Characterization of a Novel Biocatalyst System for Sulfide Oxidation Charles McComas and Kerry L. Sublette* Center for Environmental Research & Technology, University of Tulsa, 600 S. College Ave., Tulsa, Oklahoma 74104 Gary Jenneman Phillips Petroleum Co., 224 Geoscience Bldg., Bartlesville, Oklahoma 74004 Greg Bala Idaho National Engineering and Environmental Laboratory, 2525 Freemont Ave., P.O. Box 1625, Idaho Falls, Idaho 83415 It has been demonstrated that an enrichment culture dominated by Thiomicrospira sp. CVO may be cultured on H 2 S(g) as an energy source under sulfide-limiting conditions in suspended culture with nitrate as the electron acceptor. Hydrogen sulfide (10,000 ppmv) was completely removed from the feed gas and oxidized to sulfate in <3 s of gas-liquid contacting time. Maximum loading of the biomass for sulfide oxidation was observed to be 5.8 mmol H 2 S/h-g biomass protein, comparable to that reported previously for Thiobacillus denitrificans under similar conditions. However, the enrichment culture was shown to be more tolerant of extremes in pH and elevated temperature than T. denitrificans. Coupled with a reported tolerance of CVO for up to 10% NaCl, these observations suggest that a CVO-based culture is potentially a more robust biocatalyst system for sulfide oxidation than cultures based on Thiobacilli. Introduction Thermogenic or biogenic hydrogen sulfide (H 2 S) con- taminates many oil and gas reservoirs, resulting in the production of fluids that must be treated prior to use (oil and natural gas) or disposed of (produced water). Pro- duced fluids containing H 2 S are said to be “sour” and represent a serious safety hazard in the oil and gas industry as well as a major cause of corrosion in produc- tion and processing equipment. Strategies for the control of H 2 S-related hazards and corrosion include suppression of biogenesis in reservoirs and processing equipment with biocides or treatment of produced fluids to remove and dispose of H 2 S. Surface treatment methods include stripping of H 2 S from its source stream with subsequent treatment of the stripper stream, caustic washing or scrubbing, precipitation, direct chemical oxidation methods that convert H 2 S to elemental sulfur or sulfate, and direct biological oxidation (1). Biological oxidation offers the potential of a sulfide- scavenging technology that is regenerable, selective for sulfides, nonhazardous, effective at low temperatures, and economical. A biological alternative for removing H 2 S in produced gas was proposed by Sublette in 1987 (2, 3) and later by Lee and Sublette (4) for removal of sulfide from sulfide- laden water using the lithoautotrophic bacterium Thio- bacillus denitrificans. This bacterium catalyzes the oxi- dation of H 2 S to sulfate. In the presence of oxygen the reaction proceeds according to the following equation: During anoxic operation, nitrate is used as the oxidant: The maximum specific activity for sulfide oxidation by T. denitrificans under aerobic conditions was 15.1-20.9 mmol H 2 S/h-g biomass and 5.4-7.6 mmol H 2 S/h-g bio- mass under anoxic conditions (5). When H 2 S feed rates exceeded the maximum specific oxidation rate for T. denitrificans, elemental sulfur accumulated. However, this condition was reversible, and within 2 to 3 h of suspension of the H 2 S feed the accumulated sulfur was oxidized to sulfate. However, a technical barrier to the use of T. denitrificans is the buildup of sulfate, which is inhibitory at concentrations in excess of 250 mM. This inhibition is not likely specific to sulfate but the result of an increase in overall ionic strength (6). Since oilfield brines can contain dissolved solids in excess of 10%, more salt-tolerant sulfide-oxidizing strains are needed. Another technical barrier is substrate inhibition, as T. denitrifi- cans is sensitive to increasing sulfide concentrations. A sulfide-tolerant strain, T. denitrificans strain F, was selected by Sublette and Woolsey (7) and tolerates up to 56 mg/L (1.7 mM) sulfide, and recently a T. denitrificans isolate was reported by Krishnakumar and Manilal (8) to tolerate as much as 400 mg/L (12.1 mM) sulfide. * kerry-sublette@utulsa.edu. HS - + 2O 2 w SO 4 2- + H + (1) 5HS - + 8NO 3 - + 3H + w 5SO 4 2- + 4N 2 + 4H 2 O (2) 439 Biotechnol. Prog. 2001, 17, 439-446 10.1021/bp0100169 CCC: $20.00 © 2001 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/29/2001