Crude oil as a microbial seed bank with unexpected functional potentials.

It was widely believed that oil is a harsh habitat for microbes because of its high toxicity and hydrophobicity. However, accumulating evidence has revealed the presence of live microbes in crude oil. Therefore, it's of value to conduct an in-depth investigation on microbial communities in crude oil. To this end, microorganisms in oil and water phases were collected from four oil-well production mixtures in Qinghai Oilfield, China, and analyzed for their taxonomic and functional compositions via pyrosequencing and GeoChip, respectively. Hierarchical clustering of 16S rRNA gene sequences and functional genes clearly separated crude oil and water phases, suggestive of distinct taxonomic and functional gene compositions between crude oil and water phases. Unexpectedly, Pseudomonas dominated oil phase where diverse functional gene groups were identified, which significantly differed from those in the corresponding water phases. Meanwhile, most functional genes were significantly more abundant in oil phase, which was consistent with their important roles in facilitating survival of their host organisms in crude oil. These findings provide strong evidence that crude oil could be a "seed bank" of functional microorganisms with rich functional potentials. This offers novel insights for industrial applications of microbial-enhanced oil recovery and bioremediation of petroleum-polluted environments.

The genome of the moderate halophile Amycolicicoccus subflavus DQS3-9A1(T) reveals four alkane hydroxylation systems and provides some clues on the genetic basis for its adaptation to a petroleum environment.

The moderate halophile Amycolicicoccus subflavus DQS3-9A1(T) is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1(T) has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1-12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1(T) to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain.

Bacteria in crude oil survived autoclaving and stimulated differentially by exogenous bacteria.

Autoclaving of crude oil is often used to evaluate the hydrocarbon-degrading abilities of bacteria. This may be potentially useful for bioaugmentation and microbial enhanced oil recovery (MEOR). However, it is not entirely clear if "endogenous" bacteria (e.g., spores) in/on crude oil survive the autoclaving process, or influence subsequent evaluation of the hydrocarbon-degradation abilities of the "exogenous" bacterial strains. To test this, we inoculated autoclaved crude oil medium with six exogenous bacterial strains (three Dietzia strains, two Acinetobacter strains, and one Pseudomonas strain). The survival of the spore-forming Bacillus and Paenibacillus and the non-spore-forming mesophilic Pseudomonas, Dietzia, Alcaligenes, and Microbacterium was detected using a 16S rRNA gene clone library and terminal restriction fragment length polymorphism (T-RFLP) analysis. However, neither bacteria nor bacterial activity was detected in three controls consisting of non-inoculated autoclaved crude oil medium. These results suggest that detection of endogenous bacteria was stimulated by the six inoculated strains. In addition, inoculation with Acinetobacter spp. stimulated detection of Bacillus, while inoculation with Dietzia spp. and Pseudomonas sp. stimulated the detection of more Pseudomonas. In contrast, similar exogenous bacteria stimulated similar endogenous bacteria at the genus level. Based on these results, special emphasis should be applied to evaluate the influence of bacteria capable of surviving autoclaving on the hydrocarbon-degrading abilities of exogenous bacteria, in particular, with regard to bioaugmentation and MEOR. Bioaugmentation and MEOR technologies could then be developed to more accurately direct the growth of specific endogenous bacteria that may then improve the efficiency of treatment or recovery of crude oil.

Microbial communities in long-term, water-flooded petroleum reservoirs with different in situ temperatures in the Huabei Oilfield, China.

The distribution of microbial communities in the Menggulin (MGL) and Ba19 blocks in the Huabei Oilfield, China, were studied based on 16S rRNA gene analysis. The dominant microbes showed obvious block-specific characteristics, and the two blocks had substantially different bacterial and archaeal communities. In the moderate-temperature MGL block, the bacteria were mainly Epsilonproteobacteria and Alphaproteobacteria, and the archaea were methanogens belonging to Methanolinea, Methanothermobacter, Methanosaeta, and Methanocella. However, in the high-temperature Ba19 block, the predominant bacteria were Gammaproteobacteria, and the predominant archaea were Methanothermobacter and Methanosaeta. In spite of shared taxa in the blocks, differences among wells in the same block were obvious, especially for bacterial communities in the MGL block. Compared to the bacterial communities, the archaeal communities were much more conserved within blocks and were not affected by the variation in the bacterial communities.

The genome sequence of Polymorphum gilvum SL003B-26A1(T) reveals its genetic basis for crude oil degradation and adaptation to the saline soil.

Polymorphum gilvum SL003B-26A1(T) is the type strain of a novel species in the recently published novel genus Polymorphum isolated from saline soil contaminated with crude oil. It is capable of using crude oil as the sole carbon and energy source and can adapt to saline soil at a temperature of 45°C. The Polymorphum gilvum genome provides a genetic basis for understanding how the strain could degrade crude oil and adapt to a saline environment. Genome analysis revealed the versatility of the strain for emulsifying crude oil, metabolizing aromatic compounds (a characteristic specific to the Polymorphum gilvum genome in comparison with other known genomes of oil-degrading bacteria), as well as possibly metabolizing n-alkanes through the LadA pathway. In addition, COG analysis revealed Polymorphum gilvum SL003B-26A1(T) has significantly higher abundances of the proteins responsible for cell motility, lipid transport and metabolism, and secondary metabolite biosynthesis, transport and catabolism than the average levels found in all other genomes sequenced thus far, but lower abundances of the proteins responsible for carbohydrate transport and metabolism, defense mechanisms, and translation than the average levels. These traits support the adaptability of Polymorphum gilvum to a crude oil-contaminated saline environment. The Polymorphum gilvum genome could serve as a platform for further study of oil-degrading microorganisms for bioremediation and microbial-enhanced oil recovery in harsh saline environments.

Degradation of petroleum hydrocarbons (C6-C40) and crude oil by a novel Dietzia strain.

A novel bacterial strain, DQ12-45-1b, was isolated from the production water of a deep subterranean oil-reservoir. Morphological, physiological and phylogenetic analyses indicated that the strain belonged to the genus Dietzia with both alkB (coding for alkane monooxygenase) and CYP153 (coding for P450 alkane hydroxylase of the cytochrome CYP153 family) genes and their induction detected. It was capable of utilizing a wide range of n-alkanes (C6-C40), aromatic compounds and crude oil as the sole carbon sources for growth. In addition, it preferentially degraded short-chain hydrocarbons (≤C25) in the early cultivation phase and accumulated hydrocarbons with chain-lengths from C23 to C27 during later cultivation stage with crude oil as the sole carbon source. This is the first study to report the different behaviors of a bacterial species toward crude oil degradation as well as a species of Dietzia degrading a wide range of hydrocarbons.

Amycolicicoccus subflavus gen. nov., sp. nov., an actinomycete isolated from a saline soil contaminated by crude oil.

Two novel actinomycetes, designated DQS3-9A1(T) and DQS3-9A2, were isolated from a saline soil contaminated with crude oil in the Shengli Oilfield in China. On the basis of 16S rRNA gene sequence analysis, the two strains were most closely related to Mycobacterium species (92.7-94.9 % similarities), and formed a distinct lineage in the suborder Corynebacterineae . In addition, the major sugars in the cell wall, arabinose and galactose, supported the affiliation of strain DQS3-9A1(T) with members of the family Mycobacteriaceae. However, strain DQS3-9A1(T) did not contain mycolic acids and MK-8 (85.5 %) was the major menaquinone for both isolates. The major cellular fatty acids for strain DQS3-9A1(T) were C(16 : 0) (20.5 %), 10-methyl C(17 : 0) (19.3 %), 10-methyl C(18 : 0) (16.1 %), summed feature 3 (11.4 %), C(15 : 0) (11.3 %), C(17 : 0) (5.0 %) and C(17 : 1)omega8c (5.0 %). The polar lipids of strain DQS3-9A1(T) consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and an unknown glucosamine-containing phospholipid. These chemotaxonomic data indicated that strain DQS3-9A1(T) differs from the present members of the suborder Corynebacterineae. Therefore, the creation of Amycolicicoccus subflavus gen. nov., sp. nov. is proposed, with DQS3-9A1(T) (=DSM 45089(T)=CGMCC 4.3532(T)) as the type strain.

Filomicrobium insigne sp. nov., isolated from an oil-polluted saline soil.

Strain SLG5B-19(T), isolated from an oil-polluted saline soil in Gudao in the coastal Shengli Oilfield, eastern China, was Gram-negative with monoprosthecae or bipolar prosthecae and buds on the prosthecal tips. Growth occurred at NaCl concentrations between 0 and 7 % (w/v), at temperatures between 4 and 45 degrees C, and at pH 6.0-9.0. Strain SLG5B-19(T) had Q-9 as the major respiratory quinone and unsaturated C(18 : 1)omega7c as the predominant cellular fatty acid. The G+C content of the genomic DNA was 59.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SLG5B-19(T) belonged to a clade with the genera Filomicrobium and Hyphomicrobium in the class Alphaproteobacteria. However, 16S rRNA gene sequence similarities of strain SLG5B-19(T) to the phylogenetically most closely related strains, i.e. the type strains of Filomicrobium fusiforme and Hyphomicrobium zavarzinii, were 95.8 and 94.5 %, respectively. In addition, the 16S rRNA gene sequence of strain SLG5B-19(T) had 24 signature nucleotides that were identical to those of the type strain of F. fusiforme. Based on phylogenetic analysis of 16S rRNA gene sequences, strain SLG5B-19(T) could be allocated to the genus Filomicrobium. However, distinct phenotypic differences were observed between strain SLG5B-19(T) and the type strain of F. fusiforme. It is therefore proposed that strain SLG5B-19(T) represents a novel species in the genus Filomicrobium, Filomicrobium insigne sp. nov. The type strain is SLG5B-19(T) (=CGMCC 1.6497(T)=LMG 23927(T)).

Halomonas daqingensis sp. nov., a moderately halophilic bacterium isolated from an oilfield soil.

A Gram-negative, moderately halophilic, short rod-shaped, aerobic bacterium with peritrichous flagellae, strain DQD2-30(T), was isolated from a soil sample contaminated with crude oil from the Daqing oilfield in Heilongjiang Province, north-eastern China. The novel strain was capable of growth at NaCl concentrations of 1-15 % (w/v) [optimum at 5-10 % (w/v)]. Phylogenetic analyses based on 16S rRNA gene sequences showed that the novel strain belonged to the genus Halomonas in the class Gammaproteobacteria; the highest 16S rRNA gene sequence similarities were with Halomonas desiderata DSM 9502(T) (98.8 %), Halomonas campisalis A4(T) (96.6 %) and Halomonas gudaonensis CGMCC 1.6133(T) (95.1 %). The major cellular fatty acids of strain DQD2-30(T) were C(18 : 1)omega7c (43.97 %), C(19 : 0 )cyclo omega8c (23.37 %) and C(16 : 0) (14.83 %). The predominant respiratory lipoquinone was ubiquinone with nine isoprene units (Q9). The DNA G+C content was 67.0 mol%. The DNA-DNA hybridization values of strain DQD2-30(T) with the most closely related species of the genus Halomonas were 51.8 %, 28.4 % and 23.5 % for H. desiderata, H. campisalis and H. gudaonensis, respectively. Based on these analyses, strain DQD2-30(T )(=CGMCC 1.6443(T)=LMG 23896(T)) is proposed to represent the type strain of a novel species, Halomonas daqingensis sp. nov.

Halomonas shengliensis sp. nov., a moderately halophilic, denitrifying, crude-oil-utilizing bacterium.

A moderately halophilic bacterium, designated strain SL014B-85(T), was isolated from a crude-oil-contaminated saline soil from Shengli oilfield, Shandong Province, China. Cells were Gram-negative, aerobic, short rods with lateral flagella. Growth occurred at NaCl concentrations of 0-15 % (optimum 5-15 %), at 10-42 degrees C (optimum 30 degrees C) and at pH 8.0-9.0 (optimum pH 8.5). The only respiratory quinone was Q9, and the main cellular fatty acids were C(18 : 1)omega7c, C(16 : 0) and C(19 : 0) cyclo omega8c. The G+C content of the DNA was 66.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SL014B-85(T) belonged to the genus Halomonas in the Gammaproteobacteria, with highest sequence similarity of 98.1 and 97.8 % to Halomonas alimentaria DSM 15356(T) and Halomonas ventosae DSM 15911(T), respectively. DNA-DNA relatedness values were below 40 % with members of closely related Halomonas species. Results of phenotypic, biochemical and phylogenetic analyses revealed that strain SL014B-85(T) could be classified as representing a novel species of the genus Halomonas, for which the name Halomonas shengliensis sp. nov. is proposed. The type strain is SL014B-85(T) (=CGMCC 1.6444(T)=LMG 23897(T)).