Engineering Algae to Make the ‘Wonder Material’ Nanocellulose for Biofuels and More

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NEW ORLEANS, April 7, 2013 — Genes from the family of bacteria that produce vinegar, Kombucha tea and nata de coco have become stars in a project — which scientists today said has reached an advanced stage — that would turn algae into solar-powered factories for producing the “wonder material” nanocellulose. Their report on advances in getting those genes to produce fully functional nanocellulose was part of the 245th National Meeting & Exposition of the American Chemical Society (ACS), the world’s largest scientific society, being held here this week.

“If we can complete the final steps, we will have accomplished one of the most important potential agricultural transformations ever,” said R. Malcolm Brown, Jr., Ph.D. “We will have plants that produce nanocellulose abundantly and inexpensively. It can become the raw material for sustainable production of biofuels and many other products. While producing nanocellulose, the algae will absorb carbon dioxide, the main greenhouse gas linked to global warming.”

Brown, who has pioneered research in the field for more than 40 years, spoke at the First International Symposium on Nanocellulose, part of the ACS meeting. Abstracts of the presentations appear below.

Cellulose is the most abundant organic polymer on Earth, a material, like plastics, consisting of molecules linked together into long chains. Cellulose makes up tree trunks and branches, corn stalks and cotton fibers, and it is the main component of paper and cardboard. People eat cellulose in "dietary fiber," the indigestible material in fruits and vegetables. Cows, horses and termites can digest the cellulose in grass, hay and wood.

Most cellulose consists of wood fibers and cell wall remains. Very few living organisms can actually synthesize and secrete cellulose in its native nanostructure form of microfibrils. At this level, nanometer-scale fibrils are very hydrophilic and look like jelly. A nanometer is one-millionth the thickness of a U.S. dime. Nevertheless, cellulose shares the unique properties of other nanometer-sized materials –– properties much different from large quantities of the same material. Nanocellulose-based materials can be stronger than steel and stiffer than Kevlar. Great strength, light weight and other advantages has fostered interest in using it in everything from lightweight armor and ballistic glass to wound dressings and scaffolds for growing replacement organs for transplantation.

In the 1800s, French scientist Louis Pasteur first discovered that vinegar-making bacteria make “a sort of moist skin, swollen, gelatinous and slippery” — a “skin” now known as bacterial nanocellulose. Nanocellulose made by bacteria has advantages, including ease of production and high purity that fostered the kind of scientific excitement reflected in the first international symposium on the topic, Brown pointed out.

Brown recalled that in 2001, a discovery by David Nobles, Ph.D., a member of the research team at the University of Texas at Austin, refocused their research on nanocellulose, but with a different microbe. Nobles established that several kinds of blue-green algae, which are mainly photosynthetic bacteria much like the vinegar-making bacteria in basic structure; however, these blue-green algae, or cyanobacteria, as they are called, can produce nanocellulose. One of the largest problems with cyanobacterial nanocellulose is that it is not made in abundant amounts in nature. If it could be scaled up, Brown describes this as "one of the most important discoveries in plant biology."

Since the 1970s, Brown and colleagues began focusing on Acetobacter xylinum (A. xylinum), a bacterium that secretes nanocellulose directly into the culture medium, and using it as an ideal model for future research. Other members of the Acetobacter family find commercial uses in producing vinegar and other products. In the 1980s and 1990s, Brown’s team sequenced the first nanocellulose genes from A. xylinum. They also pinpointed the genes involved in polymerizing nanocellulose (linking its molecules together into long chains) and in crystallizing (giving nanocellulose the final touches needed for it to remain stable and functional).

But Brown also recognized drawbacks in using A. xylinum or other bacteria engineered with those genes to make commercial amounts of nanocellulose. Bacteria, for instance, would need a high-purity broth of food and other nutrients to grow in the huge industrial fermentation tanks that make everything from vinegar and yogurt to insulin and other medicines.

Those drawbacks shifted their focus on engineering the A. xylinum nanocellulose genes into Nobles’ blue-green algae. Brown explained that algae have multiple advantages for producing nanocellulose. Cyanobacteria, for instance, make their own nutrients from sunlight and water, and remove carbon dioxide from the atmosphere while doing so. Cyanobacteria also have the potential to release nanocellulose into their surroundings, much like A. xylinum, making it easier to harvest.

In his report at the ACS meeting, Brown described how his team already has genetically engineered the cyanobacteria to produce one form of nanocellulose, the long-chain, or polymer, form of the material. And they are moving ahead with the next step, engineering the cyanobacteria to synthesize a more complete form of nanocellulose, one that is a polymer with a crystalline architecture. He also said that operations are being scaled up, with research moving from laboratory-sized tests to larger outdoor facilities.

Brown expressly pointed out that one of the major barriers to commercializing nanocellulose fuels involves national policy and politics, rather than science. Biofuels, he said, will face a difficult time for decades into the future in competing with the less-expensive natural gas now available with hydraulic fracturing, or “fracking.” In the long run, the United States will need sustainable biofuels, he said, citing the importance of national energy policies that foster parallel development and commercialization of biofuels. The American Chemical Society is a nonprofit organization chartered by the U.S. Congress. With more than 163,000 members, ACS is the world’s largest scientific society and a global leader in providing access to chemistry-related research through its multiple databases, peer-reviewed journals and scientific conferences. Its main offices are in Washington, D.C., and Columbus, Ohio.

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Note to journalists: Please report that this research was presented at a meeting of the American Chemical Society

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Abstracts:

What we have learned about cellulose biosynthesis from Acetobacter xylinum (Gluconacetobacter xylinus): A brief history and future prospects

This presentation will briefly cover the long history of research on Acetobacter xylinum, with particular attention to the research that has yielded new ideas and data for cellulose biosynthesis in general. Also to be covered is research on cellulose biosynthesis among certain oxygen-evolving photosynthetic bacteria known as cyanobacteria. The Brown lab has achieved a functional transfer of Acetobacter xylinum cellulose synthase genes into cyanobacteria. The major goal is to secure equivalent biosynthetic capacity in Acetobacter xylinum. If this can be achieved, then a new global source of cellulose biosynthesis will be available for commercial exploitation. Revealed for the first time will be a short video of time lapse sequences showing cellulose biosynthesis in Acetobacter. The talk will conclude with a summary of the major existing problems for developing Acetobacter commercially, and what could be done to rectify these long-standing problems. Attendees will be encouraged to find avenues for open communication so that microbial nanocellulose production can come to fruition as one of the most useful natural products in polymer chemistry. Time-lapse movies of Acetobacter synthesizing cellulose are available on YouTube: http://www.youtube.com/watch?v=GP-26xWiKLc

Fabrication of a uniaxially oriented nano-fibrous film by drawing of microbial cellulose pellicle secreted by Gluconacetobacter xylinus under an oxygen-lacking environment

A drawable microbial cellulose pellicle having a minimum physical cross-linkage of the nanofibers was secreted by Gluconacetobacter xylinus cultured in a closed space of Schramm-Hestrin culture medium covered with silicone oil for preventing immediate use of the ambient oxygen gas. The crystalline structure of the fibers thus obtained was more than 90 % rich in cellulose Iacrystalline phase, which the normal culture had not provided to date. Moreover, the obtained pellicle allowed stretching at 1.5 times to provide a novel film with oriented crystalline nanofibers. The mechanical properties and thermal stability exhibited superior to widely used polymeric materials. It was also noted that the heating process induced transformation of the dominant cellulose Ia crystalline phase into Ib phase without a loss of the crystallinity and the high Young's modulus. The microbial culture under an oxygen-lacking stress could offer fabrication of a novel oriented nano-fibrous film of cellulose Ia, promising excellent potential properties.

Bacterial nanocellulose biomaterial for the sustainable earth: Beginning from development of cost-effective feedstocks

Bacterial nanocellulose (BNC) is a natural nanostructured biopolymer mainly synthesized by acetic acid bacteria. The cellulosic biopolymer has now been recognized as an excellent and eco-friendly biomaterial for various applications in many fields, such as biomedicine, food industry, cosmetics, advanced functional materials, paper-making, and textile industry. However, the present feedstocks used for BC production are either expensive or insufficient, and sometimes give low yields of BNC, which leads to high production costs. This problem would limit the scale of industrial manufacture and extensive applications of BNC. In recent years, our group has developed some cost-effective feedstocks by using konjac glucomannan, wheat straw, rice straw, corn stalk, cotton-based waste textiles, waste fiber sludge for BNC production. This paper will give a key introduction about the biorefining technology of the BNC biomaterials from these renewable biomasses and also give an overview on the extended applications of BNC in biomedical materials, dietary fiber functional food, fuel cells, and textile area.

Synthesis of silver nanoparticles templated by TEMPO-mediated oxidized bacterial cellulose nanofibers

We have prepared silver nanoparticles on the surface of bacterial cellulose (BC) nanofibers. The synthesis of silver nanoparticles incorporates 2,2,6,6-tetramethylpiperidine-1-oxyradical (TEMPO)-mediated oxidation to introduce carboxylate groups on the surface of BC nanofibers. An ion exchange of the sodium to the silver salt was performed in AgNO3 solution, followed by thermal reduction. By using oxidized BC nanofibers as a reaction template, we have prepared stable silver nanoparticles with a narrow size distribution and high density through strong ion interactions between host carboxylate groups and guest silver cations, which have been investigated by scanning electron microscopy, UV-visible spectroscopy, and a small-angle X-ray scattering method.

In situ self-assembly and hydrophobization of Gluconacetobacter bacterial cellulose

A new method to produce hydrophobized bacterial cellulose (BC, Gluconactobacter genus) is proposed. The method mainly involves surface roughening by self-assembled silica nanoparticles (7 nm), which present a strong affinity with BC ribbons upon macromolecular synthesis. The changes in BC surface roughness were followed by FESEM imaging after BC synthesis in the culture medium modified with silica nanoparticles. An increase in water contact angle of approximately 30° was observed in the modified BC; the contact angle was further increased to approx. 100° by post-treatment with perfluorocarbons. The presence and the number density of silica nanoparticles were quantified by EDS elemental analysis. The proposed method combines nanotechnology and bioengineering of bacteria to produce in-situ rough BC that is further hydrophobized for deployment in applications such as self-cleaning membranes, anti-adhesive coatings and reinforcing fibers for fluoropolymer matrices.

Nata organisms: An overview on the fermentative microbial ecosystem

The Acetobacter and Gluconacetobacter genus (both from the Acetobacteraceae family) are the most notable acetic acid producers, their intermediate metabolites being exploited biotechnologicaly for the production of vinegar, Kombucha, cocoa and nata de coco. Extensive efforts are being made to better understand the dynamic interplay of microbial populations during fermentation processes, with ample literature existing on virtually every food product currently being consumed. In the case of nata de coco, Gluconacetobacter strains have been found to play a key role in cellulose production. Despite abundant literature with isolated cellulose-producing strains, little work has been done in analysing population dynamlcs of the microbial communities. This presentation will address the microbial interplay in the production of nata de coco, with an overview of the taxonomy of the major acetic acid strains involved. An overview on the efforts and potential implications of upgrading nata de coco production through biotechnology will also be addressed.

Characterization of water-soluble exopolysaccharides from Gluconacetobacter xylinus and their impacts on bacterial cellulose formation

Gluconacetobacter xylinus (G.xylinus) is characterized by its capacity to synthesize cellulose. Some strains of this organism are also able to produce complex exopolysaccharides (EPS). In this study, we separated them based on their affinity to BC and identified the EPS which significantly impact the cellulose crystallization and ribbon assembly. The strong association between EPS and BC can also decrease the crystallinity of BC and cause the swelling behavior of individual ribbons. We propose that the bound EPS extracted from BC films could absorb onto the surface of cellulose fibrils at the early stage of cellulose synthesis. This process may change the hydrogen bonding between cellulose fibrils and impact the cellulose formation by disrupting the crystallization process.

Functional analysis of the acsD gene in the microbe Gluconacetobacter xylinus

The acsD gene is unique in showing its sole presence in the cellulose biosynthesis operon of G. xylinus. With homologous recombination, an acsD gene disruption mutation was created in the G. xylinus genome. Using light and electron microscopy, phenotypic characterization of the acsD gene mutant was investigated. Carboxymethyl cellulose alterations and lower temperature incubations indicated that the arrangements of the pores on the cell surface were altered. These mutants led to a 90% decrease in cellulose secretion even though the polymerization and the crystallization components were functional. The acsD disruption mutant lacked a functional expression of the D gene. Successful complementation of this mutant with plasmid expressing green fluorescence protein tag restored normal cellulose biosynthesis. Taking all these results together, a new model for bacterial cellulose biosynthesis has been produced and discussed.

Deuteration of bacterial nanocellulose for structural studies

Bacterial nanocellulose (BNC), the cellulose hydrogel produced by Acetobacter and Gluconacetobacter species, resembles plant cellulose in its chemical and elementary crystalline structure. The special physical and structural properties of BNC make it preferable for utilization in biomedicine and nanotechnology. In particular, its use as a biomaterial for tissue implants and wound dressings has great potential and continues to be intensively studied. We have developed methods for specific deuterium incorporation in BNC to improve its contrast in composite materials for analysis by small-angle neutron scattering. Results from these studies provide new insights into hydrated microfibrillar structure, hydrogen bonding, and carbohydrate metabolism for cellulose synthesis. This fundamental understanding of BNC structure and synthesis will assist manipulation of its special physical properties and determination of factors that endow it with superior biocompatibility for growth of mammalian tissues and composite materials. Deuterium-tagged BNC enables potential biological tracking for in situ biodegradation and enzymatic studies.

Nanoparticles and nanocomposites from renewable resources

Owing to the hierarchical structure and semicrystalline nature of polysaccharides, crystalline nanoparticles can be extracted from these naturally occurring polymers. Native cellulose is built up by smaller and mechanically stronger microfibrils consisting of alternating crystalline and non-crystalline domains. Multiple mechanical shearing actions can be used to release these microfibrils. This material is usually called microfibrillated cellulose. Longitudinal cutting of these microfibrils can be achieved by submitting the biomass to a strong acid hydrolysis treatment, allowing dissolution of amorphous domains. The ensuing nanoparticles occur as rod-like nanocrystals. Similar acidic treatment carried out on starch granules allows obtaining platelet-like nanoparticles. Impressive mechanical properties and reinforcing capability, abundance, low weight, and biodegradability of these nanoparticles make them ideal candidates for the processing of polymer nanocomposites. With a Young's modulus around 150 GPa and a surface area of several hundred m2.g-1, they have the potential to significantly reinforce polymers at low filler loadings.

Nanofibrillated cellulose vs. bacterial cellulose: Reinforcing ability of nanocellulose obtained top-down or bottom-up

The development of nanocomposites derived from renewable sources with nanocellulose as reinforcement is currently a hot research area. Nanocellulose can be obtained via two approaches; top-down or bottom-up. The top-down approach involves the disintegration of plant cellulose, such as wood fibres, using high shear forces. The bottom-up approach, on the other hand, utilises the biosynthesis of cellulose by bacteria, such the Acetobacter species. However, no engineering tests have been conducted to evaluate the effect of different types of nanocellulose on the mechanical properties of the nanocomposites. Therefore, we study the applicability of two cellulose nanofibres bacterial cellulose and a nanofibrillated cellulose derived from bleached birch pulp as reinforcement for engineering grade thermosetting polymers as a model system. The following nanocellulose properties will be juxtaposed: crystallinity, surface energy and chemistry (zeta-potential) as well as the mechanical properties and permeability of nanocellulose paper. It was found that bacterial cellulose possesses a higher degree of crystallinity (72%) and critical surface energy (57 mN m-1) compared to nanofibrillated cellulose (41% and 42 mN m-1, respectively). The surface of bacterial cellulose was also found to be more acidic compared to nanofibrillated cellulose. To compare the reinforcing ability bacterial cellulose or nanofibrillated cellulose in nanocomposites, the nanocellulose was transformed into sheets and resin infused with an epoxy-based resin. Both the mechanical properties of the nanocellulose sheets and the nanocomposites are studied and compared in this work.

Single fibrils of bacterial nanocellulose and their application potential

Bacteria-synthesized Nanocellulose (BNC) as innovative biomaterial is generally generated as an inherently stable hydrogel, characterized by a 3-dimensional network of nanofibers resulting in outstanding material properties. During the last years, this fascinating biopolymer became increasing attention as new polymer in research, but as well in technical and biomedical applications.

Depending on the kind of cultivation the BNC synthesized by strains of Gluconacetobacter can be produced in flat or non-flat form.

Using this in situ formability, unique BNC single fibrils, obtained by well-known agitated cultivation conditions, were utilized to generate novel kinds of BNC applications. Recent results from ongoing technical as well as biomedical product development based on BNC fibrils will be presented.

Composites based on bacterial cellulose: Potential for technical processes

Bacterial cellulose (BC) occurs as nanofibrillated, high-swollen three dimensional cellulose type manufactured by biotechnological processes. The fzmb GmbH Bad Langensalza (Germany) produces BC in large amounts and is highly interested in industrial applications of this biomaterial. In the past a number of publications highlighted the effects of BC in polymer matrices. Nevertheless the fundamental suitability of BC for both, industrial scale and technical uses have not been considered yet. It was shown that BC composites can be manufactured by industrial processes and that the potential of BC can be represented in these materials. For a sufficient productivity BC-regrind should be used; for high-end applications BC-sheets are appropriate. A reinforcing with BC (as sheet or as grinded powder) seems to be more useful in resins than in thermoplastics.

A perspective on the research at the University of Aveiro

In the last two decades there has been an increasing interest in the search for renewable alternatives as sources of novel materials for application in several fields such as in packaging, biomedical products and devices, as well in high technological domains. Polysaccharides and cellulose in specific, due to its abundance and mechanical properties are among the most important raw materials for the development of novel sustainable functional materials.

More recently, nanocellulose forms like bacterial cellulose gained particular attention on this context because of their unique features that can be successfully exploited in the development of novel nanocomposite materials. In fact, nanocellulose forms can impart in the resulting materials substantial improvements in mechanical and barrier properties, and, more interestingly, innovative properties such as transparency.

In this perspective, in the last few years, we have been engaged on the investigation of new applications of bacterial cellulose including transparent nanocomposite films, thermoplastic based nanocomposites, membranes for drug delivery, membranes for fuel cells and other electronic devices, paper coating formulations, among others. A global overview of the research activities developed in these domains in our group will be presented in this communication.

Nanocellulose binders for fibre preforms

Cellulosic materials such as natural fibres have distinct advantages over conventional synthetic fibres; low cost, low density, high toughness and most importantly, biodegradability. However, non-woven natural fibre mats are often produced using film-stacking method, which requires laborious work or utilising polymeric binders to create a non-woven fibre mat. We have developed a novel method of producing non-woven sisal fibre mat using bacterial cellulose as the binder. Bacterial cellulose (BC) possesses a highly crystalline structure (~90%) and high Young's modulus of about 114 GPa. This value is comparable to conventional glass fibre (70 GPa) considering that bacterial cellulose has much lower density (1.5 g cm-3) than glass fibre (~2.5 g cm-3). It is also inherantly a nano-sized material and possesses a high surface area of about 55 m2 g-1. More importantly, bacterial cellulose possesses low thermal expansion coefficient of 0.1 ´ 10-6 K-1. By binding the natural fibres with BC, enhanced stress transfer between sisal fibres can be achieved. In addition to this, the bacterial cellulose will also serve as reinforcement for the composite materials, thereby creating green bio-based nanocomposites with much improved properties. In this study, vacuum-assisted resin infusion under flexible tooling was used to produce BC-sisal fibre mat reinforced acrylated epoxidized soybean oil (AESO) composites. It was found that the tensile modulus of the BC-sisal reinforced AESO composites increased by as much as 1400% compared to neat AESO and 77% when compared to sisal-fibre reinforced composites without BC binder. The flexural modulus of the BC-sisal fibre reinforced AESO also improved by as much as 23 times when compared to neat AESO. The properties of the BC-sisal fibre mat and its composites were also assessed in terms of their visco-elastic performances, fibre-fibre and fibre-matrix stress transfer.

Production of a bacterial cellulose-cotton gauze blended composite film in a horizontal rotating bioreactor

Bacterial cellulose (BC) sheets and BC-based composite films have been developed for unique and promising medical materials especially as wound dressings. However traditional BC-film producing technology, namely static culture, is inefficient, with low productivity and difficulties in scale-up, which heavily limits commercial scale extension of BC applications. Hence a novel BC composite producing technology was developed by using a horizontal rotating bioreactor in this study. Through this technology, a BC-textile composite film consisting in a BC gel-coated textile skeleton (in this case cotton gauze) was prepared successfully and efficiently. This BC composite film fermentation system can be easily to be scaled-up and provides high volumetric efficiency, good oxygen transfer rate, and low shear stress properties. It is an efficient way to incorporate many various materials homogeneously into BC film to improve physical properties or to provide desirable functions for the film. The yield and productivity of BC, uniformity and physical properties of BC-gauze film were investigated and compared.

Bacterial cellulose aerogels: From lightweight dietary food to functional materials

Bacterial cellulose features some outstanding properties, such as high purity and high molecular weight that render this natural source an excellent candidate for a variety of applications. Bacterial cellulose aerogels were demonstrated to feature good dimensional stability upon scCO 2 drying, and during storage under humid conditions which typically causes considerable shrinkage with aerogels of comparable density obtained by dissolution and regeneration of plant cellulose. Full accessibility of the pore network after scCO 2 drying allows for their utilization in controlled release applications such as for wound treatment or skin care. Good biocompatibility renders cellulose aerogels promising cell scaffolding materials which were shown to support both growth and adipogenic or osteogenic differentiation of human bone marrow derived mesenchymal stem cells. The anisotropy with regard to morphology and mechanical characteristics of bacterial cellulose aerogels could be of particular use for applications that require specific void size, geometry, and elasticity of the void beams.

Chemical and structural characterization of bacterial cellulose produced by Gluconacetobacter sucrofermentans CECT 7291 to be applied in paper restoration

Bacterial cellulose (BC) synthesized by Gluconacetobacter sucrofermentans has a high degree of crystallinity, great resistance and biocompatibility. In previous studies we have evaluated the effect that carbon and nitrogen sources, present in culture medium, have on BC from G. sucrofermentans CECT 7291 used to restore damaged documents. The best combination was fructose plus yeast extract-corn steep liquor, with or without ethanol. In this study, the resulting BC layers, treated with several methods to wash them and remove the bacteria (65ºC for 24 hours, ampicillin, 1% NaOH plus distillated water in all cases), were characterized in terms of SEM, X- ray diffraction, FTIR, polymerization degree, SEC, static and dynamic contact angle, and mercury intrusion porosimetry. The results show enough differences to select the best method when BC is going to be generated directly on the paper to restore and when BC is used for reinforcement after it is formed and treated.

Interfaces in bacterial cellulose nanofibrous networks and composites

A range of baterial cellulose nanofibrous networks and composites have been studied during their tensile deformation using Raman spectroscopy. It is shown that the interfaces within networks can be elucidated. Cross-linking is applied to the networks through glyoxalisation, which improves stress-transfer and the wet-strength of the networks. These cross-linked networks are further consolidated in a composite within a PLA resin material. It is shown that fibre-fibre and fibre-matrix interactions can be discriminated using the Raman technique giving unique insight into stress-transfer mechanisms.

Why nanocelluloses as medical implants? An innovative approach for an unsolved problem

After decades of research and disappointments using conventional polymers as medical implants, nanocelluloses are advancing from fundamental research to becoming an exciting alternative implant material. Our Matrix Reservoir Technology for the shaping of bacterial nanocellulose (BNC) is a powerful tool for designing biomimetic surfaces, and layered and channelled three-dimensional structured bodies. The choice of the matrix material (glass, steel, synthetic polymers) is a crucial design parameter. Non-modified BNC samples and prototypes containing channels (channel diameter of 0.1mm and larger) were produced and tested in cell cultures and sheep studies. The results surpassed our expectations: BNC implants (fleeces, patches, tubes, coated structures) were effectively colonized. So we found chondrocytes and fibroblasts not only on the surfaces but also within the layer and channel structures and endothelial cells on the blood-contacting surfaces. This remarkable bioactivity, and the biomimetic characteristic confirm the high potential of BNC as medical implant material.

Biofabrication of 3D bacterial nanocellulose scaffolds with controlled micro-architecture for growth of autologous human tissue for reconstructive surgery and diagnostics

Bacterial nanocellulose (BNC) is emerging biomaterial which combines the nanofibril network with hydrogel like behavior which makes it ideal for Tissue Engineering applications. In the present study we describe a novel biofabrication method, which uses the bacterial machinery to engineer 3D BNC scaffolds with features at different length scales - ranging from the nano, subcellular to the macroscale. Examples of 3D BNC scaffolds prepared in our laboratory include multichannel BNC for preparation of microvascular structures, highly porous 3D structures for growth of cartilage tissue and combination of 3D multichannel scaffold with highly porous architecture for growth of vascularized tissue such as bone and adipose tissue. 3D BNC scaffolds have shown to support neural network development and enable stem cell differentiation. Human tissues grown on 3D BNC scaffolds show great potential of this new biomaterial-cell constructs for applications in reconstructive surgery and as in vitro model of diseases such as Alzheimer and Osteoarthritis.

Cytotoxic, genotoxic, and mutagenic evaluations in materials based on bacterial cellulose for medical devices

Over the past years, biotechnology has advanced in the development of biomaterials for tissue regeneration and for their utilization as medical devices. In this context, bacterial cellulose (BC)-based membranes have shown promising results in tissue regeneration that can be used in the healing of skin wounds, grafts, drug delivery systems and tissue engineering. Also, the Institute of Chemistry in Araraquara (IQ UNESP) has developed BC composites for utilization as contact lenses. It is intended to incorporate drugs into these contact lenses, such as ciprofloxacin and sodium diclofenac, to improve their therapeutic properties. In order to ensure the safe use of these composites by the medical device industry, it is necessary to evaluate whether those materials have cytotoxic, genotoxic and mutagenic effects. Besides cytotoxicity, it is very important to evaluate the genotoxic and mutagenic potential of these materials, because, if they provide such risks, they can induce cellular modifications in the surrounding tissue, with possible progression to tumor formation (carcinogenesis). After performing in-vitro tests such as XTT, clonogenic surveillance, comet and micronucleus, the results were submitted to statistical analyses. The information regarding the toxic potential of the materials is essential for their safe release for future use in the medical device industry.

Bacterial nanocellulose hydrogels as a versatile biomimetic platform for tissue and organ engineering

Hydrogels have been acclaimed as the new paradigm in three-dimensional cell culture and tissue engineering applications. The study of mammalian cells ex vivo and the current need to better understand cell-scaffold interaction raise the question of how cell-friendly and life-sustaining microenvironments for physiological meaningful cell aggregates can be produced. As a fully biocompatible platform, bacterial nanocellulose (BNC) resembles a physiological microenvironment favorable to healthy cell morphology and growth. It has been used in tissue engineering, and in vitro human cell therapy research. Tissue engineering involves the creation of 3D structures where cells may be seeded and cultured to achieve clinical relevant concentrations. One great challenge is to improve functional vascularization that facilitates transport of O2 and nutrients into the center of the scaffolds. In order to exploit these challenges, results concerning the use BNC as a versatile biomimetic platform for several tissue and organ engineering applications will be presented.

Studies on the hemocompatibility of bacterial nanocellulose

The hemocompatibility of bacterial nanocellulose, including interaction with platelets will be reviewed.

Towards molecular control over BNC production process

Bacterial nano-cellulose (BNC) -based product, CelMat® wound dressing production technology invented in our Institute was successfully transferred to industry. Ongoing studies concentrate on improvement of BNC-derived materials used in internal medical applications such as peripheral nerves regeneration, hernia treatment, meniscus replacement and flat skull bones fragments reconstruction. The research focuses on controlled porosity development and the application of molecular factors stimulating the tissue regeneration. BNC scaffolds in combination with brain-derived neurotrophic factor were successfully employed in vivo in peripheral nerves disorders in rats. Porous BNC with increased active surface is being subjected to preliminary biological tests on mouse fibroblasts, giving promising results. In parallel to medical applications advancement the molecular biology studies aiming to cope with microbe-specific limitations, such as long production time or spontaneous occurrence of an unproductive phenotype (Cel-), were carried out. Final conclusions from comparative transcriptomes analysis (Ga. xylinus E25 Cel+ versus Cel- phenotypes), conducted by suppression subtractive hybridization (SSH) cDNA methodology, will be presented. Particular roles in cellulose biosynthesis of differently expressed genes identified in this study are being verified by the appropriate mutants construction. Furthermore Ga. xylinus E25 genome sequencing project has completed and some important insights to signaling and regulatory pathways present in Ga. xylinus are foreseen by bioinformatics analysis. Future prospects for further BNC material improvements depend on the profound understanding of molecular processes underlying Ga. xylinus cell development, motility and protective mechanisms.

Bacterial nanocellulose as active wound dressing and drug delivery system

The biopolymer bacterial nanocellulose (BNC) has already been proven to be advantageous for various biomedical applications, e.g., tissue engineering, implant materials, skin substitutes and wound care. The use of this biomaterial as drug delivery system offers the opportunity to combine its beneficial material properties (e.g., high purity, mechanical stability and biocompatibility) with the positive effects of incorporated drugs and opens up new promising fields of application. Comparative loading and release studies with high molecular weight protein drugs [1] as well as low molecular weight antiseptics [2] were performed using BNC fleeces, partly modified by additives. Alternatively, antiseptic Ag-nanoparticles were fixed on the BNC surface in order to evaluate the full spectrum of this biomaterial as a carrier of drugs. [3] All in all, latest results from an interdisciplinary cooperation aiming for a functionalization of BNC for wound dressing and drug delivery applications will be presented.

Electrical manipulation of bacteria to control nanocellulose architecture for biomedical applications

Tissue engineering involves the seeding and attachment of human cells onto a scaffold. Of fundamental importance to the survival of most engineered tissues is gas and nutrient exchange. In nature, this is accomplished by virtue of microcirculation, which is the feeding of oxygen and nutrients to tissues and removing waste at the capillary level. Bacterial cellulose, a natural polymer, is an ideal scaffolding material for tissue engineering due to its proven biocompatibility, mechanical integrity, hydroexpansivity, and its stability under a wide range of conditions. Irreversible Electroporation (IRE) is a process in which the delivery of electrical pulses increases the transmembrane potential which leads to the creation of irrecoverable defects in the membrane thereby killing the cells. We hypothesize that by using IRE to kill the bacteria in specific locations and particular times during cellulose production, we can introduce conduits in the overall scaffold by preventing cellulose deposition at these sites.

Development of novel cellulosic-based scaffolds for articular cartilage tissue engineering by critical directional freeze drying

Bacterial cellulose (BC) had been synthesized from Gluconacetobacter xylius (ATCC-10245) strain using two different carbon source, glusoce and mannitol in Hestrin and Schramm (HS) medium. A thick gelatinous film with high water retaining capacity had been produced from the purification of BC after the cultivation period. The morphological changes were observed by FE-SEM and FTIR analysis after drying the sample in vacuum at 80°C till constant weight. A three dimensional network of fibrils with porous structure similar to extracellular matrix had been confirmed by the FE-SEM having an average fiber diameter range of 82±18 nm. According to FTIR results, the absorbance peak at 750 and 710 cm-1 indicated the presence of cellulose I a content indicating high crystallinity index compared to plant cellulose which is rich in I b content. The directional porous structure with high crystallinity index would make this material suitable for articular tissue engineering and other biomedical applications.

Modification of bacterial cellulose by adding interfering biopolymers

Novel composites of bacterial cellulose (BC) have been developed in order to expand the scope of applications. The modifying effects of added biopolymers (alginate, chitosan, Aloe vera and gelatin) on the properties of the composites were investigated. It was found that at a certain amount of the supplement, the modified BC composites displayed homogeneous structures, which exhibited a certain level of miscibility. The FTIR results demonstrated some specified interaction between the hydroxyl group of BC and the functional groups of the supplemented polymers. The supplements of these polymers significantly affected characteristics of porous structures, mechanical properties, water absorption capacity and biocompatibility. These modified composites have potential applications for a wide range of fields, including biomedical materials, tissue engineering, drug delivery, food packaging and membrane separation.

Nano- and micro-fibrillated cellulose research at VTT

Nanocellulose has been shown to be potentially very useful for a number of technical applications in the future. The key to understanding how nanocellulose will behave in different applications is to have a thorough understanding of how the structure and interactions of nanocellulose affect its function and hence its suitability for different applications. The research performed at VTT relates to the whole production chain of nanocellulose - from selection of raw materials to development of production process and modification of nanocellulose material according to the needs of various applications. Successful use of nanocellulose in different applications requires profound understanding of structural and molecular properties of nanocellulose. The most important properties are: 1) Aspect ratio, i.e., the diameter and length of the fibrils; 2) Range of polydispersity (the preparations are usually mixtures of fibrils with different size distribution); 3) Surface chemistry, specifically how the surface has been modified by adding chemical functionalities, such as charge, hydrophobic groups, etc.; and 4) Impurities and other polymers present (nanocellulose consists seldom of only cellulose, and other compounds present may have a major impact on the properties of the preparation). At the moment nanocellulose research at VTT can be divided into five main research areas: Soft matter - emulsions, encapsulation, stabilization of dispersions, hydrogels; Condensed matter - composites; Porous matter - filters, adsorbents, scaffolds; Thin films - barriers, supports and adhesive layers; and Use of nanocellulose in fibre-based structures.

Current manufacture and product parameters of tubular cellulosic implants

There are still no artificial implants for small but vital blood vessels in the heart, brain and limbs. Implants made from synthetic polymers and even products fabricated from protein- based materials are not able to fulfil the requirements. Due to the specific structure and properties, the polysaccaridic material bacterial nanocellulose (BNC) proved to be first choice for development of novel tubular implants. Current synthetic strategies to design prototypes will be presented. Valuable relevant product parameters relate lengths up to 150 mm, inner diameters of 1-6 mm, bursting strengths of more than 1000 mm Hg and suture retention strengths around 5 N. Recent three-month studies in sheep show positive key parameters: handling during surgery, biocompatibility, mechanical stability and ingrowth of endogenous cells. Remaining problems concerning the thrombosis rate and ways to overcome them are discussed.

Oxidation of g-irradiated microbial cellulose results in a bioresorbable, highly conformable novel biomaterial

Conformability to tissues and adequate mechanical strength are clinically useful properties of resorbable biomaterials used in soft tissue repair. Here we show that controlled oxidation of microbial cellulose sheets that have been pre-irradiated with g-radiation results in a resorbable and fully conformable membrane that can be rapidly rehydrated in aqueous fluids. In vitro studies showed that degradation of the resorbable membranes occurs in two major phases: (1) initial rapid degradation of about 70-80% of the entire sample followed by (2) slower degradation of an additional 5-10% which eventually levels off leaving a small amount of nonresorbable material. In vivo, prototype materials showed marked degradation at all time points, with the most rapid degradation occurring in the first 2-4 weeks. The inflammatory reaction to the test devices was mild to moderate and was most prominent at the early time points, consistent with a rapidly absorbed material.

Innovative bacterial nanocellulose medical devices: From incubator to human body

Bacterial nanocellulose (BNC) has gained significant scientific interest during last few years as an emerging biomaterial for applications as vascular grafts, scaffolds for bone regeneration, cartilage tissue repair surgical meshes and skin substitute. The unique combination of good biocompatibility with good mechanical properties makes BNC attractive for variety of applications in tissue engineering and regenerative medicine. Successful commercialization of BNC medical devices requires however reproducible device biofabrication processes and extensive preclinical in vitro and in vivo evaluation to guarantee safety and performance of this new biomaterial. Each processing step has to be developed for full-scale production in a cGMP environment. Although successful FDA approval for simple devices may be based on 510 k process, more advanced devices may require clinical trials. There is only very limited literature data on BNC long term performance in preclinical studies and only one report on clinical performance of BNC in the human body. There is also very limited literature on the effect of the purification processes on endotoxin content, tissue integration and biomechanical performance. Collaborative efforts from the BNC scientific and corporate community are highly desirable in order to promote broad use of BNC as implants and biomaterials for regenerative and reconstructive surgery.

Commercializing bacterial nanocellulose: My 15-year experiment

The presentation is about the author's personal perspective on technology commercialization based on his experience as co-founder of a bacterial cellulose company, Xylos Corporation. The various roles of the scientific/technical founder may play as the company evolves will be described. Activities in various areas including product development, regulatory compliance, intellectual property expansion, manufacturing and marketing, that contributed to the commercialization of the company's products will be described briefly. In conclusion, a method of analysis (i.e. TRIM) to be used as a guide for decision-making in commercializing a platform technology (such as bacterial nano cellulose) will be proposed.

Twenty-five years of entrepreneurial experience with bacterial cellulose

Our presentation is an account of 25 years experience of entrepreneurship, research and development of bacterial cellulose production in industrial levels, and its use in the medical field. This is not an academic work; it is a simple speech about the pleasures and pains of the effort to transform a technology and the perception of the potential applications of a new material, in patents and effective products.

Implementation of industrial production bionanocellulose material

Bowil Biotech is a biotechnology company that is working in the production of bionanocellulose materials. In 2011, the company purchased from the Technical University of Lodz, innovative know-how knowledge of production bionanocellulose, dressing materials called CelMat" and bacterial strain Gluconacetobacter xylinus E25. Bowil Biotech company purchased the right to patent and trade mark called CelMat®. Currently, work is underway on the implementation of technologies for industrial production and the creation of wide range of products of bionanocellulose material. The first production will be a medical and cosmetic products.

Bionanocellulose is a modern biomaterial formed as a result of natural processes taking place in the nature with bacteria. As a result of this process the cellulose fibers are formed in nanometer dimension. Technology of production this kind of fibers is a result of many years of research of Polish scientists of Technical University of Lodz. The result of this work is appreciated all over the world, numerous scientific papers and patents were published, in which technology of Bowil Biotech company is based on. Bionanocellulose, thanks to its properties, has been used in medical, biotechnological, cosmetic, textile, paper, electronic, acoustic, and the food industry.

Michael Bernstein m_bernstein@acs.org 504-670-4707 (New Orleans Press Center, April 5-10) 202-872-6042 Michael Woods m_woods@acs.org 504-670-4707 (New Orleans Press Center, April 5-10) 202-872-6293

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EMBARGOED FOR RELEASE: Sunday, April 7, 2013, 10 a.m. Eastern Time Note to journalists: Please report that this research was presented at a meeting of the American Chemical Society