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     

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     

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     

Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin

To identify novel, less-toxic compounds capable of inhibiting sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris and Desulfovibrio gigas in suspension cultures were exposed to several antimicrobial peptides. The bacterial peptide antimicrobials gramicidin S, gramicidin D, and polymyxin B as well as the cationic peptides indolicidin and bactenecin from bovine neutrophils decreased the viability of both SRB by 90% after a 1-h exposure at concentrations of 25–100 μg ml⁻¹. To reduce corrosion by inhibiting SRB in biofilms, the genes for indolicidin and bactenecin were expressed in Bacillus subtilisBE1500 and B. subtilis WB600 under the control of the constitutive alkaline protease (apr) promoter, and the antimicrobials were secreted into the culture medium using the apr signal sequence. Bactenecin was also synthesized and expressed as a fusion to the pro-region of barnase from Bacillus amyloliquefaciens. Concentrated culture supernatants of B. subtilis BE1500 expressing bactenecin at 3 μg ml⁻¹ decreased the viability of Escherichia coli BK6 by 90% and the reference SRB D. vulgaris by 83% in suspension cultures. B. subtilis BE1500 and B. subtilis WB600 expressing bactenecin in biofilms also inhibited the SRB-induced corrosion of 304 stainless steel six to 12-fold in continuous reactors as evidenced by the lack of change in the impedance spectra (resistance polarization) upon addition of SRB and by the reduction in hydrogen sulfide and iron sulfide in batch fermentations with mild steel. A 36-fold decrease in the population of D. vulgaris in a B. subtilis BE1500 biofilm expressing bactenecin was also observed. This is the first report of an antimicrobial produced in a biofilm for in vivo applications and represents the first application of a beneficial, genetically-engineered biofilm for combating corrosion. Received 27 October 1998/ Accepted in revised form 21 February 1999     

Effect of sulfide to nitrate ratios on the simultaneous anaerobic sulfide and nitrate removal

Present investigation deals with the effect of sulfide to nitrate (S/N) molar ratio on the simultaneous anaerobic sulfide and nitrate removal on capacity, stability and selectivity of the process. The volumetric sulfide-sulfur and nitrate-nitrogen removal rates at molar S/N ratio of 5:2 were 4.86 kg (m³ d)⁻¹ and 0.99 kg (m³ d)⁻¹, respectively, which were higher than those at S/N molar ratios of 5:5 and 5:8. Moreover, the fluctuations in the effluent at S/N ratio of 5:2 were less than those at the other two tested ratios. During the operation, the ratio of converted sulfide to converted nitrate tended to approach 5:2. The selectivity for elemental sulfur and dinitrogen was improved when the S/N molar ratio was set at 5:2 rather than 5:5 or 5:8. The process became unstable if the influent sulfide surpassed its critical concentration. The electron balance between reactants was also analyzed for different S/N molar ratios.     

Examination of thirteen petroliferous formations for hydrocarbon-utilizing sulfate-reducing microorganisms

Virgin cores and production fluids were obtained from seven wells, ranging in depth from 805 ft to 14 492 ft, and examined for the presence of sulfate-reducing bacteria (SRB) using Rosenfeld's sulfate-reducing medium modified by using crude oil in place of lactate. Cores from an additional six wells, ranging in depth from 1160 ft to 13 337 ft were tested for SRB using the modified Rosenfeld medium and API-sulfate-reducing medium. Produced waters from five of the six wells were tested also. All of the eleven produced water samples were positive for SRB while H₂S production was not detected from the core samples.     

Effects of legacy nuclear waste on the compositional diversity and distributions of sulfate-reducing bacteria in a terrestrial subsurface aquifer

The impact of legacy nuclear waste on the compositional diversity and distribution of sulfate-reducing bacteria in a heavily contaminated subsurface aquifer was examined. dsrAB clone libraries were constructed and restriction fragment length polymorphism (RFLP) analysis used to evaluate genetic variation between sampling wells. Principal component analysis identified nickel, nitrate, technetium, and organic carbon as the primary variables contributing to well-to-well geochemical variability, although comparative sequence analysis showed the sulfate-reducing bacteria community structure to be consistent throughout contaminated and uncontaminated regions of the aquifer. Only 3% of recovered dsrAB gene sequences showed apparent membership to the Deltaproteobacteria. The remainder of recovered sequences may represent novel, deep-branching lineages that, to our knowledge, do not presently contain any cultivated members; although corresponding phylotypes have recently been reported from several different marine ecosystems. These findings imply resiliency and adaptability of sulfate-reducing bacteria to extremes in environmental conditions, although the possibility for horizontal transfer of dsrAB is also discussed.     

Reduction of chromate by fixed films of sulfate-reducing bacteria using hydrogen as an electron source

The ability of sulfate-reducing bacteria (SRB) to reduce chromate, Cr(VI), was evaluated using fixed-film growth systems and H₂ as the electron source. A main objective of the experiment was to distinguish between direct enzymatic reduction and indirect reduction by hydrogen sulfide, in order to subsequently verify and control the synergy of these two mechanisms. In batch experiments with the sulfate-reducing consortium CH10 selected from a mining site, 50 mg l⁻¹ Cr(VI) was reduced in 15 min in the presence of 500 mg l⁻¹ hydrogen sulfide compared to 16 mg l⁻¹ reduced in 1 h without hydrogen sulfide. Fixed films of a CH10 population and Desulfomicrobium norvegicum were fed-batch grown in a column bioreactor. After development of the biofilm, hydrogen sulfide was removed and the column was fed continuously with a 13-mg l⁻¹ Cr(VI) solution. Specific Cr(VI) reduction rates on pozzolana were close to 90 mg Cr(VI) h⁻¹ per gram of protein. Exposure to Cr(VI) had a negative effect on the subsequent ability of CH10 to reduce sulfate, but the inhibited bacteria remained viable. Journal of Industrial Microbiology & Biotechnology (2002) 28, 154–159 DOI: 10.1038/sj/jim/7000226 Received 20 September 2000/ Accepted in revised form 13 November 2001     

Anaerobic oxidation of dimethylsulfide and methanethiol in mangrove sediments is dominated by sulfate-reducing bacteria

The oxidation of dimethylsulfide and methanethiol by sulfate-reducing bacteria (SRB) was investigated in Tanzanian mangrove sediments. The rate of dimethylsulfide and methanethiol accumulation in nonamended sediment slurry (control) incubations was very low while in the presence of the inhibitors tungstate and bromoethanesulfonic acid (BES), the accumulation rates ranged from 0.02–0.34 to 0.2–0.4 nmol g FW sediment⁻¹ h⁻¹, respectively. Degradation rates of methanethiol and dimethylsulfide added were 2–10-fold higher. These results point to a balance of production and degradation. Degradation was inhibited much stronger by tungstate than by BES, which implied that SRB were more important. In addition, a new species of SRB, designated strain SD1, was isolated. The isolate was a short rod able to utilize a narrow range of substrates including dimethylsulfide, methanethiol, pyruvate and butyrate. Strain SD1 oxidized dimethylsulfide and methanethiol to carbon dioxide and hydrogen sulfide with sulfate as the electron acceptor and exhibited a low specific growth rate of 0.010 ± 0.002 h⁻¹, but a high affinity for its substrates. The isolated microorganism could be placed in the genus Desulfosarcina (the most closely related cultured species was Desulfosarcina variabilis , 97% identity). Strain SD1 represents a member of the dimethylsulfide/methanethiol-consuming SRB population in mangrove sediments.     

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.     

Microdistribution of sulfate-reducing bacteria in sediments of a hypertrophic lake and their response to the addition of organic matter

To clarify the ecological significance of the association of sulfate-reducing bacteria (SRB) with sediment particle size, SRB utilizing lactate (l-SRB), propionate (p-SRB) and acetate (a-SRB) were examined with different sizes of sediment particles in a hypertrophic freshwater lake using the anaerobic plate count method. The numbers ofl-SRB anda-SRB were 10⁴–10⁵ colony forming units (CFU) per ml in the 0–3 cm layer and 10²–10³ CFU ml⁻¹ in the 10–13 cm layer while the numbers ofp-SRB were one or two orders lower than those ofl-SRB anda-SRB. A sediment suspension was fractionated into four fractions (<1, 1–10, 10–94 and >94 μm). The highest proportions ofl-SRB anda-SRB were found in the 10–94 μm fraction: 66–97% forl-SRB and 53–98% fora-SRB. The highest proportion ofp-SRB was found in the >94 μm fraction (70–74%). These results indicate that most SRB were associated with sediment particles. One isolate from an acetate-utilizing enrichment culture was similar toDesulfotomaculum acetoxidans, a spore-forming sulfate-reducing bacterium. When lactate and sulfate were added to sediment samples,l-SRB anda-SRB in the <10 μm-fraction grew more rapidly than those in whole sediment for the first 2 days. This result suggests that nutrients uptake by free-living and small particle-associated (<10 μm) SRB is higher than that by SRB associated with larger particles.     

Identification of sulfate-reducing bacteria in methylmercury-contaminated mine tailings by analysis of SSU rRNA genes

Sulfate-reducing bacteria (SRB) are often used in bioremediation of acid mine drainage because microbial sulfate reduction increases pH and produces sulfide that binds with metals. Mercury methylation has also been linked with sulfate reduction. Previous geochemical analysis indicated the occurrence of sulfate reduction in mine tailings, but no molecular characterization of the mine tailings-associated microbial community has determined which SRB are present. This study characterizes the bacterial communities of two geochemically contrasting, high-methylmercury mine tailing environments, with emphasis on SRB, by analyzing small subunit (SSU) rRNA genes present in the tailings sediments and in enrichment cultures inoculated with tailings. Novel Deltaproteobacteria and Firmicutes -related sequences were detected in both the pH-neutral gold mine tailings and the acidic high-sulfide base-metal tailings. At the subphylum level, the SRB communities differed between sites, suggesting that the community structure was dependent on local geochemistry. Clones obtained from the gold tailings and enrichment cultures were more similar to previously cultured isolates whereas clones from acidic tailings were more closely related to uncultured lineages identified from other acidic sediments worldwide. This study provides new insights into the novelty and diversity of bacteria colonizing mine tailings, and identifies specific organisms that warrant further investigation with regard to their roles in mercury methylation and sulfur cycling in these environments.     

Microbial control of the production of hydrogen sulfide by sulfate-reducing bacteria

A sulfide-resistant ctrain of Thiobacillus denitrificans, strain F, prevented the accumulation of sulfide by Desulfovibrio desulfuricans when both organisms were grown in liquid medium or in Berea sandstone cores. The wild-type strain of T. denitrificans did not prevent the accumulation of sulfide produced by D. desulfuricans. Strain F also prevented the accumulation of sulfide by a mixed population of sulfate-reducing bacteria enriched from an oil field brine. Fermentation balances showed that strain F stoichiometrically oxidized the sulfide produced by D. desulfuricans and the oil field brine enrichment to sulfate. These data suggest that strain F would be effective in controlling sulfide production in oil reservoirs and other environments.     

Action of glutaraldehyde and nitrite against sulfate-reducing bacterial biofilms

A continuous flow reactor system was developed to evaluate the efficacy of antimicrobial treatments against sulfate-reducing bacterial biofilms. An annular reactor operating at a nominal dilution rate of 0.5 h⁻¹ was fed one-tenth strength Postgate C medium diluted in 1.5% NaCl and was inoculated with a mixed culture enriched from oilfield-produced water on the same medium. Thin biofilms developed in this reactor after 2 days of operation. The activity of these biofilms resulted in approximately 50 mg S l⁻¹ of sulfide at steady state prior to biocide treatment. Biocide efficacy was quantified by recording the time required for sulfide production to recover following an antimicrobial treatment. In a control experiment in which pure water was applied, the time required to reach 10 mg S l⁻¹ sulfide after the treatment was 1.7±1.2 h, whereas the time to reach this level of sulfide after a pulse dose of 500 mg l⁻¹ glutaraldehyde was delayed to 61±11 h. Nitrite treatment suppressed sulfide production as long as the nitrite concentration remained above 15 mg N l⁻¹. Sulfide production recovered more rapidly after nitrite treatment than it did after glutaraldehyde treatment. Received 01 February 2002/ Accepted in revised form 13 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.     

Temperature-dependent differences in community structure of bacteria involved in degradation of petroleum hydrocarbons under sulfate-reducing conditions

Aim: The aim of this study was to characterize the microbial community involved in anaerobic degradation of petroleum hydrocarbon under low‐ and moderate‐temperature conditions. Methods and Results: Sulfate‐reducing enrichment cultures growing on crude oil and p‐xylene were established at low and moderate temperatures. Bacterial community structures of the cultures were characterized by 16S rRNA gene‐based analysis and organisms responsible for degradation of p‐xylene were investigated by analysis of the bamA gene, involved in anaerobic degradation of aromatic compounds. The PCR‐denaturing gradient gel electrophoresis analysis indicated significant differences in microbial community structures among the cultures, depending on the temperatures of incubation. Difference depending on the temperatures was also observed in the cloning analysis of the bamA gene performed on the p‐xylene‐degrading enrichment cultures. Majority of clones detected in the culture of moderate temperature were related to Desulfosarcina ovata, whereas more diverse bamA gene sequences were obtained from the culture incubated at low temperature. Conclusions: Temperature‐dependent differences in microbial community were demonstrated by the analyses of two genes. It was suggested that sulfate‐reducing bacteria of phylogenetically different groups might be involved in the degradation of petroleum hydrocarbons in different temperature environments. Significance and Impact of the Study: This study is the first report of p‐xylene‐degrading sulfate‐reducing enrichment culture at low temperature. The results of the experiments at low temperature were distinctly different from those reported in previous studies performed at moderate temperatures.     

Carriage, quantification, and predominance of methanogens and sulfate-reducing bacteria in faecal samples

AIMS: To determine carriage rates and densities of methanogens and sulfate-reducing bacteria in adults and children using molecular methods, and to also determine if a reciprocal relationship exists between these organisms. METHODS AND RESULTS: Real-time PCR was used to detect and quantify methanogens and sulfate-reducing bacteria. Real-time PCR was more sensitive than breath methane measurements. Real-time PCR assays were applied to faecal DNA samples collected from 40 children and 12 adults. Methanogens were present in 25% of the children and 42% of the adults studied, and sulfate-reducing bacteria were detected in 15% of the children and 58% of the adults. High levels of sulfate-reducing bacteria were found in two methanogenic adults. CONCLUSIONS: Carriage rates and densities of methanogens and sulfate-reducing bacteria are greater in adults than in children. Competition does not necessarily lead to the predominance of one group in the faecal microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes sensitive, molecular assays that could be used to monitor these organisms in gastrointestinal disease. Therapeutic exclusion of one group from the bowel would not necessarily lead to the expansion of the other, as there does not appear to be a reciprocal relationship between these groups.     

A common pathway of sulfide oxidation by sulfate-reducing bacteria

Abstract Pseudomonas putida strain DMB capable of growing on 3,4-dimethylbenzoic acid as the only C and energy source was isolated by enrichment techniques. It does not utilize for growth or cooxidize the other dimethylbenzoate isomers tested. 3,4-Dimethylsalicylic acid, 3,4-dimethylphenol and 3,4-dimethylcatechol were isolated and identified by nuclear magnetic resonance and mass spectra in the reaction mixture of P. putida washed cells. The detection of the two first metabolites suggests that the initial step in the degradation of 3,4-dimethylbenzoic acid is the formation of 3,4-dimethylcyclohexa-3,5-diene-1, 2-diol-1-carboxylic acid which underwent an acid-catalyzed dehydration yielding 3,4-dimethylsalicylic acid and 3,4-dimethylphenol. Further degradation proceeds through 3,4-dimethylcatechol via the meta pathway.     

Convenient method to prepare neutral sulfide solution for cultivation of phototrophic sulfur bacteria

A device is described for the preparation and storage of sterile neutral sulfide solution which is required for the repeated addition of this substrate to growing cultures of phototrophic sulfur bacteria.     

Reduction of molybdate by sulfate-reducing bacteria

Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.