红树林湿地石油降解菌群的构建

近年来海洋污染正在日趋加剧,其中石油污染尤为严重。目前,采用微生物降解是解决海洋石油污染的有效途径之一,而滨海区域的红树林湿地是石油残留聚集和降解的重要生态系统之一。为了构建降解石油的优势菌群,分别以正十六烷烃和萘为唯一碳源,通过富集培养,从受石油污染的红树林淤泥(即土壤)中分离得到2株烷烃降解菌(即Z1、Z3)和2株芳香烃降解菌(即N1、N4)。采用三因素三水平进行正交试验,优化得到降解率最高的菌株组合,并确定了各菌株的最佳投加配比。结果表明:烷烃降解菌Z1、Z3和芳香烃降解菌N1组合的菌群降解石油效果最好,当石油初始质量浓度为2.0 g·L^-1,接种量6%,30℃好氧培养72 h,石油降解率达47.3%;且当N1、Z1、Z3三种菌的投加配比为3:1:3时降解效果最佳,好氧培养72 h,石油降解率达51.2%。     

微生物降解石油烃的功能基因研究进展

微生物对石油烃的降解在自然衰减去除土壤和地下水石油烃污染的过程中发挥了重要作用。微生物通过其产生的一系列酶来利用和降解这类有机污染物,其中,编码关键降解酶的基因称为功能基因。功能基因可作为生物标志物用于分析环境中石油烃降解基因的多样性。因此,研究石油降解功能基因是分析土著微生物群落多样性、评价自然衰减潜力与构建基因工程菌的重要基础。本文主要介绍了烷烃和芳香烃在有氧和无氧条件下的微生物降解途径,重点总结了烷烃和芳香烃降解的主要功能基因及其作用,包括参与羟化作用的单加氧酶和双加氧酶基因、延胡索酸加成反应的琥珀酸合酶基因以及中心中间产物的降解酶基因等。     

海洋石油降解微生物及其降解机理

微生物修复作为一种新型环保的生物修复技术,已成为海洋石油污染生物修复的核心技术。对海洋石油降解微生物的种类即细菌、蓝藻、真菌以及藻类进行了总结,对微生物对石油烃的降解途径与降解机理进行了综述。微生物降解烷烃的过程包括末端氧化、烷基氢过氧化物以及环己烷降解3种形式。微生物对芳香烃的降解是通过芳香烃被氧化酶氧化导致苯环开环来实现的。微生物对多环芳烃的降解是在单加氧酶或双加氧酶的催化作用下被最终降解为二氧化碳和水而被分解。并对影响石油烃降解微生物的因素包括温度、营养物质等因素进行了分析。     

一株耐碱高效长链烷烃降解菌C24MT1的筛选及其降解特性

【背景】石油被称为“液体黄金”,人类的工业生产活动在利用其创造巨大社会价值的同时,也对自然环境造成了严重的污染。微生物修复技术是现阶段治理石油类污染有效的手段之一,具有经济、高效、无二次污染等优点。【目的】从受石油污染的土壤中分离高效降解长链烷烃正二十四烷的菌株,探究其降解特性及在微生物修复中的应用前景。【方法】通过形态学及16S rRNA基因测序进行菌株鉴定,采用气相色谱法检测菌株对正二十四烷的降解效果,并结合气相色谱-质谱(gas chromatography-mass spectrometer, GC-MS)分析降解中间产物以推测其潜在代谢途径。【结果】筛选到一株可高效降解正二十四烷的菌株C24MT1,经鉴定为不动杆菌属(Acinetobacter)。该菌株最适降解条件为30 °C、pH 9.0、盐度2 g/L,该条件下生长7 d对9 g/L正二十四烷的降解率高达86.63%;与此同时,菌株在强碱性环境(pH 11.0)中生长良好(OD₆₀₀为0.39)并保持较高烷烃降解率(75.38%),对极端环境具备较强的耐受能力;对降解中间产物进行分析,推断菌株代谢长链烷烃正二十四烷的途径可能包括末端氧化及次末端氧化。【结论】不动杆菌C24MT1具有良好的环境适应能力及烷烃降解能力,在后续微生物菌剂开发和石油类污染土壤的环境修复领域具有巨大的应用前景。本研究可为盐碱地区高浓度石油类污染土壤的修复提供优良菌种,并进一步丰富石油烃类生物降解的菌种资源库。     

长链烷烃降解菌的降解特性

对长链烷烃降解菌的降解能力和摄取模式进行了研究。评价14株烃降解菌利用中长链烃生长的能力,发现只有少数烃降解菌能够获得良好生长,其中Mycobacterium fortuitum514,Pseudomonas aeruginosa1785和Pseudomonas marginata766等3株菌能够高效降解C20到C33的长链烷烃。辛烷不能支持这些长链烷烃降解菌的生长,说明其烃氧化酶与Pseudomonas oleovorans的OCT质粒编码的单氧酶不同。此外,M.fortuitum不产胞外表面活性剂,而P.aeruginosa和P.marginata则是表面活性剂产生菌,然而三者在以烃为碳源生长时均显示出很高的细胞表面疏水性。根据生长现象分析3株菌采用了不同的烷烃摄取模式。     

石油烃生物降解过程的研究进展

石油烃污染物属于难降解混合物,生物修复已经成为石油烃污染环境的主要修复方法。文中简述了微生物对石油烃的间期适应过程和转运过程,并通过对部分典型石油烃成分的微生物降解机理和代谢路径的梳理和综述,阐释了石油烃生物降解过程中的菌株、基因、代谢路径等研究进展。此外,利用基因工程和代谢工程等手段,可对野生型石油烃降解菌进行改造,进一步提升其对石油烃污染环境的生物修复能力。最后,从石油烃降解菌的代谢途径改造、人工混菌体系的设计构建等角度,结合合成生物学和代谢工程的手段,提出了对石油烃降解的研究展望,以期提升对石油烃污染物的生物修复效果。     

计算机重构石油烃降解的微生物代谢途径

目的:用计算机重构石油烃降解通路,为石油污染的生物修复提供理论依据。方法:利用KEGG反应、化合物数据提取反应等式,过滤掉所有反应中的通用化合物及小分子化合物并构建反应矩阵,然后利用广度优先搜索算法在反应矩阵中搜索降解石油烃的代谢途径。结果:计算机分别重构了256 132条链烷烃降解途径和44条环己烷降解途径,以酿酒酵母作为降解石油烃的基因工程菌为例,通过限制改构菌整合的关键酶数目,分别得到了213条不需要转入关键酶的链烷烃降解通路和6条以氧化还原酶、松柏醇脱氢酶或环己醇脱氢酶和环己酮单氧酶为关键酶的环己烷降解通路,并构建相应的降解网络图,标注每个反应的酶。结论:应用计算机重构了2种石油烃降解途径,可为利用微生物对石油污染进行生物修复提供理论依据。     

微生物降解石油污染物机制研究进展

石油污染是当前紧迫的水环境问题,研究石油污染物降解机制有助于探索石油污染修复技术路径。重点介绍了微生物降解石油污染物过程中的微生物种类、降解机制和反应机理,即具有代表性的细菌、真菌和藻类,石油烃的有氧降解(链烷烃、环烷烃和芳香烃)和厌氧降解(脱氢羟基化、延胡索酸盐加成)。并对微生物降解石油组分的影响因素进行了讨论,具体包括:烃类结构(支链多结构越复杂,越难降解)、微生物种类(混合菌的生化降解能力更强)、环境因子(pH、温度、盐度、含氧量和营养物质),进一步指出了生物修复技术应用于石油污染修复治理研究中的优缺点。此外,还对现有微生物降解技术的应用做了简要概述,归纳总结现有研究中存在的问题,尝试性的提出了今后生物降解石油污染物的研究重点,即生物降解石油的机制还需进一步明确,并重点分析了生物电化学方法在降解去除石油污染物方面可行性。综述石油烃生物降解机制和反应机理,以期为生物修复水体石油污染提供参考和借鉴作用。     

石油烃污染及修复过程中的微生物分子生态学研究进展

针对环境中广泛存在的石油烃污染问题,从分子生态学的角度总结石油烃降解过程中的微生物生态学研究进展。着重介绍分子生态学的研究方法及与石油烃降解相关的降解基因和基因芯片的最新研究进展,同时对存在的问题和今后的研究方向进行总结。     

低温石油降解菌LHB16的筛选及降解特性研究

目的:筛选、鉴定低温石油降解菌并对其降解特性进行研究.方法:富集分离低温石油降解菌;采用形态学、生理生化实验和分子生物学方法进行菌种鉴定;紫外分光光度法和GC-MS检测石油降解特性.结果:自盘锦油田低温环境土样中分离到1株低温菌,命名为LHB16,该菌能以石油烃为惟一碳源和能源.经鉴定为嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia).该菌生长温度范围0℃～35℃,最适生长温度15℃.在接种量为2%(V/V),原油浓度为0.5%(W/V),振荡培养10 d时,降解率可达80.16%.石油中长链烷烃C15～C32被完全降解.传代培养数代,降解率为81.06%,降解性能稳定.结论:菌株LHB16在低温地区石油污染的生物治理中有良好的应用前景.     

Changes in iso- and n-alkane distribution during biodegradation of crude oil under nitrate and sulphate reducing conditions

Crude oil consists of a large number of hydrocarbons with different susceptibility to microbial degradation. The influence of hydrocarbon structure and molecular weight on hydrocarbon biodegradation under anaerobic conditions is not fully explored. In this study oxygen, nitrate and sulphate served as terminal electron acceptors (TEAs) for the microbial degradation of a paraffin-rich crude oil in a freshly contaminated soil. During 185 days of incubation, alkanes from n-C11 to n-C39, three n- to iso-alkane ratios commonly used as weathering indicators and the unresolved complex mixture (UCM) were quantified and statistically analyzed. The use of different TEAs for hydrocarbon degradation resulted in dissimilar degradative patterns for n- and iso-alkanes. While n-alkane biodegradation followed well-established patterns under aerobic conditions, lower molecular weight alkanes were found to be more recalcitrant than mid- to high-molecular weight alkanes under nitrate-reducing conditions. Biodegradation with sulphate as the TEA was most pronounced for long-chain (n-C32 to n-C39) alkanes. The observation of increasing ratios of n-C17 to pristane and of n-C18 to phytane provides first evidence of the preferential degradation of branched over normal alkanes under sulphate reducing conditions. The formation of distinctly different n- and iso-alkane biodegradation fingerprints under different electron accepting conditions may be used to assess the occurrence of specific degradation processes at a contaminated site. The use of n- to iso-alkane ratios for this purpose may require adjustment if applied for anaerobic sites.     

一株不动杆菌降解石油烃的特性及关键烷烃降解基因分析

【背景】Acinetobacter sp. Tust-DM21 (GenBank登录号KX390866)是本实验室前期从渤海湾海洋石油勘探船废油收集区采集的水油混合样中分离出的一株高效石油降解菌,其对短、中、长链烷烃均表现出很强的降解能力,有较好的应用前景。【目的】从应用层面探究其最佳降解条件,同时从生物信息层面探究其降解基因的作用。【方法】将其在不同温度、pH下培养144h,通过GC-MS内标法测定石油烃各组分的变化情况,计算出其最佳降解条件;同时,通过生物信息学手段确定基因组中的降解基因,每个基因分别选择7个同源基因,对它们的蛋白序列进行比较;最后对2个降解基因在0-144 h的表达情况进行了Real-time PCR分析。【结果】Acinetobacter sp. Tust-DM21最佳降解条件为35°C、pH 8.5,该条件下对石油降解率可达97.5%,其中,对长链烷烃降解率达98.5%,对环烃为81%,对芳香烃为87%;同时,研究发现基因组中含有常见烷烃降解基因alk B(GenBank登录号MH368539)和长链烷烃降解基因alm A (GenBank登录号MH357335),2个降解基因的蛋白经比较均与其同源蛋白表现出一定的相似性,同属菌的相似性最高;通过Real-timePCR发现这2个基因在0-144 h的相对表达量随时间逐步提高。【结论】Acinetobacter sp. Tust-DM21在最佳降解条件下对石油各组分都显示出了优良的降解能力,特别对长链烷烃的降解能力尤为突出;将2个降解基因的相对表达量结合该时间段的生长趋势,证明了菌株Acinetobacter sp. Tust-DM21的生长和降解与alk B和alm A基因的上调表达存在关联。     

Nutrients and oxygen alter reservoir biochemical characters and enhance oil recovery during biostimulation

Biostimulation of petroleum reservoir to improve oil recovery has been conducted in a large number of oilfields. However, the roles and linkages of organic nutrients, inorganic salts and oxygen content during biostimulation have not been effectively elucidated. Therefore, we investigated the relationships between carbon source, nitrogen source, phosphorus source, oxygen content, and microbial stimulation, oil emulsification, and oil degradation. The organic nutrients (molasses) accelerated microbial growth, and promoted oil emulsification under aerobic conditions. The added molasses also promoted metabolites production (CO₂, CH₄ and acetic acid) and microbial anaerobic hydrocarbon degradation under anaerobic conditions. (NH₄)₂HPO₄ improved gases production by neutralizing the acidic production and molasses. NaNO₃ could also improve gases production by inhibiting sulfate-reducing bacteria to adjust pH value. Oxygen supply was necessary for oil emulsification, but bountiful supply of oxygen aggravated oil degradation, leading the entire ranges of alkanes and some aromatic hydrocarbons were degraded. Core-flooding experiments showed an oil displacement efficiency of 13.81 % in test with air package injected, 8.56 % without air package injection, and 4.77 % in control test with air package injection and 3.61 % without air package injection. The results suggest that the combined effect of organic nutrients, inorganic salts and oxygen content determines microbial growth, while production of metabolites, oil emulsification and biodegradation alter the reservoir biochemical characters and influence oil recovery during stimulation.     

Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase

LadA, a long-chain alkane monooxygenase, utilizes a terminal oxidation pathway for the conversion of long-chain alkanes (up to at least C₃₆) to corresponding primary alcohols in thermophilic bacillus Geobacillus thermodenitrificans NG80-2. Here, we report the first structure of the long-chain alkane hydroxylase, LadA, and its complex with the flavin mononucleotide (FMN) coenzyme. LadA is characterized as a new member of the SsuD subfamily of the bacterial luciferase family via a surprising structural relationship. The LadA:FMN binary complex structure and a LadA:FMN:alkane model reveal a hydrophobic cavity that has dual roles: to provide a hydrogen-bond donor (His138) for catalysis and to create a solvent-free environment in which to stabilize the C4a-hydroperoxyflavin intermediate. Consequently, LadA should catalyze the conversion of long-chain alkanes via the acknowledged flavoprotein monooxygenase mechanism. This finding suggests that the ability of LadA to catalyze the degradation of long-chain alkanes is determined by the binding mode of the long-chain alkane substrates. The LadA structure opens a rational perspective to explore and alter the substrate binding site of LadA, with potential biotechnological applications in areas such as petroleum exploration and treatment of environmental oil pollution.     

Isolation,identification, and performance studies of a novel paraffin-degrading bacterium of <Emphasis Type="Italic">Gordonia amicalis</Emphasis> LH3

In this study, we describe the isolation and identification of a novel long-chain n-alkane degrading strain, Gordonia amicalis LH3. Under aerobic conditions, it utilized approximately 18.0% of paraffin (2% w/v) after 10 day of incubation, and the paraffin compositions of C₁₈∼C₂₄ alkalines were utilized preferentially. Under anaerobic conditions, paraffin utilization was approximately 1/8 that seen under aerobic conditions, and the compositions of C₃₄ and C₃₆ alkalines were utilized preferentially. The effects of salinity, temperature, and biosurfactants on paraffin degradation were also evaluated. The strain was also demonstrated to grow on oil, and decreased oil viscosity by 44.7% and degraded oil by 10.4% under aerobic conditions. Our results indicated that G. amicalis LH3 has potential applications in paraffin control, microbial enhanced oil recovery (MEOR), and the bioremediation of hydrocarbon-polluted environments.     

Biodegradation of fuel oil hydrocarbons by a mixed bacterial consortium in sandy and loamy soils

The aerobic degradation of light fuel oil in sandy and loamy soils by an environmental bacterial consortium was investigated. Soils were spiked with 1 or 0.1% of oil per dry weight of soil. Acetone extracts of dried soils were analyzed by GC and the overall degradation was calculated by comparison with hydrocarbon recovery from uninoculated soils. In sandy soils, the sum of alkanes n-C(12) to n-C(23) was degraded to about 45% within 6 days at 20 degrees C and to 27-31% within 28 days, provided that moisture and nutrients were replenished. Degradation in loamy soil was about 12% lower. The distribution of recovered alkanes suggested a preferential degradation of shorter chain molecules (n-C(12) to n-C(16)) by the bacterial consortium. Partial 16S rDNA sequences indicated the presence of strains of Pseudomonas aeruginosa, Pseudomonas citronellolis, and Stenotrophomonas maltophilia. Toxicity tests using commercial standard procedures showed a moderate inhibition of bacterial activity. The study showed the applicability of a natural microbial community for the degradation of oil spills into soils at ambient temperatures.     

Potential for Aerobic and Anaerobic Biodegradation of Petroleum Hydrocarbons in Boreal Subsurface

We studied the role of aerobic and anaerobic petroleum hydrocarbon degradation at a boreal, light-weight fuel and lubrication oil contaminated site undergoing natural attenuation. At the site, anoxic conditions prevailed with high concentrations of CH4 (up to 25% v/v) and CO2 (up to 18% v/v) in the soil gas throughout the year. Subsurface samples were obtained mainly from the anoxic parts of the site and they represented both the unsaturated and saturated zone. The samples were incubated in microcosms at near in situ conditions (i.e. in situ temperature 8 degrees C, aerobic and anaerobic conditions, no nutrient amendments) resulting in the removal of mineral oil (as determined by gas chromatography) aerobically as well as anaerobically. In the aerobic microcosms on average 31% and 27% of the initial mineral oil was removed during a 3- and 4-month incubation, respectively. In the anaerobic microcosms, on average 44% and 15% of the initial mineral oil was removed during a 12- and 10-month anaerobic incubation, respectively, and e.g. n-alkanes from C11 to C15 were removed. A methane production rate of up to 2.5 microg CH4 h(-1) g(-1) dwt was recorded in these microcosms. In the aerobic as well as anaerobic microcosms, typically 90% of the mineral oil degraded belonged to the mineral oil fraction that eluted from the gas chromatograph after C10 and before C15, while 10% belonged to the fraction that eluted after C15 and before C40. Our results suggest that anaerobic petroleum hydrocarbon degradation, including n-alkane degradation, under methanogenic conditions plays a significant role in the natural attenuation in boreal conditions.