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Removal characteristics of dissolved organic nitrogen and its bioavailable portion in post-denitrifying biofilter: effect of C/N ratio Haidong Hu, Kewei Liao, Jinju Geng, Ke Xu, Hui Huang, Jinfeng Wang, and Hong-qiang Ren Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Removal Characteristics of Dissolved Organic Nitrogen and its Bioavailable

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Portion in Post-denitrifying Biofilter: Effect of C/N Ratio

3 4 5 6 7

Haidong Hu, Kewei Liao, Jinju Geng, Ke Xu, Hui Huang, Jinfeng Wang, Hongqiang Ren*

8 9 10 11 12 13

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

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Nanjing University, Nanjing 210023, Jiangsu, PR China

15 16 17 18

*Corresponding author.

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Tel.: +86 25 89680512; fax: +86 25 89680569.

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E-mail address: [email protected] (H. Hu); [email protected] (H. Ren).

21

Notes

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The authors declare no competing financial interest.

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ABSTRACT: Addition of external carbon sources to post-denitrification biofilters (DNFs) is

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frequently used in municipal wastewater treatment plants to enhance dissolved inorganic nitrogen

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removal. However, little is known about its influence on the removal of dissolved organic nitrogen

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(DON). This study investigated the effect of the carbon-to-nitrogen (C/N) ratio (3, 4, 5 and 6) on

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the removal characteristics of DON and bioavailable DON (ABDON) in the pilot-scale DNFs

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treating real secondary effluent. Results showed that DNFs effluent DON accounted for 31.2−39.8%

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of the effluent total nitrogen. The maximum effluent DON and ABDON concentrations were both

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occurred in DNF operated at a C/N ratio of 3. There was no significant difference in effluent DON

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concentrations in DNFs at C/N ratios of 4, 5, and 6; however, effluent ABDON and DON

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bioavailability significantly decreased with C/N ratios (p ˂ 0.05, t-test). According to the chemical

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composition analysis, effluent DON at high C/N ratios tend to contain less %molecular weight ˂1

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kDa nitrogenous organic compounds and proteins/amino sugars-like nitrogenous organic formulas,

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which is likely responsible for its low bioavailability. Overall, this study indicates the benefit of a

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high C/N ratio during the DNF process in terms of controlling the DON forms that readily stimulate

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algal growth.

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INTRODUCTION

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The effluent discharged from municipal wastewater treatment plants (MWWTPs) is an

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important source of nitrogen loading to aquatic environments and thus contributes to

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eutrophication.1 With stricter effluent total nitrogen permit limits becoming more common, many

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existing MWWTPs are facing the need to upgrade.2 Due to the benefits of the small footprint and

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high efficiency, post-denitrification biofilters (DNFs) are widely used as a practical technology for

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tertiary nitrogen removal in MWWTPs.3,4 In this process, oxidized nitrogen is denitrified to

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nitrogen gas under anoxic conditions, with organic substrates as the electron donor.5

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Effluent nitrogen includes both inorganic and organic nitrogen. Dissolved organic nitrogen

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(DON) has become more important as it can account for 52% and even up to 80% of the effluent

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total nitrogen in low effluent total nitrogen MWWTPs.6,7 Moreover, recent research has shown

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that a portion of DON in treated effluent can be bioavailable to natural phytoplankton and hence

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can play an important role in nitrogen cycling.8-11 Bioavailable DON (ABDON) is the component

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of DON which supports the growth of bacteria and algae.12 Because of the importance of DON in

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MWWTPs addressing low effluent total nitrogen concentration goals, understanding the removal

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characteristics of DON and ABDON in the DNF is of great interest. So far, most research has

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focused on the performance of DNF for the removal of dissolved inorganic nitrogen, whereas little

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is known about the DON and ABDON.13-17

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Previous research suggests that the bioavailability of DON (ABDON/DON) is related to the

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chemical composition of DON.12,18 Eom et al.19 fractionated the effluent-derived DON into

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high-molecular-weight DON (>1 kDa) and low-molecular-weight DON (˂1 kDa) by ultrafiltration

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and found that low-molecular-weight DON is highly bioavailable and stimulates phytoplankton

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growth. Recently, ultra-high-resolution Fourier−transform ion cyclotron resonance mass

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spectrometry (FTICR-MS) has been used to characterize wastewater-derived DON.20,21 FTICR-MS

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can provide an unprecedented resolution of thousands of molecular formulas and, therefore, is

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capable of distinguishing between new production and degradative loss of compounds during DON

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transformation.21 Hence, this technique could possibly reveal a potential relationship between DON

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chemical molecular composition and bioavailability.22

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The availability of an electron donor, conveniently expressed in terms of the

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carbon-to-nitrogen (C/N) ratio, is the main control parameter for the DNF process.23 It has been

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observed that nitrate and/or nitrite accumulates because of insufficient carbon dosage; however, an

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overdose of the carbon source results in a waste of expensive electron sources and increases the

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effluent chemical oxygen demand (COD).15,24 Recent studies found that the C/N ratio plays an

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important role in the removal of nitrogenous organic micropollutants,25 and the production of

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soluble microbial products (SMPs)26 under anoxic conditions. SMPs, produced as a result of

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substrate metabolism and biomass decay, is an important fraction of DON.8,27 Consequently, it is

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expected that the C/N ratio affects the removal characteristics of both inorganic and organic

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nitrogen in the DNF.

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In this study, we investigated the effect of C/N ratio on the removal characteristics of DON

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and ABDON in the pilot-scale DNFs treating real secondary effluent. The chemical composition

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of DON in DNF effluent was also investigated with the purpose of facilitating a better 4

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understanding of the impact of C/N ratio on DON bioavailability (ABDON/DON). To our

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knowledge, this is the first study focusing on organic nitrogen removal during the DNF process,

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and the efforts would allow for optimization of the DNF process for both inorganic and organic

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nitrogen removal.

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MATERIALS AND METHODS

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Description of the Pilot−scale DNF. Four pilot-scale DNFs were operated in parallel at four

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different C/N ratios (3, 4, 5, and 6). Each pilot-scale DNF system consisted of three parts: a

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denitrification bed, a raw water feeding subsystem, and a backwashing subsystem (Figure S1). The

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denitrification bed was constructed as a cylinder with an inner diameter of 0.35 m and height of 3.4

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m. The bed was filled with a 0.3 m layer of pebbles and then further filled with sea sand up to 2.0 m

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in height. The media size and density of the sand were 2−3 mm and 2.54 g/cm3, respectively. DNF

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influent collected in a storage tank was pumped to the top of the filter bed. The influent used in this

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study was secondary effluent taken from the secondary settling tank of a MWWTP (an

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anaerobic-anoxic-oxic process) in Wuxi, China. The secondary effluent quality is included in Table

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1.

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Experimental Procedures and Sample Collection. The overall experiment consisted of a

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start-up period (0−70 days) followed by an operation period (71−197 days). By continuously

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operating the DNFs for two months, each DNF effluent nitrate (NO3-) became stable (Figure 1).

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Acclimatized denitrifying bacteria were thus obtained.24 The system was then maintained at the

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same operational conditions for a few more days to verify and ensure the full accomplishment of 5

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the start-up. After start-up, the influent C/N ratio was changed by changing the amount of external

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carbon source, whereas other variables were maintained as the experiment proceeded: DNF І (C/N

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= 3); DNF II (C/N = 4); DNF III (C/N = 5); and DNF IV (C/N = 6) (Table 2). Due to its high

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denitrification rate, acetate (i.e., sodium acetate) was commonly used as the external carbon

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source for DNF.13,23 Therefore, acetate was selected as the external carbon source in this study.

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After 50 days of operation, stable NO3-, total nitrogen (TN), chemical oxygen demand (COD), and

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suspended solids (SS) removal was achieved in each DNF (Figure 1 and Figures S2−S4), which

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determined the acclimation of the DNFs to be complete.28 It was maintained for another 20 days of

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operation to ensure each DNF achieved steady state. Detailed operating conditions of the DNFs

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including the filtration velocity and backwash operation are shown in Table 2.

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Throughout the experiment, samples were collected at the inlet and outlet of the DNF every

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two to seven days and analyzed for ammonium (NH4+), NO3-, nitrite (NO2-), TN, SS, and COD. To

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investigate the effect of C/N ratio on DON removal characteristics, DON samples were collected

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weekly and analyzed for DON and DON bioavailability after each DNF became stable. Among

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them, triplicate DON samples were also used for DON size fractionation and DON molecular

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composition analysis. A peristaltic pump (average flow rate < 4 mL/min) was used for continuous,

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flow-proportional sampling of the DNF effluent. Within 20−24 h after each sampling day, the

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wastewater samples were filtered through 0.45 mm cellulose acetate membranes (ANPEL, China).

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The samples were then stored in the dark, kept at 4 °C prior to the experiments, and analyzed

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within one week after collection.

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DON, ABDON, and DON Bioavailability Determination. To minimize the impact of 6

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inorganic nitrogen on the accuracy of DON measurements, the DON was measured after a dialysis

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pretreatment using cellulose ester dialysis membranes (SPECTRUM LABORATORIES, 100−500

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Da molecular weight cutoff).29,30 After the dialysis pretreatment, the concentration of DON was

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calculated as the difference between total dissolved nitrogen (TDN) and the sum of inorganic

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nitrogen species (i.e., NH4+, NO3-, and NO2-).31-33 The concentration of ABDON was determined

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by a 14−day algal growth bioassay.12 Nitrate-free wastewater was obtained by using an

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ion-exchange (Dowex 1×8 chloride form resin, Sigma-Aldrich, USA) pretreatment method.8 To

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start the bioassay, 1.5 mL of algal seeds (Selenastrum capricornutum, obtained from the

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FACHB-collection, Chinese Academy of Sciences, China) and 1 mL mixed culture bacteria

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collected from the MWWTP were added to 100 mL DON samples in a 250 mL Erlenmeyer flask.

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Each bioassay was incubated in a temperature-controlled shaker (22−25 °C) with a 12-h light/dark

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cycle. The fate of background DON introduced by inoculation was evaluated with the deionized

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water control. The ABDON concentrations relied on the change of DON in the sample before and

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after the incubation period (eq. 1),12 where DONi and DONf represent DON before and after

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incubation for test samples and DONbi and DONbf represent DON before and after incubation for

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deionized water samples. DON bioavailability was calculated according to eq. 2.34

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ABDON (mg/L) = (DONi – DONf) – (DONbi – DONbf)

DON bioavailability (%) =

୅୆ୈ୓୒ ୈ୓୒

×100%

(1)

(2)

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Chemical Composition of DON. a) DON size fractionation. DON was classified into four

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groups on the basis of its molecular weight (MW): 10 kDa DON.

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MW fractionations were conducted in a 400-mL commercial stirred cell unit (Model 8400,

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Millipore Corp. USA), employing filters with MW cutoffs of 10, 3, and 1 kDa (Millipore Corp.

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USA) which were rinsed with Milli-Q water prior to filtration. The percentages of DON in each size

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range were calculated using the method described by Lee and Westerhoff.35

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b) DON molecular composition measurement. DON molecular composition was analyzed

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using a 9.4 T Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS, Bruker,

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Germany). A standard Bruker electrospray ionization (ESI) source was used to generate negatively

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charged molecular ions. The DON in wastewater samples was concentrated and extracted for

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FTICR-MS analysis using 1 g, 6 mL functionalized styrene-divinyl-benzene polymer resin (PPL,

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Supelco, USA) solid phase extraction (SPE) cartridges. The SPE-extracted DON sample was

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dissolved in 100% LC-MS methanol (Merck Lichrosolv, Germany) and was injected into the ESI

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source at 180 µL/h using a syringe pump. An LC−MS methanol blank was injected before and after

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each sample to ensure no cross-contamination between samples.36 Masses within the mass range of

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200−700 Da were considered.37 Methodologies for FTICR-MS mass calibration and processing can

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be found elsewhere.38 The van Krevelen diagram plots molar H/C vs O/C of each distinct empirical

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formula, aligning formulas in regions that can be attributed to lipids (O/C = 0−0.2, H/C = 1.7−2.2),

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proteins/amino sugars (O/C = 0.2−0.6, H/C = 1.5−2.2), carbohydrates (O/C = 0.6−1.2, H/C =

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1.5−2.2), lignin (O/C = 0.1−0.6, H/C = 0.6−1.7), tannins (O/C = 0.6−1.2, H/C = 0.5−1.5),

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unsaturated hydrocarbons (O/C = 0−0.1, H/C = 0.7−1.5), and condensed aromatics-like organic

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matter (O/C = 0−1, H/C = 0.3−0.7).39 Regions in which compounds share common elemental ratios

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were distinguished by N/C or the modified aromaticity index.39 8

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Wastewater Analysis. NO3- and NO2- were analyzed by ion chromatography (Dionex

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ICS-1100, USA). To measure TDN, samples were oxidized to NO3− using persulfate digestion. The

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NO3− concentration in the digested solution was then quantified by ion chromatography. NH4+ was

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determined by the salicylate method.40 Detection limits for all inorganic nitrogen species in

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wastewater samples were below 0.06 mg N/L. SS, COD, total phosphorus (TP), and UV254

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absorbance were measured according to Standard Method.40 Temperature and pH were measured

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on site using an HQ40d portable multiparameter tester (HACH, USA).

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Statistical Analyses. Student's t test (t-test) and analysis of variance (ANOVA) were

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conducted by the SPSS 19.0 (IBM, Armonk, New York). A p-value of 0.05, t-test), significant

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differences in the effluent ABDON were observed between C/N ratios of 4 and 5, 4 and 6, and 5

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and 6 (p < 0.05, t-test, Figure 3b). The concentration of effluent ABDON is an important issue

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because bioavailable nitrogen-containing organic compounds can be bioavailable to natural algae,

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which can lead to oxygen consumption and support eutrophication in waters.9,41-43 One of the

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important findings of the pilot-scale DNF tests was that effluent ABDON generally decreased with

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C/N ratios (Figure 3).

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The bioavailability of DON (ABDON/DON) in DNF effluent ranged from 38% to 55%

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(Figure 3). The range of effluent DON biodegradability observed in this study is agreeable with

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the values (18−61%) previously reported in low total nitrogen wastewater effluent.8,44 The

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bioavailability of effluent DON reached to a peak of 55 ± 4% at the C/N ratio of 4 (Figure 3a).

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After increasing C/N ratio in the feed, the bioavailability of effluent DON presented a significant

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decreasing trend for other two conditions (p < 0.05, Figure 3). These decreases in bioavailability

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indicate that the chemical composition of the effluent DON changed.12,45 This hypothesis will be

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further confirmed in the following section. Taken together, the results illustrate that DNF operating

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at high C/N ratio produce effluent DON with less bioavailability leading to relatively low nutrient

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support in receiving waters.

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Effect of C/N Ratio on Effluent DON Chemical Composition. To better understand the

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effect of C/N ratio on effluent DON bioavailability (ABDON/DON), the molecular weight (MW)

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distribution and molecular composition of DON in DNF effluent at different C/N ratios were

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investigated.

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a) Molecular weight distribution. The MW distribution of effluent DON from DNFs at

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different C/N ratios is shown in Figure 4. MW˂3 kDa DON made up the greatest portion in all

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DNF effluent (62−92%), which is consistent with previous studies,46-48 implying that most DON

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consists of low MW compounds. The %MW˂1 kDa content of DON in DNF effluent at a C/N ratio

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of 6 (average: 44%) was lower than that at C/N ratios of 3, 4, and 5 (average: 58%, 68% and 45%,

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respectively). Notice that DON substances with MW˂1 kDa property are likely to have a greater

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potential for phytoplankton production than MW˃1 kDa DON.19 This is likely the reason why the 11

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bioavailability of effluent DON at the C/N ratio of 6 was less than that at C/N ratios of 3, 4, and 5

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(Figure 3). Indeed, previous research has reported that the bioavailability of DON in wastewater

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positively correlated with low MW DON.48 In this study, the general tendency of DON

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bioavailability also increased with an increase in the %MW˂1 kDa DON, although no statistical

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significance was observed (R2 = 0.283, p > 0.05, ANOVA, Figure S6).

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b) Molecular composition. The molecular composition of DON in DNF effluent at different

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C/N ratios was analyzed by an ultra-high-resolution FTICR-MS. The mass spectra was analyzed

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to determine molecular formulas containing nitrogen, which was presented using a van Krevelen

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diagram (Figure 5a). A van Krevelen diagram is a useful way to visualize a large number of

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formulas because different regions correspond to different compound classes,49 which typically

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includes lipids, proteins/amino sugars, lignin, tannins, carbohydrates, condensed aromatics, and

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unsaturated hydrocarbons.39,50 A principal component analysis (PCA) based on the classes of

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effluent DON molecular composition was conducted, as shown in Figure 5b. Variable lignin and

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proteins/amino sugars had the highest loadings on PC1 (0.81 and -0.50, respectively, Figure S7).

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The compound type of proteins/amino sugars is aliphatic and typically represents the bulk of the

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labile DON pool.22 In contrast to the proteins/amino sugars-like compounds, the lignin-like

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compounds presumably have an aromatic nature limiting their degradability.51 The possible

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refractory nature of the lignin-like materials suggests that these compounds are presumably the

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organic residues remaining after degradation of the labile fraction of influent DON in DNF.36

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According to the PCA analysis, effluent DON from DNFs at different C/N ratios differed in

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its molecular composition. PC1, indicative of a refractory (positive PC1) vs. labile (negative PC1) 12

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character of the DON, explained 97.4% of the variance, whereas PC2 only accounted for 1.8% of

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the variance. The general trend observed for PC1 was that effluent DON at a C/N ratio of 4

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featured negative PC1 values (ranging from -9.25 to -8.30) and effluent DON at C/N ratios of 3, 5,

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and 6 had positive PC1 values (ranging from 0.96 to 5.96, Figure S8). This corresponds to the

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findings which showed that the maximum effluent DON bioavailability was reached at a C/N ratio

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of 4 (Figure 3). Furthermore, compared with the effluent DON at a C/N ratio of 5 (ranging from

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0.96 to 2.28), those at a C/N ratio of 6 were more positive on the PC1 axis (ranging from 5.36 to

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5.96, Figure S8), which suggest that effluent DON at a C/N ratio of 6 contain an important pool of

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nitrogen-containing compounds that are likely refractory or only semilabile.22,36 This data is also

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consistent with our earlier results on the bioassay tests (Figure 3).

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In summary, there were compositional differences in the DON in DNF effluent at different

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C/N ratios. For the effluent DON at C/N ratios of 4, 5, and 6, those at high C/N ratios tend to

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contain less %MW˂1 kDa nitrogenous organic compounds and proteins/amino sugars-like

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nitrogenous organic formulas, which is likely responsible for its low bioavailability.

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Implication. Researchers and engineers working on DNFs have primarily focused on whether

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treated wastewater meets the total nitrogen discharge requirements and how to optimize the

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operation of DNFs to decrease the dissolved inorganic nitrogen.23,52,53 Concern is rarely paid to the

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removal of DON during the DNF process and its impact on the receiving water systems. Optimizing

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external carbon addition not only improves the NDF process efficiency but also saves chemical

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costs and reduces secondary pollution (e.g., COD).4,54 Considering the relatively high proportion of

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DON in total nitrogen in DNF effluent (31.2−39.8%, Figure 2) and the great potential of DON to 13

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stimulate phytoplankton biomass (DON bioavailability: 38−55%, Figure 3), knowing the effect of

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C/N ratio on the removal characteristics of DON and ABDON in the DNF is critical for the

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strategies to balance eutrophication risk from wastewater effluents with secondary factors such as

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cost, energy consumption, and secondary pollution. Our results showed the maximum effluent

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DON and ABDON both occurred in DNF operated at a C/N ratio of 3. The effluent DON from

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DNFs did not significantly differ when the C/N increased from 4 to 6 (p > 0.05); however, effluent

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ABDON and DON bioavailability significantly decreased with the C/N ratio (p ˂ 0.05, t-test,

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Figure 3). In addition, results of DON size fractionation analysis suggest that the effluent DON

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from the DNF with C/N 4 and 5 contain more low-WM DON, compared with that from the DNF

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with C/N 6 (Figure 4). Low-MW DON are difficult to remove by conventional tertiary treatment,

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e.g., coagulation, and tend to enter receiving waters.35 Overall, this study indicates the benefit of

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high C/N ratios during the DNF process in terms of controlling the DON forms that readily

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stimulate algal growth.

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Although acetate has been the widely used carbon source for enhancing heterotrophic

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denitrification,13,23,55 many alternatives, e.g., methanol,56 ethanol,5 and natural and synthetic

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biopolymers (woodchips, polycaprolactone, etc.),57 are also applied. Thus, determining if different

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external carbon sources would show similar results could also be important for future research on

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this topic.

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ASSOCIATED CONTENTS

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website.

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Schematic of the DNF system (Figure S1); removal of TN, COD, and SS (Figures S2−S4);

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concentration of effluent NO2- (Figure S5); relationship between DON bioavailability and %MW˂1

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kDa DON (Figure S6); PC1 and PC2 loadings and scores of the PCA (Figure S7 and S8) (PDF)

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ACKNOWLEDGMENTS

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This study was supported by the Jiangsu Natural Science Foundation (BK20160655), the

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National Science and Technology Major Project (2017ZX07204001), and the Project of Jiangsu

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Provincial Six Talent Peaks (2015−JNHB−002).

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Table 1. The quality of secondary effluent, which was used as the influent for NDFs. Average

Range

P(25th, 75th)a

nb

NO3- (mg/L)

16.08

13.62−18.41

15.22, 17.34

39

+

0.33

0.10−0.50

0.20, 0.46

39

-

NO2 (mg/L)

0.04

0.00−0.10

0.02, 0.06

39

COD (mg/L)

23.0

17.5−31.4

19.1, 26.0

39

NH4 (mg/L)

c

SS (mg/L)

12.0

9.6−14.7

10.9, 13.1

39

DON (mg/L)

1.91

1.80−2.11

1.82, 2.03

21

TPd (mg/L)

0.94

0.67−1.10

0.88, 1.03

21

UV254

1.15

1.02−1.32

1.07, 1.22

21

pH

7.47

7.33−7.65

7.39, 7.55

21

Temperature (°C)

23.9

20.9−26.5

22.5, 25.4

21

459

a

460

b

the number of measured samples.

461

c

suspended solids.

462

d

total phosphorus.

P

(25th, 75th)

is percentile, 25th and 75th.

463 464

Table 2. Operation parameters of the DNFs. Operation conditions

Start−up periods

Operation periods

(DNF І−IV)

DNF І

DNF II

DNF III

DNF IV

4

3

4

5

6

C/N ratio Filtration velocity (m/h)

7.2

a

Backwash cycle (days)

2 Air (15 L/m2·s) + Water (6 L/m2·s)

Backwash operation modes Backwash operation time (min)

6

465

a

466

accumulation of the denitrifying bacteria.

the NDFs were not backwashed for the first seven days during the start−up periods to allow

20

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Figure 1. Removal of NO3- in DNFs operated at different C/N ratios during the start-up and

469

operation periods.

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Figure 2. The proportion of DON to total nitrogen in DNF effluent at different C/N ratios under

472

steady−state conditions (n = 9).

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(a)

(b)

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Figure 3. (a) DON, ABDON and DON bioavailability (ABDON/DON) in DNF effluent at

474

different C/N ratios under steady-state conditions (n = 9). (b) Variation of effluent DON, ABDON

475

and DON bioavailability at different C/N ratios under steady-state conditions. A hexagram indicates

476

there is significant differences in the DON, ABDON or DON bioavailability at different C/N ratios

477

(p-value < 0.05, t-test).

23

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Figure 4. Percentage of each size-fractionated DON in DNF effluent at different C/N ratios under

480

steady-state conditions (n = 3).

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(a)

(b)

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Figure 5. (a) Representative van Krevelen diagram shows the molecular formulas of DON in

482

DNF effluent. The types of nitrogen-containing molecules include (1) lipids, (2) proteins/amino

483

sugars, (3) carbohydrates, (4) lignin, (5) tannins, (6) unsaturated hydrocarbons, and (7) condensed

484

aromatics.39 (b) Principal component analysis (PCA) plot of the types of effluent DON molecules

485

from DNFs at different C/N ratios under steady-state conditions. The two principal components

486

together explain 99.2% of the variance in the types of data sets. PC1 and PC2 loadings and scores

487

plot are presented in Figure S7 and S8, respectively, in the Supporting Information.

25

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ABSTRACT ART

489

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