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Applied and Environmental Microbiology, April 2002, p. 1516-1523, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1516-1523.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer

Jutta Kleikemper,1* Martin H. Schroth,1 William V. Sigler,1 Martina Schmucki,1 Stefano M. Bernasconi,2 and Josef Zeyer1

Institute of Terrestrial Ecol., Swiss Federal Institute of Technology Zürich, CH-8952 Schlieren,1 Geological Institute, Swiss Federal Institute of Technology Zürich, CH-8092 Zürich, Switzerland2

Received 21 September 2001/ Accepted 25 January 2002

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO42- reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day-1 for sulfate reduction and from 0.13 to 0.60 day-1 for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.

* Corresponding author. Mailing address: Institute of Terrestrial Ecol.—Soil Biology, Swiss Federal Institute of Technology Zürich (ETHZ), CH-8952 Schlieren, Switzerland. Phone: 41-1-633-6047. Fax: 41-1-633-1122. E-mail: kleikemper{at}ito.umnw.ethz.ch.

Applied and Environmental Microbiology, April 2002, p. 1516-1523, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1516-1523.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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