Study On The Construction Of Mutant Strain CVM Of Chlorella Pyrenoidosa By Nitrogen Ion Beam Implantation And The Efficiency Of Municipal Wastewater Treatment

Biodiesel production of microalgae through municipal wastewater cultivation could effectively reduce the costs of wastewater treatment and biodiesel production,which has become a hot spot in the research of microalgae biodiesel.In order to improve the lipid production performance of microalgae and solve the contradiction between the growth of microalgae and the accumulation of lipid in the cultivation of Chlorella pyrenoidosa in municipal wastewater,a mutant with good performance and stable genetic characteristics had been obtained by ion implantation process in this thesis.Isobaric tags for relative and absolute quantification(i TRAQ)combined with liquid chromatography tandem mass spectrometry(LC-MS/MS)technology was used to analyze differentially expressed proteins and its function by ion implantation of C.pyrenoidosa from the angle of proteomics.The mechanism for promoting the growth and lipid accumulation of C.pyrenoidosa by ion implantation at the molecular level was also explored.In addition,optimization strategy of C.pyrenoidosa from the metabolic pathway was firstly studied,and its effectiveness and reliability was further verified by experiments.Finally,the application of mutant strain in an open raceway pond was investigated.First of all,the genetic stability of the C.pyrenoidosa mutant by ion implantation was studied,and the reasons for the enhancement of growth and lipid production of the C.pyrenoidosa mutant by ion implantation were investigated.The results showed that N~+ ion implantation could significantly improve the lipid performance of C.pyrenoidosa,and the mutant strain had stable genetic characteristics.The lipid productivity of mutant in BG11 medium and wastewater were 41.39 mg/L/d and 32.5 mg/L/d,33.34% and 23.81% higher than that of the original strain.Ion implantation did not change the individual size and the fatty acid composition of C.pyrenoidosa,but promoted the number of microalgae cells produced in the unit time and the content of monounsaturated fatty acid,which was beneficial to enhance the grade of biodiesel.Secondly,using iTRAQ technology and LC-MS/MS combined proteomics research methods,the differential expression proteins of C.pyrenoidosa were detected after ion implantation mutagenesis and their functional analysis was carried out.17 differentially expressed protein were identified in microalgal cells,including 3 up-regulated proteins and 14 down regulated proteins.After combining with central carbon metabolism network and lipid metabolism pathway of C.pyrenoidosa,it was found that both chlorophyll biosynthesis of mutant and microalgae carbon sequestration efficiency was improved through upregulation of Mg-protoporphyrin IX chelatase,resulting in more production of organic matter and further leading to biomass increased.Through down-regulation of fructose two phosphate aldolase(FBA)and transketolase(TK)enhanced glycolysis and the pentose pho sphate pathway,lipid accumulation of C.pyrenoidosa was promoted.The mechanism of ion implantation for promoting C.pyrenoidosa growth and lipid productivity was revealed on the basis of proteomics.Furthermore,the optimal regulation strategy of C.pyrenoidosa metabolism pathway was also put forward,which was promoting chlorophyll synthesis and Calvin cycle.Single factor experiments and orthogonal experiments were conducted to optimize the types and input dosage of plant growth regulators and bacte ria.The reasons for plant growth regulators and bacteria to promote growth and lipid productivity of C.pyrenoidosa were discussed.The optimum formula was: 6-BA 1.5,mg/L,2,4-D 1.5,mg/L,PSB D600 0.01,W4 bacteria D600 0.02.Under this condition,the biomass and lipid productivity were 155mg/L/d and 33.61mg/L/d,20.78% and 8.04% higher than that in the blank group,respectively.The validity and reliability of the optimal regulation strategy for the growth and metabolism pathway of C.pyrenoidosa had been proved by experiments.Finally,the cultivation of mutant in the open raceway pond was investigated,and the feasibility of open-pond culture was discussed.From the fourth day,the biomass and lipid production of the mutant tended to be stable,the highest biomass productivity was 71.67mg/L/d,and the highest lipid productivity reached 17.4mg/L/d.With the expansion of cultivation scale,open-pond culture technology had a good application prospect.In conclusion,the thesis studied the mechanism of growth and lipid productivity promotion of C.pyrenoidosa by ion implantation at the protein level,which laid a solid theoretical basis for the comprehensive analysis of central carbon metabolism network and regulation of the growth/metabolism pathway of C.pyrenoidosa.Based on the function analysis of the difference proteins expression of C.pyrenoidosa after ion implantation mutation,optimization control strategy of C.pyrenoidosa growth and metabolism pathway was proposed,which played an important role in further improving the biomass and lipid productivity of C.pyrenoidosa and guiding largescale cultivation using the open-pond.