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Isolation and Characterization of Methanobacterium Espanolae Sp 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. Supernatant from 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 transfer- ring 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.
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