International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1723–1729 Printed in Great Britain Methanocalculus pumilus sp. nov., a heavy- metal-tolerant methanogen isolated from a waste-disposal site Koji Mori,1,3 Hiroyuki Yamamoto,2 Yoichi Kamagata,3 Masahiro Hatsu4 and Kazuhiro Takamizawa4 Author for correspondence: Kazuhiro Takamizawa. Tel: j81 58 293 2906. Fax: j81 58 293 2906. e-mail: tak2003!cc.gifu-u.ac.jp 1 United Graduate School of A mesophilic hydrogenotrophic methanogen, strain MHT-1T, was isolated from Agricultural Science, Gifu the leachate of a sea-based site for solid waste disposal (the port of Osaka, University, Yanagido 1-1, T Gifu 501-1193, Japan Japan). Strain MHT-1 was found to be an irregular coccus and was able to use H /CO and formate as energy sources. Acetate was required for growth. The 2 Department of 2 2 Microbiology, St Marianna optimum temperature and pH for growth were 35 mC and 65–75, respectively. University School of Strain MHT-1T was resistant to high concentrations of several heavy metals Medicine, Sugao 2-16-1, such as CdCl and CuSO . The GMC content of the DNA was 519 mol%. Analysis Miyamae, Kawasaki, 2 4 Kanagawa 216-0015, of the 16S rRNA gene revealed that the isolate was a member of the genus Japan Methanocalculus but distinct from its nearest neighbour, Methanocalculus 3 National Institute of halotolerans, there being a sequence similarity of 989%. DNA–DNA Bioscience and Human hybridization analysis revealed 51% relatedness with the DNA of Technology, 1-1 Higashi, M. halotolerans strain SEBR 4845T. The optimum NaCl concentration was 10%, Tsukuba, Ibaraki 305-8566, Japan whereas the optimum in M. halotolerans was 50%. A new species, Methanocalculus pumilus, is proposed for strain MHT-1T. The type strain is 4 Department of T T T Bioprocessing, Faculty of MHT-1 (l DSM 12632 l JCM 10627 ). Agriculture, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan Keywords: Archaea, Methanocalculus pumilus, methanogen, heavy metal, waste- disposal site INTRODUCTION containing large amounts of heavy metals (Cu, " " " 32 mmol kgV ; Pb, 9 mmol kgV ; Zn, 199 mmol kgV ) The number of waste-disposal sites is increasing. (Takamizawa et al., 1987, 1991). This evidence sug- Except for the surface layer and the area near the point gests that there is a possibility of heavy-metal-tolerant where pipes are installed for releasing gases, the sites methanogens existing in waste-disposal sites. Here, we are usually anaerobic. The gases generated by mi- describe the isolation and characterization of a crobial decomposition contain a large amount of methanogen from a heavy-metal-contaminated dis- methane, which is obviously produced from the posal site. reclaimed wastes (Zinder, 1993). This observation indicates that methanogens play an important role for mineralizing organic matter in the sites (Takamizawa METHODS et al., 1994; Qian & Barlaz, 1996). In spite of public Source of organism. Methanocalculus halotolerans SEBR regulations, wastes at the disposal sites contain heavy 4845T was purchased from Oregon Collection of Methano- metals (Ham et al., 1992; Kock et al., 1989; gens (OCM 470T ). Oleszkiewicz & Sharma, 1990). For example, the sampling site chosen for this study (i.e. the sea-based Sampling site. Leachate samples were taken from pipes (25 cm in diameter), used for monitoring sinkage and water site for the disposal of solid waste, Osaka Port, Japan) quality, at the Osaka North Port Sea-Based Solid-Waste- handles reclaimed wastes (mostly incineration ash) Disposal Site in Japan. The site was located in the sea and was an artificial island composed of northern and southern ................................................................................................................................................. sections surrounded by sea walls (Takamizawa et al., 1987, The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA sequences 1991). At the time of sampling, the southern section was reported in this paper are AB008853 and AB020530. being filled with waste mainly consisting of incineration ash. 01368 # 2000 IUMS 1723 K. Mori and others The in situ temperature was 43–57 mC and the pH was in a Olympus BHS-RFCA microscope with a UV lamp and approximately 8n0. filters (20UG-1 and 17L420). Transmission electron micro- graphs were taken using an electron microscope (model H- Enrichment and isolation. The media were prepared ac- 7000; Hitachi) operated at 75 kV. Samples were prepared cording to the method described by Sowers & Schreier " according to the protocol of Franzmann et al. (1997), with (1995). The basal medium contained (lV ): 0n75 g K#HPO%, 0 75 g KH PO ,090 g NH Cl, 0 36 g MgCl .6H O, 0 50 g slight modifications; cells at late-exponential phase were n # % n % n # # n fixed directly in cacodylate buffer supplemented with 5% Na#S;9H#O, 0n50 g cysteine;HCl, 20n0 g NaCl, 2n0 g yeast glutaraldehyde. Cells were fixed overnight at 4 C, collected extract (Oriental Yeast), 2n0 g trypticase peptone (BBL), m by centrifugation and then fixed for 3 h in cacodylate- 0n001 g resazurin, 10 ml trace-element solution (Lobo & Zinder, 1988) and 10 ml DSM 141 vitamin solution (Sowers buffered 1% osmium tetroxide. The cells were stained en & Schreier, 1995). Twenty millilitre vials (containing 10 ml bloc with 0n5% aqueous uranyl acetate and embedded in medium) sealed with butyl-rubber stoppers and aluminium resin. Ultrathin sections were prepared with a Reinchert caps were used throughout the study. After the basal medium ultramicrotome. (without reductants) containing growth substrates was Gram staining and susceptibility to lysis. The Gram reaction autoclaved, filtered solutions (0n22 µm membrane filter) of and susceptibility tests were performed as described by Na#S;9H#O, cysteine;HCl and alcohol (when necessary) Boone & Whitman (1988). were supplied. Na#CO$ solution was also added directly in stoppered vials for adjustment of the initial pH. DNA base composition and hybridization. Cells were har- Primary enrichments were initiated by inoculating leachate vested from 500 ml culture by centrifugation at 4 mC. The " samples into the sterilized basal medium containing (lV ) cell pellet was suspended in Tris-EDTA buffer (pH 8n0) and subjected to three cycles of freezing–thawing. Proteinase K 5n0 g sodium formate, 5n0 g sodium acetate and 10 ml V" methanol, as growth substrates, under an N# atmosphere was then added at 0n075 mg ml with calcium chloride and (101n29 kPa). Incubation was performed at 30, 50 and 70 mC SDS (final concentrations were 0n003 and 2n5%, respectively) (pH 7n5). and the mixture was incubated for 3 h at 37 mC (Kim et al., 1996). The DNA was purified by the method of Marmur Methanogens were isolated using the serial dilution method. " " (Marmur, 1961) and suspended in Tris-EDTA buffer (pH Ampicillin (1000 µgmlV ) or streptomycin (100 µgmlV ) 8n0). The purified DNA was hydrolysed with P1 nuclease was employed as a selective inhibitor of eubacteria in all of from the DNA GC Kit (Yamasa Shoyu) followed by alkaline these experiments (Whitman et al., 1992). After purification, phosphatase treatment (Kamagata & Mikami, 1991). The isolates were kept in the medium without antibiotic. GjC content was determined by HPLC (SCL-6B; Shim- Physiological characteristics. H \CO , sodium formate adzu). Separation was achieved at 40 mC by using a flow rate # # V" (20 mM), sodium acetate (20 mM), sodium pyruvate of 1 ml min , a column of Shim-pack CLC-ODS (Shim- (20 mM), glucose (10 mM), trimethylamine (5 mM), meth- adzu) and 5% methanol in 10 mM phosphate buffer (pH anol (20 mM), ethanol (5 mM), 1-propanol (5 mM), 2- 3n5) as the mobile phase. Each deoxyribonucleoside was propanol (5 mM), 1-butanol (5 mM) and 2-butanol (5 mM) detected by determining the absorbance at 260 nm and an were evaluated as growth substrates. Unless otherwise equimolar mixture of four deoxyribonucleosides was used as specified, preparation of media was based on the method the standard (Yamasa Shoyu). described above. Twenty millilitre vials containing 10 ml Genetic relatedness was investigated by slot-blot DNA– medium were flushed with N \CO (80:20, v\v; 101 29 kPa). # # n DNA hybridization with a random-prime labelling system When H \CO was used as the substrate, the gas phase of $# # # α 20 ml vials containing 5 ml medium was replaced with and [( - P]dCTP (Amersham Pharmacia Biotech) (Jean- thon et al., 1998). Target DNA (500 ng) denatured by 0 8M H#\CO# (80:20, v\v; 101n29 kPa). After inoculation (10%, n v\v, of medium), the vials were incubated statically or in a NaOH was slotted on to a nylon membrane (Bio-Rad) and the labelled DNA was reassociated in a solution containing shaker at 160 r.p.m. (when H#\CO# was used). In the experiment for testing the acetate requirement, to prevent 50% formamide, 0n12 M Na#HPO%,0n25 M NaCl, 7% SDS carry-over from the former culture, the cells were harvested and 1 mM EDTA. After incubation overnight at 43 mC, the by centrifugation at 4 C, washed anaerobically with sterile membranes were washed with SSC containing 0n1% SDS. m " phosphate buffer (pH 7n5) containing Na#S;9H#O(0n5glV ) Hybridization signals were detected by autoradiography. by flushing with oxygen-free N# and suspended in the basal Triplicate tests were performed for each assay and self- medium (which was free of trypticase peptone and yeast hybridization of the probe with homologous target DNA extract). This procedure was repeated three times; the was set to 100%. washed cells obtained were inoculated into the medium, and Phylogenetic analysis of 16S rRNA gene. The 16S rRNA gene H#\CO# was used as the energy source in the absence of yeast was amplified using the following primers: forward primer extract and trypticase peptone. 5h-TCYGKTTGATCCYGSCRGAG-3h (Escherichia coli Growth was monitored by measuring methane production positions 8–27) and reverse primer 5h-GGTTACCTTG- or turbidity at 660 nm, using 20-ml vials that fit into a TTACGACTT-3h (E.