Culture Conditions For Oil Accumulation And Regulation Of Lipid Synthetic Metabolism Of Chlorella Protothecoides

Biodiesel has been attracting extensive attention because of the advantages of environmentaly friend,excellent properties and renewable.However,the development of biodiesel is limited to some extent by the high production cost of its feestock.Chlorella protothecoides is regarded as one of the most potential feedstocks for biodiesel production because of its short life cycle,high oil content,and easy to scale up.What is more,the fatty acid compositions of microalgal oil are similar to those of plant oil.In this study,C.protothecoides was taken as the trial strain.Firstly,the parameters affecting oil accumulation in the autotrophic cultivation and heterotrophic cultivation were optimized.And then,the feasibility of using enzymic hydrolyzate from Glycyrrhiza uralensis residues for heterotrophic cultivation of C.protothecoides was evaluated and futher exploited using the enzymic hydrolyzate from G.uralensis residues for mixotrophic cultivation of C.protothecoides for oil production.Furthermore,the effects of the key enzymes,the ?-subunit of Acetyl-Coenzyme A Carboxylase(accD)and phosphoenolpyru-vate carboxylase(PEPC),on the oil systhesis were investigated so as to provide theoretical and technological foundation for regulation of lipid synthetic metabolism.The main work and results are summaried below.1.During the autotrophic cultivation,the antibiotic tolerance of C.protothecoides was investigated.Meanwhile,the effects of those parameters on the oil yield of C.protothecoides were tested,including volume of medium,inoculum size,nitrogen source,phosphorus source,Mg2+ concentration,pH value,illumination intensity and temperature.And then,the key affecting parameters of microalgal oil yield were exploited by Plackett-Burman Design.Then,three key parameters,including the addition of NH4HCO3,the addition of MgSO4 and pH value,were further optimized via Box-Behnken of Response Surface Methodology.The optimized conditions were NH4HCO3 addition 502 mg/L,MgSO4 addition 274 mg/L and pH value 7.4,and the highest oil yield was 0.362 g/L.2.Under the heterotrophic cultivation,the effect of those factors on the oil yield were evaluated,including the preference of carbon source,addition of glucose,nitrogen source,phosphorms source,Mg2+ concentration,pH value and temperature.Thereafter,the key factors affecting microalgal oil yield of C.protothecoides was also exploited by Plackett-Burman Design.Accordingly,three key factors of glucose additions,C/N ratio and pH value were further optimized through Box-Behnken.The optimized conditions were glucose addition 49.5 mg/L,C/N ratio 6.31 and pH value 7.36,and the highest oil yield was 1.853 g/L.3.The combination pretreatment with bio-pretreatment and mild acid pretreatment on the ligin decompostion of G.uralensis residues was examined.Based on the former treatment,the obtained G.uralensis residues was then treated with cellulase.Furthermore,the enzymatic hydrolyzate was used as medium for heterotrophic cultivation of C.protothecoides and further confirmed the feasibility of the combined pretreatment with bio-pretreatment and mild acid pretreatment of G.uralensis residues for microalge cultivation.After 120 h heterotrophic cultivation,the obtained biomass and oil yield were respectively 4.46 g/L and 1.91 g/L,which were higher than those(biomass 2.02 g/L and oil yield 0.5 g/L)of glucose as sole carbon source.4.During the mixotrophic cultivation of C.protothecoides with enzymatic hydrolyzate as medium,the effects of those factors on the biomass and oil yield were studied,including addition of enzymatic hydrolysate,nitrogen resource,phosphorus resource,Mg2+ addition,pH value and temperature.Subsequently,the key factors affecting microalgal oil yield was explored via Plackett-Burman Design.Then,the three key factors,CO2 concentration,MgSO4 and pH value were further optimized through Box-Behnken.The optimum conditions were CO2 concentration 11.75%,NaNO3 addition 0.92 mg/L and pH value 7.2,and the obtained maximun oil yield was 2.62 g/L.5.Through particle bombardment,the plasmid of pMDLHAR was imported into C.protothecoides,and cultivated on the plate with hygromycin to screen the engineered strain.Then,the engineered strain was further verified through PCR,Southern bloting,RT-PCR,and Western bloting.After autotrophic cultivation,the oil yield of the engineered strain was insignificantly different with the control.However,the profiles of fatty acids(C14:0,C16:0 and C,6:1)of the engineered strain was significantly improved,conversely,the relative content of C18:0 and C18:1 were makedly decreased.6.Through particle bombardment,the derivative plasmid was imported into the trial strain and screened the engineered strain on plate with G418.Thereafter,by heterotrophic cultivation,the activity of PEPC enzyme and protein content of the engeered strain with sen-overpression PEPC were respectively 1.6 times and 1.45 times more than those of the control,but the oil yield was decreased 34%.The activity of PEPC enzyme and protein content of the engeered strain with anti-overpression PEPC were respectively reduced 68.9%and 27%,but the oil yield was more than 1.27 times than that of the control.