| Biodiesel is a renewable energy that can replace traditional fossil fuels.Microalgae has attracted growing attention from researchers worldwide for its advantages of fast growth rate,rapid lipid accumulation,non-arable areas,so it is the best raw material for biodiesel production.Increasing the lipid production is the key to reduce the cost of biodiesel,which can be solved by two aspects: increasing the biomass of microalgae and the content of lipid in the cells.Nitrogen deprivation has been widely used to improve lipid accumulation.However,high lipid accumulation under nitrogen starvation is usually at the cost of biomass production,and leads to only a slight enhancement or even to a decrease in lipid productivity.Aiming at this problem,this paper discusses a new microalgae cultivation scheme,which can improve biomass and lipid content simultaneously.(1)This study proposed a new strategy to solve this problem by using nitrogen limitation combined with phosphorus luxury during heterotrophic cultivation.It was found that nitrogen limitation caused a slowing in growth of Chlorella regularis,resulting in biomass productivity of only 2.34 g/L versus 4.73 g/L using the control protocol(normal nutrients).When the above nitrogen limitation was combined with a phosphorus supplement(45 mg/L),biomass productivity recovered to 4.53 g/L.Extra phosphorus in microalgae cells uniquely formed polyphosphate,which offer the necessary materials and energy for growth and lipid accumulation.Growth genes analysis revealed that brassinosteroid biosynthesis,carbon and amino acid metabolism are critical pathways in biosynthesis.The microalgae lipid content reached 42.3% and lipid productivity reached 310.0 mg/(L·d),nearly double and 1.32 times that of the control,respectively.C16 and C18 fatty acids were more than 95% of the total fatty acids;both saturated and monounsaturated fatty acid compositions increased.Therefore,the cultivation of microalgae with nitrogen limitation combined with phosphorus luxury can not only improve biomass,lipid content,but also improve the quality of biodiesel.(2)The effect of different phosphorus adding mode on the growth and lipid formation of microalgae was studied under the condition of nitrogen limitation combined with phosphorus luxury.Under such condition,the results indicate that the increase in the number of phosphorus addition on the growth of microalgae had a certain degree of inhibition,such as the maximum biomass was 3.95 g/L for two times,while 3.53g/L for eight times.However,the addition of phosphorus eight times could reach a higher lipid content of 40.5%,which is 1.1 times higher than two times.Through the analysis of synthesis of lipid related gene expression,gene Unigene7398 encoding ACCase,gene CL2735.Contig2,CL73.Contig8,CL12.Contig1,Unigene7590,CL2735.Contig1 and Unigene1920 encoding DGAT,increase the phosphorus adding times can improve the expression level of lipid synthesis genes.At the end of the culture cycle,different phosphorus adding mode had no effect on the storage form of phosphorus in microalgae,mainly in the pattern of orthophosphate monoesters of the 4 protocols.In addition,addition of phosphorus several times was conducive to rapid accumulation of saturated fatty acid(mainly C16:0),and adding less time help to improve C18:0 and C18:1.Therefore,the increase in the number of phosphorus addition has no obvious effect on the increase of lipid production,but can increase the content of lipid in the cells. |
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