Mechanistic Study of Microbial Control of Hydrogen Sulfide Production in Oil Reservoirs M. Nemati, 1 G. E. Jenneman, 2 G. Voordouw 1 1 Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada, T2N 1N4; telephone: 1 (403) 2203138; fax: 1 (403) 2899311; e-mail: nemati@ucalgary.ca 2 Phillips Petroleum Company, 224 GB, Bartlesville, Oklahoma 74004, USA Received 26 October 2000; accepted 2 March 2001 Abstract: Microbial control of biogenic production of hy- drogen sulfide in oil fields was studied in a model system consisting of pure cultures of the nitrate-reducing, sul- fide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in microbial cul- tures enriched from produced water of a Canadian oil reservoir. The presence of nitrate at concentrations up to 20 mM had little effect on the rate of sulfate reduction by a pure culture of Lac6. Addition of CVO imposed a strong inhibition effect on production of sulfide. In the absence of added nitrate SRB we were able to overcome this ef- fect after an extended lag phase. Simultaneous addition of CVO and nitrate stopped the production of H 2 S imme- diately. The concentration of sulfide decreased to a neg- ligible level due to nitrate-dependent sulfide oxidation activity of CVO. This was not prevented by raising the concentration of Na-lactate, the electron donor for sul- fate reduction. Similar results were obtained with enrich- ment cultures. Enrichments of produced water with sul- fide and nitrate were dominated by CVO, whereas enrich- ments with sulfate and Na-lactate were dominated by SRB. Addition of an NR-SOB enrichment to an SRB en- richment inhibited the production of sulfide. Subsequent addition of sufficient nitrate caused the sulfide concen- tration to drop to zero. A similar response was seen in the presence of nitrate alone, although after a pro- nounced lag time, it was needed for emergence of a siz- able CVO population. The results of the present study show that two mechanisms are involved in microbial control of biogenic sulfide production. First, addition of NR-SOB imposes an inhibition effect, possibly by in- creasing the environmental redox potential to levels which are inhibitory for SRB. Second, in the presence of sufficient nitrate, NR-SOB oxidize sulfide, leading to its complete removal from the environment. Successful mi- crobial control of H 2 S in an oil reservoir is crucially de- pendent on the simultaneous presence of NR-SOB (ei- ther indigenous population or injected) and nitrate in the environment. © 2001 John Wiley & Sons, Inc. Biotechnol Bio- eng 74: 424–434, 2001. Keywords: oil reservoir; hydrogen sulfide; souring; ni- trate; Thiomicrospira sp. strain CVO; reverse sample ge- nome probing INTRODUCTION Souring, the formation of H 2 S, often results when an oil reservoir is subjected to water flooding for secondary oil recovery. Souring increases the cost of oil production and is believed to be mediated by the activity of sulfate-reducing bacteria (SRB) in the reservoir. SRB are strict anaerobes, which use hydrogen or simple organic acids or alcohols as electron donors for sulfate reduction. These are formed as end-products of fermentative metabolism or through chemi- cal transformation of buried organic matter at high tempera- ture and pressure (Shock, 1988). Recent studies have shown that hydrocarbons in petroleum may also serve as electron donors in bioreduction of sulfate. Toxicity of H 2 S, acceler- ated corrosion rates, and decreased efficiency of secondary oil recovery due to plugging of the reservoir by SRB bio- mass and precipitated metal sulfides are some of the prob- lems associated with souring. This dictates control of H 2 S production, of its release into the environment, and of its contact with iron and steel parts in tanks, pipelines, valves, and pumps. Different approaches can be used to control sulfide contamination. Sulfide production can be reduced by inhibiting SRB activity by treatment of injection water with biocides such as glutaraldehyde, cocodiamines, or molyb- dates. The use of biocide is most successful in controlling SRB in surface facilities but is of limited effectiveness in the reservoir, because chemical components in the reservoir may scavenge biocide through reaction (Reinsel et al., 1996). Elimination of sulfate from the injection water is another option to control SRB activity (Bakke et al., 1992). It is also possible to remove H 2 S from sour water by treat- ment with physicochemical methods (Montgomery et al., 1990). Finally, sulfide production can be controlled micro- bially. For this option it is important to note that oil fields at moderate subsurface depth and a moderate resident tem- perature harbor a complex microbial community. Stimula- tion and control of the metabolism of this community can prevent or revert souring (Telang et al., 1997). Addition of nitrate and/or nitrite and introduction of nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) such as Thiobacillus denitrificans in injection water have been shown to control sulfide accumulation (Jenneman et al., 1986, 1997; McIn- Correspondence to: M. Nemati Contract grant sponsors: the Natural Sciences and Engineering Research Council of Canada and the Phillips Petroleum Company. © 2001 John Wiley & Sons, Inc.