【作者】 钱欣平；

【导师】 阳永荣；

【作者基本信息】 浙江大学 ， 化学工程， 2002， 博士

【摘要】 生物表面活性剂无毒，可以生物降解，对环境影响很小，具有较低的临界胶束浓度和较高的表面活性，因此是合成表面活性剂的理想替代品。生物表面活性剂能够促进微生物对难溶有机物的利用和降解。本文的目的主要是利用分泌生物表面活性剂的微生物来降解有机污染物。 以水溶性底物甘油作为碳源，选用铜绿假单胞杆菌合成鼠李糖脂的发酵体系作为研究对象。采用多种流加方式进行发酵，实验分析细胞浓度、碳源浓度和氮源浓度对发酵过程的影响。当摇瓶培养基中含有3％甘油和4g/L硝酸钠时，接种后60h细胞浓度达到最大值4.08g/L，糖脂最终浓度为15g/L，糖脂对于甘油的产率为0.35g/g。 实验表明，氮源是细胞生长的促进因子，碳源是细胞生长的必需因子。糖脂的合成属于部分生长伴随型。底物甘油的消耗既用于细胞生长又用于产物合成。建立非结构化动力学模型，采用龙格-库塔法解方程，通过遗传算法优化模型参数。模型计算值与实验值十分吻合。分析模型，较低的细胞生长速率有利于糖脂的合成，当细胞生长速率为0.051g/L/h时，糖脂比合成速率达到最大值0.088/h。 以菜油作为唯一碳源，分析油浓度和摇床转速等对发酵过程的影响。测定鼠李糖脂作为生物表面活性剂的基础理化性质，从胶束形成的角度分析它促进有机物乳化和溶解的性能。鼠李糖脂的临界胶束浓度为58mg/L，实验条件下对菜油的增溶度约为0.22g/g，计算鼠李糖脂混合物的HLB值在8～16的范围内。在表面活性剂的作用下，发酵液的乳化能力和乳化稳定性得到提高。当菜油含量达到10％时，发酵液的乳化能力开始下降。加入少量电解质和正丁醇等有机物可以降低CMC，提高生物表面活性剂的效能。 采用不同型式的生物反应器和搅拌桨进行油发酵实验。由于生物表面活性剂的发泡特性，实验所用气升式反应器中有机物的降解结果不是十分理想。研究搅拌反应器中的流体力学和传递特性，分析操作条件对混合和传质过程的影响。由于生物表面活性剂的发泡特性限制了通气量的调节，因此搅拌转速是影响氧气体积传质系数和油滴直径的重要因素，进而影响着微生物的生长。11 浙江大学博士学位论文 研究微生物降解有机物的发酵过程，大部分生物表面活性剂是在稳定生长期合成的，因此它属于次级代谢产物。生物表面活性剂可以通过促进有机物的分散和溶解来刺激细胞对油的吸收利用。搅拌速率和油含量对降解过程影响较大，说明油滴粒径是一关键指标，细胞生长主要发生在油滴表面。 建立可以反映生物表面活性剂特殊作用，并成功模拟有机物降解过程的机理性动力学模型。模型反映两种微生物摄取有机物的方式，区分生物表面活性剂的乳化作用和增溶作用。模型是包含9个参数，涉及4个变量的微分方程组，描述了细胞生长、表面活性剂合成以及有机物的摄取过程。模型中假设乳化油的消耗主要用于细胞生长，而维持能来源于细胞对增溶在胶束中的油的代谢过程。 通过拟合间歇式搅拌反应器中的发酵数据，获得模型参数。建立的模型可以很好地解释实验现象。通过对模型的分析，证实在本实验体系中，生物表面活性剂的主要作用是降低油滴直径，促进细胞在油／水界面上的吸附。 尝试各种方式的双碳源发酵工艺。当培养基中同时含有甘油和菜油两种碳源时，细胞主要通过代谢菜油进行生长和繁殖，甘油对细胞生长的贡献不大。但是，添加甘油，使菜油的增溶状况得到明显增强，发酵液的乳化能力和乳化稳定性得到显著提高，同时菜油的降解率提高至 80％～90％。 最后，针对该项利用生物表面活性剂特殊功能的生物治理技术，考察其实际应用效果。实验结果，废油对微生物活性的影响较小。采用简单的操作工序，便可以取得理想的降解效果，其中油炸废油的降解率达到 94．8％，降解速率为 1．59g／Lth。更多还原

【Abstract】 Biosurfactants were non-toxic, natural biodegradable products, offered the advantages of little environmental impact. Biosurfactants showed low critical micelle concentrations and high surface activities and were, therefore, promising substitutes for synthetic surfactants. Biosurfactants had the potential for enhancing the bioavailability and biodegradation of insoluble hydrocarbons. This paper aimed to contribute to the use of biosurfactants producing microorganism for the degradation of organic contaminants.The production of rhamnolipids by pseudomonas aeroginosa grown on water-miscible substrates glycerol was studied. To investigate the influences of cell concentration, carbon source concentration, nitrogen source concentration, etc. on the fermentation process, we ran fermentations with different feeding profiles. In a shake flask medium containing 3% glycerol and 4 g/1 nitrate, the biomass concentration reached a maximum of 4.08 g/1 after 60 h cultivation, a final concentration of rhamnolipids over 15 g/1 was reached, giving a production yield of rhamnolipids on glycerol of 0.35 g/g.Evidence indicated that cell growth rate could be enhanced by adding nitrogen source, and the carbon source was the limiting nutrient of the cell growth. The rhamnolipids were produced in a partly growth-associated mode. The glycerol was used in both cell growth and rhamnolipids production. An unstructured kinetic model for biosurfactant production was proposed. The model parameters were estimated using genetic algorithms, coupled with a Runge-kutta method for the interpretation of integral data. The correspondence between experimental data and model prediction was good. Analyzing the model, lower cell growth rate was favorable for rhamnolipids production. The specific rhamnolipids production rate reached a maximum 0.088 /h when the cell growth rate was 0.051 g/l/h.A fermentation using vegetable oils as sole carbon was established, the influences of oil volume percentage and the rotational speed of the shaker werestudied. Some important basic physico-chemical characteristics of the rhamnolipids were measured. Through analyzing the micellar formation, evaluating the emulsionfication ability and the pseudosolubilization ability of rhamnolipids. The rhamnolipids exhibited a low critical micelle concentration of 58 mg/1. Its miceller solubility ratio for the vegetable oil was 0.22 g/g. Its hydrophile-lipophile balance was in the range of 8-16. The emulsifying power and the stability of the whole broth were increased under the effect of rhamnolipids. When the oil volume percentage reached 10%, the emulsification capacity decreased. Some salt and hydrocarbon could reduce the CMC, leading to higher effectiveness of the rhamnolipids.Various bioreactor systems and impeller types were used in the heterogenous cultivation. Because of the problems with foaming caused by the biosurfactant during the process, the results of hydrocarbon biodegradation in the air-lift fermentators in consider were not perfect. Fluid dynamic and mass transfer characteristics of the stirred tank reactor were studied. The influences of operating conditions on mixing and mass transfer processes were researched. The foaming phenomenon limited the regulation of the air flow rate, so the stirring speed was the crucial operating factor for the volumetric oxygen mass transfer coefficient and the oil drop diameter, which was important for the microbial growth.The fermentation process for the biodegradation of hydrocarbon was investigated. The profile of biosurfactant production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. It was considered that biosurfactant stimulated cell growth on oil by enhancing the dispersion and the solubility of hydrocarbon. The biodegradation was strongly influenced by the stirring speed and the oil volume percentage, so it was concluded that the oil droplet size was an important index, and the cell growth mainly occurred at the oil droplet surface.A mechanistic kinetic model更多还原