Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix S. H. Zinder, T. Anguish, and A. L. Lobo Department of Microbiology, Stocking Hall, Cornell University, Ithaca, NY 14853, USA
Abstract. An enrichment culture which converted acetate to methane at 60~ was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-I. Strain CALS-1 grew as filaments typically 2 - 5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct crosswalls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 gm long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase ceils tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-I, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60~ (doubling time = 2 4 - 2 6 h), with no growth occurring at 70 ~C and 37 ~C. The optimum pH value for growth was near 6.5 in bicarbonate/CO2 buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 m M phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 m M were inhibitory to the culture, and growth was more rapid with 1 m M 2-mercaptoethane sulfonate (coenzyme M) or I m M titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix. Key words: Methanogens - Acetate decarboxylation Methanothrix - Archaebacteria - Thermophiles
Two methanogenic genera, Methanosarcina and Methanothrix, are known to carry out the aceticlastic reaction in which acetate is decarboxylated to CI-I 4 and CO2. Unambiguous evidence for acetotrophy by pure Methanosarcina cultures was obtained in 1978 - 1980 (Mah et al. 1978; Smith and Mah 1980; Weimer and Zeikus 1979; Zinder and Mah 1979). A sheathed filamentous rod was enriched in anaerobic Offprint requests to: S. H. Zinder
acetate medium and originally named Methanobacterium soehngenii by H. A. Barker (Barker 1936) , and has since been often observed in anaerobic bioreactor sludges and acetate enrichments. The organism had eluded isolation until 1980 when Zehnder and his colleagues (Zehnder et al. 1980; Huser et al. 1982) isolated a culture using serial dilution and treatments with vancomycin, an inhibitor of eubacterial cell wall synthesis. The culture was named Methanothrix soehngenii (strain Opfikon), a generic assignment verified by 16S r R N A catalogue analysis, which showed closer affinity to Methanosarcina than to Methanobacterium (Stackebrandt et al. 1982). M. soehngenii grew as long sheathed filaments which often formed floc-like aggregates. Electron micrographs showed a striated subunit sheath similar to that of Methanospirillum hungatei, and disc-like cross walls between cells. The only substrate which supported growth was acetate, on which the doubling time was 4 - 7 days at the optimum temperature of 37~ More recently, Fathepure (1983) isolated M. soehngenii strain VNBF from an Indian sludge digestor which had similar properties to the Opfikon strain except that it appeared to grow faster. Patel (1984) isolated a mesophilic acetotroph, which he described as a new species Methanothrix concilii, on the basis of cultural characteristics and a higher value obtained for G + C ratio. The first thermophilic acetotrophic methanogen isolated was Methanosarcina sp. strain TM-1 (Zinder and Mah 1979), recently named Methanosarcina thermophita (Zinder et al. 1985). M. thermophila grew optimally near 50~ had a minimum doubling time on acetate of 12 h, significantly shorter than the mesophilic M. barkeri. Other thermophilic Methanosarcina sp. cultures have been described, some of which grow optimally near 55 ~C (Zinder et al. 1984b; Touzel et al. 1985). The first report of the existence of a thermophile resembling M. soehngenii was that of Nozhevnikova and Yagodina (1983) which described acetate enrichment cultured derived from geothermal waters and thermophilic bioreactors. The predominant organism was a gas-vacuolated sheathed rod, and cultures showed temperature optima of 6 0 - 6 4 ~ for methanogenesis from acetate. More recently, Nozhevnikova and Chudina (1985) examined an enrichment using electron microscopy and demonstrated a subunit sheath and cross walls similar to those in M. soehngenii, as well as the presence of gas vesicles. They also proposed the species epithet Methanothrix thermoacetophila, but did not claim to have an axenic culture. Ahring and Westermann (1984, 1985) isolated a thermophilic rod which grew on either acetate, H z - - C O z ,
316 or formate. The optimum temperature for methanogenesis from acetate was 60~ (doubling time = 3 days). Zinder et al. (1984a, b) described the presence of a gas-vacuolated sheathed rod resembling M. soehngenii in the thermophilic anaerobic bioreactor they were studying, and described an enrichment culture derived from that reactor. We describe here the isolation, from the enrichment previously described (Zinder et al. 1984a, b), of a thermophilic acetotrophic methanogen clearly belonging to the genus Methanothrix and describe some of its properties.
Materials and methods
Culture medium and conditions. Unless stated otherwise, the culture medium contained (g/l): NH4C1, 0.5; KzHPO4, 0.4; MgCI2 96 Ha0, 0.1; resazurin, 0.001 ; trace metal solution (Zeikus 1977 except that 0.02 g/1 NiCI2 96 H20 was added), 10 ml/1. The medium was either boiled under Nz (scrubbed of O2 by hot copper coils) or bubbled 20 rain with N2 to make it anoxic, and was transferred into an anaerobic glove box (Coy Laboratory Products, Ann Arbor, MI, USA) where it was dispensed into 120 ml serum vials which were sealed with ahiminum-crimp butyl rubber stoppers (Balch et al. 1979) purchased from Bellco Glass, Inc., Vineland, N J, USA. The vials were autoclaved at 121~ for 20 minutes. The vial headspaces were flushed with sterile O2-scrubbed 70% N2/30% CO2 (Matheson Gas Products, Peoria, IL, USA) after which Na-flushed syringes were used to add the following sterile anoxic solutions to the following final concentrations: NazS-9H20, 0.25 g/1 (ca. 1 raM); 2-mercaptoethane sulfonic acid, sodium salt (Sigma Chemical Corp., St. Louis, MO, USA), 1 mM; NaHCO3, 12raM; CaC12' 2H20, 0.1 g/l; sodium acetate, 40 raM; vitamin solution (Balch et al. 1979), 0.5ml/50ml. Cultures were routinely incubated at 60 ~C, and the pH of the medium at 60~ was 6.5. When required, cultures were grown in a similar fashion in 10 ml amounts in 27 ml butyl rubber serum stoppered tubes (Bellco), or in 200 ml amounts in rubber-stoppered 560 ml sidearm flasks (Bellco) or in 500 ml amounts in rubber stoppered one-liter bottles. Original enrichment cultures were grown in a similar medium except that it contained 0.5 g/1 cysteine HC1 (neutralized to pH 7) instead of I mM 2-mercaptoethane sulfonate and 0.1 g/1 yeast extract (Difco Laboratories, Detroit, MI, USA) instead of the vitamin solution. Brewer's fluid thioglycollate medium was purchased from Fisher Scientific, Rochester, NY, USA. Chemical and radiochemicat analyses. Gas samples were removed using 1 ml Glaspak syringes (Fisher Scientific) fitted with Mininert values (Supelco, Inc., Bellefonte, PA, USA) to maintain pressure. Methane and carbon dioxide were determined using a Gow-Mac 550 thermal conductivity gas chromatograph (Gow-Mac Inst. Co., Bound Brook, NJ, USA) using He as a carrier gas, and 14CH4and 14C02 were determined using a gas chromatograph-gas proportional counter as previously described (Zinder and Koch 1984). Aqueous acetate and formate concentrations were determined using high performance liquid chromatography (HPLC). An Altex l l 0 A HPLC pump and Knauer 71 refractive index detector were supplied by Rainin Inst. Inc., Woburn, MA, USA. A Bio-Rad HPX-87H organic acid analysis column (Bio-Rad Laboratories, Richmond, CA,
USA) was used for resolution of the components, the column was operated at room temperature, and the solvent used was 6.5 mM sulfuric acid as previously described (Zinder and Koch 1984). A 50 gl sample loop was used, and the detection limit was near 10 gM. Aqueous 14C-label was counted in ACS scintillant (Amersham, Arlington Hgts., IL, USA) with a Beckman LS-230 liquid scintillation counter (Beckman Instruments, Inc., Fullerton, CA, USA). Quench correction was by the external standard method.
Microscopy. A Zeiss Model 18 standard microscope (distributed by Micro Med Inst., Walden, NY, USA) equipped with a mercury burner for epifluorescence and an automatic camera for photography was used for phase-contrast and epifluorescence microscopy. Cells were immobilized for photography by using poly-lysine coated slides. A Zeiss 487704 filter combination and Planapochromat objectives were used for observation of F420 autofluorescence and the 487702 filter combination and Neofhiar objectives were used for autofluorescence due to methanopterin derivatives (F341). Cells prepared for negative staining were stained unfixed with 1% phosphotungstic acid. For thin sectioning, cells were fixed by a modified Ryter-Kellenberger procedure (Kellenberger et al. 1958) and the ethanol-dehydrated samples were imbedded in Spurr low-viscosity epoxy resin (Electron Microscopy Sciences, Ft. Washington, PA, USA), sectioned, and stained with 5% uranyl acetate for 15 min and 0.31% lead citrate for 5min. For samples in which we desired gas vesicles to be intact, cells were washed during the fixation procedure by filtration rather than by centrifugation. All preparations were examined with a Phillips 300 electron microscope operated at 80 kV. Images were recorded on Kodak type 4489 electron microscopy film. Cell mass determinations. Cell dry weights were determined on quadruplicate samples by filtering exponential-phase cultures through 0.2 gm Nucleopore filters as previously described (Zinder and Koch 1984). Culture optical densities were recorded at 600 nm using a Sequoia 340 digital spectrophotometer (Fisher Scientific). Temperature and p H optimum experiments. The specific growth rates for methanogenesis (#CH4) in temperature optimum experiments were calculated by measuring the doubling time for methanogenesis during the early exponential growth phase (CH~ = 1 - 5 retool/l). The pH of the medium was manipulated by removing either the 12 mM bicarbonate or 30% CO2 from the growth medium. Replacing the bicarbonate with an equimolar amount of sodium chloride resulted in a pH value of 6.1, while adding a small amount of HC1 to bicarbonate-free medium resulted in a pH value of 5.5. If CO2 was omitted from the headspace of the culture, the pH value was 8.0, and CO/partial pressures between 0 and 30% of the headspaces resulted in pH values between 6.5 and 8.0. A small amount of NaOH added to CO2-free medium increased the pH to 8.4. Methane readings were taken before the culture had produced 4 retool/1 CH4 (= 4 m M bicarbonate produced). In pH experiments performed using phosphate buffer, 20mM potassium phosphate pH 7.0 was added to the CO2/bicarbonate containing medium, and the pH was adjusted to different values using either HC1 or NaOH.
Chemicals and radiochemicals. All chemicals used were at least reagent grade. [l-~4C]sodium acetate (61.1 mCi/mmol) and [2-14C]sodium acetate (58.9 mCi/mmol) were obtained from Amersham Corp.
Isolation. A methanogenic enrichment culture of a rodshaped thermophile morphologically resembling Methanothrix soehngenii was obtained in liquid sodium acetate medium containing 0.5 g/l cysteine HC1 and 0.1 g/1 yeast extract. The enrichment was originally derived from a 10-7 most probable number (MPN) dilution of sludge from the thermophilic (58~ anaerobic bioreactor described by Zinder et al. (1984b). The enrichment culture stoichiometrically converted acetate to methane, and could be transferred indefinitely at 60~ in medium containing 40 mM sodium acetate, yeast extract, and cysteine as organic constituents. The enrichment did not contain cells resernbline Methanosarcina or a thermophilic acetate-oxidizing coculture (Zinder and Koch 1984). Colonies of the rod were only obtained in acetate agar roll tubes receiving a dilution of 10 2 or less. Therefore, initial attempts at isolation focused on treatment of the culture with 100 ~tg/ml vancomycin, followed by dilution into liquid medium, a technique used with success by Zehnder et al. (1980). It was found that addition of a vitamin solution (Balch et al. 1979) to the medium was required to allow growth of the culture in the presence of vancomycin and that yeast extract could be deleted from the medium. After two transfers of the culture in sodium acetate medium supplemented with vancomycin and vitamins, the number of bacteria with morphotypes different from Methanothrix was greatly redued, and serial 10-fold dilutions were made from this culture into liquid sodium acetate medium lacking vancomycin (triplicate tubes for each dilution). The highest dilution which showed growth was 10-7. A culture from a 10 - 7 dilution tube appeared microscopically pure, and no growth of contaminants was obtained in Brewer's thioglycolate medium or in basal liquid growth medium supplemented with 0.5 g/1 yeast extract plus 0.25 g/1 each of glucose, sucrose, cellobiose and xylose, or supplemented with 0.2 g/1 yeast extract plus 10 mM each sodium lactate and sodium sulfate. Only the Methanothrix morphotype grew in sodium acetate medium supplemented with I0 mM sodium sulfate. However, an organism resembling Methanobacterium thermoautotrophicum grew upon inoculation of the culture into basal medium with 80% H2/20% COz added to the headspace. A further 10-7 dilution of the culture eliminated the hydrogenotrophic methanogen, and no growth was obtained in the other media used to check purity. At this point the culture was deemed pure and routinely transferred in medium lacking vancomycin. It was given the designation Methanothrix sp. strain CALS-I. Morphology. Cells of Methanothrix sp. strain CALS-I were rod-shaped with square ends (Fig. 1 a, b). The cell diameter was 1 . 0 - 1 . 2 gm, and the shortest filaments were about 5 ~tm long, similar to M. soehngenii. In contrast to M. soehngenii (Huser et al. 1984), Methanothrix sp. strain CALS-I formed relatively short filaments, generally about 2 - 5 cell units long. Long filaments were rare, and culture showed opales-
cent turbidity rather than clumps. No autofluorescence was detected in cells examined by epifluorescence microscopy and excited with either 420 nm or 350 nm radiation similar to other Methanothrix cultures, but different from most other methanogens, which are autofluorescent. Motility was never observed in the culture. Negative stain (Fig. lc) and thin-section (Fig. I d - f ) electron micrographs revealed a striated sheath surrounding the cells with the striations occurring every 1 2 - 1 3 nm. The cells were separated by cross-walls (Fig. I e). There were often two crosswalls between cells, and occasionally cytoplasm between the crosswalls (micrographs not presented), similar to M. soehngenii. There was also a layer directly outside cell membrane and inside the sheath (Fig. I d). Similar to the original enrichment culture, Methanothrix sp. strain CALS-1 showed phase-bright granules (Fig. I a) which were rendered phase-dark by hydrostatic pressure (Fig. 1 b), the presumptive test for gas vesicles (Walsby 1975). Rapid pressurization of a culture to 200 kPa (ca. 2 atm) overpressure did not cause noticeable loss of the granules, while pressurization to 300 kPa caused partial loss and 400 kPa caused nearly total loss. The morphology of the gas vesicles is shown in Fig. 1 c and If. The average diameter of the vesicles was 70 nm and lengths of up to 0.5 ~tm were seen. Cultures of Methanothrix sp. strain CALS-1 tended to have more gas vesicles in stationary-phase than during growth. A band of cells floating to the top of the liquid was occasionally observed in stationary-phase cultures. These bands floated back to the top within a few hours after perturbation (S. Zinder, personal observation).
Growth. Methanothrix sp. strain CALS-I converted 40 mM sodium acetate stoichiometrically to methane (Fig. 2) when grown at 60 ~C, and 100 mM sodium acetate was converted to methane after a 1 - 3 day lag (data not presented). When a culture was supplied with 1,375 kdpm 14CH3COO- and allowed to grow out completely, 1,125 kdpm 14CH4 and 28 kdpm 14CO2 were measured, while after addition of 900kdpm CH3t4COO -, 780kdpm 14COz and 1 kdpm ~CH4 were measured, indicating that methanogenesis from acetate was via the aceticlastic reaction. Growth and methanogenesis parallelled each other (Fig. 2) and the doubling time (Td) for methanogenesis from acetate at 60 ~C on days 4 - 7 in Fig. 5 was calculated to be 24 h, while that for increase in optical density for the same period was 26 h indicating that growth and methanogenesis were tightly coupled. The growth yield measured was 1.1 __ 0.1 g dry weight per tool CH4 produced from acetate. Strain CALS-I required biotin for growth (Fig. 3A). Cultures which did not receive biotin did not grow after more than one transfer. The culture was originally grown with 1 mM Na2S and either 1 mM or 3.5 mM cysteine as reducing agents and the Td was near 36 h (Fig. 3A). It was found that replacing cysteine with 1 mM 2-mercaptoethane sulfonate (HS-Coenzyme M) allowed more rapid growth with a Td near 24 h (Fig. 3A). Increasing the sulfide to 2 mM caused the culture to lag several days (Fig. 3 B), as did decreasing the HS-Coenzyme M concentration to 0.1 raM, and the culture did not grow when sulfide was the only reductant (Fig. 3 B). HS-Coenzyme M and cysteine apparently served primarily as reductants and not as necessary nutrients (i.e. sulfur sources) since replacing them with I mM titanium(liD citrate (Zehnder and Wuhrmann 1976)
Fig. 1. a Phase-contrast micrograph of cells from an early stationary-phase culture (concentrated by filtration) of Methanothrix sp. strain CALS-1. Marker bar represents 10 gin. b Phase-contrast micrograph of cells from the same culture after exposure to hydrostatic pressure by placing the cell suspension in a syringe, inserting the syringe needle tip into a rubber stopper, and tapping briskly on the syringe plunger. Magnification the same as a. c Negative stain electron micrograph of a filament of Methanothrix sp. strain CALS-1 along side an empty sheath. Note striations (S) in the sheath, the break (B) in the empty sheath, the cross-wall (CW), the protoplasmic cylinder (PC) within the sheath, and gas vesicles (GV). Gas vesicles which have their long axis close to perpendicular to the plane of the photograph appear American football-shaped. Marker bar represents 1 gm. d Thin-section electron micrograph of Methanothrix sp. strain CALS-1 displaying the cell envelope including sheath (SH), outer layer (OL) and cell membrane (CM). Marker bar = 0.2 jxM. e Thin-section electron micrograph of Methanothrix sp. strain CALS-I displaying partially formed cross-wall. Marker bar = 0.2 ~tM. f Thin-section electron micrograph of Methanothrix sp. strain CALS-1 displaying gas vesicles in a honey-comb like arrangement and lengthwise. Cells were prepared without centrifugation to preserve intact gas vesicles. Marker bar = 1 ~tM
,,,, CH4 1 mM Cysteine / ' ~ ~ 10
Td = 24 h /
E E 10
0.1 ,_,o0 0
Td = 26 h
Fig. 2. Methanogenesis (0) and growth (9 (measured as optical measured density of cultures at 600 nm) of Methanothrix sp. strain CALS-1 at 60~ Cultures were grown in 200 ml growth medium in 560 ml sidearm flasks (sidearm diameter = 19 ram). The growth medium contained 40 mM sodium acetate reduced with 1 mM NazS/l I mM HS-Coenzyme M as described in Materials and methods allowed rapid growth (Fig. 3B). Titanium citrate was not generally used because of formation of a precipitate in the medium during growth. 2-Mercaptoethanol has been used with success as a reducing agent in methanogenic cultures (Bhatnager et al. 1984), but concentrations as low as 1 m M completely inhibited growth of Methanothrix sp. strain CALS-1 when added in conjunction with 1 m M sulfide or with sulfide and 1 m M cysteine (data not presented). Acetate was the only compound tested which supported growth and methanogenesis by strain CALS-1. Substrates tested which did not support methanogenesis included 80% H 2 - 2 0 % CO2, 24 mM methanol, 24 m M methanol plus H 2 - C O 2 , 10 mM methylamine, 10 m M methylamine plus H 2 - C O a , 10 m M trimethylamine, 10 mM trimethylamine plus H 2 - C O z , 2 0 r a M sodium formate, and 1 0 m M ethanol. H2 - CO2 did not inhibit methanogenesis from acetate (manuscript in preparation). Formate (5 mM) was not consumed by cells during or after growth on 40 m M acetate, nor was more than 0.01 retool/1 H2 produced by cells grown in the presence of formate. The optimum temperature for methanogenesis by Methanothrix sp. strain CALS-1 was near 60~ with no methanogenesis or growth detected in cultures incubated 30 days at 37~ or 70~ (Fig. 4). The optimum pH value was found to be near 6.5 (Fig. 5). In these experiments, the pH of the growth medium was varied by manipulating the bicarbonate/CO2 equilibrium. If a 20 m M phosphate buffer system was used instead, no growth was obtained below pH 7, presumably due to phosphate toxicity at lower pH values. Utilization of acetate by washed cells of Methanothrix sp. strain CALS-1 was linear when the concentration was greater than 1 mM (Fig. 6) indicating that the apparent K~ for acetate utilization was below 1 mM. Discussion The structural and physiological similarities between the culture described here and M. soehngenii as described by
1 rnM CoM+ 1 mMNa2S
' mM" sx. /
" ~ mM N ~ s
1 mM Na2S f .x~,
Fig. 3. A Effects of biotin, cysteine, and HS-Coenzyme M on methanogenesis by Methanothrix sp. strain CALS-1. All cultures were grown in serum vials containing 50 ml medium, all vials received I mM Na2S, and incubation was at 60~C. B Effects of various reducing agents on methanogenesis by Methanothrix sp. strain CALS-1
Zehnder and his colleagues (Zehnder et al. 1980; Huser et al. 1982), clearly warrants its inclusion in the genus Methanothrix. The only significant structural differences between M. soehngenii and Methanothrix sp. strain CALS-I were that strain CALS-I grew as shorter filaments and contained gas vesicles. To our knowledge, Methanothrix sp. strain CALS-1, along with the Russian Methanothrix enrichments (Nozhevnikova and Chudina 1985), is the first thermophile reported to contain gas vesicles. Gas vesicles have been reported in other archaebacteria including mesophilic Methanosarcina barkeri strains (Zhilina and Zavarzin 1979),
320 0.025 ,4
0.020 3 "-~ '32
Temperature (" C)
Fig. 4. Effects of temperature on the specific rate of methanogenesis by Methanothrix sp. strain CALS-I grown in NazS/cysteine reduced medium
Fig. 5. Effect of medium pH on the specific rate of methanogenesis (g CH~ = 0.69/doubling time) by Methanothrix sp. strain CALS-1 grown in medium in which the pH value was changed by manipulating the bicarbonate/CO2 equilibrium in NazS/cysteine reduced medium as described in Materials and methods and in the halobacteria (Walsby 1975). No mention is made of gas vesicles in any descriptions of mesophilic Methanothrix cultures, which does not preclude their existence in mesophilic strains. All of our enrichments from the bioreactor had gas vesicles, as did the enrichments of Nozhevnikova and Yagodina (1983), suggesting that gas vesicles are common in thermophilic Methanothrix strains. The only verified function of gas vesicles is in floatation (Walsby 1975). Our findings that cells of strain CALS-1 had more vesicles at the end of growth in batch culture suggests that floatation may be a response to nutrient deprivation in the thermal anaerobic environment in which strain CALS-1 evolved. Gas vesicles could not be used for floatation in the completely mixed (250 rpm) anaerobic bioreactor from
Fig. 6. Utilization of acetate by washed concentrated (t0 • ) cells of Methanothrix sp. strain CALS-1. Growing cells were washed by centrifugation in an anaerobic glove box and were resusupended in growth medium buffered at pH 6.5 with 25 mM BES (N,N-bis[2hydroxyethyl]-2-aminoethane sulfonate)
which strain CALS-1 was isolated, yet cells resembling Methanothrix sp. in the sludge often had large numbers of gas vesicles (Zinder et al. 1984 b). The cardinal temperatures for growth of Methanothrix sp. strain CALS-1 were about 20~ higher than those mesophilic strains (Huser et al. 1982; Fathepure 1983; Patel 1984). The enrichments described by Nozhevnikova and Yagodina (1983) grew optimally at 6 0 - 6 5 0 C and did not grow at 70 ~C, similar to strain CALS-1. The doubling time for strain CALS-1 ( 2 4 - 26 h) was considerably less than that reported for M. soehngenii strain Opfikon, which is often the case when comparing mesophilic and thermophilic strains of methanogens. A 24 h doubling time was claimed for M. concilii (Patel 1984), but cultures required 28 days for complete growth, similar to the Opfikon strain. Cultures of M. soehngenii, strain VNBF (Fathepure 1984) required only 15 days to grow and may represent a faster growing mesophilic Methanothrix. Similar to other described Methanothrix cultures, strain CALS-1 only used acetate as a methanogenic substrate, carried out the aceticlastic reaction, and showed a high affinity for acetate uptake. While M. soehngenii strains Opfikon and VNBF split formate to H2 and CO2, strain CALS-1 did not. Strain CALS-I required biotin for growth. Both M. soehngenii strain Opfikon and M. concilii were reported to require a vitamin mixture (Huser et al. 1982; Patel 1984), and M. soehngenii strain VNBF was reported to require yeast extract (Fathepure 1984). Biotin has been shown to be required by Mehylococcoides methylutens (Sowers and Ferry 1985) and was stimulatory to Methanosphaera stadtmanae (Miller and Wolin 1985). In our initial attempts to isolate strain CALS-I, we found that vancomycin inhibited growth of the Methanothrix enrichment in medium containing 0.1 g/1 yeast extract but that addition of a vitamin solution allowed growth of the enrichment. From an analysis of a typical batch of yeast extract (Rohde 1968), it would be expected that 0.1 g/1 yeast extract would provide 0.4 ~tg/1 biotin, while the vitamin solution
321 used would provide 20 lag/1 biotin. It is likely that vancomycin inhibited the growth of eubacteria in the enrichment which provided additional biotin thereby allowing growth of the Methanothrix cells. Methanothrix sp. strain C A L S - I showed a fairly broad pH range, being able to grow at values between p H 6 and pH 8 in bicarbonate/CO2 buffered medium. The bioreactor from which strain CALS-1 was isolated from had a p H value of 6.3 (Zinder et al. 1984b) in accord with these findings. M. soehngenii strsin Opfikon was reported not to grow at p H values below 6.8 (Huser et al. 1982). These experiments were performed using 20 m M phosphate buffer, and we found that strain C A L S - I did not grow below p H 7 when phosphate buffer was used, Recent experiments by Brumreeler et al. (1985) on a mesophilic sludge blanket reactor dominated by Methanothrix showed that the p H optimum for methanogenesis from acetate in the low-phosphate medium used was 6.6-6.8, similar to that in strain CALS-J. Methanothrix sp. stain C A L S - I is similar or identical with the methanogen in enrichments described by Nozhenikova and Chudina (1985). They proposed the species name Methanothrix thermoacetophila, but a pure culture is required for taxonomic assignment. There are significant differences between strain C A L S - I and the thermophilic rod-shaped acetotrophic methanogen, called TAM organism, isolated by Ahring and Westermann (1984, 1985). While the sheath outer layer of strain CALS-1 and other Methanothrix cultures is straight and is of constant diameter, the outer layer in TAM organism is wavy and constricts near division planes. The culture grew more slowly on acetate (Td = 3 days) and had a higher growth yield ( 1 . 7 - 2 . 3 g dry wt/mol acetate) than did strain CALS-I. Most importantly, the TAM organism was reported to grow on either H2/ CO2 or formate, substrates not used by presently known Methanothrix cultures. It is likely that the TAM organism belongs to a different or novel genus. For a valid species name for Methanothrix sp. strain CALS-1, a value for the G + C ratio of its D N A must be obtained, and a catalogue or sequence of its 16S r R N A would be desirable. However, cultures o f strain CALS-1 have not yielded sufficient quantities of either intact D N A (S. Zinder, unpublished work) or r R N A (W. Whitman, personal communication) to allow these procedures. However, preliminary results by E. Conway de Macario and A. Macario (personal comlrmnication) indicate that there is poor immunological cross reaction between M. soehngenii strain Opfikon, and Methanothrix sp. strain CALS-1, supporting the idea that strain CALS-1 should become a separate species in the genus Methanothrix.
Acknowledgements. This research was supported by U.S. Department of Energy contract no DE-AC02-81 ER10872 and grant no DE-FG02-85ER13370. A. L. Lobo was partially supported by U. S. D.A. Hatch funds. We thank W. C. Ghiorse and J. Kemp for invaluable help with the electron microscopy.
References Ahring BK, Westermann P (1984) Isolation and characterization of a thermophilic, acetate-utilizing methanogenic bacterium FEMS Microbiol Lett 25 : 47 - 52 Ahring BK, Westermann P (1985) Methanogenesis from acetate: physiology of a thermophilic acetate-utilizing methanogenic bacterium. FEMS Microbiol Lett 28: 15-- 19
Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43: 260- 296 Barker HA (1936) Studies upon the methane-producing bacteria. Arch Microbiol 7 : 4 2 0 - 438 Bhatnagar L, Henriquet M, Zeikus JG, Aubert JP (1984) Utilization of mercapto-2-ethanol as a medium reductant for determination of the metabolic response of methanogens towards inorganic sulfur compounds. FEMS Microbiol Lett 22:155 - 158 Brummeler E ten, Pol LWJ, Dolfing J, Lettinga G, Zehnder AJB (1985) Methanogenesis in an upfiow anaerobic sludge blanket reactor at pH 6 on an acetate-propionate mixture. Appl Environ Microbiol 49:1472-1477 Fathepure BZ (1983) Isolation and characterization of an aceticlastic methanogen from a biogas digester. FEMS Microbiol Lett 19:151 - 156 Huser BE, Wuhrmann K, Zehnder AJB (1982) Methanothrix soehngenii gen. nov. sp. nov., a new acetotrophic non-hydrogenoxidizing methane bacterium. Arch Microbiol 132:1-9 Kellenberger E, Ryter A, Sechaud J (1958) Electron microscope study of DNA plasms. J Biophys Biochim Cytol 4:671-678 Mah RA, Smith MR, Baresi L (1978) Studies on an acetate fermenting strain of Methanosarcina. Appl Environ Microbiol 35:1174-1184 Miller TL, Wolin MJ (1985) Methanosphaera stadtmaniae gen. nov. sp. nov.: a species that forms methane by reducing methanol with hydrogen. Arch Microbiol 141 : 116-122 Nozhevnikova AN, Chudina VI (1985) Morphology of the thermophilic acetate methane bacterium Methanothrix thermoacetophila sp. nov. Microbiol 53:618- 624 Nozhevnikova AN, Yagodina TG (1983) A thermophilic acetate methane-producing bacterium. Microbiol 51:534--541 Patel GB (1984) Characterizaton and nutritional properties of Methanothrix concilii sp. nov., a mesophilic aceticlastic methanogen. Can J Microbiol 30:1383-1396 Rohde PA (ed) (1968) BBL Manual of products and laboratory procedures. BioQuest, Division of Becton, Dickenson and Co., Cockeysville, MD Smith MR, Mah RA (1980) Acetate as sole carbon and energy source for growth of Methanosarcina strain 227. Appl Environ Microbiol 39 : 993 - 999 Sowers KR, Ferry JG (1985) Trace metal and vitamin requirements of Methanococcoides methylutens grown with trimethylamine. Arch Microbiol 142:148-151 Stackebrandt E, Seewaldt E, Ludwig W, Schleifer K-H, Husar BA (1982) The phylogenetic position of Methanothrix soehngenii elucidated by a modified technique of sequencing oligonucleotides from 16S rRNA. Zentbl Bakt Hyg I Abt Orig C 3 : 9 0 100 Touzel JP, Petroff D, Albagnac G (1985) Isolation and characterization of a new thermophiiic Methanosareina, the strain CHTI 55. Syst. Appl Microbiol 6:66-71 Walsby AH (1975) Gas vesicles. Ann Rev P1 Physiol 26:427439 Weimer PJ, Zeikus JG (1978) Acetate metabolism in Methanosarcina barkeri. Arch Microbiol 119 : 175 - 182 Zehnder AJB, Wuhrmann K (1976) Titanium (III) citrate as a nontoxic oxidation reduction buffering system for the culture of obligate anaerobes. Science 194:1165 - 1166 Zehnder AJB, Huser BA, Brock TD, Wuhrmann K (1980) Characterization of an acetate-decarboxylating, non-hydrogenoxidizing methane bacterium. Arch Microbiol 124:1 - 11 Zeikus JG (1977) The biology of methanogenic bacteria. Bacteriol Rev 41:514-541 Zhilina TN, Zavarzin TA (1979) Comparative cytology of methanosarcinae and description of Methanosarcina vacuolata sp. nov. Microbiol 48 : 279 - 285 Zinder SH, Koch M (1984) Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture. Arch Microbiol 138 : 263 - 272
322 Zinder SH, Mah RA (1979) Isolation and characterization of a thermophilic strain of Methanothrix unable to use H 2 - C O 2 for methanogenesis. Appl Environ Microbiol 38: 996-1008 Zinder SH, Anguish T, Cardwell SC (1984a) Effects of temperature on methanogenesis in a thermophilic (58 ~C) anaerobic digestor. Appl Environ Microbiol 47: 808 - 813 Zinder SH, Cardwell SC, Anguish T, Lee M, Koch M (1984b) Methanogenesis in a thermophilic anaerobic digestor: Metha-
Note added in proof
Methanothrix sp. strain CALS-1 has been deposited in the Deutsche Sammlung yon Mikroorganismen (DSM No. 3870).
nothrix sp. as an important aceticlastic methanogen. Appl Environ Microbiol 47: 7 9 6 - 807 Zinder SH, Sowers KR, Ferry JG (1985) Methanosarcina thermophila sp. nov., a thermophilic acetotrophic methane-producing bacterium. Int J Syst Bacteriol 35: 5 2 2 - 523 Received July 21, 1986/Accepted September 1, 1986