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1 Max-Planck-Institute for Marine Microbiology, Celsiusstr. 1, 28359 Bremen, Germany
2 University of Hamburg, Institute of Soil Science, Allende-Platz 2, 20146 Hamburg, Germany
Correspondence
Verona Vandieken
vvandiek{at}mpi-bremen.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Desulfovibrio frigidus strains 18T and 77 are DQ148943 and DQ148945, and that for Desulfovibrio ferrireducens strain 61T is DQ148944.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Canfield et al., 1993; Thamdrup & Canfield, 1996; Rysgaard et al., 1998; Kostka et al., 1999; Glud et al., 2000). Most sulfate-reducing bacteria are phylogenetically placed within the Deltaproteobacteria, including the genus Desulfovibrio, which comprises 42 described species. A special characteristic of some Desulfovibrio strains is the ability to reduce Fe(III) compounds without gaining energy for growth (Coleman et al., 1993; Lovley et al., 1993; Li et al., 2004). Here, we report the isolation of three novel psychrotolerant Desulfovibrio-related strains with the ability to reduce Fe(III).
Strain 61T was isolated from an enrichment culture of artificial sea-water medium (Widdel & Bak, 1992) with approximately 30 mM poorly crystalline iron oxide, 0·4 mM MgSO4.7H2O and 20 mM lactate at 10 °C, which was inoculated with surface sediment of Tempelfjorden, Station CD (78° 25·267' N 17° 08·277' E; bottom water temperature 2·8 °C). Iron oxide was replaced by ferric citrate (approx. 30 mM) for isolation in deep-agar dilution series. Cells of strain 61T were motile vibrios and the 16S rRNA gene sequence was 95·7 % similar to the sequence of Desulfovibrio hydrothermalis AM13T. The ability of Desulfovibrio desulfuricans to reduce Fe(III) for several consecutive transfers has been shown previously (Lovley et al., 1993); however, the authors suggested that the strains grew with 0·3 mM sulfate in the medium and not by Fe(III) reduction. Correspondingly, we could not determine unequivocally whether strain 61T grew by Fe(III) reduction or with 0·4 mM sulfate in the medium. Strains 18T and 77 were enriched and isolated under sulfate-reducing conditions with 28 mM sulfate, 20 mM lactate and 10 mM formate at 4 and 17 °C from sediment of Tempelfjorden, Station CC (78° 26·039' N 17° 19·722' E; bottom water temperature 3·1 °C) and Smeerenburgfjorden, Station J (79° 42·006' N 11° 05·199' E; bottom water temperature 2·3 °C), respectively. 16S rRNA gene sequencing showed that strains 18T and 77 were closely related to Desulfovibrio hydrothermalis and the novel strain 61T.
The general physiological characteristics of strains 18T, 61T and 77 were evaluated under sulfate-reducing conditions with lactate as the electron donor in a medium with a lower salt concentration (salt-water medium) (Widdel & Bak, 1992) at their respective isolation temperature. Cultures growing with alternative substrates were transferred into fresh test medium for verification. Temperature tolerance of the strains was determined in an aluminium temperature-gradient block at 12 different temperatures between –2 and 32 °C (Sagemann et al., 1998). The salt requirement was determined in media with 12 different NaCl concentrations between 0·05 and 5 % (w/v) and 10 different MgCl2.6H2O concentrations between 0·02 and 3·6 % (w/v). The pH optima of the strains were determined in media with 12 different pH values (in triplicate) that covered a range from pH 5·5 to 8·8. For all tests, growth was monitored spectrophotometrically (UV 1202; Shimadzu) by measuring optical density at 580 nm.
PCR amplification of 16S rRNA gene was performed with the primers 8F and 1492R, and the PCR product was amplified for sequence analysis with primers 8F, 341F, 518F, 534R, 1099F and 1492R (Buchholz-Cleven et al., 1997). Phylogenetic positions of the three novel strains were evaluated by using the ARB program (Ludwig et al., 2004) with the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms in combination with different sets of filters.
Strains 61T and 77 showed a vibrioid or sigmoid morphology, 2·5–5·5x0·5–0·7 µm in size, whereas cells of strain 18T were straight rods, 3·5–4·5x0·5–0·7 µm in size. Cells of all strains were motile by means of a single polar flagellum as indicated by electron microscopy (Fig. 1). Gram staining was negative for all strains.
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). The strains reduced sulfate and other sulfur compounds like sulfite, thiosulfate or elemental sulfur (Table 1). Reduction of ferric citrate or poorly crystalline iron oxide in sulfate-free medium was observed in two to four consecutive transfers for all three strains. Reduction of Fe(III) became slower with every transfer and we suggest that the strains did not conserve energy for growth. The ability for Fe(III) reduction was previously described for several species of Desulfovibrio (Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Desulfovibrio sulfodismutans, Desulfovibrio baarsii and Desulfovibrio sp. strain G-11) as well as for Desulfomicrobium baculatum and Desulfobacterium autotrophicum (Coleman et al., 1993; Lovley et al., 1993; Li et al., 2004). Growth by Fe(III) reduction has so far only been shown for the two sulfate-reducing bacteria Desulfobulbus propionicus and ‘Desulfotomaculum reducens’ (Tebo & Obraztsova, 1998; Holmes et al., 2004).
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) and were able to grow at the freezing point of sea water, –2 °C. According to their temperature range, the strains can be considered as psychrotolerant.
The DNA G+C contents were 42·0 and 43·3 mol% for strains 61T and 18T, respectively (Table 1), and were determined by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. Strains 18T and 77 were closely related to each other (99·9 % 16S rRNA gene sequence similarity) (Fig. 2), therefore we suggest that these two strains belong to the same species. 16S rRNA gene sequence similarity was 97·6 % between strains 61T and 18T and 97·5 % between strains 61T and 77 (Fig. 2). DNA–DNA hybridization was done by the DSMZ and DNA–DNA relatedness was 14·5 % between strains 61T and 18T and 18·3 % between strains 61T and 77. Therefore, we propose the description of two novel species: Desulfovibrio ferrireducens (type strain 61T) and Desulfovibrio frigidus (type strain 18T). Both strains 61T and 18T are closely related to the undescribed Desulfovibrio sp. strain Aspo3 (respectively 97·4 and 95·4 % 16S rRNA gene sequence similarity) isolated from subterranean groundwater (Pedersen et al., 1996), as well as to Desulfovibrio hydrothermalis (95·7 and 95·0 %) isolated from a deep-sea hydrothermal chimney (Alazard et al., 2003), Desulfovibrio zosterae (94·8 and 94·3 %) isolated from marine seagrass roots (Nielsen et al., 1999) and Desulfovibrio salexigens (94·6 and 95 %) (Fig. 2).
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). Desulfovibrio hydrothermalis, Desulfovibrio zosterae and Desulfovibrio salexigens are mesophiles with temperature optima at 33–37 °C (Postgate, 1984; Nielsen et al., 1999; Alazard et al., 2003), whereas strains 18T, 61T and 77 are psychrotolerant (temperature optima at 20–23 °C) and able to grow at –2 °C. As the three strains were isolated from fjord sediments with temperatures of 2–3 °C at the time of sampling, they are able to grow at the permanently low in situ temperature of Svalbard sediments.
Description of Desulfovibrio ferrireducens sp. nov.
Desulfovibrio ferrireducens [fer.ri.re.du'cens. L. n. ferrum iron; L. part. adj. reducens leading back, bringing back and in chemistry converting to a different oxidation state; N.L. part. adj. ferrireducens reducing Fe(III) to Fe(II)].
Cells are vibrioid or sigmoid, 2·5–5·5x0·7 µm in size, motile by a single polar flagellum. Gram-negative. No vitamins are required for growth. Lactate, formate, hydrogen, ethanol, propanol, fumarate and succinate are oxidized with sulfate reduction. Sulfate, thiosulfate and sulfite serve as electron acceptors. Iron compounds [Fe(III) oxide and Fe(III) citrate] are reduced without growth. Disproportionation of malate, pyruvate and fumarate. Optimum NaCl concentration is 1–2·5 %, and growth occurs between 0·7 and 4 % NaCl; for MgCl2 the optimum concentration is between 0·02 and 2·5 % and growth occurs up to a concentration of 3·5 %. pH optimum is 7·1–7·5 and pH range is 6·3–7·5. Temperature optimum is 23 °C and growth range is between –2 and 30 °C. The DNA G+C content is 42·0 mol%.
The type strain, 61T (=DSM 16995T=JCM 12925T), was isolated from a permanently cold sediment of the west coast of Svalbard.
Description of Desulfovibrio frigidus sp. nov.
Desulfovibrio frigidus (fri'gi.dus. L. masc. adj. frigidus cold, referring to growth in the permanently cold sediment of Svalbard).
Cells are rod-shaped or vibrioid, 2–5x0·7 µm in size, motile by a single polar flagellum. Gram-negative. No vitamins are required for growth. Lactate, formate, hydrogen, ethanol, fumarate and alanine are oxidized with sulfate reduction; one strain oxidizes propanol. Sulfate and sulfite serve as electron acceptors; one strain reduces elemental sulfur. Iron compounds [Fe(III) oxide and Fe(III) citrate] are reduced without growth. Disproportionation of malate, pyruvate, fumarate and glucose is possible for one or the other strain. Growth range for NaCl and MgCl2 is different for the two strains, but the optimum NaCl concentration is 2–3 %, and growth occurs at 2–3·5 % NaCl; for MgCl2 the optimum concentration is around 0·4 % and growth occurs up to a concentration of 1·9 %. pH optimum is 7·1 and pH range is 6·9–7·5. Temperature optimum is at 20–23 °C and growth range is from –2 to 25 °C. The DNA G+C content is 43·3 mol%.
Strain 18T (=DSM 17176T=JCM 12924T) is the type strain. Strain 77 is a second strain of the species. Both strains were isolated from a permanently cold sediment at the west coast of Svalbard.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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