**2. Results**

#### *2.1. The Effect of Phosphorus Deficiency on the Formation of Nonmotile Cells of H. pluvialis*

As shown in Figure 1, the total number of cells and the number of nonmotile cells in the two experimental groups both showed a trend of increasing with time. After 9 days of cultivation, the total number of cells and nonmotile cells in the two groups both reached the maximum value. The total number of cells in the control group reached 147.5 × 10<sup>4</sup> cells mL−1, which was about 59% higher than that in the P-deficiency group. The maximum number of nonmotile cells in the P-deficiency group was 37.5 × 10<sup>4</sup> cells mL−1, which was 4.17 times that of the control group. We also calculated the daily percentage growth rate of nonmotile cells, and the results showed that the percentage growth rate of nonmotile cells under phosphorus deficiency condition increased by an average of about 4.5% per day, which was 6.7 times that under normal conditions, indicating that phosphorus deficiency can significantly promote the formation of *H. pluvialis* nonmotile cells.

As shown in Figure 2 and Table 1, after 9 days of phosphorus deficiency treatment, more than 40% of the motile cells have been transformed into nonmotile cells. During this process, red pigmentation due to astaxanthin accumulation appears towards the center of nonmotile cells. By comparison, most of the cells in the control group are still motile cells in green color, with only about 6% nonmotile cells. In addition, some dead cells were observed in P-deficiency group and the cell mortality reached 9.4%, which was about 5.5% higher than that of the control group, indicating that phosphorus deficiency can increase the cell death rate.

**Figure 1.** Changes on the total cell number (**a**) and nonmotile cells number (**b**) of *H. pluvialis* in control- and P-deficiency treatment group. The results were presented as mean + SD.

**Figure 2.** The cell morphology of *H. pluvialis* on day 0 and day 9 in control- and P-deficiency treatment groups.

**Table 1.** The percentage of nonmotile cells, daily percentage growth rate of nonmotile cells, and cell mortality of controland P-deficiency treatment groups.


*2.2. The Effect of Adding NaCl on the Formation of Nonmotile Cells under Phosphorus Deficiency Condition*

Under phosphorus deficiency conditions, adding different concentrations of NaCl has significant effects on the growth, cell morphology, nonmotile cells formation rate, and cell mortality of *H. pluvialis*. As shown in Figure 3a, the total number of cells in both 0.1% NaCl and 0.4% NaCl treatment groups increased first, followed by a decrease, and then increased again. The total number of cells in the 0.2% NaCl treatment group also showed this trend. After 72 h of treatment, the total number of cells in the 0.1% NaCl and 0.2% NaCl treatment groups reached the maximum value, which were 75.0 × 10<sup>4</sup> cells mL−<sup>1</sup> and 70.0 × 10<sup>4</sup> cells mL−1, respectively. The maximum total cell number in the 0.4% NaCl treatment group appeared at the 12th h, and the value was 69.25 × 10<sup>4</sup> cells mL−1.

**Figure 3.** Changes on the total cell number (**a**) and nonmotile cells number (**b**) of *H. pluvialis* in 0.1%, 2%, and 0.4% NaCl treatment groups. The data at 61 h and 72 h in the 0.4% NaCl treatment group are not shown due to cell adhesion has affected the accurate determination of cell number. The results were presented as mean + SD.

It can be seen from Figure 3b that the number of nonmotile cells in the 0.1% NaCl and 0.2% NaCl treatment groups showed a rapid increase after 23 h of treatment. After 72 h of treatment, the number of nonmotile cells in the 0.1% NaCl treatment group reached 61.25 × 10<sup>4</sup> cells mL−1, which was about 11.4% higher than that in the 0.2% NaCl treatment group. The increase of nonmotile cells number in the 0.4% NaCl treatment group was the fastest among the three groups within 36 h of treatment, and then the increase rate slowed down. After 47 h of treatment, cell adhesion occurred in the 0.4% NaCl treatment group and accompanied by a large number of cell deaths, which made it impossible to accurately determine the cell number. Therefore, the data of the 0.4% NaCl treatment group at 61 h and 72 h were not shown in Figure 3.

As shown in Figure 4 and Table 2, after 72 h of treatment, 81.7% of the motile cells in the 0.1% NaCl treatment group had been transformed into green nonmotile cells, the daily percentage growth rate of nonmotile cells reached 27.2% day−1, and the cells was in good shape. In the 0.2% NaCl treatment group, after 36 h of treatment, the cell color gradually changed from green to orange-green due to the accumulation of carotenoids. After 72 h of treatment, 78.6% of the motile cells transformed into nonmotile cells. The daily percentage growth rate of nonmotile cells was 26.2% day−1, and 3.4% of the cells died due to stress.

When the concentration of NaCl added to the phosphorus deficiency medium reached 0.4% (*w*/*v*), it was observed that some cells were damaged after 23 h of treatment. After 36 h of treatment, carotenoids began to accumulate inside the cells. After treatment for 47 h, cell adhesion occurred and over 38% of the cells died (Table 2). By comparison, the cell mortality in 0.1% NaCl and 0.2% NaCl treatment groups were only 1.8% and 3.4%, indicating that high concentrations of NaCl can significantly increase cell mortality rate.

**Figure 4.** Morphological changes of *H. pluvialis* cells in 0.1%, 0.2%, and 0.4% NaCl treatment groups. The damaged or dead cells are indicated by arrows.

**Table 2.** The percentage of nonmotile cells, daily percentage growth rate of nonmotile cells, and cell mortality in 0.1, 0.2, and 0.4 NaCl treatment groups.


1 Obtained after 72 h of treatment. 2 Obtained after 47 h of treatment. 3 Obtained after 61 h of treatment.

#### **3. Materials and Methods**

#### *3.1. Algal Strain and Culture Conditions*

*H. pluvialis* CCMA-451 was obtained from the Center for Collections of Marine Algae in Xiamen University, China. The motile cells were grown photoautotrophically at 20 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> in liquid Bold Basal Medium (BBM) with 3 times NaNO3.

The 5-day-old green motile cells were collected by centrifugation (2000 rpm, 2 min), and transferred into a fresh phosphorus-free BBM medium at an initial optical density of 0.5 (OD680). Then NaCl was added to the cultures to adjust the concentrations to 1%, 2%, and 4% (*m*/*v*). All experiments were performed in triplicate in a 1-L glass columns (inner diameter 5 cm) at 25 ± 1 ◦C under continuous illumination (30 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1) for 3 days. Culture mixing was provided continuously by bubbling of filtered air enriched with 1.5% (*v*/*v*) CO2 at a flow rate of 100 mL min−1.
