**1. Introduction**

Aquaculture was one of the fastest-growing commercial activities in the last few decades. The production of marine organisms has broken historical records, reaching 114.5 million tonnes in 2018 [1]. In terms of the increasing storage of natural resources, the recirculating aquaculture system (RAS) has become one of the most sustainable models of marine animal aquaculture [2–4]. Due to high density cultivation with limited volumes of seawater, the wastewater from RAS usually contains high concentrations of nutrients, posing a potential risk to the surrounding environment [3,5,6]. Much research has been performed to look for bioremediation technologies that could solve this problem and ensure its environmental sustainability [7–9]. Recently, due to their low cost and high uptake efficiency, seaweeds have become a feasible alternative in the bioremediation of eutrophic wastewater [10–12]. It is very critical to select appropriate seaweed species with great nutrient demands and high economic value for RAS.

Intertidal seaweeds are subjected to cyclical immersion and emersion because of their periodic exposure to tidal fluctuations. During low tide, the intertidal seaweeds are exposed to air and experience various environmental stresses, such as drastic temperature shifts, high osmotic pressure, and desiccation [13–15]. Desiccation with different periods and frequencies is unavoidable for seaweeds growing in different vertical zones, and their

**Citation:** Li, J.; Cui, G.; Liu, Y.; Wang, Q.; Gong, Q.; Gao, X. Effects of Desiccation, Water Velocity, and Nitrogen Limitation on the Growth and Nutrient Removal of *Neoporphyra haitanensis* and *Neoporphyra dentata* (Bangiales, Rhodophyta). *Water* **2021**, *13*, 2745. https://doi.org/10.3390/ w13192745

Academic Editor: Jesus Gonzalez-Lopez

Received: 10 September 2021 Accepted: 27 September 2021 Published: 2 October 2021

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**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/).

physiological and metabolic activities, including growth and nutrient assimilation, are significantly affected [16–18]. Besides desiccation, water velocity is another important abiotic factor affecting the metabolism of seaweeds [15,19]. Adverse hydrodynamic conditions in the sea can produce different degrees of influences on the productivity of natural and cultivated seaweeds, which depends on their different environmental tolerances and nutrient utilization performances [20–23]. Similarly, under laboratory conditions, the growth and nutrient uptake of diverse seaweed species have been identified to be significantly inhibited by immoderate water velocity [24,25].

Moreover, the nutrient uptake and growth of seaweeds are greatly affected by diverse biotic factors, including the species, competition, growth phase, and nitrogen level in the algae [15,26,27]. Nitrogen and phosphorus are two essential nutrient components to be incorporated into physiological compounds that are crucial for seaweed growth and development [28]. In recent years, ambient nitrogen deficiency has been reported to cause aggravated damage to the seaweed mariculture systems due to the reduced environmental tolerance of algae caused by an undesirable internal nutrient status [29–31]. Nevertheless, as a physiological strategy in response to nutrient deprivation, it was found that the nitrogen uptake ability of seaweeds was enhanced under the condition of nitrogen limitation [32–34], and was positively associated with the degree of nitrogen limitation [35]. Therefore, the assessment of the potential of nitrogen limitation in aquaculture water purification with seaweeds is considered to have significant ecological and commercial values.

Macroalgae of the genus *Neoporphyra* are considered edible, delicious and nutritious, and were recently separated from the genus *Porphyra* [36]. *Neoporphyra* species are important commercially available marine crops in China, and have been massively cultivated because of great demand [17,37,38]. Due to their extremely high surface area to volume ratio, they are capable of the rapid assimilation of nutrients, which promotes high rates of growth in these algae [39–41]. As a result, they have been proven to contribute to the purification of seawater in aquaculture ponds and nearshore farming areas in China, with total nitrogen and PO4-P removal rates of 65.8–80.2% and 71.1–84.6% [42,43]. These facts suggest that this genus is one of the most promising candidates for bioremediation and integrated aquaculture [40,41,44].

*Neoporphyra haitanensis* and *Neoporphyra dentata* are two common *Pyropia* species inhabiting the intertidal zone of rocky shores along the coast of southern China [45]. Their optimal temperature ranges for growth are 19–23 ◦C and 17–23 ◦C, respectively [46,47], which are generally consistent with the cultivation temperature of economic marine animals such as turbot and grouper [48,49]. These provide advantageous conditions for aquaculture wastewater purification using these two species. As one of the major commercial species, *N. haitanensis* has received extensive attention for its physiological and metabolic responses to biotic and abiotic stressors for aquaculture production optimization [50–54]. *N. dentata* is a promising species for cultivation in South China, and has been farmed on an experimental scale over the past few years [46,47,55]. Nevertheless, the nutrient removal capacities and potential application in aquaculture wastewater purification have been rarely investigated in these species. Furthermore, it is very vital to understand the correlation between the nutrient removal capacities and diverse biotic and abiotic factors.

In the present study, three short-term laboratory experiments were conducted to investigate the respective effects of desiccation, water velocity and nitrogen limitation on the growth and NO3-N and PO4-P removal of *N. haitanensis* and *N. dentata*. The results of this study are expected to provide valuable information to improve aquaculture wastewater management and to assess the bioremediation potential of these two high-valued cultivars in China.

#### **2. Materials and Methods**

#### *2.1. Sample Collection and Maintenance*

Gametophytic thalli of *N. haitanensis* and *N. dentata* were collected from cultivated populations on Nanao Island, Guangdong, China (23◦28 N, 117◦06 E) in December 2018. These samples were rinsed several times with filtered seawater to remove epiphytic organisms and detritus. The surface seawater temperature at the sampling site was measured at the same time. The samples were promptly transported to the laboratory under lowtemperature conditions. Healthy thalli were then selected and cultured in several plastic tanks containing sterilized seawater. For the subsequent experiments, these thalli were maintained at 23 ◦C (the surface seawater temperature at the sampling site), with an irradiance of 100 μmol photon m–2 s–1 and a 12:12-h light/dark cycle for 2 days.

#### *2.2. Desiccation Experiment*

A culture experiment was conducted over a period of 4 days after five periods of desiccation: 0, 1, 2, 4, and 6 h of air exposure. The water loss percentages of *N. haitanensis* and *N. dentata* were 34.4% and 41.4% after 1 h of desiccation, 55.2% and 59.2% after 2 h of desiccation, 70.5% and 71.9% after 4 h of desiccation, and 77.6% and 81.2% after 6 h of desiccation, respectively. There were a total of 10 experimental treatments for each species, and each treatment was performed in three replicates. Before the culturing, 5 g thalli were randomly selected for each replicate. After being blotted dry, those thalli at 1–6 h of desiccation treatments were transferred into autoclaved Petri dishes (10 cm in diameter) containing a layer of gauze soaked with a small amount of culture medium (NO3-N: 50 mg L<sup>−</sup>1; PO4-P: 5 mg L−1), which was made using a nutrient solution and sterilized seawater from the coast of Taipingjiao, Qingdao, with a salinity of approximately 31 psu. Next, these Petri dishes were placed into incubators at 23 ◦C for 1, 2, 4, and 6 h, respectively. After desiccation, the thalli of each replicate were moved into a side-arm flask with 500 mL culture medium and GeO2, which were then gently aerated. During this experiment, a temperature of 23 ◦C, a 12:12-h light/dark cycle, and an irradiance of 100 μmol photon m–2 s–1 were maintained.

The fresh weights of all of the thalli before and after the experiment were measured after removing excess seawater on the surface. The relative growth rate (RGR; % day–1) of each replicate was calculated using the following Equation (1):

$$\text{RGR } (\% \text{ day}^{-1}) = 100 \times (\ln \text{W}\_t - \ln \text{W}\_o) / \text{t} \tag{1}$$

where Wo is the initial fresh weight, Wt is the final fresh weight, and t is the time of the culture in days.

For all of the treatments, the culture media before and after the experiment were separately collected, and the concentrations of NO3-N and PO4-P were analyzed using the cadmium column reduction method and the phosphomolybdenum blue spectrophotometric method, respectively [56,57]. The removal rates of NO3-N and PO4-P were estimated using the following Equation (2):

$$R\_{\rm N\_2P} = (C\_0 - C\_4) / C\_0 \times 100\% \tag{2}$$

where RN, P are the removal rates of NO3-N and PO4-P (%); C0 is the initial concentration of NO3-N and PO4-P (mg L−1); and C4 is the final concentration of NO3-N and PO4-P (mg L<sup>−</sup>1) after 4 days.

#### *2.3. Water Velocity Experiment*

In order to examine the effect of the water velocity on the growth and nutrient removal of these two species, they were cultured for 4 days at three water velocities (0.1, 0.2, and 0.5 m s−1) with three replicates. For this experiment, a total of 18 side-arm flasks were prepared, and each contained 500 mL culture medium with GeO2 and 5 g thalli. During the experimental period, a temperature of 23 ◦C, a 12:12-h light/dark cycle, and an irradiance of 100 μmol photon m–2 s–1 were maintained. At the end of this experiment, the calculations of the RGR and the removal rates of NO3-N and PO4-P were the same as for the desiccation experiment.
