A new mesophilic, cellulolytic species of Bacteroides was isolated from a methanogenic cellulose enrichment culture of municipal sewage sludge and is described. This species ferments only cellulose and cellobiose. The fermentation products are acetic acid, C02, H2, ethanol, and small amounts of lactic acid. The deoxyribonucleic acid base composition is 43 mol% guanine plus cytosine. The name Bacteroides cellulosolvens is proposed. Type strain WM2 is deposited in the National Research Council of Canada culture collection as strain NRCC 2944.A mesophilic coculture consisting of a cellulolytic anaerobe and a saccharolytic anaerobe was isolated from a cellulose enrichment culture started from municipal sewage sludge. These two anaerobes grew on cellobiose agar as a single, slimy colony (6). The saccharolytic member of this coculture, Clostridium saccharolyticum, has been isolated and described previously (13). In this paper, we describe the isolation and characteristics of the cellulolytic microbe and propose for it the name Bacteroides cellulosolvens. MATERIALS AND METHODSMedia. The basal medium used for isolation and maintenance of the strain which we studied had the following composition (in milligrams per liter); NaHC03, 2,000; MgSO, * 7H20, 120; CaC12 -2H20, 61; FeS04 * 7H20, 21; N(CH2COOH)3, 15; NaC1, 10; M n S O 4 * H 2 O , 5 ; cocl2 -6H20, 1; ZnSO4 7H20, 1; c u s o 4 -5H20, 0.1; AIK(S04)2 12H20, 0.1; H3B03, 0.1; Na2Mo04 * 2H20, 0.1; pyridoxine hydrochloride, 0.1; thiamine hydrochloride, 0.05; riboflavin, 0.05; nicotinic acid, 0.05; p-aminobenzoic acid, 0.05; lipoic acid, 0.05; biotin, 0.02; folic acid, 0.02; vitamin BI2, 0.005; and resazurin, l ( 7 ) . This medium was prepared by the procedure of Holdeman et al. (3) and was prereduced by the Hungate technique (3, using 250 mg of cysteine hydrochloride per liter and 250 mg of Na2S -9H20 per liter as the reducing agents. The reduced medium was dispensed under 80% N2-20% C 0 2 in 10-ml amounts into 60-1111 serum vials (11) containing preweighed amounts of insoluble substrates, such as cellulose. The vials of media were then autoclaved at 104 kPa for 15 min. The soluble substrates were filter sterilized and were injected by means of hypodermic syringes into the basal medium after the vials had been autoclaved and cooled. The final pH of all media was 7.0 * 0.2. The isolated strain was maintained at 35°C in basal medium containing approximately 0.5% (wt/vol) cellulose in the form of small squares of 4-ply facial tissue.Isolation. All isolation procedures were conducted anaerobically in serum vials or in an anaerobic chamber.Biochemical tests. The procedures of Holdeman et al. (3) were used for biochemical characterization. Selected substrates were added to the basal medium at concentrations of 1% (wthol). When a test required the addition of a fermentable substrate (e.g., sulfate reduction medium), 0.5% cellobiose was used. The test media were inoculated with 0.05 ml of NH4C1, 680; K2HP04, 296; KH2P04, 180; (NH4)2S04, 150; * Corresponding author. t National Resea...

The ultrastructure of the cells of the major component of an enriched culture of a presumed methanogen which utilized acetic acid was studied by transmission and scanning electron microscopy. The filaments were composed of Gram-positive, rod-shaped cells, 1--2 micrometer in length and about 0.5 micrometer in breadth, attached end to end. Septa between cells were complex, with a central, electron-dense sheet which had a spherical enlargement in the center separating the cell walls. The cells walls themselves were of variable thickness with a light, fluffy, thin portion on the outside and a denser, thicker portion within. They contain a series of rings stacked side by side which are composed of material that stains strongly and positively with phosphotungstate ion. The cytoplasmic membrane of these cells had an outer leaflet which stains more densely with uranium and lead ions than the inner leaflet. There were no recognizable organelles in the cytoplasm other than ribosomes. It is shown in these observations that the presumed methanogen may likely be a new species.

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Soine lipids, particularly diglycerides, cholesterol, or cholesterol derivatives, are able to induce visible birefringence in pellicles of estracellular bacterial cellulose produced by Acetobacter xylinzrnz when they are added in snlall amounts to the surface of cc~ltures. This birefringence is due to orientation of a fraction of the cellulose microfibrils, not the cells, in the pellicle. Orientation may begin with small microscopic domains which are present in pellicles of untreated cultures and some of which inay overgrow others under the influence of a lipid. How the lipids induce alignment of the microfibrils in the bacterial cult~ire is unknown but the process appears to involve an interaction between ilemly formed extracellular microfibrils and the ~nicrocrpstalline surface of the lipid. IntroductionDiffuse and weak areas of birefringence in pellicles of bacterial cellulose have been recognized for a long time but have not been investigated systernatically, probably because they were attributed to slight stretching or compression of films and were therefore only of trivial interest. Recently, two additional forms of birefringence in pellicles of cellulose from cultures of Acetobacter xylinz~m have beell investigated in more detail (Colvin 1965(Colvin , 1966 because they reflect definite orientation of extracellular cellulose microfibrils in a systenl where the factors causing alignment cannot be mechanical or hydrodynan~ic. The purpose of the present paper is to report that several classes of lipids may induce birefringence in pellicles produced by cultures of il. xylinum, t o show t h a t this birefringence is due t o partial orientation of the cellulose microfibrils, and to describe some of the properties of the cultures which are associated with this orientation. Materials and MethodsPellicles of bacterial cellulose were grown and their birefringence observed and recorded by methods previously described (Colvin 1965). Compounds were applied to the surfaces of static cultures containing 100 1111 of seeded medium per 15-cm Pctri plate either as an ethanol solution (1 ~ng/ml or a saturated solutioil if the solubility of the c o~~l p o u l~d in ethanol was below this limit) or as small crystals. When ethanol solutions were used, results were coinpared with cultures to which an equal volume of ethanol (10 drops) had been added a t the same time (12-24 11 after pouring the medium, when only a slight cloudilless had appeared without any forlllatio~l of a coherent pellicle). When crystals were used, 1-2 mg of the compound u7as dropped on surfaces of cultures im~nediately after the medium uras poured. Results were compared to plates to which nothing had been added. 'N.R.C. Contribution KO. 9472. Canadian Jourllal of Microbiology. Volume 13 (196;) Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 12/22/14 For personal use only.Sections of pellicles froin cultures were obtained as follows. Areas of interest in the pellicle were carefully dissected between crossed Polaroid filin...

The genus Acetivibrio is emended to include nonmotile, gram-negative, obligately anaerobic rods that produce acetic acid and ethanol from fermentation of carbohydrates. A new species, Acetivibrio cellulosolvens, which was isolated from a cellulose-enriched culture of sludge, is described. The colonies on agar are entire, raised, and cream colored. The cells are straight rods, gram-negative, nonsporeforming, and nonmotile. The isolate which we studied ferments cellobiose, cellulose, esculin, and salicin and does not require yeast extract or rumen fluid for growth. The major end products produced from cellobiose or cellulose are acetic acid, ethanol, COz, and Hz. Lactic acid and glucose also are produced in small amounts. The type strain of A . cellulosolvens is strains BAS (= NRC 2936).A cellulolytic anaerobe that was isolated from a heattreated, cellulose-enriched culture (8) started from sewage sludge is described. This nonsporeforming, mesophilic, rodshaped anaerobe has the ability to survive heating at 80°C for 10 min in mixed cultures, but not in pure culture. The new isolate is described as Acetivibrio cellulosolvens sp. nov. MATERIALS AND METHODSMedia. The basal medium used for isolation and culture maintenance was the same medium that was used for the development of cellulose-enriched cultures (7). This basal medium contained the following components (in milligrams per liter): NaHC03, 2.060; NH4Cl, 680; K2HPO4, 296; CaC12 -2H20, 61; FeS04 a 7Hz0, 21; N(CHZCOOH)~, 15; NaCl, 10; M n S 0 4 H z O , 5 ; CoC12 . 6 H z 0 , 1; ZnS04 -7Hz0, 1; CuS04 -5Hz0, 0.1; A1K(S04)* -12Hz0, 0.1; H3B04, 0.1; NazMo04 -2H20, 0.1; pyridoxine hydrochloride, 0.1; thiamine hydrochloride, 0.05; riboflavin, 0.05; nicotinic acid, 0.05; p-aminobenzoic acid, 0.05; lipoic acid, 0.05; biotin, 0.02; folic acid, 0.02; vitamin BIZ, 0.005; and resazurin, 1. This medium was supplemented with cellulose (type CF-11; Whatman) or tissue paper (Kleenex; KimberlyClark of Canada, Ltd.) and with 2% (wt/vol) agar (Difco Laboratories, Detroit, Mich.) for culture isolation. The pH of the medium was adjusted to 7.0 5 0.2, and the medium was reduced by using a cysteine-sodium sulfide solution and the Hungate technique (5). A gas mixture containing 80% N2 and 20% COz was used as a headspace gas. Usually, 10-ml portions of the prereduced medium were dispensed into 60-ml serum vials. All other media and reagents required for characterization tests were prepared as described by Holdeman et al. (3).Isolation and stock culture maintenance. The inoculum used for isolation was obtained from a fermentor that was started by using a cellulose-enriched culture and was heated at 80°C for 10 min (8). The cellulolytic anaerobes present in this heat-treated culture survived repeated heating at 80°C for 10 min. The inoculum was serially diluted in the basal medium. Each dilution (0.1 ml) was disseminated onto a KHzP04, 180; (NH4)zS04, 150; MgS04 * 7H20, 120; * Corresponding author.T Issued as National Research Council of Canada paper 23597. filter paper placed over agar sla...