| In recent years,environmental and energy issues have become the focus of attention in the world,and the enzymatic synthesis process of biodiesel,as one of the recognized alternatives to traditional fossil fuels,has been attracting considerable attention.The production process of Yarrowia lipolytica lipase 2(YLLIP2)is mature,and the high protein yield and high enzyme activity of the modified engineered strain via high-density fermentation can significantly reduce the production cost of the lipase,which renders it an extremely strong potential for industrial applications.Nevertheless,due to the difficulties in mixing the substrates in the liquid lipase reaction system,the negative effects of methanol and glycerol on the catalytic activity of the enzyme,the inability to satisfy the environmental conditions required for effective lipase catalysis,and the fact that the dynamic change pattern of the reaction process is still unclear,these scientific problems have led to the technical bottlenecks such as lengthy reaction time and low yield in the biodiesel synthesis catalyzed by YLLIP2,which have become the main obstacles to its application in largescale production.To overcome these shortcomings,the conditions of the liquid lipase catalyzed biodiesel synthesis from soybean oil were first explored and optimized.The optimum reaction conditions were: water content 45 wt%,1,000 U/g of lipase dosage,reaction temperature40 ℃,reaction time 24 h and p H 7.0.Under these conditions,the biodiesel yield of liquid lipase using enzyme powder could reach 91.8%.For the direct fermentation supernatant,the optimum conditions were: 1,000 U/g of lipase dosage,40 wt% of water,40 ℃ of reaction temperature,reaction time of 24 h,and the biodiesel yield attained 92%.The results of the fermentation supernatant and the liquid lipase formulated with the enzyme powder were nearly the same,indicating that the liquid enzyme catalyzed process of biodiesel synthesis by YLLIP2 has a greater application prospect.To study the dynamics within the liquid lipase reaction system,a kinetic model for the synthesis of biodiesel catalyzed by YLLIP2 in liquid form was developed with reference to the Ping-Pong Bi Bi reaction mechanism.Dynamics of each component in the reaction were sequentially examined by gas chromatography and gel permeation chromatography,and the simulated values of each parameter in the reaction equation were all obtained with COPASI software.Then,with this method,the dynamic changes of the main components in the reaction system were simulated within 120 min and 24 h,respectively.The calculated values were in good agreement with experimental ones.In addition,dynamic simulations confirmed the reaction mechanism in the liquid lipase reaction system,that is,transesterification,hydrolysis and esterification reactions occur in the reaction system at the same time.In order to further enhance the biodiesel yield of the liquid lipase reaction system,a new deep eutectic solvent(DES)was synthesized from choline chloride and glucose,and applied to the biodiesel synthesis process.When the molar ratio of choline chloride to glucose was 2:1,the DES synthesized was the most effective in enhancing the yield of biodiesel.The optimum water content of the reaction was 45 wt% at 2.19 g of soybean oil,and the optimal amount of DES was 0.5 m L.Under this condition,the biodiesel yield catalyzed by liquid lipase increased from 91.8% to 98.5% after addition of deep eutectic solvent.The dynamic changes of the major components in the reaction system after addition of DES were analyzed,and it was identified that the addition of DES enhanced the utilization efficiency of YLLIP2 for the substrates,and at the same time,the catalytic effect of YLLIP2 was also significantly improved in the recycling utilize of five batches.Consequently,biodiesel was synthesized in a 3 L ultrasonic reactor utilizing liquid lipase,and the reaction conditions were optimized.The optimum reaction conditions were:30 wt% of water content,3 wt% of enzyme addition,60 W of ultrasonic power and 1,000 rpm of stirring speed.93.3% of biodiesel yield was achieved in only 6 h under these optimized conditions.Compared with the small reaction system,the reaction time was reduced by 75%.The kinetics was further studied,and the results showed that the introduction of ultrasound enhanced the miscibility of the substrate,improved the rate of transesterification and esterification,and could effectively solve the problem of long reaction time of the liquid enzyme catalysis.The kinetic model predicted that the optimal water content was 30 wt%,and the optimal molar ratio of oil to alcohol was 1:6,which were further verified to be validated by the experimental results.Thus,it was confirmed that the kinetic model could be applied to the large-scale production system.Finally,the DES was combined with the ultrasonic reactor,and the reaction conditions were optimized.The highest biodiesel yield could reach over 99% with DES addition of 0.3L,water content of 40 wt% and oil-alcohol molar ratio of 1:4.The repeated use batches of YLLIP2 in the sonication reactor were also investigated with DES,and the biodiesel yield remained 89% after 5 repeated batches,and retained above 55% after 10 repeated batches.In summary,in this study,by combination of ultrasound technique and DES and further systematic investigation of the reaction kinetics,the problems of insufficient substrate miscibility and the negative effects from short-chain alcohols and by-product glycerol on enzyme activity were totally solved.The reaction time was significantly shortened,biodiesel yield was markedly improved,and the continuous use batches of the liquid enzyme were effectively elongated with low ultrasound power.Moreover,the strategy has the advantages of green,high efficiency,easy operation and low energy consumption,which possesses an extremely prosperous potential for future large-scale industrial production. |
PDF Full Text Request