INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1991, p. 191-196 Vol. 41, No. 2 0020-7713/91/020191-06$02.00/0 Copyright 0 1991, International Union of Microbiological Societies

Isolation and Characterization of a Novel Thermophilic Strain YOICHI KAMAGATA* AND EIICHI MIKAMI Fermentation Research Institute, Agency of Industrial Science and Technology, Tsukuba, Ibaraki 305, Japan

A novel thermophilic acetotrophic Methanosaeta strain was isolated from a thermophilic anaerobic digest or by using enrichment and serial dilution in the presence of vancomycin and neomycin. This isolate, designated Methanosaeta sp. strain P,, resembled Methanosaeta sp. strain CALS-1 morphologically;however, it occasionally formed filaments longer than 100 pm and exhibited autofluorescence. The content of coenzyme F,,, was much higher than that of Methanosaeta reference strains, and coenzyme F,,, with four glutamyl residues on the side chain was the predominant component. Furthermore, a comparative analysis of the antigenic fingerprint of strain P, with the fingerprints of reference organisms showed that this isolate was not related antigenically to the reference methanogens, including Methanosaeta sp. (“” sp.) strain CALS-1 and Methanosaeta concilii (“Methanothrix soehngenii”) Opfikon. Strain P, formed visible colonies in a deep agar medium when high cell concentrations were present. However, transfer of a colony into liquid medium resulted in no growth. Strain P, could utilize only acetate as a sole carbon and energy source. The optimum temperature and optimum pH for were near 55°C and 6.7, respectively. The specific formation rate pCH,under optimum conditions was 0.47 day-’, and the doubling time was 1.49 days. The DNA base composition was 52.7 mol% guanine plus cytosine.

Acetate is a major intermediate in the anaerobic digestion other methanogens, and thus much knowledge remains to be of organic compounds, and conversion of acetate to methane accumulated. is one of the rate-limiting reactions. Among the anaerobes, In the field of anaerobic digestion, there has been in- only some methanogens and sulfate-reducing bacteria can creased interest in high-temperature digestion because it is utilize acetate as a carbon and energy source. Of these advantageous to obtain a higher reaction rate in the conver- microorganisms, members of the genus Methanosaeta sion of organic waste to methane than the rate that occurs in (b ‘Methanothrix”) appear to be predominant and play an mesophilic processes. To elucidate the ecological, morpho- important role as acetate-consuming methanogens in anaer- logical, and physiological aspects of the role of thermophilic obic digestors, especially in up-flow anaerobic sludge blan- Methanosaeta strains in high-temperature digestion, we kets (6). Barker described a filamentous acetate-utilizing attempted to isolate a potent thermophilic Methanosaeta methanogen named Methanobacterium soehngenii about 50 strain and obtained a novel strain from a thermophilic years ago (2), but for many years no axenic cultures of anaerobic digestor. In this paper we describe the isolation Methanosaeta strains were obtained because of difficulties and characterization of a thermophilic isolate belonging to in cultivation (i.e., slow growth or no colony formation). the genus Methanosaeta and discuss several novel aspects Methanosaeta sp. was first described as an “acetate of our isolate. organism” by Zehnder et al., and these authors designated their organism Methanothrix soehngenii Opfikon (11, 28). After this description, several other mesophilic Methanosa- MATERIALS AND METHODS eta strains were isolated (8, 19, 26). Very recently, Patel and Sprott, who purified Methanothrix concilii GP6 (19), pro- Microorganisms. Methanobacterium thermoautotrophi- posed that the genus Methanothrix should be changed to the cum DSM1053 and DSM2133, barkeri genus Methanosaeta because the genus Methanothrix is DSM800, and Methanosaeta concilii DSM2139 (‘ ‘Methano- nomenclaturally invalid according to the International Code thrix soehngenii” Opfikon) and DSM3671 (= GP6) were of Nomenclature of Bacteria (13, 20). On the basis of the obtained from the Deutsche Sammlung von Mikroorganis- proposal by Patel and Sprott (20) and Touzel et al. (26), the men, Braunschweig, Federal Republic of Germany. Metha- mesophilic strains previously described (11, 12, 19, 26) nosaeta concilii U, and P, were recently isolated from should be assigned to the species Methanosaeta concilii. mesophilic anaerobic sludge digestors (12) by using a serial A thermophilic strain of the genus Methanosaeta (“Meth- dilution technique (28) in our laboratory; these strains still anothrix”) was first enriched from thermal lake mud and contained a very small number of contaminants. other sources by Nozhevnikova et al. (17, 18). Recently, Media and growth conditions. The culture medium used for Zinder et al. obtained a pure culture of thermophilic Meth- isolation and maintenance of stock cultures was medium 334 anosaeta sp. strain CALS-1 from a thermophilic (58°C) (5) of the Deutsche Sammlung von Mikroorganismen, which anaerobic digestor (30, 31). However, details of the physio- was first described by Touzel and Albagnac (25). Slight logical and biochemical characteristics of this organism have modifications were made during preparation; sodium acetate not been described well. Therefore, the study of the genus was added to a final concentration of 80 mM, and a vitamin Methanosaeta is in its infancy compared with the study of solution was prepared as described by Balch et al. (1). Unless otherwise stated, all cultivations were carried out at 55°C in 125-ml serum vials containing 30 ml of medium or in __-____ 1,000-ml bottles containing 500 ml of medium under an * Corresponding author. atmosphere containing N, and CO, (80/20, vol/vol). The

191 192 KAMAGATA AND MIKAMI INT. J. SYST.BACTERIOL. serum vials were closed with butyl rubber stoppers that were were determined by reversed-phase high-performance liquid sealed with aluminum crimping. chromatography (HPLC) (Shimadzu model LCBA system) Methanobacterium thermoautotrophicum DSM2133 and supplemented with fluorometric detection; our system was DSM1053 were cultivated at 60°C in 1,000-ml bottles con- based on the system described by van Beelen et al. (27) and taining 500 ml of medium 119 of the Deutsche Sammlung von Gorris et al. (9, 10). For the fluorometric determinations, Mikroorganismen (3,except that the fatty acid mixture was coenzyme F420 was detected by emission at 470 nm after excluded. Mixed gas (H2-CO,, 80/20, vol/vol) was supple- excitation at 400 nm. Four types of coenzyme F420 (two to mented at appropriate intervals, and the liquid and gas five glutamyl residues on the side chain) were separated from phases were mixed vigorously with a magnetic stirrer during each other by this procedure. An authentic standard of cultivation. coenzyme F420 with two glutamyl residue on the side chain Methanosarcina barkeri DSM800 was cultivated at 37°C in was purified from Methanobacterium thermoautotrophicum 1,000-ml bottles containing 500 ml of medium 120 of the DSM2133 by using the methods of Schoenheit et al. (23) and Deutsche Sammlung von Mikroorganismen (5). Eirich et al. (7). Methanosaeta concilii DSM2139 (‘ ‘Methanothrix soehn- Antigenic fingerprint. The antigenic fingerprint was deter- genii” Opfikon), DSM3671, U,, and P, were cultivated at mined as described previously by performing an indirect 37°C under the same conditions as strain P,. immunofluorescence and quantitative slide immunoenzy- Effects of pH and temperature. To determine the optimum matic assay (14-16). We used the following 18 antibody pH for growth, the pH values of medium containing 10 mM probes, which were selected from a collection of 33 heterol- acetate were adjusted to 5.7 to 8.5 (approximately 0.4-pH- ogous S-probes of reference methanogens: Methanobacte- unit intervals) by adding HCl or NaOH under a 100% N, rium formicicum MF; Methanosarcina barkeri MS; Metha- atmosphere. Duplicate cultures (1% [vol/vol] inoculum) nobacterium bryantii MoH; Methanobacterium bryantii were incubated at 55”C, and levels of methane production in MoHG; Methanosarcina barkeri RlM3; Methanospirillum the early stages of growth (within 6 days) were determined. hungatei JF1; Methanobrevibacter arboriphilus DH1; Meth- The pH changes during the early stages of cultivation were anobacterium thermoautotrophicum GC1; Methanobacte- not significant under the experimental conditions which we rium thermoautotrophicum AH; Methanosarcina barkeri used because of the low consumption of acetate. 227; Methanosarcina mazei S6; Methanosarcina barkeri W; Cultures in acetate medium (3% [voVvol] inoculum) were Methanosarcina thermophila TM1; Methanobrevibacter ar- incubated at various temperatures (30 to 70”C), and methane boriphilus AZ; Methanobrevibacter arboriphilus DC; Meth- production was analyzed. The specific methane formation anothermus fervidus V24S; Methanosaeta concilii (“Meth- rates (pcH) during the exponential phase were calculated as anothrix soehngenii”) Opfikon; and Methanosaeta sp. described by Huser et al. (11). (‘ ‘Methanothrix sp.”) strain CALS-1. The levels of antigenic Effects of antibiotics and other additives. Antibiotics and relatedness between the new isolate and the reference meth- other additives were obtained from the following sources: anogens were determined by comparing the antigenic finger- vancomycin, streptomycin, tetracycline, cycloserine, neo- prints, using a reference table (14). mycin, and bacitracin were from Sigma Chemical Co.; Determination of DNA base composition. DNA was ex- penicillin G , kanamycin , chloramphenicol, and Polypepton tracted and purified by using the methods of Beji et al. (3) were from Wako Pure Chemicals Industry, Ltd.; yeast and Saito and Miura (22). A brief sonication (10 kW, 5 s) was extract was from Difco Laboratories; and coenzyme M used after alkaline sodium dodecyl sulfate treatment to get a (mercaptoethanesulfonic acid) was from Pierce Chemical better yield. The purified DNA was hydrolyzed with P1 Co. The sludge supernatant was prepared as described nuclease (GC kit; Yamasa Shoyu Co.) followed by alkaline below. The thermophilic, suspended, digested sludge used phosphatase (from Escherichia coli; Wako Pure Chemicals for the isolation of strain P, was dispersed by brief sonica- Industry, Ltd.) as described by Tamaoka and Komagata tion and centrifuged at 10,000 x g for 30 min to obtain a clear (24). The guanine-plus-cytosine content was determined by supernatant. Each solution prepared under an N2 gas phase reversed-phase HPLC (Shimadzu model LC-6A system). was added aseptically through a sterilized filter (pore size, Separation was achieved at 50°C by using a flow rate of 1 0.22 pm) to acetate medium at the concentration indicated ml/min, a column of YMC-Pak AQ-312 (Yamamura Chemi- below (see Tables 2 and 3). The medium was inoculated with cal Institute Co.), and 5% methanol in 10 mM phosphate a preculture of the isolate (3% [vol/vol] inoculum), and the buffer (pH 3.5) as the mobile phase. Each deoxyribonucleo- amount of methane formed was determined. side was detected by determining and an equimolar Microscopy. An exponential-phase culture was observed mixture of four deoxyribonucleosides was used as the stan- by phase-contrast microscopy and epifluorescence micros- dard. copy, using an Olympus model BHS-RFK microscope. For epifluorescence microscopy, a type BP405 filter was used as RESULTS the exciter filter and a type Y455 filter was used as the suppression filter. Isolation. Digested sludge that was obtained from a ther- Analytical methods. The amount of methane produced was mophilic (55°C) anaerobic digestor in our laboratory was determined by gas chromatography by using a molecular inoculated into acetate medium containing 100 pg of vanco- sieve column connected to a Shimadzu model GC-8AIT mycin per ml and was enriched at 55°C by consecutive thermal conductivity detector. Argon was used as the carrier transfers, using 10% (vol/vol) inocula. After 10 transfers at gas. The amount of acetate was determined by gas chroma- 2-week intervals, cells resembling the cell of thermophilic tography by using a Shimalite PEG-6000 column (Shimadzu) Methanosaeta (“Methanothrix”) strains as described by connected to a Shimadzu model GC-5A flame ionization Nozhevnikova et al. (17, 18) and Zinder et al. (30, 31) were detector. predominant, and no methanogen of the Methanosarcina Determination of the contents of coenzyme F,,, and its type was observed in the culture. For further enrichment, analogs. The coenzyme F420 in cells was extracted by using the culture was serially diluted in acetate medium containing the method of Peck (21), and the coenzyme F420 contents 100 pg of vancomycin per ml and 1 pg of neomycin per ml. VOL. 41, 1991 THERMOPHILIC METHANOSAETA STRAIN 193

FIG. 1. Photomicrographs of strain P,. (a) Phase-contrast photomicrograph of strain P,. Bar = 10 pm. (b) Phase-contrast photomicro- graph of strain PT after centrifugation at 3,000 x g for 10 min. Bar = 10 pm. (c) Epifluorescence photomicrograph of strain P,. Bar = 10 pm. (d) Colonies of strain PT in deep agar medium. Bar = 0.5 mm.

Although the addition of neomycin resulted in considerable Morphological characteristics of strain PT and content of delays of growth, an almost pure culture was obtained from coenzyme F,, analogs. Strain P, cells were nonmotile, the and dilutions, and thus this procedure was straight, sheathed, gram-negative rods with flat ends (Fig. la repeated several times. The culture which we obtained and b). The mean dimensions of single cells were 0.8 by 3.0 contained no contaminants as determined by microscopy. pm, and the cells were connected to each other forming The purity of the culture was also examined by inoculating characteristic filaments which sometimes were more than samples into the following media (no contaminants grew on 100 pm long. While the filaments sedimented during cultiva- these media): (i) thioglycolate medium (Difco); (ii) thiogly- tion, they were dispersed homogeneously after gentle shak- colate medium containing 10 mM glucose; (iii) thioglycolate ing. Phase-light particles, which were readily eliminated by medium containing 10 mM cellobiose; (iv) thioglycolate centrifugation or sonication (Fig. lb), were observed in medium containing 10 mM sucrose; (v) thioglycolate me- filaments. These particles were very similar to the gas dium containing 10 mM xylose; (vi) thioglycolate medium vesicles of thermophilic Methanosaeta strains described by containing 10 mM lactate and 10 mM sulfate; (vii) thiogly- Nozhevnikova and Chudina (17) and Zinder et al. (30). colate medium containing 10 mM acetate and 10 mM sulfate; Our isolate grew on media containing 0 to 0.2 M NaCl. (viii) AC medium (Difco); (ix) acetate medium containing 10 Cells harvested in the late logarithmic phase were not lysed mM sulfate; (x) acetate medium without acetate but contain- by sodium dodecyl sulfate (O.lg/liter) when we used the ing 10 mM lactate and 10 mM sulfate; and (xi) the H,-CO, procedure described by Boone and Whitman (4). Suscepti- medium used for culture of Methanobacterium thermoau- bility to lysis under hypotonic conditions (4) was not ob- totrophicum (see Materials and Methods). The pure culture served. was designated strain P, and was used for further experi- During isolation, visible colonies developed in deep agar mentation. The stock culture was routinely maintained in tubes containing acetate medium in dilutions lower than acetate medium without antibiotics. The colonies were white to slightly yellowish and star 194 KAMAGATA AND MIKAMI INT. J. SYST.BACTERIOL.

TABLE 1. Contents of coenzyme F,,, types in methanogens a Coenzyme F4zocontent Methanogen (nmol/g [dry wt] of cells)" Total Glu-2 Glu-3 Glu-4 Glu-5 Methanosaeta sp. strain 63.5 1.9 4.9 43.8 12.9 PT Methanosaeta concilii 11 0.7 1.4 1.4 7.5 DSM2139 Methanosaeta concilii 6.1 0.4 0.5 0.6 4.6 DSM3671 Methanosaeta concilii 4.4 0.2 0.9 0.4 2.9 "A Methanosaeta concilii 3.2 ND 0.3 0.3 2.6 PM Methanosarcina barkeri 258 6.9 4.0 61.1 186 DSM800 0 4 8 4 8 12 Methanobacterium ther- 697 693 3.3 0.8 ND CULTURE TIME (DAYS) moautotrophicum CULTURE TIME (DAYS) DSM1053 Methanobacterium ther- 210 207 1.9 1.5 ND FIG. 2. (a) Conversion of acetate to methane during growth of moautotrophicum strain P,. Symbols: 0, methane; .,acetate. (b) Semilogarithmic DSM2133 plot of methanogenesis. '' ND, Not detected. Glu-2, Glu-3, Glu-4, and Glu-5 indicate the number of glutamyl residues on the side chain of coenzyme F420. methane production was observed at pH 6.1 to 7.5 (data not shown). The optimum temperature for methanogenesis was shaped (Fig. Id), presumably as a result of elongation of 55°C (Fig. 3). Neither methanogenesis nor growth occurred many filaments. The agar in which colonies were formed was at 30 or 70°C after 3 weeks of incubation. split by gas production. Transfer of colonies into a liquid Acetate medium was supplemented with organic com- medium was attempted, but no growth was observed even pounds or hydrogen (H,-CO,, 80/20), and the effects of these after prolonged incubation. additives on methane production were investigated (Table Epifluorescence microscopy showed that the cells exhib- 2). One of these additives, the supernatant of the sludge from ited blue-green autofluorescence (Fig. lc); all other Metha- which strain P, was isolated, slightly increased methanogen- nosaeta strains described previously exhibit no or very weak esis, giving a kCH,of 0.466 day-'. The calculated doubling autofluorescence (11, 12, 17, 30). To determine the content time was about 1.49 days (36 h). The addition of Polypepton of coenzyme F420, an autofluorescent coenzyme peculiar to or coenzyme M did not affect methanogenesis, and the methanogens, a cell extract was analyzed by HPLC supple- addition of 0.1% yeast extract had an inhibitory effect. mented with a fluorometric determination. Table 1shows the Effects of antibiotics. The effects of antibiotics on methane formation are shown in Table 3. Inhibition was caused by contents of coenzyme F42, types with two to five glutamyl residues on the side chain in our isolate and some reference kanamycin, tetracycline, cycloserine, neomycin, bacitracin, methanogens. The contents in the reference methanogens and chloramphenicol at the concentrations tested. Neomy- which we determined were generally in good agreement with cin at a concentration of 1 pg/ml did not have a lethal effect the values described previously (10,27), except that the level but caused a significant lag period during enrichment (see of coenzyme F,,, in Methanobacterium thermoautotrophi- above). Antigenic fingerprint. Strain P, did not react with any of 18 cum DSM2133 was slightly lower. The total coenzyme F420 content of strain P, was 6- to 20-fold higher than the contents heterologous S-probes which we used (see Materials and of the four Methanosaeta reference strains, while much Methods), including the antibodies to Methanosaeta concilii higher amounts of coenzyme F42, were detected in the Methanobacterium and Methanosarcina strains. In four Methanosaeta reference strains coenzyme F4,, with five glutamyl residues was a predominant constituent, as Gorris and van der Drift described previously (10). In contrast, strain P, contained a much more coenzyme F,,, with four glutamyl residues than coenzyme F,,, with five glutamyl residues. Growth characteristics. Strain P, utilized only acetate as a carbon and energy source. Neither methanogenesis nor growth was observed when H,-CO,, formate, methylamines, or methanol was used as a substrate. Hydrogen did not suppress methane formation from acetate. Formate was not split to H2 and CO,. A typical course of methanogenesis and acetate consumption at 55°C is shown in Fig. 2a. Methane was generated exponentially from day 4 to day 10 of culti- 0 -.20 40 60 80 vation (Fig. 2b). Strain P, converted 80 mM acetate stoi- chiometrically to methane. TEMPERATURE (OC) The optimum pH for methanogenesis at 55°C was near 6.7; FIG. 3. Effect of temperature on kCH4by strain P,. VOL. 41, 1991 THERMOPHILIC METHANOSAETA STRAIN 195

TABLE 2. Effect of additives on pCH4for strain P, his thermophilic strain CALS-1 to be 50 mol% (29). These results indicate that strain P, should be assigned to the genus Additive" kHp Methanosaeta. None ...... 0.409 & 0.006 Strain P, contained phase-light particles that were appar- ...... 0.377 0.003 HZ-CO, * ently similar to the gas vesicles of thermophilic Methanosa- Polypepton (0.1%) ...... 0.421 0.001 * eta Yeast extract (0.1%) ...... 0.199 * 0 strains (17, 30). Zinder et al. have suggested that gas Coenzyme M (2 mM) ...... 0.412 5 0.007 vesicles are common in thermophilic Methanosaeta strains Sludge supernatant (3%) ...... 0.466 2 0.003 (30). However, on rare occasions we have observed phase- Rumen fluid (8%) ...... 0.314 2 0.033 light particles even in Methanosaeta strains in mesophilic anaerobic digestors (unpublished data). Thus , the formation a Duplicate cultures were incubated at 55°C. of gas vesicles may depend on environmental or physiolog- ical conditions rather than on thermophilic growth. In contrast to the finding that long filaments are rare in Opfikon and Methanosaeta sp. strain CALS-1, as deter- Methanosaeta sp. strain CALS-1 (30), our isolate formed mined by indirect immunofluorescence and the quantitative filaments that were sometimes more than 100 km long. Long slide immunoenzymatic assay. A comparative analysis of the filaments were also observed when strain P, was cultivated antigenic fingerprint of strain P, with the fingerprints of in CALS-1 medium (data not shown), indicating that the reference organisms indicated that strain P, was not related difference in the contents of the two media was not respon- antigenically to the well-known methanogens. sible for the formation of long filaments. DNA base composition. The guanine plus cytosine compo- One of the most characteristic features of strain P, was sition of strain P, was calculated to be 52.7 mol%. For that it autofluoresced, as determined by epifluorescence comparison, DNAs were also extracted from two Methano- microscopy. In general, Methanosaeta strains, including the saeta strains by the same procedure and analyzed; the strains of thermophilic species, exhibit very weak or no guanine-plus-cytosine contents were calculated to be 51.9 autofluorescence because of the low level of coenzyme F420 and 50.3 mol% for Methanosaeta concilii DSM2139 and in the cells (10, 28). Quantification of coenzyme F420 in the DSM3671, respectively. strain P, cells by HPLC revealed that they contained a higher level of coenzyme F420 than the cells of other strains. DISCUSSION The predominant component was coenzyme F,,, with four glutamyl residues rather than coenzyme F,,, with five glu- On the basis of morphological characteristics, strain P, tamyl residues, which was the predominant coenzyme F420 was classified as an acetoclastic methanogen that probably type in other Methanosaeta strains (Table 1). belongs to the genus Methanosaeta (20). Strain P, formed a A comparative analysis of the antigenic fingerprint of sheathed structure in which many cells were arranged. It strain P, with the fingerprints of reference methanogens, could utilize only acetate as a carbon and energy source. which is a very useful way to distinguish organisms at the Sludge supernatant slightly increased growth and methano- species level, also showed the novelty of our isolate. Strain genesis, while yeast extract, peptone, and coenzyme M did P, was not antigenically related to the well-known metha- not. These characteristics were very similar to those of all nogens, including the closely related organisms Methanosa- other Methanosaeta strains described previously (8, 11, 17, eta sp. strain CALS-1 and Methanosaeta concilii Opfikon, 20, 30). We determined the DNA base composition of strain suggesting that this isolate is a new, as yet undescribed P, to estimate its chemotaxonomic location. The DNA base immunotype. On the basis of the findings described above, compositions of strain P, and Methanosaeta concilii Op- we believe that strain P, is a novel Methanosaeta strain. It fikon (=DSM2139) and GP6 (=DSM3671) were 52.7, 51.9 should be useful not only in thermophilic anaerobic digestors and 50.3 mol%, respectively. The values for strains Opfikon to enhance the activity in the digestors, in which the con- and GP6 were recently determined by Touzel et al. (26) using version of acetate to methane is slow, but also in biochem- the thermal denaturation procedure to be 52.3 and 50.2 ical analysis, particularly in studies of the function of coen- mol%. The values calculated by us for these strains were in zyme F420 in acetoclastic methanogenesis. good agreement with the values reported by Touzel et al. Methanosaeta sp. strain P, has been deposited in the Recently, Zinder determined the DNA base composition of culture collection of the Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH, Braunschweig, Germany (DSM 6194). TABLE 3. Effect of antibiotics on methanogenesis by strain P," ACKNOWLEDGMENTS Relative formation Antibiotic of methane (%) We thank Everly Conway de Macario and Albert0 J. L. Macario for the antigenic fingerprinting of our isolate. We also thank Kazu- None 100 hiro Tanaka of our laboratory for much technical advice concerning Penicillin G 500 96 anaerobic cultivation. Vancomycin 100 93 This work was supported by a grant (New Wastewater Treatment Streptomycin 100 95 System) from the Ministry of International Trade and Industry. Kanam ycin 100 4 Tetracycline 100 2 REFERENCES C ycloserine 10 34 1. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Neomy cin 10 1 Wolfe. 1979. Methanogens: reevaluation of a unique biological Bacitracin 10 <0.1 group. Microbiol. Rev. 43:260-296. C hloramphenicol 5 <0.1 2. Barker, H. A. 1936. Studies upon the methane-producing bac- " The amount of methane formed was determined after 10 days of cultiva- teria. Arch. Mikrobiol. 7:420438. tion at 55°C. The amount of methane formed in the control culture (acetate 3. Beji, A., D. Izard, F. Gavini, H. Leclerc, M. Leseine-Delstanche, medium without antibiotic) was defined as 100%. and J. Krembel. 1987. A rapid chemical procedure for isolation 196 KAMAGATA AND MIKAMI INT. J. SYST.BACTERIOL.

and purification of chromosomal DNA from gram-negative the thermophilic acetate methane-producing bacterium Metha- bacilli. Anal. Biochem. 162:18-23. nothrix thermoacetophila sp. nov. Microbiology (Eng. Transl. 4. Boone, D. R., and W. B. Whitman. 1988. Proposal of minimal Mikrobiologiya) 53:618-624. standards for describing new taxa of methanogenic bacteria. Int. 18. Nozhevnikova, A. N., and T. G. Yogodina. 1983. Morphology of J. Syst. Bacteriol. 38:212-219. thermophilic acetate methane-producing bacteria. Microbiology 5. Deutsche Sammlung von Mikroorganismen. 1983. Catalogue of (Eng. Transl. Mikrobiologiya) 51534-541. strains, 3rd ed. Gesellschaft fuer Biotechnologische Forschung 19. Patel, G. B. 1984. Characterization and nutritional properties of mbH, Braunschweig, Germany. Methanothrix concilii sp. nov., a mesophilic, aceticlastic meth- 6. Dolfing, J., A. Griffioen, A. R. W. van Neerven, and L. P. T. M. anogen. Can. J. Microbiol. 30:1383-1396. Zevenhuizen. 1985. Chemical and bacteriological composition of 20. Patel, G. B., and G. D. Sprott. 1990. Methanosaeta concilii gen. granular methanogenic sludge. Can. J. Microbiol. 31:744-750. nov., sp. nov. (“Methanothrix concilii”) and Methanosaeta 7. Eirich, L. D., G. D. Vogels, and R. S. Wolfe. 1978. Proposed thermoacetophila nom. rev., comb. nov. Int. J. Syst. Bacteriol. structure for coenzyme F42, from Methanobacterium. Biochem- 40~79-82. istry 17:45834593. 21. Peck, M. W. 1989. Changes in concentrations of coenzyme F42, 8. Fathepure, B. 1983. Isolation and characterization of an aceti- analogs during batch growth of Methanosarcina barkeri and clastic methanogen from a biogas digester. FEMS Microbiol. Methanosarcina mazei. Appl. Environ. Microbiol. 55:940-945. Lett. 19:151-156. 22. Saito, H., and K. Miura. 1963. Preparation of transforming 9. Gorris, L. G. M., A. K. Kivaisi, and H. J. M. Op den Camp. deoxyribonucleic acid by phenol treatment. Biochim. Biophys. 1989. Quantification of coenzyme F420 analogues from methano- Acta 72:619-629. genic bacteria by HPLC and fluorimetry. Biotechnol. Tech- 23. Schoenheit, P., H. Keweloh, and R. K. Thauer. 1981. Factor F,,, niques 3:239-244. degradation in Methanobacterium thermoautotrophicum during 10. Gorris, L. G. M., and C. van der Drift. 1986. Methanogenic exposure to oxygen. FEMS Microbiol. Lett. 12:347-349. cofactors in pure cultures of methanogens in relation to sub- 24. Tamaoka, J., and K. Komagata. 1984. Determination of DNA strate utilization, p. 144-150. In H. C. Dubourguier et al. (ed.), base composition by reversed-phase high-performance liquid Biology of anaerobic bacteria. H. C. Elsevier Science Publish- chromatography. FEMS Microbiol. Lett. 25:125-128. ers B.V., Amsterdam. 25. Touzel, J. P., and G. Albagnac. 1983. Isolation and characteri- 11. Huser, B. A,, K. Wuhrmann, and A. J. B. Zehnder. 1982. zation of Methanococcus mazei strain MC,. FEMS Microbiol. Methanothrix soehngenii gen. nov., sp. nov., a new ace- Lett. 16:241-245. totrophic non-hydrogen-oxidizing methane bacterium. Arch. 26. Touzel, J. P., G. Prensier, J. L. Roustan, I. Thomas, H. C. Microbiol. 132:l-9. Dubourguier, and G. Albagnac. 1988. Description of a new 12. Kamagata, Y., and E. Mikami. 1990. Some characteristics of strain of Methanothrix soehngenii and rejection of Methano- two morphotypes of Methanothrix soehngenii from mesophilic thrix concilii as a synonym of Methanothrix soehngenii. Int. J. anaerobic digesters. J. Ferment. Bioeng. 70:272-274. Syst. Bacteriol. 38:3&36. 13. Lapage, S. P., P. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, 27. van Beelen, P., A. C. Dijkstra, and G. D. Vogels. 1983. Quanti- H. P. R. Seeliger, and W. A. Clark (ed.). 1975. International tation of coenzyme F,,, in methanogenic sludge by the use of code of nomenclature of bacteria. 1975 Revision. American reversed-phase high-performance liquid chromatography and a Society for Microbiology, Washington, D.C. fluorescence detector. Eur. J. Appl. Microbiol. Biotechnol. 14. Macario, A. J. L., and E. Conway de Macario. 1983. Antigenic M67-69. fingerprinting of methanogenic bacteria with polyclonal anti- 28. Zehnder, A. J. B., B. A. Huser, T. D. Brock, and K. Wuhrmann. body probes. Syst. Appl. Microbiol. 4:451-458. 1980. Characterization of an acetate-decarboxylating, non-hy- 15. Macario, A. J. L., and E. Conway de Macario. 1985. Monoclonal drogen-oxidizing methane bacterium. Arch. Microbiol. 124:l- antibodies of predefined molecular specificity for identification 11. and classification of methanogens and for probing their ecolog- 29. Zinder, S. H. Personal communication. ical niches, p. 213-247. In A. J. L. Macario and E. Conway de 30. Zinder, S. H., T. Anguish, and A. L. Lobo. 1987. Isolation and Macario (ed.), Monoclonal antibodies against bacteria, vol. 2. characterization of a thermophilic acetotrophic strain of Meth- Academic Press, Inc., Orlando, Fla. anothrix. Arch. Microbiol. 146:315-322. 16. Macario, A. J. L., E. ConwaydeMacario, andR. J. Jovell. 1986. 31. Zinder, S. H., S. C. Cardwell, T. Anguish, M. Lee, and M. Koch. Slide immunoenzymatic assay (SIA) in hybridoma technology. 1984. Methanogenesis in a thermophilic (58°C) anaerobic di- Methods Enzymol. 121:50%525. gestor: Methanothrix sp. as an important aceticlastic methano- 17. Nozhevnikova, A. N., and V. I. Chudina. 1985. Morphology of gen. Appl. Environ. Microbiol. 47:796-807.