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

Abstract

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.

FIG. 1

(A and C) Epifluorescence photomicrographs of strains CVO (A) and FWKO B (C) grown with sulfide and nitrate (bars = 20 μm). (B and D) SEM photomicrographs of strains CVO (B) and FWKO B (D) grown on sulfide and nitrate (bar = 2 μm).

FIG. 2

(A) Growth curves and sulfide consumption rates, respectively, for CVO (○ and ●) and FWKO B (■ and □) growing at 30°C in DTA brine medium containing 2 mM sulfide and 10 mM KNO₃. Cell numbers are expressed as AODC. (B) Cell density versus sulfide consumption for CVO and FWKO B plotted from data in panel A. Linear trend lines and R² values were determined with Microsoft Excel 97 by setting the y intercept to 0.

FIG. 3

Hybridization of either labeled eubacterial (EUB 338) or labeled CVO-specific (CLI) oligonucleotide probe to nucleic acids from whole cells of bacteria known to be closely related to CVO (Fig. 4; isolate Light C is described in the text) or to nucleic acids from cells from an enrichment of sulfide-laden produced brine collected at CV Well 59-20 amended with nitrate and phosphate (culture 59-20).

FIG. 4

Phylogenetic trees of 16S rRNA sequences of strains CVO and FWKO B. (A) Tree based on pairwise homologies obtained with the GCG program PileUp. The horizontal distances reflect the pairwise sequence similarities as indicated on the scale. (B) Tree obtained by PAUP analysis with the sequence for E. coli as the outgroup and using 100 bootstrap replicates. The observed frequencies of the groups are indicated at the nodes. The horizontal distances are arbitrary. In addition to the sequences for CVO and FWKO B, the following sequences are represented in the tree: af054578, Helicobacter sp. strain UNSW1.7sp; af072333, Helicobacter sp. strain hamster B; af061104, Helicobacter typhlonicus; htu65103, Helicobacter trogontum; thmrrd, Thiomicrospira denitrificans; cj16srna, Campylobacter jejuni; cajrrdg, Campylobacter upsaliensis; af097691, Campylobacter hyointestinalis; saf1f16srna, Sulfurospirillum arcachonense; u85965, Sulfurospirillum sp. strain SM-5; gbu41564, S. barnesii; sdy13671, S. deleyianum; af038843, S. barnesii; cajrrdk, Campylobacter sp. strain CCUG 1392 CLO; dsm806, Campylobacter sp. strain DSM806 (determined in this study); dmx82931, D. multivorans; gau85964, S. arsenophilum; aobrrdc, A. butzleri; aobrrda, A. cryoaerophilus; aobrrdd, A. nitrofigilis; ecorrdb, E. coli. The strains sequenced in this study are in boldface.