## 3 Results and Discussion
The extraction yield for cultures 5-8 was calculated as the mass of astaxanthin that was extracted into ethyl acetate to the mass of astaxanthin in the biomass before the extraction with ethyl acetate (Section 2.4). The highest astaxanthin extraction yield was reached 49 h after the start of germination for the phototrophic culture 6 cultivated at ambient air with 16%, compared to 9% for the phototrophic culture 5 germinated with additional CO2. An extraction yield of 11% was reached for the mixotrophic culture 8, germinated at ambient air, and a yield of 8% for the mixotrophic culture 7, in which additional CO2 was supplied. In the first set of experiments (culture 1-4), the greatest amount of astaxanthin could be extracted under mixotrophic conditions at ambient air (culture 4). In the second set of experiments (culture 4-8), most astaxanthin was extracted under phototrophic conditions at ambient air (culture 6). However, the tendencies of the extracted amount of astaxanthin are similar within the first and second set of experiments for the cultivation conditions applied, with exception of the results of mixotrophic conditions at ambient air. Only the values under mixotrophic conditions at ambient air and 100 umol\({}^{-2}\) s\({}^{-1}\) (culture 4) and mixotrophic conditions at ambient air at 75 m\({}^{-2}\) s\({}^{-1}\) (culture 8) show a deviation when compared to the other three germination conditions. Thus, it was assumed that the change of the light intensity from 100 to 75 umol m\({}^{-2}\) s\({}^{-1}\) did not influence the tendencies obtained within the first set and second set of experiments, but that there was a problem in  
Figure 6: Time course of the biomass concentration and the astaxanthin content in the algal biomass of phototrophic culture 5 (phototrophic + CO2 + 75 μm mol m\({}^{-2}\) s\({}^{-1}\)) and culture 6 (phototrophic + ambient air + 75 μm mol m\({}^{-2}\) s\({}^{-1}\)), and mixotrophic culture 7 (mixotrophic + 4 mM glucose + CO2 + 75 μm mol m\({}^{-2}\) s\({}^{-1}\)) and culture 8 (mixotrophic + 4 mM glucose + ambient air + 75 μm mol m\({}^{-2}\) s\({}^{-1}\)). Biomass concentration: error bars show \(\pm\)SD, n = 3. Astaxanthin content: error bars show \(\pm\)SD, n = 3 at t = 0 h, t = 49 h, and t = 141 h and \(\pm\)SD, n = 2 at t = 17 h, t = 25 h, t = 41 h, t = 68 h, and t = 89 h of one biological sample; asterisks indicate significant differences (_t_-test) of measured value vs. control (t = 0 h) at \(p\) < 0.05.  
germination for mixotrophic cultivation at ambient air and 100 umol m\({}^{-2}\) s\({}^{-1}\) (culture 8) compared to mixotrophic conditions at ambient air and 75 umol m\({}^{-2}\) s\({}^{-1}\) (culture 4). To evaluate whether the cyst cell germination is enhanced under phototrophic or mixotrophic conditions at ambient air, in the third set of experiments, phototrophic (culture 11) and mixotrophic (culture 12) germination at ambient air and a light intensity of 75 umol m\({}^{-2}\) s\({}^{-1}\) was repeated. Since CO\({}_{2}\) had a negative effect on phototrophic and mixotrophic germination, it was assumed that it is not fixed by the cyst cell and that the overall photosynthetic activity of the cysts is reduced. To show that _H. pluvialis_ cyst germination can be performed independently of photosynthetic activity, two heterotrophic conditions were applied for the germination of cultures 9 and 10. Both cultures were grown in dark conditions, without light, and at ambient air. Heterotrophic culture 9 was germinated without an external carbon source and depended on internal carbon reserves such as starch, fatty acids, or triacylglycerlecrides (TAG) for energy production. Heterotrophic culture 10 was germinated with additional glucose in the BBM.