Effects of Caprolactam Wastewater on Algal Growth and Nutrients Removal by Arthrospira platensis

Caprolactam wastewater (WCP), which is generated during the production of caprolactam, contains high contents of NO3− and inorganic P and is considered to be difficult to treat. In this study, Arthrospira platensis was used to remove N and P from WCP. Culture conditions and wastewater addition were optimized to relieve the inhibition effects of WCP. The results show that A. platensis growth and photosynthetic activity were inhibited depending on WCP concentrations. The inhibition rates were enhanced as the culture time increased under batch mode. However, the fed-batch mode significantly minimized the negative impact on A. platensis, which is beneficial for removing N and P from WCP by Arthrospira. After 10 d of cultivation of A. platensis in a 25 L circular photobioreactor in fed-batch addition of WCP (1.25% mixed WCP (v/v) each day), the average biomass productivity reached 17.48 g/(m2·d), the maximum protein content was 69.93%, and the N and P removal ratios were 100%. The accumulation effect of WCP inhibition on algal growth was not observed under this culture condition. Fed-batch cultivation of A. platensis is a promising way for bioremediation of WCP with high N and P removal efficiencies and high value-added biomass production.


Introduction
Microalgae are photosynthetic organisms that convert C, N, P, and light energy into biomass and can be applied in biofuels, food, feed, and biofertilizer industries [1]. The main factors limiting the commercial mass culture of microalgae are the high production costs and water resource consumption [2].
To alleviate this issue, wastewater has been considered as a medium for microalgae cultivation [3]. Coupling wastewater treatment with microalgae cultivation is an advanced strategy for wastewater recycling that can reduce microalgae cultivation costs and remove N and P pollutants. Many studies have confirmed that microalgae can grow in different wastewaters, such as municipal wastewater [4], olive mill wastewater [5], soy sauce wastewater [6], and agro-industrial wastewater [7,8].
Caprolactam wastewater (WCP) is generated during the production of caprolactam (CPL). It is estimated that approximately 4.2 t to 4.7 t of wastewater will be generated for each ton of CPL production [9]. WCP contains NO 3 − , PO 4 3− , and organic matter [9][10][11]. WCP discharge may lead to a risk of environmental pollution.

Effects of Different Wastewater Concentrations on the Growth of A. platensis
To understand the effects of WCP on algal growth, A. platensis was cultured in diluted mixed WCP for 6 d. The mixed WCP concentrations were 12.5%, 25.0%, and 50.0%, and the culture medium without wastewater was used as a control. The culture medium containing 50% mixed wastewater contained 3.302 g/L of NaNO 3 and 0.133 g/L of KH 2 PO 4 . To avoid the influence of nutrient concentrations on algal growth, 3.30 g/L of NaNO 3 and 0.13 g/L of KH 2 PO 4 were added to the control medium, and the corresponding amounts of NaNO 3 and KH 2 PO 4 were added to the culture medium with 12.5% and 25.0% mixed wastewater to ensure that all the culture media had consistent initial concentrations of N and P. Other nutrients were then added by adding each nutrient salt or its concentrated solution, and the formulations of the four culture media are shown in Supplementary  Table S1.
An aerated column photobioreactor was used in the experiment. The bioreactor was composed of glass columns (working volume of 250 mL and inner diameter of 3 cm) and accessory equipment that maintained the culture conditions of a temperature of 30 • C, the light intensity of 320 µmol photons/(m 2 ·s), light/dark cycle of 14 h/10 h, and airflow of 250 mL/min (enriched with 2% CO 2 ). The initial optical density of the culture at 560 nm (OD 560 ) was 0.3 ± 0.05, and the corresponding biomass dry weight (DW) was approximately 0.15 g/L. The DW of the biomass was measured daily. Three parallel cultures were conducted for each treatment and control, and the experiment was repeated at least twice.

Effects of Four Main Organic Matters in WCP on the Growth of A. platensis
CHN and CHL are volatile; therefore, aerated culture is not suitable for testing the effects of these two organic matters on the growth of A. platensis. Flask cultures were used for this experiment. First, 100 mL of algal suspension with an initial optical density of 0.3 ± 0.05 (corresponding to an initial biomass DW of approximately 0.15 g/L) was incubated in 250 mL conical flasks. The basal growth medium was modified Zarrouk's medium with 6 g/L of NaHCO 3 (Supplementary Table S1). The culture was grown for 4 d at 28 • C with a light intensity of 80 µmol photons/(m 2 ·s) and a photoperiod of 14 h/10 h (light/dark). Three parallel cultures were grown for each treatment and control group.
The mouth of the flasks was sealed with sealing film, and no extra air was aerated during cultivation. The flasks were shaken by hand several times daily.

Effects of Reduced WCP on the Growth of A. platensis
To investigate whether the peroxide in WCP was the main factor that negatively affected the growth of A. platensis, reduced WCP was prepared. Na 2 SO 3 was added to the mixed WCP to reduce peroxide. The iodometric method [30] verified that the peroxide in the mixed WCP was completely reduced by 2.0 g/L of Na 2 SO 3 .
The completely reduced WCP was diluted to different concentrations (12.5%, 25.0%, and 50.0%). Nutrients were then added to the medium according to the method described in Section 2.3, and the medium without WCP was used as the control. All the culture media of the treatment and control had consistent concentrations of each nutrient, including N and P sources.
The treatment and control were cultivated as described in Section 2.3, and the biomass DW was measured at the end of the culture. Three parallel cultures were conducted for each treatment and control group.

Effects of Heat-Treated WCP on the Growth of A. platensis
The heat-treated WCP was prepared by boiling mixed WCP at 100 • C for 30 min. During the boiling process, the air was aerated into the WCP, and deionized water was added to compensate for the evaporated water. The boiled WCP was then cooled to room temperature and deionized water was added to return the initial volume of the WCP.
The heat-treated WCP was diluted to different concentrations (12.5%, 25.0%, and 50.0%), and then nutrients were added to the medium according to the method described in Section 2.3. The medium without WCP was used as the control. All the culture media of the treatment and control had consistent concentrations of each nutrient, including N and P sources.
The treatment and control were cultivated as described in Section 2.3, and the biomass DW was measured at the end of the culture period. Three parallel cultures were grown for each treatment and control group.

Effects of WCP Supply Modes on the Growth, Photosynthetic Pigments, and Biochemical Components of A. platensis
To minimize the negative impact of WCP on the growth of A. platensis, different WCP supply modes were tested. For fed-batch mode 1, 3.13% (v/v) mixed WCP was added daily 16 times, and the accumulated WCP concentration reached 50.0%. For fed-batch mode 2, 12.5% (v/v) mixed WCP was added every 4 days for 4 times, and the accumulated WCP concentration reached 50.0%. For comparison, WCP was added at the beginning to reach a concentration of 50% in batch mode, and no WCP was added for the control. The culture conditions were maintained as described in Section 2.3, but the incubation period was 16 d. During the 16 d of cultivation, the biomass of each treatment and control was harvested four times on days 4, 8, 12, and 16. The biomass DW was measured before and after harvest to calculate the net increase in biomass. After harvest, the biomass DW was maintained at a minimum of 0.3 g/L. The harvested biomass was freeze-dried, and the contents of chlorophyll a (Chl-a), carotenoid (Car), and PC, as well as biochemical components (proteins, carbohydrates, and total lipids) were determined.
Addition of 50% mixed WCP at the beginning made a NaNO 3 concentration of 3.30 g/L and a KH 2 PO 4 0.13 g/L for batch mode. To ensure consistent initial concentrations of N and P sources in the culture media of the treatment and control, 3.30 g/L of NaNO 3 and 0.13 g/L of KH 2 PO 4 were added to the culture medium of the control, 3.10 g/L of NaNO 3 and 0.12 g/L of KH 2 PO 4 were added to the culture medium of fed-batch mode 1, and 2.48 g/L of NaNO 3 and 0.10 g/L of KH 2 PO 4 were added to the culture medium of fedbatch mode 2. To maintain consistent total concentrations of N and P sources in the culture media of the treatment and control, after the first, second, and third harvest, 0.83 g/L of NaNO 3 and 0.03 g/L of KH 2 PO 4 were added to each culture medium. The formulations of the culture media and the concentrations of each nutrient after harvest are shown in Supplementary Tables S2 and S3. Three parallel cultures were grown for each treatment and control group.

Removal of N and P in WCP by A. platensis Cultivated in an Open Circular Photobioreactor
A self-designed open circular photobioreactor was used in the experiment. The structure of the open circular photobioreactor is shown in Figure 1. The depth of the algal suspension in the photobioreactor was 15 cm, and the culture volume was 25 L. Illumination of 1300 µmol photons/(m 2 ·s) was provided by LED light with a light/dark cycle of 14 h/10 h. The culture was maintained at 30 • C in a water bath. The algal suspension was mixed with two vertical blades, which provided a culture flow velocity of 45 cm/s. After inoculation, the initial OD 560 was 0.3 ± 0.05, and the corresponding biomass DW was approximately 0.15 g/L. The culture was grown for 10 d. Pure CO 2 was injected into the culture medium through a gas diffuser under the programing control of an online pH sensor to maintain the culture pH at 9.5 ± 0.1. Mixed WCP was added to the medium every day at a concentration of 1.25%, and the accumulated WCP concentration reached 12.50%. To enhance the light penetrability of the culture, when the OD 560 increased to approximately 1.6, some of the algal biomass was harvested to reduce the OD 560 to approximately 1.0. Biomass measurements were conducted before and after each harvest, and the protein content of the A. platensis biomass was measured before harvest. The experiments were performed in duplicate. 0.13 g/L of KH2PO4 were added to the culture medium of the control, 3.10 g/L of NaNO3 and 0.12 g/L of KH2PO4 were added to the culture medium of fed-batch mode 1, and 2.48 g/L of NaNO3 and 0.10 g/L of KH2PO4 were added to the culture medium of fed-batch mode 2. To maintain consistent total concentrations of N and P sources in the culture media of the treatment and control, after the first, second, and third harvest, 0.83 g/L of NaNO3 and 0.03 g/L of KH2PO4 were added to each culture medium. The formulations of the culture media and the concentrations of each nutrient after harvest are shown in Supplementary Tables S2 and S3. Three parallel cultures were grown for each treatment and control group.

Removal of N and P in WCP by A. platensis Cultivated in an Open Circular Photobioreactor
A self-designed open circular photobioreactor was used in the experiment. The structure of the open circular photobioreactor is shown in Figure 1. The depth of the algal suspension in the photobioreactor was 15 cm, and the culture volume was 25 L. Illumination of 1300 μmol photons/(m 2 ·s) was provided by LED light with a light/dark cycle of 14 h/10 h. The culture was maintained at 30 °C in a water bath. The algal suspension was mixed with two vertical blades, which provided a culture flow velocity of 45 cm/s. After inoculation, the initial OD560 was 0.3 ± 0.05, and the corresponding biomass DW was approximately 0.15 g/L. The culture was grown for 10 d. Pure CO2 was injected into the culture medium through a gas diffuser under the programing control of an online pH sensor to maintain the culture pH at 9.5 ± 0.1. Mixed WCP was added to the medium every day at a concentration of 1.25%, and the accumulated WCP concentration reached 12.50%. To enhance the light penetrability of the culture, when the OD560 increased to approximately 1.6, some of the algal biomass was harvested to reduce the OD560 to approximately 1.0. Biomass measurements were conducted before and after each harvest, and the protein content of the A. platensis biomass was measured before harvest. The experiments were performed in duplicate.

Analytical Methods
The N (NO3 − -N) and P (PO4 3− -P) concentrations in the medium were analyzed using ion chromatography [31]. Organic matter (CPL, CHN, CHL, and CHO) in the WCP was determined by gas chromatography, as described by [11].
Cell growth was estimated using the biomass DW. The DW was measured by filtering 5 mL of algal suspension on 0.45 μm pre-weighed membranes and dried at 105 °C. The inhibition ratio (I) was calculated as follows: Chl-a and Car were determined using the method described by [32]. The Chl-a and Car contents were calculated according to the following equations:

Analytical Methods
The N (NO 3 − -N) and P (PO 4 3− -P) concentrations in the medium were analyzed using ion chromatography [31]. Organic matter (CPL, CHN, CHL, and CHO) in the WCP was determined by gas chromatography, as described by [11].
Cell growth was estimated using the biomass DW. The DW was measured by filtering 5 mL of algal suspension on 0.45 µm pre-weighed membranes and dried at 105 • C. The inhibition ratio (I) was calculated as follows: Chl-a and Car were determined using the method described by [32]. The Chl-a and Car contents were calculated according to the following equations: Chl-a (mg/L) = 16.72 × OD 665.2 − 9.16 × OD 652. 4 (2) Car (mg/L) = (1000 × OD 470 − 1.63 × Chl-a)/221 The photosynthetic status of A. platensis was measured using a PAM 2500 fluorometer [33]. Phycobiliproteins were extracted with phosphate buffer, and absorbance was measured at 620 nm and 652 nm, respectively. The concentrations of PC and allophycocyanin (APC) were calculated according to the equation reported by [34].
APC (mg/mL) = A 652 − 0.208 × A 620 5.09 (5) Total phycobiliproteins (T-PC) = PC + APC The total nitrogen (TN) contents in the algal biomass were determined using a Soli TOC cube analyzer (Elementar, Germany). The total carbohydrate and lipid contents were determined using the method described by [35]. To measure the crude protein content, an empirical formula described by [36] was adopted, as follows: Crude protein (%) = 6.25 × TN(%) (6) where TN (%) is the mass fraction of N in the biomass.

Statistical Analyses
For all the aerated column cultures and flask cultures in this study, three parallel cultures were conducted for each treatment and control (n = 3), and the results were expressed as mean ± standard deviation. The groups were compared statistically using a one-way analysis of variance followed by Tukey's test at a confidence level of 95%, and differences were considered statistically significant at p < 0.05.

Effects of WCP on the Growth and Photosynthetic Performance of A. platensis
In this experiment, all the culture media of the treatment and control had consistent initial concentrations of N (3.30 g/L of NaNO 3 ) and P (0.13 g/L of KH 2 PO 4 ) to avoid the influence of the nutrient concentration on algal growth. To our knowledge, this is the first study evaluating the effects of real WCP on A. platensis. Figure 2A shows that Arthrospira grew linearly over time in the control and all three treatments, but a clear decreasing trend in the growth rate was shown as the WCP concentration increased. After 6 d of cultivation, the final biomass concentrations varied between different WCP concentrations ( Figure 2B), with the highest DW of 2.57 g/L observed in the culture without WCP addition and the lowest DW of 1.86 g/L observed in the culture with 50% WCP addition. Compared with the control, the growth of A. platensis was inhibited by 7.13%, 14.80%, and 29.57% at WCP concentrations of 12.5%, 25.0%, and 50.0% (v/v), respectively ( Figure 2C). These results suggest a dose-dependent toxic effect of WCP on the growth of A. platensis. Many studies have demonstrated that wastewater needs to be diluted before use in cultivation because of the presence of toxic substances. Jiang et al. (2015) reported that the growth of Spirulina subsalsa decreased as the wastewater increased from a monosodium glutamate factory [37]. Laohaprapanon et al. (2012) reported that blade wastewater had inhibitory effects on Desmodesmus subspicatus at the lowest concentration of 3.125% (v/v) [38]. Di Caprio et al. (2018) found that 9% (v/v) olive mill wastewater had a significant inhibitory effect on the growth of Scenedesmus sp. [39]. Park et al. (2013) reported that a municipal wastewater concentration of less than 40% had stimulatory effects on A. platensis growth, whereas an inhibitory effect was observed at concentrations greater than 40% [40]. Han et al. (2021) found that Spirulina NCU-Sm grew well in 100% raw soy sauce wastewater [6]. These results indicate that wastewater composition is an important factor affecting algal growth. In this study, metal toxicity was neglected because the concentration of heavy metal ions in the WCP was very low (data not published). Therefore, it is important to investigate other possible inhibitory factors to improve the performance of N and P removal from WCP by A. platensis.
A. platensis in the 50.0% WCP treatment was significantly lower than that in the control (p < 0.05), whereas the 12.5% and 25.0% WCP treatments had no significant effect. These results indicated that compared with lower concentrations of WCP, the harmful effect of higher concentrations of WCP on the activity of PSII was observed early. On day 6, the photosynthetic activity of A. platensis significantly decreased in all cultivations with WCP addition ( Figure 2D), thereby indicating that the low WCP concentration (12.5%) also had a significant inhibitory effect on the activity of PSII in A. platensis.  Figure 3 shows that the inhibition rate of A. platensis exposed to different concentrations of WCP changed over time. The inhibition rates of all three treatments increased as the culture time increased. Thus, inhibition was not alleviated during cultivation. The maximum quantum yield of photosystem II (PSII) (Fv/Fm) is a sensitive indicator of stress in oxygenic photosynthetic organisms [41]. To investigate the effect of WCP on the photosynthetic activity of A. platensis, variations in the maximum photochemical yield (Fv/Fm) were recorded. On the second day of culture, the Fv/Fm value of A. platensis in the 50.0% WCP treatment was significantly lower than that in the control (p < 0.05), whereas the 12.5% and 25.0% WCP treatments had no significant effect. These results indicated that compared with lower concentrations of WCP, the harmful effect of higher concentrations of WCP on the activity of PSII was observed early. On day 6, the photosynthetic activity of A. platensis significantly decreased in all cultivations with WCP addition ( Figure 2D), thereby indicating that the low WCP concentration (12.5%) also had a significant inhibitory effect on the activity of PSII in A. platensis. Figure 3 shows that the inhibition rate of A. platensis exposed to different concentrations of WCP changed over time. The inhibition rates of all three treatments increased as the culture time increased. Thus, inhibition was not alleviated during cultivation.
wastewater composition is an important factor affecting algal growth. In this study, metal toxicity was neglected because the concentration of heavy metal ions in the WCP was very low (data not published). Therefore, it is important to investigate other possible inhibitory factors to improve the performance of N and P removal from WCP by A. platensis.
The maximum quantum yield of photosystem II (PSII) (Fv/Fm) is a sensitive indicator of stress in oxygenic photosynthetic organisms [41]. To investigate the effect of WCP on the photosynthetic activity of A. platensis, variations in the maximum photochemical yield (Fv/Fm) were recorded. On the second day of culture, the Fv/Fm value of A. platensis in the 50.0% WCP treatment was significantly lower than that in the control (p < 0.05), whereas the 12.5% and 25.0% WCP treatments had no significant effect. These results indicated that compared with lower concentrations of WCP, the harmful effect of higher concentrations of WCP on the activity of PSII was observed early. On day 6, the photosynthetic activity of A. platensis significantly decreased in all cultivations with WCP addition ( Figure 2D), thereby indicating that the low WCP concentration (12.5%) also had a significant inhibitory effect on the activity of PSII in A. platensis.  Figure 3 shows that the inhibition rate of A. platensis exposed to different concentrations of WCP changed over time. The inhibition rates of all three treatments increased as the culture time increased. Thus, inhibition was not alleviated during cultivation.

Investigation of Factors Inhibiting the Growth of A. platensis
The results presented in Section 3.1 showed that all three WCP concentrations had toxic effects on the growth and photosynthetic performance of A. platensis. To establish an efficient culture process to remove N and P from WCP, it is helpful to understand which chemical components in WCP are the major factors affecting the growth of A. platensis. According to Liu et al. (2020) [11] and Ren et al. (2017) [14], there are two types of inhibitory factors in WCP. The first type is organic compounds, including raw materials for CPL production, such as CHN, CHL, intermediate products such as cyclohexanoneoxime (CHO), and end products such as CPL. The second type is peroxides. Therefore, the effects of organic matter and peroxides on the growth of A. platensis were investigated.
3.2.1. Effects of Organic Matter in WCP on the Growth of A. platensis Figure 4A shows the effects of CPL on the biomass concentration of A. platensis. After 4 d of cultivation, a biomass concentration of 0.53 g/L was obtained in the control. Similar biomass concentrations were also observed in the cultures with 100, 300, 500, and 1000 mg/L of CPL addition, thereby indicating that CPL concentrations as high as 1000 mg/L had no negative effect on the growth of A. platensis.

Effects of Peroxides in WCP on the Growth of A. platensis
Studies have shown that WCP contains peroxides [14]. If peroxides in the WCP were the main inhibiting factor, then reduced WCP would no longer inhibit the growth of A. platensis, or the inhibition would be significantly relieved. Thus, reduced WCP produced by chemical reduction using Na2SO3 was tested.
The growth curves of A. platensis exposed to different reduced WCP concentrations are shown in Figure 5A. After 6 d of cultivation, the biomass concentrations of A. platensis were 2.34, 2.13, and 1.72 g/L at 12.5%, 25.0%, and 50.0% reduced WCP, respectively ( Figure 5B). Compared with that of the control, the biomass of A. platensis was inhibited by 6.8%, 15.1%, and 31.3% at 12.5%, 25.0%, and 50.0% reduced WCP, respectively ( Figure  5C). The inhibition effect was very similar between the WCP and the reduced WCP, as shown in Figures 2 and 5. Thus, the inhibition of algal growth by WCP could not be alleviated by chemical reduction. The results implied that peroxides in the wastewater were not the main factors affecting the growth of A. platensis. CHN at a concentration as high as 1200 mg/L had no negative effect on the growth of A. platensis (p > 0.05), as demonstrated by the similar biomass concentration compared with the control ( Figure 4B). Higher CHN concentrations (1800 mg/L and 2000 mg/L) significantly (p < 0.05) inhibited the growth of A. platensis by 9.6% and 13.5%, respectively. CHL at a concentration as high as 600 mg/L had no negative effect on the growth of A. platensis (p > 0.05). Further increases in the CHL concentration to 800 mg/L and 1000 mg/L significantly (p < 0.05; Figure 4C) inhibited the growth of A. platensis by 7.3% and 11.9%, respectively. CHO at a concentration as high as 250 mg/L had no adverse effects on the growth of A. platensis (p > 0.05), whereas an increase in the CHO concentration significantly (p < 0.05) inhibited the growth of A. platensis ( Figure 4D).
In this study, the CHN, CHL, and CHO concentrations in the 50% mixed WCP were 82.3, 16.4, and 23.0 mg/L, respectively, and no CPL was detected ( Table 1). The minimum concentrations of CHN, CHL, and CHO, which had significant effects on A. platensis growth, were 1800, 800, and 500 mg/L, respectively (Figure 4). The CHN, CHL, and CHO concentrations in the 50.0% mixed WCP were far lower than those that inhibited the growth of A. platensis, and will further decreased after dilution. However, A. platensis growth was inhibited by 12.5% and 50.0% WCP (Figure 2). Thus, CHN, CHL, and CHO could not explain the inhibitory effect of WCP on A. platensis growth. In other words, the growth of A. platensis may be inhibited by other factors in WCP.

Effects of Peroxides in WCP on the Growth of A. platensis
Studies have shown that WCP contains peroxides [14]. If peroxides in the WCP were the main inhibiting factor, then reduced WCP would no longer inhibit the growth of A. platensis, or the inhibition would be significantly relieved. Thus, reduced WCP produced by chemical reduction using Na 2 SO 3 was tested.
The growth curves of A. platensis exposed to different reduced WCP concentrations are shown in Figure 5A. After 6 d of cultivation, the biomass concentrations of A. platensis were 2.34, 2.13, and 1.72 g/L at 12.5%, 25.0%, and 50.0% reduced WCP, respectively ( Figure 5B). Compared with that of the control, the biomass of A. platensis was inhibited by 6.8%, 15.1%, and 31.3% at 12.5%, 25.0%, and 50.0% reduced WCP, respectively ( Figure 5C). The inhibition effect was very similar between the WCP and the reduced WCP, as shown in Figures 2 and 5. Thus, the inhibition of algal growth by WCP could not be alleviated by chemical reduction. The results implied that peroxides in the wastewater were not the main factors affecting the growth of A. platensis.

Effects of Peroxides in WCP on the Growth of A. platensis
Studies have shown that WCP contains peroxides [14]. If peroxides in the WCP were the main inhibiting factor, then reduced WCP would no longer inhibit the growth of A. platensis, or the inhibition would be significantly relieved. Thus, reduced WCP produced by chemical reduction using Na2SO3 was tested.
The growth curves of A. platensis exposed to different reduced WCP concentrations are shown in Figure 5A. After 6 d of cultivation, the biomass concentrations of A. platensis were 2.34, 2.13, and 1.72 g/L at 12.5%, 25.0%, and 50.0% reduced WCP, respectively ( Figure 5B). Compared with that of the control, the biomass of A. platensis was inhibited by 6.8%, 15.1%, and 31.3% at 12.5%, 25.0%, and 50.0% reduced WCP, respectively ( Figure  5C). The inhibition effect was very similar between the WCP and the reduced WCP, as shown in Figures 2 and 5. Thus, the inhibition of algal growth by WCP could not be alleviated by chemical reduction. The results implied that peroxides in the wastewater were not the main factors affecting the growth of A. platensis.

Effect of Heat-Sensitive and Volatile Chemical Matters in WCP on the Growth of A. platensis
The results presented in Sections 3.2.1 and 3.2.2 showed that four organic compounds and peroxides in WCP were not the main factors affecting the growth of A.

Effect of Heat-Sensitive and Volatile Chemical Matters in WCP on the Growth of A. platensis
The results presented in Sections 3.2.1 and 3.2.2 showed that four organic compounds and peroxides in WCP were not the main factors affecting the growth of A. platensis; some unknown components must have existed and caused growth inhibition. If the unknown components that negatively impact the growth of A. platensis are heat-sensitive, then heat treatment of WCP will relieve the growth inhibition of algae. Therefore, heat-treated WCP was prepared by boiling mixed WCP at 100 • C for 30 min as described in Section 2, and then the effect of heat-treated WCP on algal growth was tested.
Arthrospira platensis was cultivated with heat-treated WCP with concentrations of 12.5%, 25.0%, and 50.0%. As shown in Figure 6, heat-treated WCP exhibited significant inhibitory effects on Arthrospira growth, and the inhibition increased as the heat-treated WCP concentration increased. Compared with that of the control, the biomass concentration of A. platensis was decreased by 9.9%, 13.6%, and 29.9% at 12.5%, 25.0%, and 50.0% heattreated WCP (Figure 6), respectively.

tested.
Arthrospira platensis was cultivated with heat-treated WCP with concentrations of 12.5%, 25.0%, and 50.0%. As shown in Figure 6, heat-treated WCP exhibited significant inhibitory effects on Arthrospira growth, and the inhibition increased as the heat-treated WCP concentration increased. Compared with that of the control, the biomass concentration of A. platensis was decreased by 9.9%, 13.6%, and 29.9% at 12.5%, 25.0%, and 50.0% heat-treated WCP (Figure 6), respectively. The growth inhibition rates of A. platensis by WCP, reduced WCP, and heat-treated WCP and the contents of the main organic matters (CHN, CHL, and CHO) in the three types of WCP are summarized in Table 2. The inhibition rates of A. platensis by heat-treated WCP were similar to those by WCP and reduced WCP, thereby suggesting that heat treatment of WCP did not relieve the negative effect of WCP. Furthermore, the chemical analysis results showed that CHN, CHL, and CHO could not be detected in the heat-treated WCP. These results confirmed that the four organic compounds, peroxides, and other heat-sensitive and volatile chemicals in WCP were not the main factors that affected the growth of A. platensis. The inhibitory effect of WCP on A. platensis might have been the result of the combined influence of multiple factors rather than just one substance.
Before the main inhibitory factors were clarified, it was difficult to completely eliminate the inhibitory effect of WCP. Nevertheless, optimizing the WCP supply mode is another way to minimize the inhibitory effect and improve the efficiency of N and P removal in WCP by A. platensis.  The growth inhibition rates of A. platensis by WCP, reduced WCP, and heat-treated WCP and the contents of the main organic matters (CHN, CHL, and CHO) in the three types of WCP are summarized in Table 2. The inhibition rates of A. platensis by heat-treated WCP were similar to those by WCP and reduced WCP, thereby suggesting that heat treatment of WCP did not relieve the negative effect of WCP. Furthermore, the chemical analysis results showed that CHN, CHL, and CHO could not be detected in the heat-treated WCP. These results confirmed that the four organic compounds, peroxides, and other heat-sensitive and volatile chemicals in WCP were not the main factors that affected the growth of A. platensis. The inhibitory effect of WCP on A. platensis might have been the result of the combined influence of multiple factors rather than just one substance. Before the main inhibitory factors were clarified, it was difficult to completely eliminate the inhibitory effect of WCP. Nevertheless, optimizing the WCP supply mode is another way to minimize the inhibitory effect and improve the efficiency of N and P removal in WCP by A. platensis.

Impact of Fed-Batch Application of WCP on A. platensis Growth
To evaluate the effect of WCP supply modes on A. platensis growth, one batch supply mode and two fed-batch supply modes were tested, as described in Section 2. Over the course of 16 d of culture, all cultivations were harvested every 4 d. The changes in the biomass DW are shown in Figure 7A. After 16 d of culture, the accumulated biomass concentration for the control, fed-batch supply mode 1, fed-batch supply mode 2, and batch supply mode were 7.07, 5.51, 5.19, and 3.88 g/L ( Figure 7B), respectively. The biomass concentration of fed-batch mode 2 was significantly higher than that of the batch supply mode, and the biomass concentration of fed-batch supply mode 1 was significantly higher than that of fed-batch supply mode 2 and the batch supply mode ( Figure 7B). Figure 7C shows the inhibition rates in the culture periods of days 0-4, 0-8, 0-12, and 0-16. These results indicated that by supplying the same amount of WCP, the fed-batch mode was more beneficial to algal growth than the batch mode. Moreover, a lower dose of each WCP addition resulted in a lower growth inhibition rate.
of each WCP addition resulted in a lower growth inhibition rate. Figure 3 shows that the growth inhibition rates of 12.5%, 25.0%, and 50.0% WCP on Arthrospira increased as the culture time increased during the 6 d culture period. The inhibition of Arthrospira by WCP showed an accumulation effect over culture time. What would happen if the culture time further increased? Figure 7C shows that the inhibition rate of the batch mode reached a maximum of 48.10% on day 12, and then decreased to 45.16% on day 16. After 12 d of culture, although the inhibition rates of the two fed-batch modes did not decrease, the upward trend of the inhibition rate began to slow. The phenomenon of the decline of inhibition rate, or the slowing of the upward trend of the inhibition rate, might indicate that under the combined effects of physical, chemical, and biological factors in algal culture, the inhibitory components in WCP could not exist stably for a long time, i.e., they were consumed as the culture time increased. Although the inhibitory components and how they are consumed are unclear, the fact that Arthrospira culture has a consumption effect on the inhibitory factors in WCP is likely beneficial for removing N and P from WCP by cultivation of Arthrospira.

Effect of WCP Supply Modes on A. platensis Photosynthesis Pigments
The pigments of photosynthetic microalgae are important indicators of algal cell growth [38]. The photosynthetic pigments of A. platensis, including Chl-a, Car, and phy-  Figure 3 shows that the growth inhibition rates of 12.5%, 25.0%, and 50.0% WCP on Arthrospira increased as the culture time increased during the 6 d culture period. The inhibition of Arthrospira by WCP showed an accumulation effect over culture time. What would happen if the culture time further increased? Figure 7C shows that the inhibition rate of the batch mode reached a maximum of 48.10% on day 12, and then decreased to 45.16% on day 16. After 12 d of culture, although the inhibition rates of the two fed-batch modes did not decrease, the upward trend of the inhibition rate began to slow. The phenomenon of the decline of inhibition rate, or the slowing of the upward trend of the inhibition rate, might indicate that under the combined effects of physical, chemical, and biological factors in algal culture, the inhibitory components in WCP could not exist stably for a long time, i.e., they were consumed as the culture time increased. Although the inhibitory components and how they are consumed are unclear, the fact that Arthrospira culture has a consumption effect on the inhibitory factors in WCP is likely beneficial for removing N and P from WCP by cultivation of Arthrospira.

Effect of WCP Supply Modes on A. platensis Photosynthesis Pigments
The pigments of photosynthetic microalgae are important indicators of algal cell growth [38]. The photosynthetic pigments of A. platensis, including Chl-a, Car, and phycobiliproteins, were measured during cultivation with different WCP supply modes ( Table 3). The WCP supply modes significantly influenced the contents of Chl-a and PC, and the batch mode had the greatest impact. For example, the Chl-a and PC contents of fed-batch mode 1 in the second growth period (day 8) were 16.38 mg/g and 98.01 mg/g, respectively. However, the contents were 12.59 mg/g and 69.52 mg/g, respectively, in the batch mode. The WCP supply modes had little effect on the Car and APC contents.
As the culture time increased, the Chl-a and PC contents in fed-batch mode 1 and fedbatch mode 2 showed a decreasing trend. In fed-batch mode 1, the Chl-a content decreased by 13.3%, 14.3%, 26.3%, and 24.6% on days 4, 8, 12, and 16, respectively, compared with the control. Moreover, the PC content decreased by 12.0%, 25.7%, 38.4%, and 36.8% on days 4, 8, 12, and 16, respectively, compared with the control. This phenomenon is consistent with the observation that the inhibition of algal growth was enhanced over time ( Figure 7C).  Table 4 shows the effect of the WCP supply mode on the biochemical composition of A. platensis. Compared with the control, WCP significantly reduced the protein content of A. platensis, whereas the batch mode had the greatest impact. The protein content in fedbatch mode 1 was 65.36%, 67.82%, 63.44%, and 58.54% on days 4, 8, 12, and 16, respectively. The protein content in fed-batch mode 2 was almost the same as that of fed-batch mode 1. The protein content in the batch mode, which was 55.11%, 59.70%, 58.01%, and 57.98% on days 4, 8, 12, and 16, respectively, was significantly lower than that of fed-batch mode 1. The results indicated that supplying WCP to Arthrospira culture in a fed-batch manner could reduce the negative effects of WCP on protein synthesis. Similar results were also observed in other studies. For example, it was found that the fed-batch process can reduce the toxic or inhibitory compound loadings in wastewater and improve the algal tolerance to high substrate concentrations [23,42]. The protein content in fed-batch mode 1 ranged from 58.54% to 67.28%, which was higher than that recorded in other reports. For example, Spirulina subsalsa had a protein content of 40.65% in 25.0% (v/v) wastewater from a monosodium glutamate factory [37]. Arthrospira platensis had a protein content of 38.13% in olive oil mill wastewater in the presence of excess nutrients [43]. Spirulina platensis was found to contain a protein content of 46.02% in municipal wastewater [44].
In contrast, the batch mode significantly increased the carbohydrate content of A. platensis. The carbohydrate content in fed-batch mode 1 was 18.80%, 15.02%, 16.65%, and 13.51% on days 4, 8, 12, and 16, respectively, and the carbohydrate content in fed-batch mode 2 was almost the same as that in fed-batch mode 1. The carbohydrate content in the batch mode was 30.37%, 22.94%, and 20.22% on days 4, 8, and 12, respectively, which was significantly higher than that of fed-batch mode 1. The total lipid content was not affected by the WCP supply modes, and remained stable during the 16 d of cultivation. A similar result was reported in a study by Jiang et al. (2015), in which the carbohydrate content increased from 3.29% to 17.69% as the wastewater concentration increased from 0.00% to 50.00%, but the wastewater had little influence on the total lipid content [37]. Table 5 shows the protein and total phycobiliprotein (T-PC) production of A. platensis affected by the WCP supply modes. The total yields of protein and T-PC of fed-batch mode 1 were 3573.38 mg/L and 745.49 mg/L, respectively. Although these yields were lower than those of the control, they were significantly higher than those of fed-batch mode 2 and the batch mode (p < 0.05). These results implied the advantage of fed-batch mode 1 in wastewater supply in WCP denitrification. Considering all the results of the effects of the WCP supply modes on the growth, photosynthesis pigments, and biochemical composition of A. platensis, the fed-batch mode of wastewater addition clearly significantly minimized the negative impact of WCP on A. platensis.

Removal of N and P from WCP by A. platensis Cultivated in a Circular Photobioreactor
To test the N and P removal efficiencies under the condition of fed-batch addition of mixed WCP, A. platensis was cultured in a 25 L circular photobioreactor (Figure 8). Mixed WCP was added daily at a concentration of 1.25%, and the culture was continued for 10 d with total WCP addition of 3.125 L (12.5%, v/v). During cultivation, the daily evaporation of water was approximately 0.7 L; 0.3125 L (1.25%) of WCP addition compensated for some of the water evaporation, and deionized water was added to compensate for the remaining water evaporation so that the culture volume was maintained at 25 L. Changes in the NO 3 − -N and inorganic P concentrations in the algal suspension are shown in Figure 8. After adding wastewater every day, the NO 3 − -N and P concentrations reached 14.26 mg/L and 0.76 mg/L, respectively. After 24 h of cultivation, NO 3 − -N and P were not detected in the algal suspension, thereby indicating that all the N and P from WCP addition were assimilated by A. platensis.
To test the N and P removal efficiencies under the condition of fed-batch addition of mixed WCP, A. platensis was cultured in a 25 L circular photobioreactor (Figure 8). Mixed WCP was added daily at a concentration of 1.25%, and the culture was continued for 10 d with total WCP addition of 3.125 L (12.5%, v/v). During cultivation, the daily evaporation of water was approximately 0.7 L; 0.3125 L (1.25%) of WCP addition compensated for some of the water evaporation, and deionized water was added to compensate for the remaining water evaporation so that the culture volume was maintained at 25 L. Changes in the NO3 − -N and inorganic P concentrations in the algal suspension are shown in Figure 8. After adding wastewater every day, the NO3 − -N and P concentrations reached 14.26 mg/L and 0.76 mg/L, respectively. After 24 h of cultivation, NO3 − -N and P were not detected in the algal suspension, thereby indicating that all the N and P from WCP addition were assimilated by A. platensis. The biomass DW was 0.15 g/L at the beginning of the culture, and the biomass increased linearly during cultivation. After 5 d of culture, the biomass reached 0.7 g/L. To avoid a high biomass density, which might slow algal growth, the biomass was harvested to reduce the DW to 0.31 g/L. After harvest, the biomass increased linearly again and reached 0.92 g/L on day 10. The overall biomass productivity reached 17.48 g/(m 2 ·d) with an average growth rate of 0.22 d −1 . The maximum protein content of Arthrospira on days 5 and 10 of culture was determined to be 67.56% and 69.93%, respectively. The high protein content resulted in high protein production of 12.03 g/(m 2 ·d). The biomass DW was 0.15 g/L at the beginning of the culture, and the biomass increased linearly during cultivation. After 5 d of culture, the biomass reached 0.7 g/L. To avoid a high biomass density, which might slow algal growth, the biomass was harvested to reduce the DW to 0.31 g/L. After harvest, the biomass increased linearly again and reached 0.92 g/L on day 10. The overall biomass productivity reached 17.48 g/(m 2 ·d) with an average growth rate of 0.22 d −1 . The maximum protein content of Arthrospira on days 5 and 10 of culture was determined to be 67.56% and 69.93%, respectively. The high protein content resulted in high protein production of 12.03 g/(m 2 ·d).
The average biomass areal productivities were 17.25, 15.60, 14.75, 17.44, and 17.44 g/(m 2 ·d) during days 0-4, 0-5, 0-6, 0-8, and 0-10, respectively. Although there were some fluctuations, the biomass productivities did not exhibit a trend of decline during cultivation and an accumulation effect of growth inhibition of WCP on A. platensis was not observed, thereby indicating that a smaller amount of WCP addition (1.25%) every day was less toxic to the algae.
Based on the changes in the NO 3 − -N and inorganic P concentrations in the culture suspension, the removal ratios (%) and removal rates [g/(m 2 ·d)] of N and P were calculated ( Table 6). The removal ratios of N and P reached 100%. Arthrospira platensis could absorb and utilize all the added N and P from the wastewater within 24 h. The ability to efficiently remove N and P from WCP might be due to the ability of luxury absorption of N and P of A. platensis. The luxury absorption of N and P by microalgae means that the amount of N and P absorbed exceeds the N and P demand in cell cycles [45,46]. The excessive absorbed N and P are stored in cells in some form to cope with the possible lack of N and P in the future. As long as the nutrient input (wastewater addition) is properly regulated, A. platensis can absorb all the N and P from the wastewater. Table 6. N, P removal rate and removal ratio, biomass and protein productivities of Arthrospira platensis cultivated in a circular photobioreactor. The average biomass and protein productivities of A. platensis, the N and P removal ratios, and the average removal rates are summarized in Table 6. The algal suspension depth of the circular photobioreactor was 15 cm, which was similar to the culture depth (approximately 20 cm) of a raceway pond. The light intensity was 1300 µmol photons/(m 2 ·s) with a 14 h/10 h light/dark cycle, and the culture temperature was maintained at 30 • C. The culture conditions were similar to those of an open raceway pond in good production season. Therefore, the N and P removal capacities of A. platensis cultivated in an open raceway pond can be calculated based on the data shown in Tables 5 and 6. Assuming an Arthrospira biomass yield of 15 g/(m 2 ·d) and a protein content of 65%, a 10,000 m 2 culture area can assimilate all the N and P from 12,000 L of WCP and produce 150 kg of biomass each day. This result illustrates the feasibility of growing Arthrospira on WCP for biomass production and N and P removal.

Conclusions
WCP significantly inhibited the growth of A. platensis, and the inhibitory effect increased as the wastewater concentration increased. Organic matter, including CHN, CHL, and CHO, and peroxides in WCP were not the main factors that induced the inhibition of alga. The inhibitory effect of WCP on A. platensis might have been the result of the combined influence of multiple factors. Fed-batch addition of wastewater significantly minimized the negative impact on A. platensis. Average biomass productivity of 17.48 g/m 2 /d and maximum protein content of 69.93% was obtained in a 25 L circular photobioreactor with daily supply of 1.25% mixed WCP, and the N and P removal ratios reached 100%. It can be concluded that A. platensis is a candidate for bioremediation of WCP with high biomass production and high N and P removal efficiencies. Thus, the removal of N and P from WCP by A. platensis is promising.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/app12010227/s1. Table S1: Culture media formulas used in the effect test of different wastewater concentrations on the growth of A. platensis. Table S2: Culture media formulas used in the effect test of WCP supply modes on the growth, photosynthetic pigments, and biochemical components of A. platensis. Table S3: Addition amount of each nutrient after harvest in the effect test of WCP supply modes on the growth, photosynthetic pigments, and biochemical components of A. platensis.
Author Contributions: Y.Y. conceived and designed the experiments, performed the laboratory work, analyzed, and interpreted the data and drafted the paper. X.L. and J.R. analyzed the wastewater samples and provided caprolactam wastewater. Y.H. and K.W. performed the laboratory work. Z.W. participated in the laboratory work and revised the manuscript. Y.G. maintained the seed culture of Arthrospira platensis. Y.L. and X.W. contributed to experimental design and data interpretation, and critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.