Nitrate-reducing, Sulfide-oxidizing Bacteria Research Articles

Simultaneous use of nitrate and calcium peroxide to control sulfide and greenhouse gas emission in sewers

The sewer system is a significant source of hydrogen sulfide (H2S) and greenhouse gases which has attracted extensive interest from researchers. In this study, a novel combined dosing strategy using nitrate and calcium peroxide (CaO2) was proposed to simultaneously control sulfide and greenhouse gases, and its performance was evaluated in laboratory-scale reactors. Results suggested that the addition of nitrate and CaO2 improved the effectiveness of sulfide control. And the combination index method further proved that nitrate and CaO2 were synergistic in controlling sulfide. Meanwhile, the combination of nitrate and CaO2 substantially reduced greenhouse gas emissions, especially the carbon dioxide (CO2) and methane (CH4). The microbial analysis revealed that the combined addition greatly stimulated the accumulation of nitrate reducing-sulfide oxidizing bacteria (NR-SOB) that participate in anoxic nitrate-dependent sulfide oxidation, while the abundance of heterotrophic denitrification bacteria (hNRB) was reduced significantly. Moreover, the presence of oxygen and alkaline chemicals generated by CaO2 facilitated the inhibition of sulfate-reducing bacteria (SRB) activities. Therefore, the nitrate dosage was diminished significantly. On the other hand, the generated alkaline chemicals promoted CO2 elimination and inhibited the activities of methanogens, leading to a decrease of CO2 and CH4 fluxes, which facilitated elimination of greenhouse effects. The intermittent dosing test showed that the nitrate and CaO2 could be applied intermittently for sulfide removal. And the chemical cost of intermittent dosing strategy was reduced by 85 % compared to the continuous dosing nitrate strategy. Therefore, intermittent dosing nitrate combined with CaO2 is probably an effective and economical approach to control sulfide and greenhouse gases in sewer systems.

Characterization of eubacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) and Next Generation Sequencing (NGS) in a desulfurization biotrickling filter using progressive changes of nitrate and nitrite as final electron acceptors

Anoxic biotrickling filters (BTFs) represent a technology with high H2S elimination capacity and removal efficiencies widely studied for biogas desulfurization. Three changes in the final electron acceptors were made using nitrate and nitrite during an operating period of 520 days. The stability and performance of the anoxic BTF were maintained when a significant perturbation was applied to the system that involved the progressive change of nitrate to nitrite and vice versa. Here the impact of electron acceptor changes on the microbial community was characterized by denaturing gel gradient electrophoresis (DGGE) and next generation sequencing (NGS). Both platforms revealed that the community underwent changes during the perturbations but was resilient because the removal capacity did not significantly change. Proteobacteria and Bacteroidetes were the main Phyla and Sulfurimonas and Thiobacillus the main nitrate-reducing sulfide-oxidizing bacteria (NR-SOB) genera involved in the biodesulfurization process.

The presence of nitrate- and sulfate-reducing bacteria contributes to ineffectiveness souring control by nitrate injection

Nitrate injection has been widely used to minimize the production of biological hydrogen sulfide in oil and gas field industry, by controlling the growth of sulfate-reducing bacteria (SRB) chemically and biologically. This study aimed to investigate the changes in the bacterial community in response to nitrate addition used to control biological souring. Specifically, we examined the effect of nitrate addition in an artificial souring experiment, using diluted crude oil as substrate and electron donor. Desulfotignum sp. was the predominant SRB under all conditions tested. Addition of nitrate at the beginning (N0) repressed the growth of SRB, as revealed by chemical and bacterial community analysis, concomitant with significant growth of the nitrate-reducing bacteria (NRB) Thalassospira sp. Nitrate addition after SRB growth (at day 28, N28) successfully remediated the sulfide produced by SRB, but no significant reduction in sulfate was observed subsequently; moreover, the bacterial communities before and after nitrate addition remained identical. Isolation of Desulfotignum YB01 (D. YB01) proved the resistance of this predominant SRB in high nitrate environment. Simultaneous reduction of sulfate and nitrate by D. YB01 was also observed in this study. Therefore, the phenomenon in the N28 experiment might be the result of the role of Arcobacter sp. which are nitrate-reducing sulfide-oxidizing bacteria, and/or the ability of Desulfotignum sp. to reduce nitrate and/or nitrite as a stress response. Thus, SRB might persist after nitrate addition, potentially causing subsequent SRB outbreaks.

Kinetics of Indigenous Nitrate Reducing Sulfide Oxidizing Activity in Microaerophilic Wastewater Biofilms

Nitrate decreases sulfide release in wastewater treatment plants (WWTP), but little is known on how it affects the microzonation and kinetics of related microbial processes within the biofilm. The effect of nitrate addition on these properties for sulfate reduction, sulfide oxidation, and oxygen respiration were studied with the use of microelectrodes in microaerophilic wastewater biofilms. Mass balance calaculations and community composition analysis were also performed. At basal WWTP conditions, the biofilm presented a double-layer system. The upper microaerophilic layer (~300 μm) showed low sulfide production (0.31 μmol cm-3 h-1) and oxygen consumption rates (0.01 μmol cm-3 h-1). The anoxic lower layer showed high sulfide production (2.7 μmol cm-3 h-1). Nitrate addition decreased net sulfide production rates, caused by an increase in sulfide oxidation rates (SOR) in the upper layer, rather than an inhibition of sulfate reducing bacteria (SRB). This suggests that the indigenous nitrate reducing-sulfide oxidizing bacteria (NR-SOB) were immediately activated by nitrate. The functional vertical structure of the biofilm changed to a triple-layer system, where the previously upper sulfide-producing layer in the absence of nitrate split into two new layers: 1) an upper sulfide-consuming layer, whose thickness is probably determined by the nitrate penetration depth within the biofilm, and 2) a middle layer producing sulfide at an even higher rate than in the absence of nitrate in some cases. Below these layers, the lower net sulfide-producing layer remained unaffected. Net SOR varied from 0.05 to 0.72 μmol cm-3 h-1 depending on nitrate and sulfate availability. Addition of low nitrate concentrations likely increased sulfate availability within the biofilm and resulted in an increase of both net sulfate reduction and net sulfide oxidation by overcoming sulfate diffusional limitation from the water phase and the strong coupling between SRB and NR-SOB syntrophic relationship.

Bioreactor performance and functional gene analysis of microbial community in a limited-oxygen fed bioreactor for co-reduction of sulfate and nitrate with high organic input

Limited-oxygen mediated synergistic relationships between sulfate-reducing bacteria (SRB), nitrate-reducing bacteria (NRB) and sulfide-oxidizing bacteria (SOB, including nitrate-reducing, sulfide-oxidizing bacteria NR-SOB) were predicted to simultaneously remove contaminants of nitrate, sulfate and high COD, and eliminate sulfide generation. A lab-scale experiment was conducted to examine the impact of limited oxygen on these oxy-anions degradation, sulfide oxidation and associated microbial functional responses. In all scenarios tested, the reduction of both nitrate and sulfate was almost complete. When limited-oxygen was fed into bioreactors, S0 formation was significantly improved up to ∼70%. GeoChip 4.0, a functional gene microarray, was used to determine the microbial gene diversity and functional potential for nitrate and sulfate reduction, and sulfide oxidation. The diversity of the microbial community in bioreactors was increased with the feeding of limited oxygen. Whereas the intensities of the functional genes involved in sulfate reduction did not show a significant difference, the abundance of the detected denitrification genes decreased in limited oxygen samples. More importantly, sulfide-oxidizing bacteria may alter their populations/genes in response to limited oxygen potentially to function more effectively in sulfide oxidation, especially to elemental sulfur. The genes fccA/fccB from nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB), such as Paracoccus denitrificans, Thiobacillus denitrificans, Beggiatoa sp., Thiomicrospira sp., and Thioalkalivibrio sp., were more abundant under limited-oxygen condition.

GeoChip-based analysis of the microbial community functional structures in simultaneous desulfurization and denitrification process

The elemental sulfur (S0) recovery was evaluated in the presence of nitrate in two development models of simultaneous desulfurization and denitrification (SDD) process. At the loading rates of 0.9kg S/(m3·day) for sulfide and 0.4kg N/(m3·day) for nitrate, S0 conversion rate was 91.1% in denitrifying sulfide removal (DSR) model which was higher than in integrated simultaneous desulfurization and denitrification (ISDD) model (25.6%). A comprehensive analysis of functional diversity, structure and metabolic potential of microbial communities was examined in two models by using functional gene array (GeoChip 2.0). GeoChip data indicated that diversity indices, community structure, and abundance of functional genes were distinct between two models. Diversity indices (Simpson's diversity index (1/D) and Shannon–Weaver index (H′)) of all detected genes showed that with elevated influent loading rate, the functional diversity decreased in ISDD model but increased in DSR model. In contrast to ISDD model, the overall abundance of dsr genes was lower in DSR model, while some functional genes targeting from nitrate-reducing sulfide-oxidizing bacteria (NR-SOB), such as Thiobacillus denitrificans, Sulfurimonas denitrificans, and Paracoccus pantotrophus were more abundant in DSR model which were highly associated with the change of S0 conversion rate obtained in two models. The results obtained in this study provide additional insights into the microbial metabolic mechanisms involved in ISDD and DSR models, which in turn will improve the overall performance of SDD process.

Reduction of Net Sulfide Production Rate by Nitrate in Wastewater Bioreactors. Kinetics and Changes in the Microbial Community

Nitrate addition stimulated sulfide oxidation by increasing the activity of nitrate-reducing sulfide-oxidizing bacteria (NR-SOB), decreasing the concentration of dissolved H2S in the water phase and, consequently, its release to the atmosphere of a pilot-scale anaerobic biore- actor. The effect of four different concentrations of nitrate (0.12, 0.24, 0.50, and 1.00 mM) was investigated for a period of 3 days in relation to sulfide concentration in two bioreactors set up at Guadalete wastewater treatment plant (Jerez de la Frontera, Spain). Physicochemical variables were measured in water and air, and the activity of bacteria implicated in the sulfur and nitrogen cycles was analyzed in the biofilms and in the water phase of the bioreactors. Biofilms were a net source of sulfide for the water and gas phases (7.22±5.3 μmol s �1 ) in the absence of nitrate dos- ing. Addition of nitrate resulted in a quick (within 3 h) decrease of sulfide both in the water and atmospheric phases. Sulfide elimination efficiency in the water phase increased with nitrate concentrations following the Michaelis-Menten kinetics (Ks=0.63 mM NO3 � ). The end of nitrate addition resulted in a recovery or increase of initial net sulfide production in about 3 h. Addition of nitrate increased the activity of NR-SOB and decreased the activity of sulfate-reducing bacteria. Results con- firmed the role of NR-SOB on hydrogen sulfide con- sumption coupled with nitrate reduction and sulfate recycling, revealing Sulfurimonas denitrificans and Paracoccusdenitrificansas NR-SOB of great importance in this process.

Modeling and Simulation of Nitrate Injection for Reservoir Souring Remediation

Summary Reservoir souring refers to the onset of hydrogen sulfide (H2S) production during waterflooding. Besides health and safety issues, H2S content reduces the value of the produced hydrocarbon. Nitrate injection is an effective method to prevent the formation of H2S. Designing this process requires the modeling of a complicated set of biogeochemical reactions involved in the production of H2S and its inhibition. This paper describes the modeling and simulation of biological reactions associated with the injection of nitrate to inhibit reservoir souring. The model is implemented in a general-purpose adaptive reservoir simulator (GPAS). To the best of our knowledge, GPAS is the first field-scale reservoir simulator that models reservoir souring treatment. The basic mechanism in the biologically mediated generation of H2S is the reaction between sulfate in the injection water and fatty acids in the formation water in the presence of sulfate-reducing bacteria (SRB). There are proposed mechanisms that describe the effect of nitrate injection on souring remediation. Depending on the circumstances, more than one mechanism may occur at the same time. These mechanisms include the inhibitory effect of nitrite on sulfate reduction, the competition between SRB and nitrate-reducing bacteria (NRB), and the stimulation of nitrate-reducing sulfide-oxidizing bacteria (NR-SOB). For each mechanism, we specify the biological species and chemical components involved and determine the role of each component in the biological reaction. For every biological reaction, a set of ordinary differential equations along with differential equations for the transport of chemical and biological species are solved. The results of reported experiments in the literature are used to find the input parameters for field-scale simulations. This reservoir simulator can then predict the onset of reservoir souring and the effectiveness of nitrate injection and helps in the design of the process. The comprehensive modeling accounts for variation in biological system characteristics and reservoir conditions that affect the production and remediation of H2S.

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 (≤10(3) 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.

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 (FeCO(3)), 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 FeCl(2) 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 (Fe(3)S(4)). Addition of nitrite to cultures of SRB, containing microbially formed FeS, was similarly effective. Nitrite reacts chemically with HS(-) to form polysulfide and sulfur (S(0)), which then transforms SRB-formed FeS to greigite, possibly via a sulfur addition pathway (3FeS + S(0) --> Fe(3)S(4)). Further chemical transformation to pyrite (FeS(2)) is expected at higher temperatures (>60 degrees 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.

Nitrate-reducing, sulfide-oxidizing bacteria as microbial oxidants for rapid biological sulfide removal

The emission of hydrogen sulfide into the atmosphere of sewer systems induces the biological production of sulfuric acid, causing severe concrete corrosion. As a possible preventive solution, a microbial consortium of nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) was enriched in a continuously stirred tank reactor in order to develop a biological technique for the removal of dissolved sulfide. The consortium, dominated by Arcobacter sp., was capable of removing 99% of sulfide. Stable isotope fractioning of the sulfide indicated that the oxidation was a biological process. The capacity of the NR-SOB consortium for rapid removal of sulfide was demonstrated by using it as an inoculum in synthetic and real sewage. Removal rates up to 52 mg sulfide-S g VSS(-1) h(-1) were achieved, to our knowledge the highest removal rate reported so far for freshwater species in the absence of molecular oxygen. Further long-term incubation experiments revealed the capacity of the bacteria to oxidize sulfide without the presence of nitrate, suggesting that an oxidized redox reserve is present in the culture.

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.

Microbiology to Help Solve Our Energy Needs

Our society depends greatly on fossil fuels, and the environmental consequences of this are well known and include significant increases of the CO(2) concentration in the earth's atmosphere. Although microbiology has traditionally played only a minor role in fossil-fuel extraction, two novel key discoveries indicate that this may change. First, the realization that oil components can be converted to methane and CO(2) by methanogenic consortia in the absence of electron acceptors (oxygen, nitrate, sulfate) explains how much of the world's oil has been biodegraded in situ. In addition to inorganic nutrients, only water is needed for these methanogenic conversions. Hence, continued methanogenic biodegradation may have shaped the heavy-oil reservoirs that are so prevalent today. The potential to exploit these reactions, for example, by in situ gasification, is currently being actively investigated. Second, injection of nitrate in oil and gas fields can lower sulfide concentrations. High sulfide concentrations, caused by the action of sulfate-reducing bacteria (SRB), are associated with increased risk of corrosion, reservoir plugging (through precipitated sulfides), and human safety. Nitrate injection into an oil field stimulates subsurface heterotrophic nitrate-reducing bacteria (hNRB) and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). Nitrite, formed by these NRB by partial reduction of nitrate, is a strong and specific SRB inhibitor. Nitrate injection has, therefore, promise in positively controlling the oil-field sulfur cycle. There is now more interest in and potential to apply petroleum microbiology than there has been in the past, allowing microbiologists to contribute to a sustainable energy future.

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.

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.

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.

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 degrees 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.

Sulfur cycling in mixed cultures of sulfide-oxidizing and sulfate- or sulfur-reducing oil field bacteria

Thiomicrospira strain CVO and Arcobacter strain FWKO B are two nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) isolated from the Coleville field in western Canada. Analysis by reverse sample genome probing (RSGP) indicates that both can be enriched from Coleville produced water samples by addition of nitrate. Neither could be enriched from waters produced from oil fields with a high resident temperature (40-60°C). In co-cultures of the two microorganisms, CVO dominated at lower and FWKO B at higher sulfide concentrations. Sulfide concentrations of up to 15 mM (480 ppm) could be controlled (i.e., reduced to lower values) by the co-culture. Mixed cultures of a sulfate-reducing bacterium (Desulfovibrio strain Lac6) and CVO or FWKO B produced considerably less sulfide than cultures of Lac6 alone, indicating that these NR-SOB can efficiently oxidize sulfide generated by sulfate reduction provided nitrate is present. The same observations were made for mixed cultures of a sulfur-oxidizing bacterium (Desulfuromonas acetoxidans) and CVO. The results indicate that successful nitrate-mediated conversion of sulfide to oxidized forms (sulfur, sulfate) in oil field production waters by NR-SOB requires the absence of high concentrations of electron donors (e.g., lactate, acetate) that can reverse the nitrate-mediated oxidation reaction.