PH混合菌对多环芳烃的吸附、摄取及生物降解

发布时间：2018-04-05 01:14

本文选题：海洋溢油　切入点：多环芳烃降解菌　出处：《中国海洋大学》2014年博士论文

【摘要】：随着国家对能源需求的增加，石油在其开采、运输以及加工炼制过程中大量进入环境，特别是海洋溢油事故频繁发生，严重威胁了海洋生物、生态环境以及人类健康。针对风化后难以被降解，且具有“三致”作用的多环芳烃，以及低温、营养物匮乏、高盐等特殊海洋环境的难题，本论文筛选多环芳烃降解菌，初步探讨了石油烃特别是多环芳烃的吸附、摄取及降解机理或规律，同时利用微生物固定化技术，制备固定化微生物，模拟海洋溢油的生物修复过程，强化去除多环芳烃，得出以下结论：（1）从胜利油田石油污染污泥中筛选分离出一组被命名为PH系列的多环芳烃降解菌（PH混合菌），，其原油降解率为60%，经鉴定： PH-1、PH-2、PH-3和PH-4分别隶属于假单胞菌属（Pseudomonas sp., KF113575），芽孢杆菌属（Bacillus sp., KF113576），苍白杆菌属（Ochrobactrum sp., KF421109）和假单胞菌属（Pseudomonas sp., KF113577）。（2）PH混合菌降解多环芳烃及原油的最适宜环境条件不同。萘：浓度800mg/L，pH=7.5，NaCl10g/L，温度35oC；菲：浓度40mg/L，pH=7，NaCl5g/L，温度30oC；芘：浓度100mg/L，pH=7，NaCl15g/L，温度30oC；原油：浓度3g/L，pH=8，NaCl7g/L，温度30oC。在萘、菲、芘和原油各自最佳条件下的生物降解率分别为：80%、30%、56%和48%。（3）PH混合菌对重金属离子具有较大的容忍性（最大容忍浓度分别为6.2mM Cu2+，2.7mM Zn2+，9.5mM Pb2+），且在重金属离子和污染底物共存的情况下，其对萘、菲、芘和原油的降解分别能达到53%、21%、32%、44%。（4）共代谢底物葡萄糖、α-乳糖、可溶淀粉、酵母粉、脲中，α-乳糖是PH混合菌降解石油烃（萘、菲、芘、原油）的最佳共代谢底物，其萘、菲、芘和原油生物降解率分别达95%、68%、60%、40%。（5）活性混合菌体表面吸附和体内摄取石油烃的稳定性顺序为：萘菲≈芘原油；加热致死混合菌体吸附多环芳烃的量为常量，而吸附原油的量为变量，且逐渐降低。（6）PH混合菌的疏水性35%，表现出较强的降解活性和乳化性能；其疏水性与底物浓度、毒性和菌浓度有关；PH混合菌分泌的膜周、膜内和胞外酶对不同的污染底物（萘、菲、芘、原油）的代谢性能不同。（7）推测细菌可能在降解有毒污染物（原油及多环芳烃）过程中通过改变细胞膜及细胞壁的通透性来摄取污染物，细胞内部主要通过细胞质（包括细胞器如核糖体和内部酶等）来降解污染物。（8）核桃壳和半焦炭对萘、菲、芘、原油的吸附符合拟二阶动力学模拟方程式，且浓度越小拟合性越好；不同载体材料对固定化微生物制备的影响因素重要性不同：以核桃壳为吸附剂制备的固定化微球，各因素影响重要性顺序及最优配比为：核桃壳（20%） CaCl2（1%） α-乳糖（0.1%）海藻酸钠（6%）；半焦炭为吸附剂：α-乳糖（0.3%）半焦炭（20%）海藻酸钠（6%） CaCl2（1%）；活性炭为吸附剂：α-乳糖（0.5%） CaCl2（5%）活性炭（20%）海藻酸钠（4%）；固定化降解菌经过5d的生物降解，其原油降解率48%较游离菌高出21%。（9）混合菌细胞存在羧基、氨基和磷酸盐等官能团；生物降解污染底物过程中要经过吸附、摄取等阶段；半焦炭经固定化处理后孔隙度变大，更易于微生物吸附。（10）生物修复过程中微生物主要消耗硝基氮；人工海水较自然海水的生物降解效果好；固定化菌较游离菌，五种烷基化芳烃降解率高，经过28d自然海水中对萘系列的降解率高于50%，菲、二苯并噻吩、芴和系列高于30%，总芳烃的降解率达63%；为取得好修复效果，应及时添加菌剂、磷源和硝基氮源。本论文研究结果对了解微生物如何吸附、摄取与降解污染底物具有重要意义，并为固定化多环芳烃降解菌剂应用于海洋溢油的生物修复的可行性提供理论与技术支持。
[Abstract]:With the increasing demand of energy , the oil has entered the environment in the process of its exploitation , transportation and processing , especially the frequent occurrence of marine oil spill , which seriously threatens marine life , ecological environment and human health . In view of the difficult degradation of marine organism , ecological environment and human health , this paper mainly discusses the adsorption , uptake and degradation mechanism or regulation of polycyclic aromatic hydrocarbon , especially polycyclic aromatic hydrocarbon , and uses microorganism immobilization technology to prepare immobilized microorganism , simulates the biological repair process of marine oil spill , strengthens the removal of polycyclic aromatic hydrocarbon , and concludes the following conclusions :

( 1 ) A group of polycyclic aromatic hydrocarbon degrading bacteria ( PH mixed bacteria ) named PH series were isolated and isolated from the oil polluted sludge of Shengli Oilfield . The degradation rate of crude oil was 60 % . The results were as follows : PH - 1 , PH - 2 , PH - 3 and PH - 4 belong to Pseudomonas sp . , KF113575 , Bacillus sp . , KF113576 , Ochrobactrum sp . , KF421109 and Pseudomonas sp . , KF113577 .

( 2 ) The most suitable conditions for degrading polycyclic aromatic hydrocarbons and crude oil by PH mixed bacteria were different : naphthalene : concentration 800mg / L , pH = 7.5 , NaCl10g / L , temperature 35oC ;
Pheni : concentration 40mg / L , pH = 7 , NaCl5g / L , temperature 30oC ;
Pyrene : concentration 100mg / L , pH = 7 , NaCl15g / L , temperature 30oC ;
Crude oil : concentration of 3 g / L , pH = 8 , NaCl7g / L and temperature of 30oC . The biodegradation rates were 80 % , 30 % , 56 % and 48 % , respectively .

( 3 ) PH mixed bacteria have greater tolerance to heavy metal ions ( 6.2 mM Cu2 + , 2.7 mM Zn2 + , 9.5 mM Pb 2 + ) , and the degradation of naphthalene , phenanthropy , pyrene and crude oil can reach 53 % , 21 % , 32 % and 44 % , respectively , under the coexistence of heavy metal ions and pollution substrates .

( 4 ) Co - metabolism substrate glucose , 伪 - lactose , soluble starch , yeast powder , urea , 伪 - lactose are the best co - metabolism substrates for the degradation of petroleum hydrocarbons ( naphthalene , phenanthropy , pyrene , crude oil ) by PH mixed bacteria . The biodegradation rates of naphthalene , phenanthropy , pyrene and crude oil reach 95 % , 68 % , 60 % and 40 % , respectively .

( 5 ) the stability sequence of the surface adsorption and the in vivo uptake of petroleum hydrocarbons by the active mixed bacteria is as follows :
The amount of adsorbed polycyclic aromatic hydrocarbons ( PAHs ) was constant , and the amount of adsorbed crude oil was variable and gradually decreased .

( 6 ) the hydrophobic 35 % of PH mixed bacteria showed strong degradation activity and emulsifying performance ;
Its hydrophobicity is related to substrate concentration , toxicity and concentration of bacteria ;
The membrane weeks , membrane and extracellular enzymes secreted by PH mixed bacteria have different metabolic properties to different contaminant substrates ( naphthalene , fip , pyrene , crude oil ) .

( 7 ) It is speculated that bacteria may ingest contaminants by altering the permeability of cell membranes and cell walls during the degradation of toxic pollutants ( crude and polycyclic aromatic hydrocarbons ) , primarily through the cytoplasm ( including organelle , such as ribosomes and internal enzymes , etc . ) .

( 8 ) The adsorption of the walnut shell and the semi - coke on naphthalene , fip , pyrene and crude oil conforms to the pseudo - second - order dynamic simulation equation , and the better the concentration is , the better the fitting property ;
The influence factors of different carrier materials on the preparation of immobilized microorganism were different : immobilized microspheres prepared by using walnut shell as adsorbent , the order of importance and the optimal proportion were as follows : walnut shell ( 20 % ) CaCl2 ( 1 % ) 伪 - lactose ( 0.1 % ) sodium alginate ( 6 % ) ;
Semi - coke as adsorbent : 伪 - lactose ( 0.3 % ) semi - coke ( 20 % ) sodium alginate ( 6 % ) CaCl2 ( 1 % ) ;
Activated carbon as adsorbent : 伪 - lactose ( 0.5 % ) CaCl2 ( 5 % ) activated carbon ( 20 % ) sodium alginate ( 4 % ) ;
After 5 days of biodegradation of immobilized degrading bacteria , the degradation rate of crude oil was 48 % higher than that of free bacteria by 21 % .

( 9 ) the mixed bacteria cell has functional groups such as carboxyl , amino and phosphate ;
in that proces of biodegradation of the polluted substrate , adsorption , uptake and the like are to be carried out ;
the porosity of the semi - coke after the immobilization treatment becomes large , and the microorganism is more easily absorbed by microorganisms .

( 10 ) the microorganism mainly consumes nitronitrogen during the biological repair process ;
the artificial seawater has better biodegradation effect than natural seawater ;
the degradation rate of the five alkylated aromatic hydrocarbons is higher than that of the free bacteria and the five alkylated aromatic hydrocarbons , and the degradation rate of the naphthalene series in the natural seawater at 28 days is higher than 50 percent , and the degradation rate of the total aromatic hydrocarbons reaches 63 percent ;
in ord to achieve good repair effect , that bacterial agent , the phosphorus source and the nitro nitrogen source should be added in a timely manner .

The results of this paper are of great significance to understand how the microorganism can adsorb , absorb and degrade the pollution substrate , and provide the theory and technical support for the feasibility of the immobilized polycyclic aromatic hydrocarbon degrading bacterial agent in the biological repair of marine oil spill .

【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：X55;X172

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