© 2022 The Authors Water Science & Technology Vol 86 No 5, 979 doi: 10.2166/wst.2022.

216

A novel sulfur-driven autotrophic denitrification coupled with bio-cathode system for

bioelectricity generation and groundwater remediation

Jianping Chenga, Dai Tanga, Zhiguo Tanga, * and Jin Guob

a
School of Mechanical Engineering, Hefei University of Technology, Hefei, Anhui Province 230009, China
b
School of Environment and Chemical Engineering, Anhui Vocational and Technical College, Hefei, Anhui Province 230011, China
*Corresponding author. E-mail: tzhiguo@hftut.edu.cn

ABSTRACT

This study explored the feasibility of treating wastewater using sulfur-driven autotrophic denitrification (SAD) coupled with the bio-cathode of
microbial fuel cell (MFC), focusing on simultaneous bioelectricity generation, denitrification, and desulphurization. A maximum output voltage
of 360 mV was obtained with a power generation cycle of 25 h when simulated wastewater with 100.0 mg/L of each NO
2

3 -N and S -S was


employed as the influent in the SAD-BMFC. Compared with solo SAD or MFC, SAD-BMFC obtained a higher NO3 -N removal rate (E12 h ¼
87.7%, E24 h ¼ 100%), and less NO
2
0
2 -N accumulation. S -S of the influent was almost completely removed, oxidized to S -S (88.6–
90.2 mg/L) and SO2

4 -S (9.8–11.4 mg/L). The reaction system achieved self-balance of acidity-alkalinity (pH 7.05–7.35). The SAD process
was the main pathway for NO
3 -N removal (80.2%) and a smaller proportion of electrons came from the bio-cathode. This study effectively
combined SAD with a bio-cathode system for simultaneous energy harvest and bio-enhanced remediation of groundwater contaminated by
both NO
2

3 -N and S -S.

Key words: bio-cathode microbial fuel cells, bioelectricity generation, sulfur-driven autotrophic denitrification

HIGHLIGHTS

• The triple performance of bioelectricity generation, denitrification and desulfurization was realized in the SAD-BMFC.
• The denitrification efficiency was improved and the power generation performance was weakened due to the addition of S2
-S.
• S2
-S was oxidized to S0-S, even SO2

4 -S.
• The self-balance of acidity-alkalinity (pH ¼ 7.05–7.29) was achieved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and
redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).

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 Water Science & Technology Vol 86 No 5, 980

GRAPHICAL ABSTRACT

INTRODUCTION
The excessive use of nitrogen fertilizer leads to nitrate pollution in groundwater, which directly affects the safety of drinking
water as it intensifies. Biological nitrogen removal is commonly used to address nitrate pollution as it is capable of removing
fixed nitrogenous compounds as harmless dinitrogen gas (N2) in an effective and economical way (Ren et al. 2020). Biological




denitrification is accomplished by aerobic nitrification (NHþ 4 -N → NO2 -N → NO3 -N) and anoxic denitrification (NO3 -N →
NO
2 -N → N2) using aerobic nitrifying bacteria and anoxic denitrifying bacteria, respectively. Although other technologies
(such as Anammox) have recently emerged and are being widely implemented (Capodaglio et al. 2016), biological nitrific/
denitrif is the most common process adopted. Microorganisms are not able to carry out efficient heterotrophic denitrification
in wastewater treatment applications where organic carbon sources are insufficient (Zhang et al. 2012; He et al. 2016).
Autotrophic denitrification can reduce NO
3 -N to N2 using electron donors (such as inorganic hydrogen (H2), elemental
sulfur (S0-S), and reduced iron (Fe0/Fe2þ)) when there is sufficient inorganic carbon (i.e., CO2, HCO
2

3 , and CO3 ) (Ashok
& Hait 2015; Francesco et al. 2019).
Bio-cathode denitrification is a new technology that combines denitrification with electrochemical technology. This pro-
cess uses hydrogen (H2) produced at the cathode via electrolysis from water as the electron source for autotrophic
denitrifying bacteria (Liu et al. 2015). This enables denitrification without an organic carbon source as the in-situ H2 can
be directly utilized by microorganisms attached to the cathode with a high utilization efficiency (Molognoni et al. 2017). How-
ever, the costs of operating and maintaining this system are high due to the low solubility of H2 in water, the large
consumption of anode carbonaceous materials by O2 produced from the electrolysis process, and the high voltage that is
required (Cecconet et al. 2020).
Microbial fuel cells (MFC) use microorganisms to catalyze anodic oxidation reactions or/and cathodic reduction
reactions to achieve energy recovery, waste recycling, and pollutant removal (Zou & He 2018). The bio-cathode of
an MFC can directly provide electrons to make up for the lack of a carbon source for denitrification and to recover
energy without applying external voltage (Zhai et al. 2018). A nitrate removal rate of 0.146 kg NO
3
3 -N/(m ·d) and a
3
maximum power density of 8 W/m have been obtained using the bio-cathode of an MFC without an organic
carbon source (Clauwaert et al. 2007). Power generation and denitrification were both affected by the configuration
of the MFC, electrode materials, separation materials, carbon nitrogen ratio (C/N), and dissolved oxygen (Guo et al.

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 Water Science & Technology Vol 86 No 5, 981

2019; Zeng et al. 2020). Researchers have used various approaches to improve NO
3 -N removal efficiency, such
as combining MFC and controlled biocathodic denitrification (CBD) (Cecconet et al. 2018a, 2018b).
Sulfur-driven autotrophic denitrification (SAD) is a process well suited for the treatment of nitrogen and sulfur-polluted
wastewater without the supplementation of organic carbon. Generally, reduced sulfur (S2
-S, S0-S, and S2 O2
3 -S) is used as
the electron donor that drives NO
3 -N denitrification with inorganic carbon sources such as CO 2 , HCO

3 , and CO2
3 under
0
anoxic conditions (Lin et al. 2018). As an electron donor, elemental sulfur (S -S) can not only obtain two electrons to form
sulfide (S2
-S), but can also lose electrons to form oxides such as SO2
4 -S, and form compounds with various valence states
such as thiosulfate (S2 O2

3 ) through covalent bonds. Simultaneous desulfurization and denitrification, in which the electron
transfer of sulfide oxidation (S2
-S → S0-S) and anoxic denitrification (NO
3 -N → NO

2 -N → N2) can be balanced, was rea-
lized by controlling the appropriate processing conditions (Wang et al. 2007). Xu et al. (2016) obtained a sulfide
removal rate of 98% and a nitrate removal rate of 96% by microorganisms under conditions that were near neutral and
weak alkali. An effective desulfurization could be achieved by adjusting the pH to near neutral (influent pH was
7.5–8.0) and weak alkali (influent pH was 8.5–9.0) over 24 h without aeration or the addition of organic matter. Utilizing
S2
-S as the electron donor, this system could not only denitrify NO
3 -N in sewage, but also controlled the S
2

concen-
tration in the effluent (Zhang et al. 2020). Notably, a novel SAD coupled with MFC (SAD-MFC) has been widely
used to treat wastewater contaminated with organic, sulfurous, and nitrogenous compounds. Specifically, sulfide rep-
resents a superior electron donor at the anode due to its very low redox potential (S2
S/H2S:
0.28 V versus
standard hydrogen electrode at pH 7.0) (Zhang et al. 2018) and its ability to transfer eight electrons per sulfur atom
in both abiotic and biotic processes (Gong et al. 2013). The produced electrons can then be transferred through external
circuits to the cathode where NO
3 -N and other oxidized pollutants can be reduced, concomitantly generating electricity
during the redox reaction (Wang et al. 2018). In practice, the SAD-MFCs obtained a coulombic efficiency of 53.0% +
2.2% at the optimal feeding total organic carbon to sulfide (TOC/S) mass ratio of 4.69 and cathodic feeding DO of
4.2 mg/L in a looped microbial fuel cell system (Guo et al. 2020). Furthermore, a summed coulombic production of
554.8 C/d was achieved with a desirable feeding S/N molar ratio of 3 in a coupled nitrifying and denitrifying sulfide
removal MFC system (Chen et al. 2019).
However, to date, the combined effects of denitrification, S2
-S removal, and bioelectricity generation in a sulfur-
driven autotrophic denitrification system coupled with bio-cathode MFC (SAD-BMFC) have not been studied. This
is despite the direct relevance as pollutants including both NO
3 -N and S
2

-S are commonly mixed together in
groundwater.
Therefore, a coupled system (SAD-BMFC) was constructed to accelerate NO
3 -N removal and spontaneously generate bio-
electricity. The removal and transformation of S2
-S in the cathode chamber was explored and the self-balancing of the
reaction system’s acidity-alkalinity was investigated. The electron sources for denitrification were analyzed. The mechanisms
of the SAD-BMFC system promoting the removal of NO
2

3 -N and S -S were revealed. This study will directly help efforts to
combine SAD with bio-cathode systems for bioelectricity generation and bio-enhanced remediation of groundwater contami-
nated by coexisting NO
2

3 -N and S -S.

EXPERIMENTAL
SAD coupled with bio-cathode microbial fuel cell (SAD-BMFC)
Methacrylate MFC reactors (Figure 1), as reported (Guo et al. 2020) in our previous study, were built with anode and cathode
compartments separated by a proton exchange membrane (Nafion 117, Dupont, USA).
The cathode chamber and anode chamber were completely symmetrical, with geometric dimensions of 12 cm  4 cm 
12 cm (length  width  height), and an effective solution volume of 400 mL. The effective area of the proton membrane
was 63.5 cm2. Stoppers were equipped to create monitoring ports, liquid inlets, and liquid outlets. Before use, the carbon
brushes were soaked in 1 mol/L sodium hydroxide solution and 1 mol/L hydrochloric acid solution for 24 h, then repeatedly
washed with deionized water until neutral and placed in deionized water for use. The anode and cathode were electrically
connected through an external copper circuit under a 1,000 Ω load (R). Connections were fixed with nonconductive
epoxy resin. A data acquisition card (DAQ Card, USB-4716, Yanhua Corp., China) was used in the system to measure the
produced voltage and data were recorded every 180 s throughout the entire experiment.

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 Water Science & Technology Vol 86 No 5, 982

Figure 1 | Schematic of the SAD-BMFC.

Inoculum and substrates

The anolyte contained the following components: carbon source (CH3COONa 0.641 g/L), buffer solution (K2HPO4·3H2O
6.57 g/L, KH2PO4 2.88 g/L), NH4Cl 0.31 g/L, 12.50 mL microelement solution, and 5.0 mL nutrient solution in a batch
mode as previously reported (Guo et al. 2020).
The catholyte was simulated wastewater containing the following components: carbon source (NaHCO3 1.0 g/L),
NaH2PO4·12H2O 0.10 g/L, 12.50 mL microelement solution, and 5.0 mL nutrient solution. Pollutants (S2
-S and NO
3 -N)
also were included. Na2S·9H2O (0.75 g/L) solution was provided as S -S, and KNO3 solution was provided as NO

2

3 -N.
The S2
-S concentrations of 100.0 mg/L was chosen considering that the concentration of S2
-S tolerance of microorganisms
was 200–300 mg/L (Tao et al. 2020). The initial addition of S2
-S was controlled within 100.0 mg/L in the solution, and the
initial NO
3 -N concentration was also limited to 100.0 mg/L, namely, the mass ratio of sulfur to nitrogen (S/N) was 1.0.
Inoculated anaerobic sludge was added to the chamber. The microorganisms of the anode and cathode chambers were
from anaerobic sludges domesticated for three months. The suspended solids (SS) and volatile suspended solids (VSS) of
the anaerobic sludge was 500.0 mg/L and 2,000.0 mg/L, respectively.

Operating conditions
The SAD-BMFC reactors adopted the sequential batch operation mode. The reactors were placed in a biochemical incubator,
and the ambient temperature was controlled to 25 + 1 °C. The mixed solution of anolyte and inoculated sludge was added to
the anode chamber, and the mixed solution of cathode liquid and inoculated sludge was added to the cathode chamber.
Before each test, the anode liquid and catholyte were bubbled with argon for deoxidization. When the voltage and
NO
3 -N removal efficiency stabilized, it was judged that the reactor had started successfully. If the output voltage of the
SAD-BMFC dropped below 50.0 mV, it was determined that the power generation cycle had ended, and the anolyte and cath-
olyte were both replaced. In each power generation cycle, the output voltage was recorded every 180 s. The electrode
potential was measured at 1, 5, 10, 15, 20, and 25 h. The NO


3 -N and NO2 -N tests were carried out at 0, 3, 6, 9, 12, 18,
and 25 h. The concentration of S2
-S in the cathode chamber was measured at the beginning of every power generation
cycle. The TDS and SO2
0
4 -S concentration in the cathode solution were tested at 25 h. S -S concentration was calculated
using sulfur element balance accounting. The pH value of the catholyte was measured at 0, 6, 12, 18, and 24 h.
To clarify the mechanisms of the SAD coupled with bio-cathode microbial fuel cell system for denitrification, the electrons
were calculated from the SAD and bio-cathode separately. The circuit was disconnected from the SAD-BMFC, or S2
-S was

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 Water Science & Technology Vol 86 No 5, 983

not added to the catholyte, allowing the estimation of SAD and BMFC electrons, respectively. The bioelectricity generation,
denitrification, desulfurization efficiency, and pH balance were compared and studied.

Analytical methods and calculations

Bioelectrochemical measurements
The output voltage (U, mV) was recorded using a multimeter data acquisition unit (PCI1713). The potentials of the cathode
and anode were measured using an Ag/AgCl electrode (þ0.195 V vs. SHE) as reference electrode.

Chemical analyses
The pH in the reactors was monitored using a pH meter (WTW pH 3210, Germany), but not regulated. NO


3 -N and NO2 -N
were measured by the ultraviolet spectrophotometric screening and colorimetric methods, respectively (APHA 2012). The
total dissolved sulfide (TDS), including H2S, HS
-S, and S2
-S were determined using the methylene blue method during
an entire electricity generation cycle (APHA 2012). Intermediate sulfides in compounds were determined using an X-ray
photoelectron spectroscope (Thermo Fisher Scientific, USA) and an ESCALAB 250 spectrometer (VG Instrument Group
Co., Ltd, UK) (Sun et al. 2009); SO2
2

4 -S and S2 O3 -S thiosulfate were determined with an ion chromatograph (HIC-Super
20A, Shimadzu, Japan). The concentration of elemental sulfur S0-S was calculated according to the sulfur element balance
(Graaff et al. 2012), which followed the Equation (1).

C(s0
s)o ¼ C(s2

s)i
C(SO2

s)o
C(TDS
s)o (1)

4

where, C(s2

s)o is the S0-S concentration of effluent after a cycle; C(s0
s)i is the initial S0-S concentration; C(SO2

s)o is the
4


SO2

4 -S concentration of effluent after a cycle; C(TDS
s)o is the total dissolved sulfide (TDS), including H2S, HS -S, and
S2
-S, of effluent after a cycle.

RESULTS AND DISCUSSION

Bioelectricity generation in the SAD-BMFC
The bioelectricity generation performance of the SAD-BMFC was shown in Figure 2(a). The bioelectricity generation cycles 1,
2, 3, 7, and 8 were obtained from the BMFC reactor. The average output voltage was about 450 mV and the average bioelec-
tricity generation cycle lasted 35 h. Cycles 4, 5, and 6 were obtained from the SAD-BMFC reactor. The average output voltage
was about 350 mV and the average power generation cycle lasted 25 h. These measurements indicated that the power gen-
eration performance of the SAD-BMFC, including the maximum output voltage and cycle time, was lower than that of

Figure 2 | Electricity production. (a) Voltage output and (b) potential change in the SAD-BMFC (the electricity production cycles marked 1, 2,


3, 7, and 8 were from the MFC, in which S2 was not added to the cathode chamber; the electricity production cycles marked 4, 5, and 6
2

were from the SAD-BMFCs, in which S was added to the cathode chamber).

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 Water Science & Technology Vol 86 No 5, 984

MFC under the same conditions. The only difference was the addition of S2
-S, which caused weakness in the power gener-
ation performance.
The electrons for NO
3 -N denitrification in the SAD-BMFC came from two pathways. Some electrons were generated from
sodium acetate in the anode as it was oxidized; these electrons then would flow to the cathode through the wire to form a
closed circuit, producing the observed output voltage of the SAD-BMFC, as shown in formula (2). The other electrons
came from S2
-S oxidation in the cathode, as shown in formula (3). All electrons and NO
3 -N were available to the cathode
denitrifying bacteria for metabolism and to complete the denitrification process, as shown in formula (4).

CH3 COO
þ 2H2 O ! 2:5CO2 þ 6e
þ 7Hþ (2)

þ
2S2
þ 3H2 O ! S0 þ SO2

4 þ 6e þ 6H (3)

2NO
þ

3 þ 12H þ 10e ! N2 þ 6H2 O (4)

Due to the addition of S2
-S in the cathode liquid, a portion of NO
3 -N was consumed and autotrophic sulfur denitrification
occurred, resulting in the reduction of NO
3 -N as an electron acceptor (An et al. 2010), which showed that the power gener-
ation performance of SAD-BMFC was not better than the MFC, indicating that the weakened output voltage and power
generation cycle of the SAD-BMFC resulted from the participation of S2
-S.

NO

3 -N removal efficiency in the SAD-BMFC
Promoted NO
2

3 -N removal in the presence of S -S
The NO
3 -N removal efficiencies of the SAD, MFC, and SAD-BMFC reactors are shown in Figure 3. After a power generation
cycle of 25 h, the NO
3 -N concentration in the SAD-BMFC reactor decreased from 100.0 mg/L to almost 0 mg/L, with a
removal rate close to 100%. The removal rate exceeded 64.7% by BMFC and 84.5% removal rate was obtained by SAD.
These results showed that the NO
3 -N removal efficiency was enhanced by the presence of S -S.
2

NO3 -N concentrations were used to estimate kinetic parameters, as shown in Table 1. A quasi first-order reaction kinetics
model was a good choice to describe NO
2
3 -N removal (R ¼ 0.954–0.987) in the SAD, MFC, and SAD-BMFC reactions,
among which there were obvious differences. The highest NO
3 -N removal efficiency was observed in the SAD-BMFC reactor
(E25 h ¼ 100%), which was 1.19 and 3.98 times higher than in the MFC (E25 h ¼ 64.7%) and SAD (E25 h ¼ 84.5%) reactors after
an entire power generation cycle. These results indicated that another electron source became available due to the addition of
S2
-S (Tong et al. 2017). SAD was the main pathway for nitrate NO
3 -N denitrification, and the application of the bio-cathode
enhanced the microbial catalytic activity, which was partially responsible for the enhanced performance. Furthermore, 87.7%

Figure 3 | Nitrate removal in three different reactors.

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 Water Science & Technology Vol 86 No 5, 985

Table 1 | Nitrate removal efficiencies (E1 h and E24 h) and simulation results using apparent first-order reaction kinetics (k and R 2) in the BMFC,
SAD, and SAD-BMFC reactors

Nitrate removal efficiencies /% Simulation results

1
Reactor no E12 h E25 h k/h R2

MFC 47.8 64.7 0.08 + 0.004 0.983

SAD 67.8 84.5 0.11 + 0.004 0.987
SAD-BMFC 87.7 100 0.13 + 0.004 0.954

of the nitrate removal (E12 h) occurred by 12 h, which indicated that denitrification mainly occurred early in the power gen-
eration cycle.

NO

2 -N accumulation
As shown in Figure 4, the NO
2 -N concentration showed a similar trend, initially increasing and then decreasing to zero, indi-
cating that there was hardly any NO
2 -N accumulation in the three models. The peak value (0.56 mg/L) of nitrite
accumulation in the SAD-BMFC reactor was 78% and 68% of the MFC (0.72 mg/L) and SAD (0.82 mg/L) reactors, and
the peak in the SAD-BMFC reactor occurred earlier (8 h) than in the MFC (9 h) and SAD (11 h) reactors. This may have
been because the denitrification efficiency was increased by access to twice as many electron donors, which reduced
NO
2 -N accumulation (Liu et al. 2019).

Analysis of S2
-S removal and transformation

Transformation of S2
-S in the solution
It can be seen from Figure 5 that the mixed sample of cathode precipitation and solution had two characteristic peaks on the
XPS spectrum. The wide peak at 160–172 eV was made up of overlapping peaks, the S2P3/2 peak at 161.5–164 eV belonging to
S(-II) (S2
-S), the S2P1/2 peak at 163.8 eV belonging to S(0) (S0-S), and the peak at 166–172 eV belonging to S(þVI) (SO2

4 -S).
That is to say, S2
-S, S0-S, and SO2
4 -S were detected simultaneously in the cathode solution of SAD-BMFC.
A large number of studies have examined the sulfide oxidation pathway, however, our understanding of sulfide is still lack-
ing. Generally speaking, the oxidation pathway of sulfide involves two typical intermediates, elemental sulfur (S0-S) and
thiosulfate (S2 O2
2

3 -S), and one final product, sulfate (SO4 -S), following the reactions described in formulas (5), (6), and

Figure 4 | Nitrite accumulation in three different reactors.

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 Water Science & Technology Vol 86 No 5, 986

Figure 5 | Characterization of sulfide in the SAD-BMFC using XPS.

(7) (Susumu et al. 1987).

5S2
þ 2NO
þ 0
3 þ 12H ! 5S þ N2 þ 6H2 O (5)

6S0 þ 2NO
2

3 þ 3H2 O ! 3S2 O3 þ N2 þ 6H
þ
(6)

2S2 O2
0 2

3 þ 2NO3 ! S þ 3SO4 þ N2 (7)

The conversion of sulfide, S2
-S, to elemental sulfur, S0-S and S2 O2
3 -S, is described in formulas (5) and (6). When there is
excess NO
3 -N in the system, autotrophic bacteria continue to use electron donors such as S0-S and S2 O2

3 -S to carry out meta-
bolic activities, so the reactions shown in formulas (5) and (6) are more likely. Of the three formulas, formula (5) is the process
of consuming Hþ and formula (6) is the process of producing Hþ, both of which affect the pH of the solution. Although for-
mula (7) does not affect the solution pH directly, it is directly dependent on formulas (5) and (6). The reaction in formula (7) is
only carried out when there is still NO
3 -N remaining in the system after the reactions in formulas (5) and (6) are complete,
that is, it is limited by the concentration of NO
3 -N, and its reaction rate is slow.
From the experimental cathode solution, only S0-S and SO2
2

4 -S were detected and no intermediate S2 O3 -S was present.
2

This was because S2 O3 -S is not exclusively a product of the sulfide oxidation reaction (formula (5)), but can also participate
in autotrophic denitrification as an electron donor to produce S0-S and SO2
4 -S (formula (7)) depending on the amounts of
2

other substances. Therefore, theoretically, the concentration of S2 O3 -S in the effluent depends on the degrees of the reac-
tions in formulas (6) and (7). In the early stage of the power generation cycle, the high pH of the cathode solution makes
the reaction in formula (6) much easier to carry out, with S2 O2
3 -S being the product of the reaction process. As the reaction
progresses, the rate at which Hþ is consumed by NO
3 -N on the bio-cathode exceeds the rate at which Hþ is produced by
SAD, which reduces the pH of the solution and weakens the inhibitory effect of OH
on formula (5). Furthermore,


S2 O2

3 -S in the influent can be utilized by autotrophic bacteria to continue to interact with NO3 -N to produce S -S and
0

2

SO4 -S in the system. Generally, its concentration in the effluent should be reduced as much as possible, that is, the reaction
in formula (6) should be inhibited and the reaction in formula (7) should be promoted. S2 O2
3 -S was not detected in this exper-
iment. The standard Gibbs energy (
240 kJ/mol;
38.0 kJ/mol) and redox potential (þ0.79 V;
0.22 V) in the two processes
of denitrification and SO2
2

4 -S differ greatly. Denitrification will inhibit the SO4 -S reduction process (Löffler et al. 1999; Cao
et al. 2017). In addition, NO3 -N is often widely used as an inhibitor of SO4 -S process, and NO


2
2

3 -N diminished SO4 -S and
2

prevented S -S accumulation under freshwater, brackish, and saltwater conditions. Sodium-, potassium-, and calcium
NO


3 -N were equally effective in curtailing sulfide formation. NO3 -N amendments control the formation of S -S in oily
2

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 Water Science & Technology Vol 86 No 5, 987

waste streams both by preventing SO2
2

4 -S reduction and by stimulating anaerobic S -S oxidation (Satoshi et al. 2002; Londry
& Suflita 1999). Therefore, in the SAD-BMFC coupling system, there is a weak SO2
4 -S reduction process in the late stage of
the reaction, which will not affect the removal of NO
3 -N.

Removal of S2
-S
As shown in Table 2, S2
-S was removed and converted after a power generation cycle of 25 h. The removal efficiency of S2
-S
reached 100% and there was no obvious TDS-S accumulation. When 100.0 mg/L of S2
-S was added into the influent of
SAD, while there was hardly any TDS-S in the effluent, the concentration of S0-S was 47.7–62.4 mg/L and that of SO2
4 -S
was 52.3–37.4 mg/L. This showed that the probability of S0-S and SO2
4 -S being generated was almost the same. Similarly,
in the SAD-BMFC, the concentration of TDS in the effluent was also zero, however, the concentration of S0-S was 88.6–
90.2 mg/L and that of SO2
2
0
4 -S was 9.8–11.4 mg/L, indicating that more S -S was transformed into S -S, and even SO4 -S.
2

2
0 0 2
0
As the oxidation rate of S -S to S -S was 3.31 times faster than that of S -S to SO4 -S, S -S accumulation would be observed
under these conditions (Yuan et al. 2019). Desulfurization and denitrification were both feasibly realized in the SAD-BMFC.
The micro voltage generated from SAD-BMFC caused the proportions of S0-S and SO2
4 -S to diverge, indicating that the
micro voltage had affected the performance of S2
-S removal by influencing the enrichment of the associated microorganisms
in the bio-cathode (Guo et al. 2020) when another electron donor was supplied. According to the reaction formulas (5) and
(6), these changes would reduce the probability of generating SO2
4 -S, which was consistent with the experimental results.

pH change in the solution

As shown in Figure 6, the pH fluctuated during the nitrate removal process. The initial pH value of the cathode solution was
7.2 + 0.2. Theoretically, the complete reduction of 1 mg NO
3 -N will consume 4.57 mg CaCO3 alkalinity in SAD (Sahinkaya
et al. 2014), and the pH value will decrease (formula (3)). The experimental results showed that the pH of the cathode sol-
ution decreased to 6.44 in 6 h, then slowly decreased to 6.22 where it remained to the end of the cycle. The reduction of

Table 2 | Sulfur balance in the MFC, SAD and SAD-BMFC/(mg/L)

Influent Effluent

2

Reactors no S -S TDS S0-S SO2

4 -S

SAD 100 0 47.7–62.4 52.3–37.4

SAD-BMFC 100 0 88.6–90.2 9.8–11.4

Figure 6 | Change in pH during nitrate removal in three different reactors.

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 Water Science & Technology Vol 86 No 5, 988

NO
þ
3 -N consumes H in the MFC (formula (4)), so the pH of the solution increased slowly, and peaked at 7.64 after 24 h of an
electricity generation cycle.
However, there was no significant fall or rise in pH value in the SAD-BMFC. The pH value decreased to 6.78 in 6 h, then
increased slowly to 7.35 by the end of the power generation cycle. This was because Hþ was generated more quickly by SAD
and the bio-cathode than it was consumed by NO
3 -N, which made the pH of the cathode solution decrease. However, as the
reaction time progressed, the S -S concentration decreased and the Hþ generated rate from the SAD and bio-cathode no
2

longer exceeded rate of NO
3 -N consumption, at which point the pH value began to rise again until reaching pH self-balance
(Park et al. 2016). The above results indicated that acid-base self-balance could effectively be realized in the SAD-BMFC.

Analysis of electron sources for denitrification

Based on the principle of SAD and MFC, electron balances were calculated. There were two electron sources in the SAD-
BMFC, i.e., sodium acetate oxidation, where electrons were transferred to an external circuit for generation of bioelectricity,



and S2
-S oxidation to S0
S and SO2
4 -S (Park et al. 2016). These electron sources were accepted by NO3 -N or NO2 -N. To

2

denitrify 100.0 mg/L NO3 -N to N2 (311C) electrons are needed, and based on the S -S and SO4 -S generated (Table 2), S2
-S
0

oxidation supplied (249C), the other electrons came from the bio-cathode.
As shown in Figure 7, the proportions of electrons provided by S2
-S oxidation were 100% and 80.2%, respectively, in the
SAD and SAD-BMFC reactors, and the corresponding proportions of electrons provided by the bio-cathode were 0% and
19.7%. The above results demonstrated that S2
-S was the main electron donor for denitrification and the bio-cathode
made a relatively small contribution of electrons, that is, in the SAD-BMFC.

Mechanism for enhanced denitrification in the SAD-BMFC

To better understand the mechanisms underlying denitrification and desulfurization in the SAD-BMFC, the fates of NO
3 -N
and S2
-S in the cathode chambers were investigated separately (Figure 8). The results showed that the SAD-BMFC removed
nitrate efficiently (E24 h ¼ 100%); 1.19 and 3.98 times more efficient, respectively, than in the MFC (E24 h ¼ 64.7%) and SAD
(E24 h ¼ 84.5%) (Table 2). Furthermore, there was hardly any NO
2 -N accumulation in the SAD-BMFC (Figure 4). According
to previous literature (Liu et al. 2019), the required electrons, which partially originated from the bio-cathode, were consumed
during the NO


3 -N or NO2 -N denitrification processes. Without additional energy, the electrons released from the oxidation
of sodium acetate in the anode spontaneously moved from low to high potential, that is, the electrons migrated through three
steps: ‘anode microorganism → anode’, ‘anode → cathode’, ‘cathode → cathode microorganism’. This enabled electrons to be

Figure 7 | Proportion of electrons derived from S2
-S in the SAD-BMFC.

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 Water Science & Technology Vol 86 No 5, 989

Figure 8 | Possible mechanisms of bio-cathode denitrification enhancement in the SAD-BMFC.

accepted by the cathode microorganisms and complete the denitrification process (Lefebvre et al. 2008). The results showed
that lower cathode potentials (
0.110 V vs. SHE, Figure 2) created more favorable conditions for anaerobic digestion com-
pared to the anode potential (þ0.350 V vs. SHE) (Logan et al. 2008), indicating that the required electrons partially originated
from the anaerobic digestion of sodium acetate.
Previous studies indicated that S2
-S could be oxidized to S0-S, S2 O2
2

3 -S even SO4 -S by denitrifying community with the
release of electrons (Park et al. 2016 and Capua et al. 2019). Similar results were obtained in our study, where S0-S and
SO2
2

4 -S were generated simultaneously by the oxidation of S -S through microbial processes under anaerobic conditions
(Figure 5 and Table 2). S2
-S from the original 100.0 mg/L was hardly found, and the generated S0-S and SO2
4 -S peaked
at 88.6 mg/L and 9.8 mg/L (Table 2). This allowed the electrons from SAD to function as electrons for denitrification.
These results confirmed the occurrence of S2
-S driven NO
3 -N denitrification in the cathode chamber.
Previous studies indicated that the complete reduction of NO
3 -N would consume alkalinity and reduce the pH value of
SAD, which would lower the denitrification efficiency (Sahinkaya et al. 2014). However, the acid-base self-balance was effec-
tively realized in the SAD-BMFC system. Specifically, the Hþ demand for NO
3 -N consumption could be provided by both the
Hþ of the SAD and the bio-cathode. The experimental results indicated that the Hþ rate provided by the SAD and bio-cathode
was greater than the NO
3 -N consumption, which made the pH of the cathode solution decrease to 6.78 in 6 h, before increas-
ing slowly to 7.35 by the end of a power generation cycle (Figure 6). This was because the S2
-S concentration decreased as
the reaction time progressed, and the Hþ generated from the SAD and bio-cathode could not make up for the NO
3 -N con-
sumption of Hþ, so the pH began to rise again until realizing pH self-balance.
The higher effluent S0-S in SAD-BMFC may be considered related to S2
-S to NO
3 -N (S/N) mass ratio. SAD process con-



sists of sequential reductive reactions from NO3 -N to NO2 -N, nitric oxide (NO-N), nitrous oxide (N2O) and then finally to
nitrogen (N2) catalyzed by specific metalloenzymes respectively (Akashdeep et al. 2021). Although other intermediates had
been not detected apart from NO


3 -N and NO2 -N. A minimum theoretical stoichiometric S/N mass ratio (g-S/g-N) of 1.4
(with S -S as an electron donor) and 1.9 (with S0-S as an electron donor) is necessary for complete reduction of NO

2

3 -N
2

to N2 (Yang et al. 2016). The S/N mass ratio was 1.0 in the manuscript. The S -S quantity was far from enough to make
formula (6), formula (7) realize, so as to higher effluent S0-S in SAD-BMFC occur.

CONCLUSIONS
The triple performance of bioelectricity generation, denitrification, and desulfurization was realized in the SAD-BMFC, how-
ever, the denitrification efficiency was improved and the power generation performance was weakened by the addition of S2
-S.
When the concentrations of NO
2

3 -N and S -S were 100.0 mg/L, a maximum output voltage of 360 mV and a cycle time of
25 h were obtained in the SAD-BMFC. NO
3 -N was removed efficiently (E12 h ¼ 87.7%; E24 h ¼ 100%), there was hardly any

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 Water Science & Technology Vol 86 No 5, 990

NO
2
0 2

2 -N accumulation; and the 100.0 mg/L S -S was oxidized to S -S (88.6–90.2 mg/L) and SO -S (9.8–11.4 mg/L). The
self-balance of acidity-alkalinity (pH ¼ 7.05–7.29) was also achieved in the SAD-BMFC. The SAD was the main pathway driv-
ing NO
3 -N denitrification, providing 80.2% of the required electrons, and the bio-cathode was partially responsible for
enhancing the denitrification efficiency.

ACKNOWLEDGEMENTS
This work was financed by the Natural National Science Foundation of Anhui Province (Grant No. 1808085MD102) and
Hefei Natural Science Foundation (Grant No. 2021045).

DATA AVAILABILITY STATEMENT

All relevant data are included in the paper or its Supplementary Information.

CONFLICT OF INTEREST
The authors declare there is no conflict.

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First received 4 May 2022; accepted in revised form 9 July 2022. Available online 15 July 2022

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