Studies On Mutation Breeding Of Trichoderma Viride By Heavy Ion Beam And Cellulase Production

With the depletion of fossil energy and the intensification of energy crisis around the world,renewable resources have been raised widespread attentions.Lignocellulose is the most abundant and renewable resource in the world,and its effective utilization could ease the global energy crisis.Cellulase is an enzyme which can efficient hydrolyze cellulose and convert to glucose,thus it was widely used in paper-making,textile,brewing,food,animal feed,environmental protection because the enzymatic hydrolysis conditions are mild and easy to control,and has no pollution to the environment.However,there are still many problems for the cellulase production,including the low activity and hydrolysis efficiency,high production cost and tedious downstream separation work of cellulase,and they were severely restricted the development of the cellulase industry.Therefore,the spores suspension of T.viride were irradiated by heavy-ion beams to study the mutagenic effectiveness and breed mutant strains with high cellulase activity,and then the fermentation process of mutant strains were optimized to improve the cellulase production.Moreover,the relation between mycelial morphology and cellulase activity were investigated using morphological engineering techniques involving the addition of microparticles,and evaluated the effects of mycelial morphology and fermentation process on cellulase activity of T.viride.The main research contents are given as followed:1.The mutagenic effectiveness and breed mutant strains of T.viride induced by heavy-ion beams irradiationT.viride My were irradiated by heavy-ion beams with different does and The results showed that the dose of half-lethal was 120 Gy,the survival rate of spores were declined to 9.43%at 400 Gy.In addition,the positive mutation rates were higher than negative mutation rates at the radiation does of 40Gy?18.5%?,160Gy?24%?,which were good for screening the positive mutants.After the irradiation of T.viride by heavy-ion beams,a mutants?My160-5?was obtained after the plate screening,24-square deep-well microliter plate screening and shaking flask fermentation rescreening.The filter paper activity?FPA?and CMCase?EG?activity of T.viride My160-5 were achieved 532.81 U/mL,875.15 U/mL after the shake flask fermentation,which increased by 20.54%and 24.4%?p<0.01?compared with the original strains,respectively.The mutant T.viride My160-5 was stable in genetics and could produce cellulase with high activity.2.Fermentation process optimization of the cellulase production by T.viride My160-5.To improve the cellulase production by T.viride My160-5,the fermentation conditions were optimized using the Plackett-Burman design?PBD?and Box-Behnken design?BBD?with response surface methodology?RSM?.The results showed that three variables?CMC-Na concentration,initial pH and peptone concentration?were selected from the nine different variables using the PBD,and then the optimal culture conditions were obtained after the analyzed with Box-Behnken design?BBD?and response surface methodology?RSM?.The optimal culture conditions were given as followed:CMC-Na 11.25 g/L,peptone 6.5 g/L,growth medium volume 20%,?NH₄?₂SO₄ 2.1 g/L,KH₂PO₄ 2g/L,Tween 80 1ml,initial pH 4.25 for 120 h of fermentation period at 30?.The observed FPA activity which was produced under optimal conditions were achieved 628.36 U/ml,which increased by 17.93%?p<0.01?compared with the original conditions.3.The regulation of mycelial morphology of T.viride My160-5 under submerged fermentation.The effects of mycelial morphology on cellulase activity and growth of T.viride My160-5 were investigated using the morphological engineering technique,which applied the function of microparticles in the fluid.The results showed that The addition of microparticles to the preculture caused a substantial change in pellet diameter,and the pellet diameter were significantly decreased with the increase of microparticles concentrations.In addition,cellulase activity were efficiently improved at the special microparticles concentration.The highest cellulase activity was achieved with 10 g/L aluminum oxide.Under these conditions,the resulting cellulase activities in the FPA and EG assays were increased by 17.1%and 13.3%,respectively,compared to that of the control.Moreover,the predicted model of pellet size change was fitted according to relation between microparticles concentrations and pellet size change.Controlling the mycelial morphology and cellulase activity by morphological engineering technique and mathematical models,which is an encouraging step towards commercial cellulase production.