Physical Characteristics and Cell-Adhesive Properties of In Vivo Fabricated Hyaluronan/Bacterial Cellulose Nanocomposites

This study attempts to clarify the basic material properties of in-vivo-fabricated hyaluronan (HA)/bacterial cellulose (BC) nanocomposites prepared previously. BC membranes (pellicles) generated by Gluconacetobacter hansenii (G. hansenii) are promising biomaterials owing to their outstanding biocompatible properties. Recently, specific demands for biomedical applications of BC have increased owing to its excellent mechanical properties. Although many techniques have been developed to improve the biofunctional properties of BC pellicles, such modifications remain limited owing to technical difficulties in the modulation of complex biosynthetic processes. Therefore, we previously developed an in vivo modification technique to produce nanocomposite pellicles composed of BC and HA (in vivo HA/BC), which are directly secreted from genetically engineered G. hansenii. In the present study, the HA extractability and content rate, physical characteristics, and cytocompatibility of in vivo HA/BC have been investigated in comparison to conventional in situ HA/BC and native BC pellicle. The results suggested that HA more strongly adsorbed to the solid BC surface of in vivo HA/BC than that of in situ HA/BC, which possibly affected the dynamic viscoelastic characteristics. Furthermore, in vivo HA/BC showed remarkably high human epidermal cell adhesion. These results indicate the great potential of in vivo modification to expand the usefulness of BC-based biomaterials.

Bacterial cellulose: characterization of a biomaterial for apparel products application

Purpose This study aims to evaluate two bacterial cellulose (BC) films as an alternative textile surface suitable for use in the manufacture of clothing prototypes. Design/methodology/approach A combination of experiments for the production and characterization of BC films with traditional techniques for sewing fabrics was carried out. BC films were produced from the bacterum Gluconacetobacter hansenii UCP1619 and from Kombucha, a consortium of microorganisms grown on sugared tea. The BC films were then purified, characterized by scanning electron microscopy (SEM) and evaluated for mechanical strength. Two clothing prototypes were developed by combining BC films with a flat fabric composed of 70% linen and 30% polyester to assess the viability of the garment for future clothing making using biomaterials. Findings The results showed that the combination of flat fabric with BC-based biomaterials is a viable alternative for the innovative use of BC films in the manufacture of apparel products, especially after optimizing the mechanical properties of the artefact. Originality/value BC application studies in the textile industry are still in their early stages, although they are attracting more and more the attention of researchers around the world. The experiments carried out in this research provide new information on the handling and application of this material in innovative products for the textile industry.

The structure of Gluconacetobacter hansenii GH 1/2008 population cultivated in static conditions on various sources of carbon

Bacterial cellulose (BC) is a natural polymer that has a number of unique properties that determine the need to synthesize large amounts of it and to search the ways to increase the productivity of strains and to optimize the nutritive media. It is known that the choice of the producer for BC synthesis has an impact on its final properties and on the productivity of this polymer production. Under static liquid phase cultivation conditions, all cellulose-producing bacteria form a uniform film on the medium surface that serves as a scaffold for cells immobilization, thus providing them with the access to the air/liquid interface, where the access to oxygen is not limited. Meanwhile, when cultivation goes under agitating conditions, most of Gluconacetobacter xylinus strains produce less cellulose in form of globules of various sizes, despite the better oxygen access. Several authors explain the lower cellulose outcome when cultivated under agitated conditions by the appearance of spontaneous mutants that do not produce cellulose in the population. It was revealed that when grown on agarized media, cellulosenon- producing mutants form colonies of a specific mucoid type, while non-mucoid phenotype cells form smooth colonies of non-mucoid type. To our knowledge, there is no published research on the impact of cultivation conditions and nutritive medium composition on the appearance of spontaneous phenotype mutations in the population of Gluconacetobacter hansenii representatives. The aim of the present research is to elucidate the impact of the carbon source on the productivity of G. hansenii strain and the appearance of cellulose-negative mutants under static cultivation conditions. We studied the strain G. hansenii GH 1/2008 (VKPM В-10547) as a BC source. Liquid phase static cultivation of G. hansenii GH 1/2008 was carried out using the modified Hestrin-Schramm (HS) medium, containing 4% of monosaccharides (glucose, fructose and galactose) or disaccharides (sucrose, maltose, lactose) as carbon sources. The occurrence of mutants was calculated considering phenotypes of colonies obtained by seeding the samples of cultural liquid and wash-offs of cells from films produced by the cultivation of the producer on modified agarized HS media. The polymer outcome was expressed as the film absolute dry weight (a.d.w.) per cultivation medium volume unit. We studied the morphology of the producer’s wild type and mutant cells by means of atomic force microscopy (AFM) (See Fig. 8). The structural organization of the produced films and gel was revealed by means of scanning electron microscopy (SEM) performed after freeze-drying. The composition of the fibers was checked acquiring FTIR Spectroscopy. We established that G. hansenii GH 1/2008 produces a dense film on media containing fructose, glucose and sucrose, while the polymer has gel consistence when grown on maltose, galactose and lactose (See Fig. 1). The maximal quantity (a.d.w.) of polymer was produced on fructose- and sucrose-containing media. The overall number of immobilized producer cells was considerably higher when grown on media with glucose, fructose and sucrose than on gels grown on those containing maltose, galactose and lactose (See Table 1). SEM imaging revealed considerable difference in the microscale organization of films and gels produced by G. hansenii GH 1/2008 on various carbon sources (See Fig. 2). Fructose-containing medium yields the densest structure with dense layers separated by 2μm thick areas filled with non-ordered BC fibrils. The microscale organization of sucrose- and glucose-based films were very similar and had a cell-like structure. In cases where the synthesized polymer had squeezable gel consistence, its microstructure was not layered but close to isotropic. The studies of gels by means of FTIR spectroscopy showed that the gels are also formed of BC molecules; the spectra were almost identical (See Fig. 4). The only difference, i.e. the intensity of the 1638 см-1 peak, can be explained by the presence of a higher amount of bound water in the latter. It is known that some strains of this species may produce glucuronic acid oligomers under unfavorable conditions, but peaks corresponding to carboxyl or carbonyl groups were not revealed in the spectra. This is the evidence that no detectable amounts of glucuronic acid were produced under conditions studied. The analysis of colonies of G. hansenii GH 1/2008 cultivated under static conditions on media containing various carbon sources revealed colonies with two dominating phenotypes: non-mucoid smooth convex colonies and mucoid flat ones (See Fig. 5). The number of cells forming smooth non-mucoid colonies on agarized media was maximal in the inoculations of cultural liquids after the cultivation on media containing fructose and sucrose, i.e. those carbon sources that demonstrated high productivity per 1L of cultural liquid (See Fig. 6). In the inoculations of the cultural liquid and wash-offs of cells immobilized on gels obtained by the cultivation on media containing galactose and lactose, the number of mucoid colonies was considerably higher (See Table 2). The clones forming mucoid type colonies did not produce BC films when reinoculated in liquid media, while those forming colonies of mucoid (smooth) type produce films on the 3rd day of cultivation (See Fig. 7). The analysis of cells shape and sizes by means of AFM did not reveal any statistically valid difference between the mutants and the wild type. The present research shows that the source of carbon is a selective factor in the formation of the inner composition of the population of clones of the bacterial cellulose producer Gluconacetobacter hansenii GH 1/2008. The proliferation of cellulosenegative cells arouses competition for the substrate with cellulose-positive cells of G. hansenii GH 1/2008 that reduces the number of the latter and the production of the exopolymer.

Hybrid Living Capsules Autonomously Produced by Engineered Bacteria

Bacterial cellulose (BC) has excellent material properties and can be produced cheaply and sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli. Here, we describe a simple approach for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii was co-cultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which were then colonized by the E. coli upon transfer to selective lysogeny broth media. We show that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, we produced capsules capable of altering their own bulk physical properties through enzyme-induced biomineralization. This novel system, based on autonomous biological fabrication, significantly expands the functionality of BC-based living materials.

Evaluation of Different Methods for Cultivating Gluconacetobacter hansenii for Bacterial Cellulose and Montmorillonite Biocomposite Production: Wound-Dressing Applications

Bacterial cellulose (BC) has received considerable attention due to its unique properties, including an ultrafine network structure with high purity, mechanical strength, inherent biodegradability, biocompatibility, high water-holding capacity and high crystallinity. These properties allow BC to be used in biomedical and industrial applications, such as medical product. This research investigated the production of BC by Gluconacetobacter hansenii ATCC 23769 using different carbon sources (glucose, mannitol, sucrose and xylose) at two different concentrations (25 and 50 g∙L−1). The BC produced was used to develop a biocomposite with montmorillonite (MMT), a clay mineral that possesses interesting characteristics for enhancing BC physical-chemical properties, at 0.5, 1, 2 and 3% concentrations. The resulting biocomposites were characterized in terms of their physical and barrier properties, morphologies, water-uptake capacities, and thermal stabilities. Our results show that bacteria presented higher BC yields in media with higher glucose concentrations (50 g∙L−1) after a 14-day incubation period. Additionally, the incorporation of MMT significantly improved the mechanical and thermal properties of the BC membranes. The degradation temperature of the composites was extended, and a decrease in the water holding capacity (WHC) and an improvement in the water release rate (WRR) were noted. Determining a cost-effective medium for the production of BC and the characterization of the produced composites are extremely important for the biomedical applications of BC, such as in wound dressing materials.

Produção e caracterização de celulose pela Glucanoacetobacter hansenii em meio contendo glicose ou manitol

RESUMO A celulose, polissacarídeo de origem vegetal, é um biopolímero abundante encontrado na natureza e, portanto, de grande valia para a ciência dos materiais em aplicações na área médica, cosmética, refinaria e outros. Como alternativa à produção tradicional de celulose, tem-se a via microbiana, que resulta numa fibra de caráter nanométrico e com boas propriedades mecânicas. Dentre os diversos micro-organismos que produzem celulose bacteriana (CB), e que apresenta resultados satisfatórios destaca-se a bactéria estritamente aeróbica e Gram-negativa conhecida como Gluconacetobacter hansenii (ATCC - 23769). Esse biopolímero apresenta enorme potencial de aplicação devido às suas propriedades térmicas, mecânicas e biocompatibilidade. Muitas pesquisas estão sendo realizadas para otimizar os processos produtivos de CB, resultando em maiores conversões com um menor custo produtivo. O presente trabalho teve como objetivo, produzir as membranas de celulose bacteriana em meio de manitol (C-MM) ou Hestrin e Schramm (C-MH), e também caracterizá-las por meio de análises de espectroscopia de infravermelho (IV), termogravimétrica (TGA), e difração de raios X (DRX). Como principais resultados, as celuloses produzidas apresentaram imagens morfológicas similares, mas no meio de manitol apresentou maior rendimento (2,09 g.L-1) na produção de CB, quando comparado ao Meio Hestrin e Schramm (HS) (1,15 g.L-1). Além disso, a celulose bacteriana produzida no meio de manitol apresentou maior cristalinidade (78%) que a produzida no meio de Hestrin e Schramm (HS) (65%). Através da análise de IV, foi possível confirmar os grupos funcionais existentes na celulose bacteriana sem a presença de quaisquer contaminantes oriundo do processo de produção. Já com relação a análise termogravimétrica, o polímero formado a partir do meio de manitol apresentou maior estabilidade térmica. Desta forma, os biofilmes produzidos nos diferentes meios apresentaram propriedades diferentes, revelando que as características poliméricas são modificadas em função do meio de crescimento bacteriano.