Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil

Abstract

A mesophilic sulfate-reducing enrichment culture growing anaerobically on crude oil was used as a model system to study which nutritional types of sulfate-reducing bacteria may develop on original petroleum constituents in oil wells, tanks, and pipelines. Chemical analysis of oil hydrocarbons during growth revealed depletion of toluene and o-xylene within 1 month and of m- xylene, o-ethyltoluene, m-ethyltoluene, m-propyltoluene, and m- isopropyltoluene within approximately 2 months. In anaerobic counting series, the highest numbers of CFU (6 x 106 to 8 x 106 CFU ml-1) were obtained with toluene and benzoate. Almost the same numbers were obtained with lactate, a substrate often used for detection of the vibrio-shaped, incompletely oxidizing Desulfovibrio sp. In the present study, however, lactate yielded mostly colonies of oval to rod-shaped, completely oxidizing, sulfate-reducing bacteria which were able to grow slowly on toluene or crude oil. Desulfovibrio species were detected only at low numbers (3 x 105 CFU ml-1). In agreement with this finding, a fluorescently labeled, 16S rRNA- targeted oligonucleotide probe described in the literature as specific for members of the Desulfovibrionaceae (suggested family) hybridized only with a small portion (<5%) of the cells in the enrichment culture. These results are consistent with the observation that known Desulfovibrio species do not utilize aromatic hydrocarbons, the predominant substrates in the enrichment culture. All known sulfate-reducing bacteria which utilize aromatic compounds belong to a separate branch, the Desulfobacteriaceae (suggested family). Most members of this family are complete oxidizers. For specific hybridization with members of this branch, the probe had to be modified by a nucleotide exchange. Indeed, this modified probe hybridized with more than 95% of the cells in the enrichment culture. The results show that completely oxidizing, alkylbenzene-utilizing sulfate-reducing bacteria rather than Desulfovibrio species have to be considered in attempts to understand the microbiology of sulfide production in oil wells, tanks, and pipelines when no electron donors other than the indigenous oil constituents are available.