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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.
r e f e r e n c e s
Biffinger, J.C., Ray, R., Little, B.J., Fitzgerald, L.A., Ribbens, M.,Finkel, S.E., Ringeisen, B.R., 2009. Simultaneous analysis ofphysiological and electrical output changes in an operatingmicrobial fuel cell with Shewanella oneidensis. Biotechnol.Bioeng. 103 (3), 524e531.
Breugelmans, P., Leroy, B., Bers, K., Dejonghe, W., Wattiez, R., DeMot, R., Springael, D., 2010. Proteomic study of linuron and3,4-dichloroaniline degradation by Variovorax sp WDL1:evidence for the involvement of an aniline dioxygenase-
wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 382
related multicomponent protein. Res. Microbiol. 161 (3),208e218.
Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D.,Huntley, J., Fierer, N., Owens, S.M., Betley, J., Fraser, L.,Bauer, M., Gormley, N., Gilbert, J.A., Smith, G., Knight, R., 2012.Ultra-high-throughput microbial community analysis on theIllumina HiSeq and MiSeq platforms. Isme J. 6 (8), 1621e1624.
Catal, T., Fan, Y.Z., Li, K.C., Bermek, H., Liu, H., 2008. Effects offuran derivatives and phenolic compounds on electricitygeneration in microbial fuel cells. J. Power Sources 180 (1),162e166.
Chen, C.Y., Chen, T.Y., Chung, Y.C., 2014. A comparison ofbioelectricity in microbial fuel cells with aerobic andanaerobic anodes. Environ. Technol. 35 (3), 286e293.
Chen, G., Cheng, K.Y., Ginige, M.P., Kaksonen, A.H., 2012. Aerobicdegradation of sulfanilic acid using activated sludge. WaterRes. 46 (1), 145e151.
Cui, D., Guo, Y.Q., Cheng, H.Y., Liang, B., Kong, F.Y., Lee, H.S.,Wang, A.J., 2012. Azo dye removal in a membrane-free up-flowbiocatalyzed electrolysis reactor coupled with an aerobic bio-contact oxidation reactor. J. Hazard. Mater. 239, 257e264.
Fernando, E., Keshavarz, T., Kyazze, G., 2014. Completedegradation of the azo dye acid orange-7 and bioelectricitygeneration in an integrated microbial fuel cell, aerobic two-stage bioreactor system in continuous flow mode at ambienttemperature. Bioresour. Technol. 156, 155e162.
Field, J.A., 2002. Limits of anaerobic biodegradation. Water Sci.Technol. 45 (10), 9e18.
Fotouhi, L., Khakpour, M., Nematollahi, D., Heravi, M.M., 2008.Investigation of the electrochemical behavior of somecatechols in the presence of 4,6-dimethylpyrimidine-2-thiol.Arkivoc 43e52.
Freguia, S., Masuda, M., Tsujimura, S., Kano, K., 2009. Lactococcuslactis catalyses electricity generation at microbial fuel cellanodes via excretion of a soluble quinone.Bioelectrochemistry 76 (1e2), 14e18.
Freguia, S., Rabaey, K., Yuan, Z.G., Keller, J., 2008. Syntrophicprocesses drive the conversion of glucose in microbial fuel cellanodes. Environ. Sci. Technol. 42 (21), 7937e7943.
Gruning, A., Beecroft, N.J., Avignone-Rossa, C., 2014. Low-potential respirators support electricity production inmicrobial fuel cells. Microb. Ecol., bioRxiv. http://dx.doi.org/10.1007/s00248-014-0518-y L. in press.
Johnson, C.W., Beckham, G.T., 2015. Aromatic catabolic pathwayselection for optimal production of pyruvate and lactate fromlignin. Metab. Eng. 28, 240e247.
Juarez-Ramirez, C., Velazquez-Garcia, R., Ruiz-Ordaz, N.,Galindez-Mayer, J., Monroy, O.R., 2012. Degradation kinetics of4-amino naphthalene-1-sulfonic acid by a biofilm-formingbacterial consortium under carbon and nitrogen limitations. J.Ind. Microbiol. Biotechnol. 39 (8), 1169e1177.
Kahng, H.Y., Kukor, J.J., Oh, K.H., 2000. Characterization of strainHY99, a novel microorganism capable of aerobic and anaerobicdegradation of aniline. FEMS Microbiol. Lett. 190 (2), 215e221.
Kan, J.J., Hsu, L., Cheung, A.C.M., Pirbazari, M., Nealson, K.H.,2011. Current production by bacterial communities inmicrobial fuel cells enriched from wastewater sludge withdifferent electron donors. Environ. Sci. Technol. 45 (3),1139e1146.
Li, G.Y., Wan, S.G., An, T.C., 2012. Efficient bio-deodorization ofaniline vapor in a biotrickling filter: metabolic mineralizationand bacterial community analysis. Chemosphere 87 (3),253e258.
Liang, B., Cheng, H., Van Nostrand, J.D., Ma, J., Yu, H., Kong, D.,Liu, W., Ren, N., Wu, L., Wang, A., 2014. Microbial communitystructure and function of nitrobenzene reduction biocathodein response to carbon source switchover. Water Res. 54,137e148.
Liu, Z., Yang, H., Huang, Z., Zhou, P., Liu, S.J., 2002. Degradation ofaniline by newly isolated, extremely aniline-tolerant Delftia spAN3. Appl. Microbiol. Biotechnol. 58 (5), 679e682.
Luo, H.P., Liu, G.L., Zhang, R.D., Jin, S., 2009. Phenol degradation inmicrobial fuel cells. Chem. Eng. J. 147 (2e3), 259e264.
Marcus, A.K., Torres, C.I., Rittmann, B.E., 2007. Conduction-basedmodeling of the biofilm anode of a microbial fuel cell.Biotechnol. Bioeng. 98 (6), 1171e1182.
Mehta-Kolte, M.G., Bond, D.R., 2012. Geothrix fermentans secretestwo different redox-active compounds to utilize electronacceptors across a wide range of redox potentials. Appl.Environ. Microbiol. 78 (19), 6987e6995.
Milliken, C.E., May, H.D., 2007. Sustained generation of electricityby the spore-forming, gram-positive, desulfitobacteriumhafniense strain DCB2. Appl. Microbiol. Biotechnol. 73 (5),1180e1189.
Mu, Y., Rabaey, K., Rozendal, R.A., Yuan, Z.G., Keller, J., 2009a.Decolorization of azo dyes in bioelectrochemical systems.Environ. Sci. Technol. 43 (13), 5137e5143.
Mu, Y., Rozendal, R.A., Rabaey, K., Keller, J., 2009b. Nitrobenzeneremoval in bioelectrochemical systems. Environ. Sci. Technol.43 (22), 8690e8695.
Pant, D., Van Bogaert, G., Diels, L., Vanbroekhoven, K., 2010. Areview of the substrates used in microbial fuel cells (MFCs) forsustainable energy production. Bioresour. Technol. 101 (6),1533e1543.
Peng, L., You, S.J., Wang, J.Y., 2010. Carbon nanotubes as electrodemodifier promoting direct electron transfer from Shewanellaoneidensis. Biosens. Bioelectron. 25 (5), 1248e1251.
Peres, C.M., Naveau, H., Agathos, S.N., 1998. Biodegradation ofnitrobenzene by its simultaneous reduction into aniline andmineralization of the aniline formed. Appl. Microbiol.Biotechnol. 49 (3), 343e349.
Philipp, B., Schink, B., 2012. Different strategies in anaerobicbiodegradation of aromatic compounds: nitrate reducersversus strict anaerobes. Environ. Microbiol. Repl. 4 (5),469e478.
Rabaey, K., Boon, N., Hofte, M., Verstraete, W., 2005. Microbialphenazine production enhances electron transfer in biofuelcells. Environ. Sci. Technol. 39 (9), 3401e3408.
Rau, J., Knackmuss, H.J., Stolz, A., 2002. Effects of differentquinoid redox mediators on the anaerobic reduction of azodyes by bacteria. Environ. Sci. Technol. 36 (7), 1497e1504.
Ringeisen, B.R., Ray, R., Little, B., 2007. A miniature microbial fuelcell operating with an aerobic anode chamber. J. PowerSources 165 (2), 591e597.
Rosenbaum, M., Cotta, M.A., Angenent, L.T., 2010. Aeratedshewanella oneidensis in continuously fed bioelectrochemicalsystems for power and hydrogen production. Biotechnol.Bioeng. 105 (5), 880e888.
Saratale, R.G., Saratale, G.D., Chang, J.S., Govindwar, S.P., 2011.Bacterial decolorization and degradation of azo dyes: a review.J. Taiwan Inst. Chem. E 42 (1), 138e157.
Schnell, S., Schink, B., 1991. Anaerobic aniline degradation viareductive deamination of 4-aminobenzoyl-coa indesulfobacterium-anilini. Arch. Microbiol. 155 (2), 183e190.
Sheludehenko, A.S., Kolomytseva, M.P., Travkin, V.A.,Akimov, V.N., Golovleva, L.A., 2005. Degradation of aniline bydelftia tsuruhatensis 14S in batch and continuous processes.Appl. Biochem. Microbiol. 41 (5), 465e468.
Srikanth, S., Marsili, E., Flickinger, M.C., Bond, D.R., 2008.Electrochemical characterization of geobacter sulfurreducenscells immobilized on graphite paper electrodes. Biotechnol.Bioeng. 99 (5), 1065e1073.
Tan, N.C.G., van Leeuwen, A., van Voorthuizen, E.M., Slenders, P.,Prenafeta-Boldu, F.X., Temmink, H., Lettinga, G., Field, J.A.,2005. Fate and biodegradability of sulfonated aromaticamines. Biodegradation 16 (6), 527e537.
wat e r r e s e a r c h 8 1 ( 2 0 1 5 ) 7 2e8 3 83
Tatusova, T., Ciufo, S., Fedorov, B., O'Neill, K., Tolstoy, I., 2014.RefSeq microbial genomes database: new representationand annotation strategy. Nucleic Acids Res. 42 (D1),D553eD559.
Wang, A.J., Cheng, H.Y., Liang, B., Ren, N.Q., Cui, D., Lin, N.,Kim, B.H., Rabaey, K., 2011. Efficient reduction of nitrobenzeneto aniline with a biocatalyzed cathode. Environ. Sci. Technol.45 (23), 10186e10193.
Wang, A.J., Cheng, H.Y., Ren, N.Q., Cui, D., Lin, N., Wu, W.M.,2012a. Sediment microbial fuel cell with floating biocathodefor organic removal and energy recovery. Front. Environ. Sci.Engin. 6 (4), 569e574.
Wang, A.J., Cui, D., Cheng, H.Y., Guo, Y.Q., Kong, F.Y., Ren, N.Q.,Wu, W.M., 2012b. A membrane-free, continuously feeding,single chamber up-flow biocatalyzed electrolysis reactor fornitrobenzene reduction. J. Hazard. Mater. 199, 401e409.
Xing, D.F., Cheng, S.A., Logan, B.E., Regan, J.M., 2010. Isolation ofthe exoelectrogenic denitrifying bacterium Comamonasdenitrificans based on dilution to extinction. Appl. Microbiol.Biotechnol. 85 (5), 1575e1587.
Xu, Q., Sun, J., Hu, Y.Y., Chen, J., Li, W.J., 2013. Characterizationand interactions of anodic isolates in microbial fuel cells
explored for simultaneous electricity generation and Congored decolorization. Bioresour. Technol. 142, 101e108.
Zhang, C.P., Li, M.C., Liu, G.L., Luo, H.P., Zhang, R.D., 2009.Pyridine degradation in the microbial fuel cells. J. Hazard.Mater. 172 (1), 465e471.
Zhang, T., Ren, H.-F., Liu, Y., Zhu, B.-L., Liu, Z.-P., 2010. A noveldegradation pathway of chloroaniline in Diaphorobacter sp.PCA039 entails initial hydroxylation. World J. Microbiol.Biotechnol. 26 (4), 665e673.
Zhang, Y., Angelidaki, I., 2014. Microbial electrolysis cells turningto be versatile technology: recent advances and futurechallenges. Water Res. 56, 11e25.
Zhou, J.Z., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soilsof diverse composition. Appl. Environ. Microbiol. 62 (2),316e322.
Zissi, U.S., Lyberatos, G.C., 1999. Kinetics of growth and anilinedegradation by Stenotrophomonas maltophilia. WaterEnviron. Res. 71 (1), 43e49.
Zuo, Y., Xing, D.F., Regan, J.M., Logan, B.E., 2008. Isolation of theexoelectrogenic bacterium Ochrobactrum anthropi YZ-1 byusing a U-tube microbial fuel cell. Appl. Environ. Microbiol. 74(10), 3130e3137.