**4. Discussion**

The goal of improving the cultivation technology of *H. pluvialis* is to maximize the production of biomass and astaxanthin. Achieving cell growth and astaxanthin accumulation simultaneously under the same cultural conditions is difficult. The major reason for this is that the rapid growth of *H. pluvialis* requires favorable environmental conditions [14–16] and the accumulation of astaxanthin requires unfavorable cultural conditions [11,17–19]. The two-stage cultivation strategy has effectively solved the contradiction between cell growth and astaxanthin accumulation. However, it fails to solve the high cell mortality during the red stage, so this technology still has a lot of room for improvement.

Previous studies have confirmed that the motile cells are susceptible to photooxidative stress and tend to die under high intensity of light conditions [9,12,20]. Compared with motile cells, nonmotile cells with thick cell walls are not only more tolerant to stress [12], but also more effectively use chemical energy accumulated during cell transformation to rapidly synthesize and accumulate astaxanthin under stress conditions [12,21,22]. Li et al. [20] suggested that using nonmotile cells as the dominant cell type in the red stage can reduce cell mortality more than 70%, while biomass and astaxanthin content can increase up to 2.12 times and 3.5 times, respectively. Therefore, by inducing motile cells to transform into nonmotile cells with more tolerance and astaxanthin accumulation ability before entering the red stage, the problem of high cell mortality is expected to be effectively solved.

The formation of nonmotile cells of *H. pluvialis* is accompanied by encystment, which is considered to be a self defense mechanism [22–24]. Previous studies have shown that the encystment of *H. pluvialis* may be related to carbon assimilation [23,25] and phosphorus plays a key role in photosynthetic carbon assimilation [26]. The phosphate translocator is an important structure in the chloroplast membrane, which can transport triose phosphate (the first sugar produced in photosynthesis) produced during the Calvin cycle to the cytoplasm for the synthesis of sucrose, and at the same time transport the released inorganic phosphate (Pi) to chloroplast matrix. When phosphorus is deficient, the Pi exchanged with triose phosphate decreases, resulting in a reduction of Pi in the chloroplast and a decrease in the ATP/ADP ratio, which affects the transport in the C3 pathway and the progress of related reactions, thereby reducing the photosynthetic rate. At the same time, triose phosphate is converted into starch due to phosphorus deficiency and stored in the chloroplast. The reduction of triose phosphate transported from the chloroplast to the cytosol further affects the synthesis of sucrose, thereby inhibiting the photosynthesis of algae and promoting the formation of encystment [14] to maintain the cell integrity, structure, and function. Although phosphorus deficiency can promote the formation of nonmotile cells, the efficiency is low. According to our results, the addition of NaCl under phosphorus deficiency conditions significantly promotes the formation of nonmotile cells. It made the daily percentage growth rate of nonmotile cells increase from 4.5% to more than 26%. We speculate that NaCl promotes the formation of nonmotile cells under the condition of phosphorus deficiency owing to the following reasons. (1) The increase of NaCl concentration affects the oxygen metabolism in algal cells, accelerates the production

of reactive oxygen species (ROS), and reduces the function of the scavenging system, which leads to the accumulation of ROS in the cells. (2) Higher salinity increases the osmotic pressure and promotes loss of water from cells, thereby affecting the normal metabolic activities of the cells. (3) The increase of NaCl concentration affects the ion homeostasis in the cells, thereby causing nutritional stress. Thus, it is necessary to carry out further physiological and biochemical studies.

We have to point out that we did not use algae cultured without NaCl for 72 h as a control. However, it doesn't seem to affect our conclusion that adding NaCl to phosphorusdeficiency medium can significantly promote the formation efficiency of *H. pluvialis* nonmotile cells. We have also determined the most efficient induction method (synergistic induction by phosphorus deficiency and 0.1% NaCl) of nonmotile cells. After three days of cultured under the optimal conditions developed in this study, more than 80% of motile cells transformed into nonmotile cells. The astaxanthin production efficiency of these nonmotile cells should be evaluated by extraction and HPLC in future. This will be profitable for the *Haematococcus* industry.

**Author Contributions:** Data curation, N.Z.; formal analysis, N.Z., Y.Z. and F.L.; funding acquisition, M.C.; investigation, F.L., Q.L., C.Q., Z.Q., Y.W. and Z.Y.; methodology, N.Z. and Y.Z.; project administration, M.C.; resources, X.H. and C.L.; writing—original draft, F.L.; writing—review & editing, F.L. and M.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was mainly supported by Xiamen Southern Ocean Technology Center of China under Grant (number 14CZP035HJ09) and partly funded by Xiamen Scientific and Technologic Projects under Grant (number 3052Z20031086 and 3052Z20123004); Marine Science Base Scientific Research Training and Scientific Research Ability Enhancement Project of Xiamen University under Grant (number J1210050); Xiamen University Training Program of Innovation and Entrepreneurship for Undergraduates under Grant (number 2016X0619); and the Special Funds for Scientific Research of Marine Public Welfare Industry under Grant (number 201305016).

**Conflicts of Interest:** The authors declare no conflict of interest.
