## 3 Results and Discussion
### Bio-Particle Distribution by Coulter Counter and Astaxanthin Recovery  
Figure 3 shows the bio-volume distribution and total bio-volume of _H. pluvialis_ bio-particles before and after French press cell treatment with different pressures (10,000-30,000 psi) and then passes (1-3). Here, \"bio-particles\" collectively refer to the ruptured cells, cell debris, and internal biomolecules that have leaked outwards, as well as the initial intact cells. In the untreated control, most of the _H. pluvialis_ cysts were of 15-60 \(\upmu\)m cell size and they exhibited a relatively large particle volume distribution (\(\sim\)4.1 \(\times\) 10\({}^{7}\)\(\upmu\)m\({}^{3}\)/mL) (Figure 3a). However, when homogenized at a pressure of 10,000 psi, the particle volumes of bio-particles in the same range significantly decreased in proportion to the increase in the number of passage: \(\sim\)1.7 \(\times\) 10\({}^{7}\)\(\upmu\)m\({}^{3}\)/mL for one pass, \(\sim\)1.3 \(\times\) 10\({}^{7}\)\(\upmu\)m\({}^{3}\)/mL for two passes, and \(\sim\)0.5 \(\times\) 10\({}^{7}\)\(\upmu\)m\({}^{3}\)/mL for three passes. On the contrary, the volumes of the bio-particles smaller than \(\sim\)15 \(\upmu\)m increased slightly when compared to the untreated control. These changes were induced by the mechanical destruction of the initial cyst cells (see Figure 1 for morphological changes). Figure 3b plotted the changes in the total bio-volumes of the bio-particles after the homogenization treatments. The total bio-volume of the homogenized bio-particles was calculated by summing the volume of each bio-particle between 15 to 60 \(\upmu\)m while considering their characteristic volume reductions. The total bio-volume of the untreated _H. pluvialis_ cysts (1.9 \(\times\) 10\({}^{9}\) m\({}^{3}\)/mL) decreased almost linearly with an increasing number of passes (1-3), irrespective of the tested pressure intensity (10,000-30,000 psi). However, no significant positive effect of the high pressures (20,000-30,000 psi) was observed as compared to the case of 10,000 psi, which is similar to that of the microscopic cell typing (Figure 2). The cell disruption efficiency could be simply derived from the degree of total volume reduction of the homogenized samples (versus the untreated control, 100%), and its maximal value was estimated to be 91.8 \(\pm\) 2.2%. This value was also almost equal to the highest value (89 \(\pm\) 0.8%) that was obtained from the microscopic cell counting in Figure 2.  
When comparing the cell disruption efficiencies that were calculated from Coulter counting and microscopic cell typing methods, a statistically good correlation (r\({}^{2}\) = 0.95) was obtained (Figure 4a). Furthermore, by the subsequent ethyl acetate extraction, astaxanthin was recovered from the _H. pluvialis_ biomass treated under various high-pressure homogenization conditions (see Figures 2 and 3). As  
Figure 3: Changes in volume distribution (**a**) and total volume (**b**) of _H. pluvialis_ bio-particles after mechanical disruptions with different pressures and passes. The bio-particles refer to all internal components as well as intact, leaky, and ruptured cyst cells. Representative particle-size-volume distributions after the 10,000 psi-homogenizations at three different passes were presented based on the Coulter counter analysis. Total volumes were calculated by summing each volume of all particles sized between 15 and 60 \(\upmu\)m.  
expected, the astaxanthin concentration increased almost linearly from 5.3 to 15.2 mg/L in proportion to the increase in cell disruption level (Figure 4b), which indicated that cell disruption is a critical factor for solvent-based astaxanthin recovery. The astaxanthin content of the _H. pluvialis_ biomass was estimated to be 1.1% (w/w). Astaxanthin from the freeze-dried _H. pluvialis_ cyst cells could also be extracted while using solely ethyl acetate, although its concentration was as low as 5.3 mg/L.