## 3 Results and Discussion
### Volatile Compounds  
Microorganisms that are cultivated for industrial applications often contain unique aromatic compounds, and the presence or absence of these compounds can affect the application and commercialization of microalga-derived products in the food and cosmetic industries [35]. Particularly, some microalgae can emit strong 'fishy' odors, which limits their commercial utilization [36]. Thus, we compared the composition and content of volatile and aromatic compounds in WT and the pigment-deficient Mutant 335.  
Our initial assessment indicated that Mutant 335 had a less 'fishy' odor than WT. To verify this and analyze the aroma component profiles of the mutants, we conducted aroma analysis that specifically compared these two strains. We first identified all volatile compounds using GC/MS (see Supplementary Table S1). The results indicate that Mutant 335 produced fewer types of volatile compounds than WT, and that the concentrations of these compounds were generally lower. Notably, the levels of aldehydes and sulfur compounds, which are known to cause 'fishy' odors, are much lower in Mutant 335. In contrast, the WT contained high concentrations of many volatile compounds such as alcohols, aldehydes, sulfur compounds, and hydrocarbons, and this was likely responsible for its strong and complex 'fishy' aroma. Photosynthesis is not possible in pigment-deficient Mutant 335, which may be a key factor in the changes observed in the produced volatile compounds. More volatile compounds are produced in oils like linoleic acid when chlorophyll is present. In principle, chlorophyll can generate singlet oxygen from triplet oxygen upon exposure to fluorescent light, which plays important roles in the formation of 2-pentylfuran, _trans_-2-heptenal, and 1-octen-3-ol in linoleic acid [37]. 1-Octen-3-ol is one of the volatile compounds found in the WT in our _Euglena_-related research but is not detected in the pigment-deficient Mutant 335. In Mutant 335, chlorophyll-photosensitized reactions do not occur, which may explain the reduced concentration and number of volatile compounds observed compared to the WT. However, the possibility of the production of a volatile compound caused by other genetic changes resulting from EMS treatment cannot be ignored; it is also limited to explain the extensive changes in the volatile compound profile caused solely by genetic variations. In the future, identification of the responsible gene in the mutants can provide a clearer understanding of the relationship between the associated phenotypes and changes in the paramylon and volatile compound content.  
Figure 7: Paramylon content of _Euglena_ WT, Mutant 333, and Mutant 335 determined by gravimetry and aniline blue staining. Data were analyzed using one-way ANOVA followed by Duncan’s test (\(p<0.05\)). Different lowercase letters at the top of the bar graph indicate statistically different between groups (\(p<0.05\)), and the same lowercase letter indicates no significant difference (\(p>0.05\)). n = 3.  
Table 2 presents the results of the volatile organic compound (VOCs) analysis for Mutant 335 and WT based GC-olfactory analysis of the 127 volatile compounds listed in Supplementary Table S1. Notably, Mutant 335 had no'mugwort' scent (1-octen-3-ol, 2-methyl-1-heptene, styrene), and lacked the 'barley tea' and 'green tea' scent (hexanal, hexyl formate, 4-ethylbenzoic acid) that was present in WT. However, compounds associated with a 'herbal' scent (2-methyl-1-undecanol) and a 'green' scent (2,4,6-Trimethyldecane) had higher concentrations in Mutant 335. This suggests that the decrease or modification in specific aroma compounds in Mutant 335 may make it suitable for various food products, such as tea, salad dressings, and herb-based foods.