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.

Characterization of 16S rRNA genes from oil field microbial communities indicates the presence of a variety of sulfate-reducing, fermentative, and sulfide-oxidizing bacteria.

Oil field bacteria were characterized by cloning and sequencing of PCR-amplified 16S rRNA genes. A variety of gram-negative, sulfate-reducing bacteria was detected (16 members of the family Desulfovibrionaceae and 8 members of the family Desulfobacteriaceae). In contrast, a much more limited number of anaerobic, fermentative, or acetogenic bacteria was found (one Clostridium sp., one Eubacterium sp., and one Synergistes sp.). Potential sulfide oxidizers and/or microaerophiles (Thiomicrospira, Arcobacter, Campylobacter, and Oceanospirillum spp.) were also detected. The first two were prominently amplified from uncultured production water DNA and represented 28 and 47% of all clones, respectively. Growth on media containing sulfide as the electron donor and nitrate as the electron acceptor and designed for the isolation of Thiomicrospira spp. gave only significant enrichment of the Campylobacter sp., which was shown to be present in different western Canadian oil fields. This newly discovered sulfide oxidizer may provide a vital link in the oil field sulfur cycle by reoxidizing sulfide formed by microbial sulfate or sulfur reduction.

Rapid comparison of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris using polymerase chain reaction amplification.

Polymerase chain reaction amplification was used to compare different regions of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris, to examine homology within the species. Six 30-base polymerase chain reaction primers and three probes were synthesized on the basis of the published nucleic acid sequence of the cytochrome c3 gene from D. vulgaris, NCIMB 8303. Amplifications were performed on genomic DNA isolated from NCIMB 8303 as well as eight other strains. Six strains, NCIMB 8302, 8305, 8306, 8311, 11779, and DSM 2119, showed amplification products of equal size and quantity to those of strain 8303. Two other strains, NCIMB 8456 and DSM 1744, either showed reduced levels or no detectable amplification products. These results were confirmed by hybridization of amplified DNA to radiolabeled probes specific for each product. DNA sequencing of a 145-bp polymerase chain reaction fragment from strains NCIMB 8302, 8303, 11779, and DSM 2119 revealed complete sequence homology between these strains, whereas slight differences were seen with strain NCIMB 8456. Amino acid sequencing of the first 20 residues of cytochrome c3 purified from strains NCIMB 8456 and 8303 also showed differences in the two proteins. In contrast to the results obtained with strain NCIMB 8456, limited homology was observed between the first 20 amino acid residues of cytochrome c3 from strain DSM 1744 and strain NCIMB 8303.

Biological effects of background radiation: mutagenicity of 40K.

The naturally occurring radioactive isotope 40K is the single largest contributor to the internal background radiation dose in living organisms. We examined cell growth and mutation rate or frequency in several strains of Escherichia coli in (i) media containing the natural content of 40K, (ii) media containing potassium from which essentially all of the 40K had been removed by isotope separation, and (iii) media highly enriched in 40K. Growth rates (doubling times) were identical in the present or absence of 40K. In more than 40 chemostat experiments, we were unable to detect any significant differences in mutation rate to bacteriophage T5 resistance or in mutation frequency to valine resistance or tryptophan prototrophy attributable to 40K. We conclude that, in the bacterial systems we have studied, 40K does not make a significant contribution to spontaneous mutation.

Cysteine synthesis by Desulfovibrio vulgaris extracts.

Extracts of Desulfovibrio vulgaris were found to contain serine transacetylase and cysteine synthase activities. When extracts were incubated with bisulfite and o-acetylserine, or acetyl coenzyme A plus L-serine, under a hydrogen atmosphere, cysteine was formed. Pyruvate served as a reductant for bisulfite reduction to sulfide and concomitantly provided the acetyl moiety for acetyl coenzyme A formation. Consequently, when extracts were incubated with pyruvate, bisulfite, and L-serine, cysteine synthesis resulted.