## 3 Results and Discussion
### Effect of Flow Rate on Seed Cysts and Cell Growth  
The impact of varying flow rates from 1.0 to 3.0 mL/min with a fixed loop number of 3 on the viability of _H. lacustris_ cysts is presented in Figure 3a. Cell viability, assessed using trypan blue staining, revealed that untreated cells showed the highest viability at  
Figure 2: Life cycle of _Haematococcus lactisris_, illustrating the three distinct cell stages during photosynthetic cultivation: cyst germination, vegetative growth, and encrystment. Various cell morphologies, including cysts, germinating cells, released daughter cells, biflagellate cells, and palmella types (I–V), were observed by light microscopy. During germination, a cyst cell undergoes repeated mitotic events and cytokinesis to produce daughter cells, eventually releasing motile cells. In this study, microfluidic collision treatment was applied to _H. lacustris_ cysts to break dormancy and enhance subsequent photosynthetic cell growth. 98.6 \(\pm\) 1.7%. However, even in controls, some cells might have experienced damage due to shear stress during agitation in the seed flask and subsequent syringe pumping [29]. Cysts exposed to lower flow rates (1.0 to 2.0 mL/min) exhibited viability similar to controls, ranging from 98.1 to 98.7%. However, at higher flow rates (2.5 and 3.0 mL/min), viability slightly decreased to 95.5-95.7%. Microscopic analysis indicated that the cysts treated at lower flow rates maintained morphology similar to controls, while those subjected to higher flow rates showed signs of cellular stress or damage, such as cell wall separation from the cytoplasm and secretion of intracellular materials (Figure S1). These findings suggest that while microfluidic collision can be used to manipulate cysts, precise control of flow rate is critical to maintaining cell viability. Interestingly, ROS levels, which are typically indicators of cellular stress [48; 49], decreased significantly with increasing flow rates from 1.0 to 3.0 mL/min, contrary to expectation (Figure 3b). ROS levels dropped from 322.5 to 200.3, which is notably lower than the control group's level of 357.4 \(\pm\) 9.7. This unexpected result might be explained by the possibility that mechanical stress from microfluidic collision induces adaptive responses in the cysts, enhancing their capacity to manage oxidative stress [50]. This hypothesis was further explored (and discussed in subsequent sections) by analyzing changes in astaxanthin content per cell after collision treatment. The reduction in ROS levels suggests that  
Figure 3: Effect of microfluidic flow rate on (**a**) cell viability and (**b**) reactive oxygen species levels in _Haematococcus lacustris_ cysts. (**c**) Time course profile of total cell number density for 72 h of photosynthetic well plate cultivation using cysts treated at different microfluidic flow rates (1.0, 1.5, 2.0, 2.5, and 3.0 mL/min). Error bars represent the mean and standard deviation of three independent experiments (\(n=6\)). Different italicized letters (\(a\)–\(e\)) indicate the significance of the differences (\(p<0.05\)). DCF: 2′,′′-dichlorofluorescein. n.s. denotes non-significance between the groups. _H. lacustris_ exhibits varied responses to abiotic stress depending on the physiological state of the cell. This complexity in cellular responses is evident when comparing our results with other studies. For example, [PERSON] et al. [38] observed a 14% increase in ROS levels in biflagellate cells of _H. lacustris_ under compressed chip conditions after 6 d of photosynthetic cultivation. Similarly, [PERSON] et al. [43] reported a gradual increase in ROS levels in greenish brown palmella cells of _H. lacustris_ that were electrochemically treated by increasing the current from 5 to 20 mA over 4 h. These varied responses underscore the need for further research to understand the mechanisms underlying stress responses in _H. lacustris_. Figure 3c depicts the time course profile of total cell number density of _H.