丝瓜络固定化米根霉催化光皮树油制备生物柴油的研究
Immobilization of Rhizopus oryzae onto Loofah Sponge as a Whole-Cell Biocatalyst to preparation of biodiesel from Comus wilsoniana fruit oil
[目的]生物柴油作为一种新型可再生能源,是化石燃料的理想替代品。它具有低硫、低碳和高十六烷含量的特点。与传统化学方法相比,生物酶法制备生物柴油不需要额外去除催化剂和盐,纯化费用低,具有很大的应用潜力,然而目前生物法中催化剂的高成本一直被认为是影响其产业化的一个重要因素,利用固定化全细胞作为生物催化剂可以避免脂肪酶的复杂纯化过程,有效降低生物催化剂的制备成本。[方法]本研究对一株高产脂肪酶的米根霉进行了固定化研究,并将其应用于生物柴油的制备。首先通过比较固载率和甲酯化效果,在生物基固定化材料丝瓜络、纳米纤维、水凝胶和椰壳活性炭中筛选出米根霉的最适固定化材料,探索其适宜的固定化条件,以获得的固定化米根霉作为全细胞催化剂催化光皮树油制备生物柴油。并进一步探讨了转酯化条件对生物柴油得率的影响。[方法]研究结果表明,绿色环保、价格低廉的丝瓜络为米根霉的最适宜固定化材料,其固载率达到 162+6%。丝瓜络是一种天然高分子材料,不但具有高孔隙率(79%-93%)和低密度(0.02-0.04 g/cm3)的纤维血管网状结构利于米根霉菌丝的粘附,而且具有优良的力学性能和坚韧的质地可以在后期应用过程中承受较大的剪切力。在 100 ml米根霉发酵培养基中加入 2.25 g丝瓜络作为固定化载体,培养 72 h即可收获效果最佳的固定化米根霉全细胞催化剂。为了提高丝瓜络固定化米根霉的催化效率,研究了培养基成分对其影响。橄榄油含有较多的不饱和脂肪酸,作为碳源具有一定的诱导作用,可有效提高米根霉中的脂肪酶表达量,以此获得的固定化米根霉催化光皮树油的甲酯得率达到 93%以上。通过评价固载率和甲酯得率,选择多聚蛋白胨为有机氮源,NaNO3 为无机氮源的复合氮源来培养及固定化米根霉细胞。同时为了提高固定化细胞的使用寿命和重复使用次数,研究了用浓度为 0.1%的戊二醛、双醛淀粉、京尼平溶液进行交联处理的可行性。结果发现,在戊二醛处理的固定化细胞中脂肪酶活性损失较少,在重复使用 6次后,转酯化效果依然保持在 80%以上。[结论]本研究获得的全细胞催化剂具有制备简单、稳定性良好、可重复利用等优点,通过优化的培养基组成也具有价格较低廉的特点,有效控制了全细胞催化剂的制备成本。
[Objective]Biodiesel,as a new renewable energy,is an ideal substitute for fossil fuels.It has the characteristics of low sulfur,low carbon and high cetane content.Compared with traditional chemical methods,the preparation of biodiesel by biological enzyme method does not need to remove extra catalyst and salt,and the purification cost is low,so it has great application potential.However,the high cost of catalyst in biological method has always been considered as an important factor affecting its industrialization.Using immobilized whole cells as biocatalyst can avoid the complicated purification process of lipase and effectively reduce the preparation cost of biocatalyst.[Method]In this study,a strain of Rhizopus oryzae with high lipase production was immobilized and applied to the preparation of biodiesel.Firstly,by comparing the immobilization rate and the methyl esterification effect,the most suitable immobilized materials of R.oryzae were screened out from the bio-based immobilized materials of loofah sponge,nanofiber,hydrogel and coconut shell activated carbon,and the suitable immobilization conditions were explored.The immobilized R.oryzae was used as a whole cell biocatalyst to catalyze the preparation of biodiesel from Comus wilsoniana fruit oil.The effect of esterification conditions on the yield of biodiesel was discussed.[Result]The results showed that loofah sponge was the most suitable immobilization material for R.oryzae,and its immobilization rate reached 162+6%.Loofah sponge is a kind of natural polymer material.It not only has a fibrous vascular network structure with high porosity(79%-93%)and low density(0.02-0.04 g/cm3),which is beneficial to the adhesion of R.oryzae,but also has excellent mechanical properties and tough texture,which can bear large shear force in the later application process.Adding 2.25 g loofah sponge to 100 ml R.oryzae fermentation medium as immobilized carrier,and culturing for 72 h,the best immobilized R.oryzae whole cell biocatalyst can be obtained.In order to improve the catalytic efficiency of R.oryzae immobilized on loofah sponge,the effects of medium components on it were studied.Olive oil contains more unsaturated fatty acids,which can be used as a carbon source to induce the expression of lipase in R.oryzae,and the methyl ester yield of immobilized R.oryzae catalyzed by it can reach over 93%.By evaluating the immobilization rate and methyl ester yield,the compound nitrogen source with polypeptone as organic nitrogen source and NaNO3 as inorganic nitrogen source was selected to cultivate and immobilize R.oryzae.At the same time,in order to improve the service life and reuse times of immobilized cells,the feasibility of crosslinking with 0.1%glutaraldehyde,dialdehyde starch and genipin solution was studied.The results showed that the loss of lipase activity was less in the immobilized cells treated with glutaraldehyde,and the transesterification effect remained above 80%after repeated use for 6 times.[Conclusion]The whole-cell biocatalyst obtained in this study has the advantages of simple preparation,good stability and reusability.The optimized medium composition also has the characteristics of low price,which effectively controls the preparation cost of the whole-cell biocatalyst.
纪淑兰;李迅;王飞;
南京林业大学化学工程学院,江苏 南京 210037;南京林业大学化学工程学院,江苏 南京 210037;南京林业大学化学工程学院,江苏 南京 210037;
Whole cell catalyst Rhizopus oryzae Loofah Sponge Biodiesel Comus wilsoniana fruit oil
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