石油降解混合菌的构建及其固定化修复污染土壤的研究

发布时间：2018-09-05 06:09

【摘要】：本研究利用前期已筛选的4株高效石油降解菌,采用响应面法构建混合菌并考察环境因素对石油降解混合菌降解石油效果的影响,分析石油降解单菌及混合菌降解产物,探讨石油烃的降解机制,并为筛选微生物提供理论基础。选取小麦秸秆、生物质炭和活性炭为吸附载体,通过吸附-包埋法对混合菌进行固定化用于室内模拟修复石油污染土壤,比较不同固定化载体土壤修复效果和微生物群落结构及分布特征。响应面优化混合菌配比结果表明,植生拉乌尔菌(Raoultella planticola)接种量为4%、粘质沙雷氏菌(Serratia marcescens)接种量为2%、蜡状芽孢杆菌(Bacillus cereus)接种量为2%、变栖克雷伯氏菌(Klebsiella variicola)接种量为2%,优化后的混合菌对石油降解率可达68.83%。混合菌对盐度和p H耐受范围广,最佳盐度0.5%,最佳p H 7.5。同时发现混合菌在5%盐度的条件下,5天后原油的降解率仍可达到40.37%,可以用于石油污染海水的修复。在以柴油为唯一碳源,等接种量培养条件下,单菌株对石油烃的降解率达到41.83%~54.87%,混合菌降解效率高于单菌株,达到64.27%。结合GC-MS分析结果,R.planticola和S.marcescens对C12~C16的直链烃碳源有降解作用;B.cereus和K.variicola对C12~C22的直链烃均有降解效果。4株单菌都可以在有氧条件下降解正十六烷,脱氢生成双键并引入羰基。B.cereus、K.variicola和混合菌还能进一步氧化十六烷生成不饱和羧酸酯并形成带甲基的支链。双键和羰基引入可能是正十六烷有氧条件下前期降解的一种新途径。采用小麦秸秆、生物质炭和活性炭3种载体对混合菌进行吸附包埋固定,结果表明固定化混合菌比游离态混合菌对石油的去除效果显著提高,可以在相对短的时间内达到相对高的去除率。生物质炭固定化混合菌对原油培养基5d降解效率高达99.76%。秸秆、生物质炭和活性炭空白载体5d原油吸附量分别为49.08%、61.48%、63.12%。充分证明固定化混合菌小球类似于吸附降解一体的微型反应器。扫描电镜图观察到生物质炭和活性炭小球内部具有大量孔隙的骨架结构,微生物在载体内部生长良好。固定化混合菌模拟修复石油污染土壤实验结果表明,固定化混合菌对原油的降解效果比游离菌好,且不同固定化载体修复效果有差异。经过42d的修复,灭菌组中秸秆固定化混合菌修复效果最好,降解率为54.92%,比单纯的游离混合菌提高了20%;未灭菌组中生物质炭固定化混合菌修复效果最好,降解率为54.19%,比游离菌提高了近30%。说明在没有土著菌群竞争情况下,秸秆通过增强土壤养分含量而增加土壤脲酶活性,对于提高混合菌石油降解率是显著影响条件;而在土著菌群存在的未灭菌组土壤中,生物质炭可以提供固定化混合菌最适宜的生长环境是显著影响条件,比秸秆提高养分对于修复土壤更重要。灭菌组秸秆固定化修复处理后期脲酶活性最高,脲酶活性最大值0.75mg?g-1?24h-1为空白对照组的3.26倍;未灭菌组生物质炭修复脲酶活性最高为1.15 mg?g-1?24h-1为空白对照组的2.05倍。土壤脲酶活性与固定化混合菌石油降解率呈正相关。土壤香侬-威纳指数与微生物多样性T-RFLP的分析表明,灭菌组固定化混合菌修复土壤种群多样性呈递减趋势,未灭菌组多样性先升高再下降;石油降解率和微生物多样性变化相吻合,石油降解率高的微生物多样性高。固定化混合菌是由厚壁菌门和变形菌门组成,在石油生物降解过程中起重要作用。在灭菌组和未灭菌组,生物质炭都是促进厚壁菌门和变形菌门生长的最优载体,使二者总和占土壤比例分别高达5.37%、3.73%。同时发现灭菌条件下,秸秆固定化载体能促进芽孢杆菌纲生长,使其占土壤比例高达1.35%;在未灭菌组,生物质炭载体最能促进?-变形菌纲生长,使其含量高达2.71%。对各样本的T-RFLP谱图进行聚类分析,结果发现灭菌组秸秆和未灭菌组生物质炭群落相似度高;生物质炭和活性炭群落相似度高;而未灭菌组秸秆固定化土壤与其它5组距离较远,群落相似度低。
[Abstract]:In this study, four strains of high-efficiency petroleum-degrading bacteria were used to construct the mixed bacteria by response surface methodology. The effects of environmental factors on the petroleum-degrading effect of the mixed bacteria were investigated. The degradation products of the single bacteria and the mixed bacteria were analyzed, and the mechanism of petroleum hydrocarbon degradation was discussed. Straw, biomass charcoal and activated carbon were used as adsorbent carriers to immobilize mixed bacteria for indoor simulated remediation of petroleum-contaminated soil. The effects of different immobilized carriers on soil remediation, microbial community structure and distribution characteristics were compared. Response surface methodology (RSM) was used to optimize the proportion of mixed bacteria. The Inoculated Amount of anticola, Serratia marcescens, Bacillus cereus, Klebsiella variicola and Klebsiella variicola was 4%, 2% and 2%, respectively. The oil degradation rate of the optimized mixed bacteria was 68.83%. The mixed bacteria had wide tolerance to salinity and P H, and the optimum salinity was 0.5% and the best was the highest. At the same time, it was found that under the condition of 5% salinity, the degradation rate of crude oil could still reach 40.37% after 5 days, which could be used for the remediation of oil-polluted seawater. According to the results of GC-MS analysis, R. planticola and S. marcescens could degrade the carbon source of C12-C16, B. cereus and K. variicola could degrade the linear hydrocarbons of C12-C22. All the four strains could degrade n-hexadecane under aerobic conditions, dehydrogenate to form double bonds and introduce carbonyl. B. cereus, K. variicola and mixed bacteria could further oxygen. Hexadecane is converted into unsaturated carboxylic esters and methyl-branched chains are formed. The introduction of double bonds and carbonyl groups may be a new way of pre-degradation of n-hexadecane under aerobic conditions. The removal efficiency of crude oil by immobilized mixed bacteria was 99.76%. The adsorption capacity of crude oil by straw, biomass carbon and activated carbon was 49.08%, 61.48% and 63.12% respectively. Scanning electron microscopy (SEM) showed that there were a lot of pores in the biochar and activated carbon spheres, and microorganisms grew well in the carrier. Immobilized mixed bacteria simulated the remediation of oil-contaminated soil. The results showed that the immobilized mixed bacteria had better degradation effect on crude oil than free bacteria, and there was no significant difference between immobilized mixed bacteria and free bacteria. After 42 days of repair, the straw immobilized mixed bacteria had the best repair effect, the degradation rate was 54.92%, which was 20% higher than that of the pure free mixed bacteria; the biochar immobilized mixed bacteria had the best repair effect, the degradation rate was 54.19%, which was nearly 30% higher than that of the free bacteria. Under the competition of Aboriginal microflora, straw increased soil urease activity by increasing soil nutrient content, which had a significant effect on improving the oil degradation rate of mixed bacteria; while in the unsterilized soil of Aboriginal microflora, biomass char could provide the most suitable growth environment for Immobilized Mixed bacteria, which was significantly affected by straw extraction. The highest urease activity was 0.75 mg? G-1? 24h-1 in the sterilized group, which was 3.26 times higher than that in the blank control group, and 1.15 mg? G-1? 24h-1 in the non-sterilized group, which was 2.05 times higher than that in the blank control group. The Shannon-Wiener index and microbial diversity T-RFLP analysis showed that the biodiversity of the immobilized mixed bacteria in the sterilized group tended to decrease, the biodiversity of the unsterilized group increased first and then decreased, and the biodegradation rate and microbial diversity were consistent, and the biodiversity of the high biodegradation rate was diverse. Immobilized mixed bacteria are composed of thick-walled phylum and deformed phylum, which play an important role in the process of petroleum biodegradation. In both sterilized and non-sterilized groups, biomass char is the best carrier to promote the growth of thick-walled phylum and deformed phylum, making the total proportion of them in soil as high as 5.37% and 3.73% respectively. Straw immobilized carriers could promote the growth of Bacillus spp. and accounted for 1.35% of the soil, while biomass carbon carriers could promote the growth of Proteus spp. and the content of Proteus spp. was as high as 2.71% in the non-sterilized group. The community similarity of charcoal and activated carbon was high, while the immobilized soil of non-sterilized group was far from the other five groups, and the community similarity was low.
【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X53;X172

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