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 Biocide treatment is commonly used to inhibit SRB, but has now been replaced by nitrate treatment on several North Sea oil fields.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.     

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.     

Molecular- and cultivation-based analyses of microbial communities in oil field water and in microcosms amended with nitrate to control H2S production

Nitrate injection into oil fields is an alternative to biocide addition for controlling sulfide production (‘souring’) caused by sulfate-reducing bacteria (SRB). This study examined the suitability of several cultivation-dependent and cultivation-independent methods to assess potential microbial activities (sulfidogenesis and nitrate reduction) and the impact of nitrate amendment on oil field microbiota. Microcosms containing produced waters from two Western Canadian oil fields exhibited sulfidogenesis that was inhibited by nitrate amendment. Most probable number (MPN) and fluorescent in situ hybridization (FISH) analyses of uncultivated produced waters showed low cell numbers (≤10³ MPN/ml) dominated by SRB (>95% relative abundance). MPN analysis also detected nitrate-reducing sulfide-oxidizing bacteria (NRSOB) and heterotrophic nitrate-reducing bacteria (HNRB) at numbers too low to be detected by FISH or denaturing gradient gel electrophoresis (DGGE). In microcosms containing produced water fortified with sulfate, near-stoichiometric concentrations of sulfide were produced. FISH analyses of the microcosms after 55 days of incubation revealed that Gammaproteobacteria increased from undetectable levels to 5–20% abundance, resulting in a decreased proportion of Deltaproteobacteria (50–60% abundance). DGGE analysis confirmed the presence of Delta- and Gammaproteobacteria and also detected Bacteroidetes. When sulfate-fortified produced waters were amended with nitrate, sulfidogenesis was inhibited and Deltaproteobacteria decreased to levels undetectable by FISH, with a concomitant increase in Gammaproteobacteria from below detection to 50–60% abundance. DGGE analysis of these microcosms yielded sequences of Gamma- and Epsilonproteobacteria related to presumptive HNRB and NRSOB (Halomonas, Marinobacterium, Marinobacter, Pseudomonas and Arcobacter), thus supporting chemical data indicating that nitrate-reducing bacteria out-compete SRB when nitrate is added.     

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     

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     

Corrosion risk associated with microbial souring control using nitrate or nitrite

Souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB) in oil reservoirs, can be controlled through nitrate or nitrite addition. To assess the effects of this containment approach on corrosion, metal coupons were installed in up-flow packed-bed bioreactors fed with medium containing 8 mM sulfate and 25 mM lactate. Following inoculation with produced water to establish biogenic H₂S production, some bioreactors were treated with 17.5 mM nitrate or up to 20 mM nitrite, eliminating souring. Corrosion rates were highest near the outlet of untreated bioreactors (up to 0.4 mm year^–1). Nitrate (17.5 mM) eliminated sulfide but gave pitting corrosion near the inlet of the bioreactor, whereas a high nitrite dose (20 mM) completely eliminated microbial activity and associated corrosion. More gradual, step-wise addition of nitrite up to 20 mM resulted in the retention of microbial activity and localized pitting corrosion, especially near the bioreactor inlet. We conclude that: (1) SRB control by nitrate or nitrite reduction shifts the corrosion risk from the bioreactor outlet to the inlet (i.e. from production to injection wells) and (2) souring treatment by continuous addition of a high inhibitory nitrite dose is preferable from a corrosion-prevention point of view.     

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     

Effects of nitrate treatment on a mixed species, oil field microbial biofilm

Biofilms of bacteria, indigenous to oil field produced water, were grown in square section, glass capillary flow cells at 45 °C. Initially, in situ image analysis microscopy revealed predominantly coccoid bacteria (length-to-width ratio measurements (l _c:w _c) of bacterial cells gave a mean value of 1.1), while chemical measurements confirmed sulphate reduction and sulphide production. After nitrate ion addition at 100 and 80 mg/l, in the two repeat experiments respectively, the dominance of rod-shaped bacteria (mean l _c:w _c = 2.8) was observed. This coincided with the occurrence of nitrate reduction in the treated flow cells. Beneficially, no significant increase in biofilm cover was observed after the addition of nitrate. The dominant culturable nitrate-reducing bacterium was Marinobacter aquaeolei. The l _c:w _c ratio measured here concurs with previously reported cell dimensions for this organism. Several Marinobacter strains were also isolated from different oil fields in the North Sea where nitrate treatment has been applied to successfully treat reservoir souring, implying that this genus may play an important role in nitrate treatment.     

Control of microbial souring by nitrate,nitrite or glutaraldehyde injection in a sandstone column

Microbial souring (production of hydrogen sulfide by sulfate-reducing bacteria, SRB) in crushed Berea sandstone columns with oil field-produced water consortia incubated at 60°C was inhibited by the addition of nitrate (NO₃) or nitrite (NO ₂ ^– ). Added nitrate (as nitrogen) at a concentration of 0.71 mM resulted in the production of 0.57–0.71 mM nitrite by the native microbial population present during souring and suppressed sulfate reduction to below detection limits. Nitrate added at 0.36 mM did not inhibit active souring but was enough to maintain inhibition if the column had been previously treated with 0.71 mM or greater. Continuous addition of 0.71–0.86 mM nitrite also completely inhibited souring in the column. Pulses of nitrite were more effective than the same amount of nitrite added continuously. Nitrite was more effective at inhibiting souring than was glutaraldehyde, and SRB recovery was delayed longer with nitrite than with glutaraldehyde. It was hypothesized that glutaraldehyde killed SRB while nitrite provided a long-term inhibition without cell death. Removal of nitrate after as long as 3 months of continuous addition allowed SRB in a biofilm to return to their previous level of activity. Inhibition was achieved with much lower levels of nitrate and nitrite, and at higher temperatures, than noted by other researchers.     

Sulfate-reducing bacterial community structure and their contribution to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions

The community structure of sulfate-reducing bacteria (SRB) and the contribution of SRB to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions were investigated by combining molecular techniques, molybdate inhibition batch experiments, and microelectrode measurements. A 16S rDNA clone library of bacteria populations was constructed from the biofilm sample. The 102 clones analyzed were grouped into 53 operational taxonomic units (OTUs), where the clone distribution was as follows: Cytophaga-Flexibacter-Bacteroides (41%), Proteobacteria (41%), low-G+C Gram-positive bacteria (18%), and other phyla (3%). Three additional bacterial clone libraries were also constructed from SRB enrichment cultures with propionate, acetate, and H₂ as electron donors to further investigate the differences in SRB community structure due to amendments of different carbon sources. These libraries revealed that SRB clones were phylogenetically diverse and affiliated with six major SRB genera in the delta-subclass of the Proteobacteria. Fluorescent in situ hybridization (FISH) analysis revealed that Desulfobulbus and Desulfonema were the most abundant SRB species in this biofilm, and this higher abundance (ca. 2–4×10⁹ cells cm^–3 and 5×10⁷ filaments cm^–3, respectively) was detected in the surface of the biofilm. Microelectrode measurements showed that a high sulfate-reducing activity was localized in a narrow zone located just below the oxic/anoxic interface when the biofilm was cultured in a synthetic medium with acetate as the sole carbon source. In contrast, a broad sulfate-reducing zone was found in the entire anoxic strata when the biofilm was cultured in the supernatant of the primary settling tank effluent. This is probably because organic carbon sources diffused into the biofilm from the bulk water and an unknown amount of volatile fatty acids was produced in the biofilm. A combined approach of molecular techniques and batch experiments with a specific inhibitor (molybdate) clearly demonstrated that Desulfobulbus is a numerically important member of SRB populations and the main contributor to the oxidation of propionate to acetate in this biofilm. However, acetate was preferentially utilized by nitrate-reducing bacteria but not by acetate-utilizing SRB.     

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).     

Inhibiting sulfate-reducing bacteria in biofilms on steel with antimicrobial peptides generated in situ

In batch and continuous fermentations, the reduction in corrosion of SAE 1018 mild steel and 304 stainless steel caused by inhibition of the reference sulfate-reducing bacterium (SRB) Desulfovibrio vulgaris by a protective, antimicrobial-producing Bacillus brevis biofilm was investigated. The presence of D. vulgaris produced a thick black precipitate on mild steel and a higher corrosion rate in batch cultures than that seen in a mono-culture of non-antimicrobial-producing Pseudomonas fragi K upon the addition of SRB to the aerobic P. fragi K biofilm. In continuous reactors, the polarization resistance R _p decreased for stainless steel and increased for mild steel upon the addition of SRB to a P. fragi K biofilm. Addition of either 200 μg/ml ampicillin, chloramphenicol, or ammonium molybdate to batch and continuous reactors after SRB had colonized the metal was ineffective in killing SRB, as inferred from the lack of change in both R _p and the impedance spectra. However, when ampicillin was added prior to SRB colonization, the growth of SRB was completely inhibited on stainless steel in continuous reactors. Prior addition of ampicillin was only able to delay the growth of SRB on mild steel in continuous reactors. External addition of the purified peptide antimicrobial agent gramicidin S prior to the addition of SRB also inhibited the growth of SRB on stainless steel in continuous reactors, and the SRB were also inhibited on stainless steel in both batch and continuous reactors by producing gramicidin S in situ in a protective biofilm when the gramicidin-S-overproducing strain Bacillus brevis 18 was used. Received: 29 October 1998 / Received revision: 18 February 1999 / Accepted: 26 February 1999     

A green triple biocide cocktail consisting of a biocide, EDDS and methanol for the mitigation of planktonic and sessile sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) cause souring and their biofilms are often the culprit in Microbiologically Influenced Corrosion (MIC). The two most common green biocides for SRB treatment are tetrakis-hydroxymethylphosphonium sulfate (THPS) and glutaraldehyde. It is unlikely that there will be another equally effective green biocide in the market any time soon. This means more effective biocide treatment probably will rely on biocide cocktails. In this work a triple biocide cocktail consisting of glutaraldehyde or THPS, ethylenediaminedisuccinate (EDDS) and methanol was used to treat planktonic SRB and to remove established SRB biofilms. Desulfovibrio vulgaris (ATCC 7757), a corrosive SRB was used as an example in the tests. Laboratory results indicated that with the addition of 10–15% (v/v) methanol to the glutaraldehyde and EDDS double combination, mitigation of planktonic SRB growth in ATCC 1249 medium and a diluted medium turned from inhibition to a kill effect while the chelator dosage was cut from 2,000 to 1,000 ppm. Biofilm removal was achieved when 50 ppm glutaraldehyde combined with 15% methanol and 1,000 ppm EDDS was used. THPS showed similar effects when it was used to replace glutaraldehyde in the triple biocide cocktail to treat planktonic SRB.     

Effect of nitrate injection on the bacterial community in a water-oil tank system analyzed by PCR-DGGE

Sulfide production by sulfate-reducing bacteria (SRB) is a major concern for the petroleum industry since it is toxic and corrosive, and causes plugging due to the formation of insoluble iron sulfides (reservoir souring). In this study, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) using two sets of primers based on the 16S rRNA gene and on the aps gene (adenosine-5-phosphosulfate reductase) was used to track changes in the total bacterial and SRB communities, respectively, present in the water-oil tank system on an offshore platform in Brazil in which nitrate treatment was applied for 2 months (15 nitrate injections). PCR-DGGE analysis of the total bacterial community showed the existence of a dominant population in the water-oil tank, and that the appearance and/or the increase of intensity of some bands in the gels were not permanently affected by the introduction of nitrate. On the other hand, the SRB community was stimulated following nitrate treatment. Moreover, sulfide production did not exceed the permissible exposure limit in the water-oil separation tank studied treated with nitrate. Therefore, controlling sulfide production by treating the produced water tank with nitrate could reduce the quantity of chemical biocides required to control microbial activities.     

Response of denitrification rate associated with wetting and drying cycles in a littoral wetland area of Lake Biwa,Japan

To clarify the relationship between denitrification activity and dry–wet levels in the littoral wetland sediments of Lake Biwa, Japan, denitrification rates and their regulating parameters (degree of dryness, redox potential, nitrate concentration) were measured on different moisture sediments. Redox potential in sediments was higher in the exposed region in contact with atmosphere than the flooded region covered with water. The nitrate concentration in interstitial waters was undetectable in the flooded region. On the other hand, concentration in the exposed region increased with increase in the degree of sediment dryness. The denitrification rate ranged from <0.001 to 0.88 μg N cm⁻³ h⁻¹ in the exposed region and increased with the increase in the degree of dryness. In the flooded region, on the other hand, no detectable rate (<0.001 μg N cm⁻³ h⁻¹) was observed. This indicates that the rates in the exposed region were mainly influenced by nitrate concentration in the interstitial waters accumulated by desiccation of sediments, whereas rates in the flooded region were strongly limited by no accumulation of nitrate in the anaerobic conditions. The potential denitrification rate, under the application condition of nitrate, ranged from 0.13 to 0.26 μg N cm⁻³ h⁻¹ in the flooded region and from 0.77 to 1.5 μg N cm⁻³ h⁻¹ in the exposed region. The potential rates in the flooded region had a tendency to be lower than those in the exposed region, implying that the number of denitrifying bacteria in the flooded region was low due to inactivation of aerobic respiration and denitrification in the denitrifying bacteria community. Kinetic parameters, maximum rate (V _max) and half-saturation constant (K _s) for denitrification were calculated on the experimental procedures of the wetting–drying cycles of sediments. Both parameters decreased by the wetting treatment and increased by the drying treatment. The fluctuation of V _max values with wetting–drying cycles indicated that the number of denitrifying bacteria was influenced by aerobic respiration and denitrification in the denitrifying bacteria community similar to the potential rates, and denitrifying enzyme was induced by the nitrate supplied by nitrification accelerated through the drying process. On the other hand, the fluctuation of K _s values implied that members of denitrifying bacteria were shifted to members of high nitrate affinity by wetting treatment and of low nitrate affinity by drying treatment.     

Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor

A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria (SRB) in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater. The wastewater was fed with ethanol as an external electron donor. Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth (i.e. 10⁸). Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity, with only two genotypes among the 26 strains obtained in the pure culture. The low genetic diversity suggests the absence of micro-niches within the reactor, which might be due to a low spatial and temporal micro-heterogeneity. The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio. The two strains differed in as many as five physiological traits, which might allow them to occupy distinct niches and thus co-exist within the same habitat. Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor. The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB, representing 30–35% and 25–35%, respectively, of the total SRB community. Desulfobulbus-like bacteria contributed for 20–25%, and the Desulfobacca acetoxidans-specific probe targeted approximately 15–20% of the total SRB. The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor.     

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.     

Acetate Production from Oil under Sulfate-Reducing Conditions in Bioreactors Injected with Sulfate and Nitrate

Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266–269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus Desulfovibrio, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.     

Biofilms: strategies for metal corrosion inhibition employing microorganisms

Corrosion causes dramatic economic loss. Currently widely used corrosion control strategies have disadvantages of being expensive, subject to environmental restrictions, and sometimes inefficient. Studies show that microbial corrosion inhibition is actually a common phenomenon. The present review summarizes recent progress in this novel strategy: corrosion control using beneficial bacteria biofilms. The possible mechanisms may involve: (1) removal of corrosive agents (such as oxygen) by bacterial physiological activities (e.g., aerobic respiration), (2) growth inhibition of corrosion-causing bacteria by antimicrobials generated within biofilms [e.g., sulfate-reducing bacteria (SRB) corrosion inhibition by gramicidin S-producing Bacillus brevis biofilm], (3) generation of protective layer by biofilms (e.g., Bacillus licheniformis biofilm produces on aluminum surface a sticky protective layer of γ-polyglutamate). Successful utilization of this novel strategy relies on advances in study at the interface of corrosion engineering and biofilm biology.