Substrate Availability,pH, and Temperature Influence Methanogenesis and Mild Steel Corrosion

Solid deposits of corroded pipelines in the Niger Delta were analyzed both chemically and microbiologically. The addition of substrate, especially acetate, significantly stimulated the methane production ranging from 0.85 to 1.60 mmole compared with 0.65 mmole of the control. Acetotrophic and hydrogenotrophic methanogens had their optimal methane production and corrosion rate at pH 5.5. All three types of methanogens produced the most methane at 37°C. Methane production by methanogens correlated strongly with corrosion rate. Pyrosequencing surveys show dominance of acetotrophic, hydrogenotrophic, and methylotrophic methanogens in the samples with no significant presence of sulfate-reducing bacteria (SRB), most likely due to the presence of the biocide, tetrakis-hydroxymethyl phosphonium sulfate. Our study shows that methanogens were one possible cause of pipeline failures in samples from the Niger Delta without the syntrophic association with SRB.     

Planktonic nitrate-reducing bacteria and sulfate-reducing bacteria in some western Canadian oil field waters

Oil fields that use water flooding to enhance oil recovery may become sour because of the production of H₂S from the reduction of sulfate by sulfate-reducing bacteria (SRB). The addition of nitrate to produced waters can stimulate the activities of nitrate-reducing bacteria (NRB) and control sulfide production. Many previous studies have focused on chemolithotrophic bacteria that can use thiosulfate or sulfide as energy sources while reducing nitrate. Little attention has been given to heterotrophic NRB in oil field waters. Three different media were used in this study to enumerate various types of planktonic NRB present in waters from five oil fields in western Canada. The numbers of planktonic SRB and bacteria capable of growth under aerobic conditions were also determined. In general, microbial numbers in the produced waters were very low (<10 ml⁻¹) in samples taken near or at wellheads. However, the numbers increased in the aboveground facilities. No thiosulfate-oxidizing NRB were detected in the oil field waters, but other types of NRB were detected in 16 of 18 produced water samples. The numbers of heterotrophic NRB were equal to or greater than the number of sulfide-oxidizing, chemolithotrophic NRB in 12 of 15 samples. These results showed that each of the oil fields contained NRB, which might be stimulated by nitrate amendment to control H₂S production by SRB. Journal of Industrial Microbiology & Biotechnology (2002) 29, 83–92 doi:10.1038/sj.jim.7000274 Received 20 February 2002/ Accepted in revised form 14 May 2002     

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     

Microbial response to reinjection of produced water in an oil reservoir

The microbial response to produced water reinjection (PWRI) in a North Sea oil field was investigated by a combination of cultivation and culture-independent molecular phylogenetic techniques. Special emphasise was put on the relationship between sulphate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB), and results were used to evaluate the possibility of nitrate treatment as a souring management tool during PWRI. Samples were collected by reversing the flow of the injection water, which provided samples from around the injection area. The backflowed samples were compared to produced water from the same platform and to backflowed samples from a biocide-treated seawater injector, which was the previous injection water treatment of the PWRI well. Results showed that reinjection of produced water promoted growth of thermophilic SRB. Thermophilic fatty acid oxidising NRB and potential nitrate-reducing sulphide-oxidising bacteria were also found. The finding of thermophilic NRB makes nitrate treatment during PWRI possible, although higher nitrate concentration will be necessary to compensate for the increased SRB activity.     

Microbial analysis of backflowed injection water from a nitrate-treated North Sea oil reservoir

Reservoir souring in offshore oil fields is caused by hydrogen sulphide (H₂S) produced by sulphate-reducing bacteria (SRB), most often as a consequence of sea water injection. Biocide treatment is commonly used to inhibit SRB, but has now been replaced by nitrate treatment on several North Sea oil fields. At the Statfjord field, injection wells from one nitrate-treated reservoir and one biocide-treated reservoir were reversed (backflowed) and sampled for microbial analysis. The two reservoirs have similar properties and share the same pre-nitrate treatment history. A 16S rRNA gene-based community analysis (PCR-DGGE) combined with enrichment culture studies showed that, after 6 months of nitrate injection (0.25 mM NO₃ ⁻), heterotrophic and chemolithotrophic nitrate-reducing bacteria (NRB) formed major populations in the nitrate-treated reservoir. The NRB community was able to utilize the same substrates as the SRB community. Compared to the biocide-treated reservoir, the microbial community in the nitrate-treated reservoir was more phylogenetically diverse and able to grow on a wider range of substrates. Enrichment culture studies showed that SRB were present in both reservoirs, but the nitrate-treated reservoir had the least diverse SRB community. Isolation and characterisation of one of the dominant populations observed during nitrate treatment (strain STF-07) showed that heterotrophic denitrifying bacteria affiliated to Terasakiella probably contributed significantly to the inhibition of SRB. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Second derivative UV absorbance analysis to monitor nitrate-reduction by bacteria in most probable number determinations

Heterotrophic and autotrophic nitrate-reducing bacteria (NRB) play important roles in many environments. These bacteria are often enumerated by most probable number (MPN) methods. Measuring NO(3)(-) depletion in the MPN cultures is the definitive way to determine the presence of NRB. Media used for MPN determinations of NRB in oil field waters usually contain high Cl(-) concentrations, matching those in the water samples. Many methods for measuring NO(3)(-) concentrations, such as ion chromatography (IC), cadmium reduction and ion electrode methods, are adversely affected by high concentrations of Cl(-) and organic compounds. A second derivative UV absorbance method proved to be a fast and reliable means for measuring NO(3)(-) depletion in MPN media used for enumerating autotrophic and heterotrophic NRB, without interferences from Cl(-) or the organic components in the latter medium. The MPN results for heterotrophic NRB determined by the second derivative UV absorbance agreed well with those determined by the production of nitrous oxide, and were often higher than those determined by measuring nitrate depletion by the diphenylamine spot test.     

Design features of offshore oil production platforms influence their susceptibility to biocorrosion

Offshore oil-producing platforms are designed for efficient and cost-effective separation of oil from water. However, design features and operating practices may create conditions that promote the proliferation and spread of biocorrosive microorganisms. The microbial communities and their potential for metal corrosion were characterized for three oil production platforms that varied in their oil-water separation processes, fluid recycling practices, and history of microbially influenced corrosion (MIC). Microbial diversity was evaluated by 16S rRNA gene sequencing, and numbers of total bacteria, archaea, and sulfate-reducing bacteria (SRB) were estimated by qPCR. The rates of ³⁵S sulfate reduction assay (SRA) were measured as a proxy for metal biocorrosion potential. A variety of microorganisms common to oil production facilities were found, but distinct communities were associated with the design of the platform and varied with different locations in the processing stream. Stagnant, lower temperature (<37 °C) sites in all platforms had more SRB and higher SRA compared to samples from sites with higher temperatures and flow rates. However, high (5 mmol L^?1) levels of hydrogen sulfide and high numbers (10⁷ mL^?1) of SRB were found in only one platform. This platform alone contained large separation tanks with long retention times and recycled fluids from stagnant sites to the beginning of the oil separation train, thus promoting distribution of biocorrosive microorganisms. These findings tell us that tracking microbial sulfate-reducing activity and community composition on off-shore oil production platforms can be used to identify operational practices that inadvertently promote the proliferation, distribution, and activity of biocorrosive microorganisms.     

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.     

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.     

Kinetic and microbial community analysis of methyl ethyl ketone biodegradation in aquifer sediments

Methyl ethyl ketone (MEK) is a common groundwater contaminant often present with more toxic compounds of primary interest. Because of this, few studies have been performed to determine the effect of microbial community structure on MEK biodegradation rates in aquifer sediments. Here, microcosms were prepared with aquifer sediments containing MEK following a massive spill event and compared to laboratory-spiked sediments, with MEK biodegradation rates quantified under mixed aerobic/anaerobic conditions. Biodegradation was achieved in MEK-contaminated site sediment microcosms at about half of the solubility (356 mg/L) with largely Firmicutes population under iron-reducing conditions. MEK was biodegraded at a higher rate [4.0 ± 0.74 mg/(L days)] in previously exposed site samples compared to previously uncontaminated sediments [0.51 ± 0.14 mg/(L days)]. Amplicon sequencing and denaturing gradient gel electrophoresis of 16S rRNA genes were combined to understand the relationship between contamination levels, biodegradation, and community structure across the plume. More heavily contaminated sediments collected from an MEK-contaminated field site had the most similar communities than less contaminated sediments from the same site despite differences in sediment texture. The more diverse microbial community observed in the laboratory-spiked sediments reduced MEK concentration 47 % over 92 days. Results of this study suggest lower rates of MEK biodegradation in iron-reducing aquifer sediments than previously reported for methanogenic conditions and biodegradation rates comparable to previously reported nitrate- and sulfate-reducing conditions.     

Heterotrophic soil respiration and soil carbon dynamics in the deciduous Hainich forest obtained by three approaches

Three different approaches were used to calculate heterotrophic soil respiration (Rh) and soil carbon dynamics in an old-growth deciduous forest in central Germany. A root and mycorrhiza exclosure experiment in the field separated auto- and heterotrophic soil respiration. It was compared to modeled heterotrophic respiration resulting from two different approaches: a modular component model of soil respiration calculated autotrophic and heterotrophic soil respiration with litter, climate and canopy photosynthesis as input variables. It was calibrated by independent soil respiration measurements in the field. A second model was calibrated by incubation of soil samples from different soil layers in the laboratory. In this case, the annual sum of Rh was calculated by an empirical model including response curves to temperature and a soil moisture. The three approaches showed good accordance during spring and summer and when the annual sums of Rh calculated by the two models were compared. Average Rh for the years 2002–2006 were 436 g C m^?2 year^?1 (field model) and 417 g C m^?2 year^?1 (lab-model), respectively. Differences between the approaches revealed specific limitations of each method. The average carbon balance of the Hainich forest soil was estimated to be between 1 and 35 g C m^?2 year^?1 depending on the model used and the averaging period. A comparison with nighttime data from eddy covariance (EC) showed that EC data were lower than modelled soil respiration in many situations. We conclude that better filter methods for EC nighttime data have to be developed.     

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     

南海莺琼盆地沉积环境中几种厌氧细菌的组成与分布

在严格的厌氧条件下,用MPN计数的方法测定了莺琼盆地东方1-1-1井垂直剖面不同沉积层的地质样品中的硫酸盐还原菌、发酵细菌的数量,并检测了产甲烷细菌,对各种菌的形态进行了观察,比较了菌数量与一些指标的关系,并对产甲烷细菌的代谢类型和产甲烷能力进行了观察。研究结果表明：硫酸盐还原菌在各样品中均存在,与沉积深度无相关性,而与样品中SO2-4的含量有一定相关性;而发酵细菌的分布也与沉积深度无相关性,而与样品中的有机质有一定的负相关性。全部样品中均检测出两种形态产甲烷细菌,即甲烷球菌（Methanococcus）和甲烷杆菌（Methanobacterium）,其营养类型为H2／CO2。     

Applying a most probable number method for enumerating planktonic, dissimilatory, ammonium-producing, nitrate-reducing bacteria in oil field waters

A most probable number (MPN) method was used to enumerate dissimilatory ammonium-producing, nitrate-reducing bacteria (DAP-NRB) in oil field waters and to determine whether they were stimulated by nitrate addition used to control hydrogen sulfide production. An ammonium production medium with 5 carbon and energy sources (acetate, glucose, glycerol, pyruvate, and succinate) and nitrate was used in a 3-tube MPN procedure to enumerate DAP-NRB. These bacteria were detected in 12 of 18 oil field water samples, but they were seldom detected in wellhead samples. Three oil field water samples were amended with nitrate in serum bottles and the numbers of different NRB were determined over a 38-day incubation time. This amendment stimulated increases in the numbers of heterotrophic NRB and autotrophic nitrate-reducing, sulfide-oxidizing bacteria, but DAP-NRB remained a minor portion of these communities. Overall, DAP-NRB were present in many of the oil field waters that were examined but their numbers were low. It appears that DAP-NRB would play a minor role in the consumption of nitrate injected into oil field waters for the control of hydrogen sulfide production.     

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor was investigated over a 10-month period in a hotel. Planktonic and sessile numbers of sulphate reducing bacteria (SRB) and heterotrophic bacteria were monitored. The corrosion rate was determined by the weight loss method. The corrosion products were analyzed by energy dispersive X-ray spectroscopy and X-ray diffraction. A mineralized, heterogeneous biofilm was observed on the coupons. Although a biocide and a corrosion inhibitor were regularly added to the cooling water, the results showed that microorganisms, such as SRB in the mixed species biofilm, caused corrosion of galvanized steel. It was observed that Zn layers on the test coupons were completely depleted after 3?months. The Fe concentrations in the biofilm showed significant correlations with the weight loss and carbohydrate concentration (respectively, p?<?0.01 and p?<?0.01).     

Microbially influenced corrosion of galvanized steel pipes in aerobic water systems

Aims: To investigate the role of heterotrophic bacteria in the corrosion of galvanized steel in the presence of water. Methods and Results: Samples were taken from corroding galvanized steel pipes conveying water for specialist applications, and heterotrophic bacteria were isolated and cultured. The majority of bacteria were Gram‐negative aerobes and included Pseudomonas sp., Bacillus pumilus, Afipia spp. and Blastobacter denitrificans/Bradyrhizobium japonicum. Zinc tolerance was assessed through growth and zinc disc diffusion experiments. In general, zinc negatively influenced growth rates. An unidentified yeast also isolated from the system demonstrated a high tolerance to zinc at concentrations up to 4 g l^?1. Coupon experiments were performed to assess corrosion by the bacteria on galvanized steel and steel coupons. The majority of isolates as pure culture biofilms (69%) accelerated corrosion of galvanized coupons, assessed as zinc release, relative to sterile control coupons (P < 0·05). Pure culture biofilms did not increase the corrosion of steel, with four isolates demonstrating protective effects. Conclusions: Pure culture biofilms of heterotrophic bacteria isolated from a corroding galvanized pipe system were found to accelerate the corrosion of galvanized steel coupons. Significance and Impact of the Study: Microbially influenced corrosion is a potential contributor to sporadically occurring failures in galvanized steel systems containing water. Management strategies should consider microbial control as a means for corrosion prevention in these systems.     

Comparison of fermentation reactions in different regions of the human colon

Colonic contents were obtained from two human sudden-death victims within 3 h of death. One of the subjects (1) was methanogenic, the other (2) was a non-CH, producer. Measurements of bacterial fermentation products showed that in both individuals short-chain fatty acids, lactate and ethanol concentrations were highest in the caecum and ascending colon. In contrast, products of protein fermentation, such as ammonia, branched chain fatty acids and phenolic compounds, progressively increased from the right to the left colon, as did the pH of gut contents. In Subject 1, cell population densities of methanogenic bacteria (MB) increased distally through the gut and methanogenic activity was lower in the right (0.78–1–18 μmol CH₄ produced/h/g dry wt contents) than in the left colon (1.34 μmol CH₄ produced/h/g dry wt contents). Methane production rates did not correlate with MB numbers.
Sulphate-reducing bacteria (SRB) were not found and dissimilatory sulphate reduction was not detected in any region of the colon. Methanogenic bacteria did not occur in subject 2, but high numbers of SRB were present throughout the gut ( ca 10⁹/g dry wt contents). Sulphate reduction rates were maximal in the ascending and transverse colons (0.24 and 0.22 μmol ³⁵SO^2–₄ reduced/h/g dry wt contents, respectively). Short-chain fatty acid production by caecal contents was up to eight-fold higher than contents from the sigmoid/rectum. These findings demonstrate significant differences in fermentation reactions in different regions of the large gut.     

Inhibition of hydrogen sulfide, methane, and total gas production and sulfate-reducing bacteria in in vitro swine manure by tannins, with focus on condensed quebracho tannins

Management practices from large-scale swine production facilities have resulted in the increased collection and storage of manure for off-season fertilization use. Odor and emissions produced during storage have increased the tension among rural neighbors and among urban and rural residents. Production of these compounds from stored manure is the result of microbial activity of the anaerobic bacteria populations during storage. In the current study, the inhibitory effects of condensed quebracho tannins on in vitro swine manure for reduction of microbial activity and reduced production of gaseous emissions, including the toxic odorant hydrogen sulfide produced by sulfate-reducing bacteria (SRB), was examined. Swine manure was collected from a local swine facility, diluted in anaerobic buffer, and mixed with 1 %?w/v fresh feces. This slurry was combined with quebracho tannins, and total gas and hydrogen sulfide production was monitored over time. Aliquots were removed periodically for isolation of DNA to measure the SRB populations using quantitative PCR. Addition of tannins reduced overall gas, hydrogen sulfide, and methane production by greater than 90 % after 7 days of treatment and continued to at least 28 days. SRB population was also significantly decreased by tannin addition. qRT-PCR of 16S rDNA bacteria genes showed that the total bacterial population was also decreased in these incubations. These results indicate that the tannins elicited a collective effect on the bacterial population and also suggest a reduction in the population of methanogenic microorganisms as demonstrated by reduced methane production in these experiments. Such a generalized effect could be extrapolated to a reduction in other odor-associated emissions during manure storage.     

A synergistic d-tyrosine and tetrakis hydroxymethyl phosphonium sulfate biocide combination for the mitigation of an SRB biofilm

Microbiologically influenced corrosion (MIC) is a major problem in various industries such as oil and gas, and water utilities. Billions of dollars are lost to microbiologically influenced corrosion (MIC) each year in the US. The key to MIC control is biofilm mitigation. Sulfate-reducing bacteria (SRB) are often the culprits. They are also involved in souring and biofouling. SRB biofilms are notoriously difficult to eradicate. Due to environmental concerns and increasing costs, better biocide treatment strategies are desired. Recent studies suggested that D: -tyrosine and some other D: -amino acids may signal biofilm dispersal. Experimental results in this work indicated that D: -tyrosine is an effective biocide enhancer for tetrakis hydroxymethyl phosphonium sulfate (THPS) that is a green biocide. Desulfovibrio vulgaris (ATCC 7757) was used in biofilm prevention and biofilm removal tests. It was found that 100?ppm D: -tyrosine alone and 50?ppm THPS alone were both ineffective against the SRB biofilm. However, when 1?ppm D: -tyrosine was combined with 50?ppm THPS, the synergy between the two chemicals successfully prevented the establishment of the SRB biofilm on C1018 mild steel coupon surfaces in batch treatment tests. It also eradicated established SRB biofilms from coupon surfaces in both 1 and 3-h shock treatment tests.     

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.