Separation And Screening Of Oil-rich Microalgae And Its Enzymatic Analysis Of Lipid Accumulation

At present,the human beings face two serious problems,which are environmental problems and the energy crisis.Therefore,developing clean and renewable energy to replace fossil fuels for reducing greenhouse gas emissions has become the focus of attention.Bioenergy as a renewable and environmentally friendly alternative energy sources play an important role to solving environmental problems and energy crisis.Among them,the biodiesel as one of the main bio-fuel is clean and can replace fossil fuel.Now the traditional oilseed crop,such as soybean,rapeseed and palm oil has been widely used in the production of biodiesel.Compared with the traditional oil crops,microalgae have great potential,and are the ideal material for biodiesel production.Based on the microalgae source of saline lakes in the Daqing,Zhaodong,and Zhalong of Heilongjiang province,the procedures of experiment contain isolation,screening and identification of microalgae.After isolation and screening,seven species strains of microalgae are obtained.Based on morphology and molecular identification,HDA01 is Chlorella sp,HDA02 is Scenedesmus obliques,HDA03 is Scenedes musdimorphus,HDA04 is Chlorella sorokiniana,HDA05 is Micractinium pusillum,HDA06 is Desmodes musintermedius,HDA07 is Chlorella pyrenoidosa.Seven species of microalgae are cultured under autotrophic and heterotrophic conditions respectively.Under autotrophic conditions,biomass of HDA03 is 0.63±0.03 g/L that is the highest.In heterotrophic conditions,biomass of HDA04 is the highest,reaching 3.95±0.05 g/L that is seven times of HDA03 under autotrophic condition.Under autotrophic condition,the highest oil content is 16.50±0.26%from HDA06.In heterotrophic conditions,the oil content of HDA04 is the highest,reaching 25.93±1.04%.Under heterotrophic conditions,fat productivity of HDA04 is the highest,reaching 472.99±18.97 mg/L·d.Considering the growth,oil content and fatty acid composition of microalgae,HDA04is selected as the starting algae strain for subsequent tests.Effect of single-factors initial pH,culture temperature,speed,inoculum size,liquid volume,glucose concentration,nitrogen,phosphorus source,magnesium ions,and iron ions on HDA04 microalgae biomass is discussed.By response surface method,optimum fermentation medium composition and fermentation conditions are known.Plackett-Burman experiment shows that glucose,initial pH and fermentation temperature have significant effects on the biomass of microalgae HDA04.Using the central composite design,the optimal culture conditions of HDA04 include initial pH value 8.89,temperature 32.91?,rotation speed 120 rpm,20%inoculum size,liquid volume l00 mL/250 mL,glucose concentration 21.46 g/L,most suitable nitrogen source peptone,K₂HPO₄·3H₂O concentration 120 mg/L,MgSO₄·7H₂O concentration 75 mg/L,the concentration of ferric citrate 12 mg/L.Under these conditions,biomassof microalgae HDA04 has a maximum value 7.869 g/L,is 1.34 times of biomass of HDA04 with the single factor test?5.89±0.16 g/L?.Under the culture conditions of nitrogen deficiency,the total fat content increases at first,and then decreases with increasing nitrogen concentration in the fermentation medium.The maximum of ACC activity is 140.20±7.50 U/L at peptone concentration of1.0 g/L,the maximum of PEPC activity is 82.56±2.71 U/L at peptone a concentration of0.5 g/L,the maximum of GPDH activity is 95.25±3.70 U/L at peptone concentration of1.5 g/L,the maximum of ACS activity is 73.17±6.27 U/L at peptone concentration of2.0 g/L,the maximum of DGAT activity is 83.54±6.36 U/L at the peptone concentration1.0 g/L,the maximum of AGP activity is 61.40±5.01 U/L at peptone concentration of1.0 g/L.Under culture conditions of phosphorus imbalance,the total fat content increases at first,and then decreases with increasing phosphorus concentration in fermentation medium.the maximum of ACC activity is 165.60±5.49 U/L at the phosphorus concentration of 120 mg/L,the maximum of PEPC activity is 88.11±1.50 U/L at the phosphorus concentration of 0 mg/L,the maximum of GPDH activity is 109.92±6.39U/L at the phosphorus concentration of 40 mg/L,the maximum of ACS enzyme activity is 94.96±2.46 U/L at the phosphorus concentration of 40 mg/L,the maximum of DGAT activity is 79.92±3.72 U/L at the phosphorus concentration of 80 mg/L,the maximum of AGP activity is 59.43±5.45 U/L at the phosphorus concentration of 0 mg/L.It was found that the procedure of microalgae lipid synthesis was affected by activity of some enzymes,where ACC,ACS,GPDH and DGAT could promote lipid synthesis,whereas AGP and PEPC restricted lipid synthesis at some extent.In this test,samples are drew from the saline alkaline lakes of Heilongjiang Province which owns the largest area of soda saline alkaline in the northern of China,and lay the foundation for the cultivation of microalgae in saline alkaline areas.At the same time,the microalgae species also are enriched,and provide a theoretical reference for the solution of the imbalance between the growth and the lipid synthesis of microalgae.