石油烃厌氧生物降解研究进展

烃是由碳和氢两种元素组成的稳定化合物,长期以来人们普遍认为烃的生物降解只能在好氧条件下进行.近年来国外的研究表明,厌氧微生物能以硝酸盐、硫酸盐、三价铁或二氧化碳等为电子受体代谢烃,烃厌氧代谢的初始反应机理主要有延胡索酸盐结合反应、羧化反应、羟基化反应和甲基化反应,但国内还未见相关报道.本文综述了烃厌氧降解微生物及其代谢机理,并以甲苯为例概述了烃厌氧生物降解过程中的起始反应酶和作用机制.结合国外烃厌氧降解的研究进展和本实验室的工作,作者也提出了自己的思考和看法.     

石油烃降解酶及其基因的结构、功能和表达调控

研究烃降解酶及其基因是进行石油微生物分子检测和工程菌构建的重要基础.本文对目前烃降解酶及其基因的结构、功能和调控机制的最新研究进展进行了总结.催化烷烃好氧降解的起始酶有几类加氧酶,膜整合甲烷单加氧酶、萘-1,2-双加氧酶和异丙苯双加氧酶的晶体结构已经被解析.烷基或芳基琥珀酸合酶催化烃厌氧代谢的主要起始反应,而Azoarcus sp.乙苯厌氧代谢起始反应由乙苯脱氢酶催化.在细菌中,烃代谢相关基因主要通过形成操纵子进行表达调控,基因转录受烃或类似物诱导,并受细胞全局调控.一些微生物由于存在多种烃代谢途径而可能具有复杂的基因调控机制.此外,生态学研究表明,环境中烃降解基因的诱导动态与实验室内纯培养分析不同.在分析石油降解工程菌构建有待解决问题的基础上,提出了烃代谢综合调控和环境中相关酶及基因诱导研究的重要性,并对未来烃降解酶及其基因在有毒物降解理论研究和生物修复上的应用进行了展望.     

石油污染土壤的微生物修复及土壤微生物活性变化

为快速有效地测定石油污染土壤中功能性微生物的活性变化,分别以石油烃、正十六烷烃、多环芳烃为自定义碳源,应用Biolog法研究油污土壤生物修复过程中石油烃、烷烃、多环芳烃降解菌的代谢活性.结果显示,向油污土壤中投加混合降解菌群进行生物强化修复处理,可以有效去除土壤中的石油烃,修复13周土壤中石油烃去除率达到42.3%;生物刺激和自然修复对土壤石油烃的去除率分别为28.3%和20.5%.Biolog测定结果表明,生物强化法修复初期的土壤微生物群落对石油烃、烷烃两种碳源的代谢能力较强,而生物刺激法修复后期的土壤微生物群落对烷烃有较强的代谢能力;不同处理的土壤微生物群落比较偏好、利用率较高的碳源是石油烃,其次是烷烃,而对多环芳烃几乎不利用;土壤中石油烃、烷烃降解菌的活性越大,土壤微生物对石油烃的去除效率越高.上述研究结果说明,通过利用Biolog法测定土壤微生物活性变化可有效指示土壤中石油烃的去除效果.     

石油污染物的微生物降解研究

以炼油厂污水池底泥中分离的3株细菌和3株真菌为供试微生物,陕北黄土(0～20 cm)制备的土壤悬浮液为土壤微生物对照,灭菌培养基为非生物降解对照,研究不同组合微生物的生长动态及对石油烃的降解率.将不同组合的微生物接种到石油烃质量浓度为10000 mg·L-1的液体培养基中,29 ℃±1 ℃,摇床连续培养50 d.于0 ～72 h内取样,进行微生物生长动态检测;于5～50 d定期取样,考察培养基中石油烃降解率的动态变化.结果表明:单独外源菌降解效果优于混合外源菌和土著微生物,25 d后土著微生物生物降解率超过混合外源菌.外源细菌、外源真菌、混合外源菌和土著微生物50 d生物降解率分别为71.54 %,60.13 %,47.26 %和51.49 %.土壤细菌对石油污染物具有较强的生长适应性,外源细菌降解效果最好.     

强化微生物修复石油污染海洋的研究进展

海洋石油污染使石油烃(Petroleum hydrocarbons,PHCs)成为海洋重要有机污染物.快速有效清除石油污染物是当前急需解决的海洋环境问题之一.利用石油烃降解菌(Petroleum hydrocarbon-degrading bacteria,PHDB)进行的原位微生物修复是一种对环境友好且较经济的清除石油污染场所和管理海洋表面溢油的有效技术.本文综述了石油烃降解菌的特性和对环境的适应性.重点分析探讨了引入外源微生物,投加生物表面活性剂、添加营养物质和增加溶解氧含量、采用微生物固定化技术等方法强化原位微生物修复石油污染海洋的机制和研究进展.展望了海洋石油污染微生物修复领域的研究前景.     

胁迫条件下极端微生物修复石油烃污染土壤研究进展

嗜冷菌、嗜盐菌、耐重金属菌、耐重油菌等极端微生物广泛存在于极地高寒、盐碱地以及存在重金属、重油等污染的毒性污染土壤中,是胁迫条件下石油烃降解与转化的重要微生物资源。文章从适应机制、降解机理、降解特性、修复实践等角度出发,综述了低温、盐碱、重金属、重油等不同胁迫条件下的石油烃污染土壤微生物修复进展。在石油烃降解机理方面,微生物细胞与油滴的附着机制尚不清楚,而生物表面活性剂的产生和作用机制已经得到了很好的研究。嗜冷菌的适冷机制与细胞膜脂类组成、冷激蛋白、冷适应蛋白、嗜冷酶、能量代谢等有关,低温(15℃)时石油烃降解效率可达70%以上。嗜盐菌具备细胞外被隔离机制和离子反向运输机制,能产生渗透压调节剂、具有独特的渗透压平衡方式,NaCl浓度为30g·L-1时石油烃的降解效率可达60%以上。石油烃降解菌对重金属的耐受机制包括生物吸附、细胞内积累、酶催化转化、生物浸出和生物矿化、氧化还原反应等过程,会影响土壤中重金属的迁移率和生物有效性,提高作物的产量和对重金属的富集。微生物吸收重油的机制包括界面张力降低、选择性堵塞、粘度降低、生物降解和润湿性改变等,对重质原油的总体降解率可达70%以上,但是对其中沥青质单一组分的历史最高生物降解率仅为48%。利用极端微生物修复极端、胁迫条件下的石油污染土壤,应加强菌种培育、未明机制探索、重油组分(沥青质和树脂)降解、风险评估、修复工艺参数优化及推广应用等工作。     

添加不同营养助剂对石油污染土壤生物修复的影响

实验室条件下研究NPK复合肥、诺沃肥和腐殖酸三种物质不同配比添加对石油污染土壤的生物修复效果.在60d的修复实验中,定期取样测定土壤含油量、总异养菌数、石油烃降解菌数和脱氢酶活性,并采用PCR-DGGE技术研究修复过程中微生物多样性变化.结果表明,NPK肥的添加和NPK肥-诺沃肥-腐殖酸复合添加能够提高土壤中微生物的数量、微生物多样性和脱氢酶活性.在含油量为84600mg.kg-1的土壤中,添加营养助剂的处理60d后石油烃降解率为31.3%—39.5%,不添加营养助剂的石油烃降解率仅为3.5%.NPK肥-诺沃肥-腐殖酸复合添加对石油烃的降解率要高于NPK肥的单独添加(高8%),其原因可能是诺沃肥-腐殖酸能够有效提高土著微生物的活性,增加微生物的多样性,增强石油烃的降解.     

植物-微生物修复石油烃污染土壤与根际微生态环境变化

石油烃作为一类持久性难降解有机污染物对土壤环境质量产生严重的危害。以天津大港油田原油污染土壤中筛选出的耐低温高效石油烃降解菌为靶细胞,以小麦、紫花苜蓿作为供试植物,利用盆栽试验,对植物-外源菌协同修复体系中的脱氢酶活性和土壤微生物多样性进行研究,分析其变化及其与石油烃降解率的关系。结果表明植物-微生物协同修复对石油烃具有较好的降解能力,其中小麦-固定化外源菌组具有最高的降解率,石油烃含量从最初的30 600 mg獉kg-1下降为24 300 mg獉kg-1,降解率为20.6%,并且其试验后期石油烃的降解率最大,远远高于其他时期,表现出良好的修复潜力。外源菌投加的初始阶段会迅速提高脱氢酶活性,然而这种影响随着降解时间延长而逐渐减弱。初期脱氢酶活性与总石油烃的降解存在较好的相关性,脱氢酶活性可以在一定程度上表征土壤石油烃的降解情况。微生物多样性与总石油烃降解也存在一定的相关性。     

基因工程菌在石油污染修复中的研究进展与前景

构建基因工程菌（genetically engineered microorganisms,GEMs）是石油污染生物修复的重要发展方向.目前,通过基因编辑、过表达和定向进化等手段改造微生物的石油污染物降解和调控途径,可以提高微生物的环境适应能力和污染物降解能力,用于石油污染物的生物降解和监测.本文概述了石油污染物降解基因工程菌的主要构建策略,包括选择和改造宿主菌、改造与优化石油污染物关键酶和代谢通路、开发微生物全细胞传感器和构建基因工程菌的自毁程序.此外,基因工程菌也可用于石油污染的酶修复、微生物菌群修复和细菌-植物联合修复.随着系统生物学和合成生物学在降解微生物中的应用,基因工程菌在石油污染修复中展现出良好的研究和应用前景.     

土壤和地下水中多环芳烃生物降解研究进展

多环芳烃是一类普遍存在于环境中的难降解的危险性"三致"有机污染物。受污染的土壤和地下水中的多环芳烃,生物降解是其归宿的主要途径。研究表明,对于土壤中低分子量多环芳烃类化合物,微生物一般以唯一碳源方式代谢;而大多数细菌和真菌对四环或四环以上的多环芳烃的降解作用一般以共代谢方式开始。文章重点论述了多环芳烃的来源、降解多环芳烃的微生物、生物降解机理、影响生物降解的因素以及生物修复方法。认为今后的研究方向是高分子量多环芳烃的降解机理与降解途径,基因工程技术在多环芳烃生物降解方面的应用,以及生物表面活性剂产生的机理及其在实际处理中的应用等。     

Degradation rates of aged petroleum hydrocarbons are likely to be mass transfer dependent in the field

Evidence for on site biodegradation may be difficult to provide at heterogeneous sites without additional experiments in controlled laboratory conditions. In this study, microbial activities measured as CO₂ and CH₄ production were compared in situ, in intact soil cores and in bottle microcosms containing sieved soils. In addition, biodegradation rates were determined by measuring the decrease in petroleum hydrocarbon concentrations at 7°C in aerobic and anaerobic conditions. Elevated concentrations of CO₂ and CH₄ in the soil gas phase indicated that both the aerobic and anaerobic microbial activity potentials were high at the contaminated site. Aerobic and anaerobic microbial degradation rates in laboratory experiments of petroleum hydrocarbons were highest in soils from the most contaminated point and degradation in the aerobic and anaerobic microcosms was linear throughout the incubation, indicating mass-transfer-dependent degradation. Different results for microbial activity measurements were obtained in laboratory studies depending on pretreatment and size of the sample, even when the environmental conditions were mimicked. These differences may be related to differences in the gas exchange rates as well as in changes in the bioavailability of the contaminant in different analyses. When predicting by modeling the behavior of an aged contaminant it is relevant to adapt the models in use to correspond to conditions relevant at the contaminated sites. The variables used in the models should be based on data from the site and on experiments performed using the original aged contaminant without any additions.     

Biodegradation and phytotoxicity assessment of phenanthrene by biosurfactant-producing Bacillus pumilus 1529 bacteria

ABSTRACT

Phenanthrene is a toxic and mutagenic pollutant that can cause severe environmental and human health issues. The bioremediation of these polyaromatic hydrocarbons (PAHs) is possible with a biosurfactant by enhancing hydrophobicity. In this study, the production of a biosurfactant by Bacillus pumilus 1529 and its effects on the phenanthrene biodegradation pathway were examined. Biosurfactant production was determined using hemolytic activity, emulsification index, and surface tension. For phenanthrene metabolite detection, samples at 0, 7, 14, and 21 incubation days were analysed by gas chromatography-mass (GC-mass) spectrometry. The results showed that Bacillus pumilus 1529 can reduce surface tension to 22.83?±?1.1?mN?m^?1. Furthermore, the GC-mass spectrometry analysis showed that 1-hydroxy-2-naphthoic acid, benzaldehyde, o-phthalic acid, and phenylacetic acid were notable phenanthrene metabolites produced during phenanthrene biodegradation. Biodegraded phenanthrene and its metabolites have a less toxic effect on the germination of safflower seeds than non-biodegraded phenanthrene. The IC50 of phenanthrene on seed germination after biodegradation was increased to approximately 113?mg?L^?1. In general, biodegradation aided by biosurfactant producing bacteria contributed to turning the toxic phenanthrene into less harmful metabolites with lower phytotoxicity effects, indicating that its application in the bioremediation of PAHs is promising.     

活性艳红X-3B的厌氧生物降解与机理

以活性艳红X-3B(RBR)为研究对象,分3个不同浓度组研究其在厌氧颗粒污泥作用下的降解性能,并采用生物降解动力学方程拟合其降解过程,同时利用GC-MS分析RBR的生物降解途径.试验结果表明,RBR在厌氧颗粒污泥中降解性能很好,去除率可达93.67%,且其降解过程符合二级反应动力学,其半衰期约为3.561 h.从GC-MS和UV-可见光谱的分析得出,RBR在厌氧颗粒污泥的作用下偶氮键断裂,且生成的芳香胺类化合物进一步降解为小分子的烃类、酚类、醇类和脂类化合物.     

Microbiological degradation of polycyclic aromatic hydrocarbons: Anthracene,benzo(K)fluoranthene,dibenzothiophene, benzo(H) quinoline and 2‐nitronaphthalene

Biodegradation experiments of various polycyclic aromatic hydrocarbons were studied with mixed bacteria culture under aerobic conditions. An easy‐to‐handle clean‐up procedure was developed for PAH and their metabolites simultaneously as well as a gc‐ms‐method to identify and quantify these compounds.

Anthracene and dibenzothiophene are completely degradable in an aqeous system, whereas biodegradation of benzo(k)fluoranthene and benzo(h)quinoline is possible only in an oil‐in‐water‐system with dodecane as cosubstrate. No degradation of nitronaphthalene was observed in aqueous systems. New metabolites are 2,3‐dihydroxybenzothiophene, hydroxybenzothiophenecarbonic acid and benzothiophenequinone for dibenzothiophene and hydroxyfluoranthenic acid for benzo(k)flouranthene. Whereas the former metabolites are degradable under the experimental conditions, the latter accumulates during the degradation experiment.

The results are important for microbiological wastewater treatment, since knowledge of biodegradation processes is indespensable for the successful treatment of PAH‐containing wastewater.     

Bioremediation of highly contaminated oilfield soil: Bioaugmentation for enhancing aromatic compounds removal

This study evaluated the effectiveness of different amendments--including a commercial NPK fertilizer, a humic substance （HS）, an organic industrial waste （NovoGro）, and a yeast-bacteria consortium--in the remediation of highly contaminated （up to 6% of total petroleum hydrocarbons） oilfield soils. The concentrations of hydrocarbon, soil toxicity, physicochemical properties of the soil, microbial population numbers, enzyme activities and microbial community structures were examined during the 90-d incubation. The results showed that the greatest degradation of total petroleum hydro- carbons （TPH） was observed with the biostimulation using mixture of NPK, HS and NovoGro, a treatment scheme that enhanced both dehydrogenase and lipase activities in soil. Introduction of exogenous hydrocarbon-degrading bacteria （in addition to biostimulation with NPK, HS and NovoGro） had negligible effect on the removal of TPH, which was likely due to the competition between exogenous and autochthonous microorganisms. None- theless, the addition of exogenous yeast-bacteria consor- tium significantly enhanced the removal of the aromatic fraction of the petroleum hydrocarbons, thus detoxifying the soil. The effect of bioaugmentation on the removal of more recalcitrant petroleum hydrocarbon fraction was likely due to the synergistic effect of bacteria and fungi.     

石油烃类污染土壤的生物修复技术研究进展

介绍了治理石油烃类污染土壤的各种生物修复技术——原位处理法、异位处理法(现场处理法、预制床法、堆制处理法、生物反应器、厌氧生物处理法)及植物修复技术的原理、研究、应用的最新进展；分析了生物修复技术的影响因素及未来的发展趋势。     

Cytochrome P450 1A activity in liver and fixed wavelength fluorescence detection of polycyclic aromatic hydrocarbons in the bile of tongue-fish (Cynoglossus acrolepidotus, Bleeker) in relation to petroleum hydrocarbons in the eastern Gulf of Thailand.

This investigation was conducted in an area of oil spill along the east coast of Thailand to examine the relations among cytochrome P450 1A activity in liver and PAHs in the bile of the tonguefish and petroleum hydrocarbons in the sediments. PAH sediment concentrations in the reference and oil spill areas were 5.03 +/- 0.42 and 0.21 +/- 0.043 microg(-1) dry weight respectively Cytochrome activity in fish liver from oil spill area was 45.40 +/- 3.50 pmoles/ min/mg protein, almost threefold higher than that from the reference sites. Flourescense detection in bile metabolites at the oil spill area, 69.8 +/- 9.9 flourescense unit was significantly higher than that at the reference sites, 22.9 +/- 5.5 and 22.2 +/- 3.5 fluorescence unit. A strong correlation was found among cytochrome P450 1A activity in liver, PAH of bile metabolites and petroleum hydrocarbons. Both cytochrome and bile metabolites activity decreased seaward varying to the distance from the oil polluted area. We concluded that both detections in tonguefish can be regarded as a complementary biomarkers for the exposure of PAHs in tropical marine environments.     

Contaminants in Muscle of Plaice and Halibut Collected From the St. Lawrence Estuary and Northwest Atlantic

Levels of aromatic hydrocarbons in muscle or plaice and halibut were determined by fluorescence, using the chrysene standard, as recommended by the International Oceanographic Commision, for the analysis of PAH in environmental extracts. Concentrations were highest in muscle of halibut collected at the most contaminated, nearshore site, in the Saguenay Fjord of the St. Lawrence Estuary, compared to other locations further from shore. Although concentrations of fluorescing compounds were not statistically different in plaice, the saturated hydrocarbons displayed unquestionably more biodegradation, with a decrease of n-alkanes and increase of branched aliphatics, at the less contaminated site. Synchronous fluorescence indicated the presence of benzenoid and biphenyl hydrocarbons in the extracted mixtures, while GC-MS-TIC analysis tentatively identified the presence of a series of benzenoid (alkyl benzenes), chlorinated (PCB and DDE), N (trialkylamines) and O (phenols) hydrocarbons. These anthropogenic compounds could derive from petroleum products, surfactants and common products used in industry and households. This study emphasizes the importance of a multispectroscopic approach when investigating complex environmental mixtures.     

Anaerobic phenanthrene biodegradation with four kinds of electron acceptors enriched from the same mixed inoculum and exploration of metabolic pathways

Anaerobic phenanthrene biodegradation enriched process was described in detail. The enriched bacterial communities were characterized under four redox conditions. The enriched archaeal communities were stated under high percentage conditions. Relatively intact pathways of anaerobic phenanthrene biodegradation were proposed. Polycyclic aromatic hydrocarbons (PAHs) are widespread and persistent contaminants worldwide, especially in environments devoid of molecular oxygen. For lack of molecular oxygen, researchers enhanced anaerobic zones PAHs biodegradation by adding sulfate, bicarbonate, nitrate, and iron. However, microbial community reports of them were limited, and information of metabolites was poor except two-ring PAH, naphthalene. Here, we reported on four phenanthrene-degrading enrichment cultures with sulfate, bicarbonate, nitrate, and iron as electron acceptors from the same initial inoculum. The high-to-low order of the anaerobic phenanthrene biodegradation rate was the nitrate-reducing conditions>sulfate-reducing conditions>methanogenic conditions>iron-reducing conditions. The dominant bacteria populations were Desulfobacteraceae, Anaerolinaceae, and Thermodesulfobiaceae under sulfate-reducing conditions; Moraxellaceae, Clostridiaceae, and Comamonadaceae under methanogenic conditions; Rhodobacteraceae, Planococcaceae, and Xanthomonadaceae under nitrate-reducing conditions; and Geobacteraceae, Carnobacteriaceae, and Anaerolinaceae under iron-reducing conditions, respectively. Principal component analysis (PCA) indicated that bacteria populations of longtime enriched cultures with four electron acceptors all obtained significant changes from original inoculum, and bacterial communities were similar under nitrate-reducing and iron-reducing conditions. Archaea accounted for a high percentage under iron-reducing and methanogenic conditions, and Methanosarcinaceae and Methanobacteriaceae, as well as Methanobacteriaceae, were the dominant archaea populations under iron-reducing and methanogenic conditions. The key steps of phenanthrene biodegradation under four reducing conditions were carboxylation, further ring system reduction, and ring cleavage.     

Isolation of petroleum and chlorinated hydrocarbons from the same extract

Sequential analysis is an extremely powerful technique for complex samples analysis. For determination of petroleum and chlorinated hydrocarbons, the same sample is divided into two parts. One part is utilized to extract the petroleum hydrocarbons and the second part is used to extract the organochlorine compounds because petroleum hydrocarbons usually appear in higher concentrations and interfering with organochlorine compounds. This analysis method required more solvent, time, and more sample volume. The analytical strategy, in this paper, involves the use of effective and efficient but simple techniques for petroleum hydrocarbons and chlorinated hydrocarbons separation. A clean‐up procedure is presented, by which these compounds are isolated from aliphatic as well as polyaromatic hydrocarbons through a combination of HPLC and adsorption chromatography on a florisil microcolumn. Mass spectrometric identification was used as confirmation method. GLC equipped with ECD and FID for organochlorine and petroleum compounds analysis, respectively.