## 3 Results and Discussion
The cell counting revealed that more zoospores were released from culture 6 compared to culture 5, which is in good accordance with the extracted amount of astaxanthin. As can be seen in Figures 4 and 5, 49 h after the start of germination, in culture 6, germinated at ambient air, 45% of the existing cells were in the zoospores stage, while in culture 5, germinated with additional CO\({}_{2}\), only 25% were found in that stage. Figure 3: Astaxanthin concentration in the ethyl acetate extract of the phototrophic culture 1 (phototrophic + CO\({}_{2}\) + 100 μmol m\({}^{-2}\) s\({}^{-1}\)) and culture 2 (phototrophic + ambient air + 100 μmol m\({}^{-2}\) s\({}^{-1}\)) and the mixotrophic culture 3 (mixotrophic + 4 mM glucose + CO\({}_{2}\) + 100 μmol m\({}^{-2}\) s\({}^{-1}\)) and culture 4 (mixotrophic + 4 mM glucose + ambient air + 100 μmol m\({}^{-2}\) s\({}^{-1}\)) at different time points after the start of germination; data obtained by single determination. In addition, as presented in Figure 6, 49 h after the start of germination the measured astaxanthin content in the biomass was higher in the phototrophic culture 6 germinated at ambient air (1.9 wt.%) than in phototrophic culture 5 (1.3 wt.%) germinated with additional CO2, which might be another reason for the higher concentration of astaxanthin extracted with ethyl acetate. Forty-nine hours after the start of germination, only slightly more astaxanthin (4.4 mg L\({}^{-1}\)) could be extracted with ethyl acetate under mixotrophic conditions at ambient air (culture 8) compared to 3.8 mg L\({}^{-1}\) with additional CO2 (culture 7). At this point in time, in cultures 7 and 8, approximately 38% of the cells were at the zoospore stage, which is slightly lower compared to phototrophic culture 6, which was approximately 45% at that stage. Thus, as shown in Figure 4, the concentration of astaxanthin extracted with ethyl acetate at this time point was lower for the mixotrophic cultures. Additional to the lower amount of released zoospores, this was also due to the lower astaxanthin content in the biomass at mixotrophic conditions with additional CO2 (culture 7, 1.1 wt.%) and ambient air (culture 8, 1.27 wt.%) compared to phototrophic conditions at ambient air (culture 6, 1.88 wt.%), 49 h after the start of germination (Figure 6). Figure 4: Astaxanthin concentration in the ethyl acetate extract of the phototrophic culture 5 (phototrophic + CO2 + 75 umol m\({}^{-2}\) s\({}^{-1}\)) and culture 6 (phototrophic + ambient air + 75 umol m\({}^{-2}\) s\({}^{-1}\)), and mixotrophic culture 7 (mixotrophic + 4 mM glucose + CO2 + 75 umol m\({}^{-2}\) s\({}^{-1}\)) and culture 8 (mixotrophic + 4 mM glucose + ambient air + 75 umol m\({}^{-2}\) s\({}^{-1}\)) at different time points after the start of germination; data obtained by single determination. Figure 5: Relative distribution of the cell stages of _H. pluvialis_ at different points in time after the start of germination for culture 5 (phototrophic + CO2 + 75 umol m\({}^{-2}\) s\({}^{-1}\)) and culture 6 (phototrophic + ambient air + 75 umol m\({}^{-2}\) s\({}^{-1}\)), and culture 7 (mixotrophic + 4 mM glucose + CO2 + 75 umol m\({}^{-2}\) s\({}^{-1}\)) and culture 8 (mixotrophic + 4 mM glucose + ambient air + 75 umol m\({}^{-2}\) s\({}^{-1}\)). The initial astaxanthin content in the cyst cells of cultures 5-8 was 1.4 wt.%. In the phototrophic culture 5 and mixotrophic culture 7, the CO2 gassing resulted in a significant reduction (_p_ < 0.05) of the intracellular astaxanthin content, to 0.66 and 0.53 wt.%, compared to its starting value 141 h after the start of the germination. In contrast, in phototrophic culture 6, which was germinated at ambient air, 49 h after the start of germination an increase of the astaxanthin content in the biomass to 1.88 wt.% was measured and then declined slightly to 1.08 wt.% at 141 h after the start of germination (Figure 6). Additionally, in the mixotrophic culture 8, the astaxanthin content of the biomass remained relatively stable and was at 1.08 wt.% 149 h after the start of germination.