Recycling of rural abandoned constructed wetlands : mariculture wastewater treatment

This study aimed to investigate the behavioral shifts of constructed wetland (CW) when the treated water was changed from domestic wastewater to mariculture wastewater. The results showed that the average removal efficiencies of ammonium nitrogen (NH4 -N), total nitrogen and chemical oxygen demand (COD) were 29.54, 46.07 and 57.15% in mariculture wastewater, respectively, which were significantly lower than those in domestic wastewater (71.35, 66.34 and 74.98%, respectively). While there was no significant difference in the removal efficiency of nitrate and phosphate (P> 0.05) between the two systems. Based on the analysis of bacterial community and adsorption properties, the results further indicated that the removal mechanism of NH4 -N between both systems was mainly due to substrate adsorption: the maximum adsorption capacity of NH4 -N on the substrate in mariculture wastewater was 5,432 mg kg , whereas that in domestic wastewater was 18,033 mg kg . In terms of bacterial communities, the dominant bacteria at the family level were Victivallaceae (18.63%) in domestic wastewater and Porphyromonadaceae (18.37%) in mariculture wastewater, which showed the significant alteration to the bacterial community. In conclusion, this study showed that conventional CW could be used for treating wastewater from land-based marine aquaculture, while the operating conditions needed to be optimized in the process of application.


INTRODUCTION
Recently, there has been growing interest in the development of intensive land-based marine aquaculture that could bring economic benefits to coastal areas with environmental sustainability (Martins et al. ). In a study on land-based Atlantic salmonin farming in China, Sun et al. () reported that only 36.5-47.8% nitrogen (N) and 20.4-38.6% phosphorus (P) in the feed were digested by fish and the residual nutrients were dissolved in drainage.
Without proper treatment, nutrient enrichment or eutrophication in aquatic environments can cause an increase in algae growth and a decrease in dissolved oxygen, which further destroys the ecological balance and structure (Liang et al. ). Therefore, the treatment of wastewater from marine aquaculture in an environmentally friendly way is crucial for sustainable intensification of aquaculture.
Many technologies have been widely employed for purification of mariculture wastewater including biological technologies and physicochemical equipment (Munavalli & Pise ; Zhao et al. ; Cao et al. ), while these approaches lead to some problems such as capital investment, energy consumption, maintenance requirements, secondary pollution and low removal efficiency (He et al. ). Apart from these methods, constructed wetland (CW) as a lower cost, environment friendly, work efficient and energy-saving technology with no production of secondary pollutants, have shown potential for the mariculture wastewater treatment (Teresa ; Wang et al. a). In addition, with the development of the urban ecological civilization construction and the progress of rural domestic wastewater interception, many CWs that were originally used for domestic wastewater treatment in rural areas are now idled. Therefore, these CWs had the feasibility of being reused to treat mariculture wastewater. However, during the transformation of the wastewater, understanding of the key purification mechanisms of CWs for specific contaminants in mariculture wastewater remains unclear. Therefore, the current study was conducted to (1) investigate and compare the removal efficiency of pollutants and substrate bacterial community of CW in treating domestic and mariculture wastewater and (2) clarify the influence mechanisms that cause and influence the changes in performance of pollutant removal during the transformation of the wastewater.

Experimental wetland system
In this study, two horizontal flow structure CW systems were constructed to treat simulated domestic wastewater (R1) and mariculture wastewater (R2). The volume of each CW was 100 L, and the inlet and outlet were arranged at the bottom and upper parts of the device, respectively.
Each CW tank was filled with zeolite (φ ¼ 4-14 mm; porosity ¼ 0.43), which was taken from the CW used to treat domestic wastewater in our research group. During the experimental period, the hydraulic loading rate of each system was set to 20 cm d À1 .
Artificial wastewater was used in this study: mariculture wastewater was made of chemical oxygen demand (COD) (glucose) 100 mg L À1 , NH þ 4 -N 25 mg L À1 , phosphate 10 mg L À1 , nitrate 30 mg L À1 and nitrite 5 mg L À1 , which were mixed with seawater; domestic wastewater used tap water and pollutants were consistent with mariculture wastewater. Water samples were collected at a fixed time from the inlet and outlet of each CW system. At the end of the experiment, substrate samples were collected from each CW system to analyze the bacterial community.

Adsorption properties of the substrate
A quantitative sterilized substrate of CW was placed in 250 mL conical flasks. 200 mL 30 mg L À1 ammonia solution and 200 mL 30 mg L À1 ammonia solution with 3% NaCl were added to two separate conical flasks, which were placed in a thermostatic oscillator at 50 r min À1 , 25 C.
Samples were taken at 0, 10, 30, 60, 120, 240, 360 and 480 min. Finally, the adsorption capacity of the substrate to NH þ 4 -N was calculated according to the residual concentration of the solution in each conical flask. Based on the experimental results, the quasi first-order model, quasi second-order model and intramolecular diffusion model were used to fit the adsorption process of the substrate for ammonium nitrogen. The three models are shown as follows: Quasi first-order model: ln (C e À C t ) ¼ ÀK 1 t þ ln C e Quasi second-order model: Intramolecular diffusion model: where C e is the amount of pollutants adsorbed by the substrate at adsorption equilibrium (mg kg À1 ); C t is the amount of pollutants adsorbed by the substrate at t (mg kg À1 ); K 1 is Quasi first-order model adsorption kinetic constant (min À1 ); K 2 is Quasi second-order model adsorption kinetic constant (kg mg À1 min À1 ) and K p is the diffusion rate constant (mg kg À1 min 1/2 ).
A quantitative sterilized substrate of CW was added to Langmuir equation: where C w is the concentration of pollutants in the solution at adsorption equilibrium (mg L À1 ); C s is the adsorption capacity per unit substrate material (mg kg À1 ); Q max is the maximum adsorption capacity per unit substrate material (mg kg À1 ); K L is the Langmuir equilibrium constant, the larger the value, the stronger the adsorption capacity (L kg À1 ); K f is the Freundlich coefficient, which represents the adsorptive capacity of the substrate (L kg À1 ) and n is a constant of the equation, which can represent the adsorptive strength of the substrate. β is the D-R adsorption constant; ε is the Polanyi potential; R is the ideal gas constant, 8.314 J mol À1 K À1 ; T is the absolute temperature (K) and E is the average adsorption energy (kJ mol À1 ).

Sample analysis
Samples were analyzed for NH þ 4 -N, nitrate, nitrite, total nitrogen, COD and phosphate. In R1, all pollutants were measured based on the national standard method (China E.P.A. ). In R2, considering the character of mariculture wastewater, the NH þ 4 -N was measured by indophenol blue spectrophotometry, COD was measured by the potassium permanganate method and other pollutants were measured based on the national standard method. In addition, as nitrite can interfere with the use of COD as an indicator of organic matter content; thus, in this study, COD correction data were applied to characterize the removal of organic matter.

Statistical analysis
In our experiment, the statistical analysis was performed using SPSS. One-way analysis of variance (ANOVA) was used to compare mean values between different treatments with the least significant difference (LSD) test at the significance level of 0.05.

Pollutant removal
The pollutant removal efficiencies in R1 and R2 are shown in Figure 1. During the transformation of the wastewater, there were significant differences (P < 0.05) in the removal efficiencies of NH þ 4 -N, total nitrogen and COD in CW. In R1, the average removal efficiencies of NH þ 4 -N, total nitrogen and COD were 71.35, 66.34 and 74.98%, respectively. In R2, the average removal efficiency of NH þ 4 -N, total nitrogen and COD were all significantly lower than R1 (P < 0.05) at 29.54, 46.07 and 57.15%, respectively.
However, the removal efficiency of phosphate and nitrate was not statistically significant between the two systems (P > 0.05). In R1, the average removal efficiencies of these two indices were 77.78 and 16.44%, and in R2, they were 75.31 and 16.78%, respectively.

Bacterial community
The relative abundances of the dominant bacterial community (>1%) at phylum, class, order and family levels in

Carbon and nitrogen metabolism pathways in CW
To reveal the changes in the degradation pathways of nitrogen and carbon in different CW systems, relevant functional enzyme-encoding genes from R1 and R2 were analyzed based on function prediction analysis (Figures 3 and 4).
The nitrogen metabolism pathways in R1 and R2 were the same and included four reduction pathways of nitrification, assimilation nitrate reduction, dissimilation nitrate     were only marginally more abundant in R1 (9.20 × 10 À3 ) than in R2 (8.02 × 10 À3 ). For example, the relative abundance of encoding genes of glucokinase was obviously higher in R1 (1.05 × 10 À3 ) than in R2 (8.32 × 10 À4 ). Notably, this phenomenon implied that the removal efficiency of COD in R1 was higher than in R2.

Adsorption properties of NH 4 þ -N
Since there was a greater significant difference in the NH þ 4 -N removal efficiencies compared with that of the other pollutant removal efficiencies between the different CW systems, the removal mechanism of NH þ 4 -N was further analyzed by substrate the adsorption properties (Table 1).
Adsorption kinetics are primarily used to describe the rate at which adsorbents adsorb solutes. The results shown in Table 1 indicated that in simulated wastewater, three models all could fit the adsorption process of NH þ 4 -N on zeolite (R 2 > 0.9, P < 0.05). However, in wastewater with 3% NaCl, the quasi second-order model could fit the adsorption process of NH þ 4 -N on zeolite (R 2 ¼ 0.91) better than the intramolecular diffusion model (R 2 ¼ 0.81) and the quasi first-order model (R 2 ¼ 0.84). Therefore, we selected the quasi second-order model to analyze the difference in adsorption between the different systems. Based on these parameters, it could find that the adsorption rate of zeolite to NH þ 4 -N in the wastewater (7.04 × 10 À5 kg mg À1 min À1 ) was lower than that in the wastewater with 3% NaCl (1.90 × 10 À4 kg mg À1 min À1 ). In addition, the quasi secondorder model assumed that the adsorption rate was controlled by chemisorption (Liu et al. ), thereby the result also indirectly explained that the adsorption process of NH þ 4 -N on zeolite in the different wastewater was chemisorption.
Adsorption isotherm describes the solute molecule concentration between the liquid phase and the solid phase when the adsorption processes of the molecules are in equilibrium at a given temperature. In this experiment, the Langmuir equation was unable to fit the data (R 2 < 0.3, P > 0.05), so we selected the Freundlich equation and D-R equation to analyze the difference in adsorption among the different systems (R 2 > 0.8, P < 0.05). As shown in Table 1, the Freundlich equation and D-R equation fitted the NH þ 4 -N data in simulated wastewater and wastewater with 3% NaCl (R 2 > 0.9, P < 0.05), respectively, and the respective K f values, n values and Q max values were ranked as follows: wastewater with 3% NaCl (K f ¼ 0.037  Table 1, the values of E were 10 kJ mol À1 in wastewater and 8.3 kJ mol À1 in simulated wastewater with 3% NaCl, respectively. Thus, in our experiment, the adsorption mechanism of the zeolite for NH þ 4 -N was chemisorption, which was consistent with the results of the adsorption kinetics simulation.

DISCUSSION
In this study, the inhibitive effects on NH þ 4 -N and total nitrogen removal were observed during treatment transformation with inhibition ratios of 41.81 and 20.27%, respectively, which are consistent with the previous studies (Liang et al. ; Fu et al. ). However, there was no significant removal difference in phosphate and nitrate which was different from other studies. For example, Gao et al. () found that the salt in mariculture wastewater could negatively influence phosphate removal in CW. Moreover, there are limited literature reviews on COD during CW for COD removal was also inhibited, and the inhibition ratio was 21.67%.
Generally, microbes play a key role in the nitrification and denitrification processes, which are widely considered as the major N removal mechanism in CWs (Vymazal ). Besides, substrate adsorption was another major way for N removal in CWs due to zeolite as the substrate in this experiment. From Figure 3, in nitrification, the relative abundance encoding genes of enzymes involved in mariculture wastewater (9.7 × 10 À5 ) were more abundant than those in domestic wastewater (4.5 × 10 À5 ). In contrast, the removal efficiency of NH þ 4 -N in mariculture wastewater was significantly lower than that of domestic wastewater.
Combining the results of adsorption properties, we believed the reason for this phenomenon was the adsorption property of NH þ 4 -N on the substrate. Natural zeolite is a kind of silicate with silica-oxygen tetrahedron and aluminaoxygen tetrahedron as its skeleton and it has large specific surface area (SSA), strong ion exchange capacity and high static adsorption capacity for NH  Table 1.
Compared with NH þ 4 -N, nitrate removal efficiency did not significantly differ between the two systems. In our research, we thought that there were two main reasons for this phenomenon: (1) low substrate adsorption capacity and (2) insufficient carbon compounds in the denitrification process. A previous study concluded that the affinity for adsorption of nitrate onto Al oxides was weak (Zhou & Haynes ). Although there was a salinity difference between the two systems, this did not affect the adsorption for nitrate on the substrate. In denitrification, theoretically, the reduction 1.0 g nitrate to nitrogen requires approximately 4.4 g of organic matter (COD). However, the organic content in both systems could not meet the requirements of denitrification. Therefore, the carbon source became a limiting factor in the denitrification process which limited the significant difference in nitrate removal efficiency between different systems. Combining these two factors, there was no difference in the nitrate removal efficiency during the transformation of the wastewater. This conclusion also could be supported by the analysis results of the bacterial community and nitrogen metabolism pathway. As shown in Figures 2 and 3, in denitrification, the relative abundance of encoding genes of enzymes in mariculture wastewater was not inhibited; several genes even had elevated expression. For example, the relative abundance of nirK, which is a key functional gene in denitrification (Jones & Hallin ) in R2 (1.4 × 10 À4 ) was greater than that in R1 (5.2 × 10 À5 ). This result indicated that there was no effect on denitrification after the transformation of the wastewater objective. In terms of total nitrogen, we thought that the NH þ 4 -N removal efficiency in mariculture wastewater was significantly lower than that in domestic wastewater, which directly led to lower total nitrogen removal efficiency in mariculture wastewater.
The removal of COD in CW mainly depends on the metabolic activities of microorganisms. As shown in Figure 2 with that in mariculture wastewater. In addition, as shown in Figure 4, relevant functional enzyme-encoding genes involved in glycolysis were more abundant in domestic wastewater than that in mariculture wastewater, which also implied that the removal efficiency of COD may be higher in domestic wastewater than that in mariculture wastewater, and this result was consistent with COD removal.
With regards to phosphate, CWs can effectively remove phosphorus through filtration, adsorption, co-precipitation, ion exchange, plant absorption and microbial decomposition by taking advantage of the physical, chemical and biological coordination (Vymazal ). However, in our experiment, we did not add the plants to the CW, and the most important point was that there was no biofilmenhanced phosphorus removal mechanism in our systems because the environmental conditions were not suitable for the growth of phosphorus accumulating organisms.
Thus, in our study, the phosphate removal mechanism mainly depended on the biosynthetic utilization and substrate adsorption. In general, the maximum phosphate uptake rate of bacteria was achieved under a higher concentration ratio of organic carbon to phosphate in the wastewater. However, the organic content in both systems was all insufficient. In addition, Zhou & Haynes () found that the relative affinities for adsorption of anions onto Al oxides often follow the order: phosphate > chloride ion. Therefore, chlorine ions in mariculture wastewater did not affect the adsorption of phosphate onto the substrate.
Considering these two factors, there was no significant difference in the phosphate removal efficiency between the two systems. Besides, zeolites are a class of crystalline aluminosilicates that have a net negative charge due to the substitution of Si (IV) by Al (III) in the Si tetrahedral (Nikolakis ). Therefore, adsorption of anions, such as phosphate, by zeolites is generally lower than that of cations (Haynes ).

CONCLUSION
Conventional CW can be applied in mariculture wastewater treatment, while the process was affected by wastewater characteristic, and it had negative effects on NH þ 4 -N, total nitrogen and COD removal efficiency. These negative effects on COD were mainly caused by salts inhibiting the function of microorganisms in the substrate which will further change the community structures; meanwhile, salts would weaken NH þ 4 -N removal efficiency by the substrate due to the competition for uptake into ion. This study provides both a new strategy for reuse of abandoned CW in rural areas and a new understanding of the removal mechanism of mariculture wastewater in CW.