The Breeding And Cell Immobilization Of Lipase-producing Strain

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 envionmental pollution.Main contents discussed in this thesis are as follows:(1)Isolation, screening and identification of lipase-producing strains: A lipase-producing stranins are isolated by the primal screening on agar plates containing olive oil. After analyzing and comparating of microorganism morphology, physiology and biochemistry propterties, the strain isolated was identified as Geotrichum Link and named Geotrichum Candidum NS3. Its initial activities of hydrolytic lipase and synthetic lipase reached 321 U/g dry cell and 0.18 U/g dry cell, respectively.(2) Induce mutation of lipase-producing strains: In order to inprove the lipase-producing activety, the strains isolated was treated by the routine UV and NTG. A strain with comparatively high lipase yield had been selected. Its hydrolytic lipase activities and synthetic lipase activities reached 490 U/g dry cell and 0.35 U/g dry cell, respectively. The strain mutated named Geotrichum Candidum NS3T3. After five generations genetic culture and testing, it showed excellent heredity stabilization.(3) Investigation of immobilization and fermentation conditions of lipase-producing strain Geotrichum Candidum NS3T3: The immobilization conditions of lipase-producing Geotrichum Candidum NS3T3 with polyurethane foam were investigated. The results showed that the cells load of polyurethane foam reached 2.32g/g under the following optimal conditions: carrier density27kg/m~3, particle size 6×6×6mm, culture time 60 hrs, and there are 73.8% cells grown been immobilized in PUF pellets. The pictures of scanning electric microscope (SEM) showed that the mycelium Geotrichum Candidum NS3T3 enlaced or filled in the holes inner and the wall of carrier (PUF), it grown nicer and attached tightness in the PUF material. By single factor experiment, its culture conditions were optimized. The test results showed: the synthetic lipase activities of immobilization cells reached 0.54U/g dry cell under the condition: inoculum quantity 12%(v/v), volume of broth 50mL/250mL, 30℃, 180r/min. The catalysis reaction for biodiesel synthesis has been carry out in packed bed column reactor with immobilization cells pellet, the test result by TLC showed that the transesterzation reaction was effective.(4) Immobilization of lipase-producing strain Candida Valida T2: The immobilization conditions of lipase-producing Candida valida T2 with polyurethane foam were investigated. The results showed that the cells load of polyurethane foam reached 1415mg/g and hydrolytic lipase activity achieved 1255U/g dry cell under the following conditions: optimal carrier density, particle size, pretreatment employed, appropriate ratio of solid carrier and fermentation liquid. The pictures of scanning electric microscope (SEM) showed that the mycelium Candida valida T2 enlaced or filled in the hole inner and the wall of carrier (PUF). Candida valida T2 grew well and colony configuration immobilized was stable. When the immobilization cells pellet was used to catalyze hydrolysis reaction of tung oil, the remained rate of enzymatic activity reached 73%, and the loss rate of the immobilized cell less than 12.5% after five repeat reactions.