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.

Identification of Gluconacetobacter xylinus LYP25 and application to bacterial cellulose production in biomass hydrolysate with acetic acid.

Bacterial cellulose (BC) is a remarkable biomacromolecule with potential applications in food, biomedical, and other industries. However, the low economic feasibility of BC production processes hinders its industrialization. In our previous work, we obtained candidate strains with improved BC production through random mutations in Gluconacetobacter. In this study, the molecular identification of LYP25 strain with significantly improved productivity, the development of chestnut pericarp (CP) hydrolysate medium, and its application in BC fermentation were performed for cost-effective BC production process. As a result, the mutant strain was identified as Gluconacetobacter xylinus. The CP hydrolysate (CPH) medium contained 30 g/L glucose with 0.4 g/L acetic acid, whereas other candidates known to inhibit fermentation were not detected. Although acetic acid is generally known as a fermentation inhibitor, it improves the BC production by G. xylinus when present within about 5 g/L in the medium. Fermentation of G. xylinus LYP25 in CPH medium resulted in 17.3 g/L BC, a 33 % improvement in production compared to the control medium, and BC from the experimental and control groups had similar physicochemical properties. Finally, the overall process of BC production from biomass was evaluated and our proposed platform showed the highest yield (17.9 g BC/100 g biomass).

A recombinant strain of Komagataeibacter xylinus ATCC 23770 for production of bacterial cellulose from mannose-rich resources.

The development of bacterial cellulose (BC) industrialization has been seriously affected by its production. Mannose/mannan is an essential component in many biomass resources, but Komagataeibacter xylinus uses mannose in an ineffective way, resulting in waste. The aim of this study was to construct recombinant bacteria to use mannose-rich biomass efficiently as an alternative and inexpensive carbon source in place of the more commonly used glucose. This strategy aimed at modification of the mannose catabolic pathway via genetic engineering of K. xylinus ATCC 23770 strain through expression of mannose kinase and phosphomannose isomerase genes from the Escherichia coli K-12 strain. Recombinant and wild-type strains were cultured under conditions of glucose and mannose respectively as sole carbon sources. The fermentation process and physicochemical properties of BC were investigated in detail in the strains cultured in mannose media. The comparison showed that with mannose as the sole carbon source, the BC yield from the recombinant strain increased by 84%, and its tensile strength and elongation were increased 1.7 fold, while Young's modulus was increased 1.3 fold. The results demonstrated a successful improvement in BC yield and properties on mannose-based medium compared with the wild-type strain. Thus, the strategy of modifying the mannose catabolic pathway of K. xylinus is feasible and has significant potential in reducing the production costs for industrial production of BC from mannose-rich biomass.

Superabsorbent bacterial cellulose film produced from industrial residue of cashew apple juice processing.

The industrial residue of cashew apple juice processing (MRC) was evaluated as an alternative medium for bacterial cellulose (BC) production by Komagataeibacter xylinus ATCC 53582 and Komagataeibacter xylinus ARS B42. The synthetic Hestrin-Schramm medium (MHS) was used as a control for growing and BC production. First, BC production was assessed after 4, 6, 8, 10, and 12 days under static culture. After 12 days of cultivation, K. xylinus ATCC 53582 produced the highest BC titer in MHS (3.1 g·L-1) and MRC (3 g·L-1), while significant productivity was attained at 6 days of fermentation. To understand the effect of culture medium and fermentation time on the properties of the obtained films, BC produced at 4, 6, or 8 days were submitted to infrared spectroscopy with Fourier transform, thermogravimetry, mechanical tests, water absorption capacity, scanning electron microscopy, degree of polymerization and X-ray diffraction. The properties of BC synthesized in MRC were identical to those of BC from MHS, according to structural, physical, and thermal studies. MRC, on the other hand, allows the production of BC with a high water absorption capacity when compared to MHS. Despite the lower titer (0.88 g·L-1) achieved in MRC, the BC from K. xylinus ARS B42 presented a high thermal resistance and a remarkable absorption capacity (14664 %), suggesting that it might be used as a superabsorbent biomaterial.

Improved production of bacterial cellulose using Gluconacetobacter sp. LYP25, a strain developed in UVC mutagenesis with limited viability conditions.

Bacterial cellulose (BC), a natural polymer synthesized by bacteria, has received considerable attention owing to its impressive physicomechanical properties. However, the low productivity of BC-producing strains poses a challenge to industrializing this material and making it economically viable. In the present study, UV-induced random mutagenesis of Gluconacetobacter xylinus ATCC 53524 was performed to improve BC production. Sixty mutants were obtained from the following mutagenesis procedure: the correlation between UVC fluence and cell death was investigated, and a limited viability condition was determined as a UVC dose to kill 99.99 %. Compared to the control strain, BC production by the mutant strains LYP25 and LYP23 improved 46.4 % and 44.9 %, respectively. Fermentation profiling using the selected strains showed that LYP25 was superior in glucose consumption and BC production, 13.8 % and 41.0 %, respectively, compared to the control strain. Finally, the physicochemical properties of LYP25-derived BC were similar to those of the control strain; thus, the mutant strain is expected to be a promising producer of BC in the bio-industry based on improved productivity.

A novel static cultivation of bacterial cellulose production from sugar beet molasses: Series static culture (SSC) system.

In bacterial cellulose (BC) production, we developed a new static cultivation system named series static culture (SSC) to eliminate air limitation problem encountered in conventional static culture (CSC). In SSC system, the fermentation broth at the bottom of BC pellicle produced in initial culture medium is transferred to the next empty sterile culture medium at the end of a certain fermentation period. This procedure was performed until BC production ceased. Fermentation experiments were carried out using Gluconacetobacter xylinus NRRL B-759 and sugar beet molasses at 30 °C and initial pH 5. Also, some quality parameters of produced BC pellicles were determined. Final pH at the stages of SSC system was higher that of the initial pH due to sugar content (sucrose) of molasses and microorganism used. Total BC production increased with increasing sugar concentration in SSC. As a result, an increase of 22.02 % in BC production was achieved using developed SSC. FT-IR spectra of all BC pellicles produced were typical spectra. The absorption bands at the relevant wavenumbers identify the mode of vibrations of the created chemical bonds arising at the BC surface such as OH, CH, H-O-H, C-O-C, and C-OH. XRD analyses showed that the crystallinity index values of BC obtained from CCS and SSC were high. The form of produced all BC pellicles is generally Cellulose I. Removal of surface moisture and depolymerisation of carbon skeleton were determined from TGA-DTA thermograms. SEM images showed that the BC samples produced had nano-sized cellulose fibrils which were aggregated in fermentation media containing molasses. Finally, the BC samples, especially in molasses media, having high mechanical strength and WHC were found.

Enhanced production of fibrous bacterial cellulose in Gluconacetobacter xylinus culture medium containing modified protein of okara waste.

In Gluconacetobacter xylinus cultivation for bacterial nanocellulose production, agro-industrial wastes, soybean residual okara, okara extracted protein, and modified okara protein, were used as a protein source. In comparison with homogenized raw okara and protein extracted from raw okara, acetic-acid modified protein provided the higher cellulose yield (2.8 g/l at 3 %w/v protein concentration) due to the improved protein solubility in the culture medium (89 %) and smaller particle size (0.2 µm) leading to facile uptake by the bacteria. Importantly, pH of the culture medium containing the modified protein measured before and after the cultivation was similar, suggesting the buffering capacity of the protein. Nanocellulose fibers were then produced densely in the network of hydrogels with high crystallinity nearly 90 %. Based on the results, economic constraints around nanocellulose production could be alleviated by valorization of okara waste, which provided enhanced sustainability.

Microbial Production of Bacterial Cellulose Using Chestnut Shell Hydrolysates by Gluconacetobacter xylinus ATCC 53524.

Bacterial cellulose (BC) is gaining attention as a carbon-neutral alternative to plant cellulose, and as a means to prevent deforestation and achieve a carbon-neutral society. However, the high cost of fermentation media for BC production is a barrier to its industrialization. In this study, chestnut shell (CS) hydrolysates were used as a carbon source for the BC-producing bacteria strain, Gluconacetobacter xylinus ATCC 53524. To evaluate the suitability of the CS hydrolysates, major inhibitors in the hydrolysates were analyzed, and BC production was profiled during fermentation. CS hydrolysates (40 g glucose/l) contained 1.9 g/l acetic acid when applied directly to the main medium. As a result, the BC concentration at 96 h using the control group and CS hydrolysates was 12.5 g/l and 16.7 g/l, respectively (1.3-fold improved). In addition, the surface morphology of BC derived from CS hydrolysates revealed more densely packed nanofibrils than the control group. In the microbial BC production using CS, the hydrolysate had no inhibitory effect during fermentation, suggesting it is a suitable feedstock for a sustainable and eco-friendly biorefinery. To the best of our knowledge, this is the first study to valorize CS by utilizing it in BC production.

Enhancing bacterial cellulose production with hypoxia-inducible factors.

Komagataeibacter xylinus is an aerobic strain that produces bacterial cellulose (BC). Oxygen levels play a critical role in regulating BC synthesis in K. xylinus, and an increase in oxygen tension generally means a decrease in BC production. Fumarate nitrate reduction protein (FNR) and aerobic respiration control protein A (ArcA) are hypoxia-inducible factors, which can signal whether oxygen is present in the environment. In this study, FNR and ArcA were used to enhance the efficiency of oxygen signaling in K. xylinus, and globally regulate the transcription of the genome to cope with hypoxic conditions, with the goal of improving growth and BC production. FNR and ArcA were individually overexpressed in K. xylinus, and the engineered strains were cultivated under different oxygen tensions to explore how their overexpression affects cellular metabolism and regulation. Although FNR overexpression did not improve BC production, ArcA overexpression increased BC production by 24.0% and 37.5% as compared to the control under oxygen tensions of 15% and 40%, respectively. Transcriptome analysis showed that FNR and ArcA overexpression changed the way K. xylinus coped with oxygen tension changes, and that both FNR and ArcA overexpression enhanced the BC synthesis pathway. The results of this study provide a new perspective on the effect of oxygen signaling on growth and BC production in K. xylinus and suggest a promising strategy for enhancing BC production through metabolic engineering. KEY POINTS: • K. xylinus BC production increased after overexpression of ArcA • The young's modulus is enhanced by the ArcA overexpression • ArcA and FNR overexpression changed how cells coped with changes in oxygen tension.

Biotropic liquid crystal phase transformations in cellulose-producing bacterial communities.

Biological functionality is often enabled by a fascinating variety of physical phenomena that emerge from orientational order of building blocks, a defining property of nematic liquid crystals that is also pervasive in nature. Out-of-equilibrium, "living" analogs of these technological materials are found in biological embodiments ranging from myelin sheath of neurons to extracellular matrices of bacterial biofilms and cuticles of beetles. However, physical underpinnings behind manifestations of orientational order in biological systems often remain unexplored. For example, while nematiclike birefringent domains of biofilms are found in many bacterial systems, the physics behind their formation is rarely known. Here, using cellulose-synthesizing Acetobacter xylinum bacteria, we reveal how biological activity leads to orientational ordering in fluid and gel analogs of these soft matter systems, both in water and on solid agar, with a topological defect found between the domains. Furthermore, the nutrient feeding direction plays a role like that of rubbing of confining surfaces in conventional liquid crystals, turning polydomain organization within the biofilms into a birefringent monocrystal-like order of both the extracellular matrix and the rod-like bacteria within it. We probe evolution of scalar orientational order parameters of cellulose nanofibers and bacteria associated with fluid-gel and isotropic-nematic transformations, showing how highly ordered active nematic fluids and gels evolve with time during biological-activity-driven, disorder-order transformation. With fluid and soft-gel nematics observed in a certain range of biological activity, this mesophase-exhibiting system is dubbed "biotropic," analogously to thermotropic nematics that exhibit solely orientational order within a temperature range, promising technological and fundamental-science applications.

Stoichiometric Analysis and Production of Bacterial Cellulose by Gluconacetobacter liquefaciens using Borassus flabellifer L. Jaggery.

The objective of the work is to examine the potential utilization of Palmyra palm jaggery (PPJ) for the enhancement of bacterial cellulose (BC) production by Gluconacetobacter liquefaciens. To evaluate the culturing condition, the production of BC fermentation was carried out in batch mode using different carbon sources namely glucose, sucrose and PPJ. PPJ in the HS medium (PHS medium) resulted maximum concentration of BC (14.35 ± 0.18 g/L) under shaking condition than other carbon sources in HS medium. The influence of different medium variables including initial pH and nitrogen sources on BC production was investigated using PHS medium under shaking condition. The maximum BC concentration of 17.79 ± 2.4 g/L was obtained in shaking condition at an initial pH of 5.6 using yeast extract as nitrogen source. Stoichiometric equation for the cell growth and BC synthesis was developed using elemental balance approach. The metabolic heat of reaction (40 kcal generated per liter of medium) was evaluated using electron balance approach. Based on the process economic analysis and the yield of BC during the fermentation, PHS medium without nitrogen source could be a promising cost-effective nutrient than HS medium. Thermal stability, crystallinity index and structural characterizations of produced BC using PPJ medium were evaluated using TGA, XRD and FTIR and the obtained results were compared with HS medium containing glucose and sucrose.

[Regulating the structure of bacterial cellulose by altering the expression of bcsD using CRISPR/dCas9].

Gluconacetobacter xylinus is a primary strain producing bacterial cellulose (BC). In G. xylinus, BcsD is a subunit of cellulose synthase and is participated in the assembly process of BC. A series of G. xylinus with different expression levels of the bcsD gene were obtained by using the CRISPR/dCas9 technique. Analysis of the structural characteristics of BC showed that the crystallinity and porosity of BC changed with the expression of bcsD. The porosity varied from 59.95%-84.05%, and the crystallinity varied from 74.26%-93.75%, while the yield of BC did not decrease significantly upon changing the expression levels of bcsD. The results showed that the porosity of bacterial cellulose significantly increased, while the crystallinity was positively correlated with the expression of bcsD, when the expression level of bcsD was below 55.34%. By altering the expression level of the bcsD gene, obtaining BC with different structures but stable yield through a one-step fermentation of G. xylinus was achieved.

Effect of Culture Conditions on Cellulose Production by a Komagataeibacter xylinus Strain.

Bacterial cellulose (BC) is an abundant biopolymer with a wide range of potential industrial applications. However, the industrial application of BC has been hampered by inefficient production. This study aims to investigate the influence of a spontaneous mutation that results in decreased cellulose production by a Komagataeibacter xylinus strain. The yields of cellulose are significantly different under different culture conditions, which imply that the shearing force is responsible for the selection of spontaneous mutants. Fermenter culture conditions under shake-flask culture conditions are further simulated. The shearing force activates the conversion of microbial cells to Cel- mutants, and the accumulation of water-soluble exopolysaccharides is observed. The Cel+ cells under agitated culture are not easily converted into Cel- mutants upon the addition of water-soluble exopolysaccharides synthesized by K. xylinus and a viscous polysaccharide, such as xanthan gum. The conversion ratio of Cel+ cells to Cel- mutants is strongly related to the shearing force and viscosity of the fermentation broth. The synthetic pathways of bacterial cellulose and water-soluble polysaccharides are independent of each other at the genetic level. However, a substrate competitive relationship between these two polysaccharides is found, which is significant in terms of the optimization of cellulose production in commercial processes.

Improved water dispersion and bioavailability of coenzyme Q10 by bacterial cellulose nanofibers.

The purpose of this study was to investigate the potential of bacterial cellulose nanofiber suspension (BCNs) as stabilizer in anti-solvent precipitation and its effect on improving bioavailability of coenzyme Q10. Bacterial cellulose (BC) was hydrolyzed by sulfuric acid followed by the oxidation with hydrogen peroxide to prepare BCNs. The suspension of BCNs-loaded CoQ10 (CoQ10-BCNs) were prepared by antisolvent precipitation. The zeta potential of CoQ10-BCNs was about -36.01 mV. The properties of CoQ10, BCNs and CoQ10-BCNs were studied by scanning electron microscopy, transmission electron microscope, Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry and thermo gravimetric analysis. The crystallinity of CoQ10 decreased in CoQ10-BCNs compared with the raw CoQ10, and CoQ10-BCNs have good physicochemical stability. In oral bioavailability studies, the area under curve (AUC) of CoQ10-BCNs was about 3.62 times higher than the raw CoQ10 in rats.

Bacterial Cellulose: Production, Characterization, and Application as Antimicrobial Agent.

Bacterial cellulose (BC) is recognized as a multifaceted, versatile biomaterial with abundant applications. Groups of microorganisms such as bacteria are accountable for BC synthesis through static or agitated fermentation processes in the presence of competent media. In comparison to static cultivation, agitated cultivation provides the maximum yield of the BC. A pure cellulose BC can positively interact with hydrophilic or hydrophobic biopolymers while being used in the biomedical domain. From the last two decades, the reinforcement of biopolymer-based biocomposites and its applicability with BC have increased in the research field. The harmony of hydrophobic biopolymers can be reduced due to the high moisture content of BC in comparison to hydrophilic biopolymers. Mechanical properties are the important parameters not only in producing green composite but also in dealing with tissue engineering, medical implants, and biofilm. The wide requisition of BC in medical as well as industrial fields has warranted the scaling up of the production of BC with added economy. This review provides a detailed overview of the production and properties of BC and several parameters affecting the production of BC and its biocomposites, elucidating their antimicrobial and antibiofilm efficacy with an insight to highlight their therapeutic potential.

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.

Bacterial cellulose/lignin nanoparticles composite films with retarded biodegradability.

In practical applications, the full biodegradability of all-biomass-based bacterial cellulose (BC) composites enhances their environmentally friendliness but results in the poor durability especially at humid conditions. This work prepared BC/lignin nanoparticles (LNPs) composite films with retarded biodegradability, which could broaden their application area. Three LNPs were fabricated using technical lignins extracted by deep eutectic solvent (DES), ethanol organosolv, soda/anthraquinone from poplar. LNPs involvement during BC fermentation showed limited influence on its productivity but significantly retarded the biodegradation of composite films. The potential inhibition mechanism was physical barrier and non-productive binding of LNPs. The BC/Soda LNPs showed much higher retarded degradation property (~58 wt% degradation) compared to BC/Organosolv LNPs and BC/DES LNPs (~85 wt% and ~ 97 wt% degradation respectively) at high enzyme loadings of 5 mg g-1 BCE. While at low enzyme loadings of 1 mg g-1 BCE, all these three composite films showed comparable retarded degradation property of ~60 wt%.

Characterization of Bacterial Cellulose Produced by Acetobacter xylinum Strain LKN6 Using Sago Liquid Waste as Nutrient Source.

<b>Background and Objective:</b> Bacterial Cellulose (BC) is an exopolysaccharide produced by bacteria with unique structural and mechanical properties and is highly pure compared to plant cellulose. This study aimed to produce novel bacterial cellulose using sago liquid waste substrate and evaluate its characteristics as a potential bioplastic.<b>Materials and Methods:</b> Production of BC by static batch fermentation was studied in sago liquid waste substrate usingAcetobacter xylinumLKN6. The BC structure was analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform infrared spectroscopy (FT-IR). Mechanical properties were measured include tensile strength, elongation at break, elasticity (Young's modulus) and Water Holding Capacity (WHC). <b>Results:</b> The BC yield from sago liquid waste as a nutrients source was achieved 12.37 g L¹ and the highest BC yield 14.52 g L¹ in sago liquid waste medium with a sugar concentration of 10% (w/v) after 14 days fermentation period. The existence of bacterial cellulose is proven by FT-IR spectroscopy analysis based on the appearance of absorbance peaks, which are C-C bonding, C-O bonding, C-OH bonding and C-O-C bonding and represents the fingerprints of pure cellulose. The mechanical properties of BC from sago liquid waste were showed a tensile strength of 44.2-87.3 MPa, elongation at break of 4.8-5.8%, Young's Modulus of 0.86-1.64 GPa and water holding capacity of 85.9-98.6 g g¹. <b>Conclusion:</b> The results suggest that sago liquid waste has great potential to use as a nutrient source in the production of bacterial cellulose and BC's prospect as the bioplastic.

Soft Magnetostrictive Actuator String with Cellulose Nanofiber Skin.

The formation of cellulose nanofibrous skin with a colloidal suspension is challenging due to the diffusion of colloidal particles and bacteria to the bulk and a limited supply of oxygen for bacteria in the liquid culture environment. A composite-actuating string was fabricated with magnetic nanoparticles (MNPs) and Gluconacetobacter xylinus in a solid matrix of hydrophobic microparticles. G. xylinus synthesizes a dense skin layer of cellulose nanofibers enclosing MNPs in the solid matrix to form an actuator string responsive to an external magnetic field. The nanofibrous actuator string is transformable to fit the diverse shapes of tubular structures in cross section due to its softness and plastic deformability, which reduce friction and stress against the walls of organ tissues. The nanofibrous skin string is bendable at an acute angle by magnetic actuation and is applicable as an endoscopic guidewire to reach a target deep inside a model kidney structure.

Production of bacterial cellulose tubes for biomedical applications: Analysis of the effect of fermentation time on selected properties.

Biosynthesis of bacterial cellulose (BC) in cylindrical oxygen permeable molds allows the production of hollow tubular structures of increasing interest for biomedical applications (artificial blood vessels, ureters, urethra, trachea, esophagus, etc.). In the current contribution a simple set-up is used to obtain BC tubes of predefined dimensions; and the effects of fermentation time on the water holding capacity, nanofibrils network architecture, specific surface area, chemical purity, thermal stability, mechanical properties, and cell adhesion, proliferation and migration of BC tubes are systematically analysed for the first time. The results reported highlight the role of culture time on key properties of the BC tubes produced, with significant differences arising from the denser and more compact fibril arrangements generated at longer fermentation intervals.