【作者】 吴苏喜；

【导师】 官春云；

【作者基本信息】 湖南农业大学 ， 作物学， 2007， 博士

【摘要】 当前,生物柴油作为环境友好、可以再生的清洁液体燃料之一,正倍受世界各国的关注和开发利用。生物柴油的生产原料主要是植物油脂。油菜由于具有许多独特的优势而成为我国发展生物柴油比较理想的原料油来源。在生物柴油的各种生产方法中,生物酶法最有前景。但现已开发的生物酶法大多存在生产成本高、酶寿命短、反应速度慢和转酯率低等瓶颈问题而难以实现产业化。因此,急需开发具有产业化前景的生物柴油酶促合成新技术。本论文从降低成本、改善酶反应体系入手,以菜籽油为原料,先建立了能快速、准确地判断生物柴油合成反应程度的气相色谱分析法,然后循序渐进地研究了有机溶剂-水双液相体系、有机溶剂微水体系以及无溶剂体系中酶促菜籽油醇解合成生物柴油的机理和工艺条件,继而创新性地开发出了醇解催化酶再生技术和菜籽油酶促交酯化技术,经过精制后获得了符合有关标准的生物柴油产品。1.建立了快速准确测定油菜生物柴油合成反应程度的气相色谱法油菜生物柴油的主要成分是棕榈酸甲酯、硬脂酸甲酯、油酸甲酯、亚油酸甲酯、亚麻酸甲酯、花生一烯酸甲酯和芥酸甲酯。实验表明,它们具有良好分离效果的气相色谱条件是:使用HP-innowax毛细管色谱柱（30m*0.25mm*0.25μm）,初始柱温170℃并保持0.5min,然后以5℃/min升温至200℃,再以15℃/min升温至240℃并保持5min。在此条件下以十三酸甲酯为内标,能快速准确地测得产品中各脂肪酸甲酯的含量和反应程度。2.研究了有机溶剂-水双液相体系中菜籽油酶促醇解的机理与工艺条件正交试验结果表明,在有机溶剂-水双液相体系中,Lipozyme TLIM酶比Novozym 435酶更有效地催化菜籽油的醇解反应;在醇油摩尔比4:1、温度40℃、甲醇分三次加入时,其最佳醇解反应条件是:溶剂（石油醚）3mL/g、TLIM酶7%（w/w）、水20%（w/w）、时间18h;醇解反应程度与各影响因素之间均存在高度显著的三阶多项式回归关系;油脂的醇解反应伴随有油脂水解负反应发生,使得反应转酯率最多只有75%左右。3.研究了有机溶剂微水体系中菜籽油酶促醇解的机理与工艺条件以Lipozyme TLIM酶为催化剂、以不同质量等级的油脂为原料、以不同短碳醇为酰基受体,研究了有机溶剂微水体系中菜籽油酶促醇解生产生物柴油的机理和工艺条件。正交实验结果表明,油脂的醇解反应伴随有油脂水解反应和脂肪酸酯化反应;四级油是比一级油、二级油和三级油更好的生物柴油原料油,乙醇是比甲醇更好的酰基受体;其最佳反应条件是:以四级油为原料、添加20%的Lipozyme TLIM酶、乙醇（醇油摩尔比4:1）分三次均量加入、于40℃下反应10h,此时的转酯率达到86.2%。4.创立了无溶剂体系中菜籽油醇解催化酶再生技术,并优化了工艺条件以四级菜籽油为原料,考察了无溶剂体系中酶种类、短碳醇种类、温度、时间以及酶再生剂种类等对醇解反应程度和酶活稳定性的影响。结果表明,固定化脂肪酶Lipozyme TLIM比Novozym 435表现出更好的醇解催化行为,乙醇是比甲醇具有更好综合优势的酰基受体,ZSJ1是比ZSJ2具有更好再生效果的酶活再生剂,30℃最有利于酶活激发并维持酶活稳定的适宜温度。对反应系统中的酶进行定期再生处理,可使反应时间缩短到9h,此时的酶促醇解转酯率达到90%以上,而且酶被重复应用15批次后的相对活力仍保持在96%以上。5.创立了菜籽油酶促交酯化制备生物柴油的新技术基于菜籽油酶促醇解的转酯率不够高、酶活稳定性不够理想、酶再生操作麻烦,创立了菜籽油酶促交酯化制备生物柴油的新技术。先考察了酶种类、酶量、底物摩尔比、温度、时间对交酯化反应的影响,然后应用二次回归正交旋转组合试验法对酶量、底物摩尔比、温度、时间等因素进行了优化设计,回归模型为:Y=91.9417+1.1125X₁+0.5458X₃—2.6490X₁²—3.3740X₂²—2.7990X₃²—2.7490X₄²研究结果表明,在交酯化反应体系中,固定化酶Lipozyme TLIM比LNovozym435表现出更好的催化行为;四级菜籽油酶促交酯化的最佳工艺条件是:40℃、Lipozyme TLIM酶量40%（w/w）、底物摩尔比14:1、反应时间12h/批;单批反应的交酯化率达91.7%,而且酶可以一批接着一批地使用而很少发生酶活损失现象,从而为酶法生物柴油产业化提供技术依据。6.研究了油菜生物柴油粗品的碱炼精制工艺条件,得到了基本符合有关生物柴油标准要求的精制产品。采用正交优化设计法得到粗品生物柴油的最佳碱炼工艺条件是:超量碱量0.35%、碱液浓度15%、碱炼初温50℃。此条件下的生物柴油精炼率为94.5%、酸价为0.40mgKOH/g。红外光谱和气相色谱分析表明,油菜生物柴油的分子结构为长链酯类结构,其化学成分中18碳和16碳脂肪酸甲酯含量占60%以上,基本符合矿物柴油替代品对化学组成和分子结构的要求。在燃料特性指标方面,除了粘度偏大、十六烷值偏小外,其余指标均符合德国生物柴油标准要求以及我国生物柴油标准（初稿）要求。可以采取加强真空精馏操作、添加十六烷值改进剂等措施来降低粘度、提高十六烷值。更多还原

【Abstract】 Biodiesel,as an environmentally friendly,renewable and clean liquid fuel,is being greatly concerned and widely developed in the world.Its main raw material is vegetable oil.Rapeseed oil,which has many unique advantages as one of the main vegetable oil in China and Hunan province,will become the better raw material of biodiesel than other oils.Enzymatic method is the most promising method in the various methods for biodiesel production.But there is a series of fatal shortcomings in the most of developed enzymatic production methods,such as high cost,slow reaction,short life of lipase,low yield of methyl ester and so on.All these problems has greatly restricted us to achieve industrialization of biodiesel produced by enzymatic method.So,it is greatly in need of developing a new technology to make the industrialization become reality.In order to lower the cost of biodiesel and improve enzyme reaction system,a series of research about enzymatic production of biodiesel from rapeseed oil has been done.At first,gas chromatography analysis method was established to rapidly and accurately judge the reaction degree.Secondly,the reaction mechanism and process conditions of enzymatic transesterification of rapeseed oil was studied in the water-bearing organic solvent system,water-little organic solvent system and solvent-free system.Thirdly,a new technology about how to renew lipase used in the solvent-free alcoholysis of rapeseed oil,as well as a new enzymatic interesterification technology,was developed.At last,the optimal refining process condition of crude biodiesel was obtained,the refined product can almost meet the demands of the relative biodiesel standard. 1.The established gas chromatography analysis methodThe main component of rapeseed oil biodiesel are palmitic ME,stearic ME, methyl oleic ME,linoleic ME,linolenic ME,cis-11-eicosenoic ME,erucic ME.The best separation condition in the GC apparatus with HP-innowax capillary column （30m* 0.25mm*0.25μm）was that the initial column temperature was 170℃keeping 0.5min,then the column was heated to 200℃by 5℃per minute,and then to 240℃by 15℃per minute and keeped for 5min.Under these conditions,the transesterification degree of rapeseed oil can be detected rapidly and accurately with methyl tridecanoate as the internal standard.2.The reaction mechanism and process conditions of enzymatic transesterification of rapeseed oil in the two liquid phase system with composition of water and organic solvent.The lipozyme TLIM lipase is more effective catalyst than Novozym 435 lipase for transesterification of rapeseed oil.When the molar ratio of methanol to oil is 4:1, the temperature is 40℃,and the methanol is added with three interval batches,the optimal reaction condition was that 3 mL/g petroleum ether,7%（w/w）TLIM and 20%（w/w）water based on the oil weight were added to react for 18h.There are highly significant third-order polynomial regression relationship between the methanolysis degree and the various affecting factors.Because of the accompanying with the hydrolysis of triglyceride,the methanolysis rate is not more than 75%.3.The reaction mechanism and process conditions of enzymatic transesterification of rapeseed oil in water-little organic solvent system.we took Lipozyme TLIM enzyme as a catalyst,rapeseed oil with different quality grades as raw materials,different short-chain alcohol as acyl acceptors in the organic solvent-little water system.The orthogonal experimental results showed that alcoholysis reaction of triglyceride was accompanied with hydrolysis of triglyceride and esterification of fatty acid.The fourth grade oil is a better biodiesel feedstock than the first,second and the third grade oil;ethanol is a better acyl acceptor than methanol. The optimal reaction condition for biodiesel production was that the fourth grade oil reacted with 20%（w/w）Lipozyme TLIM and 4 molar ethanol per molar oil for 10h at the temperature of 40℃.Under the optimal condition,a transfer rate of 86.2%was obtained.4.A new technology to renew lipase used in the solvent-free alcoholysis of rapeseed oil and its optimal process conditions.When the fourth grade rapeseed oil was used as raw material,immobilized lipase lipozyme TLIM is more effective catalyst than Novozym 435,ethanol is a better acyl receptor than methanol,ZSJ1 has the better renewing effect on the enzyme activity than ZSJ2,30℃is most appropriate temperature to stimulate enzyme activity and to keep it stable.When the enzyme in the reaction was periodically renewed,the reaction time was shortened to 9 hours,a transesterification rate of more than 90% was obtained,and the relative activity of the lipase being repeatedly used 15 batches was remained more than 96%.5.A new enzymatic interesterification technology for biodiesel productionBecause of that the transesterification rate is not high enough,enzymatic activity is not stable enough,the renewing operation is troublesome in the alcoholysis reaction, a new e enzymatic interesterification technology for biodiesel production from rapeseed oil was developed.The main factors of effecting interesterification reaction rate were the enzyme type,enzyme quantity,substrate molar ratio,temperature and time.After the enzyme type was determined as Lipozyme TLIM,other factors were optimized according to the quadratic regression orthogonal rotation test design,a regression model obtained was as following.Y=91.9417+1.1125X₁+0.5458X₃-2.6490X₁²-3.3740X₂² -2.7990X₃²-2.7490X₄²The results shows that,in the interesterification system,the immobilized enzyme Lipozyme TLIM demonstrated better catalytic behavior than Novozym 435;the best enzymatic interesterification conditions of the fourth grade rapeseed oil were:40℃, 40%（w/w）Lipozyme TLIM quantity,14:1 substrate molar ratio,the reaction time 12 hours/batch.Under the optimal condition,an interesterification rate of 91.7%was obtained,and the enzyme can be repeatedly used with little activity loss,which provided technical basis for the industrialization of enzymatic biodiesel.6.the optimal refining conditions of crude biodiesel and the properties of the refined productsThe best conditions for crude biodiesel alkali refining process was obtained by orthogonal design optimization.excess of alkali is 0.35%,alkaline concentration is 15%,initial alkali refining temperature is 50℃.Under these conditions,the biodiesel refining rate was 94.5%,and acid value of product was 0.40mg KOH/g.IR and GC analysis showed that the C18 and C16 fatty acid methyl ester content in rapeseed biodiesel account for more than 60%of total fatty acid methyl ester,the chemical composition and molecular structure could almost meet the request of mineral diesel substitutes.The characteristics inders of rapeseed oil biodiesel was according to the German biodiesel standards and Chinese biodiesel standard（draft）requirements,except the higher viscosity and the smaller cetane number.High vacuum distillation and cetane improver can be taken to lower viscosity and improve the cetane number.更多还原