Advanced oxygenation efficiency and purification of wastewater using a constant partially unsaturated scheme in column experiments simulating vertical subsurface flow constructed wetlands

STOTEN-135480; No of Pages 9 Science of the Total Environment xxx (xxxx) xxx

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Advanced oxygenation efficiency and purification of wastewater using a constant partially unsaturated scheme in column experiments simulating vertical subsurface flow constructed wetlands Xinhui Zheng a,1, Lin-Lan Zhuang b,1, Jian Zhang b,c,⁎, Xiangzheng Li b, Qian Zhao b, Xiran Song b, Cheng Dong b, Jiayi Liao b a b c

Institute of Marine Science and Technology, Shandong University, Qingdao 266237, China Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science & Engineering, Shandong University, Qingdao 266237, China State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Unsaturated strategy enhances oxygen supply in simulating constructed wetland (SCW). • Partially unsaturated SCW performed higher ammonia nitrogen removal. • Partial unsaturated SCW performs higher organic removal. • Unsaturated strategy reached the oxygen uptake of 336 ± 44 g m−3 d−1.

a r t i c l e

i n f o

Article history: Received 27 July 2019 Received in revised form 8 November 2019 Accepted 9 November 2019 Available online xxxx Editor: Jan Vymazal Keywords: High oxygen supply Ammonia nitrogen removal Organics removal Constructed wetland Unsaturated zone

a b s t r a c t The presence of sufficient dissolved oxygen (DO) in a constructed wetland (CW) is vital to the process of removing ammonia nitrogen and organics from wastewater. To achieve total nitrogen removal, which is characterised by enhanced ammonia nitrogen removal, this study offers an efficient strategy to increase the oxygen supply by establishing constant unsaturated zones and baffles in simulating constructed wetlands (SCWs). Henceforth, this strategy is addressed as a partially unsaturated SCW. A centrally located high tube was set up inside the wetland to create an unsaturated zone at a higher level. The effectiveness of the unsaturated zone to supplement the oxygen content was evaluated by comparing with controls (an unaerated SCW and an aerated SCW). The results show the chemical oxygen demand removal rate (85 ± 6%) in the partially unsaturated SCW was equivalent to that in the aerated SCW (83 ± 6%), while the ammonia nitrogen removal rate was 11 times higher compared to that of the unaerated SCW. The removal potential of the partially unsaturated SCW under different HRT (hydraulic retention time)s (12, 24, and 36 h) was examined, and the 36 h-SCW performed the best in the removal of organics and nitrogen. The mechanisms behind the unsaturated zone strategy were studied by analysing water and microbe samples along the pathway. The results from the water quality indicators and the quantitative polymerase chain reactions along the pathway showed the unsaturated zone contributed to the removal of primary

⁎ Corresponding author at: Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science & Engineering, Shandong University, Qingdao 266237, China. E-mail addresses: [email protected] (X. Zheng), [email protected] (L.-L. Zhuang), [email protected] (J. Zhang). 1 Xinhui Zheng and Lin-Lan Zhuang contributed equally to this study.

https://doi.org/10.1016/j.scitotenv.2019.135480 0048-9697/© 2019 Published by Elsevier B.V.

Please cite this article as: X. Zheng, L.-L. Zhuang, J. Zhang, et al., Advanced oxygenation efficiency and purification of wastewater using a constant partially unsaturate..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135480

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organics and ammonia nitrogen. The superior performance of unsaturated zone strategy was discussed further using the enrichment of ammonia-oxidising bacteria, mass of oxygen uptake, and baffle design. The results indicate that the amoA gene/16s rRNA gene abundance ratio and the oxygen uptake (336 ± 44 g m−3 d−1) in the partially unsaturated SCW was higher than that observed in the two controls. © 2019 Published by Elsevier B.V.

1. Introduction The concept of constructed wetlands (CWs) was first proposed as an ecological technology in the 1960s to be used for the purpose of advanced wastewater treatment. It became the favoured technology to tackle water pollutants due to its low energy demands, low maintenance costs, and excellent ecological service values. Among the contaminants in water, ammonia nitrogen is toxic to many aquatic plants and animals (Liu and von Wiren, 2017). For example, Egnew et al. (2019) showed that the 96 h-LC50 (lethal concentration of 50%) of ammonia is 33.24 mg/L for a largemouth bass. Organics, in terms of chemical oxygen demand (COD), are the carbon sources used by heterotrophic microbes. Wastewater, containing ammonia and organics, could cause serious ecological problems if discharged into waterbodies (Silva et al., 2018; Zheng et al., 2019). Hence, the careful removal of contaminants in receiving water continues to be an urgent challenge that needs to be addressed. Regarding the traditional nitrogen eliminating mechanisms, both aerobic and anaerobic microbial processes are the dominant ways used for wastewater purification in subsurface constructed wetlands, other than the physical processes of sedimentation and filtration (Saeed et al., 2016). Moreover, the dissolved oxygen (DO) content in the CW determines the efficiency of ammonia nitrogen removal and the thorough decomposition of organics (i.e. COD). Three channels through which oxygen is supplied to the CWs were mentioned before (Liu et al., 2016; Wang and Chu, 2016), including influent oxygen supply, oxygen released from plant roots, and through atmospheric reaeration. However, the DO content is usually deficient for nitrification processes because the limited DO supply is insufficient to meet the demands of the contaminant removal process. Furthermore, organic degradation has an advantage over nitrification when competing for DO. Many studies that showed relatively poor decontamination effectiveness contended that the DO content was rather low in the CWs (Matamoros et al., 2008; Ong et al., 2010a). To achieve higher efficiency in ammonia removal, the oxygen supply effect of the CWs needs to be urgently improved. Considering that DO content plays a key role in the degradation of ammonia nitrogen and organics, a number of materials were utilised in the last decade to help eliminate ammonia nitrogen and organics from wastewater in a CW system, along with the introduction of several new strategies to create an aerobic environment (Vymazal, 2007, 2011; Wu et al., 2014). Currently, the strategies related to tidal flow, drop aeration, and artificial aeration are widely used in vertical subsurface flow CWs to supplement the oxygen supply necessary for continuous wastewater treatment. The tidal flow operation consists of a drain phase and flood phase, where the wetland beds are oxygenated with air in the drain phase, while reverting back quickly to an anoxic condition at the beginning of the flood phase (Han et al., 2019). Hu et al. (2012b, 2014) pointed out that the permanence of the oxygenating property is limited after the flood phase, with a DO content of around 1.0 mg/L just after flood, which then reduces to a strictly anoxic condition 2 h after the flood in an up-tidal flow CW under room temperature. Additionally, the flood might also flush out some microorganisms during the drain process (Wu et al., 2014), which increases the turbidity of the effluents and reduces the treatment efficiency. The drop aeration oxygenating strategy generally enhances the DO content by raising the height of the influent and improving flux, leading to an increase in the DO content

in the areas near the influent of the wetland bed. Zhai et al. (2012) adopted a cascaded drop aeration approach to increase the DO concentration to 3.80 ± 0.11 mg/L in the case of a horizontal subsurface flow CW with 0.45 m water depth at a temperature of 17.3–19.1 °C, but this high DO concentration reduced to 0.31 ± 0.18 mg/L in the first 17% of the CW's flow path. The artificial aeration strategy, although effective in supplementing the oxygen content, is generally regarded as highly energy-consuming and unsuitable for economically underdeveloped areas. Austin and Nivala (2009) compared the energy requirements for the artificial aeration operation and the tidal flow operation in CWs and found that the energy requirements of the artificial aeration operation were 2.3 times higher than that of the tidal flow operation. Thus, an oxygenation strategy with low energy consumption and durability is imperative during the application process for an actual operation. To understand the supply of DO in a CW, specific attention has been given to unsaturated CWs in recent years. In unsaturated CWs, the substrate and the biofilm on the surface are not submerged by water. Instead, water flows on the surface of the substrate as a thin layer above the biofilm and helps improve the oxygen transfer rate from air to the microbes, which enhances the subsequent oxidation of ammonia and COD (Nivala et al., 2013). Studies on saturated and unsaturated CWs are implemented more often these days. Total unsaturated CWs were considered less desirable when compared to partially unsaturated CWs with respect to nitrogen removal (Pelissari et al., 2018). Furthermore, Sgroi et al. (2018) reported that partially unsaturated CWs perform better than total unsaturated CWs in terms of total nitrogen (TN) reduction, which is inconsistent with the results obtained by Saeed and Sun (2017), where the total unsaturated area shows superior removal efficiency compared to the partially unsaturated CW. Nevertheless, the mechanisms behind the oxygenating potential of unsaturated strategies were rarely mentioned. Thus, there is an urgent need for further research to provide experimental results that can act as a reference, and to understand the oxygenating and pollutant removal mechanisms. A simulating constructed wetland (SCW) with an advanced, more efficient oxygenation strategy, which uses constant unsaturated zones and baffles, was established based on the oxygen regulation mechanisms. The term ‘constant unsaturated zone’ indicates that there was a permanent unsaturated functional zone as well as a permanent saturated functional zone in the SCW. In this study, the following aspects were examined: (1) the effectiveness of oxygen supply by the constant unsaturated zone; (2) the removal potential under different HRTs (12, 24, and 36 h); (3) the mechanism behind the constant unsaturated strategy; and (4) the superiority of the constant unsaturated strategy.

2. Materials and methods 2.1. Configuration of the unsaturated constructed wetland 2.1.1. Configuration of the SCW To further understand the operating mechanisms of the constant partially unsaturated SCW and use the limited space adequately, a centrally located high tube was set up inside the wetland bed to form a baffle design, where a constant unsaturated zone was formed to promote the efficient removal of pollutants, particularly ammonia nitrogen and organics. The influent entered from the top of the central tube, flowing

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down through the inner tube, and then turned upwards and flowed out from the top of the outer ring of the wetland. Five SCW systems were established at Shandong University in Tsingtao, China (36°22′4.10″N, 120°41′16.13″E) using polythene columns in the laboratory with continuous influent and effluent patterns, as depicted in Fig. 1. The laboratory temperature was maintained at 20.0 ± 0.2 °C using an air conditioner. Three partially unsaturated SCW systems were chosen to represent the experimental groups, differing only with respect to their hydraulic retention times (HRTs), which were 12, 24, and 36 h, respectively. An unaerated SCW system and an aerated SCW system were chosen as the controls. The three partially unsaturated SCWs consisted of an outer column, which was 82 cm high and 30 cm in diameter as well as a suspended inner column, which was 90 cm high and 20 cm in diameter (Fig. 1). There was polythene base floor at the lowest side of outer column, but no base was present in the inner column, in order to enable the wastewater to flow into the system from the top of the inner column, and then flow out from the top of the outer column. The polythene columns of the unaerated SCW and the aerated SCW were similar to the outer columns of experimental systems in terms of the diameter. Both the control SCW systems had a height of 70 cm to maintain a similar bulk of substrates, and ensure that the wastewater flowed from bottom to top. An aeration disc was placed at the bottom of the aerated SCW with an aeration rate of 0.2 L/min (Zhang et al., 2018). For the experimental group, ceramsite was filled (Jing et al., 2015) (diameter of 8–10 mm, 50% porosity) for the top 50 cm of the inner column, and a quartz sand filling (diameter of 2–6 mm, 40% porosity) for the rest of the inner column and the lower 50 cm of the outer column. Perforated pipes were inserted in every SCW bed in order to collect water samples as well as monitor physical and chemical indicators in situ. According to studies conducted on nitrogen removal in vertical subsurface flow CWs, the nitrogen removal efficiency relied more on the contribution of microbes adhering to the substrate rather than the role of plants (Maltais-Landry et al., 2009; Reinhardt et al., 2006). Since the role of the operation design seemed prior to plants, Nivala et al. (2019) excluded plants in their reciprocating system to clearly highlight the superiority of the reciprocating design. Taking into consideration that the aim is to explore oxygenation efficiency of the unsaturated substrate zone without the interference of plants, no plant was grown in the SCWs in this study as well. Actually, the experiments in this study are column experiments simulating vertical subsurface flow constructed wetlands. 2.1.2. Simulated water The synthetic water used for the experiments was simulated to resemble secondary effluents from wastewater treatment plants (WWTPs) in China using tap water. The detailed influent ingredients

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dosage per litre is presented in Table 1 to compose the simulated water with the hydraulic loading rate (HLR) of 0.58, 0.29, 0.19, 0.58, 0.58 m3/m2d, the organic loading rate of (OLR) of 58, 29, 19, 58, 58 g COD/m2d, the nitrogen loading rate (NLR) of 22.6, 11.3, 7.4, 22.6, 22.6 g N/m2d, and the phosphorus loading rate (PLR) of 1.7, 0.9, 0.6, 1.7, 1.7 g P/m2d in 12 h- partially unsaturated SCW, 24 h- partially unsaturated SCW, 36 h- partially unsaturated SCW, aerated SCW and unaerated SCW respectively. Additionally, FeSO4·7H2O (10.00 mg/L) and microelement solutions (1.0 mL/L) were utilised based on Wu et al. (2011a), and the continuous influent and effluent patterns observed were applied in our experiment. In order to ensure the stability of the influent ingredients, an ultraviolet lamp was placed in the influent bucket, which was cleaned and disinfected using Javel water every day. 2.2. Water samples and analysis Effluents in each SCW were collected from the outlets every two or three days to determine the purification efficiency of the SCWs. Water samples along the flow path were collected every 10 cm, from the inlet to the outlet, to find out the changes of water quality along the path in SCWs. In the partially unsaturated SCW, the first batch of water samples along the flow path was collected through a perforated pipe at the boundary between the unsaturated zone and the saturated zone in the inner tube, and the last batch of water samples was collected from the perforated pipe at the top of the outer tube. In the unaerated and the aerated SCWs, the flow path water samples were collected through a perforated pipe from the bottom to the top. All the water samples were analysed immediately after collection. The water quality indicators, including COD, TP, TN, NH+ 4 -N, nitrite − (NO− 2 -N), and NO3 -N were characterised using the exemplar method (APHA, 2005). DO was detected in situ in the perforated pipe using a DO meter (HQ40d, Hach, USA), and pH was determined in situ in the perforated pipe using a pH-meter (SG2, METTLER TOLEDO, Switzerland). 2.3. Quantitative PCR (q-PCR) assays At the end of the experiment, substrate samples were collected every 10 cm along the flow path in the wetland bed. In the partially unsaturated SCW, samples from both the unsaturated zone and the inner/ outer saturated zone were collected at different heights. In the unaerated and the aerated SCWs, substrate samples were collected from the bottom to the top in the wetland bed. All biofilm samples were separated from the substrate using sonication (Lan et al., 2018). Genomic DNA was extracted from the biofilm samples using the E.Z.N.A. Soil DNA Kit (Omega) (Sun et al., 2018). The biofilm samples

Fig. 1. Sketch of the configured SCW systems.

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Table 1 Detailed dosage of influent ingredients in theoretical simulated water. Indicators

Simulated water concentration

Ingredients

Dosage of ingredients

Chemical oxygen demand (COD) Ammonia nitrogen (NH+ 4 -N) Nitrate (NO− 3 -N) Total phosphorus (TP)

100 mg/L 25 mg/L 14 mg/L 3 mg/L

Sucrose Ammonium sulphate Potassium nitrate Mono potassium phosphate

89.14 mg/L 117.98 mg/L 101 mg/L 13.18 mg/L

and the genomic DNA were then frozen at −20 °C. Ammonia monooxygenase (amoA) helps encode the first step of nitrification, and was, therefore, chosen as a genetic signal for the ammoniaoxidising bacteria (Dionisi et al., 2002). Nitrate reductase (narG), nitrite reductase (nirS and nirK), and nitrous oxide reductase (nosZ) were used as the functional genes of denitrification. 16s rRNA was analysed to examine the total bacterial content. The functional genes were detected using the quantitative polymerase chain reaction (q-PCR) method (Roche LightCycler 480). 2.4. Statistical analysis For the purpose of statistical analysis, all water samples and substrate samples were collected in triplicate. To estimate the statistically significant differences between the different SCWs in this study, ttests were employed and conducted using the SPSS 19.0 program (SPSS Inc. Chicago, USA). A significant difference between two samples is indicated by a p value that is b0.05 (Zhang et al., 2018). 3. Results and discussion 3.1. Overall removal performance 3.1.1. Removal performance of the partially unsaturated SCW After 30 days, the five SCWs reached a period of stability, and were then operated for another 80 days. Water quality indicators (e.g. TN, TP, and COD) were monitored every two or three days to determine the wastewater purification efficiency of the SCWs. The relevant parameters are presented in Table 2. After water flew through the unsaturated zone, the DO detected in situ at the boundary between the unsaturated zone and the saturated zone was constantly 5.08 ± 0.54 mg/L. The COD removal rate of the partially unsaturated SCW reached around 85 ± 6%, which was similar to the removal rate of the aerated SCW (83 ± 6%). The COD removal rate of the unaerated SCW was relatively low compared to the others (p b 0.05). The ammonia nitrogen removal rate of the unaerated SCW was 3.8 ± 1.9%, while the removal rate of the partially unsaturated SCW remained steady at 46.2 ± 11.9%. The ammonia nitrogen removal rate of partially unsaturated SCW was 11 times that of the value for the unaerated SCW, but was lower than the removal rate of the aerated SCW (87.3 ± 2.4%) (p b 0.05). Saeed and Sun (2017) also treated municipal wastewater that contained an ammonia nitrogen concentration of 33.4 ± 12.1 mg/L using a partially unsaturated CW. The average purification rate of ammonia nitrogen in their study was 36.3%, which was

not comparable to the results obtained from the partially unsaturated SCW in our study. In this study, the concentration of TN was calculated as a sum of the − − NH+ 4 -N, NO3 -N and NO2 -N. The TN removal rate obtained was 36.0 ± 7.2%, which was significantly lower than the removal rate obtained for the unaerated SCW as well as the aerated SCW. These results can be further explained by the unsatisfactory denitrification efficiency in the saturated zone. However, with respect to TP, the removal rate of 32.2 ± 5.4% observed in the partially unsaturated SCW was significantly higher than the removal rate observed in the unaerated (6.5 ± 4.4%) and the aerated (16.6 ± 6.6%) SCWs. Taking into account that no plants were grown in this study, the reason behind the superior phosphorus removal rate is the better adsorption performance observed in the unsaturated zone when compared to the other two saturated SCWs. Additionally, the COD and ammonia nitrogen removal in the SCWs show higher rates of efficiency when the microbes remain in an aerobic environment (Hu et al., 2012a). Furthermore, the high removal efficiency of nitrate in the unaerated SCW and the distinct removal of ammonia nitrogen in the aerated SCW were consistent with the results obtained in previous studies (Lyu et al., 2018; Wu et al., 2014). During this study, a zone above the aerated disc, which is approximately 10 cm high, in the aerated SCW got clogged at the end of the process. This could be deconstructed as a boost in aerobic microbes that interrupts the oxygen diffusion from the aerated disc to the upper zone (Ren et al., 2015). The results indicate that the partially unsaturated strategy has a strengthening effect in terms of oxygen supply, providing a stronger aerobic environment than the unaerated SCW. In engineering applications, process pump energy is the basic process of energy consumption seen in different types of SCWs. With the exception of the indispensable pumps, all other means of energy consumption are combined to achieve different goals. For instance, blowers are installed to aerate the wetlands (Austin and Nivala, 2009). Although the oxygenating effect in the partially unsaturated SCW is similar to the effect seen in the aerated SCW, the superiority of energy conservation during oxygenation by the unsaturated substrates enables the partially unsaturated SCW to develop greater long-term profits. 3.1.2. Influence of different HRTs on purification of wastewater Three partially unsaturated SCWs were studied to explore their removal potential under different HRTs of 12, 24, and 36 h. t-Tests were applied to estimate the differences between the three SCWs. With respect to the COD and NH+ 4 -N removal rates, there was no significant difference between the 12 h-SCW and the 24 h-SCW, but the removal rates for both the 12 h-SCW and the 24 h-SCW were significantly lower than

Table 2 Water quality indicators for influents and effluents across the partially unsaturated SCW, the unaerated SCW, and the aerated SCW (mean ± SD, n = 20). Indicators

COD NH+ 4 -N NO− 3 -N − NO2 -N TN TP

Influent (mg/L)

104 ± 8 24.3 ± 3.8 15.5 ± 2.2 0 39.9 ± 2.3 3.0 ± 0.2

Partially unsaturated SCW

Unaerated SCW

Effluent (mg/L)

Removal rate (%)

Effluent (mg/L)

Removal rate (%)

Effluent (mg/L)

Aerated SCW Removal rate (%)

16 ± 5 13.5 ± 3.0 14.7 ± 2.5 0.7 ± 0.6 28.8 ± 3.7 2.0 ± 0.2

85 ± 6 46.2 ± 11.9 1.9 ± 1.7 – 36.0 ± 7.2 32.2 ± 5.4

20 ± 4 24.1 ± 1.2 0.7 ± 0.4 0.3 ± 0.3 25.1 ± 1.3 2.8 ± 0.3

80 ± 5 3.8 ± 1.9 96.1 ± 1.9 – 41.6 ± 3.1 6.5 ± 4.4

17 ± 6 3.2 ± 0.5 19.5 ± 2.4 0.1 ± 0.1 22.6 ± 1.8 2.5 ± 0.4

83 ± 6 87.3 ± 2.4 −8.4 ± 5.9 – 47.3 ± 4.2 16.6 ± 6.6

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the rate observed for the 36 h-SCW (p b 0.05). In general, the removal efficiency for organics and ammonia nitrogen would be enhanced with a suitable increase in HRT (Caselles-Osorio and Garcia, 2006; Ong et al., 2010b). The significant difference observed between the 24 hSCW and the 36 h-SCW supports this general conclusion. The longer the HRT, the longer the water stays on the surface of the biofilm, which increases the mass flux potential between the inside and the outside of the biofilm under an oxygenated environment in an unsaturated zone. However, it is important to note that unusual phenomenon though the HRT was doubled, the contaminant removal rate did not show a comparative increase. Thus, when this technology is applied at a large scale, an optimal HRT should be chosen carefully to trade off the contaminant removal rate and the removal efficiency (Fig. 2). 3.2. Mechanism of degrading nitrogen and organics along the flow pathway To examine the oxygen supply and the contaminant purification effect of the partially unsaturated SCW, water quality indicators were monitored along the flow pathway in the partially unsaturated SCW, the unaerated SCW, and the aerated SCW. After 120 days, all the SCWs were closed, and microbes samples attached to the substrates along the flow pathway were collected for the q-PCR analysis. The DO concentration in the influents was 6.68 ± 0.07 mg/L for all three SCWs. In the partially unsaturated SCW, the DO concentration showed a slight decrease (5.03 ± 0.54 mg/L) in the unsaturated zone of the path between 0 cm and 50 cm, which was consistent with the results from Nivala et al. (2019). A sharp decrease (0.05 ± 0.01 mg/L) was then observed when the water entered the saturated zone between 50 cm and 90 cm, followed by a smooth increase (1.08 ± 0.16 mg/L) at the surface of the outer saturated zone due to atmospheric reaeration (Fig. 3a). In comparison, the DO concentration in the unaerated SCW showed a sharp decrease (0.21 ± 0.06 mg/L) within a length of 10 cm once the wastewater entered the SCW, and then stabilised (around 0.18 mg/L to 0.49 mg/L; Fig. 3b), while the DO concentration in the aerated SCW was observed at a range of 3–5 mg/L in the wetland bed (Fig. 3c). The DO concentration was supposed to exceed the minimum demand of 2.0 mg/L for the nitrification process, but was not supposed to surpass the maximum concentration of 0.2 mg/L for denitrification (Beebe et al., 2015). In the partially unsaturated SCW, the DO concentration distribution is able to supply diverse environments and aid in nitrogen removal processes, including nitrification, mainly in the unsaturated zone, and denitrification, mainly in the saturated zone. The DO concentration

Fig. 2. Effluent water quality indicators in three partially unsaturated SCWs (12 h-SCW, 24 h-SCW, and 36 h-SCW). The data is presented as mean ± SD. Two-sample t-tests were applied to evaluate the significance among the three SCWs; a and b indicate significant differences at the p b 0.05 level.

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distribution in the unaerated SCW and the aerated SCW can only provide a limited anaerobic/anoxic or aerobic environment. As depicted in Fig. 3(a, b, and c), the COD concentration declined continuously across the entire flow pathway in each SCW. In the partially unsaturated SCW, nitrogen transformation was observed in the unsaturated zone, where the ammonia nitrogen levels decreased and nitrate accumulated, while the overall TN removal rate clearly declined in the unsaturated zone. The decline of TN, beginning at the unsaturated zone, might be explained by the simultaneous nitrification and denitrification (SND) processes occurring due to DO diffusion in the biofilm (Cao et al., 2017). On the other hand, the nitrogen transformation process was no longer distinct in the saturated zone, except for a slight decrease in nitrate concentration. Moreover, nitrite, in general, was detected at low concentrations in each SCW (1.0 mg/L). In the unaerated SCW, the main factor influencing TN removal was nitrate degradation, which showed a sharp decline within the first 10 cm, but maintained a steady concentration along the flow pathway. No evident changes in ammonia nitrogen were detected along the entire pathway. In the aerated SCW, ammonia nitrogen conversion exhibited an overall downward trend, but nitrate conversion showed an upward trend, with the effluent nitrate being detected at an even higher level than the influent. Fig. 3 depicts the distinct potential for degradation of organics and ammonia nitrogen in the unsaturated part of the partially unsaturated SCW, along with TN removal. Although the COD was yet to undergo degradation in the saturated zone of the flow pathway, the degradation of the COD in the unsaturated zone accounted for 70% of the entire pathway (Fig. 3a), which was consistent with another study on partially unsaturated SCWs (Al-Saedi et al., 2018). During the nitrogen transformation process, functional genes (amoA, narG, nirS, nirK, nosZ) encode enzymes that perform the nitrogen transformation reactions. The related reactions in this study are as follows (Kuypers et al., 2018): NH4 þ þ O2 þ 2e− þ Hþ →NH2 OH þ H2 O ðamoAÞ;

ð1Þ

NO3 − þ 2e− þ 2Hþ →NO2 − þ H2 O ðnarGÞ;

ð2Þ

NO2 − þ e− þ 2Hþ →NO þ H2 O ðnirS&nirKÞ;

ð3Þ

N2 O þ 2e− þ 2Hþ →N2 þ H2 O ðnosZÞ:

ð4Þ

From 0 cm to 50 cm in the unsaturated zone in the partially unsaturated SCW, nitrogen-related gene abundance was observed (Fig. 4a). amoA is commonly considered as the main functional gene of the ammonia-oxidising bacteria (Dionisi et al., 2002), and its absolute abundance was observed at an average amount of 1.76 × 106 copies g−1 substrate. This was found to be more than the 4.04 × 105 copies g−1 substrate observed in the saturated zone, greater than the 3.98 × 105 copies g−1 substrate observed in the unaerated SCW, and even N8.81 × 105 copies g−1 substrate observed in the aerated SCW (Fig. 4b). Furthermore, the gene abundance of the denitrifying genes (nirK, nirS, narG and nosZ) demonstrated that the denitrifiers were also highly abundant in the unsaturated zone. A higher amount of the nirK gene compared to the nirS gene was detected in the three SCW systems indicating that the major nitrite reductase was copper nitrite reductase that is coded by the nirK gene. These results are different from the results obtained by Hu et al. (2016) and Zhi and Ji (2014), but similar to Paranychianakis et al. (2016). The SND process also occurred in the unsaturated zone in the partially unsaturated SCW, owing to the solid– liquid–gas phases that promote the oxygen mass transfer process in biofilms attached to substrates (Hu et al., 2012a). Furthermore, the solid–liquid–gas phases in the unsaturated zone allows for the growth of more biofilm layers, because of the wide range of the DO concentration gradient, which might help optimise the distribution of microbes based on their diverse demands for DO.

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Fig. 3. Dynamic changes in the COD and nitrogen concentration along the flow pathway in the partially unsaturated SCW (a), the unaerated SCW (b), and the aerated SCW (c).

In terms of the overall removal effects of the partially unsaturated SCW, the removal of organics and nitrogen mainly took place in the first 0 cm to 50 cm along the flow direction, which was in the unsaturated zone. However, even under excellent conditions of DO for denitrification (below 0.2 mg/L) (Beebe et al., 2015), there was no noticeable denitrification observed between 50 cm and 140 cm along the flow direction, when the wastewater flows into the saturated zone. This might be explained by the low C/N ratio in the saturated zone. The dominant denitrifying bacteria are considered to be heterotrophic bacteria showing an essential demand for organic carbon as an energy source. Zhi and Ji (2014) introduced an optimal C/N ratio of 12 in their tidal flow CW. After the first 50 cm in the flow pathway, the C/N ratio remaining was about 0.75, which was distinctly lower than the appropriate C/N ratio observed in previous studies (Huett et al., 2005; Jia et al., 2019; Zhi and Ji, 2014). Therefore, the primary reason for unsatisfactory

denitrification in this study is due to an insufficient carbon source. External solid carbon source seems to optimise C/N ratio effectively according to Zhuang et al. (2019), which also proved by our current experiment (unpublished). 3.3. Superiority of SCW design 3.3.1. High enrichment of ammonia-oxidising bacteria in the partially unsaturated SCW To compare the enrichment of ammonia-oxidising bacteria between the partially unsaturated SCW and the two control SCW systems, the proportion of the amoA gene abundance to the 16s rRNA gene abundance in each SCW was calculated, where gene abundance is the mean abundance along the entire flow pathway in each SCW. The results showed that the amoA gene/16s rRNA gene abundance ratio was

Fig. 4. Functional gene abundance along the flow pathway of the unsaturated zone in the partially unsaturated SCW (a) as well as the average functional gene abundance in the unaerated and the aerated SCW (b).

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1.43‱ in the partially unsaturated SCW, 0.27‱ in the unaerated SCW, and 1.24‱ in the aerated SCW. According to statistical analysis, there was no significant difference between the amoA gene/16s rRNA gene abundance ratio of the partially unsaturated SCW and the aerated SCW, both of which were significantly greater than the unaerated SCW (p b 0.05). That's to say, the enrichment of ammonia-oxidising bacteria in partially unsaturated SCW was observed higher than unaerated SCW. 3.3.2. Eco-efficient oxygen supply in the unsaturated zone To further understand the oxygen supply effect in the experimental SCW and the control group, an equation was developed to calculate oxygen uptake (OU, g m−3 d−1) in the SCW (Kim et al., 2000; Liu and Wang, 2012). The oxygen demand (OD) is usually calculated as the sum of the OD for the decomposition of organic materials and the OD for nitrification (Platzer, 1999; Wu et al., 2011b). Therefore,
OU ¼ 0:99 ðCODin−CODout Þ þ 4:23 NH4 þ −Nin−NH4 þ −Nout ; where the (CODin − CODout) is the removed mass of COD, and (NH+ 4 −3 Nin − NH+ 4 -Nout) is the mass of nitrified ammonia nitrogen (g m d−1). The results of the OU calculations for all three SCWs are presented in Table 3. The OU of the partially unsaturated SCW was significantly higher than the other SCWs (unaerated and aerated), which indicates that the unsaturated zone serves as an excellent, functional oxygen supply zone. First, the substrate pores are filled with air under the unsaturated condition. Second, the solid–liquid–gas phase in the unsaturated zone enhances the oxygen mass transfer among the biofilm layers from outside to inside by increasing the dissolved oxygen consumption. In addition, the unsaturated zone is less likely to be clogged because the materials that become clogged are usually biodegradable under an aerobic environment; the primary material that becomes clogged in anaerobic conditions are less biodegradable (Zapater-Pereyra et al., 2015). Additionally, the unsaturated zone is able to enhance oxygen uptake efficiency from the gas phase to liquid phase. In the aerated SCW, the OU was significantly lower than the OU in the partially unsaturated SCW, indicating that the air generated from the aerator does not come into contact with the biofilm entirely, as observed in the unsaturated zone. Furthermore, the upper zone of the aerated disc or the perforated pipe is likely to become clogged because of the existence of a blind oxygenation region. We had compared the energy consumption (Ce) between partially unsaturated SCW and the aerated SCW with the methods reported by Austin and Nivala (2009). The daily Ce of partially unsaturated SCW was 1.1 × 10−4 kWh and the Ce of aerated SCW was 3.3 × 10−3 kWh. Compared to the aerated SCW, the partially unsaturated SCW not only demonstrated better OU efficiency, but also consumed less energy. The partially unsaturated SCW does not require energy to support oxygen supply due to its automatic self-oxygenating property in the solid– liquid–gas phases, while the aerated SCW requires electric energy to support the blower (Austin and Nivala, 2009). In the unaerated SCW, oxygen can be replenished only by the reaeration of the atmosphere at the surface, which is much lesser than the levels observed in the partially unsaturated SCW and the aerated SCW.

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Although the unsaturated design is similar to a tidal strategy, containing a drain phase and a flood phase, the partially unsaturated SCW, with a spatially distinguishable permanent unsaturated zone and a saturated zone, surpasses a tidal strategy with a distinguishable unsaturated zone and saturated zone over time (Hu et al., 2014; Kizito et al., 2017), because the spatially unsaturated design is able to provide more permanent solution for DO supply and enrich the functional microorganisms in a more stable manner. Also, the design of spatially distinguishable permanent unsaturated zone and a saturated zone in our study has more potential for both nitrification and denitrification than conventional VFSSCWs and VFSSCWs with up flow (Vymazal, 2013, 2014). 3.3.3. Advantages of the baffle design The central higher tube inside the partially unsaturated SCW plays the role of a baffle that helps optimise the water flow path, creating a system that is similar to a plug flow reactor. Additionally, the baffle design forces the wastewater to flow through alternating environments of constant aerobic and anaerobic/anoxic conditions along the pathway. The aerobic environment is generated again specially at the end of the entire flow pathway, because of atmospheric reaeration (Ong et al., 2010a), which enables the SCW to accomplish the removal of advanced organics and nitrogen. As reported, a design of down flow and alternative drainage height in VFSSCWs consists of unsaturated zone and saturated zone which was similar in our study, while the baffle design in our study seems to show more superiorities than it (Saeed and Sun, 2017). If the C/N ratio is increased, the advantages of the TN removal process would be more significant. The partially unsaturated SCW in this study can be considered a hybrid SCW, which combines a constant unsaturated zone and a constant saturated zone, with the ability to achieve hybridised effects in terms of pollutants and water purification. Furthermore, the baffle design provides an advantage in terms of its spacesaving features compared to the conventional hybrid CWs (Saeed et al., 2019). 4. Conclusions The advanced oxygen supply method, which uses a partially unsaturated scheme, presented in this study showed excellent oxygen supply efficiency. This can be corroborated by the following data: the DO content at the end of the unsaturated zone (5.03 ± 0.54 mg/L) was much higher than the DO content in the saturated zone, the COD removal rate at the end of the unsaturated zone was found to be steady at 83 ± 5%, and the ammonia nitrogen removal rate at the end of the unsaturated zone was found to be around 45.0 ± 10.0%. The oxygen uptake was found to be 336 ± 44 g m−3 d−1 in the partially unsaturated SCW, and this value was significantly better than the value for the unaerated SCW (p b 0.05). The unsaturated scheme showed effects analogous to the aerated scheme regarding the COD removal rate, while the oxygen uptake of the unsaturated scheme was significantly higher than the aerated scheme (p b 0.05). Additionally, the lower energy consumption during the unsaturated scheme compared to the aerated scheme makes it a promising eco-efficient oxygen supply method. More efficient removal of TP without plants was also observed in the partially unsaturated SCW compared to the others. However, the low C/N ratio in saturated zone needs to be optimised in order to achieve

Table 3 Changes in COD, NH+ 4 -N, and oxygen uptake in the partially unsaturated SCW, unaerated SCW, and aerated SCW (mean ± SD). CW types

CODin (mg d−1)

CODout (mg d−1)

NH+ 4 -Nin (mg d−1)

NH+ 4 -Nout (mg d−1)

Vf (m3)

OU (g m−3 d−1)

Partially unsaturated Unaerated Aerated

4230 ± 311 4230 ± 311 4230 ± 311

1038 ± 26 994 ± 209 813 ± 169

984 ± 155 984 ± 155 984 ± 155

482 ± 59 890 ± 37 232 ± 9

0.016 0.049 0.049

336 ± 44 73 ± 12 134 ± 16

Note: Vf is the volume of the functional oxygen supplementation area in each SCW and OU is the oxygen uptake per unit volume per day.

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better TN removal efficiency. And further study combined with the plant influence mechanism will be implemented after finishing the study of partially unsaturated configuration mechanism.

Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and all the authors listed have approved the manuscript that is enclosed. The work described above is original and has not been published in other journals, in whole or in part. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant numbers- 51720105013 and 51878388), the Fundamental Research Fund of Shandong University (Grant number11440078614025), the Doctoral Natural Science Foundation of Shandong Province (Grant number- ZR2019BEE020), and the 64th China Postdoctoral Science Foundation (Grant number- 2018M640633). References Al-Saedi, R., et al., 2018. Nitrogen removal efficiencies and pathways from unsaturated and saturated zones in a laboratory-scale vertical flow constructed wetland. J. Environ. Manag. 228, 466–474. https://doi.org/10.1016/j.jenvman.2018.09.048. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. twentyfirst ed. American Public Health Association, Washington, DC. Austin, D., Nivala, J., 2009. Energy requirements for nitrification and biological nitrogen removal in engineered wetlands. Ecol. Eng. 35 (2), 184–192. https://doi.org/ 10.1016/j.ecoleng.2008.03.002. Beebe, D.A., et al., 2015. Biogeochemical-based design for treating ammonia using constructed wetland systems. Environ. Eng. Sci. 32 (5), 397–406. https://doi.org/ 10.1089/ees.2014.0475. Cao, Y., et al., 2017. The effect of dissolved oxygen concentration (DO) on oxygen diffusion and bacterial community structure in moving bed sequencing batch reactor (MBSBR). Water Res. 108, 86–94. https://doi.org/10.1016/j.watres.2016.10.063. Caselles-Osorio, A., Garcia, J., 2006. Performance of experimental horizontal subsurface flow constructed wetlands fed with dissolved or particulate organic matter. Water Res. 40 (19), 3603–3611. https://doi.org/10.1016/j.watres.2006.05.038. Dionisi, H.M., et al., 2002. Quantification of Nitrosomonas oligotropha-like ammoniaoxidizing bacteria and Nitrospira spp. from full-scale wastewater treatment plants by competitive PCR. Appl. Environ. Microbiol. 68 (1), 245–253. https://doi.org/ 10.1128/aem.68.1.245-253.2002. Egnew, N., et al., 2019. Physiological insights into largemouth bass (Micropterus salmoides) survival during long-term exposure to high environmental ammonia. Aquat. Toxicol. 207, 72–82. https://doi.org/10.1016/j.aquatox.2018.11.027. Han, Z., et al., 2019. Nitrogen removal of anaerobically digested swine wastewater by pilotscale tidal flow constructed wetland based on in-situ biological regeneration of zeolite. Chemosphere 217, 364–373. https://doi.org/10.1016/j.chemosphere.2018.11.036. Hu, Y., et al., 2012a. High rate nitrogen removal in an alum sludge-based intermittent aeration constructed wetland. Environ. Sci. Technol. 46 (8), 4583–4590. https://doi.org/ 10.1021/es204105h. Hu, Y.S., et al., 2012b. Comprehensive analysis of step-feeding strategy to enhance biological nitrogen removal in alum sludge-based tidal flow constructed wetlands. Bioresour. Technol. 111, 27–35. https://doi.org/10.1016/j.biortech.2012.01.165. Hu, Y., et al., 2014. Robust biological nitrogen removal by creating multiple tides in a single bed tidal flow constructed wetland. Sci. Total Environ. 470-471, 1197–1204. https://doi.org/10.1016/j.scitotenv.2013.10.100. Hu, Y., et al., 2016. Microbial nitrogen removal pathways in integrated vertical-flow constructed wetland systems. Bioresour. Technol. 207, 339–345. https://doi.org/10.1016/ j.biortech.2016.01.106. Huett, D.O., et al., 2005. Nitrogen and phosphorus removal from plant nursery runoff in vegetated and unvegetated subsurface flow wetlands. Water Res. 39 (14), 3259–3272. https://doi.org/10.1016/j.watres.2005.05.038. Jia, L., et al., 2019. Exploring utilization of recycled agricultural biomass in constructed wetlands: characterization of the driving force for high-rate nitrogen removal. Environ. Sci. Technol. 53 (3), 1258–1268. https://doi.org/10.1021/acs.est.8b04871. Jing, Z., et al., 2015. Practice of integrated system of biofilter and constructed wetland in highly polluted surface water treatment. Ecol. Eng. 75, 462–469. https://doi.org/ 10.1016/j.ecoleng.2014.12.015. Kim, Y.C., et al., 2000. Relationship between theoretical oxygen demand and photocatalytic chemical oxygen demand for specific classes of organic chemicals. Analyst 125 (11), 1915–1918. https://doi.org/10.1039/b007005j. Kizito, S., et al., 2017. Treatment of anaerobic digested effluent in biochar-packed vertical flow constructed wetland columns: role of media and tidal operation. Sci. Total Environ. 592, 197–205. https://doi.org/10.1016/j.scitotenv.2017.03.125. Kuypers, M.M.M., et al., 2018. The microbial nitrogen-cycling network. Nat. Rev. Microbiol. 16 (5), 263–276. https://doi.org/10.1038/nrmicro.2018.9.

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