Journal of Hazardous Materials 403 (2021) 123682

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Journal of Hazardous Materials

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The reduction of nitrobenzene by extracellular electron transfer facilitated

by Fe-bearing biochar derived from sewage sludge
Yue Lu a, b, *, Qingqing Xie a, b, Lin Tang a, b, *, Jiangfang Yu a, b, Jingjing Wang a, b,
Zhaohui Yang a, b, Changzheng Fan a, b, Shoujuan Zhang a, b
a
College of Environmental Science and Engineering, Hunan University, Changsha 410082, China
b
Key Laboratory of Environmental Biology and Pollution Control, Hunan University, Ministry of Education, Changsha 410082, China

A R T I C L E I N F O A B S T R A C T

Keywords: In this work, the incorporation of Fe-bearing sludge-derived biochar greatly enhanced both biotic and abiotic
Geobacter reduction of nitrobenzene (NB) to aniline, which was attributed to the concomitant microbial dissimilatory iron
Sewage sludge reduction. Biogenic Fe(II) produced by Geobacter sulfurreducens dominated the anaerobic reduction of NB
Biochar
following the pseudo-first-order kinetic. Besides, the increase of pyrolysis temperature from 600 to 900 ℃ to
Nitrobenzene reduction
generate biochar resulted in an accelerated removal rate of NB in Geobacter-biochar combined system. The
Extracellular electron transfer
morphology and structural characterization of biochar with G. sulfurreducens confirmed the formation of
conductive bacteria-biochar aggregates. Electrochemical measurements suggested the presence of graphitized
domains and quinone-like moieties in biochar as redox-active centers, which might play an important role in
accelerating electron transfer for microbial dissimilatory iron reduction and NB degradation. This study provides
a feasible way of using Fe-bearing sludge as a valuable feedstock for biochar generation and its application with
electrochemically active bacteria for the bioremediation of nitroaromatic compounds-polluted wastewater.

1. Introduction
Nitroaromatic compounds (NACs) originated from the industrial
manufacture of explosives, pharmaceuticals, dyes and pesticides are
ubiquitous in wastewater and groundwater environments as a result of
massive use and improper disposal (Luan et al., 2010). The mutagenic
and carcinogenic properties of NACs make it urgent to study their
environmental fate in order to deal with the severe contamination (Wu
et al., 2013). The biotic reduction of NACs with biochemical techniques
exhibits obvious superiority over traditional physical and chemical
methods in terms of avoiding secondary pollution and reducing treat­
ment costs (Wang et al., 2012). Nitro substituent groups on the aromatic
ring of NACs featuring with strong electron affinity are found to be
resistant to biological oxidation, however, they are vulnerable to elec­
trophilic attack and can be effectively reduced to degradable aniline
under anoxic conditions (Wang et al., 2011).
For the microbial anaerobic conversion of NACs, the efficiency of
electron transfer between electrochemically active bacteria (EAB, typi­
cally Geobacter and Shewanella spp.) and extracellular substances acts as a
limiting factor (Pereira et al., 2014). To promote the electron transfer,
redox mediators as a group of chemicals that can be reversibly oxidized
and reduced like catalysts are applied as electron transfer stations be­
tween the initial electron donor and the terminal electron acceptor in
multiple redox reactions (Van der Zee and Cervantes, 2009). Among
these, biochar with graphitic structure and/or quinoid-like redox active
groups was found to be capable of facilitating electron transfer and
speeding up the biotic reductive transformation of contaminants (Kappler
et al., 2014; Yu et al., 2015). For instance, Tong et al. (2014) reported that
biochar enhanced the reductive dechlorination of pentachlorophenol by
promoting extracellular electron transfer (EET) of Fe(III)-reducing and
dechlorinating bacteria in the paddy soil. Beside the biochar, Fe(II)/Fe(III)
redox couple with the extensive reduction potential was found to be
responsible for mediating redox transformation of multiple pollutants,
such as carbon tetrachloride, heavy metal, azo dye (Melton et al., 2014).
Fe(II)/Fe(III) cycling can be biotically driven by EAB that are capable of
dissimilatory iron reduction with Fe(III) oxides as terminal electron ac­
ceptors for their growth (Shi et al., 2016). Highly active species formed by
ferrous iron associated with iron minerals have been demonstrated to
induce reductive transformation of nitrobenzene (NB) in the presence of
Shewanella putrefaciens (Luan et al., 2015).
As described above, sole biochar or Fe(II)/Fe(III) redox couple as
intermediate redox reaction might have facilitated the EET process of

* Corresponding authors.
E-mail addresses: yuelu@hnu.edu.cn (Y. Lu), tanglin@hnu.edu.cn (L. Tang).

https://doi.org/10.1016/j.jhazmat.2020.123682
Received 26 May 2020; Received in revised form 4 August 2020; Accepted 6 August 2020
Available online 13 August 2020
0304-3894/© 2020 Published by Elsevier B.V.
Y. Lu et al.
EAB and affected the reductive transformation of contaminants.
Therefore, in order to further study the impact of Fe-bearing biochar on
the rate and extent of NACs reduction by EAB, iron-rich municipal
sewage sludge, an unavoidable by-product in wastewater treatment
process, is selected as a precursor for biochar production. Each year,
massive sewage sludge has been produced, as a consequence, sludge
disposal and management become an intractable environmental issue
mainly due to its complicated composition (Ho et al., 2017). In this
study, pyrolysis of iron-rich municipal sewage sludge into Fe-bearing
biochar also provides an environmentally friendly alternative to
reduce the volume of the solid residue and to eliminate inherent path­
ogens (Zhang et al., 2018; Yu et al., 2019).
Therefore, this study is a worthwhile attempt to combine Fe-bearing
biochar with G. sulfurreducens PCA (as a model EAB) for NACs reduction.
Iron-rich municipal sewage sludge was calcined into biochar at different
pyrolysis temperatures, and the impact of Fe-bearing biochar on the
transformation of NB (as a model NAC) coupled with electron transfer
process of G. sulfurreducens was investigated. Herein, we showed the Fe-
bearing biochar stimulated EET of G. sulfurreducens PCA and the
dissimilatory iron reduction enhanced the reduction of NB to aniline.
2. Materials and methods
2.1. Materials
NB and aniline were purchased from Shanghai Chemical Corp. All
chemicals utilized in this study were of analytical grade and used
without further purification.
2.2. Synthesis of sludge-derived biochar
Municipal sewage sludge cake formed by filter-press dewatering in
Xingsha Wastewater Treatment Plant (Changsha, China, 28 ◦ N, 113 ◦ E)
was collected with airtight plastic bags and directly transported to the
Key Laboratory of Environmental Biology and Pollution Control, Hunan
University, China. The sewage sludge was stored at room temperature
without any pretreatment. The method used to prepare biochar was
slightly modified from the previous report (Tang et al., 2018). The
sewage sludge was dried under ambient temperature, crushed and
screened through a 100-mesh sieve (0.15 mm). Approximately 27 g of
sieved sludge was transferred to a quartz boat and put into a tubular
reactor (SK-G08123 K, China) with a constant heating rate of 5 ℃/min
under continuous flow of nitrogen (5 mL/min). Samples were pyrolyzed
for 2 h after reaching 600 ℃ (SDB600) or 900 ℃ (SDB900), respectively.
After natural cooling to ambient temperature, the produced biochar was
washed with deionized water and ethanol till reaching neutral pH, and
then dried at 80 ℃ for 12 h in a vacuum oven. The as-prepared biochar
was stored in a desiccator for use without further modification.
2.3. Cultivation of G. sulfurreducens
G. sulfurreducens strain PCA was provided by Prof. Hanqing Yu from
the University of Science and Technology of China. The strain was
routinely cultured in modified DSMZ medium with 10 mL/L of Wolfe’s
vitamin solution, and with 20 mM acetate and 50 mM fumarate as
electron donor and electron acceptor, respectively (Li et al., 2014).
Resazurin was excluded from the medium. For further experiment,
G. sulfurreducens in the exponential growth phase were harvested by
centrifugation (5000 rpm, 5 min) in an artificial atmospheric chamber
(520 L, NO. 690321, Spilfyter) filled with nitrogen. Subsequently, the
cell pellet was resuspended and then washed twice by repeated centri­
fugation and resuspension in the DSMZ basal medium without fumarate.
After being washed, cells were then resuspended in fumarate-free me­
dium for further experiment.
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Journal of Hazardous Materials 403 (2021) 123682
2.4. Experiment set up of G. sulfurreducens cultures amended with
biochar and NB
A series of batch experiments were conducted in sterile 125 mL
serum bottles sealed with rubber stopper and aluminum caps. The
headspace was flashed with N2/CO2 (4:1) gas mixture to ensure an
anaerobic atmosphere. Acetate (20 mM) and NB (initial concentration of
100 μM) were added to 100 mL of modified DSMZ medium as electron
donor and electron acceptor, respectively. After autoclave process, two
types of sludge-derived biochars (SDB600 and SDB900) were respec­
tively added to the culture medium to realize a final concentration of 1
g/L biochar. An 5% (v/v) inoculum of G. sulfurreducens strain PCA was
spiked to reach initial OD600 of 0.035. Biotic controls without adding
biochar (only PCA cells + NB) and abiotic controls without adding PCA
cells (only SDB900/SDB600 + NB) were prepared in parallel. Another
control treatment to investigate microbial iron reduction was prepared
with PCA cells and SDB900/SDB600 but without NB addition. All serum
bottles were set up in triplicate and cultivated at 30 ℃ with 150 rpm
shaking. During the cultivation, 1 mL of sample was sacrificed for the
analysis of HCl-extractable Fe(II) and total Fe concentration following
the standard protocol (Kappler et al., 2014).
2.5. Analytical methods of NB
Another 1 mL of sample was periodically withdrawn from each bottle
and immediately filtered through a 0.22 μm filter to remove biochar and
bacteria cells for NB measurement. The analyses of NB and other
reducing products were performed by high-performance liquid chro­
matography (HPLC, 1260 Infinity, Agilent Co.) equipped with a C18
column (4 μm, 150 × 4.6 mm, Agilent Co.) at detection wavelength of
265 nm (NB) and 231 nm (aniline), respectively. The acetonitrile/water
mixture (1:1) was employed as a mobile phase with flow rate of 1.0 mL/
min at 30 ℃. The filtered liquid sample was extracted with 0.5 mL
hexane by overnight shaking. The upper organic phase (20 μL) was
harvested after centrifugation (5000 rpm, 10 min) and injected into
HPLC.
2.6. Characterization of G. sulfurreducens and biochar
Electron paramagnetic resonance (EPR, JES FA200) spectrometer
was utilized for the detection of persistent free radicals in as-prepared
sludge-derived biochar. Scanning electron microscope (SEM, Hitachi,
S4800) equipped with energy dispersive spectrometer (EDS) was
applied to observe the morphology and correlation between
G. sulfurreducens and biochar. For SEM analysis, the samples with cells
were collected by centrifugation (5000 rpm, 5 min) and fixed with 2.5%
glutaraldehyde overnight at 4 ℃. After being washed twice with 0.1 M
phosphate buffer solution, the samples were serially dehydrated with
30%, 50%, 70%, 85%, 95% and 100% ethanol, and dried in a vacuum
freeze-dryer. The BET specific surface area and pore volumes were
calculated by N2 sorption isotherms using a Quantachrome NovaWin
(NOVA 2000e) at 77 K. The crystallographic structure of biochar was
confirmed by X-ray diffraction (XRD, D8 Advance) with the 2θ scanning
range from 10 to 80◦ . The element composition was determined by X-ray
photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher)
under an Al-Kα X-ray radiation of 1486.6 eV. XPS spectra were decon­
volved using Avantage 5.52 software. Fourier transform infrared spec­
trometer (FTIR, Nicolet 5700) characterized the surface functional
groups ranging from 400 to 4000 cm− 1 wave number. The Raman
spectrum was obtained at the excitation wavelength of 532 nm using a
LabRAM HR800 spectrometer (Horiba Jobin Yvon, France).
2.7. Electrochemical measurements
Electrochemical measurements were applied to investigate the
electrochemical activity of G. sulfurreducens and sludge-derived biochar.
Y. Lu et al.
Solid samples were gathered via centrifugation at 8500 rpm for 5 min
and resuspended in phosphate buffer solution (pH 6.8, 100 mM), and the
supernatant was collected separately. Prior to the electrochemical tests,
the electrolyte (supernatant or solid samples in phosphate buffer solu­
tion) was deoxygenated by sparging pure N2 gas for 20 min and glassy
carbon electrode (GCE) was pretreated as reported previously (Peng
et al., 2010). Cyclic voltammetry (CV) and Differential pulse voltam­
metry (DPV) were tested in an electrochemical workstation (CHI 760E,
China) equipped with the three-electrode system, namely saturated
calomel electrode (SCE) as the reference electrode, the GCE as the
working electrode and Pt electrode as the counter electrode. The CV
measurement was executed from -0.7 V to 0.8 V at the scan rate of 0.01
V/s. And the DPV measurement was performed from -0.6 V to 0.4 V with
the following parameters: pulse height of 50 mV, pulse width of 0.2 s,
pulse period of 0.5 s. All measurements were conducted at ambient
temperature and the electrochemical data of each sample were collected
in triplicate.
2.8. Statistical analysis
OriginPro 8 was utilized to conduct the statistical analyses. One-way
analysis of variance (ANOVA) was performed using SPSS 20.0 (SPSS
Inc., Chicago, USA).
Fig. 1. The FTIR (a) and XRD (c) spectra of the sludge-derived biochar at pyrolysis temperature of 600 ℃ (SDB600) and 900 ℃ (SDB900). SEM pattern of
G. sulfurreducens attached to biochar (b) during NB bioreduction. SEM elemental mapping of SDB900 (red stands for Fe, blue stands for O, green stands for N) (d-g).
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Journal of Hazardous Materials 403 (2021) 123682
3. Results
3.1. Characterizations of sludge-derived biochar
Two different types of biochar were produced from municipal sewage
sludge with pyrolysis temperature of 600 ℃ (SDB600) and 900 ℃
(SDB900), respectively. FTIR spectra of sludge-derived biochar and raw
sludge were measured to investigate the surface functional groups, as
presented in Fig. 1a. The common bands ranging from 1500 to 1720 cm− 1
reflected the vibration of aromatic C=C and carbonyl C=O in quinones,
ketones and carboxylic acids, respectively (Amezquita-Garcia et al.,
2013). Also, the appearance of a small peak at 1389 cm− 1 in SDB900
reflected the C–
– C stretching in aromatic ring carbons (Peng et al., 2016).
The strong peak located at 1056 cm− 1 was assigned to C–O stretching
vibration and showed a slight change, which was attributed to the direct
combination of various forms of oxygen with adjacent carbon elements
during pyrolysis process (Ho et al., 2017; Chen et al., 2014; Pourhosseini
et al., 2018). A broad peak located at 3428 cm− 1 was assigned to the
stretching vibration of − OH groups (hydroxyl or carboxyl), and its
decrease indicated that − OH groups were gradually decomposed at
elevated temperature (Zhu et al., 2017). The two bands assigned to C–H
on aromatic ring (785 cm− 1) and − CH2– (682 cm− 1) group weakened
with increasing temperature, suggesting that the dehydrogenation reac­
tion occurred to enhance aromaticity (Tang et al., 2018; Chen et al.,
2014). Meanwhile, the two peaks with low intensity at 2855 and 2923
cm− 1 were attributed to symmetric and asymmetric stretching of
Y. Lu et al.
aliphatic − CH3, respectively, which disappeared once pyrolysis con­
ducting at higher temperature to generate biochar (Gao et al., 2019).
Therefore, presented results indicated that higher temperature led to the
decrease of surface functional groups due to the transformation of − OH
and − CH3 groups into aromatic structures by gas emission and thermal
decomposition (Tang et al., 2019). The bands around 464 cm− 1 and 557
cm− 1 corresponded to Fe2O3/Si− O− Si and Fe− OH, respectively, indi­
cating the formation of Fe oxides or Fe-O complexes as a consequence of
the interaction of iron and oxygen on the surface of biochar during py­
rolysis process. In addition, the low-intensity peak at 2366 cm− 1 was
assigned to the presence of the goethite (Fe2O3⋅H2O) (Yang et al., 2018).
The XRD pattern showed that the form of Fe oxides in sludge-derived
biochar was mainly γ-Fe2O3 (JCPDS No. 39-0238) with a minor
amount of Fe3O4 (JCPDS No. 65-3107) (Fig. 1c). Apart from Fe oxides,
carbon (JCPDS No. 26-1080), quartz (JCPDS No. 65-0466) and
aluminum oxide (JCPDS No. 10-0425) were also identified as the
Fig. 2. C1s XPS spectra of the sludge-derived biochar at pyrolysis temperature of 600 ℃ (SDB600) (a) and 900 ℃ (SDB900) (b), Fe2p XPS spectra of SDB600 (c) and
SDB900 (d). Curve-fitting of Raman spectra of SDB600 (e) and SDB900 (f).
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Journal of Hazardous Materials 403 (2021) 123682
predominant compositions in sludge-derived biochar.
XPS analysis was executed to further characterize the elemental
compositions and surficial structure of SDB600 and SDB900. The XPS
full survey presented that C, N, O, Fe, Si and Al were the main elements
of sludge-derived biochar (Fig. S1 and Table S2), consisting with the
results of XRD. Deconvolution of the C 1s spectra of SDB600 and SDB900
was fitted into four peaks, namely, aromatic C–C at 284.28-284.47 eV,
C–O at 285.41-286.08 eV and C=O at 288.25-289.31 eV (Fig. 2a and b).
Moreover, C–C bond accounted for 62.2% (SDB600) and 71.0%
(SDB900) of the total carbon content, which verified the formation of
highly carbonized and aromatic structures in sludge-derived biochar
(Zhao et al., 2018). These results were in good agreement with that of
FTIR spectra. As shown in Fig. 2d, the Fe 2p3/2 spectra of SDB900 was
deconvoluted into two peaks at 711.17 and 714.73 eV, corresponding to
γ-Fe2O3 and Fe(III), respectively (Han et al., 2007). While for SDB600,
the Fe 2p spectra shifted slightly to 711.30 and 714.98 eV (Fig. 2c).
Y. Lu et al.
FeOOH was also demonstrated to exist in both SDB900 (724.41 eV, Fe
2p1/2) and SDB600 (724.81 eV, Fe 2p1/2).
The SEM graph of SDB900 presented irregular surface with many
impurity particles and the pore distribution was not obvious, as well as
those for SDB600 (Fig. S2). Sludge-derived biochar provided a carrier for
G. sulfurreducens PCA so that the cells could closely contact the material
surface (Fig. 1b). The EDS elemental mapping analyses of SDB600
(Fig. S1b-e) and SDB900 (Fig. 1d–g) further supported the relatively
uniform distribution of O, N and Fe on sludge-derived biochar. All of the
aforementioned results suggested that iron oxides and abundant func­
tional groups (mainly − OH, C=O, C=C) formed within pyrolysis process
established the basis for imparting sludge-derived biochar with good
catalytic properties.
The carbon structures of two sludge-derived biochars were further
characterized by Raman spectroscopy. The deconvolution of Raman
spectra was carried out to further understand the structural changes of
biochars during pyrolysis process (Li et al., 2006), as shown in Fig. 2e
and f. D and G bands were typically observed in the curve-fitted Raman
spectrum of both biochars, corresponding to the disordered carbon/­
structural defects and graphitic carbon, respectively (Jiang et al., 2013).
A weak and broad 2D band centered at 2800 cm− 1 was observed for the
multilayer graphene phase structure (Li et al., 2017). As a significant
parameter, the intensity ratio of the D and G bands (ID/IG) based on the
peak area values was calculated to evaluated the degree of graph­
itization/defectiveness in carbon materials (Shao et al., 2017). The
resulting ID/IG values showed a decrease from 1.41 for SDB600 to 1.17
for SDB900. The change implied that higher graphitized degree was
generated from the conversion of amorphous carbon to ordered sp2
carbon crystallites with increasing pyrolysis temperature (Rhim et al.,
2010). The increase of the pyrolysis temperature decreased the full
width half maximum (FWHM) of the G band in SDB900 (73.83 cm− 1)
compared to that of SDB600 (88.07 cm− 1), suggesting an enhancement
of the crystallinity in biochar (Sousa et al., 2020). Meanwhile, the peak
position of the G band was shifted to higher wavenumbers with the
pyrolysis temperature increasing from 600 to 900 ℃ (Table S1). These
results demonstrated the formation of more graphitized structure in
SDB900 than that in SDB600. All results supported the increase of
graphitic content during pyrolysis treatments, which were in line with
the conclusion from previous literature (Rhim et al., 2010).
3.2. Reduction of Fe(III) with sludge-derived biochar
Compared to bacteria-free controls, the reduction extent of Fe(III) in
those two sludge-derived biochars (SDB600 and SDB900) was signifi­
cantly enhanced by the stimulation of G. sulfurreducens PCA within 10
days (p < 0.05) (Fig. 3). Both SDB600 and SDB900 inherently contained
100− 200 μM of HCl-extractable Fe(II), which was possibly originated
from iron oxides found on the surface of sludge-derived biochar. With
the addition of G. sulfurreducens PCA, the concentrations of HCl-
extractable Fe(II) increased from 160.9 ± 23.6 μM to 427.7 ± 0.6 μM
(SDB600) and 116.6 ± 6.1 μM to 487.6 ± 53.8 μM (SDB900) after
incubating for 10 days. Consistent with the previous literatures,
G. sulfurreducens PCA was capable of reducing crystalline iron oxides or
poorly crystalline iron oxides (Notini et al., 2019). Our results further
confirmed that G. sulfurreducens PCA could utilize Fe-bearing complexes
on biochars as electron acceptors to enhance the transformation of Fe
(III) to Fe(II) by EET process. The generation of biogenic Fe(II) by
G. sulfurreducens PCA was beneficial to reduce the redox potential of the
reaction system and thus facilitated the anaerobic transformation of NB.
3.3. Biochar-stimulated reduction of NB
The reduction of NB by G. sulfurreducens with the presence of sludge-
derived biochar was investigated within a time span of 15 days. Fig. 4a
and b illustrated the reduction of NB to aniline as the corresponding
product and no other reaction intermediates (e.g., nitrosobenzene or
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Journal of Hazardous Materials 403 (2021) 123682
Fig. 3. The concentrations of HCl-extractable Fe(II) from SDB600 and SDB900
with or without the presence of G. sulfurreducens PCA at each time interval.
Error bar represents the standard deviation of triplicate.
phenylhydroxylamine) were observed in HPLC chromatograms. In the
initial 2 h, the removal of NB by two sludge-derived biochars was
different and the generation of aniline was not observed (Fig. S8). Also,
no NB degradation products in the cell-free biochar medium were
observed in HPLC chromatograms, indicating that the lag period of
aniline production was mainly attributed to the NB adsorption property
of biochar. And the equilibrium concentrations of NB were approxi­
mately 24 μM and 57 μM in the presence of SDB600 or SDB900 after 2 h,
respectively. Subsequently, only negligible aniline production occurred
in the treatments amended with SDB600 or SDB900 while without
G. sulfurreducens, suggesting that sole sludge-derived biochar mainly
achieved NB removal by adsorption rather than reduction process under
anaerobic conditions. For the treatment with pure G. sulfurreducens, the
NB concentration decreased from 100 μM to 75 μM with an aniline
production of 25 μM after incubating for 15 days, illustrating the bio­
logical reduction of NB by G. sulfurreducens occurred. Compared with
the biotic or abiotic controls, the removal rate of NB was increased to
99% for G. sulfurreducens-SDB900 system and 89% for G. sulfurreducens-
SDB600 system in the end of anaerobic incubation, respectively. As re­
ported in previous study, other detected components of SDB600 and
SDB900, such as quartz and Al2O3, had no effect on the degradation of
NB (Luan et al., 2015). Therefore, in the co-existence system of
G. sulfurreducens and sludge-derived biochar, the fate of NB could be
divided into three routes, namely, i) absorption by biochar, ii) degra­
dation of NB to aniline and iii) the residual part. Interestingly, all re­
action systems exhibited mass balance during the 15-days removal of NB
(Fig. 4c and S3). It could be clearly seen that the total amounts of NB
reduced by G. sulfurreducens-SDB600 or G. sulfurreducens-SDB900 sys­
tems were 56 μM and 66 μM after 15 Day, respectively, which were
higher than that of pure cells. Thus, in addition to the biological
reduction of G. sulfurreducens itself, biogenic Fe(II) may also be involved
in the abiotic reduction of NB to aniline.
The reduction of NB in these reaction systems was fitted with the
pseudo-first-order kinetic model. As shown in Fig. 4d, the first-order rate
constants for NB removal were 0.135 ± 0.011 d− 1 and 0.230 ± 0.020
d− 1 in G. sulfurreducens with SDB600 and SDB900, respectively. These
values were 7.9- to 13.5-times higher than that of pure G. sulfurreducens
PCA (0.017 ± 0.005 d− 1). These results demonstrated that the anaerobic
Y. Lu et al. Journal of Hazardous Materials 403 (2021) 123682

Fig. 4. The adsorption and reduction of nitrobenzene (NB) (a) with the production of aniline (b) in different controls and treatments. The mass balance of NB
concentrations during the cultivation of G. sulfurreducens with the presence of SDB900 (c). NB concentrations transformed to ln([Ct]/[C0]) to calculate first-order
reduction rates (d). The initial concentration of NB was 100 μM, and the biochar concentration was 1 g/L. Error bar represents the standard deviation of triplicate.

reduction rate of NB was appreciably accelerated by sludge-derived peak around − 16 mV vs. SCE appeared in the forward scan, while its
biochars prepared with the elevated pyrolysis temperature from 600 reduction counterpart was not discernible in the reverse scan. The slight
to 900 ℃. intensity of this peak indicated that its contribution to electron transfer
was insignificant. For the DPV of pure SDB600, a minor reduction peak
at +14 mV vs. SCE appeared in the reverse scan, but its counterpart was
3.4. Impacts of biochar on the microbial electron flow route
too weak to be detected in the forward scan. Nevertheless, the redox
peaks in the combined system of SDB600 and G. sulfurreducens nega­
Differential pulse voltammetry (DPV) and Cyclic voltammetry (CV)
tively shifted to − 216 mV (reduction peak) and − 36 mV vs. SCE
were performed to characterize the electrochemical properties of
(oxidation peak) compared to pure SDB600 (Fig. S5b). The comparison
SDB600 and SDB900 before the reduction of NB. DPV with high selec­
of SDB600 and SDB900 suggested that SDB900 possessed more redox
tivity/sensitivity was applied to show the redox peaks masked by the
substances and higher electrochemical activity, which were essential to
catalytic wave in CV. DPV on pure G. sulfurreducens PCA showed a major
support EET process.
pair of redox peaks at around − 422/− 387 mV vs. saturated calomel
DPV analysis was also conducted to detect redox properties of su­
electrode (SCE) (Fig. S5a). The value of major redox peaks gave prox­
pernatant of culture medium with pure G. sulfurreducens, SDB900 or
imity to the potential reported for c-type cytochrome OmcB (− 430 mV
SDB600, respectively (Fig. 5c). No obvious redox peaks were detected in
vs. SCE) located at the outer membrane of G. sulfurreducens (Richter
fresh growth medium. An apparent reduction peak at +225 mV vs. SCE
et al., 2009). Besides, the reduction peak at − 387 mV vs. SCE had a
was detected in the supernatant of pure PCA cells, which was likely
shoulder peak at approximately − 309 mV vs. SCE and another minor
attributed to the redox protein secreted by strain PCA itself (Li et al.,
oxidation peak appeared at − 141 mV vs. SCE, which were presumably
2016). The supernatant of SDB900 and SDB600 without strain PCA
assigned to c-type cytochromes of G. sulfurreducens (Magnuson et al.,
exhibited two pairs of DPV peaks, suggesting that some redox substances
2001).
would be leached from sludge-derived biochar into the medium.
SDB900 displayed strong DPV signals near − 136 mV and − 296 mV
Additional evidences to support sludge-derived biochar as electron
vs. SCE, indicating the presence of redox structures that might act as
shuttle resulted from CV analysis (Fig. 5d). The redox couples at -273
electron shuttle for electron transfer (Fig. 5a). The reduction peak at
and − 370 mV vs. SCE were attributed to the existence of c-type cyto­
− 296 mV was broaden by the adjacent peak at − 172 mV. SDB900
chromes in G. sulfurreducens, which was confirmed to be directly
combined with G. sulfurreducens showed three distinct redox couples
involved in EET process as previously reported (Katuri et al., 2010). Both
(Fig. 5b). The pair at − 481 mV and − 448 mV vs. SCE were potentially
SDB900 and SDB600 with the presence of G. sulfurreducens displayed a
attributed to c-type cytochrome OmcZ (− 465 mV vs. SCE) or OmcS
single redox couple in CV with potentials ranging from +300 mV to
(− 456 mV vs. SCE) (Inoue et al., 2010; Jain et al., 2011). Besides, the
+500 mV vs. SCE. The range of the redox potentials resembled the
pair at − 265 mV and − 199 mV vs. SCE might represent the redox sub­
electrochemical properties of quinone/hydroquinone redox couple as
stances in the composite system, which were postulated to be c-type
previously reported (Quan et al., 2007). Compared to the pure bacterial
cytochrome or quinone-like substances (Leng et al., 2014). An additional

6
Y. Lu et al.
Fig. 5. DPV analyses of pure SDB900 (a), SDB900-G. sulfurreducens PCA combined system (b) and supernatant of different controls (c). Cyclic voltammetric analyses
of glassy carbon electrode (GCE), pure G. sulfurreducens PCA and G. sulfurreducens PCA with SDB900/ SDB600 (d). The arrows represented the scan direction of DPV.
cells, the combined system of SDB900/SDB600 and G. sulfurreducens
together had larger peak currents. The increase of peak current
demonstrated that sludge-derived biochar contributed in promoting
electron transfer from G. sulfurreducens to glassy carbon electrode (GCE).
Notably, SDB900 generated higher peak currents than SDB600, further
supporting that SDB900 exhibited higher electrical conductivity for EET.
Further EPR analysis was conducted on SDB600 and SDB900
(Fig. S6). The emergence of signals for both biochars showed that
persistent free radicals were formed in the process of sludge pyrolysis.
The g value was calculated from EPR spectrum to enable the determi­
nation of free radical type (Roden et al., 2010). SDB600 and SDB900
exhibited the same g value of 2.0031, which could be attributed to the
mixed carbon- and oxygen-centered radicals (Xu et al., 2016). However,
the EPR signal intensity markedly decreased with the increase of the
pyrolysis temperature. Similar scenarios were found in previous studies
(Zhu et al., 2018; Duan et al., 2018). These results were ascribed to the
elimination of oxygen functionalities and condensation/graphitization
of carbon clusters (Zhu et al., 2018). Herein, we deduced that electron
transfer was accelerated by highly graphitic biochars to improve both
biotic and abiotic reduction of NB.
4. Discussion
In this study, Fe-bearing biochar was generated from municipal
sewage sludge, and with G. sulfurreducens together they were jointly
responsible for the enhanced extracellular reduction of NB to produce
aniline as end product. During the cultivation, bacterial cells formed
compact aggregates with Fe-bearing biochar during the NB reduction
process, which was accordance with the results found with the SEM
micrographs. The dense aggregate was in favor of shortening the dis­
tance of EET between G. sulfurreducens and biochar to increase the rate
7
Journal of Hazardous Materials 403 (2021) 123682
of electron exchange (Kappler et al., 2014).
During the pyrolysis process, inherent iron of sludge was converted
into iron oxides (mainly γ-Fe2O3 and Fe3O4), and the Fe-bearing biochar
showed magnetic property (Fig. S7). The generation of magnetism was
beneficial to the growth of EAB like Geobacter and could stimulate EET
process (Zhou et al., 2019). Besides, the standard redox potential of Fe
(II)/Fe(III) couple was known to be +770 mV (Pereira et al., 2016).
Accordingly, both SDB900 (+263 mV vs. SCE) and SDB600 (+129 mV
vs. SCE) could theoretically act as feasible redox mediators to accept
electrons from G. sulfurreducens (− 415 mV vs. SCE) and further donate
those electrons to Fe(III).
Besides, the addition of sludge-derived biochar triggered microbial
dissimilatory iron reduction process by G. sulfurreducens, in which Fe
(III) on sludge-derived biochar could be firstly reduced to Fe(II) by
G. sulfurreducens, and those generated biogenic Fe(II) provided reactive
sites to facilitate electrons transfer to NB occurring at the cell/biochar
interface. According to previous reports, adsorbed Fe(II) species
possessed higher reductive activity than aqueous Fe(II) for NB reduction
(Luan et al., 2015; Lu et al., 2019). Therefore, biogenic Fe(II) in Geo­
bacter-biochar combined system may be the major driving factor for the
NB reduction. Besides, some biogenic Fe(II) might be quickly adsorbed
on the surface of sludge-derived biochar to decrease its redox potential,
which may explain the negative shift of redox pair in the DPV diagram of
Geobacter-biochar combined systems. Additionally, the Fe(II)/total iron
ratio remained almost constant during 15 days of cultivation (Fig. S4),
which suggested biogenic Fe(II) could be re-oxidized to Fe(III) and a
dynamic cycling of Fe(II)/Fe(III) occurred in the Geobacter-biochar
combined system during long-term reduction of NB (Lu et al., 2019).
In our study, the increase of pyrolysis temperature (from 600 ℃ to
900 ℃) resulted in the generation of more graphitized structure, which
was endowed with higher mediation activity and favored the EET of
Y. Lu et al.
G. sulfurreducens as well as the NB reduction. The results of FTIR, Raman
and EPR suggested the elevated pyrolysis temperature accelerated the
decomposition of sludge to generate the condensed aromatic structure
and increased graphitized degree. Higher graphitized degree favored
biochar with higher conductivity to promote electrons transfer from
G. sulfurreducens to contaminants. These results were in good agreement
with a previous study, where Sun et al. (2017) reported the higher py­
rolysis temperature led to an increase in the formation of graphitic
structures of biochar for rapid electron transfer. In addition, quinone
and hydroquinone couple has previously been proved to act as a surface
redox-active center, allowing biochar to accept and contribute electrons
reversibly (Yu et al., 2015; Zhang et al., 2018). Therefore, quinoid-like
groups might distribute on the surface of biochar to mediate electron
transfer in NB reduction and microbial iron reduction, which was in
good agreement with the characterization results of FTIR and XPS as
described above. Similar to our study, Saquing et al. (2016) showed that
wood biochar accelerated electron transfer from G. metallireducens to
nitrate and promoted the microbial denitrification mediated by the
charging and discharging functions of quinone/hydroquinone redox
couple.
In the end, a systematical mechanism was proposed for the biotic and
abiotic reduction of NB (Fig. 6). A relatively small portion of NB was
directly reduced by pure G. sulfurreducens, while the majority of NB was
indirectly reduced by surface-adsorbed biogenic Fe(II) produced by
G. sulfurreducens from Fe-bearing biochar. Besides, as reflected by the
electrochemical measurements, most probably c-type cytochromes of
G. sulfurreducens were involved in EET process, in which intracellular
electrons were released to external environment for dissimilatory iron
reduction or the biotic reduction of NB (Inoue et al., 2010). Fe-bearing
biochar played the role of electron transfer conductor and natural iron
source in the Geobacter-biochar combined reduction system. Besides, to
explain the mechanisms of electron transfer mediated by biochar, two
major routes were proposed. On one hand, the graphitized domains of
biochar as direct electron transfer conduit had the potential to directly
Fig. 6. Schematic of the proposed mechanism of integrated biotic/abiotic reduction of nitrobenzene (NB) by the combination of G. sulfurreducens PCA and Fe-
bearing biochar.
8
Journal of Hazardous Materials 403 (2021) 123682
mediate electron transfer. On the other hand, quinone/hydroquinone
redox couple in biochar, i.e. combining quinone groups as electron
acceptor and hydroquinones as electron donor, could form an electron
transfer conduit by redox reaction for EET. And according to our results,
the conductor mechanism by graphitized structure of biochar, instead of
the mediation of quinone moieties, played a dominant role in mediating
electron transfer during NB reduction. NB was transformed to the end
product aniline through Geobacter-biochar combined system under
anaerobic conditions, which was consistent with the results of previous
studies (Wang et al., 2011; Luan et al., 2015). Besides, aniline was
relatively less toxic and easier to be degraded than the parent com­
pounds. In order to completely deal with the NB contaminant in prac­
tical applications, subsequent treatments should be conducted for
further biodegradation and mineralization of aniline.
5. Conclusion
Our results demonstrated the addition of Fe-bearing biochar
(generated from sludge by one-step pyrolysis) significantly enhanced the
reduction of NB to aniline by G. sulfurreducens. The enhancement effect
was primarily attributed to the important role of Fe-bearing biochar in
both mediating EET of G. sulfurreducens and assisting the formation of
biogenic Fe(II) as natural iron source. Graphitized structure and
quinone-like moieties in generated biochar might act as electron shut­
tles, and thus accelerated EET process and enhanced the rate of NB
reduction. Besides, adding Fe-bearing biochar triggered microbial
dissimilatory iron reduction by G. sulfurreducens and the biogenic Fe(II)
in Geobacter-biochar combined system was the major driving factor for
the NB reduction, which might act as an important but overlooked
electron transfer route in the anaerobic environments. The great mag­
netic property of sludge-derived biochar makes it easy to separate from
reaction system, therefore, further investigation will conduct on the
regeneration property of deactivated sludge-derived biochar to improve
the reusability. Future applications of the combination of
Y. Lu et al.
microorganism and biochar should evaluate the contaminant removal in
a long-term period with the change of biochar surface structure. Such
assessments can contribute to advance the understanding on the fate of
nitroaromatic compounds in anoxic environment and help to predict the
natural attenuation at in situ fields amended with biochar.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Yue Lu: Conceptualization, Methodology, Investigation, Writing -
review & editing, Funding acquisition. Qingqing Xie: Conceptualiza­
tion, Investigation, Writing - original draft, Data curation, Formal
analysis. Lin Tang: Supervision, Project administration, Funding
acquisition, Investigation. Jiangfang Yu: Conceptualization, Formal
analysis, Methodology, Writing - review & editing. Jingjing Wang:
Investigation, Data curation, Writing - review & editing. Zhaohui Yang:
Resources, Writing - review & editing. Changzheng Fan: Resources,
Writing - review & editing. Shoujuan Zhang: Methodology, Software,
Writing - review & editing.
Acknowledgements
We thank the research group of Prof. Hanqing Yu (University of
Science & Technology of China) to provide Geobacter sulfurreducens PCA.
This work was supported by the National Natural Science Foundation of
China (Grant No. 51709100, 51679084, 51579096), the Fundamental
Research Funds for the Central Universities (531107050936), the Key
Research and Development Project of Hunan Province of China
(2017SK2241), the National Innovative Talent Promotion Program of
China (2017RA2088), and the Funds for Innovative Province Con­
struction of Hunan Province of China (2019RS3012).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jhazmat.2020.123682.
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