Enhanced bacterial cellulose production in Gluconacetobacter xylinus by overexpression of two genes (bscC and bcsD) and a modified static culture.

Bacterial cellulose (BC), a nanostructured material, is renowned for its excellent properties. However, its production by bacteria is costly due to low medium utilization and conversion rates. To enhance the yield of BC, this study aimed to increase BC yield through genetic modification, specifically by overexpressing bcsC and bcsD in Gluconacetobacter xylinus, and by developing a modified culture method to reduce medium viscosity by adding water during fermentation. As a result, BC yields of 5.4, 6.2, and 6.8 g/L were achieved from strains overexpressing genes bcsC, bcsD, and bcsCD, significantly surpassing the yield of 2.2 g/L from wild-type (WT) strains. In the modified culture, the BC yields of all four strains increased by >1 g/L with the addition of 20 mL of water during fermentation. Upon comparing the properties of BC, minimal differences were observed between the WT and pbcsC strains, as well as between the static and modified cultures. In contrast, BC produced by strains overexpressing bcsD had a denser microstructural network and exhibited demonstrated higher tensile strength and elongation-to-break. Compared to WT, BC from bcsD overexpressed strains also displayed enhanced crystallinity, higher degree of polymerization and improved thermal stability.

Cellulose biosynthesis using simple sugars available in residual cacao mucilage exudate.

Worldwide only 8% of the biomass from harvested cacao fruits is used, as cacao beans, in chocolate-based products. Cacao mucilage exudate (CME), a nutrient-rich fluid, is usually lost during cacao beans fermentation. CME's composition and availability suggest a potential carbon source for cellulose production. CME and the Hestrin and Schramm medium were used, and compared, as growth media for bacterial cellulose (BC) production with Gluconacetobacter xylinus. CME can be used to produce BC. However, the high sugar content, low pH, and limited nitrogen sources in CME hinder G. xylinus growth affecting cellulose yields. BC production increased from 0.55 ± 0.16 g L-1 up to 13.13 ± 1.09 g L-1 after CME dilution and addition of a nitrogen source. BC production was scaled up from 30 mL to 15 L, using lab-scale experiments conditions, with no significant changes in yields and production rates, suggesting a robust process with industrial possibilities.

Biotech nanocellulose: A review on progress in product design and today's state of technical and medical applications.

Biotech nanocellulose (bacterial nanocellulose, BNC) is a high potential natural polymer. Moreover, it is the only cellulose type that can be produced biotechnologically using microorganisms resulting in hydrogels with high purity, high mechanical strength and an interconnecting micropore system. Recently, the subject of intensive research is to influence this biosynthesis to create function-determining properties. This review reports on the progress in product design and today's state of technical and medical applications. A novel, dynamic, template-based technology, called Mobile Matrix Reservoir Technology (MMR Tech), is highlighted. Thereby, shape, dimensions, surface properties, and nanonetwork structures can be designed in a process-controlled manner. The formed multilayer materials open up new applications in medicine and technology. Especially medical materials for cardiovascular and visceral surgery, and drug delivery systems are developed. The effective production of layer-structured composites and coatings are important for potential applications in the electronics, paper, food and packaging technologies.

Sustainable, superhydrophobic membranes based on bacterial cellulose for gravity-driven oil/water separation.

Bacterial cellulose (BC) is a substrate material with high purity and robust mechanical strength, but due to its small pore size and relatively expensive price, it is restricted as an oil-/water separation membrane. In this study, cheaper plant cellulose needle-leaf bleached kraft pulp (NBKP) was added to BC to increase the pore size of the composite membrane, and a superhydrophobic/superoleophilic membrane was prepared for oil-/water separation. The modified membrane surface displayed a petal-like micro-structure and a water contact angle (WCA) of 162.3°, while the oil contact angle was decreased to 0°. What's more, the membrane exhibited excellent oil-/water separation under gravity, recyclability, and a separation efficiency (>95 %), and it was both pH and salt resistant. The membrane also remained durably hydrophobic after 10 separation cycles. And the separation methodology is expected to be highly energy-efficient.

In Vitro Study of a Stentless Aortic Bioprosthesis Made of Bacterial Cellulose.

PURPOSE: The paper present findings from an in vitro experimental study of a stentless human aortic bioprosthesis (HAB) made of bacterial cellulose (BC). Three variants of the basic model were designed and tested to identify the valve prosthesis with the best performance parameters. The modified models were made of BC, and the basic model of pericardium. METHODS: Each model (named V1, V2 and V3) was implanted into a 90 mm porcine aorta. Effective Orifice Area (EOA), rapid valve opening time (RVOT) and rapid valve closing time (RVCT) were determined. The flow resistance of each bioprosthesis model during the simulated heart systole, i.e. for the mean differential pressure (ΔP) at the time of full valve opening was measured. All experimental specimens were exposed to a mean blood pressure (MBP) of 90.5 ± 2.3 mmHg. RESULTS: The V3 model demonstrated the best performance. The index defining the maximum opening of the bioprosthesis during systole for models V1, V2 and V3 was 2.67 ± 0.59, 2.04 ± 0.23 and 2.85 ± 0.59 cm2, respectively. The mean flow rate through the V3 valve was 5.7 ± 1, 6.9 ± 0.7 and 8.9 ± 1.4 l/min for stroke volume (SV) of 65, 90 and 110 mL, respectively. The phase of immediate opening and closure for models V1, V2 and V3 was 8, 7 and 5% of the cycle duration, respectively. The mean flow resistance of the models was: 4.07 ± 2.1, 4.28 ± 2.51 and 5.6 ± 2.32 mmHg. CONCLUSIONS: The V3 model of the aortic valve prosthesis is the most effective. In vivo tests using BC as a structural material for this model are recommended. The response time of the V3 model to changed work conditions is comparable to that of a healthy human heart. The model functions as an aortic valve prosthesis in in vitro conditions.

Fabrication of bacterial cellulose-collagen composite scaffolds and their osteogenic effect on human mesenchymal stem cells.

Scaffold plays a critical role in stem cell differentiation and tissue regeneration. Composite scaffolds composed of bacterial cellulose (BC) and collagen (Col) in different ratios (1:1, 3:1, 5:1) were fabricated in this study. The composite scaffolds exhibit a well-organized interconnected porous structure, significantly better physical stability than Col scaffold, and more water uptake up to 400%. They were also favorable with cell attachment and growth. After osteogenic induction of umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) for 3 weeks, we found more up-regulated osteogenic markers (collagen type 1, osteocalcin, bone sialoprotein) and significantly elevated proteins and calcium deposition, particularly with BC/Col (5:1) scaffold. When PKH-26 pre-labelled MSC-loaded scaffolds were subcutaneously transplanted in a mouse model, they showed many PKH-26-labelled cells and positive signals of α-smooth muscle actin, for neovascularization in the BC/Col (5:1). The current work demonstrates that our BC/Col composites may be promising as a bone tissue-engineered scaffold.

In situ preparation of bacterial cellulose with antimicrobial properties from bioconversion of mulberry leaves.

It's long been understood that antimicrobial properties are one of the most important function in the field of biomedicine. In this thesis, we introduce a new technique to functionalize bacterial cellulose (BC) with antimicrobial properties by in situ method. We design a series of experiments on hydrolyzing mulberry leaves and exploring the methods of fermenting and purifying to obtain a BC complex with antimicrobial properties. Meanwhile, the anti-bacterial performances of MH-BC (fermented by the mulberry leaves acid hydrolysate fermentation medium) were evaluated with Escherichia coli and Staphylococcus aureus, and the experimental results showed that the MH-BC have excellent anti-bacterial activities. Considering the excellent biocompatibility of MH-BC towards hMSCs, we expect that this antibacterial functional BC composite will find potential applications in biomedicine and regenerative medicine.

Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant.

Bionanocellulose (BNC) is a clear polymer produced by the bacterium Gluconacetobacter xylinus. In our current study, "Research on the use of bacterial nanocellulose (BNC) in regenerative medicine as a function of the biological implants in cardiac and vascular surgery", we carried out material analysis, biochemical analysis, in vitro tests and in vivo animal model testing. In stage 1 of the project, we carried out physical and biological tests of BNC. This allowed us to modify subsequent samples of bacterial bionanocellulose. Finally, we obtained a sample that was accepted for testing on an animal model. That sample we define BNC1. Patches of BNC1 were then implanted into pigs' vessel walls. During the surgical procedures, we evaluated the technical aspects of sewing in the bioimplant, paying special attention to bleeding control and tightness of the suture line and the BNC1 bioimplant itself. We carried out studies evaluating the reaction of an animal body to an implantation of BNC1 into the circulatory system, including the general and local inflammatory reaction to the bioimplant. These studies allowed us to document the potential usefulness of BNC as a biological implant of the circulatory system and allowed for additional modifications of the BNC to improve the properties of this new implantable biological material.

Bacterial cellulose production by Acetobacter xylinum ATCC 23767 using tobacco waste extract as culture medium.

In this study, bacterial cellulose (BC) was synthesized by Acetobacter xylinum ATCC 23767 using tobacco waste extract (TWE) as a carbon source. Nicotine was found to be an inhibitory factor for BC synthesis, but it can be removed at pH 9.0 by steam distillation. After removing nicotine, the BC production was 2.27 g/L in TWE prepared with solid-liquid (S-L) ratio at 1:10. To further enhance the BC production, two fermentation stages were performed over 16 days by re-adjusting the pH to 6.5 at 7 days, after the first fermentation stage was completed. Using this two-stage fermentation, the BC production could reach 5.2 g/L. Structural and thermal analysis by FE-SEM, FT-IR, XRD and TGA showed the properties of BC obtained from TWE were similar to that from Hestrin-Schramm (HS) medium. Considering the huge disposal tobacco waste in China, the present study provides an alternative methodology to synthesize BC.

Efficient bioconversion from acid hydrolysate of waste oleaginous yeast biomass after microbial oil extraction to bacterial cellulose by Komagataeibacter xylinus.

Biomass acid hydrolysate of oleaginous yeast Trichosporon cutaneum after microbial oil extraction was applied as substrate for bacterial cellulose (BC) production by Komagataeibacter xylinus (also named as Gluconacetobacter xylinus previously) for the first time. BC was synthesized in static culture for 10 days, and the maximum BC yield (2.9 g/L) was got at the 4th day of fermentation. Most carbon sources in the substrate (glucose, mannose, formic acid, acetic acid) can be utilized by K. xylinus. The highest chemical oxygen demand (COD) removal (40.7 ± 3.0%) was obtained at the 6th day of fermentation, and then the COD increased possibly due to the degradation of BC. The highest BC yield on COD consumption was 38.7 ± 4.0% (w/w), suggesting that this is one efficient bioconversion for BC production. The BC structure was affected little by the substrate by comparison with that generated in classical HS medium using field-emission scanning electron microscope (FE-SEM), Fourier transform infrared, and X-ray diffraction. Overall, this technology can both solve the issue of waste oleaginous yeast biomass and produce valuable biopolymer (BC).

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3·6H2O) and iron(II) chloride tetrahydrate (FeCl2·4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 °C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young's modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Antioxidant and anti-inflammatory levan produced from Acetobacter xylinum NCIM2526 and its statistical optimization.

Levan is a homopolymer of fructose naturally obtained from both the plants and microorganisms. Along with the general properties of a biopolymer like bio-compatibility, bio-degradability, renewability, flexibility, and eco-friendliness, levan also offers some important biomedical properties such as anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-AIDS and hyperglycaemic inhibitor. In this study, we have demonstrated the microbial production of therapeutically potential levan by batch fermentation process in sucrose rich medium using Acetobacter xylinum NCIM 2526. The produced Levan was characterized using various physicochemical techniques such as FTIR, (1)H NMR, (13)C NMR spectroscopy, TGA and HPLC. The biomedical potential of the isolated A. xylinum levan for its anti-oxidant and anti-inflammatory activities was exploited in vitro. Further the present study also focused on the optimization of levan production using one factor at a time approach followed by a statistical method, central composite design (CCD) with selected variables. The yield of levan was increased significantly from 0.54 to 13.25g/L with the optimized variables.

Bacterial nanocellulose as a new patch material for closure of ventricular septal defects in a pig model.

OBJECTIVES: Current materials for closure of cardiac defects such as ventricular septal defects (VSDs) are associated with compliance mismatch and a chronic inflammatory response. Bacterial nanocellulose (BNC) is a non-degradable biomaterial with promising properties such as high mechanical strength, favourable elasticity and a negligible inflammatory reaction. The aim of this study was the evaluation of a BNC patch for VSD closure and the investigation of its in vivo biocompatibility in a chronic pig model. METHODS: Young's modulus and tensile strength of BNC patches were determined before and after blood exposure. Muscular VSDs were created and closed with a BNC patch on the beating heart in an in vivo pig model. Hearts were explanted after 7, 30 or 90 days. Macropathology, histology and immunohistochemistry were performed. RESULTS: Young's modulus and tensile strength of the BNC patch decreased after blood contact from 6.3 ± 1.9 to 3.86 ± 2.2 MPa (P < 0.01) and 0.33 ± 0.06 to 0.26 ± 0.06 MPa (P < 0.01), respectively, indicating the development of higher elasticity. Muscular VSDs were closed with a BNC patch without residual shunting. After 90 days, a mild chronic inflammatory reaction was present. Moreover, there was reduced tissue overgrowth in comparison with polyester. Proceeding cellular organization characterized by fibromuscular cells, production of extracellular matrix, neoangiogenesis and complete neoendothelialization were found. There were no signs of thrombogenicity. CONCLUSIONS: BNC patches can close VSDs with good mid-term results and its biocompatibility can be considered as satisfactory. Its elasticity increases in the presence of blood, which might be advantageous. Therefore, it has potential to be used as an alternative patch material in congenital heart disease.

Binding of dietary polyphenols to cellulose: structural and nutritional aspects.

The interactions between polyphenols and plant fibres play an important role in controlling the release of phenolic compounds from food matrices for absorption in the gastrointestinal tract. This study probed the molecular interactions of diverse polyphenols with cellulose fibres by using a pure cellulose-producing bacterial model. Alkali treatment of bacterial cellulose was an effective method for obtaining a high purity cellulose model for study of polyphenol binding. Representatives of different polyphenol classes all bound to cellulose spontaneously, rapidly, and to comparable extents (up to 60% w/w of cellulose). Langmuir binding isotherms were applied to determine quantitative aspects of the adsorption at equilibrium. The study indicated that binding was similar on a molar basis for ferulic acid, gallic acid, catechin and cyanidin-3-glucoside (but lower for chlorogenic acid), with the native charge of polyphenols a secondary factor in the interactions between polyphenols and cellulose.

Bacterial cellulose membranes used as artificial substitutes for dural defection in rabbits.

To improve the efficacy and safety of dural repair in neurosurgical procedures, a new dural material derived from bacterial cellulose (BC) was evaluated in a rabbit model with dural defects. We prepared artificial dura mater using bacterial cellulose which was incubated and fermented from Acetobacter xylinum. The dural defects of the rabbit model were repaired with BC membranes. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All animals were humanely euthanized by intravenous injection of phenobarbitone, at each time point, after the operation. Then, the histocompatibility and inflammatory effects of BC were examined by histological examination, real-time fluorescent quantitative polymerase chain reaction (PCR) and Western Blot. BC membranes evenly covered the surface of brain without adhesion. There were seldom inflammatory cells surrounding the membrane during the early postoperative period. The expression of inflammatory cytokines IL-1ß, IL-6 and TNF-α as well as iNOS and COX-2 were lower in the BC group compared to the control group at 7, 14 and 21 days after implantation. BC can repair dural defects in rabbit and has a decreased inflammatory response compared to traditional materials. However, the long-term effects need to be validated in larger animals.

3D culturing and differentiation of SH-SY5Y neuroblastoma cells on bacterial nanocellulose scaffolds.

A new in vitro model, mimicking the complexity of nerve tissue, was developed based on a bacterial nanocellulose (BNC) scaffold that supports 3D culturing of neuronal cells. BNC is extracellularly excreted by Gluconacetobacter xylinus (G. xylinus) in the shape of long non-aggregated nanofibrils. The cellulose network created by G. xylinus has good mechanical properties, 99% water content, and the ability to be shaped into 3D structures by culturing in different molds. Surface modification with trimethyl ammonium beta-hydroxypropyl (TMAHP) to induce a positive surface charge, followed by collagen I coating, has been used to improve cell adhesion, growth, and differentiation on the scaffold. In the present study, we used SH-SY5Y neuroblastoma cells as a neuronal model. These cells attached and proliferated well on the BNC scaffold, as demonstrated by scanning electron microscopy (SEM) and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Following neuronal differentiation, we demonstrated functional action potentials (APs) by electrophysiological recordings, indicating the presence of mature neurons on the scaffolds. In conclusion, we have demonstrated for the first time that neurons can attach, proliferate, and differentiate on BNC. This 3D model based on BNC scaffolds could possibly be used for developing in vitro disease models, when combined with human induced pluripotent stem (iPS) cells (derived from diseased patients) for detailed investigations of neurodegenerative disease mechanisms and in the search for new therapeutics.

Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation.

Bacterial nanocellulose (BNC) was biosynthesized by Gluconacetobacter xylinus. The surface area, physicochemical structure and morphology of the materials were characterized. Here provides a method for an efficient production of uniform BNC, which is beneficial for the fast characterization and evaluation of BNC. In vitro cytotoxicity of the materials was evaluated by the proliferation, the adhesion, the viability and the morphology of NIH/3T3 cells. Low cytotoxicity of the BNC was observed, and micrographs demonstrate a good proliferation and adhesion of NIH/3T3 cells on BNC. Large area full-thickness skin defects were made on the back of C57BL/6 mice in animal surgery. The wounds were transplanted with BNC films and the results compared to those in a control group. The rehabilitation of the wound surfaces and the pathological sections of mice were investigated and are discussed. Histological examinations demonstrated faster and better healing effect and lower inflammatory response in the BNC group than those in the control group. Preliminary results on wound dressings from BNC show a curative effect promoting the healing of epithelial tissue. BNC is a promising natural polymer with medical applications in wound dressings.

Bacterial cellulose modified using recombinant proteins to improve neuronal and mesenchymal cell adhesion.

A wide variety of biomaterials and bioactive molecules have been applied as scaffolds in neuronal tissue engineering. However, creating devices that enhance the regeneration of nervous system injuries is still a challenge, due the difficulty in providing an appropriate environment for cell growth and differentiation and active stimulation of nerve regeneration. In recent years, bacterial cellulose (BC) has emerged as a promising biomaterial for biomedical applications because of its properties such as high crystallinity, an ultrafine fiber network, high tensile strength, and biocompatibility. The small signaling peptides found in the proteins of extracellular matrix are described in the literature as promoters of adhesion and proliferation for several cell lineages on different surfaces. In this work, the peptide IKVAV was fused to a carbohydrate-binding module (CBM3) and used to modify BC surfaces, with the goal of promoting neuronal and mesenchymal stem cell (MSC) adhesion. The recombinant proteins IKVAV-CBM3 and (19)IKVAV-CBM3 were successfully expressed in E. coli, purified through affinity chromatography, and stably adsorbed to the BC membranes. The effect of these recombinant proteins, as well as RGD-CBM3, on cell adhesion was evaluated by MTS colorimetric assay. The results showed that the (19)IKVAV-CBM3 was able to significantly improve the adhesion of both neuronal and mesenchymal cells and had no effect on the other cell lineages tested. The MSC neurotrophin expression in cells grown on BC membranes modified with the recombinant proteins was also analyzed.

Small calibre biosynthetic bacterial cellulose blood vessels: 13-months patency in a sheep model.

OBJECTIVES: Many patients in need of bypass surgery lack graft material and current synthetic alternatives have poor performance. A 4 mm vascular graft composed of bacterial cellulose (BC) was developed and tested in pilot study in a large animal model. DESIGN: BC is a biopolymer made by the bacteria acetobacter xylinum. BC grafts (n = 16) with 4 cm length and 4 mm internal diameter were implanted bilaterally in the carotid arteries of eight sheep. No long-term antithrombotic therapy was administered. Patency was assessed with ultrasound. Histology, immunohistochemistry, and electron microscopy were performed after explantation. RESULTS: Fifty percent of the grafts occluded within two weeks. One animal died with patent grafts after 14 days. In the three remaining animals 5/6 grafts were patent after nine months. Two animals were followed 13 months after implantation with 3/4 grafts patent at explantation. All patent grafts had confluent endothelial-like cells. CONCLUSIONS: Biosynthetic small calibre vascular grafts made from BC can be patent for up to 13 months in sheep carotid arteries. BC is a potential material for small calibre grafts but patency in animal models needs to be improved before clinical studies can be planned.

Modulatory effect of Acetobacter xylinum cellulose on peritoneal macrophages.

INTRODUCTION: The present work aimed to assess the effect of bacterial cellulose (BC) from Acetobacter xylinum on some functions of peritoneal macrophages. MATERIALS AND METHODS: Mice peritoneal macrophages were lavaged and co-cultured with various concentrations of BC in microtiter plate. Viability of macrophages was determined by MTT assay. Nitric oxide (NO) amount was detected in culture supernatants of macrophages by Griess reagent and cytotoxicity effect of culture supernatants on WEHI-164 cells as tumor necrosis factor α (TNF-α) bioassay were done. RESULTS: MTT assay for stimulated macrophages was not different (p = 0.486) and amount of NO production in all doses was the same as unstimulated control macrophages (p = 0.057). Supernatant of macrophages stimulated with 0.01 mg/mL of BC had cytotoxic effect on WEHI-164 fibrosarcoma cells (p = 0.040). CONCLUSION: In this study, we showed that BC had no effect on macrophages viability and amount of NO production. BC suppressed the release of TNF-α from macrophages innate immune cells as indication of beneficial effect on wound healing. This data and further studies would imply use of this biomaterial for more applications.