# Marine Microalgae for Potential Lutein Production
## 7 Extraction of Lutein from Microalgae and Challenges
[70] also investigated the use of supercritical fluid extraction using CO\({}_{2}\) for the extraction of astaxanthin and lutein from disrupted red phase biomass of the _Haematococcus pluvialis_. A bench-scale reactor in a semi-batch configuration was employed. Parameters such as extraction time (20, 40, 60, 80, and 120 min), CO\({}_{2}\) flow rate (3.62 and 14.48 g min\({}^{-1}\)) temperature (50, 65, and 80 \({}^{\circ}\)C), and pressure (100, 400, and 550 bar.) were investigated. The results indicate the maximum recovery of astaxanthin and lutein obtained was 98.6% and 52.3%, respectively, at 50 \({}^{\circ}\)C and 550 bars. [PERSON] et al. [71] employed supercritical fluid for the extraction of lutein from _Scenedesmus almteriensis_. The optimisation of the main parameters affecting the extraction, such as biomass pretreatment, temperature, pressure, and CO\({}_{2}\) flow rate, was conducted. Firstly, the effect of mechanical pretreatment with diatomaceous earth (DE) and biomass mixing in the range of 0.25-1 DE/biomass, grinding speed varying between 0-600 rpm, and pretreatment time changing from 2.5 to 10 min, was evaluated on lutein extraction efficiency. Next, the influence of SFE extraction parameters such as pressure (250-550 bar), temperature (50 and 65 \({}^{\circ}\)C), and CO\({}_{2}\) flow rate (7.24 and 14.48 g min\({}^{-1}\)) on lutein recovery and purity was examined. According to the results, increases in the temperature, pressure, and CO\({}_{2}\) flow rate improved the lutein recovery and purity. The maximum lutein recovery (~98%) with a purity of ~34% was accomplished running at 65 \({}^{\circ}\)C and 550 bar with CO\({}_{2}\) flow rate of 14.48 g min\({}^{-1}\). Research conducted by [PERSON] et al. [68] in regard to utilising SFE for the extraction of lutein from _Scenedesmus_ sp. biomass found that an increase in both pressure and temperature resulted in an increase in lutein yield, but an increase in temperature resulted in increased impurity within the HPLC profile. This increase in yield was not as significant when compared to the yields from conventional extraction methods. Optimum parameters for lutein recovery yield were determined as 400 bar pressure and temperature at 70 \({}^{\circ}\)C while using ethanol as the co-solvent at a ratio of 30 mol\(\%\). These parameters resulted in lutein recovery of 76.7%, which was compared to conventional methanol extraction methods. Research carried out by [PERSON] et al. [72] involved the use of a pretreatment process using alcohol aimed at removing chlorophyll \(a\), \(b\), and _b_-carotene from _Chlorella vulgaris_. This process was developed to enhance the yield and selectivity of lutein in the extract obtained by subsequent supercritical fluid extraction. SFE was carried out in the pressure range 200 to 400 bar and the temperature range of 40 to 80 \({}^{\circ}\)C, with methanol and ethanol trialled as the co-solvents, with ethanol the most suitable for lutein extraction. Lutein yield within the extract increased with pressure but decreased with temperature. The optimal parameters for lutein recovery yield were determined as 400 bar and 40 \({}^{\circ}\)C, using ethanol as the co-solvent. Under these conditions, the maximum recovery percentage and selectivity percentage of lutein resulted in approximately 52.9 \(\pm\) 0.02% and 43.1 \(\pm\) 0.02%, respectively. [PERSON] et al. [23] aimed to extract lutein from _Chlorella pyrenoidosa_ using ultrasound-enhanced subcritical CO\({}_{2}\) extraction (USCCE). As part of this research, parameters such as pretreatment process, pressure, temperature, CO\({}_{2}\) flow rate, and ultrasonic power were examined, alongside orthogonal analysis, to study the effects of varying parameters on lutein extraction yields. The developed USCCE method was conducted as part of this study and it was compared to other common extraction methods.