石油污染土壤中石油烃微生物降解性能的研究
摘要
本文首先对石油污染土壤中石油烃含量的测定方法进行了研究,分别用石油醚、二氯甲烷、三氯甲烷和甲苯作为萃取溶剂,采用重量法分析石油污染土壤中的石油烃。并对四种不同溶剂萃取物进行了石油烃含量、四组分组成、红外光谱、结构参数等方面的对比分析,最终确定二氯甲烷为石油污染土壤中石油烃的最佳萃取溶剂。
实验室运用单因素实验、正交实验对发酵培养基和发酵控制参数进行优化,得到最优发酵条件,筛选了石油烃降解的高效菌种,并对固体菌剂进行了制备。
为获得微生物对原油各馏分降解情况,将孤东原油蒸馏得到的柴油馏分、蜡油馏分分别进行微生物降解,对柴油馏分进行GC-MS分析,并对柴油、蜡油降解产物进行FT-ICR MS分析。在柴油馏分中鉴定出O1、O2、N1等多种分子类型,其中O2相对丰度远高于O1、N1类型,该类化合物是脂肪酸。油样中脂肪酸存在明显的C16、C18优势,降解前后组成差异不明显,其中BC-I菌剂降解后,低碳数脂肪酸相对丰度似有增加。从碳数分布来看,蜡油以m/z=293(C19双环环烷酸)为中心正态分布,烷基咔唑中C5-咔唑丰度最高,苯并咔唑相对丰度很低。O2类化合物丰度很低,可见脂肪酸含量很低。DBE为4或5的O2类化合物明显占优势,对应3~4环环烷酸丰度较高。另外,将正十六烷作为微生物生长的唯一碳源进行降解以确定正构烷烃的生物代谢途径,产物经FT-IR和GC-MS分析检测到十六酸的存在,这说明正构烷烃的代谢产物是有机酸。
将微生物修复土壤技术应用于孤东石油污染区,开展一段时间的中试试验后,将从现场不同时间取得的土样进行石油烃测定分析,确定现场微生物降解的效果。
Firstly, the determination method of hydrocarbons in oil polluted soil has been studied, gravimetric method is used to extract hydrocarbon from petroleum contaminated soil. Petroleum ether, dichloromethane, chloroform and toluene are used as solvents respectively, hydrocarbon content, SARA, infrared spectra, structural parameters of extracts are compared to determine dichloromethane is the best extracting solvent.
Secondly, single-factor experiments, orthogonal design, uniform design, optimization of the liquid fermentation medium and fermentation control parameters are studied, the optimal fermentation conditions is acquired, so efficient degradation bacteria is selected.
In order to obtain the degradation situation of fractions of Gudong crude oil, GC-MS analysis of the diesel fractions,the microbial degrading products of diesel and gas oil are analyzed using FT-ICR MS. O1, O2, N1 and other molecular types are identified in diesel fraction, in which O2 compounds (fatty acids) abundance is higher than the others. Carbon number of fatty acids concentrates at 16, 18, the composition of oil samples changes scarely after degradation. The carbon number distribution of the VGO takes on a normal distribution centered m/z=293 (C19 double ring naphthenic acid), C5 alkyl carbazole has the highest abundance while benzocarbazole has lower abundance. It is also showed that fatty acid content at lower abundance and naphthenic with 3-4 rings has higher abundance. In addition, the n-hexadecane is biodegraded as the sole carbon source to determine n-alkanes to determine the metabolic pathway. The presence of palmitie acid is metabolic pathway by GC-MS, it shows that organic acid is the metabolic product of n-alkanes.
Finally, microbial remediation technology is applied in Gudong oil-field, hydrocarbons extracted from polluted soil is analyzed to determine the effect of microbial degradation of petroleum contaminated soil.
实验室运用单因素实验、正交实验对发酵培养基和发酵控制参数进行优化,得到最优发酵条件,筛选了石油烃降解的高效菌种,并对固体菌剂进行了制备。
为获得微生物对原油各馏分降解情况,将孤东原油蒸馏得到的柴油馏分、蜡油馏分分别进行微生物降解,对柴油馏分进行GC-MS分析,并对柴油、蜡油降解产物进行FT-ICR MS分析。在柴油馏分中鉴定出O1、O2、N1等多种分子类型,其中O2相对丰度远高于O1、N1类型,该类化合物是脂肪酸。油样中脂肪酸存在明显的C16、C18优势,降解前后组成差异不明显,其中BC-I菌剂降解后,低碳数脂肪酸相对丰度似有增加。从碳数分布来看,蜡油以m/z=293(C19双环环烷酸)为中心正态分布,烷基咔唑中C5-咔唑丰度最高,苯并咔唑相对丰度很低。O2类化合物丰度很低,可见脂肪酸含量很低。DBE为4或5的O2类化合物明显占优势,对应3~4环环烷酸丰度较高。另外,将正十六烷作为微生物生长的唯一碳源进行降解以确定正构烷烃的生物代谢途径,产物经FT-IR和GC-MS分析检测到十六酸的存在,这说明正构烷烃的代谢产物是有机酸。
将微生物修复土壤技术应用于孤东石油污染区,开展一段时间的中试试验后,将从现场不同时间取得的土样进行石油烃测定分析,确定现场微生物降解的效果。
Firstly, the determination method of hydrocarbons in oil polluted soil has been studied, gravimetric method is used to extract hydrocarbon from petroleum contaminated soil. Petroleum ether, dichloromethane, chloroform and toluene are used as solvents respectively, hydrocarbon content, SARA, infrared spectra, structural parameters of extracts are compared to determine dichloromethane is the best extracting solvent.
Secondly, single-factor experiments, orthogonal design, uniform design, optimization of the liquid fermentation medium and fermentation control parameters are studied, the optimal fermentation conditions is acquired, so efficient degradation bacteria is selected.
In order to obtain the degradation situation of fractions of Gudong crude oil, GC-MS analysis of the diesel fractions,the microbial degrading products of diesel and gas oil are analyzed using FT-ICR MS. O1, O2, N1 and other molecular types are identified in diesel fraction, in which O2 compounds (fatty acids) abundance is higher than the others. Carbon number of fatty acids concentrates at 16, 18, the composition of oil samples changes scarely after degradation. The carbon number distribution of the VGO takes on a normal distribution centered m/z=293 (C19 double ring naphthenic acid), C5 alkyl carbazole has the highest abundance while benzocarbazole has lower abundance. It is also showed that fatty acid content at lower abundance and naphthenic with 3-4 rings has higher abundance. In addition, the n-hexadecane is biodegraded as the sole carbon source to determine n-alkanes to determine the metabolic pathway. The presence of palmitie acid is metabolic pathway by GC-MS, it shows that organic acid is the metabolic product of n-alkanes.
Finally, microbial remediation technology is applied in Gudong oil-field, hydrocarbons extracted from polluted soil is analyzed to determine the effect of microbial degradation of petroleum contaminated soil.
引文
[1]马强,林爱军等.土壤中总石油烃污染(TPH)的微生物降解与修复研究进展.生态毒理学报,2008,3(1):1-7
[2]詹研.中国土壤石油污染的危害及治理对策.环境污染与防治,2008,30(3):91-94
[3]胡佩,蒲万芬,胡星琪.油田污染土壤微生物治理现状及研究趋势.皮革科学与工程,2004,14(6):43-45
[4]任磊.石油勘探开发中的石油类污染及其监测分析技术.中国环境监测,2004,20(3):44-47
[5]夏平,李学亚,刘斌.石油污染的生物修复.污染防治技术,2006,19(3):37-40
[6]罗洪君,王绪远等.石油污染土壤生物修复技术的研究进展.四川环境, 2007,26(3):105-109
[7] A. Salinas-Mart?′neza, M. de los Santos-Co′rdovaa, O. Soto-Cruza, E. Delgadoa, Development of a bioremediation process by biostimulation of native microbial consortium through the heap leaching technique. Journal of Environmental Management. 2008, 88:115–119
[8] Krysta Paudyn , Allison Rutter , R. Kerry Rowe , John S. Poland .Remediation of hydrocarbon contaminated soils in theCanadian Arctic by landfarming.Cold Regions Science and Technology 2008,53 :102–114
[9] Wan Namkoong, Eui-Young Hwang, Joon-Seok Park. Bioremediation of diesel-contaminated soil with composting.Environmental Pollution 2002, 119: 23–31
[10]齐永强,王红旗.微生物处理土壤石油污染的研究进展.上海环境科学,2002,21(3):177-180
[11] Salanitro JP.Crude oil hydrocarbon bioremediation and soil ecotoxicity assessment.Environ Sci Technol, 1997, 31(6):1769-1776.
[12]袁红莉,杨金水.降解石油微生物菌种的筛选及降解特性.中国环境科学,2003,23(2):157~161
[13] Rowland Ap.Effects of beach sand properties, temperature and rainfall on the degradation rates of oil in buried oil/beach sand mixtures. Environ Pollut.2000.109 (1):109-118
[14] A Haiim.Growth of hydrocarbon-utilizing isolated in chemically defined media. International&Biodegradation, 1996, 37(12):61-68
[15] Lin Q.Effects of bioremediation agents on oil degradation inmineral and sandy salt marsh sediments.Environ Technal, 1999, 20(8):825-837
[16] AyotamunoM J, Kogbara R B, Ogaji SOT, etal. Bioremediation of a Crude oil Polluted Agricultural -soil at Port Harcourt, Nigeria [J]. Appl Energy, 2006, 83:1249-1257
[17] J. Sabat'e, M. Vi?nas, A.M. Solanas, Laboratory-scale bioremediation experiments on hydrocarbon-contaminated soils. International Biodeterioration & Biodegradation 2004, 54: 19– 25
[18] Kishore Das, Ashis K. Mukherjee, Crude petroleum-oil biodegradation effciency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminatedsoil from North-East India.Bioresource Technology 2007, 98:1339–1345
[19]刘晓春,阎光绪等.微生物降解土壤中石油污染物的研究进展.污染防治技术,2007,20(6):51-54
[20]孙远军,聂麦茜等.石油降解优势菌的筛选和降解性能.水处理技术,2007,33(8):47-49,57
[21]王红旗,陈延君,孙宁宁.土壤石油污染物微生物降解机理与修复技术研究.地学前缘,2006,13(1):135-138
[22]张学佳,康志军,孙大勇.土壤中石油类污染物的自然降解.石化技术与应用,2008,26(3):273~278
[23]黄学,伍晓林等.短短芽孢杆菌和蜡状芽孢杆菌降解原油烃机制研究.石油学报,2006,27(5):95
[24]王萌,张鹏等.微生物对石油污染的降解作用.科技信息, 2009(2):14-15
[25]李华,王红旗,齐永强.土壤正构烷烃微生物降解变化实验分析.环境科学研究, 2003,16 (5):24-28
[26]苏荣国,牟伯中等.微生物对石油烃的降解机理及影响因素.化工环保, 2001,21(4):205-207
[27]齐永强,王红旗,刘敬奇,吴班.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
[28]潘学芳,倪秀珍,王浩.菌株SD-1对石油烃类降解机理的初步研究.长春师范学院学报, 2002,21(2):50
[29]王红旗,熊樱,陈延君.土壤中石油污染物微生物修复动力学和机理初探.环境化学, 2008,27(3):339-344
[30]潘学芳,王浩.微生物对石油烃类运输机理的初步研究.山东轻工业学院学报, 2003,17(1):31-33
[31]刘东,王宗贤等.渣油组分亚甲基与甲基比值和周边取代情况研究.石油大学学报(自然科学版),1999,23(6):70-72
[32]梁文杰.石油化学.第一版,山东东营:石油大学出版社,1995:150-151
[33]李超敏,王加宁,邱维忠,迟建国.高效降解石油细菌的分离鉴定及降解能力的研究.生物技术,2007, 17(4):81-82
[34]王加宁,李超敏等.降解石油污染物及石油产品的固体微生物菌剂及制备方法.中国专利:ZL200710013906.7, 2009-5-13
[35]史权,赵锁奇,徐春明,侯读杰.傅立叶变换离子回旋共振质谱仪在石油组成分析中的应用.质谱学报, 2008,29(6):368-374
[36]史权,侯读杰等.负离子电喷雾-傅里叶变换离子回旋共振质谱分析辽河原油中的环烷酸.分析测试学报, 2007,26:317-319
[37]陆小泉,史权等.碱液萃取前后原油中酸性化合物组成的高分辨质谱分析.分析化学, 2008,36(5):614-618
[38]潘学芳,梁泉峰,王浩.细菌8-A-2对石油烃类运输及正十六烷代谢产物的初步分析.农业生物技术学报, 2002,10(4):413-414
[39]冷凯良,楚晓珉,张辉珍.微生物对石油烃降解代谢产物的分析方法研究.海洋水产研究,2001,22(2):56-61
[40]刘岚铮,潘学芳.菌株SD-2对正十六烷摄取及中间代谢产物的初步研究.中国工业医学杂志, 2O03, 16(4):251-252
[41]陈延君,王红旗等.正十六烷微生物降解酶的定域和酶促降解性.环境科学研究, 2007,20(6):120-125
[42]刘清坤,王君等.嗜热地芽孢杆菌DM-2烃降解特性研究.环境科学, 2008,29(12):3554-3559
[2]詹研.中国土壤石油污染的危害及治理对策.环境污染与防治,2008,30(3):91-94
[3]胡佩,蒲万芬,胡星琪.油田污染土壤微生物治理现状及研究趋势.皮革科学与工程,2004,14(6):43-45
[4]任磊.石油勘探开发中的石油类污染及其监测分析技术.中国环境监测,2004,20(3):44-47
[5]夏平,李学亚,刘斌.石油污染的生物修复.污染防治技术,2006,19(3):37-40
[6]罗洪君,王绪远等.石油污染土壤生物修复技术的研究进展.四川环境, 2007,26(3):105-109
[7] A. Salinas-Mart?′neza, M. de los Santos-Co′rdovaa, O. Soto-Cruza, E. Delgadoa, Development of a bioremediation process by biostimulation of native microbial consortium through the heap leaching technique. Journal of Environmental Management. 2008, 88:115–119
[8] Krysta Paudyn , Allison Rutter , R. Kerry Rowe , John S. Poland .Remediation of hydrocarbon contaminated soils in theCanadian Arctic by landfarming.Cold Regions Science and Technology 2008,53 :102–114
[9] Wan Namkoong, Eui-Young Hwang, Joon-Seok Park. Bioremediation of diesel-contaminated soil with composting.Environmental Pollution 2002, 119: 23–31
[10]齐永强,王红旗.微生物处理土壤石油污染的研究进展.上海环境科学,2002,21(3):177-180
[11] Salanitro JP.Crude oil hydrocarbon bioremediation and soil ecotoxicity assessment.Environ Sci Technol, 1997, 31(6):1769-1776.
[12]袁红莉,杨金水.降解石油微生物菌种的筛选及降解特性.中国环境科学,2003,23(2):157~161
[13] Rowland Ap.Effects of beach sand properties, temperature and rainfall on the degradation rates of oil in buried oil/beach sand mixtures. Environ Pollut.2000.109 (1):109-118
[14] A Haiim.Growth of hydrocarbon-utilizing isolated in chemically defined media. International&Biodegradation, 1996, 37(12):61-68
[15] Lin Q.Effects of bioremediation agents on oil degradation inmineral and sandy salt marsh sediments.Environ Technal, 1999, 20(8):825-837
[16] AyotamunoM J, Kogbara R B, Ogaji SOT, etal. Bioremediation of a Crude oil Polluted Agricultural -soil at Port Harcourt, Nigeria [J]. Appl Energy, 2006, 83:1249-1257
[17] J. Sabat'e, M. Vi?nas, A.M. Solanas, Laboratory-scale bioremediation experiments on hydrocarbon-contaminated soils. International Biodeterioration & Biodegradation 2004, 54: 19– 25
[18] Kishore Das, Ashis K. Mukherjee, Crude petroleum-oil biodegradation effciency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminatedsoil from North-East India.Bioresource Technology 2007, 98:1339–1345
[19]刘晓春,阎光绪等.微生物降解土壤中石油污染物的研究进展.污染防治技术,2007,20(6):51-54
[20]孙远军,聂麦茜等.石油降解优势菌的筛选和降解性能.水处理技术,2007,33(8):47-49,57
[21]王红旗,陈延君,孙宁宁.土壤石油污染物微生物降解机理与修复技术研究.地学前缘,2006,13(1):135-138
[22]张学佳,康志军,孙大勇.土壤中石油类污染物的自然降解.石化技术与应用,2008,26(3):273~278
[23]黄学,伍晓林等.短短芽孢杆菌和蜡状芽孢杆菌降解原油烃机制研究.石油学报,2006,27(5):95
[24]王萌,张鹏等.微生物对石油污染的降解作用.科技信息, 2009(2):14-15
[25]李华,王红旗,齐永强.土壤正构烷烃微生物降解变化实验分析.环境科学研究, 2003,16 (5):24-28
[26]苏荣国,牟伯中等.微生物对石油烃的降解机理及影响因素.化工环保, 2001,21(4):205-207
[27]齐永强,王红旗,刘敬奇,吴班.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
[28]潘学芳,倪秀珍,王浩.菌株SD-1对石油烃类降解机理的初步研究.长春师范学院学报, 2002,21(2):50
[29]王红旗,熊樱,陈延君.土壤中石油污染物微生物修复动力学和机理初探.环境化学, 2008,27(3):339-344
[30]潘学芳,王浩.微生物对石油烃类运输机理的初步研究.山东轻工业学院学报, 2003,17(1):31-33
[31]刘东,王宗贤等.渣油组分亚甲基与甲基比值和周边取代情况研究.石油大学学报(自然科学版),1999,23(6):70-72
[32]梁文杰.石油化学.第一版,山东东营:石油大学出版社,1995:150-151
[33]李超敏,王加宁,邱维忠,迟建国.高效降解石油细菌的分离鉴定及降解能力的研究.生物技术,2007, 17(4):81-82
[34]王加宁,李超敏等.降解石油污染物及石油产品的固体微生物菌剂及制备方法.中国专利:ZL200710013906.7, 2009-5-13
[35]史权,赵锁奇,徐春明,侯读杰.傅立叶变换离子回旋共振质谱仪在石油组成分析中的应用.质谱学报, 2008,29(6):368-374
[36]史权,侯读杰等.负离子电喷雾-傅里叶变换离子回旋共振质谱分析辽河原油中的环烷酸.分析测试学报, 2007,26:317-319
[37]陆小泉,史权等.碱液萃取前后原油中酸性化合物组成的高分辨质谱分析.分析化学, 2008,36(5):614-618
[38]潘学芳,梁泉峰,王浩.细菌8-A-2对石油烃类运输及正十六烷代谢产物的初步分析.农业生物技术学报, 2002,10(4):413-414
[39]冷凯良,楚晓珉,张辉珍.微生物对石油烃降解代谢产物的分析方法研究.海洋水产研究,2001,22(2):56-61
[40]刘岚铮,潘学芳.菌株SD-2对正十六烷摄取及中间代谢产物的初步研究.中国工业医学杂志, 2O03, 16(4):251-252
[41]陈延君,王红旗等.正十六烷微生物降解酶的定域和酶促降解性.环境科学研究, 2007,20(6):120-125
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