Bacterial Nanocellulose Biosynthesis Research Articles

Bacterial nanocellulose (BNC) biosynthesis by Komagataeibacter hansenii RM-03 using agricultural waste as substrates and BNC-silver nanocomposite preparation

Bacterial nanocellulose (BNC) is a remarkable biopolymer synthesised by bacterium, exhibiting exceptional properties. However, conventional Hestrin-Schramm (HS) medium, particularly the carbon source, poses challenges of high costs and low productivity. This study explores BNC biosynthesis on a modified HS medium, employing agricultural wastes (sugarcane molasses, banana peel, and pineapple peel) as carbon sources, and compares the overall yield of BNC produced. Sugarcane molasses proved to be the most effective, yielding the highest BNC concentration (8.19 g/L) after 7 days, followed by pineapple peel (2.16 g/L) and banana peel (2.11 g/L). Extensive research was conducted to enhance properties of BNC by an environmentally friendly approach, incorporating silver nanoparticles (AgNP) utilising Momordica charantia fruit extract, resulting in a BNC-Ag nanocomposite. The synthesis involved mixing 1 mM silver nitrate (AgNO3) with 15 mL of M. charantia fruit extract to reduce Ag ions to AgNP, which was confirmed by UV-vis spectroscopy with an absorbance peak between 400 and 410 nm. Characterisation using FESEM and TEM on the synthesized BNC showed minimal impact on BNC fiber diameter from waste-derived carbon sources. XRD indicated slight variations in crystallinity index, with the highest (85%) in TSM-derived BNC. FTIR analysis revealed similar chemical profiles across all BNC, indicating cellulose formation. The BNC-Ag nanocomposite exhibited potent antibacterial activity against multi-drug resistant strains (Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis, Staphylococcus aureus) through disc diffusion method with inhibition zones up to 16.8 mm. Overall, the findings from this study contribute to the development of environmentally sustainable for the production of functional BNC materials with enhanced properties for diverse applications.

Complete genome sequence and transcriptome response to vitamin C supplementation of Novacetimonas hansenii SI1 - producer of highly-stretchable cellulose

Novacetimonas hansenii SI1, previously known as Komagataeibacter hansenii, produces bacterial nanocellulose (BNC) with unique ability to stretch. The addition of vitamin C in the culture medium increases the porosity of the membranes and their stretchability making them highly moldable. To better understand the genetic background of this strain, we obtained its complete genome sequence using a hybrid sequencing and assembly strategy. We described the functional regions in the genome which are important for the synthesis of BNC and acetan-like II polymer. We next investigated the effect of 1% vitamin C supplementation on the global gene expression profile using RNA sequencing. Our transcriptomic readouts imply that vitamin C functions mainly as a reducing agent. We found that the changes in cellular redox status are balanced by strong repression of the sulfur assimilation pathway. Moreover, in the reduced conditions, glucose oxidation is decreased and alternative pathways for energy generation, such as acetate accumulation, are activated. The presence of vitamin C negatively influences acetan-like II polymer biosynthesis, which may explain the lowered yield and changed mechanical properties of BNC. The results of this study enrich the functional characteristics of the genomes of the efficient producers of the N. hansenii species. Improved understanding of the adaptation to the presence of vitamin C at the molecular level has important guiding significance for influencing the biosynthesis of BNC and its morphology.

Biosynthesis of Bacterial Nanocellulose from Low-Cost Cellulosic Feedstocks: Effect of Microbial Producer.

Biodegradable bacterial nanocellulose (BNC) is a highly in-demand but expensive polymer, and the reduction of its production cost is an important task. The present study aimed to biosynthesize BNC on biologically high-quality hydrolyzate media prepared from miscanthus and oat hulls, and to explore the properties of the resultant BNC depending on the microbial producer used. In this study, three microbial producers were utilized for the biosynthesis of BNC: individual strains Komagataeibacter xylinus B-12429 and Komagataeibacter xylinus B-12431, and symbiotic Medusomyces gisevii Sa-12. The use of symbiotic Medusomyces gisevii Sa-12 was found to have technological benefits: nutrient media require no mineral salts or growth factors, and pasteurization is sufficient for the nutrient medium instead of sterilization. The yield of BNCs produced by the symbiotic culture turned out to be 44-65% higher than that for the individual strains. The physicochemical properties of BNC, such as nanofibril width, degree of polymerization, elastic modulus, Iα allomorph content and crystallinity index, are most notably dependent on the microbial producer type rather than the nutrient medium composition. This is the first study in which we investigated the biosynthesis of BNC on hydrolyzate media prepared from miscanthus and oat hulls under the same conditions but using different microbial producers, and showed that it is advisable to use the symbiotic culture. The choice of a microbial producer is grounded on the yield, production process simplification and properties. The BNC production from technical raw materials would cover considerable demands of BNC for technical purposes without competing with food resources.

Bacterial Nanocellulose Derived from Banana Leaf Extract: Yield and Variation Factors

Bananas are one of the most important crops worldwide. However, a large amount of residual lignocellulosic biomass is generated during its production and is currently undervalued. These residues have the potential to be used as feedstock in bio-based processes with a biorefinery approach. This work is based on the valorization of banana leaf and has the following objectives (i) to determine the effect of certain physical and environmental factors on the concentration of glucose present in banana leaf extract (BLE), using a statistical regression model; (ii) to obtain Bacterial Nanocellulose (BNC), using BLE (70% v/v) and kombucha tea as fermentation medium. In addition, the physicochemical properties of BNC were evaluated by X-ray diffraction (XRD), Fourier transform infrared (FTIR), and thermogravimetric analysis (TGA). The results indicate that storage time, location, leaf color, and petiole type are factors related to BLE concentration, which is reduced by approximately 28.82% and 64.32% during storage times of five days. Regarding BNC biosynthesis, the results indicate that the highest yield, 0.031 g/g, was obtained at 21 days. Furthermore, it was determined that the highest production rate was 0.11 gL−1h−1 at 11 days of fermentation. By FTIR, it was determined that the purification step with NaOH (3M) should be carried out for approximately two hours. This research supports the development of a circular bioeconomy around the banana value chain, as it presents a way of bioprocessing residual biomass that can be used to produce bioproducts.

Bacterial nanocellulose and softwood pulp for composite paper

Abstract: Scaling biosynthesis of bacterial nanocellulose (BNC) allowed samples of composite paper with an increased proportion of BNC to be obtained. This work aims to study BNC samples and bleached soft wood kraft pulp (BSKP) composite paper with a ratio of components varying across a wide range: 10:90, 30:70, 50:50, 60:40, 70:30, 90:10. The method of paper manufacturing was chosen based on the determinations of strength and deformation properties of composite samples with the BNC:BSKP ratio of 20:80. Surface application of BNT on BSKP handsheet provided for an increase in the strength values (tear resistance – by 37%, burst index – by 17%) and deformation characteristics (tension stiffness – by 66%, fracture work – by 8%, breaking length – by 4%) compared to a reference sample. The formation of composites is confirmed in all samples. Scanning electron spectroscopy revealed that paper composites comprise interlaced micro BSKP and nano BNC fibres. As the proportion of BNC in composites elevated, densification of the structure was observed due to an increased fraction of cross-linked nanosized elements. IR spectroscopy indicated the resemblance of cellulose structure in all samples. It was found that an increase in the degree of polymerisation of composite paper is directly proportional to an increase in the BNC amount in the samples. The filtering ability of composite paper samples against microorganisms in the culture liquid of the Medusomyces gisevii Sa-12 producer was studied. It should be noted that yeast retention is achieved with 70% BNC in the paper composite. The presented properties of the new material determine prospects for its use in filtering microorganisms.

Scale-Up of Biosynthesis Process of Bacterial Nanocellulose.

Bacterial nanocellulose (BNC) is a unique product of microbiological synthesis, having a lot of applications among which the most important is biomedicine. Objective complexities in scaling up the biosynthesis of BNC are associated with the nature of microbial producers for which BNC is not the target metabolite, therefore biosynthesis lasts long, with the BNC yield being small. Thus, the BNC scale-up problem has not yet been overcome. Here we performed biosynthesis of three scaled sheets of BNC (each having a surface area of 29,400 cm2, a container volume of 441 L, and a nutrient medium volume of 260 L and characterized them. The static biosynthesis of BNC in a semisynthetic nutrient medium was scaled up using the Medusomyces gisevii Sa-12 symbiotic culture. The experiment was run in duplicate. The BNC pellicle was removed once from the nutrient medium in the first experiment and twice in the second experiment, in which case the inoculum and glucose were not additionally added to the medium. The resultant BNC sheets were characterized by scanning electron microscopy, capillary viscosimetry, infrared spectroscopy, thermomechanical and thermogravimetric analyses. When the nutrient medium was scaled up from 0.1 to 260 L, the elastic modulus of BNC samples increased tenfold and the degree of polymerization 2.5-fold. Besides, we demonstrated that scaled BNC sheets could be removed at least twice from one volume of the nutrient medium, with the yield and quality of BNC remaining the same. Consequently, the world’s largest BNC sheets 210 cm long and 140 cm wide, having a surface area of 29,400 cm2 each (weighing 16.24 to 17.04 kg), have been obtained in which an adult with burns or vast wounds can easily be wrapped. The resultant sheets exhibit a typical architecture of cellulosic fibers that form a spatial 3D structure which refers to individual and extremely important characteristics of BNC. Here we thus demonstrated the scale-up of biosynthesis of BNC with improved properties, and this result was achieved by using the symbiotic culture.

ENGINEERING OF BIOSYNTHESIS STAGE OF BACTERIAL NANOCELLULOSE USING МEDUSOMYCES GISEVII SA-12

The extended cultivation with metabolite extraction and the cyclic cultivation of bacterial nanocellulose (BNC) have, for the first time, been examined herein. The biosynthesis of BNC has been scaled up in a 440-L vessel, which is currently the largest scale-up in the world practice.

BIOSYNTHESIS AND FUNCTIONALIZATION OF BACTERIAL NANOCELLULOSE FOR INDUSTRIAL APPLICATIONS

This study aimed at scale-up the biosynthesis of bacterial nanocellulose (BNC) and its functionalization with the production of cellulose nitrates (CNs).

Bacterial Nanocellulose Nitrates.

Bacterial nanocellulose (BNC) whose biosynthesis fully conforms to green chemistry principles arouses much interest of specialists in technical chemistry and materials science because of its specific properties, such as nanostructure, purity, thermal stability, reactivity, high crystallinity, etc. The functionalization of the BNC surface remains a priority research area of polymers. The present study was aimed at scaled production of an enlarged BNC sample and at synthesizing cellulose nitrate (CN) therefrom. Cyclic biosynthesis of BNC was run in a semisynthetic glucose medium of 10−72 L in volume by using the Medusomyces gisevii Sa-12 symbiont. The most representative BNC sample weighing 6800 g and having an α-cellulose content of 99% and a polymerization degree of 4000 was nitrated. The nitration of freeze-dried BNC was performed with sulfuric-nitric mixed acid. BNC was examined by scanning electron microscopy (SEM) and infrared spectroscopy (IR), and CN was explored to a fuller extent by SEM, IR, thermogravimetric analysis/differential scanning analysis (TGA/DTA) and 13C nuclear magnetic resonance (NMR) spectroscopy. The three-cycle biosynthesis of BNC with an increasing volume of the nutrient medium from 10 to 72 L was successfully scaled up in nonsterile conditions to afford 9432 g of BNC gel-films. CNs with a nitrogen content of 10.96% and a viscosity of 916 cP were synthesized. It was found by the SEM technique that the CN preserved the 3D reticulate structure of initial BNC fibers a marginal thickening of the nanofibers themselves. Different analytical techniques reliably proved the resultant nitration product to be CN. When dissolved in acetone, the CN was found to form a clear high-viscosity organogel whose further studies will broaden application fields of the modified BNC.

Biofabrication of bacterial nanocellulose scaffolds with complex vascular structure

Bacterial nanocellulose (BNC) has proven to be an effective hydrogel-like material for different tissue engineering applications due to its biocompatibility and good mechanical properties. However, as for all biomaterials, in vitro biosynthesis of large tissue constructs remains challenging due to insufficient oxygen and nutrient transport in engineered scaffold-cell matrices. In this study we designed, biofabricated and evaluated bacterial nanocellulose scaffolds with a complex vascular mimetic lumen structure. As a first step a method for creating straight channeled structures within a bacterial nanocellulose scaffold was developed and evaluated by culturing of Human Umbilical Vein Endothelial Cells (HUVECs). In a second step, more complex structures within the scaffolds were produced utilizing a 3D printer. A print mimicking a vascular tree acted as a sacrificial template to produce a network within the nanoporous bacterial nanocellulose scaffolds that could be lined with endothelial cells. In a last step, a method to produce large constructs with interconnected macro porosity and vascular like lumen structure was developed. In this process patient data from x-ray computed tomography scans was used to create a mold for casting a full-sized kidney construct. By showing that the 3D printing technology can be combined with BNC biosynthesis we hope to widen the opportunities of 3D printing, while also enabling the production of BNC scaffolds constructs with tailored vascular architectures and properties.

Исследование биосинтеза бактериальной наноцеллюлозы продуцентом Medusomyces gisevii Sa-12 на ферментативном гидролизате продукта щелочной делигнификации мискантуса

A high demand for bacterial nanocellulose (BNC) in various economic sectors has resulted in intensification of studies aimed at searching for ways of reducing the cost of its production. BNC on an industrial scale is obtained using synthetic nutrient media, the cost of which may amount to 30–60% of the total cost of the process. Therefore, the study of BNC biosynthesis using low-cost nutrient media, such as food and cellulosic waste and other alternative sources of raw materials, seems to be highly relevant. The biomass of miscanthus currently used as a raw material for biofuels may become such an alternative source. Miscanthus is characterised by availability, low cost and annual renewability on an industrial scale. This study was aimed at investigating the BNC biosynthesis process from the enzymatic hydrolyzate obtained by alkaline delignification of miscanthus. The delignification process involved treatment of the plant with a dilute sodium hydroxide solution at ambient pressures. Enzymatic hydrolysis of the obtained substrate was carried out in a 11L-fermenter. For the first time, BNC was successfully obtained in a nutrient medium from an enzymatic hydrolyzate produced by alkaline delignification of miscanthus. The symbiotic culture of Medusomyces gisevii Sa-12 characterised by a high adaptive potential was used as a producer. The maximum specific growth rates were 0.360 and 0.384 day -1 for yeast and acetic acid bacteria, respectively. Consumption of reducing substances was observed to proceed in two stages: the rate constant of substrate utilisation at the first and the second stages were equal to 0.464 day and 0.034 day -1 , respectively. The highest yield of BNC was 5.14% on the 14th day of cultivation, which is 1.8 times lower than that on a synthetic nutrient medium. Although the nutrient medium of the enzymatic hydrolyzate obtained by alkaline delignification of miscanthus is not biologically pure, Medusomyces gisevii Sa-12 shows a high technological potential and ability to synthesize a chemically pure BNC even under adverse conditions.

ВЛИЯНИЕ УСЛОВИЙ КУЛЬТИВИРОВАНИЯ НА БИОСИНТЕЗ БАКТЕРИАЛЬНОЙ НАНОЦЕЛЛЮЛОЗЫ

This paper describes conditions for the cultivation of bacterial nanocellulose using various primary producers. Research into parameters determining a high yield in the production of bacterial nanocellulose is highly relevant due to an increased demand in this product in various industries. Key parameters that affect the growth of cellulose-synthesizing bacteria and the biosynthesis of bacterial nanocellulose include the following: the concentration of the carbon source in the nutrient solution; aeration; cultivation temperature; active acidity level. The concentration of reducing substances in the nutrient solution can range from 6 to 100 g/l. The concentration of dissolved oxygen in the nutrient solution can be considered as a limiting factor for all cellulose-synthesizing microorganisms. It is shown that the temperature range for the bacterial nanocellulose biosynthesis can vary from 25 to 33°C for various primary producers. PH values that provide a maximal yield of bacterial nanocellulose are determined to range from 4 to 6.5 for various primary producers. The literature review has proven the importance of a case-by-case approach when selecting cultivation conditions for every primary producer so as to maximize the bacterial nanocellulose yield.

БИОСИНТЕЗ БАКТЕРИАЛЬНОЙ НАНОЦЕЛЛЮЛОЗЫ НА СРЕДАХ, ПОЛУЧЕННЫХ ИЗ ЦЕЛЛЮЛОЗОСОДЕРЖАЩЕГО СЫРЬЯ

In this research, we set out to conduct a thematic review of literature published on the processes of bacterial nanocellulose biosynthesis (BNC) using alternative culture media with the purpose of identifying key technological approaches in the field. The literature search and selection cover a period of 20 years. The analysis of the discovered patented technical solutions has shown BNC pro-duction to be a complicated and expensive process. Three main directions for reducing its costs have been identified: substitution of edible raw materials by non-edible ones; development of new engineering solutions permitting either a greater product yield or a higher BNC quality; isolation (from natural sources or creation with bioengineering technologies) of new micro-organism strains that possess a high biosynthetic action, thus enabling a higher product yield over a shorter time. A number of patents under investigation are shown to cover all three of the aforementioned directions. It is concluded that the identified directions in the BNC production can be considered promising for the development of new technical solutions.

Utilization of Post-Maceration Liquid from [i]Spartium junceum[/i] Enzymatic Retting in Biosynthesis of Bacterial Nanocellulose

Utilization of Post-Maceration Liquid from [i]Spartium junceum[/i] Enzymatic Retting in Biosynthesis of Bacterial Nanocellulose

Using In situ Dynamic Cultures to Rapidly Biofabricate Fabric-Reinforced Composites of Chitosan/Bacterial Nanocellulose for Antibacterial Wound Dressings.

Bacterial nano-cellulose (BNC) is considered to possess incredible potential in biomedical applications due to its innate unrivaled nano-fibrillar structure and versatile properties. However, its use is largely restricted by inefficient production and by insufficient strength when it is in a highly swollen state. In this study, a fabric skeleton reinforced chitosan (CS)/BNC hydrogel with high mechanical reliability and antibacterial activity was fabricated by using an efficient dynamic culture that could reserve the nano-fibrillar structure. By adding CS in culture media to 0.25–0.75% (w/v) during bacterial cultivation, the CS/BNC composite hydrogel was biosynthesized in situ on a rotating drum composed of fabrics. With the proposed method, BNC biosynthesis became less sensitive to the adverse antibacterial effects of CS and the production time of the composite hydrogel with desirable thickness could be halved from 10 to 5 days as compared to the conventional static cultures. Although, its concentration was low in the medium, CS accounted for more than 38% of the CS/BNC dry weight. FE-SEM observation confirmed conservation of the nano-fibrillar networks and covering of CS on BNC. ATR-FTIR showed a decrease in the degree of intra-molecular hydrogen bonding and water absorption capacity was improved after compositing with CS. The fabric-reinforced CS/BNC composite exhibited bacteriostatic properties against Escherichia coli and Staphylococcus aureus and significantly improved mechanical properties as compared to the BNC sheets from static culture. In summary, the fabric-reinforced CS/BNC composite constitutes a desired candidate for advanced wound dressings. From another perspective, coating of BNC or CS/BNC could upgrade the conventional wound dressings made of cotton gauze to reduce pain during wound healing, especially for burn patients.

Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants

The small size and heterogeneity of the pores in bacterial nanocellulose (BNC) hydrogels limit the ingrowth of cells and their use as tissue-engineered implant materials. The use of placeholders during BNC biosynthesis or post-processing steps such as (touch-free) laser perforation can overcome this limitation. Since three-dimensionally arranged channels may be required for homogeneous and functional seeding, three-dimensional (3-D) laser perforation of never-dried BNC hydrogels was performed. Never-dried BNC hydrogels were produced in different shapes by: (i) the cultivation of Gluconacetobacter xylinus (DSM 14666; synonym Komagataeibacter xylinus) in nutrient medium; (ii) the removal of bacterial residues/media components (0.1M NaOH; 30min; 100°C) and repeated washing (deionized water; pH 5.8); (iii) the unidirectional or 3-D laser perforation and cutting (pulsed CO2 Rofin SC ×10 laser; 220μm channel diameter); and (iv) the final autoclaving (2M NaOH; 121°C; 20min) and washing (pyrogen-free water). In comparison to unmodified BNC, unidirectionally perforated – and particularly 3-D-perforated – BNC allowed ingrowth into and movement of vital bovine/human chondrocytes throughout the BNC nanofiber network. Laser perforation caused limited structural modifications (i.e. fiber or globular aggregates), but no chemical modifications, as indicated by Fourier transform infrared spectroscopy, X-ray photoelectron scattering and viability tests. Pre-cultured human chondrocytes seeding the surface/channels of laser-perforated BNC expressed cartilage-specific matrix products, indicating chondrocyte differentiation. 3-D-perforated BNC showed compressive strength comparable to that of unmodified samples. Unidirectionally or 3-D-perforated BNC shows high biocompatibility and provides short diffusion distances for nutrients and extracellular matrix components. Also, the resulting channels support migration into the BNC, matrix production and phenotypic stabilization of chondrocytes. It may thus be suitable for in vivo application, e.g. as a cartilage replacement material.

In Situ Synthesis of Photocatalytically Active Hybrids Consisting of Bacterial Nanocellulose and Anatase Nanoparticles

Bacterial nanocellulose (BNC) is an extraordinary biopolymer with a wide range of potential technical applications. The high specific surface area and the interconnected pore system of the nanofibrillar BNC network suggest applications as a carrier of catalysts. The present paper describes an in situ modification route for the preparation of a hybrid material consisting of BNC and photocatalytically active anatase (TiO(2)) nanoparticles (NPs). The influence of different NP concentrations on the BNC biosynthesis and the resulting supramolecular structure of the hybrids was investigated. It was found that the number of colony forming units (CFUs) and the consumption of glucose during biosynthesis remained unaffected compared to unmodified BNC. During the formation of the BNC network, the NPs were incorporated in the whole volume of the accruing hybrid. Their distribution within the hybrid material is affected by the anisotropic structure of BNC. The photocatalytic activity (PCA) of the BNC-TiO(2) hybrids was determined by methanol conversion (MC) under UV irradiation. These tests demonstrated that the NPs retained their PCA after incorporation into the BNC carrier structure. The PCA of the hybrid material depends on the amount of incorporated NPs. No alteration of the photocatalyst's efficiency was found during repeated PCA tests. In conclusion, the in situ integration of photocatalytically active NPs into BNC represents an attractive possibility to extend its fields of application to porous filtering media for drinking water purification and air cleaning.