Chemosphere 189 (2017) 134e142

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Mixed sulfate-reducing bacteria-enriched microbial fuel cells for the

treatment of wastewater containing copper
Waheed Miran a, 1, Jiseon Jang a, 1, Mohsin Nawaz a, Asif Shahzad a, Sang Eun Jeong b,
Che Ok Jeon b, Dae Sung Lee a, *
a
Department of Environmental Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu, 41566, Republic of Korea
b
Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea

h i g h l i g h t s g r a p h i c a l a b s t r a c t

SRB enriched MFC system was

developed for investigating heavy
metal removal.

Ten successive batches for voltage
generation showed excellent
reproducibility.

Maximum copper tolerance concen-
tration in the biological anodic
chamber was 20 mg/L.

Among SRB, Desulfovibrio (38.1%) was
the most abundant genus at the
anode.

a r t i c l e i n f o a b s t r a c t

Article history: Microbial fuel cells (MFCs) have been widely investigated for organic-based waste/substrate conversion
Received 31 July 2017 to electricity. However, toxic compounds such as heavy metals are ubiquitous in organic waste and
Received in revised form wastewater. In this work, a sulfate reducing bacteria (SRB)-enriched anode is used to study the impact of
31 August 2017
Cu2þ on MFC performance. This study demonstrates that MFC performance is slightly enhanced at
Accepted 11 September 2017
Available online 13 September 2017
concentrations of up to 20 mg/L of Cu2þ, owing to the stimulating effect of metals on biological reactions.
Cu2þ removal involves the precipitation of metalloids out of the solution, as metal sulfide, after they react
Handling Editor: J. de Boer with the sulfide produced by SRB. Simultaneous power generation of 224.1 mW/m2 at lactate COD/SO2 4
mass ratio of 2.0 and Cu2þ of 20 mg/L, and high Cu2þ removal efficiency, at >98%, are demonstrated in the
Keywords: anodic chamber of a dual-chamber MFC. Consistent MFC performance at 20 mg/L of Cu2þ for ten suc-
Heavy metals cessive cycles shows the excellent reproducibility of this system. In addition, total organic content and
Sulfate-reducing bacteria sulfate removal efficiencies greater than 85% and 70%, respectively, are achieved up to 20 mg/L of Cu2þ in
MFC 48 h batches. However, higher metal concentration and very low pH at <4.0 inhibit the SRB MFC system.
Microbial community
Microbial community analysis reveals that Desulfovibrio is the most abundant SRB in anode biofilm at the
genus level, at 38.1%. The experimental results demonstrate that biological treatment of low-
concentration metal-containing wastewater with SRB in MFCs can be an attractive technique for the
bioremediation of this type of medium with simultaneous energy generation.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
* Corresponding author.
E-mail address: daesung@knu.ac.kr (D.S. Lee). Tremendous micro and macro industrialization with heavy
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.chemosphere.2017.09.048
0045-6535/© 2017 Elsevier Ltd. All rights reserved.
 W. Miran et al. / Chemosphere 189 (2017) 134e142
metals as an integral part of numerous activities has led to dis-
charges of large amounts of metal-containing wastewater, which
has caused severe environmental problems. Cu2þ-containing
waste, in particulardfrom many metal industries (including hy-
drometallurgy and metal finishing), mining, electroplating, battery
production and microelectronicsdhas been continuously
increasing in the influent streams reaching wastewater treatment
plants (Ma et al., 2014). Cu2þ is a toxic and non-biodegradable
inorganic heavy metal pollutant that bioaccumulates in the food
chain, creating lethal effects in many vital human organs and also in
all other organisms (Gall et al., 2015). In addition, high sulfate
content is prevalent in waste streams from metal-leaching opera-
tions. As a pollutant, sulfate corrodes metals and causes H2S-altered
toxicological effects. Extended exposure to it can also cause acute
renal failure, hepatotoxicity, increases in hippocampus superoxide
dismutase, neurological impairment, and shock (Hussain et al.,
2016). Therefore, heavy metals including Cu2þ and sulfates must
be treated efficiently before wastewater is discharged into the
environment.
Many physical and chemical methods are available for treating
wastewater containing metals (Wang and Chen, 2009), including
chemical precipitation, electrochemical treatment, reverse
osmosis, ion exchange, and evaporation recovery, which can be
often futile or incur huge expenses, particularly if the metal con-
centrations is as low as 1e100 mg/L (Ahluwalia and Goyal, 2007).
Moreover, the treatment of organics present in wastewater cannot
be achieved by these techniques alone. Therefore, economical,
effective, and eco-friendly processes are continuously sought to
remove heavy metals from wastewater and ensure their presence
in the environment is below permissible limits.
A microbial fuel cell (MFC) is a promising technology for directly
generating electricity from organics; therefore, it has huge poten-
tial to treat wastewater economically and sustainably without
involving fossil fuel-based energy (Logan and Regan, 2006; Rabaey
and Verstraete, 2005). In earlier MFC studies, Cu2þ removal/re-
covery has been studied using dual-chamber MFCs in which Cu2þ
was mainly removed in the cathodic chamber by cathode metal
reduction and organic matter in the anodic chamber was used as a
carbon source and an electron donor (Heijne et al., 2010; Pau et al.,
2015; Tao et al., 2011). In those studies, however, the cathodic
medium contained only Cu2þ without organic compounds,
whereas many waste streams contain sulfates and organic sub-
stances in addition to heavy metals like textile, paper and pulp, acid
mine drainage, and mining and mineral processing wastewater.
Therefore, the use of the microbial section of MFCs can be more
viable for the simultaneous removal of these pollutants up to
certain limits.
Microbial communities in MFCs play a very important role in
overall current generation and effective pollutant treatment
(Herna
ndez-Ferna
ndez et al., 2015). Sulfate-reducing bacteria (SRB)
were recently tested for removing organics and generating power
in MFCs, and the SRB were confirmed to have an electroactive na-
ture with the ability to perform extracellular electron transfer
(Kang et al., 2014). One of the most attractive approaches for the
treatment of metallic waste is the precipitation of metal ions in the
form of their respective sulfides, in which metal precipitation can
be aided by biological sulfate reduction to S2, HS, H2S, and other
components. Such a technique offers attractive advantages over the
conventional chemical precipitation methods. Thus, SRBs enriched
MFCs can be cost-effective due to moderate operational condition,
low sludge production, and high electric efficiency (Lovley, 2008).
Removal of heavy metals by SRB is due to the formation of highly
insoluble precipitates with biogenic sulfide. The solubility product
of most metal sulfides is extremely low, where the solubility
product constant of CuS is 1.27  1036 (White and Gadd, 2000).
135
This implies that small sulfide concentrations are required to
effectively remove metals. The additional advantage associated
with the biological sulfate reduction technique is the production of
a low amount of secondary sludge for further disposal. Thus, it is
known to be an efficient technique for removing heavy metals from
wastewater at low initial concentrations (Cabrera et al., 2006).
The aim of the present work is to develop and investigate an
MFC system to treat wastewater containing low concentrations of
Cu2þ, using mixed SRB in an anodic chamber coupled with
bioelectricity generation. The reproducibility of the MFC system is
confirmed by running many consecutive cycles at a specific feed
condition. The maximum tolerance concentration (MTC) for Cu2þ in
the anodic chamber is determined, and the mechanism of Cu2þ
removal with sulfate reduction and current generation is proposed.
The tests are also designed to differentiate between sorption and
other removal mechanisms of Cu2þ in the anodic chamber. Micro-
bial community analysis by using the Illumina Miseq platform is
also conducted to determine the shift toward SRB-dominant
cultures.
2. Materials and methods
2.1. Inoculum and culture medium
Sludge was collected from the anaerobic section of a domestic
wastewater treatment plant in Daegu, Republic of Korea. The sludge
was sieved to remove larger foreign particles and was then used for
the enrichment of SRBs. Briefly, 1-L glass bottles were seeded with
sieved anaerobic sludge and Postgate's B medium after autoclaving
at 121  C and 15 psi for 20 min. The medium contained sodium
sulfate and sodium lactate for sulfate and lactate sources, respec-
tively. Sodium bromoethane sulfonate was also initially added to
the medium to avoid methanogenic activity, which can consume
the carbon (lactate) source. Enrichment was conducted in a shaking
incubator at 130 rpm and 30  C. 70% of the medium was decanted
and was replaced weekly. The enriched medium after six cycles,
with sulfate reduction and sulfide formation indicated by black-
ening of the media, was then transferred to the anodic chamber of
the MFC for further experiments.
2.2. MFC setup and operation
The MFC was composed of two 0.2-L chambers separated by a
183 mm thick Nafion 117 proton exchange membrane. Treatment of
the Nafion membrane was conducted for performance enhance-
ment by 0.1 M H2SO4 solution, deionized water, 0.1 M H2O2 solu-
tion, and deionized water again, heating each at boiling point for
1 h. A 3.18 mm thick carbon felt (Alfa Aesar, Haverhill, USA) having
length of 5 cm and a width of 5 cm was used as the anode electrode.
A 1.0 mg/cm2, 20 wt% platinum-coated carbon cloth (Fuel Cell
Earth, Wakefield, USA) having dimensions of 5 cm  5 cm was
utilized as the cathode electrode. The anode and cathode electrodes
were connected by a titanium wire through a 500 U resistor. The
growth medium used in the anode contained per liter 500 mg
(COD) of lactate, 500 mg of K2HPO4, 1000 mg of NH4Cl, 60 mg of
FeSO4$7H2O, 100 mg of MgSO4$7H2O, and 1 mL of trace mineral
solution. A lactate COD/SO2 4 mass ratio of 2.0 was used. It is
important to mention here that lactate is used due to its superiority
as an electron donor to many others such as ethanol, H2, propio-
nate, and acetate in terms of energy and biomass yield. Thus, lactate
as the electron donor for SRB accelerates the start-up of the bio-
process. Moreover, complete oxidation of lactate by SRB produces
3 mol of bicarbonate alkalinity per mol of substrate, and sulfido-
genic lactate oxidation yields some alkalinity even if the oxidation
is incomplete, which is not the case with other substrates (Nagpal
136 W. Miran et al. / Chemosphere 189 (2017) 134e142

et al., 2000). However, lactate is too expensive substrate to use in primers with adaptors targeting V3eV4 hypervariable regions of
large-scale wastewater treatment. Therefore, an economical sub- the 16S rRNA genes e the underlined denotes adaptor sequences e
strate (suitable real wastewater) is required for practical treatment and the PCR condition was as follows: 1 cycle of 94  C for 3 min,
once a rational biomass yield has been obtained. The pH of the followed by 30 cycles of 94  C for 20 s, 56  C for 45 s, and 72  C for
growth medium was adjusted to 6.0, and it was then flushed with 45 s, and finally 1 cycle of 72  C for 3 min. The PCR products were
N2 to remove dissolved oxygen. The medium in the anode chamber purified using a PCR purification kit (Solgent, Korea). The second
was mixed gently by using a magnetic stirrer. To complete the
reduction process at the cathode, oxygen (air) as the oxidant was
supplied continuously in the cathode chamber by an air-stone
connected to an aquarium blower. The cathode chamber was fil-
led with a 0.1 M phosphate buffer at pH 7 as the cathode electrolyte.
All experiments were conducted in batch mode at a temperature of
30 ± 2  C using a water bath. Two control groups were also set for
comparison. The first group was sterile control using the dual-
chamber MFC without inoculation, and second included auto-
claved biofilm.

2.3. MFC calculations and physiochemical analysis

The output voltage (Ecell) was continuously monitored across

the resistor by using a digital multimeter (Keithley Instruments,
Inc., Cleveland, Ohio, USA) connected to a data acquisition system.
The current I in amperes was calculated using Ohm's law, I ¼ Ecell/
Rext, where Ecell is the measured voltage in volts and Rext is the
known value of the external load resistor in Ohms (U). The
maximum power density and polarization curve were determined
by adjusting the external resistance from 100,000 U to 10 U. During
this process, the external resistance changed when a stable output
voltage was recorded. The power density (mW/m2) and current
density (mA/m2) normalized by the surface area of the anodic
chamber were estimated by using the data obtained from the
current-voltage measurements.
The sulfates were analyzed by using a DX ICS-1000 ion chro-
matography unit (Dionex, USA) equipped with a conductivity de-
tector and a self-regenerating suppressor. A DX Ionpac AS14-HC
analytical column (4  250 mm) connected to a DX Ionpac AG14-HC
guard column (4  50 mm) was used. The unit was operated in
autosuppression mode with the eluent, 3.5 mM Na2CO3 þ 1 mM
NaHCO3, at a flow rate of 1.2 mL/min. The pH measurements were
conducted by using a Hach pH meter calibrated regularly with
standard pH solutions of 4.0, 7.0, and 10.0. X-Ray photoelectron
spectroscopy (XPS) was performed by using a Quantera SXM
(ULVAC-PHI, Japan) to study the biofilm composition by mainly
recording sulfur and copper XPS spectra. The XPS spectra of anode
biofilm for sulfur and copper were studied, and XPS peaks were
assigned based on the National Institute of Standards and Tech-
nology (NIST) X-ray Photoelectron Spectroscopy Database (https://
srdata.nist.gov/xps/). The supernatant was used to analyze the re-
sidual metal ion concentrations by inductively coupled plasma
optical emission spectroscopy (ICP-OES; Perkin-Elmer Optima 2100
DV).

2.4. Microbial community analysis

Sludge samples, raw sludge from wastewater treatment plant as

the initial inoculum, and MFC biofilm were collected in Eppendorf
tubes. Then, the samples were centrifuged at 12,000 rpm for
10 min, and the supernatant was discarded. The pellets obtained
were stored at 80  C until further analysis. The DNA of the sam-
ples was extracted by using the FastDNA SPIN Kit for Soil (MP
Biomedicals) according to the manufacturer's guidelines. The bac-
terial 16S rRNA genes from the genomic DNA were first amplified
using 341F (50 -TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-
Fig. 1. Voltage generation for a) increasing Cu2þ concentration, b) ten successive
CCTACGGGNGGCWGCAG-30 ) and 805R (50 -GTCTCGTGGGCTCGG- batches at 20 mg/L of Cu2þ, and c) three successive batches at 30 mg/L of Cu2þ.
AGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-30 )
 W. Miran et al. / Chemosphere 189 (2017) 134e142
PCR using the purified PCR products as templates was performed
with i5 (50 -AATGATACGGCGACCACCGAGATCTACAC-[i5]-
TCGTCGGCAGCGTC-30 ) and i7 (50 -CAAGCAGAAGACGGCATACGA-
GAT-[i7]-GTCTCGTGGGCTCGG-30 ) index primers e [i5] and [i7]
denote unique barcode sequences for Illumina Miseq sequencing.
The PCR condition was same as the first PCR except for 8 cycles of
94  C for 20 s, 56  C for 45 s, and 72  C for 45 s. The PCR products
were purified using a PCR purification kit (Solgent, Korea) and
sequenced using the Illumina Miseq platform (Roche, Germany) at
Macrogen (Korea). Then, the purified PCR products were cautiously
quantified by using an enzyme-linked immunosorbent assay reader
equipped with a Take3 multivolume plate, and a composite DNA
sample was prepared by pooling equal amounts of purified PCR
amplicons from each sample. High-throughput sequencing was
finally performed using the Illumina Miseq platform at Macrogen
(Seoul, South Korea). The Illumina Next-Generation sequencing
workflow is shown in Fig. S1. To remove the adapter sequence,
Scythe (v0.994 BETA) and Sickel software was used. Data analysis
was performed with Mothur software v. 1.39.5 following MiSeq SOP
(https://www.mothur.org/wiki/MiSeq_SOP). Briefly, sequences
<200 bp and those with ambiguous base calls were eliminated.
Operational taxonomic units (OTU) were generated, and chimeras
were removed. OTUs were defined by clustering at 3% divergence
(97% similarity). Final OTUs were taxonomically classified by using
the Silva v128 database (Quast et al., 2013). The ShannoneWeaver
and Chao1 biodiversity indices were calculated by using normal-
ized sequences (14,674) to avoid the bias of each sample.
Fig. 2. a) Cu2þ removal time profile, b) Cu2þ removal at 6 and 12 h, c) sulfate removal, and d) TOC removal.
137
3. Results and discussions
3.1. Effect of copper concentration on SRB-enriched MFC
performance
Once the MFC system enriched with SRB was stabilized by
batches of constant voltage generation using lactate as the carbon
source, its performance with increasing Cu2þ concentration was
studied in 48 h batches operation. Lactate degradation leads to
acetate and propionate formation, which acts as the electron do-
nors in the MFC after further degradation in SRB MFC systems
(Miran et al., 2017). Voltage generation showed that it was slightly
increased, approximately 10%, with a 0e20 mg/L increase in Cu2þ
concentration (Fig. 1a). This result is most likely attributed to the
stimulation of biological reactions at low metal concentrations. At
low concentrations, copper sulfide, which is formed in SRB systems
as a result of biogenic sulfide reaction with Cu2þ, likely resulted in
microbial long-distance extracellular electron transfer similar to
that used for metallic iron sulfides reported in earlier research
(Kondo et al., 2015). Moreover, in SRB systems, insoluble metal
sulfide precipitation at low metal concentration does not have the
same toxic effect of the metal on SRB owing to sulfide formation.
This is considered as the main mechanism in the precipitation of
metals and tolerance of metals by SRB at low concentrations (White
and Gadd, 2000). The MFC system performance in terms of the
consistency of voltage results was shown by running 10 consecu-
tive batches at 20 mg/L of Cu2þ (Fig. 1b). This voltage generation,
0.48 ± 0.012 V shows excellent repeatability of the SRB MFC system.
138 W. Miran et al. / Chemosphere 189 (2017) 134e142

However, when the metal concentration was increased to 30 mg/L,

the voltage began to decrease very sharply (Fig. 1c). More than
twice the voltage was dropped in its third cycle, which shows the
possible toxicity behavior of Cu2þ metal at this concentration.
Although heavy metals can promote biological reactions at low
concentrations, they are toxic to microbes at moderate or higher
concentrations and can impede such processes (Ozbelge € et al.,
2007). At higher concentrations, metals are toxic to bacteria
owing to their greater bioavailability, thus causing enzyme deac-
tivation and denaturation in addition to cell organelle rupture
(Alexandrino et al., 2011). Similar to voltage generation, the power
density curves showed maximum power of 185.2, 224.1, and
54.7 mW/m2 for batches without Cu2þ, Cu2þ at 20 mg/L, and Cu2þ
at 30 mg/L (third batch), respectively (Fig. S2). In these batches, the
internal resistances based on the linear part of polarization curve
were 380, 376.4, and 376.5 U, respectively.
In terms of Cu2þ removal (Fig. 2a and b) more than 95% was
removed during the first 6 h operation, when the Cu2þ concen-
tration was 5e20 mg/L. However, it decreased to less than 70%
when the concentration increased to 30 mg/L. Such a drop in Cu2þ
removal indicates that the activity of SRB at this concentration was
partially inhibited. Therefore, these results demonstrate that the
SRB was tolerant of at least 20 mg/L Cu2þ. Control experiments
were also conducted to ascertain the role of Cu2þ sorption in un-
inoculated MFC and bacterial autoclaved MFC. In the case of un-
inoculated MFC, the Cu2þ removal was less than 3.0%, which shows
very insignificant sorption by the bare carbon electrode. However,
the bacterial autoclaved MFC showed a result of approximately
63.5% owing to sorption in 48 h operation. SRBs are known for their
high biosorption capacity for metals because they involve a sig-
nificant capacity of metal ions for binding with SRB. For example, it
has been previously reported that 23% of Cd metal was removed by
bioprecipitation and 77% by biosorption (Pagnanelli et al., 2010). It
can be inferred from these findings that in the Cu2þ removal by SRB,
biosorption had a significant contribution.
The effect of Cu2þ concentration on sulfate reduction is similar
Fig. 3. XPS spectra of the anodic biofilm for a) sulfur and b) copper.
to current generation, where no decrease in sulfate removal was
found up to 20 mg/L of Cu2þ, and the sulfate removal was greater
than 70% (Fig. 2c). At the reactors assembly, where SRB could
adhere and form a biofilm to offer some physical protection, the was oxidized to elemental sulfur or back to sulfate. Part of the
resistance of SRB to Cu2þ at certain limits increased owing to the sulfide formed ultimately lead to insoluble CuS formation. Sulfide
reduced susceptibility of the biofilm (Bai et al., 2012). This can be oxidation is possible abiotically as well as biotically. In MFCs, it was
also true for an MFC system in which biofilm is formed on the confirmed in previous research that SRB converts sulfate to sulfide,
anode. However, at 30 mg/L of Cu2þ, it was revealed that Cu2þ then the MFC anode biofilm oxidizes the formed sulfide to
played a strong role in the inhibition of the sulfate. The treatment of elementary sulfur with electricity generation (Lee et al., 2014). The
wastewater containing Cu2þ in terms of TOC removal showed that copper peaks (Cu2p3/2 and Cu2p1/2) at 932.4 and 952.4 are also
more than 90% TOC was removed in 48 h batch studies for initial attributed to CuS formation (Fig. 3b). To further study the mecha-
Cu2þ concentrations of 0e20 mg/L (Fig. 2d). This removal was nisms of bioprecipitation of Cu2þ by SRB, the anode biofilm was
decreased to less than 80% when the Cu2þ concentration was characterized by SEM-EDS (Fig. S3). SEM showed that the anode
increased to 30 mg/L. The different ranges of Cu2þ concentrations with biofilm in comparison to the bare electrode was very well
from 10 to 50 mg/L have been found to inhibit respiration and densely layered with microbes of different forms including round,
bacterial growth during wastewater treatment (Neumegen et al., rod, and dumbbell shapes. The qualitative EDS analysis showed that
2005). multiple elements including C, Cu, S, O, and Fe were present. The
presence of sturdiest peaks of Cu and S revealed that CuS was the
3.2. Copper bioprecipitation removal mechanism major product of the bioprecipitation of Cu2þ by SRB. C, O, and, Fe
originated mainly from the electrode and the culture medium.
Two major characteristic peaks were observed on the XPS S2p Based on these results and those of earlier studies, it can be inter-
spectra of the anode biofilm (Fig. 3a). One doublet and one singlet preted that the treatment mechanism of heavy metals containing
peak were defined between 158 and 167 eV. The doublet peaks wastewater by SRB enriched MFC involves two stages. First, SRB
(S2p3/2-1/2) at 161.5 and 162.8 eV were attributed to S(-II), and the oxidized the lactate and generated hydrogen sulfide and bicar-
singlet (S2p3/2) at 163.6 eV was assigned to S(0). The second major bonate ion under anaerobic conditions. Second, biologically pro-
peak (S2p3/2) at 168.7 eV was attributed to S(þVI). These peaks are duced hydrogen sulfide reacts with dissolved heavy metals such as
consistent with the results of an earlier study involving microbe- Cu to form insoluble metal sulfide precipitates (Kieu et al., 2011;
assisted sulfide oxidation in an MFC anode (Sun et al., 2009). It Neculita et al., 2007):
can be inferred that the sulfate was reduced to sulfide, part of which
 W. Miran et al. / Chemosphere 189 (2017) 134e142

CH3CHOHCOOH þ SO2
4 / 2CH3COOH þ H2S þ 2HCO3

CH3COOH þ SO2
4 / H2S þ 2HCO3

2CH3CHOHCOOH þ 3SO2
4 / 3H2S þ 6HCO3
M2þ þ H2S / MS(s) þ 2Hþ
where M2þ ¼ metal, such as Cu2þ in this study.
It is important to mention here that valuable metals and
elemental sulfur from biologically precipitated metal sulfide can be
recovered and recycled in downstream processes (Sierra-Alvarez
et al., 2007; Xu et al., 2012). In addition, metal Sorption by SRB
also played a significant role in Cu2þ removal i.e., 63.5% Cu2þ was
removed by sorption onto SRB.
3.3. Effect of initial pH on SRB-enriched MFC performance
Considering the usually low pH wastewater containing metals
such as acid mine drainage, the effect of influent pH on the SRB-
enriched MFC performance was investigated with initial pH
decreasing from 6.0 to 3.0 when the loading of Cu2þ was 20 mg/L
and the COD/sulfate mass ratio was 2.0. The voltage generation
decreased with a decrease in pH (Fig. 4a). However, the voltage
generation decrease was less significant up to pH 4.0, i.e., the
voltage was decreased by approximately 20% when the pH
decreased from 6.0 to 4.0. This decrease was significantly higher
when the initial pH was reduced to 3.0, i.e., more than a 60%
decrease in voltage at pH 3.0 was observed in comparison to that at
Fig. 4. Effect of pH on a) voltage generation, b) Cu2þ removal at 6 and 12 h, c) sulfate removal, and d) TOC removal.
139
(1) pH 6.0. It has been reported that anode biofilms with well-known
anode respiring bacteria such as Geobacter are not resistant to
(2) acidic conditions, with growth inhibited below pH 5.8. This is
attributed to biofilms at the anode being acidified by proton pro-
(3) duction in which a proton is generated for every electron produced;
subsequent production leads to the highest proton generation of
(4) any known microbial process (Popat and Torres, 2016). High-
current density ultimately requires a high proton-transport rate.
Electrons are known to be transported efficiently and rapidly to the
anode by conduction through the biofilm matrix, whereas the
protons then need to be removed from the biofilm by diffusion. If
the proton cannot be transported out of the biofilm fast enough, the
interior of the biofilm becomes acidified; this stops the bacterial
activity for electron generation (Torres et al., 2010). However, in the
case of SRBs, alkalinity is produced that leads to an increase in the
pH, thereby resisting acidic conditions (Fig. S4). It is important to
mention here that a higher diffusion coefficient for HCO 3 produced
in SRB MFC in comparison to the generally used buffer H2PO4- can
lessen the requirement of commonly used phosphate buffers for
maintaining such an important pH at the anode. This aspect of SRBs
can be studied in more detail in future research. The effect of initial
pH on the batch treatment of Cu2þ by SRB showed that more than
90% of Cu2þ was removed over an initial pH range of 4.0e6.0 after
12 h of batch operation (Fig. 4b). However, the removal of Cu was
not effective at pH 3.0, in which less than 55% was removed after
12 h of batch operation. Sulfate removal with the decrease in pH
showed that more than 70% of sulfate was removed up to the initial
pH of 5.0 and decreased to less than 20% when the initial pH of the
batch was 3.0 (Fig. 4c). These results are in alignment with earlier
140 W. Miran et al. / Chemosphere 189 (2017) 134e142
Table 1
Summary of sequencing data and statistical analysis of bacterial communities.
Sample Total bases Read count OTUs
Initial inoculum 46,707,374 155,174 2563
Anode biofilm 47,989,634 159,434 2015
results (Bai et al., 2013). One study reported that SRBs have the
ability to reduce sulfate effectively and can generate alkalinity
when tested with an influent pH as low as 4.0; this shows that SRB
systems can be used for bioremediation of very acidic, sulfate-
polluted wastewater (Jong and Parry, 2006). More than 80% TOC
removal was observed in 48 h batch operation at initial pH levels of
4.0, 5.0 and 6.0 (Fig. 4d). However, the TOC removal was reduced to
approximately 60% when the initial pH was reduced to 3.0. This
decrease occurred through the inhibition of microbial activities at
this low pH. In SRB systems, it is known that at low pH, the
hydrogen sulfide formed exists in an undissociated form and at
very low pH may cause severe inhibition by absorbing sulfide into
the cells, ultimately resulting in damage to their proteins (Zuhair
et al., 2008).
3.4. Microbial community results
The total read counts for the initial inoculum and anode biofilm
were 155,174 and 159,434, respectively, according to the Illumina
MiSeq sequencing analysis. For the initial inoculum and anode
biofilm, 17,697 and 22,148 high-quality reads were obtained,
respectively, and were assigned to different phyla in each sample.
The rarefaction curve for the initial inoculum was steeper in com-
parison to the anode biofilm communities. However, for both
samples (Fig. S5), the rarefaction curves did not reach saturation,
which implies the very high diversity of the bacterial community in
this study. Other important statistical data are shown in Table 1.
The sequencing results showed that the bacterial community in
the initial inoculum was composed primarily of Firmicutes (42.6%),
Proteobacteria (31.7%), Bacteroidetes (11.4%), and Actinobacteria
(5.6%) along with other low-abundance phyla (Fig. 5a). A huge shift
occurred in the bacterial community in the anode biofilm owing to
the enrichment of SRB, with the phyla belonging mainly to
Fig. 5. Bacterial communities at the a) phylum and b) class levels in the initial inoculum and the anode biofilm.
Shannon index Chao1 index Q30 (%) No. of high-quality reads
4.6 9516 76.32 17,697
3.72 7832 77.78 22,148
Proteobacteria (77.2%), Bacteroidetes (10.4%), and Actinobacteria
(3.8%) in addition to several low-abundance phyla. At the class level
(Fig. 5b), dominant microbial communities in the initial inoculum
were Clostridia (35.2%), Betaproteobacteria (21.1%), Alphaproteo-
bacteria (9.3%), and Bacteroidia (8.2%). After the shift in microbial
communities, the majority of the communities at the class level at
the anode belonged to Deltaproteobacteria (45.0%), Betaproteobac-
teria (12.5%), Gammaproteobacteria (11.4%), and Alphaproteobacteria
(8.1%). It has been well established that most of the sulfate reducers
belong to the Deltaproteobacteria class (Muyzer and Stams, 2008),
which was most abundant in this study. The most abundant genus
(Fig. 6a) found at the anode biofilm was Desulfovibrio (38.1%), which
is a well-known sulfate reducer. Desulfovibrio is an important SRB
that has the tendency to remove sulfate in neutral and even acidic
conditions (Zheng et al., 2014). Desulfovibrio has also been applied
in earlier studies for bioremoval of heavy metals such as Cu2þ and
zinc (Chandraprabha et al., 2012). Moreover, Desulfovibrio strains
are believed to be incomplete oxidizers of lactate. Producing ace-
tate in almost stoichiometric amounts to that of the added sub-
strate leads to approximately 95% of the lactate used for energy
generation, and the remainder is used to produce cell material (Vita
et al., 2015). Thus far, most of the SRBs have illustrated that the
electroactive properties belong to the Desulfovibrio genus. In earlier
studies, three main mechanisms have been reported for extracel-
lular electron transfer by Desulfovibrio: (I) direct extracellular
electron transfer from the SRB bacteria to the electrode through
multihemic membrane-bound cytochrome c proteins (Kang et al.,
2014), (II) direct electron transfer by electrically conductive nano-
scale appendages produced by the SRBs (Eaktasang et al., 2016), and
(III) indirect electron transfer from SRBs to solid electrodes by
inorganic electron mediators or shuttles such as sulfate/sulfide, i.e.,
the anode electrons originate from sulfide oxidation (Zhao et al.,
2008). In the anode biofilm communities, the second most
 W. Miran et al. / Chemosphere 189 (2017) 134e142 141

Fig. 6. Bacterial communities at the a) genus level and b) SRB genera at the MFC anode.

abundant SRB genus was Desulfobulbus (3.2%). The Desulfobulbus
genus can utilize many different types of organic carbon sources
such as lactate, pyruvate, ethanol, and propanol and is known for
incomplete oxidization of organic substrates (Yamashita et al.,
2011). Electron transfer by strains belonging to the Desulfobulbus
genus to solid electrodes have also been reported involving the
indirect electron transfer mechanism (Holmes et al., 2004). In
addition to these two, SRB genera found at anode biofilm include
Desulfatirhabdium, Desulfomonile, Desulfobacteraceae (unclassified),
Desulfobacterales (unclassified), Desulfobulbaceae (unclassified),
Desulfovibrionaceae (unclassified), Desulfovibrionales (unclassified),
and Desulfuromonadales (unclassified) (Fig. 6b). Geobacter, which
has been widely reported for producing electricity, was also found
in bacterial communities with relative abundance of 0.54% at the
anode biofilm. Geobacter metallireducens, Geobacter sulfurreducens,
and Geobacter bremensis have been successfully isolated from the
anode of bioelectrochemical systems (BESs). Overall, the microbial
community analysis showed that the dominant communities found
in this study can efficiently remove heavy metals, degrade organics,
reduce sulfates, and produce electricity by extracellular electron
transfer directly or indirectly. Moreover, suspended microorgan-
isms in MFCs reactor can also play a significant role in MFCs overall
performance. The MFC systems where only direct electron transfer
is responsible for current generation; suspended microorganisms
cannot play any direct role. However, in case of mediated electron
transfers like this study (sulfide mediated), suspended microor-
ganisms also become more relevant. For instance, SRBs in the
suspended form can reduce sulfate to sulfide, a part of which
142 W. Miran et al. / Chemosphere 189 (2017) 134e142
results in CuS formation and the remaining is oxidized at the anode
to give electrons, resulting in electricity generation. In this way,
suspended microorganisms can assist in copper removal and cur-
rent generation as well.
4. Conclusions
An SRB-enriched MFC system was studied for concomitant
treatment of heavy metal, sulfate, and organics removal while
generating electricity. A high removal rate of the Cu2þ with stable
and repeatable voltage generation was achieved at a low metal
concentration up to 20 mg/L of Cu2þ. However, at a high metal
concentration and very low pH, sulfate and organics removal were
inhibited, thus resulting in an overall reduced performance by SRB.
Bioprecipitation in addition to Cu2þ sorption by the SRB biomass
made a significant impact in the removal of Cu2þ. The results of this
study highlight the potential environmental importance of SRB in
the bioremediation of wastewater containing Cu2þ at the MFC
anode.
Acknowledgments
The authors gratefully acknowledge the financial support for
this study by Human Resource Training Program for Regional
Innovation and Creativity through the Ministry of Education (ME)
and National Research Foundation (NRF) of Korea (NRF-
2014H1C1A1066929). The grants (NRF-2016R1A2B4010431, NRF-
2009-0093819) were also provided through the ME and NRF of
Korea. Additionally, support for this work was provided by an NRF
grant from the Korean government (MSIP) (NRF-
2015M2A7A1000194).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.chemosphere.2017.09.048.
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