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Oleaginous algae are promising feedstock for converting sunlight and carbon dioxide to renewablebiofuels.Nannochloropsis is a genus of unicellular photosynthetic microalgae in the class Eustigmatophyceae,ranging in size of 2-5μm and widely distributed in marine,fresh and brackish waters.They are of industrialinterest due to their ability to grow rapidly,synthesize large amounts of triacylglycerols (TAG) and highvaluepolyunsaturated fatty acids,and to tolerate broad environmental and culture conditions.Here usingthe bioprocess of nitrogen depletion induced TAG-synthesis by Nannochloropsis oceanica IMET1 strainas a model,we established single-cell Raman spectrometry (SCRS) as a rapid,sensitive,non-invasiveand reproducible approach to monitor the oil-producing bioprocess at single-cell resolution in real-time.This approach does not require lipid extraction or staining of Nile Red that are slow and invasive,thusprovided a strategy for in vivo monitoring.We acquired Raman spectra of 60 randomly selected cells (20cells per biological replicate) at each of the eight time-points over 96 hours from an isogenic population,and used for the prediction of growth status of each individual cell.SCRS showed that in the cells underN- condition,intensities of all major lipid bands,including bands representing chemical bonds attributed tochain unsaturation (1265,1656 and 3013cm-1) and saturation (1305,1445,2850 and 2890cm-1),apparentlyincreased along the cultivation time since 6h,revealing significant lipid accumulation.Notably,the 1746cm-1 Raman band,which indicates the C=O stretching vibration specifically of TAG,also occurred andincreased remarkably after 6h.In the meanwhile,intensities of protein-relative bands (e.g.1005,1200-1350,and 1600-1700cm-1) decreased in the N- group.The findings suggested a continuous TAG production bythe cells accompanied with deficiency of protein biosynthesis under a nitrogen- deficiency condition.TheSCRS was able to distinguish cells between TAG-producing and non-TAG-producing groups with >93.3%accuracy at as early as 6th hours.For the culture under nitrogen depletion,cells of different time points weredistinguished with >90.4 % accuracy.Our rapid approach has great potential in direct and high-throughputscreening of microalgae and also provides valuable information for optimization and selection of species andgrowth conditions.