Methanocalculus Taiwanensis Sp. Nov., Isolated from an Estuarine Environment
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International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1799–1806 DOI: 10.1099/ijs.0.01730-0 Methanocalculus taiwanensis sp. nov., isolated from an estuarine environment 1 Department of Botany, Mei-Chin Lai,1 Sheng-Chung Chen,1 Chin-Ming Shu,1 Ming-Shing Chiou,2 National Chung Hsing 1 1 1 1 University, Taichung, Chia-Chi Wang, Ming-Jen Chuang, Tong-Yung Hong, Chia-Chi Liu, 40227 Taiwan, Li-Jane Lai1 and Jack Jay Hua2 Republic of China 2 Food Research & Development Institute, Author for correspondence: Mei-Chin Lai. Tel: j886 4 22840419 ext. 612. Fax: j886 4 22874740. Hsinchu, Taiwan, e-mail: mclai!dragon.nchu.edu.tw Republic of China Two novel hydrogenotrophic methanogens, designated strains P2F9704aT and P2F9705, were isolated from an estuary in Eriln Shi, Taiwan. The cells of strain P2F9704aT were non-motile, irregular cocci 09–14 µm in diameter. They stained Gram-negative. The cells catabolized formate and H2MCO2 to produce methane, but did not utilize acetate, methanol, trimethylamine, ethanol or secondary alcohols as methanogenic substrates. The optimal growth parameters for strain P2F9704aT were pH 67, 37 SC and 05% NaCl. Acetate was required for cell growth even though it was not a substrate for methanogenesis. The trace element tungsten was not required but slightly stimulated the growth of strain P2F9704aT. However, tungsten extended the growth ranges relating to temperature, pH and salt. The sequences of the 16S rRNA genes of strains P2F9704aT and P2F9705 were nearly identical and possessed 991 and 985% similarity to the genes of Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, strain P2F9704aT possessed 14 and 12% DNA relatedness with respect to Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, the optimal salt concentrations, the cellular protein profiles and the molecular masses of surface-layer protein subunits of strain P2F9704aT were different from those of the other two known Methanocalculus species. On the basis of these observations, it is proposed that these two organisms should be placed in a new species, namely Methanocalculus taiwanensis. The type strain is P2F9704aT (l OCM 671T l CCRC 16182T l DSM 14663T). Keywords: archaea, methanogen, Methanocalculus, estuarine, tungsten INTRODUCTION ments such as geothermal springs, deep-sea hydro- thermal vents and hypersaline environments (Boone et Strictly anaerobic methanogens are the only archaea al., 1993a; Jones et al., 1983; Huber et al., 1982; that are truly ubiquitous. Methanogenic species have Ollivier et al., 1994; Stetter et al., 1993). been isolated from virtually every habitat in which Methanomicrobiales anaerobic biodegradation of organic compounds The order comprises three Methanomicrobiaceae Methanocorpuscula occurs, including freshwater and marine sediments, families ( , - ceae Methanospirillaceae the digestive and intestinal tracts of animals, and and ) and nine genera (Boone et al ' et al anaerobic waste digesters (Boone et al., 1993b; Ferry, ., 1993b; Rouviere ., 1992) of hydrogeno- et al 1997; Jones et al., 1987; Wolfe, 1996). Moreover, trophic methanogens (Garcia ., 2000). The family Methanomicrobiaceae isolates have also been obtained from extreme environ- contains seven genera, i.e. Methanomicrobium, Methanolacinia, Methanogenium, ................................................................................................................................................. Methanoculleus, Methanoplanus, Methanofollis and Published online ahead of print on 7 June 2002 as DOI 10.1099/ Methanocalculus. The morphology of cells in the ijs.0.01730-0. family Methanomicrobiaceae includes small rods, The GenBank accession numbers for the 16S rDNA sequences of strains highly irregular cocci, and plane-shaped cells. The cell P2F9704aT and P2F9705 are AF172443 and AF411470, respectively. walls are proteinaceous. All strains can use H#jCO# 01730 # 2002 IUMS Printed in Great Britain 1799 M.-C. Lai and others and formate as substrates of methanogensis. In addi- NaHCO$ in the medium were modified to obtain pH values tion, some can use secondary alcohols (Garcia et al., between 5n6 and 8n3. 2000). Among them is Methanocalculus, a newly Enrichment and isolation. The enrichment was begun im- described genus that was first proposed by Ollivier et mediately after the sample had been brought to the lab- al. (1998); it encompasses the irregular cocci of oratory. In an anaerobic chamber, the estuarine water Methanocalculus halotolerans, which was isolated from sample (5 ml) was added to 160 ml serum bottles that contained 45 ml MB medium with sodium formate (50 mM) an offshore oil well. Methanocalculus halotolerans " grows optimally at 5% NaCl but tolerates NaCl as the catabolic substrate. Vancomycin (100 mg l− ) was concentrations from 0 to 12%. This growth range is added to inhibit the growth of non-methanogenic organisms. the widest reported, to date, for any hydrogenotrophic The culture was incubated in the dark at room temperature methanogen (Garcia et al., 2000; Ollivier et al., 1998). for one month. A large amount of methane was detected in Recently, another Methanocalculus species (Methano- the cultures; 5 ml culture was then transferred anaerobically into a new bottle of sterile MB medium with formate. This calculus pumilus) was isolated from a waste-disposal procedure was repeated for four successive transfers. The site in Japan. It grows optimally at 1% NaCl and culture was then diluted and transferred into molten MB tolerates only concentrations up to 7% NaCl (Mori et agar, and the roll-tube technique was performed. Colonies al., 2000). Here, we report the isolation and charac- grew on the inner wall of the glass tube after 2–3 weeks. In a terization of two new Methanocalculus isolates, strains Coy anaerobic chamber, colonies were picked with dis- T P2F9704a and P2F9705, from the estuarine environ- posable, sterilized inoculation needles and transferred to ment of Eriln Shi, Taiwan. anaerobic tubes containing 5 ml MB medium. Cultures from a single colony were further incubated at 30 mC for 1–2 weeks. Two methane-producing cultures, derived from two METHODS morphological distinct colonies, were furthered purified with repeated serial dilutions with vancomycin until it was free of Source of organisms. Methanocalculus pumilus MHT-1T T T contamination by non-methanogenic bacteria. The axenic (l DSM 12632 l JCM 10627 ) was kindly provided by nature of the culture was established on the basis of Dr Koji Mori of Gifu University, Japan. Methanocalculus T T microscopic examination, the presence of a single colony halotolerans SEBR 4845 (l OCM 470 ) was obtained from type in roll tubes, and the absence of growth in anaerobically the Oregon Collection of Methanogens, USA. prepared Bacto thioglycollate medium. Sampling site. The sampling site was the estuary environ- Determination of catabolic substrates. The catabolic sub- ment at Eriln Shi, Taiwan. The water temperature in this strates tested under N#\CO# (4:1) were sodium formate subtropical environment was 30–34 mC during the summer, (100 mM), sodium acetate (50 mM), trimethylamine and the salinity at this estuary was around 1% (w\v). The (40 mM), methanol (50 mM), ethanol (48 mM), 2-propanol sample was collected in a stainless steel sampling basket. (48 mM), iso-butanol (48 mM) and 2-butanol (48 mM). H# From there, it was immediately transferred to a sterile Oak- was tested for by pressurizing the culture tubes with H# Ridge bottle that had been equilibrated overnight inside a (100%, 200 kPa). Utilization of the substrates was de- Coy anaerobic chamber with an N#\CO# ratio of 4:1. The termined in MB\W media by monitoring methane pro- sample was then transported (within 3 h) to the laboratory duction. Methane production was determined by GC with under anoxic conditions. flame-ionization detection (Lai et al., 1999). Media and culture techniques. The modified anaerobic Antibiotic susceptibility. The sensitivity of strain P2F9704aT technique of Hungate was utilized (Balch et al., 1979; to ampicillin, penicillin, spectinomycin, kanamycin, tetra- Sowers & Noll, 1995). Sterilized media were prepared under cycline and chloramphenicol (each at a concentration of " an oxygen-free N#\CO# (4:1) atmosphere. The MB medium 100 µgml− ) was tested in MB\W medium with sodium used contained the following (per litre deionized water): formate (100 mM) at 37 mC. Growth was determined from MgCl# .6H#O, 1n0 g; KCl, 0n5 g; NaCl, 5 g; CaCl# .2H#O, methane production. 0n1g;K#HPO%,0n4g;NH%Cl, 1n0 g; cysteine . HCl, 0n25 g; NaHCO$,4n0 g; yeast extract, 2 g; tryptone, 2 g; and Whole-cell protein profile and surface-layer protein study. resazurin, 0n001 g. Vitamin (Wolin et al., 1963) and trace- Whole-cell proteins were extracted from cell pellets by element (Ferguson & Mah, 1983) solutions without tung- adding loading buffer containing 4% sodium dodecyl sulfate state were each added to a final concentration of 1% (v\v). at a ratio of 1 ml buffer per OD unit. An OD unit was the The pH of the MB medium was 7n0. An MB\W medium was amount of cells found in 1 ml culture with an absorbance of made from MB medium prepared with a 1% (v\v) trace- 1n0. Surface-layer proteins were isolated according to the −" element solution containing tungstate (Na#WO%,0n3mgl ). protocol of Ko$ nig (1995). SDS-PAGE was performed as MM medium was MB medium without the addition of yeast described by Laemmli (1970). Gels were stained with extract and tryptone. All of the constituents except sulfide Coomassie blue R-250. were dissolved in water, boiled and then cooled under an Determination of growth rates. Specific growth rates were oxygen-free N#\CO# (4:1) atmosphere. The medium was calculated from the methane production, which was ana- distributed into serum bottles (Wheaton Scientific) or lysed by linear regression of the logarithm of the total Hungate tubes (Belleco Glass) under the same atmosphere. amount of methane that accumulated over time (Lai et al., The anaerobic tubes were then sealed and autoclaved at 2000). Inocula were grown under conditions similar to the 121 mC for 20 min. Sodium sulfide from a sterilized anoxic experimental conditions.