Microbial Photoelectrotrophic Denitrification as a Sustainable and

Oct 13, 2017 - School of Environmental Science and Engineering, Sun Yat-Sen University, East Campus, No. ... rate in PEDeN equaled that in OEDeN with ...
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Microbial photoelectrotrophic denitrification as a sustainable and efficient way for reducing nitrate to nitrogen Hao-Yi Cheng, Xia-Di Tian, Chuanhao Li, Shusen Wang, Shi-Gang Su, HongCheng Wang, Bo Zhang, Hafiz Muhammad Adeel Sharif, and Aijie Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02557 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Microbial photoelectrotrophic denitrification as a sustainable

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and efficient way for reducing nitrate to nitrogen

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Hao-Yi Cheng,a Xia-Di Tian,ab , Chuan-Hao Li,c Shu-Sen Wang, ab Shi-Gang Su, ab

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Hong-Cheng Wang,ab Bo Zhang,a Hafiz Muhammad Adeel Sharif,ab Ai-Jie Wang,*ab

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a

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Eco-Environmental Sciences, Chinese Academy of Sciences, No. 18 Shuangqing Road,

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Haidian District, Beijing 100085, China

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b

Key

Laboratory

of

Environmental

Biotechnology,

Research

Center

for

University of Chinese Academy of Sciences, No. 19 Yuquan Road, Shijingshan

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District, Beijing 100049, China

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c

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Campus, No. 135 Waihuan Road, Daxuecheng District, Guangzhou 510006, China

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* Corresponding Author: Tel & Fax: +86-10-62915515; Email: [email protected].

School of Environmental Science and Engineering, Sun Yat-Sen University, East

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Abstract: :

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Biological removal of nitrate, a highly-concerning contaminant, is limited when the

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aqueous environment lacks bioavailable electron donors. In this study, we

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demonstrated, for the first time, that bacteria can directly use the electrons originated

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from photoelectrochemical process to carry out the denitrification. In such

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photoelectrotrophic denitrification (PEDeN) systems (denitrification biocathode

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coupling with TiO2 photoanode), nitrogen removal was verified solely relying on the

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illumination dosing without consuming additional chemical reductant or electric

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power. Under the UV illumination (30 mW·cm-2, wavelength at 380±20 nm), nitrate

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reduction in PEDeN apparently followed the first order kinetics with the constant of

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0.13±0.023 h-1. Nitrate was found almost completely converted to nitrogen gas at the

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end of batch test. Compared to the electrotrophic denitrification systems that driven

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by organics (OEDeN, biocathode /acetate consuming bioanode) or electricity (EEDeN,

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biocathode/abiotic anode), denitrification rate in PEDeN equaled to that in OEDeN

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with COD/N ratio at 9.0 or that in EEDeN with applied voltage at 2.0 V. This study

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provides a sustainable technical approach for eliminating nitrate from water. PEDeN

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as a novel microbial metabolism may shed further light onto the role of sunlight

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played in the nitrogen cycling in certain semi-conductive and conductive minerals

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enriched aqueous environment.

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Key words: photoelectrotrophic; photoelectrochemical; denitrification; biocathode;

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selective. 2

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Introduction

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Nitrate is one of the most concerning contaminants due to its ubiquity and adverse

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effects on human health and ecosystem.1 Autotrophic denitrification, the biological

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process of transforming nitrate to the innocuous nitrogen gas with inorganic electron

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donors, has received great attention in past decades.

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denitrification does not rely on the presence of organic matter, it is particularly

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attractive during the treatment of drinking water and the secondary effluent of

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wastewater treatment plants, in which the organic form of electron donors is typically

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lacking.4, 5 Compared to the heterotrophic denitrification by adding organic electron

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donors, autotrophic denitrification is suggested to be more cost-effective and it avoids

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the risk of secondary organic pollution.2

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Because the autotrophic

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In autotrophic denitrification, hydrogen is most frequently used electron donor due

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to its innocuous nature.6 However, the direct use of hydrogen gas may lead to safety

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issues and suffer from a low H2 utilization efficiency due to its low water solubility.

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Therefore, denitrification using electrodes, which generates hydrogen through

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hydrogen evolution reactions in-situ, was developed as an alternative approach.2, 7

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After that, bacteria was found to directly receive electrons from electrode for

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denitrification without hydrogen as an electron mediator (here defined as the

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electrotrophic denitrification, EDeN).8 In this process, EDeN gains the benefit of less

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energy loss because of its occurrence at more positive potential compared to the

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hydrogen evolution.8 In the case of the denitrification driven by electrode (as the

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cathode), another electrode has to be coupled as the anode. Oxygen evolution is the 3

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main anodic reaction in most cases with the theoretical occurrence potential at 0.82 V

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(versus standard hydrogen electrode, SHE, at pH 7). Because this potential is more

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positive than that of nitrate reduction (i.e. ENO3-/NO2- is 0.43 V vs SHE at pH 7),

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electric energy input is required. Alternatively, microbial catalytic anode system can

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be employed without the power supply, where biodegradable organics are oxidized by

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anode respiration bacteria at a more negative potential (e.g. the typical Eacetate/HCO3- is

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-0.28 V vs SHE at pH 7) compared to the nitrate reduction.9, 10

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Solar energy is considered to be a clean and sustainable energy source. Conversion

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of solar energy to chemical energy is realized with the help of a semiconductor, which

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respectively provides the reducing and oxidizing power through the photoexcited

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electrons and holes under illumination.11 To date, TiO2 is the most intensively studied

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semiconductor since it is inexpensive and easily synthesized.12 Photocatalytic nitrate

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reduction has been reported in TiO2 systems,13, 14 but the selectivity of N2 formation is

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low and the over reduction of nitrate to ammonium ions is particularly undesired.

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Although the

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TiO2/Ag, TiO2/Ni-Cu, etc.,13, 15 the cost of the catalysts would inevitably increase,

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while the formation of ammonium is still not negligible.15 On the contrary, microbial

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denitrification usually has high selectivity.

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formation was reported to be at very low level or even not detected.8, 16 Considering

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that the conduction band of TiO2 (-0.5 V vs SHE at pH 7) lied at an energy level that

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is noticeably higher than that of nitrate reduction, the photo-excited electrons should

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have energy high enough to drive EDeN without additional energy input, such as

selectivity is improved by doping other metals, such as TiO2/Pt,

Regarding the EDeN, the ammonium

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power supply as in the previous study.17 Therefore, we hypothesize that combining the

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TiO2 semiconductor system with the EDeN will gain a high selectivity for reducing

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nitrate to N2 and the occurrence of this process can solely rely on the illuminating

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dose. In such a novel photoelectrotrophic denitrification (PEDeN) process, the

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generation of photoelectrons is independent of the presence of organics, which brings

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about the PEDeN particularly promising in the denitrification under organics limited

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conditions. To the best of our knowledge, the proposed PEDeN here has not been

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verified yet.

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Herein, the PEDeN was developed by constructing a photoelectrochemical cell,

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where a denitrification biocathode was connected to a TiO2 photoanode without

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voltage supply (Figure 1a). The objectives of this study are i) to demonstrate the

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PEDeN is capable of efficiently reducing nitrate to nitrogen without additional

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reductant and energy input; ii) to understand the performance of PEDeN by

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comparing with the already established electricity driven EDeN system (EEDeN, also

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known as electrochemical bioreactor of denitrification, Figure 1b) and organics driven

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EDeN system (OEDeN, also known as denitrification microbial fuel cell, Figure 1c).

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Materials and Methods

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Reactor construction. The reactor was designed as three chambers which were

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separated by cation exchange membranes (Ultrex CMI-7000,

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International, U.S.). The cathode chamber was arranged in the middle, while the two

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side chambers were set as photoanode chamber and bioanode chamber, respectively 5

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(Figure 1). The side plate of the photoanode chamber was equipped with a quartz

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glass window (6 cm in diameter) that allowed the light passage. The TiO2/Ti plate

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(surface area =16 cm2), graphite granules (3~4 mm in diameter) and carbon brush

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were employed as the electrode materials in each chamber as the photoanode,

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biocathode and bioanode, respectively. The working volumes of these three chambers

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were 230 mL, 100 mL and 180 mL respectively. The biocathode was connected to the

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photoanode in the PEDeN test while to the bioanode in the OEDeN test. A 10 Ω

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resistance was inserted into the electric circuit in series to allow the sampling of

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current. In case of the EEDeN test, the TiO2/Ti plate was replaced by a piece of

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carbon paper with the same projected area. The biocathode at this moment was

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connected to the carbon paper electrode through a DC power. The operation manners

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of this three-chamber reactor as PEDeN, OEDeN and EEDeN were schematically

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illustrated in Figure 1. Each chamber was equipped with a Ag/AgCl reference

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electrode (saturated KCl, 197 mV vs standard hydrogen electrode, SHE) to measure

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the potentials of the corresponding electrodes.

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Preparation of TiO2/Ti photoanode and denitrification biocathode. The TiO2/Ti

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photoanode was prepared by a modified sol-gel and spin-coating method as described

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previously by Dong et al.18 Briefly, 38 mL solution A (76% C2H5OH, 9% H2O and 15%

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CH3COOH) was added dropwise to 62 mL solution B (54% Ti(OC4H9)4 and 46%

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C2H5OH). Nitric acid was then added in the mixture for adjusting the pH to 1~2. This

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solution was further kept at room temperature for 24 h for the sol-gel aging.

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Subsequently, the prepared sol-gel was spin-coated in three layers on the Ti sheet and 6

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calcined for 2 h at 500 °C in a furnace (KSL-1200X, MTI KJ Group, Hefei, China).

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The denitrification biocathode was acclimated in the above-mentioned three-chamber

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reactor by coupling a bioanode which

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bioelectrochemical system using acetate as the electron donor. The active sludge

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obtained from local domestic wastewater treatment plant was used as the inoculum for

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the biocathode. The modified M9 nutrient medium (pH=7) amended by 10 mL·L-1

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Wolfe’s trace elements and KNO3 (NO3-N=35 mg·L-1) was employed as the catholyte,

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while the same solution but replacing the KNO3 by 500 mg·L-1 sodium acetate as the

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anolyte. Both of these solutions were prior deoxygenated using argon gas (purity >

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99.9%) and then were continuously fed into the chambers with a flowrate of 120

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mL·d-1. The cathode and the anode were connected through a 10 Ω resistance. Once

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the current generation was observed and kept stable, the acclimation of denitrification

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biocathode was considered to be successful.

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Operation. For the PEDeN, OEDeN and EEDeN tests, the reactors were operated as

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batch-fed mode. In the PEDeN test, the denitrification biocathode was coupled with

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the TiO2/Ti photoanode. The cathode chamber was fed with the same solution as in

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the acclimation stage except the inoculum, while the anode chamber was just fed with

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the modified M9 nutrient medium. A xenon lamp (PLS-SXE300UV, PerfectLight,

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Beijing, China) was used as the light source and equipped with a 380-nm (±20 nm)

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bandpass filter (DT380, PerfectLight, Beijing, China). The intensity of the

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illumination was adjusted to be 30 mW·cm-2, 45 mW·cm-2 and 60 mW·cm-2

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respectively. In order to understand the effect of the illumination on the denitrification,

was pre-cultured in a separate

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the xenon lamp was switched on/off to provide a light/dark condition. In parallel, the

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reactors equipped with the abiotic cathode were set to explore the role of bacteria in

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denitrification. For EEDeN and OEDeN tests, the experiments were carried out under

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dark conditions with the modified M9 nutrient medium and that amended by sodium

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acetate (0, 68, 135, 270, 405, 540 mg·L-1) as the anolyte, respectively. In PEDeN and

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EEDeN tests, the bioanode chamber was just filled with phosphate buffered solution

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to exclude the possibility of acetate diffusing to the biocathode chamber. All of the

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above-mentioned experiments were conducted with replicate reactors in a temperature

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controlled laboratory (~28°C). For each condition, the reactors were operated at least

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3 runs.

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Analysis and Calculation. The TiO2/Ti photoanode and denitrification biocathode

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were electrochemically characterized through linear scan voltammetry (LSV) and

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cyclic voltammetry (CV), respectively, using an electrochemical working station

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(model-660D, CHI Instruments Inc. Shanghai, China). The LSVs for photoanodes

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were performed from the potential of -0.5 V to 1.2 V (versus SHE) with a scan rate of

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10 mV·s-1. The CVs for denitrification biocathode were carried out with a scan rate of

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0.5 mV·s-1 in the potential window between -0.4 V and 0.8 V (versus SHE). The

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morphology of the TiO2/Ti photoanode and cathode biofilm were examined by a field

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emission scanning electron microscope (FESEM, SU8020 Hitachi, Ltd, Japan). The

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crystallographic structure of the TiO2 thin film was characterized by an X-ray

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diffraction (PANalytical PW 3050 Philips X’pert Pro). The electrode potential and the

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current flowing through the sampling resistance were recorded every 10 minutes by a 8

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data acquisition system (Model 2700, Keithley Instru. Inc., U.S.). The NO3- and NO2-

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were measured using an ion chromatograph (883 Basic IC plus. Metrohm,

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

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Switzerland). The concentration of N2O in the headspace of cathode chamber (~10

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mL) was determined using a gas chromatographic mass spectrometry as described in

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Cheng et al.19 The N2O concentration in the liquid phase was calculated according to

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that in the gas phase using Henry’s law. The reported aqueous N2O concentration was

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modified by adding those N2O in the gas phase. The NH4+ ions were detected

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according to the Nessler reagent colorimetry method. The sodium acetate was

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measured by a high performance liquid chromatography (HPLC, e2695, Waters Co.,

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U.S.) equipped with a Aminex HPX-87 column (Bio-Rad Laboratories, Inc, U.S.) at a

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wavelength of 210 nm. The current efficiency for denitrification at cathode was

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calculated as the ratio of the theoretical numbers of electron transfer basing on the

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transformation of nitrogen compounds to the observed numbers of electron transfer in

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the batch-fed experiments:

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current efficiency =

           )    ]×× [ ×(   



 !"# 



(1)

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' #   where $%& (mM) is the initial nitrate nitrogen concentration. $%& (mM)  %  %

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and $%# &% (mM) are the concentrations of nitrite nitrogen and nitrous oxide

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nitrogen, respectively at t (s) time. V (L) is the working volume of the cathode

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chamber. I (mA) is the recorded current. F is the Faraday’s constant (96485 C·mol-1).

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5 is the electron transfer number of nitrate to nitrogen gas. 2 is the electron transfer

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number of nitrate to nitrite. 4 is the electron transfer number of nitrate to nitrous 9

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oxide.

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Results

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Characterization of TiO2 photoanode and denitrification biocathode. As shown in

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Figure 2a, the sol-gel formed a film of TiO2 on Ti sheets. This TiO2 film has visible

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cracks on it, which was consistent with the previous studies and would likely provide

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a large reaction surface.20,

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crystallographic structure of the TiO2 is indexed as the anatase phase. Current

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generation as a function of electrode potential is shown as in Figure 2c. Negligible

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photocurrent was observed under dark condition, while photocurrent produced when

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the electrode potential was positively scanned to -0.25 V vs SHE and finally reached

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4.5 mA at 1.2 V vs SHE (scan rate of 10 mV·s-1). Since the performance of

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light-to-current conversion could be varied among individual prepared photoanode

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(data not shown), only those photoanodes with similar photoelectrochemical behavior

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were used for the following experiments.

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According to the XRD analysis (Figure 2b), the

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In terms of the acclimation of denitrification biocathode (Figure 2e), the current

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generation presents a lag time of two days and then rapidly increases from day 2 to

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day 8. Subsequently, the current kept stable for 4 days at around -3.5 mA. These

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results suggest the successful development of denitrification biocathode. The

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denitrification biocathode was then electrochemically characterized by the CV test

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with the scan rate at 0.5 mV·s-1. As shown in Figure 2f, an obvious reduction peak is

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observed in biocathode CV with the presence of nitrate, which has the onset potential

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and peak potential at around 0.15 V and 0 V (versus SHE), respectively. As the 10

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control, CV of abiotic cathode was also performed, which showed negligible faradaic

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current, clearly indicating the peak related to nitrate reduction in the biocathode CV is

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a result of microbial catalysis. At the end of the denitrification biocathode acclimation,

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the graphite granules were characterized by SEM, which showed the coverage of the

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biofilm (Figure 2d).

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Denitrification driven by light. Nitrate concentration as well as the photocurrent was

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monitored in the ON-OFF intermittent illumination test. As shown in Figure 3a, the

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nitrate reduction of PEDeN system is coordinated with the current generation and

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only observed under illumination. This result clearly reveals the nitrate reduction is

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capable of being driven by light. Control test by coupling the photoanode with an

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abiotic cathode was carried out under the same ON-OFF intermittent illumination

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condition, in which the dosing of illumination did not arouse current generation. As a

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result, the change of nitrate concentration at the cathode chamber was negligible.

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These observations confirm that the nitrate reduction in PEDeN system depends upon

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both of the illumination dosing at photoanode and the microbial activity at cathode.

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Upon illumination, the reduction of nitrate followed the pseudo first-order reaction

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with a kinetic constant of 0.13±0.023 h-1 (n=4, fitted according to Figure 3b). During

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the nitrate reduction, the accumulations of nitrite and nitrous oxide were observed,

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which is consistent with the previous works.22, 23 These denitrification intermediates

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depleted after approaching to a maximum concentration. In all batch tests, ammonium

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was always below the detection limit, which is 0.02 mg·L-1 (Nessler reagent

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colorimetry method), indicating the nitrate was selectively reduced to nitrogen. As 11

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shown in Figure 3c, initially photocurrent keeps at about 1.5 mA and then declines

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after 12 h. With the exhaustion of oxidized nitrogen, the photocurrent declined to zero.

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The current efficiency at cathode was calculated and its value for denitrification was

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97.02±1.4% (n=4) at the end of the batch tests. Cathode potential during the

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experiments fell into the range between 0.05 V and -0.03 V (versus SHE), which is

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more positive than the theoretical potential of hydrogen evolution and consequently

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confirms the denitrification does not rely on the hydrogen as the electron mediator. In

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addition to hydrogen evolution, other side reactions, such as the CO2 reduction to

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methane or organics, can be also excluded because they were thermodynamically

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unfavorable (i.e. the standard potentials for CO2 reduction to methane and organics at

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pH 7 are about -0.24 V vs SHE and -0.28 V vs SHE, respectively, which are more

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negative to the cathode working potential of PEDeN). Therefore, the small numbers

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of electron loss here is likely due to the consumption by microbial growth.

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As mentioned above, the photocurrent kept stable but not declined with the

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depletion of oxidized nitrogen compounds during the initial 12 hours. This suggests

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the rate-limiting factor of denitrification in the current set of PEDeN was not come

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from the cathode side but more likely due to the low electrons providing capability of

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the photoanode. In order to get insight to this issue, PEDeN was further tested under

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higher illumination intensities (45 mW·cm-2 and 60 mW·cm-2). As shown in Figure S1,

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both of the photocurrent and denitrification rate enhanced with increasing the

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illumination intensity. When the illumination density got to 60 mW·cm-2, the

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denitrification rate constant reached 0.15±0.02 h-1 (n=2), which was 36.36% higher 12

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than that observed under 30 mW·cm-2 illumination. Compared to the PEDeN operated

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at lower illumination intensity, no substantial difference of the current efficiency was

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observed at higher illumination intensity (96.86±1.8%, n=4, 60 mW·cm-2). Under

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high illumination intensity condition, the variation of photocurrent followed the

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identical trends as that of total nitrogen, indicating the denitrification rate-limiting

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factor switched to the cathode side (i.e. controlled by the available oxidized nitrogen

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compounds serving as electron acceptors).

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PEDeN verus OEDeN and EEDeN. As the reactors employed in this study have a

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three-chamber configuration, the denitrification performance in PEDeN, OEDeN and

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EEDeN can be compared using the same denitrification biocathode. In case of

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OEDeN, the denitrification biocathode was coupled by an acetate consuming

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bioanode. As shown in Figure 4a, the performance of OEDeN is sensitive to the

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COD/N (g COD/g NO3--N) ratio. The total nitrogen (TN) removal efficiency of

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OEDeN can be comparable to that of PEDeN only at COD/N ratio higher than 9.0.

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The number of electrons transferred, in terms of coulombs, to biocathode are plotted

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in Figure 4a, which also shows a positive correlation to the COD/N ratio, indicating

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the inferior TN removal efficiency of OEDeN at low COD/N ratio was due to the

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lacking of electron donors. The theoretical COD/N ratio for completely converting

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nitrate to nitrogen gas should be 2.86 in OEDeN based on the stoichiometry. However,

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the electron equivalents could be lost for the microbial assimilation or competitive

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methanogenesis at the bioanode.24 Thus, the observed COD/N can be always higher

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than the theoretical value. The COD/N ratio obtained in our study falls in the same 13

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order of magnitude as that reported by others (4.5-16 g COD to g NO3--N).10, 25

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In the EEDeN test, the TiO2/Ti photoanode was replaced by a piece of carbon paper

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with the same projected area. Without the applied voltage, the current generation was

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negligible (Figure S2b). This is because the potential of electrochemical oxygen

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evolution is more positive than that of the nitrate reduction. Although carbon dioxide

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formation at a carbon based electrode is thermodynamically possible at more negative

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potential (