in two phases. First, in the green phase, the cells are given an abundance of nutrients to promote proliferation of the cells. In the subsequent red phase, the cells are deprived of nutrients and subjected to intense sunlight to induce encystment (carotogenesis), during which the cells produce high levels of astaxanthin as a protective mechanism against the environmental stress. The cells, with their high concentrations of astaxanthin, are then harvested. Phaffia yeast Xanthophyllomyces dendrorhous exhibits 100% free, non-esterified astaxanthin, which is considered advantageous because it is readily absorbable and need not be hydrolysed in the digestive tract of the fish. In contrast to synthetic and bacteria sources of astaxanthin, yeast sources of astaxanthin consist mainly of the (3R, 3'R)-form, an important astaxanthin source in nature. Finally, the geometrical isomer, all-E, is higher in yeast sources of astaxanthin, as compared to synthetic sources. In shellfish, astaxanthin is almost exclusively concentrated in the shells, with only low amounts in the flesh itself, and most of it only becomes visible during cooking as the pigment separates from the denatured proteins that otherwise bind it. Astaxanthin is extracted from Euphausia superba (Antarctic krill) and from shrimp processing waste. 12,000 pounds of wet shrimp shells can yield a 6–8 gallon astaxanthin/triglyceride oil mixture. Biosynthesis Astaxanthin biosynthesis starts with three molecules of isopentenyl pyrophosphate (IPP) and one molecule of dimethylallyl pyrophosphate (DMAPP) that are combined by IPP isomerase and converted to geranylgeranyl pyrophosphate (GGPP) by GGPP synthase. Two molecules of GGPP are then coupled by phytoene synthase to form phytoene. Next, phytoene desaturase creates four double bonds in the phytoene molecule to form lycopene. After desaturation, lycopene cyclase first forms γ-carotene by converting one of the ψ acyclic ends of the lycopene as a β-ring, then subsequently converts the other to form β-carotene. From β-carotene, hydrolases (blue) are responsible for the inclusion of two 3-hydroxy groups, and ketolases (green) for the addition of two 4-keto groups, forming multiple intermediate molecules until the final molecule, astaxanthin, is obtained. Synthetic sources The structure of astaxanthin by synthesis was described in 1975. Nearly all commercially available astaxanthin for aquaculture is produced synthetically, with an annual turnover of over $200 million and a selling price of roughly $5000–6000 per kilo as of July 2012. The market grew to over $500 million by 2016 and is expected to continue to grow with the aquaculture industry. An efficient synthesis from isophorone, cis-3-methyl-2-penten-4-yn-1-ol