Acetivibrio Cellulolyticus Research Articles

Crystallization and preliminary X-ray analysis of Acetivibrio cellulolyticus cellulosomal type II cohesin module: two versions having different linker lengths.

The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2_S) and the second incorporating the full native 45-residue linker (CohB2_L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2_S and CohB2_L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2(1)2(1)2(1) with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 A for CohB2_S and space group P2(1)2(1)2 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 A for CohB2_L. The crystals diffracted to 2.0 and 2.9 A resolution, respectively. The asymmetric unit of CohB2_S contains three cohesin molecules, while that of CohB2_L contains two molecules.

Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes ofAcetivibrio cellulolyticusandClostridium thermocellum

We have sequenced a new gene, cel9B, encoding a family-9 cellulase from a cellulosome-producing bacterium, Acetivibrio cellulolyticus. The gene includes a signal peptide, a family-9 glycoside hydrolases (GH9) catalytic module, two family-3 carbohydrate-binding modules (CBM3c-CBM3b tandem dyad) and a C-terminal dockerin module. An identical modular arrangement exists in two putative GH9 genes from the draft sequence of the Clostridium thermocellum genome. The three homologous CBM3b modules from A. cellulolyticus and C. thermocellum were overexpressed, but, surprisingly, none bound cellulosic substrates. The results raise fundamental questions concerning the possible role(s) of the newly described CBMs. Phylogenetic analysis and preliminary site-directed mutagenesis studies suggest that the catalytic module and the CBM3 dyad are distinctive in their sequences and are proposed to constitute a new GH9 architectural theme.

Clostridium alkalicellum sp. nov., an Obligately Alkaliphilic Cellulolytic Bacterium from a Soda Lake in the Baikal Region

The first anaerobic alkaliphilic cellulolytic microorganism has been isolated from the Verkhnee Beloe soda lake (Buryatiya, Russia) with pH 10.2 and a salt content of up to 24 g/l. Five strains were characterized. Strain Z-7026 was chosen as the type strain. The cells of the isolate are gram-positive spore-forming rods. A mucous external capsule is produced. The microorganism is obligately alkaliphilic, growing in a pH range of 8.0-10.2, with an optimum at pH 9.0. Sodium ions and, in carbonate-buffered media, sodium chloride are obligately required. The microorganism is slightly halophilic; it grows at 0.017-0.4 M Na+ with an optimum at 0.15-0.3 M Na+. The metabolism is fermentative and strictly anaerobic. Cellulose, cellobiose, and xylan can be used as growth substrates. Plant and algal debris can be fermented. Lactate, ethanol, acetate, hydrogen, and traces of formate are produced during cellulose or cellobiose fermentation. Yeast extract or vitamins are required for anabolic purposes. The microorganism fixes dinitrogen and is nitrogenase-positive. It is tolerant to up to 48 mM Na2S. Growth is not inhibited by kanamycin or neomycin. Chloramphenicol, streptomycin, penicillin, ampicillin, ampiox, bacillin, novobiocin, and bacitracin suppress growth. The DNA G+C content is 29.9 mol %. According to the nucleotide sequence of its 16S rRNA gene, strain Z-7026 is phylogenetically close to the neutrophilic cellulolytic bacteria Clostridium thermocellum (95.5%), C. aldrichii (94.9%), and Acetivibrio cellulolyticus (94.8%). It is proposed as a new species: Clostridium alkalicellum sp. nov.

Growth and endoglucanase activity of Acetivibrio cellulolyticus grown in three different cellulosic substrates

The growth kinetics of Acetivibrio cellulolyticus grown in medium containing different carbon sources (cellobiose, amorphous or crystalline cellulose) was investigated. The specific growth rate was higher in cellobiose fed cultures than in the presence of the other two substrates. Endoglucanase production was greater in cultures grown on amorphous cellulose; enzyme activity increased during the stationary phase in cultures grown on crystalline cellulose.

A novel cellulosomal scaffoldin from Acetivibrio cellulolyticus that contains a family 9 glycosyl hydrolase.

A novel cellulosomal scaffoldin gene, termed cipV, was identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of Clostridium thermocellum. The cipV gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted "recognition codes" differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of C. thermocellum and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain. Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from C. thermocellum as opposed to the known scaffoldins from the mesophilic clostridia. The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.

Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage

Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage

Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage.

The nucleotide sequences of the 16S rRNA genes of Acetivibrio cellulolyticus, A. cellulosolvens, and Bacteroides cellulosolvens were determined and shown to be affiliated with the low G + C members of Gram-positive bacteria. The sequences for A. celluolyticus and A. cellulosolvens were revealed to be identical, supporting the proposal by W.D. Murray [Int. J. Syst. Bacteriol. (1986) 36, 314-316] that A. cellulosolvens be correctly classified as A. cellulolyticus. The closest relative to A. cellulolyticus is Clostridium aldrichii, related at 98.5% sequence similarity. B. cellulosolvens and A. cellulolyticus are related at 94.4% sequence similarity.

Factors affecting the utilization of steam- and explosion-decompressed aspen wood by cellulolytic anaerobes

Acetivibrio cellulolyticus and Clostridium thermocellum were unable to grow in medium containing 2 g litre −1 of untreated steam- and explosion-decompressed (SED) aspen wood as a source of carbon. When the medium was concurrently supplemented with 5 g litre −1 of Whatman CF11 cellulose powder, growth of both anaerobes, to levels comparable to media containing only CF11 cellulose, was observed. Treatment of the SED wood with ethanol-water, dioxane-water or sodium hydroxide released adhered lignin or lignin residues and rendered the SED wood suitable for the growth of both anaerobes. The results indicate that at concentrations of 4 g litre −1 or lower, growth on untreated SED wood is probably prevented because of inaccessibility of adherence sites for these bacteria, and at concentration of 4 g litre −1 or more it is also inhibited due to increased concentration of inhibitory materials such as lignin and lignin residues.

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.

Formation of esterase, xylanase and xylosidase by cellulolytic anaerobes

Three mesophilic anaerobes, namely Acetivibrio cellulolyticus, Acetivibrio cellulosolvens and Bacteroides cellulosolvens and a thermophilic anaerobe, namely Clostridium thermocellum, produced xylanase, xylosidase and esterase activities when grown on medium containing cellulose. Xylanase and xylosidase activities produced by both the mesophilic and thermophilic anaerobes were stimulated by the presence of ascorbic acid, cysteine or dithiothreitol, but differed in their stability at high temperatures, their response to the presence of calcium and magnesium and they had different temperature optima. Esterase activity from both mesophilic and thermophilic anaerobes had a pH optimum between 5.5 and 6.0 but the temperature optimum of the esterase was 37°C for mesophilic and 50°C for thermophilic anaerobes. The strains used in this study were unable to utilize glucose or xylose and their growth on xylan was limited. These anaerobes appear to produce these enzymes as a part of their cellulolytic enzyme system and degrade xylan during their growth on cellulose.

Acetivibrio cellulosolvens Is a Synonym for Acetivibrio cellulolyticus: Emendation of the Genus Acetivibrio

Acetivibrio cellulosolvens BASTwas compared with Acetivibrio cellulolyticus CD2Tand was found to be identical in all morphological and biochemical characteristics. The description of the genus Acetivibrio is emended as originally described by I. M. Robinson and A. E. Ritchie (Int. J. Syst. Bacteriol. 31:333-338, 1981) to include anaerobic, gram-negative, straight to slightly curved rods that produce mainly acetic acid, ethanol, H2 and CO2 and that are motile by means of a single flagellum or multiple flagella. It is proposed that A. cellulosolvens BASTbe correctly classified as A. cellulolyticus.

Influence of Clostridium saccharolyticum on cellulose degradation by Acetivibrio cellulolyticus*

AbstractThe interrelationship between Acetivibrio cellulolyticus and Clostridium saccharolyticum, two anaerobes recently isolated from a methanogenic cellulose‐enriched culture, was studied in an examination of the role of saccharolytic anaerobes in habitats where cellulose is converted to methane. The presence of Cl. saccharolyticum decreased cellulose degradation by A. cellulolyticus as a result of substrate competition. Nevertheless A. cellulolyticus provided essential growth factor(s) and assimilable sugars including glucose which A. cellulolyticus itself does not utilize. Cl. saccharolyticum converted sugars to acetic acid and H2, known substrates for methanogens, and ethanol and lactic acid, known substrates for sulphate reducing bacteria. In natural habitats, the presence of both methanogens and sulphate reducing bacteria is essential for the existence of cellulolytic anaerobes.

Location and kinetic properties of the cellulase system of Acetivibrio cellulolyticus

The cellulose-solubilizing and carboxymethylcellulase activities of Acetivibrio cellulolyticus were observed to exist largely in a soluble form whereas β-glucosidase activity was cell associated. Reducing agents significantly enhanced cellulose-solubilizing activity, and thiol-binding agents at micromolar concentrations resulted in almost total inhibition of this activity. Carboxymethylcellulase activity was also inhibited by these agents but to a lesser degree. Cellulose-solubilizing activity was strongly stimulated by Mg2+ and Ca2+ but these ions had a negligible effect on carboxymethylcellulase activity. Cellulase activity was not inhibited by glucose and only slightly by cellobiose. A strong preference for p-nitrophenylglucoside over cellobiose by β-glucosidase activity was observed.

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose, cellobiose and cellulose

The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N2/CO2 or N2 gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h−1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and 14C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (μ=0.03h−1) and molar growth yield (Yglucose=21.5) were considerably lower than those obtained (μ=0.17 h−1, Ycellobiose=68.9) in cellobiose media. A YATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H2, 2.64 CO2, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.

Effect of pH and Oxygen on Growth and Viability of Acetivibrio cellulolyticus

Growth, hydrogen production and cellulose digestion by Acetivibrio cellulolyticus strain CD2 were considerably greater when the culture pH was maintained at 70 than when the pH was not controlled. Furthermore, if the pH of the growth medium was controlled the number of viable organisms was 6-fold greater after 3 d incubation and 100-fold greater after 6 d incubation compared with equivalent cultures in which the pH was not controlled. The differences were due to the combined effect of low pH values and acetic acid accumulation. The number of viable organisms was 2- to 3-fold lower after 12 h incubation in substrate-free medium containing 40 mm-acetic acid at pH 55 than in the same medium at pH 70. Addition of 90 mm-acetic acid during growth in a cellobiose-containing medium lowered the growth rate by 30% and the rate of hydrogen production by 40%. Exposure of A. cellulolyticus to oxygen for up to 2 h did not affect viability measurements provided that the organisms were subsequently transferred to reduced media.

A one step process for the conversion of cellulose to ethanol using anaerobic microorganisms in mono- and co-culture

The reducing sugars, glucose, and ethanol produced during growth of the anaerobes Clostridium thermocellum and Acetivibrio cellulolyticus on cellulose were assayed. Zymomonas mobilis was grown under similar conditions and could ferment glucose to ethanol. The ethanol production by the cellulolytic bacteria alone and in co-culture with Zymomonas is described. Approximately 27% of a 1% cellulose substrate could be converted to ethanol by this co-culture.

Accumulation of an iodophilic polysaccharide during growth of Acetivibrio cellulolyticus on cellobiose

Maximum growth of Acetivibrio cellulolyticus in 1% cellobiose (w/v, added as filter sterilized solution) medium was observed after about 24h of incubation at 35°C. The metabolic end products of growth were H2, CO2, acetic acid, ethanol and glucose. Growth was adversely affected if cellobiose was autoclaved with the rest of the media ingredients. In the presence of an excess of cellobiose, the cells accumulated large quantities of an iodophilic polysaccharide (IPS). The maximum IPS accumulation (about 37% of the cell dry weight) was observed after about 12h growth under nitrogen-limiting conditions. Starvation of these cells anaerobically, in a pH 7.0 phosphate buffer for 10 h at 35°C, resulted in about 50% drop in the IPS. The results also indicated that A. cellulolyticus accumulated this iodophilic polysaccharide during growth on cellobiose but not during cultivation on cellulose.

Cellulolytic enzyme system of Acetivibrio cellulolyticus.

Polyacrylamide gel electrophoresis of the cellulolytic system from culture supernates of Acetivibrio cellulolyticus showed the presence of four major enzymes: a beta-glucosidase, an exoglucanase, and two endoglucanases. The relative proportions of these enzymes in the culture supernate were affected by the nature of the cellulosic substrate and by the length of the incubation period. The molecular weights of the cellulolytic enzymes were beta-glucosidase, 81 000; exoglucanase, 38 000; endoglucanase C2, 33 000; and endoglucanase C3, 10 400, as estimated by their electrophoretic mobilities relative to proteins of known molecular weight. Treatment of the high molecular weight endoglucanase with SDS--mercaptoethanol led to reversible dissociation of the enzyme into polypeptide subunits similar to the low molecular weight endoglucanase. Endoglucanase activity could be assayed for directly using a novel method of incorporating carboxymethyl cellulose in the polyacrylamide gels. The molecular weights and functions of these enzymes are compared with those detected in culture filtrates of various fungi.

Cellulolytic Enzyme System of Acetivibrio cellulolyticus, a Newly Isolated Anaerobe

Crude enzyme preparations from Acetivibrio cellulolyticus converted ball-milled pulp, cotton batting, filter and tissue paper, a microcrystalline cellulose, carboxymethylcellulose, cellobiose and xylan to reducing sugars. The preparations showed maximum activity between pH 5 and 6 and at a temperature between 37 and 50 °C, depending on the substrate used. The enzyme activity was fairly stable at 2 °C for 4 weeks. The saccharifying ability of the preparation was comparable to that of commercially available cellulase preparations from Aspergillus niger and Trichoderma viride.

Cellulase production by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus, an isolate from an established sewage sludge culture, degraded cellulose powder, Avicel cellulose, and cellobiose. The organism showed maximum cellulose degradation in a medium containing 10 g/L of cellulose and it could also degrade cellulose in media containing up to 75 g/L of cellulose. During the exponential growth phase, large quantities of cellulolytic enzymes were found extracellularly whereas cellobiase activity was cell associated. The crude culture supernate contained endo- and exo-glucanase activities with a pH optimum at 5.0 and a temperature optimum at 50 °C. Maximum cellulase activities were detected in 2- to 3-day-old cultures grown on 1 g/L of cellulose. Cellulose concentration above 10 g/L caused the adsorption of these enzymes to the substrate and consequently lowered their detection in the supernate. The activities at 50 °C for endoglucanase, exoglucanase, and filter paper degrading ability, expressed as micrograms of glucose equivalents released per minute per milligram of protein culture supernate, were 510, 135, and 40 respectively.