Extremophiles (2015) 19:899–907 DOI 10.1007/s00792-015-0765-y ORIGINAL PAPER Desulfonatronobacter acetoxydans sp. nov.,: a first acetate‑oxidizing, extremely salt‑tolerant alkaliphilic SRB from a hypersaline soda lake D. Y. Sorokin1,2 · N. A. Chernyh1 · M. N. Poroshina3 Received: 6 May 2015 / Accepted: 26 May 2015 / Published online: 18 June 2015 © The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Recent intensive microbiological investigation alkaliphile, growing with butyrate at salinity up to 4 M total of sulfidogenesis in soda lakes did not result in isolation of Na+ with a pH optimum at 9.5. It can grow with sulfate as any pure cultures of sulfate-reducing bacteria (SRB) able to e-acceptor with C3–C9 VFA and also with some alcohols. The directly oxidize acetate. The sulfate-dependent acetate oxida- most interesting property of strain APT3 is its ability to grow tion at haloalkaline conditions has, so far, been only shown in with acetate as e-donor, although not with sulfate, but with two syntrophic associations of novel Syntrophobacteraceae sulfite or thiosulfate as e-acceptors. The new isolate is pro- members and haloalkaliphilic hydrogenotrophic SRB. In the posed as a new species Desulfonatronobacter acetoxydans. course of investigation of one of them, obtained from a hyper- saline soda lake in South-Western Siberia, a minor component Keywords Soda lakes · Haloalkaliphilic · Acetate was observed showing a close relation to Desulfonatrono- oxidation · Sulfate-reducing bacteria (SRB) · bacter acidivorans—a “complete oxidizing” SRB from soda Desulfobacteracea lakes. This organism became dominant in a secondary enrich- ment with propionate as e-donor and sulfate as e-acceptor. A Introduction pure culture, strain APT3, was identified as a novel member of the family Desulfobacteraceae. It is an extremely salt-tolerant During the last two decades, intensive microbiological and, recently, also molecular ecology investigation of naturally Communicated by A. Oren. occurring saline, alkaline (soda) lakes resulted in a wealth of information on functional structure of natronophilic microbial Nucleotide sequence accession number: The GenBank/EMBL communities (last reviewed by Sorokin et al. 2014a). In par- accession numbers of the 16S rRNA and dsrAB gene sequences ticular, the microbial sulfur cycle received much attention as determined in this study are KP223254 and KP784416- KP784417, respectively. the most active in soda lakes (Sorokin et al. 2011). Relatively, high rates of sulfidogenesis have been detected in anaero- Electronic supplementary material The online version of this bic sediments even in hypersaline soda lakes and the actual article (doi:10.1007/s00792-015-0765-y) contains supplementary presence of SRB was confirmed by the analysis of both the material, which is available to authorized users. key functional genes of the dissimilatory sulfite reductase * D. Y. Sorokin (dsrAB) and, in some cases, also by the 16S rRNA gene anal- [email protected]; [email protected] ysis (Scholten et al. 2005; Foti et al. 2007, 2008; Mesbah and Wiegel 2012). Most of the SRB isolates and clones in soda 1 Winogradsky Institute of Microbiology, Russian Academy lakes belong to lithotrophic members of the order Desulfo- of Sciences, Prospect 60‑let Octyabrya 7/2, 117811 Moscow, Russia vibrionales (reviewed by Sorokin et al. 2011). Despite that molecular analysis demonstrated a presence of several clus- 2 Department of Biotechnology, Delft University of Technology, Delft, The Netherlands ters apparently belonging to the “complete oxidizing” SRB in the family Desulfobacteraceae, so far, only a single haloalka- 3 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Puschino, liphilic SRB, described as Desulfonatronobacter acidivorans, Russia has been found in soda lakes (Sorokin et al. 2012a). Although 1 3 900 Extremophiles (2015) 19:899–907 Fig. 1 Cell morphology (phase contrast microphotographs) of strains APT3 grown at 2 M Na+, pH 9.5 with thiosulfate as e-acceptor and propi- onate (a), butyrate (b), acetate (c) or caprylate (d) as e-donor it oxidized several volatile fatty acids (VFA) completely detected both in 16S-rRNA gene and dsrAB-targeted DGGE to CO2, no growth was observed with externally provided analyses (KF588528 and KF835258, respectively). Both acetate. Until now, the acetate oxidation at sulfate-reducing sequences clustered with Desulfonatronobacter acidivorans, conditions in soda lakes has only been demonstrated in two thus hinting at the presence of a “complete oxidizing” SRB syntrophic associations of reverse acetogenic members within in the acetate-oxidizing syntrophic culture. The present paper the clostridial family Syntrophomonadaceae and lithotrophic describes isolation and properties of this organism. SRB. At low salt concentrations, the association included ‘Candidatus Contubernalis alkalaceticum’ and Desulfona- tronum cooperativum (Zhilina et al. 2005), while ‘Candidatus Methods Syntrophonatronum acetioxidans’/Desulfonatronospira sp. ASO3-2 were able to oxidize acetate in nearly saturated soda Isolation source brines (Sorokin et al. 2014b). During the investigation of the latter at different stages of purification, a presence of a minor The source for the isolation was a syntrophic enrichment SRB component in the enrichment at 3 M total Na+ had been culture oxidizing acetate with sulfate as the e-acceptor at 1 3 Extremophiles (2015) 19:899–907 901 Fig. 2 Phylogenetic position (a) of strain APT3 based on 16S APT3 (KP223254) ●▲ rRNA gene (a), dsrA (b) and (KF588528) dsrB (c) sequence analysis. 81 clone, soda lake Bitter-1 (Kulunda Steppe) The bootstrap values above clone, soda lake Bitter-1 (Kulunda Steppe)(EF622456) 50 % from 1000 replicates are clone, hypersaline soil (India) (JX240718) shown next to the branches. The 51 er evolutionary distances were clone, alkaline Lonar Lake (India) (JQ739024) st 74 computed using the neighbour clone, haloalkaline lakes Wadi Natrun (Egypt) (DQ432412) joining and maximum likeli- clone, soda lake Tanatar-3(Kulunda Steppe) (EF622458) es clu hood methods and are in the units of the number of base clone, soda lake Tanatar-1 (Kulunda Steppe) (EF622452) 52 substitutions per site. Symbols 81 clone, soda lake Tanatar-1 (Kulunda Steppe) (EF622454) in (a): open circle, incomplete soda lak oxidizers; closed circle, com- clone, soda lake Tanatar-5 (Kulunda Steppe) (GU479049) plete oxidizers; closed triangle, Desulfonatronobacter acidivorans (GU289732) ● acetate oxidizers. Clones and 87 soda Mono Lake (California), clone ML28 (AY245462) pure cultures obtained from alkaline saline lakes are in bold Desulfosalsimonas propionicica (DQ067422) ● clone, hypersaline mat, (EU245702) Desulfatiglans anilini (EU020016) ●▲ Desulfonema limicola (U45990) ●▲ 52 Desulfococcus multivorans (AF418173) ●▲ Desulfatitalea tepidiphila (AB614135) ●▲ 71 Desulfosarcina variabilis (M34407) ●▲ Desulfatibacillum aliphaticivorans (AY184360) ●▲ Desulfoluna butyratoxydans (AB110540)○ 99 Desulfofrigus fragile (AF099065) ● 99 Desulfofrigus oceanense (AF099064) ●▲ Desulfofaba gelida (AF099063) ○ 61 Desulforegula conservatrix (AF243334) ○ Desulfatirhabdium butyrativorans (DQ146482) ● 99 Desulfobotulus sapovorans (FJ789839) ○ Desulfobotulus alkaliphilus (FJ788523)○ Desulfocella halophila (AF022936) ○ Desulfatiferula olefinivorans (DQ826724)○ 81 Desulfobacula toluolica (AJ441316) ● 97 Desulfobacula phenolica (AJ237606) ● 50 Desulfoconvexum algidum (EF442984)● 66 Desulfobacter postgatei (AF418180) ●▲ 94 Desulfospira joergensenii (X99637) ● 73 Desulfotignum phosphitoxidans (AF420288) ● 99 Desulfotignum balticum (AF418176) ●▲ 75 Desulfotignum toluenicum (NR 115992) ●▲ 0.02 pH 10 and 3 M Na+ inoculated with anaerobic sediments syntrophic acetate-oxidizing mixed culture obtained from from the hypersaline soda lake Bitter-1 in Kulunda Steppe, sulfidic sediments of hypersaline soda lake Bitter-1 in Altai, Russia (Sorokin et al. 2014b). Kulunda Steppe, Altai, Russia (Sorokin et al. 2014b) and maintained at 2–3 M total Na+, pH 10 and 30 °C. The basal Enrichment, isolation and cultivation conditions sodium carbonate-based mineral medium containing 3 M total Na+ was made by 1:1 mixing of the media contain- Anaerobic sub-enrichment with propionate as e-donor ing 2 and 4 M total Na+, pH 10, as described previously (10 mM) and sulfate (20 mM) was performed from the (Sorokin et al. 2011b). Routine cultivation was performed 1 3 902 Extremophiles (2015) 19:899–907 Fig. 2 continued (b) 98 Desulfosarcina cetonica (AAN31398) Desulfosarcina variabilis (AAK58134) Desulfonema limicola (AAC24099) BAM15573 Desulfatitalea tepidiphila EPR41197 Desulfococcus multivorans WP 028323468 Desulfatirhabdium butyrativorans APT3 (KP784416) Desulfosalsimonas propionicica (ABD46860) Desulfonatronobacter acidivorans (KF835253) Desulfatiglans anilini (WP 028320139) Desulforegula conservatrix (WP 027357910) 63 Desulfocella halophila (AAL57473) 52 Desulfobotulus sapovorans (AAC24109) Desulfatiferula olefinivorans (ABH03459) Desulfofaba gelida (AAK83201) Desulfobacter postgatei (WP 004072374) 98 Desulfobacula phenolica (AAQ05965) 100 Desulfobacula toluolica (CCK78725) 80 Desulfospira joergensenii (WP 022667229) 95 Desulfoconvexum algidum (AFJ95152) 79 Desulfotignum balticum (AAN31406) 0.02 94 Desulfotignum phosphitoxidans (AAN31400) (c) 72 clone, soda lake Tanatar-2, Kulunda Steppe (ABO28290) 74 clone, soda lake Tanatar-1, Kulunda Steppe (ABO28292) 77 clone, soda lake Elongated, Kulunda