## 3 Results
### Effects of Electrical Treatment on Intracellular Substances in N. closterium f. minutissima  
This study examines the accumulation of various nutrients in _N. closterium_ f. _minutissima_, analyzing both the total culture and their proportion in dry biomass. Nutrient content in the culture indicates the overall accumulation influenced by cell density, relevant to the nutritional value of feed products. The proportion in dry weight, however, reflects cellular metabolic changes and the microalgae's response to electrical stimulation, highlighting physiological shifts in nutrient composition.  
Different intensities of short-term and continuous electrical stimulation significantly impacted chlorophyll-\(\alpha\) content in _N. closterium_ f. _minutissima_ at both the culture and dry weight levels (\(p<0.05\)) (Figure 5a). Chlorophyll-\(\alpha\) content in the D-1 V and D-3 V groups significantly increased compared to CK, especially in the D-1 V group, which showed a 38.95% increase (\(p<0.05\)) with a concentration of 7.00 \(\pm\) 0.31 ug/mL. However, no significant changes were observed in the D-5 V and D-10 V groups. At the dry weight level, chlorophyll-\(\alpha\) in the D-1 V group increased by 39.20% (\(p<0.05\)) compared to CK, while the D-10 V group showed a significant decrease (\(p<0.05\)). For continuous stimulation, chlorophyll-\(\alpha\) content in the C-0.05 V, C-0.5 V, and C-1 V groups significantly decreased compared to C-10 V (\(p<0.05\)), with C-1 V showing a 35.79% drop compared to C-0 V. Similarly, chlorophyll-\(\alpha\) proportion in dry weight also significantly decreased in these groups (\(p<0.05\)), with C-1 V having the lowest proportion at 0.96 \(\pm\) 0.15%. Notably, the C-0 V group had the highest proportion of chlorophyll-\(\alpha\) across all groups.  
Polysaccharide content was also significantly affected by different electrical stimulation intensities (\(p<0.05\)) (Figure 5b). Although no significant difference was found, the D-3 V group had the highest polysaccharide content at the culture level, 24.61% higher than CK, while the D-10 V group showed a significant 25.26% decrease (\(p<0.05\)). In dry weight, D-10 V significantly decreased by 31.64 percentage points compared to CK.  
Protein content was significantly affected by short-term stimulation at both the culture and dry weight levels, while continuous stimulation only showed a significant effect at the culture level (\(p<0.05\)) (Figure 5c). The D-3 V group had the highest protein content at 133.02 \(\pm\) 6.08 ug/mL, whereas D-10 V significantly decreased by 27.06%. Continuous stimulation showed no significant differences.  
Figure 5: Contents and proportion in biomass dry weight of chlorophyll-\(\alpha\) (**a**), polysaccharide (**b**), protein (**c**), and lipid (**d**) in N. _closterium f. minutissima_ under DC stimulation. Different letters indicate significant differences among treatments. The column in each subfigure represents the nutrient content, and the triangle represents the proportion of each nutrient in the microalgae biomass.  
Lipid content was also significantly influenced by both short-term and continuous stimulation (\(p<0.05\)) (Figure 5d). The D-3 V group had a 67.66% increase in lipid content at the culture level compared to CK. At the dry weight level, lipid content in the D-1 V, D-3 V, and D-5 V groups significantly increased, with D-3 V reaching the highest proportion of 17.38 \(\pm\) 1.98%.  
Overall, short-term stimulation promoted nutrient accumulation, particularly lipids, with an increase followed by a decline at higher voltages, suggesting that moderate stimulation benefits nutrient accumulation, while high voltages may inhibit it. Continuous stimulation, however, resulted in lower nutrient levels across the culture, likely due to background inhibition, making its effects on nutrient accumulation unclear.