Thalassospira tepidiphila sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from seawater.

A Gram-negative, mesophilic bacterial strain, designated 1-1B(T), which degrades polycyclic aromatic hydrocarbons, was isolated from petroleum-contaminated seawater during a bioremediation experiment. A 16S rRNA gene sequence analysis indicated that the isolate was affiliated with the genus Thalassospira in the Alphaproteobacteria; the sequence was found to be most similar to those of Thalassospira profundimaris WP0211(T) (99.8 %), Thalassospira xiamenensis M-5(T) (98.2 %) and Thalassospira lucentensis DSM 14000(T) (98.1 %). However, the levels of DNA-DNA relatedness between strain 1-1B(T) and these type strains were 50.7+/-17.2, 35.7+/-17.8 and 32.0+/-21.1 %, respectively. In addition, strain 1-1B(T) was found to be distinct from the other described species of the genus Thalassospira in terms of some taxonomically important traits, including DNA G+C content, optimum growth temperature, salinity tolerance, utilization of carbon sources and fatty acid composition. Furthermore, strain 1-1B(T) and T. profundimaris were also different with regard to motility and denitrification capacities. On the basis of physiological and DNA-DNA hybridization data, strain 1-1B(T) represents a novel species within the genus Thalassospira, for which the name Thalassospira tepidiphila sp. nov. is proposed. The type strain is 1-1B(T) (=JCM 14578(T) =DSM 18888(T)).

Sulfurospirillum cavolei sp. nov., a facultatively anaerobic sulfur-reducing bacterium isolated from an underground crude oil storage cavity.

A novel facultatively anaerobic sulfur-reducing bacterium, designated strain Phe91(T), was isolated from petroleum-contaminated groundwater in an underground crude oil storage cavity at Kuji in Iwate, Japan. Cells of strain Phe91(T) were slightly curved rods with single polar flagella. Optimum growth was observed at pH 7.0 and 30 degrees C. The novel strain utilized elemental sulfur, thiosulfate, sulfite, dithionite, arsenate, nitrate and DMSO as electron acceptors with lactate as an energy and carbon source, but nitrite was not utilized. Microaerophilic growth was also observed. Fumarate, pyruvate, lactate, malate, succinate, hydrogen (with acetate as a carbon source) and formate (with acetate) could serve as electron donors. Fumarate, pyruvate and malate were fermented. The DNA G+C content was 42.7 mol%. On the basis of 16S rRNA gene sequence phylogeny, strain Phe91(T) was affiliated with the genus Sulfurospirillum in the class Epsilonproteobacteria and was most closely related to Sulfurospirillum deleyianum (sequence similarity 97 %). However, the DNA-DNA hybridization value between strain Phe91(T) and S. deleyianum was only 14 %. Based on the physiological and phylogenetic data, Phe91(T) should be classified as a representative of a novel species in the genus Sulfurospirillum; the name Sulfurospirillum cavolei sp. nov. is proposed, with Phe91(T) (=JCM 13918(T)=DSM 18149(T)) as the type strain.

Molecular characterization of resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in petroleum-contaminated soil.

PCR assays for analyzing resistance-nodulation-division transporters from solvent- and drug-resistant bacteria in soil were developed. Sequence analysis of amplicons showed that the PCR successfully retrieved transporter gene fragments from soil. Most of the genes retrieved from petroleum-contaminated soils formed a cluster (cluster PCS) that was distantly related to known transporter genes. Competitive PCR showed that the abundance of PCS genes is increased in petroleum-contaminated soil.

Sulfuricurvum kujiense gen. nov., sp. nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium isolated from an underground crude-oil storage cavity.

A facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain YK-1(T), was isolated from an underground crude-oil storage cavity at Kuji in Iwate, Japan. The cells were motile, curved rods and had a single polar flagellum. Optimum growth occurred in a low-strength salt medium at pH 7.0 and 25 degrees C. It utilized sulfide, elemental sulfur, thiosulfate and hydrogen as the electron donors and nitrate as the electron acceptor under anaerobic conditions, but it did not use nitrite. Oxygen also served as the electron acceptor under the microaerobic condition (O(2) in the head space 1 %). It did not grow on sugars, organic acids or hydrocarbons as carbon and energy sources. The DNA G+C content of strain YK-1(T) was 45 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that its closest relative was Thiomicrospira denitrificans in the 'Epsilonproteobacteria', albeit with low homology (90 %). On the basis of physiological and phylogenetic data, strain YK-1(T) should be classified into a novel genus and species, for which the name Sulfuricurvum kujiense gen. nov., sp. nov. is proposed. The type strain is YK-1(T) (=JCM 11577(T)=MBIC 06352(T)=ATCC BAA-921(T)).

Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions.

Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the epsilon subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S(2-) + 4NO(3)(-) --> SO(4)(2-) + 4NO(2)(-) (Delta G(0) = -534 kJ mol(-1)). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.

Diversity, abundance, and activity of archaeal populations in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity.

Fluorescence in situ hybridization has shown that cells labeled with an Archaea-specific probe (ARCH915) accounted for approximately 10% of the total cell count in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity. Although chemical analyses have revealed vigorous consumption of nitrate in cavity groundwater, the present study found that the methane production rate was higher than the nitrate consumption rate. To characterize the likely archaeal populations responsible for methane production in this system, fragments of 16S ribosomal DNA (rDNA) were amplified by PCR using eight different combinations of universal and Archaea-specific primers. Sequence analysis of 324 clones produced 23 different archaeal sequence types, all of which were affiliated with the kingdom EURYARCHAEOTA: Among them, five sequence types (KuA1, KuA6, KuA12, KuA16, and KuA22) were obtained in abundance. KuA1 and KuA6 were closely related to the known methanogens Methanosaeta concilii (99% identical) and Methanomethylovorans hollandica (98%), respectively. Although no closely related organism was found for KuA12, it could be affiliated with the family METHANOMICROBIACEAE: KuA16 and KuA22 showed substantial homology only to some environmental clones. Both of these branched deeply in the Euryarchaeota, and may represent novel orders. Quantitative competitive PCR showed that KuA12 was the most abundant, accounting for approximately 50% of the total archaeal rDNA copies detected. KuA1 and KuA16 also constituted significant proportions of the total archaeal rDNA copies (7 and 17%, respectively). These results suggest that limited species of novel archaea were enriched in the oil storage cavity. An estimate of specific methane production rates suggests that they were active methanogens.