Journal of Methods Microbiological Journal of Microbiological Methods 33 (1998) 255–262 A differential medium for the isolation and enumeration of sulfolane-degrading bacteria * E. Anne Greene, Phillip M. Fedorak Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada Accepted 17 June 1998 Abstract A differential medium for the detection of tetrahydrothiophene-1,1-dioxide (sulfolane)-degrading bacteria was developed. The basis of differentiation is pH; aerobic sulfolane biodegradation results in the production of acid. The medium contained 2.3 g of Plate Count Agar, 17 g of agar, 0.1 g of bromthymol blue and 1 g of sulfolane per litre of water. Agar plates were green. Bacterial growth resulted in the production of a blue colour; a yellow halo developed around colonies that used sulfolane. The sulfolane biodegradation abilities of 271 colonies isolated from the differential agar were tested in liquid medium with sulfolane as a sole carbon and sulfur source. Of 135 colonies that produced yellow halos (indicating sulfolane consumption on the plates), 93% degraded sulfolane in liquid culture. Of 136 colonies that did not produce yellow halos, 93% did not degrade sulfolane in liquid culture. This new medium was useful for the isolation and enumeration of sulfolane-degrading bacteria.  1998 Elsevier Science B.V. All rights reserved. Keywords: Aerobic; Bacteria; Biodegradation; Differential; Sulfolane; Tetrahydrothiophene-1,1-dioxide 1. Introduction the process. Because of its high water solubility, sulfolane has percolated through the soil and become Tetrahydrothiophene-1,1-dioxide (sulfolane) is a groundwater contaminant. The mobility of sul-  used as a physical solvent in the Sulfinol process, folane is considered to be very high (Luther et al., which removes H S from sour natural gas (Goar, 1998), thus, it is likely to contaminate surface waters 2  1971). The Sulfinol process is particularly effective or domestic water wells near sour gas plants. at high H S concentrations, therefore, it is often used Previous research has demonstrated that anaerobic 2 in western Canada, where natural gas reserves can sulfolane biodegradation is unlikely to contribute contain up to 35% H S. Sour gas treatment facilities significantly to attenuation of the compound at 2 have been in operation since the early 1960s (Fit- contaminated sites (Greene et al., 1998). Mixed zgerald and Richardson, 1966), and sulfolane has cultures of aerobic sulfolane-degrading bacteria, on been introduced into soils and aquifer sediments the other hand, have been demonstrated to mineralize through landfilling and spills since the inception of sulfolane to CO , sulfate and biomass (Greene and 2 Fedorak, unpublished results). Enumeration of * aerobic sulfolane-degrading bacteria at contaminated Corresponding author. Tel.: 1403 492 3670; fax: 1403 492 9234; e-mail: Phil.Fedorak@ualberta.ca sites could facilitate the estimation of the potential 0167-7012 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0167-7012(98)00059-1