## 3 Results and Discussion
### Effect of Loop Number on Seed Cysts and Cell Growth  
Figure 4 shows the effect of varying loop numbers (3-30) on _H. lacustris_ cysts at a fixed flow rate of 2.0 mL/min. The loop number correlates with cumulative mechanical stress experienced by the cysts during the microfluidic treatment. As shown in Figure 4a, _H. lacustris_ cysts treated with lower loop numbers (3 and 6) exhibited high viabilities (97.6-97.7%), comparable to the untreated controls (98.1 \(\pm\) 1.8%). However, increasing the loop number to 10 resulted in a modest reduction in viability to 95.6 \(\pm\) 0.3%. Further increases to 20-30 loops led to more significant decreases in viability (92.3-94.0%). Notably, at 80 loops, viability dropped sharply to <20%. These findings, combined with findings from varying flow rates (Figure 3a), suggest that while _H. lacustris_ cysts have robust cell walls [6, 46], excessive mechanical stress can substantially impair cell viability.  
ROS levels in _H. lacustris_ cysts treated with different loop numbers followed a similar trend as with varying flow rates (Figure 4b). All treated cysts exhibited lower ROS levels than controls, with two distinct responses observed. At lower loop numbers (3-20), ROS levels ranged from 241.4-295.5, representing a 24.6-29.8% reduction compared to the control (344.0 \(\pm\) 6.3). At 30 loops, ROS levels dropped sharply to 193.1, a 44% reduction compared to controls. Microscopic observations revealed significant cell lysis at this loop (Figure S2).  
Further analysis focused on the astaxanthin content in _H. lacustris_ cysts after treatment across loop numbers ranging from 3-30 (Figure S3). Notably, higher loop numbers (6-30) resulted in lower astaxanthin content (49.1-53.5 pg/cell) compared to the lowest loop number of 3 and untreated controls (60.1-63.8 pg/cell). This reduction in astaxanthin content suggests that _H. lacustris_ may be utilizing astaxanthin as part of its stress response to mechanical stimulation. Astaxanthin is a potent antioxidant that can be consumed to counteract oxidative stress, indicating a trade-off between stress mitigation and the retention of this valuable carotenoid. Additionally, studies are needed to explore the roles of _H. lacustris_ sophisticated enzyme systems in managing oxidative stress--such as superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase [45, 51]. Understanding these complex biological mechanisms could provide insights into how mechanical stimulation might be harnessed to break the dormancy of microalgal cysts without compromising cell viability.  
Figure 4c shows the time course profile of the total cell number density of _H. lacustri_ during 72 h of photoautotrophic cultivation in a well plate, with cysts treated at loop numbers ranging from 3-30 and a fixed flow rate of 2.0 mL/min. Across all loop numbers, treated cultures consistently exhibited higher cell density compared to controls. Cysts treated with an optimal loop number of 10 achieved the highest cell number density after 72 h (28.9 \(\pm\) 2.0 \(\times\) 10\({}^{4}\) cells/mL), representing a 44.5% increase over untreated cells. This significant improvement suggests that microfluidic collision treatment, when carefully controlled, serves as an effective stimulus to enhance the photosynthetic proliferation of _H. lacustri_ cysts.