## 3 Results and Discussion
### Screening EMS Mutants  
Colonies that survived the 72 h treatment with 0.012 M EMS were isolated, re-cultured, and subjected to identical EMS treatments until the resulting strains exhibited distinct changes in color, morphology, or motility. The sequence of three EMS treatments ultimately led to the isolation of one strain with increased motility (Mutant 333) and another strain with chlorophyll deficiency (Mutant 335). To select strains with increased motility, we used a YM agar plate that only contained 7 g/L of agar powder, leading to a soft solid agar region that did not restrict cell movement. To compare the motility of mutant strains and the WT, we dropped 10 uL of the same number of mutant and WT _Euglena_ cells (OD\({}_{68\mathrm{hm}}\) = 0.5) at the center of an agar plate and observed the cells every 2 days (Figure 2). After 3 days, the colony of Mutant 333 was much larger than that of WT, and after 7 days Mutant 333 had a colony diameter of 7.0 cm, nearly 1.3 times larger than that of the WT (5.2 cm). To investigate the relationship between mobility and cell division, the two strains (WT and Mutant 333) were grown on plates for 7 days, collected, and the cell count was measured under a microscope. The WT cells had a concentration of 1.76 \(\times\) 10\({}^{5}\)\(\pm\) 47 cells mL\({}^{-1}\) and the Mutant 333 cells had a concentration of 0.74 \(\times\) 10\({}^{5}\)\(\pm\) 205 cells mL\({}^{-1}\). Thus, Mutant 333 had increased motility, but decreased production of biomass. Because Mutant 333 was generated through random mutagenesis, its unique phenotype (increased motility and decreased growth) could be attributed to multiple mutations. We also isolated Mutant 335, which had a notable chlorophyll deficiency, after three successive EMS treatments (Figure 3). This mutant had the lowest motility and rate of cell division. Figure 1: Survival of _Euglena gracilis_ UTEX367 (WT) after treatment with different concentrations of EMS (0.008, 0.012, 0.016 M) for different durations. Data were analyzed using one-way ANOVA followed by Duncan’s test (\(p<0.05\)). Liquid culture is commonly used for the large-scale production of valuable compounds in _Euglena_. If the results of liquid culture can be inferred from the growth rate on agar plates, it would save significant time and effort in the selection of strains capable of rapidly accumulating biomass in a liquid medium among the many mutants generated under EMS treatment conditions. Thus, we used modified YM liquid medium to grow WT, Mutant 333, and Mutant 335 cells under identical conditions (Figure 4). The WT cells began to grow rapidly after about 10 days and reached a maximum absorbance at 20 days (\(\text{OD}_{680\text{nm}}=4.3\)). In contrast, Mutant 333 reached only about 50% of the WT value after 30 days. Mutant 335 exhibited a lower accumulation of biomass, presumably related to chlorophyll deficiency. Our measurements also demonstrated that the chlorophyll content was slightly greater in Mutant 333 than in WT, but that Mutant 335 had extremely low or undetectable levels of chlorophyll (Figure 5). Despite the slightly greater chlorophyll content of Mutant 333, it had the lowest growth rate under mixotrophic conditions. However, under phototrophic conditions in a static culture (no mixing), Mutant 333 had a slightly better growth rate than WT during the later stages of cultivation (Figure 6). In particular, WT had greater growth during the initial 16 days, but Mutant 333 had greater growth after 16 days. According to the Beer-Lambert law, when the absorbance is greater than 0.2, the initial light intensity decreases by 50% in the center [27]. Therefore, in various experiments optimizing cultivation conditions through continuous culture at a constant concentration, the cell density value is adjusted to not exceed an \(\text{OD}_{680\text{nm}}\) value of 0.2 [28; 29]. In the cultivation of _Euglena_, without physical agitation and with light provided from above, the mixing of internal cells during cultivation relies solely on the motility of _Euglena_.