Oil Field Souring Control by Nitrate-Reducing Sulfurospirillum spp. That Outcompete Sulfate-Reducing Bacteria for Organic Electron Donors

Nitrate injection into oil reservoirs can prevent and remediate souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Nitrate stimulates nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) and heterotrophic nitrate-reducing bacteria (hNRB) that compete with SRB for degradable oil organics. Up-flow, packed-bed bioreactors inoculated with water produced from an oil field and injected with lactate, sulfate, and nitrate served as sources for isolating several NRB, including Sulfurospirillum and Thauera spp. The former coupled reduction of nitrate to nitrite and ammonia with oxidation of either lactate (hNRB activity) or sulfide (NR-SOB activity). Souring control in a bioreactor receiving 12.5 mM lactate and 6, 2, 0.75, or 0.013 mM sulfate always required injection of 10 mM nitrate, irrespective of the sulfate concentration. Community analysis revealed that at all but the lowest sulfate concentration (0.013 mM), significant SRB were present. At 0.013 mM sulfate, direct hNRB-mediated oxidation of lactate by nitrate appeared to be the dominant mechanism. The absence of significant SRB indicated that sulfur cycling does not occur at such low sulfate concentrations. The metabolically versatile Sulfurospirillum spp. were dominant when nitrate was present in the bioreactor. Analysis of cocultures of Desulfovibrio sp. strain Lac3, Lac6, or Lac15 and Sulfurospirillum sp. strain KW indicated its hNRB activity and ability to produce inhibitory concentrations of nitrite to be key factors for it to successfully outcompete oil field SRB.     

Competitive oxidation of volatile fatty acids by sulfate- and nitrate-reducing bacteria from an oil field in Argentina

Acetate, propionate, and butyrate, collectively referred to as volatile fatty acids (VFA), are considered among the most important electron donors for sulfate-reducing bacteria (SRB) and heterotrophic nitrate-reducing bacteria (hNRB) in oil fields. Samples obtained from a field in the Neuquén Basin, western Argentina, had significant activity of mesophilic SRB, hNRB, and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). In microcosms, containing VFA (3 mM each) and excess sulfate, SRB first used propionate and butyrate for the production of acetate, which reached concentrations of up to 12 mM prior to being used as an electron donor for sulfate reduction. In contrast, hNRB used all three organic acids with similar kinetics, while reducing nitrate to nitrite and nitrogen. Transient inhibition of VFA-utilizing SRB was observed with 0.5 mM nitrite and permanent inhibition with concentrations of 1 mM or more. The addition of nitrate to medium flowing into an upflow, packed-bed bioreactor with an established VFA-oxidizing SRB consortium led to a spike of nitrite up to 3 mM. The nitrite-mediated inhibition of SRB led, in turn, to the transient accumulation of up to 13 mM of acetate. The complete utilization of nitrate and the incomplete utilization of VFA, especially propionate, and sulfate indicated that SRB remained partially inhibited. Hence, in addition to lower sulfide concentrations, an increase in the concentration of acetate in the presence of sulfate in waters produced from an oil field subjected to nitrate injection may indicate whether the treatment is successful. The microbial community composition in the bioreactor, as determined by culturing and culture-independent techniques, indicated shifts with an increasing fraction of nitrate. With VFA and sulfate, the SRB genera Desulfobotulus, Desulfotignum, and Desulfobacter as well as the sulfur-reducing Desulfuromonas and the NR-SOB Arcobacter were detected. With VFA and nitrate, Pseudomonas spp. were present. hNRB/NR-SOB from the genus Sulfurospirillum were found under all conditions.     

Containment of biogenic sulfide production in continuous up-flow packed-bed bioreactors with nitrate or nitrite

Produced water from the Coleville oil field in Saskatchewan, Canada was used to inoculate continuous up-flow packed-bed bioreactors. When 7.8 mM sulfate and 25 mM lactate were present in the in-flowing medium, H(2)S production (souring) by sulfate-reducing bacteria (SRB) was prevented by addition of 17.5 mM nitrate or 20 mM nitrite. Changing the sulfate or lactate concentration of the in-flowing medium indicated that the concentrations of nitrate or nitrite required for containment of souring decreased proportionally with a lowered concentration of the electron donor lactate, while the sulfate concentration of the medium had no effect. Microbial communities were dominated by SRB. Nitrate addition did not give rise to changes in community composition, indicating that lactate oxidation and H(2)S removal were caused by the combined action of SRB and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). Apparently the nitrite concentrations formed by these NR-SOB did not inhibit the SRB sufficiently to cause community shifts. In contrast, significant community shifts were observed upon direct addition of high concentrations (20 mM) of nitrite. Strains NO3A and NO2B, two newly isolated, nitrate-reducing bacteria (NRB) emerged as major community members. These were found to belong to the epsilon-division of the Proteobacteria, to be most closely related to Campylobacter lari, and to oxidize lactate with nitrate or nitrite as the electron acceptor. Thus the mechanism of microbial H(2)S removal in up-flow packed-bed bioreactors depended on whether nitrate (SRB/NR-SOB) or nitrite (SRB/NR-SOB as well as NRB) was used. However, the amount of nitrate or nitrite needed to completely remove H(2)S was dictated by the electron donor (lactate) concentration, irrespective of mechanism.     

Nitrite reductase activity of sulphate-reducing bacteria prevents their inhibition by nitrate-reducing,sulphide-oxidizing bacteria

Sulphate-reducing bacteria (SRB) can be inhibited by nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB), despite the fact that these two groups are interdependent in many anaerobic environments. Practical applications of this inhibition include the reduction of sulphide concentrations in oil fields by nitrate injection. The NR-SOB Thiomicrospira sp. strain CVO was found to oxidize up to 15 mM sulphide, considerably more than three other NR-SOB strains that were tested. Sulphide oxidation increased the environmental redox potential (Eh) from -400 to +100 mV and gave 0.6 nitrite per nitrate reduced. Within the genus Desulfovibrio, strains Lac3 and Lac6 were inhibited by strain CVO and nitrate for the duration of the experiment, whereas inhibition of strains Lac15 and D. vulgaris Hildenborough was transient. The latter had very high nitrite reductase (Nrf) activity. Southern blotting with D. vulgaris nrf genes as a probe indicated the absence of homologous nrf genes from strains Lac3 and Lac6 and their presence in strain Lac15. With respect to SRB from other genera, inhibition of the known nitrite reducer Desulfobulbus propionicus by strain CVO and nitrate was transient, whereas inhibition of Desulfobacterium autotrophicum and Desulfobacter postgatei was long-lasting. The results indicate that inhibition of SRB by NR-SOB is caused by nitrite production. Nrf-containing SRB can overcome this inhibition by further reducing nitrite to ammonia, preventing a stalling of the favourable metabolic interactions between these two bacterial groups. Nrf, which is widely distributed in SRB, can thus be regarded as a resistance factor that prevents the inhibition of dissimilatory sulphate reduction by nitrite.     

Mechanistic study of microbial control of hydrogen sulfide production in oil reservoirs.

Microbial control of biogenic production of hydrogen sulfide in oil fields was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in microbial cultures 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 effect after an extended lag phase. Simultaneous addition of CVO and nitrate stopped the production of H2S immediately. The concentration of sulfide decreased to a negligible 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 sulfate reduction. Similar results were obtained with enrichment cultures. Enrichments of produced water with sulfide and nitrate were dominated by CVO, whereas enrichments with sulfate and Na-lactate were dominated by SRB. Addition of an NR-SOB enrichment to an SRB enrichment inhibited the production of sulfide. Subsequent addition of sufficient nitrate caused the sulfide concentration to drop to zero. A similar response was seen in the presence of nitrate alone, although after a pronounced lag time, it was needed for emergence of a sizable 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 increasing 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 microbial control of H2S in an oil reservoir is crucially dependent on the simultaneous presence of NR-SOB (either indigenous population or injected) and nitrate in the environment.     

Gene expression analysis of the mechanism of inhibition of Desulfovibrio vulgaris Hildenborough by nitrate-reducing, sulfide-oxidizing bacteria

Sulfate-reducing bacteria (SRB) are inhibited by nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) in the presence of nitrate. This inhibition has been attributed either to an increase in redox potential or to production of nitrite by the NR-SOB. Nitrite specifically inhibits the final step in the sulfate reduction pathway. When the NR-SOB Thiomicrospira sp. strain CVO was added to mid-log phase cultures of the SRB Desulfovibrio vulgaris Hildenborough in the presence of nitrate, sulfate reduction was inhibited. Strain CVO reduced nitrate and oxidized sulfide, with transient production of nitrite. Sulfate reduction by D. vulgaris resumed once nitrite was depleted. A DNA macroarray with open reading frames encoding enzymes involved in energy metabolism of D. vulgaris was used to study the effects of NR-SOB on gene expression. Shortly following addition of strain CVO, D. vulgaris genes for cytochrome c nitrite reductase and hybrid cluster proteins Hcp1 and Hcp2 were upregulated. Genes for sulfate reduction enzymes, except those for dissimilatory sulfite reductase, were downregulated. Genes for the membrane-bound electron transferring complexes QmoABC and DsrMKJOP were downregulated and unaffected, respectively, whereas direct addition of nitrite downregulated both operons. Overall the gene expression response of D. vulgaris upon exposure to strain CVO and nitrate resembled that observed upon direct addition of nitrite, indicating that inhibition of SRB is primarily due to nitrite production by NR-SOB.     

Corrosion risk associated with microbial souring control using nitrate or nitrite

Souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB) in oil reservoirs, can be controlled through nitrate or nitrite addition. To assess the effects of this containment approach on corrosion, metal coupons were installed in up-flow packed-bed bioreactors fed with medium containing 8 mM sulfate and 25 mM lactate. Following inoculation with produced water to establish biogenic H₂S production, some bioreactors were treated with 17.5 mM nitrate or up to 20 mM nitrite, eliminating souring. Corrosion rates were highest near the outlet of untreated bioreactors (up to 0.4 mm year^–1). Nitrate (17.5 mM) eliminated sulfide but gave pitting corrosion near the inlet of the bioreactor, whereas a high nitrite dose (20 mM) completely eliminated microbial activity and associated corrosion. More gradual, step-wise addition of nitrite up to 20 mM resulted in the retention of microbial activity and localized pitting corrosion, especially near the bioreactor inlet. We conclude that: (1) SRB control by nitrate or nitrite reduction shifts the corrosion risk from the bioreactor outlet to the inlet (i.e. from production to injection wells) and (2) souring treatment by continuous addition of a high inhibitory nitrite dose is preferable from a corrosion-prevention point of view.     

Impact of nitrate-mediated microbial control of souring in oil reservoirs on the extent of corrosion

The effect of microbial control of souring on the extent of corrosion was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in an SRB consortium enriched from produced water from a Canadian oil reservoir. The average corrosion rate induced by the SRB consortium (1.4 g x m(-2) x day(-1)) was faster than that observed in the presence of strain Lac6 (0.2 g x m(-2) x day(-1)). Examination of the metallic coupons at the end of the tests indicated a uniform corrosion in both cases. Addition of CVO and 10 mM nitrate to a fully grown culture of Lac6 or the SRB consortium led to complete removal of sulfide from the system and a significant increase in the population of CVO, as determined by reverse sample genome probing. In the case of the SRB consortium addition of just nitrate (10 mM) had a similar effect. When grown in the absence of nitrate, the consortium was dominated by Desulfovibrio sp. strains Lac15 and Lac29, while growth in the presence of nitrate led to dominance of Desulfovibrio sp. strain Lac3. The addition of CVO and nitrate to the Lac6 culture or nitrate to the SRB consortium accelerated the average corrosion rate to 1.5 and 2.9 g x m(-2) x day(-1), respectively. Localized corrosion and the occurrence of pitting were apparent in both cases. Although the sulfide concentration (0.5-7 mM) had little effect on corrosion rates, a clear increase of the corrosion rate with increasing nitrate concentration was observed in experiments conducted with consortia enriched from produced water.     

Chemical and microbiological changes in laboratory incubations of nitrate amendment "sour" produced waters from three western Canadian oil fields

Nitrate addition to oil field waters stops the biogenic formation of sulfide because the activities of nitrate-reducing bacteria (NRB) suppress the activities of sulfate-reducing bacteria (SRB). In general, there are two types of NRB — the heterotrophic NRB and the chemolithotrophic NRB. Within the latter group are the nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). To date, no study has specifically addressed the roles of these different NRB in controlling sulfide concentrations in oil field produced waters. This study used different culture media to selectively enumerate heterotrophic NRB and NR-SOB by most probable number (MPN) methods. Produced waters from three sulfide-containing western Canadian oil fields were amended with nitrate as an electron acceptor, but no exogenous electron donor was added to the serum bottle microcosms. Changes in the chemical and microbiological characteristics of the produced waters were monitored during incubation at 21°C. In less than 4 days, the sulfide was removed from the waters from two of the oil fields (designated P and C), whereas nearly 27 days were required for sulfide removal from the water from the third oil field (designated N). Nitrate addition stimulated large increases in the number of the heterotrophic NRB and NR-SOB in the waters from oil fields P and C, but only the NR-SOB were stimulated in the water from oil field N. These data suggest that stimulation of the heterotrophic NRB is required for rapid removal of sulfide from oil field-produced waters. Received 25 March 2002/ Accepted in revised form 10 June 2002     

Transformation of iron sulfide to greigite by nitrite produced by oil field bacteria

Nitrate, injected into oil fields, can oxidize sulfide formed by sulfate-reducing bacteria (SRB) through the action of nitrate-reducing sulfide-oxidizing bacteria (NR-SOB). When reservoir rock contains siderite (FeCO₃), the sulfide formed is immobilized as iron sulfide minerals, e.g. mackinawite (FeS). The aim of our study was to determine the extent to which oil field NR-SOB can oxidize or transform FeS. Because no NR-SOB capable of growth with FeS were isolated, the well-characterized oil field isolate Sulfurimonas sp. strain CVO was used. When strain CVO was presented with a mixture of chemically formed FeS and dissolved sulfide (HS⁻), it only oxidized the HS⁻. The FeS remained acid soluble and non-magnetic indicating that it was not transformed. In contrast, when the FeS was formed by adding FeCl₂ to a culture of SRB which gradually produced sulfide, precipitating FeS, and to which strain CVO and nitrate were subsequently added, transformation of the FeS to a magnetic, less acid-soluble form was observed. X-ray diffraction and energy-dispersive spectrometry indicated the transformed mineral to be greigite (Fe₃S₄). Addition of nitrite to cultures of SRB, containing microbially formed FeS, was similarly effective. Nitrite reacts chemically with HS⁻ to form polysulfide and sulfur (S⁰), which then transforms SRB-formed FeS to greigite, possibly via a sulfur addition pathway (3FeS + S⁰ → Fe₃S₄). Further chemical transformation to pyrite (FeS₂) is expected at higher temperatures (>60°C). Hence, nitrate injection into oil fields may lead to NR-SOB-mediated and chemical mineral transformations, increasing the sulfide-binding capacity of reservoir rock. Because of mineral volume decreases, these transformations may also increase reservoir injectivity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Control of microbial souring by nitrate,nitrite or glutaraldehyde injection in a sandstone column

Microbial souring (production of hydrogen sulfide by sulfate-reducing bacteria, SRB) in crushed Berea sandstone columns with oil field-produced water consortia incubated at 60°C was inhibited by the addition of nitrate (NO₃) or nitrite (NO ₂ ^– ). Added nitrate (as nitrogen) at a concentration of 0.71 mM resulted in the production of 0.57–0.71 mM nitrite by the native microbial population present during souring and suppressed sulfate reduction to below detection limits. Nitrate added at 0.36 mM did not inhibit active souring but was enough to maintain inhibition if the column had been previously treated with 0.71 mM or greater. Continuous addition of 0.71–0.86 mM nitrite also completely inhibited souring in the column. Pulses of nitrite were more effective than the same amount of nitrite added continuously. Nitrite was more effective at inhibiting souring than was glutaraldehyde, and SRB recovery was delayed longer with nitrite than with glutaraldehyde. It was hypothesized that glutaraldehyde killed SRB while nitrite provided a long-term inhibition without cell death. Removal of nitrate after as long as 3 months of continuous addition allowed SRB in a biofilm to return to their previous level of activity. Inhibition was achieved with much lower levels of nitrate and nitrite, and at higher temperatures, than noted by other researchers.     

Sulfide persistence in oil field waters amended with nitrate and acetate

Nitrate amendment is normally an effective method for sulfide control in oil field-produced waters. However, this approach has occasionally failed to prevent sulfide accumulation, despite the presence of active nitrate-reducing bacterial populations. Here, we report our study of bulk chemical transformations in microcosms of oil field waters containing nitrate-reducing, sulfide-oxidizing bacteria, but lacking denitrifying heterotrophs. Amendment with combinations of nitrate, acetate, and phosphate altered the microbial sulfur and nitrogen transformations. Elemental sulfur produced by chemotrophic nitrate-reducing bacteria was re-reduced heterotrophically to sulfide. Ammonification, rather than denitrification, was the predominant pathway for nitrate reduction. The application of nitrite led to transient sulfide depletion, possibly due to higher rates of nitrite reduction. The addition of molybdate suppressed both the accumulation of sulfide and the heterotrophic reduction of nitrate. Therefore, sulfidogenesis was likely due to elemental sulfur-reducing heterotrophic bacteria, and the nitrate-reducing microbial community consisted mainly of facultatively chemotrophic microbes. This study describes one set of conditions for continued sulfidogenesis during nitrate reduction, with important implications for nitrate control of sulfide production in oil fields.     

Acetate Production from Oil under Sulfate-Reducing Conditions in Bioreactors Injected with Sulfate and Nitrate

Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266–269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus Desulfovibrio, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.     

Inhibition of microbial H<Subscript>2</Subscript>S production in an oil reservoir model column by nitrate injection

The effect of nitrate addition on microbial H2S production in a seawater-flooded oil reservoir model column with crude oil as carbon and energy source was investigated. Injection of 0.5 mM nitrate for 2.5-3.5 months led to complete elimination of H2S (initially 0.45-0.67 mM). The major decline in H2S level coincided with the first complete nitrate consumption and production of nitrite. When nitrate was excluded, H2S production resumed after approximately 2.5 months and reached previous levels after approximately 5 months. Using a fluorescent antibody technique, three populations each of sulfate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB) were monitored. SRB dominated the anoxic zone prior to nitrate addition, comprising 64-93% of the total bacterial population. The monitored NRB constituted less than 6% and no increase was observed during nitrate addition (indicating that other, unidentified, NRB populations were present). After 1-3 months without significant H2S production (3.5-5.5 months with nitrate), the SRB population collapsed, the fraction being reduced to 9-25%. The dominant SRB strain in the column, which constituted on average 94% of the monitored SRB population, was partly/completely inhibited by 50/75 microM nitrite in batch culture tests. Similar nitrite concentrations (50-150 microM) were detected in the column when the H2S level declined, indicating that nitrite inhibition was the main cause of H2S elimination. The results from this study indicate that nitrate/nitrite can be used to prevent detrimental SRB activity in oil reservoirs.     

Molecular- and cultivation-based analyses of microbial communities in oil field water and in microcosms amended with nitrate to control H2S production

Nitrate injection into oil fields is an alternative to biocide addition for controlling sulfide production (‘souring’) caused by sulfate-reducing bacteria (SRB). This study examined the suitability of several cultivation-dependent and cultivation-independent methods to assess potential microbial activities (sulfidogenesis and nitrate reduction) and the impact of nitrate amendment on oil field microbiota. Microcosms containing produced waters from two Western Canadian oil fields exhibited sulfidogenesis that was inhibited by nitrate amendment. Most probable number (MPN) and fluorescent in situ hybridization (FISH) analyses of uncultivated produced waters showed low cell numbers (≤10³ MPN/ml) dominated by SRB (>95% relative abundance). MPN analysis also detected nitrate-reducing sulfide-oxidizing bacteria (NRSOB) and heterotrophic nitrate-reducing bacteria (HNRB) at numbers too low to be detected by FISH or denaturing gradient gel electrophoresis (DGGE). In microcosms containing produced water fortified with sulfate, near-stoichiometric concentrations of sulfide were produced. FISH analyses of the microcosms after 55 days of incubation revealed that Gammaproteobacteria increased from undetectable levels to 5–20% abundance, resulting in a decreased proportion of Deltaproteobacteria (50–60% abundance). DGGE analysis confirmed the presence of Delta- and Gammaproteobacteria and also detected Bacteroidetes. When sulfate-fortified produced waters were amended with nitrate, sulfidogenesis was inhibited and Deltaproteobacteria decreased to levels undetectable by FISH, with a concomitant increase in Gammaproteobacteria from below detection to 50–60% abundance. DGGE analysis of these microcosms yielded sequences of Gamma- and Epsilonproteobacteria related to presumptive HNRB and NRSOB (Halomonas, Marinobacterium, Marinobacter, Pseudomonas and Arcobacter), thus supporting chemical data indicating that nitrate-reducing bacteria out-compete SRB when nitrate is added.     

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.Nitrate is injected into oil fields to remedy souring (34, 37, 38), the reduction of sulfate to sulfide coupled to the oxidation of oil organics that is catalyzed by resident sulfate-reducing bacteria (SRB). Nitrate acts by stimulating heterotrophic nitrate-reducing bacteria (hNRB) and sulfide-oxidizing, nitrate-reducing bacteria (soNRB), collectively referred to as NRB. The former can compete with SRB for the same oil organics, whereas the latter remove produced sulfide by oxidation to sulfur and sulfate. Both groups reduce nitrate to nitrite and then to either N₂ or ammonium by denitrification or dissimilatory nitrate reduction to ammonium (DNRA), respectively (7, 13). The produced nitrite strongly inhibits dissimilatory sulfite reductase (Dsr), the enzyme responsible for sulfide production by SRB. Hence, nitrite can be regarded as a magic bullet, which targets SRB metabolism exactly where desired. Some SRB can overcome nitrite inhibition by an Nrf-type periplasmic nitrite reductase, which reduces nitrite to ammonium, preventing its inflow into the cytoplasm, where Dsr is located (10, 12).Oil fields are ideal windows into the subsurface, allowing monitoring of produced waters for the presence of chemical compounds and microbes that are active in the sulfur and nitrogen cycles (8, 9, 26, 28, 34, 37, 38). The Enermark Medicine Hat Glauconitic C field (the Enermark field) in southeastern Alberta, Canada, produces oil from a depth of 850 m (down-hole temperature of 30°C) through produced water reinjection (PWRI) (see Fig. ​Fig.1).1). In 2007, the water plants (WPs) had an output of approximately 2,500 m³ of injection water per day, of which 25% was make-up water (MW). The latter was mostly the purified and chlorinated water from the municipal sewage plant and is the only input of sulfate (4 to 5 mM) in the system, giving the injection water an average sulfate concentration of ∼1 mM. Although oil (1,000 m³/day) has been produced through PWRI since 2000, souring did not become a problem until 2006. To control souring, a 45% (wt/wt) calcium nitrate concentrate has been injected since May 7, 2007 (week 1), as follows: (i) continuous field-wide injection of 2.4 mM nitrate at the WPs, which is still going on today, (ii) application of batches of high nitrate concentration (1 h/week; peak concentration of 760 mM) at a single injection well (IW) (14-IW) from week 33 to 101, and (iii) field-wide injection of pulses of weekly alternating high (14 mM) or low (2.4 mM) nitrate concentrations at the WPs from week 64 to 96. Continuous nitrate injection lowered the sulfide concentration, but this was followed by a recovery (39). Zero sulfide at two PWs was obtained through batchwise or pulsed injection. The results indicated that continuous injection leads to microbial zonation (39), in which hNRB grow in the near-injection wellbore region (see Fig. ​Fig.1,1, zone A) whereas SRB grow deeper in the reservoir (see Fig. ​Fig.1,1, zone B). This causes injected nitrate to be primarily reduced by hNRB through oxidation of oil components, like toluene (20), without reaching the sulfide-producing zones deeper in the reservoir.Open in a separate windowFIG. 1.Schematic representation of oil production through PWRI. The oil-water mixture pumped up at producing wells (PW) is separated, and the water is piped to a water plant, where it is mixed with make-up water. The resulting injection water is injected at injection wells. Sampling points are indicated (*). Two points of nitrate injection are indicated at the WP and at a specific IW. The Enermark field had 3 MW sources, 3 WPs, 55 IWs, and 107 PWs. Many of these are horizontal wells (not shown). The oil-producing subsurface (for the Enermark field: depth, 850 m; resident temperature, 30°C) has been divided into zones A to C, thought to harbor different microbial groups as outlined in the text.The effect of nitrate injection on aqueous sulfide concentrations emerging in produced waters from the Enermark field has thus been extensively analyzed (39). We report here on the microbial community present in these waters during nitrate injection as determined by denaturing gradient gel electrophoresis (DGGE) and on the fate of the injected nitrate by monitoring ammonium concentrations in produced and injection waters.     

Effect of nitrate and nitrite on sulfide production by two thermophilic,sulfate-reducing enrichments from an oil field in the North Sea

Thermophilic sulfate-reducing bacteria (tSRB) can be major contributors to the production of H₂S (souring) in oil reservoirs. Two tSRB enrichments from a North Sea oil field, NS-tSRB1 and NS-tSRB2, were obtained at 58°C with acetate-propionate-butyrate and with lactate as the electron donor, respectively. Analysis by rDNA sequencing indicated the presence of Thermodesulforhabdus norvegicus in NS-tSRB1 and of Archaeoglobus fulgidus in NS-tSRB2. Nitrate (10 mM) had no effect on H₂S production by mid-log phase cultures of NS-tSRB1 and NS-tSRB2, whereas nitrite (0.25 mM or higher) inhibited sulfate reduction. NS-tSRB1 did not recover from inhibition, whereas sulfate reduction activity of NS-tSRB2 recovered after 500 h. Nitrite was also effective in souring inhibition and H₂S removal in upflow bioreactors, whereas nitrate was similarly ineffective. Hence, nitrite may be preferable for souring prevention in some high-temperature oil fields because it reacts directly with sulfide and provides long-lasting inhibition of sulfate reduction.     

The influence of nitrate on microbial processes in oil industry production waters

Sulfide accumulation due to bacterial sulfate reduction is responsible for a number of serious problems in the oil industry. Among the strategies to control the activity of sulfate-reducing bacteria (SRB) is the use of nitrate, which can exhibit a variety of effects. We investigated the relevance of this approach to souring oil fields in Oklahoma and Alberta in which water flooding is used to enhance oil recovery. SRB and nitrate-reducing bacteria (NRB) were enumerated in produced waters from both oil fields. In the Oklahoma field, the rates of sulfate reduction ranged from 0.05 to 0.16 μM S day⁻¹ at the wellheads, and an order of magnitude higher at the oil–water separator. Sulfide production was greatest in the water storage tanks in the Alberta field. Microbial counts alone did not accurately reflect the potential for microbial activities. The majority of the sulfide production appeared to occur after the oil was pumped aboveground, rather than in the reservoir. Laboratory experiments showed that adding 5 and 10 mM nitrate to produced waters from the Oklahoma and Alberta oil fields, respectively, decreased the sulfide content to negligible levels and increased the numbers of NRB. This work suggests that sulfate reduction control measures can be concentrated on aboveground facilities, which will decrease the amount of sulfide reinjected into reservoirs during the disposal of oil field production waters. Journal of Industrial Microbiology & Biotechnology (2001) 27, 80–86. Received 30 January 2001/ Accepted in revised form 30 June 2001     

Dynamics of corrosion rates associated with nitrite or nitrate mediated control of souring under biological conditions simulating an oil reservoir

Representative microbial cultures from an oil reservoir and electrochemical techniques including potentiodynamic scan and linear polarization were used to investigate the time dependent corrosion rate associated with control of biogenic sulphide production through addition of nitrite, nitrate and a combination of nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB) and nitrate. The addition of nitrate alone did not prevent the biogenic production of sulphide but the produced sulphide was eventually oxidized and removed from the system. The addition of nitrate and NR-SOB had a similar effect on oxidation and removal of sulphide present in the system. However, as the addition of nitrate and NR-SOB was performed towards the end of sulphide production phase, the assessment of immediate impact was not possible. The addition of nitrite inhibited the biogenic production of sulphide immediately and led to removal of sulphide through nitrite mediated chemical oxidation of sulphide. The real time corrosion rate measurement revealed that in all three cases an acceleration in the corrosion rate occurred during the oxidation and removal of sulphide. Amendments of nitrate and NR-SOB or nitrate alone both gave rise to localized corrosion in the form of pits, with the maximum observed corrosion rates of 0.72 and 1.4 mm year⁻¹, respectively. The addition of nitrite also accelerated the corrosion rate but the maximum corrosion rate observed following nitrite addition was 0.3 mm year⁻¹. Furthermore, in the presence of nitrite the extent of pitting was not as high as those observed with other control methods.