印度洋深海沉积物石油烃降解菌分离、鉴定与多样性分析

烃类物质在海洋环境中广泛分布,深海可能含有特殊的烃降解微生物。本研究通过对西南印度洋中脊与印度洋中部深海沉积物中石油降解菌的富集培养和分离鉴定,从6个站点的样品中共分离得到800株菌,通过BOX-PCR去重复菌株后,对其中183株菌进行了16S rRNA基因序列分析,发现这些菌分属于23个属;其中,γ-变形菌纲的食烷菌属(Alcanivorax)和放线菌纲的微杆菌属(Microbacterium)占优势。此外,还发现了食烷菌属2个潜在的新种、假海栖菌属(Pseudooceanicola)1个潜在新种。高通量测序结果证明,富集菌群中γ-变形菌纲是优势菌,主要包括食烷菌属、盐单胞菌属(Halomonas)、海杆菌属(Marinobacter)等。结合可培养菌与高通量测序结果,食烷菌属、盐单胞菌属、海杆菌属、交替假单胞菌(Pseudoalteromonas)、海源菌属(Idiomarina)与微杆菌属(Microbacterium)是沉积物样品中常见的石油烃降解菌,迪茨氏菌属(Dietzia)、红球菌属(Rhodococcus),假单胞菌属(Pseudomonas)、赤杆菌属(Erythrobacter)与副球菌属(Paracoccus)等可能也参与了烃的降解。

Antarctic Psychrophile Bacteria Screening for Oil Degradation and Their Degrading Characteristics

Two hydrocarbon degrading bacteria NJ276 and NJ341 were screened from 385 Antarctic marine bacteria and their degrading characteristics were studied. Diesel oil as sole carbon source was used in this study. The results showed that the oil degradation rates of Antarctic psychrophile bacteria NJ276 and NJ341 were 23.47 % and 32.15 %, respectively, after 20 days culturation at 5 ℃, and the rates were 43.95 % and 62.47 % respectively after 20 days culturation at 15 ℃. The oil degradation abilities were enhanced remarkably with the increasing culture temperature. GC - MSs indicated the residual oil contained C15 - C21 7 alkyls after degradation by NJ276, and C16, C17 and C18 3 alkyls after degradation by NJ341. The 16S rDNA gene sequences homology and phylogenetic analysis of the two Antarctic psychrophile bacteria showed that NJ276 belonged to the described genus Pseudoalteromonas and NJ341 belonged to the genus Colwellia.

Biological Characteristics of Biosurfactantproducing Petroleum-degrader Bacterium Bacillus BS-8

The growth characteristics of petroleum-degrader BS-8（Bacillus sp.） and the factors influencing its biosurfactant production were tested; the biosurfactant releasing mode of BS-8 was speculated by measuring OD600, surface tension and oil displacement of fermentation broth; and the effects of carbon source, nitrogen source, temperature, p H, and Na Cl concentration on biosurfactant production by BS-8 were observed in this study. The results showed that the biosurfactant releasing mode of BS-8 was growth-correlated, the surface tension of BS-8 fermentation broth declined with the total bacteria increasing, and the oil displacement was positive correlated with biosurfactant content in fermentation broth; and the optimal culture conditions for effective biosurfactant production included： glucose as carbon source,yeast extract as nitrogen source, Na Cl concentration of 2%, p H of 7.0 and temperature at 30 ℃.

Response of Microbial Community to Petroleum Stress and Phosphate Dosage in Sediments of Jiaozhou Bay, China

The dynamic microcosms were used to evaluate the effect of oil spills on microbial ecological system in marine sediment and the enhancement of nutrient on the oil removal. The function and structure of microbial community caused by the oil pollution and phosphate dosage were simultaneously monitored by dehydrogenase activity assay and PCR-denaturing gradient gel electrophoresis(DGGE) techniques. The results indicated that the amount of total bacteria in all dynamic microcosms declined rapidly with incubation time. The number of petroleum-degrading bacteria and the activity of sediment dehydrogenase were gradually enhanced by petroleum in the oil-treated microcosms, while they both showed no obvious response to phosphate dosage. In comparison, phosphate spiked heterotrophic bacteria and they showed a significant increase in amount. DGGE profiles indicated that petroleum dosage greatly changed community structure, and the bacteria belonged to class Deltaproteobacteria, and phyla Bacteroidetes and Chlorobi were enriched. This study demonstrated that petroleum input greatly impacted the microbial community structure and consequently the marine sediment petroleum-degrading activity was enhanced. Phosphate dosage would multiply heterotrophic bacteria but not significantly enhance the petroleum degradation.

Degradation Characteristics and Community Structure of a Hydrocarbon Degrading Bacterial Consortium

A hydrocarbon degrading bacterial consortium KO5-2 was isolated from oil-contaminated soil of Karamay in Xinjiang, China, which could remove 56.9% of 10 g/L total petroleum hydrocarbons(TPH) at 30 ℃ after 7 days of incubation, and could also remove 100% of fluorene, 98.93% of phenanthrene and 65.73% of pyrene within 3, 7 and 9 days, respectively. Twelve strains from six different genera were isolated from KO5-2 and only eight ones were able to utilize the TPH. The denaturing gradient gel electrophoresis(DGGE) was used to investigate the microbial community shifts in five different carbon sources(including TPH, saturated hydrocarbons, fluorene, phenanthrene and pyrene). The test results indicated that the community compositions of KO5-2 in carbon sources of TPH and saturated hydrocarbons, respectively, were roughly the same, while they were distinctive in the three different carbon sources of PAHs. Rhodococcus sp. and Pseudomonas sp. could survive in the five kinds of carbon sources. Bacillus sp., Sphingomonas sp. and Ochrobactrum sp. likely played key roles in the degradation of saturated hydrocarbons, PAHs and phenanthrene, respectively. This study showed that specific bacterial phylotypes were associated with different contaminants and complex interactions between bacterial species, and the medium conditions influenced the biodegradation capacity of the microbial communities involved in bioremediation processes.

Study on Isolation, Identification of A Petroleum Hydrocarbon Degrading Bacterium Bacillus fusiformis sp. and Influence of Environmental Factors on Degradation Efficiency

A hydrocarbon degrading bacterium KL2-13 was isolated from ten sites of oil contaminated soil in the Karamay oilfield. It was identified as the Bacillusfusiformis sp. bacterium based on its morphological and physiological characteristics and the 16S rDNA sequence analysis. The factors influencing the hydrocarbon degradation by the bacterium KL2-13 were determined. The test results have showed that the hydrocarbon degrading bacterium KL2-13 requires an optimum pH range of 6-8, and the optimum inoculation quantity is 3%. The low-concentration metal ions Fe^2＋, Mg^2＋ and Ca^2＋can improve the degradation ability of the bacteria KL2-13. A too low concentration of Tween-80 does not show obvious promotion to the degrading bacterium KL2-13, and an excessively high concentration can decrease the degradation ability of the bacterium, the best dosage of which is 2%. The hydrocarbon degrading rate reached 59.07%4-0.37% under the optimum culture conditions.

Isolation and Characterization of a Thermophilic Oil-Degrading Bacterial Consortium

In this study, a thermophilic oil-degrading bacterial consortium KO8-2 growing within the temperature range of 45--65℃ （with 55℃ being the optimum temperature） was isolated from oil-contaminated soil of Karamay in Xinjiang, China. Denaturing gradient gel electrophoresis （DGGE） showed that there were nine strains included in KO8-2, which originated from the genera of Bacillus, Geobacillus and Clostridium. They all belonged to thermophilic bacteria, and had been previously proved as degraders of at least one petroleum fraction. The crude oil degraded by KO8-2 was analyzed by infrared spectrophotometry, hydrocarbon group type analysis and gas chromatography. The results indicated that the bacterial consortium KO8-2 was able to utilize 64.33% of saturates, 27.06% of aromatics, 13.24% of resins and the oil removal efficiency reached up to 58.73% at 55 ~C when the oil concentration was 10 g/L. Detailed analysis showed that KO8-2 was able to utilize the hydrocarbon components before C19, and the n-alkanes ranging from C20--C33 were signifi- cantly degraded. The ratios of nC17/Pr and nC18/Ph were 3.12 and 3.87, respectively, before degradation, whereas after degradation the ratios reduced to 0.21 and 0.38, respectively. Compared with the control sample, the oil removal efficiency in KO8-2 composting reactor reached 50.12% after a degradation duration of 60 days.

Isolation and Characterization of Microorganisms for Degradation of Minimal Oils and Fats at Low Temperatures

The isolation of microorganisms for biodegradation of minimal fats and oils at low temperatures was reported. By using rapeseed oil as a sole carbon source, six strains were isolated from five kinds of oils/fats-contaminated wastewater, soil, and active sludge. Among them, two strains which show the highest oil removal ratios were identified as Pseudomonas pseudoalcaligenes and Pseudomonas mendocina, respectively. The experiments of orthogonal impact conditions show that the optimal oil degradation condition is at pH 8.0, 5 ℃ and 100 mg/L oil. Under this condition, the rapeseed oil degradation ratios of two strains after 24 hours amount to 92.6% and 92.0% respectively, whereas the removal ratios of lard decrease to 39.5% and 54.3%.

Development and characterization of a functional microbial consortium for crude oil degradation

Crude oil-degrading microbial consortia were enriched from three oil-contaminated sites to achieve the efficient biodegradation of crude oil,especially its refractory residues.The gravimetric method was used to analyze the degradation efficiency of the enriched consortia and changes in the fractions of the crude oil.The effects of changes in environmental factors were also studied to determine the optimal oil-reducing conditions and assess the dominant bacteria of the mixed flora.Results show that all three consortia exhibit reliable crude oil-biodegradation abilities and that their mixture results in biodegradation rate are as high as(48.0±3.5)%over 30 d of incubation.The consortium mixture can degrade 11.1%of the refractory resins,79.7%of the saturated hydrocarbons,and 45.7%of the aromatics in crude oil.Neutral pH,an incubation temperature of 30℃,and low mineral salt concentrations(0.8%to 4.0%)are optimal for crude oil biodegradation.The dominant genera in the consortium mixture include Pseudomonas,Stenotrophomonas,Brucella,Serratia,Brevundimonas,and Achromobacter.The richness and diversity of the microbial community in the consortium remain stable during crude oil degradation.Therefore,microbial enrichment from multiple sources may be performed to construct a mixed consortium for crude oil pollution bioremediation.

Novel method for separation and screening of lubricant-degrading microorganisms and bacterial biodegradation

With the rapid increase of lubricant consumption, oil contamination becomes more serious. Biotreatment is an important method to remove oil contamination with some advantages. In this study, acclimatized oil- contaminated soil and used lubricating oil were sampled to isolate lubricant-degrading strains by several methods. 51 isolates were obtained and 24-well plates were employed to assess bacterial potential in high- throughput screening. The method was noted for the prominence of oil-water two-phase system with saving chemicals, shortening cycles and lessening workloads. In order to decrease inaccuracy, subculture and resting cells were inoculated into mineral salt medium with 200 μ1 oil in well plates for the cultivation at 37 ℃ for 5 and 7 days, and the biodegradation potential was characterized by the changes of oil film and cell density. With appropriate evaluation by shaking flask tests, 5 isolates were retained for their potentials with the maxi- mum biodegradation from 1500 to 2200 mg· L-1 and identified as Acidovorax dtrulli, Pseudomonos balearica, Adnetobacterjohnsonii （two isolates with different biodegradation potentials） and Addovorax avenae using 16S rRNA sequencing analysis. Also, lipase activity was determined using indicator titration and p-nitrophenyl palmitate （p-NPP） methods. The results indicated that only p-NPP was successful to test lipase activity with the range of 1.93-6.29 mg· L-1 Although these five strains could degrade 1000 mg· L-1 lubricating oil in 158-168 h, there existed distinct difference in enzyme activity, which demonstrates that lipase activity could not be used as the criterion to evaluate microbial biodegradation potential for petroleum hydrocarbons.

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 ℃ and pH 5.5. We sequenced the genome and found a single chromosome of 4800175 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.

Feasibility of Tea Saponin-Enhanced Soil Washing in a Soybean Oil-Water Solvent System to Extract PAHs/Cd/Ni Efficiently from a Coking Plant Site

Mixed contaminated brownfield sites have brought serious risks to human health and environmental safety. With the purpose of removing polycyclic aromatic hydrocarbons （PAHs） and heavy metals from a coking plant site, an innovative technology for ex-situ washing was developed in the present work. The combination of 15.0 mLL-1 soybean oil and 7.5 g L-1 tea saponin proved an effective method to extract co-pollutants from soil. After two consecutive washing cycles, the efficiency rates of removal for 3-, 4-, 5（＋6）-ring, and total PAHs, Cd, and Ni were approximately 98.2%, 96.4%, 92.3%, 96.3%, 94.1%, and 89.4%, respectively. Meanwhile, as evaluated by Tenax extraction method and metal stability indices, the residual PAHs and heavy metals after consecutive washing mainly existed in the form with extremely low bioaccessibility in the soil. Thus, in the soil after two washing cycles, there appeared limited environmental transfer risk of co-pollutants. Moreover, a subsequent precipitation method with alkaline solution and PAH- degrading strain Sphingobium sp. PHE9 inoculation effectively removed 84.6%-100% of Cd, 82.5%-91.7% of Ni, and 92.6%-98.4% of PAHs from the first and second washing solvents. The recovered solvents also exhibited a high recycling effectiveness. Therefore, the combined cleanup strategy proposed in this study proved environmentally friendly, which also played a major role in risk assessment and marlagement in mixed polluted sites.

Biodegradation of n-hexadecane by bacterial strains B1 and B2 isolated from petroleum-contaminated soil

Two hydrocarbon-biodegrading bacterial strains, B1 and B2, were isolated from petroleum-contaminated soil collected from Tianjin, China. The strains were identified as Pseudomonas aeruginosa （B 1） and Acinetobacter junii （B2）. The degradation rate of n-hexadecane by BI and B2 reached 96% and 78% respectively after 7 days, though the strains employed different mechanisms of degradation. The results showed that B2 was not able to use glucose as carbon source. B 1 could produce glyco- lipid surfactants using glucose as the carbon source, according to the results of blue agar plate analysis and thin layer chroma- tography （TLC）, and the bacterial culture of B 1 had a high oil discharge and emulsification activity. Both B I and B2 could produce biosurfactants with hexadecane as the sole carbon source, but their modes of action were different. The carbon source was found to affect the cell surface hydrophobicity. Cell surface hydrophobicity was poor with glucose as the carbon source, but enhanced when hexadecane was used as the carbon source.