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 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 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 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 and isolates of irregularly coccoid , 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. Growth requirements of Methunogenium bourgense MS2T n a Amt of CH4 produced (pmoV20 ml) after:b aa Compound(s) I added" 8 10 14 20 27 34 41 0 Days Days Days Days Days Days Days E 10( a None 1 ND" 1 111 1 Y Yeast extract 4 ND 13 32 61 162 227 w Trypticase 1 ND 2 22 2 3 z Acetate 16 ND 124 320 350 ND ND a Yeast extract + 9 ND 45 110 150 172 236 I Trypticase l- 5( Yeast extract + 165 370 ND ND ND ND ND W acetate z Trypticase + 152 321 ND ND ND ND ND acetate Yeast extract + 305 366 ND ND ND ND ND Trypticase + ( acetate a Compounds were added at concentrations of 1 gfliter; all vessels con- TEMPERATURE (*C) tained 5 g of sodium formate per liter. The basal medium contained no organic compounds in this experiment; 25 ml of a mineral solution (1) and 5 ml of a FIG. 4. Effect of temperature on methane production by trace elements solution (1) were added to each liter of basal medium. Methanogenium bourgense MS2=. Cultures were incubated for 7 Cultures were incubated at 35°C. days at pH 7.0. ND, Not determined. 300 OLLIVIER ET AL. INT. J. SYST.BACTERIOL.

TABLE 2. Comparison of strain MS2= and related species of methanogens Guanine- plus- Optimum OptimumNaCl Optimum Formate Refer- Strain cytosine temp rowth factorsa Morphology Motility catabo- content CC) pH lism ence(s) (rnol%) (mM)

MS2T 59 37 17 6.7 Acetate coccus (1-2)b - + This study Methanogenium 52 20-25 540 6.8-7.3 Acetate, yeast Coccus (2.6) + (peritrichous) + 23 cariaci JR1 extract Methanogenium 61 20-25 190 6.2-6.6 Peptone Coccus (1.3) + (peritrichous) + 23 marisnigri JR1 Methanogenium tatii 54 40-45 200 7.0 Acetate, yeast Coccus (3.0) + (peritrichous) + 26 DSM 2702 extract, or peptone Methanogenium 59 55 250 7.0 Peptone, COCCUS(1.0-1.3) - + 22 thermophilicum vitamins CR-1 Methanogenium 52 35 <3 6.5-7.0 Acetate, yeast Coccus, in - + 16 aggregans MSt extract, or aggregates (1.0) peptone Methanococcus 31 30-40 <51 8.0 None Coccus (0.5-4.0) + (polar tuft) + 1,13,24 vannielii SB Methanococcus 31 32-40 21-82 6.7-7.4 Yeast extract Coccus (0.5-3.0) + + 1 voltae PS Methanococcus 31 65 68 7.0 None Coccus (1.5) + (polar tuft) + 11 thermolithotro- phicus SN1 (= DSM 2095) Methanococcus 31 85 500 6.0 None Coccus (1.5) + (two flagellar - 14 jannaschii JAL-1 bundles) Methanococcus 33 35-39 110 6.8-7.2 None Oval (1.0-1.3 by + (weak) + 15 maripaludis JJ 1.5-1.8) Methanospirillum 45 30-37 6.6-7.4 Peptone, yeast Curved rod (0.5 + (polar tuft) + 10 hungatei JF1 extract by 7.4) Methanomicrobium 49 38-40 6.1-6.9 Rumen fluid Rod (0.7 by + (monotrichous, + 17 mobile BP 1.5-2.0) polar) Methanomicrobium 45 40 150 7.0 Acetate Rod to COCCUS - - 21 paynteri G-2000 (0.6 by 1.5-2.5) Methanoplanus 47.5 40 17 7.0 Acetate Plane (0.3 by + (polar tuft) + 25 limicola DSM 2279 1.6-2.8 by 1.5)

a Organic compounds which are necessary for growth or are greatly stimulatory. The peptone used was Trypticase peptone. * The values in parentheses are cell sizes (in micrometers). methylotrophic substrates (acetate, methanol, or methyl- unusual for Methanogenium species (3-5). For example, amines). All previously described species with these char- Methanogenium cariaci JR1 cross-reacts more strongly with acteristics are members of the families Methanomicro- antiserum to Methanospirillum hungatei than it does with biaceae and Methanococcaceae. An antigenic analysis antiserum to Methanogenium marisnigri JR1 (3, 5). For showed that strain MS2T was more closely related to mem- these reasons we believe that strain MS2T belongs in the bers of the family than to members of genus Methanogepium. the Methanococcaceae, and Methanogenium is the only Within the genus Methanogenium strain MS2T appears to genus of the Methanomicrobiaceae which contains cocci (1). be most closely related to Methanogenium aggregans. Other The closer relationship of strain MS2T to members of the members of the genus Methanogenium require elevated Methanornicrobiaceae than to members of the Meth- levels of NaCl for optimum growth (Table 2) compared with anococcaceae was corroborated by the guanine-plus- Methanogenium aggregans and strain MS2T. Also, cytosine contents of the deoxyribonucleic acid (59 mol% Methanogenium aggregans and strain MS2T have typical guanine plus cytosine for strain MS2T and 31 to 33 mol% for mesophilic temperature optima, whereas other members of the genus Methanococcus [l, 11, 14, 151). Methanogenium species have higher or lower optima. Strain Table 2 compares strain MS2T with related species of MS2T differs from Methanogenium aggregans in the follow- methanogens, including coccoid methanogens that are not ing three important ways: (i) it has a higher guanine-plus- able to use methylotrophic substrates. Based on these char- cytosine content, (ii) it does not form aggregates, and (iii) its acteristics, strain MS2T appears to be most closely related to optimum salt concentration for growth is higher. the genus Methanogenium. Members of other genera of the We propose that strain MS2T be placed in the genus Methanornicrobiaceae (Methanospirillum and Meth- Methanogenium as a new species, Methanogenium anomicrobium) exhibit morphological differences, as well as bourgense. A species description is given below. differences in guanine-plus-cytosine contents. Cells of strain Methanogenium bourgense sp. nov. Methanogenium MS2T reacted with S probe antisera prepared against these bourgense (bourg.en’.se. N.L. neu. adj., from Bourg-en- two genera, but such intergeneric cross-reactions are not Bresse, France) cells are irregular, nonmotile cocci 1to 2 pm VOL.36, 1986 METHANOGENIUM BOURGZ SP. NOV. 301 in diameter and stain gram negative. They are sensitive to philic lithotrophic methanogen. Arch. Microbiol. 132:47-50. lysis by 0.2 g of sodium dodecyl sulfate per liter. Young 12. Hungate, R. E. 1969. A roll tube method for the cultivation of colonies are circular, convex, and white; older colonies are strict anaerobes, p. 117-132. In J. R. Norris and D. W. Ribbons yellowish. H2-C02and formate are the only substrates used (ed.), Methods in microbiology, vol. 3B. Academic Press, Inc., for growth and methanogenesis. Acetate is required for New York. 13. Jones, J. B., B. Bowers, and T. C. Stadtman. 1977. Methano- growth, and yeast extract or Trypticase peptone is highly coccus vanniefii:ultrastructure and sensitivity to detergents and stimulatory. antibiotics. J. Bacteriol. 130:1357-1363. Growth is most rapid at pH 6.7, with 10 g of NaCl per liter, 14. Jones, W. J., J. A. Leigh, F. Mayer, C. R. Woese, and R. S. and at 35 to 40°C. Wolfe. 1983. Methanococcus jannaschii sp. nov., an extremely The guanine-plus-cytosine content is 59 mol%. thermophilic methanogen from a submarine hydrothermal vent. The type strain is strain MS2 (= DSM 3045). Arch. Microbiol. 136:25&261. 15. Jones, W. J., M. J. B. Paynter, andR. Gupta. 1983. Character- ACKNOWLEDGMENTS ization of Methanococcus maripaludis sp. nov., a new meth- We are grateful to E. Conway de Macario (Department of Health, anogen isolated from salt marsh sediment. Arch. Microbiol. State of New York) for performing the immunological typing of strain 135:91-97. MS2=. Ralph Robinson (Department of Microbiology and Cell 16. Ollivier, B. M., R. A. Mah, J. L. Garcia, and R. Robinson. 1985. Science, University of Florida) took the electron photomicrographs. Isolation and characterization of Methanogenium aggregans sp. We also thank Robert Sleat, Indra Mathrani, Yitai Liu, and Thomas nov. Int. J. Syst. Bacteriol. 35127-130. J. Ferguson for helpful discussions and suggestions. 17. Paynter, M. J. B., and R. E. Hungate. 1968. Characterization of This study was supported by grants 480-323-0423 and IFAS-GRI- Methanobacterium mobilis, sp. n., isolated from the bovine FIA-MCS 2171 from the Gas Research Institute and by grant rumen. J. Bacteriol. 951943-1951. DE-AT03-80-ER10684 from the U.S. Department of Energy. 18. Preston, J. F., and D. R. Boone. 1973. Analytical determination of the buoyant density of DNA in acrylamide gels after prepar- LITERATURE CITED ative CsCl gradient centrifugation. FEBS Lett. 37:321-324. 19. Price, C. W., G. B. Fuson, and H. J. Phaff. 1978. Genome 1. Balch, W. E., G. E. Fox, L. J. Magrum, and R. S. Wolfe. 1979. comparison in yeast systematics: delimitation of species within Methanogens: reevaluation of a unique biological group. Micro- the genera Schwanniomyces, Debaryomyces, and Pichia. Mi- biol. Rev. 43:260-296. crobiol. Rev. 42:161-193. 2. Baresi, L., R. A. Mah, D. M. Ward, and I. R. Kaplan. 1978. 20. Rhodes, M. E. 1958. The cytology of Pseudomanas sp. as Methanogenesis from acetate: enrichment studies. Appl. revealed by a silver-plating method. J. Gen. Microbiol. 18:639- Environ. Microbiol. 36: 186-197. 648. 3. Conway de Macario, E., A. J. L. Macario, and M. J. Wolin. 21. Rivard, C. J., J. M. Henson, hi. V. Thomas, and P. H. Smith. 1982. Antigenic analysis of and Methanomicvobium Methanobrevibacter arboriphilus. J. Bacteriol. 152:762-764. 1983. Isolation and characterization of paynteri sp. nov., a mesophilic methanogen isolated from ma- 4. Conway de Macario, E., A. J. L. Macario, and M. J. Wolin. 1982. Specific antisera and immunological procedures for char- rine sediments. Appl. Environ. Microbiol. 46:484490. acterization of methanogenic bacteria. J. Bacteriol. 149: 22. Rivard, C. J., and P. H. Smith. 1982. Isolation and character- 320-328. ization of a thermophilic marine methanogenic bacterium, Methanogenium thermophilicum 5. Conway de Macario, E., M. J. Wolin, and A. J. L. Macario. sp. nov. Int. J. Syst. Bacte- 1982. Antibody analysis of relationship among methanogenic rial. 32:430-436. bacteria. J. Bacteriol. 149:316-319. 23. Romesser, J. A., R. S. Wolfe, F. Mayer, E. Spiess, and A. 6. Corder, R. E., L. A. Hook, J. M. Larkin, and J. I. Frea. 1983. Walther-Mauruschat. 1979. Methanogenium, a new genus of Isolation and characterization of two new methane-producing marine methanogenic bacteria, and characterization of cocci: Methanogenium olentangyi, sp. nov., and Methanococ- Methanogenium cariaci sp. nov. and Methanogenium cus deltae, sp. nov. Arch. Microbiol. 134:28-32. marisnigri sp. nov. Arch. Microbiol. 121:147-153. 7. Doddema, H. J., and G. D. Vogels. 1978. Improved identification 24. Stadtman, T. C., and H. A. Barker. 1951. Studies on the of methanogenic bacteria by fluorescence microscopy. Appl. methane fermentation. X. A new formate-decomposing bacte- Environ. Microbiol. 36:752-754. rium, Methanococcus varinielii. J. Bacteriol. 61:269-280. 8. Edwards, T., and B. C. McBride. 1975. New method for the 25. Wildgruber, G., M. Thomm, H. Konig, K. Ober, T. Richiuto, isolation and identification of methanogenic bacteria. Appl. and K. 0. Stetter. 1982. Methanoplanus fimicola, a plate-shaped Microbiol. 29:54&545. methanogen representing a novel family, the Methano- 9. Ferguson, T. J., and R. A. Mah. 1983. Isolation and character- planaceae. Arch. Microbiol, 132:3 1-36. ization of an H2-oxidizing thermophilic methanogen. Appl. 26. Zabel, H. P., H. Konig, and J. Winter. 1984. Isolation and Environ. Microbiol. 45265-274. characterization of a new coccoid methanogen, Methanogenium 10. Ferry, J. G., P. H. Smith, and R. S. Wolfe. 1974. Methanospirif- tatii sp. nov. from a solfataric field on Mount Tatio. Arch. lum, a new genus of methanogenic bacteria, and characteriza- Microbiol. 137:308-315. tion of Methanospirillurn hungatii sp. nov. Int. J. Syst. Bacte- 27. Zinder, S. H., and R. A. Mah. 1979. Isolation and characteriza- rial. 24:465-469. tion of a thermophilic strain of Methanosarcina unable to use 11. Huber, H., M. Thomm, H. Konig, G. This, and K. 0. Stetter. H2-C02 for methanogenesis. Appl. Environ. Microbiol. 1982. Methanococcus thermolithotrophicus, a novel thermo- 39:996-1008.