Synthetic Medium for Cellulolytic Anaerobe,Ruminococcus albus

Some physico-chemical and heat-induced gelling properties of myosin heavy chains (MHCs) from rabbit skeletal muscle were studied. MHCs were found to be almost devoid of ATPase activity, possibly because of the absence of myosin light chains. MHCs formed precipitates below and ionic strength of 0.3, and above this ionic strength MHCs became soluble. On heating to 65°C at low concentrations of the protein, MHCs lost their solubility and formed aggregates even at high ionic conditions. Partially irreversible change in the conformation of MHC (from helical to random coil) also occurred in heated MHCs.

The heat-induced gel strength of MHCs was found to be almost equal to that of intact myosin molecules with identical protein concentrations. This suggests that the gelling potential of myosin is solely confined in MHC. Although myosin light chains do not seem to contribute to the gelling power of myosin, possibly they provide some stability to the gel if the pH is varied above the optimum value, i.e., 6.0. The addition of actin to a MHC system weakened the gel strength of the latter proportionally to the quantity of added actin.     

Increased cellulose hydrolysis by Bacteroides cellulosolvens in a simplified synthetic medium

The nutrient requirements of the cellulolytic anaerobe Bacteroides cellulosolvens were determined, and a new synthetic medium was formulated for its growth. B. cellulosolvens showed optimum cellobiose consumption and product formation in medium containing 40 mM ammonia nitrogen, 3 mM phosphate, 1 mM sulfide, 100 μM magnesium and 45 μM iron. This microbe had an essential-vitamin requirement for biotin; while zinc, manganese and copper slightly stimulated cellobiose degradation. In the new synthetic medium B. cellulosolvens was able to degrade 30% more cellulose.     

Chemically Defined Medium for Growth of Clostridium thermocellum, a Cellulolytic Thermophilic Anaerobe

Nutritional requirements of Clostridium thermocellum were examined, and a defined medium was formulated which supported reproducible growth through 10 serial subcultures.     

Trophic Links between the Acetogen Clostridium glycolicum KHa and the Fermentative Anaerobe Bacteroides xylanolyticus KHb, Isolated from Hawaiian Forest Soil

Isolate KH was obtained from Hawaiian forest soil and found to be composed of two functionally linked anaerobes, KHa and KHb. Gene analyses (16S rRNA, fhs, cooS) identified KHa as an acetogenic strain of Clostridium glycolicum and KHb as Bacteroides xylanolyticus. KHb fermented xylan and other saccharides that KHa could not utilize and formed products (e.g., ethanol and H₂) that supported the acetogenic growth of KHa.     

Isolation of a Cellulolytic Bacteroides sp. from Human Feces

An anaerobic cellulolytic bacterium, identified as a Bacteroides sp., was present in 10^-8 g of feces from only one of five human subjects.     

Symbiotic Relationship of Bacteroides cellulosolvens and Clostridium saccharolyticum in Cellulose Fermentation

In coculture, Bacteroides cellulosolvens and Clostridium saccharolyticum fermented 33% more cellulose than did B. cellulosolvens alone. Also, cellulose digestion continued at a maximum rate 48 h longer in coculture. B. cellulosolvens hydrolyzes cellulose and supplies C. saccharolyticum with sugars and a growth factor replaceable by yeast extract. Alone, B. cellulosolvens exhibited an early cessation of growth which was not due to nutrient depletion, low pH, or toxic accumulation of acetic acid, ethanol, lactic acid, H₂, CO₂, cellobiose, glucose, or xylose. However, a 1-h incubation of B. cellulosolvens spent-culture medium with C. saacharolyticum cells starved for growth factor allowed a resumption of B. cellulosolvens growth. The symbiotic relationship of this naturally occurring coculture is one of mutualism, in which the cellulolytic microbe supplies the saccharolytic microbe with nutrients, and in turn the saccharolytic microbe removes a secondary metabolite toxic to the primary microbe.     

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

Abstract 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. cellulolyticus 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.     

Anaerobe/aerobe environmental flux determines protein expression profiles of Bacteroides fragilis, a redox pathogen

The oxidation-reduction (redox) of the environment characterizes the Bacteroides fragilis pathogenic potential. Previously, using 3D confocal laser scanning microscopy, the bacteria prepared from cultures grown under oxidizing conditions (Eh(7)ca. + 100 mV) were able to penetrate into Hela cell monolayers. In contrast, when grown under reducing conditions (Eh(7)ca. - 60 mV), there were no bacteria evident within Hela cells. The influence of the anaerobe/aerobe environmental flux during the process of the anaerobe infection could be significant. In B. fragilis peritonitis, this may depend on the occurrence of aerobiosis as opposed to anaerobiosis. To this end, three clinical B. fragilis strains, two infectious and one non-infectious, were grown under oxidizing and reducing conditions; then, the outer membrane protein expressions derived from these strains were assessed, following sarcosyl extraction and SDS-PAGE. The differences between the protein profiles from these strains when cultured under oxidizing and reducing conditions were found to be statistically significant for the two infectious strains, but not for the non-infectious strain. OMP profiles under aerobic conditions compared to anaerobic conditions exhibited products with a range of apparent molecular weights suggestive of unique participation in the interaction with the host cell.     

Mechanism of Isolated Hemicellulose and Xylan Degradation by Cellulolytic Rumen Bacteria

Although certain strains of cellulolytic rumen bacteria cannot utilize isolated hemicelluloses or xylan as a source of energy, all strains examined can degrade or solubilize these materials from an 80% ethyl alcohol insoluble to a soluble form. Centrifugation and washing of the cellobiose-grown bacterial cells did not affect the rate or extent of utilization or degradation or both. When the level of a nonutilizing culture inoculum (either normal or washed) was doubled, a corresponding increase in the initial rate of degradation was observed. With a nitrogen-free medium, utilization of xylan was almost completely inhibited for a utilizing strain, whereas degradation by either type of organism was not markedly affected. Cellobiose medium cell-free culture filtrates from a nonutilizing strain were able to degrade or solubilize xylan. The percentage of degradation increased with the volume of cell-free filtrate, and all activity was lost when the filtrate was boiled. No utilization (loss in total pentose) was observed with cell-free filtrates from utilizing or nonutilizing strains. The release of free hexose from insoluble cellulose by culture filtrates from a nonutilizing strain was very limited. On the other hand, carboxymethylcellulose (CMC-70L) and cellulodextrins were degraded to an 80% ethyl alcohol soluble form by filtrates from both types of organisms. Similar enzyme activity was obtained in cell-free culture filtrates from four additional strains of cellulolytic rumen bacteria (one xylan utilizer and three nonutilizers). When the assays were carried out aerobically, CMC-70L solubilization was reduced to a much greater extent than xylan or cellulodextrin solubilization. The enzyme or enzymes responsible for the degradation of hemicellulose by cellololytic rumen bacteria unable to utilize the hemicellulose as an energy source appear to be constitutive in nature, and this activity may be a nonspecific action of a beta-1, 4-glucosidase or -cellulase.     

Effects of cellobiose and glucose on cellulose hydrolysis by both growing and resting cells of Bacteroides cellulosolvens

The cellulase system of Bacteroides cellulosolvens was subjected to both catabolite repression and feedback inhibition by cellobiose. Cellulose-solubilizing activity was 50% inhibited at a cellobiose concentration of 2.6 g/L and completely inhibited by 12 g/L. Glucose at 12 g/L (the highest concentration tested) had no effect on cellulase activity. Supplementation of B. cellulosolvens cellulase with beta-glucosidase resulted in increased conversion of cellobiose to glucose; however, a constant cellobiose pool size of approximately 7 g/L was maintained.     

Architecture of the Bacteroides cellulosolvens cellulosome: description of a cell surface-anchoring scaffoldin and a family 48 cellulase

A large gene downstream of the primary Bacteroides cellulosolvens cellulosomal scaffoldin (cipBc, now renamed scaA) was sequenced. The gene, termed scaB, contained an N-terminal leader peptide followed by 10 type I cohesins, an "X" domain of unknown structure and function, and a C-terminal S-layer homology (SLH) surface-anchoring module. In addition, a previously identified gene in a different part of the genome, encoding for a dockerin-borne family 48 cellulosomal glycoside hydrolase (Cel48), was sequenced completely, and a putative cellulosome-related family 9 glycosyl hydrolase was detected. Recombinant fusion proteins, comprising dockerins derived from either the ScaA scaffoldin or Cel48, were overexpressed. Their interaction with ScaA and ScaB cohesins was examined by immunoassay. The results indicated that the ScaB type I cohesin of the new anchoring protein binds selectively to the ScaA dockerin, whereas the Cel48 dockerin binds specifically to the type II ScaA cohesin 5. Thus, by virtue of the 11 type II ScaA cohesins and the 10 type I ScaB cohesins, the relatively simple two-component cellulosome-integrating complex would potentially incorporate 110 enzyme molecules onto the cell surface via the ScaB SLH module. Compared to previously described cellulosome systems, the apparent roles of the B. cellulosolvens cohesins are reversed, in that the type II cohesins are located on the enzyme-binding primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldin. The results underscore the extensive diversity in the supramolecular architecture of cellulosome systems in nature.     

Growth and Cellulase Formation by Cellvibrio fulvus

S ummary : The aerobic cellulolytic bacterium Cellvibrio fulvus grew on several sugars and polysaccharides, but not on highly substituted cellulose derivatives, organic acids and alcohols. Whereas no growth was obtained on long cotton fibres, it occurred on such fibres cut into small pieces, and on filter paper and chromatography powders derived from cotton. Lignin free wood pulp was rapidly degraded. The organism grew best at pH 7–8 and utilized nitrate, ammonium and some amino acids as nitrogen sources. The bacteria have cell-bound cellulase but enzyme was also found in the culture medium. Glucose repressed cellulase formation and the enzyme activity of cultures grown on cellulose was much higher than on sugars. Reducing sugar was not detected in cellulose cultures. The pH optimum for hydrolysis of carboxymethylcellulose (CMC) was 7 and the enzyme was inhibited by mercuric acetate but not by p -chloromercuribenzoate or EDTA. Fractionation of cellulase preparations from cultures grown on partially hydrolysed filter paper gave many components of different molecular weights. The activities of these components against carboxymethylcellulose and microcrystalline cellulose differed.     

Clostridium oceanicum, sp. n., a Sporeforming Anaerobe Isolated from Marine Sediments

Fourteen strains of a terminal-spored anaerobe were isolated from marine sediments obtained off the Atlantic and Pacific coasts of tropical South America. These strains are proteolytic, lecithinolytic, only slightly saccharolytic, often form cells with two spores, and appear unlike any described species of terminal-spored, proteolytic anaerobe. The name Clostridium oceanicum is suggested. The type strain (no. 25647) is deposited in the American Type Culture Collection.     

Cloning of the Thermostable Cellulase Gene from Newly Isolated Bacillus subtilis and its Expression in Escherichia coli

A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated strain. The optimum temperature of the enzyme reaction was 50°C, and CelDR retained 70% of its maximum activity at 75°C after incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain.     

Biodegradation of Chlorpyrifos by a Newly Isolated Bacillus subtilis Strain,Y242

A bacterial strain Y242 isolated from agricultural wastewater was found to be highly effective in degrading chlorpyrifos. On the basis of morphology, physiological characteristics, biochemical tests, and phylogenetic analysis of 16S rRNA sequence, the isolate was identified as Bacillus subtilis. The efficiency of the B. subtilis Y242 isolate as a chlorpyrifos degrader was examined under different culture conditions formulated according to the Plackett-Burman experimental design. It was observed that B. subtilis Y242 was able to utilize chlorpyrifos as a sole carbon and energy source and grows on a medium containing concentration up to 150 mg/L. A growth medium formulated based on the results of the Plackett-Burman experiment and supplied with 150 mg/L chlorpyrifos recorded 95.12% pesticide decomposition within 48 h. Degradation study of chlorpyrifos by B. subtilis Y242 was examined by gas chromatography–mass spectrometer (GC-MS) and high-performance liquid chromatography (HPLC). These results suggest that B. subtilis Y242 will be potentially useful in the cleanup of contaminated pesticide waste in the environment.     

Desulfurization of Dibenzothiophene and Diesel Oils by a Newly Isolated Gordona Strain, CYKS1

A dibenzothiophene (DBT)-desulfurizing bacterial strain was isolated and identified as Gordona strain CYKS1. Strain CYKS1 was found to transform DBT to 2-hydroxybiphenyl via the 4S pathway and to be able to also use organic sulfur compounds other than DBT as a sole sulfur source. Its desulfurization activity was susceptible to sulfate repression. Active resting cells for desulfurization could be prepared only in the early growth phase. When two types of diesel oils, middle distillate unit feed (MDUF) and light gas oil (LGO) containing various organic sulfur compounds including DBT, were treated with resting cells of strain CYKS1 for 12 h, the total sulfur content significantly decreased, from 0.15% (wt/wt) to 0.06% (wt/wt) for MDUF and from 0.3% (wt/wt) to 0.25% (wt/wt) for LGO. The newly isolated strain CYKS1 is considered to have good potential for application in the biodesulfurization of fossil fuels.