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Stimulation of oxygen to bioanode for energyrecovery from recalcitrant organic matter anilinein microbial fuel cells (MFCs)

Hao-Yi Cheng a,b,1, Bin Liang a,1, Yang Mu c, Min-Hua Cui b, Kun Li b,Wei-Min Wu d, Ai-Jie Wang a,b,*

a Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy

of Sciences, Beijing 100085, PR Chinab State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (SKLUWRE, HIT),

Harbin 150090, PR Chinac CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science & Technology

of China, Hefei 230026, PR Chinad Department of Civil and Environmental Engineering, William & Cloy Codiga Resource Recovery Center, Center for

Sustainable Development & Global Competitiveness, Stanford University, Stanford, CA 94305-4020, USA

a r t i c l e i n f o

Article history:

Received 12 December 2014

Received in revised form

5 May 2015

Accepted 6 May 2015

Available online 12 May 2015

Keywords:

Microbial fuel cell

Oxic bioanode

Aniline

Oxygen

Energy recovery

* Corresponding author. Key Laboratory ofAcademy of Sciences, Beijing 100085, PR Chi

E-mail addresses: waj0578@hit.edu.cn, aj1 These authors contributed equally to thi

http://dx.doi.org/10.1016/j.watres.2015.05.0120043-1354/© 2015 Elsevier Ltd. All rights rese

a b s t r a c t

The challenge of energy generation from biodegradation of recalcitrant organics in mi-

crobial fuel cells (MFCs) is mainly attributed to their persistence to degradation under

anaerobic condition in anode chamber of MFCs. In this work, we demonstrated that

electricity generation from aniline, a typical recalcitrant organic matter under anaerobic

condition was remarkably facilitated by employing oxygen into bioanode of MFCs. By

exposing bioanode to air, electrons of 47.2 ± 6.9 C were recovered with aniline removal

efficiency of 91.2 ± 2.2% in 144 h. Limited oxygen supply (the anodic headspace was initially

filled with air and then closed) resulted in the decrease of electrons recovery and aniline

removal efficiency by 52.5 ± 9.4% and 74.2 ± 2.1%, respectively, and further decline by

respective 64.3 ± 4.5% and 82.7 ± 1.0% occurred under anaerobic condition. Community

analysis showed that anode biofilm was predominated by several aerobic aniline degrading

bacteria (AADB) and anode-respiration bacteria (ARB), which likely cooperated with each

other and finally featured the energy recovery from aniline. Cyclic voltammetry indicated

that anodic bacteria transferred electrons to anode mainly through electron shuttle. This

study provided a new sight to acquaint us with the positive role of oxygen in biodegra-

dation of recalcitrant organics on anode as well as electricity generation.

© 2015 Elsevier Ltd. All rights reserved.

Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinesena. Tel./fax: þ86 10 62915515.wang@rcees.ac.cn (A.-J. Wang).s work.

rved.

Fig. 1 e Hypothesis of facilitating energy recovery from

aromatic amine with assistant of oxygen.

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 3 73

1. Introduction

In past decade, microbial fuel cells (MFCs) have been tested as

a promising technique for wastewater treatment with energy

recovery. However, most successful studies were performed

with easily biodegradable organics (e.g., volatile fatty acids

and carbohydrates) to generate extracellular electrons (Pant

et al., 2010). The energy recovery from recalcitrant organics,

especially the aromatic and heterocyclic compounds, is still

difficult in MFCs. The challenges are mainly attributed to that

these compounds persisted in anaerobic condition which is

normally employed in anode chamber of MFC. Although

anode might replace oxygen as an alternatively terminal

electron acceptor, similar as nitrate and sulfate, that enabled

the microbial anaerobic utilization of recalcitrant organics

(Philipp and Schink, 2012; Zhang et al., 2009), it cannot fulfill

the function of oxygen that activates the oxygenases which

are essential for the ring structure cleavage (Field, 2002).

Hence, the energy recovery from the recalcitrant organics

does not always work in MFCs (Catal et al., 2008).

Aromatic amines are typical recalcitrant compounds under

anaerobic condition (Field, 2002), and persist in MFC bioanode,

as they were detected as final products in MFC feeding with

azo compounds (Fernando et al., 2014). In addition to anode

side involved degradation, the cathode of MFC or MEC (mi-

crobial electrolysis cell, operated with applying voltage) was

also proposed to reductively degrade azo/nitro compounds.

When their daughter products, aromatic amines, went

through bioanode, further degradation was inactive as well

(Cui et al., 2012; Wang et al., 2012b). Therefore, despite of

which operation mode, additional aerobic treatment was

required to further mineralize the produced amines, which

could lead to the increase in operational and capital costs. If

aromatic amines are available as electron donors in the bio-

anode, energy efficient mineralization of their parent com-

pounds in a single MFC or MEC would be possible.

For years, bioanode has been operated under anaerobic

condition because oxygen is considered as a competitive

electron acceptor. Deterioration of current production did

occur when easily degrading electron donors, such as acetate,

glucose etc., are used in the presence of oxygen (Chen et al.,

2014; Ringeisen et al., 2007). However, other studies also re-

ported that oxygen stimulated bacterial growth or helped in

the expansion of substrate utilization range, and conse-

quently enhanced the current generation capability in MFCs

(Biffinger et al., 2009; Rosenbaum et al., 2010). In case of aro-

matic amine, their degradation is much more flexible and

more efficient under aerobic condition than anaerobic condi-

tion (Kahng et al., 2000; Tan et al., 2005). Several pure or mixed

bacterial cultures use various aromatic amines as sole carbon

and energy sources (Chen et al., 2012; Juarez-Ramirez et al.,

2012; Sheludehenko et al., 2005). Organic acids, such as py-

ruvate or succinate, were found as intermediates during aer-

obic aniline degradation (Li et al., 2012; Liu et al., 2002). These

organic acids can easily serve as electron donor for bioanode

(Kan et al., 2011;Milliken andMay 2007). Thus, we hypothesize

that a) the energy extraction or recovery from aromatic amine

removal would be facilitated by introducing oxygen to bio-

anode in MFC because oxygen helps biotransformation or

conversion of aromatic amines to non-ring organic products

that can be utilized by ARB; and b) the delivery of oxygen

should be controlled at a desired level to reduce the electrons

loss through oxygen as the competitive electron acceptor. The

hypothesis is illustrated in Fig. 1.

In this study, we selected aniline as the model aromatic

amine. The MFC performance with aniline as sole electron

donor was evaluated and compared under oxic and anaerobic

anodic conditions in terms of anode biofilm acclimation,

electricity conversion capability and aniline removal effi-

ciency. As our results indicated that power generation was

enhanced in MFC with oxic bioanode (O-bioanode), cyclic

voltammetry (CV) and high-throughput 16S rRNA gene based

Illumina MiSeq sequencing were employed to investigate the

electron transfer mechanism and the microbial community

structure of the O-bioanode. Furthermore, the role of oxygen

in current generation from aniline was also discussed.

2. Materials and methods

2.1. MFC construction and operation

In this study, MFC reactor consisted of two identical glass

chambers (175 mL for each) that were modified from the

commercial serumbottles and separated by a cation exchange

membrane with a working area of 7 cm2 (Ultrex CMI-7000,

Membranes International, U.S.) as shown in SI, Fig. S1. Car-

bon brushes (about 50 cm3 in apparent volume), binding car-

bon fibers (T700 3000K, Toray Co., Ltd., Japan) into two twisted

titanium wires with 1 mm in diameter (Baoji Lixing Titanium

Group Co., Ltd., China), were punctured through the rubber

stopper and deployed into both of the chambers as anode and

cathode. One tube connected with valve was also inserted

through the rubber stopper into the top of anode chamber to

meet the requirement of various operation conditions as

explained below. All contacted parts of the rubber stopper to

the titanium wires, tubes and the chambers were well sealed

by the epoxy glue. A saturated calomel electrode (SCE, 247 mV

vs standard hydrogen electrode, SHE.) was employed as the

reference electrode to measure anode potential, with all po-

tentials reported vs SHE in the study.

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 374

In the anode biofilm acclimation stage, 100 mL aniline

amended (2.8 ± 0.1 mM, as sole electron donor) nutrient me-

dium (50 mM PBS, 0.2 g/L NH4Cl, 0.13 g/L KCl, 10 mL/L Wolf'svitamins, 10mL/LWolf's trace elements, pH¼ 7)was filled into

the anode chamber, which was then inoculated with active

sludge (15 mL) obtained from a domestic wastewater treat-

ment plant, Harbin, China, and the effluent (15 mL) from a

volatile fatty acid feeding MEC. As a result, 130 mL aqueous

working volume and 45 mL headspace were provided in the

anode chamber. The cathode chamber was filled with 130 mL

PBS (50 mM, pH ¼ 7) amended with potassium ferricyanide

(100 mM) as electron acceptor. Subsequently, the anode and

cathode were connected via a 1000U resistor unless otherwise

specified. The generated current was recorded using a data

acquisition system (Model 2700, Keithley Instru. Inc., U.S.),

which automatically converted the measured voltage to cur-

rent based on the ohm's law. The anode solution was

completely replaced by 130 mL aniline amended nutrient

medium once the current decrease was observed in each

experiment.

The current generation capabilities of MFCs with bioanode

in the presence or absence of oxygen were evaluated by

switching on and off the air gas valve of anode chamber (see

SI, Fig. S1). In order to achieve anaerobic condition, anolyte

was flushed with nitrogen gas (purity > 99.9%) for 20 min prior

to each run. For the control, MFCswere set up eitherwith open

circuit or without inoculation. To further explore the impact of

oxygen on the O-bioanode, after the repeated current gener-

ation was achieved, a MFC with the O-bioanode was then

switched to anaerobic mode (i.e., the anodic headspace was

filled with only nitrogen gas) or oxygen supply limited mode

(i.e., the anodic headspace was filled with air and then the

headspace was closed by turning off gas valve to cut up

further oxygen invasion from outside source). At the end of

MFC run under oxygen supply limited condition, the gas

pressure in anodic headspace was measured by a U type

pressure gauge (±20 KPa, Shanghai Tianlei Instrument Co.,

Ltd., China), in which the gas valve was connected to the

pressure gauge and then was opened.

All of the experimentsmentioned abovewere performed at

ambient temperature (~26 �C) in triplicate reactorswhichwere

operated at least 3 runs in each series.

2.2. Electrochemical analysis

Polarization tests were performed when the maximum cur-

rent output was observed in each experiment. MFCs were

operated under open circuit condition for 2 h prior to the test,

and then sequentially connected to various resistances with

decreasing order (each for 15 min) until obtaining the

maximum power density according to the calculations based

on the external resistance and the detected output voltage.

Cyclic voltammetry (CV) was analyzed using an electro-

chemical working station (model-660D, CHI Instruments Inc.

U.S.) equipped with 3-electrode system and SCE as reference

electrode. When the MFC anode was used as the working

electrode, the MFC cathode was employed as the counter

electrode. Before starting CV, external circuit of MFC was

opened for 2 h, and then scanning from �0.243e0.252 V at the

rate of 1 mV/s. To further explore the electron transfer

mechanism involved in the O-bioanode, CVs were also per-

formed in a 15 mL glass cell which was filled with 10 mL PBS

buffer solution, catechol amended nutrient medium, fresh

anolyte, or anolyte supernatant prepared at the end of runs.

Here, a glassy carbon electrode (diameter: 3 mm, working

area: ~7 mm2) and a graphite rod (4 mm in diameter) were

used as the working and counter electrode, respectively. Prior

to the CV tests, the electrolyte was deoxygenated by flushing

N2 gas for 20 min and the glassy carbon electrode was pre-

treated as described previously (Peng et al., 2010). All electro-

chemical analysis was conducted at ambient temperature

(~26 �C).

2.3. Chemical analyses and calculations

Samples taken from the MFC anode chamber were filtered

through 0.22 mmfilters. Aniline concentrationwas determined

using an HPLC as described previously (Wang et al., 2011).

Dissolved oxygen (DO) in anolyte was measured using a DO

sensor (FDO-925, WTW GmbH, Germany) that was inserted

into the anode chamber through a short connected glass tube

with inner diameter of 16 mm (see SI, Fig. S1). Concentration

of total organic carbon (TOC) in anolyte was measured by a

TOC analyzer (Fusion TOC, Tekmar-Dohrmann, U.S.).

Power density (P, W/m3), aniline degradation efficiency

(EAN, %), cumulative electric charge through the external cir-

cuit (Qi, C), and coulombic efficiency (CE, %) were calculated to

evaluate the performance of MFCs as followings:

P ¼ I2 � RVa

(1)

EAN ¼ CoAN � CAN

CoAN

� 100% (2)

where I is the current (A), R is the external resistance (ohm), Va

is the apparent anode volume (m3), C0AN is the initial aniline

concentration in anolyte (mM) and CAN is the detected aniline

concentration at different run time (mM); and

Qi ¼Xn

i¼1Ii � ti (3)

CE ¼ 1000� Qi�CoAN � CAN

�� V � 28� F� 100% (4)

where Ii (A) is the current at time ti (s), V is anodic liquid vol-

ume (L), F is the Faraday's constant (96,485 C/mol electron),

and 28 is the theoretic electrons transfer number when the

organic carbon of aniline is completely mineralized to CO2 as

following equation.

C6H5NH2 þ 12H2O/6CO2 þNHþ4 þ 27Hþ þ 28e� (5)

2.4. Microbial morphology and community analysis

Anode biofilmmorphology was examined by using a scanning

electronmicroscope (SEM) (Helios NanoLab 600i, FEI Company,

U.S.). Samples taken from the anode were pre-treated as

described previously (Wang et al., 2012a). Anode biofilms

genomic DNA was extracted according to a previous method

with some modifications (Zhou et al., 1996). DNA purity and

quantity were determined by a PicoGreen using FLUOstar

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 3 75

Optima (BMGLabtech, Jena, Germany) andby aNano-DropND-

1000 Spectrophotometer (NanoDrop Technologies Inc., Wil-

mington, DE, U.S.). The primers 515F (50-GTGCCAGCMGCCGCGGTAA-30) and 806R (50-GGAC-TACHVGGGTWTCTAAT-30) which targeted V4 hypervariable

regions of bacterial 16S rRNA genes were selected (Caporaso

et al., 2012). Both forward and reverse primers were tagged

with adapter, pad and linker sequences and each barcode

sequence was added to the reverse primer. The detailed in-

formation for the used primers was described in SI (Table S1).

PCR amplification, PCR products purification and quantifica-

tion, sequencing with Illumina MiSeq platform (at Institute for

Environmental Genomics of the University of Oklahoma, Nor-

man, OK, U.S.) and data analysis were described elsewhere

(Liang et al., 2014).

3. Results and discussion

3.1. Bioanode acclimation with aniline as sole electrondonor in the presence or absence of oxygen

When anodewas exposed to air (Fig. 2), after 50-hr inoculation

the current started increasing and reached to a peak value of

0.205 V at hr 132. The similar current generation profiles were

then observed when the anolyte was completely refilled with

aniline as sole electron donor, indicating that the O-bioanode

was successfully developed. The short lag time obtained here

might be attributed to electrochemically active bacteria

already existing in the inoculum. Compared to the O-bio-

anode, the MFC with anaerobic anode produced much lower

current especially after anolyte was refilled (Fig. 2). The dra-

matic current drop here was likely due to that some biode-

gradable substrates originated from the inoculum were

removed by replacing the anolyte. The recorded current of the

MFC with the anaerobic anode was only 0.02 mA with aniline

as sole electron donor and quite close to that observed in the

abiotic anode control, implying that the acclimation of bio-

anode was not successful under anaerobic condition.

Fig. 2 e Current profiles of bioanode acclimation by

exposing to air or not. Arrow: Renewing anolyte with

aniline as sole electron donor (aniline concentration was

2.8 ± 0.1 mM).

Afterwards, we tested the development of bioanode under

anaerobic condition for one month, but failure since no

notable increase of current was observed (data not shown).

The degradation of aniline under anaerobic condition was

reported with starting at the transformation to 4-

aminobenzoate through carboxylation, which was then acti-

vated to 4-aminobenzoyl-CoA (Schnell and Schink, 1991). The

standardGibbs freeenergyof thefirst reactionwasestimated to

be 49.28 kJ/mol (>0, details described in SI) and implementation

of the second reaction required to consume ATP energy, indi-

cating the initial stage of aniline utilization by bacteria was not

thermodynamically favorable. This was consistent with the

observed failure of bioanode acclimation with aniline as sole

substrate in the absence of oxygen in this study.

3.2. Effect of oxygen on MFC performance with the O-bioanode

MFC reactors with the mature O-bioanode were operated

under different oxygen exposure conditions, including head-

space oxygen exposure (i.e., the gas vale of anode chamber

kept opening to connect the anode headspace to outside at-

mosphere), limited oxygen supply (i.e., the anode headspace

was filled with air initially and then closed by turning off the

gas valve) and no oxygen (anaerobic) in the anode chamber to

examine the impact of introducing oxygen on energy gener-

ation and aniline degradation.

When the anode was exposed to outside atmosphere, the

currentwas generated alongwith the degradation of aniline in

MFCs. Aniline removal reached up to 91.2 ± 2.2% after 144 h

and the peak current obtained was around 0.15 mA (Fig. 3).

After aniline degradation for 144 h, electrons of 47.2 ± 6.9C

were recovered and CE was calculated as 5.1 ± 0.5% (Table 1).

The polarization curve was carried out when the current

reached up to the peak in the independent runs and the

maximum power density achieved to 1.71 ± 0.5 W/m3 (Table

1). The dissolved oxygen (DO) concentration was measured

as 0.61 ± 0.17 mg/L at the beginning of the runs and decreased

to around 0.08 mg/L after 6 h (SI, Fig. S2).

Under the limited oxygen supply condition, no additional

oxygen entered the reactor headspace except initially filled

oxygen. The anodic headspace contained about 0.376 mmol

oxygen (assuming oxygen content was 21%, details of calcu-

lation is described in SI) at the beginning. As shown in Fig. 3b,

aniline removal was fast in the initial 24 h, but then became

slower. The final aniline removal efficiency was 23.5 ± 2.3%

(Table 1). Compared to the oxygen exposure condition, the

recovered electrons and the maximum power density were

decreased by 52.5 ± 9.4% and 16.9 ± 2.8%, respectively (Table

1). At the end of the run, the pressure loss in the headspace

was 17.7 ± 1.2 KPa, which equals gas loss by 0.322 ± 0.02mmol

(calculation is in SI) or is close to the oxygen contained in the

headspace at the beginning, suggesting that oxygen was

basically consumed during the aniline degradation process.

Additionally, DO concentration was only 0.01 mg/L at the end

of this run. This value was much lower than that measured

when the anode always exposing to air (~0.08mg/L) (SI Fig. S2),

indicating that the limitation of energy recovery and poor

aniline removal efficiency were resulted from the insufficient

oxygen supply.

Fig. 3 e Current generation (a) and time course of aniline

degradation (b) in MFCwith the O-bioanode under different

operation conditions. Before each condition, the anolyte

was completely renewed by fresh aniline amended

(2.8 ± 0.1 mM) nutrient medium. Current data reported

here was one of the triplicate settings for each condition.

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 376

When oxygen was completely removed and MFC anode

was operated anaerobically, further decline of aniline removal

efficiency and energy recovery were observed as expected.

During the run, only 15.8 ± 1.1% of aniline was removed after

144 h (Table 1). The recovered electrons and the maximum

power density were also much lower, which decreased by

64.3 ± 4.5% and 78.9% ± 1.5%, respectively, compared to that

under oxygen exposure condition (Table 1). The acclimation of

bioanode under anaerobic condition failed as mentioned

above. However, current output was observed in MFCwith the

matured O-bioanode. This result suggested that current

Table 1 e Performance of MFCs with the O-bioanodeunder different operation conditions.

Pmax

(W/m3)EAN (%) Qi (C) CE (%)

Anaerobic 0.36 ± 0.3 15.8 ± 1.1 16.6 ± 0.8 11.9 ± 0.5

Limited oxygen

supply

1.42 ± 0.4 23.5 ± 2.3 22.1 ± 4.2 11.3 ± 2.4

Oxygen exposure 1.71 ± 0.5 91.2 ± 2.2 47.2 ± 6.9 5.1 ± 0.5

generation from aniline under anaerobic condition seemed

possible once bioanode was acclimated, although the pro-

duced current was much lower. Here, endogenous respiration

of biofilm might provide energy to activate the initial ther-

modynamic unfavorable step of anaerobic aniline

degradation.

Although the above results clearly showed the positive

effect of introducing oxygen on energy recovery from aniline,

the effect of oxygen as a competitive electron acceptor was

still concerned. Based on the results of this study, the DO

concentration was only 0.08 mg/L, indicating that the supply

of oxygen was enough but also limited even the headspace

was opened. When the MFC was operated under limiting ox-

ygen supply condition (the headspace was closed), CE of

11.3 ± 2.4% was obtained. Under this condition, the oxygen

consumed (0.322 mmol) theoretically equals the calculated

aniline consumption of 0.046 mmol (or 0.354 mM) using the

Equation (6), which accounted to 53.8% of the total removed

aniline and indicated that the electrons flowed to oxygen was

about 5 times as much as that to current. Compared to MFC

operated under oxygen exposure condition, limiting oxygen

supply resulted in the increase of CE by 2.2-fold (Table 1),

which suggested that careful controlling oxygen supply could

be a strategy to improve CE. Additionally, as oxygen could

compete with anode as electron acceptor, electrons flowing to

anodewere just expected to be preferredwhen the anilinewas

closed to the anode. In this study, the apparent volume ratio of

anode to anolyte was only 39%, indicating that aniline

removed in most part of the anolyte was due to aerobic

degradationwith oxygen as final electron acceptor rather than

anode respiration. Optimization of CE is also expected by

increasing the volume ratio of anode to anolyte.

C6H5NH2 þ 7O2 þHþ/6CO2 þNHþ4 þ 2H2O (6)

3.3. Cyclic voltammetry of the O-bioanode

When the potential of the O-bioanode was swept positively,

current increased with a two-step profile at the potential of

�0.204 V and �0.041 V, respectively, and finally reached the

plateau (~2.5 mA) at 0.12 V (Fig. 4a). First derivative CV (DCV)

clearly showed that the thermodynamic driven activity for

oxidation had two potential windows approximately centered

at �0.155 V and 0.030 V, respectively (Fig. 4b). To further

explore the possible involved electron transfer mechanism,

the anolyte sample taken at the end of run was centrifuged

and the supernatant was analyzed by CV on a glassy carbon

electrode. As shown in Fig. 4c, two pairs of redox peaks were

observed, which had the midpoint potential of �0.027 V and

0.063 V, respectively. Compared to the DCV of the O-bioanode,

the potential window of these two redox peaks

(�0.10 Ve0.14 V) was consisted with that of the thermody-

namic driven activity at higher centered potential, indicating

that current production at higher potential may involve in the

mechanism of electrons shuttle. Since no redox peaks were

observed at potential below �0.1 V in supernatant CV, the

electron transfer at lower potential was more likely governed

by certain outermembrane-bound proteinwith redox activity.

As the working potentials of anode during MFC run were al-

ways over 0.3 V (see SI, Fig. S3), electron transfer through both

Fig. 4 e (a) CV of the O-bioanode; (b) DCV of the O-bioanode;

(c) CV of glassy carbon electrode in anolyte supernatant

taken at the end of run. Insert (c): subtracting by CV in fresh

aniline medium. CV of the O-bioanode was performed

when current output reached plateau. DCV of the O-

bioanode was obtained through first derivative analysis

from the O-bioanode CV. Scan rates were 1 mV/s and

10 mV/s for the O-bioanode CV and glassy carbon electrode

CV, respectively.

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 3 77

pathways was thermodynamically possible in this study.

Higher current increase was aroused by thermodynamic

driven activity centered at 0.030 V compared to that centered

at �0.155 V, indicating that extracellular electron transfer of

the O-bioanode was mainly involved in electrons shuttle.

CVs were also performed on the fresh anolyte (Fig. 4c), as

well as the nutrient medium amended by catechol (interme-

diate in aniline aerobic degradation) that was reported to

produce redox peak in CV (Fotouhi et al., 2008) (see SI, Fig. S4).

Because no obvious peak was detected at relative potential

window in these two control tests, the aforementioned two

pairs of redox peaks obtained in supernatant were likely

aroused by some microbial excreted redox compounds.

However, it was still not clear the type of electron mediators,

although the potential was in the range of the quinoid type

(�0.3 Ve0.3 V) (Rau et al., 2002). Additionally, most of reported

microbial produced electron mediator just showed one pair of

redox peak in CV and some other revealed two-pair, such as

riboflavin (Freguia et al., 2009; Mehta-Kolte and Bond, 2012;

Rabaey et al., 2005). Thus, it would be also interesting to un-

derstand if the two redox peaks come from single compound

or two independent compounds in future study.

3.4. Microbial community analysis of the anode biofilm

Illumina sequencing method was employed to analyze the

microbial community of biofilm on the O-bioanode (3 repli-

cates) as well as the inoculum (3 replicates). Over 20,000 high-

quality sequences for each sample were obtained. Compared

to the inoculum, the operational taxonomic units (OTUs)

numbers (richness, at 97% cutoff), Chao 1 estimator, Shannon-

Weaver index (H), invsimpson index (1/D) and Simpson

evenness in the anode biofilms were significantly decreased

(Table S2), indicating a relatively narrow diversified microbial

community was selected and developed to adapt the specific

anodic condition.

Hierarchical clustering analysis clear showed the signifi-

cant shift of microbial community structure from the inoc-

ulum to the anode biofilm (Fig. 5a). Based on the community

identification, Proteobacteria (45.57 ± 0.16%) and Bacteroidetes

(24.41 ± 0.47%) were the dominant groups in the inoculum,

whereas significant decrease of Bacteroidetes (to 7.85 ± 1.71%,

P ¼ 0.002) with the obvious increase of Proteobacteria (to

73.97 ± 6.89%, P ¼ 0.019) was observed in the biofilm samples.

The mainly enriched bacteria in the anode biofilm were

Acidobacteria, Chlorobi and Lentisphaerae which accounted to

5.74 ± 1.32% (P ¼ 0.072), 3.05 ± 5.24% and 2.06 ± 0.84%

(P ¼ 0.051) of the total sequences, respectively (Fig. 5b).

At genus level, we observed distinct predominant bacterial

components in the biofilm compared to the inoculum (Table 2,

relative abundance �1%). Several ARB were obviously

enriched in the dominant population, including Comamonas

(12.95 ± 5.21%), Aquamicrobium (7.85 ± 1.96%), Geothrix

(4.51 ± 0.92%), Geobacter (3.59 ± 1.43%) and Ochrobactrum

(0.99 ± 0.31) as reported by others (Mehta-Kolte and Bond,

2012; Srikanth et al., 2008; Xing et al., 2010; Xu et al., 2013;

Zuo et al., 2008). Geobacter sp. was the most widely studied

ARB or electrogens, which transferred electron to anode relied

on their outer-membrane bound cytochrome c. Cyclic vol-

tammetry analysis revealed that Geobacter sp. cloned bio-

anode had a characteristic thermodynamic driven activity

wave with the center potential at �0.15 V (Srikanth et al.,

2008), which was quite close to that observed in the present

study at lower centered potential. Although Geothrix fermen-

tans was well defined to secrete two types of electron media-

tors to feature its extracellular electron transfer, neither of

themmatched the putative electronmediators in this work by

potential (Mehta-Kolte and Bond, 2012). Notably, Comamonas,

the most abundant microbes in the anode biofilm, can

Fig. 5 e Hierarchical cluster analysis of bacterial communities in the anode biofilm and inoculum (a). Taxonomic

classification of 16S rRNA gene sequences from the anode biofilm and inoculum at the phylum level (b). The OTU numbers

were logarithmically transformed for the hierarchical cluster analysis. Brighter red coloring indicates higher OTU

abundances as shown in the colorbar. Relative abundance of each phylum was defined as the percentage of the same

phylum to the corresponding total sequences. Some phyla were <1% abundances and unclassified 16S rRNA gene

sequences were summarized as others. (For interpretation of the references to color in this figure caption, the reader is

referred to the web version of this article.)

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 378

produce current and degrade aniline aerobically (Peres et al.,

1998; Xing et al., 2010) and consequently may metabolize an-

ilinewith the assistant of oxygen and competitively use anode

as the terminal electron acceptor. Some other bacteria with

the ability to degrade aromatic amine aerobically were also

significantly enriched in the dominant population, such as

Variovorax (4.56 ± 0.62%), Stenotrophomonas (2.15 ± 0.82%), and

Diaphorobacter (2.20 ± 1.04%) (Table 2) (Breugelmans et al.,

2010; Zhang et al., 2010; Zissi and Lyberatos, 1999), which

were likely to participate in the aniline metabolism as well.

3.5. Possible mechanism involved in electricityproduction from aniline by MFC with the O-bioanode

As shown in Fig. 2, current production was lagged behind the

aniline degradation in the presence of oxygen. Dramatic in-

crease of current was usually observed after closing the circuit

for 12e24 h in each run. Although oxygen could play the role

as competitive electron acceptor and thus may inhibit current

output, it cannot explain such a long lag time as DO had

already decreased to around 0.08 mg/L in 6 h and showed a

stable trend in the following reaction period (see SI, Fig. S2).

Therefore, current generationmay not be directly from aniline

butmore likely from themetabolites of aniline degradation. In

order to provide evidences to support this assumption, the

change of TOC in addition to aniline was also monitored

during the experiments. As shown in Fig. 6, themeasured TOC

was always higher than the value calculated from aniline

(TOCAN) during the experiment. The difference between them

(i.e., TOCother) was accumulated initially and then reduced,

indicating the production of organic metabolites along with

aniline degradation. Compared to open circuit condition, cir-

cuit closing led to lessmetabolite accumulation, implying that

the produced metabolites were utilized through anode respi-

ration. Similar result was reported by Freguia et al. More COD

accumulation in open circuit condition than that in close

circuit condition was observed when the syntrophic interac-

tion of fermentative bacteria and anode-respiration bacteria

(ARB) was studied by using glucose as the substrate (Freguia

et al., 2008).

Further evidence can be found in Fig. 7 in which MFC

produced obvious higher currentwhen circuit was closed after

initially opening for 48 h (MFCO-C) compared to that was al-

ways operated by closing circuit (MFCC). According to

NernsteMonod model, current has positive correlation to

working potential and the concentration of electron donors

(Marcus et al., 2007). Since the working potential of MFCO-C

was lower than that of MFCC when producing higher current

Table 2 e Phylogenetic classification of the 16S rRNA gene sequences (relative abundance ≥1% at genus level) in the anodebiofilm and initial inoculum. The significant difference of dominant genera between biofilm and inoculum were analyzedby the two-tailed unpaired t-test (n ¼ 3). Any P value <0.1 was bolded.

Phylum Class Family Genus (%) Biofilm Inoculum P value

Proteobacteria b-proteobacteria Comamonadaceae Comamonas 12.95 ± 5.21 0.85 ± 0.02 0.057

Proteobacteria a-proteobacteria Phyllobacteriaceae Aquamicrobium 7.85 ± 1.96 0.01 ± 0.01 0.020

Proteobacteria b-proteobacteria Rhodocyclaceae Shinella 5.84 ± 2.95 0.02 ± 0.00 0.076

Proteobacteria g-proteobacteria Xanthomonadaceae Dokdonella 4.91 ± 0.94 1.63 ± 0.06 0.026

Proteobacteria b-proteobacteria Comamonadaceae Simplicispira 4.63 ± 2.05 0.35 ± 0.03 0.069

Proteobacteria b-proteobacteria Comamonadaceae Variovorax 4.56 ± 0.62 0.14 ± 0.04 0.006

Acidobacteria Holophagae Holophagaceae Geothrix 4.51 ± 0.92 0.44 ± 0.06 0.016

Proteobacteria b-proteobacteria Comamonadaceae Petrimonas 3.89 ± 2.20 0.00 ± 0.00 0.092

Proteobacteria d-proteobacteria Desulfuromonadales Geobacter 3.59 ± 1.43 0.03 ± 0.00 0.050

Proteobacteria a-proteobacteria Brucellaceae Ochrobactrum 0.99 ± 0.31 0.00 ± 0.00 0.031

Proteobacteria b-proteobacteria Comamonadaceae Acidovorax 2.21 ± 0.71 0.77 ± 0.03 0.072

Proteobacteria b-proteobacteria Comamonadaceae Diaphorobacter 2.20 ± 1.04 0.33 ± 0.02 0.090

Proteobacteria g-proteobacteria Xanthomonadaceae Stenotrophomonas 2.15 ± 0.82 0.01 ± 0.01 0.045

Proteobacteria a-proteobacteria Bradyrhizobiaceae Bosea 2.14 ± 0.61 0.03 ± 0.00 0.027

Lentisphaerae Lentisphaeria Victivallaceae Victivallis 2.06 ± 0.84 0.01 ± 0.01 0.051

Proteobacteria b-proteobacteria Alcaligenaceae Bordetella 2.03 ± 1.21 0.00 ± 0.00 0.102

Proteobacteria b-proteobacteria Sphaerotilus_f Aquabacterium 1.77 ± 0.86 0.22 ± 0.04 0.088

Proteobacteria d-proteobacteria Desulfovibrionaceae Desulfovibrio 1.64 ± 0.78 0.22 ± 0.01 0.088

Proteobacteria b-proteobacteria Rhodocyclaceae Thauera 1.43 ± 2.01 0.16 ± 0.02 0.390

Acidobacteria Holophagae Holophagaceae Holophaga 1.11 ± 0.51 0.05 ± 0.01 0.070

Proteobacteria ε-proteobacteria Helicobacteraceae Sulfurovum 0.00 ± 0.00 2.62 ± 0.02 <0.001

Proteobacteria b-proteobacteria Nitrosomonadaceae Nitrosomonas 0.00 ± 0.00 2.55 ± 0.09 <0.001

Proteobacteria b-proteobacteria Rhodocyclaceae Dechloromonas 0.63 ± 0.52 1.96 ± 0.14 0.040

Bacteroidetes Sphingobacteria Chitinophagaceae Terrimonas 0.02 ± 0.02 1.92 ± 0.11 <0.001

Bacteroidetes Sphingobacteria Chitinophagaceae Ferruginibacter 0.00 ± 0.00 1.24 ± 0.07 <0.001

Proteobacteria b-proteobacteria Rhodocyclaceae Ferribacterium 0.00 ± 0.00 1.23 ± 0.07 0.001

Bacteroidetes Sphingobacteria Saprospiraceae Haliscomenobacter 0.00 ± 0.00 1.19 ± 0.02 <0.001

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 3 79

(Fig. 7b), this higher current output should be attributed to

more electron donors existing in the anolyte. As suggested by

Fig. 6, both of the aniline and the putative metabolites have

higher concentration at hr 48 in MFCO-C than that in MFCC.

However, aniline didn't likely result in this higher current,

because even with higher aniline concentration (e.g. at hr 24),

the output current inMFCCwas still low. Therefore, the higher

Fig. 6 e Change of TOC with the mineralization of aniline

under close circuit condition (c) and open circuit condition

(B). TOCAN is calculated based on aniline concentration

(TOC ¼ 72/93.1 £ aniline concentration). TOCother was

obtained though TOC subtracting by TOCAN.

current output in MFCO-C should be contributed by the more

accumulated metabolites, which indicated that the accumu-

latedmetabolites in aerobic aniline degradation can be further

converted to current. Moreover, there was no lag time for

current output after the circuit was reclosed, suggesting these

metabolites were already available for anode respiration.

Additionally, some dominant ARB in the biofilm commu-

nity, such as Geobacter and Geothrix, don't contain aniline

dioxygenase and catechol dioxygenase, according to the an-

notated proteins from the reported genomes in the National

Center for Biotechnology Information (NCBI) database

(Tatusova et al., 2014). Nonetheless, they were enriched and

accounted for 8.1% of the community. Their survivals likely

thank to that oxygen helped the ring cleavage of aniline and

produced some non-ring organics as the available substrates.

We further evaluated how the O-bioanode responded to

the non-ring metabolite of aerobic aniline degradation under

anaerobic condition. Here we used pyruvate, a non-ring

metabolite in aniline degradation through catechol 2,3-

dioxygenase pathway which was suggested to be followed

by Comamonas, the most abundant bacteria in the biofilm

community of O-bioanode (Peres et al., 1998). As shown in

Fig. S5 (see SI), current was rapidly generated after closing the

circuit. This suggested that the anodic bacteria had been

capable of using pyruvate for current generation and this

capability was O2 independent. We are aware that the non-

ring metabolites involved in our O-bioanode could be more

diverse than pyruvate, as the mixed culture was used in this

work. However, since only the ring cleavage of aniline in

aerobic aniline degradation is O2-dependent, the observation

Fig. 7 e Current output (a) and the corresponding potential

of bioanode (b) in MFC with circuit initially opening and

then reclosing (MFCO-C). Arrow represent the circuit was re-

closed to a 1000 U resistance at hr 48. MFCC was operated

by always closing the circuit to a 1000 U resistance as the

control.

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 380

of O2-independent current production from pyruvate implied

that once aniline is converted to non-ring metabolites with

oxygen, the metabolites would easily serve as electron donor

for anode respiration. Further study should be carried out to

identify the involved metabolites and therefore understand

this synthropic interaction in depth.

The morphology of the O-bioanode is showed in Fig. 8. The

dense biofilm with average thickness of approximately 20 mm

was formed on the anode surface, which was believed to pro-

vide amoreanoxic condition insideandconsequently favor the

terminal electron transfer to anode other than oxygen.

3.6. Outlook

In this study, we demonstrated that aniline served as the sole

electron donor by introducing oxygen into anode chamber,

and energy recovery from aniline degradationwas achieved in

MFC. The results have conceptually proved that introducing

oxygen into bioanode enhances degradation of recalcitrant

organics rather than causes unfavorable condition for the

competition of electrons as widely assumed previously. For

electricity generation from complex compounds, syntrophic

interaction by anodic consortium was suggested as a strategy

to utilize the complex substrates as electron donors (Gruning

et al., 2014). In case of aniline as substrate, due to difficulty of

anaerobic degradation of aniline, introducing oxygen to anode

chamber can stimulated the activities of AADB to break down

aniline into intermediates which are then utilized by the ARB

to finally facilitate the current output under anaerobic con-

dition. In addition to aniline, many other aromatic amines

have the same problem for anaerobic degradation. We expect

that they can be converted to intermediates which serve as

electron donor resource if oxygen is introduced. In addition,

although several compounds with cyclic structure, such as

phenol and pyridine, were reported to produce current under

anaerobic condition, the acclimation stage requires the addi-

tion of co-substrate and poor columbic efficiencies (<10%)

were achieved, which is similar to that obtained in this work

(Luo et al., 2009; Zhang et al., 2009). Using O-bioanode may be

also promising for these compounds when lacking co-

substrate.

To date, MFC has been developed to diverse types of bio-

electrochemical systems (BESs), which have various func-

tions, such as hydrogen production, desalination, resource

recovery and recalcitrant contaminants reduction (Zhang and

Angelidaki, 2014). The concept of O-bioanode could also be

utilized in the BESs when the target electron donors are not

easy to be biodegraded under anaerobic condition.

In our previous work, we demonstrated that BES was a

promising pretreatment technology for nitrobenzene con-

taining wastewater, which involved in the efficient trans-

formation of nitrobenzene to aniline in the cathode side (Wang

et al., 2011). However, external organic carbon was needed for

bioanode to drive the cathode reaction, although the require-

ment had alreadymuch lower than anaerobic biologic process

(Mu et al., 2009b). As energy recovery from aniline has been

proved in this study, we propose a self-maintenancemode for

nitrobenzene reduction. In this mode, the aniline produced

fromnitrobenzene is fed back toO-bioanode as electrondonor,

and is mineralized. Based on the stoichiometry, self-

maintenance nitrobenzene reduction required the CE of ani-

line achieving 21.5% (nitrobenzene reduction to aniline

required 6 electrons, while the oxidation of aniline could pro-

vide 28 electrons in maximum). In addition to nitrobenzene,

the reduction of azo dye in BES also produced aromatic amines

(Mu et al., 2009a; Saratale et al., 2011). The self-maintenance

reduction feasibility of several azo dyes, previously investi-

gated in BES cathode, was evaluated by the same way as

nitrobenzene-aniline system.As shown inTable 3, CEs of these

amines to support their parent azo dyes reduction are much

lower, indicating that this proposed operation mode is more

feasible for azo dye compounds if their daughter amine prod-

ucts would be produced as electron donor at the O-bioanode.

The proposed approach should be examined in future study.

Based on above discussion, how to increase CE is a key issue

to further improve the O-bioanode. Besides the strategies to

control oxygen supply and to increase the apparent volume

ratio of anode to anodic solution as discussed in Section 3.2,

other effort can be also carried out by decoupling aerobic ani-

line degradation and the anode respiration. However, the key

challengehere ishowtopreventmineralizationof thenon-ring

metabolites of aniline through TCA cycle, as this step is often

Fig. 8 e Scanning electron micrograph of the O-bioanode. Left: magnified 1000 times. Right: magnified 15000 times.

Table 3 e The required coulombic efficiencies of aromatic amines as electron donor to achieve the self-maintenancereduction of their parent contaminants.

Parentcontaminants

e-needed Reduced products Max. e-donating CE for self -maintenancereduction (%)

Nitrobenzene 6 Aniline 28 21.5

Acid orange 7 4 Sulfanilic acid & 1-amino-2-naphthol 69 5.8

Methyl orange 4 Sulfanilic acid & dimethyl-4-phenylenediamine 63 6.3

Orange G 4 Aniline & 1-amino-2-naphthol-7,8-disulfonic acid 70 5.7

Nitrogen and sulfur groups was not calculated to donate electrons.

wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 3 81

quite fast. Employing the engineered strains could be one

possible choice, as it could prevent these intermediates

entering TCA cycle if certain genes were deleted (Johnson and

Beckham, 2015). Regarding of aerobic aniline degradation, py-

ruvate and succinate are respective TCA intermediates when

the aniline conversion follows the catechol 2,3-dioxygenase or

1,2-dioxygenase pathway. According to the Equations (7) and

(8), 1 mol of pyruvate and succinate can provide 10 mol and

14 mol of electrons, respectively. Assuming that 50% of them

are converted to current through the anode respiration, CEs of

aniline as electron donor in this decoupled operation manner

can be respectively up to 18% and 25%.

3HCO�3 þ 10e� þ 12Hþ/C3H3O

�3 þ 6H2O (7)

4HCO�3 þ 14e� þ 16Hþ/C4H4O

�4 þ 8H2O (8)

4. Conclusions

This study demonstrated that the presence of oxygen, a previ-

ously considerednegative factor inMFCbioanode, benefited the

energy generation from aniline with efficient aniline removal.

Several AADB andARBwere found to dominate the community

of the anode biofilm. Oxygen facilitated the ring cleavage of

anilinewith the assistance of aerobic aniline degrading bacteria

(AADB), therefore, anilinewasconversed to themetabolites that

wereutilizedforenergygenerationandmineralization toCO2by

anode-respiration bacteria (ARB). Cyclic voltammetry indicated

that extracellular electron transfer in the O-bioanode was

mainly conducted via electron shuttle mediated by certain mi-

crobial excreted redox compounds.

Acknowledgments

We gratefully acknowledge the financial support by Natural

Science Foundation of China (Grant No.51222812, 31370157

and 21407164), National Science Foundation for Distinguished

Young Scholars (Grant No. 51225802), Hundred Talents Pro-

gram of the Chinese Academy of Sciences (29BR2013001) and

the Jiangsu Key Laboratory of Chemical Pollution Control and

Resources Reuse in NJUST (Grant No. 30920140122008). Dr.

Wei-Min Wu was a Guest Professor at Harbin Institute of

Technology during this study.

Appendix A. Supplementary data

Supplementary data related to this article can be found at

http://dx.doi.org/10.1016/j.watres.2015.05.012.

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