Clostridium cellulolyticum sp. nov., a Cellulolytic, Mesophilic: Species from Decayed Grass

Abstract

A new cellulolytic Clostridium species that was isolated from decayed grass is described. The colonies produced by this organism on cellulose agar are circular, translucent, and unpigmented and have undulate margins. The cells of this bacterium are gram-positive straight to slightly curved rods 3 to 6 km long by 0.6 to 1 pm wide and are motile; they form round, terminal spores 1.5 pm in diameter. A variety of carbohydrates are fermented by this mesophilic anaerobe. The major fermentation products from cellulose are carbon dioxide, hydrogen, ethanol, acetate, lactate, and formate. The deoxyribonucleic acid base composition is 41 mol% guanine plus cytosine. The type strain of Clostridium cellulolyticum sp. nov. is strain Hlo (= ATCC 35319).Anaerobic cellulolytic bacteria that take part in the degradation of cellulosic materials have been isolated from manure, rumen contents, and soil. In this report we describe the isolation and characterization of a new species of Clostridiurn from compost. MATERIALS AND METHODSMedia and culture techniques. The cornposition of the medium which we used was the same as the composition of the cellulose-yeast extract-salts medium (CM medium) described by Weimer and Zeikus (15), except that Na2S was omitted, the cysteine hydrochloride concentration was 1 g/liter, and the cellulose MN300 concentration was 7.5 g/liter.For solid media CM medium was supplemented with 20 g of agar (Difco Laboratories, Detroit, Mich.) per liter. The pH was adjusted to 7.5 with NaOH. This medium was prereduced by the Hungate technique (5) and was dispensed under a constant flow of N2 in 8-ml amounts (4.5-ml amounts for solid medium) into anaerobic culture tubes (16 by 125 mm; Bellco Glass, Inc., Vineland, N.J.).For enrichment cultures, we used anaerobic tubes (25 by 142 mm; Bellco) that contained 25 ml of medium.Carbohydrate media were prepared by replacing cellulose with filter-sterilized solutions of carbohydrates to give final concentrations of 7.5 g/liter. The cultures were incubated at 35°C without shaking.The anaerobic culture techniques of Hungate (5), as modified by Bryant (l), were used throughout this work.All cultures were inoculated and incubated with nitrogen as the gas.Isolation of strain HloT. Strain HloT (T = type strain) was obtained from cultured decayed grass by enrichment and isolation.Enrichment cultures were initiated by inoculating 1 to 2 g of grass from compost into anaerobic culture tubes (25 by 142 mm; Bellco) containing 25 ml of prereduced medium. After 1 week of incubation at 35"C, the cultures were heated at 80°C for 10 min, and 1 ml of each culture was then transferred to 8 ml of prereduced liquid medium.After a few days of incubation, cultures that produced * Corresponding author.discrete gas bubbles and solubilized cellulose were selected for isolation in solid medium. Serial dilutions were incubated in cellulose agar roll tubes. After 3 to 5 days, colonies which produced cleared zones of cellulose digestion were transferred individually into the liquid medium by means of a be...

Clostridium sufflavum sp. nov., isolated from a methanogenic reactor treating cattle waste

“…The novel strain may form spores only rarely during growth on soluble saccharides, although thermotolerant cells did occur in cultures grown on filter paper. The rare formation of spores has also been reported for C. cellulolyticum (Petitdemange et al, 1984).…”

“…The next most closely related species were also cellulolytic members of Clostridium cluster III, namely Clostridium termitidis DSM 5398 T (sequence similarity of 96.2 %) (Hethener et al, 1992), Clostridium papyrosolvens DSM 2782 T (96.1 %) (Madden et al, 1982), Clostridium josui FERM P-9684 T (95.9 %) (Sukhumavasi et al, 1988) and Clostridium cellulolyticum ATCC 35319 T (95.5 %) (Petitdemange et al, 1984) (Fig. 2).…”

Characterization of endoglucanase A from Clostridium cellulolyticum

“…In particular, their mesophilic character can be an advantage for industrial processes such as the treatment of urban and industrial refuse or the production of chemicals. For several years, therefore, we have been investigating the cellulolytic system of Clostridium cellulolyticum (7,8,10,11,20). Once the characterization of the bacteria (20) and its physiological study were well under way (10,11), we began to study its cellulolytic components by using a molecular genetics approach (7).…”

“…For several years, therefore, we have been investigating the cellulolytic system of Clostridium cellulolyticum (7,8,10,11,20). Once the characterization of the bacteria (20) and its physiological study were well under way (10,11), we began to study its cellulolytic components by using a molecular genetics approach (7). Up to now, we have identified five genes coding for components of the cellulolytic system of C. cellulolyticum; one of these, celCCA, coding for endoglucanase A from C. cellolyticum (EGCCA), has been sequenced (8).…”

Engineered microbial consortium for the efficient conversion of biomass to biofuels: A preliminary study

“…It is rod shaped with dimensions 1.0-2.0 × 4.0-5.0 µm, motile, does not sporulate, does not produce capsules, intracellular lipids or glycogen, optimal pH range for growth is 6 to 7.0, optimal temperature Clostridium cellulolyticum ATCC 35319 formerly identified as strain H 10 was isolated in the fall of 1982 from decayed grass compost packed for 3 to 4 months at the Université de Nancy, France. it is a Gram-positive, straight to slightly curved rods that are 3 to 6 µm long by 0.6 to 1.0 µm wide, with a mean G + C content of 41% and forms spores in cultures of cellulose media 3 or more days old (Petitdemange et al, 1984). C. cellulolyticum produces several cellulases, which are re-grouped into an extracellular enzymatic complex called cellulosomes and cellulolytic activities allow the release of soluble cellodextrins from cellulose, which in return permits microbial growth (Desvaux, 2005).…”