Enhancing nitrogen removal via the complete autotrophic nitrogen removal over nitrite process in a modified single-stage tidal flow constructed wetland

Ecological Engineering 103 (2017) 170–179

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Enhancing nitrogen removal via the complete autotrophic nitrogen removal over nitrite process in a modified single-stage tidal flow constructed wetland Zhen Wang ∗ , Menglu Huang, Ran Qi, Yumeng Zhang School of Resources and Environment, Anhui Agricultural University, Hefei 230036, China

a r t i c l e

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Article history: Received 12 October 2016 Received in revised form 4 March 2017 Accepted 1 April 2017 Keywords: Tidal flow constructed wetland (TFCW) Shunt ratio Complete autotrophic nitrogen removal over nitrite (CANON) Anammox Nitrogen transformation

a b s t r a c t This study attempts to achieve a high-rate nitrogen removal via the complete autotrophic nitrogen removal over nitrite (CANON) process in a modified single-stage tidal flow constructed wetland (TFCW) with step-feeding, and nitrogen transformation pathways in the TFCWs treating domestic wastewater were explored under shunt ratio constraints. Shunt ratio significantly affected nitrogen transformation pathways in the TFCWs throughout the experiment. The anammox bacteria were enriched most effectively at the shunt ratio of 1:1, and then the initiation of a CANON process was accomplished in the TFCW under the appropriate limited oxygen condition. The mean TN removal rate reached up to (127.00 ± 13.78) mg (L d)−1 correspondingly. It could be concluded that autotrophic nitrogen removal via CANON process developed in the TFCW with optimized microenvironment that developed as a result of appropriate shunt ratio. The optimal shunt ratio of the TFCW for nitrogen removal was 1:1. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Constructed wetlands (CWs) have proven to be an efficient ecological technology for the treatment of various kinds of contaminated waters. In comparison with conventional treatment systems, constructed wetlands are more easily maintained and operated, require reduced input, and consume less energy (Vymazal, 2010). Nevertheless, nitrogen removal in CWs exhibited substantial fluctuations and was often unsatisfactory (Sun et al., 2005). Therefore, the nitrogen removal capacity of CWs must be improved because nitrogen is a major contributor to water eutrophication. Classical nitrogen removal route, known as conventional nitrification-denitrification, was once thought to be the major nitrogen removal route in subsurface flow CWs (Saeed and Sun, 2012). Nevertheless, this route is often impaired in a CW system due to either lack of organics or inadequate dissolved oxygen (DO). Apart from conventional nitrification-denitrification process, the autotrophic nitritation-anammox process also exists in CWs as an unconventional pathway, which requires less oxygen, eliminates the need for organics, generates less sludge, and reduces greenhouse gas emission (Sun and Austin, 2007). Although distinct conditions are required for nitritation (aerobic) and anam-

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.ecoleng.2017.04.005 0925-8574/© 2017 Elsevier B.V. All rights reserved.

mox (anoxic) respectively, it is feasible to integrate these two autotrophic nitrogen conversion processes within a single aerobic reactor under oxygen-limiting situation, referred as complete autotrophic nitrogen removal over nitrite (CANON) (Sliekers et al., 2002). Hence, enhancing nitrogen removal via the CANON process will be conducive to realize effective nitrogen removal in a singlestage CW when treating wastewater with low C/N ratio. As a passively-aerated biofilm system, CWs possess natural advantages (limited oxygen supply, redox stratification and high biomass retention, etc.) to facilitate the CANON process. Although nitrogen removal via CANON process has been reported in several studies with different types of CWs (Dong and Sun, 2007; Hu et al., 2014; Sun and Austin, 2007; Tao and Wang, 2009; Tao et al., 2011), achieving stable and high-rate autotrophic nitrogen conversion is still a challenge in such systems. One major challenge is that it is extremely difficult to control oxygen supply and maintain appropriate level of DO in CWs. On the other hand, CWs are usually operated with a relative low nitrogen load, which is unfavorable to maintaining anammox (Joss et al., 2009). Recently, tidal flow constructed wetlands (TFCWs) has been proposed to enhance the removal effect of nitrogen because its oxygen supply can be greatly strengthened by the “tidal” operation (Wu et al., 2011). As mentioned, the level of DO in bed has substantial impacts on activities of nitritation and anammox in a CW. Hence, appropriate microenvironment in the system should be created for CANON process on the premise that the TFCW receives some mod-

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ifications. Hu et al. (2014) reported that oxygen supply in TFCWs could be weakened with adoption of up-flow mode instead of the original down-flow mode, which could trigger CANON process in the system. It was found in our previous study that ananmox bacteria in TFCW could be enrichment to some extent by adopting step-feeding with up-flow mode, which proved to be conducive to enhance nitrogen removal (Wang et al., 2017). Hence, it was expected that step-feeding with up-flow mode could be an important controlling factor for the CANON route, since this modification may have significant impact on the nitrogen conversion pathway. As we know, step-feeding has been broadly demonstrated in conventional activated sludge process as an effective option to enhance TN removal by stepwise introduction of the influent to the nitrified liquid, thus making more efficient use of the influent carbon source for denitrification (Puig et al., 2004; Tang et al., 2007). Until recently, some studies are carried to investigate step-feeding in improving nitrogen removal in CWs and this setup is subsequently proved to be effective on TN removal in the systems (Hu et al., 2012; Fan et al., 2013). Since step-feeding is usually achieved by the installation of a shunt pipe in one location of a CW (Wang et al., 2014), the shunt ratio becomes a crucial parameter which can play a key role in forming effective anoxic conditions that are favorable for reducing oxidized-N to improve removal TN. Nevertheless, few efforts have been made to achieve satisfactory NH4 + -N and TN removal in one single-stage TFCW via the CANON process, and the regulation of shunt ratio still remains unclear as the single-stage TFCW with the adoption of step-feeding is used in the degradation of wastewater that contains relative lower organic carbon concentration. So, it is quite necessary to qualify and evaluate the effect of the shunt ratio in TFCW on wastewater treatment as shunt ratio can affect functional microorganisms involved in nitrogen transformation by changing the oxygen transfer rate, and more attempts should also be made to investigate nitrogen removal mechanisms at the molecular level in the system. Given this, the single-stage TFCWs with adoption of a modified step-feeding mode were established in our study, which was expected to enhance nitrogen removal via the CANON process. A comparison study of the modified TFCW at six different shunt ratios to treat domestic wastewater was carried out. Nitrogen transformation and treatment performances of the TFCW were investigated under shunt ratio constraints. For the TFCW, the absolute abundance of genes involved in nitrogen removal and their ecological associations were assessed to evaluate the relationship between the shunt ratio and the microbial community performing nitrogen removal.

2. Materials and methods 2.1. System descriptions TFCWs were constructed at the greenhouse of Anhui Agricultural University (AHAU) in Hefei, China. Each single-stage TFCW (a polyethylene tank with a diameter of 20 cm and depth of 80 cm) was filled with 70 cm of oyster shell (particle size: 2–5 mm) as the substratum layer, as well as 10 cm of gravel (particle size: 10–15 mm) as the bottom under-drainage layer. The bed had a total volume of 25.12 L and a working volume of 10.50 L (initial porosity 41.80%). A “”-shaped perforated inlet pipe was installed on the top of each TFCW, whereas another perforated pipe was installed at the bottom of the system as the shunt pipe. A vertical perforated PVC pipe (80 cm in length and 3.5 cm in diameter) was inserted into the bed in the middle of each TFCW to measure the physical and chemical parameters of wastewater in situ. Four orifices (25 mm internal diameter for each), which were used for collecting substratum samples, were respectively excavated in a line at different depths (14,

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28, 42, and 56 cm) of side wall below the top of the bed. Every orifice was sealed by a rubber plug. Four reeds (initial height of approximately 30 cm) were planted in each system, and each of them has a main stem and two or three new shoots.

2.2. Experimental conditions The TFCWs received schoolyard domestic sewage from AHAU after anaerobic pretreatment. In the process of anaerobic treatment, most of the biodegradable organic substrate was consumed, resulting in a low BOD5 /N ratio (≈0.88) in the wastewater. The water quality parameters of the sewage were as follows: TSS, (65.12 ± 24.51) mg L−1 ; COD, (79.40 ± 17.72) mg L−1 ; BOD5 , (37.83 ± 12.57) mg L−1 ; NH4 + -N, (35.54 ± 1.80) mg L−1 ; NO2 − -N, (2.80 ± 0.57) mg L−1 ; NO3 − -N, (1.72 ± 0.18) mg L−1 ; TN, (42.97 ± 2.85) mg L−1 ; TP, (13.64 ± 2.39) mg L−1 ; and pH, (7.74 ± 0.58). Each TFCW was operated in a modified step-feeding mode in order to enhance nitrogen removal via the CANON process. Specifically, the entire operation cycle, which occurred every 6 h, could be divided into four phases in chronological order (as shown in Fig. 1): (a) Feeding Phase: part of wastewater (via the inlet pipe) was loaded to the TFCW in batch mode (t = 10 min), meanwhile, the rest of wastewater (via the shunt pipe) was continuously fed to the TFCW in 10 min by a peristaltic pump, in order to regulate DO level in the system, the wastewater via the shunt pipe was pumped in an up-flow pattern; (b) Flood Phase: since the ending of Feeding Phase, the whole bed was kept saturated for a certain period of time (t = 240 min); (c) Drain Phase: all the wastewater was drained out rapidly (t = 10 min) via the outlet pipe installed at the bottom of the TFCW. (d) Rest Phase: the whole bed was allowed to “rest” (unsaturated) for a while (t = 100 min). Ten liters of wastewater were added to each TFCW for each cycle [corresponding to an HLR of 1.27 m3 (m2 d)−1 ], including the wastewater added to the TFCW through the shunt pipe. The shunt ratio was defined as the ratio between the inflow volume through the shunt pipe and the inflow volume through the inlet pipe. In our study, six different shunt ratios were adopted: 0:1, 1:4, 1:3, 1:2, 1:1 and 2:1. Notably, the TFCWs with a shunt ratio of 0:1 were regarded as the control group with an operation mode representing that of conventional TFCWs. Prior to the experiments, all the TFCWs with a shunt ratio of 0:1 were fed wastewater for three months to allow the development of plants and biofilms in the bed. Thereafter, The experimental period lasted for 616 days and was divided into six periods: (1) Period A (shunt ratio of 0:1), lasted for 91 d; (2) Period B (shunt ratio of 1:4), lasted for 93 d; (3) Period C (shunt ratio of 1:3), lasted for 93 d; (4) Period D (shunt ratio of 1:2), lasted for 123 d; (5) Period E (shunt ratio of 1:1), lasted for 123 d; and (6) Period F (shunt ratio of 2:1), lasted for 93 d. Since the TFCWs were placed indoors, influents and effluents ranged in temperature from 21 to 26 ◦ C throughout the study (data not shown), namely water temperature did not vary significantly over the 616 days of operation, which assured comparison of the shunt ratios across the six periods.

2.3. Analytical procedure Water samples were collected in triplicate once every three days from the inlet and outlet of each TFCW and analyzed immediately. Substratum samples were collected from each TFCW at least two times during each period. During the sampling event, substrate samples excavated from the sampling orifices were mixed evenly, placed in an ice incubator, and immediately sent to the laboratory for the follow-on experiments.

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Fig. 1. Schematic of the modified single-stage TFCW during a typical cycle (including Feeding Phase, Flood Phase, Drain Phase, and Rest Phase).

2.3.1. Water quality analyses Water quality analyses were conducted for temperature, pH, DO, TSS, COD, BOD, TN, NH4 + -N, NO2 − -N, NO3 − -N, and TP. The analyses (including TSS, COD, BOD, TN, NH4 + -N, NO2 − -N, NO3 − -N, and TP) were performed according to standard methods for assessing water and wastewater (APHA, 2002), pH values of the water samples were determined by a digital pH meter (PB-10, Sartorius, Germany), water temperature and DO were all measured in situ at the midpoint of the water depth from the vertical perforated pipe, using a portable HACH HQ30d Multi-Parameter analyzer. 2.3.2. Fluorescent in situ hybridization (FISH) Ammonium-oxidizing bacteria (AOB) and anammox bacteria populations at each operational period were identified by the FISH technique. All the biomass samples from the TFCWs were collected and fixed in 4% paraformaldehyde solution, and FISH was conducted according to Amann et al. (1990). The oligonucleotide probes were purchased as Cy3-, Cy5-, and fluorchromes fluorescein isothiocyanate (FITC)-labelled derivatives (Invitrogen, California, USA). Probes NEU653 and Amx820 were used to target Nitrosomonas-like AOB bacteria (Mobarry et al., 1996) and anammox bacteria genera “Candidatus Brocardia” and “Candidatus

Kuenenia”, respectively (Vázquez-Padín et al., 2010). Fluorescence signals were recorded with a LSM 710 confocal laser scanning microscope (CLSM) (Carl Zeiss, Inc., Germany). The CLSM images were analyzed by the standard software for the LSM 710.

2.3.3. Quantification of the nitrogen functional genes Soil DNA kits (D5625-01; Omega, USA) were used to extract and purify total genomic DNA. The genomic DNA extracted from the soil samples was detected by 1% agarose gel electrophoresis and stored at −20 ◦ C. The abundance of bacterial 16S rRNA, archaeal 16S rRNA, and nitrogen functional genes (amoA, nxrA, narG, nirS, nirK, nosZ, and anammox bacteria gene 16S rRNA) were determined by real-time PCR utilizing SYBR-green and a MyiQ2 Real-Time PCR Detection System (Bio-Rad, USA). Each 20-␮L reaction mixture included 10 ␮L of SYBR Green I PCR master mix (Applied Biosystems, USA), 1 ␮L of template DNA (sample DNA or plasmid DNA for standard curves), 0.5 ␮L of forward primers, 0.5 ␮L of reverse primers, and 8 ␮L of sterile water. Each amplification consisted of 40 cycles. The primer sequences and real-time PCR protocol were adopted from those reported by Ji et al. (2012).

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2.4. Statistical analysis Removal rates and transformation or accumulation rates of each form of nitrogen were calculated according to the formulas reported by Zhi and Ji (2014). One-way ANOVA was performed using SPSS 21.0. The threshold of significance was p < 0.05. Differences between means were evaluated using Duncan’s test. Pearson’s correlational analysis was used to identify significant linear relationships. Stepwise regression models were built to determine the quantitative response relationships between nitrogen transformation rates and functional genes using SPSS 21.0.

3. Results and analysis 3.1. Overall performance of TFCW under shunt ratio constraints As mentioned in Section 2.2, the low concentrations of TSS and organics were contained in the sewage owing to the anaerobic pretreatment. Further, there was little biodegradable organics in the sewage as can be seen in the low BOD influent concentration. Even so, effective removal of TSS, BOD, and COD were still achieved when the TFCW was operated under shunt ratio constraints, and variation of the shunt ratio did not reduce the rates at which TSS, BOD, and COD were removed (Table 1). Besides, Table 1 shows that an ideal effect on TP removal could also be observed at each shunt ratio, which should be attributed to the significant phosphorus adsorption capacity of oyster shell used in the TFCWs (Wang et al., 2013). Shunt ratio significantly affected nitrogen transformation and treatment performances of the TFCW. Fig. 2 shows that, the DO concentration at the beginning of Flood Phase reached up to (4.33 ± 0.31) mg L−1 as the TFCW was operated with the shunt ratio of 0:1, and most DO could be depleted during this phase. Simultaneously, a high NH4 + -N removal rate [(123.68 ± 4.92) mg (L d)−1 ] was observed on this occasion, whereas the TN removal rate was only (8.92 ± 1.07) mg (L d)−1 . Nitrogen in the effluent existed mainly in the form of NO3 − -N [(32.49 ± 2.49) mg L−1 ] and the ratio of nitrate production to ammonium consumption was (1.05 ± 0.09). The results demonstrated that a nitrification (conversion of NH4 + -N to NO3 − -N), which is often the limiting step for eliminating nitrogen from wastewater in treatment wetlands (Sun et al., 2003), occurred at a greater rate in the TFCW operated with a shunt ratio of 0:1. Generally, reaeration capacity of CWs has a great impact on the oxidation of NH4 + -N (Wu et al., 2015). Compared to the traditional CWs, the capacity of oxygen supply can reach up to 450 g (m2 d)−1 in TFCWs (Wu et al., 2011), which remarkably improves the removal effect of NH4 + -N. Nevertheless, the system could not provide suitable conditions to complete conversion of oxidized nitrogen to gaseous nitrogen forms, owing to the lack of organic carbon and the predominant aerobic environment. Correspondingly, no anammox bacteria (Amx 820, oligonucleotide probe special for anammox bacteria strains belonging to genus Candidatus brocardia and Candidatus kuenenia) were detected in the TFCWs by FISH analysis (data not shown). As the shunt ratio increased from 1:4 to 1:3, the DO concentration at the beginning of Flood Phase deceased from (3.60 ± 0.20) to (2.97 ± 0.12) mg L−1 , and the TN removal rates [(12.58 ± 1.34) mg (L d)−1 and (15.48 ± 3.57) mg (L d)−1 , respectively] were still low during Period B and Period C even though the higher NH4 + -N removal rates [(130.14 ± 7.10) mg (L d)−1 and (121.79 ± 8.68) mg (L d)−1 , respectively] obtained, indicating a feeble conversion of ammonium into nitrogen gas either through anammox or a denitrification process. Also no visible signals of anammox bacteria were apparent by FISH analysis during these two stages. Noticeably, nitrite clearly started to accumulate as Period C began, indicating an inhibition of nitrite oxidizing bacteria (NOB)

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under oxygen-limiting situation because of the adoption of stepfeeding. Generally, the inhibition of NOB can be achieved by using of limited aeration, which has been proven to be a strong controlling factor for partial nitrification (Peng and Zhu, 2006). The nitrite accumulation rate (NAR) reached the peak value of 77.96% on day 250 as the TFCWs operated during Period C. Fig. 2 also shows that, the ratio of nitrate production to ammonium consumption ranged from (1.05 ± 0.09) to (0.46 ± 0.14) as the shunt ratio increased from 0:1 to 1:3, which was different from the standard stoichiometric molar ratios of the CANON process (Sliekers et al., 2002). Thereafter, the shunt ratio continued to increase in order to create a stricter oxygen-limiting environment in the TFCW, and thus TN removal was improved partly in the TFCW since the level of DO within the bed could be optimized with the shunt ratio increased. Since the beginning of Period D, the DO concentration at the beginning of Flood Phase deceased to (1.69 ± 0.24) mg L−1 , and the TN removal rates began to increase on the promise that a high NH4 + N removal rate [(118.18 ± 6.69) mg (L d)−1 ] was maintained. NAR started to drop rapidly and the nitrite concentration of the effluent changed from 16.49 mg L−1 on day 280–5.25 mg L−1 on day 400. Notably, the TFCWs with the shunt ratio of 1:1 had the best nitrogen removal performances, the mean TN and NH4 + -N removal rates were (127.00 ± 13.78) and (124.77 ± 4.37) mg (L d)−1 , respectively. Correspondingly, the DO concentration at the beginning of Flood Phase remained at (1.03 ± 0.23) mg L−1 in the circumstances. In addition to the maximal TN removal rate [0.279 mg (L d)−1 ] obtained by Sun and Austin (2007), our finding was higher than other wetlands treating wastewater reported in the literatures [0.0024–0.084 mg (L d)−1 ] (Dong and Sun, 2007; Hu et al., 2014; Tao and Wang, 2009; Tao et al., 2011; Wen et al., 2013), even though types of wastewater in the studies are different. Specifically, our result was close to that reported by Third et al. (2001), who started up the CANON process in two different reactor types (sequencing batch reactor and chemostat) [0.120 mg (L d)−1 ]. As seen in Fig. 3, visible signals of oligonucleotide probes specific for anammox bacterial strains belonging to genus Candidatus brocardia and Candidatus kuenenia, were observed in the biofilm by FISH analysis as the TFCWs operated with the shunt ratios of 1:2, 1:1, and 2:1. Further, the CLSM images of the hybridized samples indicated that the AOB strains of Nitrosomonas spp. coexisted on the biofilm with the anammox bacterial strains of genus Candidatus brocardia and Candidatus kuenenia which existed in the internal anoxic layer of biomass. It is well understood that redox stratification within the microbial aggregate (due to oxygen transfer resistance) is the major cause of the CANON process: AOB is active in the outer layer of the aggregate and producing nitrite for anammox bacteria, which exist in the inner layers and are protected from oxygen in the bulk liquid (Van Hulle et al., 2010). These findings inferred that the CANON process in a single-stage TFCW could be significantly enhanced since the system operated with an appropriate shunt ratio. The stoichiometric molar ratios of nitrate production to ammonium consumption were showed a regular distribution after the appearance of anammox bacteria (Fig. 2). And the molar ratios approached gradually to 0.11 as Period E began, indicating the CANON process had been established in the TFCWs during Period E. Besides, we should also note that both TN and NH4 + -N removal rates decreased since Period F began, and the mean TN and NH4 + -N removal rates during the period deceased to (99.84 ± 14.51) and (84.43 ± 13.76) mg (L d)−1 , respectively. During Period F, the DO concentration at the beginning of Flood Phase deceased to (0.54 ± 0.20) mg L−1 , the molar ratios of nitrate production to ammonium consumption remained at (0.087 ± 0.054), and this represented a slight deviation from the standard ratio of CANON process. The data indicated that the nitrite produced by the Flood Phase might be insufficient for its consumption in the anam-

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Table 1 TSS, COD, BOD, and TP removal of TFCW under shunt ratio constraints. Influent

−1

TSS/mg L COD/mg L−1 BOD/mg L−1 TP/mg L−1

65.12 ± 24.51 79.40 ± 17.72 37.83 ± 12.57 13.64 ± 2.39

Effluent Shunt ratio 0:1

1:4

1:3

1:2

1:1

2:1

3.77 ± 1.01 20.60 ± 1.23 3.26 ± 2.21 0.74 ± 0.15

3.87 ± 0.78 23.38 ± 3.24 4.25 ± 1.28 0.63 ± 0.11

3.80 ± 0.72 22.28 ± 2.16 3.40 ± 1.74 0.74 ± 0.08

4.38 ± 0.84 25.16 ± 3.71 4.10 ± 1.47 0.70 ± 0.23

4.18 ± 1.21 21.43 ± 1.07 3.44 ± 2.06 0.77 ± 0.14

4.70 ± 1.39 23.94 ± 2.45 3.94 ± 2.17 0.83 ± 0.13

Fig. 2. Nitrogen transformation and treatment performances of the TFCW under shunt ratio constraints, and variations of DO concentrations at the beginning and end of Flood Phase in TFCW under shunt ratio constraints (Period A: shunt ratio of 0:1, Period B: shunt ratio of 1:4, Period C: shunt ratio of 1:3, Period D: shunt ratio of 1:2, Period E: shunt ratio of 1:1, Period F: shunt ratio of 2:1).

mox process owing to the obstruction of oxygen transfer caused by the shunt ratio of 2:1.

In the TFCW system, nitrogen and phosphorus from the influent could also act as nutrients for the development of the plants,

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Fig. 3. FISH analysis of substratum samples from the TFCWs: (A1 –A3 ) hybridization of biomass with Cy5-labelled Amx820 (red) on day 340, day 493, and day 586; (B1 –B3 ) hybridization of the same biomass with FITC-labelled NEU653 (green) on day 340, day 493, and day 586; (C1 –C3 ) simultaneous hybridization of biomass with Cy5-labelled Amx820 (red) and FITC-labelled NEU653 (green) on day 340, day 493, and day 586. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and all reeds survived and reproduced well without obvious symptoms of toxicity or nutrient deficiency till the experiments ended, indicating that there was no obvious adverse effect on the plant growth under the shunt ratio constraints. However, owing to the less quantity of plants in the lab-scale experimental device, plant uptake on nitrogen removal was neglected and oxygen secretion of rhizosphere in the system was also limited (data not shown). Overall, the optimal shunt ratio for nitrogen removal by the TFCWs used in this study was 1:1. On this occasion, high-rate autotrophic nitrogen removal via CANON process could be achieved in a single-stage TFCW.

3.2. Variation in the absolute abundance of nitrogen functional genes in TFCW under shunt ratio constraints The absolute abundance of key functional genes (i.e. bacterial 16S rRNA, anammox 16S rRNA, amoA, nxrA, narG, napA, nirK, nirS, qnorB, and nosZ), which involved in nitrogen transformation, were quantified during each period (Fig. 4). Adequate microbial biomass is considered as the guarantee to ensure the purifying capacity of a CW system (Vymazal, 2010). Fig. 4 shows that, the absolute abundance of bacterial 16S rRNA in the TFCW, which ranged from 1.16 × 109 copies g−1 to 1.32 × 109 copies g−1 throughout the experiment, provided guarantee for the development of CANON process in the system.

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Fig. 4. Absolute abundance of nitrogen transformation functional genes in the TFCW under shunt ratio constraints (Period A: shunt ratio of 0:1, Period B: shunt ratio of 1:4, Period C: shunt ratio of 1:3, Period D: shunt ratio of 1:2, Period E: shunt ratio of 1:1, Period F: shunt ratio of 2:1).

amoA is a marker of NH4 + -N oxidation to NO2 − -N (Dionisi et al., 2002), whereas nxrA is a marker of NO2 − -N oxidation to NO3 − -N (Poly et al., 2008). The anammox 16S rRNA gene, a marker of anammox, is involved in NH4 + -N transformation (Mulder et al., 1995). As shown in Fig. 4, after the acclimation of our TFCW, the absolute abundance of amoA, nxrA, and anammox 16S rRNA remained relatively stable throughout Period A. And the absolute abundance of the three functional genes were 6.53 × 105 , 1.78 × 104 , and 5.26 × 102 copies g−1 , respectively, at the end of Period A. The absolute abundance of amoA and nxrA, which were all much greater than that of anammox 16S rRNA during Period A, were still maintained at a high level during Period B. Notably, nxrA exhibited a variation trend similar to that of amoA, only with decreased abundance. As the system progressed from Period A to Period B, the absolute abundance of nxrA ranged from 1.81 × 104 copies g−1 on day 31–1.72 × 104 copies g−1 on day 184. The associated fluctuating pattern shown by amoA and nxrA was due to similar environmental adaptations and ecological interactions by AOB and NOB (Ji et al., 2012), while the corresponding lower abundance of nxrA in comparison with that of amoA should be attributed to the nitrification reaction process and the competitive advantages of AOB. Just unlike the above two functional genes, the absolute abundance of anammox 16S rRNA was kept at a low level before Period B ended. The finding suggested that aerobic oxidation of ammonia to nitrate was

the dominant NH4 + -N removal pathway in the TFCW during the two periods, and nitrification proceed at a greater rate with little nitrite accumulation. Nevertheless, almost no anammox occurred in the system, mainly due to the accumulation of nitrate and the overhigh DO concentration in the bed (Zhou et al., 2014; Yin et al., 2016). Since the shunt ratio was greater than 1:3, the absolute abundance of nxrA dropped rapidly as Period D began, owing to the inhibition of oxygen supply in the TFCW. However, the absolute abundance of amoA had no significant changes during Period D and Period E. On the other hand, the absolute abundance of anammox 16S rRNA increased from 4.40 × 103 copies g−1 on day 217–3.74 × 105 copies g−1 on day 463 and was maintained at a relatively high level until the end of Period E. It is a prerequisite for the anammox process that the antecedent nitrification process stops at nitrite by AOB (partial nitrification), i.e. the oxidation of nitrite to nitrate carried out by NOB has to be avoided (Miao et al., 2016). The finding indicated that partial nitrification could be achieved by regulating DO concentration in the bed with the adoption of shunt ratio, as low DO (≈1 mg L−1 ) has been proven to be the strong controlling factors for nitritation (Chen et al., 2016). Furthermore, the enrichment of anammox bacteria demonstrated that CANON process could be developed within a single stage TFCW under oxygen-limiting situation. Thus CANON process was enhanced to become a primary nitrogen elimination pathway gradually since the beginning of Period D. As the shunt ratio increased to 2:1, the absolute abundance of amoA and anammox 16S rRNA decreased significantly after the system uptime exceeded 556 d, indicating the excessive shunt ratio had an adverse impact on CANON process within the TFCW. Nitritation (i.e. the oxidation of NH4 + -N to NO2 − -N) should be inhibited since oxygen transport in the system was critically obstructed caused by the excessive shunt ratio, which ultimately hindered the CANON process due to the ruin of the microbial system involved in the process in the TFCW on that occasion. Fig. 4 shows the absolute abundance of narG, napA, nirK, nirS, qnorB, and nosZ, which are involved in denitrification. The denitrification pathway from nitrate to nitrogen gas can be divided into four steps as follows (Zhu and Chen, 2011): NO3 − -N → NO2 − -N → NO → N2 O → N2 As both narG and napA are involved in the first step of denitrification, the absolute abundance of each gene was summed (narG + napA) to represent the potential for conversion of NO3 − N into NO2 − -N (Bru et al., 2007). nirK and nirS, which are involved in the second denitrification step, are regarded as markers of the conversion of NO2 − -N into NO (Yan et al., 2003). qnorB is involved in the third denitrification step and regarded as a marker of the conversion of NO into N2 O (Braker and Tiedje, 2003). nosZ is involved in the last denitrification step and regarded as a marker of the conversion of N2 O into N2 (Stres et al., 2004). Owing to the appropriate enhancement of the anaerobic/anoxic environment in the TFCW, the absolute abundance of narG, napA, nirK, nirS, qnorB, and nosZ increased slightly during Period D and Period E; however, these changes were not significant because of the poor denitrification performance of the system, which was attributed to a lack of organics owing to a low BOD5 /N ratio (≈0.88) in the wastewater. Generally, in order to achieve denitrification of 1 g NO3 − -N to N2 , the organic material equivalent of 2.86 g BOD is  needed. It was found that a C/N ratio (measured as mg BOD5 : mg NOx − -N) of less than 2.3 would limit denitrification rates (Wang et al., 2014). Hence, nitrogen in the wastewater was difficult to be removed by conventional nitrification-denitrification process in the TFCW. Besides, the absolute abundance of narG, napA, nirK, nirS, qnorB, and nosZ decreased from the beginning of Period F because of the

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Table 2 Quantitative response relationships between nitrogen transformation rates and functional genes (n = 20). Equations −8

−3

v(TN) = 95.45 + 13.72·(anammox 16S rRNA + amoA/bacterial 16S rRNA) + 6.10 × 10 ·bacteria 16S rRNA − 3.42 × 10 ·nxrA v(NH4 + -N) = 71.31 + 1.04 × 105 ·(amoA/bacterial 16S rRNA) + 7.43·[(amoA + anammox 16S rRNA)/bacterial 16S rRNA] v(NO3 − -N) = −19.84 + 3.18 × 106 ·(nxrA/bacterial 16S rRNA) + 3.34 × 102 ·[(nxrA + anammox)/bacterial 16S rRNA] − 2.10 × 10−4 ·(narG + napA) v(NO2 − -N) = −1.27 + 9.89 × 105 ·(amoA/bacterial 16S rRNA) − 29.62·[(amoA + anammox 16S rRNA)/bacterial 16S

R2

p-Value

0.960 0.972 0.913 0.969

0.003 0.001 0.007 0.005

rRNA] − 1.20 × 107 ·(nxrA/bacterial 16S rRNA)

adverse impact on nitrogen transformation microbial system in the TFCW caused by the excessive shunt ratio.

3.3. Quantitative response relationships between nitrogen transformation rates and nitrogen functional genes In order to identify key functional genes that determine nitrogen transformation rates in the TFCW under shunt ratio constraints, a series of stepwise regression models were built to offer a linear quantitative measure of the relationships between changes in gene expression and nitrogen transformation rates. Ten nitrogen functional genes were used as candidate variables in stepwise regression analysis to associate their expression levels with nitrogen transformation rates. Functional gene groups that might illuminate the dynamics of nitrogen transformation processes were also introduced as variables in the stepwise regression analysis (as ratios or summations of the expression levels of different functional genes). Four regression equations describing changes in gene expression and the transformation rates of TN, NH4 + -N, NO3 − -N, and NO2 − -N yielded high R2 values ranging from 0.913 to 0.972 (Table 2). As shown in Table 2, the largest part of the variation in TN transformation rate was explained by three significant variables: (amoA + anammox 16S rRNA)/bacterial 16S rRNA, bacterial 16S rRNA abundance, and nxrA. (amoA + anammox 16S rRNA)/bacterial 16S rRNA was positively associated with the TN transformation rate, revealing that the CANON process was the main route for TN removal. Moreover, this variable also indicated that changes in the relative abundance, rather than the absolute abundance, of anammox 16S rRNA and amoA were responsible for their relationship with the TN transformation rate. The positive relationship between bacterial 16S rRNA abundance and the TN transformation rate indicated that effective TN removal from the TFCW was dependent to some extent on the presence of an adequate microbial biomass. Considering that nxrA is primarily involved in NO2 − -N oxidation, nxrA, which represents the extent of NO3 − -N accumulation, was negatively associated with the TN transformation rate. This variable could be explained by the finding that the activity of anammox could be inhibited owing to the NO3 − -N accumulation (Zhou et al., 2014). The equation indicated that TN removal from the TFCW relied largely on the CANON process. The transformation rate of NH4 + -N was determined by assessing (amoA/bacterial 16S rRNA), and [(amoA + anammox 16S rRNA)/bacterial 16S rRNA]. Both of the variables were positively associated with the NH4 + -N transformation rate. amoA is involved in NO2 − -N production; therefore, changes in amoA abundance could reflect NO2 − -N accumulation caused by nitritation. Increased NO2 − -N accumulation is associated with enhanced NH4 + -N transformation. [(amoA + anammox 16S rRNA)/bacterial 16S rRNA] indicated that the CANON process was also responsible for NH4 + -N removal, as NH4 + -N is directly involved in the CANON process. Therefore, the main NH4 + -N transformation pathways included the nitrification process and the coupling of the CANON process with NO2 − -N produced by nitritation/denitrification. The NO3 − -N accumulation rate was collectively determined by (nxrA/bacterial 16S rRNA), [(nxrA + anammox)/bacterial 16S rRNA],

and (narG + napA). The first two variables, which were positively associated with the NO3 − -N accumulation rate, denoted NO3 − -N production. As nxrA and anammox are involved in the nitrification process and the CANON process, respectively, which are the processes contributing to NO3 − -N production. While (narG + napA) was negatively associated with the NO3 − -N accumulation rate, as narG and napA are involved in NO3 − -N consumption in the denitrification process. The main pathway for NO3 − -N removal should be denitrification. Although anammox could remove NO3 − -N when NO3 − -N was converted to NO2 − -N by narG/napA, Wang et al. (2012) has demonstrated that aerobic ammonium oxidation, rather than nitrate reduction, provides a direct local source of nitrite for anammox in the suboxic zone. The largest part of the variation in the NO2 − -N accumulation rate was jointly determined by (amoA/bacterial 16S rRNA), [(amoA + anammox 16S rRNA)/bacterial 16S rRNA], and (nxrA/bacterial 16S rRNA). (amoA/bacterial 16S rRNA), which denoted NO2 − -N accumulation, was positively related with the NO2 − -N accumulation rate. The other two variables, which represented NO2 − -N consumption via the CANON and nitrification processes, respectively, was negatively related to the NO2 − -N accumulation rate. This equation indicated that NO2 − -N transformation in the system was due mainly to the nitrification process and the CANON process, suggesting an ecological and functional interaction between the nitrifying microorganisms and anammox.

4. Discussion It could be concluded that different nitrogen transformation processes could be coupled in the TFCW at the molecular level to collaboratively contribute to nitrogen transformation, and certain nitrogen functional genes acted as key factors regulating the transformation rate of each form of nitrogen. Specifically, the abundance of functional genes that play a significant role in the proper functioning and maintenance of wetland systems is mainly dependent on environmental parameters, wastewater properties, and operating conditions. Changes in pollutant removal rates can be caused by changes in one or two factors that are closely related to the abundance of genes involved in nitrogen transformation (Truu et al., 2009). Therefore, variations in nitrogen transformation rates throughout the experiment could be attributed to fluctuations in the absolute abundance of nitrogen functional genes caused by differences in the microenvironment (e.g. the distribution of DO and organics) of the bed under shunt ratio constraints. When the TFCW operated with adoption of a step-feeding mode, the microenvironment of the bed could be optimized by regulating the shunt ratio so as to facilitate the occurrence of the CANON process in the system. TN removal in the TFCW with the shunt ratio of 1:1 relied largely on the CANON process by amoA and anammox 16S rRNA, and the main NH4 + -N transformation pathway was the coupling of the CANON process with NO2 − -N produced by nitritation. On the other hand, NOx − -N transformation in the system was due mainly to coupling of nitrification and anammox. Notably, denitrification was inhibited and denitrifying microorganisms could not contribute to NOx − -N transformation in the TFCWs owing to the lack of organic carbon.

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Consequently, we developed a single-stage constructed wetland for nitrogen removal that achieved a high TN removal rate via CANON process. The successful development of a single-stage CW for autotrophic nitrogen removal demonstrates the potential of such systems to replace hybrid systems that combine various types of CWs, as well as to reduce the costs of aeration and capital construction, especially for the low C/N ratio wastewater treatment. Also note that there will be several issues need to be further explored if this technique is put into practice. One major issue is the effect of wetland plants on nitrogen transformation. In general, presence of plants is essential for CWs in terms of improving nitrogen removal performances (Leverenz et al., 2010; Herouvim et al., 2011), thus the effect of plants on nitrogen transformation (especially the CANON process) can’t be neglected as the TFCWs with step-feeding were applied in the practical engineering. So, the issue needs to be further exploded in the follow-up study. Another issue is the effect of temperature on the nitrogen transformation in CWs. Literatures report that temperature at a higher or lower level has adverse effects on nitrogen removal due to the fluctuations in the activities of the nitrogen transforming bacterial (Liu et al., 2016; Gonzalez-Martinez et al., 2016). In the practical engineering, CWs usually operate under the outdoor conditions, and the variation of temperature may lead to the instability of the microbial system involved in the CANON process. Hence, this issue should also be noticed. Overall, this is a primary report of study, more information on optimizing CANON process in CWs still need to be going on. 5. Conclusions Shunt ratio significantly affected nitrogen transformation pathways in the TFCW with step-feeding. The anammox bacteria were enriched most effectively at the shunt ratio of 1:1, and then initiation of the CANON process was accomplished in the TFCW under an appropriate limited oxygen condition. The mean TN removal rate reached up to (127.00 ± 13.78) mg (L d)−1 correspondingly. It could be concluded that autotrophic nitrogen removal via CANON process developed in the TFCW with optimized microenvironment that developed as a result of appropriate shunt ratio. The optimal shunt ratio of the TFCW for nitrogen removal was 1:1. Acknowledgments This research was sponsored by the National Natural Science Foundation of China (51508002), the Natural Science Foundation of Anhui Province (1508085QE99), and the Youth Fund Project of Anhui Agricultural University (YJ2015-20). References APHA, 2002. Standard Methods for the Examination of Water and Wastewater. American Public Health Assoiation, Washington, DC., USA. Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., Stahl, D.A., 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56 (6), 1919–1925. Braker, G., Tiedje, J.M., 2003. Nitric oxide reductase (norB) genes from pure cultures and environmental samples. Appl. Environ. Microbiol. 69 (6), 3476–3483. Bru, D., Sarr, A., Philippot, L., 2007. Relative abundances of proteobacterial membrane-bound and periplasmic nitrate reductases in selected environments. Appl. Environ. Microbiol. 73 (18), 5971–5974. Chen, Z., Wang, X., Yang, Y., Mirino Jr., M.W., Yuan, Y., 2016. Partial nitrification and denitrification of mature landfill leachate using a pilot-scale continuous activated sludge process at low dissolved oxygen. Bioresour. Technol. 218, 580–588. Dionisi, H.M., Layton, A.C., Harms, G., Gregory, I.R., Robinson, K.G., Sayler, G.S., 2002. Quantification of Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR. Appl. Environ. Microbiol. 68 (1), 245–253.

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