*3.1. Effect of Desiccation on Growth and Nutrient Removal*

The results of the two-way ANOVA showed that the RGRs of *N. haitanensis* and *N. dentata* were significantly affected by desiccation, but they did not significantly differ between the two species (Figure 1; Table 1). A significant interaction between desiccation and the species was not detected. The RGRs of the two species decreased significantly with increasing desiccation periods from 0 to 6 h.


**Table 1.** Analysis of variance (two-way ANOVA) examining the effects of desiccation on the *RGR* and NO3-N and PO4-P removal rates of *N. haitanensis* and *N. dentata*, and the comparison of these parameters between the two species.

**Figure 1.** The RGRs and NO3-N and PO4-P removal rates of *N. haitanensis* and *N. dentata* cultured for 4 days under five desiccation periods. Different capital and small letters indicate statistical significance ( *P* < 0.05) among species and desiccation periods, respectively. The data represent the mean ± SE (n = 3 replicates).

The NO3-N removal rates of *N. haitanensis* and *N. dentata* were significantly affected by desiccation, but they did not significantly differ between the two species. A significant interaction between desiccation and the species was detected. The NO3-N removal rates of *N. haitanensis* and *N. dentata* decreased significantly with increasing desiccation periods, with ranges of 87.2–72.4% and 89.2–67.1%, respectively.

The PO4-P removal rates of *N. haitanensis* and *N. dentata* were not significantly affected by desiccation, and also did not significantly differ between the two species. A significant interaction between desiccation and the species was not detected. The PO4-P removal rates of *N. haitanensis* and *N. dentata* ranged from 88.9 to 98.0%, and from 90.9 to 98.2%, respectively.

#### *3.2. Effect of the Water Velocity on Growth and Nutrient Removal*

The RGRs of *N. haitanensis* and *N. dentata* were significantly affected by the water velocity, and also differed significantly between the two species (Figure 2; Table 2). A significant interaction between the water velocity and species was detected. The RGRs of *N. haitanensis* and *N. dentata* at 0.1 and 0.2 m s−<sup>1</sup> were significantly greater than those at 0.5 m s<sup>−</sup>1. The RGR of *N. dentata* at 0.2 m s−<sup>1</sup> was significantly greater than that at 0.1 m s<sup>−</sup>1. The RGRs of *N. haitanensis* were significantly greater than those of *N. dentata* at all three water velocities.

The NO3-N removal rates of *N. haitanensis* and *N. dentata* were significantly affected by the water velocity, but they did not significantly differ between the two species. A significant interaction between the water velocity and species was detected. The NO3-N removal rates of *N. haitanensis* and *N. dentata* at 0.1 (*N. haitanensis*: 86.2%; *N. dentata*: 80.5%) and 0.2 m s−<sup>1</sup> (*N. haitanensis*: 83.7%; *N. dentata*: 89.9%) were significantly greater than those at 0.5 m s−<sup>1</sup> (*N. haitanensis*: 44.6%; *N. dentata*: 48.4%). There were no significant differences in the NO3-N removal rates between 0.1 and 0.2 m s−<sup>1</sup> for both species.

**Figure 2.** The RGRs and NO3-N and PO4-P removal rates of *N. haitanensis* and *N. dentata* cultured for 4 days under three water velocities. Different capital and small letters indicate statistical significance (*P* < 0.05) among species and water velocities, respectively. The data represent the mean ± SE (n = 3 replicates).

**Table 2.** Analysis of variance (two-way ANOVA) examining the effects of the water velocity on the *RGR* and NO3-N and PO4-P removal rates of *N. haitanensis* and *N. dentata*, and the comparison of these parameters between the two species.


The PO4-P removal rates of *N. haitanensis* and *N. dentata* were significantly affected by the water velocity, and also differed significantly between the two species. A significant interaction between the water velocity and species was detected. The PO4-P removal rate of *N. haitanensis* at 0.1 m s−<sup>1</sup> (86.9%) was significantly greater than those at 0.2 (70.3%) and 0.5 m s−<sup>1</sup> (64.8%). The PO4-P removal rates of *N. dentata* at 0.1 (62.4%) and 0.2 m s−<sup>1</sup> (65.9%) were significantly greater than that at 0.5 m s−<sup>1</sup> (45.4%). The PO4-P removal rate of *N. haitanensis* was significantly greater than those of *N. dentata* at 0.1 and 0.5 m s<sup>−</sup>1.

#### *3.3. Effect of Nitrogen Limitation on Growth and Nutrient Removal*

The RGRs of *N. haitanensis* and *N. dentata* were significantly affected by nitrogen limitation, and also differed significantly between the two species (Figure 3; Table 3). A significant interaction between nitrogen limitation and the species was detected. After

4 day nitrogen recovery, the RGRs of *N. haitanensis* at 2 and 3 day nitrogen limitation were significantly greater than that at 1 day nitrogen limitation. There were no significant differences in the RGRs among the three nitrogen limitation levels for *N. dentata*. The RGR of P. dentata was significantly greater than that of *N. haitanensis* at each nitrogen limitation level.

**Figure 3.** The RGRs and NO3-N and PO4-P removal rates of *N. haitanensis* and *N. dentata* cultured for 4 days after three different periods of nitrogen limitation. Different capital and small letters indicate statistical significance (*P* < 0.05) among species and nitrogen limitation periods, respectively.The data represents the mean ± SE (n = 3 replicates).


**Table 3.** Analysis of variance (two-way ANOVA) examining the effects of nitrogen limitation on the *RGR* and NO3-N and PO4-P removal rates of *N. haitanensis* and *N. dentata*, and the comparison of these parameters between the two species.

The NO3-N removal rates of *N. haitanensis* and *N. dentata* were significantly affected by nitrogen limitation, but they did not significantly differ between the two species. A significant interaction between nitrogen limitation and species was detected. After 4 day nitrogen recovery, the NO3-N removal rate of *N. haitanensis* at 3 day of nitrogen limitation (91.8%) was significantly greater than those at 1 day (78.4%) and 2 day of nitrogen limitation (80.0%). The NO3-N removal rate of *N. dentata* increased significantly from 1 to 3 day nitrogen limitation (1 day: 72.9%; 2 day: 82.0%; 3 day: 93.3%).

The PO4-P removal rates of *N. haitanensis* and *N. dentata* were significantly affected by nitrogen limitation, and differed significantly between the two species. A significant interaction between nitrogen limitation and the species was detected. After 4 day of nitrogen recovery, the PO4-P removal rate of *N. haitanensis* at 1 day of nitrogen limitation (80.3%) was significantly greater than those at 2 (64.4%) and 3 day of nitrogen limitation (59.5%). The PO4-P removal rates of *N. dentata* at 1 (68.9%) and 2 day of nitrogen limitation (62.4%) were significantly greater than that at 3 day of nitrogen limitation (48.3%). The PO4-P removal rates of *N. haitanensis* were significantly greater than those of *N. dentata* at 1 and 3 day of nitrogen limitation.

#### **4. Discussion**

Intertidal seaweeds usually experienced the stress of desiccation with different periods originating from tidal alternation because they are essentially marine organisms [15,16]. Several studies have demonstrated that desiccation significantly affects the growth and nutrient uptake of *Neoporphyra* species under laboratory conditions [17,18]. Similarly, in the present study, we found that different degrees of desiccation exhibited a significant inhibitory effect on the growth and nitrogen removal of *N. haitanensis* and *N. dentata.* This inhibitory effect intensified significantly with the increase of the desiccation period. This finding is supported by Cao et al. [58], showing that, due to the artificial reduction of the air exposure period, the thalli of *Neoporphyra yezoensis* (formerly *Pyropia yezoensis*) from cultivated populations were significantly larger than those from wild populations in the surrounding area. Li et al. [18] also documented that periodical dehydration could reduce the relative growth rate of *N. yezoensis* by 7–10% in different salinity conditions. However, the effects of desiccation on the growth and nutrient uptake of seaweeds varied from species to species, which is coordinated with their vertical distribution patterns [16,59,60]. Thomas et al. [61] found that the upper-shore species *Pelvetiopsis limitata* and *Fucus distichus* (Phaeophyceae) achieved their maximum nitrate uptake following severe desiccation that inhibited the nitrate uptake in the low-shore species *Gracilaria pacififica* (Rhodophyta). Kim et al. [16] suggested that species in the intertidal zone that have longer exposure times may have a higher time-use efficiency than subtidal species in terms of their nitrate uptake and growth. On the other hand, the impact of desiccation on seaweeds appears to be associated with their water-retaining abilities caused by structure features. For example, desiccation appeared to have no significant influences on the growth and photosynthesis of *Ulva linza* (Chlorophyta) and *Gloiopeltis furcata* (Rhodophyta) because of their internal hollow cavities being conductive to water retention [60,62]. The two *Neoporphyra* species used in this study are monolayer or bilayer membranous thalli with a weak water-retaining capacity, which may partially contribute to the inhibition of nutrient removal under desiccation stress.

Although the water loss percentages of the two experimental species in this study were as high as approximately 80% after 6 h of desiccation, they were still alive. After a 96-h culture, both species effectively recovered the capacity of nutrient absorption. Particularly, the removal rates of PO4-P were not significantly different among the desiccation periods from 0 to 6 h. These results suggest that *N. haitanensis* and *N. dentata* possess extremely strong resilience to high desiccation stress, and thus can adapt well to changeable oceanic conditions. Similarly, Gao and Wang [63] examined the effect of single dehydration on the physiological features of *N. yezoensis,* and found that the photosynthetic activity of *N. yezoensis* that lost 86% of its cellular water could be fully restored after 30 min of rehydration. Therefore, the periodical emersion of seeded nets has been widely applied in *Pyropia/Neopyropia/Neoporphyra* field aquaculture to kill most fouling organisms, including diatoms and macroalgal spores [64]. Additionally, *N. dentata* appears to be slightly more sensitive to desiccation stress than *N. haitanensis*, even though their vertical distribution

ranges are similar [45]. This may be closely associated with the fact that *N. haitanensis* has a thicker thallus (65–110 μm) with local double-layer cell tissue, whereas *N. dentata* only has a very thin single-layer thallus (30–55 μm) [45,65].

Water motion is a critical physical process in the marine environment that can transport the inorganic carbon and nutrients required for the growth and survival of marine macrophytes [66]. It has been demonstrated that an increased water velocity can reduce the diffusion boundary layer along the algae surface, and can thereby enhance its nutrient uptake and growth until a saturating velocity is reached [67,68]. Nevertheless, in this study, the growth of *N. dentata* exhibited a significant increase from 0.1 to 0.2 m s−1, but was significantly inhibited at 0.5 m s−1. Similarly, *N. haitanensis* had a significant lower RGR at 0.5 m s−<sup>1</sup> compared with lower water velocities. Their removal capacities of NO3-N and PO4-P were also inhibited at a high water velocity of 0.5 m s−1. Analogous growth and physiological inhibition caused by an over-high water velocity has also been reported for the brown algae *Laminaria digitata* and *Sargassum siliquastrum* (Phaeophyceae) [68,69]. Furthermore, Yang [70] found a clear inverse correlation between the cell growth and chlorophyll synthesis of *Chlorella* (Chlorophyta) species and water velocity. All of these results suggested that over-high water velocity can greatly restrict the photosynthetic and nutrient accumulation activities of algae, and can even cause direct damage to algae membranes. Furthermore, *N. haitanensis* appeared to exhibit a greater tolerance and less sensitivity to variable hydrodynamic conditions than *N. dentata*. This may still be correlated with their different structure traits. It is conceivable that thicker and more solid algae can better resist the negative impact of external forces.

In seaweed species, nitrogen can be accumulated for growth demands and stored in its inorganic form in the cellular reserve pool [15,19]. As an ecological adaption to nitrogen deficiency stress, stored nitrogen can be remobilized to support seaweed survival without an ongoing inorganic nitrogen supply [33,71]. Their physiological and metabolic activities, including growth, photosynthesis, and protein and nucleic acid synthesis, will be suppressed by nitrogen deficiency below the critical concentration [72,73]. However, nitrogen replenishment quickly reverted the nitrogen accumulation and metabolism of algae, with the reestablishment of the nitrogen reserve pool and growth promotion [33,50,74]. In this study, the nitrogen removal capacities of both species after nitrogen recovery were positively correlated with the nitrogen limitation period, exhibiting a compensatory response in nitrogen accumulation. Similar phenomena have been documented for floating *Sargassum horneri* (Phaeophyceae) and *Agarophyton tenuistipitatum* (formerly *Gracilaria tenuistipitata*) (Rhodophyta), showing that nitrogen limitation and then recovery significantly increased their ammonium uptake rates in comparison with a control without nitrogen limitation [34,75]. These results suggest that nitrogen limitation can lead to a deficit status of the nitrogen reserve pool. In an effort to maintain life and restore health, the algae in worse conditions must possess a stronger ability to replenish nitrogen when subjected to nitrogen resupply. Curiously, the phosphorus removal in both species did not show the compensatory responses consistent with nitrogen removal. As we known, in seaweed species, there are interactions among different nutrients, which are required in a certain ratio [19]. Perini and Bracken [76] confirmed that nitrogen availability may mediate the ability of marine primary producers to access phosphorus. Therefore, we suspect that particularly rapid nitrogen accumulation may have a negative effect on phosphorus accumulation, which is supported by a finding regarding the phosphorus absorption inhibition by an ambient high nitrogen concentration in *Skeletonema costatum* (Bacillariophyta) [77]. Further studies should be carried out to test our hypothesis.

Under the same nitrogen limitation and recovery conditions, *N. haitanensis* and *N. dentata* showed similar NO3-N removal rates, whereas the RGRs of *N. haitanensis* were significantly lower than those of *N. dentata*. Liu et al. [74] demonstrated that nitrogen deprivation and then recovery notably promoted the growth of *Asparagus schoberioides* (formerly *Gracilaria lemaneiformis*) (Rhodophyta), but significantly inhibited the accumulation of phycoerythrin and chlorophyll a. In contrast, Friedlander et al. [78] reported

that after nitrogen limitation and recovery, although the external nitrogen concentration was sufficient, *Gracilaria conferta* (Rhodophyta) did not grow significantly, probably due to more internal organic synthesis. All of these findings suggest that seaweeds under nitrogen starvation show a species-specific nitrogen utilization strategy when encountering nitrogen resupply. As mentioned above, in comparison with *N. dentata*, *N. haitanensis* with a thicker thallus and more complex structure appears to require more nitrogen resources for the synthesis of the necessary organic compounds rather than growth, resulting in the significantly lower RGR and deeper color observed in our experiment.

#### **5. Conclusions**

Dealing with eutrophic wastewater is a major challenge for aquaculture production and water environment protection. According to the data of this study, *N. haitanensis* and *N. dentata* appear to play an important role in the removal of inorganic nitrogen and phosphorus from aquaculture wastewater, and therefore are likely to be used as efficient and environmentally friendly remediation tools. *N. dentata* is a more ideal candidate because of its greater environmental resistance. Our results show that desiccation, water velocity and nitrogen limitation are three significant variables affecting the growth and nutrient removal of both species. *N. haitanensis* and *N. dentata* showed different nitrogen usage strategies after nitrogen limitation and recovery. These findings provide valuable information for developing and improving wastewater treatment technologies for RAS. Moreover, apart from being a biological filter, these two *Neoporphyra* species can be harvested as aquaculture byproducts to increase economic income owing to their excellent growth capacities. Due to the limited physiological data in this study, further experiments measuring multiple parameters are warranted to comprehensively assess the application feasibility of wastewater purification using *Neoporphyra* species.

**Author Contributions:** Conceptualization, methodology, and writing—original draft preparation, J.L. and X.G.; writing—review and checking, X.G.; data curation, project administration, and funding acquisition, J.L.; investigation, G.C. and Y.L.; validation and resources, Q.G.; formal analysis, J.L. and Q.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the National Key R&D Program of China (No. 2020YFD0900201).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data of this study are available from the corresponding author upon reasonable request.

**Acknowledgments:** We are grateful to Weizhou Chen of Shantou University for providing *N. haitanensis* and *N. dentata* as the experimental materials.

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

#### **References**

