No abstract

and RKU-10 T were 468 and 461 mol %. Phenotypic characteristics and 16S rDNA sequences of the isolates were similar to those of Thermotoga maritima and Thermotoga neapolitana, both being hyperthermophilic bacteria isolated from hydrothermal fields. However, the isolates differed from these species in their minimum growth temperatures, utilization of some sugars, sensitivity to rifampicin and the effects of elemental sulfur and thiosulfate on growth. The low levels (less than 31 %) of DNA reassociation between any two of these hyperthermophilic Thermotoga strains indicated that the isolates were novel species. Analysis of the gyrB gene sequences supported the view that the isolates were genotypically different from these reference species. The isolates were named Thermotoga petrophila sp. nov., with type strain RKU-1 T (l DSM 13995 T l JCM 10881 T ), and Thermotoga naphthophila sp. nov., with type strain RKU-10 T ( l DSM 13996 T l JCM 10882 T ).

The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.

The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58°C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 ؋ 10 3 to 4.6 ؋ 10 4 cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. Dominant Thermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50°C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0.01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.

A hyperthermophilic archaeon strain, KODI, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100°C, and the optimal temperature was 950C. The anaerobic strain was an So-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110°C, with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90°C for 2 h, and the half-life of enzymatic activity at 100°C was 60 min. The proteolytic activity was significantly inhibited byp-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.