Recently, Nature published a review characterizing the rapid development in global aquaculture in the past two decades and highlighting the integration of aquaculture in the global food system (Naylor et al., 2021). It was expected that aquaculture would contribute 52% of the world total fishery production in 2025 (Ramli et al., 2020). Inevitably, the fast expansion of largescale intensive culture activities has brought about adverse effects, especially pollutions on the aquaculture ecosystem itself and the ambient environment (Liu and Su, 2017). According to statistics, aquaculture pollution represented above 20% of the total nutrient input into freshwater in some provinces of China (Naylor et al., 2021). It was reported by Ramli et al. (2020) that over 50% of feed nitrogen turned into waste in the aquaculture system, instead of being assimilated by cultured organisms (Ramli et al., 2020). And, the extensive use of antibiotics and chemicals further aggravated the excessive accumulation of nitrogenous waste in aquaculture ecosystems, which could not be ignored (Luo et al., 2018). It has been generally accepted that nitrogen is one of the main pollution sources (Yuan et al., 2021).

Improving the ecological status of aquaculture system has become a growing concern worldwide, in particular with reducing nitrogen in wastewater effluent to diminish the occurrence of eutrophication (Mohsenpour et al., 2021). To achieve the nitrogen concentration in wastewater effluent that is in compliance with the environmental quality standards for surface water of China (GB 3838-2002), various measures have been exerted in the intensive aquaculture practices. Among them, bioremediation is considered as an innovative and optimistic technology owing to its cost-effectiveness and environmental compatibility, with a diverse range of microorganisms, including fungi, bacteria, and microalgae (Bilal and Iqbal, 2020).

Unicellular microalgae, the microscopic eukaryotic organisms with short life cycles, are capable of generating biomasses via photosynthesis (Dai et al., 2022). For one thing, as a robust source of pigments, proteins, essential fatty acids, etc., microalgae have been regarded as a promising feed additive in aquaculture to boost the growth, immunity, and meat coloration/quality of aquatic animals (Ye et al., 2022). In many independent studies, certain microalgae belonging to genera Nitzschia, Chaetoceros, Nannochloropsis, Pavlova, Chlorella, Isochrysis, Cyclotella, Thalassiosira, Porphyridium, and Phaeodactylum are famous for great potentials for use in aquaculture (Pahl et al., 2010; Haas et al., 2016; Ding et al., 2021; Ye et al., 2022). For another thing, microalgae have received much interest in implementing them in wastewater treatment, profiting from their ability to utilize nutrition sources in wastewater (Saeed et al., 2022). Extensive studies have evaluated many microalgae species in treating a range wastewater types, including municipal, brewery, agricultural, and industrial effluents (Choi, 2016; Prandini et al., 2016). Thereof, Scendesmus sp., Chlorella sp., Spirulina sp., and Chaetoceros muelleriare were widely used in the treatment of aquaculture wastewater, with varying nitrogen absorption efficiencies (Godoy et al., 2012; Ansari et al., 2017; Ahmad et al., 2020). In brief, due to the potential for nitrogen uptake and more importantly the nutritional value of algal biomass generated, microalgae have been referred as one of the ideal bio-absorbents used in wastewater purification (Mohsenpour et al., 2021). However, most of these attempts were performed independent of one another under different conditions and did not focus on the nutritional properties of microalgae. Thus, to effectively couple nitrogen removal with nutritional value, selecting the proper microalgae species is the key in this field.

Nannochloropsis sp., Cyclotella sp., and Conticribra weissflogii (previously known as Thalassiosira weissflogii) are well-known as feed ingredients or live diets for aquaculture. It was reported that a 50 % fish oil replacement by Nannochloropsis sp. meal had positive effects on the growth performance and nutrient utilization of juvenile sea bass (Haas et al., 2016). Moreover, the disease resistance of gilthead seabream (Sparus aurata) was enhanced with Nannochloropsis sp. as functional ingredient in low fishmeal diets (Jorge et al., 2019). Several studies showed that C. weissflogii contributed to the improvement of the growth performance of shrimps (Litopenaeus vannamei), emphasizing the survival rate of juveniles (Godoy et al., 2012). Similar study was also conducted with Cyclotella sp. to investigate its nutritional value as an alternative feed for aquaculture (Pahl et al., 2010). Compared with their feed potential, there are very few reports on the assessment of nitrogen removal capacity for aquaculture wastewater treatment with these three algae, especially Cyclotella sp. and C. weissflogii. Only a few attempts have been made to improve the water quality of aquaculture system by aid of Nannochloropsis sp. (Ding et al., 2021), while ignoring analyses on their nutritional properties.

Over the years, our team has been committed to separation of various diet microalgae and exploration of their nutritional properties (Yang et al., 2015; Liao et al., 2017). In our previous work, Nannochloropsis oceanica and two diatom strains (Cyclotella atomus and C. weissflogii) were screened and characterized from the rearing water of the shellfish hatchery (27°19′27″N, 120°13′0.16″E) in Fuding city, Fujian province, China. Here, the comprehensive analyses on growth performances, absorption efficiencies of nitrate-N ( NO 3 − ), and nutritional properties were conducted in N. oceanica, C. atomus, and C. weissflogii.

The demand for innovative wastewater treatment techniques has been growing in many nations, in particular with nitrogen removal. Microalgal cultivation using aquaculture wastewater is a promising technology for integrated nitrogen removal and subsequent biomass accumulation. Apart from improving water quality, microalgae have demonstrated to be an excellent living diet, which is the principal advantage of incorporating them into wastewater treatment (Mohsenpour et al., 2021). However, several practical challenges still hinder the industrial application of microalgae in this field, which need to be addressed (Mohsenpour et al., 2021). One such challenge is the availability of superior microalgal strains with optimal nitrogen removal capacity and nutritional properties. Many species of microalgae, e.g., N. oceanica, C. atomus, and C. weissflogii, have been applied in aquaculture as live diets. However, estimation of nitrogen removal capability and concomitant nutritional properties of the three species have been rarely reported. It is necessary to comprehensively analyze their growth performances, nitrogen removal capacities, and accumulations of sugar, protein, lipid, and fatty acid, which have been carried out in this study.

As referred above, nitrogen is one of the main pollution sources of aquaculture wastewater. The inorganic nitrogen forms in aquaculture wastewater comprise of nitrate, nitrite, nitric acid, ammonium, etc. (Wuang et al., 2016). Nitrate-N is the most oxidized and the most thermodynamically stable form. It is hence more common to find nitrate-N in aquaculture environments (Goncalves et al., 2017). Nitrate-N is also the main concern of this study and chosen as the nitrogen source with two concentrations, both of which exceeded the limit of the environmental quality standards for surface water of China (GB 3838-2002). For the selected three strains, there were significant differences observed in the maximum cell density, specific growth rate, biomass concentration, Fv/Fm, total chlorophyll and Car content between two groups. These results indicated nitrate-N concentration had a substantial effect on microalgal growth and photosynthetic performance. This has been a broad consensus proved in many microalgae (Ma et al., 2016). For instance, it was observed that Chlorella zofingiensis showed rapid growth with sufficient nitrogen, whereas growth inhibition under nitrogen limitation condition (Zhu et al., 2014). Additionally, the nitrogen removal rates were in line with the growth curves of the three microalgal strains in this study ( Figures 1 , 2 ), clearly reflecting the importance of nitrogen for microalgal biomass generation and confirming the feasibility of removing nitrogen from wastewater with them.

Fv/Fm is a sensitive indicator of plant photosynthetic performance (Jägerbrand and Kudo, 2016). Generally, lower values of Fv/Fm may at least partly reflect stress, photoinhibition, or downregulation of photosynthesis (Jägerbrand and Kudo, 2016). Fv/Fm has been used as an indicator of environmental stress in algae, including nutrient depletion. For example, studies in Scenedesmus obliquus, Chlorella sorokiniana, and Ankistrodesmus falcatus demonstrated that Fv/Fm values obviously decreased with nutrient depletion (Ansari et al., 2017). Another study also indicated that the negative effect of ultraviolet radiation on Isochrysis galbana was partly supported by the decline of Fv/Fm (Cao et al., 2019). In the present study, photosynthetic efficiencies of the three strains were monitored in terms of Fv/Fm. The decline of Fv/Fm values was indicative of nitrogen consumption in both groups, which was further confirmed by analyses of nitrate-N utilization ( Figure 2 ). It is worth mentioning that Fv/Fm of N. oceanica still maintained at a high level (0.78 and 0.73), even though there was almost no residual nitrate-N in cultural water at the end of culture process ( Figure 2 ). However, Fv/Fm values of C. atomus and C. weissflogii decrease by a much wider margin, compared to that of the first day. It can be speculated that N. oceanica has better adaptability towards nitrogen depletion compared to other two selected strains.

In the current study, N. oceanica, C. atomus, and C. weissflogii removed 90% of nitrate-N within five days and 99% of that within seven days, showing splendid nitrogen removal potentials. Nevertheless, the nitrogen absorption efficiencies were quite different from one another. Analyzing the average amount of nitrate-N absorbed by each cell daily, C. weissflogii gained the largest value, followed successively by C. atomus and N. oceanica. This was likely due to species specificity. Similarly in a comparative study by Ansari et al. (2017), A. falcatus showed stronger nitrate-N absorption capacity than S. obliquus and C. sorokiniana. An additional aspect was that the absorption efficiency in NC group was significantly higher than that in NW group at the same timepoint, applicable for all three microalgae. We speculated that it was mainly caused by the discrepancy in nitrogen concentration. In two independent studies of Wang et al. (2010) and Cabanelas et al. (2013), Chlorella sp. both showed an improved nitrogen absorption efficiency when treating wastewater with higher nitrogen concentration, similar to the results observed in this study.

Indeed, apart from microalgal species and nitrogen concentration, there are many other factors influencing nitrogen absorption efficiency. One of them is the preference of nitrogen species. The assimilation of nitrate-N requires previous reduction into ammonium, in a two-step process catalyzed by nitrate reductase and nitrite reductase, while ammonium assimilation does not (Goncalves et al., 2017). Therefore, most algae prefer ammonium as the nitrogen source rather than nitrate-N, as less energy is needed (Ramli et al., 2020). Nannochloropsis oculata, Stigeoclonium nanum, and Chlorella vulgaris fall into this category (Ansari et al., 2017; Ramli et al., 2017). There are, of course, some exceptions. For example, diatoms prefer nitrate-N over ammonium (Ramli et al., 2020). Other factors, such as light intensity and temperature, are also deemed closely correlated with nitrogen uptake by microalgae (Goncalves et al., 2017). Considering that nitrate-N is the main nitrogen form in aquaculture wastewater, this study was conducted with nitrate-N as the only nitrogen source under the same light intensity and temperature. Further investigation is needed to ascertain the impacts of other factors.

Nitrogen, one of fundamental macronutrients, is required for the accumulation of nutritional components (Zarrinmehr et al., 2020). It was reported that many microalgal species could transform protein to lipid or sugar as energy reserve component when encountering nitrogen-depletion (Huo et al., 2011). For example, studies in N. oceanica, I. galbana, and Scenedesmus sp. have demonstrated that nitrogen depletion triggered more lipids (Pancha et al., 2014; Jia et al., 2015; Zarrinmehr et al., 2020). Nitrogen starvation was thus proposed to be an effective technique for lipid production in microalgae (Yaakob et al., 2021). On the other hand, limitation of nitrogen availability also seemed to be the most promising way of accumulating sugars in microalgae (Chen et al., 2013). Zarrinmehr et al. (2020) found that the sugar content in I. galbana was enhanced by 47% under nitrogen-deficient condition. Cultivating Spirulina sp. in nitrogen-reduction medium caused the dramatical elevation in sugars (Braga et al., 2018). Additionally, there are many reports showing that nitrogen bears directly on the fatty acid profile. The common view is that low nitrogen leads to more saturated fatty acid and monounsaturated fatty acid, while high nitrogen is more conducive to the synthesis of polyunsaturated fatty acid (Zhang et al., 2021). Our results further confirmed this view. Another notable aspect was C20:5, one famous polyunsaturated fatty acid (Zhang et al., 2021). The content of C20:5 changed drastically with nitrogen concentration, especially in N. oceanica and C. weissflogii.

Over the past decades, abundant studies have clearly shown that the nutritional requirements of aquatic animals depend on different species (fish, shrimp, shellfish, etc.) and different ages (e.g., larva or juvenile) (Ayisi et al., 2017; <xr rid="r106">Li et al., 2021</xr>). The appropriate supply of nutrition is beneficial for the growth, development, and production performance of aquatic animals. According to literatures, the concentration of nitrogen in aquaculture wastewater ranges from 2-110 mg·L⁻¹ (Ansari et al., 2017). Since the nutritional properties of microalgae varies with nitrogen concentration, the selection of suitable microalgae species should be taken into consideration in practical application for assimilating nitrogen and simultaneously producing biomass as diet for various aquatic animals. Nitrogen concentration is one of key factors in the coupling system of wastewater treatment and biomass production of microalgae. The present study was designed to estimate the growth performances, absorption efficiencies of nitrate-N, and biochemical compositions of three diet microalgae with synthetic medium. However, Microalgal treatment of real wastewater exhibited many complexities, mainly resulting from the increased physiological stresses caused by variable nutrient levels as well as high concentrations of organics in real wastewater. In the follow-up studies, the effects of other nutrient (e.g., phosphorus) on the potentials of diet microalgae for wastewater treatment will be investigated. Furthermore, cultivation with real wastewater without any additional nutrients will be carried out.

In this study, N. oceanica, C. atomus, and C. weissflogii were cultivated with two initial nitrate-N concentration, designated as NC (13.85 mg·L^–1) and NW (5 mg·L^–1) groups, respectively. Two nitrate-N concentrations both exceeded the limit of the environmental quality standards for surface water of China (GB 3838-2002). For the selected three strains, significant differences were observed in the maximum cell density, specific growth rate, biomass concentration, Fv/Fm, total chlorophyll and Car content between two groups. Judging from Fv/Fm, N. oceanica has better adaptability towards nitrogen depletion compared to other two selected strains. At the end of cultivation, the biomass concentration of N. oceanica, C. atomus, and C. weissflogii in NC group was 1.59, 1.29, and 1.25 times that in NW group, respectively. In addition, the three strains all showed excellent nitrogen removal potentials. The nitrate-N absorption efficiencies in NC group were all significantly higher than that in NW group at the same timepoint. From the average amount of nitrate-N absorbed by each cell daily, C. weissflogii gained the largest value, followed successively by C. atomus and N. oceanica. Further analyses on nutritional properties demonstrated lower nitrate-N was conductive to the synthesis of soluble sugar, total lipid, and saturated fatty acid, while higher nitrate-N resulted in more soluble protein and polyunsaturated fatty acid. To sum up, N. oceanica, C. atomus, and C. weissflogii all possessed strong nitrogen removal capacity. And their growth performance and nutritional properties were closely related with nitrogen concentration.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

LZ and JX designed the experiment. LZ analyzed the data and drafted the manuscript. JH and JX revised the paper. SM and YZ measured the microalgal growth and nitrogen removal capacity. YW contributed to the analyses of nutritional properties. All authors contributed to the article and approved the submitted version.