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Methanothrix Soehngenii and Rejection of Methanothrix Concilii As a Synonym of Methanothrix Soehngenii

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Methanothrix Soehngenii and Rejection of Methanothrix Concilii As a Synonym of Methanothrix Soehngenii INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,Jan. 1988, p. 30-36 Vol. 38, No. 1 0020-7713/881010030-07$02.00/0 Copyright 0 1988, International Union of Microbiological Societies Description of a New Strain of Methanothrix soehngenii and Rejection of Methanothrix concilii as a Synonym of Methanothrix soehngenii JEAN PIERRE TOUZEL,l* GERARD PRENSIER,, JEAN LOUIS ROUSTAN ,l ISABELLE THOMAS,l HENRI CHARLES DUBOURGUIER,l AND GUY ALBAGNAC' Institut National de la Recherche Agronomique, Station de Technologie Alimentaire,' and lnstitut National de la Santt et de la Recherche MPdicale, Unite' 42,2B.P. 39, F-59651 Villeneuve d'Ascq Ce'dex, France A new mesophilic strain of Methanothrix, strain FE, was highly purified from the sludge of an anaerobic digester after enrichment on sodium acetate and is described. Strain FE was compared with other strains of Methanothrix, Methanothrix soehngenii strain OpfikonT (= DSM 2139T) (T = type strain) and Methanothrix concilii strain GP6T (= DSM 3671T). The differences within the strains were mainly related to their requirement for yeast extract. The three strains were found to be similar in their deoxyribonucleic acid guanine-plus-cytosinecontents (50.2 to 52.6 mol %) and showed 100 % deoxyribonucleic acid-deoxyribonucleic acid homology. For these reasons we propose to recognize synonymy, reject the name Methanothrix concilii Patel 1985, 35223, and assign this organism to the species Methanothrix soehngenii Huser, Wuhrmann and Zehnder 1983, 33:439. Methanothrix soehngenii was first isolated by serial dilu- The sterile media were inoculated with 2% (vol/vol) portions tion from a continuous culture on acetate starting from of previous cultures. anaerobically digested sludge in Switzerland (17, 40; B. A. Enrichment. Enrichment cultures were made on 50 mM Huser, thesis, Eidgenossischen Teschnischen Hochschule, sodium acetate in BCYT medium without HEPES. Transfers Zurich, Switzerland, 1981). This bacterium was unique at were made at 60- to 10-week intervals. In the first transfers, that time among the methane bacteria because of its ability a Methanosarcina-like organism was observed in the enrich- tlo split acetate into methane and carbon dioxide and its ments. After six transfers, the medium was supplemented inability to use other methanogenic substrates, especially with vancomycin, and the resulting culture was purified by H,-C02. It was first described as a slow-growing bacterium repeated serial dilutions. with doubling times ranging from 5 to 8 days. This bacterium Growth rate determinations and pH range. Methane was was positioned in the family Methanosarcinaceae by 16s measured as previously described (37) by using nitrogen as ribosomal ribonucleic acid oligonucleotide sequencing (33). In 1984 (27), Patel described the isolation of Methanothrix an internal standard. Growth rates were determined by concilii, which differed from Methanothrix soehngenii calculating the slope of the logarithm of the methane content mainly by deoxyribonucleic acid (DNA) base composition as a function of time. Slopes were computed by using the and nutritional requirements. In this paper we characterize a least-squares method. new strain of Methanothrix, strain FE, and compare it with The pH range for growth was tested by using two different the two mesophilic type strains of the genus. buffering systems (carbonate-CO, and PIPES). When the carbonate buffering system was used, the pH values were MATERIALS AND METHODS obtained by using different concentrations of potassium Sources of strains. Strain FE was initially purified from the bicarbonate. They were checked with a pH meter. When sludge of an industrial mesophilic anaerobic contact digester PIPES buffer was used, the chosen pH values were adjusted treating wastewaters from the vegetable-canning industry with 10 N NaOH by using a pH meter at 35°C under an (39). Methanothrix soehngenii strain OpfikonT (= DSM N,-CO, gas stream before the tubes were filled. 2139T) (T = type strain) and Methanothrix concilii strain Substrate determinations. Acetate was determined by gas- CiP6T (= DSM 3671T) were obtained from the Deutsche liquid chromatography (37), and formate was measured by Slammlung von Mikroorganismen, Gottingen, West Ger- the colorimetric method of Sleat and Mah (32). many. All of the strains used for immunological studies were Effect of antibiotics. Antibiotics were assayed in duplicate also obtained from the Deutsche Sammlung von Mikroor- in 25-ml penicillin vials containing 10-ml cultures and were ganismen, except for Methanosarcina sp. strains CHTI55, added from filter-sterilized stock solutions as soon as meth- MST-A1, and MC3, which were isolated in our laboratory, ane production started. The final concentrations used were 1 and Methanobrevibacter arboriphilicus AZ and Methano- mg/ml for ampicillin and kanamycin and 0.1 mg/ml for sarcina barkeri MST, which were gifts from J. G. Zeikus. D-cycloserine and penicillin G. Media and culture conditions. Cultivation of methanogens Influence of hydrogen. Assays were performed in 125-ml was done on BCYT medium as previously described (37). vials containing 50-ml cultures. After inoculation, the vials The medium was buffered with 50 mM HEPES (N-2-hydroxy- were pressurized with 1 atm (101.29 kPa) of overpressure of ethylpiperazine-N'-2-ethanesulfonicacid) or PIPES [pipera- HZ-CO, (80120). zine-l,4-bis(2-ethanesulfonic acid)] unless otherwise indi- Microscopy. Photomicrographs were taken with a Nachet cated. The anaerobic gas was an N,-CO, mixture (85:15). NS 400 microscope equipped with Nomarsky optics. Trans- mission electron microscopy was done as previously de- * Corresponding author. scribed (19). For scanning electron microscopy, the tech- 30 VOL.38, 1988 METHANOTHRIX SOEHNGENII 31 nique of Costerton (8) was used, except that the mixtures were denatured by heating in boiling water for 10 thiocarbohydrazide step was omitted. min. Hybridizations were conducted for 18 h at 35°C. The Immunology. Antisera against Methanothrix sp. strain FE, single-stranded DNAs were eliminated by incubating the Methanosarcina barkeri strains MS and 227, Methanosar- preparation for 1 h at 30°C in the presence of a suitable cina mazei strains S6 and MC3, Methanosarcina thermo- dilution of S1 nuclease, and the hybrids were precipitated phila strain TM1, Methanosarcina sp. strains CHTI55 and with an equal volume of 10% trichloroacetic acid, collected MST-A1, “Methanosarcina vacuolata,” Methanobrevi- on Whatman GF/F fiberglass disks, washed five times with bacter arboriphilicus strain AZ, and Methanospirillum ice-cold trichloroacetic acid and then with ethanol, dried, hungatei strain JF1 were elicited in rabbits by using the and counted in the presence of 10 ml of Insta-gel scintillation method of Conway de Macario et al. (7). The standard mixture. hyperimmunization method was also used for Methanothrix Chemicals. All chemicals were of analytical grade and sp. strain FE. All of the sera were titrated by indirect were obtained from E. Merck AG, Darmstadt, West Ger- immunofluorescence and were used at the S dilution (I. many, except as described below. Yeast extract and Bio- Thomas, thesis, UniversitC des Sciences et Techniques de Trypcase were obtained from Bio-Merieux, Charbonnihre- Lille-Flandres-Artois, Lille, France, 1986). les-Bains, France; HEPES and PIPES were obtained from Protein polyacrylamide gel electrophoretic patterns. Protein Janssen Chimica, Beerse, Belgium; phenol was obtained patterns were obtained as previously described (3 l), except from BDH Chemicals Ltd., Poole, England; and hydroxyl- that an isocratic 15% polyacrylamide gel was used and apatite (DNA grade Bio-Gel HTP) was obtained from Bio- staining was done after picric acid fixation (34). Rad Laboratories, Richmond, Calif. Vancomycin was ob- Guanine-plus-cytosine contents of DNAs. After chemical tained from Eli Lilly & Co., Indianapolis, Ind., and the other lysis of cells by the method of Beji et al. (3), the DNA was antibiotics were obtained from Sigma Chemical Co., St. purified by the Marmur procedure. Briefly, cells were lysed Louis, Mo. The nick translation kit and deoxyribonuclease by using sodium dodecyl sulfate (SDS) (final concentration, S1 (S1 nuclease) from Aspergillus oryzae were obtained from 2.8%) under alkaline conditions (0.03 N NaOH). Lysis Bethesda Research Laboratories, Herblay, France, salmon occurred very rapidly after addition of the SDS solution, and sperm DNA was obtained from Calbiochem, France Bio- the pH of the lysate was adjusted to below 8.2 no more than chem, Meudon, France, and [1’,2’,5’,-3H]deoxycytidine 5’- 30 s after SDS addition in order to limit any alkaline triphosphate (ammonium salt) was obtained from Amersham hydrolysis of the DNA. France, Les Ulis, France. Insta-gel was obtained from The thermal denaturation procedure was used to deter- Packard Instruments, Rungis, France. mine the guanine-plus-cytosine content. The DNA was dissolved in saline citrate solution (0.15 M NaCl plus 0.015 RESULTS M trisodium citrate 2-hydrate; pH brought to 7 with 0.1 N HCl). Absorbance at 260 nm and cell temperature were Characteristics of strain FE. (i) Purification. All attempts to digitally recorded at 1-min intervals with a model SP 800 obtain colonies of Methanothrix from the enrichments were spectrophotometer (Kontron, Zurich, Switzerland) equipped unsuccessful. Even incubations on roll tubes for more than 6 with a model PD 415 temperature programmer (Huber, months failed. After six transfers on 50 mM acetate at 4- to Offenburg-Elgersweier,
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