| Traditional fossil fuels are increasingly consumed,and it is urgent to develop renewable energy.Biodiesel as a renewable energy can be used as an ideal substitute for fossil fuels.The oil in algal cells is a good raw material for biodiesel production,and the production of biodiesel from microalgae is attracting more and more attention.In this study,three strains of Monoraphidium sp.were isolated from freshwater lakes in Yunnan plateau,and the Monoraphidium sp.QLY-1 was selected as the research object.The two-stage cultivation strategy of heterotrophic cultivation and photo-chemical modulator induction were used in this study.Light induced binding to nitrogen deficiency,high salt,betaine and melatonin conditions are effects on the growth,biomass,oil accumulation,chlorophyll a,protein,carbohydrate,reactive oxygen species?ROS?,fatty acid composition,key enzyme activities and related gene expression levels of diphthophyllum,and their correlation with oil synthesis.Based on the above results,this paper provided a two step cultivation strategy for a rapid improvement in biomass and lipid of microalgae.The effects of different culture conditions and chemical inducers on the lipid accumulation were examined,and the enhancement of the lipid synthesis in Monoraphidium sp.QLY-1 on the levels of molecular and protein were discussed,which presents certain reference for investigation of gene levels in microalgae.The experiment results are summarized as follows:?1?Three algal strains were isolated from Yilong lake and Qilu lake and named Monoraphidium sp.QLY-1,Monoraphidium sp.QLZ-3 and Monoraphidium sp.YLY-2 based on its morphological and 18S rRNA molecular phylogenetic characterization.With the analysis and comparision of the growth,lipid content and fatty acids composition in this alga under autotrophic condition.Lastly,the alga Monoraphidium sp.QLY-1 was selected as our reseaech object based on its higher growth(0.878 g L-1)and lipid content?52.79%?.?2?Application of a two step strategy comprising heterotrophic cultivation and photo-chemical modulator induction to cultivate Monoraphidium sp.QLY-1.The alga was cultivated heterotrophically to achieve high biomass concentration(5.54 g L-1)with a lipid content of 22.47%,then diluted and transferred to autotrophic condition,the lipid content was achieved to 36.68%.Under autotrophic condition,the lipid content was further enhanced by 1.21,1.32,and 1.29 folds in algal cells treated with nitrogen deficiency,20 g L-1 NaCl,and 5 mM glycine betaine,respectively.Furthermore,the maximum lipid content?48.54%?and lipid productivity(121.27 mg L-1 d-1)were obtained in treatments with 20 g L-1 NaCl and 5 mM GB,respectively.?3?The lipid content was increased from 37.58%to 49.6%under 1?M melatonin addition and photoinduction conditions,and the maximal lipid content?49.6%?increased by 1.32 folds than that of the control.However,the protein and carbohydrate contents decreased rapidly from 27.21%to 17.96%and from 33.4%to17.71%,respectively.Moreover,the ROS contents in the control group and the experimental group increased by 1.17 and 1.15 times on induction day 3,respectively,but the maximum ROS content in the experimental group reduced 19.26%than the control group.?4?Under the combination of photoinduction and melatonin conditions,the activities of ACCase and ME increased significantly,and ACCase activity increased by 30.25%and 24.33%on the induction day 1 and 3,respectively.However,the activity of PEPCase was reduced significantly.In addition,the expression levels of accD and me were increased by 1.98 and 1.12 times under the induction of melatonin,respectively.The pepc and rbcL expression levels were reduced in different degrees.In conclusion,this chapter analyzed the mechanism of the melatonin induces lipid synthesis in Monoraphidium sp.QLY-1 based on the levels of protein and molecular,and provides a certain theory basis for application of genetic engineering in improving the lipid accumulation in microalgae. |
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