Geobacillus strains that have potential value in microbial enhanced oil recovery.

Bacteria from the genus Geobacillus are generally obligately thermophilic, with a unique bioenergy production capacity and unique enzymes. Geobacillus species were isolated primarily from hot springs, oilfields, and associated soils. They often exhibit unique survival patterns in these extreme oligotrophic environments. With the development of the microbial resources found in oilfields, Geobacillus spp. have been proven as valuable bacteria in many reports related to oilfields. After the isolation of Geobacillus by culture methods, more evidence was found that they possess the abilities of hydrocarbon utilization and bioemulsifier production. This paper mainly summarizes some characteristics of the Geobacillus species found in the oilfield environment, focusing on the inference and analysis of hydrocarbon degradation and bioemulsifier synthesis based on existing research, which may reveal their potential value in microbial enhanced oil recovery. It also provides references for understanding microbes in extreme environments.

Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China.

A rod-shaped, spore-forming, thermophilic, chemoorganotrophic, aerobic or facultatively anaerobic bacterial strain, 1017T, was isolated from production water sampled at the Dagang oilfield (PR China), and was characterized by using a polyphasic approach. The strain is capable of anaerobic glucose fermentation. Nitrate is reduced to nitrite. Optimal growth was observed at 60-65 °C, at pH between pH 7.0 and 7.5, and with 1-2 % (w/v) NaCl. The major cellular fatty acids were iso-C17 : 0, anteiso-C17 : 0, iso-C15 : 0, iso-C16 : 0 and C16 : 0. The predominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analysis based on the 16S rRNA, gyrB and parE gene sequences indicated that the isolate belonged to the genus Geobacillus and was most closely related to Geobacillus thermoleovorans KCTC 3570T (99.5, 96.1 and 97.9 % sequence similarity, respectively). Genome sequencing revealed a genome size of 3.57495 Mb and a DNA G+C content of 51.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 1017T and G. thermoleovorans KCTC 3570T were 95.9 and 64.9 %, respectively. Results of phylogenomic metrics analysis of the genome and 1172 core genes of strain 1017T and its physiological and biochemical characteristics confirmed that strain 1017T represented a novel species of the genus Geobacillus, for which the name Geobacillusproteiniphilus sp. nov. is proposed. The type strain is 1017T (=VKM B-3132T=KCTC 33986T).

Geobacillus yumthangensis sp. nov., a thermophilic bacterium isolated from a north-east Indian hot spring.

A thermophilic, spore-forming, rod-shaped bacterium isolated from the Yumthang hot spring in North Sikkim, India was subjected to taxonomic studies. The thermophilic bacterial isolate was designated as strain AYN2T. Cells were Gram-stain-positive, aerobic, motile, rod-shaped, catalase-positive and methyl red-negative. Strain AYN2T was able to grow in the pH range from 6 to 10 (optimum, pH 7.5-8.0), at 40-70 °C (60 °C) and in NaCl concentrations of 0-4 % (1 %). The major cellular fatty acids were iso-C15 : 0 (12.8 %), iso-C16 : 0 (13.9 %) and iso-C17 : 0 (13.8 %). No matches were found in the rtsba6 Sherlock libraries. The G+C content of the genomic DNA was 42.11 mol%. Based on phylogenetic analysis of the 16S rRNA gene sequences, strain AYNT showed highest sequence similarity to the type strain of Geobacillus toebii (96 %). However, the phenotypic properties of strain AYN2T were clearly distinct from those of G. toebii and related species. On the basis of polyphasic analysis, strain AYN2T represents a novel species in the genus Geobacillus, for which the name Geobacillus yumthangensis sp. nov. is proposed. The type strain is AYN2T(MTCC=12749=KCTC=33950= JCM 32596).

Characterization of the facial microbiome in twins discordant for rosacea.

Previously, we determined that genetic and environmental factors contributed equally towards rosacea in twins. To assess an environmental factor, we characterized the malar cheek bacterial microbiome from twins discordant for rosacea. We found no significant difference in facial microbiome alpha and beta diversity between related twins discordant for rosacea. However, the relative percentage abundance of Gordonia and Geobacillus, low-abundant genera, was positively and negatively associated with rosacea severity, respectively. Our data demonstrate a significant correlation between facial microbiome and severity of rosacea in genetically matched twins and importantly that overall microbiome composition is largely unchanged.

Characteristics of Newly Isolated Geobacillus sp. ZY-10 Degrading Hydrocarbons in Crude Oil.

An obligately thermophilic strain ZY-10 was isolated from the crude oil in a high-temperature oilfield, which was capable of degrading heavy crude oil. Phenotypic and phylogenetic analysis demonstrated that the isolate should be grouped in the genus Geobacillus, which shared thd highest similarity (99%) of the 16S rDNA sequence to Geobacillus stearothermophilus. However, the major cellular fatty acid iso-15:0 (28.55%), iso-16:0 (24.93%), iso-17:0 (23.53%) and the characteristics including indole production, tolerance to NaN3 and carbohydrate fermentation showed some difference from the recognized species in the genus Geobacillus. The isolate could use tridecane, hexadecane, octacosane and hexatridecane as sole carbon source for cell growth, and the digesting rate of long-chain alkane was lower than that of short-chain alkane. When the isolate was cultured in the heavy crude oil supplement with inorganic salts and trace yeast extract, the concentration of short-chain alkane was significantly increased and the content of long-chain alkane was decreased, suggesting that the larger hydrocarbon components in crude oil were degraded into shorter-chain alkane. Strain ZY-10 would be useful for improving the mobility of crude oil and upgrading heavy crude oil in situ.

Geobacillus icigianus sp. nov., a thermophilic bacterium isolated from a hot spring.

A Gram-reaction-positive, motile, thermophilic spore-forming strain, G1w1(T), was isolated from a hot spring of the Valley of Geysers, Kamchatka (Russia). Based on data from the present polyphasic taxonomic study, including phylogenetic analysis of 16S rRNA and spo0A gene sequences, the strain is considered to represent a novel species of the genus Geobacillus, for which the name Geobacillus icigianus sp. nov. is proposed. The type strain is G1w1(T) ( = VKM B-2853(T) = DSM 28325(T)).

Purification studies on a thermo-active amidase of Geobacillus pallidus BTP-5x MTCC 9225 isolated from thermal springs of Tatapani (Himachal Pradesh).

An intracellular aliphatic amide degrading inducible thermo-active amidase produced by Geobacillus pallidus BTP-5x MTCC 9225 was purified to apparent homogeneity using anion exchange and gel filtration chromatography, giving a yield of 6.7 % and a specific activity of 30.49 units mg(-1). The purified protein migrated as a single band of estimated molecular mass of 158 kDa (homo-tetramer) in 8 % polyacrylamide gel electrophoresis and ∼38.5 kDa in 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Optima of pH and temperature varied widely in broad pH range (pH 6-9) and temperature range (45-70 °C). The purified amidase was stable up to 6 h at 50 °C, with a t (1/2) of 7 h at 55 °C. The multimeric nature of the holozyme (tetramer) contributed to protection of the enzyme against thermal denaturation. The enzyme showed resistance to metal chelating agents (EDTA, 8-hydroxyquinoline, and sodium azide), explaining its non-metallic nature, and is strongly inhibited by thiol reagents that means cysteine is involved in catalysis. The amidase of G. pallidus BTP-5x preferentially hydrolyzed only small aliphatic amides and has a narrow substrate spectrum. The K (M) value for acrylamide is 10.54 mM, V (max) 45.19 µmol(-1) min(-1) mg(-1) protein, and k (cat) 4.29 min(-1). The sequence of amino acids of the purified enzyme MRHGDISSSHDTVGI appears similar to thermophilic amidases. Sequence analysis of the amidase gene showed that the enzyme is 347 amino-acid-long with a molecular weight of 38.4 kDa (as observed in SDS-PAGE), theoretical pI 5.38, and show strong similarity to thermostable amidases, possessing unique restriction sites.

Geobacillus subterraneus subsp. aromaticivorans subsp. nov., a novel thermophilic and alkaliphilic bacterium isolated from a hot spring in Sirnak, Turkey.

A new thermophilic spore-forming strain Ge1(T) was isolated from the Guclukonak hot spring in Sirnak, Turkey. The strain was identified by using a polyphasic taxonomic approach. Strain Ge1(T) was Gram-positive, spore-forming, alkaliphilic rod-shaped, motile, occurring in pairs or filamentous. Growth was observed between 30 and 65°C (optimum 60°C) and at pH 5.5-10.0 (optimum pH 9.0). It was capable of utilizing starch, growth was observed at 0-3% NaCl (w/v) and was positive for catalase and urease. The major cellular fatty acids were iso-C(15:0) and iso-C(17:0), and the predominant lipoquinone found was menaquinone MK7 type. The DNA G+C content of the genomic DNA of strain Ge1(T) was 52.0%. Comparative 16S rRNA gene sequence studies showed that the isolate belonged to the genus Geobacillus. The DNA-DNA hybridization mean values between the representative strain Ge1(T) and the closely related species G. subterraneus, G. thermodenitrificans, G. thermocatenulatus, G. vulcani and G. thermoleovorans were 69.3%, 57%, 37%, 27% and 26%, respectively. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Ge1(T). Based on these results, we propose assigning a novel subspecies of Geobacillus subterraneus, to be named as Geobacillus subterraneus subsp. aromaticivorans subsp. nov. with the type strain Ge1(T) (DSM 23066 (T)= CIP 110341(T)).