*6.2. Biomass Extraction*

Microalgal biomass is usually processed via solvent extraction, to render carotenoid extracts—with typical contents of 25% [45]; this can be used directly in the formulation of supplements, or undergo further multistep purification— encompassing hydrolysis to release hydroxylated carotenoids from the accompanying fatty acids, and final recrystallization to polish the product. 

Obtaining a carotenoid-rich oleoresin from microalgae—dried or in wet paste form, is a more straightforward task; such extracts may then be subjected to classical processes to obtain purer lutein [45,74] that may successfully compete with that extracted from marigold. 

## 6.2.1. Organic Solvent-Mediated Extraction

Solvent extraction usually resorts to hexane—and has advantages over alkaline treatment because all lutein and zeaxanthin are converted to their free forms, while carboxylic acids and chlorophylls remain in the aqueous phase [45]; this method has been optimized for *S. almeriensis* [74]. Extraction was maximized with a 1:1 (v/v) ratio of hexane to sample, and the optimal number of extraction steps was typically six— which led to 95% recovery of lutein. Less conventional solvents—e.g., ethyl lactate, have been recently proposed [76] for plant matter at large, but can in principle be applied also to microalgae. 

A significant improvement would be to eliminate the drying step of microalgal biomass prior to extraction; Fernández-Sevilla *et al*. [77] have accordingly proposed a modification of a previous approach [74] that can handle wet biomass paste (*ca*. 20% DW), based on an extraction phase composed by hexane/ethanol/water and KOH—which simultaneously effects an alkaline treatment to saponify susceptible lipids and extract the intended carotenoids. 

Another enhancement is the accelerated solvent extraction methodology, which uses a special type of contactor to circulate solvent at high pressure through a tightly packed bed of biomass. However, high temperatures are required (over 60 °C, and usually as high as 170 °C) to lower the viscosity of the solvent, which leads to formation of pheophorbide from the microalgal chlorophylls that are of a major toxicological concern. In any case, extraction with hexane or ethanol allows easy solvent removal afterwards, as well as high-content lutein extracts [45]. 

For selective extraction of free astaxanthin from red encysted *Haematococcus* sp., an alternative procedure has been designed that resorts to dodecane and methanol [75]; it consists of dodecane-mediated extraction of the crude mixture, followed by extraction with methanol. The first stage did not require previous cell harvesting, and separation of the dodecane-rich phase from the culture medium containing cell debris proceeded rapidly via plain settling. In the second stage, the free astaxanthin in the former extract was selectively solubilized in methanol along with saponification of astaxanthin esters—thus leading to a total recovery of astaxanthin above 85%. 
