## 3 Results and Discussion  
### Morphological Changes by Mechanical Disruption  
The effect of French press cell treatment (10,000 psi, 1 pass) on mature _H. pluvialis_ cysts were evaluated morpho-dynamically while using a combination of light and fluorescence microscopy (Figure 1). In the phase-contrast mode of optical microscope, the intact _H. pluvialis_ cysts showed significant amounts of astaxanthin (bright red) and chlorophyll (green) inside the cells. The high-pressure homogenization could partially damage or completely rupture the cell walls of the cysts, resulting in significant releases of the cytoplasmic constituents into the surrounding medium. The _H. pluvialis_ cysts could be largely categorized into intact, leaky, and ruptured types, depending on their degree of destruction (see Figure 2 for their distribution). Three different fluorescent probes of Calcofluor white, Nile red, and FITC were used under fluorescence microscopy, respectively, in order to simultaneously visualize the cell wall and cytoplasmic polysaccharides (blue), neutral lipids (yellow), and proteins (green) of _H. pluvialis_ cyst cells. Figure 1 shows astaxanthin (magenta) and chlorophyll (red) auto-fluoresce without chemical staining. The total carbohydrate content of the _H. pluvialis_ cysts that were incubated under the outdoor conditions while using a CO\({}_{2}\)-rich (~3.5%, v/v) power-plant flue gas was estimated to be 12% (w/w). With the Calcofluor white dye, the polysaccharide fractions of the cell wall and the cytoplasmic contents were stained blue under the DAPI filter. The intact encysted cells showed a continuous blue color on their cell walls, because the _H. pluvialis_ cell wall is mostly composed of mannan and cellulose polysaccharides [21; 22]. However, significant dispersion of the blue-colored cell wall and cytoplasmic polysaccharides were observed after mechanical disruption. Nile-red-stained neutral lipids are usually detected as cytoplasmic yellow-colored droplets in oleaginous microalgal species, such as _Chlorella_[23] and _Nannotchioropsis_[24], under fluorescent microscopy. The total lipid content of the _H. pluvialis_ cyst cell was as high as 40% (w/w) when estimated in terms of FAME amount by GC. This is similar to the typical lipid compositions of _H. pluvialis_ (32-37% (w/w)) harvested from the red cultivation stage [25]. However, it should be noted that, in _H. pluvialis_ cysts, astaxanthin biomolecules co-exist inside the lipid body mostly in mono- and di-ester forms with long-chain fatty acids [26; 27]. Therefore, under the present fluorescence microscopy condition, the former (magenta) and latter (yellow) cannot be clearly distinguished from the intact and leaky cell types. However, interestingly, after almost the complete rupture of the _H. pluvialis_ cyst cells, several yellow-colored lipid droplets appeared from the dispersed cytoplasm. The protein fractions from _H. pluvialis_ cells fluoresced green after the FITC staining. As expected, the intact cysts showed a relatively uniform green distribution in the cytosol, except for lipids, astaxanthin, and chlorophyll. However, following the mechanical treatment, the intensity of green color was significantly reduced. This might be mainly attributed to the significant loss of water-soluble proteins in the medium. Overall, the degree of dispersion of the internal constituents, including astaxanthin, increased in proportion to the degree of cell rupture. This resulted in the effective recovery of astaxanthin from the robust _H. pluvialis_ cysts by subsequent solvent extraction (see next sections). Figure 1: Microscopic images of _H. pluvialis_ cysts after the high-pressure homogenization treatment (10,000 psi and one pass). Cell shapes were classified into three types (i.e., intact, leaky, and ruptured) considering their morphological changes by the mechanical breakages. See colored arrows for each cell type.