【作者】 杨国君；

【导师】 李华钟； 华兆哲；

【作者基本信息】 江南大学 ， 环境工程， 2004， 硕士

【摘要】 烃类的摄取是石油污染土壤和水体生物修复过程中的关键,目前主流的两种烷烃被摄取模式是:(1)微生物细胞与比其小得多的被溶解、似溶解的烷烃颗粒作用,然后进行摄取,即微生物被诱导产生生物表面活性剂,通过生物表面活性剂的增溶作用摄取烷烃;(2)微生物与比细胞大的烷烃颗粒直接接触,进而进行摄取,即微生物以直接接触油滴方式摄取烷烃。研究发现自然界中烷烃的摄取绝大多数采用第一种模式。第二种模式研究较少,还没有形成共识。本论文将研究证明烷烃被摄取时,两种不同摄取模式的存在,主要研究内容如下:1.根据微生物在油水两相的分布特点,从被石油污染的土壤中筛选得到一株亲水性很强的细菌和一株亲油性很强的细菌,通过生理生化实验和16S rDNA鉴定均为铜绿假单胞菌,亲水性很强的铜绿假单胞菌表示为P.aeruginosa S,亲油性很强的铜绿假单胞菌表示为P.aeruginosa Y。2.优化了两株菌在煤油培养基中的生长条件。在单因素实验的基础上,设计L9(34)正交实验,进行极差和方差分析。结果表明影响P.aeruginosa Y菌体生长量的因素主次顺序为:煤油添加量,天然有机物的添加,氮源和接种量。P.aeruginosa Y生长的优化培养基组成为:氮源为1.5%的尿素,煤油量为14%,添加0.1%的蛋白胨,pH7.0,其它同无机盐培养基。优化培养条件为:接种量10%,装液量50mL/250mL,37℃下200r/min摇瓶培养。影响P.aeruginosa S菌体生长量的因素主次顺序为:温度,煤油添加量,天然有机物的添加和接种量。温度是影响P.aeruginosa S菌体生长量的显著性因素。P.aeruginosa S生长的优化培养基组成为:煤油量为6%,添加0.1%的牛肉膏,pH7.0,其它同无机盐培养基。优化培养条件为:接种量7%,装液量30mL/250mL,32℃下200r/min摇瓶培养。优化后P.aeruginosa Y的菌体量从1.9g/L提高到3g/L,P.aeruginosa S的菌体量从3.3g/L提高到4.34g/L。3.研究了两菌株对烷烃的降解,它们对混合烷烃的降解效果要比单一烷烃好。分析了它们对十六烷的降解途径,GC分析胞内存在十六烷和十六酸,GC分析胞外存在脂肪酸,HPLC分析胞外存在小分子有机酸,得知十六烷末端甲基通过单加氧酶的作用生成十六醇,然后通过两步脱氢作用先后生成十六醛和十六酸,十六酸按照β-氧化途径逐步脱去两个分子碳后变成短链酸,短链酸进一步生成乙酰辅酶A,进入三羧酸循环,最终彻底被降解成二氧化碳和水。4.综上实验,研究探讨了两株菌摄取烷烃的不同模式。在生长方面:两株菌在LB培养基中生长时,在达到生长稳定期后A600值没有差异,而在煤油培养基中生长时生长速度有显著差别。在细胞表面性质方面:P.aeruginosa Y的亲油性很好,MATH值平均约为0.994,P.aeruginosa S的亲油性很差,MATH值平均约为0.007。在溶液乳化方面: P.aeruginosa Y培养基的表面张力降幅比P.aeruginosa S小14mN m-1左右;P.aeruginosa Y的菌体主要集中在油相,水相没有乳化现象,P.aeruginosa S的菌体主要集中在油水<WP=6>界面,水相中乳化程度很高;测菌液的乳化力,P.aeruginosa Y接近零,P.aeruginosa S约为25%;P.aeruginosa Y在添加不同浓度的表面活性抑制剂EDTA后生长没有明显差异,而P.aeruginosa S则明显受到抑制。从而推知两菌株对烷烃具有不同的摄取机制,P.aeruginosa Y对烷烃的摄取主要是以直接接触油滴方式摄取烷烃的,而P.aeruginosa S对烷烃的摄取依赖于其自身合成的表面活性剂,烷烃的摄取是通过生物表面活性剂的增溶作用完成的。更多还原

【Abstract】 Hydrocarbon uptake is key factor in the bioremediation of soil and water polluted by petroleum. Two modes of hydrocarbon transport to microbial cells are generally considered: (1) interaction of cells with “solubilized/pseudosolubilized” or “accommodated” hydrocarbon droplets much smaller than the cells, it means microbes uptake the hydrocarbon emulsified with the biosurfactant produced by the cells (2) direct contact of cells with large hydrocarbon drops, it means microbes uptake hydrocarbon by the direct contact. It was found that major microbes in natural uptake hydrocarbon with the first mode. As to the second mode, the research of it was little and the existence of it was not confirmed. The existence of two modes of hydrocarbon uptake will be verified in this paper, the main study results are as follows:1. A hydrophilic bacterium and a hydrophobic bacterium were screened from soil polluted by petroleum according to the distribution character in oil-water system. They both belonged to Pseudomonas aeruginosa identified by morphological, biological tests and 16S rDNA. The hydrophilic bacterium was named as P.aeruginosa S; the hydrophobic bacterium was named as P.aeruginosa Y.2. The flask culture conditions for growth of P. aeruginosa in kerosene medium were investigated and optimized. The factors affected the growth of P.aeruginosa Y from major to minor were: kerosene volume, natural organism, nitrogen source, inoculation. The optimal kerosene medium for growth of P.aeruginosa Y was: CO(NH2)2 1.5g/L, kerosene 14%, peptone 0.1g/L, pH7.0 in mineral salt medium. The optimal culture conditions of P.aeruginosa Y were: inoculation 10%, shaking speed 200 r/min in 50mL/250mL flask, growth temperature 37℃. The factors affected the growth of P.aeruginosa S from major to minor were: temperature, kerosene volume, natural organism, inoculation. The optimal kerosene medium for growth of P.aeruginosa S was: kerosene 6%, beef extracts 0.1g/L, pH7.0 in mineral salt medium. The optimal culture conditions of s were: inoculation size 7%, shaking speed 200 r/min in 30mL/250mL flask, growth temperature 32℃. After optimum, the dry cell weight of P.aeruginosa Y increased from 1.9g/L to 3.0g/L and the dry cell weight of P.aeruginosa S increased from 3.3g/L to 4.34g/L.3. The biodegradation of hydrocarbon by two bacteria was studied. The mixed hydrocarbon was better biodegraded than single hydrocarbon by two bacteria. The biodegradation process of hexadecane was analyzed, the intracellular hexadecane and hexadecanoic acid were checked by GC, the extracellular fatty acid was checked by GC, and the extracellular organic acids were checked by HPLC. It could be known that hexadecane turned to hexadecanoic alcohol by momoterminal, hexadecanoic alcohol was oxygenated to hexadecanoic aldehyde and hexadecanoic acid, hexadecanoic acid was β-oxygenated to short-chain fatty acids, then the fatty acids were degraded into AcCoA, which switched to TCA,and was degraded to carbon dioxide and water in the end.4. According to the former experiments, the different modes of hydrocarbon uptake were studied in two bacteria. Growth: they both grew well in LB medium and the value of A600 was almost same in stationary phase, however, the growth velocity of them was different in kerosene medium, the growth of P.aeruginosa S was better than P.aeruginosa Y. Hydrophilicity: P.aeruginosa Y owned good hydrophobic character, the value of MATH was 0.994, P.aeruginosa S owned good hydrophilic character, the value of MATH was <WP=8>0.007. Emulsification: the decrease of surface tension in P.aeruginosa Y kerosene medium was less 14 mN m-1 than that in P.aeruginosa S kerosene medium; the main cells of P.aeruginosa Y were aggregated in oil phase, there was no obvious emulsification in water phase, the main cells of P.aeruginosa S were aggregated in oil-water phase, there was apparent emulsification in water phase; the emulsified ability was measured, the value of P.aeruginosa Y was nearly zero and the value of P.aeruginosa S was about 25%; the growth ability of更多还原