The reutilisation of Dunaliella salina carotenogenesis medium, after microalgal biomass separation by centrifugation, was assessed. The wastewater had an NaCl concentration between 174 g dm(-3) and 254 g dm(-3) and an average total organic matter concentration of 1540 mg dm(-3) ash-free dry weight, of which 41% (w/v) was glycerol. The biological treatment was established at laboratory scale and batch operations used halophilic bacteria from the wastewater itself. The wastewater was supplemented with NH4+, PO43-, K+ and Mg2+ ions to enhance growth. The effect of each ion added per se was initially investigated and a response surface methodology (RSM) used to identify the optimal conditions for maximisation of glycerol removal from the wastewater, which was considered to be the main objective. Addition of NH4+ ions alone achieved 79% glycerol removal compared with only 59% in the absence of supplement, after 8 days incubation. The combined addition of ions ([NaCl] =214g dm [Mg2+] = 114 mg dm(-3), [K+] = 131 mg dm(-3), [NH4+] = 113 mg dm(-3), [PO43-] = 40 mg dm(-3)) increased glycerol removal from the wastewater such that, after 2 days incubation, no residual glycerol was apparent in cultures. These ion combinations enabled the halophilic bacteria to efficiently remove glycerol from the wastewater and consequently reduce organic matter. This treated wastewater should be appropriate for reutilisation as a carotenogenesis medium for beta -carotene production from D salina.