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.     

蟋蟀后肠纤维素降解细菌的分离与鉴定

近年来,具有农业、能源和环保价值的昆虫微生物种类和基因得到了开发,昆虫肠道微生物展示了其巨大的应用潜力,本研究旨在从蟋蟀后肠分离和鉴定纤维素降解细菌。首先采用羧甲基纤维素钠液体培养基对蟋蟀后肠中的微生物进行富集培养,然后使用羧甲基纤维素钠固体培养基分离和筛选单菌落,再通过16S rRNA测序对纤维素降解细菌进行分子鉴定,最后通过刚果红染色来进一步分析细菌降解纤维素的能力。从蟋蟀后肠中共分离出20株纤维素降解细菌,16S rRNA基因测序结果显示来自肠杆菌属(Enterobacter)9株,不动杆菌属(Acinetobacter)7株,克雷伯氏菌属(Klebsiella)2株,鞘氨醇杆菌属(Sphingobacterium)1株和葡萄球菌属(Staphylococcus)1株。刚果红染色试验结果显示,克雷伯氏菌属两株PDSCDXS_2B和8B,鞘氨醇杆菌属PDSCDXS_7C和不动杆菌属PDSCDXS_12C具有较高的纤维素降解能力。这是首次从蟋蟀后肠分离和筛选出来具有纤维素降解能力的细菌,为昆虫源纤维素降解细菌的研究提供了微生物资源。     

Electron microscopic study of the methylcellulose-mediated detachment of cellulolytic rumen bacteria from cellulose fibers

The presence of methylcellulose prevents the attachment of cellulolytic rumen bacteria to cellulose fibers. The addition of methylcellulose to pure cultures of these organisms in which the cells are already adherent to cellulose causes their detachment from this insoluble substrate and the inhibition of their growth. Methylcellulose is not used as a carbon source by these organisms and has no effect on their growth when glucose and cellobiose are the carbon sources. Attached cells of Bacteroides succinogenes orient themselves in the plane of the individual cellulose fibers and their methylcellulose-induced detachment, which is complete (almost 100%), leaves grooves where the cellulose has been digested. Attached cells of Ruminococcus albus colonize the cellulose in a looser and less regular pattern and their almost complete methylcellulose-induced detachment leaves less regular pits in the cellulose surface. On the other hand, attached cells of Ruminococcus flavefaciens colonize the cellulose surface in a random orientation by means of a discernible exopolysaccharide network, and their less complete methylcellulose-induced detachment leaves no residual impressions on the cellulose surface. These data support the suggestion that bacterial attachment is necessary for the digestion of highly ordered crystalline cellulose, and that cellulolytic species differ in the nature of their attachment to this insoluble substrate and in the nature of their enzymatic attack. Methylcellulose is an effective agent for detaching major rumen cellulolytic bacteria from their cellulosic substrate.     

Nutritional Interdependence Among Rumen Bacteria During Cellulose Digestion In Vitro

A study has been made of the promoting effect of starch on cellulose digestion by mixed rumen bacteria in a cellulose-urea medium. Starch supplementation of the medium promoted the growth of bacteria that required neither amino acids (AA) nor branched-chain fatty acids (BrFA). The growth of these bacteria was followed by the growth of AA-dependent bacteria, AA- or BrFA-dependent bacteria, BrFA-producing bacteria, and finally, BrFA-dependent cellulolytic bacteria. Population changes of these bacterial groups corresponded with a cross-feeding of AA and BrFA and the overall disappearance of cellulose. The data suggest that the nutritional interdependence among rumen bacteria affects the rate of cellulose digestion.     

New photoantimicrobial films composed of porphyrinated lipophilic cellulose esters

Porphyrinated cellulose laurate esters have been prepared in homogeneous DMA/LiCl medium by "one-pot, two-step" reactions starting from cellulose, protoporphyrin IX, and lauric acid and using a TsCl/Pyridine system. The plastic films obtained after casting were shown to display photobactericidal activity against Gram positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. This new photobactericidal polymer has potential for industrial, medical, or household applications.     

Unravelling carbon metabolism in anaerobic cellulolytic bacteria

Carbon metabolism in anaerobic cellulolytic bacteria has been investigated essentially in Clostridium thermocellum, Clostridium cellulolyticum, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. While cellulose depolymerization into soluble sugars by various cellulases is undoubtedly the first step in bacterial metabolisation of cellulose, it is not the only one to consider. Among anaerobic cellulolytic bacteria, C. cellulolyticum has been investigated metabolically the most in the past few years. Summarizing metabolic flux analyses in continuous culture using either cellobiose (a soluble cellodextrin resulting from cellulose hydrolysis) or cellulose (an insoluble biopolymer), this review aims to stress the importance of the insoluble nature of a carbon source on bacterial metabolism. Furthermore, some general and specific traits of anaerobic cellulolytic bacteria trends, namely, the importance and benefits of (i) cellodextrins with degree of polymerization higher than 2, (ii) intracellular phosphorolytic cleavage, (iii) glycogen cycling on cell bioenergetics, and (iv) carbon overflows in regulation of carbon metabolism, as well as detrimental effects of (i) soluble sugars and (ii) acidic environment on bacterial growth. Future directions for improving bacterial cellulose degradation are discussed.     

斜纹夜蛾幼虫肠道细菌分离鉴定及其功能初步分析

昆虫肠道微生物对其寄主的生长发育、营养代谢、免疫以及农药抗性等方面都发挥着重要作用。为研究斜纹夜蛾Spodoptera litura幼虫肠道细菌的多样性,并为其功能验证做准备,本文利用传统微生物分离纯培养方法从斜纹夜蛾4龄幼虫肠道中共分离鉴定得到10株细菌,分别为属于变形菌门(Proteobacteria)的脱氮假单胞菌(Pseudomonas denitrificans),不动细菌(Acinetobacter sp.),肺炎克雷伯氏菌(Klebsiella pneumoniae)和肠杆菌(Enterobacter sp.);属于厚壁菌门(Firmicutes)的鸡葡萄球菌(Staphylococcus gallinarum),蒙氏肠球菌(Enterococcus mundtii),蜡样芽胞杆菌(Bacillus cereus)和枯草芽胞杆菌(Bacillus subtilis)以及放线菌门(Actinobacteria)的微杆菌(Microbacteriums sp.)和乳酪棒杆菌(Corynebacterium casei)。变形菌门和厚壁菌门是斜纹夜蛾肠道可培养细菌中的优势菌群。功能验证实验表明肠杆菌具备纤维素降解能力,微杆菌具备很强的苯酚降解能力。本研究为未来深入研究斜纹夜蛾肠道微生物的功能提供了方向和菌株材料。     

瘤胃纤维素降解细菌的研究进展

反刍动物瘤胃是自然界中最有效的纤维素降解系统，其纤维素降解能力主要源于寄居于其中的纤维素降解细菌、真菌和原虫。其中，瘤胃纤维素降解细菌因数量庞大、种类繁多以及代谢途径丰富，在木质纤维素降解及利用方面发挥着重要作用。本文综述了国内外瘤胃纤维素降解细菌的种类，分析了瘤胃纤维素降解细菌的特性；阐述了瘤胃纤维素降解细菌通过纤维小体对纤维素的降解过程，以及瘤胃微生物之间的相互作用和相互制约关系；简述宏组学技术在开发新纤维素降解菌和新纤维素酶方面的应用，旨在为进一步研究纤维素降解细菌的降解机理，开发新的纤维素菌种和酶资源提供新的思路。     

Adhesion and growth rate of Clostridium cellulolyticum ATCC 35319 on crystalline cellulose.

The rate of tritiated-thymidine incorporation into DNA was used to estimate Clostridium cellulolyticum H10 growth rates on Avicel cellulose, taking into consideration both the unattached cells and the cells adhered to the substrate. The generation time on cellobiose calculated from the data on cell density (4.5 h) agreed well with the generation time calculated by tritiated-thymidine incorporation (3.8 h). Growth on Avicel cellulose occurred when bacteria were adhered to their substrate; 80% of the biomass was detected on the cellulose. Taking into consideration attached and free bacteria, the generation time as determined by thymidine incorporation was about 8 h, whereas by bacterial-protein estimation it was about 13 h. In addition to the growth rate of the bacteria on the cellulose, the release of adhered cells constituted an important factor in the efficiency of the cellulolysis. The stage of growth influenced adhesion of C. cellulolyticum; maximum adhesion was found during the exponential phase. Under the conditions used, the end of growth was characterized by an acute release of biomass and cellulase activity from the cellulose. An exhaustion of the accessible cellulose could be responsible for this release.     

Nanofibrous microfiltration membrane based on cellulose nanowhiskers

A multilayered nanofibrous microfiltration (MF) membrane system with high flux, low pressure drop, and high retention capability against both bacteria and bacteriophages (a virus model) was developed by impregnating ultrafine cellulose nanowhiskers (diameter about 5 nm) into an electrospun polyacrylonitrile (PAN) nanofibrous scaffold (fiber diameter about 150 nm) supported by a poly(ethylene terephthalate) (PET) nonwoven substrate (fiber diameter about 20 μm). The cellulose nanowhiskers were anchored on the PAN nanofiber surface, forming a cross-linked nanostructured mesh with very high surface-to-volume ratio and a negatively charged surface. The mean pore size and pore size distribution of this MF system could be adjusted by the loading of cellulose nanowhiskers, where the resulting membrane not only possessed good mechanical properties but also high surface charge density confirmed by the conductivity titration and zeta potential measurements. The results indicated that a test cellulose nanowhisker-based MF membrane exhibited 16 times higher adsorption capacity against a positively charged dye over a commercial nitrocellulose-based MF membrane. This experimental membrane also showed full retention capability against bacteria, for example, E. coli and B. diminuta (log reduction value (LRV) larger than 6) and decent retention against bacteriophage MS2 (LRV larger than 2).     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces

ABSTRACT: BACKGROUND: The synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation. In this study, we analyzed both the production of cellulose by all known members of the Rhizobiaceae and the diversity of Rhizobium celABC operon predicted to be involved in cellulose biosynthesis. We also investigated the involvement in cellulose production and biofilm formation of celC gene encoding an endoglucanase (CelC2) that is required for canonical symbiotic root hair infection by Rhizobium leguminosarum bv. trifolii. RESULTS: ANU843 celC mutants lacking (ANU843DeltaC2) or overproducing cellulase (ANU843C2+) produced greatly increased or reduced amounts of external cellulose micro fibrils, respectively. Calcofluor-stained cellulose micro fibrils were considerably longer when formed by ANU843DeltaC2 bacteria rather than by the wild-type strain, in correlation with a significant increase in their flocculation in batch culture. In contrast, neither calcofluor-stained extracellular micro fibrils nor flocculation was detectable in ANU843C2+ cells. To clarify the role of cellulose synthesis in Rhizobium cell aggregation and attachment, we analyzed the ability of these mutants to produce biofilms on different surfaces. Alteration of wild-type CelC2 levels resulted in a reduced ability of bacteria to form biofilms both in abiotic surfaces and in planta. CONCLUSIONS: Our results support a key role of the CelC2 cellulase in cellulose biosynthesis by modulating the length of the cellulose fibrils that mediate firm adhesion among Rhizobium bacteria leading to biofilm formation. Rhizobium cellulose is an essential component of the biofilm polysaccharidic matrix architecture and either an excess or a defect of this "building material" seem to collapse the biofilm structure. These results position cellulose hydrolytic enzymes as excellent anti-biofilm candidates.     

The <Emphasis Type="Italic">Fibrobacteres</Emphasis>: an Important Phylum of Cellulose-Degrading Bacteria

The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognised as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted molecular approaches have demonstrated that novel centres of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degradation beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a “third” way of attacking the most abundant form of organic carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilise membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecology and potential as a source of novel glycosyl hydrolases of biotechnological importance.     

Statistical analysis of optimal culture conditions for Gluconacetobacter hansenii cellulose production

AIM: The purpose of this study was to analyse the effects of different culture parameters on Gluconacetobacter hansenii (ATCC 10821) to determine which conditions provided optimum cellulose growth. METHODS AND RESULTS: Five culture factors were investigated: carbon source, addition of ethanol, inoculation ratio, pH and temperature. jmp Software (SAS, Cary, NC, USA) was used to design this experiment using a fractional factorial design. After 22 days of static culture, the cellulose produced by the bacteria was harvested, purified and dried to compare the cellulose yields. The results were analysed by fitting the data to a first-order model with two-factor interactions. CONCLUSIONS: The study confirmed that carbon source, addition of ethanol, and temperature were significant factors in the production of cellulose of this G. hansenii strain. While pH alone does not significantly affect average cellulose production, cellulose yields are affected by pH interaction with the carbon source. Culturing the bacteria on glucose at pH 6.5 produces more cellulose than at pH 5.5, while using mannitol at pH 5.5 produces more cellulose than at pH 6.5. The bacteria produced the most cellulose when cultured on mannitol, at pH 5.5, without ethanol, at 20 degrees C. Inoculation ratio was not found to be a significant factor or involved in any significant two-factor interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings give insight into the conditions necessary to maximize cellulose production from this G. hansenii strain. In addition, this work demonstrates how the fractional factorial design can be used to test a large number of factors using an abbreviated set of experiments. Fitting a statistical model determined the significant factors as well as the significant two-factor interactions.     

Fungal and other β-d-glucosidases — Their properties and applications

β-d-Glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) has been described in a variety of fungi and bacteria. Its function — to catalyse the hydrolysis of cellobiose, and aryl and alkyl β-d-glucosides — depends upon the nature of its source. Recent interest in this enzyme centres on its role in the enzymatic hydrolysis of cellulose. The rate and extent of cellulose hydrolysis can be increased by supplementing commercial cellulases with immobilized β-d-glucosidase, which has high stability and can be recovered and reused. The current state of β-d-glucosidase biotechnology is described.     

微生物发酵产木聚糖酶研究进展

木聚糖是植物半纤维素的主要成分,是自然界中仅次于纤维素的可再生资源。木聚糖酶是一类重要的木糖苷键水解酶酶系,可将木聚糖逐次降解为低聚木糖及木糖,在饲料、造纸、食品和生物转化等行业应用广泛。目前利用微生物发酵生产木聚糖酶的研究很多,菌种涉及到细菌、真菌等,其发酵生产木聚糖酶的工艺、产量及特性也各有不同,对此进行了综述,并展望了木聚糖酶发酵生产的研究方向。     

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.