Abstract

In order to study the characteristics of nitrous oxide (N2O) production and hydroxylamine (NH2OH) variation under oxic conditions, concentrations of NH2OH and N2O were simultaneously monitored in a short-cut nitrification sequencing batch reactor (SBR) operated with different influent ammonia concentrations. In the short-cut nitrification process, N2O production was increased with the increasing of ammonia concentration in influent. The maximum concentrations of dissolved N2O-N in the reactor were 0.11 mg/L and 0.52 mg/L when ammonia concentrations in the influent were 50 mg/L and 70 mg/L respectively. Under the low and medium ammonia load phases, the concentrations of NH2OH-N in the reactor were remained at a low level which fluctuated around 0.06 mg/L in a small range, and did not change with the variation of influent NH4+-N concentration. Based on the determination results, the half-saturation of NH2OH in the biochemical conversion process of NH2OH to NO2-N was very small, and the value of 0.05 mg NH2OH-N/L proposed in the published literature was accurate. NH2OH is an important intermediate in the nitrification process, and the direct determination of NH2OH in the nitrification process was beneficial for revealing the kinetic process of NH2OH production and consumption as well as the effects of NH2OH on N2O production in the nitrification process.

INTRODUCTION

Nitrous oxide (N2O) is a potent greenhouse gas, and has a large negative effect on the environment (IPCC 2013). According to the report published by the United Nations Environment Programme, 0.2 Tg N2O-N were emitted from wastewater to the atmosphere every year (UNEP 2013). The biological wastewater treatment process has been identified as an anthropogenic source responsible for the increase of N2O in the atmosphere (UNEP 2013). It is generally accepted that N2O can be produced during both aerobic nitrification and anoxic denitrification in biological nutrient removal processes (Ding et al. 2016). In heterotrophic denitrification, the mechanisms of N2O production and loss are quite clear and uncontested. N2O is produced in nitric oxide (NO) reduction as an intermediate, and finally is reduced to N2 by heterotrophic denitrifiers. However, mechanisms of N2O production in nitrification are quite complicated. In nitrification, ammonia (NH3) is converted to nitrate (NO3) via hydroxylamine (NH2OH) and nitrite (NO2). Two types of bacteria are responsible for this conversion process, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). N2O production and emission can be stimulated by the increase of NO2 (Wang et al. 2016a), and decreased NO2 accumulation can reduce the N2O generation (Chen et al. 2014). AOB have been identified as the main contributor for N2O production in nitrification process (Chen et al. 2014).

N2O can be produced by chemical breakdown of the unstable nitrosyl radical (NOH) during the oxidation of NH2OH (Poughon et al. 2001). Meanwhile, reduction of NO produced from the oxidation of NH2OH can also produce N2O (Stein 2011). Moreover, the electrons released from the oxidation of NH2OH were used to sustain ammonium oxidation and satisfy the cell's reductant needs including reduction of CO2 and generation of a proton gradient using oxygen (O2) as terminal electron acceptor (Arp & Stein 2003). Generally speaking, NH2OH is an important intermediate formed in the oxidation of ammonia/ammonium (NH3/NH4+) to nitrite (NO2) by AOB (Vajrala et al. 2013), and it has a direct relationship with N2O production in biological wastewater treatment processes (Kim et al. 2010).

However, due to short-lived and extremely reactive properties of NH2OH (Liu et al. 2014), determination of NH2OH in aqueous solution is not an easy task. As a result, experimental studies of effects of NH2OH on N2O production during nitrification process are scarce. Furthermore, the lack of the quantitative relation between NH2OH and N2O production caused great difficulties in understanding the mechanism of N2O production in the nitrification process.

In this study, a short-cut nitrification process with different influent ammonia concentrations was operated for more than 6 months, and concentrations of NH2OH and N2O were monitored simultaneously for understanding the characteristics of N2O production and NH2OH variation. Also through the relationships between NH4+, NH2OH and NO2, the half-saturation constant of NH2OH oxidation in the nitrification process is discussed and further understandings of the effects of NH2OH on N2O production can be obtained.

MATERIAL AND METHODS

The short-cut nitrification SBR

A laboratory-scale sequencing batch reactor (SBR) was made from transparent plexiglas with a working volume of 10 L. The schematic diagram of the reactor is shown in Figure 1. The diameter and the height were 200 mm and 400 mm respectively. The reactor was stirred with a rectangular mixing paddle. The air was supplied to the reactor by an air compressor through a diffuser placed at the bottom of the reactor, and the air flow was controlled by an air flow meter. The dissolved oxygen (DO) concentration in the reactor was maintained in the range of 0.4 mg/L–0.5 mg/L. The temperature of the SBR was controlled at 30 °C ± 2 °C by a water jacket. The drainage ratio of the reactor was 0.5.

Figure 1

The schematic diagram of the partial nitrification SBR process.

Figure 1

The schematic diagram of the partial nitrification SBR process.

The seeding sludge was taken from the oxic unit of the fourth wastewater treatment plant of Xi'an city, Shaanxi Province, China.

Operating conditions

Three phases including cultivation phase (CP), medium ammonia load phase (MAP) and low ammonia load phase (LAP), were operated under different conditions in this study. The influent NH4+-N concentrations of CP, MAP and LAP were 110, 70 and 50 mg/L respectively. The chemical oxygen demand (COD) to NH4+-N (C/N) ratios of the influent in all operation phases were maintained at 2 by changing the COD concentrations in the influent of each operation phase.

In the CP, the sludge concentration of the reactor was ∼3,800 mg/L, and the corresponding sludge retention time (SRT) was 18 d. In the MAP, the sludge concentration of the reactor was ∼3,300 mg/L, and the corresponding SRT was 15 d. In the LAP, the sludge concentration of the reactor was ∼3,100 mg/L, and the corresponding SRT was 15 d. The sludge concentrations and SRTs of the different phases are listed in Table 1.

Table 1

The sludge concentrations and SRTs of different phases

 Cultivation phase (n = 8)Medium ammonia load phase (n = 5)Low ammonia load phase (n = 5)
Sludge concentrations (mg/L) 3,877 ± 111 3,344 ± 75 3,172 ± 72 
SRT (d) 18 15 15 
 Cultivation phase (n = 8)Medium ammonia load phase (n = 5)Low ammonia load phase (n = 5)
Sludge concentrations (mg/L) 3,877 ± 111 3,344 ± 75 3,172 ± 72 
SRT (d) 18 15 15 

The influent quality and compositions of the three operating phases are shown in Table 2. In addition, NaHCO3, MgSO4·7H2O, CaCl2 and trace elements were also contained in influent and their dosages were unchanged during the three phases. The dosages of NaHCO3, MgSO4·7H2O and CaCl2 in the influent were 950.0, 50.0 and 20.0 mg/L respectively. The use of trace elements was the same as in Cheong & Hansen (2008).

Table 2

Influent quality and main compositions of the three operating phases

Operating phaseCompoundsConcentrationCODNH4+-NPO43-P
Cultivation CH3COONa 256 mg/L 200 mg/L   
NH4HCO3 564.3 mg/L  100 mg/L  
Na3PO4 21.2 mg/L   4 mg/L 
Medium ammonia CH3COONa 179.2 mg/L 140 mg/L   
NH4HCO3 395.0 mg/L  70 mg/L  
Na3PO4 21.2 mg/L   4 mg/L 
Low ammonia CH3COONa 128 mg/L 100 mg/L   
NH4HCO3 282.2 mg/L  50 mg/L  
Na3PO4 21.2 mg/L   4 mg/L 
Operating phaseCompoundsConcentrationCODNH4+-NPO43-P
Cultivation CH3COONa 256 mg/L 200 mg/L   
NH4HCO3 564.3 mg/L  100 mg/L  
Na3PO4 21.2 mg/L   4 mg/L 
Medium ammonia CH3COONa 179.2 mg/L 140 mg/L   
NH4HCO3 395.0 mg/L  70 mg/L  
Na3PO4 21.2 mg/L   4 mg/L 
Low ammonia CH3COONa 128 mg/L 100 mg/L   
NH4HCO3 282.2 mg/L  50 mg/L  
Na3PO4 21.2 mg/L   4 mg/L 

Each phase operated with a cyclic duration of 4 hours, including a sequence of filling, aeration and mixing, settle, draw and idle. The operation procedures of the three operating phases are shown in Figure 2. In each operation phase, DO concentration was detected by real-time monitoring. Aeration duration of each operation phase was not fixed, and aeration was immediately stopped when obvious increase of DO concentration in the reactor was detected.

Figure 2

The operation procedures of the three operating phases.

Figure 2

The operation procedures of the three operating phases.

Analytical methods

COD, NH4+-N, NO3-N, NO2-N, N2O-N, NH2OH-N, DO concentration, pH value and temperature during the experiments were monitored. COD, NH4+-N, NO3-N and NO2-N were monitored according to standard methods (APHA 1998). DO concentrations, temperatures and pH values in the reactor were measured by a DO meter (Hach) and pH meter (Rex, ShangHai).

N2O-N concentration profiles in the reactor were measured using Clark-type microelectrodes continuously. The N2O 500 microelectrode and the microsensor monometer were purchased from Unisense (Aarhus, Denmark). After polarization, the N2O microelectrode responded linearly in the range of 0.0 to 22.0 mg N2O/L. The microelectrode was calibrated with the two-point method according to the instruction provided by Unisense. By establishing the relationship between the reading voltages and the corresponding two N2O-N concentrations, a calibration curve can be acquired. The dissolved concentrations of N2O-N can be obtained by substituting reading voltages into the calibration curve.

NH2OH-N concentrations were measured by the spectrophotometric method proposed by Hu et al. (in press). Under acidic condition, NH2OH can be stabilized as NH3OH+. The spectrophotometric method was based on oxidation of NH2OH to N2O by Fe(III) using ferric ammonium sulfate (NH4Fe(SO4)2) as oxidation agent (Bengtsson et al. 2002). NH2OH was determined through formation of ferrion, a tris complex of 1,10-phenanthroline with Fe(II). Ferrion is a red-colored octahedral complex ion, and is soluble and stable in aqueous solution in the pH range of 2–9 (Adhikamsetty et al. 2008). By eliminating the interference of phosphate by the numerical method proposed by Hu et al. (in press), the spectrophotometric method can be used to determine NH2OH-N concentrations in biological wastewater treatment processes.

Nitrite accumulation rate

Nitrite accumulation rate (NAR) was calculated by:
formula
(1)
where CNitrite and CNitrate represent NO2-N and NO3-N concentrations in the reactor, mg/L.

RESULTS AND DISCUSSION

Performances of the SBR process under different operating phases

Figure 3 shows the performance of the process in the CP. In CP, parts of NH4+-N in influent were converted to NO2-N at the beginning, and the NAR was lower than 40%. After 12 days, the NAR was dramatically increased and the majority of NH4+-N in influent was converted to NO2-N. After 22 days, the NO2-N concentration in the effluent of the reactor was higher than 33.0 mg/L and the NO3-N concentration in the effluent of the reactor was lower than 2.0 mg/L. Consequently, the NAR was higher than 95%.

Figure 3

The performances of the short-cut nitrification SBR process in the CP.

Figure 3

The performances of the short-cut nitrification SBR process in the CP.

When the short-cut nitrification process was successively achieved in the SBR, the operation of the reactor was changed to MAP and then LAP. The operation periods of both MAP and LAP lasted for 1 to 2 months for achieving the steady performance. After the reactor achieved steady state in the LAP and the MAP, measuring campaigns were carried out in each operation phase. The performances of the short-cut nitrification SBR process during continuous 9 days of the steady states under the LAP and the MAP are shown in Figure 4.

Figure 4

The steady performances of the short-cut nitrification SBR process in the MAP and LAP.

Figure 4

The steady performances of the short-cut nitrification SBR process in the MAP and LAP.

In the MAP and the LAP, all NO3-N concentrations in effluent were lower than 2.0 mg/L, and NARs were always higher than 90%. Under the three operation phases, the SBR achieved stable short-cut nitrification and high NARs.

In order to achieve the short-cut nitrification quickly, high influent NH4+-N concentration (110 mg/L), high temperature (30 °C), high pH value (∼8.0) and low DO concentration (0.4–0.5 mg/L) were adopted in the CP in this study. Also, real-time monitoring for DO concentration in the reactor was carried out, which eliminated excessive air supply. A broad range of operating parameters and factors are essential for achieving short-cut nitrification, such as pH, DO, temperature and free ammonia (FA) (Sinha & Annachhatre 2007). It is generally accepted that FA inhibits the activity of nitrite oxidoreductase (NOR) (Anthonisen et al. 1976; Yang & Alleman 1992) or NOB (Ahn et al. 2011). Also, most of the published literature related to nitrite accumulation using pH as a decisive factor stated that pH in the range of 7.5–8.5 was most suited to inhibit the nitrite oxidizers (Sinha & Annachhatre 2007). FA and pH had a great effect on nitrite accumulation. According to the study of Anthonisen et al. (1976), the ratio of NH4+-N to NH3-N would decrease with the increasing of pH value; consequently, the activity of NOR would become more inhibited as the content of FA would increase when pH was increased. Temperature also was an important parameter for nitrite accumulation. Although, short-cut nitrification or partial nitrification can be fulfilled at low temperature (Gu et al. 2012), the short-cut nitrification process can be more easily achieved at high temperature (30–35 °C) or mild temperature (20–30 °C) (Gu et al. 2012). Furthermore, according to the study carried out by Guo et al. (2009), a stable short-cut nitrification process was achieved for a long time in the SBRs operated with high DO (above 3.0 mg/L on average) and low DO (0.4–0.8 mg/L) concentrations, and DO was not the crucial factor for achievement of short-cut nitrification. However, AOB seemed to be more robust towards low DO than were NOB (Sinha & Annachhatre 2007) and nitrite oxidation was strongly inhibited by low DO (<0.5 mg/L) (Hanaki et al. 1990). Thus, low DO concentration was beneficial for quick achievement of short-cut nitrification.

From the ecological concept, r-strategist microorganisms can grow quickly on easily available substrate (Andrews & Harris 1986). During CP, the residual NH4+-N concentrations were relatively high at the end of the cycle, which was beneficial for the enrichment of an r-strategist AOB population (Terada et al. 2013). Furthermore, Nitrospira sp. and Nitrobacter sp. are widely regarded as the two major types of NOB present in biological wastewater treatment plants (Wang et al. 2016b). Oxygen affinity of Nitrospira sp. belonging to r-strategist was low; however, that of Nitrobacter sp. belonging to K-strategist was high (Regmi et al. 2014). High DO level could provide competitive advantage for AOB over Nitrospira sp., and DO limitation can suppress the growth of Nitrobacter sp. (Wang et al. 2016b). Low DO levels were beneficial for washing out of Nitrobacter sp. (Wang et al. 2016b). In this study, low DO concentrations were maintained in the reactor, which made AOB dominant in the reactor instead of NOB. Consequently, high NARs were obtained.

According to the operation results, the operation strategy for achieving the short-cut nitrification in this study was effective. The short-cut nitrification was completely achieved on day 22 after the start-up of the SBR. Also, as NOB were washed out from the system in the CP, the short-cut nitrification process did not deteriorate when the operation phase was shifted to the LAP and the MAP from the CP.

N2O production and NH2OH variation in typical cycles of LAP and MAP

Figure 5 shows the variation profiles of different contaminations in the reactor during three typical cycles of the LAP and the MAP.

Figure 5

Concentration variations of different contaminants in typical cycles of the short-cut nitrification SBR process under the MAP and LAP.

Figure 5

Concentration variations of different contaminants in typical cycles of the short-cut nitrification SBR process under the MAP and LAP.

Variation trends of COD, NH4+-N, NO2-N, NO3-N, PO43−-P, NH2OH-N, dissolved N2O-N and NAR in the LAP and the MAP were almost same, except that the maximum dissolved N2O-N concentration in MAP was almost five times higher than that in LAP. During the typical cycles, COD concentrations in the reactor under two phases decreased to the lowest content within 20 min with the effects of dilution and oxidation. Also, NH4+-N concentrations were decreased linearly, NO2-N concentrations were increased linearly, and NO3-N concentrations in cycle durations were lower than 2.0 mg/L. Correspondingly, NARs in whole cycle durations were higher than 90%, which meant the short-cut nitrification process was dominant in the SBR. Specifically, the NH2OH-N concentrations fluctuated around 0.06 mg/L in a small range during all monitoring operation cycles under the LAP and the MAP, and dissolved N2O-N concentrations were increased in more than half of the cycle durations, and decreased in the latter part of the cycle durations.

In all operation phases, C/N ratio of the influent was maintained at 2 which did not inhibit the nitrification process (Campos et al. 1999), and consequently a stable short-cut nitrification process was successively achieved. During the short-cut nitrification process, N2O production was suppressed to some extent during the first 30 min of operation cycles in LAP and MAP owing to the storage of COD to PHB which used for the reduction of NO2-N (Zhao et al. 2016) and NO production (Pocquet et al. 2016). At the latter part of the monitoring cycle durations, the concentrations of N2O-N were decreased rapidly as the NH4+-N in influent was closed to depletion.

In order to distinguish the differences in nitrification reaction rates before and after the maximum dissolved N2O-N concentration in the reactor, the concentrations of NH4+-N and NO2-N were divided into two groups respectively in each operation phase. The variations of nitrification reaction rates are shown in Figure 6.

Figure 6

Nitrification rate variations before and after the maximum concentration of dissolved N2O-N.

Figure 6

Nitrification rate variations before and after the maximum concentration of dissolved N2O-N.

In Figure 6, NH4+-N (I) and NH4+-N (D) represent the concentrations of NH4+-N corresponding to the increase and the decrease periods of N2O-N concentration in the reactor respectively. NO2-N (I) and NO2-N (D) represent the concentrations of NO2-N corresponding to the increase and the decrease periods of N2O-N concentration in the reactor respectively. Before the peak concentrations of dissolved N2O-N in operation cycles under two phases, the oxidation rates of NH4+-N and the production rates of NO2-N were higher than those after the peak concentrations of dissolved N2O-N, which can be explained by the effects of low NH4+-N concentration on the nitrification reaction rate.

These phenomena indicated that N2O production was decreased in the latter period of the monitoring cycles. During nitrification, two pathways were considered to be the major processes responsible for N2O emissions, namely AOB denitrification process, also called nitrifier denitrification, and incomplete hydroxylamine oxidation by the hydroxylamine oxidoreductase (Pocquet et al. 2016). AOB denitrification was favored by limited oxygen conditions (Tallec et al. 2006) such as the DO level in this study. In the AOB denitrification process, NH4+-N can be converted to NO and NO2-N via NH2OH (Ni et al. 2011; Pocquet et al. 2016), and NH2OH can enhance the N2O emission (Kim et al. 2010). Based on the determination, concentrations of NH2OH-N in all monitoring cycles were around 0.06 mg/L, and NH2OH did not accumulate under two NH4+-N concentrations (50 mg/L and 70 mg/L). As the presence of high concentration of NH2OH are toxic to AOB (Vajrala et al. 2013), it was speculated that maybe there was a detoxification mechanism in bacteria for avoiding accumulation of NH2OH. Consequently, NH2OH in the nitrification process cannot accumulate to a high level.

From the perspective of biochemical reaction dynamics, NH2OH might be the limitation substrate for the nitrification reaction because of its low concentration. However, according to Figure 6, although production rates of NO2-N were smaller than consumption rates of NH4+-N in monitoring cycles under the LAP and the MAP, the differences between them were not distinct, which meant the low NH2OH-N concentration in the two operation phases did not obviously result in the reduction of the production rate of NO2-N. According to the determination results of NH2OH-N and the toxicity of high NH2OH concentration in nitrification, it was reasonable that the half-saturation of NH2OH in the biochemical conversion process of NH2OH to NO2 was very small. The value of estimated by Law et al. (2012) (0.05 mg NH2OH-N/L) was more accurate than the estimated values used by Ni et al. (2011) (2.4 mg NH2OH-N/L) and Pocquet et al. (2016) (0.9 mg NH2OH-N/L).

CONCLUSIONS

In a short-cut nitrification SBR, the characteristics of N2O production and NH2OH variation were studied in this study.

Combined with real-time monitoring of DO, the short-cut nitrification process can be quickly achieved with the operation parameters of high influent NH4+-N concentration, high temperature, high pH value and low DO concentration. The amount of N2O production was increased with the increasing of the influent NH4+-N concentration. Furthermore, according to the determination results of NH2OH-N concentrations, the half-saturation constant of NH2OH in the biochemical conversion process of NH2OH to NO2-N was very small, and the value of 0.05 mg NH2OH-N/L of in the published literature was accurate.

ACKNOWLEDGEMENTS

This work was supported by the Shaanxi Province Science & Technology Development Program (Grant No. 2014K15-03-02); the Fundamental Research Funds for the Central Universities (Grant No. 310828171004; Grant No. 310829161004); National Training Programs of Innovation and Entrepreneurship for Undergraduates (Grant No. 201610710081) and the National Natural Science Foundation of China (Grant No. 51778057).

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