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Chaoyun Chen, Andreas Harst, Wuxin You, Jian Xu, Kang Ning, Ansgar Poetsch

• \(\bf Background:\) \(\textit {Nannochloropsis oceanica}\) belongs to a large group of photoautotrophic eukaryotic organisms that play important roles in fixation and cycling of atmospheric CO\(_{2}\). Its capability of storing solar energy and carbon dioxide in the form of triacylglycerol (TAG) of up to 60% of total weight under nitrogen deprivation stress sparked interest in its use for biofuel production. Phenotypes varying in lipid accumulation among an \(\textit {N. oceanica}\) population can be disclosed by single-cell analysis/sorting using fluorescence-activated cell sorting (FACS); yet the phenomenon of single cell heterogeneity in an algae population remains to be fully understood at the molecular level. In this study, combination of FACS and proteomics was used for identification, quantification and differentiation of these heterogeneities on the molecular level. \(\bf Results:\) For \(\textit {N. oceanica}\) cultivated under nitrogen deplete (−N) and replete (+N) conditions, two groups differing in lipid content were distinguished. These differentiations could be recognized on the population as well as the singlecell levels; proteomics uncovered alterations in carbon fixation and flux, photosynthetic machinery, lipid storage and turnover in the populations. Although heterogeneity patterns have been affected by nitrogen supply and cultivation conditions of the \(\textit {N. oceanica}\) populations, differentiation itself seems to be very robust against these factors: cultivation under +N, −N, in shaker bottles, and in a photo-bioreactor all split into two subpopulations. Intriguingly, population heterogeneity resumed after subpopulations were separately recultivated for a second round, refuting the possible development of genetic heterogeneity in the course of sorting and cultivation. \(\bf Conclusions:\) This work illustrates for the first time the feasibility of combining FACS and (prote)-omics for mechanistic understanding of phenotypic heterogeneity in lipid-producing microalgae. Such combinatorial method can facilitate molecular breeding and design of bioprocesses.