## 3 Results and Discussion
### Cell Type Distribution by Microscopic Counting  
Figure 2 shows the relative cell type distribution of _H. pluvialis_ cysts after various high-pressure homogenization treatments (pressure intensity, 10,000-30,000 psi; pass, 1-3 times). The morphological cell types were calculated by sorting over 100 cells while using the improved Neubauer counting chamber under microscopic observation (also see Figure 1). After the homogenization in one pass at a pressure of 10,000 psi, the number of intact cyst cells accounted for ca. 45%, whereas the rest of the initial cysts became either leaky or ruptured completely (cell disruption, 54.8 \(\pm\) 0.3%). Here, the cell disruption (%) was estimated by subtracting the % ratio of both the leaky and ruptured cells from the initial cells (100%). The cell disruption degree significantly increased to 80.2 \(\pm\) 6.0% and 89 \(\pm\) 0.8%, respectively, when the algal-suspension was homogenized once and twice more at the same pressure. On the contrary, at higher pressures of 20,000-30,000 psi, the cell disruption efficiency of _H. pluvialis_ cysts did not improve when compared to that of the 10,000 psi treatment even if the passage number was increased from 1 to 3. The cell disruption efficacy of the high-pressure homogenization process can be influenced by the algal cell concentration, as well as the homogenizer specification (loading pressure and number of passage) [12]. Lowering the cell dosage might be considered in order to improve the cell disruption rate to more than 90%. However, it should be noted that this approach is not generally recommended in terms of large-scale biorefining of _H. pluvialis_ biomass on an industrial scale [8].  
Although the high-pressure homogenization has been recommended as a relatively efficient and scalable method among various physicochemical cell disruption techniques for large-scale algal biorefining [8,12], studies on _H. pluvialis_ cyst cells are very limited. [PERSON] et al. [19] reported that, when compared to ionic liquid-based extractions, the 30,000 psi-fixed high-pressure homogenization resulted in a high yield of 24 pg astaxanthin/cell from wet _H. pluvialis_ cysts that were cultured under small-scale laboratory conditions. However, the cell disruption rate and key operating parameters (especially pressure intensity and number of passages) were not elucidated. [PERSON] et al. [17] obtained the highest protein extraction yield (40%) from lyophilized _H. pluvialis_ biomass while using a high-pressure cell disruptor that was fixed at 2700 bar (39,150 psi), as compared to manual grinding, alkaline treatment, and ultrasonication treatments. Moreover, they reported that, under the same condition, the maximal protein recovery efficiency showed a decreasing trend in accordance with the increasing order of cell wall rigidity among the tested algal strains: _Arthrospira platensis < Porphidium crucentum < Chlorella vulgaris < Nannochloropsisulata < H. pluvialis_. Detailed investigation into cell physiology, cell wall biology, and physical strength of _H. pluvialis_ cells is required in order to develop an  
Figure 2: Cell type distribution of _H. pluvialis_ cysts after various high-pressure homogenization treatments (pressure, 10,000–30,000 psi; passage, 1–3). The cell types were classified into intact, leaky, and ruptured according to the morphological features that are based on the microscopic observation (Figure 1).  
economical high-pressure homogenization process. In fact, this insight would create high potentialities for a synergistic or novel process that utilizes various existing biological-physicochemical treatments.