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Applied and Environmental Microbiology, February 2007, p. 1239-1247, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.02264-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Situ Biosurfactant Production by Bacillus Strains Injected into a Limestone Petroleum Reservoir

Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma,¹ School of Petroleum and Geological Engineering, University of Oklahoma, Norman, Oklahoma²

Received 25 September 2006/ Accepted 6 December 2006

Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO₂ and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 ± 0.01 h^–1), carbon balances (107% ± 34%), biosurfactant production rates (0.02 ± 0.001 h^–1), and biosurfactant yields (0.015 ± 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.

Published ahead of print on 15 December 2006.