Mesophilic cellulolytic clostridia from freshwater environments

Eight strains of obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from mud of freshwater environments. The isolates (C strains) were rod-shaped, gram negative, and formed terminal spherical to oval spores that swelled the sporangium. The guanine plus cytosine content of the DNA of the C strains ranged from 30.7 to 33.2 mol% (midpoint of thermal denaturation). The C strains fermented cellulose with formation primarily of acetate, ethanol, CO(2), and H(2). Reducing sugars accumulated in the supernatant fluid of cultures which initially contained >/=0.4% (wt/vol) cellulose. The C strains resembled Clostridium cellobioparum in some phenotypic characteristics and Clostridium papyrosolvens in others, but they were not identical to either of these species. The C strains differed from thermophilic cellulolytic clostridia (e.g., Clostridium thermocellum) not only in growth temperature range but also because they fermented xylan and five-carbon products of plant polysaccharide hydrolysis such as d-xylose and l-arabinose. At 40 degrees C, cellulose was degraded by cellulolytic mesophilic cells (strain C7) at a rate comparable to that at which C. thermocellum degrades cellulose at 60 degrees C. Substrate utilization and growth temperature data indicated that the C strains contribute to the anaerobic breakdown of plant polymers in the environments they inhabit.     

Bacterial strains from human feces that reduce CO2 to acetic acid.

We used dilutions of fecal suspensions from a human volunteer to enrich cultures for bacteria that reduce CO2 to acetate in the colon. The soluble enrichment substrates used were glucose, methanol, formate, and vanillate, which were used with a gas phase that contained 80% N2 and 20% CO2. The gaseous enrichment substrates used were 80% H2-20% CO2 and 50% CO-50% CO2. We isolated three different strains that produced acetate from CO2. One strain produced acetate from methanol, vanillate, H2-CO2, glucose, and other sugars. The other two strains did not form acetate from methanol or vanillate. Both of the latter strains formed acetate from glucose and other sugars, but only one of these strains formed acetate from H2-CO2. Both of these strains cometabolized formate. However, none of the enrichment cultures or pure cultures used CO or formate as a substrate for growth. The two strains that produced acetate from H2 and CO2 grew slowly when the gases alone were used as substrates, but they rapidly cometabolized H2 and CO2 when they were grown with organic substrates. The ability of all of the strains to produce acetate from CO2 and/or other one-carbon precursors was verified by determining the radioactivity of the methyl and carboxyl groups of the acetate formed after growth with 14CO2 or other radioactively labeled one-carbon precursors.     

Detrimental effect of cellulose oxidation on cellulose hydrolysis by cellulase

To effectively convert complex and recalcitrant biomass carbohydrates to simple platform sugars useful for fuel and chemicals production, mechanical or chemical pre-treatments are often required to make the carbohydrates more accessible for enzymatic hydrolysis. Due to their harsh conditions, some pre-treatments might negatively affect enzymatic hydrolysis because of events such as cellulose oxidation. To study how oxidative modification may impact cellulose's reactivity toward hydrolysis by cellulases, we prepared three cellulose substrates by cupric ion and hypochlorite oxidations, and subjected the derived celluloses to hydrolysis by various cellobiohydrolases from glycoside hydrolase families 6 and 7, and one cellulolytic Hypocrea jecorina extracellular enzyme mixture. We observed a profound decrease of enzymatic hydrolysis on the oxidized celluloses. The effect was attributed to the interference, from oxidized functional groups in cellulose, on its binding/activation in the active pocket/tunnel of cellobiohydrolases. Potential implication of the observed effect from cellulose oxidation on pre-treatment optimization and cellulase improvement was discussed.     

Cellulose degradation by a new isolate from sewage sludge, a member of the Bacteroidaceae family.

A mesophilic anaerobe, a member of the Bacteroidaceae family (NRC2248), isolated from a cellulose-enrichment culture, digested untreated Whatman cellulose powder and HCl-treated cotton battings while producing hydrogen, carbon dioxide, cellobiose, glucose, and acetic acid as the sole volatile acid. This organism also utilized cellobiose as carbon and energy source but did not utilize glucose. It grew well in synthetic medium containing ammonium salts as nitrogen source and having a pH value of 7.0-7.1 and an Eh value of -160mV or lower. The nutrient requirements of this organism were found to be similar to those of other anaerobes except for Na2S which inhibited cellulose degradation in concentrations above 0.75 mM. Best cellulose degradation occurred under an atmosphere of 80% N2-20% CO2. Use of H2 or 80% H2-20% CO2 as headspace gas inhibited growth. Although accumulation of acetic acid in about 15-16 mM concentrations inhibited the further formation of H2, CO2, and acetic acid in the broth, it did not stop the degradation of cellulose. The results indicate that this organism has the ability to grow in media containing up to 20 g/L of cellulose and to produce industrially important and easily separable end products from cellulose.     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium.

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

Assessing the Performance of Bacterial Cellulases: the Use of <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Paenibacillus</Emphasis> Strains as Enzyme Sources for Lignocellulose Saccharification

Plant biomass offers a renewable and environmentally favorable source of sugars that can be converted to different chemicals, second-generation ethanol, and other liquid fuels. Cellulose makes up approximately 45 % of the dry weight of lignocellulosic biomass. Prior to the enzymatic hydrolysis of cellulose, lignin and hemicellulose must be structurally altered or removed, at least in part, by chemical and/or physical pretreatments. However, the high cost and low efficiency of the enzymatic hydrolysis prevent the process from being economically competitive. For this reason, it is necessary to find enzymes suitable for this type of process, with higher specific activities and greater efficiency. Members of the Bacillus and Paenibacillus genera have been traditionally used for the production of many enzymes for industrial applications. Cellulases produced by both genera have shown activity on soluble and crystalline cellulose and high thermostability and/or activity over a wide pH spectrum. In this review, the most recent information about the characterization of cellulolytic enzymes obtained from new strains of the Bacillus and Paenibacillus genera are reviewed. We focused on the variety of isoenzymes produced by these cellulolytic strains, their optimal production and reaction conditions, and their kinetic parameters and biotechnological potential.     

合成气厌氧发酵生产有机酸和醇的研究进展

合成气来自于煤、石油、生物质和有机废物的气化,其主要成份为CO、H2和CO2。研究发现某些厌氧菌能利用合成气生成乙醇、乙酸、丁醇和丁酸等燃料和化学品。由于生物转化所具有的优势,合成气厌氧发酵被认为是一项极具潜力和竞争力的技术,在生物质及有机废物的利用方面将发挥重要作用。对厌氧发酵合成气生产有机酸和醇的研究进展,包括利用合成气产有机酸和醇的微生物,合成气发酵的代谢途径和关键酶（一氧化碳脱氢酶/乙酰辅酶A合成酶）及用于合成气发酵的反应器等进行了综述,并对该项技术的发展提出了一些建议。     

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.

     

Regulation of hexose phosphate metabolism in Acetobacter xylinum

The metabolism of glucose and fructose was studied in resting succinate-grown cells of Acetobacter xylinum. From fructose only cellulose and CO(2) were formed by the cells, whereas from glucose, gluconate was formed much more rapidly than these two products. The molar ratio of sugar converted into cellulose to sugar converted into CO(2) was significantly greater than unity for both hexoses. The pattern of label retention in the cellulose formed by the cells from specifically (14)C-labelled glucose, fructose or gluconate corresponded to that of hexose phosphate in a pentose cycle. On the other hand, the isotopic configuration of cellulose arising from variously singly (14)C-labelled pyruvate did not agree with the operation of a pentose cycle on gluconeogenic hexose phosphate. Readily oxidizable tricarboxylic acid-cycle intermediates such as acetate, pyruvate or succinate promoted cellulose synthesis from fructose and gluconate although retarding their oxidation to CO(2). The incorporation into cellulose of C-1 of fructose was greatly increased in the presence of these non-sugar substrates, although its oxidation to CO(2) was greatly diminished. It is suggested that the flow of hexose phosphate carbon towards cellulose or through the pentose cycle in A. xylinum is regulated by an energy-linked control mechanism.     

Bioconversion of Cellulose to Acetate with Pure Cultures of Ruminococcus albus and a Hydrogen-Using Acetogen

Bioconversion of cellulose to acetate was accomplished with cocultures of two organisms. One was the cellulolytic species Ruminococcus albus. It ferments crystalline cellulose (Avicel) to acetate, ethanol, CO(inf2), and H(inf2). The other organism (HA) obtains energy for growth by using H(inf2) to reduce CO(inf2) to acetate. HA is a gram-negative coccobacillus that was isolated from horse feces. Coculture of R. albus with HA in batch or continuous culture alters the fermentation products formed from crystalline cellulose by the ruminococcus via interspecies H(inf2) transfer. The major product of the fermentation by R. albus and HA coculture is acetate. High concentrations of acetate (333 mM) were obtained when batch cocultures grown on 5% cellulose were neutralized with Ca(OH)(inf2). Continuous cocultures grown at retention times of 2 and 3.1 days produced 109 and 102 mM acetate, respectively, when fed 1% cellulose with utilization of 84% of the substrate.     

Photobactericidal plastic films based on cellulose esterified by chloroacetate and a cationic porphyrin

The synthesis and characterisation of pyridinium porphyrinic chloroacetyl cellulose ester chlorides, where photosensitizing agents are covalently bounded to the polymeric chain, is presented in this paper. First, cellulose was homogenously converted into chloroacetate cellulose ester in DMAc/LiCl solvent by using chloroacetyl chloride. The complete substitution of cellulose was achieved using 7equiv of chloroacetyl chloride for a 2h reaction at room temperature. The absence of base did not prove detrimental to reaction. The grafting of monopyridyltritolylporphyrin onto chloroacetate cellulose ester was then realised by alkylation of the photosensitizer in DMF. These new plastic films were found to be thermostable up to 55 degrees C; higher temperatures led to progressive deacetylation. First results of their photobactericidal activity against Staphylococcus aureus and Escherichia coli strains are very encouraging. Such materials could find applications in medical environments as an alternative to overcome the rampant bacterial multiresistance to classical antibiotics.     

Progress in biocatalysis with immobilized viable whole cells: systems development,reaction engineering and applications

Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.     

Analysis of behavior using genetical genomics in mice as a model: from alcohol preferences to gene expression differences.

Most familial behavioral phenotypes result from the complex interaction of multiple genes. Studies of such phenotypes involving human subjects are often inconclusive owing to complexity of causation and experimental limitations. Studies of animal models argue for the use of established genetic strains as a powerful tool for genetic dissection of behavioral disorders and have led to the identification of rare genes and genetic mechanisms implicated in such phenotypes. We have used microarrays to study global gene expression in adult brains of four genetic strains of mice (C57BL/6J, DBA/2J, A/J, and BALB/c). Our results demonstrate that different strains show expression differences for a number of genes in the brain, and that closely related strains have similar patterns of gene expression as compared with distantly related strains. In addition, among the 24 000 genes and ESTs on the microarray, 77 showed at least a 1.5-fold increase in the brains of C57BL/6J mice as compared with those of DBA/2J mice. These genes fall into such functional categories as gene regulation, metabolism, cell signaling, neurotransmitter transport, and DNA/RNA binding. The importance of these findings as a novel genetic resource and their use and application in the genetic analysis of complex behavioral phenotypes, susceptibilities, and responses to drugs and chemicals are discussed.     

Molecular engineering of the cellulosome complex for affinity and bioenergy applications

The cellulosome complex has evolved to degrade plant cell walls and, as such, combines tenacious binding to cellulose with diverse catalytic activities against amorphous and crystalline cellulose. Cellulolytic microorganisms provide an extensive selection of domains; those with affinity for cellulose, cohesins and their dockerin binding partners that define cellulosome stoichiometry and architecture, and a range of catalytic activities against carbohydrates. These robust domains provide the building blocks for molecular design. This review examines how protein modules derived from the cellulosome have been incorporated into chimaeric proteins to provide biosynthetic tools for research and industry. These applications include affinity tags for protein purification, and non-chemical methods for immobilisation and presentation of recombinant protein domains on cellulosic substrates. Cellulosomal architecture provides a paradigm for design of enzymatic complexes that synergistically combine multiple catalytic subunits to achieve higher specific activity than would be obtained using free enzymes. Multimeric enzymatic complexes may have industrial applications of relevance for an emerging carbon economy. Biocatalysis will lead to more efficient utilisation of renewable carbon-fixing energy sources with the added benefits of reducing chemical waste streams and reliance on petroleum.     

Microbial CO conversions with applications in synthesis gas purification and bio-desulfurization

Recent advances in the field of microbial physiology demonstrate that carbon monoxide is a readily used substrate by a wide variety of anaerobic micro-organisms, and may be employed in novel biotechnological processes for production of bulk and fine chemicals or in biological treatment of waste streams. Synthesis gas produced from fossil fuels or biomass is rich in hydrogen and carbon monoxide. Conversion of carbon monoxide to hydrogen allows use of synthesis gas in existing hydrogen utilizing processes and is interesting in view of a transition from hydrogen production from fossil fuels to sustainable (CO2-neutral) biomass. The conversion of CO with H2O to CO2 and H2 is catalyzed by a rapidly increasing group of micro-organisms. Hydrogen is a preferred electron donor in biotechnological desulfurization ofwastewaters and flue gases. Additionally, CO is a good alternative electron donor considering the recent isolation of a CO oxidizing, sulfate reducing bacterium. Here we review CO utilization by various anaerobic micro-organisms and their possible role in biotechnological processes, with a focus on hydrogen production and bio-desulfurization.     

Enzymatic degradation of cell wall and related plant polysaccharides

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.