Reducing N2O Emission from a Domestic-Strength Nitrifying Culture

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Reducing N2O Emission from a Domestic-Strength Nitrifying Culture by Free Nitrous Acid-Based Sludge Treatment Dongbo Wang, Qilin Wang, Andrew Elohim Laloo, and Zhiguo Yuan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00660 • Publication Date (Web): 13 Jun 2016 Downloaded from http://pubs.acs.org on June 15, 2016

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Environmental Science & Technology

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Reducing N2O Emission from a Domestic-Strength Nitrifying Culture by Free

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Nitrous Acid-Based Sludge Treatment

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Dongbo Wang, Qilin Wang, Andrew Elohim Laloo, Zhiguo Yuan*

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Advanced Water Management Centre (AWMC), The University of Queensland, QLD 4072, Australia

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Corresponding author

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Tel: +61-7-33654374

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Fax: +61-7-33654726

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E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: The increase of nitrite in the domestic-strength range is generally recognized to stimulate

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nitrous oxide (N2O) production by ammonia-oxidizing bacteria (AOB). It is found in this study, however,

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that N2O emission from a mainstream nitritation system (cyclic nitrite: 25-45 mg N/L), which was established

12

by free nitrous acid (FNA)-based sludge treatment, was not higher but much lower than that from the initial

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nitrifying system with full conversion of NH4+-N to NO3--N. Under dissolved oxygen (DO) levels of 2.5-3.0

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mg/L, N2O emission from the nitritation stage was 76% lower than that from the initial stage.

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DO level was reduced to 0.3-0.8 mg/L, N2O emission from the nitritation stage was still 40% lower. The

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mechanism investigation showed that FNA treatment caused a shift of the stimulation threshold of nitrite on

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N2O emission. At the nitritation stage, the maximal N2O emission factor occurred at ~16 mg N/L nitrite.

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However, it increased with increasing nitrite in the range of 0-56 mg N/L at the initial stage. FNA treatment

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decreased the biomass-specific N2O production rate, suggesting the enzymes relevant to nitrifier

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denitrification were inhibited.

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community diversity but increased the AOB and denitrifiers abundances.

Even when

Microbial analysis revealed that FNA treatment decreased the microbial

TOC Art

22

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INTRODUCTION

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Nitrous oxide (N2O), which is a potent greenhouse gas and a main sink for stratospheric ozone, can be

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produced in wastewater treatment plants (WWTPs) during microbial nitrification and denitrification.1,2

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Although both heterotrophic denitrifiers and ammonia-oxidizing bacteria (AOB) can generate N2O as a

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byproduct, there are increasing evidences showing that the latter is a more important contributor.2-4

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understanding the mechanisms and developing mitigation strategies of N2O production by AOB attracted

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much attention in the past few years.5-8

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Thus,

According to the current understanding, the three pathways proposed for N2O production by AOB are: (i)

2


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nitrifier (or AOB) denitrification, (ii) biological oxidation of hydroxylamine (NH2OH), and (iii) chemical

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oxidation of NH2OH.9-12 Among them, nitrifier denitrification is a dominant process in most cases.13,14

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amount of N2O generated by AOB, particularly from the nitrifier denitrification pathway, is reported to largely

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depend on the nitrite level, as denitrifying genes are sensitive to nitrite concentration.12,14

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nitrite concentration is usually thought to lead to increased N2O emission.1,2,15

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reported that nitrite accumulation at high-strength levels (e.g., 500-1000 mg N/L) showed inhibitory effect on

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N2O emission, likely due to the inhibition of the AOB denitrification pathway by a high level of nitrite,6,12,16

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all previous studies demonstrated that the increase of nitrite in the domestic-strength range (e.g., 0-50 mg N/L)

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resulted in stimulating effect on N2O emission.1,2,12,14 For example, Law et al. showed that the specific N2O

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production rate increased from 0.1 to 0.9 mg N2O-N/h/g VSS (volatile suspended solids) with the increase of

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nitrite concentration from 0 to 50 mg N/L.12

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rate and N2O emission factor increased as nitrite level increased from 3 to 50 mg N/L.14

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important implications to the operation of WWTPs.

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The

The increase of

Although some publications

Peng et al. found that both the biomass specific N2O production These findings have

It is increasingly recognized that the function of WWTPs should be transformed from waste removal to

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resource recovery.17,18

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energy recovery simultaneously and is therefore considered as the most promising technology for future

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WWTPs.19-21

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nitritation (i.e. the conversion of approximately 50% of the ammonium to nitrite), and anaerobic ammonium

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oxidation (Anammox).21 One example for the integration of these units into a mainstream deammonification

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process has been detailed in our previous publication.22

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nitritation stage.23

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the domestic-strength range will inevitably occur in the nitritation stage.

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understanding, N2O emission from the mainstream deammonification process is postulated to be much higher

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than that from the conventional nitrification-denitrification process due to the accumulation of

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domestic-strength nitrite. This becomes one of the major concerns of this energy-saving technology. Thus,

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methods that can effectively mitigate the increase of N2O production or even decrease the N2O production by

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AOB with nitrite accumulation in the domestic-strength range will have huge significance to the application of

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the mainstream deammonification process, and the relevant mechanism will also enhance the current

Mainstream deammonification can enable desirable nutrient removal and maximal

Mainstream deammonification consists of upfront separation of organic carbon, partial

In such a system, N2O emission mainly occurs in the

Domestic sewage generally contains ~50 mg/L ammonium, thus nitrite accumulation in

3


According to the current


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understanding regarding N2O production by AOB. Our recent studies demonstrated that stable nitritation can be achieved in domestic-strength wastewater

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by free nitrous acid (FNA)-based sludge treatment.22

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reactor treating synthetic wastewater containing ammonium at 57 mg N/L without organic carbon.

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Compared with the initial nitrifying stage with full conversion of NH4+-N to NO3--N, 25-45 mg N/L of nitrite

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accumulated in the nitritation stage.22

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stage was supposed to be higher than that from the initial stage.

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this nitritation reactor was not higher but much lower, compared to that is the initial stage with full conversion

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of ammonium to nitrate. FNA, the protonated species of nitrite, was previously found to bring inhibitory

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effect on N2O reduction in both nitrifying and denitrifying cultures.24,25

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FNA-based sludge treatment on N2O emission from domestic-strength nitrifying culture has not been

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investigated.

This method was experimentally verified in a nitrifying

According to previous findings, N2O emission from the nitritation Nevertheless, the measured N2O emission in

To date, however, the role of

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The aim of this study was to understand the underlying mechanisms of how FNA-based sludge treatment

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reduces N2O emission from a domestic-strength nitrifying culture with the accumulation of nitrite. Firstly,

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N2O emission between the initial full nitrifying stage and the subsequent nitritation stage was compared at

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dissolved oxygen (DO) concentrations of 2.5-3.0 mg/L. Then, the mechanisms responsible for the lowered

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N2O emission in the nitritation stage with substantial nitrite accumulation were investigated.

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N2O emission from the nitritation reactor at a lower DO level (0.3-0.8 mg/L) was studied. This is the first

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study showing that N2O emission can be decreased with the increase of nitrite accumulation in the

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domestic-strength range.

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production by AOB and erases one of the main concerns with the practical application of the mainstream

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deammonification process.

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MATERIALS and METHODS

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Initial Operation of the Mainstream Reactor with Fully-Nitrifying.

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a working volume of 11 L was conducted as the mainstream reactor at a temperature controlled room (22 ±

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1℃).

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operated with four 6-h cycles daily. Each cycle comprised a 90 min aerobic feeding phase, a 210 min

Finally, the

The finding reported here challenges the regular recognition relevant to N2O

In this study, a lab-scale reactor with

The seed sludge was taken from a municipal WWTP in Brisbane, Australia.

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The reactor was

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aerobic phase, a 50 min settling phase, and a 10 min decanting phase. During the feeding phase, 5 L

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synthetic medium was pumped into the reactor.

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ammonium (~57 mg/L), which was prepared by the following components (per liter): 0.3104 g of NH4HCO3

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(60 mg NH4+-N), 0.33 g NaHCO3, 0.035 g MgSO4•7H2O, 0.072 g of NaH2PO4•H2O, 0.184 g of NaCl, 0.029 g

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KCl, and 0.3 mL of a trace element stock that was detailed previously.26

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synthetic medium used in this study contained no organic carbon source in order to minimize the denitrifiers’

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impacts on N2O production. In the feeding and aerobic phases, DO and pH were respectively controlled at

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2.5-3.0 mg/L and 7.5 with programmed logic controllers. Except for the settling and decanting phases, the

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reactor was continuously mixed with a magnetic stirrer. During the decanting phase, 5 L of the supernatant

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was discharged from the reactor, which resulted in a hydraulic retention time of 13.2 h. No sludge wasting

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was operated. By measuring the total suspended solids (TSS) concentrations in the reactor and in the

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effluent, it was estimated that sludge retention time was approximately 12 d during stable operation.

The synthetic medium contained a domestic level of

It should be emphasized that the

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To investigate the effect of domestic-strength nitrite variation on N2O production at the initial nitrifying

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stage, batch tests were performed after the mainstream reactor achieved steady-state operation. Seven levels

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of initial nitrite concentration (i.e., 0, 10, 20, 30, 40, 50, and 60 mg N/L) were tested by adding different

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volumes of NaNO2 stock solution (4 g/L). All batch tests were operated in triplicate at room temperature (22

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± 1℃) in a batch reactor with a working volume of 1 L.

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from the mainstream reactor at the end of the aerobic phase (before settling). The mixture was centrifuged

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(5000 rpm for 5 min), washed twice with the synthetic medium mentioned above, and re-suspended in the

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synthetic medium with a final volume of 1 L before being transferred into the batch reactor. Each test lasted

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for 45 min. During the entire period, pH was maintained at 7.5 with a programmed logic controller while

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DO was manually controlled at 2.5-3.0 mg/L by the use of a gas mixture of N2 and air.13,14

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are identical to those applied to the main reactor.

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Nitritation Established by Sludge Treatment with FNA at 1.82 mg N/L.

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mainstream reactor operation was the same as in the initial stage except that 2750 mL of sludge was

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withdrawn daily from the mainstream reactor at the end of the aerobic phase just before settling. The sludge

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withdrawn was first thickened to 130 mL and then transferred into the FNA treatment unit operated at room

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temperature (22 ± 1℃). Afterwards, a NaNO2 stock solution (60 g N/L) was added into the unit, resulting in

For each test, 200 mL mixed liquor was withdrawn

5


These conditions

During this stage, the


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a NO2--N concentration of 750 mg N/L. pH in the unit was maintained at 6.0 all the time with the addition of

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HCl controlled by a programmed logic controller. The temperature, pH, and nitrite concentration applied

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give a calculated FNA concentration of 1.82 mg N/L. After 24 h treatment, the FNA-treated sludge was

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manually returned to the mainstream reactor. The FNA level, treatment time, and sludge treatment ratio

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applied in the FNA treatment unit were chosen to establish nitritation in the main reactor.22

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d for the mainstream reactor to achieve stable nitritation, and then the following batch tests were performed.

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It took about 24

Batch tests were conducted under initial nitrite concentration of 0, 10, 20, 30, 40, 50, and 60 mg N/L

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before and after 24 h FNA-treatment.

For each nitrite level, 200 mL mixed liquor was withdrawn from the

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mainstream reactor, centrifuged, washed twice with the synthetic medium, and re-suspended in the synthetic

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medium with a final volume of 1 L before being transferred into the batch reactor. All operations of the

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batch tests were the same as described above. After N2O measurement, the mixture was centrifuged, washed,

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and re-suspended in the synthetic medium with a final volume of 130 mL before being transferred into a FNA

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treatment unit. All the operations of FNA treatment unit applied here were also the same as those applied

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above. After 24 h treatment, FNA in the mixture was removed through washing, and then the N2O emission

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from this treated biomass was also measured. The procedure of the measurement was the same as that

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applied in other batch tests described above.

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N2O Emission under DO Level of 0.3-0.8 mg/L. As low DO conditions are generally considered to benefit

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partial nitritation but increase N2O emission,13,21 the FNA-based N2O reduction method was finally examined

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at a lower DO level (0.3-0.8 mg/L). The mainstream reactor and the FNA treatment unit in this stage were

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operated the same as those in the section “Nitritation Established by Sludge Treatment with FNA at 1.82 mg

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N/L” except that the DO concentration in the mainstream reactor was reduced from 2.5-3.0 mg/L to 0.3-0.8

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mg/L. After operation for 28 d the mainstream reactor reached stable performance, and then the N2O

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measurement and cycle studies described above were performed.

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Analytical Methods.

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from the reactors using a syringe and filtered immediately through disposable Millipore filter units (0.22 mm

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pore size). The levels of chemical oxygen demand (COD), TSS, and VSS were analyzed in accordance with

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standard methods.28

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QuikChem8000 Flow Injection Analyzer (Lachat Instrument, Milwaukee, Wisconsin). N2O concentration in

For the analyses of ammonium, nitrite, and nitrate, mixture samples were taken out

The concentrations of ammonium, nitrite, and nitrate were measured using a Lachat

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liquid phase was determined online using a N2O microsensor (N2O-100), as detailed in the literature.27

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N2O concentration was measured with a URAS 26 infrared photometer (Advance Optima Continuous Gas

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Analyser AO2020 series, ABB), with a measuring range of 0-100 ppmv and a detection limit of 1.0 ppmv.

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The detailed method was described in our previous publication.12

Gas

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The diversity of microbial community between the initial stage and the nitritation stage was assayed by

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the amplicon sequencing. Sludge samples from the reactor were withdrawn and centrifuged for 5 min at

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10000 rpm. The total genomic DNA was extracted from the withdrawn sludge using the FastDNA Kit (BIO

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101, Vista, CA) according to the manufacture’s instruction. The community profiling was performed on the

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Illumina MiSeq platform using the universal primers 926F (5’- AAACTYAAAKGAATTGRCGG - 3’) and

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the 1392R (5’-ACGGGCGGTGWGTRC - 3’). These primers amplify the V6 region of the small subunit

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(SSU) ribosomal RNA of prokaryotes (16S). The raw sequencing data was analyzed using Quantitative

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Insights Into Microbial Ecology. The combined reads were classified into their operational taxonomic units

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(OTUs) at 97% similarity against the Green Genes database.

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RESULTS and DISCUSSION

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Comparison of N2O Emission from the Mainstream Reactor between the Initial Nitrifying and the

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FNA-Established Nitritation Stages. During the stable operation of the initial stage, it was measured that

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0.34 ± 0.03 mg/L of NH4+-N, 1.36 ± 0.42 mg/L of NO2--N, and 55.6 ± 1.7 mg/L of NO3--N were in the effluent,

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suggesting that almost full nitrification was achieved in this stage (Table S1, Supporting Information).

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During the stable operation of the FNA-established nitritation stage, 0.79 ± 0.28 mg/L of NH4+-N, 34.9 ± 0.5

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mg/L of NO2--N, and 23.9 ± 0.9 mg/L of NO3--N were determined in the effluent (Table S1, Supporting

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Information). It can be found that the conversion of NH4+-N was unaffected whereas the conversion of

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NO2--N was partially suppressed by FNA treatment. The main reason was that FNA treatment substantially

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reduced the abundance of nitrite oxidizing bacteria (NOB), especially that of Nitrospira sp..22

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Figure 1 shows the profiles of nitrogen compounds and N2O during a typical cycle at the steady-states of

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initial and nitritation stages under the same DO level (2.5-3.0 mg/L). At the initial nitrifying stage, although

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substantial nitrite was accumulated in the first 180 min (the highest nitrite level of 16.6 mg/L was measured at

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180 min), the accumulated nitrite was almost fully oxidized to nitrate in the remaining aerobic period (Figure

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1A-I), confirming again that ammonium was fully converted to nitrate at the end of the aerobic period.

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Compared with the initial nitrifying stage, partial nitrification was achieved after FNA treatment was

172

implemented. In the feeding period, ammonium concentration increased gradually accompanied by the

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decreases of nitrite and nitrate. Afterwards, ammonium decreased gradually, with concurrent increases in the

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nitrite and nitrate levels. At the end of the aerobic period, ammonium was completely oxidized, and 32.4 mg

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N/L of nitrite and 23.5 mg N/L of nitrate were measured, respectively. feeding 120

50 40 Ammonium Nitrite Nitrate

30 20 10

Gas phase Liquid phase

60

120

180

240

300

360

Time (min) feeding

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1 60 0.8

40

0.6

20

A-II

30 20 10

Gas phase N2O (ppmv)

40

60

120

180 240 Time (min)

feeding

50 Ammonium Nitrite Nitrate

0.4 0

50

settling/ decanting

aerobic

aerobic

300

360

settling/ decanting

40

60

120

180

30

0.3

Gas phase Liquid phase

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0.2

10

0.1

300

360

0 0

60

120

180 240 Time (min)

300

360

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B-I 0

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Figure 1. Profiles of nitrogen compounds and N2O during a typical cycle at steady-state of the initial

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stage (A) and nitritation stage (B) under the same DO level (2.5-3.0 mg/L).

Time (min)

240

0.5 0.4

0

0

1.6

1.2

0

A-I 0

Nitrogen compound (mg N/L)

settling/ decanting

1.4

100

0

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aerobic

Liquid phase N2O (mg N/L)

60

140

Liquid phase N2O (mg N/L)

settling/ decanting

aerobic


Nitrogen compound (mg N/L)

feeding

B-II

180

The cyclic nitrite concentration varied between 0.98 and 16.6 mg/L in the initial stage while it varied

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between 24.7 and 32.7 mg/L in the nitritation stage (Figure 1A-I and 1B-I). According to the current

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understandings, it is expected that N2O emission from the latter will be higher than that from the former.

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Nevertheless, experimental results showed that both the gas and liquid phase N2O levels in the latter were 8


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much lower than those in the former, which is contrast with the results reported previously.

For example, the

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highest gas and liquid phase N2O levels were respectively 114.6 ppmv and 1.0 mg N/L in the initial stage

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whereas the corresponding data in the nitritation stage were 39.0 ppmv and 0.3 mg N/L (Figure 1A-II and

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1B-II). Further calculation showed that the N2O emission factor (the N2O nitrogen emitted/the ammonium

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nitrogen converted) in the initial and nitritation stages was 5.5 ± 0.3% and 1.3 ± 0.1%, respectively, indicating

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that the N2O emitted from the former stage was 4.2-fold that from the latter stage.

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al.28 also found that N2O emission in a nitrification-denitrification reactor with the nitrite pathway established

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by sludge treatment with FNA did not increase in comparison to that from a control reactor without nitrite

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accumulation.

In the literature, Wang et

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Apart from the N2O emission factor, the N2O profiles in the two stages also exhibited different shapes.

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In the initial stage, the N2 O emission peak occurred around the end of the feeding period (Figure 1A-II).

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However, the peak of N2O emission appeared at the beginning of the feeding period in the nitritation stage

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(the total N2O emission in the peak period represented 0.3% of the total influent nitrogen, Figure 1B-II). In

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fact, the shapes of the gaseous N2O concentration profiles in the first 30 min in the two cases are very similar

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(a small peak was also observed in the initial stage, accounting for 0.2% of the total influent nitrogen, Figure

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1A-II), suggesting that they were likely caused by the same mechanism. We hypothesize that the initial sharp

200

rise in N2O emission was due to the stripping of the N2O accumulated during the proceeding settling and

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decanting phases. Although the dissolved N2O data showed little increase (Figure 1B-II) or even a slight

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decrease in N2O concentrations in these phases, it should be noted that the sensor was placed in the clear water

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zone and therefore did not measure the N2O concentration in the sludge blanket, where N2O production was

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expected to occur due to e.g. continued AOB metabolism and endogenous heterotrophic denitrification.29

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the nitritation stage (Figure 1B-II), the gaseous N2O concentration decreased consistently following the initial

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peak. In contrast, the gaseous N2O concentration in the initial nitrification stage continued to increase after a

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minor drop following the first peak. The substantial difference between the two stages following the initial

208

peak was likely caused by the different N2O production rates by AOB in the two cases. As a minor

209

observation, there appeared to be a secondary N2O peak in the gaseous N2O concentration profile in the

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nitritation stage. This was presumably caused by the cessation of liquid volume increase in the reactor.

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During the feeding period, the liquid ‘hold up’ of N2O increased with the liquid volume increase, causing an 9


In


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N2O emission rate that is lower than the N2O production rate. Such an N2O sink does no longer exist after

213

feeding.

214

1A-II), this would be much less pronounced due to a much N2O emission rate. Hence, such a peak was not

215

visible in Figure 1A-II. The experiments performed in this work therefore focus on the likely mechanisms

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responsible for the decreased N2O emissions in the FNA-established nitritation stage.

217

Mechanisms of Decreased N2O Emission in the FNA-Established Nitritation Stage. As mentioned in the

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“Materials and Methods” section, the FNA-treated sludge was returned into the mainstream reactor manually

219

at the nitritation stage. Although both the gas and liquid N2O in the normal cycle showed similar profiles to

220

those in the cycle with FNA-treated sludge return, the N2O emitted from the latter was lower (Figure 2). It

221

was calculated that the N2O emission factor was 1.33 ± 0.07% in the former whereas the corresponding value

222

was 1.15 ± 0.06% in the latter, suggesting that the returned FNA-treated sludge reduced N2O emission.

While such an effect should have also affected the N2O profile in the nitrification cycle (Figure

50

0.5 Cycle II


40

0.4 Sludge withdrawn at aerobic end and replaced with FNA-treated sludge

30

0.3 Gas phase

20

0.2

Liquid phase

10

0.1

0

0 0

60

120

180

240

300

360 420 Time (min)

480

540

600

660

720

223 224

Figure 2. Cyclic N2O profiles in the mainstream reactor before (Cycle I) and after (Cycle II) the return of

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the FNA-treated sludge (DO: 2.5-3.0 mg/L).

226

Two major differences between the normal cycle (i.e., Cycle I in Figure 2) and the cycle with

227

FNA-treated sludge return (i.e., Cycle II in Figure 2) are: 1) nitrite level in Cycle II was higher than that in

228

Cycle I due to the high nitrite level contained in the FNA-treated sludge (Figure S1, Supporting Information);

229

and 2) a part of sludge in the mainstream reactor was replaced by the same amount of FNA-treated sludge

230

(Figure 2). Therefore, the following mechanism studies mainly focused on these two points.

231

To investigate whether FNA treatment caused changes of the nitrite impact on N2O emission, the effect of

10


Liquid phase N 2O (mg N/L)

Cycle I

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different nitrite levels on N2O emission factor at the steady-state of the initial stage and the nitritation stage

233

under the same DO level (2.5-3.0 mg/L) was first studied. It was found that the N2O emission factor

234

increased with increasing nitrite concentration before FNA treatment was implemented (i.e., in the initial stage,

235

Figure 3A).

236

4.5 ± 0.3 to 6.5 ± 0.4%, which was in agreement with other reports.14

237

stage, the N2O emission factor showed a different response to nitrite level.

238

~6 to 16 mg/L the N2O emission factor increased from 0.97 ± 0.06 to 1.50 ± 0.09%. However, further

239

increase of nitrite did not increase but decreased the N2O emission factor. It appears that FNA treatment

240

caused a threshold shift of nitrite stimulation on N2O emission, with the highest N2O emission factor

241

appearing around 16 mg/L of nitrite after FNA treatment.

With the increase of nitrite level from ~6 to ~56 mg/L, the N2O emission factor increased from In the FNA-established nitritation With the increase of nitrite from

242 243

Figure 3. Effect of nitrite level on N2O emission factor at steady-state in the initial stage (A) and the

244

nitritation stage (B) under the same DO level (2.5-3.0 mg/L).

245

It should be noted that the N2O emission factor in the nitritation stage was much lower than that in the

246

initial stage at all nitrite levels (Figure 3). Moreover, the highest N2O emission factor was 1.50 ± 0.1%

247

(determined at ~16 mg/L nitrite) in the nitritation stage while the lowest one reached 4.5 ± 0.3% (determined

248

at ~6 mg/L nitrite) in the initial stage. All these results indicate that FNA treatment largely inhibited the N2O

249

emission.

250

specific N2O production rate, and the results are shown in Figure 4.

251

production rate measured in both stages showed similar patterns with the N2O emission factor against nitrite.

252

In the initial stage, the specific N2O production rate increased from 2.61 ± 0.11 to 3.57 ± 0.30 mg N/h/g VSS

To express the N2O emission more accurately, N2O emission was also transformed to biomass It can be seen that the specific N2O

11



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with the increase of nitrite from ~6 to ~56 mg/L. In comparison, the highest specific N2O production rate

254

was measured as 0.87 ± 0.07 mg N/h/g VSS at ~16 mg/L nitrite in the nitritation stage, and further increases

255

of the nitrite level decreased the biomass specific N2O production rate. Also, the biomass specific N2O

256

production rate in the nitritation stage was much lower at all nitrite levels, compared with that in the initial

257

stage.

258 259

Figure 4. Effect of nitrite level on biomass specific N2O production rate at steady-state in the initial

260

stage (A) and the nitritation stage (B) under the same DO level (2.5-3.0 mg/L). Table 1. Batch Results of N2O Emission Factor and Biomass Specific N2O Production Rate before and after FNA Treatment under the same DO level (2.5-3.0 mg/L) a N2O emission factor

Biomass specific N2O production rate

(%)

(mg N/h/g VSS)

Average nitrite level (mg/L)

Before treatment

After treatment

Before treatment

After treatment

~6

0.97 ± 0.06

0.45 ± 0.05

0.50 ± 0.04

0.24 ± 0.04

~16

1.50 ± 0.09

0.72 ± 0.07

0.87 ± 0.07

0.42 ± 0.04

~26

1.10 ± 0.07

0.52 ± 0.04

0.55 ± 0.08

0.26 ± 0.05

~36

0.91 ± 0.04

0.41 ± 0.05

0.48 ± 0.03

0.24 ± 0.03

~46

0.95 ± 0.05

0.46 ± 0.06

0.51 ± 0.04

0.25 ± 0.03

~56

0.90 ± 0.06

0.43 ± 0.04

0.49 ± 0.05

0.24 ± 0.04

a

Biomass used was taken from the mainstream reactor at the steady-state nitritation stage.

Results are the averages and standard

deviations from triplicate tests.

261

The above analysis revealed that the N2O emission was reduced by FNA treatment. To verify this

262

directly, we further compared the N2O emission at different nitrite levels before and after FNA treatment using

263

the biomass withdrawn from the mainstream reactor in the steady-state nitritation stage. Table 1 summarizes 12


Page 13 of 23


264

the results of the batch tests. It was found that both the N2O emission factor and the biomass specific N2O

265

production rate significantly decreased by FNA treatment (p < 0.05) at all nitrite levels investigated. FNA

266

treatment implemented in this study not only resulted in the shift of nitrite stimulation threshold on N2O but

267

also severely inhibited both the N2O emission factor and the biomass specific N2O production rate. This

268

explains why the FNA-established nitritation stage showed decreased N2O emission, despite of the substantial

269

nitrite accumulation.

270

To reveal the details of how FNA treatment reduced the N2O production, changes of nitrogen compounds,

271

N2O, soluble COD, and total COD in the FNA treatment unit with the time of FNA treatment were also

272

determined (Table S2, Supporting Information). It was found that no significant variation was determined in

273

ammonium, nitrite, and nitrate at any time investigated (p > 0.05). Similar observation was also made on

274

total COD variation. These results suggested that both the nitrification and denitrification were severely

275

inhibited by the FNA treatment.

276

when FNA was added into the unit (Figure S2, Supporting Information). Afterwards, it maintained at very

277

low levels (0-0.1 mg N/L) during the remainder of the treatment period, indicating N2O emission from the

278

FNA treatment reactor was negligible as this reactor was not aerated.

279

presence of FNA heavily inhibited the enzymes relevant to N2O production from both nitrification and

280

denitrification.

281

Liquid N2O in the FNA treatment unit sharply decreased in the first 2 h

All these facts indicate that the

It has been reported that FNA can significantly affect enzymes of microbes especially the nitrite

282

reductase NirK, the enzyme responsible for N2O production.30

283

microorganisms to turn on or off these particular enzymes to defend against its toxicity. 30

284

FNA may directly react with the enzymes as well. 30

285

homotrimeric structure with two distinct Cu-binding sites.31

286

copper-containing enzymes.30

287

change, which might be the reasons for the decreased N2O production. In the literature, it was reported that

288

FNA was the main environmental parameter that controlled the expression of NirK in N. europaea., and the

289

level of NirK at higher FNA concentrations was lower than that at lower FNA concentrations.30,32

290

FNA treatment unit, it was observed that soluble COD increased with the FNA treatment time (Table S2,

291

Supporting Information), possibly due to cell lysis and disruption of extracellular materials.33,34

On one hand, FNA may force On the other hand,

NirK is a copper-containing enzyme, which folds a FNA could bind to the active sites of

As a result, NirK may be either inactivated or undergone a conformational

13


In the

It is known


Page 14 of 23

292

that nitrification and denitrification relevant enzymes such as NirK are cell membrane-bound enzymes which

293

are protected by extracellular polymeric substances.

294

by the biocidal effect of FNA, membrane-bound enzymes are vulnerable to FNA.

295

Effect of FNA Treatment on Microbial Community. The structure of microbial community and the

296

abundance of functional bacteria are relevant to N2O production. Thus the microbial community in the

297

initial and the FNA-established nitritation stages was compared by using the amplicon sequencing. The

298

bacterial sequences in the initial stage were much higher than those in the FNA-established nitritation stage

299

(Figure S3, Supporting Information), suggesting that FNA treatment reduced the bacterial diversity.

300

known that FNA has a strong biocidal effect on microorganisms in activated sludge at parts per million levels.

301

Pijuan et al.35 showed that sludge treatment with 1-2 mg N/L of FNA for 24−48 h killed 50-80% of the cells in

302

sludge, which was consistent with the results obtained in this work.

When extracellular polymeric substances are disrupted

303

304

14


It is

Page 15 of 23


305

Figure 5. Genus level distributions of bacterial populations at steady-state of the initial stage (A) and

306

nitritation stage (B) under the same DO level (2.5-3.0 mg/L).

307

Figure 5 illustrates the genus level distributions of bacterial populations in the two stages. In the initial

308

stage, three types of AOB, namely Nitrosomonas sp., Nitrosospira sp., and Nitrosovibrio sp. were detected,

309

which accounted for 26.7%, 2.5%, and 0.5% of the total bacterial sequences, respectively. The genus of

310

Nitrosomonas was the dominant AOB at this stage, accounting for 89.9% of the total AOB. In the nitritation

311

stage, Nitrosovibrio sp. was completely washed out from the reactor.

312

Nitrosospira sp. decreased to 0.7% of the total bacterial sequences whereas the amount of Nitrosomonas sp.

313

increased to 34.8% of the total bacterial sequences.

314

Nitrosomonas sp. accounted for 98.2% of the total AOB. The results indicated that Nitrosomonas sp. could

315

tolerate the biocidal effect from FNA treatment at 1.82 mg N/L whereas Nitrosospira sp. and Nitrosovibrio sp.

316

seemed to be more sensitive to the FNA toxicity. All the three types of AOB were documented to be able to

317

produce N2O, but their production abilities were different.36-38

318

production rate of Nitrosospira sp. was 2 amol/cell/h whereas the corresponding value of Nitrosomonas sp.

319

reached 58 amol/cell/h.

320

Nitrosomonas sp. was > 9-fold higher than that of Nitrosovibrio sp..38

321

reports, it could be postulated that the initial stage should produce less N2O in comparison to the nitritation

322

stage since the latter had a higher abundance of Nitrosomonas sp..

323

stage was 4.2-fold of that in the latter stage (Figure 1). This paradox suggested that the N2O production by

324

Nitrosomonas sp. in the nitritation stage was severely inhibited by FNA treatment.

Meanwhile, the abundance of

This suggested that the relative abundance of

Shaw et al.37 showed that the aerobic N2O

Remde and Conrad demonstrated that the aerobic N2O production rate of According to the previous literature

However, the N2O emitted in the former

325

From Figure 5, it can also be seen that Nitrospira sp. and Nitrobacter sp., the main NOB present in

326

WWTPs, accounted for 6.6% and 5.0%, respectively, of the total bacterial sequences in the initial stage.

327

FNA treatment completely eliminated Nitrospira sp. and significantly reduced the abundance of Nitrobacter

328

sp. to 3.3%, which was the reason for the substantial accumulation of nitrite obtained at the nitritation stage.

329

Apart from nitrifiers, there are also a lot of other bacteria detected in both stages.

330

Lysobacter, Pseudomonas, and Algoriphagus were determined in both stages. Previous publications pointed

331

out that these bacteria could produce and secrete proteases.39-41

332

degrade proteins, which is the main component of soluble microbial products generated during biomass

For example, the genera of

These proteases are important enzymes to

15



Page 16 of 23

333

growth and decay.42

334

though no organic carbon source was included in the synthetic medium. In the initial stage, four types of

335

heterotrophic denitrifiers, Acidovorax sp.,43 Rhodanobacter sp.,44 Ottowia sp.,45 and Stenotrophomonas sp.,46

336

were found in the mainstream reactor. However, this number increased to seven in the nitritation stage

337

(Figure 5). Moreover, the relative abundance of denitrifiers also increased from 17.1% (the initial stage) to

338

34.5% (the nitritation stage). It can therefore be suggested that denitrifiers in the nitritation stage contributed

339

more to N2O emission than those in the initial stage. It was reported that FNA treatment accelerated the

340

disruption of extracellular polymeric substances as well as cell envelop.33

341

substrates would be released for the growth of denitrifiers (Table S2, Supporting Information), which may be

342

the reason for the increased abundance of denitrifiers.

343

N2O Emission under DO Level of 0.3-0.8 mg/L. As shown in Figure 5B, although Nitrospira sp. was

344

completely washed out by FNA treatment, there was still 3.3% of Nitrobacter sp. present in the mainstream

345

reactor. It is reported that Nitrobacter sp. are r-strategists with a low oxygen affinity, suggesting that low DO

346

levels are beneficial for its elimination.19

347

stimulated N2O emission.13,14

348

increased from 2.4 ± 0.1% to 10.6 ± 1.7% as DO concentration decreased from 3.0 mg/L to 0.2 mg/L. Thus,

349

the N2O emission in the FNA-established nitritation reactor under a lower DO level (i.e., 0.3-0.8 mg/L) was

350

finally determined.

The degraded substrates can be supplied for the growth of heterotrophic bacteria,

As a result, more soluble

However, previous publications showed that low DO levels

For example, Peng et al.13 demonstrated that the N2O emission factor

Table 2. Performance of the Mainstream Reactor in Steady-state Operation of Nitritation Phases under

the DO Level of 0.3-0.8 mg/L a N2O emission factor (%)

3.3 ± 0.1

Effluent NH4+-N (mg/L)

0.88 ± 0.17

Effluent NO2--N (mg/L)

43.6 ± 1.1

Effluent NO3--N (mg/L)

10.8 ± 0.6

Effluent soluble COD (mg/L)

6±1

TSS (mg/L)

171 ± 5

VSS (mg/L)

157 ± 6

a

Results are the averages and standard deviations from triplicate measurements.

16


Page 17 of 23


351

Table 2 summarizes the performance of the mainstream reactor at the DO level of 0.3-0.8 mg/L. It can

352

be seen that the decrease of DO concentration lead to higher accumulation of nitrite, as expected. During the

353

stable operation, effluent nitrite concentration increased to 43.6 ± 1.1 mg/L while effluent nitrate decreased to

354

10.8 ± 0.6 mg/L. Additionally, ammonium conversion was unaffected by the decrease of DO level. At this

355

DO level, an emission factor of 3.3 ± 0.1% of N2O was measured (cyclic profiles of gas and liquid N2O are

356

shown in Figure S4, Supporting Information).

357

mg/L, the reduced DO level caused an increase in the N2O emission factor. However, the N2O emission

358

factor measured at the low DO level was still much lower than that measured at the initial full nitrification

359

stage (3.3 ± 0.1% vs 5.5 ± 0.3%). Thus, it can be expected that reduced N2O emission from the mainstream

360

deammonification

361

nitrification-denitrification process.

362

Implications for Mainstream Deammonification. To date, all the previous studies have shown that the

363

increase of nitrite in domestic-strength range (e.g., 0-50 mg N/L) would cause increasing emission of N2O by

364

AOB.1,2,12,14

365

domestic-strength nitrifying culture with the increased nitrite level using FNA treatment, which fill a

366

recognition gap relevant to N2O production by AOB. This was experimentally demonstrated by use of a

367

nitrifying reactor treating synthetic wastewater containing domestic-strength ammonium under two DO levels.

368

Although previous investigations found that FNA present in wastewater bioreactors stimulated N2O emission

369

in both nitrifying and denitrifying cultures,24,25 this work revealed that sludge treated by FNA has a lower N2O

370

production rate.

process

can

be

Compared with the nitritation stage at a DO level of 2.5-3.0

achieved,

as

compared

with

that

from

the

conventional

This study reveals for the first time that reduced N2O emission can be achieved from a

371

The finding obtained in this work may have important implications to the operation of future WWTPs.

372

With the growing energy crisis worldwide, WWTPs are increasingly considered as facilities for energy

373

recovery rather than merely for waste removal,47 which leads to an ongoing paradigm shift in the operation of

374

WWTPs from waste removal to both nutrient removal and energy recovery.

375

requires much less energy, as compared with the conventional nitrification-denitrification technology,

376

enabling maximal carbon source recovery and desirable nutrient removal concurrently. Thus, it is widely

377

considered the most promising technology for WWTPs in future,19-21 attracting much attention recently.

378

Since domestic sewage usually contains ~50 mg/L ammonium, nitrite accumulation in this range is inevitable 17


Mainstream deammonification


Page 18 of 23

379

in the nitritation phase. Although both the nitritation and Anammox processes can produce N2O, the former

380

is a more important contributor.23,48

381

mainstream deammonification process is expected to be much higher in comparison to that from the

382

conventional nitrification-denitrification process, which raises concerns. The FNA-based N2O mitigation

383

strategy, as demonstrated in this work, potentially mitigates this concern. Based on the results obtained in

384

this study, 53.8 t (50 mg/L × 105 m3/d × 0.5 × 5.9% × 365 d) N2O will be emitted per year from the nitritation

385

phase of a mainstream deammonification WWTP (Q: 105 m3/d; ammonium: ~50 mg/L) if 50% conversion of

386

influent ammonium is achieved by conventional methods.

387

× 105 m3/d × 0.5 × 1.1% × 365 d) if the 50% ammonium conversion is achieved by the FNA treatment based

388

approach, as introduced in the current work.

389

emission would result in an increase of 30% in the carbon footprint of wastewater treatment.2

390

huge municipal wastewater daily treated worldwide, this FNA-based N2O mitigation method have significant

391

benefits.

According to the current understandings, N2O emission from the

However, this value will be only 10.0 t (50 mg/L

It should be emphasized that an increase of 1% in N2O Due to the

392

Extensive efforts have been dedicated to mitigating N2O emission from WWTPs, but almost all the

393

strategies proposed have been developed for the conventional nitrification-denitrification process and

394

sidestream deammonification.1,2

395

mitigating N2O emission from the mainstream deammonification process so far, largely due to the fact that

396

mainstream deammonification is a recently proposed investigation. This work therefore fills this technology

397

gap and provides for engineers a technical reserve for future operation of WWTPs.

398

waste-generated, renewable compound that can be in-situ produced in WWTPs as a byproduct of wastewater

399

treatment by nitritation of the anaerobic digestion liquor, which makes it easily obtained in WWTPs.49

400

using this chemical to treat sludge, the number of NOB is reduced, the growth of AOB is retained, and the

401

N2O emission by AOB is reduced. This implies that the FNA-based N2O mitigation method is not only

402

economically viable but also technically feasible for mainstream deammonification.

403

has so far only been tested at bench-scale. Its technical performance and economic evaluation require to be

404

further evaluated at real-world situations in the future.

405

ACKNOWLEDGEMENTS

To the best of our knowledge, no effective method has been documented for

18


FNA is a

By

However, this strategy

Page 19 of 23


406

The authors acknowledge the Australian Research Council for funding support through Discovery Project

407

DP120102832. Dr. Qilin Wang acknowledges the support of Australian Research Council Discovery Early

408

Career Researcher Award (DE160100667) and the Philanthropic Grant for Early Career Engineering

409

Researcher (GE12015).

410

ASSOCIATED CONTENT

411

Supporting Information Available

412

This file contains Tables S1-S2 and Figures S1-S4.

413

Table S1: Performance of the mainstream reactor at steady-state in the initial stage and the nitritation

414

stage under the same DO level (2.5-3.0 mg/L). Table S2: Variations of total COD, soluble COD, ammonium,

415

nitrite, and nitrate in the FNA treatment reactor.

416

Figure S1: Cyclic profiles of nitrogen compounds in the mainstream reactor before (Cycle I) and after

417

(Cycle II) the FNA-treated sludge was returned (DO: 2.5-3.0 mg/L). Figure S2: Representative profile of

418

liquid N2O in the FNA treatment unit reactor. Data were measured at steady-state nitritation stage (DO:

419

2.5-3.0 mg/L). Figure S3: The shift of microbial diversity from the initial phase (on the right) to the nitritation

420

phase, which shows a greater reduction in the microbial population (A), and the details of microbial

421

composition (B). Figure S4: Cycle profile of N2O at steady-state nitritation stage under the DO level of

422

0.3-0.8 mg/L. A: Gas phase; B: Liquid phase.

423

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Reducing N2O Emission from a Domestic-Strength Nitrifying Culture

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