Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.

Liquid-crystalline ordering in bacterial cellulose produced by Gluconacetobaсter hansenii on glucose-containing media.

This research is dedicated to the studies of the microscale morphology of bacterial cellulose (BC) obtained by means of static cultivation of Gluconacetobacter hansenii GH-1/2008. We found that the microscale morphology depended on the BC production rate that was varied by using different glucose concentrations in the cultivation medium. It was revealed that at higher production rates, BC fibrils were aligned in a liquid-crystalline-like (LC-like) order. The observed helical alignment was always left-handed. The half-periods of the helix varied from 50 µm to 150 µm depending on the cultivation conditions. The mechanical and water absorption properties of the obtained BC pellicles were measured. The former correlated mainly with the density of the samples; the latter were the best for films with layered structure, where the BC had segregated into fleece sheets separated by gaps with low density of fibrils.

Systematic optimization of exopolysaccharide production by Gluconacetobacter sp. and use of (crude) glycerol as carbon source.

The usage of polysaccharides as biodegradable polymers is of growing interest in the context of a sustainable and ecofriendly economy. For this, the production of exopolysaccharides (EPS) by Gluconacetobacter sp. was investigated. Glycerol as carbon source revealed to be beneficial compared to glucose. In addition, pure glycerol could be substituted by a crude glycerol waste stream from biodiesel production. Systematic analysis of the peptone and phosphate concentrations in glycerol-based media indicated a strong effect of peptone. Optimized parameters resulted in a titer of 25.4 ± 2.4 g/L EPS with a productivity of 0.46 ± 0.04 g*(L*h)-1. With decreasing peptone, a variation in the monomer ratios was observed. An accompanying change in molecular size distribution indicated the production of two different polysaccharides. Intensified analysis revealed the main polysaccharide to be composed of glucose (Glc), galactose (Gal), mannose (Man) and glucuronic acid (GlcA), and the minor polysaccharide of Gal, Man, ribose (Rib).

Effect of chia seed mucilage/bacterial cellulose edible coating on bioactive compounds and antioxidant activity of strawberries during cold storage.

This study aimed to investigate the effect of chia seed mucilage (CSM) - bacterial cellulose nano-fiber (CNF) edible coating on bioactive compounds and antioxidant enzyme activity of strawberries. Strawberries were coated with CSM containing 0.6 and 8.0% (w/w) of CNF. The content of total phenol, flavonoids, anthocyanin, ascorbic acid, protein content, antioxidant activity and the activity of polyphenol oxidase, peroxidase, superoxide dismutase and phenylalanine ammonia-lyase enzymes were evaluated. The use of CSM - CNF edible coatings further preserved the phenolic, flavonoid, ascorbic acid and antioxidant activity of strawberries, and this effect was more evident in the CSM-coated sample containing CNF; However, the accumulation of anthocyanins in the coated samples was lower than the control sample. The activity of polyphenol oxidase and peroxidase enzymes, which lead to the degradation of phenolic compounds and brown color in the product, was also effectively controlled by the edible coating.

Enzymatic and Chemical Cross-Linking of Bacterial Cellulose/Fish Collagen Composites-A Comparative Study.

This article compares the properties of bacterial cellulose/fish collagen composites (BC/Col) after enzymatic and chemical cross-linking. In our methodology, two transglutaminases are used for enzymatic cross-linking-one recommended for the meat and the other proposed for the fish industry-and pre-oxidated BC (oxBC) is used for chemical cross-linking. The structure of the obtained composites is characterized by scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, and Fourier transform infrared spectroscopy, and their functional properties by mechanical and water barrier tests. While polymer chains in uncross-linked BC/Col are intertwined by H-bonds, new covalent bonds in enzymatically cross-linked ones are formed-resulting in increased thermal stability and crystallinity of the material. The C2-C3 bonds cleavage in D-glucose units, due to BC oxidation, cause secondary alcohol groups to vanish in favor of the carbonyl groups' formation, thus reducing the number of H-bonded OHs. Thermal stability and crystallinity of oxBC/Col remain lower than those of BC/Col. The BC/Col formation did not affect tensile strength and water vapor permeability of BC, but enzymatic cross-linking with TGGS improved them significantly.

Underwater Superoleophobic Matrix-Formatted Liquid-Infused Porous Biomembranes for Extremely Efficient Deconstitution of Nanoemulsions.

Wettability is one of the most critical interfacial properties of any surface. Surfaces with special wettability such as superwetting or superantiwetting are being intensively explored for their wide-ranging applicability by a biomimetic exploration of unusual wetting phenomena in nature. This study provides a green water-infused superoleophobic composite membrane by boosting bacteria nanocellulose growth on a reinforcement fibrous substrate. It was shown that this versatile antifouling membrane is capable of removing water from surfactant-stabilized oil-in-water micro/nanoemulsions and helps to isolate the oil fraction with very high filtration efficiency. The renewable membrane based on bacteria nanocellulose matrices can vastly improve current technologies by cultivating a naturally occurring soft materials approach with lubricious conformal interfaces to effectively and simply cover suitable surfaces.

Surface Interactions between Bacterial Nanocellulose and B-Complex Vitamins.

The interactions between films of bacterial nanocellulose (BNC) and B complex vitamins were studied using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). Thin films of BNC were generated in situ by QCM-D, followed by real-time measurements of the vitamin adsorption. The desorption of vitamins was induced by rinsing the system using phosphate buffers at a pH of 2 and 6.5, emulating gastric conditions. Changes in frequency (which are proportional to changes in adsorbed mass, ∆m) detected by QCM-D were used to determine the amounts of vitamin adsorbed and released from the BNC film. Additionally, changes in dissipation (∆D) were proven to be useful in identifying the effects of the pH in both pristine cellulose films and films with vitamin pre-adsorbed, following its changes during release. The effects of pH on the morphology of the vitamin-BNC surfaces were also monitored by changes in rugosity from images obtained by atomic force microscopy (AFM). Based on this data, we propose a model for the binding phenomena, with the contraction on the relaxation of the cellulose film depending on pH, resulting in an efficient vitamin delivery process.

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.

Silk sericin-enhanced microstructured bacterial cellulose as tissue engineering scaffold towards prospective gut repair.

As a first step towards the production of functional cell sheets applicable for the regeneration of gut muscle layer, microstructured bacterial cellulose (mBC) was assessed for its ability to support the growth of enteric nervous system (ENS) and gut smooth muscle cells (SMCs). To improve the cellular response, mBC was modified with silk sericin (SS) which has renowned abilities in supporting tissue regeneration. While SS did not impair the line structures imparted to BC by PDMS templates, similarly to the patterns, it affected its physical properties, ultimately leading to variations in the behavior of cells cultured onto these substrates. Enabled by the stripes on mBC, both SMCs and ENS cells were aligned in vitro, presenting the in vivo-like morphology essential for peristalsis and gut function. Interestingly, cell growth and differentiation remarkably enhanced upon SS addition to the samples, indicating the promise of the mBC-SS constructs as biomaterial not only for gut engineering, but also for tissues where cellular alignment is required for function, namely the heart, blood vessels, and similars.

Hybrid and biocompatible cellulose/polyurethane nanocomposites with water-activated shape memory properties.

Water-activated shape memory bacterial cellulose/polyurethane nanocomposites were prepared by the immersion of bacterial cellulose (BC) wet membranes into waterborne polyurethane (WBPU) dispersions for different times. The high affinity between the hydrophilic BC and water stable polyurethane led to the coating and embedding of the BC membrane into the WBPU, facts that were confirmed by FTIR, SEM and mechanical testing of the nanocomposites. The mechanical performance of the nanocomposites resulted enhanced with respect to the neat WBPU, confirming the reinforcing effect of the BC membrane. An improvement of the shape fixity ability and faster recovery process with the presence of BC was observed. In 3 min, the nanocomposite with highest BC content recovered the 92.8 ± 6.3% of the original shape, while the neat WBPU only recovered the 33.4 ± 9.6%. The obtained results indicated that 5 min of impregnation time was enough to obtain nanocomposites with improved mechanical performance and fast shape recovery for potential biomedical applications. The present work provides an approach for developing environmentally friendly and biocompatible BC/polyurethane based materials with enhanced mechanical and shape memory properties.

Poly(glycidyl methacrylate)/bacterial cellulose nanocomposites: Preparation, characterization and post-modification.

Nanocomposites composed of poly(glycidyl methacrylate) (PGMA) and bacterial cellulose (BC) were prepared by the in-situ free radical polymerization of glycidyl methacrylate (GMA) inside the BC network. The resulting nanocomposites were characterized in terms of structure, morphology, water-uptake capacity, thermal stability and viscoelastic properties. The three-dimensional structure of BC endowed the nanocomposites with good thermal stability (up to 270 °C) and viscoelastic properties (minimum storage modulus = 80 MPa at 200 °C). In addition, the water-uptake and crystallinity decreased with the increasing content of the hydrophobic and amorphous PGMA matrix. These nanocomposites were then submitted to post-modification via acid-catalysed hydrolysis to convert the hydrophobic PGMA into the hydrophilic poly(glyceryl methacrylate) (PGOHMA) counterpart, which increased the hydrophilicity of the nanocomposites and consequently improved their water-uptake capacity. Besides, the post-modified nanocomposites maintained a good thermal stability (up to 250 °C), viscoelastic properties (minimum storage modulus = 171 MPa at 200 °C) and porous structure. In view of these results, the PGMA/BC nanocomposites can be used as functional hydrophobic nanocomposites for post-modification reactions, whereas the PGOHMA/BC nanocomposites might have potential for biomedical applications requiring hydrophilic, swellable and biocompatible materials.

Rational design of a scalable bioprocess platform for bacterial cellulose production.

Bacterial cellulose (BC) has been gaining importance over the past decades as a versatile material that finds applications in diverse industries. However, a secured supply is hindered by the slow production rate and batch-to-batch variability of the yield. Here, we report a rational approach for characterising the BC production process using Design of Experiment (DoE) methodology to study the impact of different parameters on desired process attributes. Notably, we found that the carbon source used for bacterial growth significantly impacts the interplay between the process variables and affects the desired outcomes. We therefore, propose that the highest priority process outcome in this study, the yield, is a function of the carbon source and optimal reactor design. Our systematic approach has achieved projected BC yields as high as ∼40 g/L for Gluconacetobacter hansenii 53582 grown on sucrose as the carbon source compared to the widely reported yields of ∼10 g/L.

Dyeing of bacterial cellulose films using plant-based natural dyes.

The aim of this work was to test the use of plant-based natural dyes on bacterial cellulose (BC) to add aesthetic value to dyed pellicles while maintaining the mechanical properties. Natural pigments from Clitoria ternatea L. and Hibiscus rosa-sinensis were tested. The commercial ARAQCEL RL 500 was also used for comparison purposes. The behavior of biocellulose regarding dye fixation, rehydration, tensile strength, and elasticity was evaluated in comparison to the dried biomaterial, showing that dyeing is a process that can be performed on hydrated BC. Dyeing the BC films through an innovative process maintained the crystallinity, thermal stability and mechanical strength of the BC and confirmed the compatibility of the membrane with the dyes tested, from the observed Scanning Electron Microscopy (SEM) morphology of nanofibers. Dyed biomaterial can be applied to various products, as confirmed by the results of the mechanical tests. As environmental awareness and public concern regarding pollution increase, the combination of natural dyes and BC pellicles can produce an attractive new material for the textile industry.

Enhanced anti-obesity effects of bacterial cellulose combined with konjac glucomannan in high-fat diet-fed C57BL/6J mice.

This study aimed to investigate the effects of supplementation with bacterial cellulose (BC), konjac glucomannan (KGM) and combined BC/KGM fiber on high-fat (HF)-diet-induced obesity in C57BL/6J mice. The results showed that combined supplementation with BC/KGM in HF-fed mice was more efficient in reducing body weight, lowing serum lipid profiles and suppressing insulin resistance than single supplementation with BC or KGM. Moreover, supplementation with combined BC/KGM fiber more efficiently alleviated HF-diet-induced liver injury by decreasing hepatic steatosis in comparison with supplementation with BC or KGM alone. Furthermore, supplementation with combined BC/KGM fiber in HF-fed mice had a more positive effect on obesity-associated hepatic inflammation by reducing levels of TNF-α and IL-6 and suppressing the protein expression of Nrf-2/ARE in comparison with supplementation with BC or KGM alone. Consumption of these dietary fibers, especially mixed BC/KGM, resulted in an improved antioxidant defense system and reduced lipid peroxidation in the liver by increasing the activity of antioxidant enzymes and reducing the formation of MDA in the liver. Moreover, supplementation with these fibers regulated the levels of leptin and adiponectin and inhibited the protein expression of PPARγ by reducing the size of cells in the adipose tissue of HF diet-fed mice. Therefore, fiber supplementation (especially with combined BC/KGM) efficiently inhibited HF-induced obesity in mice by reducing insulin resistance, liver injury and inflammation, enhancing the antioxidant defense system and regulating the secretion of adipocytokines and adipogenesis-associated proteins.

Activated carbon monoliths derived from bacterial cellulose/polyacrylonitrile composite as new generation electrode materials in EDLC.

Bacterial cellulose (BC) gel is synthesized by static culture process at the interface between air and medium. The solvent-exchanged BC gel is incorporated into polyacrylonitrile (PAN) copolymer solution under heating at 90 °C and subsequent cooling gives bacterial cellulose-polyacrylonitrile composite (BC-PAN) monolith. The BC-PAN monolith is carbonized at 1000 °C with physical activation in the presence of CO2 to obtain the activated carbon monolith, BC-PAN-AC, with large surface area and high microporosity. Unique morphologies are observed for BC gel which is propagated to the BC-PAN monolith and restored in BC-PAN-AC. The BC nanofibers remain entwined throughout the porous skeleton of the PAN backbone and the entangled structure helps in retaining the continuity of the matrix of BC-PAN-AC and reduce the grain boundary impedance for electrical conduction. Cyclic voltammetry shows that these activated carbons are good electrode materials in electric double layer capacitors (EDLC) with capability of high-speed charging and discharging.

Molecular study of wound healing after using biosynthesized BNC/Fe3O4 nanocomposites assisted with a bioinformatics approach.

BACKGROUND: Molecular investigation of wound healing has allowed better understanding about interaction of genes and pathways involved in healing progression. OBJECTIVES: The aim of this study was to prepare magnetic/bacterial nanocellulose (Fe3O4/BNC) nanocomposite films as ecofriendly wound dressing in order to evaluate their physical, cytotoxicity and antimicrobial properties. The molecular study was carried out to evaluate expression of genes involved in healing of wounds after treatment with BNC/Fe3O4 films. STUDY DESIGN MATERIALS AND METHODS: Magnetic nanoparticles were biosynthesized by using Aloe vera extract in new isolated bacterial nanocellulose (BNC) RM1. The nanocomposites were characterized using X-ray diffraction, Fourier transform infrared, and field emission scanning electron microscopy. Moreover, swelling property and metal ions release profile of the nanocomposites were investigated. The ability of nanocomposites to promote wound healing of human dermal fibroblast cells in vitro was examined. Bioinformatics databases were used to identify genes with important healing effect. Key genes which interfered with healing were studied by quantitative real time PCR. RESULTS: Spherical magnetic nanoparticles (15-30 nm) were formed and immobilized within the structure of BNC. The BNC/Fe3O4 was nontoxic (IC50>500 µg/mL) with excellent wound healing efficiency after 48 hours. The nanocomposites showed good antibacterial activity ranging from 6±0.2 to 13.40±0.10 mm against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa. The effective genes for the wound healing process were TGF-B1, MMP2, MMP9, Wnt4, CTNNB1, hsa-miR-29b, and hsa-miR-29c with time dependent manner. BNC/Fe3O4 has an effect on microRNA by reducing its expression and therefore causing an increase in the gene expression of other genes, which consequently resulted in wound healing. CONCLUSION: This eco-friendly nanocomposite with excellent healing properties can be used as an effective wound dressing for treatment of cutaneous wounds.

Influence of chemical and physical conditions in selection of Gluconacetobacter hansenii ATCC 23769 strains with high capacity to produce bacterial cellulose for application as sustained antimicrobial drug-release supports.

AIMS: Obtain varieties of Gluconacetobacter hansenii from original strain ATCC 23729 with greater efficiency to produce bacterial cellulose (BC) membrane with better dry mass yield for application as support of sustained antimicrobials' drug release. METHODS AND RESULTS: Application of different chemical and physical conditions (pH, temperature and UV light exposure) to obtain different G. hansenii varieties with high capacity to produce BC membranes. Characterization of the G. hansenii variants was performed by scanning electron microscopy (SEM) and optical microscopy of the colony-forming units. BC membrane produced was characterized by SEM, infrared spectroscopy and X-ray diffraction. The BC produced by variants isolated after incubation at 35°C showed elevated dry mass yield and high capacity of retention and sustained release of ceftriaxone antibiotic with the produced BC by original G. hansenii ATCC 23769 strain subjected to incubation at 28°C and with commercial BC. CONCLUSION: The application of different chemical and physical conditions constitutes an important method to obtain varieties of micro-organisms with dissimilar metabolism advantageous in relation to the original strain in the BC production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the importance of in vivo studies for the application, in medicine, of BC membranes as support for antimicrobial-sustained release for the skin wound treatment.

Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of Nitrogenase From Gluconacetobacter diazotrophicus.

Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3). In its most prevalent form, it consists of two proteins, the catalytic molybdenum-iron protein (MoFeP) and its specific reductase, the iron protein (FeP). A defining feature of nitrogenase is that electron and proton transfer processes linked to substrate reduction are synchronized by conformational changes driven by ATP-dependent FeP-MoFeP interactions. Yet, despite extensive crystallographic, spectroscopic, and biochemical information on nitrogenase, the structural basis of the ATP-dependent synchronization mechanism is not understood in detail. In this chapter, we summarize some of our efforts toward obtaining such an understanding. Experimental investigations of the structure-function relationships in nitrogenase are challenged by the fact that it cannot be readily expressed heterologously in nondiazotrophic bacteria, and the purification protocols for nitrogenase are only known for a small number of diazotrophic organisms. Here, we present methods for purifying and characterizing nitrogenase from a new model organism, Gluconacetobacter diazotrophicus. We also describe procedures for observing redox-dependent conformational changes in G. diazotrophicus nitrogenase by X-ray crystallography and electron paramagnetic resonance spectroscopy, which have provided new insights into the redox-dependent conformational gating processes in nitrogenase.

Conductive bacterial cellulose-polyaniline blends: Influence of the matrix and synthesis conditions.

Bacterial cellulose/polyaniline (BC/PANi) blends present a great potential for several applications. The current study evaluates the impact of using different BC matrixes (drained, freeze-dried and regenerated) and different synthesis conditions (in situ and ex situ) to improve the inherent properties of BC, which were monitored through FTIR-ATR, EDX, XRD, SEM, AFM, swelling, contact angle measurement and IGC. The employment of in situ polymerization onto drained BC presented the most conductive membrane (1.4 × 10-1 S/cm). The crystallinity, swelling capacity, surface energy and acid/base behavior of the BC membranes is substantially modified upon PANi incorporation, being dependent on the BC matrix used, being the freeze-dried BC blends the ones with highest crystallinity (up to 54%), swelling capacity (up to 414%) and surface energy (up to 75.0 mJ/m2). Hence, this work evidenced that the final properties of the BC/PANi blends are greatly influenced by both the BC matrixes and synthesis methods employed.

Evaluation of ATR-FTIR for analysis of bacterial cellulose impurities.

This research evaluated the utility of using the large amount of spectral data obtained during attenuated total reflection Fourier-transform infrared spectrophotometry (ATR-FTIR) analysis of dried biocellulose (BC) to estimate the type and concentration of potential bacterial cells impurities present in the BC. Pre-cleaned BC was contaminated with known concentrations of representative nucleic acid, lipid, and protein impurities, as well as whole bacterial cells. These impurity standards were then subjected to ATR-FTIR analysis, and the resulting spectral data were used to develop models to estimate the concentrations of impurities in differentially processed BC. Results indicated that ATR-FTIR is a useful tool for estimating impurities in BC, and may also be applicable for measurement of levels of non-cellulose biomolecules added to BC for various purposes.