INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY , Apr. 1990, p. 117-122 Vol. 40, No. 2 OO20-7713/90/020117-06$02.OO/O Copyright 0 1990, International Union of Microbiological Societies

Transfer of Methanogenium bourgense, Methanogenium marisnigri, Methanogenium olentangyi, and Methanogenium thermophilicum to the Genus Methanoculleus gen. nov., Emendation of Methanoculleus marisnigri and Methanogenium, and Description of New Strains of Methanoculleus bourgense and Met h a noc u 11 e us ma ris n igri GLORIA M. MAESTROJUAN,l DAVID R. BOONE,l* LUYING XUN,2t ROBERT A. MAH,, AND LANFANG ZHANG' Department of Environmental Science and Engineering, Oregon Graduate Center, Beaverton, Oregon 97006-1999, and School of Public Health, University of California, Los Angeles, California 9O01ij2

Two strains of Methanogenium bourgense, strains MS2T(T = type strain) and LX1, were characterized, and, based in part on previously published DNA hybridization data, this species was transferred to a new genus, Methanoculleus, as Methanoculleus bourgense comb. nov. Methanogenium marisnigri JRIT and a new strain of Methanogenium marisnigri, strain ANS, were also characterized. This species was also transferred to the genus Methanoculleus as MethanocuUeus marisnigri comb. nov. et emend., and its description was emended to indicate that the species has a temperature optimum near 40°C, is halotolerant, and is slightly alkaliphilic; in contrast, the previous description of this organism indicates that it has a temperature optimum of 20 to 25"C, is halophilic, and is slightly acidophilic. We also propose the transfer of two other phylogenetically related species, Methanogenium thermophilicum and Methanogenium olentangyi, to the genus Methanoculleus as Methanoculleus thermophilicum and Methanoculleus olentangyi, respectively. Methanogenium cariaci JRIT was also further characterized, and its description is emended.

The results of studies of diffusion of H, and formate from (16), Methanogenium olentangyi (9,and Methanogenium bulk solutions to methanogenic cells suggest that the small organophilum (14). DNA hybridization data indicate deep size of many methanogens may be advantageous in scaveng- divisions among these species, with Methanogenium ing small concentrations of these substrates (1). However, marisnigri, Methanogenium thermophilicum, Methanoge- the isolation and characterization of small coccoid methano- nium bourgense, and Methanogenium olentangyi being gens from digestors and other natural methanogenic environ- distinct from other members of the genera Methanogenium ments are fairly recent developments (5, 9, 12, 15, 16, 18, and Methanocorpusculum (15). Sequences have been de- 21), perhaps because these methanogens grow slowly and termined for the 16s rRNAs of six of these organisms, are sensitive to detergents and physical and osmotic stress. Methanoplanus limicola, Methanogenium organophilum, Colonies may often be found in roll tube media inoculated Methanogenium cariaci, Methanomicrobium mobile, Meth- with high dilutions if a small amount of salt is included in the anogenium marisnigri, and Methanogenium thermophilicum media and the incubation period is sufficiently long (4). (P. E. Rouvi&re and C. R. Woese, manuscript in prepara- The genus Methanogenium is a genus of coccoid, meth- tion). As determined by both evolutionary distance and anogenic archaeobacteria which was originally described as maximum parsimony analyses, these six sequences (which marine (12). Since the original description, a number of new are all more than 90% related to one another) fall into two species of coccoid methanogens have been isolated and distinct, but specifically related, phylogenetic groups. One placed in the genus Methanogenium because they appeared comprises the first four organisms. The other includes Meth- to fit there better than in other genera. The inclusion of these anogenium marisnigri and Methanogenium thermophilicum organisms led to an informal broadening of the genus de- (Rouviere and Woese, in preparation). This finding is in scription. Recently, the genus Methanocorpusculum, a new accord with the DNA hybridization data (15), which indicate genus in the family Methanomicrobiaceae, was described that Methanogenium marisnigri, Methanogenium thermo- (18). Methanogenium aggregans was transferred to that philicum, Methanogenium bourgense, and Methanogenium genus (15), two new species, Methanocorpusculum bavari- olentangyi should be separated into a new genus. Therefore, cum and Methanocorpusculum sinense, were described, and we propose that this group of organisms should be trans- a new family for the genus was proposed (19). Currently, ferred to a new genus, Methanoculleus, with Methanocul- there are the following seven valid species of the genus leus bourgense as the type species. Methanogenium: Methanogenium cariaci (12), Methanoge- nium marisnigri (12), Methanogenium thermophilicum (ll), MATERIALS AND METHODS Methanogenium bourgense (9), Methanogenium tationis Media and culture techniques. We used the anaerobic techniques of Hungate (6), with syringes and serum tubes. * Corresponding author. MG medium was similar to MS medium (2) but also con- t Present address: Department of Biochemistry and Bacteriology, tained 2.5 g of NaCl per liter and 5 mmol of sodium acetate University of Idaho, Moscow, ID 83843. per liter, and 0.5 g of 2-mercaptoethanesulfonic acid re-

117 118 MAESTROJUAN ET AL. INT.J. SYST.BACTERIOL. placed the cysteine. This medium also contained 7 g of nium olentangyi RC/ERT (= OGC 52T), Methanogenium NaHCO, per liter, which was added after boiling to remove tationis DSM 2702T (= OGC 43T), Methanocorpusculum O,, or it contained 4 g of NaOH per liter and was equili- aggregans MSt (= OGC 21), and Methanocorpusculum brated with an N,-CO, mixture (7:3) without boiling to parvum XI1 (= OGC 63) were obtained from the Collection remove dissolved 0, (8). The final pH was 7.2 to 7.3. of Methanogenic Archaeobacteria at the Oregon Graduate Enrichment medium was the same as MG medium but Center. The authenticities of our cultures of Methanogenium without cysteine or 2-mercaptoethanesulfonate and with the marisnigri JRIT and Methanogenium cariaci were confirmed peptone and yeast extract concentrations reduced to 0.5 by electrophoretic comparison with additional cultures ghiter. Routinely, the catabolic substrate was 100 mM for- kindly supplied by Hans Hippe of the Deutsche Sammlung mate, and cultures were incubated at 37°C without shaking. von Mikroorganismen. For growth on H,-C02, media (with an N,-CO, gas phase) The inoculum from which we isolated strain AN8 (= OGC were inoculated and then pressurized with pure H, to a 51 = DSM 4552) was effluent from an anaerobic pig manure partial pressure of 101 kPa. The resulting cultures were digestor in Spain, and the inoculum from which we isolated incubated with orbital shaking (120 rpm with a 27-mm stroke strain LX1 was effluent from a sewage sludge digestor at the radius) and were frequently repressurized to 101 kPa above Hyperion Wastewater Treatment Plant in Los Angeles, atmospheric pressure with an H,-C02 mixture (3:l). The Calif. ability to use soluble catabolic substrates was tested by G+C content of the DNA. To determine the guanine- inoculating the following media: media supplemented with plus-cytosine (G+C) content of DNA, late-log-phase cul- 20 mM formate, media supplemented with 20 mM formate tures were cooled to 4°C and were harvested by centrifuga- plus the substrate to be tested at a concentration of 10 mM, tion at 5,000 X g for 15 min. DNA was isolated by a media supplemented with the substrate to be tested at a modification of the Marmur method (7), and the density was concentration of 10 mM, and controls without a catabolic determined by ultracentrifugation in CsCl gradients (10). substrate. Measurement of the methane produced by these DNAs from Micrococcus lysodeikticus (density assumed to cultures indicated whether the substrate was inhibitory and be 1.731 g/cm3; Sigma Chemical Co., St. Louis, Mo.) and whether it could be catabolized. Media at various pH values Clostridium perfringens (density assumed to be 1.691 g/cm3; were prepared by equilibrating medium with gas at various Sigma) were used as standards. The G+C content of the CO? partial pressures. For pH values higher than 8.1, we DNA was calculated by using the formula of Schildkraut et equilibrated the medium with 100% N, and added a sterile, al. (13). anoxic NaOH solution to adjust the pH. For pH values lower Analytical techniques. Proteins were analyzed by poly- than 6.5 we equilibrated the medium with 100% CO, and acrylamide gel electrophoresis as described previously (8). added sterile, anoxic HC1 to adjust the pH. The pH was Specific growth rates were determined from an analysis of checked after autoclaving and equilibration at the growth methane production, and methane was quantified by gas temperature and again after the growth of cultures for chromatography (8). We used a type 02filter set (Carl Zeiss, determination of the optimal pH for growth, During mea- Inc., Thornwood, N.Y .) for epifluorescence microscopy to surement of specific growth rates at various pH values, the distinguish colonies likely to be methanogenic based on their pH did not change more than 0.2 pH unit during growth; the blue-green fluorescence, pH values indicated in Fig. 2 are the initial pH values. Media with various Na+ concentrations were prepared, or the Na+ RESULTS AND DISCUSSION content of MG medium was adjusted by adding a sterile, anoxic 1 or 4 M NaCl solution to individual tubes. When Isolation of strain AN8. Serial dilutions of the effluent from additions to media were a significant fraction of their vol- an anaerobic digestor of pig manure in MG enrichment ume, we corrected the salt concentration for the volume of medium supplemented with 100 mM formate produced meth- solution added. Requirements for organic growth factors ane with an inoculum of as little as 0.1 nl of the digestor were determined by cultivating organisms in media for at effluent. An isolate from such an enrichment culture was least three successive transfers (5% [vol/vol] inocula) in named strain AN8. This culture contained a single morpho- order to prevent the carry-over of nutrients. For mass logical type (coccoid, epifluorescent cells). The cells did not cultivation, cells were grown in 2-liter bottles modified with grow in MG medium when formate was omitted, in fluid 20-mm serum tops. Tops from serum tubes or bottles cannot thioglycolate medium, or in MG medium supplemented with be safely annealed to the hard glass of the larger bottles, so formate and incubated aerobically. The culture appeared to we obtained serum style fittings (Bellco Glass, Inc., Vine- be pure and was deposited in the Collection of Methanogenic land, N.J.) of Corning type 7740 glass; large serum style Archaebacteria at the Oregon Graduate Center as strain bottles may be obtained by writing to D.R.B. OGC 51 and in the Deutsche Sammlung von Mikroorganis- Cultures were isolated from enrichment cultures by serial men as strain DSM 4552. dilution in MG medium and inoculation into molten (45°C) Isolation of strain LX1. Strain LX1 was isolated by using MG medium solidified with 18 g of purified agar per liter the method used for strain AN8 (4). Within 1 week, colonies prepared as roll tubes. Colonies were picked by using a appeared in tubes which received larger inoculum volumes Pasteur pipette with a drawn-out tip (6) and were transferred (as small as 100 nl of the original sample for each sample); to liquid medium. The resulting liquid culture was immedi- these colonies were made up of epifluorescent organisms ately diluted in MG medium and again inoculated into roll which appeared to be members of the genus Methanobacte- tubes. This process was repeated until a single colony type rium. After 2 to 3 weeks of incubation, colonies were remained and then one additional time to ensure that the detected in tubes which received much smaller inocula (as culture was axenic. small as 10 pl). These subsurface colonies were small (diam- Sources of cultures and inocula. Methanogenium bourg- eter, <1 mm), clear, cream colored, spherical, entire, and ense MS2T (= OGC lST), Methanogenium bourgense LX1 epifluorescent. One of these colonies was purified and de- (= OGC 24) (4), Methanogenium cariaci JRIT (= OGC 49T), posited in the Collection of Methanogenic Archaebacteria at Methanogenium marisnigri JRIT (= OGC 56T), Methanoge- the Oregon Graduate Center as strain OGC 24. Several VOL. 40, 1990 METHANOCULLEUS GEN. NOV. 119

FIG. 1. Electrophoretic comparison of cellular proteins of methanogens. (A) Photograph of the gel. (B) Diagram of locations and densities of visible bands. Lane 1, Molecular weight standards (molecular weights, 180,000, 116,000, 84,000, 58,000, 48,500, 36,500, and 26,600); lane 2, strain AN8; lane 3, Methanoculleus marisnigri (Methanogenium marisnigri) JRIT; lane 4, Methanoculleus olentangyi (Methanogenium olentangyi) RC/ERT; lane 5, Methanoculleus bourgense (Methanogenium bourgense) MS2T; lane 6, Methanoculleus bourgense (Methano- genium bourgense) LX1; lane 7, Mefhanogenium cariaci JRIT; lane 8, strain AN8. strains isolated from Hyperion sludge were similar in colony Electrophoretic analysis of whole-cell proteins. We exam- and cell morphology to strain LX1 and were not further ined the proteins of strain AN8 and Methanogenium bourg- characterized. Microscopic examination of the purified cul- ense LX1 by performing an electrophoretic analysis (8) and ture revealed onIy small, epifluorescent cells. No motility compared them with the proteins of all of the strains listed in was seen in wet mounts. DNA-DNA hybridization studies of Materials and Methods. Figure 1 shows a comparison of the strain LX1 indicated that it belongs in the species Methano- organisms whose proteins appear to be most similar to those genium bourgense. of strain AN8. The greatest differences occurred in the Morphology of strain AN$. Strain AN8 colonies were lighter-staining bands. Figure 1B is a drawing which indi- circular with entire edges and cream colored, attaining a cates the locations of bands which were seen in the gel. The maximum diameter of 1 mm after 10 days of incubation. general similarities (among Methanogenium species) of the Individual cells were coccoid and 0.8 to 1.4 bm in diameter. darker bands confirm the results of other electrophoretic The cells stained gram negative. We were unable to obtain analyses (16) and limit the utility of this method for studying electron micrographs because the cells lysed upon fixation. the phylogeny of this group. G+C content of strain ANS. The G+C content of strain Methanogenium bourgense LX1 appears to be most sim- AN8 DNA was 61.7 f 1 mol%. ilar to Methanogenium bourgense MS2T, confirming its 120 MAESTROJUAN ET AL. INT.J. SYST.BACTERIOL. placement in that species based on DNA-DNA hybridization data (15). Similarities between strain LX1 and Methanoge- nium olentangyi RC/ER~are also apparent, in agreement with DNA sequence similarity data (15) which show a very close phylogenetic relationship between Methanogenium olentangyi and Methanogenium bourgense. Strain AN8, Methanogenium marisnigri JRIT, and Methanogenium bourgense MS2T were all very similar in their protein patterns. However, many other characteristics of strain AN8, including G + C content and physiological characteris- tics (see below), such as halotolerance, indicate that strain AN8 is most closely related to Methanogenium marisnigri JRIT and is a reference strain of that species. Physiology of Methanogenium marisnigri ANS, Methanoge- nium bourgense LXl, and related type strains. (i) Catabolic ....- .... substrates. All of the strains tested (Methanogenium maris- 05 1 nigri JRIT and AN8, Methanogenium bourgense MS2T and Sodium concentratlon (M) LX1, Methanogenium olentangyi, Methanogenium tationis, FIG. 2. Effect of Na+ concentration on specific growth rates of and Methanogenium cariaci) grew well in MG medium at some methanogens grown in MG medium containing 100 mM 37°C with formate as the substrate. This is the first report of formate. Vertical bars indicate standard deviations. formate catabolism by Methanogenium olentangyi. In the original study of this organism, Corder et al. (5) tested formate catabolism in medium without CO,, so the high pH nogenium marisnigri AN8 grew in mineral medium (MG of the medium may have inhibited growth. The more rapid medium without organic constituents) containing 100 mM growth of Methanogenium marisnigri JRIT at 37 than at 25°C formate and 5 mM acetate (specific growth rate, 0.047 h-'), appears to conflict with the initial report of this organism even after three transfers, or in mineral medium containing (12); this difference may be due to adaptation of the strain 100 mM formate and 2 g of yeast extract per liter (0.042 h-'). since its isolation or to the different methods used for Either acetate or some other substance(s) present in yeast determining optimum environmental conditions. extract was required for growth, and the specific growth rate Strain AN8 grew poorly and inconsistently on H,-CO, as was most rapid in medium containing 5 mM acetate and the catabolic substrate, apparently due to inhibitory effects either 2 g of yeast extract per liter (0.070 h-'), 2 g of of shaking rather than an inability to catabolize H,, as Trypticase peptones per liter (0.073 h-'), or 2 g of Trypticase described previously for Methanogenium thermophilicum peptones per liter and 2 g of yeast extract per liter (0.073 (17). When cultures of strain AN8 catabolizing formate were h-'). Strain AN8 did not grow in mineral medium containing incubated with shaking, growth was likewise poor and 100 mM formate and 2 g of Trypticase peptones (BBL inconsistent. Microbiology Systems, Cockeysville, Md.) per liter. Metha- Zellner and co-workers (18-20) showed that a number of nogenium marisnigri JRITgrows in mineral medium contain- alcohols are catabolized by methanogens, especially species ing peptones as the sole organic addition (12). of the genus Methanocorpusculum. None of the methano- (iv) Effects of salinity. We tested the effect of Na+ on gens discussed below catabolized 2,3-butanediol, malate, or growth and found that none of the strains tested was strongly primary alcohols (methanol, ethanol, 1-propanol, 1-butanol, halophilic (Fig. 2); all grew well (at least 50% as fast as the and 1-pentanol). Methanogenium cariaci JRIT and AN8, maximum observed specific growth rate) in medium without Methanogenium marisnigri JRIT, Methanogenium tationis, added NaCl, although the growth rates of some strains at the and Methanogenium bourgense MS2T grew on the second- lowest salt concentrations were very slightly suboptimal ary alcohols tested (2-propanol, 2-butanol, 2-pentanol, and (Fig. 2). All of these strains were halotolerant to some 3-pentanol), except that neither Methanogenium marisnigri degree; Methanogenium cariaci appeared to be the most JRIT, strain AN8, nor Methanogenium cariaci JRIT catab- halotolerant in that it grew as well in the presence of 1 M olized 2-pentanol. In contrast, Methanogenium bourgense Na+ as it did in medium containing lower salt concentra- LX1 and Methanogenium olentangyi did not catabolize tions. Methanogenium marisnigri JRIT and AN8 grew much secondary alcohols. Our findings confirmed those of Widdel better at lower Na+ concentrations (0.1 to 0.4 M) than at an et al. (14) that Methanogenium marisnigri uses secondary Na+ concentration of 1 M, although they did grow at Na+ alcohols. concentrations near 1 M. For comparison, Methanogenium Growth of Methanogenium cariaci JRIT on formate re- tationis is much less halotolerant, growing well in the portedly was inhibited in Na,CO,-buffered media (12), but presence of 0.3 M Na+ but not in the presence of 0.5 M Na+ we found that our strain (strain OGC 4gT) grew well on (16). Methanogenium bourgense MS2T produces methane formate in our C0,-bicarbonate-buffered medium. most rapidly at Na+ concentrations less than about 0.3 M, (ii) Growth rates, In MG medium supplemented with although specific growth rates were not determined (9). formate the specific growth rates were 0.128 h-' for Metha- (v) Optimum pH. Methanogenium marisnigri AN8 grew nogenium marisnigri JRIT, 0.157 h-' for strain AN8, 0.099 best at pH values near 7.9 (Fig. 3). The other strains tested h-' for Methanogenium bourgense MS2T, 0.073 h-' for had pH optima above pH 7. The previously reported opti- Methanogenium tationis, 0.072 h-' for Methanogenium mum for Methanogenium marisnigri JRIT is lower than the bourgense LX1, 0.080 h-' for Methanogenium cariaci, and value which we found, but our values were determined by 0.035 h-' for Methanogenium olentangyi RC/ERT. Metha- measuring specific growth rates, whereas in the previous nogenium olentangyi RC/ERT grew more rapidly on H,-CO, study Romesser et al. (12) recorded maximum optical den- (0.062 h-') than on formate. sities measured during incubation. It is possible that condi- (iii) Growth requirements for organic compounds. Metha- tions leading to most rapid growth allowed cultures to grow VOL.40, 1990 METHANOCULLEUS GEN. NOV. 121

0.15- (vii) G+C content of strain LX1 DNA. The G+C content of Methanogenium bourgense LX1 DNA was 62 mol%. Methanoculleus marisnigri Transfer of Methanogenium bourgense to the genus Metha- noculleus gen. nov. as the type species. The results of DNA hybridization studies (15) indicate that four species in the genus Methanogenium (Methanogenium marisnigri, Metha- nogenium bourgense , Methanogenium olentangyi, and Methanogeniurn thermophilicum) form a coherent phyloge- netic group which is distinct from the type species, Metha- nogenium cariaci. The results of our analyses of whole-cell proteins were consistent with this finding. The cells of these species are smaller than those of Methanogenium cariaci and Methanogenium tationis, as shown by Zabel et al. (16). We propose the transfer of Methanogeniurn bourgense to a new genus, Methanoculleus, as the type species of that 5 6 7 8 9 genus. PH Methanoculleus (Me.tha.no.cul‘le.us. M. L. n. metha- FIG. 3. Effect of pH on specific growth rates of some methano- num, methane; Gr. n. culleus, bag; M. L. masc. n. Metha- gens grown in MG medium containing 100 mM formate. Vertical noculleus, methane[-producing] bag) species are irregular bars indicate standard deviations. coccoids which form methane from H,-CO,, from formate, and sometimes from alcohols. Cells are 1to 2 pm in diameter completely before the first determination of optical density, and occur singly or in pairs. Some cells appear to have and cultures may have lysed after growth was complete, as flagella, but motility has not been observed. Cells have a has been reported for Methanogenium bourgense (9). We protein cell wall which is sensitive to lysis by detergents; often observed a stringy precipitate in old cultures, which cells stain gram negative. Organic growth factors are re- appeared to indicate lysis of cells. The proposed minimal quired. The G+C content of the DNA is 54 to 62 mol%. The standards for descriptions of methanogens (3) suggest mea- type species is Methanoculleus bourgense comb. nov. surement of specific growth rates to determine optimal Methanoculleus bourgense comb. nov. Methanoculleus conditions, as we used in our study. The pH optima of bourgense (bourg.en’se. N. L. neut. adj. bourgense, from Methanogenium tationis and Methanogenium bourgense Bourg-en-Bresse, France) strains are coccoid methanogens MS2T are near 7 (16) and 6.7 (9), respectively, although which grow on H,-CO,, on formate, and sometimes on specific growth rates were not measured in the latter deter- secondary alcohols; acetate is a growth factor. Cells are 1 to mination. 2 pm in diameter and grow best at mesophilic temperatures, (vi) Optimal temperature. The optimal growth tempera- at neutral pH values, and at Na+ concentrations below 0.3 tures of all of the strains tested were similar (Fig. 4). The M. Recognized habitats are limited to anaerobic digestors. optimal growth temperature of Methanogenium bourgense Strain MS2 (= DSM 3045 = OGC 15) is the type strain, and MS2T was 37°C. The optimal growth temperatures of Me- strain LX1 (= OGC 24) is a reference strain. thanogenium cariaci and Methanogeniurn marisnigri were Transfer of Methanogenium marisnigri to the genus Metha- previously reported to be 20 to 25°C (12). However, in that noculleus as Methanoculleus marisnigri comb. nov. et emend. study Romesser et al. did not measure specific growth rates, The results of our physiological studies, in agreement with and our findings are confirmed by the results of another previously published data (16), indicate that the description study (16) in which specific growth rates were also mea- of Methanogenium marisnigri should be emended. DNA sured. It is possible that the discrepancies were due to hybridization studies and analysis of whole-cell proteins differences in methods or to an adaptation of the cultures to indicate that this species should be transferred to the genus growth at higher temperatures. Methanoculleus as Methanoculleus marisnigri comb. nov. et emend. Methanoculleus marisnigri (markni’gri. L. gen. n. maris, of the sea; L. adj. niger, black; M. L. neut. n. 0.20 marisnigri, Black Sea) strains are coccoid methanogens which grow on H,-CO,, on formate, and sometimes on E secondary alcohols; peptones or yeast extract is required as f 0.15 a growth factor(s). Cells are 1 to 2 km in diameter and grow best at mesophilic temperatures, at slightly alkaline pH values (7 to 8), and at Na+ concentrations below 1 M. Recognized habitats include marine sediments and anaerobic 0 digestors. Strain JR1 (= DSM 1498 = OGC 56) is the type 5a strain, and strain AN8 (= OGC 51 = DSM 4552) is a t% t% 0.05 reference strain. Transfer of Methanogenium thermophilicum and Methano- genium olentungyi to the genus Methanoculleus as Methano- culleus thermophilicus and Methanoculleus olentangyi, respec- O 20 30 40 50 60 tively. Methanogenium thermophilicum UCLA has 94% Temperature (C) DNA sequence similarity with the type strain of this species, FIG. 4. Effect of temperature on specific growth rates of some Methanogeniurn thermophilicum CR-l(l7). Because Metha- methanogens grown in MG medium containing 100 mM formate. nogenium thermophilicum UCLA and Methanogenium olen- Vertical bars indicate standard deviations. tangyi RC/ERT are phylogenetically closer to Methano- 122 MAESTROJUAN ET AL. INT.J. SYST.BACTERIOL.

culleus bourgense MS2T than to Methanogenium cariaci 4. Boone, D. R., and L. Xun. 1987. Effects of pH, temperature, and JRIT, we propose the transfer of these two species to the nutrients on propionate degradation by a methanogenic enrich- genus Methanoculleus as Methanoculleus thermophilicus ment culture. Appl. Environ. Microbiol. 53:1589-1592. and Methanoculleus olentangyi, respectively. 5. Corder, R. E., L. A. Hook, J. M. Larkin, and J. I. Frea. 1983. Isolation and characterization of two new methane-producing Emendation of the genus Methanogenium and the species cocci: Methanogenium olentangyi, sp. nov., and Methanococ- Methanogenium cariaci. After the transfer of the species of cus deltae, sp. nov. Arch. Microbiol. 134:28-32. the new genus Methanoculleus out of the genus Methano- 6. Hungate, R. E. 1969. A roll tube method for cultivation of strict genium, the characteristics of the remaining species conform anaerobes, p. 117-132. In R. Norris and D. W. Ribbons (ed.), more closely to some aspects of the original description of Methods in microbiology, vol. 3B. Academic Press, Inc., New the genus. However, the results of our physiological studies York. and those of Zabel et al. (16) indicate that Methanogenium 7. Johnson, J. L. 1985. DNA reassociation and RNA hybridisation cariaci JRIT is substantially different from the original of bacterial nucleic acids, p. 33-74. In Methods in microbiology, description, especially with regard to the temperature opti- vol. 18. Academic Press, Inc. (London), Ltd., London. 8. Liu, Y., D. R. Boone, and C. Choy. 1990. Methanohalophilus mum (near 37"C), and indicate that the descriptions of the oregonense sp. nov., a methylotrophic methanogen from an species Methanogenium cariaci and the genus Methanoge- alkaline, saline aquifer. Int. J. Syst. Bacteriol. 4O:lll-116. nium should be emended. 9. Ollivier, B. M., R. A. Mah, J. L. Garcia, and D. R. Boone. 1986. Species of Methanogenium gen. emend. are irregular Isolation and characterization of Methanogenium bourgense sp. coccoids which form methane from H,-C02, from formate, nov. Int. J. Syst. Bacteriol. 36:297-301. and sometimes from alcohols. The cells are 0.5 to 3 pm in 10. Preston, J. F., and D. R. Boone. 1973. Analytical determination diameter and occur singly or in pairs; they are nonmotile, of buoyant density of DNA in acrylamide gels after preparative although flagella may be observed. Good growth occurs at a CsCl gradient centrifugation. FEBS Lett. 37:321-324. wide range of Na+ concentrations, from less than 0.05 M to 11. Rivard, C. J., and P. H. Smith. 1982. Isolation and characteri- more than 1 M. Optimal temperatures for growth are 37 to zation of a thermophilic marine methanogenic bacterium, Me- thanogenium thermophilicum sp. nov. Int. J. Syst. Bacteriol. 45°C. The cells have a protein cell wall and are sensitive to 32:430436. lysis by anionic detergents; cells stain gram negative. Or- 12. Romesser, J. A., R. S. Wolfe, F. Mayer, E. Spiess, and A. ganic growth factors are required. The G+C content of the Walther-Mauruchat. 1979. Methanogenium, a new genus of DNA is 47 to 54 mol%. These organisms are found in marine marine methanogenic bacteria, and characterization of Metha- and freshwater environments and exhibit various degrees of nogenium cariaci sp. nov. and Methanogenium marisnigri sp. halotolerance, but are not strongly halophilic. The type nov. Arch. Microbiol. 121:147-153. species is Methanogenium cariaci. 13. Schildkraut, C. R., J. Marmur, and P. Doty. 1962. Determina- Strains of Methanogenium cariaci sp. emend. are coccoid tion of the base composition of deoxyribonucleic acid from its methanogens which grow on H,-CO,, on formate, and buoyant density in CsC1. J. Mol. Biol. 4430443. 14. Widdel, F., P. E. Rouvihre, and R. S. Wolfe. 1988. Classification sometimes on secondary alcohols; acetate and yeast extract of secondary alcohol-utilizing methanogens including a new are required as growth factors. Cells are 1 to 3 Fm in thermophilic isolate. Arch. Microbiol. 150:477481. diameter and grow best at mesophilic temperatures (up to 15. Xun, L., D. R. Boone, and R. A. Mah. 1989. Deoxyribonucleic 45"C), neutral pH values, and Na+ concentrations at or acid study of Methanogenium and Methanocorpusculum spe- below about 1 M. The recognized habitat is marine sedi- cies, emendation of the genus Methanocorpusculum, and trans- ments. fer of Methanogenium aggregans to the genus Methanocorpus- culum, as Methanocorpusculum aggregans comb. nov. Int. J. ACKNOWLEDGMENTS Syst. Bacteriol. 39:lOP-lll. 16. Zabel, H. P., H. Konig, and J. Winter. 1984. Isolation and We thank Jane Boone for helpful discussions and Carl Woese and characterization of a new coccoid methanogen, Methanogenium Pierre Rouvi&re for discussions of sequence similarities of 16s tatii spec. nov. from a solfataric field on Mount Tatio. Arch. rRNAs. We also thank JosC Fiestas, Instituto de la Grasa y sus Microbiol. 137:308-315. Derivados, Seville, Spain, for help, including supplying the swine 17. Zabel, H. P., H. Konig, and J. Winter. 1985. Emended descrip- waste digestor samples. tion of Methanogenium thermophilicum, Rivard and Smith, and This study was supported by contract 5086-260-1303Eas part of a assignment of new isolates to this species. Syst. Appl. Micro- joint program on methane from biomass funded by the Gas Research biol. 6:72-78. Institute and the University of Florida Institute of Food and 18. Zellner, G., C. Alten, E. Stackebrandt, E. Conway de Macario, Agricultural Sciences and by Solar Energy Research Institute sub- and J. Winter. 1987. Isolation and characterization of Methano- contract XX-8-18079-1under Department of Energy prime contract corpusculum parvum, gen. nov., spec. nov., a new tungsten DE-AC02-83CH10093. requiring, coccoid methanogen. Arch. Microbiol. 147:13-20. 19. Zellner, G., E. Stackebrandt, P. Messner, B. J. Tindall, E. LITERATURE CITED Conway de Macario, H. Kneifel, U. B. Sleyter, and J. Winter. Boone, D. R., R. L. Johnson, and Y. Liu. 1989. Diffusion of the 1989. Methanocorpusculaceae fam. nov., represented by Me- interspecies electron carriers H, and formate in methanogenic thanocorpusculum parvum, Methanocorpusculum sinense ecosystems, and implications in the measurement of K, for H2 spec. nov., and Methanocorpusculum bavaricum spec. nov. or formate uptake. Appl. Environ. Microbiol. 55:1735-1741. Arch. Microbiol. 151 :3 81-390. Boone, D. R., and R. A. Mah. 1987. Effects of calcium, 20. Zellner, G., and J. Winter. 1987. Secondary alcohols as hydro- magnesium, pH, and extent of growth on the morphology of gen donors for C0,-reduction by methanogens. FEMS Micro- Methanosarcina mazei S-6.Appl. Environ. Microbiol. 54:1699- biol. Lett. 44:323-328. 1700. 21. Zhao, Y., D. R. Boone, R. A. Mah, J. E. Boone, and L. Xun. Boone, D. R., and W. B. Whitman. Minimal standards for the 1989. Isolation and characterization of Methanocorpusculum description of methanogenic bacteria. Int. J. Syst. Bacteriol. labreanum sp. nov. from the LaBrea Tar Pits. Int. J. Syst. 38:212-219. Bacteriol. 39:lO-13.