Iron Robustly Stimulates Simultaneous Nitrification and

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Iron Robustly Stimulates Simultaneous Nitrification and Denitrification Under Aerobic Conditions Hong Chen, Xuhao Zhao, Yuying Cheng, Mingji Jiang, Xiang Li, and Gang Xue Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04751 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Environmental Science & Technology

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Iron Robustly Stimulates Simultaneous Nitrification and

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Denitrification Under Aerobic Conditions

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Hong Chen1,2, Xuhao Zhao1, Yuying Cheng1, Mingji Jiang1, Xiang Li1,2, Gang Xue*

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(1School of Environmental Science and Engineering, Donghua University, 2999 North

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Renmin Road, Songjiang District, Shanghai, 201620, China

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2

Jiangsu Tongyan Environm Prod Sci & Technol Co Lt, Yancheng, 224000, China)

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*Corresponding author

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E-mail: [email protected]

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Tel: 86-21-67792537

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Fax: 86-21-67792522

1

ACS Paragon Plus Environment


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Abstract

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Simultaneous nitrification and denitrification (SND) is a promising single-reactor biological

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nitrogen-removal method. Activated sludge with and without iron scrap supplementation

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(Sludge-Fe and Sludge-C, respectively) was acclimated under aerobic condition. The total

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nitrogen (TN) content of Sludge-Fe substantially decreased from 25.0±1.0 to 11.2±0.4 mg/L, but

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Sludge-C did not show the TN-removal capacity. Further investigations excluded a chemical

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reduction of NO3--N by iron and a decrease of NH4+-N by microbial assimilation, and the

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contribution of SND was verified. Moreover, the amount of aerobic denitrifiers, such as bacteria

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belonging to the genera Thauera, Thermomonas, Rhodobacter and Hyphomicrobium, was

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considerably enhanced, as observed through Miseq Illumina sequencing method. The activities of

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the key enzymes ammonia monooxygenase (AMO) and nitrite oxidoreductase (NXR), which are

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associated with nitrification, and periplasmic nitrate reductase (NAP) and nitrite reductase (NIR),

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which are related to denitrification, in Sludge-Fe were 1.23-, 1.53-, 3.60- and 1.55-fold higher

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than those in Sludge-C, respectively. In Sludge-Fe, the quantity of the functional gene NapA

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encoding enzyme NAP, which is essential for aerobic denitrification, was significantly promoted.

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The findings indicate that SND is the primary mechanism underlying the removal of TN and that

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iron scrap can robustly stimulate SND under aerobic environment.

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Introduction

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Nitrogen is one of the main causes of waterbody degradation because excess nitrogen loading

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to waterways leads to increased eutrophication and associated water quality impacts.1, 2 To reduce

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the amount of nitrogen entering waterbodies, nitrogen removal from wastewater has become a

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focus of wastewater treatment plants (WWTP), and biological removal methods using activated

2


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sludge are often applied because of their low cost and high efficiency.

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In traditional biological treatment processes, nitrogen removal primarily occurs in separate

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anoxic and aerobic phases and is generally conducted in separate bioreactors or by different

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aeration intervals. With the observation of dissimilative nitrogen loss during the aerobic stage in

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the wastewater treatment process, the nitrification and denitrification can occur concurrently in a

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single reactor under aerobic conditions, which is often referred as simultaneous nitrification and

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denitrification (SND) process. In recent years, the discovery of aerobic denitrifiers can explain the

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SND process from microbial perspective, and many bacteria strains owing aerobic denitrification

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capacity are isolated and characterized. Aerobic denitrifiers have promising potential for nitrogen

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removal in a single aerobic reactor. It was reported that the coal-based ethylene glycol industry

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wastewater with high concentrations of nitrate and nitrite could be treated when an aerobic reactor

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was bioaugmented with a Pseudomonas strain;3 A continuous-upflow submerged biofilter

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inoculated with Pseudomonas stutzeri X31 worked effectively to remove nitrate under an aerobic

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atmosphere;4 When bioaugmented with the aerobic denitrifier Microvirgula aerodenitrificans, the

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nitrate produced during the aerobic nitrifying-phosphate uptake phase was reduced concurrently;5

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Piggery wastewater treatment using Alcaligenes faecalis strain No. 4 exhibited simultaneous

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aerobic nitrification and denitrification process.5 The approach of bioaugmentation with special

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aerobic denitrifiers shows a total nitrogen removal capacity as mentioned above. However, few

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pilot or full-scale applications have been carried out so far. The likely reason is that the abundance

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of the seeded strain is getting lower with the operation time, causing the nitrogen removal

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efficiency unstable.3 Moreover, the methods currently used for aerobic denitrifiers isolating or

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enriching include intermittent aeration, bromothymol blue (BTB) medium separation and 3



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cultivation using denitrification medium,6 which are difficult to realize in an actual wastewater

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treatment plant for functional strains enriching. Consequently, if a simple method increasing the

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abundance of aerobic denitrifiers in the WWTP itself could be found, the SND would become

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more practicable and reliable in future engineering application.

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During SND process, NH4+-N is successively oxidized to NH2OH, NO2--N and NO3--N via

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catalysis by the enzymes of ammonia monooxygenase (AMO), hydroxylamine oxidoreductase

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(HAO) and nitrite oxidoreductase (NXR), respectively in nitrification process. In the subsequent

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aerobic denitrification stage, the produced NO3--N is catalytically reduced to NO2--N by enzyme

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of periplasmic nitrate reductase (NAP).6 Then, the enzyme nitrite reductase (NIR) catalyzes the

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one-electron reduction of nitrite into nitric oxide, and the enzymes nitric oxide reductase (NOR)

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and nitrous oxide reductase (NosZ) reduce nitric oxide and nitrous oxide, respectively. Notably,

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the membrane nitrate reductase (NAR), which catalyzes the nitrate reduction under anaerobic

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condition, is seriously suppressed by oxygen, while NAP can be expressed under both aerobic and

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anaerobic conditions,7, 8 and it is essential for aerobic denitrification.9 Moreover, the detection of

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NapA gene, which encodes the NAP enzyme, also suggests that aerobic denitrification can occur.10,

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11

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process are summarized in Figure 1.12, 13

In this study, the nitrogen species transformations and the corresponding enzymes in SND

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NO3

-

NosZ

-

NO2

N2 2e-

HAO Hemec

NH3

Heme c

1e

NIR

Fe/S NapA

NapA Fe/S NO2-

-

Cyt c

N2O

NapB

2e-

NO

NH2OH NxrA Fe-S 2H+

NapG 4Fe/S

NxrB Fe-S

UQ Cu/Fe

Tat complex

b UQH2

NapH

UQ 2Fe/S

UQH2

AMO Holo-NapA

Fe/S

4Fe/S NapF

NOR (Fe)

(Fe-S) 2eFAD 2eSuccinate

Apo-NapA NapD

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2H+

2H+

Cyt c1

UQH2

(Fe-S)

UQ

Cyt b

UQH2

2H+

UQ

NADH Dehydrogenase Periplasm (Fe-S)

2H+

FP

2H+ Cyt bc1 Complex

Cytoplasm NADH/H+

NAD+

Fumarate

Electron transfer

Nitrogen species transformation

Proton transfer

Other substances transformation

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Figure 1. The nitrogen species transformations and the relevant enzymes participating in

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nitrification and denitrification.

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It can be seen from Figure 1 that large amounts of metalloproteins (such as cytochrome and

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dehydrogenase) and certain functional nitrification and denitrification enzymes (i.e. AMO, NXR,

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NAP and NOR) contain iron acting as the active center. In addition, other indispensable metal

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element such as copper is contained in enzymes of AMO, NirK, and NosZ.12 Furthermore, iron

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can be used as electron acceptor in the electron transport chains and prosthetic groups of

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oxidoreductases, thereby enhancing the metabolic activity of the microbial community.

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Researchers have found that the iron plays active role in bacteria for nitrogen utilization or

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removal. The isolated strains such as Klebsiella pneumoniae, Pseudomonas aeruginosa and

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Bacillus licheniformis can use the NH4+-N and NO3--N concurrently in aerobic condition, and the

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removal efficiency was significantly promoted after Fe2+ was added to the medium.9, 14, 15 When

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using Paracoccus versutus LYM to simultaneously remove NH4+-N and NO3--N, some NO2--N

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was left during the SND process, and Fe2+ adding benefited bacterial growth and NO2--N

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reduction.16 After growing Paracoccus pantotrophus P16 in medium supplemented with 1.5 uM

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Fe3+, the enzymatic activity of nitrate and nitrite reductase showed a 2.6- and 1.7- fold increase.17

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In addition, adding Fe3+ of 50 mg/L obtained a considerable increasing in TN and NH4+-N removal 5



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during the nitrification-denitrification process in a submerged membrane bioreactor.18 In our

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previous study, it was confirmed that iron scrap could improve the nitrification efficiency in the

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aerobic stage and the dissolved ferric ions in the nitrification liquid were beneficial for the

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subsequent anoxic denitrification process during the advanced nitrogen removal treatment from

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dyeing wastewater.19 Ma found that iron shaving pretreatment could remarkably enhance the

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organic pollutants and nutrient removal efficiency in the following biological treatment system.5

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Therefore, the iron supplementation could enhance the inherent nitrogen removal capacity of the

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bacteria strains. Figure 1 revealed that the NAP system of periplasmic nitrate reduction consists of

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several proteins rich in Fe/S cluster. Moreover, NAP system can be found almost extensively in

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the phylum proteobacteria,12 which is commonly detected in WWTP under both aerobic and

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anaerobic conditions. In general, the aerobic system in WWTP presents nitrification. When iron is

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added in the aerobic nitrification system, whether the iron supplementation affects the aerobic

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nitrification performance? Can the enzymes relevant to denitrification especially NAP be

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stimulated by iron and then initiate the aerobic denitrification, making SND occur in its original

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aerobic nitrification system? Furthermore, what is the role of iron in this process if SND was

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started successfully?

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In this study, the probable effect of iron on SND stimulation was investigated. The easily

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available and low-cost iron scrap was supplemented to a universal aerobic activated sludge system,

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and the nitrogen removal performance was detected. After acclimation for several days, it was

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found amazingly that the system with iron supplementation showed TN removal ability, whereas

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the control system without iron addition only performed nitrification. Therefore, the TN removal

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behavior and primary factors underlying its removal efficiency were discussed. Then, the 6


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mechanisms of TN removal induced by iron were interpreted based on the possibilities of NO3--N

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chemical reduction with iron, the NH4+-N utilization by microbe assimilation, and the verification

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of SND process. Finally, the shift in microbial community structure was analyzed via Miseq

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Illumina sequencing method. The key enzymatic activity correlated with nitrification and

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denitrification were tested, and the functional genes encoding denitrification enzymes were

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measured via real-time quantitative polymerase chain reaction (qPCR) assays.

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Materials and Methods

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Iron. Iron scrap from lathe cutting waste with lengths of 5~10 cm and a spiral bending shape

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was used in this study (Figure S1, see Supporting Information (SI)). Before the experiment, the

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iron scrap was immersed and washed in NaOH (0.2 M) for 24 h to remove the surface oil. After

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the iron scrap was dissolved by nitric acid, its composition was measured by inductively coupled

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plasma optical emission spectrometry (ICP-OES, PerkinElmer Optima 2100 DV, USA), and the

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results are shown in Table S1 in SI.

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Set-up and Operation of the Parent Reactor. Reactors (Reactor-C and Reactor-Fe) with inner

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pore plate and working volumes of 1 L were used (Figure S2 in SI). The seeding activated

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sludge, obtained from the Songjiang WWTP in Shanghai, was centrifuged and washed three times

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using 0.9% NaCl solution and then added to the Reactor-C and Reactor-Fe along with a mixed

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liquid suspended solids (MLSS) concentration of approximately 3000 mg/L. According to the

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results of our previous study,19 the 60 g/L of iron scrap was put on the inner pore plate of

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Reactor-Fe to avoid the influence of mixture stirring. Reactor-C without iron adding was set as the

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control. During the operation period, synthetic wastewater (described in SI) was used. Briefly, the

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influent COD using acetic acid was 300 mg/L, and the initial NH4+-N or TN concentration was 25

7



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mg/L. The pH value was adjusted to 7.5 using 2 M NaOH or 2 M HCl. The reactors were

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maintained in room temperature and constantly mixed using magnetic stirrer with stirring rate of

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700 r/min except during the settling and decanting period. Air was provided to the reactors by an

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aerator using an on/off control system and the dissolved oxygen (DO) concentration was

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maintained at around 3 mg/L. During the acclimation period, one reaction cycle was set as 24 h.

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After 22 h aeration period, the mixture was settled for 1 h, the supernatant of 750 mL was

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discharged for 5 min and 55 min idle period was followed. Then fresh synthetic wastewater of 750

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mL was supplemented during the initial 5 min of next cycle, leading a hydraulic retention time

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(HRT) of 30.6 h. At the end of aerobic stage of one cycle every day, sludge mixture of 100 mL

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was discharged, and the sludge retention time (SRT) was 10 d. The nitrogen removal of these two

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reactors was determined every day during the acclimation period. Reactor-C and Reactor-Fe were

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constructed in triplicate. After the reactors reached a stable nitrogen removal performance, the

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sludges from Reactor-C and Reactor-Fe were named as Sludge-C and Sludge-Fe, and were used

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for further investigation.

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Batch Experiments for Optimal Operation Conditions Determination. Batch experiments

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using Sludge-Fe were conducted to find the optimal operation condition from the aspects of pH

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value, DO concentration and C/N ratio. When considering the optimal pH value for TN removal,

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Mixture of 400 mL withdrawn from parent Reactor-Fe before the end of aerobic stage was

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centrifuged at 4000 rpm for 5 min, washed with 0.9% NaCl solution for 3 times, and resuspended

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in 100 mL of 0.9% NaCl solution before being divided into 4 batch reactors. The 25 mL of the

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resuspended sludge and 25 mL synthetic wastewater (containing 0.5 mL concentrated solution,

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0.027 mL trace-element solution and 0.027 mL acetic acid, see SI) were fed into each reactor. 8


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Distilled water was then added to make the final volume of the mixture in each reactor to be 100

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mL, resulting in the influent COD and NH4+-N concentrations were 300 and 25 mg/L, respectively.

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Before experiment, the initial pH values were adjusted to 6, 7, 8 and 9 by using 4 M NaOH or HCl.

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During the experiment, the DO concentration was maintained at 3 mg/L as parent reactors, and the

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mixture was constantly stirred. The TN concentration was detected every hour, and the optimal pH

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was determined based on the TN removal efficiency. Similarly, the DO concentrations were

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controlled as 1, 2, 3, 4 and 5 mg/L in different reactors when taking account of the optimal DO

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value, meanwhile the pH value was set as the optimal one, and the COD and NH4+-N were set as

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300 and 25 mg/L, respectively. Based on the optimal pH value and DO concentration, setting the

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NH4+-N concentration as 25 mg/L, changing the initial acetic acid dosages to make the C/N ratios

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as 0, 4, 8, 10, 12, 14, 16 and 18, the optimal C/N ratio was determined.

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Other Analytical Methods. The measurements of enzymatic activities of AMO, NXR, NAP and

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NIR are described in SI. The analysis of the microbial community was conducted by Illumina

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MiSeq sequencing technique for it can generate a large number of sequences. The samples for

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sequencing were withdrawn from triplicate reactors and mixed completely to homogenize the

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samples for DNA extraction and microbial analysis (in SI). The real-time qPCR assay of the

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denitrification-related genes and the specific oxygen uptake rate measurements for Sludge-C and

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Sludge-Fe were performed according to the literature with modifications (see SI).20 The COD,

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NH4+-N, NO3--N, NO2--N, TN, MLSS and mixed liquid volatile suspended solid (MLVSS)

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analyses were conducted according to standard methods.21 The amounts of Fe3+ and Fe2+ were

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determined by the phenanthroline spectrophotography method using a UV-vis spectrophotometer

180

(PerkinElmer, Lambda 35). 9



All tests were performed in triplicate, and the results are expressed as the

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Statistical Analysis.

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mean ± standard deviation. An analysis of variance (ANOVA) was performed to test the

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significance of the results, and p0.05), implying that the contribution of assimilation to TN removal

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in the Sludge-Fe system could be ignored. Because the assimilation and SOURs had no obvious

323

difference, the cell growth rate and the biomass concentration of Sludge-Fe and Sludge-C should

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be similar when using NH4+-N as nitrogen source. However, after finishing the domestication

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process, the MLSS and MLVSS of Sludge-Fe were 4320±122 and 2600±85 mg/L, which were

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litter higher than those in Sludge-C, with MLSS and MLVSS of 3560±120 and 2350±60 mg/L. 17



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Here, the TN removal occurred in a single reactor (Reactor-Fe) during the aerobic stage, which

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meant that nitrification and denitrification might occur simultaneously, and NO3--N could be used

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for assimilation as well by Sludge-Fe during denitrification process, causing the biomass

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concentration of Sludge-Fe to be higher. Therefore, SND might have been initiated in the

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Sludge-Fe system, and it was further demonstrated as following.

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Contribution of SND to TN Removal. To verify the NO2--N or NO3--N can be denitrified by

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Sludge-Fe, NO2--N or NO3--N at concentration of 25 mg/L was used to replace the NH4+-N in the

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influent, and their concentrations with the reaction time were detected. 30 -

Using NO2 -N as influent

Concentration (mg/L)

25

-

-

NO2 -N

NO3 -N

-

Using NO3 -N as influent

20

-

NO3 -N

15 10 5 0 0

1

2

3

4

5

6

Time (h)

335 336

Figure 6. Concentration variation of NO2--N and NO3--N with reaction time. Error bars represent

337

the standard deviations of triplicate tests.

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As shown in Figure 6, when using NO3--N as the influent, the concentration decreased with

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the time, demonstrating that the NO3--N could be directly denitrified. As to NO2--N, 25 mg/L of

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NO2--N was totally removed and approximate 13 mg/L NO3--N was produced. It could be

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speculated that the NO2--N could be directly reduced, and then the remaining NO2--N was

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oxidized to NO3--N; additionally, the NO2--N could also be initially oxidized to NO3--N and then

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the produced NO3--N was denitrified. Both two possible pathways could totally remove the 18


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NO2--N and cause the final NO3--N concentration to decrease. Since the NO2--N could be easily

345

oxidized to NO3--N in both pathways, it was more likely that the aerobic denitrification began with

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NO3--N reduction, and the enzyme discussion below would further demonstrate it. Moreover, the

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experiment using NO3--N and NO2--N as influent was conducted using Sludge-C, and the results

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indicated that almost all the influent NO2--N was transformed to NO3--N, and the influent NO3--N

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concentration remained constant during the entire reaction period. In conclusion, nitrification

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occurred in both Sludge-C and Sludge-Fe while denitrification process only appeared in Sludge-Fe,

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which confirmed that SND might be the main reason for the TN removal found in Sludge-Fe.

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Shift in the Microbial Community. Microorganisms play key roles on the nitrogen species

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transformation in biological nitrogen removal process. To further explore the effect of iron scrap

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on microbial structure and verify the contribution of SND to TN removal, a comprehensive

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investigation of the microbial community and the structural differences between Sludge-C and

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Sludge-Fe was conducted using Illumina MiSeq sequencing technique. The results showed that

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the microbial diversity and richness of the Sludge-Fe system were slightly higher than those of the

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Sludge-C system (Table S4, SI). Twenty-two phyla were detected in both reactors, and there were

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8 primary ones including Proteobacteria, Bacteroidetes, Planctomycetes, Acidobacteria,

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Nitrospirae, Chlorobi, Armatimonadetes and Firmicutes, which accounted for 98.5% (Sludge-C)

361

and 98.6% (Sludge-Fe) of the total reads (shown in Figure S6, SI). It was reported that aerobic

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denitrifiers mainly belonged to Proteobacteria,6 and it was the predominant phylum found in both

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Sludge-C and Sludge-Fe. Although there was no significant difference on phyla level, the structure

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and composition of microbial species were changed remarkably between Sludge-C and Sludge-Fe.

365

Venn analysis showed that only 1562 operational taxonomic units (OTUs), or 34.3% of the total 19



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OTUs (4560, 3% distance), were shared by Sludge-C and Sludge-Fe (Figure S7, SI). Moreover, to

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explain the variation in the nitrogen removal capacity, which was likely attributed to microbes and

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the functional evolution of the microbial community, the abundance of microbes relating with

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denitrification was explored at genus level as shown in Table 1.

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Table 1. Abundance of bacteria in Sludge-C and Sludge-Fe related with nitrification and

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denitrification at genus level Function

Genus

Affiliated phylum

Nitrification

Nitrospira

Denitrification

Abundance (%) Sludge-C

Sludge-Fe

Nitrospirae

0.955

1.774

Dechloromonas

Proteobacteria

2.283

4.576

Thauera

Proteobacteria

1.422

1.945

Thermomonas

Proteobacteria

0.018

1.524

Rhodobacter

Proteobacteria

0.003

0.091

Hydrogenophaga

Proteobacteria

0.039

0.087

Hyphomicrobium

Proteobacteria

0.009

0.015

Thiothrix

Proteobacteria

0.003

0.011

Fusibacter

Firmicutes

0.000

0.121

Flavobacterium

Bacteroidetes

0.009

0.030

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Although both Sludge-C and Sludge-Fe presented high nitrification efficiency, the genus

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Nitrospira relating to nitrification was enhanced in Sludge-Fe (1.774% versus 0.995% in

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Sludge-C). The microbes associated with denitrification, including Dechloromonas,27,

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Thauera,29 Thermomonas,30 Rhodobacter,31 Hydrogenophaga,32 Hyphomicrobium,33 Thiothrix,34

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Fusibacter35 and Flavobacterium,36 were all remarkably increased, and their total amount

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occupied 3.786% of the total bacterial in Sludge-C, while the total proportion increased to 8.4% in

378

Sludge-Fe. Among these denitrification bacteria, Thauera, Thermomonas, Rhodobacter and

379

Hyphomicrobium have been reported to act as aerobic denitrifiers,29-31, 33 and their total abundance

380

increased prominently in the presence of iron scrap (3.575% in Sludge-Fe versus 1.452% in the

381

Sludge-C), especially the abundance of genus Thermomonas remarkably increasing from 0.018% 20


28

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to 1.524%, and genus Rhodobacter improving from 0.003% to 0.091%. Obviously, the microbial

383

community in the Sludge-Fe system was shifted to a direction favoring for denitrification, which

384

was in accord with the TN removal observed in Reactor-Fe.

385

Key Enzymatic Activity and Functional Genes. The iron scrap has been verified to

386

influence the bacteria community structure which was associated with the SND appearance. The

387

biological nitrogen removal is directly related to the bacterial activity, which is commonly

388

reflected and determined by the corresponding enzymes. The observation of aerobic denitrification

389

in Sludge-Fe system implied that denitrification-related enzymes might have been activated and

390

subsequently taken part in denitrification process. The nitrate reduction can be catalyzed by

391

enzyme of NAP or NAR; NAP can be expressed under both aerobic and anaerobic conditions

392

while NAR is inhibited by oxygen.7, 8 Therefore, NAP is essential for nitrate reduction in aerobic

393

denitrification process. Besides NAP, the other enzymes participating in aerobic denitrification are

394

almost the same as those of the anaerobic process, including NIR, NOR and NosZ. In this study,

395

the activities of AMO and NXR, which are the main enzymes responsible for nitrification,37 and

396

NAP and NOR, which are crucial for denitrification, were measured.38

397

As shown in Figure 7(a), when the enzyme activity of Sludge-C was set to 100%, the AMO

398

and NXR activities in Sludge-Fe increased to 123±6% and 153±5%, respectively, suggesting that

399

iron was beneficial for nitrification. However, their nitrification efficiencies had no remarkable

400

difference perhaps because Sludge-C already had the excellent nitrification capacity. With respect

401

to the denitrification enzymes, the iron scrap supplementation increased the NAP activity of

402

Sludge-Fe by 3.60±0.15-fold compared with that of Sludge-C. Meanwhile, the activity of NIR was

403

155±7% higher in Sludge-Fe than that in Sludge-C. The promotion of activities of key enzymes 21



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404

especially the denitrification enzymes directly related with the aerobic denitrification, which

405

showed good agreement with the TN removal in Sludge-Fe. Sludge-C

a

Sludge-Fe

700

Key Genes Relative Quantity (%)

Relative Enzyme Activity (%)

400 350 300 250 200 150 100 50 0

AMO

406

Nitrification

NXR

NAP

NIR

b

Sludge-C Sludge-Fe

600 500 400 300 200 100 0

NapA

NirK

NirS

Denitrification

407

Figure 7. Comparing the enzyme activity (a) and relative quantity of functional genes (b) in

408

Sludge-Fe and Sludge-C. Error bars represent the standard deviations of triplicate tests.

409

It is well-known that the enzymes secreted by denitrifiers are encoded and controlled by the

410

corresponding functional genes. Since the notable difference of these two sludges was that

411

Sludge-Fe had aerobic denitrification ability while Sludge-C had not, the functional genes

412

encoding the denitrification key enzymes were further quantified by real-time qPCR. The

413

detection of the NapA gene, which encodes enzyme NAP responsible for NO3--N reduction, has

414

often been applied as the proof of aerobic denitrification in mixed liquor samples.10 The genes

415

NirK and NirS encode the enzyme responsible for reducing NO2--N to NO. It was indicated from

416

Figure 7(b) that the relative quantities of the NapA, NirK and NirS genes in Sludge-Fe were 2.24-,

417

6.87- and 5.45- fold higher than those of Sludge-C, respectively. The up-regulated genes suggested

418

that more aerobic denitrification genes regulated the synthesis and expression of corresponding

419

NAP and NIR enzymes when iron scrap was added, which led to the TN removal in Sludge-Fe.

420

In this study, that the SND process could be initiated by iron scrap adding in the aerobic

421

nitrification activated sludge system was demonstrated. After the iron scrap supplementation, the 22


Page 23 of 28


422

abundance of aerobic denitrifiers was enhanced. The activities of key enzymes relating to

423

biological nitrogen removal were increased, and the functional genes encoding denitrification

424

enzymes were up-regulated. Although the method shows a promising future in nitrogen removal,

425

more investigations are required before putting it into the engineering, such as increasing the

426

influent nitrogen concentration, improving the TN removal efficiency, and reducing the carbon

427

source demanding. Furthermore, the mechanism of SND robustly initiated by iron scrap rather

428

than Fe2+ or Fe3+ should be deeply interpreted in future study.

429

430

AUTHOR INFORMATION

431

Corresponding Author

432

*Phone: 86-21-67792537; fax: 86-21-67792522; e-mail: [email protected].

433

ACKNOWLEDGMENT

434

This work was financially supported by National Natural Science Foundation of China (Grant

435

no. 51508081), the Natural Science Foundation of Jiangsu Province (BK20160431) and the

436

Fundamental Research Funds for the Central Universities (2232015D3-23).

437

ASSOCIATED CONTENT

438

Supporting Information Available.

439

methods including the composition of synthetic wastewater, the MiSeq Sequencing method, the

440

detection of specific oxygen uptake rate (SOUR), and the determination of activities of enzymes

441

AMO, NXR, NAP and NIR. This information is available free of charge via the Internet at

442

http://pubs.acs.org/.

443

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TOC

555 556

Activated sludge Iron scrap

Sludge-C

400 Relative Enzyme Activity (%)

Air

Sludge-Fe

350 300 250 200 150 100 50 0 NAP

TN was found to remove

Concentration (mg/L)

25

NIR

Denitrification key enzyme

+

NH4 -N -

NO3 -N

20

The quantity of functional gene NapA was increased

TN 15 10

The abundance of aerobic denitrifiers was enhanced

5 0 0

1

2

3

4

5

6

Time (h)

SND is simulated by iron scrap under aerobic condition 557 558

28


Iron Robustly Stimulates Simultaneous Nitrification and

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