Biodiesel Production Catalyzed By Recombinant Rhizomucor Miehei Lipase-displaying Pichia Pastoris Whole-cell

Biodiesel is a sort of bioenergy, which is green, renewable and environmental friendly. It has become a research hotspot around the world for it could alleviate the crisis brought by the depletion of petroleum resources and environmental pollution. Compared with chemical methods, enzymatic biodiesel production has become a prime interest in recent works owing to mild reaction conditions and low energy consumption; it is also environmentally friendly. However, the preparation of immobilized lipases involving tedious purification and meticulous immobilization steps, often results in loss of activity and increased cost, which are considered the main hurdles in the industrialization of lipase-catalyzed biodiesel production. At present, these problems are expected to be solved by using whole-cell catalyst technology. In this study, Rhizomucor miehei lipase-displaying Pichia pastoris was used as the catalyst. It owned the characteristics of the immobilized enzyme, and its production process is simple, the cells were collected after centrifugation, followed by lyophilization, then can be used as biocatalysts for the biodiesel production without immobilization.Freeze-drying process of lipase-displaying yeast whole-cell was studied, results showed that the esterification activity and transesterification activity of RML-displaying whole-cell can be enhanced by Ca2+ and trehalose, which solved the problem of lipase activity loss during the freeze-drying process. The influential factors on the reaction were studied. The optimal reaction conditions were: Isooctane was the most suitable reaction medium, 2 volume ratio of isooctane to oil, 55oC of temperature, 40% (w/w, based on the oil weight) of whole-cell, 4.5:1 of molar ratio of methanol to oil, 0.66 of water activity and each 1.5 molar equivalent of methanol to oil added in a three-step procedure at the reaction time of 0, 24 and 48 h, respectively. Under these optimized conditions, a methyl esters (ME) yield reached 83.14% after 72 h. The RML-displaying P. pastoris whole-cell biocatalyst exhibited quite a good operational stability, and more than 80.46% of its original activity was retained after 10 batches reaction at 55oC.After single factor optimization, temperature, catalyst amount and molar ratio of methanol to oil were the important factors. The central composite design (CCD) of the response surface methodology (RSM) was employed, and the effects of five-level-three-factor and their reciprocal interactions were assessed. Using response surface methodology Minitab15 software, transesterification process was optimized. And the optimized reaction conditions: a reaction temperature of 52oC, whole-cell amount of 48% (w/w, based on weight of oil), and an methanol-to-oil molar ratio of 5.8:1, further verification experiments with biodiesel yield of 89.17% , which was close to the predicted biodiesel yield (88.00%), and higher than the best one (83.14%) achieved before optimization.Based on RSM, reaction parameters of biodiesel synthesis were optimized in a 500 ml stirred reactor. And the optimal congditions were: reaction volume 200 ml, whole-cell 60%(w/w, based on oil weight), each 1/3 equivalent of enzyme added in a three-step procedure at the reaction time of 0, 12 h and 36 h, speed 300 rpm, 3A molecular sieve 12 g. Under these conditions, ME yield reached 86.88%, which was close to the level of shaking level, indicating that RML-displaying P. pastoris whole-cell could efficiently catalyze the transesterification of soybean oil and methanol in a expaned reaction system.In this study, the frying oil as raw material, was used for biodiesel production catalyzed by RML-displaying P. pastoris whole-cell, and the main reaction factors were optimized. The optimal transesterification conditions were: petroleum ether as the optimal reaction medium, ratio of petroleum ether to the frying oil 2.5:1 (v/v), whole-cell amount 60% (based on the quality of frying oil), molar ratio of methanol to oil 4.5:1, each 1.5 molar equivalent of methanol to oil added in a three-step procedure at the reaction time of 0, 24 h and 48 h, after reaction of 72 h, the ME yield could reach 80.06%.Refinement and separation methods of raw biodiesel were studied, and purified biodiesel could be obtained by the rotary evaporation. Compared with appearance, analysis of major composition by GC and quality of raw product and purified biodiesel, the quality of purified biodiesel is better than raw biodiesel. These results further proved that the whole-cell biocatalyst is promising for the production of biodiesel on an industrial scale.