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without intermittent aeration under different organic loading rates
Accepted Manuscript Nitrogen removal and N2O emission in subsurface wastewater infiltration systems with/without intermittent aeration under different organic loading rates Yingying Jiang, Yafei Sun, Jing Pan, Shiyue Qi, Qiuying Chen, Deli Tong PII: DOI: Reference:
S0960-8524(17)31251-8 http://dx.doi.org/10.1016/j.biortech.2017.07.135 BITE 18555
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
6 June 2017 20 July 2017 23 July 2017
Please cite this article as: Jiang, Y., Sun, Y., Pan, J., Qi, S., Chen, Q., Tong, D., Nitrogen removal and N2O emission in subsurface wastewater infiltration systems with/without intermittent aeration under different organic loading rates, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.135
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Nitrogen removal and N2O emission in subsurface wastewater infiltration systems with/without intermittent aeration under different organic loading rates Yingying Jiang#, Yafei Sun#, Jing Pan∗, Shiyue Qi, Qiuying Chen, Deli Tong College of Life Science, Shenyang Normal University, Shenyang 110034, China. Corresponding to: Jing Pan, Tel: +86 24 86593328, Fax: +86 24 86592584, E-mail
∗
address: [email protected] #
These authors contributed equally to this study and share first authorship.
Abstract: Nitrogen removal, N2O emission and nitrogen removal functional gene abundances in intermittent aerated (IA) and non-aerated (NA) subsurface wastewater infiltration systems (SWISs) under different organic loading rates (OLRs) were investigated. Aeration successfully created aerobic condition at 50 cm depth and did not change anoxic or anaerobic condition at 80 and 110 cm depths under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d). Meanwhile, aeration enhanced COD, NH4+-N, TN removal and the enrichment of nitrogen removal functional genes (amoA, nxrA, napA, narG, nirS, nirK, qnorB and nosZ) compared to NA SWIS. High COD removal rate of 91.6%, TN removal rate of 85.9% and low N2O emission rate of 32.4 mg/(m2 d) were obtained in IA SWIS under OLR of 16.5 g BOD/(m2 d). Intermittent aeration is a sensible strategy to achieve high OLR, low N2O emission, satisfactory organic matter and nitrogen removal performance for SWISs. Keywords: N2O; Subsurface wastewater infiltration system; Intermittent aeration; Nitrogen removal functional genes; Organic loading rate 1
1. Introduction Subsurface wastewater infiltration system (SWIS) has been proved as a promising technology for decentralized domestic wastewater treatment due to its efficient organics and phosphorus removal performance and low operation cost (Li et al., 2011a; Pan et al., 2016). Previous studies reported TN removal rates were only around 50% in conventional SWISs which remained as a major challenge for SWISs (Li et al., 2011b; Kong et al., 2014; Pan et al., 2015). Nitrification/denitrification is generally considered as the primary way for nitrogen removal. Nitrification demands aerobic condition while denitrification occurs in anoxic or anaerobic environment, which could not be fulfilled simultaneously. The effluent quality was not satisfied and far above the discharge standards when conventional SWISs were applied under high organic pollutant loading (Li et al., 2011a). SWISs would emit considerable amount of N2O in treating wastewater (Kong et al., 2002). Researches on N2O emission have been increasing worldwide due to its global warming potential accounting for approximately 5% of the total greenhouse effect (Tol et al., 2002). N2O emission was influenced by various parameters and environmental conditions such as dissolved oxygen (DO), carbon availability and water temperature (Jørgensen et al., 2012). Nitrification/denitrification processes involve several functional genes, including ammonia monooxygenase (amoA), nitrite oxidoreductase (nxrA), periplasmic nitrate reductase (napA), membrane-bound nitrate reductase (narG), nitrite reductase (nirK/nirS), nitric oxide reductase (qnorB) and nitrous oxide reductase (nosZ) (Ji et al., 2012). Nitrogen removal, N2O emission and 2
nitrogen removal functional genes were dependent on influent pollutant loading (Ma et al., 2008; Wang et al., 2015; Fei et al., 2016). Intermittent aeration was an effective strategy for conventional SWISs, which increased TN removal by creating favorable condition for nitrification/denitrification simultaneously (Pan et al., 2015). So far, there have been no reports about the effects of influent organic loading rate (OLR) on nitrogen removal, N2O emission and nitrogen removal functional gene abundances in SWISs with/without intermittent aeration. Therefore, the main purposes of this paper were: (1) to assess the characteristics of matrix DO levels along the SWISs with/without intermittent aeration under different OLRs; (2) to investigate the effects of OLR and intermittent aeration on nitrogen removal performance, N2O emission and nitrogen removal functional gene abundances; (3) to determine optimal operational schemes for high nitrogen removal and low N2O emission in SWISs. This study will not only improve effluent quality and reduce greenhouse gas emission of SWISs, but also will be in favor of lessening water quality deterioration and greenhouse effect. 2. Material and methods 2.1. System description and operation Pilot systems made of organic glass column were operated indoors, which were 1.2 m in height and 0.5 m in diameter. The matrix was 20% coal slag and 80% brown soil mixed evenly by weight ratio. The mixed matrix comprised of 187.6±3.4 m2/kg of surface area and (1.89±0.2)×10-3 cm/s of hydraulic conductivity. Distributing pipe was installed at 50 cm depth below the surface in each infiltration system. Artificial 3
intermittent aerated (IA) SWIS was composed of aerated unit which consisted of air compressor, air tube and micro-bubble diffuser at the depth of 40 cm. 15 cm of gravel (10~20 mm, diameter) wrapped up the distributing pipe and micro-bubble diffuser to protect clogging and diffuse air. DO electrodes were placed in the center of each SWIS at 50, 80 and 110 cm depths. 10 cm of deep gravel (10~20 mm, diameter) was prepared at the bottom to support infiltration system and evenly distributed the treated water. The treated wastewater was gathered at the outlet. The indoor temperature was 20±1℃ during the period of the experiment. Domestic wastewater was pretreated in a septic tank prior to being flowed into each SWIS continuously under a hydraulic loading of 0.08 m3 /(m2 d). The ranges of wastewater from the septic tank after pretreatment were as follows: pH 6.9-7.3, COD 172.3-286.4 mg/L, BOD 142.8-224.2 mg/L, TN 34.3-42.6 mg/L, TP 3.1-5.8 mg/L, NH4+-N 32.7-41.5 mg/L. Four runs were arranged, with OLR gradually elevated from 5.3 (a) to 10.9 (b), 16.5 (c) and 21.8 (d) g BOD/(m2 d). Each run lasted for 60 days. Four aerated/non-aerated cycles were used in intermittent aerated SWIS every day. In each aerated/non-aerated cycle, the system was aerated for one hour with an airflow rate of 2±0.2 L/min, and then had five hours interval without aeration. The aeration would begin at 0:00 AM, 6:00 AM, 12:00 PM and 6:00 PM, respectively. 2.2. Sample collection and analytical methods Water samples were taken from influent and effluent to analyze COD, NH4+-N and TN concentrations according to the standard methods every 5 days (APHA, 2003). Statistical checks were made at significant differences of 0.05 for all analyses using 4
SPSS 12.0 (n=12). Gas sampling was done using a closed static chamber. Five gas samples were collected at 0, 30, 60, 90 and 120 min after enclosure at the same time of the day between 9:00 AM and 11:00 AM every 5 days. N2O concentration was analyzed by gas chromatography. Agilent 6890N equipped with an electron capture detector and a Poropak Q column and used 40 mL/min argon-containing 5% methane as the carrier gas. Temperature of the detector and oven was set at 300℃ and 120℃, respectively. After determining the concentration of N2O, N2O emission rate was calculated by the following equation (Nakano et al., 1995). Flux = (dC/dt)×(1/A)×(13.5M)×[0.0225×0.082×(T+273)×1000]-1 where Flux is the N2O emission rate (mg/(m2 h)); dC/dt is the slope of best-fit line for plot of gas concentration inside the chamber and time data points (mg/h); A is the section area of the gas chamber (m2); M is the molecular weight of N2O; and T is the air temperature inside the chamber (℃). N2O conversion ratio is the quality percentage of nitrogen convert to N2O occupied in TN. Matrix samples were collected from sampling ports after each experiment. Four samples were taken from each layer and blended (5.0 g). After each on-site collection, the samples were stored in an ice incubator, and subsequently brought back to laboratory for analyses. Soil DNA kits (Omega, D5625-01) were used to extract and purify total genomic DNA from the samples. Extracted genomic DNAs were detected by 1% agarose gel electrophoresis, and preserved at -20℃ freezer until further use. Quantitative analysis was made on the target fragments of the following functional 5
genes: amoA, nxrA, narG, napA, nirK, nirS, qnorB and nosZ. The protocol and parameters for each target gene according to previous study (Pan et al., 2017). The standard samples were diluted to yield a series of 10-fold concentrations, and were subsequently used for qPCR standard curves. The coefficient of determination (R2) for all standard curves were greater than 0.99. Functional gene abundances involved in nitrogen removal were quantitated by qPCR technique according to Ji et al. (2012). qPCR was performed on a Roche Lightcycler 480 Real-Time PCR detection system (Roche Diagnostics, Meylan, France) in final 20 µL volume reaction mixtures containing the following components: 10 µL SYBR Green I PCR master mix (Applied Biosystems, USA), 1 µL template DNA (sample DNA or plasmid DNA for standard curves), forward and reverse primers, and sterile water. qPCR was performed in a three-step thermal cycling procedure. Each qPCR was performed in 40 cycles and followed by a melting curve analysis. Sterile water was used as a negative control and the data obtained from the qPCR were normalized to copies per gram of biological carrier in the SWISs. 3. Results and discussion 3.1. DO profiles in an aerated/non-aerated cycle DO concentrations less than 0.2 mg/L are commonly interpreted as anaerobic environment and more than 2.0 mg/L are deemed as aerobic environment. DO concentrations in the range of 0.2-0.5 mg/L represent anoxic environment (Wang et al., 2006). Fig. 1 shows DO distribution in an aerated/non-aerated cycle in aerated and non-aerated (NA) SWISs under different OLRs. DO concentrations were below 1.23, 6
0.26 and 0.02 mg/L at 50, 80 and 110 cm depths in NA SWIS under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), respectively. It could conclude that non-aerated SWIS was under anoxic or anaerobic condition. The prevalent environment in conventional SWISs was anoxic or anaerobic below distributing pipe because oxygen from air diffused to the matrix was limited (Fan et al., 2013b; Pan et al., 2016). DO concentrations decreased with OLR increasing in NA and IA SWISs because more oxygen was consumed by biological oxidation of excessive organic matter. DO concentrations at 50 cm depth of IA SWIS were higher than that in NA SWIS. DO concentrations enhanced in aerated period, but declined slowly in non-aerated period. High aeration-initiated DO values may be decreased by the degradation of nutrients and organic matter, which may interpret the decreasing tendency in non-aerated period. Under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), DO concentrations were more than 5.2 mg/L at 50 cm depth during aeration and as high as 2.0 mg/L when supplementary aerations were turned off. At 80 and 110 cm depths, DO concentrations were below 0.44 and 0.07 mg/L during aeration, and were below 0.30 and 0.03 mg/L without aeration, respectively. Aerobic condition was effectively created at 50 cm depth and anoxic or anaerobic condition was not changed at 80 and 110 cm depths in IA SWIS by intermittent aeration under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d). Under OLR of 21.8 g BOD/(m2 d), DO concentrations were lower than 2.0 and 1.0 mg/L at 50 cm depth after aeration shut down for 140 and 220 minutes, respectively. The reason was more oxygen consumed due to the mineralization of high influent organic matter. Intermittent aeration could not develop aerobic 7
environment in upper layer of the matrix under high OLR (21.8 g BOD/(m2 d)) in this experimental condition. [here insert Fig. 1] 3.2. COD, NH4+-N and TN removal performance Average effluent concentrations and removal rates of COD, NH4+-N and TN in NA and IA SWISs during the experimental period are shown in Fig. 2. There were significant differences in NA and IA SWISs under different OLRs. NA SWIS achieved average COD removal rates of 81.5%, 73.4%, 62.3% and 44.1%, with effluent COD concentrations of 33.9, 48.5, 69.1 and 102.4 mg/L under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), respectively. With the increase of OLR, oxygen deficiency developed more obviously, resulted in decrease of COD removal rate in NA SWIS. This result was in agreement with other studies (Fan et al., 2013a; Fei et al., 2016). Average effluent COD concentrations of NA SWIS were significantly higher than that of IA SWIS under the same OLR (P<0.05). Average COD removal rates were 97.2%, 94.6% and 91.6% under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d) in IA SWIS, respectively. Increasing OLR hardly affected the efficiency of organic matter degradation under OLR between 5.3 and 16.5 g BOD/(m2 d), which was significantly different from that in NA SWIS. Previous studies informed that the suitable range of OLRs were 1.5-6.0 g BOD/(m2 d) for conventional SWISs in treating domestic wastewater (Oladoja and Ademoroti, 2006). Organic loading resistance ability of IA SWIS was much higher because of the application of intermittent aeration. Fan et al. (2013b) and Wu et al. (2015) reported that sufficient oxygen supply would greatly 8
enhance the performance of aerobic biochemical oxidation. Decomposition of organic matter occurred mostly in the upper matrix of the SWISs (Li et al., 2011a). Aerobic condition in the upper matrix enhanced by intermittent aeration and thus facilitated aerobic removal of organic matter. IA SWIS achieved average COD removal rate of 81.1% and effluent COD concentration of 34.5 mg/L under OLR of 21.8 g BOD/(m2 d). Oxygen availability was considered as one of the main rate-limiting factors for organics degradation in SWISs (Fan et al., 2013b). Intermittent aeration did not develop favorable DO environment (Fig. 1) under high OLR of 21.8 g BOD/(m2 d) in this experimental condition, which restricted the degradation of organic matter. [here insert Fig. 2] Nitrification plays an important role in nitrogen removal which is an aerobic chemo-autotrophic microbial process (NH4+→NO2 -→NO3-) and occurs only when oxygen is presented in a high enough concentration (Albuquerque et al., 2009). As shown in Fig. 2, average effluent NH4 +-N concentrations in NA SWIS were significantly higher than that of IA SWIS under the same OLR (P<0.05). It is widely accepted that DO concentrations above 1.5 mg/L are essential for better nitrification (Fan et al., 2013b). DO concentrations were lower than 1.2 mg/L at all depths in NA SWIS under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), which led to anoxic and anaerobic environment in the matrix, therefore NH4+-N removal was seriously restricted. As OLR increased from 5.3 to 21.8 g BOD/(m2 d), average NH4+-N removal rate dropped from 73.3% to 30.7%. Increased organic matter consumed more available DO and further limited the activity of autotrophic ammonia oxidation 9
bacteria through greater deficit of oxygen, thus resulted in low nitrification. Artificial aeration is the most effective strategy to guarantee sufficient oxygen supply in SWISs, especially when dealing with high organic pollutant loading wastewater (Hu and Zhao, 2012; Wu et al., 2015). As shown in Fig. 2, nitrification rates were significantly enhanced in IA SWIS. Average effluent NH4 +-N concentrations were 3.1, 3.6 and 3.8 mg/L under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), and average NH4+-N removal rates were 91.6%, 90.3% and 89.7%, respectively. DO concentrations were above 2.0 mg/L in the upper layer through artificial aeration in IA SWIS, which facilitated achieving high NH4 +-N removal rate above 89% even under high OLR of 16.5 g BOD/(m2 d). Intermittent aeration did not supply enough oxygen for nitrification (Fig. 1) under high OLR of 21.8 g BOD/(m2 d) in this experimental condition, thus inhibited nitrification. With increasing OLR, more oxygen would demand owing to the requirement of mineralization of excess influent organic matter. Nitrification could transform nitrogen into various forms. Nevertheless, it could not achieve nitrogen removal from wastewater. Nitrified nitrogen must be processed through anaerobic and heterotrophic microbial denitrification (NO3-→NO2-→NO-→N2O→N2) to be removed from wastewater permanently. Insufficient organic carbon source and excess oxygen could restrict denitrification completion (Fan et al., 2013b). As shown in Fig. 2, TN removal rates were far from satisfactory in NA SWIS and average effluent TN concentrations were 13.3, 16.7, 22.3 and 29.5 mg/L under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), respectively. Average TN removal rate dropped from 66.4% to 25.5% with OLR 10
increasing from 5.3 to 21.8 g BOD/(m2 d), which was similar to the variation of NH4 +-N removal rate. NA SWIS could not achieve high NH4 +-N removal without adequate DO concentration, which would adversely restrict denitrification due to insufficient supply of NO3--N as electron acceptors. Average TN removal rates in IA SWIS were significantly higher than that of NA SWIS under the same OLR (P<0.05). Average TN removal rate was enhanced from 81.6% under OLR of 5.3 g BOD/(m2 d) to 84.3% under OLR of 10.9 g BOD/(m2 d), to 87.1% under OLR of 16.5 g BOD/(m2 d) in IA SWIS. After effective nitrification, NO3--N and NO2--N as electron accepters could not be removed unless sufficient organic carbon was supplied as electron donor. Carbon deficiency became the main limiting factor for TN removal (Fan et al., 2013a). In this study, high OLR could supply more organic matter as electron donor for efficient denitrification, and achieved the highest TN removal rate under OLR of 16.5 g BOD/(m2 d). However, average TN removal rate of IA SWIS decreased to 56.1% under OLR of 21.8 g BOD/(m2 d) due to lower nitrification. According to the results, OLR variations of 5.3-16.5 g BOD/(m2 d) were recommended for high COD, NH4+-N and TN removal performances in intermittent aerated SWISs. When compared to other enhancing SWISs, average COD, NH4+-N and TN removal efficiencies of intermittent aerated SWIS under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d) in this study were higher than those in intermittent operated SWIS (91.5% for COD, 86.2% for NH4+-N, 80.8% for TN, Li et al., 2015), in bioaugmentation SWIS (83.8% for COD, 75.0% for NH4+-N, 61% for TN, Zou et al., 2009) and in 11
shunt distributing wastewater SWIS (84.8% for COD, 87.7% for NH4+-N, 70.1% for TN, Li et al., 2011b). 3.3. N2O emission N2O is an intermediate product of nitrification/denitrification processes in SWISs (Kong et al., 2002). Denitrification is generally regarded as the dominant process responsible for the N2O emission (Tiedje, 1982). Fig. 3 shows N2O conversion ratio and emission rate of each SWIS. N2O conversion ratios of NA and IA SWISs (0.44-0.98%) were consistent with previous studies (Johansson et al., 2003; Li et al., 2017). N2O emission rates under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d) were as follows: 42.1, 38.5, 32.4, 22.5 mg/(m2 d) for IA SWIS, and 36.7, 33.6, 26.3, 18.8 mg/(m2 d) for NA SWIS, respectively. Under the same OLR, N2O emission rates of IA SWIS were higher than that of NA SWIS. Low N2O emission rate of NA SWIS was attributed to low nitrification, which restricted denitrification process. Denitrification is highly dependent on nitrification, an aerobic chemoautotrophic process, which produces nitrate from ammonium. Intermittent aeration developed appropriate oxygen condition for nitrification/denitrification processes in IA SWIS. N2O emission rate of NA SWIS decreased with the increase of OLR. The reason for NA SIWS was increased organic matter consumed more available oxygen and further limited nitrification process. In IA SWIS, N2O emission rate decreased with OLR increasing within the range of 5.3 and 16.5 g BOD/(m2 d). The upper matrix was sufficient in oxygen by intermittent aeration which enhanced the nitrification of NH4 +-N and aerobic biochemical oxidation of COD. However, carbon source of lower 12
part was not enough which inhibited part of N2O from being reduced to N2 before released to the atmosphere under low OLR. Higher OLR could supply more carbon source for N2O to N2 reduction, therefore N2O emission rate decreased. Previous study concluded that sufficient carbon source supplied after efficient nitrification could greatly reduce N2O emission (Wang et al., 2014). Low N2O emission rate of IA SWIS under OLR of 21.8 g BOD/(m2 d) was due to low nitrification. According to the results, OLR of 16.5 g BOD/(m2 d) were recommended for low N2O emission in intermittent aerated SWISs. [here insert Fig. 3] 3.4. Functional gene abundances involved in nitrogen removal Nitrogen removal functional genes are shown in Fig. 4, which are involved in nitrification/denitrification processes. The amoA and nxrA genes are the marker of oxidizing NH4+-N to NO2--N and oxidizing NO2--N to NO3--N (Dionisi et al., 2002), respectively. The abundances of amoA and nxrA decreased along the wastewater flow direction in NA and IA SWISs, which followed the same trend as DO. The life activity, growth and enrichment of ammonia-oxidizing bacteria are related to DO (Fan et al., 2013b). Ammonia oxidizing bacteria (AOB) oxidize NH4+-N to NO2--N and provide substrate for nitrite-oxidizing bacteria (NOB) to oxidize NO2--N to NO3--N, which might explain lower abundance of nxrA compared to amoA. With OLR increasing, the abundances of amoA and nxrA decreased in NA SWIS. OLR increased by 5 g BOD/(m2 d), the abundances of amoA and nxrA decreased one order of magnitude at 50 cm depth. Previous studies also found that high organic matter reduced the growth 13
of AOB and NOB in conventional infiltration systems (Fan et al. 2013a). In IA SWIS, the abundances of amoA and nxrA were significantly higher than those of NA SWIS under the same OLR (P<0.05) at 50 cm depth. Sufficient oxygen supply could greatly improve the number of nitrification bacteria and enzyme activities involved in NH4 +-N transformation under high OLR (Pan et al., 2015). DO concentrations of IA SWIS (DO>2.0 mg/L) were improved through aeration at 50 cm depth under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), which was favorable for the nitrification bacteria. The abundances of amoA and nxrA were the lowest under OLR of 21.8 g BOD/(m2 d) in IA SWIS. The reason was more oxygen consumed due to the degradation of high influent organic matter which restricted enzyme activities involved in NH4+-N transformation. [here insert Fig. 4] NO3--N to NO2--N reduction, catalyzed by the key gene narG and napA, is the first reaction process in denitrification. NO2--N to NO reduction is the second reaction process, which is catalyzed by gene of nirS and nirK. NO to N2O reduction is the third process, which is catalyzed by qnorB. N2O to N2 reduction catalyzed by nosZ is the last reaction. As seen from Fig. 4, the abundances of napA, narG, nirS, nirK, qnorB and nosZ declined with OLR increasing in NA SWIS similar to other study (Sun et al., 2017). OLR increased by 5 g BOD/(m2 d), the abundances of napA, narG, nirS, nirK, qnorB and nosZ decreased one order of magnitude at 80 and 110 cm depths. Less NO3- as the substrate for anaerobic denitrification due to low nitrification decreased the abundances of napA, narG, nirS, nirK, qnorB and nosZ in NA SWIS. In IA SWIS, 14
the abundances of napA, narG, nirS, nirK, qnorB and nosZ increased with OLR increasing under OLR range of 5.3 and 16.5 g BOD/(m2 d). More organic matter provided good nutritional condition for the growth and enrichment of anaerobic denitrifying bacteria with OLR increasing in IA SWIS after nearly complete nitrification with aeration, in parallel to enhancing the enrichment of nitrogen removal functional genes. The level of nosZ gene expression is a key factor determining whether N2O is converted to N2 or released into the atmosphere as a greenhouse gas (Thomson et al., 2012). Previous study confirmed that increasing nosZ gene abundance promoted N2O reduction (Harter et al., 2014). More organic matter improved the conversion of N2O to N2 under higher OLR, which could further explain the lowest N2O emission in IA SWIS under OLR of 16.5 g BOD/(m2 d). 4. Conclusion Intermittent aeration created appropriate oxygen condition for nitrification and denitrification under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), which increased COD, NH4 +-N, TN removal, and enhanced the abundances of amoA, nxrA, napA, narG, nirS, nirK, qnorB and nosZ genes. Increasing OLR affected COD, NH4+-N and TN removal more in non-aerated SWIS than that in intermittent aerated SWIS. High COD removal rate of 91.6%, TN removal rate of 85.9% and low N2O emission rate of 32.4 mg/(m2 d) were obtained in IA SWIS under OLR of 16.5 g BOD/(m2 d). Acknowledgements This research is financially supported by the National Natural Science Foundation of China (No.41001321), (No.41471394); Liaoning BaiQianWan Talents Program 15
[2015(45)]; Shenyang Science and Technology Project (17-231-1-93); Natural Science Foundation of Liaoning (2015010585-301); Ecology and Environment Research
Center
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Oladoja, N.A., Ademoroti, C.M.A., 2006. The use of fortified soil-clay as on-site system for domestic wastewater purification. Water Res. 40, 613-620. Pan, J., Fei, H.X., Song, S.Y., Yuan, F., Yu, L., 2015. Effects of intermittent aeration on pollutants removal in subsurface wastewater infiltration system. Bioresour. Technol. 191, 327-331. Pan, J., Qi, S.Y., Sun, Y.F., Jiang, Y.Y., Zhao, N., Huang, L.L., Sun, Y.N., 2017. Nitrogen removal and nitrogen functional gene abundances in three subsurface wastewater infiltration systems under different modes of aeration and influent C/N ratios. Bioresour. Technol. 10.1016/j.biortech.2017.05.112. Pan, J., Yuan, F., Zhang, Y., Huang, L.L., Fei, H.X., Cheng, F., Zhang, Q., 2016. Nitrogen removal in subsurface wastewater infiltration systems with and without intermittent aeration. Ecol. Eng. 94, 471-477. Sun, Y.F., Pan, J., Qi, S.Y., Fei, H.X., 2017. Effects of hydraulic loading rate and aeration mode on nitrogen removal and nitrogen functional gene abundances in subsurface wastewater infiltration systems. Water Sci. Technol. 76, 210-217. Thomson, A. J., Giannopoulos, G., Pretty, J., 2012. Biological sources and sinks of nitrous oxide and strategies to mitigate emissions. Philos. Trans. R. Soc. London. 367, 1157-1168 Tiedje, J.M., 1982. Denitrification. In: Page, A.L. (Ed.), Methods of Soil Analysis. Part 2, second ed. American Society of Agronomy, Madison, Wisconsin, USA, 1011-1024. Tol, R.S.J., 2002. Estimates of the damage costs of climate change. Part 1: benchmark 19
estimates. Environ. Resour. Econ. 21, 47-73. Wang, H., Ji, G.D., Bai, X., 2015. Enhanced long-term ammonium removal and its ranked contribution of microbial genes associated with nitrogen cycling in a lab-scale multimedia biofilter. Bioresour. Technol. 196, 57-64. Wang, X.J., Xia, S.Q., Chen, L., Zhao, J.F., Renault, N.J., Chovelon, J.M., 2006. Nutrients removal from municipal wastewater by chemical precipitation in a moving bed biofilm reactor. Process Biochem. 41, 824-828. Wang, Z., Liu, C.X., Liao, J., Liu, L., Liu, Y.H., Huang, X., 2014. Nitrogen removal and N2O emission in subsurface vertical flow constructed wetland treating swine wastewater: Effect of shunt ratio. Ecol. Eng. 73, 446-453. Wu, H.M., Fan, J.J., Zhang, J., Ngo, H.H., Guo, W.S., Hu, Z., Liang, S., 2015. Decentralized domestic wastewater treatment using intermittently aerated vertical flow constructed wetlands: Impact of influent strengths. Bioresour. Technol. 176, 163-168. Zou, J.L., Dai, Y., Sun, T.H., Li, Y.H., Li, G.B., Li, Q.Y., 2009. Effect of amended soil and hydraulic load on enhanced biological nitrogen removal in lab-scale SWIS. J. Hazard. Mater. 163, 816-822.
Figure Captions Fig. 1 DO profiles in an aerated/non-aerated cycle in SWISs with intermittent aeration (IA) and without aeration (NA).
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Fig. 2 NH4+-N, TN and COD removal performance in SWISs without aeration (NA), with intermittent aeration (IA) under organic loading rate (a) 5.3 g BOD/(m2 d), (b) 10.9 g BOD/(m2 d), (c) 16.5 g BOD/(m2 d), (d) 21.8 g BOD/(m2 d).
Fig. 3 N2O emission rate and conversion ratio in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate (OLR) a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
Fig. 4 Abundances of nitrogen removal functional genes in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
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Fig. 1 DO profiles in an aerated/non-aerated cycle in SWISs with intermittent aeration (IA) and without aeration (NA).
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Fig. 2 NH4+-N, TN and COD removal performance in SWISs without aeration (NA), with intermittent aeration (IA) under organic loading rate (a) 5.3 g BOD/(m2 d), (b) 10.9 g BOD/(m2 d), (c) 16.5 g BOD/(m2 d), (d) 21.8 g BOD/(m2 d). 23
Fig. 2 NH4+-N, TN and COD removal performance in SWISs without aeration (NA), with intermittent aeration (IA) under organic loading rate (a) 5.3 g BOD/(m2 d), (b) 10.9 g BOD/(m2 d), (c) 16.5 g BOD/(m2 d), (d) 21.8 g BOD/(m2 d). 24
Fig. 3 N2O emission rate and conversion ratio in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate (OLR) a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
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Fig. 4 Abundances of nitrogen removal functional genes in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
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Highlights Intermittent aerated SWISs were effective in treating high strength wastewater. Intermittent aeration improves TN removal at higher OLR up to 16.5 g BOD/(m2 d). 16.5 g BOD/(m2 d) was recommended for SWISs with 4 aerated/non-aerated cycles a day. Aeration raised amoA, nxrA, narG, napA, nirK, nirS, qnorB and nosZ abundances.
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S0960-8524(17)31251-8 http://dx.doi.org/10.1016/j.biortech.2017.07.135 BITE 18555
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
6 June 2017 20 July 2017 23 July 2017
Please cite this article as: Jiang, Y., Sun, Y., Pan, J., Qi, S., Chen, Q., Tong, D., Nitrogen removal and N2O emission in subsurface wastewater infiltration systems with/without intermittent aeration under different organic loading rates, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.135
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Nitrogen removal and N2O emission in subsurface wastewater infiltration systems with/without intermittent aeration under different organic loading rates Yingying Jiang#, Yafei Sun#, Jing Pan∗, Shiyue Qi, Qiuying Chen, Deli Tong College of Life Science, Shenyang Normal University, Shenyang 110034, China. Corresponding to: Jing Pan, Tel: +86 24 86593328, Fax: +86 24 86592584, E-mail
∗
address: [email protected] #
These authors contributed equally to this study and share first authorship.
Abstract: Nitrogen removal, N2O emission and nitrogen removal functional gene abundances in intermittent aerated (IA) and non-aerated (NA) subsurface wastewater infiltration systems (SWISs) under different organic loading rates (OLRs) were investigated. Aeration successfully created aerobic condition at 50 cm depth and did not change anoxic or anaerobic condition at 80 and 110 cm depths under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d). Meanwhile, aeration enhanced COD, NH4+-N, TN removal and the enrichment of nitrogen removal functional genes (amoA, nxrA, napA, narG, nirS, nirK, qnorB and nosZ) compared to NA SWIS. High COD removal rate of 91.6%, TN removal rate of 85.9% and low N2O emission rate of 32.4 mg/(m2 d) were obtained in IA SWIS under OLR of 16.5 g BOD/(m2 d). Intermittent aeration is a sensible strategy to achieve high OLR, low N2O emission, satisfactory organic matter and nitrogen removal performance for SWISs. Keywords: N2O; Subsurface wastewater infiltration system; Intermittent aeration; Nitrogen removal functional genes; Organic loading rate 1
1. Introduction Subsurface wastewater infiltration system (SWIS) has been proved as a promising technology for decentralized domestic wastewater treatment due to its efficient organics and phosphorus removal performance and low operation cost (Li et al., 2011a; Pan et al., 2016). Previous studies reported TN removal rates were only around 50% in conventional SWISs which remained as a major challenge for SWISs (Li et al., 2011b; Kong et al., 2014; Pan et al., 2015). Nitrification/denitrification is generally considered as the primary way for nitrogen removal. Nitrification demands aerobic condition while denitrification occurs in anoxic or anaerobic environment, which could not be fulfilled simultaneously. The effluent quality was not satisfied and far above the discharge standards when conventional SWISs were applied under high organic pollutant loading (Li et al., 2011a). SWISs would emit considerable amount of N2O in treating wastewater (Kong et al., 2002). Researches on N2O emission have been increasing worldwide due to its global warming potential accounting for approximately 5% of the total greenhouse effect (Tol et al., 2002). N2O emission was influenced by various parameters and environmental conditions such as dissolved oxygen (DO), carbon availability and water temperature (Jørgensen et al., 2012). Nitrification/denitrification processes involve several functional genes, including ammonia monooxygenase (amoA), nitrite oxidoreductase (nxrA), periplasmic nitrate reductase (napA), membrane-bound nitrate reductase (narG), nitrite reductase (nirK/nirS), nitric oxide reductase (qnorB) and nitrous oxide reductase (nosZ) (Ji et al., 2012). Nitrogen removal, N2O emission and 2
nitrogen removal functional genes were dependent on influent pollutant loading (Ma et al., 2008; Wang et al., 2015; Fei et al., 2016). Intermittent aeration was an effective strategy for conventional SWISs, which increased TN removal by creating favorable condition for nitrification/denitrification simultaneously (Pan et al., 2015). So far, there have been no reports about the effects of influent organic loading rate (OLR) on nitrogen removal, N2O emission and nitrogen removal functional gene abundances in SWISs with/without intermittent aeration. Therefore, the main purposes of this paper were: (1) to assess the characteristics of matrix DO levels along the SWISs with/without intermittent aeration under different OLRs; (2) to investigate the effects of OLR and intermittent aeration on nitrogen removal performance, N2O emission and nitrogen removal functional gene abundances; (3) to determine optimal operational schemes for high nitrogen removal and low N2O emission in SWISs. This study will not only improve effluent quality and reduce greenhouse gas emission of SWISs, but also will be in favor of lessening water quality deterioration and greenhouse effect. 2. Material and methods 2.1. System description and operation Pilot systems made of organic glass column were operated indoors, which were 1.2 m in height and 0.5 m in diameter. The matrix was 20% coal slag and 80% brown soil mixed evenly by weight ratio. The mixed matrix comprised of 187.6±3.4 m2/kg of surface area and (1.89±0.2)×10-3 cm/s of hydraulic conductivity. Distributing pipe was installed at 50 cm depth below the surface in each infiltration system. Artificial 3
intermittent aerated (IA) SWIS was composed of aerated unit which consisted of air compressor, air tube and micro-bubble diffuser at the depth of 40 cm. 15 cm of gravel (10~20 mm, diameter) wrapped up the distributing pipe and micro-bubble diffuser to protect clogging and diffuse air. DO electrodes were placed in the center of each SWIS at 50, 80 and 110 cm depths. 10 cm of deep gravel (10~20 mm, diameter) was prepared at the bottom to support infiltration system and evenly distributed the treated water. The treated wastewater was gathered at the outlet. The indoor temperature was 20±1℃ during the period of the experiment. Domestic wastewater was pretreated in a septic tank prior to being flowed into each SWIS continuously under a hydraulic loading of 0.08 m3 /(m2 d). The ranges of wastewater from the septic tank after pretreatment were as follows: pH 6.9-7.3, COD 172.3-286.4 mg/L, BOD 142.8-224.2 mg/L, TN 34.3-42.6 mg/L, TP 3.1-5.8 mg/L, NH4+-N 32.7-41.5 mg/L. Four runs were arranged, with OLR gradually elevated from 5.3 (a) to 10.9 (b), 16.5 (c) and 21.8 (d) g BOD/(m2 d). Each run lasted for 60 days. Four aerated/non-aerated cycles were used in intermittent aerated SWIS every day. In each aerated/non-aerated cycle, the system was aerated for one hour with an airflow rate of 2±0.2 L/min, and then had five hours interval without aeration. The aeration would begin at 0:00 AM, 6:00 AM, 12:00 PM and 6:00 PM, respectively. 2.2. Sample collection and analytical methods Water samples were taken from influent and effluent to analyze COD, NH4+-N and TN concentrations according to the standard methods every 5 days (APHA, 2003). Statistical checks were made at significant differences of 0.05 for all analyses using 4
SPSS 12.0 (n=12). Gas sampling was done using a closed static chamber. Five gas samples were collected at 0, 30, 60, 90 and 120 min after enclosure at the same time of the day between 9:00 AM and 11:00 AM every 5 days. N2O concentration was analyzed by gas chromatography. Agilent 6890N equipped with an electron capture detector and a Poropak Q column and used 40 mL/min argon-containing 5% methane as the carrier gas. Temperature of the detector and oven was set at 300℃ and 120℃, respectively. After determining the concentration of N2O, N2O emission rate was calculated by the following equation (Nakano et al., 1995). Flux = (dC/dt)×(1/A)×(13.5M)×[0.0225×0.082×(T+273)×1000]-1 where Flux is the N2O emission rate (mg/(m2 h)); dC/dt is the slope of best-fit line for plot of gas concentration inside the chamber and time data points (mg/h); A is the section area of the gas chamber (m2); M is the molecular weight of N2O; and T is the air temperature inside the chamber (℃). N2O conversion ratio is the quality percentage of nitrogen convert to N2O occupied in TN. Matrix samples were collected from sampling ports after each experiment. Four samples were taken from each layer and blended (5.0 g). After each on-site collection, the samples were stored in an ice incubator, and subsequently brought back to laboratory for analyses. Soil DNA kits (Omega, D5625-01) were used to extract and purify total genomic DNA from the samples. Extracted genomic DNAs were detected by 1% agarose gel electrophoresis, and preserved at -20℃ freezer until further use. Quantitative analysis was made on the target fragments of the following functional 5
genes: amoA, nxrA, narG, napA, nirK, nirS, qnorB and nosZ. The protocol and parameters for each target gene according to previous study (Pan et al., 2017). The standard samples were diluted to yield a series of 10-fold concentrations, and were subsequently used for qPCR standard curves. The coefficient of determination (R2) for all standard curves were greater than 0.99. Functional gene abundances involved in nitrogen removal were quantitated by qPCR technique according to Ji et al. (2012). qPCR was performed on a Roche Lightcycler 480 Real-Time PCR detection system (Roche Diagnostics, Meylan, France) in final 20 µL volume reaction mixtures containing the following components: 10 µL SYBR Green I PCR master mix (Applied Biosystems, USA), 1 µL template DNA (sample DNA or plasmid DNA for standard curves), forward and reverse primers, and sterile water. qPCR was performed in a three-step thermal cycling procedure. Each qPCR was performed in 40 cycles and followed by a melting curve analysis. Sterile water was used as a negative control and the data obtained from the qPCR were normalized to copies per gram of biological carrier in the SWISs. 3. Results and discussion 3.1. DO profiles in an aerated/non-aerated cycle DO concentrations less than 0.2 mg/L are commonly interpreted as anaerobic environment and more than 2.0 mg/L are deemed as aerobic environment. DO concentrations in the range of 0.2-0.5 mg/L represent anoxic environment (Wang et al., 2006). Fig. 1 shows DO distribution in an aerated/non-aerated cycle in aerated and non-aerated (NA) SWISs under different OLRs. DO concentrations were below 1.23, 6
0.26 and 0.02 mg/L at 50, 80 and 110 cm depths in NA SWIS under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), respectively. It could conclude that non-aerated SWIS was under anoxic or anaerobic condition. The prevalent environment in conventional SWISs was anoxic or anaerobic below distributing pipe because oxygen from air diffused to the matrix was limited (Fan et al., 2013b; Pan et al., 2016). DO concentrations decreased with OLR increasing in NA and IA SWISs because more oxygen was consumed by biological oxidation of excessive organic matter. DO concentrations at 50 cm depth of IA SWIS were higher than that in NA SWIS. DO concentrations enhanced in aerated period, but declined slowly in non-aerated period. High aeration-initiated DO values may be decreased by the degradation of nutrients and organic matter, which may interpret the decreasing tendency in non-aerated period. Under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), DO concentrations were more than 5.2 mg/L at 50 cm depth during aeration and as high as 2.0 mg/L when supplementary aerations were turned off. At 80 and 110 cm depths, DO concentrations were below 0.44 and 0.07 mg/L during aeration, and were below 0.30 and 0.03 mg/L without aeration, respectively. Aerobic condition was effectively created at 50 cm depth and anoxic or anaerobic condition was not changed at 80 and 110 cm depths in IA SWIS by intermittent aeration under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d). Under OLR of 21.8 g BOD/(m2 d), DO concentrations were lower than 2.0 and 1.0 mg/L at 50 cm depth after aeration shut down for 140 and 220 minutes, respectively. The reason was more oxygen consumed due to the mineralization of high influent organic matter. Intermittent aeration could not develop aerobic 7
environment in upper layer of the matrix under high OLR (21.8 g BOD/(m2 d)) in this experimental condition. [here insert Fig. 1] 3.2. COD, NH4+-N and TN removal performance Average effluent concentrations and removal rates of COD, NH4+-N and TN in NA and IA SWISs during the experimental period are shown in Fig. 2. There were significant differences in NA and IA SWISs under different OLRs. NA SWIS achieved average COD removal rates of 81.5%, 73.4%, 62.3% and 44.1%, with effluent COD concentrations of 33.9, 48.5, 69.1 and 102.4 mg/L under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), respectively. With the increase of OLR, oxygen deficiency developed more obviously, resulted in decrease of COD removal rate in NA SWIS. This result was in agreement with other studies (Fan et al., 2013a; Fei et al., 2016). Average effluent COD concentrations of NA SWIS were significantly higher than that of IA SWIS under the same OLR (P<0.05). Average COD removal rates were 97.2%, 94.6% and 91.6% under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d) in IA SWIS, respectively. Increasing OLR hardly affected the efficiency of organic matter degradation under OLR between 5.3 and 16.5 g BOD/(m2 d), which was significantly different from that in NA SWIS. Previous studies informed that the suitable range of OLRs were 1.5-6.0 g BOD/(m2 d) for conventional SWISs in treating domestic wastewater (Oladoja and Ademoroti, 2006). Organic loading resistance ability of IA SWIS was much higher because of the application of intermittent aeration. Fan et al. (2013b) and Wu et al. (2015) reported that sufficient oxygen supply would greatly 8
enhance the performance of aerobic biochemical oxidation. Decomposition of organic matter occurred mostly in the upper matrix of the SWISs (Li et al., 2011a). Aerobic condition in the upper matrix enhanced by intermittent aeration and thus facilitated aerobic removal of organic matter. IA SWIS achieved average COD removal rate of 81.1% and effluent COD concentration of 34.5 mg/L under OLR of 21.8 g BOD/(m2 d). Oxygen availability was considered as one of the main rate-limiting factors for organics degradation in SWISs (Fan et al., 2013b). Intermittent aeration did not develop favorable DO environment (Fig. 1) under high OLR of 21.8 g BOD/(m2 d) in this experimental condition, which restricted the degradation of organic matter. [here insert Fig. 2] Nitrification plays an important role in nitrogen removal which is an aerobic chemo-autotrophic microbial process (NH4+→NO2 -→NO3-) and occurs only when oxygen is presented in a high enough concentration (Albuquerque et al., 2009). As shown in Fig. 2, average effluent NH4 +-N concentrations in NA SWIS were significantly higher than that of IA SWIS under the same OLR (P<0.05). It is widely accepted that DO concentrations above 1.5 mg/L are essential for better nitrification (Fan et al., 2013b). DO concentrations were lower than 1.2 mg/L at all depths in NA SWIS under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), which led to anoxic and anaerobic environment in the matrix, therefore NH4+-N removal was seriously restricted. As OLR increased from 5.3 to 21.8 g BOD/(m2 d), average NH4+-N removal rate dropped from 73.3% to 30.7%. Increased organic matter consumed more available DO and further limited the activity of autotrophic ammonia oxidation 9
bacteria through greater deficit of oxygen, thus resulted in low nitrification. Artificial aeration is the most effective strategy to guarantee sufficient oxygen supply in SWISs, especially when dealing with high organic pollutant loading wastewater (Hu and Zhao, 2012; Wu et al., 2015). As shown in Fig. 2, nitrification rates were significantly enhanced in IA SWIS. Average effluent NH4 +-N concentrations were 3.1, 3.6 and 3.8 mg/L under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), and average NH4+-N removal rates were 91.6%, 90.3% and 89.7%, respectively. DO concentrations were above 2.0 mg/L in the upper layer through artificial aeration in IA SWIS, which facilitated achieving high NH4 +-N removal rate above 89% even under high OLR of 16.5 g BOD/(m2 d). Intermittent aeration did not supply enough oxygen for nitrification (Fig. 1) under high OLR of 21.8 g BOD/(m2 d) in this experimental condition, thus inhibited nitrification. With increasing OLR, more oxygen would demand owing to the requirement of mineralization of excess influent organic matter. Nitrification could transform nitrogen into various forms. Nevertheless, it could not achieve nitrogen removal from wastewater. Nitrified nitrogen must be processed through anaerobic and heterotrophic microbial denitrification (NO3-→NO2-→NO-→N2O→N2) to be removed from wastewater permanently. Insufficient organic carbon source and excess oxygen could restrict denitrification completion (Fan et al., 2013b). As shown in Fig. 2, TN removal rates were far from satisfactory in NA SWIS and average effluent TN concentrations were 13.3, 16.7, 22.3 and 29.5 mg/L under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d), respectively. Average TN removal rate dropped from 66.4% to 25.5% with OLR 10
increasing from 5.3 to 21.8 g BOD/(m2 d), which was similar to the variation of NH4 +-N removal rate. NA SWIS could not achieve high NH4 +-N removal without adequate DO concentration, which would adversely restrict denitrification due to insufficient supply of NO3--N as electron acceptors. Average TN removal rates in IA SWIS were significantly higher than that of NA SWIS under the same OLR (P<0.05). Average TN removal rate was enhanced from 81.6% under OLR of 5.3 g BOD/(m2 d) to 84.3% under OLR of 10.9 g BOD/(m2 d), to 87.1% under OLR of 16.5 g BOD/(m2 d) in IA SWIS. After effective nitrification, NO3--N and NO2--N as electron accepters could not be removed unless sufficient organic carbon was supplied as electron donor. Carbon deficiency became the main limiting factor for TN removal (Fan et al., 2013a). In this study, high OLR could supply more organic matter as electron donor for efficient denitrification, and achieved the highest TN removal rate under OLR of 16.5 g BOD/(m2 d). However, average TN removal rate of IA SWIS decreased to 56.1% under OLR of 21.8 g BOD/(m2 d) due to lower nitrification. According to the results, OLR variations of 5.3-16.5 g BOD/(m2 d) were recommended for high COD, NH4+-N and TN removal performances in intermittent aerated SWISs. When compared to other enhancing SWISs, average COD, NH4+-N and TN removal efficiencies of intermittent aerated SWIS under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d) in this study were higher than those in intermittent operated SWIS (91.5% for COD, 86.2% for NH4+-N, 80.8% for TN, Li et al., 2015), in bioaugmentation SWIS (83.8% for COD, 75.0% for NH4+-N, 61% for TN, Zou et al., 2009) and in 11
shunt distributing wastewater SWIS (84.8% for COD, 87.7% for NH4+-N, 70.1% for TN, Li et al., 2011b). 3.3. N2O emission N2O is an intermediate product of nitrification/denitrification processes in SWISs (Kong et al., 2002). Denitrification is generally regarded as the dominant process responsible for the N2O emission (Tiedje, 1982). Fig. 3 shows N2O conversion ratio and emission rate of each SWIS. N2O conversion ratios of NA and IA SWISs (0.44-0.98%) were consistent with previous studies (Johansson et al., 2003; Li et al., 2017). N2O emission rates under OLR of 5.3, 10.9, 16.5 and 21.8 g BOD/(m2 d) were as follows: 42.1, 38.5, 32.4, 22.5 mg/(m2 d) for IA SWIS, and 36.7, 33.6, 26.3, 18.8 mg/(m2 d) for NA SWIS, respectively. Under the same OLR, N2O emission rates of IA SWIS were higher than that of NA SWIS. Low N2O emission rate of NA SWIS was attributed to low nitrification, which restricted denitrification process. Denitrification is highly dependent on nitrification, an aerobic chemoautotrophic process, which produces nitrate from ammonium. Intermittent aeration developed appropriate oxygen condition for nitrification/denitrification processes in IA SWIS. N2O emission rate of NA SWIS decreased with the increase of OLR. The reason for NA SIWS was increased organic matter consumed more available oxygen and further limited nitrification process. In IA SWIS, N2O emission rate decreased with OLR increasing within the range of 5.3 and 16.5 g BOD/(m2 d). The upper matrix was sufficient in oxygen by intermittent aeration which enhanced the nitrification of NH4 +-N and aerobic biochemical oxidation of COD. However, carbon source of lower 12
part was not enough which inhibited part of N2O from being reduced to N2 before released to the atmosphere under low OLR. Higher OLR could supply more carbon source for N2O to N2 reduction, therefore N2O emission rate decreased. Previous study concluded that sufficient carbon source supplied after efficient nitrification could greatly reduce N2O emission (Wang et al., 2014). Low N2O emission rate of IA SWIS under OLR of 21.8 g BOD/(m2 d) was due to low nitrification. According to the results, OLR of 16.5 g BOD/(m2 d) were recommended for low N2O emission in intermittent aerated SWISs. [here insert Fig. 3] 3.4. Functional gene abundances involved in nitrogen removal Nitrogen removal functional genes are shown in Fig. 4, which are involved in nitrification/denitrification processes. The amoA and nxrA genes are the marker of oxidizing NH4+-N to NO2--N and oxidizing NO2--N to NO3--N (Dionisi et al., 2002), respectively. The abundances of amoA and nxrA decreased along the wastewater flow direction in NA and IA SWISs, which followed the same trend as DO. The life activity, growth and enrichment of ammonia-oxidizing bacteria are related to DO (Fan et al., 2013b). Ammonia oxidizing bacteria (AOB) oxidize NH4+-N to NO2--N and provide substrate for nitrite-oxidizing bacteria (NOB) to oxidize NO2--N to NO3--N, which might explain lower abundance of nxrA compared to amoA. With OLR increasing, the abundances of amoA and nxrA decreased in NA SWIS. OLR increased by 5 g BOD/(m2 d), the abundances of amoA and nxrA decreased one order of magnitude at 50 cm depth. Previous studies also found that high organic matter reduced the growth 13
of AOB and NOB in conventional infiltration systems (Fan et al. 2013a). In IA SWIS, the abundances of amoA and nxrA were significantly higher than those of NA SWIS under the same OLR (P<0.05) at 50 cm depth. Sufficient oxygen supply could greatly improve the number of nitrification bacteria and enzyme activities involved in NH4 +-N transformation under high OLR (Pan et al., 2015). DO concentrations of IA SWIS (DO>2.0 mg/L) were improved through aeration at 50 cm depth under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), which was favorable for the nitrification bacteria. The abundances of amoA and nxrA were the lowest under OLR of 21.8 g BOD/(m2 d) in IA SWIS. The reason was more oxygen consumed due to the degradation of high influent organic matter which restricted enzyme activities involved in NH4+-N transformation. [here insert Fig. 4] NO3--N to NO2--N reduction, catalyzed by the key gene narG and napA, is the first reaction process in denitrification. NO2--N to NO reduction is the second reaction process, which is catalyzed by gene of nirS and nirK. NO to N2O reduction is the third process, which is catalyzed by qnorB. N2O to N2 reduction catalyzed by nosZ is the last reaction. As seen from Fig. 4, the abundances of napA, narG, nirS, nirK, qnorB and nosZ declined with OLR increasing in NA SWIS similar to other study (Sun et al., 2017). OLR increased by 5 g BOD/(m2 d), the abundances of napA, narG, nirS, nirK, qnorB and nosZ decreased one order of magnitude at 80 and 110 cm depths. Less NO3- as the substrate for anaerobic denitrification due to low nitrification decreased the abundances of napA, narG, nirS, nirK, qnorB and nosZ in NA SWIS. In IA SWIS, 14
the abundances of napA, narG, nirS, nirK, qnorB and nosZ increased with OLR increasing under OLR range of 5.3 and 16.5 g BOD/(m2 d). More organic matter provided good nutritional condition for the growth and enrichment of anaerobic denitrifying bacteria with OLR increasing in IA SWIS after nearly complete nitrification with aeration, in parallel to enhancing the enrichment of nitrogen removal functional genes. The level of nosZ gene expression is a key factor determining whether N2O is converted to N2 or released into the atmosphere as a greenhouse gas (Thomson et al., 2012). Previous study confirmed that increasing nosZ gene abundance promoted N2O reduction (Harter et al., 2014). More organic matter improved the conversion of N2O to N2 under higher OLR, which could further explain the lowest N2O emission in IA SWIS under OLR of 16.5 g BOD/(m2 d). 4. Conclusion Intermittent aeration created appropriate oxygen condition for nitrification and denitrification under OLR of 5.3, 10.9 and 16.5 g BOD/(m2 d), which increased COD, NH4 +-N, TN removal, and enhanced the abundances of amoA, nxrA, napA, narG, nirS, nirK, qnorB and nosZ genes. Increasing OLR affected COD, NH4+-N and TN removal more in non-aerated SWIS than that in intermittent aerated SWIS. High COD removal rate of 91.6%, TN removal rate of 85.9% and low N2O emission rate of 32.4 mg/(m2 d) were obtained in IA SWIS under OLR of 16.5 g BOD/(m2 d). Acknowledgements This research is financially supported by the National Natural Science Foundation of China (No.41001321), (No.41471394); Liaoning BaiQianWan Talents Program 15
[2015(45)]; Shenyang Science and Technology Project (17-231-1-93); Natural Science Foundation of Liaoning (2015010585-301); Ecology and Environment Research
Center
Director
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Shenyang
Normal
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Figure Captions Fig. 1 DO profiles in an aerated/non-aerated cycle in SWISs with intermittent aeration (IA) and without aeration (NA).
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Fig. 2 NH4+-N, TN and COD removal performance in SWISs without aeration (NA), with intermittent aeration (IA) under organic loading rate (a) 5.3 g BOD/(m2 d), (b) 10.9 g BOD/(m2 d), (c) 16.5 g BOD/(m2 d), (d) 21.8 g BOD/(m2 d).
Fig. 3 N2O emission rate and conversion ratio in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate (OLR) a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
Fig. 4 Abundances of nitrogen removal functional genes in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
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Fig. 1 DO profiles in an aerated/non-aerated cycle in SWISs with intermittent aeration (IA) and without aeration (NA).
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Fig. 2 NH4+-N, TN and COD removal performance in SWISs without aeration (NA), with intermittent aeration (IA) under organic loading rate (a) 5.3 g BOD/(m2 d), (b) 10.9 g BOD/(m2 d), (c) 16.5 g BOD/(m2 d), (d) 21.8 g BOD/(m2 d). 23
Fig. 2 NH4+-N, TN and COD removal performance in SWISs without aeration (NA), with intermittent aeration (IA) under organic loading rate (a) 5.3 g BOD/(m2 d), (b) 10.9 g BOD/(m2 d), (c) 16.5 g BOD/(m2 d), (d) 21.8 g BOD/(m2 d). 24
Fig. 3 N2O emission rate and conversion ratio in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate (OLR) a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
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Fig. 4 Abundances of nitrogen removal functional genes in two SWISs with intermittent aeration (IA) and without aeration (NA) under organic loading rate a: 5.3 g BOD/(m2 d), b: 10.9 g BOD/(m2 d), c: 16.5 g BOD/(m2 d), d: 21.8 g BOD/(m2 d).
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Highlights Intermittent aerated SWISs were effective in treating high strength wastewater. Intermittent aeration improves TN removal at higher OLR up to 16.5 g BOD/(m2 d). 16.5 g BOD/(m2 d) was recommended for SWISs with 4 aerated/non-aerated cycles a day. Aeration raised amoA, nxrA, narG, napA, nirK, nirS, qnorB and nosZ abundances.
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