Screen Of Strains Which Produce Lipase And The Study Of Immobilized Lipases Used On Biodiesel

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.