# Marine Microalgae for Potential Lutein Production
## 6 Microalgae Cultivation for Commercial Lutein Production and Challenges
In addition to the light strategy, the addition of 4.5 g L\({}^{-1}\) of calcium nitrate increased lutein productivity (3.06 mg L\({}^{-1}\) d\({}^{-1}\)), while the addition of 1.5 g L\({}^{-1}\) of calcium nitrate increased the lutein content to 2.45 mg g\({}^{-1}\). Finally, compared to a batch cultivation system, a fed-batch cultivation strategy had 11-fold higher lutein productivity (4.96 mg L\({}^{-1}\) d\({}^{-1}\)), which was the highest productivity that was achieved compared to all strategies. In the study by [PERSON] et al. [52], a two-stage cultivation strategy was applied, where heterotrophy with different temperatures was followed by photoinduction to improve biomass and lutein production in _Scenedesmus incrassatulus_. In the initial stage, where different nitrogen sources and temperatures were applied, the highest lutein content was observed using urea plus vitamins at 30 \({}^{\circ}\)C, during the second stage, after 24 h of photoinduction, lutein content increased seven times. According to the comparison between autotrophic and heterotrophic growth, [PERSON] et al. [52] concluded that lutein productivity was 1.6 times higher compared to autotrophic cultivation. Hence, heterotrophic and/or mixotrophic growth for lutein production can be suggested as a better cultivation method compared to autotrophic growth. [PERSON] et al. [53] observed different types of media and concentrations of sodium acetate and nitrate on _Chlorella sorkiniana_ FZU60 to improve mixotrophic growth and lutein production, where highest lutein content (9.57 mg g\({}^{-1}\)) and productivity (11.57 mg L\({}^{-1}\) d\({}^{-1}\)) were obtained in BG-11 medium supplemented with 1 g L\({}^{-1}\) acetate and 0.75 g L\({}^{-1}\) nitrate. Moreover, pulse feeding with 1 g L\({}^{-1}\) acetate every 48 h led to the alternation between mixotrophy and photoinduction, which resulted in a lutein production of 33.6 mg L\({}^{-1}\). In the study by [PERSON] et al. [54], _Chlorella protothecoides_ was cultivated heterotrophically (40 g L\({}^{-1}\) glucose and 3.6 g L\({}^{-1}\) urea) with nitrogen limitation in a fed-batch culture, which was followed by an increased lutein production without drastically reducing the biomass amount. Afterwards, this N-limited fed-batch culture was successfully scaled up from 3.7 to 30 L, and temperature stress was applied. Higher temperature (32 \({}^{\circ}\)C) for 84 h resulted in a 19.9% increase in lutein content but a 13.6% decrease in biomass amount, as compared to the fed-batch culture (30 L) without any treatment. In the study by [PERSON] et al. [55], the lutein content of _Auxencholorella protothecoides_ SAG 211-7a was observed under heterotrophic conditions, where the Taguchi Orthogonal Array method, a statistical technique for screening and optimisation of medium components at small-scale, was applied to select the six independent variables (yeast extract, KH\({}_{2}\)PO\({}_{4}\), MgSO\({}_{4}\).7H\({}_{2}\)O, CaCl\({}_{2}\).2H\({}_{2}\)O, EDTA, and pH) that affect lutein production. Sucrose, yeast extract, MgSO\({}_{4}\).7H\({}_{2}\)O, and EDTA were selected as the significant factors affecting lutein production. According to the experimental results, 1303 \(\pm\) 25.32 ug L\({}^{-1}\) lutein was produced when the medium was supplemented with 14 g L\({}^{-1}\) sucrose, 3 g L\({}^{-1}\) yeast extract, 0.8 g L\({}^{-1}\) MgSO\({}_{4}\).7H\({}_{2}\)O, and 0.76 g L\({}^{-1}\) EDTA. In conclusion, compared to all strategies mentioned above, the best approach for a microalgae cultivation system for maximum lutein productivity is a fed-batch cultivation strategy with different light and temperature stress depending on the selected freshwater culture and salinity for marine culture, since the maximum level of lutein in the cell is dependent on the salinity [56]. There is no commercial production system for lutein from microalgae yet; however, some outdoor productions of _Muriellopsis_ sp. and _Scenedesmus_ sp. at a pilot scale have been created, where _Muriellopsis_ sp.