Examination of Photo-, Mixo-, and Heterotrophic Cultivation Conditions on _Haematococcus pluvialis_ Cyst Cell Germination  
[PERSON]  
1 Bothermodynamics, TUM School of Life Sciences, Technical University of Munich,  
Maximus-von-Imhof-Forum 2, 85354 Freising, Germany; [EMAIL_ADDRESS]  
1  
[PERSON]  
1 Bothermodynamics, TUM School of Life Sciences, Technical University of Munich,  
Maximus-von-Imhof-Forum 2, 85354 Freising, Germany; [EMAIL_ADDRESS]  
1  
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In the final stage of cyst formation, _H. pluvialis_ develops a rigid and resistant cell wall that consists of a trilaminar sheath, secondary wall, and the tertiary wall (Figure 1d) [7]. In principle, _H. pluvialis_ can grow under phototrophic, heterotrophic, or mixotrophic conditions. When growing phototrophically, sunlight is used as energy and CO\({}_{2}\) as a carbon source [8]. With heterotrophic growth, microalgae take up organic molecules such as glucose or acetate [9; 10] as their main source of nutrition, and no light is used during cultivation [11; 12]. Glucose was used in several studies for the cultivation of _H. pluvialis_[13; 14; 15]. Mixotrophic cultivation is a variant of heterotrophic growth, where the organic carbon and CO\({}_{2}\) are assimilated simultaneously and respiratory and photosynthetic metabolism operate concurrently [16]. In literature, mixotrophic cultivation of vegetative _H. pluvialis_ cells was done, using the sugars ribose, mannose, fructose, xylose, glucose, sodium acetate, glycerol, lactose, or sucrose at a concentration of 1.33 g L\({}^{-1}\) (corresponds to 7.38 mM glucose) [17]. On an industrial scale, the upstreaming of _H. pluvialis_ is done phototrophically in the form of a two-step process using closed photobioreactors. Open ponds are rarely used for the green phase of biomass production and are usually just used for the second step of cell stressing. In the first process step (Figure 1a,b), the algal biomass is cultivated phototrophically to high cell concentrations under optimal growth conditions, with a sufficient supply of nutrients such as nitrate and phosphate [18], passing with CO\({}_{2}\)[19], and artificial lighting, especially by the red fraction of the light [20; 21]. On an industrial scale, natural sunlight is usually used for cultivation [1]. Under so-called \"stress conditions\", nitrate and phosphate deficiency and light stress, and especially the blue light spectrum [21; 22], the astaxanthin synthesis is initiated and further enhanced by CO\({}_{2}\) gassing [19] in the second process step (Figure 1c,d). Astaxanthin accumulation is accompanied by the formation of a resistant cyst cell wall, which impedes direct and efficient astaxanthin extraction. Thus, a complex downstream process is required, including centrifugation, mechanical cell wall disruption, and spray drying; finally, astaxanthin is extracted with supercritical CO\({}_{2}\)[23]. As a result, energy-intensive mechanical processes such as bead mills or high-pressure homogenization are required for the industrial cell wall disruption of the cyst cells. Up to three cycle repetitions are needed to achieve sufficient cell wall disruption efficiency using high-pressure homogenization [24]. Germination of _H. pluvialis_ cysts represents an interesting alternative to mechanical cell wall disruption. When the cysts are exposed to growth conditions and sufficient nutrients are added, the mother cell begins to divide, forms a sporangium (Figure 1e), and releases zoospores (Figure 1f) containing astaxanthin. After a certain time, these also lose their flagella, round off, and form astaxanthin-containing non-motile aplanospores (Figure 1g) [25].