[Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Haloarchaeon Halogranum amylolyticum.

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1 mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.

[Microbial diversity in shengli petroleum reservoirs analyzed by T-RFLP].

Recent investigations on the microbial ecology of oil reservoirs in a variety of locales indicated that these habitats harbor various assemblages. In this study, a cultured-independent molecular technique, Terminal Restriction Fragment Length Polymorphism (T-RFLP), was used to analyze the microbial diversity of an injection well (S12-ZHU) and three related production wells (S12-4, S12-5 and S12-19) in the ShengLi oilfield (Shandong province, China). The 16S rRNA genes were amplified by PCR with the 5'carboxy-fluorescein (5-FAM)-labelled universal forward primers (27F for bacteria and 21F for archaea) and a universal reverse primer (1495R). Then the 16S rRNA genes were digested with restriction enzymes (Hae III and Hha I) and analyzed by using an automated DNA sequencer. The Shannon-Wiener Diversity index, based on the T-RFLP profiles, indicated that the bacterial and archaeal species richness in the injection well was higher than those of the production ones. The similarity coefficient showed the microbial community similarity among the four samples was 22.4%-30.8% (Bacteria) and 20.8%-34.5% (Archaea), respectively. According to the analysis by TAP T-RFLP program, species belonging to Pseudomonas, Marinobacter and Methanosarcina as well as some uncultured archaeon were supposed to be the dominant bacteria in all four samples. Thus, this study indicates that T-RFLP is useful for analysis of the microbial diversity in petroleum reservoirs.

[Diversity of halophilic archaea in hypersaline lakes of Inner Mongolia, China].

The aims of this work were to explore the diversity of halophilic archaea in hypersaline lakes of Inner Mongolia, China and to collect novel halophilic archaea. One hundred and sixty-five halophilic archaea were isolated from the three different types of hypersaline lakes (Erliannor, shangmatala and Xilin soda lake) in Inner Mongolia. By analysis of the restriction patterns of amplified 16S rDNA (ARDRA) with the enzyme Afa I and Hae II, respectively, the isolates were clustered into 14 genotypes, and the representatives of each genotype were randomly chosen for the determination of 16S rDNA sequence. The phylogenetic analysis revealed that all of the isolates were clustered into 10 groups: Halorubrum, Natronococcus, Natronorubrum, Haloterrigena, Halorhabdus, Halobiforma, Haloarcula, Haloferax and other two unknown groups. Dominant isolates were related to Halorubrum spp. in all three lakes. Some of the isolates studied showed less affiliation with known taxa ( <98% sequence similarity) and may represent novel taxa. Two isolates HXH33 and HSH33 showed very less affiliation with known genus ( < 93% sequence similarity) and may represent two new genera. These results suggest that diverse archaea exist in and the unknown archaea thrive in the hypersaline lakes of Inner Mongolia.

[Preliminary research on bacterial diversity of Parece Vela Basin, Pacific Ocean by culture-independent method].

The environmental DNA was directly extracted from the sediment in Parece Vela Basin, Pacific Ocean, at a depth of 5010 m. Bacterial 16S rRNA gene library of 32 clones was generated using bacterial universal primers and 16S rDNA sequences were analyzed phylogenetically. 17 phylotypes were obtained. The library was dominated by gamma-Proteobacteria, alpha-Proteobacteria and marine uncultured bacteria. Sixty-two percent of the cloned sequences was highly related to the known bacteria in the genus Halomonas, Alcanivorax, Pseudomonas, Acinetobacter, Pseudoalteromonas (> 96% sequence similarity), while some of the cloned sequences showed less affiliation with known taxa (< 94% sequence similarity) and may represent novel taxa.

Purification and characterization of a monofunctional catalase from an alkaliphilic Bacillus sp. F26.

An alkaline catalase has been purified and characterized from a slightly halophilic and alkaliphilic bacterium Bacillus sp. F26. The purification was performed with a four step procedure consisting of ammonium sulfate precipitation, ion exchange, gel filtration and hydrophobic interaction chromatography, and finally achieved a 58.5-fold-purifying over the crude extract. The purified catalase was composed of two identical subunits with a native molecular mass of 140 kD. The native enzyme showed the typical Soret band appearing at 408 nm. The pyridine hemochrome spectrum indicated the presence of protoheme IX as the prosthetic group. The apparent Km value for enzyme activity on H2O2 was calculated to be 32.5 mmol/L. The activity of this catalase was not reduced by dithionite but was strongly inhibited by cyanide, azide, and 3-amino-1,2,4-triazole (the specific inhibitor of monofunctional catalase). No peroxidase activity of this enzyme was detected when using o-dianisidine, diaminobenzidine (DAB) and p-phenylenediamine as electron donor. Moreover, the N-terminal sequence of this catalase exhibited substantial similarity to the monofunctional catalase subgroup rather than catalase-peroxidase or Mn-catalase one. Therefore, we characterize the purified catalase as a monofunctional catalase. Besides, this monofunctional catalase was thermosensitive and its activity exhibited pH-independent over pH 5-9 but showed a sharp maximum at pH 11. An activity half-life of approximately 49 h was measured when the enzyme was incubated at 20 degrees C and pH 11. To our knowledge, pH 11 is the most alkaline condition for optimum catalysis and enzyme stability among the catalases reported up to now. Furthermore, this monofunctional catalase also showed excellent halo-alkali-stability with a half-life of approximately 90 h at 0.5 mol/L NaCl and pH 10.5. On the other hand, so far as we know, the characterized catalase is the first dimeric monofunctional catalase from alkaliphiles and is also the first monofunctional catalase derived from a natural soda lake, which could partially reflect the oxidative stress response in the corresponding environment.

[Amphibacillus haojiensis sp. nov.--a novel alkaliphilic and slight halophilic bacterium from Haoji Soda Lake in Inner Mongolia Autonomous Region, China].

An alkaliphilic and slightly halophilic Gram-positive rod, designated strain F10, was isolated from Haoji soda lake in Inner Mongolia Autonomous Region, China. Strain F10 grew optimally in the presence of 3% (W/V) NaCl, pH 9.5 and 37 degrees C. It was facultatively anaerobic, motile with peritrichous flagella, sporulating, catalase- negative. The (G + C) content of the DNA was 40 mol%. The cell wall contained meso-DAP. Phylogenetic analyses based on 16S rDNA sequences comparisons indicate that strain F10 was a member of the genus Amphibacillus. DNA-DNA similarity of less than 30% with the described species of Amphibacillus supported the view that this organism represented a new species of the genus Amphibacillus. The name Amphibacillus haojiensis sp. nov. is proposed.