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 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 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 bacteria because of its ability a -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, West Germany). The melting tem- 6-week intervals in BCYT medium containing 200 mg of perature was computed by using the method of the median, vancomycin per liter, the culture was vigorously shaken, as described by Ferragut and Leclerc (12). serially diluted, and inoculated into fresh medium. The last DNA homologies. All of the DNA samples were sonicated growing dilution (lo-’) was then submitted to a further at 0°C for 1 min with a Labsonic 1510 sonicator (Braun, similar dilution step. In the final culture tube, a vibrio, Melsungen, West Germany); a 10-W energy setting and a possibly sulfate reducing, was occasionally observed to- 4-mm probe were used. The sonication procedure was gether with the cells of Methanothrix. All attempts to repeated four times after 1-min resting periods. After nick eradicate the vibrio by serial dilution upon addition of either translation, the labeled DNA was separated from excess molybdate, Hibitane, or vancomycin were unsuccessful, so nucleotide by chromatography on Sephadex G-50 on a spun the Methanothrix culture was maintained in an almost pure column as described by Maniatis et al. (21). state without addition of antibiotics. Elimination of S1 nuclease-resistant duplexes was per- (ii) Morphology and ultrastructure. Strain FE cells are rods formed by chromatography on an hydroxylapatite column in that are 0.65 to 0.85 pm wide and 1.5 to 2.5 pm long and a Pasteur pipette (bed dimensions, 0.6 by 5 cm). The DNA occur in filaments or trichomes of various lengths (Fig. 1and sample was denatured by heating for 10 min in a boiling 2). The cells stained gram negative. In unstirred cultures, bath. The phosphate concentration was brought to about 0.1 filaments up to several hundred unit cells long were common M, and the sample was deposited on the top of the column. (Fig. 1). They tended to aggregate in bundles, and they Fractions (5 drops) eluted by phosphate buffer (0.12 M, formed cottonlike pellets. pH 6.8) were monitored for radioactivity and pooled. The In longitudinal thin sections, a well-defined sheath struc- pooled fractions were freeze-dried and dissolved in 0.1 ml ture was observed; it consisted of a serial array of hooplike of water. Phosphate buffer was changed for TES [tris subunits. Septa arose directly from the sheath and appeared (h ydroxymet h y 1)aminomethane-ethylenediaminetetraacetate- to be extensions of the hoops (Fig. 3). Double septation and saline) buffer by spun-column chromatography on Sephadex even triple septation were commonly observed, leading to G-50. fragile areas where the filament could easily break apart. The The reassociation reaction mixtures contained 5 pl(O.010 central part of the septum was clearly asymmetrical, and the to 0.025 pg; 6,000 to 12,000 cpm) of labeled DNA, 100 pl(40 filament thus appeared to be oriented (Fig. 3A and C). pg) of unlabeled DNA, 35 pl of water, and 280 pl of a buffer Granular inclusions in the cytoplasm have been character- containing 600 mM NaCl, 15 mM ethylenediaminetetraace- ized by Pellerin et al. (30) as being glycogen. tate (pH 8), 15 mM tris(hydroxymethy1)aminomethane buffer Scanning electron microscopy revealed a typical bamboo (pH 8), 0.0225% SDS, and 75% formamide. The DNA shape for the filaments. This appearance was due to the 32 TOUZEL ET AL. INT. J. SYST.BACTERIOL. shrinking of the cytoplasm, whereas the septa retained their full size (Fig. 2). (iii) Substrates for growth and methane formation. Growth and methane formation were only observed on acetate. Other substrates tested were methanol, H,-CO,, methyl- amines, formate, ethanol, fumarate, propionate, butyrate, and benzoate. Acetate was converted into methane even in the presence of hydrogen. Neither hydrogen nor formate inhibited methane formation from acetate. Moreover, for- mate added at a concentration of 70 mM remained un- changed after growth on acetate. (iv) Temperature and pH range for growth. Growth was observed in the pH range from 6.5 to 7.75. No growth occurred at pH 6 and 8. Growth at pH 6.5 occurred with a marked lag phase. Strain FE was a true mesophilic organism, growing opti- mally at 35°C. No growth was observed at 25 and 40°C within 4 months. (v) Effect of sulfur compounds. Methane formation by s8train FE was completely inhibited by 5 mM molecular s,ulfur, sulfite, hyposulfite, and pyrosulfite. However, growth FIG. 2. Scanning electron micrograph of trichomes of Methano- and methane formation were observed with sulfate (at con- thrix sp. strain FE. Bar = l Fm. centrations up to 200 mM) and thiosulfate (at concentrations LIPto 150 mM). Comparison of the three strains. (i) Kinetic data. Growth extract was also tested. Two controls were run in standard rates were determined for the three strains on the same BCYT medium supplemented with 0.5 g of yeast extract per medium in 500-ml anaerobic flasks containing 100 ml of liter. In addition, cultures grown without yeast extract were HEPES-buffered BCYT medium. Because of the low inoc- also reinoculated onto the standard medium to check culture ulation ratio and the low solubility of methane in water, the viability. amount of methane coming from the inoculum was negligi- Only Methanothrix soehngenii strain OpfikonT grew with- ble. In this case, exponential methane production was as- out yeast extract up to the third transfer with apparently no sumed to be the result of exponential growth. Growth rates effect on the doubling time. Strain FE grew at the first were within the same order of magnitude. The mean values inoculation with a lengthened doubling time and failed to off the doubling times for Methanothrix sp. strain FE, Meth- grow when it was transferred for the second time. Moreover, a'nothrix soehngenii strain OpfikonT, and Methanothrix con- cultures grown without yeast extract failed to grow when cilii strain GP6T were 86.3, 57.2, and 78.1 h, respectively. they were reinoculated into the normal medium. Methano- (ii) Effect of yeast extract. The requirement for yeast thrix concilii strain GP6T did not grow without yeast extract. extract was assayed in duplicate in 25-ml vials containing 10 A high level of yeast extract completely inhibited the growth nil of medium without yeast extract. The inoculum was of Methanothrix concilii strain GP6T. The growth of strain depleted in yeast products by subculturing three times on the FE and the growth of Methanothrix soehngenii strain Opfi- same medium (2% inoculum) in order to eliminate the konT were severely inhibited by 6 g of yeast extract per liter, carry-over. The effect of a high level (6 g/liter) of yeast with strain FE less affected. The high level of yeast extract induced the development of the contaminant bacteria when they were present and a change in Methanothrix morphol- ogy. Under these conditions, no filaments were formed. (iii) Influence of hydrogen. Hydrogen was added to the gas phase of the cultures as soon as growth started, as evidenced by the appearance of methane in the headspace. Time courses for methane production were obtained from two independent assays for each strain and two controls. Meth- ane production was apparently affected by the presence of hydrogen only with strain FE; however, the inhibition was not complete under the conditions used for the assay. (iv) Effect of antibiotics. Under our experimental condi- tions, the growth of the three strains was inhibited only by kanamycin. Neither ampicillin nor penicillin had any signif- icant effect. Inhibition by D-cycloserine was only transient and had disappeared by 25 days after addition of the antibi- otic. (v) DNA base compositions and DNA-DNA hybridizations. The DNA base compositions of the three strains were similar (Table 1). The value for Methanothrix concilii strain GP6T was significantly different, but the range of values for the FIG. 1. Bundles of Methanothrix sp. strain FE in a wet mount group was only 2 mol%. (Nomarsky optics). In this preparation the septation of the cells Compared with Methanothrix soehngenii strain OpfikonT, within trichomes is clearly visible. Bar = 10 km. the percentages of DNA-DNA hybridization for Methano- VOL. 38. 1988 METHANOTHRIX SOEHNGENII 33

FIG. 3. Thin sections of Methanothrix sp. strain FE. Note the asymmetrical aspect of the cell spacers (A) and the triple spacer structure entrapping a nonviable cytoplasmic portion (C). The fine structure of the sheath and the cytoplasmic membrane appears in the tangential section (B), as well as in the longitudinal (D) and transverse (E) sections. Bar = 0.2 pm. 34 TOUZEL ET AL. INT. J. SYST.BACTERIOL. thrix concilii strain GP6T and Methanothrix sp. strain FE were 101.7 and 101.8%, respectively. (vi) Polyacrylamide gel electrophoresis protein patterns. The electrophoretic protein patterns of the three strains were identical (Fig. 4). (vii) Immunology. When the immunization procedure of Conway de Macario et al. (7) for methanogens was used, the S titers and the T titers varied within ranges of 1:lOO to 1:400 and 12300 to 1:3,200, respectively, for all of the strains used as antigens except Methanothrix sp. strain FE; for strain FE tlhe corresponding titers were only 1:40 and 1:400. Using the standard hyperimmunization method resulted in enhance- ment to 1:lOO and 1300 for the titers of Methanothrix sp. strain FE antiserum. S dilutions of this serum gave only slight cross-reactions against Methanosarcina mazei anti- gens. This S probe did not react with Methanosarcina barkeri strains MS, 227, 3 (= DSM 805), and UBS, Metha- nosarcina mazei strains S6 and MC3, Methanosarcina ther- mophila strain TM1, Methanosarcina sp. strains CHTI55 and MST-A1, “Methanosarcina vacuolata,” Methanobrevi- bacter arboriphilicus strains AZ and DH1, Methanospirillum hungatei strain JF1, and Methanobacterium thermoautotro- phicum strains Delta H and Marburg. However, weak cross- reactions with Methanothrix soehngenii strain OpfikonT and Methanothrix concilii strain GP6T were observed when the two sera were used at the 1:lOO dilution. Conversely, none of FIG. 4. Protein electrophoretic patterns of the three Methano- the antisera prepared against the non-Methanothrix cultures thrix strains. l, Methanothrix sp. strain FE; 2, Methanothrix concilii gravecross-reactions with the three Methanothrix strains. strain GP6T; 3, Methanothrix soehngenii strain OpfikonT. however, its morphology and its ability to use H,-CO, DISCUSSION clearly exclude this strain from the genus Methanothrix. Isolation. Neither type strain of Methanothrix was able to Ecology. Numerous observations have confirmed the oc- grow on solid media, so culture purification was attained by currence of Methanothrix-like organisms in mesophilic en- serial dilution with addition of an antibiotic (27, 40). The richments (6, 13, 22, 24). In digesters (7, 9, 10, 14, 16, 39, reason why colonies could not be obtained is not yet clear. Methanothrix spp. have been observed as the major aceto- The toxicity of components of agar to some bacteria from the clastic methanogenic species. In these digesters, acetate soil is well known. On the other hand, the viability of the concentrations are generally less than 1mM. In addition, the strains may be affected by brief exposure to the temperature bacterial retention times in fixed-film reactors or sludge bed required for maintaining the agar in a melted state. It is reactors are assumed to be more than 50 days. Assuming noteworthy that a Methanothrix strain, strain VNBF, was that Monod kinetics are valid under these conditions, Meth- able to form colonies within 2 to 3 weeks on roll tubes (11); alqothrix would outcompete Methanosarcina species due to however, this strain was not deposited in a collection. the lower affinity constant for acetate of the former (36). Thermophilic Methanothrix sp. strain CALS-1, recently Thermophilic Methanothrix-like strains have been ob- isolated by Zinder et al. (41), was also purified by dilution served in enrichments from various habitats (flooded soil, into liquid medium, although colonies could be obtained in ooze, water, and decaying algal mats from thermal environ- acetate agar roll tubes receiving a dilution of or less. ments) (26). One strain has been purified from the mud of a On the other hand, the inability to grow on solidified media thermal lake (25) and was named “Methanothrix thermo- favors the presence of contaminants. The presence of con- acetophila.” A thermophilic strain of Methanothrix was taminating bacteria was confirmed in Methanothrix soehnge- observed in an anaerobic digester fed lignocellulosic waste nii strain OpfikonT and in Methanothrix sp. strain FE. In arid was studied by Zinder et al. (42); its isolation and both cases, the contamination was due to a vibrio (H. Hippe, characterization have been described recently (41). Another personal communication). Difficulties in isolating sheathed thermophilic acetoclastic rod-shaped methanogen, strain bacteria are not uncommon (15). In fact, “the presence of a T.4M, was described by Ahring and Westermann (1, 2); sheath . . . enables the bacteria to attach themselves to solid surfaces” (23) and seems to play a role in the capture of TABLE 1. Guanine-plus-cytosine contents of the three nutrients. The sheath could also be involved in the adhesion Methanothrix strains of other bacteria to filaments. Although such contaminants are present at very low No. of OD sd Hyper- Guanine-plus- levels, their occurrence may render difficult any comparison Strain denatu- ODS,oa chromicity temp cytosine GC rations shift (%) (“‘I content (mol%) between strains, particularly when minimal requirements or very active enzymes are studied. However, contamination is OpfikonT 6 1.75 23.61 90.83 (0.16)’ 52.29 (0.39)’ less troublesome when we study biochemical features, such FE 6 1.66 35.81 91.96 (0.27) 52.61 (0.55) as the presence of glycogen (30), 16s ribosomal ribonucleic GP6T 3 1.88 32.82 89.97 (0.19) 50.20 (0.47) acid oligonucleotide sequences (33), DNA base composition, ‘‘ ODz,,,/ODz,o, Optical density at 260 nm/optical density at 280 nm. and polyacrylamide gel electrophoresis protein patterns (this ’’ The numbers in parentheses are confidence intervals at 95% probability. work). VOL. 38. 1988 METHANOTHRIX SOEHNGENII 35

Characterization. As determined by either light micros- sheath to chemical solubilization (28). In this case, DNA is copy or transmission electron microscopy (4, 5, 17, 28, 40; easily recovered by the Marmur precipitation method. Huser, Ph.D. thesis), the structures of the three strains are The guanine-plus-cytosine value for Methanothrix concilii not significantly different. Acetate is the only substrate used. was about 10 mol% lower than the previously published The splitting of formate remains a controversial matter. value (27), and the mean guanine-plus-cytosine values for Formate reportedly is split into H, and CO, by Methanothrix the three strains were ranged from 50.2 to 52.3 mol%. These soehngenii (Huser, Ph.D. thesis) and Methanothrix concilii values, which were obtained in the same laboratory by using (27). However, we were unable to demonstrate any formate Escherichia coli DNA as a standard, do not differ from each splitting in the presence of acetate for any of the three strains other by more than 3 mol%. According to Triiper and (data not shown). The use of different media or the require- Kramer (38), and considering the phenotypic and genotypic ment for adaptation could explain these discrepancies. similarities of the three strains, these organisms have to be Kinetic data showed that the three strains are slow- considered members of the same species. growing organisms (doubling times, 57 to 86 h). These results Accordingly, we recognize Methanothrix sp. strain FE as are in contrast with the 24-h doubling time reported for a new strain of Methanothrix soehngenii. In this study we Methanothrix concilii by Patel (27). The antibiotics chosen also found that Methanothrix concilii strain GP6T is not were those used by both Huser et al. (17) and Patel (27). The significantly different from strains OpfikonT and FE, and effect of antibiotics was estimated from methane formation therefore Methanothrix concilii is a synonym of Methano- rather than from growth, as described by Pecher and Bock thrix soehngenii. (29), because of the greater sensitivity of methane determi- An axenic culture of Methanothrix soehngenii strain Op- nations compared with either turbidity or dry weight mea- fikonT (= DSM 2139T) will be available soon from the surements. Because methane production was compared with Deutsche Sammlung von Mikroorganismen (A. J. B. control values, the observed effects were merely related to Zehnder, Agricultural University, Wageningen, The Nether- an inhibition of growth. The effect of antibiotics was found to lands, personal communication), so we propose that strain be strongly time dependent. The use of different culture Opfikon, which was the first strain isolated and described, media in different laboratories further complicates the com- has to be maintained as the type strain for the genus and the parison of data. For example, Patel (27) found that Metha- species. nothrix concilii was completely inhibited by D-cycloserine at a concentration of 0.1 mglml, whereas we found that inhibi- ACKNOWLEDGMENTS tion was about 70% for 18 days after antibiotic addition and disappeared by 25 days. Methanothrix soehngenii strain We thank A. J. B. Zehnder, H. Hippe, and E. Conway de Macario OpfikonT was less susceptible to D-cycloserine, penicillin, for valuable comments. We gratefully acknowledge the skilled assistance of F. Fontaine and M. Kubaczka. and ampicillin in our study than in a previous study (17). In This work was supported in part by Region Nord/Pas-de-Calais. fact, only kanamycin significantly inhibited the three strains. This antibiotic has been shown to inhibit polypeptide elon- LITERATURE CITED gation by cell-free systems in methanogens and other archae- 1. Ahring, B. K., and P. Westermann. 1984. Isolation and charac- bacteria (20). terization of a thermophilic, acetate-utilizing methanogenic bac- As previously described (27, 40), hydrogen did not influ- terium. FEMS Microbiol. Lett. 2547-52. ence methane formation in either Methanothrix soehngenii 2. Ahring, B. K., and P. Westermann. 1985. from strain OpfikonT or Methanothrix concilii. Only a slight acetate: physiology of a thermophilic, acetate-utilizing meth- inhibition of methane formation in the presence of hydrogen anogenic bacterium. FEMS Microbiol. Lett. 28:15-19. was observed with strain FE. 3. Beji, A., D. Izard, F. Gavini, H. Leclerc, M. Leseine-Delstanche, Immunology. Cross-reactions showed that the anti-Metha- and J. Krembel. 1987. A rapid procedure for isolation and nothrix purification of chromosomal DNA from gram negative bacilli. sp. strain FE sera were strain specific; this is in Anal. Biochem. 162:18-23. contrast to anti-Methanosarcina sera, which have no strain 4. Beveridge, T. J., B. J. Harris, G. B. Patel, and G. D. Sprott. specificity (7; Thomas, Ph.D. thesis). This specificity was 1986. Cell division and filament splitting in Methanothrix con- also found by E. Conway de Macario (State of New York cilii. Can. J. Microbiol. 32:779-786. Department of Health, Albany, personal communication) 5. Beveridge, T. J., G. B. Patel, B. J. Harris, and G. D. Sprott. when he studied sera against Methanothrix soehngenii Op- 1986. The ultrastructure of Methanothrix concilii, a mesophilic fikonT, Methanothrix concilii GP6T, and thermophilic Meth- aceticlas tic me thanogen. Can. J. Microbiol. 32:703-710. anothrix sp. strain CALS-1. 6. Boone, D. R., and M. P. Bryant. 1980. Propionate-degrading . The morphology and the pH and temperature bacterium, Syntrophomonas wolinii sp. nov., gen. nov., from methanogenic ecosystems. Appl. Environ. Microbiol. 40:62& ranges of the three strains are very similar. Acetate is the 632. only substrate for growth and methane formation for the 7. Conway de Macario, E., A. J. L. Macario, and M. J. Wolin. three strains. Minor differences were found in the effects of 1982. Specific antisera and immunological procedures for char- antibiotics, in the effects of hydrogen on growth, and in the acterization of methanogenic bacteria. J. Bacteriol. 149:32& requirement for yeast extract. In addition, protein patterns 328. which are recognized as useful taxonomic characters (18) are 8. Costerton, J. W. 1980. Some techniques involved in study of identical for the three strains. adsorption of microorganisms to surfaces, p. 403432. In G. Disruption by a French press usually results in a shearing Bitton and K. C. Marshall (ed.), Adsorption of microorganisms of the DNA which cannot be purified by the Marmur to surfaces. John Wiley & Sons, Inc., New York. 9. Dubourguier, H. C., G. Prensier, E. Samain, and G. Albagnac. precipitation procedure. In such a case the phenol-hydrox- 1985. Granular methanogenic sludge: microbial and structural ylapatite method has to be used. The DNA is obtained analysis, p. 542-551. In W. Palz, J. Coombs, and D. 0. Hall rapidly and efficiently in a very pure form (data not shown). (ed.), Energy from biomass. Proceedings of the 3rd European Alternatively, lysis can be obtained by brief exposure to Communities Conference, Venice, Italy. Elsevier Applied Sci- SDS under alkaline conditions, using the method of Beji et ence Publishers, Amsterdam. al. (3), despite the extreme resistance of the Methanothrix 10. Dubourguier, H. C., E. Samain, G. Prensier, and G. Albagnac. 36 TOUZEL ET AL. INT. J. SYST.BACTERIOL.

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