Nitrite reductase activity of sulphate-reducing bacteria prevents their inhibition by nitrate-reducing, sulphide-oxidizing bacteria.

Sulphate-reducing bacteria (SRB) can be inhibited by nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB), despite the fact that these two groups are interdependent in many anaerobic environments. Practical applications of this inhibition include the reduction of sulphide concentrations in oil fields by nitrate injection. The NR-SOB Thiomicrospira sp. strain CVO was found to oxidize up to 15 mM sulphide, considerably more than three other NR-SOB strains that were tested. Sulphide oxidation increased the environmental redox potential (Eh) from -400 to +100 mV and gave 0.6 nitrite per nitrate reduced. Within the genus Desulfovibrio, strains Lac3 and Lac6 were inhibited by strain CVO and nitrate for the duration of the experiment, whereas inhibition of strains Lac15 and D. vulgaris Hildenborough was transient. The latter had very high nitrite reductase (Nrf) activity. Southern blotting with D. vulgaris nrf genes as a probe indicated the absence of homologous nrf genes from strains Lac3 and Lac6 and their presence in strain Lac15. With respect to SRB from other genera, inhibition of the known nitrite reducer Desulfobulbus propionicus by strain CVO and nitrate was transient, whereas inhibition of Desulfobacterium autotrophicum and Desulfobacter postgatei was long-lasting. The results indicate that inhibition of SRB by NR-SOB is caused by nitrite production. Nrf-containing SRB can overcome this inhibition by further reducing nitrite to ammonia, preventing a stalling of the favourable metabolic interactions between these two bacterial groups. Nrf, which is widely distributed in SRB, can thus be regarded as a resistance factor that prevents the inhibition of dissimilatory sulphate reduction by nitrite.

Impact of nitrate-mediated microbial control of souring in oil reservoirs on the extent of corrosion.

The effect of microbial control of souring on the extent of corrosion was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in an SRB consortium enriched from produced water from a Canadian oil reservoir. The average corrosion rate induced by the SRB consortium (1.4 g x m(-2) x day(-1)) was faster than that observed in the presence of strain Lac6 (0.2 g x m(-2) x day(-1)). Examination of the metallic coupons at the end of the tests indicated a uniform corrosion in both cases. Addition of CVO and 10 mM nitrate to a fully grown culture of Lac6 or the SRB consortium led to complete removal of sulfide from the system and a significant increase in the population of CVO, as determined by reverse sample genome probing. In the case of the SRB consortium addition of just nitrate (10 mM) had a similar effect. When grown in the absence of nitrate, the consortium was dominated by Desulfovibrio sp. strains Lac15 and Lac29, while growth in the presence of nitrate led to dominance of Desulfovibrio sp. strain Lac3. The addition of CVO and nitrate to the Lac6 culture or nitrate to the SRB consortium accelerated the average corrosion rate to 1.5 and 2.9 g x m(-2) x day(-1), respectively. Localized corrosion and the occurrence of pitting were apparent in both cases. Although the sulfide concentration (0.5-7 mM) had little effect on corrosion rates, a clear increase of the corrosion rate with increasing nitrate concentration was observed in experiments conducted with consortia enriched from produced water.

Control of biogenic H(2)S production with nitrite and molybdate.

The effects of the metabolic inhibitors, sodium nitrite and ammonium molybdate, on production of H(2)S by a pure culture of the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6 and a consortium of SRB, enriched from produced water of a Canadian oil field, were investigated. Addition of 0.1 mM nitrite or 0.024 mM molybdate at the start of growth prevented the production of H(2)S by strain Lac6. With exponentially growing cultures, higher levels of inhibitors, 0.25 mM nitrite or 0.095 mM molybdate, were required to suppress the production of H(2)S. Simultaneous addition of nitrite and molybdate had a synergistic effect: at time 0, 0.05 mM nitrite and 0.01 mM molybdate, whereas during the exponential phase, 0.1 mM nitrite and 0.047 mM molybdate were sufficient to stop H(2)S production. With an exponentially growing consortium of SRB, enriched from produced water of the Coleville oil field, much higher levels of inhibitors, 4 mM nitrite or 0.47 mM molybdate, were needed to stop the production of H(2)S. The addition of these inhibitors had no effect on the composition of the microbial community, as shown by reverse sample genome probing. The results indicate that the efficiency of inhibitors in containment of SRB depends on the composition and metabolic state of the microbial community.

Mechanistic study of microbial control of hydrogen sulfide production in oil reservoirs.

Microbial control of biogenic production of hydrogen sulfide in oil fields was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in microbial cultures enriched from produced water of a Canadian oil reservoir. The presence of nitrate at concentrations up to 20 mM had little effect on the rate of sulfate reduction by a pure culture of Lac6. Addition of CVO imposed a strong inhibition effect on production of sulfide. In the absence of added nitrate SRB we were able to overcome this effect after an extended lag phase. Simultaneous addition of CVO and nitrate stopped the production of H2S immediately. The concentration of sulfide decreased to a negligible level due to nitrate-dependent sulfide oxidation activity of CVO. This was not prevented by raising the concentration of Na-lactate, the electron donor for sulfate reduction. Similar results were obtained with enrichment cultures. Enrichments of produced water with sulfide and nitrate were dominated by CVO, whereas enrichments with sulfate and Na-lactate were dominated by SRB. Addition of an NR-SOB enrichment to an SRB enrichment inhibited the production of sulfide. Subsequent addition of sufficient nitrate caused the sulfide concentration to drop to zero. A similar response was seen in the presence of nitrate alone, although after a pronounced lag time, it was needed for emergence of a sizable CVO population. The results of the present study show that two mechanisms are involved in microbial control of biogenic sulfide production. First, addition of NR-SOB imposes an inhibition effect, possibly by increasing the environmental redox potential to levels which are inhibitory for SRB. Second, in the presence of sufficient nitrate, NR-SOB oxidize sulfide, leading to its complete removal from the environment. Successful microbial control of H2S in an oil reservoir is crucially dependent on the simultaneous presence of NR-SOB (either indigenous population or injected) and nitrate in the environment.

Characterization of a novel biocatalyst system for sulfide oxidation.

It has been demonstrated that an enrichment culture dominated by Thiomicrospira sp. CVO may be cultured on H2S(g) as an energy source under sulfide-limiting conditions in suspended culture with nitrate as the electron acceptor. Hydrogen sulfide (10,000 ppmv) was completely removed from the feed gas and oxidized to sulfate in <3 s of gas-liquid contacting time. Maximum loading of the biomass for sulfide oxidation was observed to be 5.8 mmol H2S/h-g biomass protein, comparable to that reported previously for Thiobacillus denitrificans under similar conditions. However, the enrichment culture was shown to be more tolerant of extremes in pH and elevated temperature than T. denitrificans. Coupled with a reported tolerance of CVO for up to 10% NaCl, these observations suggest that a CVO-based culture is potentially a more robust biocatalyst system for sulfide oxidation than cultures based on Thiobacilli.

Isolation and characterization of strains CVO and FWKO B, two novel nitrate-reducing, sulfide-oxidizing bacteria isolated from oil field brine.

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O(2)). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO(2) as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO(2). Both strains grow at temperatures between 5 and 40 degrees C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.

Effect of nitrate injection on the microbial community in an oil field as monitored by reverse sample genome probing.

The reverse sample genome probe (RSGP) method, developed for monitoring the microbial community in oil fields with a moderate subsurface temperature, has been improved by (i) isolation of a variety of heterotrophic bacteria and inclusion of their genomes on the oil field master filter and (ii) use of phosphorimaging technology for the rapid quantitation of hybridization signals. The new master filter contains the genomes of 30 sulfate-reducing, 1 sulfide-oxidizing, and 16 heterotrophic bacteria. Most have been identified by partial 16S rRNA sequencing. Use of improved RSGP in monitoring the effect of nitrate injection in an oil field indicated that the sulfide-oxidizing, nitrate-reducing isolate CVO (a Campylobacter sp.) becomes the dominant community component immediately after injection. No significant enhancement of other community members, including the sulfate-reducing bacteria, was observed. The elevated level of CVO decayed at most sampling sites within 30 days after nitrate injection was terminated. Chemical analyses indicated a corresponding decrease and subsequent increase in sulfide concentrations. Thus, transient injection of a higher potential electron acceptor into an anaerobic subsurface system can have desirable effects (i.e., reduction of sulfide levels) without a permanent adverse influence on the resident microbial community.

Mechanisms of microbial movement in subsurface materials.

The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster than mutants defective only in flagellar synthesis. Motile, nonchemotactic mutants penetrated the cores faster than did the chemotactic parental strain. This, plus the fact that a chemotactic galactose mutant penetrated cores saturated with peptone medium at the same rate with or without a galactose gradient, indicates that chemotaxis may not be required for bacterial penetration through unconsolidated porous media. The effect of gas production on bacterial penetration was studied by using motile and nonmotile E. coli strains together with their respective isogenic non-gas-producing mutants. No differences were observed between the penetration rates of the two motile strains through cores saturated with peptone medium with or without galactose. However, penetration of both nonmotile strains was detected only with galactose. The nonmotile, gas-producing strain penetrated cores saturated with galactose-peptone medium five to six times faster than did the nonmotile, non-gas-producing mutant, which indicates that gas production is an important mechanism for the movement of nonmotile bacteria through unconsolidated porous media. For motile strains, the penetration rate decreased with increasing galactose concentrations in the core and with decreasing inoculum sizes. Also, motile strains with the faster growth rates had faster penetration rates. These results imply that, for motile bacteria, the penetration rate is regulated by the in situ bacterial growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nitrate on biogenic sulfide production.

The addition of 59 mM nitrate inhibited biogenic sulfide production in dilute sewage sludge (10% [vol/vol]) amended with 20 mM sulfate and either acetate, glucose, or hydrogen as electron donors. Similar results were found when pond sediment or oil field brines served as the inoculum. Sulfide production was inhibited for periods of at least 6 months and was accompanied by the oxidation of resazurin from its colorless reduced state to its pink oxidized state. Lower amounts of nitrate (6 or 20 mM) and increased amounts of sewage sludge resulted in only transient inhibition of sulfide production. The addition of 156 mM sulfate to bottles with 59 mM nitrate and 10% (vol/vol) sewage sludge or pond sediment resulted in sulfide production. Nitrate, nitrite, and nitrous oxide were detected during periods where sulfide production was inhibited, whereas nitrate, nitrite, and nitrous oxide were below detectable levels at the time sulfide production began. The oxidation of resazurin was attributed to an increase in nitrous oxide which persisted in concentration of about 1.0 mM for up to 5 months. The numbers of sulfate-reducing organisms decreased from 10 CFU ml sludge to less than detectable levels after prolonged incubation of oxidized bottles. The addition of 10 mM glucose to oxidized bottles after 14.5 weeks of incubation resulted in rereduction of the resazurin and subsequent sulfide production. The prolonged inhibition of sulfide production was attributed to an increase in oxidation-reduction potential due to biogenic production of nitrous oxide, which appeared to have a cytotoxic effect on sulfate-reducing populations.

Isolation of halotolerant, thermotolerant, facultative polymer-producing bacteria and characterization of the exopolymer.

Over 200 bacterial strains were selected for anaerobic growth at 50 degrees C and extracellular polysaccharide production in a sucrose-mineral salts medium with NaNO(3) and up to 10% NaCl. The predominant cell type was an encapsulated gram-positive, motile, facultative sporeforming rod similar to Bacillus species. Strain SP018 grew and produced the polysaccharide on a variety of substrates at salinities up to 12% NaCl. Good polymer production only occurred anaerobically and was optimal between 4 and 10% NaCl. The ethanol-precipitated SP018 polymer was a charged heteropolysaccharide that contained glucose, mannose, arabinose, ribose, and low levels of allose and glucosamine. The SP018 polymer showed pseudoplastic behavior, was resistant to shearing, and had a higher viscosity at dilute concentrations and at elevated temperatures than xanthan gum. High-ionic-strength solutions reversibly decreased the viscosity of SP018 polymer solutions. The bacterium and the associated polymer have many properties that make them potentially useful for in situ microbially enhanced oil recovery processes.

Method for detection of microorganisms that produce gaseous nitrogen oxides.

A method was developed to detect NO- or N(2)O-producing bacteria in solid or liquid medium by their ability to oxidize the redox indicator resazurin from its reduced colorless form to its oxidized pink form. The method was sensitive to as little as 35 nM N(2)O or 0.5 nM NO. Ninety-one percent of the colonies that oxidized resazurin on plates also produced N(2)O in slant cultures. Forty-four percent of the colonies that did not oxidize resazurin did produce N(2)O. This percentage was reduced to 15% when colonies in which the coloration was difficult to discern were picked to slants to determine whether they oxidized the slant. The production of N(2)O preceded the oxidation of resazurin by liquid cultures of Escherichia coli and a sludge isolate. With the denitrifying sewage isolate, the disappearance of N(2)O was followed by the return of resazurin to its reduced state. Wolinella succinogenes was found to produce small amounts of N(2)O from NO(3), which resulted in a transient oxidation of resazurin.

Effect of Sterilization by Dry Heat or Autoclaving on Bacterial Penetration through Berea Sandstone.

A study was undertaken to determine why bacteria could penetrate lengths of consolidated sandstone (Berea) faster when the sandstone was sterilized by autoclaving than when dry heat (150 degrees C, 3 h) was used. Changes in permeability, porosity, and pore entrance size of the rock as a result of autoclaving were not sufficient to explain the differences in penetration times observed, but electron dispersion spectroscopy and electron microscopy of the rock revealed changes in mineral composition and clay morphology. Autoclaved cores contained more chloride than dry-heated cores, and the clays of autoclaved cores were aggregated and irregularly shaped. Therefore, the decreases in bacterial penetration rates caused by autoclave sterilization were probably the result of a change in surface charge of the pores of the rock and of a reduction in surface area of clays available for adhesion. The results implied that dry-heat sterilization was preferable to autoclaving when examining biotic and abiotic interactions in a native-state rock model.

Anaerobic Production of a Biosurfactant by Bacillus licheniformis JF-2.

Bacillus licheniformis JF-2 anaerobically produced a biosurfactant when grown in a glucose-mineral salts medium containing yeast extract and NaNO(3). Surface tension of the medium was reduced from 70 to 74 mN/m to as low as 28 mN/m due to the production of an anionic biosurfactant.

Microbial Penetration through Nutrient-Saturated Berea Sandstone.

Penetration times and penetration rates for a motile Bacillus strain growing in nutrient-saturated Berea sandstone cores were determined. The rate of penetration was essentially independent of permeabilities above 100 mdarcys and rapidly declined for permeabilities below 100 mdarcys. It was found that these penetration rates could be grouped into two statistically distinct classes consisting of rates for permeabilities above 100 mdarcys and rates for those below 100 mdarcys. Instantaneous penetration rates were found to be zero order with respect to core length for cores with permeabilities above 100 mdarcys and first order with respect to core length for cores with permeabilities below 100 mdarcys. The maximum observed penetration rate was 0.47 cm . h, and the slowest was 0.06 cm . h; however, these rates may be underestimates of the true penetration rate, since the observed rates included the time required for growth in the flask as well as the core. The relationship of penetration time to the square of the length of the core suggested that cells penetrated high-permeability cores as a band and low-permeability cores in a diffuse fashion. The motile Enterobacter aerogenes strain penetrated Berea sandstone cores three to eight times faster than did the nonmotile Klebsiella pneumoniae strain when cores of comparable length and permeability were used. A penetration mechanism based entirely on motility predicted penetration times that were in agreement with the observed penetration times for motile strains. The fact that nonmotile strains penetrated the cores suggested that filamentous or unrestricted growth, or both, may also be important.