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International Journal of Systematic and Evolutionary Microbiology (2000), 50, 25–33 Printed in Great Britain Desulfotomaculum alkaliphilum sp. nov., a new alkaliphilic, moderately thermophilic, sulfate-reducing bacterium E. Pikuta,1 A. Lysenko,1 N. Suzina,2 G. Osipov,4 B. Kuznetsov,3 T. Tourova,1 V. Akimenko2 and K. Laurinavichius2 Author for correspondence: E. Pikuta. Tel: ­7 95 135 0421. Fax: ­7 095 135 65 30. e-mail: pikuta!inmi.host.ru 1 Institute of Microbiology, A new moderately thermophilic, alkaliphilic, sulfate-reducing, Russian Academy of chemolithoheterotrophic bacterium, strain S1T, was isolated from a mixed Sciences, prospekt 60-let Octiabria, 7/2, Moscow, cow/pig manure with neutral pH. The bacterium is an obligately anaerobic, 117811, Russia non-motile, Gram-positive, spore-forming curved rod growing within a pH 2 Institute of Biochemistry range of 8<0–9<15 (optimal growth at pH 8<6–8<7) and temperature range of and Physiology of 30–58 SC (optimal growth at 50–55 SC). The optimum NaCl concentration for Microorganisms, growth is 0<1%. Strain S1T is an obligately carbonate-dependent alkaliphile. Russian Academy of Sciences, Pushchino, Russia The GMC content of the DNA is 40<9 mol%. A limited number of compounds are utilized as electron donors, including H Macetate, formate, ethanol, lactate and 3 Centre ‘Bioengineering’, 2 Russian Academy of pyruvate. Sulfate, sulfite and thiosulfate, but not sulfur or nitrate, can be used Sciences, Moscow, Russia as electron acceptors. Strain S1T is able to utilize acetate or yeast extract as T 4 Academician Yu. Isakov sources of carbon. Analysis of the 16S rDNA sequence allowed strain S1 Scientific Group, Russian (¯ DSM 12257T) to be classified as a representative of a new species of the Academy of Medical genus Desulfotomaculum, Desulfotomaculum alkaliphilum sp. nov. Sciences, Moscow, Russia Keywords: Desulfotomaculum alkaliphilum sp. nov., alkaliphile, thermophile, sulfate-reducing bacterium INTRODUCTION Bacillus and Clostridium) can be isolated from neutral soils (Horikoshi & Akiba, 1982). At present, the biodiversity of sulfate-reducing bac- The incubation of cow}pig manure, of neutral pH, in teria (SRB) comprises 29 genera and more than 100 a high-carbonate medium at moderately high tem- species, differing in their metabolism of substrates and perature (55 C) led to an intense sulfidogenesis, which distribution in various ecosystems. Among them, only m indicated the presence of thermophilic, alkaliphilic two species belonging to the domain Archaea can grow sulfate-reducers. Since such SRB have not been at acidic pH (from 4 5to65), whereas the majority of ± ± reported previously, we attempted to isolate this SRB are neutrophiles, which grow optimally at pH bacterium. values close to 7±0. However, two novel mesophilic SRB isolated recently from soda lakes of Africa and Central Asia, Desulfonatronovibrio hydrogenovorans METHODS and Desulfonatronum lacustre, can grow at alkaline pH Source of the isolate. Mixed cow}pig manure samples were (from 8 to 10±2) (Zhilina et al., 1997; Pikuta et al., collected at several farms in the Moscow region. The pH of 1998). The alkaliphily of these bacteria is not sur- samples ranged from 6±0to7±0. The samples were stored at prising, since they were isolated from high-carbonate 4 mC. waters, of high salinity and high pH. Nevertheless, Media and cultivation. Enrichment, isolation and cultivation alkaliphiles (mainly representatives of the genera of SRB was performed in a modified medium (Zhilina et al., 1997) under an atmosphere of 100% nitrogen. The basal −" ................................................................................................................................................. medium contained (g l ): KH#PO%,0±2; MgCl#.6H#O, 0±1; Abbreviations: MTS, maximum topological similarity; SRB, sulfate- KCl, 0±2; NH%Cl, 1±0; Na#CO$,0±5; NaHCO$,8±0; Na#SO%, reducing bacteria. 5±0; NaCl, 5±0; Na#S.9H#O, 0±5; yeast extract, 0±01; and The GenBank accession number for the 16S rDNA sequence of strain S1T is sodium formate, 5±0. The medium was supplemented AF097024. with 10 ml vitamin solution (Wolin et al., 1963) and 01055 # 2000 IUMS 25 E. Pikuta and others " 1 ml trace element solution containing (mg 200 ml− Electron acceptors. The utilization of various electron water): MnCl#.4H#O, 750; Fe(NH%)(SO%)#.12H#O, 400; acceptors was studied in basal medium containing formate −" FeSO%.7H#O, 200; CoCl#.6H#O, 200; ZnSO%.7H#O, 200; (5 g l ) as an electron donor. Electron acceptors were added NiCl#, 100; CuSO%.5H#O, 20; AlK(SO%)#.12H#O, 20; in the form of autoclaved or filter-sterilized stock solutions. H$BO$,20;Na#MoO%.2H#O, 20; and 5 ml concentrated The final concentrations (in mM unless stated) of electron HCl. After autoclaving, the pH of the medium at 50 mC was acceptors were: Na#SO%,20;Na#SO$,5;Na#S#O$,10; 8 7–9 0. NaNO , 10; benzenesulfonate, 5; fumarate, 10; and el- ± ± $ ! " emental sulfur (S ), 2 g l− . Unless otherwise noted, enrichment and pure cultures were grown in 10 ml medium in 15 ml Hungate tubes under an Antibiotic susceptibility. Sensitivity to antibiotics was de- atmosphere of N# (100%). All transfers and samplings of termined by transferring an exponentially growing culture cultures were performed with syringes. The medium was into the basal medium containing filter-sterilized antibiotics −" sterilized at 120 mC for 30 min. All incubations were carried at a concentration of 100 µgml (bacitracin and chloram- −" out at 50–55 mC. Upon cultivation on 3% (w}v) Difco agar phenicol) or 250 µgml (penicillin, ampicillin, streptomycin in Hungate tubes, carbonate solution was added to 4 ml and vancomycin). Antibiotic-containing cultures were pre- previously sterilized, carbonate-free medium. incubated for 12 h at 37 mC and subsequently incubated for 2 weeks at 50–55 mC. Growth measurements. Bacterial growth was measured by direct cell counting under an MBI-3 phase-contrast micro- Analytical procedures. The volatile products of pyruvate scope, by measuring sulfide produced from sulfate in the fermentation were quantified by using a Pye Unicam 304 gas growth medium (Tru$ per & Schlegel, 1964) or by measuring chromatograph equipped with a flame-ionization detector the increase in OD'!! (Spekol 11). and a Chromosorb 101 column (0±9m¬3±3 mm) kept at 160 mC. The carrier gas was CO#. Light microscopy. The morphology of living cultures was examined by using an MBI-3 phase-contrast microscope. Lipid analysis. Lipids were extracted from cell biomass that was dried in a stream of helium and then under a vacuum. Electron microscopy To 30 mg of dry biomass, 200 µlofa5±4 M solution of (i) Ultrathin sections. Bacterial cells were pre-fixed with 1±5% anhydrous HCl in methanol was added, and the mixture was glutaraldehyde in 0±05 M cacodylate buffer (pH 7±2) at 5 mC heated at 70 mC for 2 h. The methyl esters of fatty acids and for 1 h, washed three times with the buffer and fixed with 1% aldehyde derivatives obtained were extracted twice with OsO% in the same buffer at 20 mC for 4 h. After dehydration, 100 µl hexane. The extract was dried and silylated in the preparations were embedded in epoxy resin Epon 812 20 µl N,O-bis(trimethylsilyl)trifluoroacetamide for 15 min at (Fluka), and ultrathin sections were mounted on copper 65 mC. A 1 µl portion of the reaction mixture was analysed grids and contrasted with 3% uranyl acetate in ethanol and with a model HP-5985B GC-MS system (Hewlett Packard) then with lead citrate (Reynolds, 1963). equipped with a capillary column (25¬0±25 mm) consisting of fused quartz containing an Ultra-1 nonpolar methyl- (ii) Electron-microscopic cryofractography. Bacterial cells were silicone phase. The temperature program was run from " frozen in liquid propane cooled by a surrounding vessel of 150 C (2 min isotherm) to 250 Cat5 C min− and then m m " m liquid nitrogen using attachments to a JEE-4X set-up for from 250 to 300 mCat10mC min− . Data processing was vapour-phase deposition and fractured when a vacuum of −% carried out with an HP-1000 computer by using the standard 3¬10 Pa and a temperature of ®100 mC were reached programs of the GC-MS system (Hewlett Packard). (Fikhte et al., 1973). The surface of the fracture was contrasted with a platinum}carbon mixture at an angle of DNA analysis. The G­C content of DNA was determined 45 m and fixed by deposition of a carbon layer at an angle of by the thermal denaturation method using the DNA of 90 m. Ultrathin sections and replicas were examined in a Escherichia coli K-12 as a standard (Owen et al., 1969). JEM-100 B electron microscope at an accelerating voltage of DNA–DNA hybridization was carried out as described by 60 kV. De Ley et al. (1970). The genome size was determined from renaturation rates (Gillis et al., 1970). Effects of pH, temperature and NaCl concentration on growth. The effect of pH on growth was determined at 16S rRNA sequencing. For amplification and sequencing of 16S rRNA genes, DNA was obtained by standard phenol} 50 mC. The pH of the basal medium was adjusted to defined chloroform extraction. The 16S rRNA genes were selectively values with sterile 6M solutions of HCl or NaOH at 50 mC amplified by PCR with the following primers: 5 -AGAG- under a stream of N# and measured using a pH meter « TTTGATCCTGGCTCAG-3 (forward) and 5 -TACGGT- calibrated at 50 mC. The temperature range for growth was « « TACCTTGTTACGACTT-3 (reverse). PCR was carried determined in basal medium at pH 8±8. The effect of NaCl on « out in 50 µl standard ammonium sulfate buffer [17 mM growth was determined in basal medium containing 0±0, 0±5, " (NH ) SO , 67 mM Tris}HCl, 200 µg BSA ml− ,1mM 1±0, 2±0, 3±0, 5±0 and 7±0% (w}v) NaCl.

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