Isolation of Sulfate-Reducing Bacteria from the Terrestrial Deep Subsurface and Description of Desulfovibrio cavernae sp. nov.

Abstract

Deep subsurface sandstones in the area of Berlin (Germany) located 600 to 1060 m below the surface were examined for the presence of viable microorganisms. The in situ temperatures at the sampling sites ranged from 37 to 45 degrees C. Investigations focussed on sulfate-reducing bacteria able to grow on methanol and triethylene glycol, which are added as chemicals to facilitate the long-term underground storage of natural gas. Seven strains were isolated from porewater brines in the porous sandstone. Three of them were obtained with methanol (strains H1M, H3M, and B1M), three strains with triethylene glycol (strains H1T, B1T, and B2T) and one strain with a mixture of lactate, acetate and butyrate (strain H1-13). Due to phenotypic properties six isolates could be identified as members of the genus Desulfovibrio, and strain B2T as a Desulfotomaculum. The salt tolerance and temperature range for growth indicated that the isolates originated from the indigenous deep subsurface sandstones. They grew in mineral media reflecting the in situ ionic composition of the different brines, which contained 1.5 to 190 g NaCl x l(-1) and high calcium and magnesium concentrations. The Desulfovibrio strains grew at temperatures between 20 and 50 degrees C, while the Desulfotomaculum strain was thermophilic and grew between 30 and 65 degrees C. The strains utilized a broad spectrum of electron donors and acceptors. They grew with carbon compounds like lactate, pyruvate, formate, n-alcohols (C1-C5), glycerol, ethylene glycol, malate, succinate, and fumarate. Some strains even utilized glucose as electron donor and carbon source. All strains were able to use sulfate, sulfite and nitrate as electron acceptors. Additionally, three Desulfovibrio strains reduced manganese oxide, the Desulfotomaculum strain reduced manganese oxide, iron oxide, and elemental sulfur. The 16S rRNA analysis revealed that the isolates belong to three different species. The strains H1T, H3M and B1M could be identified as Desulfovibrio indonesiensis, and strain B2T as Desulfotomaculum geothermicum. The other Desulfovibrio strains (H1M, H1-13, and B1T) showed identical 16S rDNA sequences and similarities as low as 93% to their closest relative, Desulfovibrio aminophilusT. Therefore, these isolates were assigned to a new species, Desulfovibrio cavernae sp. nov., with strain H1M as the type strain.