A new anaerobic, mesophilic, spore-forming cellulolytic bacterium is described. Cellulose is cleared within 24 to 48 h around colonies formed in cellulose agar roll tubes. Cells stain gram negative and are nonmotile rods which form oblong spores either centrally or subterminally in a clostridial swelling. Colonies are irregular with an opaque edge and a center devoid of both vegetative cells and spores. Cellulose, xylan, pectin, cellobiose, glucose, maltose, galactose, sucrose, lactose, and mannose serve as substrates for growth. H2, C02, acetate, butyrate, formate, and lactate are produced during fermentation of cellulose or cellobiose. The temperature and pH for optimum growth are 37°C and 7.0, respectively. The DNA composition is 26 to 27 mol% guanine plus cytosine. This bacterium resembles "Clostridium lochheadii" in morphological and some biochemical characteristics but is not identical to it. The name Clostridium cellulovorans sp. nov. is proposed. The type strain is 743B (ATCC 35296).

Thermobacteroides proteolyticus sp. nov. was isolated from a methanogenic enrichment culture inoculated from a thermophilic digestor (55°C) that was fermenting tannery wastes and cattle manure. The cells were anaerobic, gram-negative, nonsporeforming, nonmotile rods that were 0.5 pm wide and 1 to 6 pm long. At the end of logarithmic growth, they were pleomorphic, with some filamentous cells. The deoxyribonucleic acid base composition was 45 mol% guanine plus cytosine. The temperature optimum was 63°C (growth range 35, to 75°C); the pH optimum was 7.5 (growth range, pH 5.0 to 8.5). The growth substrates used included yeast extract, peptone, casein, gelatin, and Trypticase peptone. Fructose, glucose, maltose, sucrose, and mannose were weakly used as growth substrates; however, addition of yeast extract and either rumen fluid or Trypticase peptone stimulated utilization of these carbohydrates. Acetate, H2, and C 0 2 were the major products of growth in medium containing gelatin or glucose. The cells were resistant to kanamycin. The type strain is strain BT (= ATCC 35242).We report the isolation and characterization of a new thermophilic anaerobic species obtained from a methanogenic enrichment inoculated from a thermophilic digestor that was operated on tannery wastes and cattle manure. This organism stains gram negative; it is a nonsporulating bacterium which uses proteins and sugars as substrates. We propose the name Thermobacteroides proteolyticus sp. nov .for this organism. MATERIALS AND METHODSInoculum and enrichment. Tannery wastes and cattle manure were digested under thermophilic conditions (55°C) at the Institut de Recherche de Chimie Appliqude, Vert le Petit, France. Digestor samples were inoculated into a 50% rumen fluid medium made up in the salt solution of Balch et al. (1) with formate and H2-C02 as methanogenic substrates. When methanogenesis was complete, the culture was maintained by inoculation of fresh medium with 5% (vol/vol) transfers. The most numerous methanogenic bacterium obtained from the enrichment was isolated and tentatively identified as Methanobacterium thermoautotrophicum .Isolation procedures. After several transfers of the thermophilic methanogenic enrichment culture, agar plates were incubated for isolation of methanogenic and nonmethanogenic bacteria. One predominant non-methanogenic colony type appeared at high dilutions on agar medium having the same composition as the enrichment medium. This colony type was picked and isolated in axenic culture by inoculation into agar medium.Culture techniques. The anaerobic culture techniques of Hungate (5) , and 1,000 ml of Milli-Q-deionized water (conductivity, 5.9 $3 . m-'). The medium was adjusted to pH 7.0 with 10 M KOH and boiled under O2-free N2 until the resazurin was reduced. Medium was cooled and dispensed into serum tubes (10 ml/tube) that were stoppered with butyl rubber stoppers (Bellco Glass, Inc., Vineland, N.J.) and sealed with aluminum crimp closures (Wheaton Scientific, Millville, N.J.). After removal from the anaerobic ch...

An anaerobic bacterium which produced acetate from H2 and C 0 2 was isolated. The rod-shaped cells were not lysed by KOH, did not hydrolyze ~-alanine-4-nitroanilide, and stained gram negative. However, the cell wall did not resemble a gram-negative wall in structure; it was comprised of two layers. The cells were motile by means of three or four peritrichous flagella. Yeast extract was required for both chemoorganotrophic and chemolithotrophic growth; yeast extract, glucose, maltose, or H2-C02 could serve as a substrate for growth. Strain NOT-3T (T = type strain) grew best at 37°C and pH 7.6 to 7.8. The deoxyribonucleic acid base composition was 36.8 mol% guanine plus cytosine. Strain NOT-3 (= ATCC 35199) is named Acetoanaerobium noterae gen. nov., sp. nov. and is the type strain of this new species.Acetate production by H2-dependent C02 reduction was first demonstrated in enrichment cultures (9). Subsequently, Clostridium aceticum, which grows readily in the presence olf H2 and COz, was isolated by Wieringa (20). Acetate is the only product formed by this organism. The original culture oif Wieringa was presumably lost for many years but was recently revived from an old endospore preparation (5). Isolates similar to C. aceticum have been described by other workers (1,14). Anaerobic Hz-oxidizing acetogenic bacteria can be found in a number of environments (6). Other species with this property have been described in the genera Acetobacterium (2,4), Acetogenium (ll), Eiibacterium (18), and Clostridiirm (19), including thermophilic species (11,19).A sediment sample taken from an oil exploration drilling site was examined for the presence of methanogens. High dilutions of the sample showed H2 uptake and acetate production without methanogenesis. A bacterium which produced acetate from H2 and C 0 2 was isolated from these dilutions. This organism (strain NOT-3T [= ATCC 35199T]) (T = type strain) is named Acetoanaerobium noterae gen. nov., sp. nov.(A brief report of this work appeared previously [Sleat, Mah, and Robinson, Abstr. Annu. Meet. Am. SOC. Microbiol. 1983Microbiol. , 154, p. 1481 MATERIALS AND METHODS Bacterial strains. Strain NOT-3T was isolated from sediment of the Notera 3 oil exploration drilling site in the Hula swamp area of Galilee, Israel. The sediment pH was 8.0. Polyethylene bottles were filled with sediment samples, shipped to the laboratory, and stored under 02-free N2 at 4°C.Culture medium. The culture medium used was prepared by using the techniques of Hungate (10). This medium contained (per liter) 0.4 g of K2HP04 * 3H20, 1.0 g of NH,CI, 0.45 g of NaCI, 2.0 g of yeast extract (Difco L,aboratories, Detroit, Mich.), 0.15 g of L-cysteine hydrochloride, and 0.001 g of resazurin; 10 ml of a trace metal solution (8) and 10 ml of vitamin solution (5) were added per liter. The medium was adjusted to pH 7.0 with 4 N NaOH, dispensed under a gas phase of either H2-C02 (4:l) or hl2-CO2 (4:1), and autoclaved. Before inoculation the pH * Corresponding author. 10 was adjusted (usually to 8.0) with a sterile ...

Methanosarcina mazei S6 and LYC were used to study the structure and differentiation of the aggregating methanogens. Cultures harvested under various conditions are described at the ultrastructural level. Cells of strain S6 are enclosed by a layer 12 nm thick in contact with the plasma membrane. In sarcinal colonies, cells are held in close association by a fibrous matrix up to 60 nm thick. Colony maturation was examined in strain S6 over a period of 1 year. Changes occurred in the shape and staining of individual cells. Also, various inclusion bodies were observed that either persist throughout colony maturation or are only found at certain growth stages. Two types of cores that are composed of double membranes in M. mazei S6 are described. One has a 90-nm diameter and contains electron-dense granules similar to those found in the cytoplasm. The other core type does not contain granules, is more numerous, and is found in older cultures. Two life cycles are described for M. mazei based on electron microscope examinations. A complex life cycle involving the release of single cells is described with two variations for strains S6 and LYC. When released cells of strain S6 are placed in fresh medium they can repeat the cycle. In addition, a limited cycle is described for both strains of M. mazei. This limited cycle contains the only sarcinal morphotypes observed in M. barkeri. When M. mazei S6 remains in the limited cycle and does not disaggregate in stationary phase, several types of possible resting forms are found.