Coupled Sulfur and Iron(II) Carbonate-Driven Autotrophic

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Coupled sulfur and iron(II) carbonate-driven autotrophic denitrification for significantly enhanced nitrate removal Tingting Zhu, Hao-Yi Cheng, Lihui Yang, Shigang Su, Hongcheng Wang, Shusen Wang, and Ai-Jie Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b06865 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Coupled sulfur and iron(II) carbonate-driven autotrophic

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denitrification for significantly enhanced nitrate removal

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Tingting Zhu a , Hao-yi Cheng a *, Lihui Yang a,b, Shigang Su a,b, Hongcheng Wang a,b,

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Shusen Wang a,b, Ai-jie Wang a,b, *

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a.Key Laboratory of Environmental Biotechnology, Research Center for Eco-

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Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, P. R. China.

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b.University of Chinese Academy of Sciences, Beijing, 100049, P. R. China.

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*Corresponding author: Key Laboratory of Environmental Biotechnology, Research

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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

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100085, P.R. China; E-mail address: [email protected] (Ai-Jie Wang) &

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[email protected] (Hao-Yi Cheng).

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Abstract

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Sulfur-based denitrification process has attracted increasing attentions because it

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does not rely on the external addition of organics and avoids the risk of COD exceeding

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the limit. Traditionally, limestone is commonly employed to maintain a neutral

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condition (SLAD process), but it may reduce the efficiency as the occupied zone by

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limestone cannot directly contribute to the denitrification. In this study, we propose a

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novel sulfur-based denitrification process by coupling with iron(II) carbonate ore

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(SICAD system). The ore was demonstrated to play roles as the buffer agent and

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additional electron donor. Moreover, the acid produced through sulfur driven

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denitrification was found to promote the Fe(II) leaching from the ore and likely extend

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the reaction zone from the surface to the liquid. As a result, more biomass was

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accumulated in the SICAD system compared with the controls (sulfur, iron(II)

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carbonate ore and SLAD systems). Owing to these synergistic effects of sulfur and

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iron(II) carbonate on denitrification, SICAD system showed much higher

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denitrification rate (up to 720.35 g∙N/m3∙d) and less accumulation of intermediates

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(NO2- and N2O) than the controls. Additionally, sulfate production in SICAD system

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was reduced. These findings offer great potential of SICAD system for practical use as

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a highly efficient post-denitrification process.

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1. Introduction

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Heterotrophic denitrification is the conventional and most widely adopted method

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for the treatment of secondary effluent. However, this process requires carbon sources

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as electron donors1, which increases the operational cost2 and leads secondary pollution.

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The accurate addition of the carbon source is difficult3. Therefore, organic independent-

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autotrophic denitrification driven by hydrogen, sulfur or reduced iron species has

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gained increasing attentions4. Thereinto, element sulfur driven denitrification is relative

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highly efficient and holds the advantages of cost-effective and safety. The overall

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reaction for sulfur-based autotrophic denitrification is described as in Eqs (1), which

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can be seen as an acid producing process.

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55S0+50NO3-+38H2O+20CO2+4NH4+ →4C5H7O2N+55SO42-+25N2+64H+

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In practice, limestone is commonly employed into the sulfur based denitrification

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system. It serves as a pH buffering agent and the source of inorganic carbon for bacteria

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growth5, which is known as sulfur-limestone autotrophic denitrification (SLAD).

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Although limestone is an effective external alkalinity source, it cannot play a role as

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electron donor. Therefore, the occupied zone by limestone will not contribute to the

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denitrification and may limit the efficiency of the system. Additionally, as shown in

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Eqs (1), sulfate can be produced during the denitrification (7.54 mg SO42- per mg NO3--

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N), which should be controlled in case of meeting certain standards. For example, the

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sulfate limit in drinking water standard is 250 mg/L in China. That means the permit

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concentration of NO3--N in the target treating water cannot over 33 mg/L. If there is

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some background value of sulfate in the water, the permit concentration will be even

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lower. This may limit the application of this technology.

(1)

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Nitrate-dependent anaerobic ferrous oxidation (NAFO) process has been

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intensively explored using Fe(II) as an electron donor to convert nitrate into nitrogen

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gas under anoxic conditions6-8. Iron(II) carbonate (FeCO3)9-11 is one of the most

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abundant Fe(II)-bearing minerals in nature and plays an important role in iron and

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carbon cycling. Recent research12 found that iron(II) carbonate ore (siderite, FeCO3 as

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the main component) can also drive the conversion of nitrate to nitrogen gas. The

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overall reaction of denitrification driven by iron(II) carbonate is described as

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follows11,12: 5FeCO3+NO3-+8H2O → 5Fe(OH)3+0.5N2+4CO2+HCO3-

(2)

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This process produces alkalinity and carbonate, which could act as pH buffer agent

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and the inorganic carbon sources for bacterial growth12. If the iron(II) carbonate was

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introduced into the sulfur driven denitrification system, it likely plays a similar role as

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the limestone but avoids the increase of hardness. In addition to the function of buffer

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agent13, iron(II) carbonate ore can work as an additional electron donor6,14-17 and

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directly contribute to the denitrification. For this reason, the denitrification efficiency

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is expected to be higher than the conventional SLAD process in which the occupation

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of limestone can forms an inactive zone of denitrification in the system. Another

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expected advantage is the decline of the sulfate production, as partial nitrate removal

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will follow the iron(II) driven denitrification route. To our best knowledge, the

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combination of element sulfur and iron(II) carbonate ore to enhance the denitrification

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efficiency has not been reported yet.

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In this study, a sulfur-iron(II) carbonate autotrophic denitrification (SICAD)

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system was investigated in reactors packed with element sulfur and siderite particles.

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The denitrification performance of SICAD system was studied by continuously feeding

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nitrate containing solution at different hydraulic retention time (HRT) conditions and

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compared with SLAD, sulfur alone, and iron(II) carbonate alone systems in terms of

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nitrate removal rate, N intermediates distribution and pH. The measurement of Fe(II)

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leaching and mössbauer analysis were performed to explore the roles and the fate of

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iron in the system. High-throughput 16S rRNA gene based Illumina MiSeq sequencing

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were employed to understand the microbial communities in different systems. The main

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objective of this study are (i) to verify the SICAD system has superior denitrification

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performance compared to the SLAD, sulfur alone and iron(II) carbonate alone systems

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and (ii) to reveal the possible synergistic effects of sulfur and iron(II) carbonate that

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result in a high denitrification efficiency in the SICAD system.

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2. Materials and methods

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2.1 Reactor setup

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Sulfur (radius 1.5 to 3.5 mm) was packed into an up-flow column reactor (radius

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10.5 cm × 30 cm) with a working volume of 1 L (hereafter referred to as SAD system)

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by connecting a gas-tight bag. Iron(II) carbonate (radius 1.0 to 1.5 mm), a mixture of

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sulfur with iron(II) carbonate and a mixture of sulfur with limestone (radius 2.0 to 4.0

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mm) were also packed into up-flow column reactors (hereafter referred to as ICAD

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system, SICAD system and SLAD system, respectively). The volume ratio of the

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mixture was 1:1 and the packed volume of particles was 0.5 L in these four systems.

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Sulfur came from a chemical plant (ShanDong, China), and the sulfur content was

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98%±0.5%. Natural iron(II) carbonate was acquired from a mine (GuiZhou, China),

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and the ferrous content was 86%±0.5%. The limestone came from Sinopharm shares,

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and the purity was 98%±0.2%.

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2.2 Inoculation and operation

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The systems were inoculated with activated sludge (50 ml, 10 g TSS/L) from Gao

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Beidian sewage treatment plant (Beijing, China) and autotrophic denitrification

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bacterial (20 mL, 17 g TSS/L) from previous sulfur packed-denitrifying bed reactors,

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which had been operated for 3 months. The synthetic wastewater was as follows (g/L):

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NaNO3 0.121, Na2S2O3 1.5, NaHCO3 0.1, NH4Cl 0.002, Na2HPO4 0.15, MgCl2·7H2O

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0.5, CaCl2·2H2O 0.01 and Wolfe’s trace element solution14 (1 ml/L). The systems were

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recirculated to allow biofilm establishment. After inoculating for 15 days, the systems

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were fed in continuous mode under hydraulic retention time (HRT) of 24 h. In the

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influent, the Na2S2O3 and NH4Cl were removed, and the concentration of NaHCO3 is

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0.02 g/L. When the stable performance of nitrate removal was achieved, the systems

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were operated continuously with HRTs from 12 h to 0.5 h to evaluate the effect of

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SICAD on the denitrification performance. The pH of the influent was controlled at

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7.3±0.2 by adding 1 M HCl or NaOH solutions. The dissolved oxygen (DO)

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concentration in the feed was always maintained at less than 0.5 mg-O2/L and the

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systems were operated in a temperature controlled laboratory, approximately 30±5oC.

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2.3 Analytical methods

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The samples were collected through a 0.45-μm membrane filter. Anions (nitrite,

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nitrate, sulfite and sulfate) were measured with an ion chromatograph (883 Basic IC

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plus, Metrohm, Swizerland) equipped with a Metrosep A Supp 5-250 column (Metrohm,

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Swizerland). The concentration of N2O was determined using gas chromatography-

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mass spectrometry as described in Cheng et al15. The N2O concentration in the liquid

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phase was calculated according to Henry's law. The concentration of Fe2+ and sulfide

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were measured regularly according to standard methods16. The total Fe concentration

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was determined by inductively coupled-optical emission spectroscopy (ICP-OES,

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PerkinElmer-OPTIMA 8300). The free nitrous acid (FNA) concentration was

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calculated

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Ka=5.05×10-4 at 30.0oC (Ka=e-2300/T, T in degrees Kelvin)17.

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2.4 The X-ray diffraction analysis (XRD)

based

on

the

relationship

[FNA]=46/14×[NO2-]/(Ka×10pH)

with

and Mössbauer spectroscopy

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The composition of iron(II) carbonate was determined by XRD analysis (X'Pert

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PRO MPD, PANalytical B.V., Netherlands) using CuKα radiation, which was shown

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in Figure S1. The composition of the precipitate that formed on the iron carbonate was

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determined by Mössbauer spectroscopy (WissEl-WA-260) at 78 K. Samples were taken

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from the surface of the iron(II) carbonate in SICAD system and washed with deionized

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water. Then, the samples were percolated through a 0.22-μm membrane filter after

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centrifugation. To minimize oxidation by O2, the treated membrane was sealed between

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two layers of Kapton tape under anaerobic conditions. The sample detection was

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identical to the procedures reported in Peretyazhko et al18. The data analysis procedures

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for Mössbauer spectra followed a report by Morris et al19.

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2.5 Scanning electron microscopy (SEM)

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SEM images were obtained using a SU-8020 scanning microscope (Hitachi, Japan)

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to get additional visual insight into the microorganisms on the particle surface. Particles

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were randomly collected from four systems, and put in a tube, respectively. The sample

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preparation was identical to the procedures reported in Wang et al20, which was

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presented in the Supplemental Material.

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2.6 Microbial community analysis and adenosine triphosphate (ATP)

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The samples for the microbial community analysis were taken at the end of

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experiment. Particles from the top, middle, and bottom sections of each system were

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pre-treated as described previously21. It was performed in triplicate. The collected

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mixture of particles were taken for total genomic DNA extraction using the protocol of

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the FastDNA SPIN Kit for soil (Sangon Biotech Co., Ltd, Shanghai, China) according

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to the manufacturer’s instructions. All extracted DNA were stored at -80oC for further

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amplifications. The primers 515F (5’-GTGCCAGCMGCCGCGG-3’) and 806R (5’-

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GGACTACHVGGGTWTCTAAT-3’) which target V4 hypervariable regions of

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microorganism 16S rRNA genes were selected22, and tagged with paired barcode

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sequence (12 mer) for pooling of multiple samples in one sequencing run. The details

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for 16S rRNA gene PCR amplification are available in the Supplemental Material. All

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primers were synthesized by Sangon Biotech (Shanghai, China).The raw sequencing

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data were generated with a MiSeq sequencing machine in fastq format. All sequencings

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were clustered at a 97% similarity level, and taxonomic assignments were made online

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by analysing the MiSeq data on the Usearch 7.0 platform (http://drive5.com/uparse/).

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The beta diversity of the microbial communities was described using the Principal

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coordinate analysis (PCoA) and heatmap analysis. The raw sequence files were

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submitted to the NCBI Sequence Read Archive database and assigned accession No.

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SRP172574.

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Filters (i.e. sulfur and siderite particles) were collected from top, middle and

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bottom sites of each system. The samples of three different parts were mixed for ATP

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and protein analysis. All ATP and protein measurements were performed in triplicate,

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and procedures were performed under sterile conditions. The collected particles were

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soaked in 10 ml sterile water homogenized by vortexing for 5 min at full speed to wash

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the bacterial on the particles. Then the solution was installed in a new tube and

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centrifuged at 8000r for 10 min and remove the supernatant. ATP was measured using

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the BacTiter-GloTM Microbial Cell Viability Assay (G8231, Promega Corporation,

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Dübendorf, CH) and a GloMax 20/20 Luminometer (Turner BioSystems, Sunnyvale,

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CA, USA). We applied the optimized protocol described by Hammes et al23 for ATP

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analysis, comparing with the manufacture’s protocol for this product. Total ATP was

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measured as follows Zhang et al24, which was added in the Supplemental Material. A

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conversion factor of 1.75×10-10 nmol ATP per cell was applied to calculate the bacterial

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cell number24, 25. Protein analysis was performed as the methods of ATP pre-treatment.

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Then the protocol of the Nuclear and Cytoplasmic Protein Extraction Kit (Sangon

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Biotech Co., Ltd, Shanghai, China) according to the manufacturer’s instructions was

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using for protein analysis, described previously by Zhou26.

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3. Results

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3.1 The nitrate removal performance

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To compare the nitrate removal performances among different autotrophic

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denitrification systems, artificial wastewater contained 20 mg/L of NO3--N was input

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to all of these systems as influent for treating. As shown in Figure 1 (A), the nitrate

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removal performance in ICAD system is worse, which was 72.29% and 40.85% at

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longer HRT of 12 h and 6 h, respectively. The nitrate removal performance ranged from

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91.31% to 97.42% in SAD, SICAD and SLAD system, respectively. The difference

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among these three systems of nitrate removal performance seemed minor. When the

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HRT was decreased to or even less 3 h, the SICAD system had an obviously better

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performance on nitrate removal than other systems. At a HRT of 3 h, the nitrate removal

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efficiency in the SICAD system was 92.6±2.5%, which was much higher than those in

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SAD system (61.0±3.2%), ICAD system (21.9±4.9%) and SLAD system (74.3±4.4%).

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It was apparent that the existence of both sulfur and iron(II) carbonate helped SICAD

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system to resist the negative shock of decreasing HRT (from 3 h to 0.5 h) compared

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with the other systems. At a shorter HRT of 0.5 h, the nitrate removal rate in SICAD

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system was 80.3±3.9%, which was higher than that in SAD system (33.2±3.8%), IAD

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system (10.9±5.2%) and SLAD system (56.1±5.1%).

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Figure 1 3.2 Total nitrogen (TN) removal and intermediate accumulation

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To further investigate the denitrification process in different systems, the TN

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removal performance was shown in Figure 1 (B). The biological denitrification is an

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effective approach to remove nitrate (NO3--N), where NO3--N serves as an electron

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acceptor and is sequentially reduced to nitrite (NO2--N), nitrous oxide (N2O) and

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nitrogen gas (N2). Due to the absence of NH4+-N in the artificial influent, organic

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nitrogen and hydroxylamine were excluded. Hence, calculated total nitrogen (TN)

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included NO3--N, NO2--N and N2O. The TN removal performance in the SICAD system

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(Figure 1 (B)) was higher than that in the other systems at different HRTs. At a HRT of

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0.5 h, the TN removal rate in the SICAD system was 720.35 g∙N/m3∙d. It was a 3.30-

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fold improvement over that in SAD system, 11.30-fold and 1.50-fold higher than that

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in ICAD system and SLAD system, respectively. The intermediates produced during

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the denitrification process were studied as well, which was shown as the form of

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nitrogen balance in Figure 2. At a HRT of 0.5 h, the ratio of intermediates accumulation

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to total nitrogen was 2.06% in SICAD system, 10.86% in sulfur system, 4.10% in

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iron(II) carbonate system and 7.15% in SLAD system. This suggested that the SICAD

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system had the lowest accumulation of intermediates and performed the highest TN

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removal.

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Figure 2

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As shown in Table 1, the produced NO2--N and soluble N2O during denitrification

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process in different systems were investigated. The effect of different particle (i.e.

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sulfur and iron(II) carbonate) on the NO2--N and N2O accumulation during the

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denitrification process were significant. The N2O concentration in the SICAD systems

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was the lowest, ranging from 0 to 0.006 mg/L. In comparison, it was 130-folder higher

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in SAD system, 2.5-folder higher in ICAD system and 5-folder higher in the SLAD

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system, respectively. It illustrated that N2O release could be reduced by SICAD. In

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addition, the accumulated nitrite in the SICAD system was the lowest as well, ranging

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from 0 to 0.41 mg/L, while it increased 7.24-fold in SAD system, 2.00-fold in the ICAD

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system and 3.41-fold in the SLAD system, respectively. It suggested that SICAD could

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also reduce the NO2- accumulation. Table 1

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3.3 Sulfate production

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As shown in Figure 3 (A), the sulfate production during per nitrate removal in

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SICAD system was less than that in SAD and SLAD systems. The sulfate production

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ranged from 5.67 to 6.92 g/gN in SICAD system, 6.15 to 7.92 g/gN in SAD system and

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6.78 to 7.98 g/gN in SLAD system. Sulfite and sulfide had not been detected during the

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entire denitrification process. It was shown that the SICAD system could decrease

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sulfate production during per nitrate removal, which was helpful to control the sulfate

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release to the water considering the sulfate limit of 250 mg/L for drinking water. This

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was attributed to a portion of the nitrate was involved to the NAFO process, resulting

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in a lower sulfate production than sulfur-based denitrification. The produced sulfate in

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SAD and SLAD system was lower than expectation based on the reaction stoichiometry

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(1) with most of the applied HRTs (ie. 12, 1.5 and 0.5 h). This result can be attributed

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to the incomplete denitrification, as reported in other researches27. The reduction of

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nitrate in the presence of sulfur can be represented as reactions (6) depending on

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whether the end product is nitrogen gas or nitrite:

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S0+3NO3-+H2O→SO42-+3NO2-+2H+

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S0+2NO2-→SO42-+N2

253 254

(6) Figure 3

3.4 The fate of iron(II) carbonate in SICAD and ICAD systems

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To investigate the contributions of iron(II) carbonate on the autotrophic

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denitrification, the fate of iron(II) carbonate was investigated. It was seen that SICAD

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promoted ferrous leaching from iron(II) carbonate. This result was consistent with the

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highest nitrate removal in SICAD system, as shown in Figure 3 (B). At a HRT of 0.5 h,

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the ferrous leaching was 0.86 mg/L in SICAD system, which was higher than that in

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ICAD system (0.14 mg/L). Based on the sulfate production and ferrous leaching, the

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contributions of iron(II) carbonate and sulfur on nitrate removal in SICAD system were

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calculated, as shown in Table S1. At a HRT of 0.5 h, the contribution of iron(II)

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carbonate and sulfur on autotrophic denitrification was 42% and 58%, respectively. The

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contribution of iron(II) carbonate increased from 8% to 42% along with the decrease of

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HRT from 12 h to 0.5 h. Shen and Zhao et al 11,13 studied that the iron(II) products of

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NADF process were ferric compounds, such as Fe(OH)3. As shown in Figure 3 (B), the

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concentration of ferric compounds was the difference between the total iron and ferric

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leaching, which was small. The total iron leaching in SICAD system was higher than

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Fe(II) by 0.0087 mg/L at HRT of 0.5 h. It indicated that ferric compounds in the effluent

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of SICAD system was low, which might be attributed to that a part of the produced

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iron(III) was absorbed on the surface of particles.

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To further investigate the fate of iron(II) carbonate, Mössbauer spectroscopy was

273

used to analyze the surface morphology of the iron(II) carbonate particles. The relative

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proportions of the areas covered by doublets and sextets to the total area of the

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Mössbauer spectrum were equivalent to the relative abundance of Fe in a particular

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crystal lattice site to that of the total Fe. The iron carbonate contained only 10% of

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high-spin Fe(II). The other 90% of the Fe in this sample was either high-spin Fe(III)

278

or low-spin Fe(II), as identified by isomer shift. The aggregates produced on the

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surface of iron(II) carbonate particles in SICAD system did not show reflections and

280

had a low blocking temperature of 77 K in the Mössbauer spectrum (Figure 3 (C)).

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This result was in agreement with that was the characteristic of ferrihydrite (88.75)

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and Ferroalluaudite (11.25%), which is [Fe2+Fe3+2(PO4)2(OH)2·8(H2O)]28.

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3.6 Microbiology analysis

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The SEM images revealed that visible microbes formed on the surfaces of particles

285

at these four systems in Figure S2. In order to further study the bacteria attached on

286

the particles, the ATP and protein on the particles in the four systems were listed in

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Table 2. The bacterial cell numbers were derived from the ATP measurements29, 30.

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The ATP content was 7.11×108 cell/mm2 on sulfur particles in SICAD system, which

289

was 9.11-fold and 4.08-fold higher than that in SAD and SLAD systems, respectively.

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The ATP content was 0.52×108 cell/mm2 on iron(II) carbonate particles in SICAD

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system, which was 13-fold higher than that in ICAD system. This suggested that the

292

bacterial cell numbers were highest in SICAD system compared with the other systems.

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The results of biofilm production were consistent with the nitrate removal

294

performance, as shown in Figure S3. Bacterial carbon storage in polymers has been

295

previously reported to represent 34-65% of the total biomass31. Hence, the protein

296

content was quantified by the biomass, which was shown in Table 2. The protein

297

content on the sulfur particles and iron(II) carbonate were 0.425 mg/mm2 and 0.476

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mg/mm2 in SICAD system, while the protein in SAD, ICAD and on the surface of

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sulfur in SLAD system was 0.12, 0.025 and 0.263 mg/mm2, respectively. It

300

demonstrated that SICAD improved the biomass production in order to enhance the

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nitrate removal.

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Table 2

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Regarding microbial community structure, the main difference at the phylum level

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among the five particles (i.e. sulfur-particles in SAD, SICAD and SLAD system, iron(II)

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carbonate-particles in ICAD and SICAD) was the phylum Proteobateria,

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Ignavibacteriae, Chlorobi, Chloroflexi and Bacteroidetes. Proteobacteria (72.32%)

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and Chlorobi (9.26%) were the dominant groups on the sulfur-particles in SICAD

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system, whereas the decreases of Proteobateria (63.92%, 64.82% and 56.71%) and

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Chlorobi (7.71%, 4.52% and 31.31%) abundances were observed in SAD, ICAD and

310

SLAD systems, respectively.

311

Figure 4

312

At the genus level, the main Proteobacteria composition was Thiobacillus, which

313

had a higher relative abundance (approximately 44.74%) in SAD system than that in

314

other systems (Figure 4 (B)). Thiobacillus are species of autotrophs that could use sulfur

315

as electron donor. As expected, the high abundance of Thiobacillus in sulfur system

316

was consistent with previous research on sulfur-based bioreactor. Chlorobaculum were

317

reported to oxidize sulfide and elemental sulfur, generating sulfate as the terminal

318

oxidation product32. The abundance of Chlorobaculum reached 31.16% in SLAD

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system, which was higher than that in SICAD (8.68%) and SAD systems (7.71%). The

320

results illustrate that Thiobacillus and Chlorobaculum were the dominant bacteria for

321

nitrate removal in SLAD system. In SICAD system, Thiobacillus (26.42% on the sulfur

322

particles and 34.63% on the iron(II) carbonate particles, p<0.01) and Ferritrophicum

323

(9.20% on the sulfur particles and 7.56% on the iron(II) carbonate particles, p<0.01)

324

were the dominant groups. Some researchers found that some kinds of Thiobacillus also

325

could use iron(II) as electron donor6,33. Therefore, these Thiobacillus that could use

326

sulfur and iron(II) might play an important role in SICAD system. Ferritrophicum, a

327

neutrophilic ferrous oxidizing bacteria (FeOB), had been reported to oxidize iron(II) to

328

iron(III) using nitrate as an electron acceptor under anoxic conditions34,35. In addition

329

to Fe2+, FeOB can use H2 and sulfide as electron donors and nitrate can serve as an

330

electron acceptor36,37. It suggested that the Ferritrophicum was responsible for nitrate

331

reduction.

332

Furthermore, the known iron-reducing bacterial genera Geothrix6,38 (p < 0.01)

333

were only found in SICAD and ICAD systems. It accounted for 6.12% and 6.38% on

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the sulfur particles and iron(II) carbonate particles in SICAD system. It might be

335

important for the ferrous and ferric cycle. Microbial community structures were

336

different from each other at different reactors based on the heatmap analysis (Figure 4

337

(C)) and the PCoA analysis (Figure 4 (D)).

338

4. Discussion

339

4.1 Possible mechanism of enhanced nitrate removal efficiency in SICAD system

340

In this study, the enhanced nitrate removal by coupling sulfur with iron(II)

341

carbonate was verified (Figure 5). Firstly, the nitrate removal improvement might be

342

the synergistic influence of SICAD on reaction process and biomass. Some

343

researchers6,34 studied the FeOB could use iron(II) carbonate ore as electron donor for

344

denitrification. The low solubility of minerals might affect the nitrate removal40-42, but

345

the electron transfer rate between FeOB and the minerals also might result in the lower

346

nitrate removal rate43 Rakshit et al.43 found that a homogeneous reaction involving

347

dissolved Fe(II) in equilibrium with the solid Fe(II) and NO2- could occur in addition to

348

the heterogeneous reaction with solid Fe(II). In this study, the acid produced by sulfur

349

denitrification might improve the release of Fe2+ and extend the reaction zone for the

350

bacteria on iron(II) carbonate from a solid phase to a solid-liquid phase (Figure 5). It

351

was verified by the higher ferrous leaching in SICAD system. Additionally, the report

352

by Straub44 about nitrate-dependent Fe(II) oxidation observed that Thiobacillus

353

denitrificans could couple denitrification with anaerobic oxidation of Fe(II) in common

354

minerals. Thus, Thiobacillus denitrificans could be used for sulfur-based denitrification

355

and ferrous iron-based denitrification. However, Thiobacillus denitrificans cannot be

356

enriched without an additional electron donor6. In addition, the kinetics of sulfur

357

denitrification was limited by electron transfer through the sulfur oxidizing bacteria in

358

direct contact with sulfur or soluble sulfur species45. Therefore, sulfur might act as an

359

electron donor to promote the growth of FeOB in SICAD system. Iron(II) carbonate

360

might promote the electron transfer by enrichment of FeOB in order to enhance the

361

nitrogen removal efficiency. This conclusion was verified by the higher biomass

362

production in SICAD system than other control systems (Table 2).Therefore, the

363

synergistic influence of SICAD on extend the reaction zone and higher biomass led to

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a much higher nitrate removal rate in SICAD system than other control systems.

365

Secondly, the improved nitrate removal could be resulted from the difference in

366

the pH between SICAD (6.80±0.1 to 7.24±0.1) and other systems. The optimum pH for

367

the dominant bacteria of Thiobacillus is 6.8-7.446. The pH ranged from 6.80 to 7.24 in

368

SICAD system. While the pH was not controlled, it was acidic (6.2±0.1 to 6.7±0.1) in

369

SAD system and alkaline (7.40±0.2 to 7.80±0.2) in ICAD system. The lower nitrate

370

removal was attributed to the unsuitable pH, because most biological activity of

371

denitrifiers was repressed at acidic or alkaline conditions. Although the pH ranged from

372

7.23±0.2 to 7.60±0.3 in SLAD system, limestone could not provide larger reaction

373

surface for denitrifiers as electron donor, compared with iron(II) carbonate in SICAD

374

system. It was consistent with the ATP and protein results (Table 2). The ATP and

375

protein of iron(II) carbonate particles in SICAD system was higher than that of

376

limestone particles in SLAD system.

377

Figure 5

378

Additionally, the redox potential of the S0/SO42- (-0.35 mv)47 couple is lower than

379

the Fe(II)/Fe(III) (+200 mv)48 couple in standard conditions47. It indicated that sulfur

380

might oxidize Fe(III) to Fe(II) in thermodynamically. Some studies had investigated

381

that many microorganisms could reduce Fe(III) to Fe(II) using an assortment of electron

382

donors, such as acetate, lactate and H2. The most notable examples include: Geobacter

383

spp.49, Shewanella spp.50 and Geothrix fermentans51. Sulfur as the function of carbon

384

source might proceed Fe redox cycling in SICAD system. The discovery of Geothrix

385

has drawn attention in this study. Geothrix could release one or more electron-shuttling

386

compounds that promote Fe(III) oxide reduction and transfer electrons from the cell to

387

Fe(III) oxide52. The redox cycling of Fe electron shuttles secreted by Geothrix from

388

iron-oxide minerals may contribute to microbial cooperative catabolism. This indicated

389

that the iron cycle process might further promote the electrons transfer between two

390

solids (ie. Sulfur and iron(II) carbonate particles ) or two bacteria by electron shuttles.

391

In sum, the possible synergistic influence of SICAD on the enhanced nitrate

392

removal might be attributed to (1) the acid produced through sulfur driven

393

denitrification was found to promote the Fe(II) leaching from the ore and likely extend

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the reaction zone from the surface to the liquid. As a result, more biomass was

395

accumulated in the SICAD system compared with the controls (sulfur, iron(II)

396

carbonate ore and SLAD systems). (2) circumneutral pH might be benefit for

397

denitrification bacteria activity (3) SICAD might bring out the redox cycling of Fe in

398

order to promote the transfer of electrons between two solids or two bacteria.

399

4.2 Low accumulation of N2O in SCIAD

400

In particular, the synergistic effect of SICAD not only improved nitrate removal

401

but also decreased NO2--N accumulation and the N2O release. A relationship exists

402

between N2O release, nitrite accumulation and pH was shown in Table 2. NO2--N and

403

N2O accumulation was likely caused by that nitrite reduction rate was lower than nitrate

404

reduction rate and N2O reduction rate was lower than nitrite reduction rate. The

405

synergistic effect of SICAD might improve the denitrification rate (nitrate reduction,

406

nitrite reduction and N2O reduction) compared with other systems. Hence, it led to

407

lowest NO2--N and N2O accumulation in SICAD system. Previous studies had shown

408

that both nitrite and pH also influence N2O reduction. Low pH was suggested to be an

409

important influential factor leading to N2O accumulation as an inter-mediate during

410

denitrification53. Hence, the N2O accumulation was highest in sulfur system. In addition,

411

FNA which was jointly determined by nitrite and pH, was likely a strong inhibitor of

412

N2O reduction54,55. At different HRTs, N2O accumulation increased with the decrease

413

of HRTs from 1.5 h to 0.5 h. The pH from 6.70±0.10 to 6.32±0.2, and the highest value

414

was 0.784 mg/L in SAD system. Then with the decrease of HRT form 1.5 h to 0.5 h,

415

the N2O production decreased to 0.12 mg/L. The tendency of the N2O concentration

416

with increment firstly was because the N2O accumulation was limited by the incomplete

417

of denitrification with the decreasing of HRT. The decreases of the nitrite reduction

418

rates at pH of 6.32 (HRT of 1.5 h) and 6.20 (HRT of 0.5 h) were likely due to FNA

419

inhibitions. At pH 6.32 and 6.20, this would give rise to FNA concentrations of 0.004

420

and 0.01 mg HO2-N/L at room temperature. These concentrations were in the range of

421

FNA that had previously been found to inhibit nitrite reduction56. Zhou et al.56 showed

422

that nitrite reduction by a EBPR sludge was inhibited by 40% at an FNA concentration

423

of 0.01 mg HO2-N/L. Compared with SAD system, FNA concentration and NO2-

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production were lower in SLAD system leading to less N2O accumulation. In ICAD

425

system, the lower nitrate reduction rate might result in the lower NO2- production and

426

N2O accumulation.

427

4.3 Advantages of SICAD in environmental application

428

SICAD decreased sulfate release compared with SAD and SLAD systems, which

429

would reduce the risk of exceeding sulfate discharge limit. In all sulfur based

430

denitrification processes (ie. Sulfur or SLAD reactor), sulfate formation leads to high

431

sulfate content in treated effluent57. It should be controlled in case of meeting certain

432

standards. The allowable limit of sulfate for drinking water was set of 250 mg/L.

433

Theoretically, around 33 mg/L NO3--N could be denitrified without exceeding the

434

above limit. If there was some background value of sulfate in the water, the control of

435

excess sulfate production is a serious challenge. According to the sulfate formation per

436

nitrate removal (Figure 3 (A)), approximately 33.24, 36.92 and 32.74 mg/L NO3--N

437

could be denitrified in SAD, SICAD and SLAD system, respectively. Combining

438

autotrophic and heterotrophic denitrification is an effective strategy to control sulfate

439

formation. Hence, the 3.68 mg/L (=36.92-33.24) NO3--N could be denitrified by 9.63

440

mg/L methanol as additional carbon sources. It would need an accurate dosing of

441

methanol. If not, it would result in the secondary pollution due to the COD exceeding

442

the limit. With the decrease of HRT, the contribution for denitrification of iron(II)

443

carbonate increased from 8% to 42% and the contribution of sulfur decreased from 92%

444

to 58% in SICAD system. It suggested that at shorter HRTs, sulfur played little role in

445

denitrification in SICAD system, which would be suitable for practical application as a

446

highly efficient post-denitrification process.

447

In this study, the synergistic effect of SICAD could improve nitrate reduction,

448

decrease N2O release and produce less sulfate products. The study of autotrophic

449

denitrification in regard to coupling sulfur and iron(II) carbonate has received little

450

attention. This study revealed both the feasibility and mechanism of SICAD, offering

451

greater potential and feasibility for practical applications for secondary effluent

452

purification. However, several important research gaps and challenges exist for further

453

advancing sulfur-iron(II) carbonate-based cleanup technology. The presence of ferric

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hydroxide as a product of the NAFO process indicated that phosphorus can be removed

455

by adsorption. Studying the synergistic effect of SICAD may also be important to gain

456

further insight into simultaneous phosphorus and nitrogen removal. However, the ferric

457

hydroxide wrapped on the surface of iron(II) carbonate may influence ferrous

458

contacting with contaminants, which should be further investigated.

459

Acknowledgments

460

The research was supported by the National Science Foundation of China

461

(No.21707156 and No. No. 51878652) and Funding for the 63th China postdoctoral

462

Science Foundation(No.Y8H1C91704).

463

References

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Figure Captions

619

Figure 1 Nitrate removal performances of four systems (A) and TN removal performances of four

620

systems (B) with different electron donors (SAD, ICAD, SICAD and SLAD) at different HRTs

621

Figure 2 Nitrogen balance of nitrogen compounds (left axis) with different electron donors

622

(SAD (A), ICAD (B), SICAD (C) and SLAD (D)) at different HRTs (green is nitrogen, yellow is

623

N2O-N, blue is NO2--N and slash is NO3--N) The right axis is N2O production (mg/L)

624

Figure 3 The sulfate production during per nitrate removal (A), Ferrous leaching (B) and

625

Mössbauer spectra of substances on the iron(II) carbonate particles in SICAD system

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626

at 77 K (C).

627

Figure 4 The classification of 16s rRNA gene sequences from the microbial

628

communities on the filter surfaces in the four reactors at the phylum level (A). The

629

microbial communities on the filter surfaces in the four reactors at the genus level

630

(relative abundance ≥1%) (B). Heatmap (C) and PCoA (D) analyses of the microbial

631

communities

632

Figure 5 Possible metabolic path of sulfur coupled with iron(II) carbonate-driven

633

autotrophic denitrification between microorganisms

634

Table Captions

635

Table 1 pH,

636

donors at different HRTs

637

Table 2 The bacterial cell numbers derived from the ATP and protein measurements

638

Supporting Information (SI)

639

Figure S1 Natural iron(II) carbonate XRD patterns.

640

Figure S2 SEM images of biofilms on the surface of the filter in SAD system (a and b);

641

ICAD system (c and d); SICAD system (e and f); and SLAD system (g and h). (Left:

642

magnified 6000 times. Right: magnified 30000 times.)

643

Figure S3 Biomass production on the particles during per TN removal.

644

Table S1 The contributions of sulfur and iron(II) carbonate to the enhanced nitrate

645

removal in SICAD system

646

Table S2 List of some previous studies on S-based denitrification and nitrate-dependent

647

anaerobic oxidation processes to remove TN

NO3--N

removal, FNA, NO2- and N2O accumulation with different electron

648

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649

650

651 652 653

Figure 1 Nitrate removal performances of four systems (A) and TN removal performances of four

654

systems (B) with different electron donors (SAD, ICAD, SICAD and SLAD) at different HRTs

655 656

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657

Table 1 pH, NO3--N removal ,FNA, NO2- and N2O accumulation with different electron

658

donors at different HRTs HRT(h)

Reactors

12

6

3

1.5

0.5

6.70±0.10

6.50±0.20

6.45±0.1

6.32±0.2

6.20±0.1

18.26

18.30

12.41

9.31

6.63

FNA

0

123×10-5

188×10-5

397×10-5

910×10-5

NO2--N

0

0.57

0.77

1.21

2.97

N2O

0.002

0.235

0.551

0.784

0.120

pH

7.80±0.2

7.67±0.2

7.62±0.1

7.41±0.3

7.40±0.2

14.46

8.16

4.38

3.44

2.17

0

4.2×10-5

7.8×10-5

20.3×10-5

22.3×10-5

pH --N

NO3 Sulfur

iron(II) carbonate

--N

NO3

FNA NO2--N

0

0.28

0.47

0.76

0.82

N2O

0.005

0.006

0.007

0.015

0.003

pH

7.24±0.1

7.11±0.1

7.01±0.1

6.82±0.2

6.80±0.1

--N

NO3 SICAD

SLAD

19.48

19.45

18.52

16.44

15.42

FNA

0

3.6×10-5

4.9×10-5

1.2×10-5

44.5×10-5

NO2--N

0

0.06

0.07

0.01

0.41

N2O

0.001

0.001

0

0

0.006

pH

7.60±0.3

7.55±0.2

7.39±0.2

7.3±0.1

7.23±0.2

NO3--N

19.02

18.29

14.87

14.12

11.22

FNA

0

0

12.8×10-5

3.91×10-5

57.2×10-5

0

0

0.46

0.11

1.41

0.003

0.001

0.010

0.014

0.030

--N

NO2

N2O

659 660

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662

663 664

Figure 2 Nitrogen balance of nitrogen compounds (left axis) with different electron donors

665

(SAD (A), ICAD (B), SICAD (C) and SLAD (D)) at different HRTs (green is nitrogen, yellow is

666

N2O-N, blue is NO2--N and slash is NO3--N) The right axis is N2O production (mg/L)

667 668

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669

670

671 672

Figure 3 The sulfate production during per nitrate removal (A), Ferrous leaching (B)

673

and Mössbauer spectra of substances on the iron(II) carbonate particles in SICAD

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Environmental Science & Technology

system at 77 K (C).

675

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Page 28 of 32

Table 2 The bacterial cell numbers derived from the ATP and protein measurements iron(II) carbonate

Sulfur in SICAD

0.78×108

0.04×108

7.11×108

±0.02×108

±0.13×107

0.12±0.03

0.025±0.008

Sulfur

ATP cell/mm2 Protein mg/mm2

iron(II) carbonate

Sulfur in SLAD

Limestone in SLAD

0.52×108

1.74×108

0.25×108

±0.37×108

±0.14×108

±0.56×108

±0.07×108

0.425±0.05

0.476±0.19

0.263±0.23

0.132±0.05

in SICAD

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679 680

681

682 683 684 685 686

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687 688

689

690 691 692 693 694 695 696 697 698

Figure 4 The classification of 16s rRNA gene sequences from the microbial communities on the filter surfaces in the four reactors at the phylum level (A). The microbial communities on the filter surfaces in the four reactors at the genus level (relative abundance ≥ 1%) (B). Heatmap (C) and PCoA (D) analyses of the microbial communities

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699 700

701 702 703

Figure 5 Possible metabolic path of sulfur coupled with iron(II) carbonate-driven

704

autotrophic denitrification between microorganisms

705

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190x135mm (96 x 96 DPI)

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