INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1986, p. 297-301 Vol. 36. No. 2 0020-7713/86/020297-05$02.OO/O Copyright 0 1986, International Union of Microbiological Societies Isolation and Characterization of Methanogenium bourgense SP nov. BERNARD M. OLLIVIER,lt ROBERT A. MAH,l* J. L. GARCIA,2 AND DAVID R. BOONEl Division of Environmental and Occupational Health Sciences, School of Public Health, University of California at Los Angeles, Los Angeles, California 90024l; and Ofice de la Recherche Scientijique et Technique Outre-Mer, Laboratoire de Microbiologie, Universitt? de Provence, Marseille 13331 Cedex 3, France2 Methane-producing bacterial strain MS2T (T = type strain) was isolated from a tannery by-products enrichment culture inoculated with sewage sludge. This methanogen was a non-motile, irregular coccoid organism (diameter, 1 to 2 pm) which used H2-C02and formate as methanogenic substrates. Acetate was required for growth but did not serve as a methanogenic substrate; yeast extract and Trypticase peptone were highly stimulatory. Growth occurred through the pH range from 5.5 to 8.0, with optimum growth at pH 6.7. The optimum temperature for growth was 37°C. The deoxyribonucleic acid base composition was 59 mol% guanine plus cytosine. The physiological and antigenic properties of this isolate place it in the genus Methanogenium. The organism is named Methanogenium bourgense sp. nov. The genus Methanogenium contains the largest number of N2. After cooling, the medium was placed into an anaerobic species and isolates of irregularly coccoid methanogens, chamber (Coy Laboratory Products, Ann Arbor, Mich.) and including both thermophilic (9, 16, 22) and mesophilic (6, 23, dispensed in 20-ml portions into 60-ml serum bottles 26) members. Although most species of Methanogenium (Wheaton Scientific Co., Millville, N.J.) which were prein- have been obtained from marine sediments, three isolates cubated in the glove box for 24 h prior to use. The bottles were not. “Methanogenium olentangyi” was isolated from were stoppered with butyl rubber closures (Bellco Glass, freshwater sediments (6), Methanogenium tatii was isolated Inc., Vineland, N.J.), flushed with N2-C02 (4:1), and sealed from a solfataric field (26), and Methanogenium aggregans with aluminum crimp seals. The serum vials were autoclaved was isolated from an anaerobic digestor (16). All species of at 121°C for 20 min. A 0.4-ml portion of 1% (wthol) Methanogenium use H2-C02 and formate as methanogenic Na2S . 9H20and 0.3 ml of 10% (wthol) Na2C03were added substrates; “Methanogenium olentangyi” uses H2-CO2 but to each vessel. The final pH was 7.0. not formate (6). No other substrates are catabolized. In this For roll tube media, 15 g of agar per liter was added after paper we describe the isolation and characteristics of a new the medium was brought to a boil. The medium was then mesophilic species of Methanogenium. This species is cooled, placed into a glove box, and dispensed in 4.5-ml named Methanogenium bourgense sp. nov. portions into serum tubes, The tubes were sealed with butyl rubber stoppers, removed from the anaerobic chamber, and MATERIALS AND METHODS flushed with N2-C02 (4:l). A 0.1-ml portion of 1% Enrichment and isolation. The inoculum for the methano- Na2S - 9H20 and 0.08 ml of 10% Na2C03 were added just genic enrichment culture was obtained from a digestor prior to inoculation. fermenting tannery by-products which was originally inocu- Deoxyribonucleic acid base composition. Deoxyribonucleic lated with digested sewage sludge from Bourg, or Bourg-en- acid was extracted and purified by the method of Price et al. Bresse, France. The 20-ml liquid enrichment culture (19), and the base content was determined from the buoyant contained formate as a methanogenic substrate and an density in a CsCl gradient (18). N2-CO2 gas phase and was incubated in 60-ml serum bottles Microscopy. Transmission electron micrographs were pre- at 37°C. The culture was maintained by weekly transfers of pared by fixing samples in glutaraldehyde and osmium 2 ml of culture (10% inoculum) to fresh medium. After 1 tetroxide and enrobing them in Spurr plastic. month the enrichment culture was diluted and used to Analytical techniques. The average values for duplicate inoculate roll tube medium for isolation. An axenic culture vessels are reported below. Each experiment was done at was obtained by using the anaerobic techniques of Hungate least twice. Methane was analyzed by gas chromatography (12). as described previously (2). Absorbance was measured at Media. The medium used contained 1.0 g of NH4C1, 0.4 g 580 nm by using a Spectronic 21 spectrophotometer (Bausch of K2HP04 - 3H20, 0.1 g of MgC12 6H20, 0.5 g of L- & Lomb, Inc., Rochester, N.Y .). cysteine hydrochloride, 5.0 g of sodium formate, 1.0 g of sodium acetate, 1.0 g of Trypticase peptone (BBL Microbi- RESULTS ology Systems, Cockeysville, Md.), 1.0 g of yeast extract Isolation. The enrichment culture was aseptically diluted (Difco Laboratories, Detroit, Mich.), and 1 mg of resazurin and used to inoculate roll tube media. Methanogenic colo- in 1liter of Milli-Q-deionized water (conductivity, 5.9 FS/m). nies identified by using ultraviolet epifluorescence (8) as The medium was adjusted to pH 7.0 with 10 M KOH and modified by Doddema and Vogels (7) appeared after 7 days boiled for 10 min while the vessel was flushed with 02-free of incubation. Colonies were picked and used to inoculate liquid medium. After growth (as measured by methanogen- * Corresponding author. esis) the cultures were diluted and inoculated into roll tubes. t Present address: Office de la Recherche Scientifique et Tech- Even after several such transfers, the methanogenic bacte- nique Outre-Mer, Laboratoire de Microbiologie, Universitd de rium was always contaminated with a motile, nonmeth- Provence, Marseille 13331 Cedex 3, France. anogenic rod-shaped organism. This nonmethanogenic con- 297 :298 OLLIVIER ET AL. INT. J. SYST.BACTERIOL. Morphology. Epifluorescent colonies that were 1 to 2 mm in diameter appeared after 1 month in roll tubes containing formate. After 1 week, the colonies were only 0.1 mm in diameter. The irregular coccoid cells (Fig. 1) occurred singly or in pairs during exponential growth. Lysis occurred imme- diately after exponential growth. The cells were not motile. Flagella were never observed by using the staining proce- dures of Rhodes (20) or by electron microscopy. Transmis- sion electron microscopy disclosed a thin cell wall (Fig. 2). One cell which received a grazing section revealed a regu- larly arrayed layer. The organism was lysed in the presence of 0.02% sodium dodecyl sulfate. Optimum growth conditions. In all experiments, sodium formate was the methanogenic substrate which we used. Growth was determined by measuring methane production. The level of absorbancy was proportional to the amount of methane produced (data not shown). The optimum pH for growth, as determined from CH4 production after 7 days of incubation, was 6.7 (Fig. 3). The optimum growth tempera- ture was determined from CH4 production at pH 7.0 after 7 days of incubation (Fig. 4). The isolate was a mesophile. The optimum concentration of added NaCl was less than 1% (wthol), as determined in complex medium (Fig. 5). The complex medium with no NaCl addition contained 1.5 g of inorganic salts per liter and 86 mM Na+ in sodium formate FIG. 1. Phase-contrast photomicrograph of Methanogeniurn and sodium acetate. This medium supported near-optimal bourgense MS2T. Bar = 10 Fm. growth with no NaCl addition (Fig. 5). Good growth with no NaCl addition also occurred when the sodium salts added to taminant was eliminated by treating the liquid culture with the medium (sodium formate, sodium acetate, and Na2C03) D-cycloserine and penicillin (27) and inoculating roll tube were replaced with H2, calcium acetate, and KHC03. medium without antibiotics. Culture purity was tested by Substrates and growth requirements. Only formate and microscopically examining cultures, by inoculating complex H2-C02were used as methanogenic substrates; neither meth- medium containing sugars to test for heterotrophic bacteria, anol, acetate, nor trimethylamine supported growth or and by examining preparations for uniform colony morphol- methanogenesis. Nevertheless, acetate was required for ogy in roll tube dilutions. growth on formate (Table 1).Under these conditions, form- FIG. 2. Transmission electron micrograph of Methanogeniurn bourgense MS2=. Bar = 1 Fm. VOL. 36, 1986 METHANOGENIUM BOURGI SP. NOV. 299 180 - 120 J 60 - 0 0 '.-.-. PH 0 10, 20 30 40 FIG. 3. Effect of pH on methane production by Methunogenium NaCl (g/L) bourgense MS2T. Cultures were incubated for 7 days at 37°C. FIG. 5. Effect of NaCl concentration\, on methane production by Methanogeniurn bourgense MS2T. Cultures were incubated at 37°C and pH 7.0. ate was metabolized within 20 days. Growth occurred, but at a slower rate, when acetate was replaced by yeast extract. In the presence of acetate, either yeast extract or Trypticase other methanogens (3-5). The Methanomicrobium mobile S peptone was highly stimulatory (Table 1). Acetate supported probe antiserum gave a 1+ reaction, and negative reactions optimal growth at concentrations between 0.5 and 1.0 g/liter were obtained with Methanococcus vannielii and Methano- (Table 2). The generation time at 37°C was 32 h with formate coccus voltae S probe antisera. A very weak reaction, below or 18 h with H2-CO2. the level usually considered 1+, was recorded for the S Immunological determinations. E. Conway de Macario probe antisera prepared against Methanogenium marisnigri, determined the antigenic fingerprint of strain MS2T (T = Methanogenium cariaci, and Methanospirillurn hungatei. type strain) by using antisera (S probes) prepared against DISCUSSION Strain MS2T had a coccoid morphology and grew and produced methane from H2-C02 or formate but not from 15C TABLE 1.