**1. Introduction**

Astaxanthin is a keto-carotenoid that has a wide range of applications in aquaculture, food, cosmetics, and human health due to its strong antioxidant and coloring functions [1,2]. Currently, there are mainly two types of astaxanthin on the market: artificial synthetic astaxanthin and natural astaxanthin [3]. Artificial synthetic astaxanthin accounts for about 95% of the market and is mainly used in aquaculture [2]. Although synthetic astaxanthin has the advantages of lower cost and price, it has not been approved for human consumption due to possible safety issues [3]. In contrast, natural astaxanthin is significantly better than artificial synthetic astaxanthin in terms of stability, antioxidant activity, absorption effect and biosafety, and has been approved by China, the United States, Japan, and some EU countries for aquaculture, dietary supplements, cosmetic ingredients, and other uses [2,4]. However, due to technical limitations in the production of raw materials, the global production of natural astaxanthin is low, resulting in high market prices [5,6], and the retail prices of nutraceutical grade astaxanthin have even reached US \$100,000 per kilogram [7].

*Haematococcus pluvialis* is the most competitive natural source for commercial astaxanthin production and the global annual production capacity is about 800 tons [8], which is

**Citation:** Li, F.; Zhang, N.; Zhang, Y.; Lian, Q.; Qin, C.; Qian, Z.; Wu, Y.; Yang, Z.; Li, C.; Huang, X.; et al. NaCl Promotes the Efficient Formation of *Haematococcus pluvialis* Nonmotile Cells under Phosphorus Deficiency. *Mar. Drugs* **2021**, *19*, 337. https:// doi.org/10.3390/md19060337

Academic Editors: Masashi Hosokawa and Hayato Maeda

Received: 25 May 2021 Accepted: 10 June 2021 Published: 13 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

still far behind the 10,000-ton for spirulina and 1000-ton for chlorella. The high cell death rate during the production process is the main reason for the low productivity of *H. pluvialis* astaxanthin [9,10]. The accumulation of biomass and the synthesis of astaxanthin are two important factors that must be considered in the production of astaxanthin in *H. pluvialis*. However, culture conditions suitable for rapid cell growth and culture conditions for astaxanthin accumulation are mutually exclusive [4]. A two-stage culture strategy is widely adopted by *H. pluvialis* industry [9], because it is the most effective strategy for solving the contradiction between cell fast growth and astaxanthin accumulation at present [11]. In actual production, however, when the vegetative cells are transferred from the first stage (green stage) to the second stage (red stage), a large number of cell deaths occur due to high intensity of light in combination with nutrient depletion stress. As a result, the overall astaxanthin productivity is very low [9]. Therefore, the reduction of cell mortality in the red stage has become the key to increasing the overall astaxanthin production of *H. pluvialis*.

The cells of *H. pluvialis* usually go through vegetative green stage, intermediate palmella stage and cys<sup>t</sup> stage, in which several types of cells are distinguished: motile cells, nonmotile palmella cells, and haematocysts (aplanospores) [4]. Previous studies found that the nonmotile cells of *H. pluvialis* were more tolerant to photooxidative stress than motile cells [12]. Using nonmotile cells as the main cell type for the astaxanthin production can significantly reduce the cell mortality and increase astaxanthin productivity in the red stage [13]. However, there are few reports on the induction of *H. pluvialis* nonmotile cells formation. In this study, we first investigated the effect of phosphorus deficiency on the formation of nonmotile cells of *H. pluvialis*, and then the effect of NaCl on the formation of nonmotile cells under the conditions of phosphorus deficiency. Our results showed that the addition of NaCl effectively promoted the formation of nonmotile cells under the condition of phosphorus deficiency. The work provides the most cost-efficient method for the preparation of *H. pluvialis* nonmotile cells so far, and this is of grea<sup>t</sup> significance for improving the production of *H. pluvialis* astaxanthin by using cell regulation technology.
