Heterotrophic And Mixotrophic Cultivation Of Microalgae Under Nitrogen Starvation For Biodiesel Production

With the rapid consumption of crude oil,alternative biofuels provided by various sustainable technologies have become more economically attractive,while transesterification of microalgae lipid to biodiesel is one of the promising green technologies.Heterotrophic cultivation of microalgae is attracting more attention due to its high lipid production efficiency.Nitrogen has been widely studied as a strategy to stimulate lipid accumulation in microalgae,but it always leads to a decrease of the lipid productivity due to the low growth rate.Sufficient phosphorus supply combined with nitrogen starvation can improve the lipid productivities of Chlorella vulgaris and Scenedesmus obliqus under autotrophic cultivation,while its role in heterotrophic culture is unclear.In this study the heterotrophic cultivation of C.vulgaris NIES-227 fed with glucose was investigated systematically using six media types;combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion.It was found that a high yield of fatty acids(0.88 of fed glucose-COD)and a high content of fatty acid methyl esters(FAMEs)(89%of dry weight)were obtained under nitrogen starved conditions.The dominant fatty acid(>50%)was methyl oleate(C18:1),a desirable component for biodiesel synthesis.FAME content under nitrogen starved conditions was significantly higher than under nitrogen sufficient conditions,while phosphorus had no significant influence,indicating that nitrogen starvation was the real "fatty acids trigger" in heterotrophic cultivation.S.obliquus NIES-2280 was cultivated heterotrophically with acetate as the carbon source.The effects of nitrogen deficiency and different phosphorus supply levels on biodiesel production by S.obliquus were investigated.It was found that S.obliquus could make good use of assimilated acetate for fatty acid accumulation.Fatty acid contents of algae in nitrogen deficiency media increased to 38-48%after 6-day cultivation.Interestingly,the productivity of fatty acid methyl esters(FAMEs)under nitrogen starved conditions increased fourfold than that under nitrogen sufficient conditions.Moreover,FAME productivity could be further enhanced by a sufficient phosphorus supply rather than under P limitation or P deficiency conditions,and the highest FAME productivity was 55.9 mg·L-1·d-1.Furthermore,the conversion yields of acetate to fatty acids(COD based)in nitrogen starvation media(18-28%)were much higher than those in nitrogen sufficient media(?7%).C.vulgaris was also cultivated heterotrophically with acetate in batch systems.The effects of nitrogen deficiency and different phosphorus supply levels on biodiesel production were investigated.It was found that C.vulgaris could assimilate acetate and accumulate fatty acids simultaneously.The highest FAME(Fatty acid methyl ester)content(56%)of algae was obtained in nitrogen deficient media.Interestingly,the productivity of fatty acids under nitrogen starved conditions was three times greater than that under nitrogen sufficient conditions.Moreover,FAME productivity was further enhanced to 66 mg L-1 d-1 by a sufficient phosphorus supply but there was no effect caused by phosphorus limitation.Furthermore,the conversion yields of acetate to fatty acids(COD-based)in nitrogen starved media were also three times higher than those in nitrogen sufficient media.FAME productivity of C.vulgaris under nitrogen starvation was not improved by controlling the pH at stable values(7.5,8.0,and 8.5),and even decreased when pH was controlled at 7.0 and 9.0.Whether lipid productivity of microalgae from mixotrophic culture can exceed the sum of those from autotrophic and heterotrophic culture is still uncertain and nitrogen starvation has been demonstrated capable of enhancing both lipid content and lipid productivity of microalgae.For the first time,fatty acid production of S.obliquus under nitrogen starvation in autotrophic,heterotrophic,and mixotrophic cultures was investigated and compared in this study.It was found that both the biomass and fatty acid productivities of mixotrophic algae cells exceeded the combination of autotrophic and heterotrophic cells when acetate was adopted as the organic carbon source,and fatty acid productivity from mixotrophic culture was 1.64 times greater than from the combination of autotrophic and heterotrophic cultures.Moreover,the fatty acid yield from mixotrophic culture was almost two times greater than from heterotrophic culture.Proteomics analysis revealed that the activity of the TCA cycle was improved in mixotrophic culture when compared with heterotrophic culture,leading to more fatty acid synthesis in S.obliquus cells.These findings can simplify the downstream extraction process,such as the extrusion of oil from soybeans.Moreover,this study indicates a great potential to combine wastewater treatment with biodiesel production via C.vulgaris and S.obliquus which can significantly improve biodiesel productivity and COD utilization under nitrogen starvation coupled with sufficient phosphorus supply.