Study On Environmental Regulation And Molecular Mechanism Of Lipid Accumulation In Microalgae

Due to the depletion of fossil fuels and the prodution of environmental pollution problems on their combution,biodiesel is receiving widespread attention.Microalgae have emerged as one of the most promising feedback for biodiesel owing to higher photosynthetic efficiency and higher lipid content.Moreover,compared with traditional terrestrial oil plants,microalgae can grow in place aways farmlands.However,because of higher production costs,the production of microalgae for biodiesel has not been undertaken commercial scale.In study,10 strains of microalgae was selected as research object,and the biomass,lipid content and lipid yield were compared,a faster growth,higher lipid content of the Ankistrodesmus gracilis CJ09was obtained,and were performed for total nutrient analysis,optimization the method of lipid extraction based on the highest content of lipid,the effects of environmental factors on the growth and lipid accumulation of A.gracilis CJ09 were invistigated,furthermore,transcriptome analysis was performed on A.gracilis CJ09 and which provided a reference basis for the genetic modification of A.gracilis CJ09.The main research results are summarized as follows:（1）10 strains of microalgae was selected as research object,and the biomass,lipid content and lipid yield were compared,a faster growth,higher lipid content of the advantages of microalgae was obtained.The results show that the biomass,lipid content and lipid yield of A.gracilis CJ09 was 1.92±0.06 g/L,23.58±0.46%and0.45±0.007 g/L.compared with other microalgae,it have emerged as one of the most promising feedback for biodiesel.Therefore,A.gracilis CJ09 was selected for follow-up research.（2）Through total nutrient analysis,it was found that Ankistrodesmus gracilis CJ09 contains rich nutritional components.Total sugar,protein,oil,moisture,ash and crude fiber accounted for 7.02%,17.99%,23.68%,9.14%,10.92%and 21.59%of the dry weight of algae powder,respectively.Ankistrodesmus gracilis CJ09 contains a variety of fatty acids,and C16 and C18 short-chain fatty acids（97.59%）are mainly contained in the cellulite cells,and C18:1（n-9）accounts for 20.11%,which can be used to produce relatively high performance biodiesel.The content of vitamins VB1,VB3 and VE of A.gracilis CJ09 were 17.84 mg/100g,14.26 mg/100g and 9.22mg/100g,respectively,other vitamins are low in content.There is a rich content of amino acids in A.gracilis CJ09,including 8 essential amino acids and two umami amino acids.There is a rich content of mineral elements in A.gracilis CJ09,among which sodium,potassium,calcium,magnesium,manganese,zinc and iron are 729.32mg/kg,4539.20 mg/kg,6183.81 mg/kg,14384.02 mg/kg,1265.55 mg/kg,641.48mg/kg and 8459.05 mg/kg respectively.It can be used as a source of mineral elements in animals.（3）In order to explore the value of lipid utilization in Ankistrodesmus gracilis CJ09,the ultrasonic-assisted method was selected to research based on the highest content of lipid in different lipid extraction methods.The Box-Behnken response surface design was used to optimize the lipid extraction conditions.the optimize extraction conditions were:extraction power 521.12 W,extraction temperature48.86℃,extraction time 50 min,liquid-to-material ratio 30 m L/g,extraction times 3.Six groups of experiments showed that the lipid extraction rate was 26.07±0.78%.The lipid extracted by ultrasonic assisted extraction was methylated and analyzed by GC-MS,the results show that the major fatty acid composition were C16 and C18short-chain fatty acids（98.56%）and not much different from algae powder,and the relative content of C18:1（n-9）were 16.20%,It was slightly lower than algae powder.（4）The cultivation conditions of Ankistrodesmus gracilis CJ09 were optimized,and single-factor test was performed to reveal the effects of different mass concentrations of Na NO₃,K₂HPO_4,Na₂CO_3,Ferric ammonium citrate,Ca Cl₂·2H₂O and p H on the growth and lipid accumulation of A.gracilis CJ09,The results showed that higher biomass and higher lipid yield were obtained when the mass concentration of Na NO₃,K₂HPO₄,Na₂CO_3,Ferric ammonium citrate,Ca Cl_2`2H₂O were 0.75g/L,0.02 g/L,0.08 g/L,12 mg/L,0.018 g/L,and the optimum p H was 7.5,respectively.（5）Transcriptome analysis was performed on Ankistrodesmus gracilis CJ09,a total of about 45 million raw reads were obtained,after filtering,the Q20 value was94.31-96%,and the Q30 value was 83.84-86.8%,the result show that the error rate of base error detection was low,and the accuracy of sequencing results was high.Trinity software was used to assembl and cluster to remove redundancy for the clean reads,and 66005 unigenes were obtained.Unigenes was used to compare with seven databases for annotation,finally 53009 Unigene was Commented in NR database（80.31%）,17915 Unigene was Commented in NT database（27.14%）,35198 Unigene annotations was Commented in Swiss Prot（53.33%）,37607 Unigene was Commented in KOG database（56.98%）,42155 Unigene was Commented in KEGG database（63.87%）,45583 Unigene was Commented in GO database（69.06%）,and 44712Unigene was Commented in Pfam database（67.74%）,the annotated Unigene was classified by GO,KOG and KEGG Pathway,and the pathway of lipid biosynthesis,strach biosynthesis,and thiamine biosynthesis were described for the genetic engineering of improving lipid yield in microalgae.The result provides a theoretical basis for lipid anabolism gene level regulation on A.gracilis CJ09.