Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions

Abstract

Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the epsilon subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S(2-) + 4NO(3)(-) --> SO(4)(2-) + 4NO(2)(-) (Delta G(0) = -534 kJ mol(-1)). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.

FIG. 1.

Neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic positions of strains YK-1, YK-2, YK-3, and YK-4 in the ɛ subclass of the class Proteobacteria. Desulfovibrio desulfuricans was used as the out-group. Accession numbers of the sequences retrieved from the databases are given in parentheses. The numbers at the branch nodes are bootstrap values (per 100 trials); only values greater than 50 are shown. The scale bar indicates 0.026 substitutions per site.

FIG. 2.

Changes in ion concentrations (nitrate, ○; nitrite,⋄; sulfate, ▵) during chemolithotrophic growth of YK-1 (□) in MBM medium supplemented with bicarbonate (2 mM) at 25°C under the N₂ atmosphere, where sulfide (0.5 mM) was an electron donor and nitrate (1 mM) was an electron acceptor. Datum points and bars are means and standard deviations, respectively (n = 3).

FIG. 3.

Growth of strain YK-1 in the presence of an excess amount of crude oil. Media used were modified forms of MBM, and growth conditions are shown in the table below the panels. (A) Growth curve. (B) Changes in sulfate concentration. Panel B also presents sulfate concentrations under culture condition 1 without inoculation with YK-1 (⋄). Datum points and bars are means and standard deviations, respectively (n = 3).