石油降解菌群的构建及其固定化技术研究

发布时间：2018-08-24 21:02

【摘要】：随着海洋石油的不断开采及远洋运输业的发展,海洋石油污染越来越突出,给海洋环境及人类的生存发展带来了极大的困扰,同时也造成了资源的极大浪费。治理石油污染势在必行。与物理和化学处理方法相比,生物处理技术由于安全、高效、经济、无二次污染、不需大型设备等优势,得到人们的重视。因此,利用微生物处理石油污染已成为当今世界的热点。本文主要研究寻求一种适合海洋的环境友好型的固定化菌剂来修复海洋的石油污染,主要包括柴油/原油降解菌的筛选、降解条件的优化、固定化载体的筛选、固定化菌群降解原油前后原油组分变化的分析和固定化菌群在实际海水中石油降解特性分析。得到的主要结论如下：(1)分别以柴油和原油为唯一碳源,从深圳市蛇口港和盐田港海域中筛选到50余株高效降解菌。针对柴油和原油,各选择5株石油降解率较高的菌株,通过多维复配试验筛选出降解率最高的1种组合,再通过正交试验优化各菌的接种量,最终构建了由C1-8、C2-10和C3-13组成的高效柴油降解菌群CQ1及由S1-38、S1-30和S2-13组成的高效原油降解菌群SQ1。通过单因素实验研究了环境因素对菌群降解柴油/原油的影响。结果表明：当温度、pH、转速和石油浓度分别为30℃、7.6、220 r/min和20 g/L时,菌群CQ1对柴油的降解率最高可达到60.5%,CQ1对柴油的降解随培养时间的增加有所升高,在培养3-9 d内增幅较为明显,7d时降解率即达到60%以上,此后柴油的降解率仍有小幅增长,但变化不大。当温度、pH、转速和原油浓度分别为30℃、7.6、200r/min和20 g/L时,菌群SQ1对原油的降解率可达73.52%,菌群SQ1对原油的降解率随培养时间的增加有所升高。(2)通过生理生化试验及16S rRNA基因序列分析对石油降解菌进行了初步鉴定,以柴油为唯一碳源筛选到的菌株主要分布于微杆菌属(Microbacterium)、假单胞菌属(Pseudomonas)、棒杆菌属(Corynebacterium)、苍白杆菌(Ochrobactrum)以及剑菌属(Ensifer)等。以原油为唯一碳源筛选到的菌株主要分布于副球菌属(Paracoccus)、拉布伦茨氏菌属(Labrenzia)、鞘氨醇盒菌属(Sphingopyxis)、假单胞菌属(Pseudomonas)以及迪茨氏菌属(Dietzia)等。其中,构成柴油降解菌群CQ1的3株菌C1-8鉴定为微杆菌(Microbacterium sp.)、C2-10剑菌(Ensifer sp.)C3-13变异棒杆菌(Corynebacterium sp.);构成原油降解菌群SQl的3株菌S1-30鉴定为变异棒杆菌(Corynebacterium sp.),S1-38为迪茨氏菌(Dietzia sp.), S2-13为拉布伦茨氏菌(Labrenzia sp.)。(3)通过对最佳载体的筛选、载体的投加量及固定化的时间的优化,初步研究了固定化SQ1对原油的降解特性。结果表明：秸秆和无纺布是菌群SQ1良好的固定化载体,秸秆和无纺布固定化SQ1对原油的降解率分别达到76.85%和73.08%,均高于单独投加SQl菌液。秸秆固定化SQ1中最佳的载体投加量和固定化时间分别为15.0g/L(干重)和36 h;无纺布固定化SQ1中最佳的载体投加量和固定化时间分别为15.0g/L(干重)和36h。2种固定化菌剂对原油的降解效果均随培养时间增加而有所升高,第9d时达到最高,分别为73.08%和76.85%。(4)通过施加不同的无机氮源(尿素、KNO3、(NH4) 2SO4、NH4Cl)和磷源(K2HPO3、KH2PO3),研究了固定化SQ1对实际海水中原油的去除效果及其降解特性。结果表明：对固定化SQ1(秸秆)降解原油促进作用最为明显的氮源为NH4Cl,磷源为KH2PO3,当NH4C1投加量为5.08g/L,KH2PO3投加量为1.14g/L时,原油降解率可达66.05%,高于不投加营养盐的处理53.42%。(5)通过GC-FID/GC-MS法分析了柴油或原油生物降解后,各烷烃组分的残留情况。结果表明,柴油生物降解体系中,菌群CQl能够降解大部分C11-C27之间的正构烷烃,其中对C21-C27等中链烷烃降解率可以达到100%,CQ1对各烷烃组分的去除情况显著好于构成菌群的单一菌株,表现出明显的协同作用。石油降解生物体系中,3株细菌和固定化SQl都能够降解C8-C35之间的正构烷烃,其中对C20-C24和C26-C35等中链、长链烷烃降解率可以达到100%。相比游离菌的SQ1,固定化SQ1对烷烃的降解效果更好,固定化菌群SQl对原油总烷烃的去除率为97.71%。其中,除了Pr、Py和C18,固定化SQ1对其余正烷烃的降解率是100%,显示出强大的烷烃降解能力。本研究为微生物法去除海洋油污的奠定了良好的理论基础,并提供了有效的菌株资源,其所制备的固定化菌剂在海洋溢油的生物修复中具备广阔的应用前景。
[Abstract]:With the continuous exploitation of offshore oil and the development of ocean transportation, the pollution of offshore oil is becoming more and more serious, which has caused great trouble to the marine environment and the survival and development of human beings, and also caused a great waste of resources. The advantages of high efficiency, economy, no secondary pollution and no need of large-scale equipment have attracted people's attention. Therefore, the use of microorganisms to treat oil pollution has become a hot spot in the world. The main conclusions are as follows: (1) Using diesel oil and crude oil as the sole carbon source, the crude oil was separately screened from Shekou Port and Yantian Port in Shenzhen. More than 50 strains of high-efficiency degrading bacteria were selected for diesel oil and crude oil. Five strains with high petroleum degrading rate were selected respectively. One combination with the highest degrading rate was screened out by multidimensional compound test. The inoculation quantity of each strain was optimized by orthogonal test. Finally, the high-efficiency diesel degrading bacteria CQ1 composed of C1-8, C2-10 and C3-13 and the groups of S1-38, S1-30 and S2-13 were constructed. The results showed that when the temperature, pH, rotational speed and petroleum concentration were 30, 7.6, 220 r/min and 20 g/L respectively, the highest degradation rate of CQ1 to diesel oil could reach 60.5%, and the degradation rate of CQ1 to diesel oil increased with the culture time. When the temperature, pH, rotational speed and crude oil concentration were 30, 7.6, 200 r/min and 20 g/L respectively, the degradation rate of crude oil by SQ1 could reach 73.52%, and the degradation rate of crude oil by SQ1 could reach 73.52%. (2) Petroleum-degrading bacteria were preliminarily identified by physiological and biochemical tests and 16S rRNA gene sequence analysis. The strains isolated from diesel oil were mainly distributed in Microbacterium, Pseudomonas, Corynebacterium and Ochrobactrum. The strains isolated from crude oil were mainly distributed in Paracoccus, Labrenzia, Sphingopyxis, Pseudomonas and Dietzia. Among them, the three strains of CQ1, C1-8, were identified as micro-organisms. Microbacterium sp., Ensifer sp., Corynebacterium sp. C3-13, three strains of SQl were identified as Corynebacterium sp., S1-38 as Dietzia sp., S2-13 as Labrenzia sp. (3) By screening the best carrier, the carrier was identified as Corynebacterium sp. The results showed that straw and non-woven fabrics were good immobilization carriers for SQ1, and the degradation rates of SQ1 immobilized on straw and non-woven fabrics were 76.85% and 73.08% respectively, which were higher than that of SQl immobilized on straw. The optimum carrier dosage and immobilization time were 15.0 g/L (dry weight) and 36 h, respectively. The optimum carrier dosage and immobilization time were 15.0 g/L (dry weight) and 36 h. The effect of immobilized SQ1 on the removal of crude oil from seawater was studied by applying different inorganic nitrogen sources (urea, KNO3, (NH4) 2SO4, NH4Cl) and phosphorus sources (K2HPO3, KH2PO3). When the dosage of KH2PO3 was 1.14g/L and 8g/L, the degradation rate of crude oil reached 66.05%, which was higher than that of the treatment without nutrients (53.42%). Among them, the degradation rate of C21-C27 and other medium-chain alkanes can reach 100%. The removal of each alkane component by CQ1 is significantly better than that by a single strain, showing obvious synergistic effect. Compared with free bacteria SQ1, immobilized SQ1 had better effect on the degradation of alkanes. The removal rate of total alkanes from crude oil by immobilized SQl was 97.71%. Among them, except Pr, Py and C18, the degradation rate of immobilized SQ1 was 100%, showing a strong ability to degrade alkanes. The immobilized microbial agents have broad application prospects in the bioremediation of marine oil spills.
【学位授予单位】：广东工业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;X55

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