## 3 Results and Discussion  
### Examination of Culture Media Recipes  
In our study, we have two different seed cultures, the grown in the modified SOT incl. NaHCO\({}_{3}\) and excl. NaHCO\({}_{3}\), and each was used for Case 1, 2, and 3, respectively. We observed that the cells were actively grown in a NaHCO\({}_{3}\)-enriched medium (modified SOT), as aggregated in the filaments with a well-developed spiral form. Figure 6 compares the cell growth status of the seed cultures during the 30 incubation days (Day 14 and 28). Figure 6 shows a large population of _S. maxima_ in a high concentration. Each cell exhibits a clear three-dimensionally left-handed (LH) open helicoidal structure, and the numbers of cells were exponentially multiplied after 28 days. However, resulting cultures in the SOT excl. NaHCO\({}_{3}\) (Figure 6) showed a severely lower cell concentration. The sparse cell population and linear form of filament aggregates indicates that the cell growth was limited and remained unsettled in the medium. Even after 28 days, we observed that the cell structures were not able to develop LH coils vividly and the cell density on Day 28 appeared only as good as that on Day 14 in the NaHCO\({}_{3}\)-inclusive SOT. From [52; 53], we may suppose that _Spiulating_ proliferation was affected by proper nutrient balance such as nitrogen and carbon more critically than light intensity or temperature. Particularly, bicarbonate is served as an external inorganic substance for the carbonic anhydrase reaction to derive CO\({}_{2}\) for photosynthesis. This result supports the previous findings [47; 54], and Figure 6 reveals that NaHCO\({}_{3}\) deficiency significantly reduced the growth rate (below the average cell reproducibility of 25% per day [55]). Figure 7 exhibits the results of the _S. maxima_ growth in three different media during 20 days. As shown in Table 1, the liquid solution temperature was measured to be constant all the way at -29 to -30 C under an average light intensity of 6842.5 lux. It was observed that Case 3 was almost translucent in the beginning and Case 2 appeared slightly thicker in  
Figure 6: Microscopy (\(\times\)150) of the seed cultures in different media: (**a**) SOT, (**b**) SOT excl. NaHCO\({}_{3}\) (Day 14 (**left**), Day 28 (**right**)). visual solution concentration than 1 until the first 5 days, even though their initial cultures were identical. In Table 2, we identified that Case 1 became optically denser, exhibiting characteristic green coloration after Day 7. However, after Day 12, such difference was related, while the solution density of Case 3 was the thinnest. This observation was confirmed by the measurement of gaps in optical density (O.D.) between 1 and 2, which was about 21.2 and 16.0% on Day 10 and 20, respectively. Suboptimal growth such as the markedly lagging development finds that the nutritional state of the parent culture is considerably important for the early-stage promotion of cultivation. Nevertheless, in this test, the NaHCO\({}_{3}\)-exclusive medium performed well to proliferate the _S. maxima_ culture after a certain period of time. This result suggests that, in enclosed reactors, the insufficient phototrophic growth due to the lack of bicarbonate can be supplemented by automatic external aeration, CO\({}_{2}\) dissolution thereof in water. It is certain that CO\({}_{2}\) is poorly soluble compared to NaHCO\({}_{3}\), and the carbon limitation constrains microalgal growth [56]. However, [PERSON] et al. [57] find that constant aeration with artificial illumination improves _S. maxima_ growth, and, despite such a positive impact of bicarbonate addition on the rapid cultivation, [PERSON] et al. [58] reveal that the growth rate is not linearly related to the NaHCO\({}_{3}\) content. Also, note that increased cell mobility attributed to the artificially aerated stirring yields reduced intercellular shading, which can lead to better light penetration and photosynthesis.