Molecular and culture-based methods were used to investigate the microbial diversity in produced water obtained from the high-temperature Troll oil formation in the North Sea. 16S rRNA gene libraries were generated from total community DNA, using universal archaeal or bacterial oligonucleotide primer sets. Sequence analysis of 88 clones in the bacterial library indicated that they originated from members of Firmicutes (48 sequences), Bacteroidetes (17 sequences), delta-Proteobacteria (15 sequences), Spirochaetes (5 sequences), Thermotogales (2 sequences) and gamma-Proteobacteria (1 sequence). Twenty-two sequences in the archaeal library were close relatives to members of the genera Methanococcus (18 sequences), Methanolobus (3 sequences) and Thermococcus (1 sequence). Most of the bacterial sequences shared less than 95% identity with their closest match in GenBank, indicating that the produced water harbours a unique community of novel bacterial species or genera. Members of the thermophilic genera Thermosipho, Thermotoga, Anaerophaga and Thermovirga were isolated. The Troll formations are not injected with sea water. Thus, dramatic changes of the in situ conditions have been avoided, and a common source of continuous contamination from injection water can be excluded. However, the majority of the organisms detected in the gene libraries were most closely related to cultivated organisms with optimum temperatures for growth well below the in situ reservoir temperature (70 degrees C), indicating that produced water from the Troll platform harbours a substantial amount of non-indigenous organisms. This was confirmed by the isolation of a number of mesophilic and moderately thermophilic organisms that were unable to grow at reservoir temperature.

Rod-shaped, thermophilic bacteria with a sheath-like outer structure ('toga') were isolated from hot oilfield water of a North Sea oil reservoir. One of the isolates, designated SJ9ST, is an obligately anaerobic, sheathed, Gramnegative, fermentative bacterium capable of reducing elemental sulfur to hydrogen sulfide and tolerating high salt concentrations. The optimum growth conditions for this isolate are 58-60 "C and pH 605-79 with 3 4 % NaCl and 0.7% MgSO, .7H,O in the medium. Vitamins are required for growth. Growth is stimulated by yeast extract. Cells of strain SJ95T vary in size from 1-2 to 40-50 pm in length and are motile with a subpolar flagellation. Cells grown on xylan have xylanase activity, presumably associated with the toga, and glucose isomerase activity was detected in xylose-grown cells. The DNA G+C content is 31 and 34 mol%, determined by the thermal denaturation and HPLC methods, respectively. Phylogenetically, strain SJ95T is most closely related to Petrotoga miotherma with a 97.7% similarity level between their 165 rDNA sequences. The DNA-DNA reassociation value between the two DNAs was 35.6 YO. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain SJ95T (= DSM 106743 is proposed as the type strain of a new species, Petrotoga mobilis. It can be readily distinguished from P. miotherma by its motility.

A mesophilic, Gram-negative, rod-shaped, marine, propionate-oxidizing sulfate reducer (strain M I 6 9 was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 31 OC, pH 68-7.2 and l*5-2.Ooh(w/v) NaCl and 0-1-0*3% (w/v) MgCl,bH,O in the medium. The growth yield with sulfate was 4 6 g cell biomass (mol propionate 0xidized)'l. Strain M16l is nutritionally related to members of the genus Desulfobulbus, but differs in that it has no vitamin requirement and is able to utilize fumarate and malate as carbon and energy sources. Hydrogenase activity measured as hydrogen uptake was mainly membrane-bound and varied with the growth substrate. Highest activity [28 pmol min'l (mg protein )

The role of Asp-328 and Ile-329 as a cofactor discrimination site of the NAD-dependent isocitrate dehydrognase (NAD-IDH) from Pyrococcus furiosus has been verified by replacing these residues with Lys and Tyr, respectively, which are the corresponding residues in NADP-IDH from Escherichia coli. The Asp-328-Lys mutant showed dual coenzyme specificity, whereas introduction of the double mutation, Asp-328-Lys/Ile-329-Tyr shifted the cofactor preference from NAD to NADP. NADP-dependent P. furiosus IDH retained thermostability and thermoactivity compared with NAD-IDH.

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