Microbial sulfite oxidation coupled to nitrate reduction in makeup water for oil production.

Bisulfite is used as an oxygen scavenger in waters used for oil production to prevent oxygen-mediated pipeline corrosion. Analysis of nitrate-containing water injected with ammonium bisulfite indicated increased concentrations of ammonium, sulfate and nitrite. To understand the microbial process causing these changes, water samples were used in enrichments with bisulfite and nitrate. Oxidation of bisulfite, reduction of nitrate, change in microbial community composition and corrosivity of bisulfite were determined. The results indicated that the microbial community was dominated by Sulfuricurvum, a sulfite-oxidizing nitrate-reducing bacterium (StONRB). Plating of the enriched StONRB culture yielded the bacterial isolate Sulfuricurvum sp. TK005, which coupled bisulfite oxidation with nitrate reduction to form sulfate and nitrite. Bisulfite also induced chemical corrosion of carbon steel at a rate of 0.28 ± 0.18 mm yr-1. Bisulfite and the generated sulfate could serve as electron acceptors for sulfate-reducing microorganisms (SRM), which reduce sulfate and bisulfite to sulfide. Nitrate is frequently injected to injection waters to contain the activity of SRM in oil reservoirs. This study suggests an alternative bisulfite injection procedure: Injection of nitrate after the chemical reaction of bisulfite with oxygen is completed. This could maintain the oxygen scavenger function of bisulfite and SRM inhibitory activity of nitrate.

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs.

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 µmol CH4 g-1 oil d-1 , orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC-MS and FTICR-MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.

Microbially Enhanced Oil Recovery by Alkylbenzene-Oxidizing Nitrate-Reducing Bacteria.

Microbially enhanced oil recovery (MEOR) of heavy oil and bitumen is challenging because light hydrocarbons, which can feed resident microbial communities are present in low concentrations, if at all. We have recently shown that increasing the toluene concentration of heavy oil by aqueous injection followed by injection of nitrate boosts the activity of toluene-oxidizing nitrate-reducing bacteria in heavy oil-containing sand pack columns, giving production of residual oil in place (ROIP). In the current work we found that ethylbenzene is as effective as toluene. Microbial community analyses indicated Thauera and Pseudomonas to be main components of nitrate-containing batch and continuous cultures, regardless whether ethylbenzene or toluene was used as the electron donor. Biomass from batch cultures grown with heavy oil amended with ethylbenzene or toluene and nitrate or biomass from continuous cultures grown on ethylbenzene or toluene and nitrate had similar MEOR activity. Increasing the concentration of injected biomass from continuous cultures increased the fraction of ROIP recovered both in the absence and in the presence of nitrate. Nitrate increased the fraction of ROIP recovered by about 2-fold by increasing the concentration of biomass in the columns. Emulsification of oil by surface-adhering biomass and blocking of aqueous flow channels by oil emulsion droplets are proposed as a possible mechanism of hydrocarbon- and nitrate-mediated MEOR. Pure isolates Thauera sp. NS1 and Pseudomonas sp. NS2, which used both ethylbenzene and toluene, were obtained but did not offer improved MEOR compared to the use of batch and continuous cultures.

Effect of selected biocides on microbiologically influenced corrosion caused by Desulfovibrio ferrophilus IS5.

The marine bacterial strain Desulfovibrio ferrophilus IS5, known for its lithotrophic growth ability to use metallic iron as a sole electron donor and for causing corrosion of steel, was used in the current study. Four commonly used biocides in the oil and gas industry, namely tetrakis(hydroxymethyl) phosphonium sulfate (THPS), glutaraldehyde (GLUT), benzalkonium chloride (BAC), and GLUT/BAC were selected to study their efficacy in controlling carbon steel corrosion in the presence of this strain. Incubations containing strain IS5 and low carbon steel coupons were prepared in the presence and absence of the four biocides, and these were monitored using both electrochemical methods (electrochemical impedance spectroscopy, linear polarization resistance and potentiodynamic polarization) and surface analyses (scanning electron microscopy, confocal measurements, optical microscopy, and profilometry) to assess the biofilm/metal interactions. When THPS, BAC, and GLUT/BAC treatments were applied, minimal corrosion was measured by all methods. In contrast, severe pitting was observed in the presence of 50 ppm GLUT, similar to what was observed when D. ferrophilus IS5 was incubated in the absence of biocide, suggesting that GLUT alone may not be effective in controlling MIC in marine environments. This study also showed that the use of non-destructive electrochemical methods is effective for screening for real time biocide selection and monitoring of the impact of chemicals post-dosage in oil and gas operations.

Comparison of Nitrate and Perchlorate in Controlling Sulfidogenesis in Heavy Oil-Containing Bioreactors.

Control of microbial reduction of sulfate to sulfide in oil reservoirs (a process referred to as souring) with nitrate has been researched extensively. Nitrate is reduced to nitrite, which is a strong inhibitor of sulfate-reducing bacteria (SRB). Perchlorate has been proposed as an alternative souring control agent. It is reduced to chlorate (ClO3 -) and chlorite (ClO2 -), which is dismutated to chloride and O2. These can react with sulfide to form sulfur. Chlorite is also highly biocidal. Here we compared the effectiveness of perchlorate and nitrate in inhibiting SRB activity in medium containing heavy oil from the Medicine Hat Glauconitic C (MHGC) field, which has a low reservoir temperature and is injected with nitrate to control souring. Using acetate, propionate and butyrate as electron donors, perchlorate-reducing bacteria (PRB) were obtained in enrichment culture and perchlorate-reducing Magnetospirillum spp. were isolated from MHGC produced waters. In batch experiments with MHGC oil as the electron donor, nitrate was reduced to nitrite and inhibited sulfate reduction. However, perchlorate was not reduced and did not inhibit sulfate reduction in these incubations. Bioreactor experiments were conducted with sand-packed glass columns, containing MHGC oil and inoculated with an oil-grown mesophilic SRB enrichment. Once active souring (reduction of 2 mM sulfate to sulfide) was observed, these were treated with nitrate and/or perchlorate. As in the batch experiments, 4 mM nitrate completely inhibited sulfide production, while partial inhibition occurred with 1 and 2 mM nitrate, but injection of 4 mM perchlorate did not inhibit sulfate reduction and perchlorate was not reduced. The enriched and isolated PRB were unable to use heavy oil components, like alkylbenzenes, which were readily used by nitrate-reducing bacteria. Hence perchlorate, injected into a low temperature heavy oil reservoir like the MHGC, may not be reduced to toxic intermediates making nitrate a preferable souring control agent.

Effect of long term application of tetrakis(hydroxymethyl)phosphonium sulfate (THPS) in a light oil-producing oilfield.

Samples of (I) produced waters, (II) central processing facility (CPF) waters and (III) pipeline solids were collected from a light oil-producing field. The biocide, tetrakis(hydroxymethyl)phosphonium sulfate (THPS) was routinely used in the CPF. Samples monitoring indicated that THPS was effective in microbial control but also increased concentrations of sulfate and phosphate in transitioning from Type I to Type II waters. Type II waters had high concentrations (up to 60 mM) of acetate but low most probable numbers (MPNs) of acid-producing and sulfate-reducing bacteria, indicating the presence of active biocide, as high MPNs were found in Type I waters. Solids had high phosphate and high MPNs, indicating that THPS was inactive. Solids had oil and an anaerobic community dominated by Acetobacterium, which may contribute to conversion of oil to acetate. The presence of THPS prevented the use of this acetate in Type II waters, where it accumulated to unusually high concentrations.

Synergy of Sodium Nitroprusside and Nitrate in Inhibiting the Activity of Sulfate Reducing Bacteria in Oil-Containing Bioreactors.

Sodium nitroprusside (SNP) disrupts microbial biofilms through the release of nitric oxide (NO). The actions of SNP on bacteria have been mostly limited to the genera Pseudomonas, Clostridium, and Bacillus. There are no reports of its biocidal action on sulfate-reducing bacteria (SRB), which couple the reduction of sulfate to sulfide with the oxidation of organic electron donors. Here, we report the inhibition and kill of SRB by low SNP concentrations [0.05 mM (15 ppm)] depending on biomass concentration. Chemical reaction of SNP with sulfide did not compromise its efficacy. SNP was more effective than five biocides commonly used to control SRB. Souring, the SRB activity in oil reservoirs, is often controlled by injection of nitrate. Control of SRB-mediated souring in oil-containing bioreactors was inhibited by 4 mM (340 ppm) of sodium nitrate, but required only 0.05 mM (15 ppm) of SNP. Interestingly, nitrate and SNP were found to be highly synergistic with 0.003 mM (1 ppm) of SNP and 1 mM (85 ppm) of sodium nitrate being sufficient in inhibiting souring. Hence, using SNP as an additive may greatly increase the efficacy of nitrate injection in oil reservoirs.

Use of carbon steel ball bearings to determine the effect of biocides and corrosion inhibitors on microbiologically influenced corrosion under flow conditions.

A consortium of sulfate-reducing bacteria consisting mostly of Desulfovibrio, Desulfomicrobium, and Desulfocurvus from oil field produced water was cultivated in a chemostat, receiving medium with 20 mM formate and 10 mM sulfate as the energy and 1 mM acetate as the carbon source. The chemostat effluent, containing 5 mM sulfide and 0.5 mM of residual acetate, was passed through 1-ml syringe columns with 60 carbon steel ball bearings (BBs) of 53.6 ± 0.1 mg each at a flow rate of 0.8 ml/h per column. These were treated every 5 days with 1.6 ml of 300 ppm of glutaraldehyde (Glut), tetrakis(hydroxymethyl)phosphonium sulfate (THPS), benzalkonium chloride (BAC), or Glut/BAC, a mixture of Glut and BAC. Alternatively, BBs were treated with 33% (v/v) of a water-soluble (CR_W) or an oil-soluble (CR_O1 or CR_O3) corrosion inhibitor for 20 s after which the corrosion inhibitor was drained off and BBs were packed into columns. The effluent of untreated control columns had no acetate. Treatment with the chemically reactive biocides Glut and THPS, as well as with Glut/BAC, gave a transient increase of acetate indicating decreased microbial activity. This was not seen with BAC alone indicating it to be the least effective biocide. Relative to untreated BBs (100%), those treated periodically with Glut, THPS, BAC, or Glut/BAC had a general weight loss corrosion rate of 91, 81, 45, and 36% of the untreated rate of 0.104 ± 0.004 mm/year, respectively. Single treatment with corrosion inhibitors decreased corrosion to 48, 2, and 1% of the untreated rate for CR_W, CR_O1 and CR_O3, respectively. Analysis of the distribution of corrosion rates from the weight loss of individual BBs (N = 120) indicated the presence of a more slowly and a more rapidly corroding group. BAC treatment prevented emergence of the latter, and this quaternary ammonium detergent appeared most effective in decreasing corrosion not because of its biocidal properties, but because of its corrosion inhibitory properties.

Aerobic microbial taxa dominate deep subsurface cores from the Alberta oil sands.

Little is known about the microbial ecology of the subsurface oil sands in Northern Alberta, Canada. Biodegradation of low molecular weight hydrocarbons by indigenous microbes has enriched high molecular weight hydrocarbons, resulting in highly viscous bitumen. This extreme subsurface environment is further characterized by low nutrient availability and limited access to water, thus resulting in low microbial biomass. Improved DNA isolation protocols and increasingly sensitive sequencing methods have allowed an in-depth investigation of the microbial ecology of this unique subsurface environmental niche. Community analysis was performed on core samples (n = 62) that were retrieved from two adjacent sites located in the Athabasca Oil Sands at depths from 220 to 320 m below the surface. Microbial communities were dominated by aerobic taxa, including Pseudomonas and Acinetobacter. Only one core sample microbial community was dominated by anaerobic taxa, including the methanogen Methanoculleus, as well as Desulfomicrobium and Thauera. Although the temperature of the bitumen-containing subsurface is low (8°C), two core samples had high fractions of the potentially thermophilic taxon, Thermus. Predominance of aerobic taxa in the subsurface suggests the potential for in situ aerobic hydrocarbon degradation; however, more studies are required to determine the functional role of these taxa within this unique environment.

Effect of Thermophilic Nitrate Reduction on Sulfide Production in High Temperature Oil Reservoir Samples.

Oil fields can experience souring, the reduction of sulfate to sulfide by sulfate-reducing microorganisms. At the Terra Nova oil field near Canada's east coast, with a reservoir temperature of 95°C, souring was indicated by increased hydrogen sulfide in produced waters (PW). Microbial community analysis by 16S rRNA gene sequencing showed the hyperthermophilic sulfate-reducing archaeon Archaeoglobus in Terra Nova PWs. Growth enrichments in sulfate-containing media at 55-70°C with lactate or volatile fatty acids yielded the thermophilic sulfate-reducing bacterium (SRB) Desulfotomaculum. Enrichments at 30-45°C in nitrate-containing media indicated the presence of mesophilic nitrate-reducing bacteria (NRB), which reduce nitrate without accumulation of nitrite, likely to N2. Thermophilic NRB (tNRB) of the genera Marinobacter and Geobacillus were detected and isolated at 30-50°C and 40-65°C, respectively, and only reduced nitrate to nitrite. Added nitrite strongly inhibited the isolated thermophilic SRB (tSRB) and tNRB and SRB could not be maintained in co-culture. Inhibition of tSRB by nitrate in batch and continuous cultures required inoculation with tNRB. The results suggest that nitrate injected into Terra Nova is reduced to N2 at temperatures up to 45°C but to nitrite only in zones from 45 to 65°C. Since the hotter zones of the reservoir (65-80°C) are inhabited by thermophilic and hyperthermophilic sulfate reducers, souring at these temperatures might be prevented by nitrite production if nitrate-reducing zones of the system could be maintained at 45-65°C.

Biocide-mediated corrosion of coiled tubing.

Coiled tubing corrosion was investigated for 16 field water samples (S5 to S20) from a Canadian shale gas field. Weight loss corrosion rates of carbon steel beads incubated with these field water samples averaged 0.2 mm/yr, but injection water sample S19 had 1.25±0.07 mm/yr. S19 had a most probable number of zero acid-producing bacteria and incubation of S19 with carbon steel beads or coupons did not lead to big changes in microbial community composition. In contrast other field water samples had most probable numbers of APB of 102/mL to 107/mL and incubation of these field water samples with carbon steel beads or coupons often gave large changes in microbial community composition. HPLC analysis indicated that all field water samples had elevated concentrations of bromide (average 1.6 mM), which may be derived from bronopol, which was used as a biocide. S19 had the highest bromide concentration (4.2 mM) and was the only water sample with a high concentration of active bronopol (13.8 mM, 2760 ppm). Corrosion rates increased linearly with bronopol concentration, as determined by weight loss of carbon steel beads, for experiments with S19, with filtered S19 and with bronopol dissolved in defined medium. This indicated that the high corrosion rate found for S19 was due to its high bronopol concentration. The corrosion rate of coiled tubing coupons also increased linearly with bronopol concentration as determined by electrochemical methods. Profilometry measurements also showed formation of multiple pits on the surface of coiled tubing coupon with an average pit depth of 60 µm after 1 week of incubation with 1 mM bronopol. At the recommended dosage of 100 ppm the corrosiveness of bronopol towards carbon steel beads was modest (0.011 mm/yr). Higher concentrations, resulting if biocide is added repeatedly as commonly done in shale gas operations, are more corrosive and should be avoided. Overdosing may be avoided by assaying the presence of residual biocide by HPLC, rather than by assaying the presence of residual surviving bacteria.

Control of Sulfide Production in High Salinity Bakken Shale Oil Reservoirs by Halophilic Bacteria Reducing Nitrate to Nitrite.

Microbial communities in shale oil fields are still poorly known. We obtained samples of injection, produced and facility waters from a Bakken shale oil field in Saskatchewan, Canada with a resident temperature of 60°C. The injection water had a lower salinity (0.7 Meq of NaCl) than produced or facility waters (0.6-3.6 Meq of NaCl). Salinities of the latter decreased with time, likely due to injection of low salinity water, which had 15-30 mM sulfate. Batch cultures of field samples showed sulfate-reducing and nitrate-reducing bacteria activities at different salinities (0, 0.5, 0.75, 1.0, 1.5, and 2.5 M NaCl). Notably, at high salinity nitrite accumulated, which was not observed at low salinity, indicating potential for nitrate-mediated souring control at high salinity. Continuous culture chemostats were established in media with volatile fatty acids (a mixture of acetate, propionate and butyrate) or lactate as electron donor and nitrate or sulfate as electron acceptor at 0.5 to 2.5 M NaCl. Microbial community analyses of these cultures indicated high proportions of Halanaerobium, Desulfovermiculus, Halomonas, and Marinobacter in cultures at 2.5 M NaCl, whereas Desulfovibrio, Geoalkalibacter, and Dethiosulfatibacter were dominant at 0.5 M NaCl. Use of bioreactors to study the effect of nitrate injection on sulfate reduction showed that accumulation of nitrite inhibited SRB activity at 2.5 M but not at 0.5 M NaCl. High proportions of Halanaerobium and Desulfovermiculus were found at 2.5 M NaCl in the absence of nitrate, whereas high proportions of Halomonas and no SRB were found in the presence of nitrate. A diverse microbial community dominated by the SRB Desulfovibrio was observed at 0.5 M NaCl both in the presence and absence of nitrate. Our results suggest that nitrate injection can prevent souring provided that the salinity is maintained at a high level. Thus, reinjection of high salinity produced water amended with nitrate maybe be a cost effective method for souring control.

The Effectiveness of Nitrate-Mediated Control of the Oil Field Sulfur Cycle Depends on the Toluene Content of the Oil.

The injection of nitrate is one of the most commonly used technologies to impact the sulfur cycle in subsurface oil fields. Nitrate injection enhances the activity of nitrate-reducing bacteria, which produce nitrite inhibiting sulfate-reducing bacteria (SRB). Subsequent reduction of nitrate to di-nitrogen (N2) alleviates the inhibition of SRB by nitrite. It has been shown for the Medicine Hat Glauconitic C (MHGC) field, that alkylbenzenes especially toluene are important electron donors for the reduction of nitrate to nitrite and N2. However, the rate and extent of reduction of nitrate to nitrite and of nitrite to nitrogen have not been studied for multiple oil fields. Samples of light oil (PNG, CPM, and Tundra), light/heavy oil (Gryphon and Obigbo), and of heavy oil (MHGC) were collected from locations around the world. The maximum concentration of nitrate in the aqueous phase, which could be reduced in microcosms inoculated with MHGC produced water, increased with the toluene concentration in the oil phase. PNG, Gryphon, CPM, Obigbo, MHGC, and Tundra oils had 77, 17, 5.9, 4.0, 2.6, and 0.8 mM toluene, respectively. In incubations with 49 ml of aqueous phase and 1 ml of oil these were able to reduce 22.2, 12.3, 7.9, 4.6, 4.0, and 1.4 mM of nitrate, respectively. Nitrate reduced increased to 35 ± 4 mM upon amendment of all these oils with 570 mM toluene prior to incubation. Souring control by nitrate injection requires that the nitrate is directed toward oxidation of sulfide, not toluene. Hence, the success of nitrate injections will be inversely proportional to the toluene content of the oil. Oil composition is therefore an important determinant of the success of nitrate injection to control souring in a particular field.

Biodegradation of isopropanol and acetone under denitrifying conditions by Thauera sp. TK001 for nitrate-mediated microbially enhanced oil recovery.

Amendment of reservoir fluid with injected substrates can enhance the growth and activity of microbes. The present study used isopropyl alcohol (IPA) or acetone to enhance the indigenous anaerobic nitrate-reducing bacterium Thauera sp. TK001. The strain was able to grow on IPA or acetone and nitrate. To monitor effects of strain TK001 on oil recovery, sand-packed columns containing heavy oil were flooded with minimal medium at atmospheric or high (400psi) pressure. Bioreactors were then inoculated with 0.5 pore volume (PV) of minimal medium containing Thauera sp. TK001 with 25mM of acetone or 22.2mM of IPA with or without 80mM nitrate. Incubation without flow for two weeks and subsequent injection with minimal medium gave an additional 17.0±6.7% of residual oil in place (ROIP) from low-pressure bioreactors and an additional 18.3% of ROIP from the high-pressure bioreactors. These results indicate that acetone or IPA, which are commonly used organic solvents, are good substrates for nitrate-mediated microbial enhanced oil recovery (MEOR), comparable to glucose, acetate or molasses, tested previously. This technology may be used for coupling biodegradation of IPA and/or acetone in waste streams to MEOR where these waste streams are generated in close proximity to an oil field.

Use of Acetate, Propionate, and Butyrate for Reduction of Nitrate and Sulfate and Methanogenesis in Microcosms and Bioreactors Simulating an Oil Reservoir.

Acetate, propionate, and butyrate (volatile fatty acids [VFA]) occur in oil field waters and are frequently used for microbial growth of oil field consortia. We determined the kinetics of use of these VFA components (3 mM each) by an anaerobic oil field consortium in microcosms containing 2 mM sulfate and 0, 4, 6, 8, or 13 mM nitrate. Nitrate was reduced first, with a preference for acetate and propionate. Sulfate reduction then proceeded with propionate (but not butyrate) as the electron donor, whereas the fermentation of butyrate (but not propionate) was associated with methanogenesis. Microbial community analyses indicated that Paracoccus and Thauera (Paracoccus-Thauera), Desulfobulbus, and Syntrophomonas-Methanobacterium were the dominant taxa whose members catalyzed these three processes. Most-probable-number assays showed the presence of up to 107/ml of propionate-oxidizing sulfate-reducing bacteria (SRB) in waters from the Medicine Hat Glauconitic C field. Bioreactors with the same concentrations of sulfate and VFA responded similarly to increasing concentrations of injected nitrate as observed in the microcosms: sulfide formation was prevented by adding approximately 80% of the nitrate dose needed to completely oxidize VFA to CO2 in both. Thus, this work has demonstrated that simple time-dependent observations of the use of acetate, propionate, and butyrate for nitrate reduction, sulfate reduction, and methanogenesis in microcosms are a good proxy for these processes in bioreactors, monitoring of which is more complex.IMPORTANCE Oil field volatile fatty acids acetate, propionate, and butyrate were specifically used for nitrate reduction, sulfate reduction, and methanogenic fermentation. Time-dependent analyses of microcosms served as a good proxy for these processes in a bioreactor, mimicking a sulfide-producing (souring) oil reservoir: 80% of the nitrate dose required to oxidize volatile fatty acids to CO2 was needed to prevent souring in both. Our data also suggest that propionate is a good substrate to enumerate oil field SRB.

Nitrate-Mediated Microbially Enhanced Oil Recovery (N-MEOR) from model upflow bioreactors.

Microbially Enhanced Oil Recovery (MEOR) can enhance oil production with less energy input and less costs than other technologies. The present study used different aqueous electron donors (acetate, glucose, molasses) and an aqueous electron acceptor (nitrate) to stimulate growth of heterotrophic nitrate reducing bacteria (hNRB) to improve production of oil. Initial flooding of columns containing heavy oil (viscosity of 3400cP at 20°C) with CSBK (Coleville synthetic brine medium) produced 0.5 pore volume (PV) of oil. Bioreactors were then inoculated with hNRB with 5.8g/L of molasses and 0, 10, 20, 40, 60 or 80mM nitrate, as well as with 17mM glucose or 57mM acetate and 80mM nitrate. During incubations no oil was produced in the bioreactors that received 5.8g/L of molasses and 0, 10, 20, 40 or 60mM nitrate. However, the bioreactors injected with 5.8g/L of molasses, 17mM glucose or 57mM acetate and 80mM nitrate produced 13.9, 11.3±3.1 and 17.8±6.6% of residual oil, respectively. The significant production of oil from these bioreactors may be caused by N2-CO2 gas production. Following continued injection with CSBK without nitrate, subsequent elution of significant residual oil (5-30%) was observed. These results also indicate possible involvement of fermentation products (organic acids, alcohols) to enhance heavy oil recovery.

Electron transfer pathways of formate-driven H2 production in Desulfovibrio.

The potential of sulfate-reducing bacteria (SRB) as biocatalysts for H2 production from formate was recently demonstrated, but the electron transfer pathways involved were not described. In the present work, we analyzed the H2 production capacity of five Desulfovibrio strains: Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio fructosivorans, and Desulfovibrio gigas. D. vulgaris showed the highest H2 productivity (865 mL Lmedium (-1)), and D. gigas the lowest one (374 mL Lmedium (-1) of H2). The electron transfer pathways involved in formate-driven H2 production by these two organisms were further investigated through the study of deletion mutants of hydrogenases (Hases) and formate dehydrogenases (Fdhs). In D. vulgaris, the periplasmic FdhAB is the key enzyme for formate oxidation and two pathways are apparently involved in the production of H2 from formate: a direct one only involving periplasmic enzymes and a second one that involves transmembrane electron transfer and may allow energy conservation. In the presence of selenium, the Hys [NiFeSe] Hase is the main periplasmic enzyme responsible for H2 production, and the cytoplasmic Coo Hase is apparently involved in the ability of D. vulgaris to grow by converting formate to H2, in sparging conditions. Contrary to D. vulgaris, H2 production in D. gigas occurs exclusively by the direct periplasmic route and does not involve the single cytoplasmic Hase, Ech. This is the first report of the metabolic pathways involved in formate metabolism in the absence of sulfate in SRB, revealing that the electron transfer pathways are species-specific.

Implications of Limited Thermophilicity of Nitrite Reduction for Control of Sulfide Production in Oil Reservoirs.

UNLABELLED: Nitrate reduction to nitrite in oil fields appears to be more thermophilic than the subsequent reduction of nitrite. Concentrated microbial consortia from oil fields reduced both nitrate and nitrite at 40 and 45°C but only nitrate at and above 50°C. The abundance of the nirS gene correlated with mesophilic nitrite reduction activity. Thauera and Pseudomonas were the dominant mesophilic nitrate-reducing bacteria (mNRB), whereas Petrobacter and Geobacillus were the dominant thermophilic NRB (tNRB) in these consortia. The mNRB Thauera sp. strain TK001, isolated in this study, reduced nitrate and nitrite at 40 and 45°C but not at 50°C, whereas the tNRB Petrobacter sp. strain TK002 and Geobacillus sp. strain TK003 reduced nitrate to nitrite but did not reduce nitrite further from 50 to 70°C. Testing of 12 deposited pure cultures of tNRB with 4 electron donors indicated reduction of nitrate in 40 of 48 and reduction of nitrite in only 9 of 48 incubations. Nitrate is injected into high-temperature oil fields to prevent sulfide formation (souring) by sulfate-reducing bacteria (SRB), which are strongly inhibited by nitrite. Injection of cold seawater to produce oil creates mesothermic zones. Our results suggest that preventing the temperature of these zones from dropping below 50°C will limit the reduction of nitrite, allowing more effective souring control. IMPORTANCE: Nitrite can accumulate at temperatures of 50 to 70°C, because nitrate reduction extends to higher temperatures than the subsequent reduction of nitrite. This is important for understanding the fundamentals of thermophilicity and for the control of souring in oil fields catalyzed by SRB, which are strongly inhibited by nitrite.

Use of Homogeneously-Sized Carbon Steel Ball Bearings to Study Microbially-Influenced Corrosion in Oil Field Samples.

Microbially-influenced corrosion (MIC) contributes to the general corrosion rate (CR), which is typically measured with carbon steel coupons. Here we explore the use of carbon steel ball bearings, referred to as beads (55.0 ± 0.3 mg; Ø = 0.238 cm), for determining CRs. CRs for samples from an oil field in Oceania incubated with beads were determined by the weight loss method, using acid treatment to remove corrosion products. The release of ferrous and ferric iron was also measured and CRs based on weight loss and iron determination were in good agreement. Average CRs were 0.022 mm/yr for eight produced waters with high numbers (10(5)/ml) of acid-producing bacteria (APB), but no sulfate-reducing bacteria (SRB). Average CRs were 0.009 mm/yr for five central processing facility (CPF) waters, which had no APB or SRB due to weekly biocide treatment and 0.036 mm/yr for 2 CPF tank bottom sludges, which had high numbers of APB (10(6)/ml) and SRB (10(8)/ml). Hence, corrosion monitoring with carbon steel beads indicated that biocide treatment of CPF waters decreased the CR, except where biocide did not penetrate. The CR for incubations with 20 ml of a produced water decreased from 0.061 to 0.007 mm/yr when increasing the number of beads from 1 to 40. CRs determined with beads were higher than those with coupons, possibly also due to a higher weight of iron per unit volume used in incubations with coupons. Use of 1 ml syringe columns, containing carbon steel beads, and injected with 10 ml/day of SRB-containing medium for 256 days gave a CR of 0.11 mm/yr under flow conditions. The standard deviation of the distribution of residual bead weights, a measure for the unevenness of the corrosion, increased with increasing CR. The most heavily corroded beads showed significant pitting. Hence the use of uniformly sized carbon steel beads offers new opportunities for screening and monitoring of corrosion including determination of the distribution of corrosion rates, which allows estimation of the probability of high rate events that may lead to failure.

Metagenomic Analysis Indicates Epsilonproteobacteria as a Potential Cause of Microbial Corrosion in Pipelines Injected with Bisulfite.

Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in pipelines transporting brackish subsurface water used in the production of bitumen by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has indicated that SBS addition increased the fraction of the sulfate-reducing bacteria (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed by reduction of aqueous protons at the iron surface, or use low potential electrons from iron and aqueous protons directly for sulfate reduction. In order to reveal the effects of SBS treatment in more detail, metagenomic analysis was performed with pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in microbial community composition and in affiliated hynL genes for the large subunit of [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely catalyzed reduction of sulfur (S(0)) originating from the metabolism of bisulfite with cathodic H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel (Fe(0)). Overall this accelerates reaction of of S(0) and Fe(0) to form FeS, making this class a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger function.