Yuehui She

Microbial diversity in long-term water-flooded oil reservoirs with different in situ temperatures in China

Water-flooded oil reservoirs have specific ecological environments due to continual water injection and oil production and water recycling. Using 16S rRNA gene clone library analysis, the microbial communities present in injected waters and produced waters from four typical water-flooded oil reservoirs with different in situ temperatures of 25°C, 40°C, 55°C and 70°C were examined. The results obtained showed that the higher the in situ temperatures of the oil reservoirs is, the less the effects of microorganisms in the injected waters on microbial community compositions in the produced waters is. In addition, microbes inhabiting in the produced waters of the four water-flooded oil reservoirs were varied but all dominated by Proteobacteria. Moreover, most of the detected microbes were not identified as indigenous. The objective of this study was to expand the pictures of the microbial ecosystem of water-flooded oil reservoirs.

Permanent draft genome sequence of Bacillus flexus strain T6186-2, a multidrug-resistant bacterium isolated from a deep-subsurface oil reservoir.

Previous studies suggest that antibiotic resistance genes have an ancient origin, which is not always linked to the use of antibiotics but can be enhanced by human activities. Bacillus flexus strain T6186-2 was isolated from the formation water sample of a deep-subsurface oil reservoir. Interestingly, antimicrobial susceptibility testing showed that this strain is susceptible to kanamycin, however, resistant to ampicillin, erythromycin, gentamicin, vancomycin, fosfomycin, fosmidomycin, tetracycline and teicoplanin. To explore our knowledge about the origins of antibiotic resistance genes (ARGs) in the relatively pristine environment, we sequenced the genome of B. flexus strain T6186-2 as a permanent draft. It represents the evidence for the existence of a reservoir of ARGs in nature among microbial populations from deep-subsurface oil reservoirs.

Permanent draft genome sequence of Geobacillus thermocatenulatus strain GS-1.

Geobacillus thermocatenulatus strain GS-1 is a thermophilic bacillus having a growth optimum at 60°C, capable of degrading alkanes. It was isolated from the formation water of a high-temperature deep oil reservoir in Qinghai oilfield, China. Here, we report the draft genome sequence with an estimated assembly size of 3.5Mb. A total of 3371 protein-coding sequences, including monooxygenase, alcohol dehydrogenase, aldehyde dehydrogenase, fatty acid-CoA ligase, acyl-CoA dehydrogenase, enoyl-CoA hydrogenase, hydroxyacyl-CoA dehydrogenase and thiolase, were detected in the genome, which are involved in the alkane degradation pathway. Our results may provide insights into the genetic basis of the adaptation of this strain to high-temperature oilfield ecosystems.

Genomovar assignment of Pseudomonas stutzeri populations inhabiting produced oil reservoirs.

Oil reservoirs are specific habitats for the survival and growth of microorganisms in general. Pseudomonas stutzeri which is believed to be an exogenous organism inoculated into oil reservoirs during the process of oil production was detected frequently in samples from oil reservoirs. Very little is known, however, about the distribution and genetic structure of P. stutzeri in the special environment of oil reservoirs. In this study, we collected 59 P. stutzeri 16S rRNA gene sequences that were identified in 42 samples from 25 different oil reservoirs and we isolated 11 cultured strains from two representative oil reservoirs aiming to analyze the diversity and genomovar assignment of the species in oil reservoirs. High diversity of P. stutzeri was observed, which was exemplified in the detection of sequences assigned to four known genomovars 1, 2, 3, 20 and eight unknown genomic groups of P. stutzeri. The frequent detection and predominance of strains belonging to genomovar 1 in most of the oil reservoirs under study indicated an association of genomovars of P. stutzeri with the oil field environments.

Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains.

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301T) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.中文概要目 的对一株地层水分离的石油降解菌Ochrobactrumintermedium2745-2进行生理生化特性的研究、全基因组测序以及比较基因组研究。创新点首次对一株分离自地层水的石油降解菌O. intermedium2745-2进行了生理生化特性研究以及基因组测序, 从基因组角度解释菌株2745-2对石油的降解能力。通过菌株2745-2与同种其他临床分离菌株的比较基因组学分析, 表明2745-2仍具有多种与致病性相关的基因。方 法通过微生物富集培养的方法从油井的地层水中分离石油降解微生物, 通过聚合酶链反应(PCR)扩增16S 核糖体RNA(rRNA)序列进行比较和分析确定菌株的分类地位属于O. intermedium(图1)。采用Illumina HiSeq2000 对菌株2745-2进行高通量测序, 采用Velvet 1.2.07 和RASTserver 分别进行数据组装和注释(表1)。PHAST寻找基因组中的噬菌体相关序列(图4 和表2)。通过BLAST+和BRIG 对环境分离菌株(2745-2)和临床分离菌株(M86、229E 和LMG3301T)进行基因组比较(表3、表4 和图5)。结 论首次对一株环境分离的O. intermedium(2745-2)进行全基因组测序, 揭示具有多种与石油降解相关的基因。通过环境分离菌株(2745-2)与临床分离菌株(M86, 229E 和LMG3301T)的基因组比较分析, 表明2745-2 仍具有多种致病性相关的基因。

Genome sequence of Brevibacillus agri strain 5-2, isolated from the formation water of petroleum reservoir

Brevibacillus agri strain 5-2 was isolated from the formation water of a deep oil reservoir in Changqing Oilfield, China. This bacterium was found to have a capacity for degrading tetradecane, hexadecane and alkanesulfonate. To gain insights into its efficient metabolic pathway for degrading hydrocarbon and organosulfur compounds, here, we report the high quality draft genome of this strain. Two putative alkane 1-monooxygenases, one putative alkanesulfonate monooxygenase, one putative alkanesulfonate transporter, one putative sulfate permease and five putative sulfate transporters were identified in the draft genome. The genomic data of strain 5-2 may provide insights into the mechanism of microorganisms adapt to the petroleum reservoir after chemical flooding.

Dynamics of a microbial community during an effective boost MEOR trial using high-throughput sequencing

Using 454 pyrosequencing of 16S rRNA gene amplicons, microbial communities in samples of injection water and production water during a serial microbial enhanced oil recovery (MEOR) field trial in a water flooded high pour point oil reservoir were determined. There was a close microbial community compositional relationship between the injection water and the successful first round MEOR processed oil reservoir which was indicated by the result of 43 shared dominant operational taxonomic units detected in both the injection water and the production water. Alterations of microbial community after the injection of boost nutrients showed that microbes giving positive responses were mainly those belonging to the genera of Comamonas, Brevundimonas, Azospirillum, Achromobacter, Pseudomonas, and Hyphomonas, which were detected both in the injection water and in the production water and usually detected in oil reservoir environments or associated with hydrocarbon degradation. Additionally, microbes only dominant in the production waters were significantly inhibited with a sharp decline in their relative abundance. Based on these findings, a suggestion of re-optimization of the boost nutrients, targetting the microbes co-existing in the injection water and the oil reservoir and having survival ability in both surface and subsurface environments, rather than simple repeats for the subsequent in situ MEOR applications was proposed.

Analysis of Seven Polyacrylamide(PAM)-Degrading Bacterias Using PCR-DGGE Method

Seven polyacrylamide(PAM)-degrading bacteria were isolated from waste water treated pool of Henan(P.R.China) oil field, six of which were isolated from the sample of sludge, one from the sample of waste water. Sequences and phylogenetic analysis of 16 S rDNA clones of the seven bacteria showed that four were affiliated with Actinobacteria, two with Alphaproteobacteria, one with Bacillus. PCR-DGGE profile was used to analyze the environmental dominant status of the seven bacteria. DGGE bands of five bacteria can be found in the lane of DNA from original water or sludge samples. The result provided valuable information about PAM waste treatment of He Nan Henan oil field.

Indigenous Microbial Diversity in Conglomerate Oil Reservoirs Analyzed by PCR-DGGE

Recent investigations of the microbial communities in an oil reservoir using culture-independent molecular techniques has indicated that this environment contains many uncultured assemblages, and that the cultured bacteria may represent very minor components of the true microbial diversity. In this study, 16S rDNA based PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) was used to monitor the microbial diversity in an injection well (12#9-11) and two related production wells (12#9-9S, 13#11-8) in the KELAMAYI oilfield (Xin Jiang Autonomous Region) . The DGGE gel profiles showed significant difference between the microbial community structure of injection and production wells. The bacterial species richness in the injection well was higher than those of the production ones. Unweighted Pair Group Method Clustering (UPGMC) showed the similarity between the injection well and two production wells was 30% and 20% respectively, whereas the similarity between the two production wells was 54%. In addition, the dominant bands in the DGGE gel were excised and analyzed by nucleotide sequence analysis. This analysis of 10 excised bands showed that the dominant microorganisms in all samples had high sequence identity with rRNA genes from uncultured bacteria in the GenBank database (NCBI). Nine sequences of excised band were affiliated with alpha, gamma, delta, epsiv-Proteobacteria, and one was related to Bacteroides. Two bands exhibited 100% identity to sequences of Pseudomonas stutzeri and Aminobacter sp. cox, respectively. Three bands showed relatively low identity (92% to 93%) to cultured bacteria in the GenBank database, indicating possible new taxa. This work analyzed the composition of the microbial community in KELAMAYI oilfield, which provides important data which may facilitate the isolation of new bacteria from this special underground environment. It also provides useful information for understanding the mechanism of MEOR and applying MEOR in practical oil production in KELAMAYI oilfield.