Increased production of bacterial cellulose as starting point for scaled-up applications

Bacterial cellulose is composed of an ultrafine nanofiber network and well-ordered structure; therefore, it offers several advantages when used as native polymer or in composite systems.

In this study, a pool of 34 acetic acid bacteria strains belonging to Komagataeibacter xylinus were screened for their ability to produce bacterial cellulose. Bacterial cellulose layers of different thickness were observed for all the culture strains. A high-producing strain, which secreted more than 23 g/L of bacterial cellulose on the isolation broth during 10 days of static cultivation, was selected and tested in optimized culture conditions. In static conditions, the increase of cellulose yield and the reduction of by-products such as gluconic acid were observed. Dried bacterial cellulose obtained in the optimized broth was characterized to determine its microstructural, thermal, and mechanical properties. All the findings of this study support the use of bacterial cellulose produced by the selected strain for biomedical and food applications.

     

Ultrafine cellulose fibers produced by Asaia bogorensis, an acetic acid bacterium

The ability to synthesize cellulose by Asaia bogorensis, a member of the acetic acid bacteria, was studied in two substrains, AJ and JCM. Although both strains have identical 16S rDNA sequence, only the AJ strain formed a solid pellicle at the air-liquid interface in static culture medium, and we analyzed this pellicle using a variety of techniques. In the presence of cellulase, glucose and cellobiose were released from the pellicle suggesting that it is made of cellulose. Field emission electron microscopy allowed the visualization of a 3D knitted structure with ultrafine microfibrils (approximately 5-20 nm in width) in cellulose from A. bogorensis compared with the 40-100 nm wide microfibrils observed in cellulose isolated from Gluconacetobacter xylinus, suggesting differences in the mechanism of cellulose biosynthesis or organization of cellulose synthesizing sites in these two related bacterial species. Identifying these differences will lead to a better understanding of cellulose biosynthesis in bacteria.     

Bacterial cellulose-based materials and medical devices: current state and perspectives

Bacterial cellulose (BC) is a unique and promising material for use as implants and scaffolds in tissue engineering. It is composed of a pure cellulose nanofiber mesh spun by bacteria. It is remarkable for its strength and its ability to be engineered structurally and chemically at nano-, micro-, and macroscales. Its high water content and purity make the material biocompatible for multiple medical applications. Its biocompatibility, mechanical strength, chemical and morphologic controllability make it a natural choice for use in the body in biomedical devices with broader application than has yet been utilized. This paper reviews the current state of understanding of bacterial cellulose, known methods for controlling its physical and chemical structure (e.g., porosity, fiber alignment, etc.), biomedical applications for which it is currently being used, or investigated for use, challenges yet to be overcome, and future possibilities for BC.     

Silicone rubber membrane bioreactors for bacterial cellulose production

Cellulose production byAcetobacter pasteurianus was investigated in static culture using four bioreactors with silicone rubber membrane submerged in the medium. The shape of the membrane was flat sheet, flat sack, tube and cylindrical balloon. Production rate of cellulose as well as its yield on consumed glucose by the bacteria grown on the flat type membranes was approximately ten-fold greater than those on the non-flat ones in spite of the same membrane thickness. The membrane reactor using flat sacks of silicone rubber membrane as support of bacterial pellicle can supply greater ratio of surface to volume than a conventional liquid surface culture and is promising for industrial production of bacterial cellulose in large scale.     

Optimizing the Production of Bacterial Cellulose in Surface Culture: Evaluation of Substrate Mass Transfer Influences on the Bioreaction (Part 1)

The interest in cellulose produced by bacteria from surface cultures has increased steadily in recent years because of its potential for use in medicine and cosmetics. Unfortunately, the low yield of the production process has limited the commercial usefulness of bacterial cellulose. This series of three papers dealing with the production of bacterial cellulose using (batch) surface culture, firstly present a complete and complex analysis of the overall system, which allows a fundamental optimization of the production process to be performed. This material has many applications but the low yield of the process limits its commercial usefulness. In part 1, the effect of the rate of mass transfer of substrate on the microbial process, which is characterized by the growth of the bacteria, product formation, and the utilization of the substrate by the bacteria, is studied. A fundamental model for the diffusion of glucose through the growing cellulose layer is proposed and solved. The model confirmed that the increase in diffusional resistance is indeed significant but other factors will also need to be taken into account.     

AMPHIESMAL ULTRASTRUCTURE OF DINOFLAGELLATES: A REEVALUATION OF PELLICLE FORMATION1

The ultrastructure of the amphiesma during pellicle formation was investigated in two species of Dinophyceae, Amphidinium rhynchocephalum Anissimowa and Heterocapsa niei (Loeblich) Morrill & Loeblich using thin sections. In both species the amphiesma consists of an outermost membrane (i.e. the plasma membrane) underlain by amphiesmal vesicles. In A. rhynchocephalum the latter appear empty whereas each amphiesmal vesicle in H. niei contains a thecal plate and a thin, amorphous layer (dark-staining layer) located between, the thecal plate and the inner amphiesmal vesicle membrane. When cells of both taxa are carefully fixed, amphiesmal vesicles are always separate entities (i.e. the sutures are undisrupted). During ecdysis the following amphiesmal components are shed: the plasma membrane, the outer amphiesmal vesicle membrane, and in H. niei the thecal plates. The inner membranes of the amphiesmal vesicles then fuse with each other and form a continuous membrane (termed pellicle membrane) that remains tightly oppressed to an underlying amorphous layer (pellicular layer). In A. rhynchocephalum the pellicular layer is already present in vegetative non-ecdysed cells, whereas in H. niei it forms during ecdysis beneath the pellicle membrane. During ecdysis in H. niei, material from the dark-staining layer precipitates on the outer surface of the pellicle membrane, where it forms a characteristic honeycomb pattern. The new observations are incorporated into a revised model of pellicle formation in dinoflagellates and contrasted with earlier proposals.     

The nucleoid‐associated protein Fis directly modulates the synthesis of cellulose,an essential component of pellicle–biofilms in the phytopathogenic bacterium Dickeya dadantii

Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface‐associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle–biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase‐regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid‐associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co‐ordinate basic metabolism, virulence and modifications of lifestyle.     

Production of bacterial cellulose from <Emphasis Type="Italic">Enterobacter amnigenus</Emphasis> GH-1 isolated from rotten apple

Bacterial cellulose finds novel applications in biomedical, biosensor, food, textile and other industries. The optimum fermentation conditions for the production of cellulose by newly isolated Enterobacter amnigenus GH-1 were investigated. The strain was able to produce cellulose at temperature 25–35°C with a maximum at 28°C. Cellulose production occurred at pH 4.0–7.0 with a maximum at 6.5. After 14 days of incubation, the strain produced 2.5 g cellulose/l in standard medium whereas cellulose yield in the improved medium was found to be 4.1 g/l. The improved medium consisted of 4% (w/v) fructose, 0.6% (w/v) casein hydrolysate, 0.5% (w/v) yeast extract, 0.4% (w/v) disodium phosphate, and 0.115% (w/v) citrate. Addition of metal ions like zinc, magnesium, and calcium and solvents like methanol and ethanol were found to be stimulatory for cellulose production by the strain. The strain used natural carbon sources like molasses, starch hydrolysate, sugar cane juice, coconut water, coconut milk, pineapple juice, orange juice, and pomegranate juice for growth and cellulose production. Fruit juices can play important role in commercial exploitation of bacterial cellulose by lowering the cost of the production medium.     

Effect of Heat, Acidification, and Chlorination on Salmonella enterica Serovar Typhimurium Cells in a Biofilm Formed at the Air-Liquid Interface

Bacterial biofilms have great significance for public health, since biofilm-associated microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents and treatments. To date most attention has focused on biofilms that arise from the colonization of solid-liquid or solid-air interfaces. It is of interest that colonization of the interface between air and liquid, which can be selectively advantageous for aerobic or facultative aerobic bacteria, has been rarely studied, although it may present a major problem in industrial aquatic systems. In this work we investigated the role of a biofilm at the interface between air and liquid (pellicle) in the susceptibility of Salmonella enterica serovar Typhimurium to stress conditions. For a control we used a mutant that had lost its ability to synthesize cellulose and thin aggregative fimbriae and thus did not produce the pellicle. Resistance of bacteria from the pellicle to heat, acidification, and chlorination was compared to resistance of planktonic cells from the logarithmic and stationary phases of growth. Pellicle cells were significantly more resistant to chlorination, and thus the surrounding matrix conferred protection against the reactive sodium hypochlorite. However, the stress management of pellicle cells in response to heat and low pH was not enhanced compared to that of stationary-phase cells. A long-period of incubation resulted in endogenous hydrolysis of the pellicle matrix. This phenomenon provides a potential new approach to combat microbial cells in biofilms.     

Effect of heat, acidification, and chlorination on Salmonella enterica serovar typhimurium cells in a biofilm formed at the air-liquid interface

Bacterial biofilms have great significance for public health, since biofilm-associated microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents and treatments. To date most attention has focused on biofilms that arise from the colonization of solid-liquid or solid-air interfaces. It is of interest that colonization of the interface between air and liquid, which can be selectively advantageous for aerobic or facultative aerobic bacteria, has been rarely studied, although it may present a major problem in industrial aquatic systems. In this work we investigated the role of a biofilm at the interface between air and liquid (pellicle) in the susceptibility of Salmonella enterica serovar Typhimurium to stress conditions. For a control we used a mutant that had lost its ability to synthesize cellulose and thin aggregative fimbriae and thus did not produce the pellicle. Resistance of bacteria from the pellicle to heat, acidification, and chlorination was compared to resistance of planktonic cells from the logarithmic and stationary phases of growth. Pellicle cells were significantly more resistant to chlorination, and thus the surrounding matrix conferred protection against the reactive sodium hypochlorite. However, the stress management of pellicle cells in response to heat and low pH was not enhanced compared to that of stationary-phase cells. A long-period of incubation resulted in endogenous hydrolysis of the pellicle matrix. This phenomenon provides a potential new approach to combat microbial cells in biofilms.     

A rapid screening method for bacteria degrading polycyclic aromatic hydrocarbons

Aims:  A rapid procedure was developed to screen for bacteria that are able to grow on polycyclic aromatic hydrocarbons (PAHs).
Methods and Results:  A drop of ethyl ether-dissolved PAH is spread on a sterilized cellulose acetate/nitrate filter lying on the top of a mineral salts agarose plate. After the evaporation of ethyl ether, a serially diluted sample is spread over the filter and incubated. Subsequently, the PAH degrading bacteria can be counted and isolated.
Conclusions:  This procedure is a simple method for screening bacterial isolates for the ability to grow with PAHs.
Significance and Impact of the Study:  This technique is rapid to screen and/or count PAH-degrading bacteria and is also used to streak cultivation without disrupting the PAH layer on plate.     

Synthesis of bacterial celluloses in multiwalled carbon nanotube-dispersed medium

Carbon nanotubes are considered to be the ideal multi-functional filler, although there is some debate regarding their toxicity for bio-related applications. The bacteria, Gluconacetobacter xylinum, which produce bacterial cellulose, were cultured in a multiwalled carbon nanotube (MWCNT) dispersed Hestrin and Schramm (HS) medium by shaking incubation. The MWCNTs were functionalized with polyethylene glycol to prepare a stable MWCNT-dispersed HS medium and its stability was characterized by measuring the transmittance of a pulsed near infrared light. To investigate the toxicity of the MWCNTs to bacteria, we also introduced a green fluorescent protein gene into the bacteria and observed the fluorescence via confocal microscopy to confirm the presence of live bacteria in the MWCNT-dispersed HS medium. On the bases of the electron microscopy observations, a substantial number of MWCNTs were found to be well-dispersed and attached to the surface of the bacterial cellulose fibrils.     

Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant–pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air–liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.     

Role of contact in bacterial degradation of cellulose

Abstract Bacterial cells can adhere to cellulose fibres, but it is not known if cell-to-fibre contact is necessary for cellulose degradation. This problem was explored using aerobic cellulolytic bacteria, including known species and new isolates from soil. These were tested on plates containing Avicel, Solka floc, CF11 cellulose, carboxymethyl cellulose, or phosphoric acid-treated cellulose. Cellulose degradation was measured both by formation of clearing zones and by growth when cellulose was the only carbon source. The bacteria tested were either inoculated directly on the cellulose-containing agar, or separated from it by a pure agar layer or by membrane filters (not containing cellulose). Even when separated from the cellulose-containing agar all strains grew well. Clearing zones, best seen in phosphoric acid-treated cellulose, were larger under colonies separated from cellulose by an agar layer than under those in direct contact with cellulose. Such zones could also appear under filters. Our results show that bacterial degradation of cellulose does not depend on cell-to-fibre contact and suggest that when cellulose is at a greater distance from the cell, the removal of end products reduces catabolite repression of cellulose formation.     

Physico-Mechanical Properties of Chemically Treated Bacterial (Acetobacter xylinum) Cellulose Membrane

Bacterial cellulose obtained through fermentation by the Acetobacter xylinum is of superior functional quality in comparison to plant cellulose. Various alkali treatment methods were used to process bio-chemically complex pellicle into a clean cellulose membrane/sheet. The effect of potassium hydroxide, sodium carbonate and potassium carbonate was found to be milder on the final cellulose product in contrast to the widely used sodium hydroxide treatment. These novel treatment methods also caused improvement in the tensile strength of the membranes in comparison to sodium hydroxide. The overall quality of the 0.1 M sodium carbonate- and potassium carbonate-treated cellulose was superior, as the membranes displayed maximum tensile strength and elongation next to the native membrane. The low tensile strength of sodium hydroxide-treated membrane is attributed to its higher swelling characteristics in alkali. Further, the low swelling property of sodium carbonate- and potassium carbonate-treated membranes resulted in their high oxygen transmission rates (low oxygen barrier). Hunter lab colour parameters were determined to assess the effect of different alkali treatments on the colour characteristics of the membranes. Further, based on the high mechanical strength and comparatively low oxygen transmission rates, the processed cellulose membranes may find application as a bio- packaging material for controlled atmosphere packaging, where hydrophilic membranes with high oxygen barrier and water vapour permeation are desirable.     

Morphological analysis of young and old pellicles of Salmonella Typhimurium

A wide variety of microorganisms are able to form biofilms at the interface between air and liquid (pellicles). In this study changes during the maturation of the pellicle of Salmonella Typhimurium were analysed and the role of cellulose in the pellicle structure and morphology evaluated. The morphology of both sides of the pellicle was characterised using atomic force microscopy and scanning electron microscopy. Overall, there was a marked difference in the morphology of the water-facing (WF) and air-facing (AF) biofilm surfaces. While the AF side appeared to be uniform, and extensively covered with an exocellular coating, cells in the WF side were distributed into clusters and were less covered. However, the similarity in size and shape of single cells from both sides of the pellicle may indicate that the bacterial cells across the pellicle have a similar physiological status. During maturation, porous structures with multiple cracks and channels were created in the pellicle, leading to disintegration. By comparison with the structure of pellicles of a cellulose-deficient mutant, it was demonstrated that the observed disintegration of mature pellicles probably occurred in part by self-hydrolysis of components of the matrix.     

The Ultrastructure of Brood Pouch Formation in Tokophrya infusionum

SYNOPSIS Asexual reproduction in Tokophrya infusionum is by internal budding, whereby a ciliated, motile embryo is formed inside the sessile, non-ciliated parent in a specialized structure, the brood pouch. The process of embryogenesis and brood pouch formation was studied with the electron microscope using synchronized cultures. Reproduction begins with invagination of the pellicle and plasma membrane in the apical region of the adult. Early invagination is characterized by the presence of numerous microtubules beneath the plasma membrane or epiplasmic layer of the invaginating membranes. These microtubules apparently are important in formation of the brood pouch for colchicine blocks embryogenesis during the early stages. When the embryo is completed, it is ejected from the brood pouch thru the birth pore, an opening which is the site of the initial invagination and is present thruout embryogenesis. Theories of brood pouch formation are reviewed and discussed in light of the present investigation.     

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.