發(fā)布時(shí)間：2018-04-05 01:14

  本文選題：海洋溢油　切入點(diǎn)：多環(huán)芳烴降解菌　出處：《中國(guó)海洋大學(xué)》2014年博士論文

【摘要】：隨著國(guó)家對(duì)能源需求的增加，石油在其開采、運(yùn)輸以及加工煉制過程中大量進(jìn)入環(huán)境，特別是海洋溢油事故頻繁發(fā)生，嚴(yán)重威脅了海洋生物、生態(tài)環(huán)境以及人類健康。針對(duì)風(fēng)化后難以被降解，且具有“三致”作用的多環(huán)芳烴，以及低溫、營(yíng)養(yǎng)物匱乏、高鹽等特殊海洋環(huán)境的難題，本論文篩選多環(huán)芳烴降解菌，初步探討了石油烴特別是多環(huán)芳烴的吸附、攝取及降解機(jī)理或規(guī)律，同時(shí)利用微生物固定化技術(shù)，制備固定化微生物，模擬海洋溢油的生物修復(fù)過程，強(qiáng)化去除多環(huán)芳烴，得出以下結(jié)論： （1）從勝利油田石油污染污泥中篩選分離出一組被命名為PH系列的多環(huán)芳烴降解菌（PH混合菌），，其原油降解率為60%，經(jīng)鑒定： PH-1、PH-2、PH-3和PH-4分別隸屬于假單胞菌屬（Pseudomonas sp., KF113575），芽孢桿菌屬（Bacillus sp., KF113576），蒼白桿菌屬（Ochrobactrum sp., KF421109）和假單胞菌屬（Pseudomonas sp., KF113577）。 （2）PH混合菌降解多環(huán)芳烴及原油的最適宜環(huán)境條件不同。萘：濃度800mg/L，pH=7.5，NaCl10g/L，溫度35oC；菲：濃度40mg/L，pH=7，NaCl5g/L，溫度30oC；芘：濃度100mg/L，pH=7，NaCl15g/L，溫度30oC；原油：濃度3g/L，pH=8，NaCl7g/L，溫度30oC。在萘、菲、芘和原油各自最佳條件下的生物降解率分別為：80%、30%、56%和48%。 （3）PH混合菌對(duì)重金屬離子具有較大的容忍性（最大容忍濃度分別為6.2mM Cu2+，2.7mM Zn2+，9.5mM Pb2+），且在重金屬離子和污染底物共存的情況下，其對(duì)萘、菲、芘和原油的降解分別能達(dá)到53%、21%、32%、44%。 （4）共代謝底物葡萄糖、α-乳糖、可溶淀粉、酵母粉、脲中，α-乳糖是PH混合菌降解石油烴（萘、菲、芘、原油）的最佳共代謝底物，其萘、菲、芘和原油生物降解率分別達(dá)95%、68%、60%、40%。 （5）活性混合菌體表面吸附和體內(nèi)攝取石油烴的穩(wěn)定性順序?yàn)椋狠练啤周旁�；加熱致死混合菌體吸附多環(huán)芳烴的量為常量，而吸附原油的量為變量，且逐漸降低。 （6）PH混合菌的疏水性35%，表現(xiàn)出較強(qiáng)的降解活性和乳化性能；其疏水性與底物濃度、毒性和菌濃度有關(guān)；PH混合菌分泌的膜周、膜內(nèi)和胞外酶對(duì)不同的污染底物（萘、菲、芘、原油）的代謝性能不同。 （7）推測(cè)細(xì)菌可能在降解有毒污染物（原油及多環(huán)芳烴）過程中通過改變細(xì)胞膜及細(xì)胞壁的通透性來攝取污染物，細(xì)胞內(nèi)部主要通過細(xì)胞質(zhì)（包括細(xì)胞器如核糖體和內(nèi)部酶等）來降解污染物。 （8）核桃殼和半焦炭對(duì)萘、菲、芘、原油的吸附符合擬二階動(dòng)力學(xué)模擬方程式，且濃度越小擬合性越好；不同載體材料對(duì)固定化微生物制備的影響因素重要性不同：以核桃殼為吸附劑制備的固定化微球，各因素影響重要性順序及最優(yōu)配比為：核桃殼（20%） CaCl2（1%） α-乳糖（0.1%）海藻酸鈉（6%）；半焦炭為吸附劑：α-乳糖（0.3%）半焦炭（20%）海藻酸鈉（6%） CaCl2（1%）；活性炭為吸附劑：α-乳糖（0.5%） CaCl2（5%）活性炭（20%）海藻酸鈉（4%）；固定化降解菌經(jīng)過5d的生物降解，其原油降解率48%較游離菌高出21%。 （9）混合菌細(xì)胞存在羧基、氨基和磷酸鹽等官能團(tuán)；生物降解污染底物過程中要經(jīng)過吸附、攝取等階段；半焦炭經(jīng)固定化處理后孔隙度變大，更易于微生物吸附。 （10）生物修復(fù)過程中微生物主要消耗硝基氮；人工海水較自然海水的生物降解效果好；固定化菌較游離菌，五種烷基化芳烴降解率高，經(jīng)過28d自然海水中對(duì)萘系列的降解率高于50%，菲、二苯并噻吩、芴和系列高于30%，總芳烴的降解率達(dá)63%；為取得好修復(fù)效果，應(yīng)及時(shí)添加菌劑、磷源和硝基氮源。 本論文研究結(jié)果對(duì)了解微生物如何吸附、攝取與降解污染底物具有重要意義，并為固定化多環(huán)芳烴降解菌劑應(yīng)用于海洋溢油的生物修復(fù)的可行性提供理論與技術(shù)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 , 偽 - lactose , soluble starch , yeast powder , urea , 偽 - 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 % ) 偽 - lactose ( 0.1 % ) sodium alginate ( 6 % ) ;
Semi - coke as adsorbent : 偽 - lactose ( 0.3 % ) semi - coke ( 20 % ) sodium alginate ( 6 % ) CaCl2 ( 1 % ) ;
Activated carbon as adsorbent : 偽 - 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 .

【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：X55;X172

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