Chemotaxis Toward Crude Oil by an Oil-Degrading Pseudomonas aeruginosa 6-1B Strain

The chemotactic properties of an oil-degrading Pseudomonas aeruginosa strain 6-1B, isolated from Daqing Oilfield, China, have been investigated. The strain 6-1B could grow well in crude oil with a specific rhamnolipid biosurfactant production. Furthermore, it exhibits chemotaxis toward various substrates, including glycine, glycerol, glucose, and sucrose. Compared with another oil-degrading strain, T7-2, the strain 6-1B presented a better chemotactic response towards crude oil and its vital component, n-alkenes. Based on the observed distribution of the strain 6-1B cells around the oil droplet in the chemotactic assays, the potential chemotaxis process of bacteria toward crude oil could be summarized in the following steps: searching, moving and consuming.     

高效降解石油外生菌根真菌的室内筛选

对7个外生菌根真菌菌株在不同石油浓度培养基中的生长及其对石油的降解作用进行了研究。结果表明:(1)不同菌株在同一石油浓度培养基中生长速度不同,同一菌株在不同石油浓度培养基中的生长速度亦不同。菌株010和菌株025在不同石油浓度培养基中生长速度均较其他菌株快。菌株010的菌丝干重随着石油浓度的增加而增加,当石油质量浓度为500 mg/L时,菌丝干重达到最大值,高于对照5.27%,石油对其生长产生了促进作用;当石油质量浓度为700 mg/L时,菌丝干重低于对照,随着石油质量浓度的升高,菌株生长呈下降趋势。石油质量浓度为100 mg/L时,菌株025生长最慢,菌丝干重低于对照9.1%。随着石油浓度的增加,菌株生长加快,当石油质量浓度为500 mg/L时,菌丝干重高于对照;当石油质量浓度为700 mg/L时,菌株025菌丝干重最高,高于对照25.65%。随着石油浓度的再度升高,菌株生长呈下降趋势。(2)不同菌株在同一石油浓度下对石油的降解能力不同,同一菌株在不同石油浓度下对石油的降解能力亦不同。菌株025对石油的降解能力最强,对石油的降解能力随着石油浓度的升高而提高。当培养基中石油质量浓度为900 mg/L时,菌株025对石油的降解率最高,达到73.65%。(3)菌株0100、09、035、LH004的菌丝生物量与对石油的降解能力呈正相关。     

Estimation of the hydrocarbon-oxidizing activity of microorganisms

A method allowing the microorganisms growing on substrates with a low water solubility (oil, fuel oil, resins, and asphalthenes) to be isolated and counted was developed. An advantage of the method is its ability to estimate the oil-utilizing activity of each strain visually according to the decolorized zones formed during its growth on oil products. The sizes of these zones indicate which oil-degrading strain is most active.     

一株油脂降解菌的筛选鉴定及降解效果分析

为了筛选分离得到一株具有油脂降解能力的菌株,同时探究菌株的特性和降解能力。以屠宰场污染土作为菌源,通过梯度驯化法最终筛选分离得到能够将橄榄油作为单一碳源生长的降解菌。随后通过形态特征观察、Biolog生理生化测试以及16S rRNA基因序列比对分析鉴定,实验菌株为革兰氏阴性菌,属于无色杆菌属（Achromobacter sp.）,在构建的系统发育树上与Achromobacter pulmonis聚为一支。综合运用紫外分光光度法和高效液相色谱法检测,测得实验菌株培养4~5 d时对橄榄油的降解率可以达到90%,同时测得菌株降解油脂的最适pH和最适温度分别为7.5和35 ℃,该菌株在盐浓度低于40 g·L-1环境中降解率较高。此外实验结果表明,实验菌株对各类型油脂均具有较高的降解效率,具有广泛的应用前景。     

一株表面活性剂产生菌的分离及抑菌活性

【目的】研究分离得到的表面活性剂产生菌的产表面活性剂能力、分类地位和抑菌活性。【方法】采用血平板、油平板进行表面活性剂产生菌的分离,以排油圈法进行表面活性的测定;通过生理生化特性和16S rDNA序列相似性分析对BS1菌株进行初步鉴定;利用对峙培养法和菌丝生长、孢子囊形成、孢子萌发的抑制率测定研究其抑菌活性。【结果】从石油污染土壤中分离到的BS1菌株可产生表面活性剂,在分类学地位上属于假单胞菌属(Pseudomonas sp.)。BS1菌体、发酵上清液、挥发性物质对12种供试病原真菌均表现出一定的抑制作用。BS1菌体、发酵上清液对大豆疫霉菌(Phytophthora sojae)的抑制率最大,分别达到65.31%和95.93%。发酵上清液通过影响大豆疫霉菌菌丝生长、孢子囊形成、孢子萌发等方式抑制病原菌的正常生长,稀释20倍的发酵上清液依然具有明显的抑制作用。BS1菌株产生的挥发性物质对大豆菌核菌(Sclerotinia sclerotiorum)的抑菌效果最好,抑制率达到84.25%。【结论】BS1菌株在产生表面活性剂的同时,还具有生物防治作用潜力。     

Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid

AIMS: The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). METHOD AND RESULTS: An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. CONCLUSIONS: The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.     

一株糖脂表面活性剂产生菌的筛选及干酪根降解

【目的】从油页岩环境中筛选可降解油页岩干酪根的产生物表面活性剂菌株。【方法】从抚顺油页岩矿废水样品中用血平板法初筛,排油圈法、乳化法和表面张力法复筛,获得产生物表面活性剂菌株。对目标菌株进行生理生化鉴定、16S r RNA基因序列和系统发育分析,用薄层色谱鉴定其发酵液表面活性成分,优化产表面活性剂的培养条件,初步考察其对油页岩干酪根的降解能力。【结果】筛选到一株产糖脂表面活性剂菌株B-1,初步鉴定为Pseudomonas sp.,该菌株有良好的排油和乳化能力以及较低的表面张力,可利用烷烃、不饱和脂肪酸和糖类作为碳源。在30-34°C范围内添加0.3%Na Cl的葡萄糖培养基(p H 7.0)中该菌生长旺盛,发酵液表面张力最低为27 m N/m。菌株B-1在添加一定量葡萄糖的无机盐培养基中作用30 d后对干酪根的降解率为2.85%,高于不添加葡萄糖无机盐培养基对照组的降解率(1.04%)。【结论】菌株B-1是一株性能良好的产糖脂表面活性剂细菌,有降解干酪根的潜力。     

Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation

Among 25 crude oil-degrading bacteria isolated from a marine environment, four strains, which grew well on crude oil, were selected for more study. All the four isolated had maximum growth on 2.5% of crude oil and strain BC (Pseudomonas) could remove crude oil by 83%. The drop collapse method and microtiter assay show that this strain produces more biosurfactant, and its biofilm formation is higher compared to other strains. Bacterial adhesions to crude oil for strains CS-2 (Pseudomonas), BC, PG-5 (Rhodococcus) and H (Bacillus) were 30%, 46%, 10% and 1%, respectively. Therefore, strain H with a low production of biosurfactant and biofilm formation had showed the least growth on these compounds. PCR analysis of these four strains showed that all isolates had alk-B genes from group (III) alkane hydroxylase. All isolate strains could utilize cyclohexan, octane, hexadecane, octadecan and diesel fuel oil; however, the microtiter plate assay showed that strain BC had more growth, respiration and biofilm formation on octadecan.     

海绵中石油降解微生物的分离筛选及降解特性研究

从大连湾原油污染海域生长的海绵中分离筛选到一株原油降解菌OA58,根据其生长形态、培养特征、16S rRNA序列相似性比对分析和生化指标检测,初步鉴定为脱叶链霉菌（Streptomyces exfoliatus）。同时,考察了该链霉菌对原油的降解效果,OA58能以原油为唯一碳源生长,在人工海水培养基中,14 d内对原油（初始浓度为1 g/L）的平均降解率为83%,是一株具有开发潜力的原油降解放线菌。     

Starvation survivability of Thermococcus strains isolated from Japanese oil reservoirs

The starvation survivability of seven Thermococcus strains isolated from four Japanese oil reservoirs was compared with that of Thermococcus strains from marine hydrothermal fields. 16S rDNA analyses showed the isolates to be closely related to Thermococcus litoralis. Growth of the isolates was dependent on amino acids, which were present at low concentrations in the oil reservoirs. At 80 degrees C in the formation water, strain CKU-1 from the oil reservoir showed a higher starvation survivability than strain KS-1 from the marine hydrothermal field. Crude oil did not affect the starvation survivability of strain CKU-1, but it reduced that of strain KS-1. These results indicate that strain CKU-1 could survive longer than stain KS-1 under the conditions of an oil reservoir. At 90 degrees C in artificial seawater without organic nutrients, the half-lives of the isolates were between 7.7 and 25.1 days. However, those of the strains from marine hydrothermal fields, except Thermococcus litoralis and Thermococcus chitonophagus, were less than 1.0 day. The higher starvation survivability is probably important for the hyperthermophiles to continue to exist in a hot subterranean oil reservoir where the supply of nutrients seems to be limited.     

Sphingobacterium bambusaue及其紫外诱变菌株的石油降解功能

【目的】研究Sphingobacterium bambusaue及其紫外诱变菌株的石油降解功能。【方法】紫外诱变后筛选石油降解高效菌株; 以不同石油浓度、pH值及盐浓度优化培养条件, 用重量法检测高效菌株石油降解率。【结果】发现菌株S. bambusaue在石油降解培养基中培养5 d的石油降解率为25.86%, UV诱变高效菌株IBFC2009-S3培养5 d的石油降解为42.85%, 比始发菌株提高65.7%; UV诱变菌株IBFC2009-S3的优化培养条件为石油浓度0.5 g/L、pH值7.0以及NaCl质量浓度为10 g/L, 其石油降解率可达50.51%。【结论】首次报道S. bambusaue具有石油降解功能; 紫外诱变获得的菌株S3的石油降解能力较强。     

Surfactant activity of a naphthalene degrading Bacillus pumilus strain isolated from oil sludge

We studied the growth, biosurfactant activities and petroleum hydrocarbon compounds utilisation of strain 28-11 isolated from a solid waste oil. The isolate was identified as Bacillus pumilus. It grew well in the presence of 0.1% (w/v) of crude oil and naphthalene under aerobic conditions and utilised these substances as carbon and energy source. The capacity of strain 28-11 to emulsify crude oil and its ability to remove hydrocarbons looks promising for its application in environmental technologies.     

Isolation and characterization of a sulfur-oxidizing chemolithotroph growing on crude oil under anaerobic conditions

Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the epsilon subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities. In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan. 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity). Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor. Oxygen also supported its growth only under microaerobic conditions. Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction. Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources. A typical stoichiometry of its energy metabolism followed an equation: S(2-) + 4NO(3)(-) --> SO(4)(2-) + 4NO(2)(-) (Delta G(0) = -534 kJ mol(-1)). In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible. When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth. On the contrary, YK-1 could not utilize crude oil as a carbon source. These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds. Based on its physiology, ecological interactions with other members of the groundwater community are discussed.     

Aerobic Microbial Cometabolism of Benzothiophene and 3-Methylbenzothiophene

A culture enriched by growth on 1-methylnaphthalene was used to study the aerobic biotransformations of benzothiophene and 3-methylbenzothiophene. Neither of the sulfur heterocyclic compounds would support growth, but they were transformed by the culture growing on 1-methylnaphthalene or glucose or peptone. Cometabolism of benzothiophene yielded benzothiophene-2,3-dione, whereas that of 3-methylbenzothiophene yielded 3-methylbenzothiophene sulfoxide and the corresponding sulfone. The identities of the dione and sulfone were verified by comparison with authentic standards. The identity of the sulfoxide was surmised from gas chromatography-mass spectrometry and gas chromatography- Fourier transform infrared spectroscopy results. Oxidation preferentially occurred at carbons 2 and 3 in benzothiophene, but when carbon 3 was substituted with a methyl group, as in 3-methylbenzothiophene, the sulfur atom was oxygenated. The predominant microorganism in the enrichment culture was a Pseudomonas strain, designated BT1, which mineralized aromatic but not aliphatic hydrocarbons. This isolate cometabolized benzothiophene and 3-methylbenzothiophene. There was no evidence that it could metabolize 3-methylbenzothiophene sulfone. When 3-methylbenzothiophene was added to Prudhoe Bay crude oil, the sulfur heterocycle was oxidized to its sulfoxide and sulfone by strain BT1 as it grew on the aromatic hydrocarbons in the crude oil. Benzothiophene-2,3-dione was found to be chemically unstable when incubated with Prudhoe Bay crude oil. Thus its formation from benzothiophene in the presence of crude oil could not be determined.     

高产不饱和油脂海鞘真菌桔青霉Asc-2-4的诱变选育

诱变育种是获得高产菌株,实现微生物工业化生产油脂的重要措施。以前期获得的高产不饱和油脂菌株桔青霉（Penicillium citrinum）Asc-2-4为出发菌株,利用丙二酸建立快速筛选高产不饱和脂肪酸突变菌的方法,通过紫外线 氯化锂复合诱变得到1株高产油脂突变菌Asc-2-4-1,油脂含量比出发菌株提高了92.98%。经过初步的培养基无机盐优化,其油脂得率和不饱和脂肪酸产量达到了7.10 g/L和3.84 g/L,与Asc-2-4相比,分别提高了84.42%和77.78%。结果表明,通过复合诱变选育技术可选育出高产突变菌株,选育的Asc-2-4-1可望作为产油微生物被开发利用。     

Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.     

Screening and Evaluation of Biosurfactant-Producing Strains Isolated from Oilfield Wastewater

The six biosurfactant-producing strains, isolated from oilfield wastewater in Daqing oilfield, were screened. The production of biosurfactant was verified by measuring the diameter of the oil spreading, measuring the surface tension value and emulsifying capacity against xylene, n-pentane, kerosene and crude oil. The experimental result showed three strains (S2, S3, S6) had the better surface activity. Among the three strains, the best results were achieved when using S2 strain. The diameter of the oil spreading of the biosurfactant produced by S2 strain was 14 cm, its critical micelle concentration (CMC) was 21.8 mg/l and the interfacial tension between crude oil and biosurfactant solution produced by S2 strain reduced to 25.7 mN/m. The biosurfactant produced by S2 strain was capable of forming stable emulsions with various hydrocarbons, such as xylene, n-pentane, kerosene and crude oil. After S2 strain treatment, the reduction rate of oil viscosity was 51 % and oil freezing point reduced by 4 °C.     

Isolation of Ashbya gossypii mutant for an improved riboflavin production targeting for biorefinery technology

AIMS: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. METHODS AND RESULTS: Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 g l(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 g l(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wild-type strain. CONCLUSIONS: The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. SIGNIFICANCE AND IMPACT OF STUDY: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.     

Isolation and characterization of crude-oil-degrading bacteria from oil-water mixture in Dagang oilfield,China

Isolating novel crude-oil-degrading bacteria from oil-water mixture of oil production well and evaluating their degradation capacities are vitally important in the remediation of oil-polluted environments and crude oil exploitation. According to the molecular screening with degenerate primers of alkane hydroxylase gene (alk B), a strain Acinetobacter sp. LS-1 with alk B gene was isolated. This strain exhibited a 99.9% similarity with genus Acinetobacter. This alk B gene which is one of the key enzymes of metabolic process was identified. This gene sequence showed 98% similarity of its nucleotide and related amino acids to the genus Marinobacter but exhibited below 70% similarity to the genus Acinetobacter. This phylogenetic analysis indicated that alk B may have been transferred horizontally between bacteria. The isolated strain could utilize crude oil as the sole carbon, achieving a high degradation (70.3%) in 7 days. Microcalorimetric analysis of the metabolic processes for hexadecane degradation also demonstrated the ability of this strain to utilize hydrocarbons. Thus, this strain enables to degrade hydrocarbons as the sole carbon source from the gene level, combined with material and energy metabolism. These findings will benefit this strain in the remediation of oil-polluted environments and oil exploitation.