石油烃厌氧降解菌的筛选及其降解特性研究
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摘要
近年来,石油污染对环境、人类的生产和生活造成了严重的影响,石油污染的治理,特别是土壤和地下水的生物修复技术已经成为了研究热点。生物修复技术包括原位和异位生物修复,对于石油污染源比较分散的土壤和地下水环境,采用原位生物修复具有明显的优势,由于土壤和地下水的缺氧特性,特别是在高温干旱等极端环境下,使得原位厌氧生物修成为可能。本文首先通过分析16个月原位强化修复石油污染土壤的理化性质和纵向分布特征,探讨厌氧降解在原位修复过程中的作用;其次,采集克拉玛依油田的污染土壤样品,选择合适的电子受体,筛选土著嗜热石油烃厌氧降解菌,并对可培养的单菌进行了分子生物学鉴定,确定了厌氧降解混合菌KLA14-2的最佳培养条件;对混合菌KLA14-2进行环境影响因素考察,分析两种不同性质原油的降解特性;最后,在底物降解范围和群落结构稳定性研究的基础上,选择单环芳烃甲苯和多环芳烃萘为模式物,考察KLA14-2对甲苯和萘的降解特性,利用紫外、红外和GC-MS等方法对甲苯和萘代谢产物进行了分析,推测可能的代谢途径,得到以下几个结论:
(1)经16个月原位强化修复后各土层的石油烃得到一定程度的去除,表层土饱和分和芳香分去除率最高;底层土IN-1处于缺氧环境,存在硫酸盐还原和反硝化作用,使得土壤pH值从7.86±0.03降低至7.27±0.03,土壤总氮从2.53±0.13g/kg降低至0.77±0.04 g/kg;厌氧菌数量与胶质和沥青质去除率之间成正相关关系,对于污染源较为分散的污染区域,采用原位生物强化修复时可以考虑引入厌氧修复;
(2)克拉玛依石油污染土壤硫酸盐含量丰富,硫酸盐适合做为石油烃厌氧降解菌的电子受体;经过初筛和复筛,得到一组稳定、降解效果较好的厌氧降解混合菌KLA14-2;经多次分离、纯化共获得四株可培养厌氧单菌,分子鉴定表明KLA14-2-1和KLA14-2-3分别属于地衣芽胞杆菌属(Bacillus licheniformis)和热嗜淀粉芽孢杆菌属(Bacillus thermoamylovorans),而KLA14-2-2和KLA14-2-4只能初步判断分别与可培养的脱硫肠状菌属(Desulfotomaculum)和铁细菌的亲缘关系较近,4株单菌的原油降解能力均较差,混合菌对原油的降解具有优势;
(3)过低的接种量不利于KLA14-2降解原油,最佳接种量为2%;混合菌可以在50~60℃的温度下保持较好的原油降解能力,最适pH值范围为6~8,低于CMC浓度的表面活性剂(50mg/L)可以一定程度上促进混合菌KLA14-2对原油的降解;硫酸盐还原作用对厌氧降解原油的贡献最大,在三种电子受体共同存在时,三价铁能够促进厌氧降解混合菌KLA14-2的降解,硝酸根会抑制KLA14-2的降解;KLA14-2对不同性质原油具有不同的降解特性,能够利用稠油中的胶质等难降解组分;
(4)混合菌的底物利用能力要强于单菌,其中混合菌KLA14-2对低浓度的8种石油烃化合物均具有较好的利用能力;PCR-ARDRA分析结果表明KLA14-2种群结构复杂,可培养和分离的微生物只占到了21.1%;在原油条件下,混合菌KLA14-2的群落结构变得更加简单,优势菌的种类发生变化,含量也由63.75%提高到73.75%;
(5)混合菌KLA14-2对萘的耐受能力要差于甲苯,萘和甲苯的厌氧降解符合非竞争性底物抑制模型,各种混合电子受体条件下KLA14-2对甲苯和萘的降解能力普遍好于单一的电子受体,一定量的外加碳酸氢盐可加速萘的降解进程,对甲苯影响不大;
(6)KLAL14-2厌氧降解甲苯主要代谢机理为苯甲基琥珀酸盐合成反应和苯甲酸盐合成反应,代谢产物有苯甲基琥珀酸和苯甲酸;厌氧降解萘的主要代谢机理为羧基化反应和甲基化反应,主要代谢中间产物有2-萘酸、萘基-2-甲基琥珀酸、5,6,7,8-四氢-2-萘酸、十氢萘酸、cis-2-羧基环已醋酸等。
In recent years, the oil pollution casused serious impact on the environment and human activities, especially the bioremediation technology used in oil polluted soil and groundwater has become a hot research topic. Bioremediation techniques include in situ and ex-situ bioremediation, and the in situ bioremediation has a great advantage in the contaminated area which polluted dispersively or the groundwater environment. Increasing evidence indicates that biodegradation of hydrocarbons takes place also in anoxic conditions. This opens new perspectives for the in situ treatment of contaminated soil and groundwater where reducing conditions below the surface limited the usefulness of O2 as an electron acceptor which could be supplied to stimulate the degradation of petroleum hydrocarbons. Under reducing conditions, other options have to be evaluated for enhancing the in situ biodegradation of organic contaminants. In this paper, firstly, the vertical distribution of physical and chemical properties of the oil contaminated soil with 16 months in situ bioremediation was studied, to analyze the role of anaerobic degradation during the in situ bioremediation process; Secondly, the Karamay oil polluted soil samples were collected, and the appropriate electronic accepter was isolated, then the thermophilic anaerobic hydrocarbon degrading bacteria which used crude oil as carbon source were isolated, and the pure degrading strain was identified by molecular biology, the optimize culture condition of the mixed culture KLAL14-2 was obtained. In addition, the environment factors effected on the mixed culture KLA14-2 was studied, and the degradation characteristics of KLA14-2 on the two different kinds of crude oil was analyzed; Finally, the range of substrate degradation and the stability of community structure were studied, the degrading characteristics of toluene and naphthalene which presented the monocyclic aromatics hydrocarbons and polycyclic aromatic hydrocarbons were investigated, and possible the metabolic pathway was speculated through ultraviolet, infrared and GC-MS methods.
The experiment results showed:
(1) In-situ bioaugmentation strategy was used for bioremediation of oil contaminated soil with 16 months, the oil of each soil layer was degraded in a certain extent, the removal rates of saturate and aromatic component of the surface soil was highest; As sulfate reduction and nitrate reduction took place in subsurface soil, the pH reduced from 7.86±0.03 to 7.27±0.03, and the TN reduced from 2.53±0.13 g/kg to 0.77±0.04 g/kg; The removal rate of resin and asphaltene was related to the anaerobic bacterial count. The anaerobic bioremediation strategy should be considered, when the in-situ remediation was applied in the oil contaminated area which polluted dispersively;
(2) The sulfate content of Karamy oil contaminated soil was abundant, the sulfate was suitable for anaerobic hydrocarbon degrading bacteria as electron acceptor; after primary and secondary screening, the stable and high hydrocarbon degrading rate mixed culture KLA14-2 was obtained. Crude oil degradation experiments of the four pure degrading bacteria (KLA14-2-1, KLA14-2-2, KLA14-2-3 and KLA14-2-4 which were isolated from the mixed culture KLA14-2) showed that these pure strain had a low crude oil removal ability, the single hydrocarbon degrading bacteria in crude oil degrading is not appropriate; Molecular identification results showed that KLA14-2-1 and KLA14-2-3 belong to Bacillus licheniformis and Bacillus thermoamylovorans respectively, and KLA14-2-2 and KLA14-2-4 could only be confirmed have a close genetic relationship of the Desulfotomaculum and iron bacteria respectively;
(3) Lower inoculum inhibited the degrading, and 2% inoculum was more appropriate, the mixed culture KLA14-2 could maintain a high oil removal rate when the temperate was above 50℃; The optimal pH range was 6-8, and the concentration of surfactant (50mg/L) which lower than the CMC could promote the oil degradation of mixed culture KLAL14-2; Sulfate-reducing have the mainly effect on the anaerobic oil degradation process, ferric iron could promote the degradation of KLA14-2, and under the nitrate-reduction condition inhibited the degradation; KLA14-2 had vary degrading characteristics with different kinds of oil, could degraded the resin component of heavy oil;
(4) Substrate utilization ability experiments showed that the mixed culture were better than the pure bacteria, the KLA14-2 was ability to use 8 kinds of low concentration hydrocarbon substrates as carbon source; The PCR-ARDRA analysis showed that the KLA14-2 had a complex population structure, the culturable bacteria could be only to 21.1%, and the population structure of KLA14-2 used crude oil as carbon source was different with the original structure, the type of dominant bacteria were changed and the content increased from 63.75% to 73.75%;
(5) The tolerance of mixed culture KLAL14-2 on naphthalene was worse than toluene, the anaerobic degradation of naphthalene and toluene was fitted to a non-competitive inhibition model, the toluene or naphthalene degradation ability of KLA14-2 on mixed electron acceptor conditions was better than each single electron acceptor conditions, the addition of a certain amount bicarbonate could accelerate the process of degradation of naphthalene, but toluene had little effect;
(6) The major metabolic mechanism of KLA14-2 for degrading toluene were benzylsuccinate synthesis and benzoate synthesis, the major metabolic production were benzylsuccinate and benzoic acid; and the major metabolic mechanism of KLA14-2 for degrading naphthalene were carboxylation and methylation, the major metabolic production were 2-naphthoic acid, naphthyl-2-methylsuccinate, 5,6,7,8– tetrahydro -2-naphthoic acid, decalin acid and cis-2-carboxycyclohexylacetate.
(1)经16个月原位强化修复后各土层的石油烃得到一定程度的去除,表层土饱和分和芳香分去除率最高;底层土IN-1处于缺氧环境,存在硫酸盐还原和反硝化作用,使得土壤pH值从7.86±0.03降低至7.27±0.03,土壤总氮从2.53±0.13g/kg降低至0.77±0.04 g/kg;厌氧菌数量与胶质和沥青质去除率之间成正相关关系,对于污染源较为分散的污染区域,采用原位生物强化修复时可以考虑引入厌氧修复;
(2)克拉玛依石油污染土壤硫酸盐含量丰富,硫酸盐适合做为石油烃厌氧降解菌的电子受体;经过初筛和复筛,得到一组稳定、降解效果较好的厌氧降解混合菌KLA14-2;经多次分离、纯化共获得四株可培养厌氧单菌,分子鉴定表明KLA14-2-1和KLA14-2-3分别属于地衣芽胞杆菌属(Bacillus licheniformis)和热嗜淀粉芽孢杆菌属(Bacillus thermoamylovorans),而KLA14-2-2和KLA14-2-4只能初步判断分别与可培养的脱硫肠状菌属(Desulfotomaculum)和铁细菌的亲缘关系较近,4株单菌的原油降解能力均较差,混合菌对原油的降解具有优势;
(3)过低的接种量不利于KLA14-2降解原油,最佳接种量为2%;混合菌可以在50~60℃的温度下保持较好的原油降解能力,最适pH值范围为6~8,低于CMC浓度的表面活性剂(50mg/L)可以一定程度上促进混合菌KLA14-2对原油的降解;硫酸盐还原作用对厌氧降解原油的贡献最大,在三种电子受体共同存在时,三价铁能够促进厌氧降解混合菌KLA14-2的降解,硝酸根会抑制KLA14-2的降解;KLA14-2对不同性质原油具有不同的降解特性,能够利用稠油中的胶质等难降解组分;
(4)混合菌的底物利用能力要强于单菌,其中混合菌KLA14-2对低浓度的8种石油烃化合物均具有较好的利用能力;PCR-ARDRA分析结果表明KLA14-2种群结构复杂,可培养和分离的微生物只占到了21.1%;在原油条件下,混合菌KLA14-2的群落结构变得更加简单,优势菌的种类发生变化,含量也由63.75%提高到73.75%;
(5)混合菌KLA14-2对萘的耐受能力要差于甲苯,萘和甲苯的厌氧降解符合非竞争性底物抑制模型,各种混合电子受体条件下KLA14-2对甲苯和萘的降解能力普遍好于单一的电子受体,一定量的外加碳酸氢盐可加速萘的降解进程,对甲苯影响不大;
(6)KLAL14-2厌氧降解甲苯主要代谢机理为苯甲基琥珀酸盐合成反应和苯甲酸盐合成反应,代谢产物有苯甲基琥珀酸和苯甲酸;厌氧降解萘的主要代谢机理为羧基化反应和甲基化反应,主要代谢中间产物有2-萘酸、萘基-2-甲基琥珀酸、5,6,7,8-四氢-2-萘酸、十氢萘酸、cis-2-羧基环已醋酸等。
In recent years, the oil pollution casused serious impact on the environment and human activities, especially the bioremediation technology used in oil polluted soil and groundwater has become a hot research topic. Bioremediation techniques include in situ and ex-situ bioremediation, and the in situ bioremediation has a great advantage in the contaminated area which polluted dispersively or the groundwater environment. Increasing evidence indicates that biodegradation of hydrocarbons takes place also in anoxic conditions. This opens new perspectives for the in situ treatment of contaminated soil and groundwater where reducing conditions below the surface limited the usefulness of O2 as an electron acceptor which could be supplied to stimulate the degradation of petroleum hydrocarbons. Under reducing conditions, other options have to be evaluated for enhancing the in situ biodegradation of organic contaminants. In this paper, firstly, the vertical distribution of physical and chemical properties of the oil contaminated soil with 16 months in situ bioremediation was studied, to analyze the role of anaerobic degradation during the in situ bioremediation process; Secondly, the Karamay oil polluted soil samples were collected, and the appropriate electronic accepter was isolated, then the thermophilic anaerobic hydrocarbon degrading bacteria which used crude oil as carbon source were isolated, and the pure degrading strain was identified by molecular biology, the optimize culture condition of the mixed culture KLAL14-2 was obtained. In addition, the environment factors effected on the mixed culture KLA14-2 was studied, and the degradation characteristics of KLA14-2 on the two different kinds of crude oil was analyzed; Finally, the range of substrate degradation and the stability of community structure were studied, the degrading characteristics of toluene and naphthalene which presented the monocyclic aromatics hydrocarbons and polycyclic aromatic hydrocarbons were investigated, and possible the metabolic pathway was speculated through ultraviolet, infrared and GC-MS methods.
The experiment results showed:
(1) In-situ bioaugmentation strategy was used for bioremediation of oil contaminated soil with 16 months, the oil of each soil layer was degraded in a certain extent, the removal rates of saturate and aromatic component of the surface soil was highest; As sulfate reduction and nitrate reduction took place in subsurface soil, the pH reduced from 7.86±0.03 to 7.27±0.03, and the TN reduced from 2.53±0.13 g/kg to 0.77±0.04 g/kg; The removal rate of resin and asphaltene was related to the anaerobic bacterial count. The anaerobic bioremediation strategy should be considered, when the in-situ remediation was applied in the oil contaminated area which polluted dispersively;
(2) The sulfate content of Karamy oil contaminated soil was abundant, the sulfate was suitable for anaerobic hydrocarbon degrading bacteria as electron acceptor; after primary and secondary screening, the stable and high hydrocarbon degrading rate mixed culture KLA14-2 was obtained. Crude oil degradation experiments of the four pure degrading bacteria (KLA14-2-1, KLA14-2-2, KLA14-2-3 and KLA14-2-4 which were isolated from the mixed culture KLA14-2) showed that these pure strain had a low crude oil removal ability, the single hydrocarbon degrading bacteria in crude oil degrading is not appropriate; Molecular identification results showed that KLA14-2-1 and KLA14-2-3 belong to Bacillus licheniformis and Bacillus thermoamylovorans respectively, and KLA14-2-2 and KLA14-2-4 could only be confirmed have a close genetic relationship of the Desulfotomaculum and iron bacteria respectively;
(3) Lower inoculum inhibited the degrading, and 2% inoculum was more appropriate, the mixed culture KLA14-2 could maintain a high oil removal rate when the temperate was above 50℃; The optimal pH range was 6-8, and the concentration of surfactant (50mg/L) which lower than the CMC could promote the oil degradation of mixed culture KLAL14-2; Sulfate-reducing have the mainly effect on the anaerobic oil degradation process, ferric iron could promote the degradation of KLA14-2, and under the nitrate-reduction condition inhibited the degradation; KLA14-2 had vary degrading characteristics with different kinds of oil, could degraded the resin component of heavy oil;
(4) Substrate utilization ability experiments showed that the mixed culture were better than the pure bacteria, the KLA14-2 was ability to use 8 kinds of low concentration hydrocarbon substrates as carbon source; The PCR-ARDRA analysis showed that the KLA14-2 had a complex population structure, the culturable bacteria could be only to 21.1%, and the population structure of KLA14-2 used crude oil as carbon source was different with the original structure, the type of dominant bacteria were changed and the content increased from 63.75% to 73.75%;
(5) The tolerance of mixed culture KLAL14-2 on naphthalene was worse than toluene, the anaerobic degradation of naphthalene and toluene was fitted to a non-competitive inhibition model, the toluene or naphthalene degradation ability of KLA14-2 on mixed electron acceptor conditions was better than each single electron acceptor conditions, the addition of a certain amount bicarbonate could accelerate the process of degradation of naphthalene, but toluene had little effect;
(6) The major metabolic mechanism of KLA14-2 for degrading toluene were benzylsuccinate synthesis and benzoate synthesis, the major metabolic production were benzylsuccinate and benzoic acid; and the major metabolic mechanism of KLA14-2 for degrading naphthalene were carboxylation and methylation, the major metabolic production were 2-naphthoic acid, naphthyl-2-methylsuccinate, 5,6,7,8– tetrahydro -2-naphthoic acid, decalin acid and cis-2-carboxycyclohexylacetate.
引文
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