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.

The production of bacterial cellulose in Gluconacetobacter xylinus regulated by luxR overexpression of quorum sensing system.

Quorum sensing is a mechanism that facilitates cell-to-cell communication. Through signal molecular density for signal recognition, which leads to the regulation of some physiological and biochemical functions. Gluconacetobacter xylinus CGMCC 2955, which produces bacterial cellulose (BC), synthesizes the LuxR protein belonging to the LuxI/LuxR type QS system. Here, a luxR overexpression vector was transformed into G. xylinus CGMCC 2955. The overexpression of luxR increased the yield of BC by 15.6% after 16 days static culture and reduced the cell density by 15.5% after 120-h-agitated culture. The glucose was used up by G. xylinus-pMV24-luxR at 72-h-agitated fermentation, which 12 h earlier than the wild-type (WT). The total N-acylhomoserine lactones (AHL) content of the luxR-overexpressing strain and the WT strain attained 1367.9 ± 57.86 mg/L and 842.9 ± 54.22 mg/L, respectively. The C12-HSL and C14-HSL contents of G. xylinus-pMV24-luxR were 202 ± 21.66 mg/L and 409.6 ± 0.91 mg/L, which were significantly lower than that of WT. In contrast, C6-HSL showed opposite results. The difference of AHL content proved that overexpression of luxR improved the binding of AHL and showed preference for some specific AHL. The metabolic results demonstrated that upon glucose exhaustion, the consumption of gluconic acid was promoted by luxR overexpression, and the content of D- ( +)-trehalose, an antiretrograde metabolite, increased significantly. KEY POINTS: • The overexpression of luxR increased the yield of bacterial cellulose • The content of signal molecules was significantly different • Differential metabolites were involved in multiple metabolic pathways.

Tailoring bacterial cellulose structure through CRISPR interference-mediated downregulation of galU in Komagataeibacter xylinus CGMCC 2955.

Diverse applications of bacterial cellulose (BC) have different requirements in terms of its structural characteristics. culturing Komagataeibacter xylinus CGMCC 2955, BC structure changes with alterations in oxygen tension. Here, the K. xylinus CGMCC 2955 transcriptome was analyzed under different oxygen tensions. Transcriptome and genome analysis indicated that BC structure is related to the rate of BC synthesis and cell growth, and galU is an essential gene that controls the carbon metabolic flux between the BC synthesis pathway and the pentose phosphate (PP) pathway. The CRISPR interference (CRISPRi) system was utilized in K. xylinus CGMCC 2955 to control the expression levels of galU. By overexpressing galU and interfering with different sites of galU sequences using CRISPRi, we obtained strains with varying expression levels of galU (3.20-3014.84%). By testing the characteristics of BC, we found that the porosity of BC (range: 62.99-90.66%) was negative with galU expression levels. However, the crystallinity of BC (range: 56.25-85.99%) was positive with galU expression levels; galU expression levels in engineered strains were lower than those in the control strains. Herein, we propose a new method for regulating the structure of BC to provide a theoretical basis for its application in different fields.

Applications of cellulose and chitin/chitosan derivatives and composites as antibacterial materials: current state and perspectives.

The bacterial infections have always a serious problem to public health. Scientists are developing new antibacterial materials to overcome this problem. Polysaccharides are promising biopolymers due to their diverse biological functions, low toxicity, and high biodegradability. Chitin and chitosan have antibacterial properties due to their cationic nature, while cellulose/bacterial cellulose does not possess any antibacterial activity. Moreover, the insolubility of chitin in common solvents, the poor solubility of chitosan in water, and the low mechanical properties of chitosan have restricted their biomedical applications. In order to solve these problems, chemical modifications such as quaternization, carboxymethylation, cationization, or surface modification of these polymers with different antimicrobial agents, including metal and metal oxide nanoparticles, are carried out to obtain new materials with improved physiochemical and biological properties. This mini review describes the recent progress in such derivatives and composites with potential antibacterial applications.

Identification of Quorum-Sensing Molecules of N-Acyl-Homoserine Lactone in Gluconacetobacter Strains by Liquid Chromatography-Tandem Mass Spectrometry.

Many Gram-negative bacteria can regulate gene expression in a cell density-dependent manner via quorum-sensing systems using N-acyl-homoserine lactones (AHLs), which are typical quorum-sensing signaling molecules, and thus modulate physiological characteristics. N-acyl-homoserine lactones are small chemical molecules produced at low concentrations by bacteria and are, therefore, difficult to detect. Here, a biosensor system method and liquid chromatography-tandem mass spectrometry were combined to detect and assay AHL production. As demonstrated by liquid chromatography-tandem mass spectrometry, Gluconacetobacter xylinus CGMCC No. 2955, a Gram-negative acetic acid-producing bacterium and a typical bacterial cellulose (BC) biosynthesis strain, produces six different AHLs, including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. Gluconacetobacter sp. strain SX-1, another Gram-negative acetic acid-producing bacterium, which can synthesize BC, produces seven different AHLs including N-acetyl-homoserine lactone, N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, N-3-oxo-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-dodecanoyl-homoserine lactone, and N-tetradecanoyl-homoserine lactone. These results lay the foundation for investigating the relationship between BC biosynthesis and quorum-sensing systems.

Metabolic flux analysis of Gluconacetobacter xylinus for bacterial cellulose production.

Metabolic flux analysis was used to reveal the metabolic distributions in Gluconacetobacter xylinus (CGMCC no. 2955) cultured on different carbon sources. Compared with other sources, glucose, fructose, and glycerol could achieve much higher bacterial cellulose (BC) yields from G. xylinus (CGMCC no. 2955). The glycerol led to the highest BC production with a metabolic yield of 14.7 g/mol C, which was approximately 1.69-fold and 2.38-fold greater than that produced using fructose and glucose medium, respectively. The highest BC productivity from G. xylinus CGMCC 2955 was 5.97 g BC/L (dry weight) when using glycerol as the sole carbon source. Metabolic flux analysis for the central carbon metabolism revealed that about 47.96 % of glycerol was transformed into BC, while only 19.05 % of glucose and 24.78 % of fructose were transformed into BC. Instead, when glucose was used as the sole carbon source, 40.03 % of glucose was turned into the by-product gluconic acid. Compared with BC from glucose and fructose, BC from the glycerol medium showed the highest tensile strength at 83.5 MPa, with thinner fibers and lower porosity. As a main byproduct of biodiesel production, glycerol holds great potential to produce BC with superior mechanical and microstructural characteristics.

Fabrication of bacterial cellulose with TiO2-ZnO nanocomposites as a multifunctional membrane for water remediation.

Superhydrophilic/underwater superoleophobic (SUS) membrane technology has attracted extensive attention for water purification. However, the fabrication of multifunctional membranes to satisfy the complex wastewater treatment is still a big challenge. In this work, bacterial cellulose (BC) based multifunctional SUS membranes were designed for water purification. Membranes were prepared by blending BC nanofibers with TiO2 nanoparticles (NPs), and further modified by the in situ growth of ZnO-NPs. The composite membranes showed oil/water (o/w) separation under a small driving pressure (0.2-0.3 bar) with a flux rate of 8232.81 ± 212 L m-2h-1 and with a high separation efficiency (>99.9%). Membranes could also separate oil-in-water emulsion with a separation flux of 1498 ± 74 L m-2h-1 and with high efficiency (99.25%). Moreover, the composite membrane exhibited photocatalytic activity under visible light with a high efficiency (>92%). The composite membranes were also investigated for antibacterial activity against Gram-positive and Gram-negative bacterial strains. This work may inspire the fabrication of next-generation multifunctional membranes for wastewater treatment, particularly oily wastewater, dyes and microbial contaminated water.

In situ regulation of bacterial cellulose networks by starch from different sources or amylose/amylopectin content during fermentation.

Bacterial cellulose (BC) is a promising biopolymer, but its three-dimensional structure needs to be controllable to be used in multiple fields. BC has some advantages over other types of cellulose, not only in terms of purity and properties but also in terms of modification (in situ modification) during the synthesis process. Here, starches from different sources or with amylose/amylopectin content were added to the growth medium to regulate the structural properties of BC in-situ. The obtained BC membranes were further modified by superhydrophobic treatment for oil-water separation. Starches alter the viscosity of the medium, thus affecting bacterial motility and cellulose synthesis, and adhere to the microfibers, limiting their further polymerization and ultimately altering the membrane porosity, pore size, and mechanical properties perpendicular to the BC fibril layer direction. The average pore diameter of the BC/PS membrane increased by 1.94 times compared to the initial BC membrane. The chemically modified BC/PS membrane exhibited super-hydrophobicity (water contact angle 167°), high oil-water separation flux (dichloromethane, 23,205 Lm-2 h-1 MPa-1), high separation efficiency (>97%). The study provides a foundation for developing methods to regulate the network structure of BC and broaden its application.

Preparation and characterization of antibacterial bacterial cellulose/chitosan hydrogels impregnated with silver sulfadiazine.

Hydrogels with pH sensitivity and stable mechanical and antibacterial properties have many desirable qualities and broad applications. A hydrogel based on bacterial cellulose and chitosan, impregnated with silver sulfadiazine (<1% w/w), was prepared using glutaraldehyde as the crosslinking agent. The presence of SSd was confirmed by Fourier transform infrared spectroscopy. Micropore size, swelling ratio, pH- sensitivity, and gram positive and negative antibacterial properties were studied by disk diffusion and colony forming unit. X-ray diffraction confirmed the presence of amorphous and crystalline regions in the hydrogel matrix following addition of SSd. The elemental composition, morphology, and mechanical properties of the hydrogels were characterized. Incorporation of SSd into bacterial cellulose-chitosan hydrogels significantly improved their mechanical and antibacterial properties. The antibacterial activity against E. coli and S. aureus was evaluated and SSd-BC/Ch hydrogels are more toxic to S. aureus than to E. coli. We use FESEM to observe bacterial morphology before and after exposure to SSd-BC/Ch hydrogels. The BacLight LIVE/DEAD membrane permeability kit is used to evaluate the membrane permeability of bacteria. These antibacterial hydrogels have many promising applications in food packaging, tissue engineering, drug delivery, clinical, biotechnological, and biomedical fields.

Bacterial cellulose and its potential for biomedical applications.

Bacterial cellulose (BC) is an important polysaccharide synthesized by some bacterial species under specific culture conditions, which presents several remarkable features such as microporosity, high water holding capacity, good mechanical properties and good biocompatibility, making it a potential biomaterial for medical applications. Since its discovery, BC has been used for wound dressing, drug delivery, artificial blood vessels, bone tissue engineering, and so forth. Additionally, BC can be simply manipulated to form its derivatives or composites with enhanced physicochemical and functional properties. Several polymers, carbon-based nanomaterials, and metal nanoparticles (NPs) have been introduced into BC by ex situ and in situ methods to design hybrid materials with enhanced functional properties. This review provides comprehensive knowledge and highlights recent advances in BC production strategies, its structural features, various in situ and ex situ modification techniques, and its potential for biomedical applications.

Designing of bacterial cellulose-based superhydrophilic/underwater superoleophobic membrane for oil/water separation.

The oil/water (o/w) separation is a global challenge because of the increasing water contamination by oil spill accidents, and oil-containing wastewater produced by food, textile, and petrochemical industries. In this study, we have developed bacterial cellulose (BC) based superhydrophilic/underwater superoleophobic (SUS) membrane for o/w separation. The membrane was designed through a facile method by blending BC nanofibers with silica microparticles (SiO2-MPs), which was further modified by bio-inspired polydopamine (PDA) coatings. The composite membrane exhibited SiO2-MPs dependent o/w separation with a high separation efficiency of >99.9 % and a high flux rate of ∼10,660 Lm-2 h-1 while applying a small negative pressure (0.3-0.5 bar). The membrane with different content of SiO2-MPs also showed the potential to separate oil-in-water emulsion with the highest oil rejection of 98.2 % and the highest flux rate of ∼1250 Lm-2 h-1 on an ultra-low pressure <0.1 bar. Moreover, the membrane showed antifouling properties, recyclability, and stability in harsh conditions.

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.

Fabrication of Bacterial Cellulose-Based Dressings for Promoting Infected Wound Healing.

Bacterial cellulose (BC) holds several unique properties such as high water retention capability, flexibility, biocompatibility, and high absorption capacity. All these features make it a potential material for wound healing applications. However, it lacks antibacterial properties, which hampers its applications for infectious wound healings. This study reported BC-based dressings containing ε-polylysine (ε-PL), cross-linked by a biocompatible and mussel-inspired polydopamine (PDA) for promoting infectious wound healing. BC membranes were coated with PDA by a simple self-polymerization process, followed by treating with different contents of ε-PL. The resulted membranes showed strong antibacterial properties against tested bacteria by both in vitro and in vivo evaluations. The membranes also exhibited hemocompatibility and cytocompatibility by in vitro investigations. Moreover, the functionalized membranes promoted infected wound healing using Sprague-Dawley rats as a model animal. A complete wound healing was observed in the group treated with functionalized membranes, while wounds were still open for control and pure BC groups in the same duration. Histological investigations indicated that the thickness of newborn skin was greater and smoother in the groups treated with modified membranes in comparison to neat BC or control groups. These results revealed that the functionalized membranes have great potential as a dressing material for infected wounds in future clinical applications.

Development and antibacterial activities of bacterial cellulose/graphene oxide-CuO nanocomposite films.

The absence of antibacterial activity of bacterial cellulose (BC) restricts its applications in the biomedical field. To introduce antimicrobial properties into BC, we studied the synthesis, structure, and antimicrobial properties of a novel nanocomposite film comprising BC, graphene oxide (GO), and copper-oxide (CuO) nanosheets. The nanocomposite film was synthesized by incorporating GO-CuO nanohybrids into BC matrix through homogenized blending. The CuO nanosheets, with a length range of 50 nm-200 nm and width range of 20 nm-50 nm, which were uniformly grown on the GO along with even distribution of GO-CuO nanohybrids on the surface of the cellulose fibers. The nanocomposites displayed better antibacterial activity against gram-positive than gram-negative bacteria. BC/GO-CuO nanocomposites showed higher antibacterial activity than BC/CuO. We also elucidated the mechanism of antibacterial activity of the nanocomposites. Further, the nanocomposites exhibited biocompatibility towards mice fibroblast cells. The nanocomposites might serve as an excellent source for development of antibacterial materials.

A Lambda Red and FLP/FRT-Mediated Site-Specific Recombination System in Komagataeibacter xylinus and Its Application to Enhance the Productivity of Bacterial Cellulose.

Komagataeibacter xylinus has received increasing attention as an important microorganism for the conversion of several carbon sources to bacterial cellulose (BC). However, BC productivity has been impeded by the lack of efficient genetic engineering techniques. In this study, a lambda Red and FLP/FRT-mediated site-specific recombination system was successfully established in Komagataeibacter xylinus. Using this system, the membrane bound gene gcd, a gene that encodes glucose dehydrogenase, was knocked out to reduce the modification of glucose to gluconic acid. The engineered strain could not produce any gluconic acid and presented a decreased bacterial cellulose (BC) production due to its restricted glucose utilization. To address this problem, the gene of glucose facilitator protein (glf; ZMO0366) was introduced into the knockout strain coupled with the overexpression of the endogenous glucokinase gene (glk). The BC yield of the resultant strain increased by 63.63-173.68%, thus reducing the production cost.

A facile construction of bacterial cellulose/ZnO nanocomposite films and their photocatalytic and antibacterial properties.

Bacterial cellulose (BC) has numerous excellent properties but the absence of antibacterial activity restricts its applications in biomedical field. Therefore, in order to introduce the antibacterial characteristics into BC; herein, a facile method for incorporation of ZnO nanoparticles (ZnO-NPs) is presented. BC films were first immersed in zinc nitrate solution, followed by treating with NaOH solution, the BC loaded ZnO nanocomposite films were dried by a sheet former instrument at 80 °C for 20 min. The obtained BC/ZnO nanocomposites were characterized by different techniques. XRD results showed the hexagonal wurtzite structure of ZnO-NPs while FE-SEM results displayed the particle size of ZnO-NPs was ranging from 70 to 100 nm. Thermogravimetric study revealed the thermal stability of nanocomposite films. The nanocomposite exhibited photocatalytic activity and revealed 91% degradation of methyl orange (MO) under UV-irradiation within 2 h. Moreover, the nanocomposites demonstrated significant UV-blocking properties and showed antibacterial activities against tested Gram-positive and Gram-negative bacterial strains. This work provides a simple and novel method for the synthesis of BC/ZnO nanocomposite as a functional biomaterial.

Development of bacterial cellulose/chitosan based semi-interpenetrating hydrogels with improved mechanical and antibacterial properties.

In the present work, novel bacterial cellulose (BC) and chitosan (CS) semi-interpenetrating network (semi-IPN) hydrogels were prepared via blending the slurry of BC with CS solution followed by cross-linking with glutaraldehyde. The structure and properties of BC-CS hydrogels were characterized by different techniques including; FTIR, XRD, FE-SEM, TGA and rotational rheometry. The results indicated cross-linking of chitosan chains by glutaraldehyde while BC was physically connected to network forming semi-IPN hydrogels. Microscopic study of cross-sectional freeze-dried hydrogels showed microporous openings. BC-CS hydrogels exhibited higher thermal stability than pure BC film or CS hydrogel alone. The rheological results presented significant mechanical properties of semi-IPN hydrogels. Moreover, the hydrogels showed antibacterial properties against tested Gram-positive and Gram-negative bacteria. The antibacterial properties were dependent on the ratio of BC to CS. Hydrogels with 20% BC to CS reduced the viable colonies by ~88%. The development of this new class of BC-CS antibacterial, mechanically strong and stable soft-material could be a promising candidate for antibacterial applications.

Reusable ternary PVA films containing bacterial cellulose fibers and ε-polylysine with improved mechanical and antibacterial properties.

The combination of high mechanical properties, antibacterial activity and a green synthesis of the polyvinyl alcohol (PVA) based films remains challenging. This study presents a ternary system of PVA films containing bacterial cellulose (BC) and epsilon-polylysine (ε-PL) by a green solution casting method. The prepared composite films showed more than 99% antibacterial properties against both Staphylococcus aureus and Escherichia coli bacteria. Moreover, the films were collected after a single use and were reused twice, which still exhibited strong antibacterial activity. The films showed thermal stability and higher mechanical properties as compared to pure PVA films. In addition, the cytotoxicity of the films was evaluated by MTT assay against NIH 3T3 mouse fibroblast cells. The results showed no toxicity of the films towards tested cells. We believe that these antibacterial films may find applications in active food packaging and biomedical fields.

Preparation and characterization of a photocatalytic antibacterial material: Graphene oxide/TiO2/bacterial cellulose nanocomposite.

In this study, bacterial cellulose (BC) was used as a matrix to synthesize graphene oxide/Titanium dioxide (GOTiO2)-based hybrid materials. It was indicated by X-ray diffraction and selected area electron diffraction that the crystal structure of GOTiO2 was a mixed phase containing anatase and rutile. TiO2 nanoparticles were of 10-30nm diameters and densely anchored on graphene oxide sheets. Superior photocatalytic performance of the GOTiO2 was achieved under near UV excitation. The photocatalytic efficiency was optimized through controlling an appropriate calcined temperature. The obtained GOTiO2 nanoparticles were filled into porous BC matrix (GOTiO2/BC), and the photocatalytic properties of GOTiO2 nanoparticles were well maintained. Consistent with photocatalytic performance of TiO2, GOTiO2/BC generated reactive oxygen species after near ultraviolet irradiation. No dark cytotoxicity was observed at the long incubation time. In parallel, following exposure of Staphylococcus aureus cells to GOTiO2 and irradiation, a significant decrease in cell viability, as well as an increased production of reactive oxygen species was observed, which induced cellular death. The results indicated that GOTiO2/BC possess an excellent photodynamic antibacterial activity.

Improvement of antimicrobial activity of graphene oxide/bacterial cellulose nanocomposites through the electrostatic modification.

Graphene oxide (GO) has an attracting and ever-growing interest in various research fields for its fascinating nanostructures. In this study, bacterial cellulose (BC) was used as a matrix to synthesize GO-based materials by a mechanical mixing method. The modification of GO with PEI significantly improved the bonding force between GO nanofillers and BC matrix. The morphology of the nanocomposites had a significant effect on the mechanical properties, hydrophilic properties as well as the antibacterial activity. After the modification, the GO-PEI/BC showed a strong antimicrobial effect on Saccharomyces cerevisiae due to the effective direct contacts between the nanofillers of the composites and the cell surfaces. This study demonstrates that the morphology of the nanocomposites has a great effect on physiochemical properties and the interactions between the microorganism and the nanocomposites.