## 3 Results and Discussion
### Examination of Culture Media Recipes
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Factor** & \multicolumn{2}{c}{**1**} & \multicolumn{2}{c}{**2**} & \multicolumn{2}{c}{**3**} \\ \hline
**Date** & **A** & **B** & **A** & **B** & **A** & **B** \\ \hline pH & 10.01 & 9.41 & 8.69 & 8.83 & 7.92 & 8.16 \\ \hline Medium temperature (\({}^{\circ}\)C) & 30.1 & 29.2 & 30.2 & 29.0 & 30.1 & 29.1 \\ \hline Lighting intensity (lux) & \multicolumn{5}{c}{6842.5 \(\pm\) 869.0 (Min: 5948, Max: 7629)} \\ \hline CO\({}_{2}\) supply (ppm) & \multicolumn{5}{c}{732.5 \(\pm\) 44.5} \\ \hline (Lab) Air temperature (\({}^{\circ}\)C) & \multicolumn{5}{c}{32.0 \(\pm\) 0.3} \\ \hline (Lab) Air humidity (\%) & \multicolumn{5}{c}{98 \(\pm\) 0.7} \\ \hline O.D. & 0.52 & 0.81 & 0.41 & 0.68 & 0.37 & 0.49 \\ \hline \hline \end{tabular} Note: A–Day 10, B–Day 20.  
\end{table} 
Table 1: Medium treatments: Cultivation environment. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**No.** & **Day 9** & **Day 14** & **Day 30** \\ \hline
**1** & & & & \\ \hline
**2** & & & & \\ \hline
**3** & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Magnified cell growth status on Day 9, 14, and 30 (\(\times\)200). Figure 7: Observation of _Spirulina_ growth in different medium treatments (–Day 20): ��⃝1 Standard SOT (injection of the initial cells cultured in SOT), ⃝2 Sodium bicarbonate (NaHCO\({}_{3}\))-free SOT (injection of the initial cells cultured in SOT), and ⃝3 SOT excl. NaHCO\({}_{3}\) (injection of the initial cells cultured in NaHCO\({}_{3}\)-free SOT). Typically, as a unicellular organism, _Spirulina_ exponentially grow from the 4 th to 15 th days, if nutrient and light conditions are met. The coloration of the bottles in Figure 7 indicates that the culture in Case 1 experienced an exponential growth phase from Day 5 to 14, whereas that in Case 2 underwent longer (2-3 day delay) and smooth growth phases. However, Case 3 showed highly lagged biological development. Even though they appeared thriving enough and became stationary after Day 20, the O.D. of Case 3 was only about 60% or less of O.D. 1 (Table 1). In general, the pH values successively increase as _Spirulina_ grows and phototrophic degradation decreases medium alkalinity. A notable feature in the NaHCO\({}_{3}\)-free SOT cultivation (2 and 3) was that the low pH values (8.69 and 7.92) due to the deficient bicarbonate metabolism increased by 2-3% on Day 20. This demonstrates that _S. maxima_ continued to grow (or maintained metabolic activity at the least), slowly though, in the carbon-limited environment. It is visible that cell propagation from the parental filaments still occurred after 14 days. In Figure 8a, the microscopy of Case 1 on Day 14 shows that the divided cells started to grow due to the relatively abundant HCO\({}_{3}\)\({}^{-}\) availability. The typical size of _Spirulina_ appears bigger on a cellular scale due to its helical shape (8-9 \(\upmu\)m in diameter and 100-200 \(\upmu\)m in trichome length [50]), comparing to other species such as the spherical Chlorella cells (2-10 \(\upmu\)m [58]). Figure 8 shows that the filaments increased in length (50-60 \(\upmu\)m) and morphed to structure the typical spirals. However, in all cases, the cell spirals were not entirely skewed to develop the helix geometry during this process. In Case 3, it seems conceivable that carbon starvation in both the parent cultivation and growth medium resulted in poor cell growth because only few cell spin-offs were visible even in a stationary mature stage (Day 30, Figure 9). Figure 8: Magnified (\(\times\)1000) observation of _S. maxima_ cell growth: (**a**) Day 4, (**b**) Day 14.