Isolation and Characterization of Methanobacterium Espanolae Sp

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INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1990, p. 12-18 Vol. 40, No. 1 0020-7713/90/010012-07$02.00/0 Copyright 0 1990, International Union of Microbiological Societies

Isolation and Characterization of Methanobacterium espanolae sp. nov. , a Mesophilic, Moderately Acidiphilic Methanogen? G. B. PATEL,l* G. D. SPROTT,l AND J. E. FEIN2 Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada, KlA OR6,l and Diversified Research Laboratories Ltd., Toronto, Ontario, Canada, M4 W 2L2,

Bacterial strain GnT (T = type strain), a nonmotile, nonsporeforming, mesophilic, methanogenic bacterium, was isolated from the primary sludge obtained from the waste treatment facility of a major kraft pulp mill in Canada. Single cells were 6.0 by 0.8 pm and stained gram positive. Growth and methane production occurred only with H,-CO, as the substrate. Acetate, formate, propionate, butyrate, pyruvate, methanol, or trimethylamine could not serve as a sole source of carbon and energy for growth. The optimum pH for growth was between 5.6 and 6.2; consistent growth and methane production were not observed below pH 4.68. The optimum temperature for growth was 35"C, and little or no growth was observed during incubation at 15 and 50°C. Kanamycin and bacitracin were severe inhibitors of growth and methanogenesis, whereas 100 pM bromoethanesulfonicacid caused 30 % inhibition. Supernatantfrom primary sludge enhanced growth by about 10%. The DNA base composition was 34 mol% guanine plus cytosine. On the basis of physiological characteristics, indirect immunofluorescence typing, and DNA-DNA hybridization studies, the isolate is named Methanobacterium espanolae sp. nov.

The optimum pH for growth of most pure cultures of mg of vitamin B12. The pH of the mixed ingredients (except methanogenic bacteria isolated to date lies between 6.0 and cysteine-Na,S and Na,CO,) was adjusted to 4.5 with 5 N 8.0, with the majority of cultures having an optimum close to HC1. The medium was then reduced with cysteine-Na,S, pH 7.0 (23,25). Methanogenic bacteria with optimum growth Na,CO, was added, and 10-ml portions were dispensed into pH values greater than 8.0 have been reported recently (27). 60-ml serum vials under 80% H2-20% CO, (5). The postau- Although methanogenic activity has been detected in acidic toclaved (121"C, 15 min) pH of the medium was 5.5 k 0.1. ecosystems, such as bogs and peats (11,24,25), at pH values Similarily, SA medium with an initial pH of 5.0 and S close to 4.0, methanogenic isolates from these ecosystems medium (the same as SA medium but without acetic acid) have been unable to grow at pH values below 5.3 (25). with pH values of 5.5 and 5.0 were prepared by appropriate Moreover, no pure culture of a methanogen that is able to pH adjustment before reduction of the media. Unless stated grow at pH values below 5.0 has been reported previously, otherwise, all tests were done in duplicate, with 10-ml to our knowledge. The isolation and study of acid-tolerant portions of media in 60-ml serum vials, and were repeated at methanogens was undertaken as part of a program on least once. The averages of duplicate tests are reported biomass degradation in anaerobic, acidic environments, with below. the purpose of extending the potential of anaerobic treat- Isolation, stock cultures, and inoculum. A 10-ml sludge ment for disposal of acidic, organic, industrial wastes. In this sample that was collected anaerobically from the anoxic paper we describe the isolation and characterization of a zone of the primary sludge of the E. B. Eddy Forest methanogen that is able to grow and produce methane at pH Products Ltd. (Espanola, Ontario, Canada) bleach kraft mill values below 5.0. was put into a 60-ml serum vial under 100% NZ. The cellulolytic and methanogenic activity of this mixed culture MATERIALS AND METHODS was maintained (incubation temperature, 35°C) by transferring it (lo%, vol/vol) to fresh primary sludge (under N,, Media. The composition of SA medium, which was used supplemented with cysteine-Na,S and 5 mM NH,Cl) every 6 for isolation and stock culture maintenance, was as de- weeks. This primary sludge enrichment culture was inocu- scribed previously (5) except for an increase in the cysteine- lated into S and SA media (80% H,-20% CO,) at pH 5.0 and Na,S concentration. This medium contained (per liter) 2,500 5.5. After we detected large quantities (lo%, vol/vol) of CH, mg of CH,COONa - 3H@, 480 mg of Na,CO,, 450 mg of gas in a headspace gas analysis, the methanogenic cultures (NH,),SO,, 290 mg of K,HPO,, 180 mg of KH,PO,, 250 mg were maintained in similar media by transferring them (10% of cysteine hydrochloride, 250 mg of Na,S - 9H,O, 120 mg of vol/vol) at 1-week intervals. Such a culture from SA medium MgSO, . 7H@, 60 mg of CaC1, . 2H,O, 54 mg of NaCl, 21 (pH 5.0) was serially diluted into SA broth (pH 5.0) and mg of FeSO, . 7H,O, 15 mg of N (CH,COOH),, 5 mg of plated onto SA agar (pH 5.5) (SA medium supplemented MnSO,.H,O, 1 mg of CoCl2.6H,O, 1 mg of with 2.2% [wt/vol] Noble agar [Difco Laboratories, Detroit, ZnSO, . 7H,O,1 mg of resazurin, 0.1 mg of CuSO, . 5H,O, Mich.]). The agar medium was prepared just as the broth 0.1 mg of AlK(SO,), . 12H,O, 0.1 mg of H,BO,, 0.1 mg of medium was, but the prereduced and autoclaved medium Na,MoO, . 2H,O, 0.1 mg of pyridoxine hydrochloride, 0.05 (20-ml portions in 60-ml vials) was poured into petri plates mg of thiamine hydrochloride, 0.05 mg of riboflavin, 0.05 mg inside an anaerobic chamber (Coy Manufacturing Co., Ann of nicotinic acid, 0.05 mg of p-aminobenzoic acid, 0.05 mg of Arbor, Mich.) containing a 5% CO,-lO% H2-85% N, atmo- lipoic acid, 0.02 mg of biotin, 0.02 mg of folic acid, and 0.05 sphere. After overnight drying in the chamber, the plates were streaked, introduced into Brewer anaerobic jars which * Corresponding author. were then flushed out with 80% H,-20% CO,, and incubated t National Research Council of Canada paper 30887. at 35°C. Representative colonies from agar plates were

12 VOL. 40, 1990 METHANOBACTERIUM ESPANOLAE SP. NOV. 13 transferred into vials containing SA broth (pH 5.5) inside the captoethanesulfonic acid, and glutathione (each at a concen- anaerobic chamber. Methanogenic broth cultures were seri- tration of 5 mM) or Na,S (1 mM) to serve as a sole source of ally diluted and plated for colony picking. This procedure sulfur for the growth of strain GP9T (T = type strain) was was repeated several times until culture purity was estab- determined as described previously (19) by using appropri- lished. ately modified SA medium (pH 5.5) reduced with 5 ml of Stock cultures of methanogenic isolates were maintained titanium (111) citrate per liter (0.40 mM Ti3+). in SA broth at pH 5.5 and 5.0 by transferring them (lo%, The vitamin requirements of strain GBTwere determined voYvol) every 7 to 10 days into fresh media at the appropri- by comparing growth and CH, production in SA medium ate initial pH. The stock culture vials were repressurized (pH 5.5) containing all of the vitamins (see above) with every 4 days by injecting 80% H,-20% CO,. growth and CH, production in vitamin-free medium. Unless stated otherwise, the inoculum for tests consisted Inhibitors and enhancers of growth. The effects of antibi- of 1% (voVvol) of a 1-week-old culture on SA medium (pH otics and other potential inhibitors and enhancers of growth 5.5) which was anaerobically and aseptically concentrated 10 were studied by supplementing SA broth (pH 5.5) (gas times into the appropriate medium to avoid carry-over of the phase, 80% H,-20% CO,) with filter-sterilized, anaerobic nutrients or compounds under investigation. All incubations (stored under N,) stock solutions of test compounds. Meth- were static, except when indicated otherwise. ane production was assayed weekly, and A660 was deter- Temperature. The optimum temperature for growth and mined after 3 weeks of incubation at 35°C. The vial head- methane production was determined in SA broth at pH 5.5 space was repressurized twice a week and flushed out once by incubating preparations at 15 to 55°C. Methane produc- a week. tion was monitored at 1- to 4-day intervals as described DNA base composition. Cells from the mid-exponential previously (17). The vial headspace was repressurized (69 growth phase in SA medium (pH 5.5) were harvested, kPa) or flushed out (3 min each) and repressurized with 80% washed with 10 mM saline-EDTA (pH 7.0), and lysed by H2-20% C02 after every 2 to 4 days of incubation. This repeatedly freezing and thawing in liquid nitrogen. The DNA procedure assured adequate substrate availability and was from the lysate was extracted and purified by using the necessary to maintain a constant pH. The pH was measured method of Marmur (16). The DNA base composition was initially, at 7 days, and at the end of incubation. The specific determined by the thermal denaturation (T,) procedure of growth rate at each incubation temperature was calculated Mandel et al. (15), using the De Ley method (6) for calcu- from the steepest slope of methane production (plotted on a lating the G+C content. DNA isolated from Pichia kluyveri semilogarithmic scale) between 3 and 16 days of incubation. (G+C content, 30.0 mol%) was used as the standard. pH. The pH requirements for growth at 35°C were deter- Immunological fingerprinting. The antigenic fingerprint of mined from specific growth rates based on CH, production, strain GP9T was determined by using antibody S probes (13) as described above for temperature studies, by using SA of 10 methanogens. The antigenic relatedness of strain GP9T broth (20 ml per 160-ml serum vial) adjusted to various pH to the 10 reference cultures was determined by quantitative values. Growth at the midpoint and growth at the end of comparison of antigenic fingerprints as described by Macario incubation were monitored by measuring the A,, (1-cm light and Conway de Macario (14). path) of aseptically withdrawn samples of cultures. The Cell protein analyses. Whole-cell and cell surface proteins H,-CO, was replenished and the methane was analyzed as of strain GP9T were compared with those of Methanobacte- described above for the temperature studies. The pH in the rium bryantii M.o.H.~(= DSM 863T = NRC 2152T) and test vials was verified anaerobically at the onset and at the M.0.H.G (= DSM 862) by sodium dodecyl sulfate (SDS)- conclusion of the experiment by using a specialized glass polyacrylamide gel electrophoresis. Strain GP9T was culti- vessel (18) and after 4 days by a drop assay in which we used vated to the mid-exponential growth phase in SA medium a Cardy C-1 pH meter (Horiba Instruments, Inc., Irvine, (pH 5.5). Similarily, strains M.o.H.~and M.0.H.G were Calif.). The drop assay was also used to measure the pH cultivated in SA medium (pH 7.0). Washed cells of each before and after the H,-CO, in the vials was replenished. strain were suspended in deionized distilled water and lysed The pH was maintained at 20.1 U during incubation. by two passages through a French pressure cell (69,000 kPa). Growth substrates. The ability of some of the generally Debris was removed by centrifugation (16,000 X g, 5 min), reported methanogenic substrates (concentration, 10 mM) to and samples of the supernatants were then subjected to support growth was determined in SA broth (pH 5.5) pre- SDS-polyacrylamide gel electrophoresis by using conditions pared under 80% N,-20% CO,. Test substrates were filter described previously (22). The gels were stained by using the sterilized, stored under N,, and added to autoclaved media silver reagent (Bio-Rad Laboratories Ltd., Mississauga, via syringe injection. Methane production was determined Ontario, Canada). The standard proteins used were bovine after 1 and 3 weeks of incubation, and A660 was determined albumin, ovalbumin, carbonic anhydrase, soybean trypsin after 3 weeks of incubation. The suitability of 2-propanol(lO inhibitor, and lysozme. For strains GP9T, M.o.H.~,and mM) or 2-butanol(lO mM) to serve as a hydrogen donor was M.0.H.G the amounts of protein loaded onto the lanes were determined after 1 week of incubation in SA medium (pH 14.3, 14.6, and 16.7 pg, respectively. 5.5) (N,-C0,gas phase). For comparison of surface proteins, washed whole cells of Incorporation of formate into cell carbon, methane, and the strains (110 pg [cell dry weight] of strain GP9T; 137 pg CO, during growth on H,-CO, was determined in 10 ml of [cell dry weight] of strain M.o.H.~;102 pg [cell dry weight] SA medium (pH 5.5) supplemented with 10 mM cold sodium of strain M.0.H.G) were suspended in 1 ml of the sample formate and 5 pCi (1 Ci = 37 GBq) of H14COONa (New loading buffer (see above) (0.031 M Tris-hydrochloride [pH England Nuclear Corp., Boston, Mass.). Incorporation of 6.81, 12.5% glycerol, 0.625% [wt/vol] SDS, 0.625% 2-mer- the label into cell biomass, CH,, and CO, in test vials and in captoethanol, 0.0006% bromophenol blue). The suspension control vials inoculated with heat-killed cells was deter- was heated for 10 min at 110°C. No lysis occurred as mined by using the methods described previously (17). determined by phase-contrast microscopic examination. Sulfur sources and vitamin requirements. The ability of Samples (50 1.1) of supernatant from a 16,000-X-g centrifu- cysteine hydrochloride, thioglycolate, methionine, 2-mer- gation of a heated suspension, which represented surface 14 PATEL ET AL. INT. J. SYST. BACTERIOL. proteins, were analyzed by SDS-polyacrylamidegel electrophoresis as described above. Protein was measured by using the dye binding assay of Peterson (20) and bovine serum albumin as the standard. DNA-DNA homology. Cells of strain GP9T in the mid- exponential growth phase were harvested from SA medium (pH 5.5) and lysed by two passages through a French pressure cell (69,000 kPa). The DNA was extracted and purified by the Marmur (16) method. DNAs from Methano- bacterium bryantii M.o.H.~and M.0.H.G grown to the mid-exponential growth phase in SA medium (pH 7.0) were similarly purified. 32P-labeledDNAs from strains M.o.H.~ FIG. 1. Phase-contrast microscopy of strain GBT. Bar = 4 pm. and M.0.H.G were purified from cultures grown in SA medium modified to contain a lowered phosphate content (0.1 mM) and supplemented with dipotassium hydrogen 32Pi Cell lysis. Cells of strain GP9= from the mid- or late- (Amersham Corp., Oakville, Ontario, Canada) at a concen- exponential growth phase did not lyse when they were tration of 0.5 rnCi/100 ml of medium. DNA-DNA hybridiza- suspended in a hypotonic solution or in SDS (0.1 g/liter) as tion was performed by using the direct binding membrane described by Boone and Whitman (4). Cells could be lysed method (8). rapidly at pH 8.0 in 50 mM NH,HCO, buffer containing 2% (wthol) SDS and 32.4 mM dithiothreitol. Temperature. Based on the specific growth rates (Fig. 2) RESULTS derived from CH, production in SA medium at different Isolation. The pH of the primary sludge used to start the temperatures, the optimum growth temperature was about A660 enrichment cultures was 6.8 k 0.2. Maintenance of this 35°C. This conclusion was also supported by the sludge under anaerobic conditions (pH 6.8) resulted in the measurements done at the end of incubation. The initial pH solubulization of a major portion of the sludge, with concur- of 5.5 was found to be maintained (+ 0.1U)at all incubation rent methane production. Transfer of this inoculum into temperatures between 15 and 50°C. At 45"C, CH, production synthetic S and SA media at pH 5.0 and 5.5, with H,-CO, as increased gradually for 4 days, and then it levelled off the a substrate, resulted in substantial methane production A,,, at the end of incubation indicated that cells had lysed within a few weeks at 35°C. This indicated the presence of a out. Negligible amounts of CH, were produced at 15°C. hydrogenotrophic methanogenic population (4H2 + CO, + Growth and CH, production were not detected at 50°C. In a CH, + 2H20) that was active at low pH values. However, separate experiment we verified that growth (A66o)and CH, aceticlastic methanogenic activity (CH,COOH + CH, + production rates were coupled during logarithmic growth at CO,) was not evident at low pH values since negligible 35°C. amounts of CH, were detected upon inoculation into SA pH, The optimum pH for growth and CH, production at media at pH 5.5 when the H,-CO, in the headspace was 35°C in SA broth was between pH 5.6 and 6.2, as deduced replaced by 80% N2-20% CO,. Microscopic observation of from specific growth rates (Fig. 3) measured from CH, the culture in SA medium (pH 5.0) (H,-CO,) revealed a production between 3 and 16 days of incubation. This predominance of rod-shaped bacteria. Serial dilutions in pH optimum was corroborated by the A,, measured at the end 5.0 broth of this culture, containing hydrogenotrophic meth- of incubation. At pH 6.1 the maximum CH, production rate anogens, were streaked onto SA agar (pH 5.5). Agar medium was used at pH 5.5 rather than at pH 5.0 to overcome problems of agar consistency (softness). Visible colonies 0.03a were usually observed after 7 to 10 days of incubation. Most of the colonies from lo-, dilutions were similar, and representative colonies were transferred into SA broth (pH 5.0), serially diluted, and plated onto agar. This procedure was repeated a few more times until culture purity was estab- lished by colony morphology and microscopic observation 0.025 of cultures transferred into synthetic and complex media

A containing yeast extract, tryptone, and glucose. Four strains r were isolated; these organisms were similar in appearance '5 and in their pH and temperature profiles. Therefore, only 2 one of these organisms, strain GP9T, was characterized in detail and is described below. 0.020 Surface colonies of strain GP9T were 0.5 to 1.0 mm in diameter after 7 to 14 days of incubation in SA agar (pH 5.5). The colonies were circular, had entire margins, were slightly raised at the center, and were creamy to light yellow in color. The cells were rod shaped and 0.8 pm wide by 3 to 22 pm long (Fig. 1) in pH 5.5 media. The average single cell was 0.0 15 about 6 pm long, but longer cells with no evidence of 15 20 25 30 35 40 45 septation were seen under certain growth conditions. Typi- Incubation ('CC) cally, the rods consisted of one to three cells in a chain. The FIG. 2. Optimum growth temperature of strain GBT cultivated cells stained gram positive and were nonmotile, and no in SA medium (pH 5.5). Specific growth rates (p) based on methane flagella were observed by electron microscopy. production are plotted. VOL. 40, 1990 METHANOBACTERIUM ESPANOLAE SP. NOV. 15

0.025 TABLE 1. Effect of potential inhibitors and enhancers on growth and CH, production of strain GP9T Maximum Cumula- l------CH, pro- tive CH, Test compound" duction production Am rate (%)b (%)b (%Ib 0.020 None (control) 100 100 100 Yeast extract (0.2%, wthol) 68 76 84 Tryptone (0.2%, wt/vol) 55 63 61 Lagoon supernatant (lo%, vol/vol) 115 111 110 Methyl viologen (5 pM) 96 94 94 0.0 15. Benzyl viologen (5 pM) 78 80 77 Chloroform (50 pM)" 44 25 35 Potassium arsenate (100 pM) 91 91 103 Sodium cyanide (100 pM) 12 11 21 Bromoethanesulfonic acid (100 pM) 62 72 75 0.0 1OL Bacitracin (0.01 mg/ml) 95 98 90 4.5 5.0 5.5 6.0 6.5 7.0 Bacitracin (0.1 mg/ml) 0 0 0 Penicillin G (0.1 mg/ml) 101 103 101 PH Penicillin G (1.0 mg/ml) 60 70 58 FIG. 3. Optimum growth pH of strain GP9= cultivated at 35°C in Vancomycin (0.1 mg/ml) 86 88 92 SA media at different pH values. Specific growth rates (p) based on Vancomycin (1.O mg/ml) <1 <1 <1 methane production are plotted. Kanamycin (0.01 mg/ml) 68 72 88 Kanamycin (0.1 mg/ml) 5 9 19 D-Cycloserine (0.1 mg/ml)d 50 38 22 was 210 pmoV2O ml of culture per day, and the maximum ~~ ~~~ specific growth rate, as determined from A,, measurements " SA medium (pH 5.5) (H2-CO,) (10 ml per 60-ml vial) was supplemented as (data not shown), was similar (0.021 h-) to that obtained indicated. Cultures were incubated for 3 weeks at 35°C. from CH, production data at pH 6.2 (Fig. 3). Growth and Data are percentages of SA medium control results, which were as follows: maximum CH4 production rate, 95 Fmol per day per 10 ml; CH, production were observed at pH 4.68, but not at pH 4.3. cumulative CH, production after 3 weeks, 1,758 pmoYlO ml; final Am, 0.40. Comparison of CH? production rates in 2-(N-cyclohexyl- There was a 1.5- to 2-week lag period. amino)-ethanesulfonicacid-buffered media at pH 7.7 and 6.1 There was a 1-week lag period. indicated that the maximum CH4 production rate during growth at pH 7.7 was 45% of that at pH 6.1 (data not shown). At a growth pH of 5.5 at 35"C, a maximum specific growth containing no alcohol. The alcohols, at the concentrations rate of 0.07 h-' (i.e., a mass doubling time of about 10 h) was used, were not inhibitory to growth and methane production achieved when the rate of H2-C02 transfer into the medium of strain GP9T cultivated under an H,-C02 gas phase. was increased by incubating the preparation with shaking at Sulfur sources and vitamin requirements. Addition of a 100 rpm in a model G24 shaker-incubator (New Brunswick titanium (111) citrate reducing solution (5 muliter) caused a 10 Scientific Co., Inc., Edison, N.J.). The corresponding max- to 20% inhibition of the maximum CH, production rate of imum CH, production rate achieved was 1,296 FmoV20 ml strain GP9= in SA medium (pH 5.5) that was reduced with of culture per day. Under these growth conditions, a net cysteine-Na,S. Various sulfur sources were investigated in increase in A,, of 0.90 was achieved after 4 days of medium reduced with titanium (111) citrate. Only cysteine incubation (1 U of A,, = 0.5 mg [cell dry weight]/ml). hydrochloride or N+S could serve as a sole sulfur source for Substrates. Formate, propionate, butyrate, pyruvate, growth. The maximum CH, production rate with Na,S (1 methanol, ethanol, isopropanol, or trimethylamine (each at a mM) was comparable to that with cysteine-Na,S, and with concentration of 10 mM) could not support growth or CH, cysteine hydrochloride (5 mM) the maximum CH, produc- production (SA medium [pH 5.51 under an 80% N,-20% CO, tion rate was 90% of that with N+S. No growth occurred in gas phase). At the concentrations tested, none of these the presence of sulfate as the sole sulfur source. compounds caused significant inhibition of growth or meth- The growth (A,,) of strain GP9T in vitamin-free SA ane production in strain GP9T cultures growing on H2-CO,. medium (pH 5.5) after 7 days of incubation was 56% of the The only substrate that supported growth was H,-CO,. growth in medium containing all of the vitamins. This However, during growth on H,-C02 in the presence of observation was confirmed even after five consecutive trans- formate, a small amount of formate carbon was incorporated fers into vitamin-free medium. Therefore, vitamins are not into cell biomass (2.2 nmoVmg [cell dry weight]). The essential for growth but are stimulatory. disappearance of label from the growth medium containing Inhibitors and enhancers of growth. The effects on strain [14C]formate was accounted for by incorporation into CH,, GP9T of supplementing SA medium (pH 5.5) (80% H2-20% CO,, and cell biomass (representing 61, 27, and 12% of the CO,, incubation at 35°C) with potential inhibitors and en- label, respectively). The labeled formate in vials inoculated hancers of growth of methanogens are shown in Table 1. with heat-killed cells was not metabolized. The mechanism Whereas primary sludge supernatant was slightly stimula- of formate utilization under our test conditions is not known. tory, yeast extract and tryptone were somewhat inhibitory. Measurable growth (increase in A660) of strain GP9= was Although benzyl viologen caused about 20% inhibition of not detected in SA media (pH 5.5) (N,-CO, gas phase) growth and CH, production, a similar concentration of containing either 10 mM 2-propanol or 10 mM 2-butanol as a methyl viologen was not as inhibitory. Bromoethanesulfonic hydrogen donor. However, the low levels of methane pro- acid caused about 30% inhibition. Among the antibiotics duction in 2-propanol- and 2-butanol-containing media (14 tested, bacitracin and kanamycin were potent inhibitors of and 17 pmol per vial, respectively) were about five times growth. Table 1 also shows that the maximum CH, produc- higher than the level (3 pmol per vial) in control medium tion rates, final A,, values, and cumulative CH, levels all 16 PATEL ET AL. INT. J. SYST. BACTERIOL.

FIG. 4. SDS-polyacrylamidegel electrophoresis analysis of cell extracts (A) and surface proteins (B) of strain GBTand Methanobacte- rium bryantii M.o.H.~and M.0.H.G. The arrows indicate regions of dissimilarity. demonstrated very similar trends with respect to the effects more, based on this criterion it is more closely related to of various compounds on growth and methanogenesis. strain M.0.H.G than to strain M.o.H.~ The presence of 1% (wtlvol) NaCl in SA medium (pH 5.5) Cell protein analyses. A comparison of whole-cell extract caused a 30% decline in the maximum CH, production rate, (Fig. 4A) and cell surface proteins (Fig. 4B) of strain GWT and no growth or CH, production was observed in the and Methanobacterium bryantii M.o.H.~and M.Q.H.G by presence of 5% NaCl (data not shown). SDS-polyacrylamide gel electrophoresis indicated that these DNA base composition. The DNA isolated from strain three strains have similar protein profiles except for certain GP9T had a ratio of A,,, to A,,, of 1.9, indicating that it was regions (Fig. 4, arrows). relatively free of protein contamination. The DNA base DNA-DNA homology. Results of DNA-DNA homology composition determined from the melting point (Tm,83.2"C) studies indicated (Table 2) a homology value of less than was calculated to be 33.7 mol% G+C. 30% between Methanobacterium bryantii strains M.o.H.~ Immunological fingerprinting. The partial antigenic finger- and M.0.H.G. Also, the levels of DNA homology between print of strain GP9T was defined with antibody S probes of 10 newly described strain GWT and strains M.o.H.~and reference methanogens (Methanobrevibacter smithii PS, M.0.H.G were less than 55%. Methanobacterium formicicum MF, Methanobacterium bryantii M.o.H.~and M.o.H.G, Methanobrevibacter smithii DISCUSSION ALI, Methanobacterium thermoautotrophicum GC1 and I AHT, Methanobrevibacter arboriphilus DH1, AZ, and DC). Methane production at pH values below 5.0 has been Strain GP9T reacted weakly (1+) with S probes of strain reported in some ecosystems (11, 24, 25). However, pure M.o.H.~,reacted 3+ with S probes of strain M.o.H.G, and culture isolates of methanogens from these environments did not react with the other probes. Therefore, strain GBT were not able to grow at pH values below 5.3, although some is antigenically more closely related to Methanobacterium methane (2% of that at the optimum pH of 6.0) was produced bryantii than to any of the other organisms tested. Further- at pH 3.0 (25). Methanococcus jannaschii, a deep-sea ther- VOL.40, 1990 METHANOBACTERIUM ESPANOLAE SP. NOV. 17

TABLE 2. Percentages of DNA-DNA homology" Methanobacterium bryantii M.o.H.~(29) and to that (33.2 mol% as determined by the bouyant density method) of % of DNA-DNA homology with ["PIDNA from: Methanobacterium bryantii M.0.H.G (1). However, DNA- Filter DNA from: DNA homology studies, as well as the indirect immunoflu- Strain Strain orescence reaction, showed that strain GP9T is distinct from M.o.H.~ M.0.H.G Methanobacterium bryantii. Moreover, our data confirmed Methanobacterium bryantii M.o.H.~ 100.0 16.0 2 1.9' the observation of Konig (12) that strains M.0.H. (the type Methanobacterium bryantii M.0.H.G 23.8 2 2.5 100.0 strain of the species) and M.0.H.G of Methanobacterium Strain GWT 47.3 2 3.8 54.8 2 1.3 bryantii exhibit a low degree (less than 30%) of DNA-DNA homology. Strains with DNA-DNA homology values below a A value of 100 indicates homologous pairs. Mean 2 standard error (data from two separate [32P]DNA preparations, 60% (9) or below 70% (4) are generally considered to be with each assay performed in triplicate). distinct species. Therefore, strain M.0.H.G should perhaps be a distinct Methanobacterium species. Similarly, significant differences observed at the molecular level between mophilic methanogen, was reported to grow at pH 5.2, the Methanobacterium thermoautotrophicum AHT and another most acidic pH tested (10). We believe that strain GP9T is the strain of this species, strain Marburg, have been used to first axenic methanogenic culture that has been demon- suggest that these two strains do not belong to the same strated to grow at pH 5.0 and below. Moreover, the optimum species (7). Methanobacterium ivanovii has been described growth pH is in the moderately acidic range (pH 5.6 to 6.2). as a species that is distinct from Methanobacterium bryantii Similar to strain GP9T, there are other methanogens that largely on the basis of indirect immunofluorescence reac- can use only H,-CO, as a growth and methanogenic sub- tions and differences in the total cellular protein profiles, the strate (3, 7, 26, 27). Like the recently described organism temperature requirements for growth, and the G+C content "Methanobacterium palustre" and Methanobacterium bry- difference of 3.9 mol% (7). antii M.o.H.~and M.0.H.G (29), strain GP9T can utilize Strain GWT exhibits less than 50% DNA-DNA homology 2-propanol or 2-butanol as a hydrogen donor for methane with Methanobacterium bryantii M.o.H.~Based on this production from CO,. degree of homology, strain GP9T can be considered a distinct On the basis of its characteristics, strain GP9T belongs to species. By the same criterion, this organism is also different the order Methanobacteriales (1).The DNA base composi- from strain M.0.H.G. The weak (1+) indirect immunofluo- tion of strain GP9T is in the range (30 to 50 mol% G+C) rescence reaction of strain GP9T with S probe anti-Metha- usually observed for species of the genus Methanobacterium nobacterium bryantii M.o.H.~furthur supports its classifi- (2, 3, 12, 23). The morphology and indirect immunofluores- cation as a species distinct from Methanobacterium bryantii. cence reaction of strain GP9T indicate that it is more closely Moreover, strain GP9T significantly differs from Methano- related to members of the genus Methanobacterium than to bacterium bryantii in its growth pH profile. The latter members of the genus Methanobrevibacter. The similarities organism has an optimum growth pH of 6.9 to 7.2 (1) and in the total cellular and cell surface protein profiles of strain cannot grow at or below pH 5.0 (21). On the basis of its GWT and Methanobacterium bryantii M.o.H.~ and characteristics, strain GP9T is described below as a new M.0.H.G also indicate that strain GBTis a member of the species of Methanobacterium, and we propose the name genus Methanobacterium, but not necessarily a strain of the Methanobacterium espanolae for this organism. species Methanobacterium bryantii. These results support The type strain is strain GP9. A species description is the identification of strain GP9T as a Methanobacterium given below. species; however, strain GP9T is a mesophile and therefore Methanobacterium espanolae sp. nov. Methanobacterium differs fionnthe thermophilic species (2, 3, 26, 28) of the espanolae (es. pa. no' lae. L. gen. fem. n. espanolae, of genus Methanobacterium. Strain GP9T also differs from the Espanola, named after the town of Espanola, Ontario, mesophilic species Methanobacterium formicicum and Canada). Stains gram positive. Nonspore forming, nonmo- Methanobacterium uliginosum in its growth pH require- tile rods. Cells are often present in chains. ments, growth substrates, and DNA base composition (12, Optimum pH, 5.6 to 6.2; minimum pH, 4.7. Grows at pH 23). Strain GP9T is distinct from Methanobacterium ivanovii values of 7.7 or higher (higher pH values not tested). on the basis of the optimum growth pH of 7.0 to 7.4 (pH Optimum temperature, ca. 35°C. No growth at 15 or 50°C. range, 6.5 to 8.2) and optimum growth temperature of 45°C H,-CO, is the only methanogenic substrate used for of the latter organism (7). Moreover, unlike strain GP9T, growth. Methanobacterium ivanovii resists inhibition by bromoeth- The G+C content of the DNA is 34.0 mol% (as determined anesulfonic acid at concentrations up to 1mM (7). The DNA by the T, method). base composition of Methanobacterium ivanovii (7) is 36.6 The type strain of Methanobacterium espanolae is strain mol% G+C (T, method). Strain GP9T differs from acid- GP9. This strain has been deposited with the National tolerant isolate 0-1, which was obtained from peatlands (29, Research Council of Canada culture collection as strain as well as from all of the presently well-characterized NRC 5912. mesophilic pure cultures of methanogens, in its ability to The source of strain GP9T was methanogenic enrichment grow at pH values below 5.0. Moreover, the indirect immu- from the anoxic zone of primary sludge from a bleach kraft nofluorescence reaction of isolate 0-1 was 3+ with anti- mill. Methanobrevibacter arboriphilus DH1 (25), whereas strain GBTgave no reaction (i.e., strain 0-1 is more closely related ACKNOWLEDGMENTS to members of the genus Methanobrevibacter). Detailed We appreciate the technical assistance of M. Moore and D. characteristics of strain 0-1 are not currently available. Agulnik (summer student assistants) and B. J. Agnew. The assis- The G+C content of strain GP9T (33.7 mol%) was similar tance of Ken Jarrell (Queen's University, Kingston, Ontario, Can- to that (30.8 mol% as determined by the T, method; 32.7 ada) in the determination of the T, of isolated DNA and the mol% as determined by the bouyant density method) of assistance of Everley Conway de Macario (New York State Depart- 18 PATEL ET AL. INT.J. SYST.BACTERIOL. ment of Health, Albany) in the immunological fingerprinting of body probes. Syst. Appl. Microbiol. 4:451458. strain GP9= are also appreciated. The assistance of R. Cook (E. B. 14. Macario, A. J. L., and E. Conway de Macario. 1985. Monoclonal Eddy Forest Products Ltd., Espanola, Ontario, Canada) and the antibodies of predefined molecular specificity for identification assistance of H. Black (Diversified Research Laboratories Ltd., and classification of methanogens and for probing their ecolog- Toronto Ontario, Canada) in the collection of primary sludge ical niches, p. 213-247. In A. J. L. 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