## 3 Results and Discussion
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 \(p<0.05\). The highest astaxanthin extraction yield of 50% was reached for germination at heterotrophic conditions and additional external glucose (culture 10), 49 h after the start of germination. The second highest extraction yield was 45% at heterotrophic conditions without an external glucose source (culture 9). Based on the large difference in the concentration of astaxanthin extracted with ethyl acetate, 24.8 mg L\({}^{-1}\) compared to 15 mg L\({}^{-1}\) (Figure 7), and the number of released zoospores of 82% to 62% (Figure 8) for heterotrophic culture with glucose in comparison without glucose, an even more significant difference of the yields would have been expected. However, due to the higher astaxanthin content measured in the biomass at heterotrophic conditions with external glucose (culture 10), 49 h after the start of germination, in comparison to without external glucose (culture 9), the yields almost converge. Significantly lower yields were reached with 22% and 16% for the mixotrophic (culture 12) and phototrophic (culture 11) germination conditions, respectively. In the previous set of experiments, the highest yield, the highest concentration of astaxanthin extracted into ethyl acetate, and the number of released zoospores were reached at heterotrophic conditions with additional glucose (culture 10). These conditions were repeated in the following set of experiments as culture 13. Additionally, a heterotrophic culture with external glucose and twice the nitrate concentration was germinated (culture 14) to rule out a limitation of the germination due to nitrate deficiency. In addition, it was found in the previous set of experiments that zoospore formation was faster under phototrophic and mixotrophic conditions (culture 11 and 12) compared to heterotrophic conditions (culture 9 and 10). However, the maximum number of released zoospores and the extracted amount of astaxanthin in ethyl acetate was higher at heterotrophic conditions compared to mixotrophic or phototrophic conditions. For that reason, two cultures were germinated; mixotrophic and heterotrophic cultivation conditions were combined. These two cultures were cultivated at a light intensity of 75 umol m\({}^{-2}\) s\({}^{-1}\) until nitrate was consumed to accelerate the zoospores formation, and further cultivated at heterotrophic conditions (without light) to reach a high total number of released zoospores. One of these cultures was germinated at double nitrate content of the medium (culture 16), while the other culture was germinated without additional nitrate (culture 15). As shown in Figure 10, a doubling of the nitrate content in heterotrophic germination conditions hardly affected the concentration of astaxanthin extracted in ethyl acetate, as can be seen when comparing culture 13 (12.9 mg L\({}^{-1}\)) and culture 14 (12.6 mg L\({}^{-1}\)). When mixotrophic and heterotrophic germination conditions were combined, the doubling of the nitrate content showed an influence on the concentration of astaxanthin extracted into ethyl acetate. There, 49 h after the start of germination, the concentration of astaxanthin extracted with ethyl acetate was 17.1 mg L\({}^{-1}\) for double nitrate concentration (culture 16) and 8.4 mg L\({}^{-1}\) when no additional nitrate was added (culture 15). Why the combination of mixotrophic and heterotrophic germination conditions at a nitrate concentration of the BBM (culture 15) with 8.4 mg L\({}^{-1}\) was worse compared to the double nitrate concentration (culture 16) with 17.1 mg L\({}^{-1}\) and the heterotrophic culture 13 (12.9 mg L\({}^{-1}\)) and culture 12 (12.6 mg L\({}^{-1}\)) cannot be clarified in this context.