Study On The Preparation Of Biodiesel From Woody Plant Oils Catalyzed By Immobilized Recombinant Lipase

In this dissertation, the extraction and refining of woody plant oils, preparation of immobilized lipase for catalyzing transesterification of the oils, optimization of transesterification conditions, and properties of biodiesel products were systematically investigated. The main results are as follows:1. The extraction yield of crude oil from the kernel of Jatropha curcas L. seed and the seed of Pistacia chinensis were 60% and 35.0%, respectively. The extracted oils were degummed and discolored prior to use as the raw material for the transesterification reactions.2. Conditions related to cell growth and lipase expression sueh as the cultural medium variety, fermentation time and methanol concentration were investigated in a 10L fermentor based on the fermention conditions of shaker flasks. The effect of fed-batch with glycerol on the production and growth of engineering strain and with methanol as the inducers of lipase were compared. The lipase activity reached at 90U/ml after induction for 96h with increasing the velocity and concentration of methanol.3. The immobilization of R. royzae lipase was conducted with different supports. It was found that the anion resin D301 is a proper matrix for immobilization of R. royzae. The optimal conditions for the preparation of immobilized lipase were as follows: absorption time 4h at 28℃or 10h at 4℃, enzyme amount 1200U/g resin, cross-lingking concentration 0.5% and cross-lingking time 20 min at 110 rpm. The optimal activity of the immobilized lipase is 160U/g under these conditons.4. The transesterification conditions of refined soybean oil catalyzed by the immobilized lipase were discussed. The proper conditions were as follows: enzyme amout 24U/g oil, oil/alcohol molar ratio 1:4.8, water content 60% based on oil mass, temperature 37°C, and reaction time 48h. The maximum methyl esters (ME) yield could reach 92% under the conditions. The immobilized lipase could be repeatedly used for 7 times in the production of biodiesel without apparent loss of lipase activity.5. The optimal alcoholysis conditions for woody plant oils were as follows: enzyme amont 24U/g oil, oil/alcohol molar ratio 1:4.7, water content (on water mass) 40% for Jatropha curcas oil and 20% for Pistacia chinensis seed oil, temperature 37°C, and reaction time 48h. The maximum methyl esters (MEs) yield could reach 94% and 92%, respectivly. The immobilized lipase could be repeatedly used for 5 times in the production of biodiesel without apparent loss of lipases activity.6. The main properties of the prepared biodiesel, such as density, kinematic viscosity, heat value, flash point and cetane number, are well consistant with the current American and Chinese biodiesel standards.