Ammonium concentrations in produced waters from a mesothermic oil field subjected to nitrate injection decrease through formation of denitrifying biomass and anammox activity.

Community analysis of a mesothermic oil field, subjected to continuous field-wide injection of nitrate to remove sulfide, with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes indicated the presence of heterotrophic and sulfide-oxidizing, nitrate-reducing bacteria (hNRB and soNRB). These reduce nitrate by dissimilatory nitrate reduction to ammonium (e.g., Sulfurospirillum and Denitrovibrio) or by denitrification (e.g., Sulfurimonas, Arcobacter, and Thauera). Monitoring of ammonium concentrations in producing wells (PWs) indicated that denitrification was the main pathway for nitrate reduction in the field: breakthrough of nitrate and nitrite in two PWs was not associated with an increase in the ammonium concentration, and no increase in the ammonium concentration was seen in any of 11 producing wells during periods of increased nitrate injection. Instead, ammonium concentrations in produced waters decreased on average from 0.3 to 0.2 mM during 2 years of nitrate injection. Physiological studies with produced water-derived hNRB microcosms indicated increased biomass formation associated with denitrification as a possible cause for decreasing ammonium concentrations. Use of anammox-specific primers and cloning of the resulting PCR product gave clones affiliated with the known anammox genera "Candidatus Brocadia" and "Candidatus Kuenenia," indicating that the anammox reaction may also contribute to declining ammonium concentrations. Overall, the results indicate the following: (i) that nitrate injected into an oil field to oxidize sulfide is primarily reduced by denitrifying bacteria, of which many genera have been identified by DGGE, and (ii) that perhaps counterintuitively, nitrate injection leads to decreasing ammonium concentrations in produced waters.

Sulfide remediation by pulsed injection of nitrate into a low temperature Canadian heavy oil reservoir.

Sulfide formation by oil field sulfate-reducing bacteria (SRB) can be diminished by the injection of nitrate, stimulating the growth of nitrate-reducing bacteria (NRB). We monitored the field-wide injection of nitrate into a low temperature (approximately 30 degrees C) oil reservoir in western Canada by determining aqueous concentrations of sulfide, sulfate, nitrate, and nitrite, as well as the activities of NRB in water samples from 3 water plants, 2 injection wells, and 15 production wells over 2 years. The injection water had a low sulfate concentration (approximately 1 mM). Nitrate (2.4 mM, 150 ppm) was added at the water plants. Its subsequent distribution to the injection wells gave losses of 5-15% in the pipeline system, indicating that most was injected. Continuous nitrate injection lowered the total aqueous sulfide output of the production wells by 70% in the first five weeks, followed by recovery. Batchwise treatment of a limited section of the reservoir with high nitrate eliminated sulfide from one production well with nitrate breakthrough. Subsequent, field-wide treatment with week-long pulses of 14 mM nitrate gave breakthrough at an additional production well. However, this trend was reversed when injection with a constant dose of 2.4 mM (150 ppm) was resumed. The results are explained by assuming growth of SRB near the injection wellbore due to sulfate limitation. Injection of a constant nitrate dose inhibits these SRB initially. However, because of the constant, low temperature of the reservoir, SRB eventually grow back in a zone further removed from the injection wellbore. The resulting zonation (NRB closest to and SRB further away from the injection wellbore) can be broken by batch-wise increases in the concentration of injected nitrate, allowing it to re-enter the SRB-dominated zone.

Competitive, microbially-mediated reduction of nitrate with sulfide and aromatic oil components in a low-temperature, western Canadian oil reservoir.

Fields from which oil is produced by injection of sulfate-bearing water often exhibit an increase in sulfide concentration with time (souring). Nitrate added to the injection water lowers the sulfide concentration by the action of sulfide-oxidizing, nitrate-reducing bacteria (SO-NRB). However, the injected nitrate can also be reduced with oil organics by heterotrophic NRB (hNRB). Aqueous volatile fatty acids (VFAs; a mixture of acetate, propionate, and butyrate) are considered important electron donors in this regard. Injection and produced waters from a western Canadian oil field with a low in situ reservoir temperature (30 degrees C) had only 0.1-0.2 mM VFAs. Amendment of these waters with nitrate gave therefore only partial reduction. More nitrate was reduced when 2% (v/v) oil was added, with light oil giving more reduction than heavy oil. GC-MS analysis of in vitro degraded oils and electron balance considerations indicated that toluene served as the primary electron donor for nitrate reduction. The differences in the extent of nitrate reduction were thus related to the toluene content of the light and heavy oil (30 and 5 mM, respectively). Reduction of nitrate with sulfide by SO-NRB always preceded that with oil organics by hNRB, even though microbially catalyzed kinetics with either electron donor were similar. Inhibition of hNRB by sulfide is responsible for this phenomenon. Injected nitrate will thus initially be reduced with sulfide through the action of SO-NRB. However, once sulfide has been eliminated from the near-injection wellbore region, oil organics will be targeted by the action of hNRB. Hence, despite the kinetic advantage of SO-NRB, the nitrate dose required to eliminate sulfide from a reservoir depends on the concentration of hNRB-degradable oil organics, with toluene being the most important in the field under study. Because the toluene concentration is lower in heavy oilthan in light oil, nitrate injection into a heavy-oil-producing field of low temperature is more likely to succeed in containing souring.

Cometabolic degradation of polychlorinated biphenyls at low temperature by psychrotolerant bacterium Hydrogenophaga sp. IA3-A.

A biphenyl-utilizing bacterium isolated from polychlorinated biphenyls (PCBs)-contaminated soils grew on tryptic soy at temperatures between 4 and 40 degrees C. The Gram-negative rod bacterium formed yellow colonies on nutrient agar and it denitrified nitrate to nitrogen. Analysis of cellular fatty acids showed that it was most closely related to Hydrogenophaga taeniospiralis. At 5 degrees C, biphenyl-grown cells cometabolically degraded di- and trichlorinated isomers of PCBs in 10 ppm of Aroclor 1248. At 30 degrees C, PCBs that were removed included a congener with four chlorine substituents. At 5 degrees C, cells transformed 2,4'-dichlorobiphenyl (2,4'-DCB) and accumulated ortho-chlorinated meta-cleavage product as a stable metabolite. Analysis of extracts of culture supernatant by gas chromatography-mass spectrometry indicated that products of transformation of 2,4'-DCB included 2- and 4-chlorobenzoic acid (2- and 4-CBA), suggesting that (chloro)biphenyl-degrading upper-pathway enzymes of the bacterium are active at low temperature. The bacterium Hydrogenophaga sp. IA3-A is a PCB-degrading psychrotolerant strain.

Isolation and characterization of a biphenyl-utilizing psychrotrophic bacterium, Hydrogenophaga taeniospiralis IA3-A, that cometabolize dichlorobiphenyls and polychlorinated biphenyl congeners in Aroclor 1221.

A psychrotrophic bacterium isolated from polychlorinated biphenyls (PCBs)-contaminated soil grew on biphenyl as sole carbon and energy source, and actively cometabolized PCBs at low temperature. Analysis of cellular fatty acids indicate that the bacterium is most closely related to Hydrogenophaga taeniospiralis. Resting cells incubated with 10 ppm of Aroclor 1221 at 5 or 30 degrees C for 48 h removed all mono-, most di-, and several trichlorobiphenyls. At 5 degrees C, removal of MCBs (monochlorobiphenyls) was between 63 to 89%, DCBs (dichlorobiphenyls) was between 30 to 78%, and TCBs (trichlorobiphenyls) was between 30 to 75%. At 30 degrees C, removal of MCBs was 100%, DCBs was between 30 to 100%, and TCBs was between 27 to 59%. Congeners with two or more ortho chlorine were generally resistant to degradation. However, removal of di-ortho plus para-substituted congeners at 30 degrees C and not at 5 degrees C, suggest that the presence of a para-chlorine enhanced the cometabolism of these congeners at 30 degrees C. Furthermore, after 72 h, resting cells removed 68 and 83% of 500 microM of 2,4'-dichlorobiphenyl (2,4'-DCB) and, 35 and 44% of 500 microM of 2,3-dichlorobiphenyl (2,3-DCB) at 5 and 30 degrees C, respectively. Analysis of metabolites by GC-MS indicates that the cometabolized 2,3-DCB was completely recovered as 2,3-chlorobenzoic acid (2,3-CBA), while the cometabolized 2,4'-DCB was not completely recovered as chlorobenzoic acid. To our knowledge, it is the first strain of Hydrogenophaga taeniospiralis found to degrade an organic pollutant, and also the first psychrotrophic strain of a member of the genus Hydrogenophaga to grow on biphenyl or cometabolize PCBs at low temperature. Results suggest that the bacterium has potential use in the bioremediation of PCB-contaminated sites in cold regions.