Enrichment and identification of biosurfactant-producing oil field microbiota utilizing electron acceptors other than oxygen and nitrate

• Two putative biosurfactant producers were enriched in anoxic conditions.
• Fermentative metabolism appeared to lead to biosurfactant production.
• Surface tension reduction was sufficient for enhancement of gas-driven oil recovery.
• Analysis of cpn60 amplicons was more informative than analysis of 16S rDNA amplicons.

Microorganisms indigenous to an oil reservoir were grown in media containing either sucrose or proteins in four steel vessels under anoxic conditions at 30 °C and 8.3 MPa for 30 days, to enrich biosurfactant producers. Fermentation of substrate was possible in the protein-containing medium and either fermentation or respiration through reduction of sulfate occurred in the sucrose-containing medium. Growth of microorganisms led to 3.4–5.4-fold surface tension reduction indicating production of biosurfactants in amounts sufficient for enhancement of gas-driven oil recovery. Analysis of sequenced cpn60 amplicons showed that Pseudomonas sp. highly similar to biosurfactant producing P. fluorescens and to Pseudomonas sp. strain TKP predominated, and a bacterium highly similar to biosurfactant producing Bacillus mojavensis was present in vessels. Analysis of 16S rDNA amplicons allowed only genus-level identification of these bacteria. Thus, cpn60-amplicon analysis was a more relevant tool for identification of putative biosurfactant producers than 16S rDNA-amplicon analysis.