# Electric Stimulation of Astaxanthin Biosynthesis in _Haematococcus pluvialis_
## 3 Results and Discussion
### CV Analysis  
To characterize the bioelectrochemical reactions occurring between the carbon electrode and the _H. pluvialis_ cells, a cyclic voltammogram was constructed based on the reaction occurring in a single-chamber electrochemical reactor with three electrodes (i.e., counter, working, and reference) and analyzed using a potentiostat (Figure 5). Similar to that in the electric stimulation process described above, green vegetative _H. pluvialis_ cells cultured for 5 days in the photobioreactor were tested. The current density-voltage relationship varied significantly in the presence of algal cells under the applied reduction/oxidation potentials. For the algal cells, both reduction and oxidation current densities showed an increasing trend relative to that in the cell-free solution (abiotic control) within the voltage range of \(-0.5\) to \(+0.5\) V (vs. Ag/AgCl). Particularly, the oxidation current density increased only slightly (from \(+0.1\) (abiotic) to \(+0.15\)\(\mu\)A cm\({}^{-2}\) (biotic)) at \(+0.5\) V, whereas the reduction current density increased significantly (from \(-0.1\) (abiotic) to \(-0.4\)\(\mu\)A cm\({}^{-2}\) (biotic)) at \(-0.5\) V. This suggests that even at the same voltage, significantly different oxidation and reduction environments can be created for the algal cells (Figures 3 and 4). The lysis and degradation of _H. pluvialis_ cells during the cathodic current treatment (Figure 3) might be attributed to the formation of strong ROS in excess at the carbon cathode. The in situ production of H\({}_{2}\)O\({}_{2}\) via the cathodic 2 e\({}^{-}\) oxygen reduction reaction (ORR, O\({}_{2}\) + 2 H\({}^{+}\) + 2 e\({}^{-}\)\(\rightarrow\) H\({}_{2}\)O\({}_{2}\)) generates potent hydroxyl radicals (-OH), which can damage algal cells [19; 27]. Specifically, carbon-based electrodes have been reported to effectively induce the 2 e\({}^{-}\) ORR reaction [28].  
Figure 6 shows a hypothetical model for electric treatment of _H. pluvialis_ in the two-chamber bioelectrochemical system. Only a limited number of studies have been conducted on the electric treatment of microalgae, and detailed bioelectrochemical mechanisms are yet to be identified [18; 19; 26; 29]. Electrical stimulation can be used to induce the synthesis of various microbial products biologically by electron transfer from the electrode or non-biologically by oxidizing chemicals generated from the electrodes [25; 30]. Biological stimulation involves the direct interaction between membrane-associated enzymes and the electrode surface or the indirect transfer of electrons to the microorganism through electron mediators such as hydrogen. As a non-biological stimulant, ROS is known to induce the biosynthesis of neutral lipids and/or astaxanthin in various algal species, including _Chlorella_ and _Haematococcus_[2; 19]. The characterization and identification of the mechanism underlying the electric stimulation of astaxanthin production in _H. pluvialis_ should be investigated in future studies.  
Figure 5: Cyclic voltammograms of the carbon electrode in the presence (biotic, red line) and absence (abiotic, black line) of _Haematococcus pluvialis_ cells in a single-chamber electrochemical reactor with three electrodes. The scan rate was 10 mV s\({}^{-1}\).