A Novel Process Of Biodiesel Synthesis Directly Catalyzed By Fermented Solid From Burkholderia Cepacia

Comparing with chemical-catalysis, enzymatic-catalysis for biodiesel synthesis hasbecome the priority alternative, owing to its advantages of mild reaction conditions, lessmethanol consumption, oil stocks adaptability and environment friendly. However,enzymatic catalysis also shows some disadvantages, such as alcohols inactivation, poorthermal stability and high cost. Up to now, lipase transesterification for biodieselproduction always follows several steps: lipase submerged fermentation, separation,purification, drying, immobilization and then lipase catalysis for biodiesel synthesis.Thistraditional enzymatic transesterification for biodiesel production is not only complicatedbut also expensive. Therefore, it’s very urgent for us to exploit a novel process forbiodiesel production using lipase fermented solid. This work reported to produce thelipase from Burkholderia cepacia G63by solid-state fermentation (SSF) using sugarcanebagasse and sunflower seed cake as substrates. After lyophilization and delipidatation, thewhole fermented solid was directly used to catalyze the ethanolysis of soybean oil forbiodiesel production in tert-butanol system. This novel biocatalyst is highly competitivedue to its low production costs. Note that it might be possible to reduce its costs evenfurther, if the lyophilisation can be replaced with a mild air drying. Interestingly, lipaseproduction by SSF can reduce the water usage and be environmental friendly incomparison with submerge fermentation. SSF conditions, performance properties offermented solid, optimization of biodisel synthesis, operational stability and reactionkinetics were investigated. The main experimental results are:1. Preparation and performace properties of fermented solid from B. cepacia G63.Investigations were carried out to produce the lipase from Burkholderia cepacia G63bySSF using sugarcane bagasse and sunflower seed cake as substrates. Results showed thatusing olive oil emulsion as inducer, enzymatic activity of the fermented solid increasedfrom23.9U/gds to72.3U/gds, which was2.8-fold of that reported in literature. Theoptimum temperature and pH, short-chain alcohol tolerance, and metal ions effects aresimilar to free G63lipase. SDS-PAGE analysis showed the products of SSF andsubmerged fermentation are similar, and the molecular weight of lipase is37KDa. 2. Optimization of biodiesel production directly catalyzed by fermented solid from B.cepacia G63. Firstly, single factor experiments were employed to investigate the effects ofcrucial operational parameters on biodiesel yield, such as reaction medium, ethanol/oilmolar ratio, water concentration, fermented solid adding amount, temperature, andtert-butanol amount. Under the single-factor experiments, the biodiesel yield of88.6%was obtained. Then, the order of the above-mentioned factors was analyzed usingPlackett-Burman design: alcohol/oil molar ratio> temperature> fermented solid addingamount> water concentration> tert-butanol amount. Finally, interactions between thosesignificant factors were optimized with response surface methodology. The optimalparameters are: alcohol/oil molar ratio4.295:1, temperature44.15℃, fermented soliddosage1.611g, water content was4.589g, tert-butanol amount20%(based on oilvolume). Under the optimized conditions, the biodiesel yield of92.7%was achieved.Fermented solid could attain more than66.9%of original activity after repeated usage for288h, and biodiesel yield by fermented solid was above80%for soybean oil, tallow oil,cooking oil, and corn oil.3. Kinetics of enzymatic transesterification for biodiesel production by fermentedsolid. Based on Ping Pong Bi Bi mechanism, the kinetics model was firstly proposed forthe enzymatic transesterification of biodiesel production by fermented solid. From thevalue of Ki (0.34mol/L), the substrate alcohol showed the inhibitory effect. According tothe Arrhenius’equation, the activation energy of reaction was calculated to be37.95kJ/mol, which was higher than that of the immobilization PS lipase (17.66kJ/mol),indicating the fermented solid showed better thermal tolerance. The limited step fromdiglyceride to monoglyceride was elucidated based on the GC analysis of the intermediateproducts.