摘要
本文以筛选含氮杂环化合物高效降解菌并应用于石油污染土壤的生物修复为目标,进行了含氮杂环化合物吡啶、喹啉降解菌的筛选实验,对所筛高效降解菌进行环境优化实验,自然富集培养物的驯化和人工构建混合菌群,利用固定化技术构建微生物反应器,最终将微生物反应器应用于石油污染土壤的生物修复实验。
通过大量实验,最终筛选出4株吡啶和喹啉高效降解菌,进行了形态特征、生理生化特征和DNA鉴定。对影响菌株生长和降解的主要因素,如pH值、温度、转速、接种量、碳氮源利用性、底物浓度等进行单因素、正交实验考察。自然富集菌群对吡啶和喹啉的降解效果和单菌株相当,且驯化时间短,操作更为简单。人工构建的优势菌群结构简单,降解效果不理想。
采用固定化技术强化菌株降解效果和提高菌株对环境的适应性。对所选载体材料,及相应的包埋剂、交联剂种类浓度、pH值和交联时间等因素进行考察,以构建微生物反应器。实验证明,微生物反应器对环境条件有较宽适应性和较强降解能力;其反应速率和降解率远优于未固定菌株;对于高浓度底物,仍表现出理想的降解效果,且重复使用效果稳定。应用微生物反应器进行东营石油污染土壤生物修复模拟试验,20d吡啶降解完全,40d喹啉降解96.5%,效果理想。
In order to screening strains with high biodegradation efficiency of nitrogenous heterocylic compounds and applying strains to oil contaminated soil, optimizing the conditions of stains, construction mix culture and domesticating strains, utilizing immobilization technology for bioreactor were investigated in this thesis.
Quinoline and pyridine degrading bacterias were isolated by enrichment from 45 soil samples from various areas of China, especially polluted site by petroleum. Four high efficiency strains were selected for the biodegradation study. An alignment of the DNA sequences of the 16S rRNA gene of the bacterium with DNA sequences, available in GenBank database indicates that strain 2-13 is closest designated as Flexibacter giganteus, strain 4-11 is designated as Thiobacillus intermedius, strain Q5 is designated as Lactobacillus fermentum, strain Q24 is designated as Arthrobacter flavescens. The primary conditions were reviewed, such as pH value, temperature, rotation speed, inoculum size, carbon and nitrogen sources, the concentration of target etc.The initial mix culture after domesticated had excellent degradation as single strains, but shorter domestication period and easy manipulation. Artificial constructed mixed strains don’t have ideal effect, only 43.2%~57.1%.
After immobilizing, the strains capacity of degradation was extremely enhanced. Compared with the untreated, the immobilized strains have a wide range on pH value and temperature, and higher degradability. At initial concentration 1500mg?L-1 of quinoline, the degrading rate in the bioreactor Q5 in 70 hours treatment coulde reach to 86.5%, and had steady effect.Bioreactor Q5 were appled to biodegradate oil contaminated soil in Shengli Oilfield, pyridine was biodegradated completely after 20ds, and the degradation rate of quinoline reach to 96.5% after 40d.
引文
[1] 夏北成.环境污染物生物降解.北京:化学工业出版社,2002:155～163
[2] 梁文杰.石油化学.东营:石油大学出版社,1996:42～48
[3] 孙丽娟,李咏梅,顾国维.含氮杂环化合物的生物降解研究进展.四川环境,2005;24(1):61～64
[4] 郑广宏.含氮杂环有机化合物缺氧生物降解特性及机理研究:[硕士学位论文],同济大学,上海:2003
[5] Meyer, S. H. Steinhart. Effects of heterocyclic PAHs (N, S, O) on the biodegradation of typical tar oil PAHs in a soil/compost mixture. Chemosphere, 2000; 40(2000): 359~367
[6] 姚珺.焦化废水中有机污染物经厌氧酸化后对好氧生物降解性能的影响.中国环境科学,1998;18(3):276～279
[7] 吴立波.自固定化高效菌种强化处理焦化废水研究.中国给水排水,1999;1(15):1～5
[8] Wangjianlong. Biodegradation of quinoline by gel immobilized Burkholderia sp. Chemosphere, 2001; 44: 1041～1046
[9] 金志刚,张彤,朱怀兰.污染物生物降解.上海:华东理工大学出版社,1997:41～46
[10] 韩力平,王建龙,刘恒,等.固定化细胞流化床反应器处理难降解有机物喹啉的试验研究.环境科学,2001;22(l):78～80
[11] 宋秀兰.固定化胶质红环菌在好氧条件下降解吲哚的研究.环境科学学报,2001;21(4):510～512
[12] 刘佐才,全向春,韩利平,等.喹啉的生物降解动力学.物理化学学报,2000;6(7):663～666
[13] 张晓健,雷晓玲,何苗,等.好氧生物处理对焦化废水中有机物的去除.环境保护,1994;8:7～10
[14] 李咏梅,顾国维,赵建夫.焦化废水中几种含氮杂环化合物缺氧降解机理.同济大学学报,2001;29(6):720～723
[15] 何苗,张晓建,雷晓玲,等.厌氧-缺氧/好氧工艺与常规活性污泥法处理焦化废水的比较.给水排水,1997;23(6):31～33
[16] 陈艳丽,章非娟.焦化废水生物脱氮反硝化段菌相分析.中国给水排水,1996;12(4):30～32
[17] 王红旗,齐永强,吴班,等.土壤中石油污染物微生物降解过程中各石油烃组分的演变规律.环境科学学报,2003;23(6):834～836
[18] WR. Modeling of the breakdown and the mobilization of hydrocarbons in unsaturated soil layers. Proceedings, 1976: 99～112
[19] JE, B. , C. DG. Effect of co-occurring aromatic hydrocarbons on the degradation of individual polycyclic aromatic hydrocarbons in marine sediment slurries. Appl. Environ. Mircrobiol, 1988; 54: 1649～1655
[20] AB. Centrifuge modeling of flow and contaminant transport through partially saturated soils. Kingston: Queen's University. 1991, 1121～1131
[21] 赵东风,赵朝成,王联社,等.石油类污染物在土壤中的迁移渗透规律.石油大学学报(自然科学版),2000;24(3):64～66
[22] 刘晓艳,纪学雁,李兴伟,等.石油类污染物在土壤中迁移的实验研究进展.土壤,2005;37(5):482～486
[23] 耿春香,路帅.西北地区土壤中石油类污染物的垂直渗透规律.环境污染与防治,2003;25(1):61～62
[24] 段云霞.生物通风(BV)法去除土壤中石油污染物的研究:[硕士学位论文],天津大学,天津:2004
[25] 李颖.石油污染土壤的生物修复:[硕士学位论文],北京化工大学,北京:2004
[26] S. S. Radwan. Enhanced remediation of hydrocarbon contaminated desert soil fertilized with organic carbons. International Biodeterioration & Biodegradation, 2001; 46: 129～132
[27] R.Boopathy. Bioremediation of explosives contaminated soil. International Biodeterioration & Biodegradation, 2000; 46: 29～36
[28] 陈玉成.土壤污染的生物修复.环境科学动态,1999;2:7～11
[29] N. Vasudevan, P. Rajaram. Bioremediation of oil sludge-contaminated soil. Environment International, 2001; 26: 409～411
[30] 常志州,何加骏.水势与用氮量对土壤中石油降解的影响.农村生态环境, 1997;13(1):21～24
[31] 何良菊,魏德州.土壤微生物处理石油污染的研究.环境科学进展,1999;7(3):110～115
[32] 丁克强,骆永明.生物修复石油污染土壤.土壤,2001;4:179～184
[33] ZZ, C., W. Richard. Organic bulking agents for enhancing oil bioremediation in soil. Biorem J., 1998; 1(3): 173～180
[34] Robert LR, et al. Volatilization of crude oil from soil a mended with bulking agents. Soil Science, 1998; 163(2): 87～92
[35] 叶小梅,常志州.调理剂对污泥中石油降解速率的影响.环境导报,1999;2:21～22
[36] 张海荣,李培军.四种石油污染土壤生物修复技术研究.农业环境保护,2001;20(2):78～80
[37] 高枫,张心平.用全 DNA 转化法构建多功能石油降解菌.南开大学学报(自然科学),1999;32(3):158～162
[38] H. Pieper, D. Engineering bacteria for bioremediation Current Opinion in Biotechnology. 2000; 11: 262～270
[39] 朱利中,冯少良.混合表面活性剂对多环芳烃的增溶作用及影响因素.环境科学学报,2002;22(6):774～778
[40] 叶为民,孙风慧.土壤石油污染的生物修复技术.上海地质,2002;4:22～25
[41] P. P. E. Carrier, F. A. Mesania. Enhanced biodegradation of creosote contaminated siol. Environmental oils, Frenseniu Joural of Analytical Chemistry, 1996; 356(6): 378～384
[42] 张丽芳,姜承志.表面活性剂对不同石油降解菌除油影响的研究.沈阳工业学院学报,2001;20(4):79～83
[43] K. S. M. Rahman, Thahira J. Rahman, Y.Kourkoutas, et al. Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients. Bioresource Technology, 2003; 90(2): 159~168
[44] 崔明超,李丽,陈繁忠.喹啉及其衍生物微生物降解研究进展.上海环境科学,2003;22(1):52～56
[45] 汤惠中,李明.紫外二元光度法测定废水中 2-甲基吡啶.化工环保,1991;11:104～117
[46] 李亚新.紫外分光光度法测定焦化废水的主要污染物.中国给水排水,2001;17(1):54～56
[47] 东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001
[48] 刘秀花,梁峰,刘茵,等.河南省土壤中芽孢杆菌属资源调查.河南农业科学,2006;8:67～71
[49] 何玉财,刘幽燕,童张法,等.微生物降解氰化物.化工科技,2004;12(2):58～62
[50] 范志明,柯明.催化裂化汽油中硫醇性硫和碱性氮化物分布规律的考察.石油大学学报(自然科学版),1998;22(5):86～89
[51] 古昌红,傅敏,丁培道.超声波降解吡啶溶液.化学研究与应用,2003;15(3):387～389
[52] 于丽华,钟俊波,沈昱.Al 2 O3 / TiO2的制备及光催化降解吡啶.大连铁道学院学报,2003;25(1):85～87
[53] 刘佐才,全向春,韩利平,等.喹啉的生物降解动力学.物理化学学报,2000;16(7):663～666
[54] 岑沛霖,蔡谨.工业微生物学.北京:化学化工出版社,2000:87～89
[55] 陈华.难降解有机废水的高效优势菌群的研究:[硕士学位论文],南京理工大学,南京:2004
[56] 黄廷林,柴蓓蓓,宁亚平.低残油土壤中高效降油菌的筛选分离及其营养平衡.环境工程,2006;23(3):7～9
[57] 邵宝林,龚国淑,张世熔,等.横断山北部高山区不同生态条件下土壤微生物数量及其与生态因子的相关性.生态学杂志,2006;25(8):885～890
[58] 金文标,宋莉晖,吴东平,等.油污土壤治理中细菌对原油的降解作用.油气田地面工程,2000;19(1):75～876
[59] 张明才.黄河三角洲柽柳群落土壤微生物多样性及其生态系统功能的研究:[硕士学位论文],山东大学.济南:2000
[60] 刘彬彬,张峰,冯晓西.对降解喹啉的厌氧生物反应器中重要功能菌群的鉴定.生态学报,2006;26(5):1390～1395
[61] 张永明.固定化细胞处理有机废水及其生物反应器的研究:[博士学位论文],华东理工大学,上海:2000
[62] 潘晶.摇蚊幼虫微生物防治的基础研究:[硕士学位论文],哈尔滨工业大学,哈尔滨:2003
[63] 欧阳藩.氧化亚铁硫杆菌的固定化及其在矿物浸出中的应用研究:[博士学位论文],中国科学院过程工程研究所,北京:2004
[64] 张志刚.固定化混合菌种降解 2,6-二叔丁基苯酚研究:[博士学位论文],同济大学,上海:2005
