Biodiesel produced from microalgal lipids is being considered as a potential source of renewable energy. However, a number of hurdles will have to be overcome if such a process is to become practical. One important factor is the volumetric production of biomass and lipid that can be achieved. The marine alga Nannochloropsis gaditana is under study since it is known to be highly oleaginous and has a number of other attractive properties. Factors that might be important in biomass and lipid production by this alga are light intensity, inoculum size and CO2. Here we have carried out for the first time a RSM-DOE study of the influence of these important culture variables and define conditions that maximize biomass production, lipid content (BODIPY (R) fluorescence) and total lipid production. Moreover, flow cytometry allowed the examination on a cellular level of changes that occur in cellular populations as they age and accumulate lipids. (C) 2014 Elsevier Ltd. All rights reserved.