international Journal of Systematic Bacteriology (1 999), 49, 1 63 1-1 643 Printed in Great Britain Psychrophi Iic sulf ate-reducing bacteria isolated from permanently cold Arctic marine sediments : description of Desulfofrigus oceanense gen. nov., sp. nov., Desulfofrigus fragile sp. nov., Desulfofaba gelida gen. nov., sp. nov., Desulfotalea psychrophila gen. nov., sp. nov. and Desulfotalea arctica sp. nov. Christian Knobtauch, Kerstin Sahm and Bo B. Jcbrgensen Author for correspondence: Christian Knoblauch. Tel: +49 421 2028 653. Fax: +49 421 2028 690. e-mail : [email protected] Max-PIa nc k-I nst it Ute for Five psychrophilic, Gram-negative, sulfate-reducing bacteria were isolated Mari ne M icro bio logy, from marine sediments off the coast of Svalbard. All isolates grew at the in Celsiusstr. 1, 28359 Bremen, Germany situ temperature of -1.7 "C. In batch cultures, strain PSv29l had the highest growth rate at 7 "C, strains ASV~~~and LSv54l had the highest growth rate at 10 "C, and strains LSv21Tand LS~514~had the highest growth rate at 18 "C. The new isolates used the most common fermentation products in marine sediments, such as acetate, propionate, butyrate, lactate and hydrogen, but only strain ASv26' was able to oxidize fatty acids completely to CO,. The new strains had growth optima at neutral pH and marine salt concentration, except for LSv54l which grew fastest with 1O/O NaCI. Sulfite and thiosulfate were used as electron acceptors by strains ASv26'. PSv29l and LSv54l, and all strains except PSv29' grew with Fe3+(ferric citrate) as electron acceptor. Chemotaxonomy based on cellular fatty acid patterns and menaquinones showed good agreement with the phylogeny based on 165 rRNA sequences. All strains belonged to the 6 subclass of Proteobacteria but had at least 9% evolutionary distance from known sulfate reducers. Due to the phylogenetic and phenotypic differences between the new isolates and their closest relatives, establishment of the new genera Desulfotalea gen. nov., Desulfofaba gen. nov. and Desulfofrigus gen. nov. is proposed, with strain ASv26' as the type strain of the type species Desulfofrigus oceanense sp. nov., LSv21Tas the type strain of Desulfofrigus fragile sp. nov., PSv2gTas the type strain of the type species Desulfofaba gelida sp. nov., LSvW as the type strain of the type species Desulfotalea psychrophila sp. nov. and LSv514l as the type strain of Desulfotalea arctica sp. nov. Keywords: sulfate-reducing bacteria, psychrophiles, chemotaxonomy, Arctic sediment, Svalbard INTRODUCTION organic carbon remineralization in marine sediments (Jargensen, 1982; Canfield et al., 1993; Nedwell et al., Sulfate reducers are responsible for up to 50% of the 1993). Acetate, propionate, lactate, butyrate and hydrogen, which are the major end-products of fer- ., .. .. , , , .. , . , . , , , .. .. , ,. , . .. .. .. .. , ., ., . , , , , ., . , , , , ., , , , , , ., . , , , , , , ., . , , ., .. , ., .. .. .. .. .. .. .. .. .. .. .. mentation, constitute their most important carbon and Abbreviations: ECL, equivalent chain-length; MK, menaquinone. energy substrates (Sorensen et al., 198 1 ; Christensen, The GenBank accession numbers for the 165 rDNA sequences of Desulfotalea arctica LSv5 14T, Desulfotalea psychrophila LSV~~~,Desulfo- 1984; Parkes et al., 1989). According to their nutrition, faba gelida PSv2gT, Desulfofrigus oceanense ASV~~~and Desulfofrigus sulfate-reducing bacteria can be separated into two fragile LSv2l are AF099061-AF099065, respectively. distinct groups. Lactate, hydrogen and propionate are 01129 0 1999 IUMS 1631 C. Knoblauch, K. Sahm and B. B. Jerrgensen the typical substrates for incompletely oxidizing medium (Widdel & Bak, 1992). The medium contained sulfate-reducing bacteria, which are mainly repre- (g 1-I) : NaC1, 26.4; MgSO, .7H,O, 6.8 ; MgC1,. 6H,O, 5.7; sented by Desulfovibrio and Desulfobulbus species. The CaC1,.2H,O, 1.5; KBr, 0.09; and KC1, 0.7. After auto- main end-product of their catabolism is acetate which claving, the medium was cooled under a gas mixture of CO,/N, (10/90, v/v) and the following components were they oxidize The major of do not further. substrates added: 50 ml of a NH,Cl (5 g 1-I) and KH,PO, (4 g 1-I) completely oxidizing sulfate-reducing bacteria like solution, 1 ml nonchelated trace element solution, 1 ml Desulfobacter, Desulfobacterium, Desulfococcus and selenite/tungstate solution, 1 in1 vitamin solution (modified Desulfosarcina strains, are fatty acids which are solution 6, with an additional 4 mg folic acid and 1.5 mg oxidized to CO, (Holt et al., 1994). Phylogenetically, lipoic acid per 100 ml), 1 ml thiamin solution, 1 ml vitamin most sulfate reducers belong to the 6 subclass of B,, solution, 1 ml riboflavin solution (25 mg 1-1 in 25 mM Proteobacteria. phosphate buffer, pH 3.2), 30 ml bicarbonate solution (1 M), 1 ml resazurin solution (1 g 1-l) and 1 ml sodium sulfide The natural environment of most sulfate reducers is solution (1 M). If necessary, the pH was adjusted with HCl cold, since 90% of the sea floor has temperatures or NaOH to 7.1-7.3. The medium was dispensed under an below 4 "C (Levitus & Boyer, 1994). Like other benthic atmosphere of COJN, (10/90, v/v) into sterile serum bottles bacteria, sulfate reducers must therefore be able to that were closed with black butyl rubber stoppers or into grow at low temperatures. However, nearly all the sterile 15 ml Hungate tubes. Before inoculating the medium, known isolates are mesophiles with a temperature dithionite (final concentration 150 pM) and the desired optimum at or above 30 "C and unable to grow below carbon source were added from sterile stock solutions. The dilution series were inoculated on board ship and trans- 4°C (Widdel & Bak, 1992). It was unclear whether ported back at 4 "C. In our laboratory, they were incubated those sulfate reducers active at low in situ temperatures at five different temperatures between 0 and 20 "C. For the are closely related to the known mesophiles or whether isolation of pure cultures, the modified deep agar dilution they represent members of new genera and species. technique (Isaksen & Teske, 1996) was applied, which Pure cultures were needed to understand their metab- protects temperature-sensitive organisms from overheating. olism and temperature adaptation as well as their Agar (Noble; DIFCO) was washed five times with distilled phylogeny. The first moderately psychrophilic sulfate- water (Widdel & Bak, 1992) before use. After three to four reducing species, Desulforhopalus vacuolatus, was iso- subsequent agar dilution series, 30 different pure cultures lated by Isaksen & Teske (1996) from a temperate were isolated from the 0, 4 and 10 "C enrichments. Stock estuary. In polar environments with permanent cultures were kept at the temperature used for isolation and temperatures around 0 "C, low-temperature-adapted transferred every 3-4 weeks to fresh medium. For the characterization of pure cultures, the saltwater medium should be the dominant organisms. The aim bacteria (Widdel & Bak, 1992) with a lower concentration of major the study isolate of present was to and describe the salts was used. This medium contained (g 1-I): NaCl, 20; most abundant low-temperature-adapted sulfate redu- Na,SO,, 4; MgC1,. 6H,O, 3 ; CaCl, .2H,O, 0.1 5; KBr, 0-09; cers from polar sediments. Special attention was paid and KCl, 0.5. After autoclaving, the medium was prepared to organisms oxidizing acetate, propionate, lactate and as described above. To prevent damage of temperature- butyr ate . sensitive cells, great care was taken to protect enrichments and pure cultures from temperatures above those used for isolation. METHODS Physiology and metabolism. The salt requirement for growth was monitored in media with 15 different NaCl concen- Sources of organisms. Arctic marine sediments at Svalbard trations between 0.2 and 5.8% (w/v) or 16 different were sampled in 1995 on a cruise with the RV 'Jan Mayen'. MgC1,. 6H,O concentrations between 0-0 and 7.0 Oh (w/v). Strains LSv21T, ASV~~~and PSV~~~ originated from The concentrations of all other salts, except the one being Hornsund sediment (76'58.2' N, 15'34.5' E) with a bottom tested, were kept constant. The vitamin demand of the water temperature of 2.6 'C. Strains LSV~~~and LS~514~ different strains was tested for at least ten subsequent were isolated from Storfjord sediment (77'33.0' N, transfers on medium without vitamins. The pH optimum 19'05.0' E) with a bottom water temperature of - 1.7 "C. was tested using media adjusted to 12 different pH values Further information about sampling sites are given in Glud between 4.9 and 9-1. The pH was adjusted in triplicate tubes et al. (1998). Desulforhopalus vacuolatus strain 1tklF' (= with HCl or NaOH and the tubes were inoculated. The DSM 9700T) was kindly provided by Kai Finster, Arhus, initial pH was measured in one tube and the remaining tubes Denmark; Desulfovibrio giganteus (DSM 4123) was were incubated. Sulfide was measured periodically during obtained from the Deutsche Sammlung von Mikro- the following six months. Growth with different electron organismen und Zellkulturen (DSMZ), Braunschweig, donors was tested with sulfate as electron acceptor. Tubes Germany. without an electron donor were inoculated and served as negative controls. Sulfide was measured periodically during Enrichment, isolation and cultivation. Sediment samples were the following year. Growth tests on different electron collected with a multicorer and subsampled directly on the acceptors were made in sulfate-free medium which was deck of the ship at an ambient temperature of 2-7 "C. supplied with the carbon source used for isolation of the Subcores were sliced in an anaerobic glove bag and samples tested strain and either thiosulfate (10 mM), elemental from five sediment depths between the surface and 30cm sulfur, nitrate (5 mM), nitrite (2 mM), iron(II1) oxy- were transferred to 90 ml sterile artificial seawater medium. hydroxide or iron(II1) citrate (30 mM). Amorphous iron(II1) These samples were suspended for 2 min with a vortex mixer oxyhydroxide was prepared by titration of an acidic FeC1, and further diluted in 15 ml Hungate tubes containing 10 ml solution (0.5 M) with NaOH (2 M) to pH 7.0.