【作者】 姚伟静；

【导师】 霍丹群；

【作者基本信息】 重庆大学 ， 微生物学， 2007， 硕士

【摘要】 随着现代石油工业的发展,石油的环境污染问题越来越突出,微生物修复技术被公认为是治理石油污染的较好方法,研究发现,获得高效、广谱、稳定、适应性强的石油降解菌株,以及配套治理工艺是利用该技术进行石油污染治理的关键。该论文从长期被石油烃类污染的土壤中经过富集培养、驯化分离筛选出一株降解能力超过60%的菌株Q3作为研究的优良石油烃降解菌株。其主要研究内容及实验结果如下:(1)通过对菌株Q3进行柴油降解能力的评价,及形态学观察和部分生理生化试验鉴定,该菌为假单胞菌属。(2)将菌株Q3进行三种方法的紫外诱变实验表明,用氯化锂诱导和经15W紫外灯照的复合诱变方法得到的菌株Q3-3对石油烃的降解率达71.2%,其最佳照射时间为30S,最高存活率达8%。诱变菌株对不同浓度石油烃的降解效果实验表明,在石油浓度为10000mg/L时,菌株Q3-3和菌株Q3降解率分别为35.7%和23.6%,诱变后菌株比诱变前提高12.1%,利用紫外复合诱变能有效的选育出高效石油烃降解菌株。(3)应用响应面方法对培养基进行优化,全因子实验能对各因子的效应进行评价并能有效地找出了影响石油烃降解率的主效:分别为硝酸氨(氮源)和磷酸盐(磷源)。中心组合实验能快速的对主要影响因素进行优化,并找出了最佳值,分别为硝酸氨的最佳质量浓度在1.2g/L,磷酸盐的最佳质量浓度在0.35g/L。菌株在优化培养基中对石油烃降解率和原培养基中对石油烃降解率分别为76.6%、71.2%,优化培养基对石油烃的降解率比原培养基提高了5.4%。(4)对影响降解菌株生长和石油降解效率的相关因素进行单因素研究,试验结果表明,微生物降解石油烃的最适条件为:接种量为10%,温度为28℃,初始pH为7-8,摇床速度为180r/min。在最适生长条件下,菌株生长和石油烃降解具有一致性。(5)表面活性剂Tween80、SDS、APG、JFC对石油烃生物降解的影响及机理研究。菌株的生长随时间的变化表明:在四种表面活性剂浓度为200mg/L时,对菌株的生长没有造成影响,而当四种表面活性剂浓度为600mg/L时菌株的生长均出现了延迟期变长,生长迟缓的现象。从经济实用的角度考察复配表面活性剂对石油烃生物降解的增溶作用表明:三种非离子表面活性剂和阴离子表面活性剂各比例复配制剂对石油烃的降解率均有不同程度的提高,最佳配比为Tween-80:SDS=1:1; JFC:SDS=2:1;APG:SDS =4:1。混合表面活性剂对石油烃产生协同增溶作用的主要机理:阴离子表面活性剂与非离子表面活性剂复配形成混合胶束和混合吸附层,使原来带负电荷的表面活性剂离子间的排斥作用减弱,胶束更易形成,复配表面活性剂的CMC值较单一表面活性剂有较大程度的降低,使得较低的表面活性剂浓度就能够对石油烃类污染物的降解起到促进作用。规则溶液理论适合于本研究,利用规则溶液理论计算复配表面活性剂(Tween-80:SDS=1:1)的理论临界胶束浓度为6.3mg/L与实际测定的数据相符。更多还原

【Abstract】 The contamination caused by petroleum becomes increasingly serious as the development of petroleum industry, which not only destroys ecological balance, but also threatens human health. Efforts have been made to find better way in resolving this problem by more and more countries. As a stable, economic and reliable way, microbiological remediation technology has been widely accepted to remediate oil in soil in which oil-degrading microorganisms are of great importance.In this paper, several oil-degrading strains have been isolated from oil-contaminated environment. Identifying work was conducted and culture condition was optimized to improve the degradation ability of the strain. The results are as follow:One of stain which we name it as Q3 has been identified to be Pseudomonas. as Observing the morphological characteristics, and doing some physiological and biochemical experiments, one oil-degrading strain,named as Q3, has been identified as Pseudomonas. It can degrade over 60% oil in the soil.The experimental results of ultraviolet irradiation indicate that the higher degradation rate can be obtained by lithium chloride induction and 15W uviol lamp shining. The optimum shining time is 30s, and the highest survival rate reaches 8%.The degradation rate of the mutated strain Q3-3 is 35.7% when the concentration of petroleum is 10000mg/L. Compared with 23.6% of Q3, it is raised by 12.1%. That is to say the high effective oil-degrading strain can be selected by ultraviolet complex induction of mutation.Experiments also prove that response surface methodology is an effective way to optimize the constitute of medium. Ammonium nitrate and phosphate as two major factors that influence the degradation rate are determined by doing full factorial experiments. And their optimum concentrations are also determined with central assembly experiments. They are 1.2g/L and 0.35 g/L, respectively. The degradation rate of strains growing in the optimized medium and that of strains growing in the original medium are 76.6% and 71.2%, respectively. That is the rate is raised by5.4%. And the optimum condition for the oil degradation by Q3 in shaking flask is: the quantity of inoculation is 10%, the initial pH of the incubation media is 7-8, the incubation temperature is 28℃, and the shaker speed is 180r/min.After respectively adding surfactants Tween80, SDS, APG and JFC, conditions of strain growth indicate that it is no effective when the concentration is 200mg/L, but when it increases to 600mg/L, the lag phases of these strains all grow longer and the growth retardation appears .Different matching of three nonionic wetting agents with anionic surfactant has different effect on the degradation rate. The optimum matching is: Tween-80: SDS=1:1, JFC: SDS=2:1, APG: SDS =4:1.The major mechanism of solubilization of mixing surfactants is: mixing micelle and adsorption layer weaken the exclusion effect among negative ions. That makes micelle forming easy. Compared with the CMC value of single surfactant, that of surfactant mixtures is largely lowered. So surfactant mixtures with lower concentration can promote oil-degrading. he theory of regular solution, that is applied to bibasic surfactant non-ideal solution, is also suitable for this research.更多还原