## 4 Discussion
### Mechanism of Electrical Stimulation Promoting Microalgal Growth  
The results of the electrostimulation experiments on the culture medium indicate that the growth-promoting effect of electrical stimulation on _Nitzschia closerium_ f. _minutissima_ is achieved through direct action rather than indirectly affecting the microalgae growth via the culture medium. The cyclic voltammetry (CV) curves show differences in the redox peaks, suggesting changes in the electrochemical properties related to the metabolic activity of diatom cells under electrical stimulation. These results collectively indicate that electrical stimulation regulates the physiological metabolic activity of _N. closerium_ f. _minutissima_. The variations in nutrient levels within the cultures further confirm that electrical stimulation significantly influences the metabolic activity of _N. closerium_ f. _minutissima_, possibly by affecting the activity or expression of specific enzymes. For example, [PERSON] et al. 's studies have shown that electrical stimulation significantly increases the expression of the key enzyme EgGSL2 during starch synthesis in _Euglena gracilis_, thereby enhancing starch production [12]. This suggests that electrical stimulation may have a similar effect on the metabolism of _N. closerium_ f. _minutissima_. Notably, chlorophyll a, as a key pigment in photosynthesis, partially reflects the photosynthetic capability of microalgae [2; 30]. However, the chlorophyll a content in the D-1 V group is significantly higher than in other groups, and this group also reaches the highest cell density, indicating that electrical stimulation may enhance the synthesis of chlorophyll a,thereby improving the photosynthetic level and accelerating cell growth. This finding partially explains the differences in the reduction peaks observed in the CV curves. Additionally, electrical stimulation may directly or indirectly influence the cell division process of microalgae, which in turn affects other physiological activities. Growth curves and growth rate measurements indicate that electrical stimulation promotes the growth of _N. closterium_ f. _minutissima_, although no significant differences in biomass dry weight are observed. [PERSON] et al. 's studies have shown that continuous electrical stimulation inhibits cell division in _Euglena gracilis_, resulting in increased biomass accumulation and cell enlargement [12]. This suggests that electrical stimulation may inhibit the division process in _N. closterium_ f. _minutissima_, leading to a decrease in cell volume and the consumption of organic material. This phenomenon reinforces the notion that increases in various nutrient contents are primarily due to the regulatory effects of electrical stimulation on metabolic pathways rather than overall growth promotion. However, the metabolic pathways of microalgae are complex, and further research is required to elucidate the specific effects of electrical stimulation on the steps of nutrient synthesis and the associated enzymes and genes. Electron microscopy observations reveal that under brief electrical stimulation at 10 V, _N. closterium_ f. _minutissima_ cells exhibit wavy folds, while cells subjected to continuous stimulation at 1 V display some degree of shrinkage. This indicates that electrical stimulation affects the permeability of the cell wall and membrane of _N. closterium_ f. _minutissima_. Coupled with cell size measurements, these morphological changes suggest that the cells exhibit trends of both enlargement and shrinkage under electrical stimulation, phenomena that have been reported in previous studies and are commonly associated with changes in microalgae metabolic activity [10; 11; 21]. This suggests that electrical stimulation may influence transmembrane transport by altering membrane properties, thereby affecting cellular metabolism. However, this study did not directly assess the extent of cell damage in each group; rather, it inferred that the damage in all groups was less than that observed under D-10 V or C-1 V conditions.