Abstract
BackgroundNannochloropsis oceanica belongs to a large group of photoautotrophic eukaryotic organisms that play important roles in fixation and cycling of atmospheric CO2. 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 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.ResultsFor 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 single-cell 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 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.ConclusionsThis 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.
Highlights
Population heterogeneity is a common phenomenon in microbial populations
fluorescence-activated cell sorting (FACS) sorting of +N and –N N. oceanica subpopulations FACS control experiments were performed to check for autofluorescence, population separation and artifacts
FACS analyses were conducted as depicted in Fig. 1, and the FACS analysis results of +N N. oceanica cells in Fig. 2a revealed no subpopulations in unstained cells, as well as for cells prepared in 15% DMSO without Nile Red
Summary
Population heterogeneity is a common phenomenon in microbial populations. It has been appreciated that certain environmental conditions as well as fluctuations in the microenvironment can elicit phenotypic heterogeneity in isogenic microbial cultures. Phenotypic heterogeneity occurs under conditions relevant to biotechnological processes, stress adaptation and biofilm formation: after continuous fed batch cultivation, an E. coli culture was found to consist of three subpopulations, one containing healthy cells, one containing cells with permeabilized membranes and dead cells [1, 2]. Phenotypes varying in lipid accumulation among an 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. Combination of FACS and proteomics was used for identification, quantification and differentiation of these heterogeneities on the molecular level
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