## 3 Results and Discussion
### Influence of Glucose on the H. pluvialis Cyst Cell Germination  
In microalgae, glucose is processed to generate energy for cell growth and maintenance via glycolysis within the cell [41]. During \(H\). _pluvialis_ cyst germination, glycolysis and fatty acid oxidation provide the primary energy and precursor for a variety of biosynthetic pathways. Starch, which is the most widely used storage polysaccharide in algae, is the carbohydrate source during germination and zoospore formation [42; 43]. It has been  
Figure 12: Time course of the biomass concentration and the astaxanthin content in the algal biomass of heterotrophic culture 13 (heterotrophic + 4 mM glucose + ambient air) and culture 14 (heterotrophic + 4 mM glucose + 2 xnitrate + ambient air), and culture 15 (mizo-/heterotrophic + 4 mM glucose + ambient air + (75 \(\mu\)mol m\({}^{-2}\) s\({}^{-1}\))) and culture 16 (mizo-/heterotrophic + 4 mM glucose + 2 xnitrate + ambient air + (75 \(\mu\)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.  
shown in phototrophic _H. pluvialis_ cyst germination that the genes and enzymes involved in the 8-oxidation of fatty acids and glycolysis show upregulation and downregulation to different degrees during germination. Stored starch is degraded via the upregulated \(\alpha\)-amylase during germination, providing dextrin and maltose for glucose metabolism [42]. It was shown that the germination was enhanced at mixotrophic in comparison to the phototrophic conditions, at otherwise identical parameters. The results were confirmed for CO\({}_{2}\) under mixotrophic conditions (culture 3 and 7) compared to phototrophic conditions (culture 1 and 5), and for ambient air under mixotrophic conditions (culture 4 and 12) compared to phototrophic conditions (culture 2 and 11). The mixotrophic conditions at ambient air (culture 8) compared to the phototrophic conditions at ambient air (culture 6) might present an exception, which might be due to the state of the cyst cells at the beginning of germination. Additionally, the results suggest that the heterotrophic germination of _H. pluvialis_ cysts is enhanced by adding external glucose into the medium (culture 10), compared to germination, which is based on the mobilization of intracellular stored carbon sources only (culture 9) (Figure 8). The sugar analysis, described in Section 2.7, detects the reaction of simple sugars, oligosaccharides, polysaccharides, and their derivatives, including methyl esters with free-reducing groups with phenol and sulphuric acid, which react to give an orange-brown color [36]. Although the heterotrophic culture 9 was germinated without an external carbon source, a sugar concentration of approximately 150 mg L\({}^{-1}\) could be measured in the medium at the start of germination, which is probably due to extra polymeric saccharides of the cyst cells (Figure 13). For both heterotrophic cultures, a decrease in glucose concentration was observed 25 h after the start of germination, which indicates active glucose consumption. The increase of the sugar concentration after 41 h in both cultures is most probably attributed to the presence of extra polymeric saccharides, parts of the mannose-containing cyst cell wall, and hexoses of the cell-matrix of the zoospores [7], which might be partly dissolved in the aqueous supernatant and detected using the analytical method for the sugar quantification. In all heterotrophic cultures examined, the astaxanthin content in the biomass remained relatively constant (\(p>0.05\)). A possible reason for the stable astaxanthin content in the biomass could be the increased supply of glucose for glycolysis that results in an increased carbon flow into the citric acid cycle.