## 3 Results and Discussion  
### EMS Mutagenesis  
EMS is widely used in the development of microalgae strains due to its advantages in simple and efficient mutant generation. There is increasing attention devoted to microalgae an eco-friendly feedstock for biodiesel production because their photosynthesis can increase biomass and lead to the accumulation of abundant lipids. For example, EMS mutations in _Chlorella vulgaris_ resulted in enhanced photosynthetic efficiency under high light conditions (200 \(\upmu\)mol photons m\({}^{2}\) s\({}^{-1}\)) and a 44.5% increase in biomass production [18]. In another study, the application of EMS mutation to _Botryococcus braunii_ AARL G036 enhanced biomass, lipid, and hydrocarbon productivity by maximizing lipid (up to 48.6%) and hydrocarbon (up to 71.6%) accumulation levels in the biomass [20]. Meanwhile, various studies are actively being conducted to develop oxygen-resistant hydrogenase for biohydrogen production [21]. However, very few studies have used EMS mutagenesis with _Euglena_. The typical concentration of EMS used for the mutagenesis of microalgae is 0.5 to 3 M, and EMS is then typically washed off after 1 to 4 h [22; 23; 24; 25]. But our preliminary application of this standard method with 0.5 to 3 M EMS completely killed all _Euglena_ cells. Therefore, we aimed to develop a method for transforming and screening _Euglena_ by treating it with EMS at a 10-fold lower concentration for 72 h, which resulted in a survival rate to below 5%. Long-term treatment with EMS is a critical strategy for increasing the frequency of mutation induction, allowing for the acquisition of a diverse range of mutants [26]. Although prolonged EMS exposure enhances the probability of inducing specific mutations, excessive exposure may compromise cell survival. Therefore, it is crucial to balance concentration and duration to maximize mutation efficiency depending on the species. In addition, we applied a serial EMS treatment, in which the surviving colonies were re-cultured after one EMS treatment, and then re-treated with the same level of EMS until the desired phenotype (altered motility or pigment content) was observed. This method increased the number of mutant cells that could be used for experiments. Thus, we first treated _Euglena_ cells with different concentrations of EMS (0, 0.008, 0.012, or 0.016 M) and then observed cell motility under a microscope for the following 5 days (Table 1). A 2 h treatment at all tested concentrations had no effect on motility. However, beginning at 24 h, the cells had decreased motility according to the concentration of EMS and time of treatment. After 48 hours, _Euglena_ activity (expressed as its motility) was not observed in the 0.016 M EMS condition, and almost all cells were rounded and coiled. In the condition treated with 0.012 M EMS, activity began to decrease rapidly after 48 hours, and no activity was observed at this concentration after 96 hours. Cells treated with  
0.008 M EMS for 96 h had slightly lower activity than the control, but more than 20% of these cells were still actively moving. In addition to monitoring changes in cell activity, we measured cell survival by determining CFUs (Figure 1). As a result, treatment with 0.008 EMS led to a survival rate of 85.19% at 2 h, but the survival rate was 11.11% on the fourth day. Treatment with 0.016 M EMS led to complete cell mortality on day 2, and treatment with 0.012 M EMS led to a survival rate of 3.33% on day 3. Other studies that examined EMS mutagenesis of microalgae reported survival rates from 0.142% to 26% [23; 24; 25]. In this study, _Euglena_ showed a survival rate of 3.33% when treated with 0.012 M EMS for 72 hours (Figure 1). Interestingly, the reduction in _Euglena_ motility (Table 1) and survival rate (Figure 1) shows a similar pattern, proportional to the EMS treatment concentration and duration. After treatment with 0.016 M EMS for 48 hours, _Euglena_ motility was not observed under the microscope (Table 1), and no _Euglena_ colonies were detected (Figure 1).