dressings are an important segment of the medical and pharmaceutical market for the treatment of

surgical or non-surgical wounds. Given the recent advances in the area of biomaterials, the use of bacterial

cellulose (BC), specially to produce high-quality dressings has outstanding due to its unique properties,

such as purity, hydrophilicity, porosity, mechanical resistance, flexibility and biocompatibility (1). However,

traditional BC dressings present only a passive character in wound healing processes. In order to produce

a bioactive dressing, BC structure can be chemical modified (by oxidation with NaIO4) to allow the

immobilization of enzymes, such as papain known to promote the wound debridement (removal of the

necrotic tissue) and accelerate the wound healing (2). Therefore, the aim of this work was to evaluate the

properties of BC bioactive dressings obtained through the covalent immobilization of papain on BC

membranes. The results showed that the BC bioactive dressings were non-cytotoxic to human fibroblasts

and keratinocytes (cell viability of 89.8 % and 86.5 %, respectively) and non- haemolytic when in contact

with blood. Moreover, the moisture vapor transmission rate for the curative (2678 ± 181 g/m2. 24h) was

similar to commercial curative highly permeable (300-3000 g/m2. 24h/Opsite Post-op® and Hydrocoll®).

The fluid absorption capacity was above 100% of its weight, being considered a high absorbent material.

Microbiologic assays showed that the material presented bacteriostatic activity against gram-negative

bacteria. The in vitro drug release was conducted using Franz diffusion cells and showed a liberation

profile of papain around 78.2% in 24h. These properties indicate that this BC bioactive dressing could acts

in the preventing infections, maintaining ideal moisture (absorbing excess exudate), promoting gas

exchange, as well as acting by removing necrotic tissue, especially in chronic wounds, facilitating the

granulation process, the contraction of the wound bed and accelerating tissue reconstitution.

References

(1) Shah, N., Ul-Islam, M., Khattak, W. A., Park, J. K. (2013). Overview of bacterial cellulose composites: a multipurpose advanced material. Carbohydrate Polymers. 98(2):1585-1598

(2) Dutra, J. A. P., Carvalho, S. G., Zampirolli, A. C. D., Daltoé, R. D., Teixeira, R. M., Careta, F. P., Villanova, J. C. O. (2017). Papain wound dressings obtained from poly (vinyl alcohol)/calcium alginate blends as new pharmaceutical dosage form: Preparation and preliminary evaluation. European Journal of Pharmaceutics and Biopharmaceutics. 113:11-23



3-4th October, 2019


P35: Towards Texture Control of Bacterial Cellulose

Sabio, L.*, González, A., Ramírez-Rodríguez, G.B., Delgado-López, J.M. & Domínguez-Vera, J.M Department of Inorganic Chemistry, Facultad de Ciencias, Universidad de Granada.

Avda. Fuente Nueva s/n 18071 Granada, Spain. *[email protected] ; [email protected]

Abstract

Crystallinity of bacterial cellulose (bacteria-free) has been traditionally related to culture conditions,

treatments for eliminating bacteria, and drying methods (e.g. lyophilization) (1,2). However, crystallinity

of cellulose biofilm, which contains the native bacteria, has been little explored so far. It has not been

established if the treatment (i.e. ethanol immersion, water boiling, NaOH washes at 90ºC, and

neutralization) can tune the texture of the material (3).

The texture dramatically affects the properties of a material (mechanical properties, absorption,

reactivity…). In the case of cellulose, controlled degradation is essential since it serves as a scaffold in

tissue engineering. Therefore, tuning the texture of bacterial cellulose is key to improve its biomedical

applications.

We have produced pellicles from Acetobacter xylinum under static and dynamic conditions and further

explored the impact of different treatments on the texture of cellulose. The chemical composition and

structure of cellulose pellicles have been in-depth characterized by spectroscopy (FTIR and Raman), X-ray

diffraction (XRD), and scanning electron microscopy (SEM) both in the presence and absence of bacteria.

Under specific conditions, we have found that bacterial cellulose becomes textured. SEM images show

highly ordered cellulose fibers in the XY plane and XRD patterns confirm such preferential orientation

along the fibril axis (b-axis).

References

(1) Keshk, S. (2014). Bacterial Cellulose production and its Industrial Applications. Journal of Bioprocessing and Biotechniques. 4:2 (2) Moon, R. J., Martini, A., Nairn, J., Simonsen, J., & Youngblood, J. (2011). Cellulose nanomaterials review: structure, properties and nanocomposites. Chemical Society Reviews, 40(7), 3941-3994. (3) Fang, L. & Catchmark, J.M. Cellulose (2014) 21: 3965.




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P36: Spray-dried redispersible bacterial nanocellulose microspheres

M. Rosas1,2, P. Cerrutti3,4, V. Bucalá1,2, V. Ramírez Rigo1,2, M. L. Foresti2,3*

1 Department of Chemical Engineering, PLAPIQUI, Universidad Nacional del Sur, Bahía Blanca, Argentina 2 National Scientific and Technical Research Council (CONICET), Argentina. 3 Institute of Technology in Polymers and Nanotechnology (ITPN), Engineering Faculty, Universidad de Buenos Aires, Buenos Aires, Argentina. Tel/fax: 0054 11 52850285. 4 Department of Chemical Engineering, Engineering Faculty, Universidad de Buenos Aires.

*[email protected]

Abstract

The economic and technical disadvantages associated with the wet storage and transport of

nanocelluloses trigger the development of simple methods able to produce nano-scaled cellulose

products with lower water content (1). However, the irreversible aggregation of cellulose nanofibrils

during dehydration (hornification) still poses a problem for the production of dried nanofibrillated

celluloses. In this context, different alternatives have been proposed to produce redispersible

nanofibrillated cellulose (mainly from vegetal origin), including chemical modification and the

incorporation of additives during drying that can hinder hydrogen bond formation among cellulose

nanofibers. With this aim, additives such as sodium chloride (2), carboxymethyl cellulose (3),

cetyltrimethylammonium bromide (4), poly (vinyl-alcohol) (1), and maltodextrin (5), have been used.

In the current contribution, bacterial nanocellulose (BNC) microspheres were produced by spray drying of

a BNC aqueous suspension using mannitol as drying adjuvant. The feed properties and operating

conditions (i.e. BNC suspension solid content, mannitol:BNC ratio, air inlet temperature, atomization air

flow rate, drying air flow rate and liquid feed flow rate), were all adjusted to obtain a powdered product

with a yield of 78 wt%. Scanning electron microscopy images of the recovered powder evidenced the

obtention of sphere-shaped particles with diameters not higher than 10 m. Particle size distribution

(PSD) results obtained by laser diffraction further indicated a unimodal and narrow PSD as the span value

obtained was 1,03. The volume mean diameter of the particles was 6,36 ± 0,11 μm. Besides, 10, 50 and

90 wt% of the particles exhibited diameters below 3,58 ± 0,03 μm, 5,91 ± 0,08 μm and 9,67 ± 0,21 μm,

respectively (results from three replicates).

The spray-dried BNC microspheres were evaluated in terms of their redispersibility in water upon

magnetic stirring and ultrasonication. Results indicated that whereas in magnetically stirred aqueous

suspensions BNC spheres showed some deformation but still retained their integrity, mild ultrasonication

during short periods of time (5-15 min) caused BNC microspheres to break down into loose BNC

nanofibers.

The BNC microspheres obtained using mannitol, a naturally occurring sugar alcohol widely used in food

and medical applications, have a vast potential for redispersible BNC production.



3-4th October, 2019


References

(1) Velásquez-Cock, J., Gómez, BE., Posada, P., Serpa, A., Gómez Hoyos, C., Gañán, P., Zuluaga, R. (2018) Poly (vinyl alcohol) as a capping agent in oven dried cellulose nanofibrils. Carbohydrate Polymers 179:118-125.

(2) Missoum, K., Bras, J., Belgacem, M. N. (2012). Water redispersible dried nanofibrillated cellulose by adding sodium chloride. Biomacromolecules. 13:4118–4125.

(3) Butchosa, N., Zhou, Q. (2014). Water redispersible cellulose nanofibrils adsorbed with carboxymethyl cellulose. Cellulose. 21:4349–4358.

(4) Fairman, E. (2014). Avoiding aggregation during drying and rehydration of nanocellulose. University of Maine.

(5) Velásquez-Cock, J., Gañán, P., Gómez Hoyos, C., Posada, P., Castro, C., Dufresne, A., Zuluaga, R. (2018). Improved redispersibility of cellulose nanofibrils in water using maltodextrin as a green, easily removable and non-toxic additive. Food Hydrocolloids. 79:30-39.


3-4th October, 2019


P37: Optimization of bacterial nanocellulose fermentation using lignocellulosic residues and

development of novel BNC-starch composites

Soares da Silva, F.A.G.1,*; Dourado, F.1,2; Ferreira, E.1; Poças, F.3; Gama, M.1 1CEB - Centre of Biological Engineering, University of Minho, Gualtar, Braga, Portugal; 2Satisfibre, S.A. Portugal 3CBQF - Centre of Biotechnology and Fine Chemistry – Associated Laboratory, Faculty of Biotechnology, Catholic University of Portugal, Rua Arquiteto Lobão Vital 172, 4200-374 Porto, Portugal *[email protected]

Abstract

In papermaking industry, significant fraction of fibres that cannot be re-utilized are wasted, which raise

economic and environmental concerns[1]. On the other hand, development of renewable polymeric

materials became a priority for the sustainability of several industries. Bacterial nanocellulose (BNC), a

biopolymer extruded by Gluconacetobacter xylinus as a 3D nanofibrillar network, provide interesting

properties as high porosity, high water retention, biocompatibility, non-toxicity and biodegradability [2].

These properties have sustained promising applications in the biomedical field, papermaking, composites

and foods. However, large-scale BNC production remains a challenge, due to ineffective fermentation

systems and high operating costs [2-3]. Therefore, the production of BNC through lignocellulosic residues

has been studied. Recycled-paper-sludge (RPS) composed of small fibres with 40% of carbohydrates were

hydrolysed and used as a carbon source in culture media formulation. Then, a Response Surface

Methodology (RSM) optimization with RPS was assessed in order to maximize BNC production, through

static fermentation with K. hansenii ATCC 53582. Overall, the results suggest that RPS had potential to be

an alternative carbon source for BNC production with a maximum BNC yield of 5 g/L.

BNC produced as described above was then used for the development of novel green thermoplastic

nanocomposites, combined with starch. When mixed with water and glycerol (with heat and shear), starch

undergoes spontaneous destructuring, forming thermoplastic starch (TPS). In particular to food packaging

applications, BNC has remained unexploited in spite of being considered to have enormous potential [4-

5]. In this work, two approaches for composite production were assessed. Firstly, BNC 3D membrane was

filled with biodegradable bio-based thermoplastic starch (TPS), where the production was achieved in a

two-step process: impregnation of TPS in the BNC membrane, followed by drying. Different thicknesses

of BNC membrane were studied (1-5 mm) as two impregnation time (24h;72h). The second approach

consisted on the use of glycerol-TPS as matrix, where different concentrations (0.05 -0.5% w/v) of

cellulose (Plant (PC) and BNC) was added. TPS-BNC and TPS-PC films were prepared by solution casting

method. All nanocomposites manufactured were then characterized in terms of mechanical properties,

morphology and permeability to water vapor (WVT). Overall, enhanced mechanical and barrier properties

were obtained with BNC-TPS composites. In comparison to TPS-BNC and TPS-PC films, higher young

modulus and tensile strength was obtained with the BNC-TPS composites. Being longer and thinner, the

BNC fibres offer greater mechanical resistance than the ordinary TPS-cellulose composites.



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References: [1] Gomes, D., L. Domingues and M. Gama (2016). "Valorizing recycled paper sludge by a bioethanol production process with cellulase recycling." Bioresour. Technol. 216: 637-644. [2] Campano, C., Balea, A., Blanco, A., Negro, C., 2016. Enhancement of the fermentation process and properties of bacterial cellulose: a review. Cellulose. 23(1): 57-91. [3] Rodrigues, A. C.; Fontão, A. I.; Coelho, A., Leal, M., da Silva, F. A. S., Wan, Y., Dourado, F., Gama, M., (2018). Response surface statistical optimization of bacterial nanocellulose fermentation in static culture using a low-cost medium. New Biotechnol., 49, 19-27. [4] Pelissari, F. M., Ferreira, D. C., Louzada, L. B., dos Santos, F., Corrêa, A. C., Moreira, F. K. V., & Mattoso, L. H. (2019). Starch-Based Edible Films and Coatings: An Eco-friendly Alternative for Food Packaging. In Starches for Food Application (pp. 359-420). Academic Press. [5] Bharimalla, A. K., Deshmukh, S. P., Vigneshwaran, N., Patil, P. G., & Prasad, V. (2017). Nanocellulose-polymer composites for applications in food packaging: Current status, future prospects and challenges. Polymer-Plastics Technology and Engineering, 56(8), 805-823.


3-4th October, 2019


P38: Synthesis and characterization of oxidized bacterial cellulose through electrochemical

methods: its biodegradability and potential as hemostatic material

E.C. Queirós1,2, S. Pinheiro1, J. E. Pereira3, J. Prada3, I. Pires3, P. Parpot1,2, M. Gama1 1CEB - Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal; 2CQUM - Centre of Chemistry, University of Minho, 4710-057 Braga Portugal; 3CECAV - Veterinary and Animal Research Centre, University of Trás-os-Montes and Alto Douro, Quinta de Prados 5000-801, Vila Real, Portugal

Abstract

Bacterial cellulose (BC) is a biocompatible material with high purity, high crystallinity, high degree of

polymerization and high-water content [1,2]. It could be applied in several fields, being the biomedical

field the most relevant to this work where biodegradability is a key requirement. BC may be chemically

modified through its hydroxyl groups, e.g., by oxidation, becoming reabsorbable and acquiring new

features, such as hemostatic behaviour. Oxidation of BC membranes was achieved through electrolysis

using tetramethylpiperidine-1-oxyl (TEMPO) radical. TEMPO is able to perform selective oxidation of the

primary hydroxyl groups, meaning that only C6 is oxidized into carboxyl groups [3].

BC membranes were characterized by FT-IR, SEM, XRD and 13C-NMR. The degree of oxidation was

evaluated by titration of the carboxyl groups and the hemostatic behaviour was investigated through

whole blood coagulation tests. Both in vitro and in vivo biodegradability of oxidized membranes was

evaluated. In vitro biodegradability was assessed in ultra-pure water after 3, 7, 14 and 63 days of

incubation while in vivo biodegradability was studied in a rat model, for 3, 14 and 56 days.

FT-IR spectra showed an increase on the absorption band around 1628 cm-1 attributed to the carboxylic

acid vibration, as compared to non-oxidized membranes, revealing the formation of carboxylic acid groups

[4]. SEM images revealed that the morphology of the membranes was not changed by the oxidation [5].

XRD patterns for all the oxidized samples were very similar to non-oxidized ones, suggesting that the

crystal structure was preserved [6]. 13C-RMN results showed that the signal around 62 ppm

corresponding to superficial C6 primary hydroxyl group decreased after the oxidation, while the peak

around 174.6 ppm assigned to carboxylate groups appeared after oxidation, confirming the selective

oxidation of C6 [7]. In vitro results showed that almost no degradation occurred on non-oxidized

membranes demonstrating the relevance of the oxidation on the improvement on BC biodegradability.

The hemostatic behaviour of the membranes evaluated through the whole blood clotting times assay

demonstrated that, contrarily to non-oxidized membranes, the oxidized ones exhibited hemostatic

activity [8]. In vivo biodegradability and biocompatibility of oxidized membranes was evaluated.

Membranes were implanted subcutaneously and the inflammatory response was studied. The obtained

results showed that there were no microscopic evidences of inflammation and even after 56 days of

implantation, the oxidized membranes did not degrade completely. Nevertheless, oxidized membranes

revealed good biocompatibility [9].

References [1] Huang Y, Zhu C, Yang J, Nie Y, Chen C, Sun D. Cellulose. 2014;21(1):1–30.


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[2] Reiniati I, Hrymak AN, Margaritis A. Crit Rev Biotechnol. 2017;37(4):510–24. [3] Parpot P, Servat K, Bettencourt AP, Huser H, Kokoh KB. Cellulose. 2010;17(4):815–24. [4] da Silva Perez D, Montanari S, Vignon MR. Biomacromolecules. 2003;4(5):1417–25. [5] Lu C, Chen SY, Zheng Y, Zheng WL, Xiang C, Wang HP. Nano-Scale Materials, Materials Processing and Genomic Engineering. Trans Tech Publications; 2014. p. 90–4. (Materials Science Forum; vol. 789). [6] Shi X, Cui Q, Zheng Y, Peng S, Wang G, Xie Y. RSC Adv. 2014;4:60749-60756. [7] Lai C, Zhang S, Chen X, Sheng L. Cellulose. 2014;21(4):2757–72. [8] Leitão A, Gupta S, Silva J, Reviakine I, Gama M. Colloids and Surfaces B: Biointerfaces. 2013; 111: 493-502 [9] Helenius G, Backdahl H, Bodin A, Nannmark U, Gatenholm P, Risberg B. J Biomed Mater Res A. 2006; 76(2):431-8


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AUTHORS LIST


3-4th October, 2019


- A – E -

Adam Junka

Agnieszka Tercjak

Aloña Retegui Miner

Amritpal Singh

Ana Cristina da Costa Rodrigues

Ana M. Hernandez Arriaga

Anett Kondor

Anna Laromaine Sagué

Anna Roig

Berit Karl

Carla Vilela

Carlos Pascoal Neto

Dagmar Fischer

Dana Kralisch

Daniela Filipa da Silva Fonseca

Daniela Martins

Elvira Maria Correia Fortunato

Eugénia Cristina Queirós Teixeira

- F - J -

Fábia Karine Andrade

Falk Liebner

Feng Hong

Fernando Dourado

Francisco de Almeida Garrett Soares da Silva

Gary Cass

Haibin Yuan

Haiyong Ao

Henriette Monteiro Cordeiro de Azeredo

Hernane da Silva Barud

Hiroyuki Kono

Inder Saxena

Irene Anton-Sales

Ives Bernardelli de Mattos

Jie Wang

Joaquin Caro-Astorga

- K – O -

Kamil Palmowski

Karol Fijalkowski

Karolina Ludwicka

Katarzyna Kubiak

Kenji Tajima

Koon-Yang Lee

Laura Sabio

Leire Urbina Moreno

Liu Jinzhi

M.Auxiliadora Prieto Jiménez

Małgorzata Ryngajłło

Marcia Margarete Meier

Maria Laura Foresti

Marzena Jedrzejczak-Krzepkowska

Mengxia Peng

Morsyleide de Freitas Rosa

Náyra de Oliveira Frederico Pinto

Orlando Rojas

- P – T -

Paulina Jacek

Piotr Siondalski

Quanchao Zhang

Ryoko Maruyama (Ryota Kose)


3-4th October, 2019


Sara Isabel Laiginha Silvestre

Simone Bottan (+1: Francesco Robotti)

Soledad Roig (Anna Roig)

Stanisław Bielecki

Tom Ellis

- U – Z -

Uwe Beekmann (+1 dinner)

Verena Andree

Vivianne Candy-Goosens

Xie Jing (Quanchao)

Yang Shanshan (Quanchao)

Yizao Wan

Zhihuan Huang (Quanchao)

4th international symposium on bacterial … · joaquin caro-astorga, imperial college london, uk...

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