Abstract

Nitrogen removal and N2O emission of a biochar-sludge amended soil wastewater infiltration system (SWIS) with/without intermittent aeration under different influent COD/N ratios was investigated. Nitrogen removal and N2O emission were affected by influent COD/N ratio. Under a COD/N ratio between 1:1 and 15:1, average chemical oxygen demand (COD), NH4+-N and total nitrogen (TN) removal rates decreased with COD/N ratio increase in non-aerated SWISs amended with/without biochar-sludge; an increasing COD/N ratio hardly affected COD and NH4+-N removal in a biochar-sludge amended SWIS with intermittent aeration; the N2O emission rate decreased with COD/N ratio increase in the studied SWISs. The biochar-sludge amended SWIS with intermittent aeration achieved high COD (92.2%), NH4+-N (96.8%), and TN (92.7%) removal rates and a low N2O emission rate (10.6 mg/(m2 d)) under a COD/N ratio of 15:1, which was higher than those in non-aerated SWISs amended with/without biochar-sludge. Combining the biochar-sludge amended SWIS with intermittent aeration enhanced the number of nitrifying bacteria, denitrifying bacteria, nitrate reductase activities, nitrite reductase activities, and improved the abundance of nitrogen removal functional genes under a high influent COD/N ratio. The results suggested that the joint use of intermittent aeration and biochar-sludge in a SWIS could be an effective and appropriate strategy for improving nitrogen removal and reducing N2O emissions in treating high COD/N ratio wastewater.

INTRODUCTION

With the rapid economic development, urbanization and population growth of China, domestic wastewater discharges exceeded 53.52 billion tons in 2015. Decentralized domestic wastewater especially from vast rural areas and small towns was the main contributor (Wu et al. 2015). In such communities, a system with low construction and operation costs, easy maintenance and high pollutant removal efficiency, such as the soil wastewater infiltration system (SWIS), may be more desirable.

SWIS has proven to be a good treatment method for decentralized wastewater treatment according to integrated mechanisms of chemical, physical and biological reactions as it passes through the unsaturated soil in infiltration system (Wang et al. 2010). However, limited nitrogen removal (merely around 50% for total nitrogen (TN) removal and generally 60–80% for NH4+-N removal) remains as a major challenge for conventional SWISs because of insufficient oxygen supply and lack of biodegradable organics (Li et al. 2011b; Sun et al. 2018). Many studies have been carried out to improve nitrogen removal in SWISs, such as intermittent operation (Li et al. 2011b), amended original soil (Li et al. 2017) and configuration styles (Li et al. 2011a; Lloréns et al. 2011). Nitrification and denitrification are widely acknowledged to be the main nitrogen removal mechanisms (Wang et al. 2010; Wu et al. 2015). Two aspects can be easily artificially regulated to enhance nitrogen removal in the SWIS. One is to develop a favorable aerobic or anaerobic environment, the other is to supply an extra carbon source into the infiltration system after an efficient nitrification process (Pan et al. 2016). Intermittent aeration has been proved to be an effective method to improve nitrification (Wu et al. 2015; Yang et al. 2016). In order to improve nitrogen removal, an extra carbon source should be added into the infiltration system to strengthen denitrification (Pan et al. 2016). Soluble carbon sources, such as methanol (Gómez et al. 2000) and hydrolyzed sludge (Zhou et al. 2018), have acted as external carbon sources to boost denitrification. However, operating costs increased and effluent water quality with soluble carbon sources fluctuated. One alternative is solid carbons such as biochar and sludge. Biochar is a product of pyrolysis of agricultural biomass waste and has been recognized as a multifunctional material for environmental applications (Zhou et al. 2017). Furthermore, the addition of biochar to wastewater treatment can improve effluent quality and reduce greenhouse gas emissions (Sun et al. 2018). Sludge is the byproduct of wastewater treatment, which is a heterogeneous mixture of organic matter, micro-organisms, colloids and cations. Many investigations have proved biochar could be a potential carbon source for denitrification (Zhou et al. 2018). Kadam et al. (2008) and Li et al. (2011b) have detected that sludge would produce high biomass concentration and significantly improve chemical oxygen demand (COD) and TN removal. Sun et al. (2018) revealed that biochar and sludge could reduce N2O emission and enhance pollutant removal performance in SWISs.

Influent COD/N ratio could affect the nitrification and denitrification functions of microorganisms, consequently, which would be one of the specific parameters for N2O emission from biological nitrogen removal (Wu et al. 2009; Zhou et al. 2017). Recently, studies have been conducted to study the impact of the influent COD/N ratio in SWISs. Pan et al. (2017) concluded that nitrogen removal increased with increasing influent COD/N ratio in intermittent aerated SWISs. Li et al. (2017) indicated that a low COD/N ratio was most beneficial for reducing N2O emissions in conventional SWISs. Nevertheless, the effect of influent COD/N ratio on nitrogen removal and the N2O emission of novel biochar-sludge amended SWISs with/without intermittent aeration still remains unclear.

Therefore, the main objectives of this study were: (1) to evaluate the effect of influent COD/N ratio on COD removal, NH4+-N removal, TN removal and N2O emission in biochar-sludge amended SWISs with/without intermittent aeration; (2) to investigate the effect of influent COD/N ratio on microbial populations, enzyme activities and functional genes involved in nitrogen removal in biochar-sludge amended SWISs with/without intermittent aeration.

MATERIAL AND METHODS

SWISs description and operation

The study was carried out in Shenyang Normal University, Liaoning, China. Three parallel microcosm SWISs were constructed for treating synthetic domestic wastewater with the same dimensions (120 cm in height and 50 cm in diameter). The schematic diagram of the experimental SWISs is shown in Figure 1. A distributing pipe was installed at 50 cm depth below the surface in each infiltration system. 10 cm of deep gravel (10–20 mm in diameter) was prepared at the bottom to support the infiltration system and evenly distribute the treated water. The treated wastewater was collected in the lower part of each system near the outlet. SWIS A was composed of an aerated unit, which consisted of an air compressor, air tube and micro-bubble diffuser at a depth of 40 cm. The micro-bubble diffuser and distributing pipe were surrounded by gravel (10–20 mm in diameter) to prevent clogging and diffuse air. Sampling ports were installed at 50, 80 and 110 cm depth from the top of each SWIS to test microbial populations, enzyme activities and functional genes involved in nitrogen removal. SWIS C was filled with 80% brown earth and 20% coal slag by weight ratio. Infiltration beds of SWIS A and B were 80% brown earth, 10% sludge and 10% biochar by weight ratio. The brown earth was collected from the top 20 cm from Shenyang Ecological Station. The coal slag was from a furnace, 4–8 mm in diameter, used to improve the permeability and absorption area of the matrix. Sludge was collected from the sludge-dewatering unit of Shenyang Beibu wastewater treatment plant, and air dried after being centrifuged. Corn straw was carbonized under anaerobic conditions with a 15 h slow pyrolysis, at a temperature ramp of 10 °C/min to a maximum temperature of 500 °C. The matrix components were mixed in a blender five times with 15 min/time to ensure uniformity.

Figure 1

Schematic diagram of three soil wastewater infiltration systems (SWISs) (SWIS A: intermittent aeration with biochar and sludge; SWIS B: no intermittent aeration with biochar and sludge; SWIS C: no intermittent aeration without biochar and sludge). (1) High-level tank; (2) liquid flow meter; (3) infiltration system body; (4) gas flow meter; (5) air compressor; (6) distributing pipe; (7) perforated diffuser; (8) 80% brown earth and 20% coal slag; (9) 80% brown earth, 10% sludge and 10% biochar; (10) gravel; (11) outlet; (12) sampling port.

Figure 1

Schematic diagram of three soil wastewater infiltration systems (SWISs) (SWIS A: intermittent aeration with biochar and sludge; SWIS B: no intermittent aeration with biochar and sludge; SWIS C: no intermittent aeration without biochar and sludge). (1) High-level tank; (2) liquid flow meter; (3) infiltration system body; (4) gas flow meter; (5) air compressor; (6) distributing pipe; (7) perforated diffuser; (8) 80% brown earth and 20% coal slag; (9) 80% brown earth, 10% sludge and 10% biochar; (10) gravel; (11) outlet; (12) sampling port.

To determine the influence of COD/N ratio on removal performance and N2O emission, the COD/N (namely chemical oxygen demand/TN concentration) ratio of influent was manipulated by changing glucose to create four COD/N ratios (1:1, 5:1, 10:1 and 15:1). Accordingly, synthetic wastewater was used with different influent COD/N ratios by mixing the following components in tap water: 54, 212, 386, 529 mg/L glucose, 163 mg/L (NH4)2SO4, 11 mg/L KH2PO4, 10 mg/L NaNO3, 10 mg/L ZnSO4·7H2O, 10 mg/L FeSO4, 10 mg/L MgSO4, 10 mg/L MnSO4 and 10 mg/L CaCl2. The characteristics of the influents with different influent COD/N ratios are shown in Table 1. Temperature was 20 ± 1 °C. Wastewater was continuously fed into each SWIS under the hydraulic loading of 0.1 m3/(m2 d). Four operation schemes were arranged in each SWIS, with influent COD/N ratio gradually elevated from 1:1 to 5:1, 10:1 and 15:1. Each operation scheme lasted for 60 days. SWIS A was subjected to aeration with an airflow rate of 2.0 ± 0.2 L/min, which had four aerated/non-aerated cycles every day. In each cycle, the system was subjected to aeration for an hour and then had a five hour interval without aeration. The aeration would begin at 0:00 AM, 6:00 AM, 12:00 PM and 6:00 PM, respectively.

Table 1

Characteristics of influent (mean)

Parameter Influent COD/N ratio
 
1:1 5:1 10:1 15:1 
COD (mg/L) 41.6 134.2 245.5 487.9 
NH4+-N (mg/L) 37.6 38.2 37.9 37.9 
NO3-N (mg/L) 3.5 3.9 3.6 4.7 
TN (mg/L) 41.1 42.1 41.5 42.6 
TP (mg/L) 3.5 3.7 3.6 3.8 
Parameter Influent COD/N ratio
 
1:1 5:1 10:1 15:1 
COD (mg/L) 41.6 134.2 245.5 487.9 
NH4+-N (mg/L) 37.6 38.2 37.9 37.9 
NO3-N (mg/L) 3.5 3.9 3.6 4.7 
TN (mg/L) 41.1 42.1 41.5 42.6 
TP (mg/L) 3.5 3.7 3.6 3.8 

Sampling and analytical methods

Water samples of influent and effluent in the three systems were used to analyze the treatment performance of organics and nitrogen every 5 days according to the standard methods (APHA 2003).

Gas sampling was collected using the static stationary chamber. Gas samples were collected at 0, 20, 40 and 60 min after enclosure between 9:00 and 10:00 AM every 5 days. N2O concentration was analyzed by Agilent 6890N gas chromatography, which equipped with an electron capture detector and a Poropak Q column and used 40 mL/min argon-containing 5% methane as the carrier gas. The temperature of the detector and oven was set at 300 °C and 120 °C, respectively. After determining the concentration of N2O, the N2O emission rate was calculated by the following equation (Li et al. 2017).  
formula
where dC/dt is the slope of the best-fit line for the 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 (°C). The N2O conversion ratio is the quality percentage of nitrogen converted to N2O occupied in TN of influent.

Matrix samples were collected from sampling ports (at 50, 80 and 110 cm depths) to investigate microbial populations, enzyme activities and functional gene abundances involved in nitrogen removal in each SWIS after each influent COD/N ratio experiment. Nitrifying and denitrifying bacteria were counted using the most probable number (MPN) calculation (Li et al. 2011a). Nitrate reductase (NR) activities and nitrite reductase (NIR) activities in the matrix were measured according to the method of Abdelmagid & Tabatabai (1987). Functional gene abundances involved in nitrogen removal were quantified by the quantitative polymerase chain reaction (qPCR) technique according to Ji et al. (2012).

Statistical analysis

All experimental data were expressed as means of triplicates. All statistical analyses were performed by software SPSS 12.0. Two-sample t-tests were used to estimate the significance of differences between means. For all tests, differences were considered statistically significant only if P < 0.05.

RESULTS AND DISCUSSION

Overall treatment performance

Figure 2 and Table 2 show the effluent water qualities and removal rates of the three SWISs. Effluent COD concentration in SWIS B and C increased with the increase of influent COD/N ratio. Average effluent COD concentrations were 8.7, 24.6, 101.3 and 294.6 mg/L, with average removal rates of 79.1%, 67.3%, 58.7% and 39.6% for various influent COD/N ratios in SWIS C. A high COD removal rate was achieved in the conventional SWIS when influent COD/N ratios were low (1:1 and 5:1). Conventional SWISs were always efficient in organic matter removal under low COD/N ratio (Song et al. 2016; Pan et al. 2017). Organic matter could be degraded by aerobic and anaerobic bacteria in SWISs and aerobic heterotrophic bacteria played an important role in the aerobic degradation of organic matter (Wu et al. 2015). Previous study showed that the prevailing conditions in conventional SWISs were anoxic or anaerobic below the distribution pipe because air diffusion to the soil matrix was limited (Wang et al. 2010). However, the limitation of oxygen became more obvious with the increase of the COD/N ratio, resulted in a decrease in the COD removal rate in SWIS C. This result was in accordance with other studies (Fan et al. 2013; Song et al. 2016). Average COD removal rates were 89.2%, 81.6%, 80.1% and 69.6% for biochar-sludge amended SWIS B, which were higher than those of SWIS C without biochar-sludge. The possible reasons are that biochar has a highly porous structure and large surface area, which provides enough space for microbial growth and reproduction. Sludge has a high biomass concentration. Former studies concluded biochar played an important role in the reduction of COD in a wastewater land treatment system (Zhou et al. 2017) and a matrix amended with sludge could improve COD removal in SWISs (Sun et al. 2018). However, average COD removal rates were 97.1%, 94.3%, 93.9% and 92.2% under the experimental influent COD/N ratios in SWIS A, respectively. Increasing the COD/N ratio hardly affected the efficiency of COD degradation, which was different from that of SWIS B and C. This was largely attributed to the intensified oxygen supply generated by intermittent aeration in SWIS A, which was consistent with a previous study (Yang et al. 2016). Yang et al. (2016) aimed to improve treatment performance of the SWIS using micro-power aeration and concluded that aerobic conditions were achieved by intermittent aeration, resulting in stable and satisfactory removal efficiencies of COD (81.1–94.8%). Fan et al. (2013) reported that sufficient oxygen supply could greatly increase the performance of aerobic biochemical oxidation and improve COD removal under high influent COD/N ratio. This study indicated that sufficient oxygen supply could greatly increase organic matter degradation for biochar-sludge amended SWISs, with average COD removal rates above 92%, even under an influent COD/N ratio of 15:1.

Table 2

COD, NH4+-N, TN removal rates in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1

  SWIS A SWIS B SWIS C 
Removal rate (%) Influent COD/N ratio of 1:1 
COD 97.1 89.2 79.1 
NH4+-N 99.5 84.3 69.9 
TN 11.0 81.1 67.5 
Influent COD/N ratio of 5:1 
COD 94.3 81.6 67.3 
NH4+-N 99.2 80.1 54.7 
TN 56.7 77.3 49.1 
Influent COD/N ratio of 10:1 
COD 93.9 80.1 58.7 
NH4+-N 98.9 70.5 33.3 
TN 84.3 66.1 27.6 
Influent COD/N ratio of 15:1 
COD 92.2 69.6 39.6 
NH4+-N 96.8 39.7 14.5 
TN 92.7 37.1 11.9 
  SWIS A SWIS B SWIS C 
Removal rate (%) Influent COD/N ratio of 1:1 
COD 97.1 89.2 79.1 
NH4+-N 99.5 84.3 69.9 
TN 11.0 81.1 67.5 
Influent COD/N ratio of 5:1 
COD 94.3 81.6 67.3 
NH4+-N 99.2 80.1 54.7 
TN 56.7 77.3 49.1 
Influent COD/N ratio of 10:1 
COD 93.9 80.1 58.7 
NH4+-N 98.9 70.5 33.3 
TN 84.3 66.1 27.6 
Influent COD/N ratio of 15:1 
COD 92.2 69.6 39.6 
NH4+-N 96.8 39.7 14.5 
TN 92.7 37.1 11.9 
Figure 2

COD removal performance in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

Figure 2

COD removal performance in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

As shown in Figure 3 and Table 2, average effluent NH4+-N concentrations were 11.3, 17.3, 24.6 and 32.4 mg/L for different influent COD/N ratios (1:1, 5:1, 10:1 and 15:1) in SWIS C, which were higher than those of biochar-sludge amended SWIS B under the same influent COD/N ratio. Nitrification occurs with aerobic conditions. Most conventional SWISs fail to achieve an efficient nitrification as a result of disadvantageous anoxic or anaerobic conditions (Fan et al. 2013). However, for biochar-sludge amended SWIS B, average NH4+-N removal rates were 89.6%, 82.8%, 70.5% and 39.7% for different influent COD/N ratios, which were higher than those of SWIS C due to the adsorption of biochar and nitrifying bacteria provided by sludge. The porous structure of the biochar provided favorable conditions for the growth of microbial communities, such as ammonia-oxidizing bacteria and nitrifying bacteria, which contributed to higher NH4+-N removal (Zhou et al. 2018). The nitrification performance of non-aerated SWIS B and C decreased with the increase of influent COD/N ratio. Higher oxygen would be demanded due to the requirement for mineralization of the excess influent carbon source with the COD/N ratio increase. Moreover, the excessive organic matter could further inhibit the activity of autotrophic ammonia oxidation bacteria by dissolved oxygen (DO) competition in non-aerated systems. Biochar-sludge amended SWIS A with intermittent aeration achieved an average NH4+-N removal rate of more than 96% even under an influent COD/N ratio of 15:1, which was significantly higher than that of SWIS B and C (P < 0.05). Nitrification, as the main process for NH4+-N removal, was an aerobic chemo-autotrophic microbial process, which was the limiting step for nitrogen removal in conventional SWISs due to insufficient DO (Pan et al. 2017). Intermittent aeration highly improved the oxygen supply in SWIS A, which facilitated nitrification. A previous study also found that more nitrifying bacteria involved in nitrogen removal were detected in intermittent aerated SWISs than non-aerated SWISs (Fei et al. 2017). Increasing the COD/N ratio hardly affected NH4+-N removal in SWIS A, which was different from that in SWIS B and C. The result was consistent with Song et al. (2016) and Fan et al. (2013). Song et al. (2016) and Fan et al. (2013) investigated the effects of COD/N ratios on pollutant removal in infiltration systems with/without intermittent aeration and concluded that increasing the COD/N ratio hardly affected NH4+-N removal in intermittent aeration systems. Because of the efficient oxygen supply, excessive organic matter and other oxygen-demanding nutrients were not a limitation for the nitrification process (Fan et al. 2013).

Figure 3

NH4+-N, TN removal performance and the effluent concentrations of NO3-N, NO2-N in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

Figure 3

NH4+-N, TN removal performance and the effluent concentrations of NO3-N, NO2-N in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

Average TN removal rates were 67.5%, 49.1%, 27.6% and 11.9% in SWIS C, respectively (Table 2). The effluents were still dominated by a high concentration of NH4+-N due to limited nitrification. Conventional SWIS C could not achieve high NH4+-N removal in anoxic or anaerobic conditions, which would greatly inhibit denitrification due to the insufficient supply of NO3-N as electron accepters. Average effluent TN concentrations of SWIS B were 5.9, 7.6, 12.9 and 25.7 mg/L for different influent COD/N ratios, which were lower than those of SWIS C under the same influent COD/N ratio. Many studies reported that biochar could facilitate denitrification (Ding et al. 2010; Zhou et al. 2017). Sludge provided denitrifying bacteria (Li et al. 2011b). In addition, biochar and sludge, as carbon rich materials, were also potential carbon sources that could facilitate denitrification under low COD/N ratios of 1:1 and 5:1. Due to more DO depletion with increasing organic matter concentration, the average TN removal rate dropped from 67.5% to 11.9% for SWIS C and from 81.1% to 37.1% for SWIS B with the influent COD/N ratio increasing from 1:1 to 15:1. Average effluent TN concentrations were 37.2, 18.3, 6.5 and 3.1 mg/L under influent COD/N ratios of 1:1, 5:1, 10:1, and 15:1 in SWIS A, respectively. Influent COD/N ratio affected TN removal in the three SWISs. As seen from Figure 3 and Table 2, average effluent NO3-N concentrations were much higher than others under low COD/N ratio of 1:1 and 5:1, which led to low TN removal rates of only 11.0% and 56.7% in SWIS A, respectively. Intermittent aeration well developed aerobic conditions in the upper matrix and anoxic or anaerobic conditions in the subsequent cycle simultaneously, which facilitated nitrification in the previous studies (Pan et al. 2016). After effective nitrification under aerobic conditions, NO3-N and NO2-N as electron accepters could not be removed permanently unless sufficient organic carbon was supplied as the electron donor (Wu et al. 2015). Carbon deficiency then became the key limiting factor for TN removal in aerated reactors (Song et al. 2016). As the influent COD/N ratio increased from 5:1 to 10:1 and 15:1, the average effluent NO3-N concentration decreased from 17.9 to 5.8 and 1.8 mg/L. A high COD/N ratio could supply sufficient organic matter as an electron donor for complete denitrification and achieve high TN removal (Fan et al. 2013). High-efficiency nitrification and denitrification simultaneously occurred in SWIS A under a high influent COD/N ratio. A high COD/N ratio effectively eliminated the accumulation of NO3-N. The average TN removal rate reached 92.7% in SWIS A, which was higher than that of other intermittent aerated SWISs (89.6–89.7%) under the same influent COD/N ratio of 15:1 due to the addition of biochar-sludge (Song et al. 2016; Pan et al. 2017). A previous study proved that adding biochar and sludge to SWISs was effective to remove TN (Sun et al. 2018). The results suggest that intermittent aeration combined with biochar-sludge addition could be a feasible method for conventional SWISs to achieve high COD, NH4+-N and TN removal under a high influent COD/N ratio.

N2O emission

N2O is a significant contributor to global warming and the destruction of the ozone layer, which accounts for approximately 5% of the total greenhouse effect. During wastewater treatment in SWISs, N2O is the byproduct of incomplete nitrification and an intermediate product of incomplete denitrification (Li et al. 2017). As can be seen in Figure 4, average N2O conversion ratios of the three SWISs (0.14–0.49%) were consistent with previous studies (Li et al. 2017; Jiang et al. 2017). Li et al. (2017) and Jiang et al. (2017) investigated N2O emission of non-aeration and intermittent aeration SWISs and concluded that N2O conversion ratios were in the range of 0.1–0.76%, respectively. Average N2O emission rates under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1 were as follows: 38.4, 36.7, 31.2, 19.4 mg/(m2 d) for SWIS C; 31.2, 29.5, 25.1, 16.8 mg/(m2 d) for SWIS B; 21.3, 18.9, 13.8, 10.6 mg/(m2 d) for SWIS A, respectively. The N2O emission rate decreased gradually with increasing influent COD/N ratio in SWIS A, B and C. The high concentration of organic matter under a high influent COD/N ratio consumed more available DO and further limited the activity of autotrophic ammonia oxidation bacteria through greater deficit of oxygen, which resulted in low nitrification and denitrification in SWIS B and C, leading to a decrease in the N2O emission rate. The result of the conventional SWIS was not consistent with previous study (Li et al. 2017). The probable reason was different operational conditions. N2O production was closely correlated to the conditions of pH, temperature and especially oxygenation (Wu et al. 2009). In SWIS A, the high influent COD/N ratio could supply more carbon source for N2O to N2 reduction after efficient nitrification, therefore the N2O emission rate decreased. The average N2O emission rate in SWIS B was lower than that of SWIS C under the same influent COD/N ratio, which agreed with Zhou et al. (2017) and Sun et al. (2018). Biochar addition could reduce or suppress N2O emission (Zhou et al. 2017). Therefore, use of biochar-sludge as a substrate could be an effective method to reduce N2O emission for conventional SWIS. The average N2O emission rate in SWIS A was significantly lower than that of SWIS B under the same influent COD/N ratio (P < 0.05). Intermittent aeration could significantly reduce N2O emission for biochar-sludge amended SWIS. Zhou et al. (2017) found that using biochar and intermittent aeration could reduce N2O emission in infiltration systems.

Figure 4

N2O emission in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

Figure 4

N2O emission in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

In view of COD removal, NH4+-N removal, TN removal and N2O emission, an influent COD/N ratio of 15:1 was recommended for a biochar-sludge amended SWIS with intermittent aeration.

Microbial populations and enzyme activities involved in nitrogen removal

The number of nitrifying bacteria, denitrifying bacteria, NR activities and NIR activities are shown in Table 3. The number of nitrifying bacteria declined with increasing depth in non-aerated and aerated SWISs; in comparison, the number of denitrifying bacteria increased with increasing depth. With the influent COD/N ratio increasing, the number of nitrifying bacteria at 50 cm depth declined in the non-aerated SWIS B and C because of the disadvantageous oxygen conditions. The COD/N ratio had hardly any influence on the number of nitrifying bacteria at 50 cm depth of aerated SWIS A. Under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1, the number of nitrifying bacteria in SWIS B was 102 times higher than that of SWIS C at 50 cm depth due to SWIS B being amended with biochar-sludge. Under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1, the number of nitrifying bacteria in SWIS A was 10, 102, 103 and 104 times higher than that in SWIS B at 50 cm depth, respectively. The oxidative conditions of the upper matrix were improved through intermittent aeration, which was favorable for the growth and reproduction of nitrifying bacteria in SWIS A. A former study also found that more nitrifying bacteria and other viable bacteria were detected in intermittent aerated systems than non-aerated systems by FISH (fluorescence in situ hybridization analysis) (Fan et al. 2013). At 80 cm and 110 cm depths, the number of denitrifying bacteria declined in SWIS B and C, while increasing in SWIS A with the COD/N ratio increasing. Under influent COD/N ratios of 10:1 and 15:1, the number of denitrifying bacteria in SWIS A was 10, and 104 times higher than that of SWIS B, and 104 and 106 times higher than that of SWIS C at 80 cm and 110 cm depths, respectively. The nitrification process was better and the carbon source was sufficient in SWIS A under influent COD/N ratios of 10:1 and 15:1, which was beneficial to the growth and reproduction of denitrifying bacteria.

Table 3

Microbial populations and enzyme activities involved in nitrogen removal of three SWISs (mean)

Depth 50 cm
 
80 cm
 
110 cm
 
Item SWIS A SWIS B SWIS C SWIS A SWIS B SWIS C SWIS A SWIS B SWIS C 
Influent COD/N ratio of 1:1 
NNBa 6.0 × 109 6.2 × 108 9.5 × 106 5.0 × 103 6.0 × 103 8.0 × 102 3.5 × 103 2.5 × 103 2.5 × 102 
NDBb 2.5 × 102 3.5 × 103 2.5 × 102 3.5 × 104 4.5 × 107 3.5 × 106 6.5 × 104 6.7 × 107 7.0 × 106 
NRAc 0.54 0.78 0.43 0.86 1.67 1.32 0.95 1.85 1.51 
NIRd 0.19 0.47 0.13 0.25 0.89 0.85 0.32 0.94 0.92 
Influent COD/N ratio of 5:1 
NNBa 4.5 × 109 2.0 × 107 7.5 × 105 4.5 × 103 2.5 × 103 4.5 × 102 3.0 × 103 1.7 × 103 2.0 × 102 
NDBb 3.0 × 102 4.5 × 103 3.0 × 102 8.0 × 105 1.1 × 107 4.5 × 105 5.0 × 105 2.5 × 107 6.5 × 105 
NRAc 0.76 0.64 0.45 1.17 1.52 1.02 1.32 1.63 1.13 
NIRd 0.36 0.35 0.15 0.33 0.69 0.48 0.48 0.52 0.56 
Influent COD/N ratio of 10:1 
NNBa 6.5 × 109 6.5 × 106 9.5 × 104 4.0 × 103 1.7 × 103 2.5 × 102 2.5 × 103 1.4 × 103 1.7 × 102 
NDBb 4.0 × 102 3.5 × 102 2.5 × 102 2.5 × 107 4.5 × 106 6.5 × 103 6.5 × 107 5.2 × 106 5.0 × 103 
NRAc 0.93 0.58 0.48 2.14 1.28 0.86 2.56 1.31 0.89 
NIRd 0.51 0.26 0.14 0.17 0.57 0.26 1.20 0.62 0.37 
Influent COD/N ratio of 15:1 
NNBa 5.5 × 109 2.5 × 105 4.5 × 103 1.4 × 103 1.4 × 103 1.7 × 102 1.4 × 103 1.1 × 103 1.1 × 102 
NDBb 4.5 × 102 4.5 × 102 4.5 × 102 2.5 × 108 3.5 × 104 8.5 × 102 4.5 × 108 3.7 × 104 9.5 × 102 
NRAc 0.98 0.47 0.32 2.98 1.05 0.63 3.14 1.14 0.72 
NIRd 0.68 0.21 0.18 1.34 0.37 0.18 1.43 0.55 0.21 
Depth 50 cm
 
80 cm
 
110 cm
 
Item SWIS A SWIS B SWIS C SWIS A SWIS B SWIS C SWIS A SWIS B SWIS C 
Influent COD/N ratio of 1:1 
NNBa 6.0 × 109 6.2 × 108 9.5 × 106 5.0 × 103 6.0 × 103 8.0 × 102 3.5 × 103 2.5 × 103 2.5 × 102 
NDBb 2.5 × 102 3.5 × 103 2.5 × 102 3.5 × 104 4.5 × 107 3.5 × 106 6.5 × 104 6.7 × 107 7.0 × 106 
NRAc 0.54 0.78 0.43 0.86 1.67 1.32 0.95 1.85 1.51 
NIRd 0.19 0.47 0.13 0.25 0.89 0.85 0.32 0.94 0.92 
Influent COD/N ratio of 5:1 
NNBa 4.5 × 109 2.0 × 107 7.5 × 105 4.5 × 103 2.5 × 103 4.5 × 102 3.0 × 103 1.7 × 103 2.0 × 102 
NDBb 3.0 × 102 4.5 × 103 3.0 × 102 8.0 × 105 1.1 × 107 4.5 × 105 5.0 × 105 2.5 × 107 6.5 × 105 
NRAc 0.76 0.64 0.45 1.17 1.52 1.02 1.32 1.63 1.13 
NIRd 0.36 0.35 0.15 0.33 0.69 0.48 0.48 0.52 0.56 
Influent COD/N ratio of 10:1 
NNBa 6.5 × 109 6.5 × 106 9.5 × 104 4.0 × 103 1.7 × 103 2.5 × 102 2.5 × 103 1.4 × 103 1.7 × 102 
NDBb 4.0 × 102 3.5 × 102 2.5 × 102 2.5 × 107 4.5 × 106 6.5 × 103 6.5 × 107 5.2 × 106 5.0 × 103 
NRAc 0.93 0.58 0.48 2.14 1.28 0.86 2.56 1.31 0.89 
NIRd 0.51 0.26 0.14 0.17 0.57 0.26 1.20 0.62 0.37 
Influent COD/N ratio of 15:1 
NNBa 5.5 × 109 2.5 × 105 4.5 × 103 1.4 × 103 1.4 × 103 1.7 × 102 1.4 × 103 1.1 × 103 1.1 × 102 
NDBb 4.5 × 102 4.5 × 102 4.5 × 102 2.5 × 108 3.5 × 104 8.5 × 102 4.5 × 108 3.7 × 104 9.5 × 102 
NRAc 0.98 0.47 0.32 2.98 1.05 0.63 3.14 1.14 0.72 
NIRd 0.68 0.21 0.18 1.34 0.37 0.18 1.43 0.55 0.21 

aNumber of nitrifying bacteria (cfu/g), bnumber of denitrifying bacteria (cfu/g), cnitrate reductase activity (mg/(g d)), dnitrite reductase activity (mg/(g d)).

In the process of denitrification, dissimilatory NR catalyzed the first step by reducing NO3-N to NO2-N and NIR catalyzed the second step by reducing NO2-N to N2O or N2. As shown from Table 3, NR and NIR activities were influenced by matrix depth, and the depth for NR and NIR activities from high to low was 110, 80 and 50 cm under the same COD/N ratio in aerated and non-aerated SWISs. Under influent COD/N ratios of 10:1 and 15:1, the NR and NIR activities of SWIS A were higher than those of SWIS B and C at 80 and 110 cm depths, which was consistent with the number of denitrifying bacteria. Oxidative conditions were enhanced by intermittent aeration at 50 cm depth, which encouraged nitrification here. Moreover, more NO3-N, NO2-N and carbon source could be obtained in the subsequent matrix, which strengthened the activities of denitrifying bacteria and enhanced the enzyme activities involved in nitrogen removal in SWIS A under high influent COD/N ratio. Pan et al. (2016) reported the number of nitrifying bacteria were in positive correlation with NH4+-N removal, and Li et al. (2011b) proved denitrifying bacteria and NIR activities were in positive correlation with TN removal in SWISs. The results of microbial populations and enzyme activities involved in nitrogen removal could further explain the high removal of NH4+-N and TN in SWIS A under high influent COD/N ratios.

Functional genes involved in nitrogen removal

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) functional genes are involved in the microbiological nitrification and denitrification processes (Wang et al. 2015). Figure 5 shows the abundances of nitrogen removal functional genes in three SWISs. The amoA and nxrA genes are often regarded as the marker of oxidizing NH4+-N to NO2-N and oxidizing NO2-N to NO3-N, respectively (Ji et al. 2012). The abundances of amoA and nxrA decreased along the flow direction in SWIS A, B and C, which followed the same trend as DO. With influent COD/N ratio increasing, the abundances of amoA and nxrA declined in the upper layer of non-aerated SWISs because of the disadvantageous oxygen conditions. However, the influent COD/N ratio had no influence on the abundances of amoA and nxrA in aerated SWIS A within the range of this study, which was consistent with NH4+-N removal. Physiological activities of aerobic ammonia oxidizing bacteria are related to DO. High DO concentration enhanced the abundances of amoA and nxrA (Sun et al. 2018). The abundances of amoA and nxrA at 50 cm depth in SWIS B were higher than those in SWIS C under the same COD/N ratio because of the use of biochar-sludge as the substrate. Biochar-sludge as a substrate could improve NH4+-N removal and enhance the abundances of amoA and nxrA at 50 cm depth in SWISs (Sun et al. 2018). In SWIS A, the abundances of amoA and nxrA at 50 cm depth were significantly higher than those in non-aerated SWISs under the same COD/N ratio (P < 0.05). It could further explain the high removal of NH4+-N in SWIS A under a high influent COD/N ratio.

Figure 5

Abundances of nitrogen functional genes in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

Figure 5

Abundances of nitrogen functional genes in three SWISs under influent COD/N ratios of 1:1, 5:1, 10:1 and 15:1.

NO3-N to NO2-N reduction, the first reaction process in denitrification, is catalyzed by the key genes narG and napA. NO2-N to NO reduction is the second reaction process in denitrification, which is catalyzed by the genes nirS and nirK. NO to N2O reduction is the third process in denitrification, which is catalyzed by the qnorB gene. N2O to N2 reduction, catalyzed by the nosZ gene, is the last reaction in the denitrification process (Ji et al. 2012). As seen from Figure 5, the abundances of napA, narG, nirS, nirK, qnorB and nosZ in aerated SWIS A were significantly higher than those in non-aerated SWIS B and C at 80 and 110 cm depths under influent COD/N ratios of 10:1 and 15:1 (P < 0.05). Aeration enhanced DO concentrations in SWIS A, which was favorable for nitrification, and a high COD/N ratio would supply more carbon source for denitrification (Song et al. 2016). The abundances of napA, narG, nirS, nirK, qnorB and nosZ declined with the COD/N ratio increase at 80 and 110 cm depths in non-aerated SWIS B and C. However, the abundances of napA, narG, nirS, nirK, qnorB and nosZ increased with the COD/N ratio increase in SWIS A within the range of this study. More organic matter and nutrients provided good nutritional conditions for the growth and enrichment of anaerobic denitrifying bacteria, with the COD/N ratio increasing in SWIS A after nearly complete nitrification with aeration, in parallel to enhancing the enrichment of the six genes. It could further explain the high removal of TN in SWIS A under high influent COD/N ratio.

CONCLUSIONS

Biochar-sludge amended SWIS with intermittent aeration enhanced the number of nitrifying bacteria, denitrifying bacteria, nitrate reductase activities, nitrite reductase activities and improved the abundance of nitrogen removal functional genes (amoA, nxrA, narG, napA, nirK, nirS, qnorB and nosZ) under high influent COD/N ratios, which achieved high COD (92.2%), NH4+-N (96.8%), and TN (92.7%) removal rates and a low N2O emission rate (10.6 mg/(m2 d)) under an influent COD/N ratio of 15:1. Nitrogen removal increased and N2O emission decreased with increasing influent COD/N ratio, which indicated that biochar-sludge amended SWIS with intermittent aeration could be a valuable strategy for treating high COD/N ratio wastewater.

ACKNOWLEDGEMENTS

This research is supported by the National Natural Science Foundation of China No. 41001321, No. 41471394); Liaoning BaiQianWan Talents Program [2015(45)]; Shenyang Science and Technology Project (18-013-0-43); National college students innovation and entrepreneurship training program (201810166030).

REFERENCES

REFERENCES
Abdelmagid
H. M.
&
Tabatabai
M. A.
1987
Nitrate reductase activity of soils
.
Soil Biology and Biochemistry
19
,
421
427
.
American Public Health Association (APHA)
2003
Standard Methods for the Examination of Water and Wastewater
.
American Public Health Association/American Water Works Association
,
Washington, DC
.
Ding
Y.
,
Liu
Y. X.
,
Wu
W. X.
,
Shi
D. Z.
,
Yang
M.
&
Zhong
Z. K.
2010
Evaluation of biochar effects on nitrogen retention and leaching in multi-layered soil columns
.
Water Air and Soil Pollution
213
,
47
55
.
Fan
J.
,
Wang
W.
,
Zhang
B.
,
Guo
Y.
,
Ngo
H. H.
,
Guo
W.
,
Zhang
J.
&
Wu
H.
2013
Nitrogen removal in intermittently aerated vertical flow constructed wetlands: impact of influent COD/N ratios
.
Bioresource Technology
143
,
461
466
.
Fei
H. X.
,
Pan
J.
,
Tong
D. L.
,
Huang
L. L.
,
Yu
L.
&
Sun
Y. F.
2017
Effect of COD/N ratio on removal performances in two subsurface wastewater infiltration systems
.
Water Environment Research
8
,
693
702
.
Gómez
M. A.
,
González-López
J.
&
Hontoria-García
E.
2000
Influence of carbon source on nitrate removal of contaminated groundwater in a denitrifying submerged filter
.
Journal of Hazardous Materials
80
,
69
80
.
Kadam
A.
,
Oza
G.
,
Nemade
P.
,
Dutta
S.
&
Shanlcar
H.
2008
Municipal wastewater treatment using novel constructed soil filter system
.
Chemosphere
71
,
975
981
.
Li
Y. H.
,
Li
H. B.
,
Xu
X. Y.
,
Xiao
S. Y.
,
Wang
S. Q.
&
Xu
S. C.
2017
Field study on N2O emission from subsurface wastewater infiltration system under variable loading rates and drying-wetting cycles
.
Water Science and Technology
76
,
2158
2166
.
Lloréns
M.
,
Pérez-Marín
A. B.
,
Aguilar
M. I.
,
Sáez
J.
,
Ortuño
J. F.
&
Meseguer
V. F.
2011
Nitrogen transformation in two subsurface infiltration systems at pilot scale
.
Ecological Engineering
37
,
736
743
.
Song
S. Y.
,
Pan
J.
,
Wu
S. W.
,
Guo
Y. J.
,
Yu
J. X.
&
Shan
Q. X.
2016
Effects of COD/N ratios on pollutants removal in the subsurface wastewater infiltration systems with/without intermittent aeration
.
Water Science and Technology
73
,
2662
2669
.
Sun
Y. F.
,
Qi
S. Y.
,
Zheng
F. P.
,
Huang
L. L.
,
Pan
J.
,
Jiang
Y. Y.
,
Hou
W. Y.
&
Xiao
L.
2018
Organics removal, nitrogen removal and N2O emission in subsurface wastewater infiltration systems amended with/without biochar and sludge
.
Bioresource Technology
249
,
57
61
.
Wang
X.
,
Sun
T. H.
,
Li
H. B.
,
Li
Y. H.
&
Pan
J.
2010
Nitrogen removal enhanced by shunt distributing wastewater in a subsurface wastewater infiltration system
.
Ecological Engineering
36
,
1433
1438
.
Wu
J.
,
Zhang
J.
,
Jia
W. L.
,
Xie
H. J.
,
Gu
R. R.
,
Li
C.
&
Gao
B. Y.
2009
Impact of COD/N ratio on nitrous oxide emission from microcosm wetlands and their performance in removing nitrogen from wastewater
.
Bioresource Technology
100
,
2910
2917
.

Author notes

These authors contributed equally to this study and share first authorship.