Flagellates of Haematococcus pluvialis accumulate secondary carotenoids (mainly astaxanthin and its esters) under nitrogen deficiency in combination with exposure to stronger light. Our cultivation scheme allows the characterization of metabolic activities during the accumulation process without interference by formation of the resting state (aplanospores). The highest rate of secondary carotenoid biosynthesis (0·046 h−1) was observed almost simultaneously with the highest rates of cell division (0·027 h−1) and of chlorophyll biosynthesis (0·015 h−1) after 1·5 days of exposure to nitrogen limitation and higher irradiance. Photosynthetic activity was studied by oxygen evolution and chlorophyll fluorescence measurements; changes in protein components of the photosynthetic apparatus were quantified by gel electrophoresis and immunoblot analysis. Accumulation of secondary carotenoids was accompanied by an increase in photosynthetic productivity expressed on a unit chlorophyll basis. Changes in the photosynthetic apparatus occurring mainly during the first 2 days of exposure to nitrogen limitation and stronger light can be interpreted as acclimation to the increased cultivation irradiance. Photoprotective action of secondary carotenoids as a ‘sunshade’ was demonstrated in red flagellates exhibiting a lower blue-light-induced decrease in photosystem II efficiency as compared with red actinic light.

The combination of nitrogen deprivation and increased cultivation light intensity resulted in the synthesis of secondary carotenoids in flagellates of Haematococcus lacustris. The pigment pattern was characterized by high-pressure liquid chromatography analysis during the accumulation period and in response to inhibitors of carotenoid biosynthesis (diphenylamine, norflurazon and tetcyclacis). Diphenylamine treatment resulted in (i) a decrease in ketocarotenoids and (ii) an accumulation of β-carotene and zeaxanthin due to inhibition of the β-carotene oxygenase. Our results indicate that astaxanthin synthesis in H. lacustris follows the biosynthetic pathway elucidated in the marine bacterium Agrobacterium aurantiacum.