STABLE ISOTOPE RATIOS PROVIDE INSIGHTS INTO MECHANISMS OF MICROBIAL SOURING CONTROL IN OIL FIELDS USING NITRATE OR NITRITE

During bacterial sulfate reduction (BSR), sulfate-reducing bacteria (SRB) exhibit isotope fractionation whereby isotopically light 32S and 16O in sulfate are preferentially metabolized leaving the remaining sulfate enriched in 34S and/or 18O. Sulfide production during BSR is harmful in many environments and can be controlled by stimulating nitrate or nitrite reducing bacteria (NRB). Chemoorganotrophic NRB may utilize the same organic compounds that SRB use as electron donors for energy generation and microbial growth allowing NRB out-compete SRB. Chemolithotrophic NRB that oxidize sulfide as their electron donor can control BSR by direct sulfide removal. Both mechanisms achieve net removal of SRB electron donors with nitrate or nitrite, thus concentrations of organics, sulfate, sulfide, nitrate, nitrite, etc. do not indicate which mechanism is operating in experimental systems. We hypothesized that stable isotope ratios of sulfur (in sulfide and sulfate) and oxygen (in sulfate) would indicate an operative mechanism. Biocompetitive exclusion of SRB by NRB should result in typical Rayleigh type isotope fractionation with partial BSR determined by nitrate or nitrite doses. Isotope fractionation when chemolithotrophic sulfide oxidizing NRB are involved should be more complex due to the occurrence of successive rounds of partial BSR and partial sulfide oxidation. Nitrate or nitrite were added in stepwise amounts to packed-bed bioreactors with established biofilms of SRB, resulting in sulfide elimination. Sulfate del 34S values in the media were always around +6‰, with increasing nitrate or nitrite doses resulting in sulfate enriched in 34S (up to +16‰) and sulfide depleted in 34S (down to -14‰). At high nitrate or nitrite doses sulfide was eliminated and sulfate del 34S values returned to +6‰, consistent with a Rayleigh fractionation pattern. Sulfate del 18O values in media were always around +8‰. Unlike sulfur, del 18O levels did not vary significantly, regardless of nitrate or nitrite treatment. Chemolithotrophic oxidation of sulfide should incorporate water-oxygen resulting in a del 18O value of -18‰ in newly formed sulfate. Constant del 18O values of sulfate around +8‰ suggest that competition for electron donors between chemoorganotrophic NRB and SRB resulted in the exclusion of SRB at high nitrate or nitrite doses in these experiments.