## 4 Conclusions  
This study successfully established an EMS-based mutagenesis method for _Euglena gracilis_ by comparing the effects of different mutagenesis conditions. We induced mutations using three sequential treatments with EMS at a concentration of 0.012 M over 72 h, and isolated one strain with increased motility (Mutant 333) and another strain with chlorophyll deficiency (Mutant 335). In addition, the changes in cell division in the mutants were similar on agar plates and a liquid medium. This demonstrates the feasibility of isolating strains of _Euglena_ that have altered motility and growth rate on plates, without the need for a liquid culture. Mutant 333 had a 1.4 times lower paramylon content than the WT, and Mutant 335 (which lacks chlorophyll) had 1.72 times more than WT. Moreover, our analysis of volatile substances showed that Mutant 335 had lower levels of aldehydes and sulfur compounds than the WT, so that it had a less 'fishy' smell and was therefore more suitable for applications in the food industries.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/microorganisms13020370/s1](https://www.mdpi.com/article/10.3390/microorganisms13020370/s1)]([https://www.mdpi.com/article/10.3390/microorganisms13020370/s1](https://www.mdpi.com/article/10.3390/microorganisms13020370/s1)), Table S1: Concentrations (\(\mu\)g/100 g) of volatile compounds in _Euglena_ WT and _Euglena_ Mutant 335 based on GC/MS.  
**Author Contributions:** Conceptualization, E.-C.S. and J.-H.K.; Investigation, Y.B.; Data curation, J.-Y.K.; Writing--original draft, J.-Y.K.; Supervision, J.-H.K.; Funding acquisition, J.-H.K. All authors have read and agreed to the published version of the manuscript.  
\begin{table}
\begin{tabular}{l l l l l} \hline \hline \multirow{2}{*}{**Volatile Compound**} & \multicolumn{2}{c}{**RT (\({}^{\mathrm{1}}\))**} & \multicolumn{2}{c}{**Relative Intensity**} & \multirow{2}{*}{**Odor Description**} \\ \cline{2-2} \cline{4-5}  & **(min)** & & **Mutant 335** & & **WT** \\ \hline
**Alcohols** & & & & \\
1-Hexanol & 10.29 & 0 & 1 & Green \\
1-Octen-3-ol & 13.83 & 0 & 2 & Mugwort \\
2-Methyl-1-undecanol & 20.23 & 2 & 0 & Herbaceous \\
**Aldehydes** & & & & \\ Hexanal & 7.99 & 0 & 1 & Green tea \\
**Acid and esters** & & & & \\ Hexyl formate & 10.21 & 0 & 2 & Barley tea \\
4-Ethylbenzoic acid & 11.31 & 0 & 2 & Green tea \\
**Hydrocarbons** & & & & \\
2-Methyl-1-heptene & 10.34 & 0 & 1 & Mugwort \\ Styrene & 10.98 & 0 & 1 & Mugwort \\
2,6-Dimethyloctane & 12.33 & 0 & 2 & Roasted \\
2,4,6-Trimethyldecane & 21.84 & 2 & 1 & Green \\
1,3-Bis(1,1-dimethylethyl)-benzene & 22.03 & 1 & 1 & Herbaceous \\ \hline \hline \end{tabular} \({}^{(\mathrm{1})}\) RT: retention time.  
\end{table}
Table 2: Odor intensity in _Euglena_ WT and _Euglena_ Mutant 335 based on GC olfactometry.  
**Funding:** This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1 IIA3055799) and the Field Technology Research and Development Program funded by Korea South-East Power Co. (KOEN).  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.  
**Conflicts of Interest:** The authors declare no conflicts of interest.