Research On The Lipid Accumulation Characteristics Of Chlorella Pyrenoidosa Under Mixotrophic Cultivation

In recent years,clean and renewable microalgae energy has been widely concerned due to the challenge of energy crisis and global warming.Oleaginous microalgae has the advantages of high photosynthesis efficiency,fast growth rate and high lipid yield,and is considered as the ideal raw material for preparing biodiesel.However,high costs of water resources,nutrients and energy consumption caused by the microalgae culture and collection process limit the industrialization development of microalgae energy.Usage of sewage for microalgae culture can not only reduce the cost of cultivation effectively,but also complete the sewage treatment.In this paper,to investigate the growth,lipid accumulation characteristics and usage of organic carbon and nutrients Chlorella pyrenodosa(C.pyrenoidosa)under the mixotrophic cultivation with sodium acetate,sodium acetate and ammonium-nitrogen and simulated anaerobic fermentation wastewater(ABR)and the effluents of domestic sewage treatment(DW),the Nile red-three dimensional fluorescence spectroscopy(3D-EEM)and fluorescence regional integration(FRI)was proposed and optimized.The relationships between the utilization of organic carbon and nutrients,growth and the accumulation of lipid were also analyzed.Meanwhile,the fatty acids and intracellular biochemical components were analyzed and compared by gas chromatography mass spectrometry(GC-MS)and Fourier transform infrared spectroscopy(FT-IR).The results of 3D-EEM showed that the excitation(?Ex)and the emission(?Ex)of neutral lipid(NL)were 525 nm and 580 nm,respectively,and the fluorescence region was(?Ex/?Em=480-540 nm/550?600 nm).The optimized conditions were as follows:the reaction time was 2 min,the OD680 was 0.2-0.5,and the optimal final concentration(or volume fraction)of Nile red and dimethyl sulfoxide was 0.5?g/mL and 10%,respectively.As the additional organic carbon under the mixotrophic cultivation,sodium acetate can not only coordinate the contradiction between the growth and lipid accumulation of C.pyrenoidosa,but also promote the accumulation of biomass,neutral lipid(NL)and total lipid production,and both of them are significantly higher than that of control samples(P<0.05).After 12 days' cultivation,the NL content per unit cells,biomass and total lipid yield in 5 g/L group were 4.5,2.1 and 3.1 times than that of control in autotrophic mode.Mixotrophic cultivation with sodium acetateand ammonium-nitrogen could not only adjust the pH of algal suspensions from 8.5 to 9.5,but also promote biomass and lipid productivity.As the increasing of NH4+-N concentrations,both of them first increased and then decreased,and reached the maximum level from 0 to 50 mg/L.The increase of sodium acetate concentration could enhance the ability of absorption and tolerance for high concentrations of NH4+-N,and improve the accumulation of NL.When sodium acetate was increased from 0 to 2 and 5 g/L,the utilization of 500 mg/L NH4+-N increased from 4.73%to 43.06%and 47.74%.Under the 10 g/L sodium acetate,algal cells could grow mixotrophically with 500 mg/L of NH4+-N,and continuously accumulate large amounts of NL and reach the maximum after 12 days(3.05 mg/109cells).C.pyrenoidosa could grow mixotrophically in waste water,achieving the pollutants removal and accumulation of biomass and lipid.After 12 days' inoculation,algae in ABR group grew rapidly and utilized 93.7%and 80.9%of COD and NH4+-N,respectively.After 30 days' cultivation,the NL in the ABR group was 1.09 mg/109 cells,which was 2.2 and 1.5 times than that of control and DW groups,respectively.The GC-MS results showed that the fatty acids of C.pyrenoidosa were mainly C16 and C18.Under the mixotrophic,the saturated fatty acids C16:0 and low unsaturated fatty acids C18:1,C18:2 were the main components,while the content of polyunsaturated fatty acids C18:3 was relative lower compared with the control samples under the autotrophic cultivation.Moreover,microalgal lipid under the mixotrophic cultivation has the potential to be developed into biodiesel due to lower unsaturation,favourable oxidation resistance,a certain low-temperature fluidity and combustion quality.