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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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Environmental Science & Technology

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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

6

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

9

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

11

c

12

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

14 15 16 17 18 19 20 21 22 23 1

ACS Paragon Plus Environment


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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

2, 3

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

4


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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


Membranes


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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

7



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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.

21

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

272

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,

285

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

290

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

291

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

292

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

294

potential (

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