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).

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.

Cell-Laden 3D Hydrogels of Type I Collagen Incorporating Bacterial Nanocellulose Fibers.

There is a growing interest in developing natural hydrogel-based scaffolds to culture cells in a three-dimensional (3D) millieu that better mimics the in vivo cells' microenvironment. A promising approach is to use hydrogels from animal tissues, such as decellularized extracellular matrices; however, they usually exhibit suboptimal mechanical properties compared to native tissue and their composition with hundreds of different protein complicates to elucidate which stimulus triggers cell's responses. As simpler scaffolds, type I collagen hydrogels are used to study cell behavior in mechanobiology even though they are also softer than native tissues. In this work, type I collagen is mixed with bacterial nanocellulose fibers (BCf) to develop reinforced scaffolds with mechanical properties suitable for 3D cell culture. BCf were produced from blended pellicles biosynthesized from Komagataeibacter xylinus. Then, BCf were mixed with concentrated collagen from rat-tail tendons to form composite hydrogels. Confocal laser scanning microscopy and scanning electron microscopy images confirmed the homogeneous macro- and microdistribution of both natural polymers. Porosity analysis confirmed that BCf do not disrupt the scaffold structure. Tensile strength and rheology measurements demonstrated the reinforcement action of BCf (43% increased stiffness) compared to the collagen hydrogel while maintaining the same viscoelastic response. Additionally, this reinforcement of collagen hydrogels with BCf offers the possibility to mix cells before gelation and then proceed to the culture of the 3D cell scaffolds. We obtained scaffolds with human bone marrow-derived mesenchymal stromal cells or human fibroblasts within the composite hydrogels, allowing a homogeneous 3D viable culture for at least 7 days. A smaller surface shrinkage in the reinforced hydrogels compared to type I collagen hydrogels confirmed the strengthening of the composite hydrogels. These collagen hydrogels reinforced with BCf might emerge as a promising platform for 3D in vitro organ modeling, tissue-engineering applications, and suitable to conduct fundamental mechanobiology studies.

Production efficiency and properties of bacterial cellulose membranes in a novel grape pomace hydrolysate by Komagataeibacter melomenusus AV436T and Komagataeibacter xylinus LMG 1518.

The microbial production of cellulose using different bacterial species has been extensively examined for various industrial applications. However, the cost-effectiveness of all these biotechnological processes is strongly related to the culture medium for bacterial cellulose (BC) production. Herein, we examined a simple and modified procedure for preparing grape pomace (GP) hydrolysate, without enzymatic treatment, as a sole growth medium for BC production by acetic acid bacteria (AAB). The central composite design (CCD) was used to optimise the GP hydrolysate preparation toward the highest reducing sugar contents (10.4 g/L) and minimal phenolic contents (4.8 g/L). The experimental screening of 4 differently prepared hydrolysates and 20 AAB strains identified the recently described species Komagataeibacter melomenusus AV436T as the most efficient BC producer (up to 1.24 g/L dry BC membrane), followed by Komagataeibacter xylinus LMG 1518 (up to 0.98 g/L dry BC membrane). The membranes were synthesized in only 4 days of bacteria culturing, 1 st day with shaking, followed by 3 days of static incubation. The produced BC membranes in GP-hydrolysates showed, in comparison to the membranes made in a complex RAE medium 34 % reduction of crystallinity index with the presence of diverse cellulose allomorphs, presence of GP-related components within the BC network responsible for the increase of hydrophobicity, the reduction of thermal stability and 48.75 %, 13.6 % and 43 % lower tensile strength, tensile modulus, and elongation, respectively. Here presented study is the first report on utilising a GP-hydrolysate without enzymatic treatment as a sole culture medium for efficient BC production by AAB, with recently described species Komagataeibacter melomenusus AV436T as the most efficient producer in this type of food-waste material. The scale-up protocol of the scheme presented here will be needed for the cost-optimisation of BC production at the industrial levels.

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.

Improvement in bacterial cellulose production by co-culturing Bacillus cereus and Komagataeibacter xylinus.

Bacterial cellulose (BC) is a bio-produced nanostructure material widely used in biomedical, food, and paper-manufacturing industries. However, low production efficiency and high-cost have limited its industrial applications. This study aimed to examine the level of improvement in BC production by co-culturing Bacillus cereus and Komagataeibacter xylinus. The BC yield in corn stover enzymatic hydrolysate was found to be obviously enhanced from 1.2 to 4.4 g/L after the aforementioned co-culturing. The evidence indicated that acetoin (AC) and 2,3-butanediol (2,3-BD) produced by B. cereus were the key factors dominating BC increment. The mechanism underlying BC increment was that AC and 2,3-BD increased the specific activity of AC dehydrogenase and the contents of adenosine triphosphate (ATP) and acetyl coenzyme A (acetyl-CoA), thus promoting the growth and energy level of K. xylinus. Meanwhile, the immobilization of BC could also facilitate oxygen acquisition in B. cereus under static conditions. This study was novel in reporting that the co-culture could effectively enhance BC production from the lignocellulosic enzymatic hydrolysate.

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.

Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin-Schramm medium.

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch-1404 and hydroxypropyl starch-1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.

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.

Economical Optimization of Industrial Medium Culture for Bacterial Cellulose Production.

The competitiveness of bacterial cellulose (BC) production with plant cellulose can be achieved by production on cost-effective media. It was found that the bacterial cell number ratio of BC to culture medium increases over time so that from the fourth day, the entrapped cell number in the cellulose network exceeds the suspended cells. Optimization based on 23-full factorial showed that inoculum development at 50 rpm and the main culture process under static conditions significantly increases BC production. A cost-effective culture medium containing molasses (ML) and corn steep liquor (CSL) was developed based on the same C/N ratio to HS medium, with 7.24 g/l cellulose at C/N ratio 12.6 is competitive with maximum production 8.7 g/L in HS medium. The BC production cost was reduced about 94% using the proposed cheap and locally available medium containing ML and CSL, while BC mechanical properties increased by about 50%.

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.

Polyethylene terephthalate hydrolysate increased bacterial cellulose production.

A major challenge to large-scale production and utilization of bacterial cellulose (BC) for various applications is its low yield and productivity by bacterial cells and the high cost of feedstock. A supplementation of the classical expensive Hestrin and Schramm (HS) medium with 1 % polyethylene terephthalate ammonia hydrolysate (PETAH) resulted in 215 % high yield. Although the physicochemical properties of BC were not significantly influenced, the BC produced in 1 % PETAH-supplemented HS medium showed a higher surface area, which showed 1.39 times higher adsorption capacity for tetracycline than BC produced in HS medium. The 1 % PETAH-supplemented HS medium respectively enhanced the activity of α-UDP-glucose pyrophosphorylase and α-phosphoglucomutase by 30.63 % and 135.24 % and decreased the activity of pyruvate kinase and phosphofructokinase by 40.34 % and 52.63 %. The results of this study provide insights into the activation mechanism of Taonella mepensis by PETAH supplementation for high yield and productivity of BC.

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.

Characterization of the Putative Acylated Cellulose Synthase Operon in Komagataeibacter xylinus E25.

Bacterial cellulose is a natural polymer with an expanding array of applications. Because of this, the main cellulose producers of the Komagataeibacter genus have been extensively studied with the aim to increase its synthesis or to customize its physicochemical features. Up to now, the genetic studies in Komagataeibacter have focused on the first cellulose synthase operon (bcsI) encoding the main enzyme complex. However, the role of other accessory cellulose operons has been understudied. Here we aimed to fill this gap by performing a detailed analysis of the second cellulose synthase operon (bcsII), which is putatively linked with cellulose acylation. In this study we harnessed the genome sequence, gene expression and protein structure information of K. xylinus E25 and other Komagataeibacter species to discuss the probable features of bcsII and the biochemical function of its main protein products. The results of our study support the previous hypothesis that bcsII is involved in the synthesis of the acylated polymer and expand it by presenting the evidence that it may also function in the regulation of its attachment to the cell surface and to the crystalline cellulose fibers.

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.

Silicone polyether surfactant enhances bacterial cellulose synthesis and water holding capacity.

The versatility and unique properties of bacterial cellulose (BC) motivate research into enhancing its synthesis. Here a silicone polyether surfactant (SPS) was synthesized and tested as a non-nutritional additive to the cultivation media of Komagataeibacter xylinus. The addition of SPS to the Hestrin-Schramm (HS) medium resulted in a concentration-dependent decrease in surface tension from 59.57 ± 0.37 mN/m to 30.05 ± 0.41 mN/m (for 0.1% addition) that was correlated with an increased yield of BC, up to 37% wet mass for surfactant concentration close to its critical micelle concentration (0.008%). Physicochemical characterization of bacterial cellulose obtained in presence of SPS, showed that surfactant is not incorporated into BC structure and has a moderate effect on its crystallinity, thermal stability. Moreover, the water holding capacity was enhanced by over 40%. Importantly, obtained BC did not affect L929 murine fibroblast cell viability. We conclude that SPS provides an eco-friendly approach to increasing BC yield in static culture, enabling more widespread industrial and biomedical applications.

[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.