## 3 Results and Discussion
### Effect of Microfluidic Collision on Biomass and Astaxanthin Production  
To assess the impact of microfluidic collision on microalgal biomass and astaxanthin production, _H. lacustris_ cysts treated under the optimal microfluidic condition (10 loops at 2.0 mL/min, as identified in well plate experiments) were cultured in 120 mL flask setups for 30 d, compared to 2 mL well plates for 3 d, while maintaining consistent photosynthetic conditions across both systems. Figure 6 shows the time course profiles of total cell number density during photosynthetic flask cultivation. During the initial 3 days, microfluidic collision treatment resulted in a modest increase of 3-10% in cell density compared to the controls. This increase became more significant after 5 d, with treated cultures achieving an 18-30% higher cell density than controls. By the end of the 30 d flask cultivation, the treated _H. lacustris_ cultures reached a total cell density of 32.0 \(\pm\) 1.1 \(\times\) 10\({}^{4}\) cells/mL, an 18.2% increase over the control (27.1 \(\pm\) 0.3 \(\times\) 10\({}^{4}\) cells/mL). While this increase was smaller than the 44.5% observed in the 3 d well plate cultures (Figure 4c), this discrepancy likely reflects differences in photosynthetic culture conditions--such as light transfer, CO\({}_{2}\) supply, nutrient mixing, and shear stress--between well plate and flask cultures [55]. Despite these variations, the results from Figures 3c, 4c and 6 consistently demonstrate the positive impact of microfluidic collision treatment on the photosynthetic cell growth of _H. lacustris_ across different culture systems. Consistent with increased cell density, Figure 7a shows that the final biomass concentration after 30 d in treated _H. lacustris_ cultures (1.48 \(\pm\) 0.09 g cell/L) was 17.0% higher than in the controls (1.27 \(\pm\) 0.09 g/L), indicating that enhanced biomass production is a direct result of the improved cell growth facilitated by microfluidic collision treatment. Interestingly, the astaxanthin content per cell remained stable at 19.2-19.4 mg/g cell, regardless of treatment (Figure 7b), suggesting that the mechanical collision of cysts does not directly affect the metabolic pathways for astaxanthin synthesis in _H. lacustris_. However, due to the increased biomass, astaxanthin productivity in treated cultures (0.95 \(\pm\) 0.07 mg/L/d)  
Figure 6: Time course profile of total cell number density during 30 d of photosynthetic flask cultivation of _Haematococcus lacustris_ cysts treated with the optimal microfluidic collision conditions (10 loops at 2.0 mL/min). Error bar represents the standard deviations of three independent experiments (\(n=6\)). was 21% higher than that in the controls (0.79 \(\pm\) 0.09 mg/L/d) (Figure 7c). These findings indicate that while mechanical collision treatment enhances overall astaxanthin production by promoting initial germination and subsequent photosynthetic cell growth, it does not alter astaxanthin content per cell. The biochemical mechanisms and regulation of germination in _H. lacustris_ remain incompletely understood [56]. Previous studies have shown that cyst germination primarily depends on nitrogen levels and light intensity [57]. For example, [PERSON] et al. [19] reported an increase in germination efficiency from 50 to 65% after 4 d when nitrate concentration was raised from 1 to 2 mM under 150 umol photons/m\({}^{2}\)/s. [PERSON] et al. [20] highlighted the importance of light wavelengths in cyst germination, observing higher efficiency under blue light compared to white or red light. However, nitrogen supplementation inhibits astaxanthin synthesis, reducing overall astaxanthin productivity [24; 27]. Managing light conditions poses challenges due to the need for artificial lighting systems and their associated energy demands [23]. This study is the first to investigate a mechanostimulation strategy for germination in _H. lacustris_ (see next section for detailed discussion). Given the complex life cycle and cellular heterogeneity of _H.