# Electric Stimulation of Astaxanthin Biosynthesis in _Haematococcus pluvialis_
## 3 Results and Discussion  
### H. pluvialis Life Cycle and Astaxanthin Induction  
Figure 2 shows the changes in the cell number density and shape of _H. pluvialis_ upon culturing for 25 days in the photosynthetic photobioreactor. To enhance astaxanthin production in _H. pluvialis_ cells, 1 mM nitrate was used as the nitrogen source. The cell number density of _H. pluvialis_ increased almost linearly with time after a short delay period of 2 days (Figure 2a). However, after 15 days of culture, as the nitrate content was depleted, the growth rate of _H. pluvialis_ decreased considerably and the culture soon entered a stationary growth phase.  
Figure 2: Time course profile of changes in the cell number density (**a**) and morphology (**b**) of _Haematococcus pluvialis_ cells during photobioreactor cultivation for 25 days. The life cycle of _H. pluvialis_ is characterized by five types of distinguishable cellular morphologies based on microscopic observation: (1) astaxanthin-rich asexual aplanospore; (2) dividing aplanospore with internal differentiated zooids; (3) green vegetative biflagellate cell; (4) green vegetative non-motile palmella; and (5) astaxanthin-accumulating palmella cell. The asterisk (*) indicates the growth stage of _H. pluvialis_ cells used for the electric stimulation experiment. Scale bars in the optical images represent 10 \(\upmu\)m.  
The growth pattern of _H. pluvialis_ cells during the 25 day cultivation period could be largely divided into two different stages: vegetative growth (cell division and enlargement), and encrystent with astaxanthin induction. More specifically, based on the morphological characteristics observed using optical microscopy, the life cycle of the microorganism was characterized by five distinct cell types (Figure 2b). When the red cysts (cell-type #1) harvested from the seed flask culture were inoculated in fresh medium contained in the photobioreactor, the cells began to germinate within 6 h. A dividing cyst (cell-type #2) with the internally differentiated zooids was split open, releasing daughter cells into the external environment. The biflagellate motile cells released (cell type #3) grew rapidly and became the predominant cell type during the vegetative growth stage. With cell aging, sequentially, the motile cells lost their flagella, expanded in volume, and formed non-motile palmella cells (cell-type #4). Under conditions of stress, such as nitrogen depletion, intracellular astaxanthin biosynthesis was induced in the palmella cells, which led to brownish-green coloration in the cells (cell-type #5). Eventually, these cells were transformed into asexual aplanospores enriched in astaxanthin with a characteristic bright red color (cell type #1).  
After the 25 day cultivation period, the specific astaxanthin productivity of _H. pluvialis_ in the photobioreactor was estimated to be 2.4 \(\pm\) 0.5 mg-g-dcw\({}^{-1}\cdot\)d\({}^{-1}\). This result is comparable to that of _H. pluvialis_ cells cultured in nitrogen-free medium for 14 days (ca. 2.1 mg\(\cdot\)g-dcw\({}^{-1}\cdot\)d\({}^{-1}\)) [24]. However, as specified above, nitrogen depletion requires a significantly long processing time and cannot be considered ideal for designing an economical _H. pluvialis_ biorefinery process.