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.