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Effect of pH on nitrous oxide production and emissions from a partial nitritation reactor under oxygen-limited conditions
Accepted Manuscript Title: Effect of pH on nitrous oxide production and emissions from a partial nitritation reactor under oxygen-limited conditions Author: Yongtao Lv Kai Ju Lei Wang Xiaoqiang Chen Rui Miao Xueling Zhang PII: DOI: Reference:
S1359-5113(16)30029-0 http://dx.doi.org/doi:10.1016/j.procbio.2016.02.017 PRBI 10631
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
6-2-2016 25-2-2016 27-2-2016
Please cite this article as: Lv Yongtao, Ju Kai, Wang Lei, Chen Xiaoqiang, Miao Rui, Zhang Xueling.Effect of pH on nitrous oxide production and emissions from a partial nitritation reactor under oxygen-limited conditions.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.02.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of pH on nitrous oxide production and emissions from a partial nitritation reactor under oxygen-limited conditions Yongtao Lva, Kai Jua, Lei Wanga*, Xiaoqiang Chenb, Rui Miaoa, Xueling Zhanga
a
School of Environmental and Municipal Engineering, Xi’an University of Architecture and
Technology, Xi’an, 710055, China b
Institute of Water and Environmental Research, Faculty of Infrastructure Engineering, Dalian
University of Technology, Dalian, 116024, China
Corresponding author: Lei Wang, School of Environmental and Municipal Engineering, Xi’an
*
University of Architecture and Technology, Xi’an, 710055, China. Tel.: +86 29 82202729; Fax: +86 29 82202729 E-mail address: [email protected]
1
Graphical Abstract pH -1000
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NO2
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Research highlights
In a PN reactor, N2O emissions decreased with the increase in initial pH.
N2O production inside sludge aggregates were quantified by using microelectrodes.
A decrease in pH led to increased N2O emissions from denitrification.
ABSTRACT The effect of pH on nitrous oxide (N2O) emissions from a laboratory-scale partial nitritation sequencing batch reactor under oxygen-limited conditions was investigated from both macro- and microscopic viewpoints. During the aeration period of a single cycle, N2O emissions decreased when the initial pH increased from 7.5 to 8.5. By application of microelectrodes, N2O production was observed inside entire sludge aggregates, and it increased with decreasing pH from 8.5 to 7.0. At pH 8.0 and 8.5, N2O was mainly produced in the outer layer (< 1000 m) of sludge aggregates, where 2
nitritation mainly occurred. At pH 7.0 and 7.5, N2O production was mainly observed in the inner layer (> 1000 m), where the dissolved oxygen was almost depleted, revealing that the dominant pathway here was denitrification. Under oxygen-limited conditions, a decrease in pH led to increased N2O emissions from denitrification pathway.
Keywords: partial nitritation; sequencing batch reactor; nitrous oxide; pH; microelectrodes
INTRODUCTION Nitrous oxide (N2O) is often emitted from biological nitrogen removal (BNR) processes [1-4], which is problematic as N2O is a powerful greenhouse gas with a much stronger greenhouse effect than carbon dioxide (about 300-fold) [1]. It is generally accepted that AOB are the major contributors to N2O emissions in BNR processes [5,6]; therefore, N2O emissions in partial nitritation (PN) reactors are of growing concern. N2O emissions are affected by process parameters (e.g., dissolved oxygen (DO) concentration, pH and substrate concentrations) in BNR systems [7-10]. Low DO concentrations, high NO2– concentrations and variation in influent NH4+ concentrations have been identified as promoting N2O formation [6,7,11]. The relationship between pH and N2O emission in BNR process has also been reported [12,13]. Pan et al. [12] found that N2O accumulated at a low pH value during denitrification by methanol utilizing denitrifiers. Law et al. [13] obtained the maximum N2O emission rate at pH 8.0 in a PN system, and found that N2O emission correlated with the ammonium oxidation rate (AOR). In previous studies, the N2O emission rate and dynamic characteristics were well studied [7,11-13], whereas the N2O production at micro-scale were seldom characterized. Theoretically, N2O is first produced inside the microbial aggregates and then is emitted to the atmosphere. Therefore, measurement of N2O production and transformation inside sludge aggregates might reveal the most plausible pathways of N2O production. Microelectrodes are one of the most suitable tools for microenvironmental measurements [14,15]. Satoh et al. [15] studied nitrification activities changes within biofilms under different operating conditions. Rathnayake et al. [16] observed that N2O was mainly produced in the outer layer of a granule where AOB showed high
3
activities, and reflected that AOB might be responsible for N2O production. However, variation in N2O production pathways with operating conditions has not previously been reported. In this study, a laboratory-scale SBR fed with a synthetic inorganic wastewater was operated for PN under oxygen-limited conditions. The N2O emissions were first investigated from a macroscopic viewpoint. Then, microelectrodes were employed to quantify the microenvironment and N transformation inside the microbial aggregates from a microscopic viewpoint. The microbial activity obtained was analyzed for correlation with the N2O production, aiming to explore how pH affects N2O production and emissions.
Materials and methods Experimental setup A laboratory-scale SBR with a working volume of 4 L was used for PN. One cycle consisted of a 5 min filling period, a 320 min aerating period, a 30 min settling period, and a 5 min drawing period. The drawn volume was 2.0 L, making the exchange volume 50%. Bulk liquid (200 mL) was removed each day providing a sludge retention time of 20 d. The mixed liquor suspended solid (MLSS) was around 3000 mgL–1 during the period of this study. During the aeration period, a mass flow controller was used to keep a constant air flow rate (0.32 L min–1), and the average DO concentration in the bulk liquid was 0.33 0.06 mg L–1 5 min after aeration. The reactor temperature was maintained at 27 1 C using a water jacket.
Biomass and synthetic wastewater In previous study, PN was successfully initiated and steadily operated by inoculating with conventional sludge, and characteristics of N2O emissions were investigated [17]. The same biomass was used for further exploration of the effect of pH on N2O emissions, as described below. The synthetic wastewater contained NH4Cl (N source), NaHCO3 (C source and buffer), and trace elements. The concentrations of NH4+ and NaHCO3 were 600 mg N L–1 and 5400 mg L–1, respectively. Trace element solutions were added as described by Ju et al. [17]. HCl (0.5 mol L–1) and NaOH (0.5 mol L–1) were used to adjust to different initial pH values of 7.5, 8.0, and 8.5.
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Chemical analysis NH4+-N, volatile suspended solids, and total suspended solids were determined according to the standard methods of APHA [18]. Nitrate-nitrogen (NO3–-N) and NO2–-N were determined using ion chromatography (761 Compact IC, Metrohm, Herisau, Switzerland). The pH and the DO concentration were directly monitored using a pH meter (Unisense, Aarhus, Denmark) and a DO meter (HQ25d, Colorado, USA), respectively.
DNA extraction, PCR-DGGE, cloning and sequencing The mixed liquor was collected from the SBR and the total DNA was extracted using a bacterial genomic mini extraction kit (Sangon, China). Universal primers F357 (with GC clamp) and R518 were used for the PCR amplification [19]. The DGGE analysis, cloning and sequencing was determined according to Lv et al. [20]. N2O measurements The N2O concentration in the off-gas was measured by a gas chromatograph (PE600, PerkinElmer, USA) with an electron capture detector and a Porapak Q column (GDX-101, Gansu, China) using 30 mL min–1 high-purity N2 as the carrier gas [17].
Microsensor measurements NH4+, NO2−, pH, DO, and N2O microelectrodes were used for microenvironmental measurements. The first four microelectrodes were manufactured and their performance was previously shown to be stable [21]. They were constructed and calibrated before measurements according to the methods of de Beer et al. [14]. The N2O microelectrode was purchased from Unisense, Denmark. A three-point calibration (pure water, 50% N2O solution, and saturated N2O solution) was carried out before each measurement. Sludge flocs were sampled from the PN reactor and placed very gently just above the nylon net in a flowing chamber (Fig. 1) [22]. The microelectrode penetrated through the sludge aggregates under settling conditions. To obtain steady-state profiles, the sludge aggregates were left for 30 min before profile measurements started, and each microelectrode measurement was made at least three times. As the flocs had a symmetrical structure, the microelectrode only had to penetrate to half the depth of the flocs to reflect the entire substrate distribution. 5
The medium used in the chamber for microprofile measurements consisted of NH4Cl (4.2 mg N L–1), NaNO2 (4.2 mg N L–1), and NaHCO3 (37.8 mg L–1), and the different pH values were controlled by adding HCl (0.5 mol L–1) and NaOH (0.5 mol L–1). To provide the oxygen-limited condition, the DO concentration of this medium was maintained below 1 mg L–1 through purging with N2. Because the pH of the PN reactor varied in the range 6.8-8.5, four different pH values (7.0, 7.5, 8.0, and 8.5) were selected for microprofile measurements.
Net volumetric rate calculations The net volumetric rates were calculated according to mass transport equation [23]: C ( z, t ) / t Ds 2C ( z, t ) / z 2 Q( z ) P( z )
(1)
where C(z,t) is substrate concentration (mmol·m–3) at time t and depth z, respectively. Q and P are consumption and production rate (mmol·m–3·s–1), respectively. Ds is the effective diffusion coefficient (m2 s–1); Ds values of 1.38 10–9, 1.25 10–9, and 2.10 10–9 m2 s–1 were used for the calculations of NH4+, NO2−, and N2O, respectively, at 25 C [3,16]. When steady state was achieved, the left expression is zero. Eq. (1) can be reduced to: Ds 2C ( z ) / z 2 Q( z ) P( z )
(2)
Defining R(z) = Q(z)-P(z) is the net volumetric rate (mmol m–3 s–1). Using Euler’s formula for numeric integration, the following equation can be obtained as:
C / zn1 C / zn h Rn / Ds
(3)
where h is the step size (100 m). According to the concentration gradient, the following equation can be obtained as:
Cn 1 Cn h C / zn
(4)
Substituting ∂C/∂zn with Eq. (3), it can be calculated as:
Rn1 Ds [(Cn1 Cn ) / h C / zn1 ] / h
.
(5)
6
Results Effect of pH on PN performance The reactor was operated for at least one week under each pH value. In the initial pH range 7.5–8.5, the change in pH had almost no effect on the PN (Fig. 2). The concentration of NH4+ in the influent was 598 12 mg N L–1, and the SBR converted around half of the NH4+–N to NO2−–N. The effluent concentrations of NH4+ and NO2− were 283 16 and 297 22 mg N L–1, respectively. The molar ratio of NO2− to NH4+ in the effluent was 1.05 0.12, which is suitable as a feed for the anammox process. The NO3– concentration in the effluent was 28.40 2.46 mg N L–1, indicating low activity of nitrite-oxidizing bacteria (NOB).
Microbial community structure On day 25 the microbial community was investigated using PCR and DGGE analyses. Nitrosomonas sp., known AOB, were detected, which is in agreement with observations from other PN systems [24-27]. Although no organic matter was fed to the reactor, Comamonas sp., Flavobacteria sp. and Acidovorax sp., known heterotrophs with denitrifying functionality [28,29], were also detected. The organic compounds might be derived from biomass decay [30]. It is worth noting that NOB were not detected, although nitrification was observed in the reactor. The lack of detection of NOB means that they accounted for < 9% of the bacterial population [31].
Effect of pH on N2O emissions in a single cycle The effect of initial pH on N transformation in a single cycle is shown in Fig. 3. For all pH values, the DO concentration decreased and maintained around 0.3 mg L–1. The pH decreased with processing of the PN, but at initial pH values of 8.0 and 7.5, for an initial processing time of 40 min, a slight increase in pH was observed, which was because of CO2 stripping [32]. The concentration of NH4+ decreased and that of NO2– increased almost linearly, and at the end of the aeration process, the molar ratio of NO2– to NH4+ ions was nearly 1:1. The AOR showed that they increased from 0.144 to 0.158 and 0.176 mg NH4+-N min–1 g–1 MLSS with pH decrease from 8.5 to 8.0 and 7.5, indicating that the activities of AOB increased with decreased initial pH. This was in agreement with Van Hulle et al. [33] who found that AOB activity decreased when the pH increased above 7.23. The concentration of NO3– ions increased slowly from around 10 up to 30 mg N L–1. 7
During the initial 50 min of aeration time, a peak N2O emission was observed after an aeration time of 10 min. The maximum N2O concentrations of 1.43, 1.24, and 0.84 mg L–1 were obtained for initial pH values of 7.5, 8.0, and 8.5, respectively. From an operating time of 50 min onward, the trend in N2O emissions in the off-gas changed. At initial pH values of 8.5 and 8.0, the N2O concentrations increased slowly from 0.22 to 0.44 mg L–1 and from 0.31 to 0.55 mg L–1, respectively, while at pH 7.5, the N2O concentrations were maintained at around 0.6 mg L–1. At pH of 7.5, 8.0 and 8.5, 63.74, 40.59 and 34.72 mg of N2O emission was obtained in a cycle, respectively. This indicated that N2O emissions decreased with the initial increase in pH from 7.5 to 8.5 under oxygen-limited conditions.
Nitrogen transformation and N2O production inside the sludge aggregates The concentration profiles from microsensor measurements are shown in Fig. 4 (A–D). The trends in the microprofiles within the sludge flocs were similar at different pH values. Oxygen was mainly consumed in the outer layer, decreasing from a concentration of around 30 mol L–1 to below 10 mol L–1 at a depth of 500 m. Therefore, oxygen-limited conditions formed, similar to those encountered in the SBR during aeration. With increasing depth, the NH4+ concentration decreased and the NO2– concentration increased, which was mainly observed in the outer layer (< 1000 m). The pH decreased inside the flocs. At different pH values, the concentration of N2O increased with increasing penetration depth, and it increased by 104.85, 83.38, 71.44, and 62.49 mol L–1 for pH values of 7.0, 7.5, 8.0, and 8.5, respectively. Thus, on increasing the pH, the production of N2O decreased, which was in agreement with the N2O emissions observed in the reactor. The net volumetric consumption or production rates were calculated, and these are shown in Fig. 4 (E–H). At different pH values, the NH4+ consumption coincided with the NO2– production, especially in the outer layer (< 1000 m), revealing that a nitritation reaction had occurred. N2O production was observed inside the entire floc, while the trends were different for different pH values. At pH values of 8.0 and 8.5, the N2O was mainly produced in the outer layer (< 1000 m), where NH4+ oxidation mainly occurred, and with an increase in depth, the net N2O production rate decreased. However, at pH values of 7.0 and 7.5, in the outer layer, the net volumetric rate of N2O production increased with increasing depth, and a higher N2O production rate was observed in the inner layer (> 1000 m), where DO was almost depleted. 8
Discussion The net volumetric rate revealed that N2O was produced in the whole sludge aggregates, but the microbial reactions were different in the outer and inner layers. In the outer layer (< 1000 m), NH4+ consumption coincided with the production of NO2–, indicating an active zone of nitritation. However, N2O production pathway in the outer layer could not be determined because both denitrification and nitrification could occur under oxygen-limited conditions. In the inner layer (> 1000 m), where the DO was almost depleted, under such an anoxic condition the denitrification pathway was the main source of N2O production. N2O produced outside the nitritation zone was also observed by Rathnayake et al. [16] inside stratified granules, and the question of how small amounts of NO2– were produced in such an anoxic zone needs further investigation. Identifying the microbial community structure was crucial to demonstrating the mechanism of biological reactions in the sludge aggregates. In this study, AOB of Nitrosomonas sp. were detected, which can reduce NO2– to produce N2O or N2 [34,35], i.e., nitrifier denitrification. Denitrifying heterotrophs of Betaproteobacteria and Flavobacteria were also detected. Gabarró et al. [36]. demonstrated that most Betaproteobacteria are capable of denitrifying N2O to N2, whereas most Bacteroidetes, including Flavobacteria, could not denitrify N2O because of the absence of the nosZ gene. Therefore, both AOB (Nitrosomonas sp.) and heterotrophic denitrifiers (Flavobacteria sp.) could increase N2O production under anoxic conditions, but it is difficult to determine which are responsible for the denitrification pathway of N2O production in this study. To determine how N2O production were affected by pH, N2O production in the outer and inner layers was quantified at different pH values and summarized in Table 1. At pH 8.0 and 8.5, N2O was mainly produced in the outer layer, accounting for 61.3 and 60.5%, respectively, of the total N2O produced (Table 2). In contrast, at pH values of 7.0 and 7.5, N2O production in the outer layer accounted for only 31.2 and 31.8%, respectively, indicating that denitrification was the dominant pathway for N2O production. Thus, it can be concluded that with decreasing pH, the denitrification pathway of N2O production becomes more favored, which is in agreement with the study by Wrage et al. [37]. This is probably the main reason why the peak value of N2O emission in a single cycle of an SBR decreased with the increase in initial pH values. In the later phase of the aeration period (from 50 min onward), the trend in N2O emissions at different pH values changed. At initial pH values of 8.0 and 8.5, N2O emission increased as the reaction 9
proceeded. This can be explained by the increased N2O produced via the denitrification pathway. As the NH4+ oxidation progressed, more NO2– was produced and the pH simultaneously decreased, both of which can lead to enhanced N2O production from denitrification [38]. However, at initial pH 7.5, no obvious increase in N2O emission was observed, which is probably because pH affects the two pathways of N2O production differently. On the one hand, a lower pH promotes denitrification, leading to more N2O being produced; on the other hand, AOB activities decreased when the pH decreased below 7.23 [33], which might lower the N2O production.
Conclusions Under oxygen-limited conditions, the effect of initial pH on N2O emissions in a laboratory-scale PN SBR showed that AOB activity increased with decreasing pH from 8.5 to 8.0 and 7.5, and resulted in more N2O emission. By using microelectrodes, N2O production was quantified inside microbial aggregates. The result demonstrated that a decrease in pH from 8.5 to 7.0 led to increased N 2O production from the denitrification pathway.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No.51108367), Housing and urban and Rural Construction Department of Shaanxi Province (No.2015-K61), and the Innovative Research Team of Xi’an University of Architecture and Technology.
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Fig. 1 The schematic diagram of the chamber for microprofile measurements
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Time(days) Fig. 2 Time course of the partial nitrification performance at different initial pH levels
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Time (min)
Time (min)
500
150
-1
150
7.5
DO concentration (mgL )
100
1.2
0.0 350
pH DO
8.0
1.2
7.5
0.8
7.0
0.4
6.5 0
50
100
150
200
250
300
-1
50
8.0
DO concentration (mgL )
0
pH DO -1
1.5
pH
400
-1
(A)
Conecentration (mg N2OL )
-1
Conecentration (mgNL )
500
1.6
(B)
DO concentration (mgL )
8.5
2.0
ammonium nitrite nitrate gas N2O
0.0 350
Time (min)
Time (min)
Fig. 3 Variation in nitrogen and process parameters during a partial nitritation cycle at different pH levels (A and B correspond to initial pH of 8.5, C and D correspond to initial pH of 8.0, E and F correspond to initial pH of 7.5)
15
pH 7
-1000
(A)
8
DO
(E)
+
NO2
pH
NH4
N2O
0
Depth (m)
9 _
1000
2000
N2O _
NO2
3000
+
NH4 0
100 200 300 -1 Concentration (molL )
400
-12
-6
0
6
12
Rate (molcm h ) -3
-1
pH -1000
6
7
(B)
N2O
8
pH
9
(F)
+
_
NO2 NH4
DO
Depth (m)
0
1000
2000
N2O _
NO2 +
3000
NH4 0
100
200
300
400 -1
Concentration (molL )
-12
-6
0
6
Rate (molcm h ) -3
16
-1
12
pH 6
7
8
9
-1000
(C) N O 2
(G)
pH _
DO
NO2
0 +
Depth (m)
NH4
1000
2000
N2O _
NO2 3000
+
NH4 0
100
200
300
400
-12
-6
0
6
12
Rate (molcm h )
-1
-3
Concentration (molL )
-1
pH 6
7
8
9
-1000
0 DO
(H)
+
(D)
_
NO2
NH4
pH
N2O
1000
2000
N2O _
NO2
3000
+
NH4 0
100
200
300 -1
Concentration (molL )
400
-12
-6
0
6
12
Rate (molcm h ) -3
-1
Fig. 4 (A–D) Steady-state microprofiles of DO, pH, NH4+, NO2−, and N2O inside the sludge aggregates (A, B, C, and D correspond to pH levels of 7.0, 7.5, 8.0, and 8.5, respectively). (E–H) Net volumetric production or consumption rates of NH4+, NO2−, and N2O inside the sludge aggregates (E, F, G, and H correspond to pH levels of 7.0, 7.5, 8.0, and 8.5, respectively)
17
Table 1 Quantification of N2O production inside the sludge aggregates N2O production in the outer N2O
production
in
layer (0–1000 m)
layer(1000–3000 m)
Concentration
Concentration
the
inner Total N2O production
pH (mol L–1)
Ratio (%)
(mol L–1)
Concentration Ratio (%)
(mol L–1)
Ratio (%)
8.5
37.7 ± 2.5
60.5 ± 3.7
24.6 ± 5.9
39.5 ± 12.2
62.3 ± 10.8
100
8.0
43.6 ± 16.2
61.3 ± 3.4
27.5 ± 7.6
38.7 ± 9.9
71.2 ± 12.7
100
7.5
26.4 ± 2.4
31.8 ± 7.5
56.6 ± 7.1
68.2 ± 19.5
82.9 ± 11.3
100
7.0
32.6 ± 6.2
31.2 ± 2.4
71.8 ± 16.2
68.8 ± 5.6
104.3 ± 21.3
100
18
S1359-5113(16)30029-0 http://dx.doi.org/doi:10.1016/j.procbio.2016.02.017 PRBI 10631
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
6-2-2016 25-2-2016 27-2-2016
Please cite this article as: Lv Yongtao, Ju Kai, Wang Lei, Chen Xiaoqiang, Miao Rui, Zhang Xueling.Effect of pH on nitrous oxide production and emissions from a partial nitritation reactor under oxygen-limited conditions.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.02.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of pH on nitrous oxide production and emissions from a partial nitritation reactor under oxygen-limited conditions Yongtao Lva, Kai Jua, Lei Wanga*, Xiaoqiang Chenb, Rui Miaoa, Xueling Zhanga
a
School of Environmental and Municipal Engineering, Xi’an University of Architecture and
Technology, Xi’an, 710055, China b
Institute of Water and Environmental Research, Faculty of Infrastructure Engineering, Dalian
University of Technology, Dalian, 116024, China
Corresponding author: Lei Wang, School of Environmental and Municipal Engineering, Xi’an
*
University of Architecture and Technology, Xi’an, 710055, China. Tel.: +86 29 82202729; Fax: +86 29 82202729 E-mail address: [email protected]
1
Graphical Abstract pH -1000
(A)
7
8
pH
NO2
DO
_
+
NH4
(E)
N2O
0
Depth (m)
9
1000
2000
N2O _
NO2
3000
+
NH4 0
100 200 300 -1 Concentration (molL )
400
-12
-6
0
6
12
Rate (molcm h ) -3
-1
Research highlights
In a PN reactor, N2O emissions decreased with the increase in initial pH.
N2O production inside sludge aggregates were quantified by using microelectrodes.
A decrease in pH led to increased N2O emissions from denitrification.
ABSTRACT The effect of pH on nitrous oxide (N2O) emissions from a laboratory-scale partial nitritation sequencing batch reactor under oxygen-limited conditions was investigated from both macro- and microscopic viewpoints. During the aeration period of a single cycle, N2O emissions decreased when the initial pH increased from 7.5 to 8.5. By application of microelectrodes, N2O production was observed inside entire sludge aggregates, and it increased with decreasing pH from 8.5 to 7.0. At pH 8.0 and 8.5, N2O was mainly produced in the outer layer (< 1000 m) of sludge aggregates, where 2
nitritation mainly occurred. At pH 7.0 and 7.5, N2O production was mainly observed in the inner layer (> 1000 m), where the dissolved oxygen was almost depleted, revealing that the dominant pathway here was denitrification. Under oxygen-limited conditions, a decrease in pH led to increased N2O emissions from denitrification pathway.
Keywords: partial nitritation; sequencing batch reactor; nitrous oxide; pH; microelectrodes
INTRODUCTION Nitrous oxide (N2O) is often emitted from biological nitrogen removal (BNR) processes [1-4], which is problematic as N2O is a powerful greenhouse gas with a much stronger greenhouse effect than carbon dioxide (about 300-fold) [1]. It is generally accepted that AOB are the major contributors to N2O emissions in BNR processes [5,6]; therefore, N2O emissions in partial nitritation (PN) reactors are of growing concern. N2O emissions are affected by process parameters (e.g., dissolved oxygen (DO) concentration, pH and substrate concentrations) in BNR systems [7-10]. Low DO concentrations, high NO2– concentrations and variation in influent NH4+ concentrations have been identified as promoting N2O formation [6,7,11]. The relationship between pH and N2O emission in BNR process has also been reported [12,13]. Pan et al. [12] found that N2O accumulated at a low pH value during denitrification by methanol utilizing denitrifiers. Law et al. [13] obtained the maximum N2O emission rate at pH 8.0 in a PN system, and found that N2O emission correlated with the ammonium oxidation rate (AOR). In previous studies, the N2O emission rate and dynamic characteristics were well studied [7,11-13], whereas the N2O production at micro-scale were seldom characterized. Theoretically, N2O is first produced inside the microbial aggregates and then is emitted to the atmosphere. Therefore, measurement of N2O production and transformation inside sludge aggregates might reveal the most plausible pathways of N2O production. Microelectrodes are one of the most suitable tools for microenvironmental measurements [14,15]. Satoh et al. [15] studied nitrification activities changes within biofilms under different operating conditions. Rathnayake et al. [16] observed that N2O was mainly produced in the outer layer of a granule where AOB showed high
3
activities, and reflected that AOB might be responsible for N2O production. However, variation in N2O production pathways with operating conditions has not previously been reported. In this study, a laboratory-scale SBR fed with a synthetic inorganic wastewater was operated for PN under oxygen-limited conditions. The N2O emissions were first investigated from a macroscopic viewpoint. Then, microelectrodes were employed to quantify the microenvironment and N transformation inside the microbial aggregates from a microscopic viewpoint. The microbial activity obtained was analyzed for correlation with the N2O production, aiming to explore how pH affects N2O production and emissions.
Materials and methods Experimental setup A laboratory-scale SBR with a working volume of 4 L was used for PN. One cycle consisted of a 5 min filling period, a 320 min aerating period, a 30 min settling period, and a 5 min drawing period. The drawn volume was 2.0 L, making the exchange volume 50%. Bulk liquid (200 mL) was removed each day providing a sludge retention time of 20 d. The mixed liquor suspended solid (MLSS) was around 3000 mgL–1 during the period of this study. During the aeration period, a mass flow controller was used to keep a constant air flow rate (0.32 L min–1), and the average DO concentration in the bulk liquid was 0.33 0.06 mg L–1 5 min after aeration. The reactor temperature was maintained at 27 1 C using a water jacket.
Biomass and synthetic wastewater In previous study, PN was successfully initiated and steadily operated by inoculating with conventional sludge, and characteristics of N2O emissions were investigated [17]. The same biomass was used for further exploration of the effect of pH on N2O emissions, as described below. The synthetic wastewater contained NH4Cl (N source), NaHCO3 (C source and buffer), and trace elements. The concentrations of NH4+ and NaHCO3 were 600 mg N L–1 and 5400 mg L–1, respectively. Trace element solutions were added as described by Ju et al. [17]. HCl (0.5 mol L–1) and NaOH (0.5 mol L–1) were used to adjust to different initial pH values of 7.5, 8.0, and 8.5.
4
Chemical analysis NH4+-N, volatile suspended solids, and total suspended solids were determined according to the standard methods of APHA [18]. Nitrate-nitrogen (NO3–-N) and NO2–-N were determined using ion chromatography (761 Compact IC, Metrohm, Herisau, Switzerland). The pH and the DO concentration were directly monitored using a pH meter (Unisense, Aarhus, Denmark) and a DO meter (HQ25d, Colorado, USA), respectively.
DNA extraction, PCR-DGGE, cloning and sequencing The mixed liquor was collected from the SBR and the total DNA was extracted using a bacterial genomic mini extraction kit (Sangon, China). Universal primers F357 (with GC clamp) and R518 were used for the PCR amplification [19]. The DGGE analysis, cloning and sequencing was determined according to Lv et al. [20]. N2O measurements The N2O concentration in the off-gas was measured by a gas chromatograph (PE600, PerkinElmer, USA) with an electron capture detector and a Porapak Q column (GDX-101, Gansu, China) using 30 mL min–1 high-purity N2 as the carrier gas [17].
Microsensor measurements NH4+, NO2−, pH, DO, and N2O microelectrodes were used for microenvironmental measurements. The first four microelectrodes were manufactured and their performance was previously shown to be stable [21]. They were constructed and calibrated before measurements according to the methods of de Beer et al. [14]. The N2O microelectrode was purchased from Unisense, Denmark. A three-point calibration (pure water, 50% N2O solution, and saturated N2O solution) was carried out before each measurement. Sludge flocs were sampled from the PN reactor and placed very gently just above the nylon net in a flowing chamber (Fig. 1) [22]. The microelectrode penetrated through the sludge aggregates under settling conditions. To obtain steady-state profiles, the sludge aggregates were left for 30 min before profile measurements started, and each microelectrode measurement was made at least three times. As the flocs had a symmetrical structure, the microelectrode only had to penetrate to half the depth of the flocs to reflect the entire substrate distribution. 5
The medium used in the chamber for microprofile measurements consisted of NH4Cl (4.2 mg N L–1), NaNO2 (4.2 mg N L–1), and NaHCO3 (37.8 mg L–1), and the different pH values were controlled by adding HCl (0.5 mol L–1) and NaOH (0.5 mol L–1). To provide the oxygen-limited condition, the DO concentration of this medium was maintained below 1 mg L–1 through purging with N2. Because the pH of the PN reactor varied in the range 6.8-8.5, four different pH values (7.0, 7.5, 8.0, and 8.5) were selected for microprofile measurements.
Net volumetric rate calculations The net volumetric rates were calculated according to mass transport equation [23]: C ( z, t ) / t Ds 2C ( z, t ) / z 2 Q( z ) P( z )
(1)
where C(z,t) is substrate concentration (mmol·m–3) at time t and depth z, respectively. Q and P are consumption and production rate (mmol·m–3·s–1), respectively. Ds is the effective diffusion coefficient (m2 s–1); Ds values of 1.38 10–9, 1.25 10–9, and 2.10 10–9 m2 s–1 were used for the calculations of NH4+, NO2−, and N2O, respectively, at 25 C [3,16]. When steady state was achieved, the left expression is zero. Eq. (1) can be reduced to: Ds 2C ( z ) / z 2 Q( z ) P( z )
(2)
Defining R(z) = Q(z)-P(z) is the net volumetric rate (mmol m–3 s–1). Using Euler’s formula for numeric integration, the following equation can be obtained as:
C / zn1 C / zn h Rn / Ds
(3)
where h is the step size (100 m). According to the concentration gradient, the following equation can be obtained as:
Cn 1 Cn h C / zn
(4)
Substituting ∂C/∂zn with Eq. (3), it can be calculated as:
Rn1 Ds [(Cn1 Cn ) / h C / zn1 ] / h
.
(5)
6
Results Effect of pH on PN performance The reactor was operated for at least one week under each pH value. In the initial pH range 7.5–8.5, the change in pH had almost no effect on the PN (Fig. 2). The concentration of NH4+ in the influent was 598 12 mg N L–1, and the SBR converted around half of the NH4+–N to NO2−–N. The effluent concentrations of NH4+ and NO2− were 283 16 and 297 22 mg N L–1, respectively. The molar ratio of NO2− to NH4+ in the effluent was 1.05 0.12, which is suitable as a feed for the anammox process. The NO3– concentration in the effluent was 28.40 2.46 mg N L–1, indicating low activity of nitrite-oxidizing bacteria (NOB).
Microbial community structure On day 25 the microbial community was investigated using PCR and DGGE analyses. Nitrosomonas sp., known AOB, were detected, which is in agreement with observations from other PN systems [24-27]. Although no organic matter was fed to the reactor, Comamonas sp., Flavobacteria sp. and Acidovorax sp., known heterotrophs with denitrifying functionality [28,29], were also detected. The organic compounds might be derived from biomass decay [30]. It is worth noting that NOB were not detected, although nitrification was observed in the reactor. The lack of detection of NOB means that they accounted for < 9% of the bacterial population [31].
Effect of pH on N2O emissions in a single cycle The effect of initial pH on N transformation in a single cycle is shown in Fig. 3. For all pH values, the DO concentration decreased and maintained around 0.3 mg L–1. The pH decreased with processing of the PN, but at initial pH values of 8.0 and 7.5, for an initial processing time of 40 min, a slight increase in pH was observed, which was because of CO2 stripping [32]. The concentration of NH4+ decreased and that of NO2– increased almost linearly, and at the end of the aeration process, the molar ratio of NO2– to NH4+ ions was nearly 1:1. The AOR showed that they increased from 0.144 to 0.158 and 0.176 mg NH4+-N min–1 g–1 MLSS with pH decrease from 8.5 to 8.0 and 7.5, indicating that the activities of AOB increased with decreased initial pH. This was in agreement with Van Hulle et al. [33] who found that AOB activity decreased when the pH increased above 7.23. The concentration of NO3– ions increased slowly from around 10 up to 30 mg N L–1. 7
During the initial 50 min of aeration time, a peak N2O emission was observed after an aeration time of 10 min. The maximum N2O concentrations of 1.43, 1.24, and 0.84 mg L–1 were obtained for initial pH values of 7.5, 8.0, and 8.5, respectively. From an operating time of 50 min onward, the trend in N2O emissions in the off-gas changed. At initial pH values of 8.5 and 8.0, the N2O concentrations increased slowly from 0.22 to 0.44 mg L–1 and from 0.31 to 0.55 mg L–1, respectively, while at pH 7.5, the N2O concentrations were maintained at around 0.6 mg L–1. At pH of 7.5, 8.0 and 8.5, 63.74, 40.59 and 34.72 mg of N2O emission was obtained in a cycle, respectively. This indicated that N2O emissions decreased with the initial increase in pH from 7.5 to 8.5 under oxygen-limited conditions.
Nitrogen transformation and N2O production inside the sludge aggregates The concentration profiles from microsensor measurements are shown in Fig. 4 (A–D). The trends in the microprofiles within the sludge flocs were similar at different pH values. Oxygen was mainly consumed in the outer layer, decreasing from a concentration of around 30 mol L–1 to below 10 mol L–1 at a depth of 500 m. Therefore, oxygen-limited conditions formed, similar to those encountered in the SBR during aeration. With increasing depth, the NH4+ concentration decreased and the NO2– concentration increased, which was mainly observed in the outer layer (< 1000 m). The pH decreased inside the flocs. At different pH values, the concentration of N2O increased with increasing penetration depth, and it increased by 104.85, 83.38, 71.44, and 62.49 mol L–1 for pH values of 7.0, 7.5, 8.0, and 8.5, respectively. Thus, on increasing the pH, the production of N2O decreased, which was in agreement with the N2O emissions observed in the reactor. The net volumetric consumption or production rates were calculated, and these are shown in Fig. 4 (E–H). At different pH values, the NH4+ consumption coincided with the NO2– production, especially in the outer layer (< 1000 m), revealing that a nitritation reaction had occurred. N2O production was observed inside the entire floc, while the trends were different for different pH values. At pH values of 8.0 and 8.5, the N2O was mainly produced in the outer layer (< 1000 m), where NH4+ oxidation mainly occurred, and with an increase in depth, the net N2O production rate decreased. However, at pH values of 7.0 and 7.5, in the outer layer, the net volumetric rate of N2O production increased with increasing depth, and a higher N2O production rate was observed in the inner layer (> 1000 m), where DO was almost depleted. 8
Discussion The net volumetric rate revealed that N2O was produced in the whole sludge aggregates, but the microbial reactions were different in the outer and inner layers. In the outer layer (< 1000 m), NH4+ consumption coincided with the production of NO2–, indicating an active zone of nitritation. However, N2O production pathway in the outer layer could not be determined because both denitrification and nitrification could occur under oxygen-limited conditions. In the inner layer (> 1000 m), where the DO was almost depleted, under such an anoxic condition the denitrification pathway was the main source of N2O production. N2O produced outside the nitritation zone was also observed by Rathnayake et al. [16] inside stratified granules, and the question of how small amounts of NO2– were produced in such an anoxic zone needs further investigation. Identifying the microbial community structure was crucial to demonstrating the mechanism of biological reactions in the sludge aggregates. In this study, AOB of Nitrosomonas sp. were detected, which can reduce NO2– to produce N2O or N2 [34,35], i.e., nitrifier denitrification. Denitrifying heterotrophs of Betaproteobacteria and Flavobacteria were also detected. Gabarró et al. [36]. demonstrated that most Betaproteobacteria are capable of denitrifying N2O to N2, whereas most Bacteroidetes, including Flavobacteria, could not denitrify N2O because of the absence of the nosZ gene. Therefore, both AOB (Nitrosomonas sp.) and heterotrophic denitrifiers (Flavobacteria sp.) could increase N2O production under anoxic conditions, but it is difficult to determine which are responsible for the denitrification pathway of N2O production in this study. To determine how N2O production were affected by pH, N2O production in the outer and inner layers was quantified at different pH values and summarized in Table 1. At pH 8.0 and 8.5, N2O was mainly produced in the outer layer, accounting for 61.3 and 60.5%, respectively, of the total N2O produced (Table 2). In contrast, at pH values of 7.0 and 7.5, N2O production in the outer layer accounted for only 31.2 and 31.8%, respectively, indicating that denitrification was the dominant pathway for N2O production. Thus, it can be concluded that with decreasing pH, the denitrification pathway of N2O production becomes more favored, which is in agreement with the study by Wrage et al. [37]. This is probably the main reason why the peak value of N2O emission in a single cycle of an SBR decreased with the increase in initial pH values. In the later phase of the aeration period (from 50 min onward), the trend in N2O emissions at different pH values changed. At initial pH values of 8.0 and 8.5, N2O emission increased as the reaction 9
proceeded. This can be explained by the increased N2O produced via the denitrification pathway. As the NH4+ oxidation progressed, more NO2– was produced and the pH simultaneously decreased, both of which can lead to enhanced N2O production from denitrification [38]. However, at initial pH 7.5, no obvious increase in N2O emission was observed, which is probably because pH affects the two pathways of N2O production differently. On the one hand, a lower pH promotes denitrification, leading to more N2O being produced; on the other hand, AOB activities decreased when the pH decreased below 7.23 [33], which might lower the N2O production.
Conclusions Under oxygen-limited conditions, the effect of initial pH on N2O emissions in a laboratory-scale PN SBR showed that AOB activity increased with decreasing pH from 8.5 to 8.0 and 7.5, and resulted in more N2O emission. By using microelectrodes, N2O production was quantified inside microbial aggregates. The result demonstrated that a decrease in pH from 8.5 to 7.0 led to increased N 2O production from the denitrification pathway.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No.51108367), Housing and urban and Rural Construction Department of Shaanxi Province (No.2015-K61), and the Innovative Research Team of Xi’an University of Architecture and Technology.
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[29] Schloe K, Gillis M, Hoste B, Pot B, Vancanneyt M, Mergaert J, Swings J, Biedermann J, Süssmut R. Polyphasic characterization of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains. Syst Appl Microbiol 2000; 23: 364-372. [30] Kindaichi T, Tsushima I, Ogasawara Y, Shimokawa M, Ozaki N, Satoh H, Okabe S. In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms. Appl Environ Microbiol 2007; 73: 4931-4939. [31] Straub KL, Buchholz-Cleven BEE. Enumeration and detection of anaerobic ferrous iron-oxidizing, nitrate-reducing bacteria from diverse European sediments. Appl Environ Microbiol 1998; 64: 4846-4856. [32] Wett B, Rauch W. The role of inorganic carbon limitation in biological nitrogen removal of extremely ammonia concentrated wastewater. Water Res 2003; 64: 1100-1110. [33] Hulle SWHV, Volcke EI, Teruel JL, Donckels B, van Loosdrecht MCM, Vanrolleghem PA. Influence of temperature and pH on the kinetics of the Sharon nitritation process. J Chem Technol Biotechnol 2007; 82: 471-480. [34] Bock E, Schmidt I, Stuven R. Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonia or hydrogen as electron donors and nitrite as electron acceptor. Arch Microbiol 1995; 163: 16–20. [35] Sutka RL, Ostrom NE, Ostrom PH, Breznak JA, Gandhi H, Pitt AJ, Li F. Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances. Appl Environ Microbiol 2006; 72: 638-644. [36] Gabarró J, Hernández-del Amo E, Gich F, Ruscalleda M, Balaguer MD, Colprim J. Nitrous oxide reduction genetic potential from the microbial community of an intermittently aerated partial nitritation SBR treating mature landfill leachate. Water Res 2013; 47: 7066-7077. [37] Wrage N, Velthof GL, van Beusichem ML, Oenema O. Role of nitrifier denitrification in the production of nitrous oxide. Soil Biol Biochem 2001; 33: 1723-1732. [38] Lu HJ, Chandran K. Factors promoting emissions of nitrous oxide and nitric oxide from denitrifying sequencing batch reactors operated with methanol and ethanol as electron donors. Biotechnol Bioeng 2010; 106: 390-398.
13
Fig. 1 The schematic diagram of the chamber for microprofile measurements
750
5
pH=8.0
pH=7.5
600
4
-
-
+
450
+
NO3-NEff
NO2-N/NH4 -N
NH4 -NInf
+
3
-
NO2-NEff
+
NH4 -NEff
NO2-N/NH4 -N in effluent
300
2
150
1
-
-1
concentration(mg N L )
pH=8.5
0
0
0
3
6
9
12
15
18
21
24
27
Time(days) Fig. 2 Time course of the partial nitrification performance at different initial pH levels
14
300 1.0 200 0.5 100
0 200
250
300
0.8
7.0
0.4
0.0 350
6.5
2.0
8.5
0
50
100
300 1.0 200 0.5 100
0 100
150
200
250
300
pH
1.5
-1
400
50
1.6
8.0
1.2
7.5
0.8
7.0
0.4
0.0 350
6.5
2.0
8.5
0
50
100
300 1.0 200 0.5 100
0 150
200
250
300
pH
1.5
-1
400
100
150
200
250
0.0 350
300
1.6
(F) Conecentration (mg N2OL )
-1
Conecentration (mgNL )
ammonium nitrite nitrate gas N2O
50
0.0 350
Time (min)
(E)
0
300
pH DO
Time (min)
500
250
(D) Conecentration (mg N2OL )
-1
Conecentration (mgNL )
ammonium nitrite nitrate gas N2O
(C)
0
200
Time (min)
Time (min)
500
150
-1
150
7.5
DO concentration (mgL )
100
1.2
0.0 350
pH DO
8.0
1.2
7.5
0.8
7.0
0.4
6.5 0
50
100
150
200
250
300
-1
50
8.0
DO concentration (mgL )
0
pH DO -1
1.5
pH
400
-1
(A)
Conecentration (mg N2OL )
-1
Conecentration (mgNL )
500
1.6
(B)
DO concentration (mgL )
8.5
2.0
ammonium nitrite nitrate gas N2O
0.0 350
Time (min)
Time (min)
Fig. 3 Variation in nitrogen and process parameters during a partial nitritation cycle at different pH levels (A and B correspond to initial pH of 8.5, C and D correspond to initial pH of 8.0, E and F correspond to initial pH of 7.5)
15
pH 7
-1000
(A)
8
DO
(E)
+
NO2
pH
NH4
N2O
0
Depth (m)
9 _
1000
2000
N2O _
NO2
3000
+
NH4 0
100 200 300 -1 Concentration (molL )
400
-12
-6
0
6
12
Rate (molcm h ) -3
-1
pH -1000
6
7
(B)
N2O
8
pH
9
(F)
+
_
NO2 NH4
DO
Depth (m)
0
1000
2000
N2O _
NO2 +
3000
NH4 0
100
200
300
400 -1
Concentration (molL )
-12
-6
0
6
Rate (molcm h ) -3
16
-1
12
pH 6
7
8
9
-1000
(C) N O 2
(G)
pH _
DO
NO2
0 +
Depth (m)
NH4
1000
2000
N2O _
NO2 3000
+
NH4 0
100
200
300
400
-12
-6
0
6
12
Rate (molcm h )
-1
-3
Concentration (molL )
-1
pH 6
7
8
9
-1000
0 DO
(H)
+
(D)
_
NO2
NH4
pH
N2O
1000
2000
N2O _
NO2
3000
+
NH4 0
100
200
300 -1
Concentration (molL )
400
-12
-6
0
6
12
Rate (molcm h ) -3
-1
Fig. 4 (A–D) Steady-state microprofiles of DO, pH, NH4+, NO2−, and N2O inside the sludge aggregates (A, B, C, and D correspond to pH levels of 7.0, 7.5, 8.0, and 8.5, respectively). (E–H) Net volumetric production or consumption rates of NH4+, NO2−, and N2O inside the sludge aggregates (E, F, G, and H correspond to pH levels of 7.0, 7.5, 8.0, and 8.5, respectively)
17
Table 1 Quantification of N2O production inside the sludge aggregates N2O production in the outer N2O
production
in
layer (0–1000 m)
layer(1000–3000 m)
Concentration
Concentration
the
inner Total N2O production
pH (mol L–1)
Ratio (%)
(mol L–1)
Concentration Ratio (%)
(mol L–1)
Ratio (%)
8.5
37.7 ± 2.5
60.5 ± 3.7
24.6 ± 5.9
39.5 ± 12.2
62.3 ± 10.8
100
8.0
43.6 ± 16.2
61.3 ± 3.4
27.5 ± 7.6
38.7 ± 9.9
71.2 ± 12.7
100
7.5
26.4 ± 2.4
31.8 ± 7.5
56.6 ± 7.1
68.2 ± 19.5
82.9 ± 11.3
100
7.0
32.6 ± 6.2
31.2 ± 2.4
71.8 ± 16.2
68.8 ± 5.6
104.3 ± 21.3
100
18