This study investigated the cultivation of the marine alga Nannochloropsis gaditana in a medium based on desalination concentrate (DC) with an optimal concentration of 75% DC, under three trophic conditions and four photoperiod schedules. N. gaditana produced a peak biomass concentration (1.25gL−1) under mixotrophic culture condition and a photoperiod of 16L:08D. N. gaditana cells compensate to different light-dark regimes producing different amounts of protein (17.9–44.8%). The intracellular lipid content in N. gaditana cells increased both under autotrophic conditions with a 16L:08D cycle (16.7%), and under mixotrophic conditions with a 08L:16D cycle (15.7%). In heterotrophic culture, N. gaditana cells were rich in polyunsaturated fatty acids (46.0%). This study demonstrates an alternative approach to enhancing intracellular lipid content of the marine alga N. gaditana by modifying the photoperiod, trophic conditions and stress-salinity-conductivity with the use of a DC-based medium.