## 3 Results and Discussion  
The results and discussion of the germination experiments are presented, comparing the influence of CO\({}_{2}\) and ambient air on the phototrophic and mixotrophic germination at 100 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\) (culture 1 to culture 4) and 75 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\) (culture 5 to culture 8). In the third set of experiments (culture 9 to culture 12), the germination was performed at heterotrophic conditions, once with the addition of an external glucose source to the BBM and once without external glucose, and compared to a mixotrophic and phototrophic culture. In the last set of experiments (culture 13 to culture 16), the influence of nitrate on heterotrophic cultivation was investigated and a combination of mixo- and heterotrophic cultivation conditions was done. Evaluation of Phototrophic, Mixotrophic, and Heterotrophic Cultivation Conditions on the Germination of H. pluvialis Cyst Cells  
In the first set of experiments, the influence of CO\({}_{2}\) on the germination of phototrophic and mixotrophic cultures was examined. Here, culture 1 (phototrophic + CO\({}_{2}\) + 100 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\)), culture 2 (phototrophic + ambient air + 100 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\)), culture 3 (mixotrophic + 4 mM glucose + CO\({}_{2}\) + 100 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\)), and culture 4 (mixotrophic+ 4 mM glucose + ambient air + 100 umol m\({}^{-2}\) s\({}^{-1}\)) were germinated. As described in Section 2.4, astaxanthin was extracted with ethyl acetate from the algal broth in regular time intervals to evaluate the germination efficiency. As shown in Figure 3, CO\({}_{2}\) had a negative effect on germination in both, phototrophic and mixotrophic cultures, in comparison to ambient air. Forty-one hours after the start of germination under phototrophic conditions (culture 2), which was germinated at ambient air, 7.2 mg L\({}^{-1}\) astaxanthin could be extracted into the solvent, whereas only 2.8 mg L\({}^{-1}\) could be extracted under phototrophic conditions (culture 1), where additional CO\({}_{2}\) was gassed. As presented in Figure 3, a similar tendency could be seen under mixotrophic conditions, where 4.5 and 8.9 mg L\({}^{-1}\) were extracted into ethyl acetate when germinated with additional CO\({}_{2}\) (culture 3) and at ambient air (culture 4), respectively. In addition, glucose showed a positive effect on the germination, as more astaxanthin was extracted in mixotrophic culture 3 (4.5 mg L\({}^{-1}\)) compared to phototrophic culture 1 (2.8 mg L\({}^{-1}\)), and mixotrophic culture 4 (8.9 mg L\({}^{-1}\)) compared to phototrophic culture 2 (7.2 mg L\({}^{-1}\)). In previous studies, it was shown that more zoospores are released from the cyst cells at higher light intensities in comparison to lower ones. However, the degradation of astaxanthin within the cells was also higher at higher light intensities compared to lower light intensities [25; 29]. To counteract a possible degradation of intracellular astaxanthin, identical germination conditions as for culture 1 to culture 4 were applied in the next set of experiments (culture 5 to culture 8), but at a lower light intensity of 75 umol m\({}^{-2}\) s\({}^{-1}\) compared to 100 umol m\({}^{-2}\) s\({}^{-1}\). In addition to the extraction with ethyl acetate, the cells were counted, and the biomass concentration and the astaxanthin content of the biomass were determined. CO\({}_{2}\) and ambient air had a similar effect on the phototrophic germination of culture 5 (phototrophic + CO\({}_{2}\) + 75 umol m\({}^{-2}\) s\({}^{-1}\)) and culture 6 (phototrophic + ambient air + 75 umol m\({}^{-2}\) s\({}^{-1}\)) as in the first set of experiments. As presented in 4, 7.0 mg L\({}^{-1}\) astaxanthin could be extracted into ethyl acetate at the phototrophic conditions at ambient air (culture 6) compared to 2.8 mg L\({}^{-1}\) with additional CO\({}_{2}\) (culture 5), 49 h after the start of germination.