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Int J Syst Evol Microbiol 57 (2007), 1089-1094; DOI  10.1099/ijs.0.64593-0
© 2007 International Union of Microbiological Societies

Desulfurispora thermophila gen. nov., sp. nov., a thermophilic, spore-forming sulfate-reducer isolated from a sulfidogenic fluidized-bed reactor

Anna H. Kaksonen1, Stefan Spring2, Peter Schumann2, Reiner M. Kroppenstedt2 and Jaakko A. Puhakka1

1 Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, Tampere, Finland
2 DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

Correspondence
Anna H. Kaksonen
anna.kaksonen{at}tut.fi


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A thermophilic, Gram-positive, endospore-forming, sulfate-reducing bacterium was isolated from a sulfidogenic fluidized-bed reactor treating acidic metal- and sulfate-containing water. The strain, designated RA50E1T, was rod-shaped and motile. The strain grew at 40–67 °C (optimum growth at 59–61 °C) and pH 6.4–7.9 (optimum growth at pH 7.0–7.3). The strain tolerated up to 1 % NaCl. Sulfate, sulfite, thiosulfate and elemental sulfur were used as electron acceptors, but not nitrate, nitrite or iron(III). Electron donors utilized were H2/CO2 (80 : 20, v/v), alcohols, various carboxylic acids and some sugars. Fermentative growth occurred on lactate and pyruvate. The cell wall contained meso-diaminopimelic acid and the major respiratory isoprenoid quinone was menaquinone MK-7. Major whole-cell fatty acids were iso-C15 : 0 and iso-C17 : 0. Strain RA50E1T was distantly related to representatives of the genera Desulfotomaculum, Pelotomaculum, Sporotomaculum and Cryptanaerobacter. On the basis of 16S rRNA gene sequence data, the strain cannot be assigned to any known genus. Based on the phenotypic and phylogenetic features of strain RA50E1T, it is proposed that the strain represents a novel species in a new genus, for which the name Desulfurispora thermophila gen. nov., sp. nov. is proposed. The type strain of Desulfurispora thermophila is RA50E1T (=DSM 16022T=JCM 14018T).

Abbreviations: DMA, dimethyl acetal; FBR, fluidized-bed reactor; SRB, sulfate-reducing bacteria

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RA50E1T is AY548776.


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The previously described thermophilic sulfate-reducing bacteria (SRB) have been classified within the genera Desulfotomaculum (Campbell & Postgate, 1965Go), Thermodesulfobacterium (Zeikus et al., 1983Go), Thermodesulfovibrio (Henry et al., 1994Go), Thermodesulforhabdus (Beeder et al., 1995Go), Thermodesulfobium (Mori et al., 2003Go), Thermodesulfatator (Moussard et al., 2004Go) and Desulfovirgula (Kaksonen et al., 2007Go). Spore-forming SRB include members of the genera Desulfotomaculum (Campbell & Postgate, 1965Go), Desulfosporosinus (Stackebrandt et al., 1997Go), ‘Desulfosporomusa’ (Sass et al., 2004Go) and Desulfovirgula (Kaksonen et al., 2007Go). Recently, SRB have been isolated from sulfidogenic fluidized-bed reactors (FBR) treating acidic metal-containing wastewater (Kaksonen et al., 2004cGo). This paper describes one of the isolates, a novel thermophilic spore-forming SRB, strain RA50E1T.

Strain RA50E1T was isolated from a sulfidogenic laboratory-scale FBR after over 600 days of continuous flow operation at 35 °C (Kaksonen et al., 2004cGo). The FBR had been inoculated with enrichment cultures originating from methanogenic granular sludge (Neson Oy, Jokioinen, Finland) and sediments from Outokumpu's Pyhäsalmi mine, Finland (Kaksonen et al., 2003bGo). The performance and microbiology of the FBR has been described previously (Kaksonen et al., 2003aGo, bGo, 2004aGo, bGo, cGo). The isolation of strain RA50E1T was performed at 50 °C using modified Postgate growth medium (pH 7.0–7.5) (Kaksonen et al., 2004cGo) and anaerobic roll-tubes solidified with 1.5 % agar. For chemotaxonomic analysis and DNA isolation, the strain was cultured at 50 °C in modified DSM medium 641 containing lactate as electron donor. The medium was supplemented with 1 ml selenate-tungstate solution (DSM medium 385) l–1 and sodium dithionate (25 mg l–1) was used as the reducing agent instead of Na2S.

The isolate was examined by phase-contrast microscopy (Zeiss Axioskop 2) and photomicrographs were obtained using the agar slide technique as described previously (Kaksonen et al., 2004cGo). Flagellum staining was performed as described by Heimbrook et al. (1989)Go and cells were examined for flagella using an Axiophot light microscope (Zeiss). Spore formation by the strain was determined microscopically and by testing for growth after heat treatment (80 °C for 20 min). The Gram type of the cells was determined by both Gram staining and the KOH test (Gregersen, 1978Go).

The effects of temperature, pH and NaCl concentration on growth were determined as described previously using lactate as electron donor in modified DSM medium 641 (Kaksonen et al., 2006aGo). The ability of the strain to utilize various electron donors (1–20 mM) was tested in a medium containing 20 mM sulfate. The utilization of various electron acceptors (10 mM) was studied using lactate (10 mM) as electron donor. Amorphous iron(III) oxyhydroxide was formed by neutralizing FeCl3 solution to pH 7 with NaOH. The cultures were incubated for 1–2 weeks. Electron donor utilization was assessed by bacterial growth (OD at 660 nm; Shimadzu UV-1601 spectrophotometer or Ultrospec II LKB Biochrom 4050 UV/visible spectrophotometer), hydrogen sulfide production or substrate conversion. Hydrogen sulfide production was determined spectrophotometrically and substrate conversion was determined by GC as described previously (Kaksonen et al., 2004cGo). Ferrous iron was determined colorimetrically (Shimadzu UV-1601) with ferrozine (Stookey, 1970Go). Concentrations of sulfate, sulfite, thiosulfate, nitrate and nitrite were determined by ion chromatography (Dionex DX-120).

Diaminopimelic acid isomers were detected in cell-wall hydrolysates by TLC as described previously (Rhuland et al., 1955Go; Kaksonen et al., 2006bGo). Respiratory isoprenoid quinones were extracted and analysed according to the methods described by Collins & Jones (1981)Go, Monciardini et al. (2003)Go and Groth et al. (1996)Go by using HPLC and electron impact MS (Kaksonen et al., 2006bGo). Methyl esters of cellular fatty acids were obtained by saponification, methylation, extraction and base wash, as described previously (Kämpfer & Kroppenstedt, 1996Go; Kroppenstedt, 1985Go; Miller, 1982Go). The fatty acid methyl ester mixtures were separated by GC (Hewlett Packard 5890 Series II) as described previously (Kaksonen et al., 2006bGo).

For 16S rRNA gene sequencing, cells were pelleted from 1.5 ml liquid culture at 10 000 g for 1 min, washed with PBS (7.2 mM Na2HPO4, 2.8 mM NaH2PO4, 130 mM NaCl, pH 7.2) and resuspended in 50 µl PBS. 16S rRNA genes were amplified by direct lysis PCR using primers 27F and 1492R (Kaksonen et al., 2004cGo). The reaction mixtures contained 25 µl HotStarTaq master mix (Qiagen), 1 µM each of both primers, 1 µl cell suspension and 23 µl sterile distilled water. Thermal cycling was carried out with a Minicycler (MJ Research) as follows: initial denaturation and activation of HotStarTaq at 95 °C for 15 min followed by 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 50 °C for 2 min and primer extension at 72 °C for 2 min, and final extension at 72 °C for 10 min. PCR products were purified with a QIAquick PCR Purification kit (Qiagen) and visualized by gel electrophoresis using ethidium-bromide-stained (0.2 mg l–1) 1 % agarose gels. The 16S rRNA genes were sequenced using primers 27F, 518R, 704F, 787R, 1100R and 1241F (Kaksonen et al., 2004cGo) in separate sequencing reactions. Sequencing was performed using the ABI PRISM Big-Dye Terminator Ready Reaction kit on an ABI 373 automated sequencer or on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) operated by the Australian Neuromuscular Research Institute (ANRI) or Royal Perth Hospital DNA sequencing service. The DNA sequence chromatograms were analysed and single primer sequences were compiled using the BioEdit sequence alignment editor (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic affiliations of the sequences were estimated initially using the program BLAST (http://www.ncbi.nlm.nih.gov/blast/) (Altschul et al., 1990Go). A phylogenetic tree of the 16S rRNA gene sequence of strain RA50E1T (1545 bp between Escherichia coli positions 28 and 1491) and related species was constructed using distance matrix and neighbour-joining methods in ARB. The robustness of the phylogeny was tested by bootstrap analysis with 1000 iterations.

Genomic DNA for G+C content determination was released by rupturing cells using a French pressure cell (Thermo Spectronic) followed by chromatographic purification on hydroxyapatite (Cashion et al., 1977Go). The DNA was hydrolysed with P1 nuclease and the nucleotides were dephosphorylated with bovine alkaline phosphatase (Mesbah et al., 1989Go). The G+C content of the resulting deoxyribonucleosides was determined by reversed-phase HPLC (Shimadzu) and calculated from the dG : dT ratio (Tamaoka & Komagata, 1984Go; Mesbah et al., 1989Go).

Cells of strain RA50E1T were straight or slightly curved rods, 0.6–1.0 µm in diameter and 2.0–9.0 µm in length (Fig. 1Go). The size of the cells was dependent on growth medium and temperature. The strain formed oval spores, which were able to germinate after a heat shock. Spores were located centrally or subterminally. Sporulation did not cause significant swelling of the cells. Cells were motile with two or more flagella and Gram-positive as determined by both Gram staining and the KOH test. The temperature, pH and NaCl ranges for growth of strain RA50E1T are shown in Table 1Go. The operating temperature of the FBR (35 °C) was lower than the minimum temperature required for growth of RA50E1T. Therefore, the strain was probably not active in the FBR, but rather survived as spores in the biofilm.


Figure 1
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  		Fig. 1. Phase-contrast micrograph of cells of strain RA50E1T. Bar, 10 µm.

 

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  		Table 1. Characteristics of strain RA50E1T and some of its closest sulfate-reducing relatives

Strains: 1, RA50E1T; 2, Desulfotomaculum thermocisternum ST90T (unless indicated, data from Nilsen et al., 1996Go); 3, Pelotomaculum thermopropionicum SIT (Imachi et al., 2002Go); 4, Sporotomaculum hydroxybenzoicum BTT (Brauman et al., 1998Go); 5, Cryptanaerobacter phenolicus LR7.2T (Juteau et al., 2005Go). NR, Not reported. All strains have rod-shaped cells and none reduce nitrate.

 
RA50E1T was able to use sulfate, sulfite, thiosulfate and elemental sulfur as electron acceptors, but not nitrate, nitrite or iron(III). Electron donors utilized by RA50E1T included H2/CO2 (80 : 20, v/v), lactate (20 mM), formate, butyrate, isobutyrate, pentanoate, heptanoate, pyruvate, crotonate, mannose, glucose, fructose, myo-inositol, ethanol, propanol and butanol (each at 10 mM), pentanol and alanine (each at 5 mM), hexanoate and octanoate (each at 2 mM), decanoate, dodecanoate, tetradecanoate and hexadecanoate (each at 1 mM) and nonanoate (0.5 mM). A number of electron donors (e.g. lactate, pyruvate, hexanoate, fructose, myo-inositol, butanol and alanine) were oxidized incompletely to acetate, but no significant acetate production was observed during the sulfidogenic oxidation of octanoate and nonanoate. RA50E1T did not utilize acetate, propionate, benzoate, malate, malonate, fumarate, succinate, tartrate, citrate, methanol, arabinose, xylose, sucrose, lactose, maltose, galactose, cellobiose, trehalose or glutamate (each at 10 mM), glycine (5 mM) or 4-hydroxybenzoate (2 mM) as electron donors. RA50E1T fermented lactate and pyruvate.

The cell wall of strain RA50E1T contained meso-diaminopimelic acid and the major respiratory isoprenoid quinone was menaquinone MK-7. The whole-cell fatty acid composition of strain RA50E1T is shown in Table 2Go. Significant proportions of iso-branched saturated fatty acids were found, the major fatty acids being iso-C15 : 0 and iso-C17 : 0 (Table 2Go). Strain RA50E1T contained larger amounts of iso-C17 : 0 and less C16 : 0 fatty acid than representatives of related genera (Table 2Go). Additionally, RA50E1T contained iso-C15 : 1, cyclopropane fatty acid (C17 : 0 cyc) and dimethyl acetals (DMA; iso-C15 : 0 DMA, C16 : 0 DMA and iso-C17 : 0 DMA), which were not detected in the selected representatives of related genera (Table 2Go). The G+C content of the total DNA of strain RA50E1T was 53.5 mol% (Table 1Go).


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  		Table 2. Whole-cell fatty acid compositions (%) of strain RA50E1T and related type strains

Strains: 1, RA50E1T; 2, Desulfotomaculum thermocisternum DSM 10259T (Imachi et al., 2002Go); 3, Pelotomaculum thermopropionicum SIT (Imachi et al., 2002Go); 4, Cryptanaerobacter phenolicus LR7.2T (Juteau et al., 2005Go). DMA, Dimethyl acetal; c, cis isomer; cyc, cyclopropane.

 
The 16S rRNA gene of strain RA50E1T contained several regions of variable length that did not align well with sequences of related species and probably represent inserts. Based on 16S rRNA gene sequences, strain RA50E1T was distantly related to representatives of the genera Desulfotomaculum, Pelotomaculum, Sporotomaculum and Cryptanaerobacter (Fig. 2Go). The closest relatives within these genera were Desulfotomaculum thermocisternum ST90T (89.3 % 16S rRNA gene similarity in pairwise comparison using BioEdit), obligately syntrophic Pelotomaculum schinkii HHT (88.7 %), Sporotomaculum hydroxybenzoicum DSM 5475T (85.1 %) and Cryptanaerobacter phenolicus LR7.2T (84.4 %).


Figure 2
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  		Fig. 2. Phylogenetic tree generated using distance matrix and neighbour-joining methods based on the 16S rRNA gene sequences of strain RA50E1T and related taxa. Archaeoglobus veneficus SNP6T (GenBank accession no. Y10011) was used as outgroup (not shown). Numbers at nodes represent bootstrap percentages based on 1000 samplings. Bar, 0.05 changes per nucleotide position.

 
Strain RA50E1T exhibited many features that are typical of members of the genera Desulfotomaculum and Pelotomaculum, such as rod-shaped, Gram-positive, spore-forming cells and MK-7 as the major menaquinone (Table 1Go). Members of the genus Pelotomaculum do not reduce sulfate, whereas the genus Desulfotomaculum harbours a collection of SRB that vary significantly with respect to their phylogeny and physiological capacities, such as complete against incomplete oxidation of substrates. Based on the phylogenetic tree (Fig. 2Go), there are species of the genus Desulfotomaculum that seem to be more divergent from each other than from the novel isolate or from representatives of the genera Pelotomaculum, Sporotomaculum and Cryptanaerobacter. Therefore, the genus Desulfotomaculum rather represents a ‘supergenus' consisting of several ‘subgenera’. Considering 16S rRNA gene sequencing, the genus Desulfotomaculum could be reclassified into several genera. On the basis of 16S rRNA gene sequence data, strain RA50E1T cannot be assigned to any genus with a validly published name. Based on its phylogenetic and phenotypic distinctiveness, it is proposed that strain RA50E1T represents a novel species in a new genus, for which the name Desulfurispora thermophila gen. nov., sp. nov. is proposed.

Description of Desulfurispora gen. nov.
Desulfurispora (De.sul.fu.ri.spo'ra. L. pref. de from; L. n. sulfur sulfur; Gr. fem. n. spora a seed and, in biology, a spore; N.L. fem. n. Desulfurispora a spore-forming organism that reduces sulfur compounds).

Cells are rod-shaped, spore-forming, Gram-positive, strictly anaerobic and thermophilic. Sulfate and other sulfur compounds are used as electron acceptors. Optimal growth occurs at neutral pH. The cell wall contains meso-diaminopimelic acid and MK-7 is the major menaquinone. Major cellular fatty acids are iso-C15 : 0 and iso-C17 : 0. Phylogenetic position based on 16S rRNA gene sequencing is in the Gram-positive bacteria. The type species is Desulfurispora thermophila.

Description of Desulfurispora thermophila sp. nov.
Desulfurispora thermophila (ther.mo.phi'la. Gr. fem. n. thermê heat; Gr. adj. philos loving; N.L. fem. adj. thermophila heat-loving).

In addition to the properties given in the description of the genus, the following properties are observed. Cells are motile and 0.6–1.0x2.0–9.0 µm. Growth occurs at 40–67 °C (optimum 59–61 °C), pH 6.4–7.9 (optimum pH 7.0–7.3) and NaCl concentration 0–1 % (optimum 0 % NaCl). Sulfate, sulfite, thiosulfate and elemental sulfur are used as electron acceptors, but nitrate, nitrite and iron(III) are not. Electron donors include H2/CO2 (80 : 20, v/v), lactate, formate, butyrate, isobutyrate, pentanoate, heptanoate, pyruvate, crotonate, mannose, glucose, fructose, myo-inositol, ethanol, propanol, butanol, pentanol, alanine, hexanoate, octanoate, decanoate, dodecanoate, tetradecanoate, hexadecanoate and nonanoate. Fermentative growth occurs on lactate and pyruvate.

The type strain is RA50E1T (=DSM 16022T=JCM 14018T), isolated from a sulfidogenic FBR treating acidic metal-containing wastewater. The genomic DNA G+C content of the type strain is 53.5 mol% (as determined by HPLC).


   ACKNOWLEDGEMENTS
 
This work was supported by the Finnish Funding Agency for Technology and Innovation, Outokumpu Oyj, Finland, the Finnish Graduate School in Environmental Science and Technology, the Academy of Finland and the European Commission (BioMinE contract 500329 and a grant for the DSMZ large scale facility). Annukka Hämäläinen, Esther Schüler, Anika Vester and Marlen Jando are acknowledged for technical assistance.


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Right arrow Articles by Puhakka, J. A.

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