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\begin{document}

\title{Impact of anodophilic biofilm bioelectroactivity on denitrification
behavior of single-chamber air-cathode microbial fuel cell in steady
state}



\author[1]{Wenjuan  Zhao}%
\author[1]{YiZhao Gao}%
\author[1]{Yongli  Zhao}%
\author[1]{Xiaoya Deng}%
\author[1]{Jun Shao}%
\author[1]{Shuiliang Chen}%
\affil[1]{Jiangxi Normal University}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Generally, high bioelectroactivity of anodophilic biofilm favors high
power generation of microbial fuel cell (MFC), however, it is not clear
whether it can promote denitrification of MFC synchronously. In this
study, the impact of anodophilic biofilms bioelectroactivity on
denitrification behavior of single-chamber air-cathode MFC (SAMFC) in
steady state was studied for the first time. Anodophilic biofilms of
various bioelectroactivity were acclimated at conditions of open circuit
(OC), Rext of 1000\selectlanguage{greek}Ω \selectlanguage{english}and 20\selectlanguage{greek}Ω \selectlanguage{english}(denoted as SAMFC-OC, SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and
SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}respectively) and run for 100 days in the presence of
nitrate. Electrochemical tests and microbial analysis results showed
that the anode of the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}delivered higher oxidation and
denitrification current response and had a higher abundance of
electroactive bacteria, like Geobacter, Pseudomonas and Comamonas, which
possessed bidirectional electron transfer function, demonstrating a
higher bioelectroactivity of the anodophilic biofilm. Moreover, these
electroactive bacteria favored the accumulation of denitrifers, like
Thauera and Alicycliphilus, probably by consuming trace oxygen through
catalyzing oxygen reduction. The SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}not only delivered a 61.7\%
higher power than the SAMFC-1000\selectlanguage{greek}Ω, \selectlanguage{english}but also achieved a stable and high
denitrification rate constant (kDN) of 1.9, which was 50\% and 40\%
higher than that of the SAMFC-OC and SAMFC-1000\selectlanguage{greek}Ω, \selectlanguage{english}respectively. It could
be concluded that the high bioelectroactivity of the anodophilic
biofilms not only favored high power generation of the SAMFC, but also
promote the growth of denitrifers at the anodes and strengthened
denitrification. This study provided an effective method and important
theoretical basis for enhancing power generation and denitrification
performance of the SAMFC synchronously.%
\end{abstract}%



\sloppy


\textbf{Impact of} \textbf{anodophilic biofilm}

\textbf{bioelectroactivity on denitrification behavior of single-chamber
air-cathode microbial fuel cell in steady state}

Wenjuan Zhao\emph{\textsuperscript{1}} , YiZhao
Gao\emph{\textsuperscript{1}} , Yongli Zhao\emph{\textsuperscript{1}} ,
Xiaoya Deng\emph{\textsuperscript{1}} , Jun
Shao\emph{\textsuperscript{1,2}} and Shuiliang
Chen\emph{\textsuperscript{1,2}}
11\textsuperscript{*}\emph{Corresponding author.: Tel.:
+86-791-881207382, Fax: +86-791-88120536;E-mail: slchenjxnu@jxnu.edu.cn
(S. Chen).}

\emph{\textsuperscript{1} Department of Chemistry and Chemical
Engineering and Nanofiber Engineering Center of Jiangxi Province,
Jiangxi Normal University, 99 Ziyang Road, 330022 Nanchang, China.}

\emph{\textsuperscript{2} Key Laboratory of Functional Small Molecules
for Ministry of Education, Jiangxi Normal University, 99 Ziyang Avenue,
Nanchang 330022, China;}

\textbf{Abstract}

Generally, high bioelectroactivity of anodophilic biofilm favors high
power generation of microbial fuel cell (MFC), however, it is not clear
whether it can promote denitrification of MFC synchronously. In this
study, the impact of anodophilic biofilms bioelectroactivity on
denitrification behavior of single-chamber air-cathode MFC (SAMFC) in
steady state was studied for the first time. Anodophilic biofilms of
various bioelectroactivity were acclimated at conditions of open circuit
(OC), R\textsubscript{ext} of 1000\selectlanguage{greek}Ω \selectlanguage{english}and 20\selectlanguage{greek}Ω \selectlanguage{english}(denoted as SAMFC-OC,
SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}respectively) and run for 100 days in the
presence of nitrate. Electrochemical tests and microbial analysis
results showed that the anode of the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}delivered higher
oxidation and denitrification current response and had a higher
abundance of electroactive bacteria, like\emph{Geobacter} ,
\emph{Pseudomonas} and \emph{Comamonas,} which possessed bidirectional
electron transfer function, demonstrating a higher bioelectroactivity of
the anodophilic biofilm. Moreover, these electroactive bacteria favored
the accumulation of denitrifers, like\emph{Thauera} and
\emph{Alicycliphilus} , probably by consuming trace oxygen through
catalyzing oxygen reduction. The SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}not only delivered a 61.7\%
higher power than the SAMFC-1000\selectlanguage{greek}Ω, \selectlanguage{english}but also achieved a stable and high
denitrification rate constant\emph{(k\textsubscript{DN}} ) of 1.9, which
was 50\% and 40\% higher than that of the SAMFC-OC and SAMFC-1000\selectlanguage{greek}Ω,
\selectlanguage{english}respectively. It could be concluded that the high bioelectroactivity of
the anodophilic biofilms not only favored high power generation of the
SAMFC, but also promote the growth of denitrifers at the anodes and
strengthened denitrification. This study provided an effective method
and important theoretical basis for enhancing power generation and
denitrification performance of the SAMFC synchronously.

\textbf{Keywords:} Denitrification, single-chamber air-cathode microbial
fuel cell, bioelectroactivity, anodophilic biofilms, steady state

\textbf{INTRODUCTION}

As one of newly developed microbial electrochemical technologies,
microbial fuel cell (MFC) attracts special attentions due to its double
functions of waste removal and electrical energy recovery from
wastewater. MFC utilizes the electrode respiration of exoelectrogens
(also referred to as electroactive microorganisms and anode respiring
bacteria) (Borole et al., 2011; Logan, 2009; Lovley, 2012) to convert
the pollutants from water to available electrical energy or resources
(Falk Harnisch \& Schroder, 2010; Rabaey \& Verstraete, 2005). Based on
the electrode respiration of the exoelectrogens, multiple pollutants in
the wastewater can be synergistically removed. As one of important
pollutants in wastewater that usually cause serious water
eutrophication, nitrate can serve as electron acceptor and is
(bio)electrochemical reduced at the cathode of MFC (Falk Harnisch \&
Schroder, 2010; Li, Yu, \& He, 2014), this provide a new idea for
nitrate removal. The simultaneous organic oxidation at bioanode and
bioelectrochemical denitrification (BEDN) at biocathode was firstly
performed in a dual-chamber MFC (Clauwaert et al., 2007), in which
autotrophic denitrifers at cathode conducted BEDN by uptaking electrons
from the organic oxidation of exoelectrogens at the anode. However, the
autotrophic denitrifers usually reproduced~slowly and thus led to a poor
denitrification performance (H. Huang et al., 2019; Patureau, Zumstein,
Delgenes, \& Moletta, 2000). Recently, it had been found that the
exoelectrogens enriched at the anode of MFC could not only conduct
organics oxidation, but also be able to catalyze cathodic oxygen
reduction (Ka Yu Cheng, Ho, \& Cord-Ruwisch, 2010) and
bioelectrochemical denitrification (BEDN) (K. Y. Cheng, Ginige, \&
Kaksonen, 2012). It meant that the BEDN could also be carried out at the
anode of MFC. It had been reported that exoelectrogens can transfer
electrons directly to denitrifers by direct attachment or through
electrode (Liang et al., 2019). The heterotrophic environment of
bioanode could facilitate the reproduction of the exoelectrogens thus
would substantially increase the denitrification performance of MFC.
Anodic BEDN was firstly conducted in dual-chamber MFC (Zhang et al.,
2013), however, the use of ion-exchange membrane decreased the
performance of MFCs on nitrate removal. Moreover, the high operating
cost sourced from the use of ion-exchange membrane also limits the
application of dual-chamber MFC in practical wastewater treatment(Haobin
Huang et al., 2018). In comparison, single-chambered air-cathode (SAMFC)
would be more favorable for practical wastewater treatment due to the
merits of membrane-less configuration, low internal resistance, low
cost. In SAMFC, the BEDN mainly occurs at bioanode (Sukkasem, Xu, Park,
Boonsawang, \& Liu, 2008), might be due to the presence of dissolved
oxygen which restrict the enrichment of denitrifers on the air-cathode.

In recent years, many investigations on BEDN of the SAMFC have been
advanced in recent years. (H. Huang et al., 2019; Haobin Huang et al.,
2018; Sukkasem et al., 2008). In one study, Liu et al compared the
denitrification performance under electricity generation (EG) (with
loads of 1000 and 270 \selectlanguage{greek}Ω\selectlanguage{english}) and open circuit (OC) conditions and found that
the denitrification activity of the SAMFC under current generation
conditions was slightly lower than that under OC state. They thought
that the possible reason was attributed to the electron competition
between the electrode and nitrate under the EG condition (Sukkasem et
al., 2008). However, it should be noted that this study was based on
transient state, in which the operation of SAMFC under specific
condition was in a relatively short time, e.g. several hours, and
difficult to reflect the real denitrification performance of SAMFC. In
another study, Huang et al also compared the denitrification performance
of SAMFC under the EG (with load of 1000 \selectlanguage{greek}Ω\selectlanguage{english}) and OC conditions in steady
state and achieved the opposite result that the denitrification
performance under the EG was higher than that under OC state (H. Huang
et al., 2019). Generally, MFC with lower external resistance favored the
acclimation of anodophilic biofilms with higher bioelectroactivity, thus
favors higher power generation, however, it is not clear whether it can
promote denitrification of MFC synchronously? How does the
bioelectroactivity of anodophilic biofilms affect the denitrification
behavior of the SAMFC in steady state?

In this study, we studied the impact of anodophilic biofilms
bioelectroactivity on the denitrification behavior of the SAMFC in
steady state for the first time, trying to achieve high power generation
and denitrification performance of SAMFC synchronously. Anodophilic
biofilms of the SAMFC with various bioelectroactivity was acclimated at
conditions of open circuit, R\textsubscript{ext} of 1000\selectlanguage{greek}Ω \selectlanguage{english}and 20\selectlanguage{greek}Ω
\selectlanguage{english}(denoted as SAMFC-OC, SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}respectively) and run
for 100 days in the presence of nitrate. The anode potentials, voltage
of the SAMFCs were monitored in the whole operation process, then the
bioelectroactivity of the anodophilic biofilms, microbial community and
denitrification performance of the SAMFCs were analyzed and compared.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1. SAMFC construction and microorganism acclimation}

Cube-shaped plexiglass with a volume of 84 mL was used as the setup of
SAMFCs. The anodes were graphite brushes with 2 cm in diameter and 2.5
cm in length. The air-cathodes were fabricated by rolling method as
previously described (Liu et al., 2014) using activated carbon as oxygen
reduction reaction catalyst. Nine SAMFCs were built and divided into
three groups with three in each group. The three groups of SAMFCs run at
conditions of open-circuit (OC), 1000\selectlanguage{greek}Ω \selectlanguage{english}load and 20\selectlanguage{greek}Ω \selectlanguage{english}load (denoted as
SAMFC-OC, SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-20\selectlanguage{greek}Ω\selectlanguage{english}), respectively. These SAMFCs were
inoculated from SAMFC that inoculated with domestic wastewater from
treatment plant (Qingshan, Nanchang) and run for more than one year. The
SAMFCs were firstly operated by feeding 50 mM phosphate buffer solution
(PBS) containing 1.64 g\selectlanguage{ngerman}·L\textsuperscript{-1} sodium acetate (equal to
1280 mg·L\textsuperscript{-1} COD) for 30 days to acclimate
exoelectrogens onto the anodes, then run by feeding the 50 mM PBS
containing 1.64 g·L\textsuperscript{-1} and extra 1.21
g·L\textsuperscript{-1} sodium acetate (200
mg·L\textsuperscript{-1}NO\textsubscript{3}\textsuperscript{-}-N) to
acclimate denitrifers. The detailed acclimation and characterization
process of the SAMFCs is shown in Figure S1.

All the SAMFCs were operated in fed-batch mode at a constant temperature
of 35±1°C. During the acclimation process, the anode potentials and cell
voltages were automatically recorded every 5 min using a data
acquisition system (digital multimeter, Keithley 2700, USA) connected to
a personal computer. Medium was refreshed when the anode potential rose
to over 0 V. All the electrode potentials were measured versus the
Ag/AgCl reference electrode (+0.198 V versus SHE, saturated KCl). The
concentrations of NO\textsubscript{3}\textsuperscript{-}-N in the SAMFCs
were measured at the 20\textsuperscript{th} (t\textsubscript{20}),
40\textsuperscript{th} (t\textsubscript{40}),
60\textsuperscript{th}(t\textsubscript{60}), 80\textsuperscript{th}
(t\textsubscript{80}), and 100\textsuperscript{th}
(t\textsubscript{100}) day, respectively. The microbial electrocatalytic
activity and community analyses of the microorganisms at the anodes were
conducted at the initial (t\textsubscript{0}), 20\textsuperscript{th}
(t\textsubscript{20}) and 100\textsuperscript{th} (t\textsubscript{100})
day, respectively. Microbial community analysis can damage the anode
biofilm, therefore one of the three SAMFCs in each group was used for
NO\textsubscript{3}\textsuperscript{-}-N concentration, CV
characterization and final microbial community analysis, while the other
two SAMFCs were only used for microbial community analysis.

\textbf{2.2 Measurements and calculations}

The concentrations of NO\textsubscript{3}\textsuperscript{-}-N in the
SAMFCs were measured regularly with the time interval of 20 days. For
example, the first and second denitrification tests were performed at
t\textsubscript{20} and t\textsubscript{40} respectively. During the
whole period for denitrifers biofilm acclimation, the concentrations of
sodium nitrate keep at 1.21 g L\textsuperscript{-1}, which is
corresponding to the NO\textsubscript{3}\textsuperscript{-}-N
concentration of 200 mg L\textsuperscript{-1}. In order to obtain
optimal C/N ratio, three different concentrations of sodium acetate of
0.82 g L\textsuperscript{-1} (640 mg L\textsuperscript{-1} COD), 1.64 g
L\textsuperscript{-1} (1280 mg L\textsuperscript{-1} COD) and 2.05 g
L\textsuperscript{-1} (1600 mg L\textsuperscript{-1} COD) were used to
control the C/N weight ratio of 1.2, 2.4 and 3, respectively. The
denitrification tests under different C/N ratios were performed at
t\textsubscript{20}. Three different SSAMFCs were sampled at 1h, 2h, 3h,
4h and 5h and immediately filtered through disposable Millipore filter
with the pore size of 0.22\selectlanguage{greek}μ\selectlanguage{english}m for the analyses of nitrate, nitrite at the
same condition simultaneously. NO\textsubscript{3}\textsuperscript{-}-N
concentration measurements were conducted by applying a
spectrophotometer (SpectroDirect, Lovibond, Gremany) using standard
methods.

Denitrification rate (r\textsubscript{DN}) was fitting by pseudo-first
order reaction with respect to concentration and calculated as follows
according to Jia et al(Jiang et al., 2018),\(\text{Ln}\left(\frac{C_{t}}{C_{0}}\right)=-\text{Kt\ }\), where
C\textsubscript{0} is the initial concentration of nitrogen in the form
of nitrate (NO\textsubscript{3}\textsuperscript{-}-N) (mg/L), t is the
time, C\textsubscript{t} is the NO\textsubscript{3}\textsuperscript{-}-N
econcentration(mg/L) at t h. K is the observed reaction rate constant.

\textbf{2.3 Bioelectroactivity analysis anodophilic biofilms}

The bioelectroactivity of the anodophilic biofilms in the SAMFCs at the
period of initial (t\textsubscript{0}), 20\textsuperscript{th} day
(t\textsubscript{20}) and 100\textsuperscript{th} day
(t\textsubscript{100}) was analyzed by cyclic voltammetry (CV) using
potentiostatically-controlled three-electrode system, in which the anode
served as the working electrode, the air-cathode served as the counter
electrode and an Ag/AgCl electrode served as the reference electrode.
The CV measurements were controlled by a potentiostat (CHI1040B,
Shanghai Chenhua Instrument Co., Ltd, China) and carried out in the
potential range from +0.4 V to -0.5V at scanning rate of 1 mV
s\textsuperscript{-1}. All the SAMFCs were set at open-circuit condition
for 1h before CV implementation.

\textbf{2.4 Microbial community analysis}

Biofilm samples were also collected from the anodes of SAMFCs at the
stages of t\textsubscript{0}, t\textsubscript{20} and
t\textsubscript{100} by using a 10-mL syringe with a 0.9\selectlanguage{ngerman}×150 mm
stainless steel needle for microbial community analysis. High-throughput
sequencing analysis based on all bacterial 16S rRNA was performed to
know the microbial communities present in the biofilms as described in
ref. (Chen et al., 2019) , detailed procedure see supplemental
information.

\textbf{3. RESULTS AND DISCUSSION}

\textbf{3.1 Power generation and bioanode potential variation of the
SAMFCS}

Figure 1A show that the SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC generate a
voltage of 0.093, 0.525 and 0.698V during nearly one month operation
process. The SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}generated a steady power of 0.432 mW, which is
61.7\% higher than the SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}with 0.267 mW (Figure 1B). Moreover,
the SAMFC acclimated under load of 20\selectlanguage{greek}Ω \selectlanguage{english}could generated a maximum power
density of 976.6 mW m\textsuperscript{-2} without power overshoot, while
that acclimated under load of 1000\selectlanguage{greek}Ω \selectlanguage{english}delivered a power of only 637 mW
m\textsuperscript{-2} with an obvious power overshoot (Figure 1C).

The potentials of the SAMFC bioanodes during the whole acclimation
process shown in Figure 2 display that, after running for about one
week, the potentials of the SAMFC anodes change periodically with
periodical refresh of the medium containing acetate. Once the medium is
refreshed, the anode potential drops rapidly and then level off at a
negative value due to the bioelectrochemical oxidation of acetate.
Figure 2C displays that the anodes of the SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}SAMFC-1000 and
SAMFC-OC could achieve a negative potential of -100, -450 and -480 mV,
respectively. When the acetate is depleted, the anode potential
increases and approaches the cathode potential (about 0 V) gradually.
Figure 2B, 2D and 2E show that the SAMFCs fed with media containing both
acetate and nitrate substrate display similar trend of anode potential
variation to that fed with media containing only acetate substrate.
However, a conspicuous potential platform appears in the curves of
anodes fed with medium containing both acetate and nitrate (Figure 2D
and 2E). Once the medium is refreshed, the anode potential of the
SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC firstly drops from about 0V to
negative and level off at -0.09, -0.369 and -0.386 V, respectively, for
about 3.6 h. The evident platform of the potential curve might be a
consequence of concurrent acetate oxidation and nitrate reduction at the
anodes of the SAMFCs, which result in a mixed anode potential. Figure
2B, 2D and 2E reveal that the potential platform periodically exists in
every cycle during denitrifers acclimatization. Subsequently, the anode
potentials of the SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC drop again and
level off at -0.11, -0.45 and -0.5V, respectively (Figure 2D), which are
basically equal to that of the SAMFCs fed with media containing only
acetate. This potential decline is believed to be caused by the
depletion of nitrate. The nitrate concentration in a complete cycle at
the 40\textsuperscript{th} day is measured and shown in Figure 1G, it
confirms that the nitrate is depleted in about 3 to 4 h in all the
SAMFCs, which is well matched with the duration of the anode potential
platform (Figure 2F). Thereby, the potential decline could be a signal
of nitrate depletion and would be used for monitoring the nitrate
concentration in wastewater. Finally, the anode potentials of SAMFC-20\selectlanguage{greek}Ω
\selectlanguage{english}increases gradually to positive potential values (Figure 2D and 2E) due
to the depletion of acetate, which is similar to that of acclimation of
exoelectrogens by feeding medium containing only acetate (Figure 2C).

\textbf{3.2 Bioelectroactivity of anodophilic biofilms}

After acclimation of the SAMFCs in medium containing acetate for 30
days, the bioelectroactivity of the biofilms enriched at the bioanodes
are measured by CVs and the results are shown in Figure 3. Figure 3A1
shows that all the CVs of anodophilic biofilms acclimated in the
presence of acetate (turnover condition) present the typical positive
sigmoidal shape (Liang et al., 2019; Pous et al., 2016), indicating that
exoelectrogens are established successfully in all the SAMFCs. Obvious
differences are observed from the CVs of different SAMFC bioanodes. The
CVs of the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}bioanodes display a high bioelectrocatalytic
oxidation current of 7.8 mA, which is much higher than that of the
SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}(5.5 mA) and SAMFC-OC (0.3 mA), respectively. These results
indicate that the biofilms enrich at the bioanodes of the SAMFC-20\selectlanguage{greek}Ω
\selectlanguage{english}possesses higher bioelectrocatalytic activity than that of the
SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC. It has been reported that the
bioelectrocatalytic activity of exoelectrogens at the anode is highly
associated with the anode potential and current density (Aelterman,
Freguia, Keller, Verstraete, \& Rabaey, 2008; Zhao \& Chen, 2018).
Higher bioelectrocatalytic activity could be obtained at comparatively
more positive anode potential and higher current density (Aelterman et
al., 2008; Hong, Call, Werner, \& Logan, 2011; Jiang et al., 2018),
because electron transfer from bacteria to anode is accelerated
substantially when SAMFC operated under lower external resistances (Zhu,
Tokash, Hong, \& Logan, 2013). In this study, the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}could
deliver comparatively more positive anode potential and higher current
density than the SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC, thereby a much higher anodic
electrocatalytic activity of exoelectrogen biofilms are enriched the
bioanodes. Furthermore, the high current density in the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}enable
anode-respiring bacteria to outcompete aerobic heterotrophs for
substrate to release much more electrons (Ren, Zhang, He, \& Logan,
2014), thus SAMFC operated under 20\selectlanguage{greek}Ω \selectlanguage{english}is an efficient means to enrich
exoelectrogens. The CVs in Figure 3A2 show that the exoelectrogens
enriched in medium containing only acetate substrate could also catalyze
BEDN, which is in accordance to that of previous reports (K. Y. Cheng et
al., 2012) .

When the nitrate is supplied to the SAMFCs accompanying with the
acetate, denitrifers are enriched at the bioanodes after acclimating for
a period time. The CVs of the anodophilic biofilms at the
20\textsuperscript{th} day are performed in the PBS medium containing
only nitrate and shown in Figure 3B2. They all display a negative
sigmoidal shape which is extremely accordance with that in ref. (Pous et
al., 2016) , proving that anodophilic biofilms could successfully
catalyze nitrate reduction (K. Y. Cheng et al., 2012). Notably, the CVs
of the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}anode display a more negative bioelectrocatalytic
reduction current response of-1.15 mA, which is four and eight times as
that of the SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}(-0.28 mA) and SAMFC-OC (-0.13 mA) bioanodes,
respectively, representing a higher BEDN performance. The CVs of the
anodophilic biofilms at the 20\textsuperscript{th} day shown in Figure
2B1 display typical positive sigmoidal shape but with increased
bioelectrocatalytic oxidation peak current of 11.5, 6.6 and 1.3 mA for
the SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC, respectively, indicating that
the bioelectrocatalytic activity of exoelectrogens at the anodes is
enhanced even in the presence of denitrifers, indicating the good
coexistence of exoelectrogens and denitrifers. Moreover, a higher
negative denitrification current is observed for all the SAMFCs at the
100\textsuperscript{th} day (Figure 3C2), demonstrating an increase of
BEDN performance with increase of acclimation time. In addition, the
enrichment of denitrifers at the anode result in a distinctive ``duck
head'' shape (Figure 3B1 and 3C1) in the CVs of the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}anodes
measured in the presence of acetate. The CVs measured under non-turnover
conditions (absence of substrate) can further characterize the
electrochemical features and extracellular electron transfer (EET) modes
of the biofilm. The non-turnover CVs of the anodophilic biofilm
acclimated by feeding media containing acetate shown in Figure 3A3
present two major clear redox systems with the formal potential ranged
from -0.260 to -0.350 V, which is reported to be related to membrane
bound c-type cytochromes and also highly consistent with the CV
characteristic that described in the literature(Fricke, Harnisch, \&
Schroder, 2008; F. Harnisch \& Freguia, 2012). The two major redox
systems at the formal potentials of -0.256 V and -0.360 V in SAMFC-20\selectlanguage{greek}Ω
\selectlanguage{english}are basically equal to that of -0.260 V and -0.350 V in SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}as
well as -0.263 V and -0.347 V in SAMFC-OC. The similar two major clear
redox systems with the formal potential ranged from 0.235 to 0.370 V can
also be observed in the non-turnover CVs of anodophilic biofilm
acclimated by feeding media containing both acetate and nitrate (Figure
3B3 and 3C3), hinting that the bioelectrochemical features and EET modes
of the exoelectrogen biofilms is preserved even in the presence of
denitrifers.

\textbf{3.3 Denitrification performance of SAMFCs}

The denitrification performance of the SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}SAMFC-1000 and
SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}at different stages is investigated. The C/N ratio of the
feeding media is firstly optimized by feeding PBS containing 1.64 g
L\textsuperscript{-1} acetate but different nitrate concentration.
Figure S2 shows that when the C/N ratio is over 2.4, the SAMFCs achieved
the optimum denitrification performance, which was basically in
accordance well with the value of 2.2 reported in literature (Dhamole,
Nair, D'Souza, Pandit, \& Lele, 2015). The denitrification performance
tests in following are based on the media with a C/N ratio of 2.4.
Figure 4 shows that all the SAMFCs display comparatively poor
denitrification performance at initial stage. But a gradual enhancement
of denitrification performance was observed along with the rise of
acclimation time. For example, In SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}the initial nitrate
concentration of 200 mg
L\textsuperscript{-1}NO\textsubscript{3}\textsuperscript{-}-N drops to
127, 110, 95, 66 and 56 mg L\textsuperscript{-1} at the
20\textsuperscript{th}, 40\textsuperscript{th}, 60\textsuperscript{th},
80\textsuperscript{th}and 100\textsuperscript{th} day, respectively, in
one hour (Figure 4A1, B1, C1, D1 and E1). The decrease of
NO\textsubscript{3}\textsuperscript{-}-N concentration from 200 mg
L\textsuperscript{-1} to lower than 30 mg L\textsuperscript{-1} takes
about 4h at the 20\textsuperscript{th}, 40\textsuperscript{th} day, but
3h at the 80\textsuperscript{th} and 100\textsuperscript{th} day. The
similar NO\textsubscript{3}\textsuperscript{-}-N concentration variation
trend could also be observed in SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC. This gradual
enhancement of denitrification rate with the rise of acclimation time in
all the SAMFCs is probably ascribed to the gradual maturation of
denitrifying biofilms at the anodes which could find the proof from
microbial community analysis results in Figure S4.

As shown in Figure 4A2, the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}possess a
low\emph{k\textsubscript{DN}} of 1.07, which is lower than that of the
SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}(of 1.26) at the 20\textsuperscript{th} day. However, after
acclimation for another 20 days, the \emph{k\textsubscript{DN}} of the
SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}increases greatly and is up to 1.57, which surpass that of the
SAMFC-1000 of about 1.28 at the 40\textsuperscript{th} day (Figure 4B2).
Moreover, Figure 4B2, C2, D2 and E2 reveal that the denitrification rate
of the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}outperforms that of the SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC from
the 40\textsuperscript{th} day and achieves a stable denitrification
rate with a high \emph{k\textsubscript{DN}} of 1.9 after acclimation for
80 days, which is much higher than that of SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC with
similar \emph{k\textsubscript{DN}} of 1.3. The slightly
lower\emph{k\textsubscript{DN}} in SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}at the
20\textsuperscript{th}day compared with that in SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}is probably
due to the competition of electrons between the denitrification of the
denitrifers in the anode and oxygen reduction (ORR) in the air-cathode.
In the primary stage of operation, the electrons released by the
exoelectrogens at the bioanodes are tend to be accepted by the
air-cathode for ORR in the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}due to the higher electrode
potential than that in the SAMFC-1000\selectlanguage{greek}Ω, \selectlanguage{english}thus shows slightly lower
denitrification rate. The SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}facilitates the accumulation of
exoelectrogens which usually are capable of bidirectional electron
transfer including release electrons by oxidation of acetate, and accept
electrons by reduction of oxygen and denitrification. Therefore, the
exoelectrogens can enhance the denitrification performance of SAMFC by
(a) direct bioelectrochemical denitrification and (b) promoting the
accumulation of denitrifers through catalyzing oxygen reduction to
remove the trace oxygen diffused from air-cathode.

\textbf{3.4} \textbf{Microbial community analysis}

The sum of the relative abundance of the denitrifers at the anodes of
SAMFCs is summarized in table S3 and displayed in Figure 5. A great
increase on the total abundance of denitrifers can be observed during
the 100 days acclimation. The total relative abundance of denitrifers
increases from 41.6\selectlanguage{ngerman}±6.9\%, 42.4±9.2\% and 32.6±9.4\% for the SAMFC-20\selectlanguage{greek}Ω,
\selectlanguage{english}SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC at the 20\textsuperscript{th} day, to
65.5\selectlanguage{ngerman}±9.8\%, 48.3±10.1\% and 48.9±8.2\% for the SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}SAMFC-1000\selectlanguage{greek}Ω
\selectlanguage{english}and SAMFC-OC at the 100\textsuperscript{th} day, respectively. In fact,
exoelectrogens with the feature of bidirectional electron transfer such
as \emph{Geobacter} , \emph{Pseudomonas} and \emph{Comamonas} are the
contributor for BEDN, they can not only release electrons by the organic
oxidation, but also enable denitrification by accepting electrons from
electrode (Gregory, Bond, \& Lovley, 2004; Yu, Wu, Cao, Gao, \& Yan,
2015). It has to be noted that \emph{Geobacter} in the anodes of
SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}with relative abundance of 4.2\% and 2.1\% at
the initial stage survive in the denitrification process and remains
2.5\% and 0.6\% at the 100\textsuperscript{th} day,
respectively.\emph{Pseudomonas} in the anode of SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}and
SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}with the relative abundance of 2.2\% and 1.5\% at the
initial stage is preserved during the 100 days acclimation, and became
2.3\% and 1.3\% at the 100th day, respectively. A high relative
abundance of 10\% \emph{Pseudomonas}is enriched in the SAMFC-OC at the
initial stage, but could not be detected at the 100\textsuperscript{th}
day. The successful inheritance of \emph{Geobacter} and
\emph{Pseudomonas} from anterior biofilm indicated that exoelectrogens
could coexist well with denitrifers during denitrification process
despite a tiny drop of the relative abundance was observed. In addition,
\emph{Comamonas} that has already existed in acetate-acclimated biofilm
is typical exoelectrogens equipped with the ability of denitrification
(H. Huang et al., 2019; Liang et al., 2019). The \emph{Comamonas} at the
initial stage has a low relative abundance of 1\textasciitilde{}2\% in
all SAMFCs, its proportion in the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}rises remarkably to 14.0\%
after acclimation for 100 days in the presence of nitrate, but there is
no obvious change in the SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and SAMFC-OC. Thus, the notable
enrichment of \emph{Comamonas} in SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}might highly contribute to
the consumption of nitrate. Most critically, the sum of relative
abundance for \emph{Geobacter} ,\emph{Pseudomonas} and \emph{Comamonas}
at the 100th day is 18.9\% in SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}which is three times higher
than that in SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}(5.7\%) and nearly five times higher than that
in SAMFC-OC (3.8\%). The high relative abundance of exoelectrogens in
the anodophilic biofilms is considered to be the key factor to endow the
SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}with excellent denitrification performance. As shown in Figure
S4, in one aspect the exoelectrogens can directly catalyze the
bioelectrochemical denitrification, in another aspect the exoelectrogens
are also able to catalyze oxygen reduction reaction, thus they could
consume the trace oxygen diffused from the air-cathode and thus promote
the propagation of denitrifers, e.g. \emph{Alicycliphilu} s. That is why
a high relative abundance of \emph{Alicycliphilu} s (18.2\selectlanguage{ngerman}±1.8 \%) could
be observed in the SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}at the 100\textsuperscript{th} day (as
shown in Figure 4), which is much higher than that in the SAMFC-1000
(9.8\selectlanguage{ngerman}±1.9\%) and SAMFC-OC (5.5±2.1\%).

\textbf{4. CONCLUSION}

The impact of anodophilic biofilms bioelectroactivity on denitrification
behavior of single-chamber air-cathode microbial fuel cell (SAMFC) in
steady state was studied. Results showed that the denitrification rates
of the SAMFCs increased with time and achieved a stable level after
running for about 80 days. The SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}showed lower denitrification
rate than the SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}at the 20\textsuperscript{th} day, but
surpassed it after running for over 40 days. The SAMFC-20\selectlanguage{greek}Ω \selectlanguage{english}not only
generated 61.7\% higher power than the SAMFC-20\selectlanguage{greek}Ω, \selectlanguage{english}but also achieved a
50\% and 40\% higher \emph{k\textsubscript{DN}} than the SAMFC-1000\selectlanguage{greek}Ω \selectlanguage{english}and
SAMFC-OC, respectively. The enhanced power generation and
denitrification performance was attributed to the high
bioelectroactivity of the anodophilic biofilms, which not only promote
current generation, but also improved BEDN facilitated the enrichment of
other denitrifers. This study provides an important theoretical basis
for enhancing power generation and denitrification performance of SAMFC
synchronously, would promote its practical application for wastewater
treatment.

\textbf{ACKNOWLEDGEMENT}

The authors wish to thank the support from the National Science and
technology award cultivation project of Jiangxi Province (20203AEI010)
and Natural Science Foundation of Jiangxi Province, China
(20192ACB20014).

\textbf{CONFLICT OF INTERESTS}

The authors declare that there is no conflict of interests.

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