# Electric Stimulation of Astaxanthin Biosynthesis in _Haematococcus pluvialis_
## 3 Results and Discussion
### Electric Induction of Astaxanthin Production Using Cathodic and Anodic Currents  
Microbial electro-stimulation can be performed by applying either a cathodic or anodic current in a two-chamber electrochemical system (Figure 1). Anodic current stimulation is useful for controlling intracellular redox balance, such as NAD(P)H/NAD(P)\({}^{+}\) balance, in bacteria [25]. Meanwhile, cathodic current treatment has been reported to stimulate the accumulation of reduced biomolecules, such as TAGs and fatty acids (particularly in _Chlorella_), via electron transfer and/or reactive oxygen species (ROS) generation from the cathode [19]. In this study, since a mild cathodic current was considered to act as a novel stress inducer for algal astaxanthin biosynthesis, alterations in the morphology and astaxanthin content of _H. pluvialis_ were evaluated in the cathodic chamber of the bioelectrochemical system previously optimized for _Chlorella_ lipids [19]. Biflagellate motile _H. pluvialis_ cells cultured for 5 days in the photobioreactor were harvested, resuspended in 0.1 M phosphate buffer (pH 7.0), and electro-treated for 2 days at a cathodic current of 3 mA (voltage: 2 V). After incubation for 2 days, regardless of the electric treatment applied, most vegetative _H. pluvialis_ cells were transformed into brown or brownish-green non-motile palmella cells, indicating that astaxanthin biosynthesis was induced, albeit at low levels, within the cells. However, the astaxanthin content of the cells treated at 2 V reduced by 57%, a significant reduction compared to that in the untreated control (4.4 \(\pm\) 0.1 mg g-dcw\({}^{-1}\)). Furthermore, some _H. pluvialis_ cells were damaged (Figure 3). This suggests that unlike in case of _Chlorella_[19], the cathodic current environment negatively affects the viability and astaxanthin-synthesizing activity of _H. pluvialis_ cells. Considering the severe cell damage induced, we switched from cathodic to anodic current for electric treatment while maintaining other bioelectrochemical parameters. Figure 4 shows the changes in the optical morphology and astaxanthin content of _H. pluvialis_ cells treated for 2 days under anodic current at 2, 4, and 6 V. The untreated _H. pluvialis_ cells remained green for 1 day and turned partially brownish-green after 2 days. Conversely, the electro-treated _H. pluvialis_ cells showed a significant color change to brown on day 1, indicating the relatively rapid induction of astaxanthin biosynthesis than that in the control. Contrary to the cathodic current results (Figure 3), no significant cell degradation and disintegration were observed at the applied voltages of 2-6 V. Instead, unique morphological changes, such as separation of the cell wall and cytoplasm and s biomaterials (possibly cell wall lysates) occurred in the electro-treated _H. pluvialis_ cells, which tended to worsen as the voltage increased from 2 to 6 V. Nevertheless, at an optimal voltage of 4 V (current intensity: 30 mA), the astaxanthin content was 36.9% higher (6.0 \(\pm\) 1.1 mg g-dcw\({}^{-1}\)) than that in the untreated control. This result implies that anodic current treatment stimulated astaxanthin accumulation in _H. pluvialis_. However, at a higher voltage of 6 V (current intensity: 81 mA), the astaxanthin content of _H. pluvialis_ cells decreased compared to that achieved at 4 V, indicating that an excessively high current does not necessarily induce astaxanthin production in _H. pluvialis_ cells. The anodic stimulation results (Figure 4) further suggested that algal astaxanthin induction is highly dependent on the current intensity and duration of treatment. Current-duration relationships and optimization (such as long treatment durations at low current intensities, short treatment durations at high current intensities, or a gradual increase/decrease in current intensity with the treatment duration) could be studied in greater detail in further investigation for designing practical induction processes.