石油污染土壤生物修复专题共52页文档
石油污染土壤的微生物修复技术 综述
石油污染土壤的微生物修复技术摘要：石油是现代工业的重要能源，但各种泄露事故造成越来越严重的环境污染，并且石油对土壤的污染有着与独特的特征。
解决土壤石油污染的方法有物理修复、化学修复和生物修复，其中石油污染土壤的微生物修复技术以其高效、环保等优点备受关注。
选择具有高效降解能力的微生物是治理石油污染的关键环节，目前主要集中在土著微生物的分离筛选、构建高效降解能力的微生物菌群、以及构建基因工程菌。
生物强化技术是通过接种高效降解菌株(群) 或增加营养盐、表面活性剂等方式提高微生物代谢活动以去除污染物的过程。
石油污染土壤的微生物修复技术已经被用于工程实践，按修复的地点又可分为原位生物修复和异位生物修复。
本文在最后对石油污染土壤的微生物修复技术进行了展望。
关键词：石油污染土壤，微生物，修复技术Bioremediation Technology of Soil Contaminatedby PetroleumAbstract：Oil is the important modern industrial energy, but a variety of leak caused serious environmental pollution, and oil pollution of the soil has its’unique characteristics. To solve the oil contamination in soil, physical repair、chemical remediation and bioremediation have applied, microbial remediation of petroleum contaminated soil with its high efficiency、environmental protection and other advantages of concern. Effectively degrade microorganisms are the key aspects of the treatment of oil pollution, mainly concentrated in the indigenous microbial screening, constructing of microorganism flora with high efficiency degradation, and constructing genetic engineering bacteria. Bioaugmentation technology is efficient degradation by vaccination strains (group) or to increase the nutrient, a surface active agent to improve the microbial metabolic activity to remove pollutants in the process. Microbial remediation of petroleum contaminated soil has been used in engineering practice, according to the location of the repair, it can be divided into in situ bioremediation and ectopic bioremediation. Article in last prospect microbial remediation of petroleum contaminated soil.Key words: Soil contaminated by petroleum，Microorganism ，remediation technology一、石油污染土壤特征1.1土壤污染土壤污染是指人类活动所产生的物质(污染物)，通过多种途径进入土壤生态系统，其数量和速度超过了土壤容纳的能力和土壤净化速度的现象。
石油污染土壤生物修复专题
2.2.1 处 理 工 艺
原位处理工艺: 原位处理工艺:
(1)工艺路线和处理过程相对简单 , 不需要复 工艺路线和处理过程相对简单, 工艺路线和处理过程相对简单 杂的设备; 杂的设备; (2)处理费用较低; 处理费用较低; 处理费用较低 (3) 被处理土壤不需要搬运,对周围环境影响 被处理土壤不需要搬运, 生态风险小. 小,生态风险小.
3.2 ISB的前提条件 的前提条件
(In situ Bioremediation of the Soil Contaminated by Petroleum Hydrocarbons)
籍国东
前
言
当今世界,石油已成为人类最主要的能源之一, 当今世界 石油已成为人类最主要的能源之一, 石油已成为人类最主要的能源之一 大量的石油及其加工品进入土壤, 大量的石油及其加工品进入土壤,造成土壤的石 油污染,给人类乃至整个生物圈带来危害, 油污染,给人类乃至整个生物圈带来危害,从而 成为世界性的环境问题. 成为世界性的环境问题.在对石油污染治理的研 究中,生物修复作为一项潜在的高效, 究中,生物修复作为一项潜在的高效,低成本的 清洁技术正日益受到重视.近十余年来, 清洁技术正日益受到重视.近十余年来,国际上 对生物修复的研究更加活跃, 对生物修复的研究更加活跃,有些国家已将此技 术用于污染土壤的实际修复, 术用于污染土壤的实际修复,并取得了显著的成 果.
原位处理简单,经济,但处理时间长. 原位处理简单,经济,但处理时间长.在 长期的处理过程中, 长期的处理过程中 , 污染物可能会扩散到 深层土壤和地下水中, 深层土壤和地下水中 , 因而该技术适用的 对象为被污染时间较长且情况已经基本稳 定的土壤或面积广阔的区域. 定的土壤或面积广阔的区域.
生物方法修复被石油污染的土壤
环境工程微生物报告（设计）题目：生物修复石油污染土壤院系：专业：姓名：指导教师：完成日期：学号：介绍了国内外在修复石油污染土壤方面的生物修复工艺：原位生物修复和异位生物修复。
通过筛选高效降解菌治理受石油污染的土壤，并通过实验确定其降解特性。
指出生物修复方法作为一种费用低、效果好、对环境影响低、无二次污染的方法，是今后治理石油对土壤污染的最可行的方法。
关键词：石油污染；土壤；生物修复；降解菌石油是非常宝贵的燃料、润滑剂和化工原料。
与煤相比，它具有价格低廉，常温下为液体而易于开采、运输、使用，含杂质少、污染低等优点，因而得到广泛的应用。
世界发达国家在能源消费中，石油均占60%以上，而中国大约为20% 。
石油在开采、炼制、贮运、使用等过程中，原油和各种石油制品进入环境而造成污染，这已成为一种普遍的现象，并在生态环境和社会环境方面造成一定的影响。
据估计全世界每年约有1×109 t石油及其产品通过各种途径进入地下水、地表水及土壤中，其中我国有60多万t。
据2007年掌握的资料，我国部分石油化工区土壤残油高达10000 mg/kg，是临界值(200 mg/kg)的50多倍，每年新污染土壤1×108 kg。
石油烃类大量溢出，应当尽可能予以回收，但有的情况下回收很困难，即使尽力回收，仍会残留一部分，对环境（土壤、地面和地下水）造成污染。
其进入土壤后，会破坏土壤结构，土壤中的石油侵蚀土层，使之盐碱化、沥青化、板结化，使土壤的透水性降低、透水量下降，影响植物生长，被污染的土壤还可对地表水和地下含水层造成二次污染；许多研究表明，一些石油烃类成分在粮食中积累，影响粮食的品质，并通过食物链，进入动物体内，对哺乳类动物及人类有致癌、致畸、致突变的“三致”作用，被荷兰Ir F A M DeHan及奥地利W M Stidiani等科学家比喻为“化学定时炸弹(Chemical Time．Bomb)”。
因此，维持土壤的安全性至关重要，已成为目前国际上环境科学研究的热点之一。
生物修复石油污染土壤-丁克强 骆永明
2001年第4期土壤・179・生物修复石油污染土壤1丁克强骆永明（中国科学院南京土壤研究所南京210008）摘要本文概述了石油污染土壤的生物降解机制，分析了生物修复土壤污染技术及其影响因子，提出了强化生物修复的措施及其在我国的应用前景。
关键词生物修复；石油污染土壤石油污染土壤的生物修复[1]，是指利用微生物及其他生物，将存在于土壤的石油污染物降解成二氧化碳和水或转化成为无害物质的工程技术系统。
生物修复技术是传统的生物处理方法的发展，与物理、化学修复污染土壤技术相比，具有多种优点[2]：①成本低于热处理及物理化学方法；②不破坏植物生长所需的土壤环境；③污染物氧化完全，没有二次污染；④处理效果好，对低分子量的污染物去除率非常高；⑤可原位处理，操作简单。
本文结合近年国内外的最新研究成果，对生物修复技术在处理石油污染土壤的研究状况进行综述。
１ 石油烃生物学降解的机理 １．１ 微生物对烃的吸收 Bringman[3]等认为，分解烃微生物是吸附在土粒上的。
它们和烃液滴的相互作用是在土壤孔隙里的油水界面上进行的。
有的烃能直接溶于细胞膜的亲脂区而进入膜内；另一些则是微生物在油滴界面处先生成表面活性剂使之溶解后吸收，气态烃要溶于水后才能被吸收[4]。
１．２ 烷、烯、炔烃的生物降解烷烃降解的生化机理是β-氧化和充氧作用[5]。
研究最多的正烷烃的降解途径如图1。
在绝大多数情况下，正烷烃的生物降解最初是由与甲烷一氧化酶类似的复杂的一氧化酶系统酶促进行的。
在此过程中，烷烃氧化成相应的伯醇[6]。
后者在β-氧化酶、丁基脱氧酶和硫酸酯酶的作用下，经由醛而转化成羧酸。
Mckenna等人[7]认为，羧酸很容易通过β-氧化，降解成少两个碳链长度的乙酰基CoA。
后者再进入三羧循环，分解成CO2和H2O，并释放出能量；或再进入其它生化过程。
关于烷烃降解过程中链烯是不是中间产物的问题仍存在着争论[8]。
Pareck[9]等人发现，正十六碳烷嫌气细菌能将十六烷转化成相应的醇和烯。
石油污染土壤的生物修复技术综述
石油污染土壤的生物修复技术综述石油是现代社会不可或缺的能源之一，然而石油开采、运输和使用不可避免地导致了大量的环境污染，尤其是土壤污染。
石油污染土壤的修复是环境保护的重要课题之一。
传统的物理和化学方法虽然能够实现石油的去除，但往往会对土壤质量造成更大的破坏，并且费时费力。
相比之下，生物修复技术因其高效、经济、环保等优势，逐渐成为石油污染土壤修复的热门方法。
生物修复技术基于微生物的能力和活性，通过利用微生物来分解、转化和去除石油污染物，从而恢复土壤的生态功能。
根据微生物修复的作用机制，可以将生物修复技术分为生物降解、生物吸附和生物转化三类。
生物降解是指利用微生物产生的酶类和代谢能力，将石油污染物降解为无害的物质。
微生物包括细菌、真菌和芽孢杆菌等。
细菌在石油污染土壤的降解中起到了关键作用，如石油降解菌属于常见的生物降解细菌，可以利用石油为主要碳源进行生长繁殖，并产生一系列酶类来分解石油中的各种有机化合物。
真菌通过产生分泌物和菌丝的吸附作用，能够有效地将土壤中的石油污染物吸附并降解掉。
生物吸附是指微生物通过胞外多糖、菌丝纤维等结构，将石油污染物吸附在其表面，从而实现石油的去除。
吸附材料主要包括生物胶体、细菌菌体和藻类等。
以微藻为材料的生物吸附剂具有较大的比表面积和吸附能力，可以吸附大量的石油污染物。
利用生物祖细胞也可以提取到胞外多糖，具有良好的吸附性能。
生物转化是指利用微生物的能力将石油污染物转化为无害的物质。
这种转化过程主要是通过微生物降解产生的代谢产物进行的。
石油污染土壤中的石油烃类可以被细菌降解成酸类、醇类和酶类等物质，这些物质一方面可以被其他微生物利用，进一步降解石油污染物；这些物质本身也具有一定的环境容忍度和无毒性。
还有一些微生物可以利用石油污染物作为能源和碳源，将其转化为生物质等有用物质。
石油污染土壤的生物修复技术主要包括生物降解、生物吸附和生物转化三类。
这些技术在实际应用中不仅具有高效、经济和环保的特点，还能够有效恢复土壤的生态功能，降低土壤污染的风险。
石油污染生物修复
• 异位生物修复
将受污染的土壤，搬动或输送到它处进行的生物修复 处理，这里的搬动和输送是有一定限度的，而且更强调人 为控制和创造更加优化的降解环境。
石油污染土壤的原位生物修复
原位处理简单，经济，但处理时间 长。在长期的处理过程中，污染物可能 会扩散到深层土壤和地下水中，因而该
技术适用的对象为被污染时间较长且情
土壤石油污染来源
• • • • • 溢油和泄漏； 污水灌溉； 油页岩矿渣堆放； 大气沉降； 药剂等的施用。
石油污染土壤的影响危害
• • • • 对土壤理化性质的影响； 对土壤微生物的影响； 对土壤酶的影响； 对农作物的影响。
土壤石油污染生物修复的主要类型
• 原位生物修复
受污染的土壤不做搬运或输送而在原位污染地进行的 生物修复处理，其恢复过程主要依赖于被污染地自身微生 物的自然降解能力和人为创造的合适降解条件。
ห้องสมุดไป่ตู้
阿尔伯塔省的焦油砂矿面积达14.1万平方公里，石油储量位居全球第 三，20%的焦油砂便可提炼出1740亿桶石油。 虽然造成可怕的环境破坏，但面对巨大的经济利益，加拿大政府很难 对开采焦油砂说“不”。据估计，2010年至2035年的焦油砂开采有望每年 为加拿大的GDP贡献2.3万亿加元，同时提供63万个就业岗位。
况已经基本稳定的土壤或面积广阔的区 域。
原位生物修复的特点
(1)工艺路线和处理过程相对简单，不 需要复杂的设备；
(2)处理费用较低；
(3)被处理土壤不需要搬运，对周围环
境影响小，生态风险小。
原位生物修复的前提条件
（1） 存在具有代谢活性的微生物； （2） 有较大的降解速率，并能将污染物浓度降低到 符合环保标准； （3） 过程中生成的中间产物必须没有毒性； （4） 污染场地必须不含对降解菌种有抑制作用的物质； （5） 污染物必须能够被微生物利用； （6）污染场地的条件必须有利于微生物生长并保持活性； （7） 技术费用必须尽可能低。
石油土壤污染的生物修复技术
石油土壤污染的生物修复技术当今世界,石油已成为人类最主要的能源之一，大量的石油及其加工品进入土壤，造成土壤的石油污染，给人类乃至整个生物圈带来危害，从而成为世界性的环境问题。
在对石油污染治理的研究中，生物修复作为一项潜在的高效、低成本的清洁技术正日益受到重视。
近十余年来，国际上对生物修复的研究更加活跃，有些国家已将此技术用于污染土壤的实际修复，并取得了显著的成果。
石油污染是指石油开采、运输、装卸、加工和使用过程中，由于泄漏和排放石油引起的污染，主要发生在海洋。
石油漂浮在海面上，迅速扩散形成油膜，可通过扩散、蒸发、溶解、乳化、光降解以及生物降解和吸收等进行迁移、转化。
油类可沾附在鱼鳃上，使鱼窒息，抑制水鸟产卵和孵化，破坏其羽毛的不透水性，降低水产品质量。
油膜形成可阻碍水体的复氧作用，影响海洋浮游生物生长，破坏海洋生态平衡，此外还可破坏海滨风景，影响海滨美学价值。
石油污染防治，除控制污染源，防止意外事故发生外，可通过围油栏、吸收材料、消油剂等进行处理。
石油对环境的污染可分为两个方面：一是油气污染大气环境，表现为油气挥发物与其它有害气体被太阳紫外线照射后，发生物理化学反应，生成光化学烟雾，产生致癌物和温室效应，破坏臭氧层等。
二是地下油罐和输油管线腐蚀渗漏污染土壤和地下水源，不仅造成土壤盐碱化、毒化，导致土壤破坏和废毁，而且其有毒物能通过农作物尤其是地下水进入食物链系统，最终直接危害人类。
本文主要介绍石油土壤污染的生物修复技术。
石油污染土壤的影响危害有：（1）对土壤理化性质的影响；(2)对土壤微生物的影响；（3）对土壤酶的影响;(4)对农作物的影响。
20世纪80年代以前．治理石油烃污染土壤还仅限于物理和化学方法，即热处理和化学浸出法。
热处理法是通过焚烧或煅烧，可净化土壤中大部分有机污染物。
但同时亦破坏土壤结构和组分，且价格昂贵而很难实施。
化学浸出和水洗也可以获得较好的除油效果。
但所用的化学试剂的二次污染问题限制了其应用。
石油污染土壤的植物微生物联合修复完整版
石油污染土壤的植物微生物联合修复HEN system office room 【HEN16H-HENS2AHENS8Q8-HENH1688】石油污染土壤的植物-微生物联合修复摘要: 由于土壤石油污染的日益严重, 其治理技术受到了世界各国的普遍关注, 而生物修复技术具有无二次污染、高效、费用较低等特点,是最具应用前景的土壤修复技术之一。
本文着重介绍植物-微生物联合修复技术的研究进展, 并对该领域今后的研究重点进行了展望。
关键词: 石油污染土壤, 生物修复, 植物-微生物联合修复近年来, 随着石油的勘探开发和区域经济不断发展, 我国石油污染问题日益凸显, 土壤污染日趋严重。
在石油的大规模开采、冶炼、运输、使用和处理过程中, 不可避免地造成石油废弃物对土壤的污染, 这些污染物从各个层面上威胁着生态环境、食品安全和人类健康: 引起土壤理化性质的变化, 导致土壤微生物群落、微生物区系的变化, 进而破坏土壤生态系统; 影响植物生长发育, 导致粮食减产, 长期食用生长于农业土地上的植物及其产品还会影响人类的健康; 土壤吸附的石油会随着雨水、灌溉用水慢慢下渗到地下含水层中, 从而污染地下水, 进而构成对人类生存的不良胁迫。
因此, 石油污染土壤的治理势在必行。
1 石油污染土壤的修复技术石油污染土壤的修复技术主要有物理法、化学法和生物法3 种方法, 物理法和化学法虽可以产生一定实效, 但因存在消极处置、费用昂贵或者二次污染严重等明显缺陷, 现在一般仅作为生物治理方法的辅助手段[ 1] ; 而生物法修复以其处理费用低、效果好又可避免二次污染等优势逐渐被人们所重视。
2 石油污染土壤的生物修复技术生物修复( bioremediat io n) 是利用生物对环境污染物的吸收、代谢、降解等功能, 在环境中对污染物的降解起催化的作用, 即加速去除环境污染物的过程, 可以消除或减弱环境污染物的毒性, 可以减少污染物对人类的健康和生态系统的风险[ 2]。
原油污染土壤的生物修复
ORIGINAL PAPERBiodegradation of petroleum sludge and petroleum polluted soil by a bacterial consortium:a laboratory studyG.D.Gojgic-Cvijovic •ic •T.M.Solevic •V.P.Beskoski •M.V.Ilic •L.S.Djokic •T.M.Narancic •M.M.VrvicReceived:23June 2010/Accepted:6May 2011/Published online:22May 2011ÓSpringer Science+Business Media B.V.2011Abstract This article presents a study of the efficiency and degradation pattern of samples of petroleum sludge and polluted sandy soil from an oil refinery.A bacterial consortium,consisting of strains from the genera Pseudomonas ,Achromobacter ,Bacillus and Micromonospora ,was isolated from a petroleum sludge sample and characterized.The addition of nitrogen and phosphorus nutrients and a chemical surfactant to both the samples and bioaug-mentation to the soil sample were applied under laboratory conditions.The extent of biodegradation was monitored by the gravimetric method and analysis of the residual oil by gas chromatography.Over a 12-week experiment,the achieved degree of TPH (total petroleum hydrocarbon)degradation amounted to 82–88%in the petroleum sludge and 86–91%in the polluted soil.Gas chromatography–mass spectrometry was utilized to determine the biodegradability and degradation rates of n -alkanes,isoprenoids,steranes,diasteranes and -plete degradation of the n -alkanes and isoprenoids fractions occurred in both the samples.In addition,the intensities of the peaks corresponding to tricyclic terpenes and homohopanes were decreased,while significant changes were also observed in the distri-bution of diasteranes and steranes.Keywords Isoprenoids ÁMixed culture Án -alkanes ÁPetroleum sludge ÁSteranes ÁSurfactant ÁTerpanesIntroductionPetroleum production,refining,transportation and use contribute highly to environmental pollution.The contamination of soil with petroleum hydrocarbons causes a significant decline in its quality and such soils become unusable.In order to control the environmental risks caused by petroleum products,bioremediation as an environmentally friendly tech-nology has been established and applied,especially biostimulation and bioaugmentation of the easily to be degraded petroleum hydrocarbons (e.g.,alkanes).Electronic supplementary material The online version of this article (doi:10.1007/s10532-011-9481-1)containssupplementary material,which is available to authorized users.G.D.Gojgic-Cvijovic (&)Áic ÁT.M.Solevic ÁV.P.Beskoski ÁM.V.IlicDepartment of Chemistry,Institute of Chemistry,Technology and Metallurgy,University of Belgrade,Njegoseva 12,P.O.Box 473,Belgrade,Serbia e-mail:ggojgic@chem.bg.ac.rsL.S.Djokic ÁT.M.NarancicInstitute of Molecular Genetics and Genetic Engineering,University of Belgrade,Vojvode Stepe 444a,P.O.Box 23,Belgrade,SerbiaM.M.VrvicFaculty of Chemistry,University of Belgrade,Studentski Trg 12-16,Belgrade,SerbiaBiodegradation (2012)23:1–14DOI 10.1007/s10532-011-9481-1The primary mechanism for the elimination of hydrocarbons from contaminated sites is biodegrada-tion by natural populations of microorganisms.To maximize the process in bioremediation technologies, two main approaches have been explored:biostimu-lation,in which nutrients are added to stimulate the intrinsic hydrocarbon degraders,and bioaugmenta-tion,in which microbial strains with specific degrad-ing abilities are added to work cooperatively with normal indigenous soil microorganisms(Alvarez and Illman2006).Petroleum and petroleum products are complex mixtures consisting of thousands of compounds that are usually grouped into four fractions:aliphatics,aro-matics,nitrogen–oxygen–sulphur(NSO)compounds and asphaltenes.Asphaltenes are generally solvent-insoluble and resistant to biodegradation.Aliphatic hydrocarbons consist of normal alkanes(n-alkanes), branched alkanes(isoalkanes)and cyclic alkanes(naph-thenes).Isoalkanes,naphthenes and aromatics are much less biodegradable than n-alkanes.According to Perry (1984),the susceptibility of hydrocarbons to micro-bial attack is ranked in the following order:n-alkanes [isoalkanes[low molecular weight aromatics[ naphthenes.A large number of microorganisms(bacteria,fungi and some algae)that are capable of using petroleum hydrocarbons as the sole source of carbon and energy have been described(Das and Chandran2011).It is generally accepted that a single microorganism is not capable of degrading all compounds from such mixtures.Mixed cultures not only have broad substrate specificity but also degradation could be achieved in a system of cooxidation and commen-salism.Although there have been many reports on crude oil biodegradation,only a small number of the studies focused on the decomposition of heavy petroleum products or waste.Similarly,the number of strains degrading branched chain(Namio et al.2005)and n-alkanes with more than30C atoms is limited (Wentzel et al.2007)in comparison with the number of light n-alkane degraders.In recent years,the study of the ability of defined microbial consortium/mixed cultures to degrade hydrocarbons of petroleum products or waste,such as petroleum sludge(PS)(Rahman et al.2003; Vasudevan and Rajaram2001),lubricating oil(Wongsa et al.2004),turbine oil(Ito et al.2008)mashine oil (Zvyagintseva et al.2001)or engine base oil(Koma et al.2003)has become of special interest.The purpose of the present study was to isolate and characterize a mixed culture capable of degrading PS and polluted sandy soil from an oil refinery,and to optimize the degradation process by enhancement of the consortium by nutrient and surfactant addition. Furthermore,the goal was to determine the extent of degradation by comparing specific fractions of hydrocarbons in the residual oil.Materials and methodsSubstrates for biodegradationThe samples are taken from the Oil Refinery Novi Sad,Serbia.Thefirst sample was PS from former storage fuel tank N8and the second sample was polluted soil(S)in the vicinity of the tank N8.Microbiological methodsMicrobial enumerationThe number of microorganisms was determined by the method of a serial dilution(10-2–10-10CFU ml-1) on agar plates.All determinations were conducted in triplicate.For bacteria nutrient agar was used,for yeasts and moulds malt agar with streptomycin (100mg l-1),and for hydrocarbon-degrading micro-organisms the mineral salts medium,MM(Lo¨ser et al. 1998)with2000ppm diesel fuel(DM).Isolation of microorganismsThe bacteria utilized for the preparation of consor-tium were isolated from sample PS.The medium consisted of mineral salt solution(NPK solution, 0.1%NH4NO3,0.025%K2HPO4)inoculated with PS sample(20%,w/v)and incubated during4weeks aerobically with shaking at200rpm at28°C.Every week a new portion of NPK solution was added to restore initial volume of solution and depleted nutrients.After4weeks,the cultures were spread onto DM agar plates with diesel fuel as the C source.Pure cultures were obtained from selected colonies by repeated inoculation on DM medium,nutrient agar,McConkey agar,Pseudomonas isolation agar and casein-starch agar.Identification of strainsThe identification of isolated strains was achieved by API tests(Biomeriex),fatty acid methyl ester (FAME)composition and by sequence analysis of 16S rRNA genes.The API50CH/E,API20NE and Coryne kits (Biomerieux)were utilized as described in the manufacturer’s instructions.Then,the data were interpreted with apiweb TM software.For FAME analysis,bacteria were cultivated on rotary shaker on tryptone soy broth(TSB).FAMEs were prepared according to the procedure described in MIDI Technical Note101(Haack et al.1994)and analysed by gas chromatography/mass spectrometry (GC–MS).Gas chromatograph/mass spectrometer system Ag-ilent7890A–5975C inert XL EI/CI was used.Column was HP-5ms(30m90.25mm90.25l m),gas helium and oven program:50°C for0min,then 4.3–285°C for5min.For FAME identification, standard bacterial acid methyl esters(BAME,Supe-lco)and NIST5a.L database were used.Analyses of16S rRNA gene sequencesThe genomic DNA of each bacterium was extracted as previously described(Hopwood et al.1985).The 16S rRNA genes were amplified by PCR using27F (50-AGAGTTTGATCMTGGCTCAG-30;Lane1991), and1492R(50-CGGCTACCTTGTTACGACTT-30; Liu et al.2007)primers and amplified fragments were sequenced using Applied Biosystems3130 genetic analyser(Foster City,USA).Taxonomic analysis was conducted by the GenBank basic local alignment search tool(BLAST)program,and16S rRNA gene sequences of analysed strains were deposited in NCBI GenBank under accession num-bers JF826526-30.Specific characterization of strainsThe growth of isolated strains on different hydrocar-bons as the sole C source was tested on MM with 200ppm of substrate in test tubes on rotary shakers incubated at28°C for15days.The tubes(10ml)were inoculated with100l l suspension(106CFU ml-1)of tested strain.After15days,growth was monitored by the increase in CFU ml-1in the nutrient agar plates.The tolerance to the presence of heavy metals was assayed by testing growth on Mueller–Hinton agar supplemented with increasing concentration of CuSO4Á5H2O,Cd(CH3COO)2Á2H2O,NiSO4Á6H2O and Cr(NO3)3Á9H2O by the method described by Nieto et al.(1989).Biodegradation experimentBiodegradation assays were carried out in500ml Erlenmeyerflasks containing100ml of MM and 10%(w/v)of samples PS or S.Pure cultures of isolated strains grown on TSB were centrifuged and suspended in physiological saline to106CFU ml-1.0.5ml of each culture was employed to inoculate the flasks containing S sample in bioaugmentation treat-ments.BioSolveÒ(Westford Chemical Corporation, USA)was added as surfactant(3%,v/v).All exper-iments were conducted in triplicate.The control flasks were prepared with the addition of2%HgCl2. Cultures were incubated on rotary shaker at200rpm at28°C.The duration of the experiment was3months. Every week,sterile water was added to theflasks to restore initial volume of solution.Content of phos-phate in fermentation broths was determined every week during experiment.If it was low,NPK solution was added.Specifically,NPK solution was added after1,2,3,4,6and8weeks to the PS sample,and after1,2,4,and6weeks to the S sample.Also, surfactant was added not only in the beginning but also after4and8weeks of study.The process was monitored by the determination of viable bacterial count,the phosphorus content in the culture broth,the pH and total petroleum hydrocarbon(TPH)content.Chemical methodsThe content of total petroleum hydrocarbons(TPH) in the samples was determined by DIN EN ISO method14345:2004.The total content of carbon and nitrogen was determined with Vario EL III CHNS/O Elemental Analyzer,hydrocarbon fractions were separated byWalker’s method(Walker et al.1975),and phospho-rus and carbonate contents in samples were deter-mined according to the recommended methods (Rump1999).The contents of Fe,Ni and V were determined by inductively coupled plasma-atomic emission spectroscopy(ICP-AES)using iCAP6500 Duo according to EPA method3051(1995).ChromatographyThe GC analysis of TPH samples was performed on the instrument Agilent4890D with FID detector.The following protocol was used:Column was HP-1MS (30m90.25mm);carrier hydrogen,injector tempera-ture250°C;initial temperature40°C for9min;followed by4°C min-1from40to65°C;9°C min-1from65to 286°C;and isothermal at285°C during12min.Polycyclic alkanes of sterane and triterpane types in THP samples were analysed by GC–MS.A Agilent 6890gas chromatograph,fitted with HP-5MS column (30m90.25mm90.25l m;temperature was lin-early programmed from60°to285°at3°C min-1),-was coupled to a Agilent5975B mass spectrometer with electron ionization.Helium was used as the carrier gas(flow rate,l ml min-1).n-Alkanes and isoprenoids were identified from m/z71,steranes and diasteranes from m/z217and terpanes from m/z191 ion chromatograms obtained from analysis in single ion monitoring mode(SIM).The most relevant peaks have been identified based on the organic geochem-ical literature data(Peters et al.2005)or on the basis of total mass spectra using data base NIST5a.L. Mineralogical analysisThe minerals in the samples were detected and determined semiquantitatively by roentgen diffrac-tion analysis powder technique using X-ray diffrac-tometer Philips(The Netherlands)type PW1050/00 (CuK a1Ni-filtered radiation;preparations—native, with glycerol and ignited).The results were inter-preted using Material Phases Data System program and ASTM Joint Committed on Powder Diffraction Standards cardfiles.Statistical analysisIn order to evaluate the effects of surfactant addition and bioaugmentation on the biodegradation of the samples PS and S,a two-way analysis of variance (ANOVA)with repeated measurements was per-formed for each sample.Two factors included:(1)the addition of surfactant for the S sample bioaugmen-tation or their combination,and(2)time intervals(4, 8or12weeks)for both measured parameters(degree of TPH degradation and number of microorganisms). Additional tests(post hoc tests for the S sample and t test for the PS sample)were employed tofind statistically significant differences between the par-ticular treatments.The analyses were performed using the software SPSS11.5program.The normality of distribution was checked using Kolmogorov–Smirnov test.The tests were considered statistically significant if P\0.05.ResultsThe substrates used in this article were selected in the preliminary experiments during the testing of the bioremediation potential of different samples with high hydrocarbon content originating from the refin-ery.With biostimulation and aeration,in one group of samples(PS),the degradation rate was satisfactory with a large number of microorganisms over several weeks of experiment,whereas in the second group of samples(S)the biodegradation was quickly stopped due to a drastic drop of the number of microorgan-isms(approximately to102CFU ml-1).Since bio-availability may represent a limiting factor for biodegradation,it had to be determined whether the key cause was insufficient solubility of the hydrocar-bon substrate,which could be resolved by means of adding surfactants,or a lack of microorganisms with specific degradation activities.Chemical,microbiological and mineralogical characteristics of samplesTPH content of sample PS was412.5g kg-1(54% saturated hydrocarbons,27%aromatic hydrocarbons and21%NSO and asphaltens).TPH content of sample S was103.2g kg-1(60%saturated hydro-carbons,20%aromatic hydrocarbons,20%NSO and asphaltenes).Other chemical and microbiological characteristics of the samples employed in the experiments are presented in Table1.On the basis of X-ray diffraction analysis,the following minerals were present(in the order of decreasing abundance):in PS quartz,feldspar and clay minerals(illite),calcite and dolomite,and for sample S—quartz and dolomite in equal quantities,and then as minor components—calcite,cristobalite,feldspar, reflections of zeolite,gypsum,pyrolusite and clay minerals(mica,illite,chlorite and montmorillonite).Although both samples used for biodegradation contain high concentrations of hydrocarbon([10%), there are significant differences between them in the type of matrix.According to the mineralogical composition,the S sample belongs to sandy soils. The N and P content is low in both the samples, hence,the addition of N and P nutrients is a basic technique(Alvarez and Illman2006)for stimulating growth of autochthonous microorganisms.Microbiological analysis of the samples indicates the presence of bacteria and fungi as well as degraders of hydrocarbons in the samples,but in relatively small numbers.The cause may lie in a high concentration of contaminants,low water content in the PS sample and the matrix effect in the S sample. Isolation and taxonomic identificationof the bacterial strainsBacterial strains used to prepare a mixed culture-consortium were isolated as pure cultures from the PS sample.An inspection of colonies grown on nutrient agar and selective media and comparison of morpho-logical,physiological and biochemical characteristics determined thefive bacterial types as dominant.Two types are Gram positive sporogenous rods,including one beta-haemolytic,Voges Proskauer negative (labelled as NS026)and the other non-haemolytic, Voges Proskauer positive(NS032).The third type represents Gram positive reddish-orange colonies with branching mycelium(NS094).The others are Gram negative rods,oxidase positive,out of which one produces afluorescent pigment(NS009)and the other one does not(NS014).According to physiological-biochemical proper-ties(API tests),the strains were preliminary iden-tified as Bacillus cereus1(NS026),Bacillus licheniformis(NS032),Arthrobacter sp.(NS094), Pseudomonasfluorescens(NS009)and Achromo-bacter denitrificans(NS014).Thefinal identification was performed by sequence analysis of the16S ribosomal RNA gene.16S rDNA sequence similarity of the isolated strains with corresponding sequence of type strains in NCBI database was shown in Table2.For the strains identified by API tests as Bacilllus,Pseudomonas and Achromobacter confir-mations were obtained.Molecular characterization indicated that the strain labelled as NS0094belongs to the genus Micromonospora.The isolated bacteria were additionally character-ized by the determination of the fatty acid methyl ester profiles(Table3).The Gram negative strains show the presence of acids with cyclopropane structure and of12-hydroxy acid in the strain Pseudomonas.The branched chain fatty acids dom-inate in the Gram positive strains.Taxonomic position of the isolated strains is in accordance with thefinding from the previously published articles(Arvanitis et al.2008;Cameotra and Singh2008;Verma et al.2006;Vasudevan and Rajaram2001),in which Pseudomonas,Acinetobac-ter,Bacillus,Rhodococcus,Arthrobacter and Cory-nebacterium are the main hydrocarbon degraders isolated from the PS and waste.The consortium members grow on the majority of the selected hydrocarbons(Table4)as the sole source of carbon,and show tolerance to the heavy metals in the range 2.5–25mmol l-1(Table5), which indicates a broad capacity for the degradation and ability to survive.Table1Initial composition of samples used for bioremediationParameter(unit)PS SWater content,%419.3pH(1:2.5in H2O)7.97.7 CaCO3,% 2.78 4.25C total36.738.17N total0.38NDP2O5,g kg-10.0490.009 Loss on ignition,%53.8116.32 Fe,g kg-110.85 2.10V,mg kg-12911Ni,mg kg-16016 Bacteria,CFU g-17.091047.59104 Hydrocarbon degraders,CFU g-1 5.991037.39103 Fungi,CFU g-1 3.09104 6.89103Calculated on soil dry massND not determinedBiodegradationThe course of biodegradation in the S and PS samples is presented in Figs.1and 2.The effect of surfactant addition on both the samples as well as bioaugmen-tation on the S sample was studied.The process was followed by determining the number of bacteria in the suspension (Figs.1a,2a)as well as through the decrease of the TPH content (Figs.1b,2b).The experiment was conducted in triplicate,and statistical analysis of obtained data was performed.Statistically significant differences between treatment groups over time were found for the both samples and for both measured parameters:degree of degradation and number of microorganisms.Moreover,the significant differences observed changed over the time course of the study,which was a clear indication that there were interactions between factors included in the study.The results shown in Figs.1a and 2a clearly indicated that biostimulation (addition of NPK)in the first week of the experiment led to an abrupt increase of the number of microorganisms in both the samples.In the PS sample,due to its high content of organic substances (high C/N/P),the consumption of phos-phates was significantly higher,and during the first month a further portion of NPK had to be added weekly,which,albeit with fluctuations,led to the growth of a stable microbial population (Fig.1a).Over this time interval (4weeks),the addition of surfactants had no statistically significant effect on the number of bacteria in the suspension,but in contrast,the differences in the degree of degradationTable 2Taxonomic identification of the isolated bacterial strains StrainGene bankaccession numberSequence alignment Nearest phylogenetic neighbour (Gene bank accession number)No.ofnucleotides a Identity,%b NS009JF826528142899P.grimontii CFML 97-514(NR 025102)NS014JF826529131098 A.spanius LMG 5911(NR 025686)NS026JF826526143795 B.cereus JCM 2152(AB598737)NS032JF826527140299 B.licheniformis CICC 10101(AY859477)NS094JF826530131496M.aurantiaca DSM 43813(NR 026279)a The number of 16S rDNA nucleotides used for the alignment,attached as Supplementary material bThe percentage identity with the 16S rDNA sequence of the nearest phylogenetic neighbourTable 3Cellular fatty acid composition of isolated strains,%of total detected Fatty acid a NS026NS032NS094NS009NS014i 12:0ND b ND 0.55ND ND 12:0ND ND 0.40 6.840.47122OH ND ND ND 6.3ND 123OH ND ND ND 0.98ND i 13:09.15ND 5.70ND ND a 13:0ND ND 2.15ND ND 13:00.65ND ND ND ND i 14:0 3.61ND 1.82ND ND 14:0 3.620.29 2.630.25 2.44i 15:030.0529.3711.79ND ND a 15:09.5833.63 4.44ND ND 15:0ND ND 0.84ND ND i 16:0 6.56 3.18 5.62ND ND R 16:1c0.68ND 1.5411.0516.5216:026.289.4928.0746.0342.13cy 17:0ND ND ND 18.6133.61i 17:08.4515.328.51ND ND a 17:0 1.09ND 6.83ND ND 17:0NDND 1.77ND ND R 18:1cND ND 13.149.370.9718:00.270.58 3.970.57 2.8918:010methyl ND ND 0.24ND ND cy 19:0NDNDNDND0.36aFatty acids are designated in terms of the total number of carbon atoms:number of double bonds.The prefixes a and i indicate anteiso and iso branching,cy refers to cyclopropane fatty acids,OH indicated the presence of hydroxyl group b not determined,values \0.20%are omitted cR total sum of monounsaturated acidof the TPH were statistically significant(Fig.1b). Thus,in thefirst month,the TPH decreased from the initial412g kg-1to331(19.6%),and with the addition of surfactant even down to154g kg-1TPH (62.6%).The effect of surfactants on the growth of the microorganisms became visible in the period between week4and8of the experiment,probably not only because the obtained biomass had a lot of available organic substance at its disposal but also because the surfactant could serve as an additional C source. Therefore,the total number of bacteria in the suspension in week6attained4.69109CFU ml-1. After8weeks,the number of microorgan-isms dropped,but by that time70–80%of the hydrocarbons had already been degraded.At the8th and12th weeks of the study,statistically significant differences were found between mean values of number of microorganisms,but not in degradation efficiency.Compared to the PS,a lower maximum density of the biomass(*106–107CFU ml-1)in the S samples (Fig.2)was attained throughout the entire study because of the lower content and availability of the organic substance.It must be emphasised that the addition of surfactants greatly increased the avail-ability of the hydrocarbons,hence,over the course of the entire experiment,in the tests with the surfactant, the number of microorganisms was higher by almost two orders of magnitude,and consequently,the degradation rate increased as well.Therefore,in the number of microorganisms and the degradation efficiency,there were statistically significant differ-ences between the mean values of both paired treatments(without and with surfactant,treatments 1and2,3and4,Fig.2)in4th and8th week. However,at the12th week,statistically significant differences were found only between treatments1 and2and only in the degree of degradation.The addition of the bacterial consortium to the S sample helped to maintain the number of the viable bacterial population.After8weeks,similar to the PSTable4Growth of isolated strains on selected hydrocarbon as the sole C sourceBacillus sp. NS026Bacillussp.NS032Micromonosporasp.NS094Pseudomonassp.NS009Achromobactersp.NS014Diesel fuel?a???? n-Hexane????? n-Hexadecane????? n-Octadecane????? Benzene????? Toluene??–––Xylene–?–––Phenantrene–??––Pyrene????? Etylbenzene??–––Octylbenzene????? Hexadecylbenzene????? Sodium-benzoate???–? 2-Phenylphenol????–Dibenzothiophene????? a?viable growth observed in1910-5dilutionTable5Tolerance to metal ions,mmol l-1Ni2?Cu2?Cr3?Cd2?Bacillus sp.NS02625 2.525 1.25Bacillus sp.NS0325 2.5 2.5 1.25Micromonospora sp.NS0945 2.55 2.5Pseudomonas sp.NS009525525Achromobacter sp.NS01412.512.5 2.512.5Numbers in table represent maximum concentration of metalsalts in Mueller–Hinton plates where visible growth isobservedsample,the number of microorganisms decreased.However,the addition of the bacterial consortium had no effect on bacterial numbers at this time,irrespec-tive of the surfactant addition.The reduced number of microorganisms after 8weeks in all treatments may be the result not only of decreased availability of any remaining hydrocarbons but also the accumulation of toxic metabolites.As indicated previously,for the S sample,the main goal was to study the effects of bioaugmentation alone and in combination with the added surfactant on the degree of degradation.Biostimulation and biostimulation with added surfactant (treatments 1,2)finally achieved hydrocarbon degradation of 40–45%and 50–60%,respectively (Fig.2b).On the otherhand,bioaugmentation (repeated inoculation with consortium biomass,treatments 3and 4)led to continual decrease in TPH values,which finally reached much lower values of 86.4–91.3%of the starting levels.In degradation efficiency,bioaugmentation with surfactant addition (treatment 4,Fig.2b)was found to be statistically significantly different at alltimesFig.1Degradation of the PS sample a Growth of bacteria treatment:biostimulation (filled square ),biostimulation ?sur-factant (shaded square ),Values are the means from three replicates,error bars represent minimum and maximum values (n =3)Statistically significant differences,P \0.05,between treatments at the 8th and 12th week were found.b Reduction of TPH content treatment:biostimulation (filled square ),biosti-mulation ?surfactant (shaded square ).Bars represent mean values ±standard deviation (n =3)Statistically significant differences,P \0.05,between treatments at the 4th week werefoundFig.2Degradation of the S sample a Growth of bacteria treatment: 1.biostimulation (filled triangle ), 2.biostimula-tion ?surfactant addition (open triangle ), 3.biostimula-tion ?bioaugmentation (filled square ) 4.biostimulation ?bioaugmentation ?surfactant (open square )Values are the means from three replicates,error bars represent minimum and maximum values (n =3)The arrows point to the addition of the bacterial consortium.At the 4th week,statistically significant differences between treatments 1and 3,2and 4were not found (P [0.05).At the 12th week,statistically significant differences between all treatments were not found (P [0.05).b Reduction of TPH content treatments:1.biostimulation (filled square ),2.biostimulation ?surfactant (shaded square ),3.biostimulation and bioaugmentation (cross line square )4.biostimulation,bioaugmentation ?surfactant (horizontal line square )Bars represent mean values ±stan-dard deviation (n =3)At the 4th week,statistically significant differences between treatments 1and 3,2and 3were not found (P [0.05).At the 8th week,statistically significant differences between treatments 2and 3were not found (P [0.05).At the 12th week,statistically significant differences between treat-ments 3and 4were not found (P [0.05)。
石油污染土壤的生态风险评价和生物修复
1、不同地区、不同类型石油污染土壤的生态效应存在差异，如何根据实际 情况制定更为有效的治理策略仍需深入研究。
2、尽管微生物修复、植物修复和动物修复等方法在实验条件下显示出一定 效果，但实际应用中仍面临许多挑战，如治理效率、稳定性等问题需要进一步解 决。
3、生物修复过程中，不同生物体系之间的相互作用机制尚不明确，如何充 分发挥各生物体系的优势，提高整体治理效果需要进一步探讨。
然而，针对石油污染土壤的生态风险评价和生物修复还存在一定的挑战。例 如，评价标准不统一、监测指标不完善、修复技术成本较高等。因此，未来需要 加强以下方面研究：1）制定更为严格的评价标准和技术规范；2）发掘高效降解 石油污染物的微生物种类及其作用机制；3）结合新兴技术手段（如基因编辑 等），优化生物修复技术；4）
一、石油污染土壤背景
石油作为一种重要的能源物质，在全球范围内被广泛使用。然而，石油的生 产、运输和加工过程中，可能会发生泄漏、事故等，导致石油及其衍生物进入土 壤，形成石油污染。石油污染土壤会影响土壤功能，对植物、动物和人类健康产 生负面影响。因此，研究石油污染土壤的生态效应及生物修复具有重要的现实意 义。
2、深入探讨微生物修复、植物修复和动物修复等方法的实际应用效果和局 限性，优化治理方案以提高治理效率和稳定性。
3、研究各生物体系在生物修复过程中的相互作用机制，充分发挥各生物体 系的优势，提升整体治理效果。
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二、石油污染土壤的生态效应
1、土壤生物多样性减少
石油污染对土壤生物多样性产生显著影响。大量研究表明，石油污染会导致 土壤中微生物、昆虫、植物等物种数量减少，生物多样性降低。这些生物的减少 会进一步影响土壤生态系统的稳定性和功能。
2、土壤质量下降
石油污染会改变土壤理化性质，如土壤有机质、氮、磷等元素的含量，导致 土壤质量下降。此外，石油中的有害物质还会影响土壤酶的活性，抑制土壤呼吸， 破坏土壤结构，使土壤变得干旱、硬结，不利于植物生长。