Biodiesel Production Based On Cotton Seed Oil Catalyzed By Lipase From A. Niger Li-38

To prepare the biocatalyst for biodiesel production, the strain which could produce lipase was screened and the characteristics of lipase were investigated. The catalyzing system was built to converse the cotton seed oil to biodiesel when the immobilized lipase was as catalyst. The results were following:A new strain, Li-38 from Xinjiang was screened which could express lipase massively. The strain of Li-38 is a member of Aspergillus niger after identified by morphology and 18SrDNA gene sequenes, and named Aspergillus niger Li-38(A. niger Li-38).The study of fermentation characteristic indicated that the cell growth of A. niger and lipase production were partially coupled. The study of fermentation characteristic indicated that the optimal cultivation parameters for temperature and pH were determined as 45℃and 5.5, respectively; the optimized nitrogen source was 1% (w/v) of (NH4)2SO4, 3% (w/v) of peptone and carbon source was 1.5% (w/v) of rap oil; the concentration of metal ions were 0.01 % (w/v) of K2HPO4, 0.01 % (w/v) of MgS04·7H2O, 0.01% (w/v)of CaCl₂, 0.01% (w/v) of NaCl₂ respectively. Under the optimized conditions, activity of lipase reached the highest yield of 84.9 U·ml^-1.Three mutant methods including UV radiating, DES treatment and microwave radiating were applied in the treatment to spore and protoplast of A. niger Li-38. The results showed that the effect of the compound treatment of UV radiating and DES on them was better. The optimal mutant parameters for spore of A. niger Li-38 were determined as the concentration of 1%DES+UV,the corresponding mutant times were 20min + 20s,15min + 30s,10min + 40s,10min + 50s,5min + 60s respectively. Preparation conditions for protoplast were optimized, which were determined as 1% of lywallzyme, 0.5% of snail enzyme, 1% of cellulose, and treatment time 40min. The optimal mutant parameters for protoplast of A. niger Li-38 were determined as the concentration of 0.2%DES+UV,the corresponding mutant times were 30min+10s,20min +20s,15min + 30s,10min +40s respectively. One mutant strain whose activity of lipase was improved 38% reached 125.36 U·ml^-1. The genetic stability of mutant strain MLi-38 was good.Research about the characteristic of lipase showed the following results. The optimal reaction temperature and pH were 50℃and 5.5 respectively, and the enzyme was relatively stable under 70℃and pH 5.5⁵.0. In all of the metal ions tested, Na+,Mg²⁺,Ca²⁺and K⁺ have activation effect, while Fe²⁺,Zn²⁺ and Cu²⁺ have inhibition effect on the enzyme and Mn²⁺ almost has no effect. When cotton seed was as substrate, the activation energy and inactivation energy of the catalyzing reaction of lipase were 9.8 kJ·mol– 1 and 87.975 kJ·mol– 1. The study also displayed that Km and Vmax were 9.706 mmol·L^-1 and 12.11μmol·L^-1·min^-1.Chitosan was as immobilizing material for the immobilized lipase and the optimal parameters for immobilization were determined that the concentration of glutaraldehyde was 0.015% and the cross-linking time was 50min. The optimal reaction parameters for biodiesel production in the transesterification of cotton seed oil and methanol were determined that t-butyl alcohol was as the reaction media, the water concentration was 0.6%, the reaction temperature was 8%, the ratio of methanol and fatty acid was 0.3. Batch-addition of methanol technology was adopted, which methanol was added in 0 h, 7 h and 16 h. After reaction for 28h, the highest rate of transesterification reached 89.1%. The research for the characteristics of cross-linked immobilized lipase showed that its storage stability was better. After 12d at room temperature, the activity of lipase also retained 80% and 30 d, 46.8%. The immobilized lipase was stable at 30-70℃, pH 5.5-6.5. After immobilized lipase utilized for seven times, the ratio of transesterification was over 80%.