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...

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The metabolism of strain H10, a cellulolytic mesophilic Clostridium sp., was studied on glucose and cellobiose as energy and carbon sources. The main products of fermentation of both sugars were acetate, lactate, and ethanol. At low sugar levels, molar growth yields were better for cellobiose than for glucose. In both cases, an inhibition of growth was observed between 1 and 2 g/liter and a total inhibition after the latter concentration. Inhibition was not the result of low pH due to acid formation; growth under static pH conditions was limited in the same way. On the other hand, acetate and lactate had no inhibitory effect when added at concentrations equal to the final titers. Concomitant with the inhibition of growth was a change in metabolic pathways for sugar concentrations between 1 and 2 g/liter, i.e., the production of lactate was higher. After complete inhibition of growth, an accumulation of carbohydrates which were neither glucose nor cellobiose was observed.

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