## 2 Materials and Methods
### Astaxanthin Extractability Analysis  
Astaxanthin can be extracted directly from the zoospores into ethyl acetate without prior mechanical cell disruption or drying [26]. Several solvents, including ethyl acetate, methyl-_tert_-butyl ether, n-heptane, butan-1-ol, dichloromethane, have been evaluated for the extraction of astaxanthin from germinated zoospores in our previous work [26]. Ethyl acetate showed the largest extraction yield; it is classified as a green solvent and was therefore chosen as an extraction solvent [32, 33]. Consequently, the algal broth's extractability can be used as an indicator for the germination of cyst cells or the number of released zoospores. Therefore, liquid-liquid extraction was carried out as a single determination at a different time point after germination, to determine the amount of extractable astaxanthin into ethyl acetate. Two milliliters of algal culture were transferred into a 15 mL falcon tube and mixed with 2 mL ethyl acetate for 60 min using a Multi Bio RS-24 shaker from Biosan (Riga, Latvia). The samples were then centrifuged at 5500 rpm \begin{table}
\begin{tabular}{l l} \hline \hline
**Name** & **Germination Conditions** \\ \hline culture 1 & phototrophic + CO\({}_{2}\) + 100 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ culture 2 & phototrophic + ambient air + 100 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ culture 3 & mixotrophic + 4 mM glucose + CO\({}_{2}\) + 100 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ culture 4 & mixotrophic + 4 mM glucose + ambient air + 100 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ \hline culture 5 & phototrophic + CO\({}_{2}\) + 75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ culture 6 & phototrophic + ambient air + 75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ culture 7 & mixotrophic + 4 mM glucose + CO\({}_{2}\) + 75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ culture 8 & mixotrophic + 4 mM glucose + ambient air + 75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ \hline culture 9 & heterotrophic + \"no external carbon source\" + ambient air \\ culture 10 & heterotrophic + 4 mM glucose + ambient air \\ culture 11 & phototrophic + ambient air + 75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ culture 12 & mixotrophic + 4 mM glucose + ambient air + 75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\) \\ \hline culture 13 & heterotrophic + 4 mM glucose + ambient air \\ culture 14 & heterotrophic + 4 mM glucose + 2 nmixture + ambient air \\ culture 15 & mixo-/heterotrophic + 4 mM glucose + ambient air + (75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\)) \\ culture 16 & mixo-/heterotrophic + 4 mM glucose + 2 nmixture + ambient air + (75 \(\upmu\)mol m\({}^{-2}\) s\({}^{-1}\)) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Applied germination conditions, each culture represents one biological sample. with a Sigma 3-16 KL centrifuge from Sigma GmbH (Germany) for 15 min. The absorbance of the extracted carotenoids in the solvent was measured once with the SPECORD 50 Plus spectrophotometer from Analytik Jena (Thuringia, Germany) at 478 nm, and the astaxanthin concentration was calculated using a calibration curve. The astaxanthin content of the biomass was determined in triplicate (n = 3) at the beginning of germination (t = 0 h), at the time of the largest zoospore release (t = 49 h), and at the end of the series (t = 141 h) for each biological sample: cultures 5-16. For cultures 5-16, a double determination of the astaxanthin content of the biomass was additionally performed for the remaining times (t = 17 h, 25 h, 41 h, and 68 h). The astaxanthin concentration in ethyl acetate after the liquid-liquid extraction was obtained by a single determination for all cultures 1-16. The yield Y corresponds to the quotient of the astaxanthin content in the solvent after, to that in the biomass before extraction, and was calculated for the points in time examined, as presented in the following equation.