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

Abstract

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.

FIG. 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.

FIG. 2.

Average activity of hNRB (A) and soNRB (B) in produced waters from the Enermark field as a function of time. The averages are for 11 PWs. The standard deviation indicates the distribution of values observed at each time point. The line drawn is a moving average for two time points.

FIG. 3.

DGGE of bacterial 16S amplicons obtained from microbial community DNAs isolated from the Enermark field. (b to i) DNAs from produced waters collected on 15 May 2007. The number of the production well is indicated at the bottom. (j to o) DNAs from hNRB enrichments grown from produced waters collected on 19 December 2007. (p to x) DNAs from soNRB enrichments grown from produced waters collected on 19 December 2007 (p to r) or 10 July 2007 (s to x). Lane a is a mixture of amplicons from markers. Only the band for Thauera sp. strain N2 is visible. All bands tagged with a number were isolated from the gel and sequenced.

FIG. 4.

Phylogenetic tree of anammox sequences retrieved from the Enermark field. DNA was isolated from 15-PW produced water and amplified with anammox-specific primers. The PCR product was cloned, and 16 clones were sequenced. Of these, 11 were closely associated with known anammox species, as shown. “Brocadia anammoxidans,” “Brocadia fulgida,” “Kuenenia stuttgartiensis,” “Jettenia asiatica,” “Anammoxoglobus propionicus,” and “Scalindua brodae” are “Candidatus” species.

FIG. 5.

(A and B) Ammonium concentrations in injection water as a function of time for water leaving 1-WP (A) or water injected at 14-IW (B). (C) Relation between ammonium concentrations at 1-WP and 14-IW, which are connected by a pipeline (transit time, 1 day). (D) Nitrate concentration at 1-WP. A period of increased nitrate injection is marked (↔). The line in this panel has been drawn based on data presented previously (39).

FIG. 6.

Nitrate (A) and ammonium (B) concentrations at producing well 13-PW as a function of time. Nitrate broke through in week 41 due to injection of batches of high nitrate concentration at neighboring injector 14-IW from week 33 to week 101 (↔). Increased concentrations of nitrite and sulfate and zero sulfide were also observed at 13-PW during this period, as shown elsewhere (39).

FIG. 7.

(A to K) Ammonium concentration at individual PWs as a function of time. (L) Production water volume-averaged ammonium concentration for 11 PWs, calculated as described in the text. Lines representing a moving average for two adjacent data points and a linear best fit are shown. A period of increased field-wide nitrate injection is indicated (↔).

FIG. 8.

Change in the concentrations of nitrate (▪), nitrite (▴), and ammonium (⧫) with time in serum bottles containing 100 ml of medium with 3 mM VFA and 4 mM nitrate and inoculated with 8 ml of produced water as indicated.