【作者】 张振乾；

【导师】 官春云；

【作者基本信息】 湖南农业大学 ， 作物学， 2009， 博士

【摘要】 生物柴油是由大豆油、菜油等植物油或动物脂肪通过低碳醇在催化剂作用下形成的长链脂肪酸单酯类物质,具有不受地理、环境等因素的影响,可减少对石油市场的依赖等优点而受到越来越多的关注。原料的价格、供应和廉价的脂肪酶是影响酶法生产生物柴油的关键因素。为解决这些问题,在本研究中,筛选了25株脂肪酶产生菌并对其中效果较好的2个脂肪酶进行催化生产生物柴油的研究,同时研究了原料的影响,为筛选合适的原料提供了参考。研究内容和结果如下:1.筛选得到25株脂肪酶菌种,其中8株活性较高,对其中活性较好的3株进行,并种类鉴定、最佳培养条件确定等研究。产气肠杆菌（Enterobacter aerogenes）以1.0%的葡萄糖和1.5%的NaHCO₃为C源,1.5%的蛋白胨为N源,添加0.1%MgSO₄.7 H₂O,起始pH 9.0,装液量50mL/100 mL,120 rpm,30℃培养48 h,酶活达34.16 U/mL;加入1.0%的橄榄油,酶活可提高到66.31 U/mL。该脂肪酶在30℃,pH 8.0时酶催化活性较高。成团肠杆菌（Enterobacter agglomerans）以2%的乳糖作为碳源,1.5%的牛肉浸膏和1.0%的酵母膏作为氮源,添加0.1%MgSO₄·7 H₂O,初始pH 7.0,120 r/min,30℃培养48 h时产酶活力达39.09 U/mL。发酵液在30℃,180 r/min时催化性能较强。在反应体系中含水量达91%以上时,仍可催化芝麻油生产生物柴油,产率达54.51%。乙酸钙不动杆菌（Acinetobacter calcoaceticus）,以1.5%的麦芽糖作为碳源,2.5%的NH₄Cl为氮源,初始pH 7.0,接种量1.0%,装液量40 mL/100 mL,120 r/min下30℃培养24 h,酶活达9.07 U/mL。2.确定了气相色谱内标法测定生物柴油方法以十七酸甲酯为内标,依据菜籽油组分含量不同,改进了气相色谱内标法测定生物柴油方法,建立了棕榈酸甲酯、硬脂酸甲酯、油酸甲酯、亚油酸甲酯、亚麻酸甲酯和花生一烯酸甲酯的线性方程,使各个组分同时处于测量范围内,确保了测定结果的准确性。各脂肪酸甲酯含量在各自线性范围内的相关系数r≥0.999、平均回收率在97.91%-100.45%,精密度高,试验标准偏差在0.28%-2.22%,重复性好。该体系可用于菜籽油合成生物柴油产率测定,也可用于原料成分影响研究和原料的筛选研究。3.脂肪酶固定化的研究对产气肠杆菌和成团肠杆菌发酵液得到的沉淀进行冷冻干燥,采用硅藻土固定化。产气肠杆菌:硅藻土用量与酶粉用量为10:1,20℃下吸附2 h,得到的固定化酶的活性最大（1111.1 U/g）。该固定化酶在30℃,pH 8.0条件下催化性和稳定性较好。成团肠杆菌:硅藻土用量与酶粉用量为15.625:1,15℃吸附1.5 h,得到的固定化酶的活性最大（666.7 U/g）。该固定化酶在30℃,pH 8.0条件下催化性和稳定性较好。4.有机溶剂体下催化生产生物柴油硅藻土固定化产气肠杆菌脂肪酶用量为1000 U,乙醇为酰基受体（醇:油摩尔比为4:1,2次等量加入）,5 mL正己烷,180 r/min,35℃,反应48 h,转化率达92.91%。同时还发现,水对该反应有抑制作用,随着其含量增加,转化率降低;油酸对该反应有促进作用,而芥酸有不利影响。硅藻土固定化成团肠杆菌脂肪酶用量为1000 U,甲醇为酰基受体,甲醇与油摩尔比为3:1,3次等量加入,5 mL正己烷,180 r/min,35℃反应48 h,转化率到91%。该反应体系可用于催化大豆油和葵花子油生产生物柴油,产率分别为88.57%和83.51%。该体系也可催化含水原料生物柴油,在反应体系中水含量达25%时,仍可获得61.97%的转化率。该研究取得了如下创新:（1）筛选得到3株活性较高的脂肪酶产生菌,进行了分类鉴定和培养条件优化:（2）改进了气相色谱内标法测定生物柴油方法,纠正了前人该方面研究的不足;（3）在有机溶剂体系下催化生产生物柴油的研究,并得到较高的产率,证实本研究得到脂肪酶的有效性,也为原料筛选提供了参考。更多还原

【Abstract】 Biodiesel is produced from soybean oil,rapeseed oil and other vegetable oils or animal fats by transesterification with methanol,has attracted considerable attention as a renewable energy.It can not be affected by geographical,environmental and other factors,and can reduce the dependence on the oil market,so it becomes more and more and more attractive.As the price and adequate supplies of raw materials are the bottleneck of the development of biodiesel,low-cost lipase is also a key factor of enzymatic biodiesel, so,a large number of strains which produce lipase were screened and different rapeseed oils were tested in this study to resolve these problem.The main points were as follows,1.25 strains produced lipase were screened and 8 of them have higher activity.Three strains with high lipase productivity were identified;the optimum culture conditions were achieved.The enzyme activity of Enterobacter aerogenes can reach 34.16 U/mL under the optimum conditions which were:initial pH 9.0,agitating at 30℃for 40 h,with NaHCO₃ and glucose as carbon sources,peptone as nitrogen source,0.1%（w/v） MgSO₄·7H₂O.When adding 1.0%（w/v） olive oil to the culture,the enzyme activity can reach 66.31 U/mL,which has increased by 56.5%compared to that obtained by using 2.0%（w/v） olive oil as the sole carbon source.With a preliminary study,the characteristic of this enzyme were found:the optimum reaction temperature is 30℃, the best reaction pH was 8.0.Under the optimum conditions determined,the enzyme activity of Enterobacter agglomerans can reached to 39.09 U/mL,and the optimal conditions were found to be: initial pH 7.0,agitating at 30℃for 48 h,with 2.0%（w/v） lactose as carbon sources, 1.5%（w/v） beef extract and 1.0%（w/v） yeast extract as nitrogen source,0.1%（w/v） MgSO₄·7H₂O.Olive oil,sesame oil and tea oil as raw materials can be catalyzed on biodiesel by the lipase of this strain under the conditions of 30℃and 180 rpm.And the yield reached to 54.51%with sesame oil as raw material,even when there contained 92.4%（w/v） water in the starting materials.The optimum culture conditions of Acinetobacter calcoaceticus were:initial pH 7.0, agitating at 30℃for 24 h,1.5%（w/v） maltose,2.5%（w/v） NH₄Cl,0.1%（w/v） MgSO₄·7H₂O.The optimal medium loading volume was 40 mL/100 mL and the inoculation volume is 1.0%,the enzyme activity reached 9.07 U/mL.2.A determination method of fatty acid methyl esters in biodiesel by GC was developed and establishedA determination method of fatty acid methyl esters in biodiesel by GC was developed and established and the impact of various components of biodiesel was studied.The Linear Equations of the fatty acid methyl esters（methyl palmitate, methyl stearic acid methyl,methyl oleate,methyl linoleate,methyl linolenate,methyl cis-11-eicosenoate） was established based on their different content for the first time, which used methyl heptadecanoate as an internal standard.When the sample of the fatty acid methyl esters was in their own linear range respectively,the linearity relative coefficient was more than 0.999,the average recoveries was from 97.91%to 100.45%,and the relative standard deviations were from 0.28%to 2.22%in the precision experimental,while the present of rapeseed oil has little affection to the determination system.The synthesis reaction degree can be judged accurately by comparing the total content of the fatty acid methyl esters in reaction system,and the determination results are creditable.This method can be used for the determination of biodiesel yield and the exact content of the various components.At the same time it can be used to the screening of raw materials and the effect of different components.3.The study of immobilized lipasesThe lipase of Enterobacter aerogenes and Enterobacter agglomerans were immobilized by diatomite in this study.The lipase of Enterobacter aerogenes was immobilized by diatomite in this study. When the dosage of enzyme powder compared with diatomite is 10:1,the adsorption time is 2 h,and the adsorption reaction was operated on 20℃,the greatest activity （1111.1 U/g） was obtained.Under the conditions of 30℃,pH 8.0,the immobilized lipase has better catalytic effects and good stability.The lipase of Enterobacter agglomerans was immobilized by diatomite in this study.When the dosage of enzyme powder compared with diatomite is 15.625:1,the adsorption time is 1.5 h,and the adsorption reaction was operated on 15℃,the greatest activity（666.7 U/g） was obtained.Under the conditions of 30℃,pH 8.0,the immobilized lipase has better catalytic effects and good stability.4.The transesterification reaction were studied under organic solvents systemThe dosage of diatomite immobilized lipase of Enterobacter aerogenes is 1000 U, ethanol as acyl receptors（alcohol:oil molar ratio is 4:1,2 times add）,5 mL n-hexane, 180 r/min,35℃,48 h,and the conversion rate of 92.91%.At the same time,we found that the conversion rate decrease with the water content increase,lower conversion rate;it is in favor of high oleic acid content in the production of biodiesel, thus erucic acid have adverse effects.The dosage of immobilized lipase of Enterobacter agglomerans is 1000 U, methanol as acyl receptors（alcohol:oil molar ratio is 3:1,3 times add）,5 mL n-hexane,180 r/min,35℃,48 h,and the conversion rate was 91%.This reaction system can catalyze soybean oil and sunflower oil too,and the conversion rate was 83.51%and 88.57%respectively.It is in favor of high oleic acid content in the production of biodiesel,thus erucic acid have adverse effects.With 25%water content, there also has a conversion rate of 61.97%,so it can be used on waste oils to produce biodiesel.Achievements of this study:1.Three strains with high lipase productivity were selected and identified,the optimum culture conditions were achieved;2.A determination method of fatty acid methyl esters in biodiesel by GC was established and developed; 3.Immobilized lipases catalyzed biodiesel were studied in organic solvents system and obtained higher yield.It tested that the lipases screened in this study can be used on biodiesel production.更多还原