Science of the Total Environment 645 (2018) 471–481

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Arsenic removal mediated by acidic pH neutralization and iron

precipitation in microbial fuel cells
Eduardo Leiva a,b,⁎, Enzo Leiva-Aravena b,d, Carolina Rodríguez b, Jennyfer Serrano c, Ignacio Vargas b,d
a
Departamento de Química Inorgánica, Facultad de Química, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Macul, 7820436, Santiago, Chile
b
Departamento de Ingeniería Hidráulica y Ambiental, Pontificia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Macul, 7820436, Santiago, Chile
c
Escuela de Biotecnología, Universidad Mayor, Camino La Pirámide 5750, Huechuraba, 8580745, Santiago, Chile
d
CEDEUS, Centro de Desarrollo Urbano Sustentable, El Comendador 1916, Providencia, Santiago 7520245, Chile

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

• Arsenic and iron concentrations were

removed in microbial fuel cell systems.
• Biofilms on electrodes were tolerant to
low pH and high concentrations of
metals.
• pH neutralization favored the formation
of Fe minerals, and subsequent As re-
moval.
• XRD analysis suggests the precipitation
of Amorphous Fe minerals.

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

Article history: High concentrations of arsenic (As) in natural waters are a growing concern worldwide. In northern Chile, fluvial
Received 17 February 2018 systems enriched in As from natural and anthropogenic sources have been found to contain microbial communities
Received in revised form 22 June 2018 with exoelectrogenic activity. Previous work performed with Microbial Fuel Cells (MFCs) resulted in a neutralizing
Accepted 29 June 2018
microbial community developed from a consortium extracted from northern Chile. Considering that the formation
Available online xxxx
of iron minerals, which have been reported as good As sorbents, would be favored by pH neutralization, the use of
Editor: F.M. Tack neutralizing MFCs could result in a sustainable alternative for Fe and As removal. In this work, we quantified the re-
moval of As and Fe from acidic waters in air-cathode single-chamber MFCs. Our results show a removal ~80% of As
Keywords: and Fe and, simultaneously, a pH neutralization from ~3.7 to ~7.2. Additionally, non-MFC experiments indicate that
Arsenic removal the removal of As and Fe is dependent only on the activity of the microbial community developed during MFC op-
iron removal eration and not on the MFC electrochemical performance. In addition, scanning electron microscopy with energy
Microbial fuel cells dispersive X-ray spectroscopy (SEM-EDS) analysis showed spatial associations between Fe and As on the surface
Iron oxyhydroxides of cathodes, suggesting the idea that iron oxyhydroxides formation would be associated with the higher oxygen con-
Acid mine drainage
centration near the cathodes. Powder X-ray diffraction (XRD) analysis showed the dominance of iron amorphous
minerals, which may be favoring the removal of As. These results indicate that acid/metal-tolerant bacteria favor
pH neutralization and consequently the removal of Fe and As by processes of surface sorption and/or As-Fe co-pre-
cipitation. Furthermore, these findings expand the possible MFC applications to the simultaneous removal of Fe and
As from acidic waters, enabling its use as an energetically sustainable bioremediation alternative.
© 2018 Published by Elsevier B.V.

⁎ Corresponding author at: Departamento de Química Inorgánica, Facultad de Química, Departamento de Ingeniería Hidráulica y Ambiental, Escuela de Ingeniería, Pontificia Universidad
Católica de Chile, Vicuña Mackenna 4860, Macul, Santiago, Chile.
E-mail address: ealeiva@uc.cl (E. Leiva).

https://doi.org/10.1016/j.scitotenv.2018.06.378
0048-9697/© 2018 Published by Elsevier B.V.
472 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481

1. Introduction
Arsenic (As) in natural and drinking water is a major worldwide
concern because of its great impact at very low concentrations
(Kumar et al., 2004; Smedley and Kinniburgh, 2002). As is a carcinogen
and has high toxic effects on human health (Cheng et al., 2011; Engel
and Lamm, 2008; Farrell et al., 2001; Wu et al., 1989). For instance, in
northern Chile, high concentrations of As profoundly affects the quality
of water resources and creates serious health risks for the use and con-
sumption of water (Caceres et al., 2005; Leiva et al., 2014; Pizarro et al.,
2010). One possible origin of this problem is the release of As from min-
erals like arsenopyrite (FeAsS) and enargite (Cu3AsS4), which can be ac-
celerated by natural conditions (e.g. pH, electrical conductivity,
microbial activity) (Nordstrom et al., 2000; Pizarro et al., 2010;
Smedley and Kinniburgh, 2002; Wang and Mulligan, 2006). Similarly,
mining discharges have been shown to significantly increase the dis-
solved concentrations of As and iron (Fe) in rivers of northern Chile,
such as the Elqui, Camarones, Loa, and Lluta rivers (Leiva et al., 2014;
Oyarzun et al., 2004; Romero et al., 2003; Valenzuela et al., 2009).
In recent decades, the development of new methodologies to treat
waters contaminated with As has received significant attention (Amin
et al., 2006; Ballinas et al., 2004; Binbing et al., 2002; Borho and
Wilderer, 1996; Johnston and Heijnen, 2001; Khan et al., 2002; Kumar
et al., 2004; Leupin and Hug, 2005; Mohan and Pittman, 2007; Ning,
2002). Particularly, the use of Fe oxides to adsorb As has been studied
as one of the inexpensive alternatives (Dixit and Hering, 2003; Mohan
and Pittman, 2007). This is based on the association that has been ob-
served between As and Fe minerals (e.g. oxyhydroxides) in natural en-
vironments (Belzile and Tessier, 1990; Smedley and Kinniburgh, 2002).
Furthermore, it has been stated that the adsorption of As onto Fe min-
erals plays a key role in the control of As concentrations in the aqueous
phase and in its speciation (Dixit and Hering, 2003; Vitre et al., 1991).
Regarding sorption mechanisms, they are dependent on the oxidation
state of As and also on the mineralogy of Fe oxyhydroxides (Bissen
and Frimmel, 2003). Additionally, it has been observed that the forma-
tion of these minerals can be favored by neutralization of pH, which is
commonly acidic for waters contaminated with As, Fe and other metals
(Burrows et al., 2017; Seo et al., 2017).
Microbial Fuel Cell (MFC) is a promising technology for renewable
energy generation (Chaudhuri and Lovley, 2003; Logan et al., 2006;
Rabaey and Verstraete, 2005). This technology uses microorganisms,
known as electrochemically active microorganisms (EAM), as catalysts
to oxidize organic or inorganic electron donors and then, exogenously
transfer electrons towards an electrode (anode), which is externally
connected to another electrode (cathode), where the reduction of a
final electron acceptor occurs (Chaudhuri and Lovley, 2003; Logan et
al., 2006). In recent years, there have been advances in different aspects
of MFC research, such as the reactor architecture (Roy et al., 2016) or
electrode materials (ElMekawy et al., 2017; Aryal et al., 2017) which en-
abled the development of biotechnological applications for the removal

Fig. 1. Conceptual model and experimental design used in the study. Microorganisms extracted from Fe-As-rich surface sediments located in a hydrothermal area in northern Chile were
enriched and used to inoculate SC-MFC reactors. As and Fe removal was evaluated in SC-MFC reactors and in batch reactors (non-MFC) inculcated with electrode biofilms (Anode and Cathode).
 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481 473

Table 1 remove As from polluted waters but to couple this process to the gener-
Chemical and electrical performance of the MFC reactors under operation. Measurements ation of bioenergy. Thus, the aim of the present study was to examine
of chemical parameters were carried out after a two-day cycle, whereas cell voltage was
measured every 10 min with a data acquisition system.
the As removal capacity of MFC systems operated with synthetic acidic
waters enriched with Fe and As. This will allow for the design and oper-
Reactor pH Maximum cell voltage (mV) ation of appropriate abatement technologies for As-contaminated wa-
Initial medium 3.7 ± 0.2 ters and will contribute to improving the quality of several water
R1a (with As and Fe) 7.2 ± 0.2 2.0 ± 0.12 supply sources.
R2a 7.7 ± 0.1 0.8 ± 0.15

R1 was operated with acidic medium (pH = 3.7) enriched with Fe and As, while R2 was 2. Materials and methods
operated only with acidic medium.
a
The data correspond to averages and standard deviations of two reactors (duplicates).
2.1. Study site and sediment sampling

In the upper section of the Azufre River sub-basin, in the Altiplano of

of various pollutants, including some heavy metals (Shantaram et al.,
2005; Tao et al., 2014; Ter Heijne et al., 2010). Thus, MFCs have been
used, with different mechanisms and efficiencies, to remove different
dissolved metals from electrolytes, such as gold, silver, cobalt, copper,
mercury, chromium, vanadium, among others (Choi and Hu, 2013;
Hao et al., 2016; Tao et al., 2012; Tao et al., 2011; Ter Heijne et al.,
2010; Wang et al., 2011; Zhang et al., 2009; B. Zhang et al., 2012;
Zhang et al., 2015).
To date, research on MFC-like applications for As treatment includes
only a few studies. Most of them have approached to it through the
microbial oxidation of As(III) to As (V), using either heterotrophic
(Li et al., 2016) or autotrophic (Pous et al., 2015; Nguyen et al., 2016;
Nguyen et al., 2017) microorganisms. The study of Li et al. (2016)
used an SC-MFC to achieve the complete oxidation of 200 μg/L of As
(III) in seven days. Other studies have used different bioelectrochemical
systems with a polarized anode to facilitate the arsenite oxidation by
chemolithoautotrophic pure or mixed cultures (Pous et al., 2015;
Nguyen et al., 2016; Nguyen et al., 2017). However, none of these stud-
ies have actually removed the As content in the water, but they only
have focused in changing its oxidation state due to the easier immobili-
zation of As(V) in comparison to As(III) (Fazi et al., 2016). On the other
hand, Xue et al. (2013) used an SC-MFC coupled to another electro-
chemical cell, where more than a 90% removal of the As content was
achieved after 2 h of treatment. This removal was associated by the au-
thors to the coordinated action of iron (Fe) oxidized species and hydro-
gen peroxide (H2O2) generated during the system operation inside the
second cell, resulting in the formation of Fe-As precipitates (Xue et al.,
2013).
The use of MFCs as a neutralizing technology (Leiva et al., 2016),
emerged as an opportunity not only to form Fe oxyhydroxides and
the Andes Mountains, a specific site was selected for sampling fluvial
sediments and microbial samples for using as inocula in single-chamber
microbial fuel cells (SC-MFC). In this area, hydrothermal waters emerge
with low pH (b2) and high concentrations of As(III) and Fe in the aque-
ous phase (Leiva et al., 2014).
Fe-As-rich surface sediments were used to inoculate MFCs for
laboratory experiments. Undisturbed sediments (5 cm depth)
used as microbial sources were collected from the field campaigns
using 1 L High-Density Polyethylene (HDPE) bottles from a
specific sampling point. The specific sampling point is located in prox-
imity to a hydrothermal source where Fe-oxyhydroxides precipitate
continuously by oxidation of ferrous iron (Fe2+). These waters have
high concentrations of dissolved As, which after few meters are
discharged into the Azufre river. Fe-As-rich sediments at this point are
characterized by low pH (3.7), high dissolved sulfate concentrations
(2500 mg L−1), high concentrations of Fe and As (542.7 mg g−1 and
7.4 mg g−1 respectively) (Leiva et al., 2014).
After collection, sediment samples were kept in darkness at 4 °C
until processing and analysis. Further, on-site water analyses were per-
formed to characterize the natural system and determine growth condi-
tions of the microorganisms. These included temperature (PHC301,
Hach), pH (PHC301, Hach), dissolved oxygen (DO) (LDO101, Hach),
and electrical conductivity (CDC401, Hach) (Hq40d Multi, Hach, Love-
land, CO, USA).
2.2. Microbial fuel cell reactors
Microorganisms extracted from Fe-As-rich surface sediments
were enriched in batch reactors and then inoculated into air-cathode
membrane-less SC-MFC reactors (Fig. 1). MFCs consisted of an anode

a b
100 100
R1 SC-MFC
90 Abiotic Control 90

80 80

70 70
As removal %
Fe removal %

60 60

50 50

40 40
30
30

20 20 R1 SC-MFC
Abiotic Control
10 10

0 0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
Time (d) Time (d)

Fig. 2. Total dissolved Fe (a) and As (b) removal in SC-MFC reactors. A marked removal of Fe and As was observed from the second day of operation. The abiotic control does not evidence
removal of dissolved Fe and As during the operation. The metal removal data are the mean of two reactors (average of two replicates). The data are presented as the mean ± standard error
of the mean (error bars).
474 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481

and a cathode placed on opposite sides of a cylindrical plastic cham- a

ber with 4 cm long by 3 cm in diameter. The internal volume of the 100
chamber was 28 mL and the surface area of both electrodes was
90
7.1 cm 2 . The anode electrode was made of carbon felt without a
catalyst, while the cathode was constructed with a flexible carbon- 80

sCOD consuption %
cloth electrode containing 0.5 mg/cm2 of Pt catalyst. For each reac- 70
tor, anode and cathode were externally connected across a 1 kΩ
60
resistor.
The SC-MFCs were operated with synthetic acidic water as electro- 50
lyte, using pyruvate as an electron donor and atmospheric oxygen as
40
electron acceptor. Two different acidic media were prepared: one
enriched with arsenate (As5+) and ferric iron (Fe3+), and another one 30

without enrichment. The latter contained (per liter): sodium pyruvate 20

(1.3 g); NH4Cl (0.31 g); KCl (0.13 g); vitamins (5 mL, 1000×); metal R1 SC-MFC
10
salts (12.5 mL, 1000×). The enriched medium contained also 20 mg of Abiotic Control
Fe3+ (added as FeCl3) and 5 mg of As+5 (added as Na2HAsO4·7H2O). 0
The pH of both media was adjusted to 3.7 ± 0.1 with 0.5 M HCl. 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60

SC-MFCs were operated for 60 days as batch reactors at room Time (d)
temperature (~20 °C) and the voltage was recorded during operation
time with a data acquisition system (2700; Keithley Instruments, b
10
Cleveland, OH, USA). In addition, pH, soluble chemical oxygen pH R1 SC-MFC
demand (sCOD) consumption, total dissolved As (AsT) and total dis- pH Abiotic Control
9
solved Fe (FeT) removal were monitored over time. Batch-operation
cycles lasted 2 to 3 days, due to the medium replenishment made ac- 8
cording to electron donor consumption. An abiotic control was oper-
ated to check that metal removal occurs mediated by biological 7
activity.
pH

6
2.3. Batch experiments and cultures
5

Experiments were conducted in 50 mL sterile centrifuge tubes at

4
37 °C with continuous agitation for 21 days using microbial commu-
nities grown in anodes and cathodes as inoculum (Fig. 1). The initial 3
cell concentration was set at ~6 × 108 cells mL−1, which was deter-
mined according to cell counting made with a Neubauer chamber. 2
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
The culture medium was the same used in SC-MFC reactors. The pH
was adjusted to 3.7 with HCl (0.5 M) and the removal of FeT and Time (d)
AsT was reported over time. The pH inside the reactors was
monitored every 12 h, whereas FeT and AsT concentrations were c 4.0
monitored every two days. Similarly, an abiotic control for each EC R1 SC-MFC
Electrical conductivity mS cm-1

experiment (culture medium without microorganisms) was simul- 3.5 EC Abiotic control
taneously carried out in order to discriminate the metal removal as-
sociated with abiotic and physical processes. 3.0

2.4. Physicochemical analyses 2.5

2.0
pH, electrical conductivity, sCOD consumption, AsT and FeT removal
were monitored in water samples. Measurements of pH (PHC301,
1.5
Hach) and electrical conductivity (CDC401, Hach) were determined im-
mediately after sample collection from SC-MFCs and batch reactors. 1.0
sCOD consumption was determined colorimetrically in a HACH spectro-
photometer DR/2010 with reactor digestion method (USEPA Method 0.5
8000) (Jirka and Carter, 1975). FeT concentrations were determined col-
orimetrically in a Hach spectrophotometer DR/2010 using FerroVer® 0.0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
reagent (Hach). AsT concentrations were determined using Inductively
Time (d)
Coupled Plasma Optical Emission Spectrometry (ICP-OES).
Fig. 3. sCOD consumption (a), pH value at the end of a cycle (b), and electrical-
2.5. Light microscopy observation and solid phase analysis by Powder X-ray conductivity value at the end of a cycle (c) in SC-MFC reactors. The sCOD consumption
diffraction and scanning electronic microscopy coupled with energy-dis- is expressed as a percentage of removal related to the initial sCOD concentration. The
persive X-ray spectroscopy initial sCOD concentration of all MFC reactors was 872 ± 74 mg L−1. The removal of
sCOD falls on day 44 but increases again from day 46 of operation. pH neutralization
was observed in SC-MFC reactors. The data are the mean of two reactors (average of
The surface of electrodes was examined at the end of the two replicates). The data are presented as the mean ± standard error of the mean (error
operation of SC-MFCs were examined by light microscopy (CX31; bars).
Olympus, Tokyo, Japan). Specifically, the precipitated solids on
cathodic biofilms were observed under a light microscope at 4×
and 10× magnifications.

Additionally, scanning electronic microscopy coupled with energy-
dispersive X-ray spectroscopy (SEM-EDS) were used to analyze the
morphology of the biofilms grown on the electrodes (anode and cath-
ode), and the compositions of the mineral phases deposited on the cath-
ode and anode surfaces. The observation was performed using a
scanning electron microscope (JSM-IT300LV; JEOL Ltd., Tokyo, Japan).
The preparation of the electrode samples for SEM-EDS analysis was con-
ducted according to previously reported procedures (Park and Zeikus,
2003). Briefly, sections from the anodes and cathodes electrodes were
fixed using a 2% glutaraldehyde solution in a sodium cacodylate buffer
(0.2 M pH 7.2) for ~4 h to hold biofilms attached to electrode surfaces.
After that, the samples were dehydrated stepwise in a series of water/
ethanol solutions (50, 70, 80 and 100%) by 5 min with changes with
100% ethanol at each step. The samples were then treated for 45 min
with a critical-point dryer (Autosamdri-815; Tousimis, Rockville, MD,
USA) and coated with silver in a desktop sputterer (Desk V; Denton Vac-
uum, Moorestown, NJ, USA). Finally, for SEM-EDS observation, each
sample was analyzed in 12 points for observation of the surface topog-
raphy and compositional analysis.
Solid phases precipitated over electrodes in MFCs were also charac-
terized by Powder X-ray diffraction (XRD). XRD mineralogic analysis
was performed on solid samples freeze-dried for 2 days. XRD was per-
formed on a Bruker D8 Advance (Laboratory of Crystallography and X-
ray Diffraction- Faculty of Physical and Mathematical Sciences/
Universidad de Chile) with monochromatized CuKa radiation (40 kV,
30 mA, λ = 1.5604 Å) and the spectra were collected between scatter-
ing angles (2θ) of 5° and 70°.
3. Results and discussion
3.1. pH neutralization and voltage generation
Under batch operation, a similar increase in the pH inside of both
MFCs was observed, independently of the medium enrichment with
Fe and As. After two days of operation (1 cycle), the pH was neutralized
from an initial value of ~3.7 to ~7.7 in the case of the MFC operated with
the non-enriched medium, and to ~7.2, in the case of the MFC with the
enriched medium (Table 1). On the other hand, pH neutralization was
not observed under abiotic conditions (data not shown). Metals, pres-
ent as either dissolved species or precipitates, can have a negative effect
on the growth and metabolic activity of microorganisms (Gadd and
Griffiths, 1977; Mazidji et al., 1992; Nies, 1999). Interestingly, the
a b
100
Anodic biofilm
90 Cathodic biofilm
Abiotic Control
80
70
Fe removal %
60
50
40
30
20
10
0 0
0 4 8 12 16 20 24 28 32
Time (d)
Fig. 4. Total dissolved Fe (a) and As (b) removal in batch reactors inoculated with electrode biofilms. Biofilms of cathode and anode were used as inocula for cultures with synthetic acidic
water enriched with arsenate (As+5) and ferric iron (Fe3+). The Fe and As removal behavior was similar in both biofilms (anode and cathode). Batch experiments were performed at room
temperature (~20 °C), with an initial cell concentration of ~6 × 108 cells mL−1. The abiotic control does not evidence removal of dissolved Fe and As during the operation. The metal
removal data are the mean of two reactors (average of two replicates). The data are presented as the mean ± standard error of the mean (error bars).
E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481 475
microorganisms used in this study are able to grow, form biofilms on
electrodes and even maintain their role in pH neutralization, previously
reported (Leiva et al., 2016), when concentrations of Fe and As reached
values over 10 mg L−1 and 1 mg L−1, respectively. This metal-tolerance
is probably associated with the natural growth conditions of these mi-
crobes, that is, the AMD-affected environment where the initial micro-
bial inocula came from. A decrease of microbial diversity has been
reported for communities developed inside MFCs in the presence of
As (Li et al., 2016). This can be associated mainly with the high toxicity
that As can present to many microorganisms (Hamamura et al., 2013;
Hassan et al., 2015). Regarding tolerance to As(V), the As species used
in our medium, it has different expressions including detoxification-sys-
tem reduction, dissimilatory reduction and a methylation-demethyla-
tion system (Crognale et al., 2017). In case of dissimilatory reduction,
it has been associated with oxidation of a great variety of substrates, in-
cluding some sugars and small organic acids (Macy et al., 2000;
Niggemyer et al., 2001). Therefore, it seems possible that some microor-
ganisms that use neither oxygen nor the MFC anode as electron accep-
tor could be using As(V) as an electron acceptor.
Regarding electrical performance, SC-MFCs using the medium
enriched with Fe and As showed an increase in the voltage generation
in comparison with those operated with the non-enriched medium.
The maximum cell voltage for an operation cycle with enriched medium
reached 2.0 mV, which is about 2.5 times greater than the value ob-
tained with the non-enriched medium. Additionally, the voltage gener-
ation for the abiotic control reached 0.2 ± 0.05 mV, showing that
voltage generation, for both cases, occurs due to microbial activity inside
the MFC. However, the voltage levels are low if they are compared to
more efficient systems in electricity generation in MFC reactors, which
reach levels close to 200–500 mV (Feng et al., 2008; Liu and Logan,
2004; Raghavulu et al., 2009). These latter values are difficult to com-
pare to ours, because they were obtained under conditions of neutrality
and with microbial communities enriched from systems more com-
monly used as EAM inocula sources, such as sludges from sewage treat-
ment plants. On the other hand, coulombic efficiency values obtained by
these reactors were 5% at the most, which indicates that most of the en-
ergy coming from the degradation of pyruvate may have another desti-
nation. Possible destinations are related to the use of oxygen as an
electron acceptor by anodic microorganisms (Liu and Logan, 2004), be-
sides other energy losses such as overpotentials, ohmic and internal
losses that can affect the electrochemical performance of an MFC
(Logan et al., 2006).
100
Anodic biofilm
90 Cathodic biofilm
Abiotic Control
80
70
As removal %
60
50
40
30
20
10
36 40 44 48 52 56 60 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
Time (d)
476 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481

3.2. As and Fe removal in MFC reactors a

100
Anodic biofilm
MFC reactors showed Fe and As removal efficiencies of above 80% 90 Cathodic biofilm
and 60%, respectively (Fig. 2). Similar removal values have been re- Abiotic Control
80
ported for MFCs operated with vanadium and copper (Hao et al.,

sCOD consuption %
2016; B. Zhang et al., 2012). Changes observed in the removal of Fe 70
and As observed for different cycles, may be a consequence of variations
60
in voltage generation, pH increase value and/or precipitation of Fe
oxyhydroxides in a particular operation cycle. Simultaneously, sCOD re- 50
moval resulted above 60% after a two-day operation cycle (Fig. 3a),
40
while a marked pH neutralization (from ~3.7 to ~7.2) was also observed,
as was reported above (Fig. 3b). By the end of the operation period, a de- 30
crease in sCOD removal is observed. This may be explained by the accu-
20
mulation of Fe and As on the biofilms, which can act as a passivating
layer affecting the microbial growth, as it has been previously reported 10
for MFCs operating with Fe-containing electrolytes (Ter Heijne et al., 0
2011). For its part, the electrical conductivity is relatively stable inside 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
the MFC reactors (Fig. 3c). Additionally, we observed that the Fe re- Time (d)
moved from the aqueous phase precipitated as Fe minerals on the ca-
thodic electrode. These results indicate that the neutralizing ability b
of the microbial consortium inhabiting the cell promotes the re- 10
Anodic biofilm
moval of metals from contaminated waters. As it has been previously
9
Cathodic biofilms
stated, the neutralization of pH favors the formation of Fe-
Abiotic Control
oxyhydroxides, and hence the removal of dissolved Fe (Burrows et
8
al., 2017; Seo et al., 2017). Thus, the increase in the medium pH
added to the presence of dissolved oxygen near the cathode would
7
explain the precipitation of Fe minerals. These precipitates can, in
turn, control the removal of As by co-precipitation and/or surface ad-
pH

6
sorption processes (Xue et al., 2013). Furthermore, under
circumneutral conditions, the sorption of As onto Fe-oxyhydroxides 5
is favored because for As3+ occurs between pH 4–7, while for As5+
is optimal between pH 6–9 (Dixit and Hering, 2003; Manning et al., 4
1998; Vitre et al., 1991). Therefore, the pH increase would likely be
the key factor for the As removal process. 3
As it has been mentioned, changes in the medium conditions
(e.g. pH) can possibly favor the metal precipitation. Other 2
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
mechanisms involved in the removal of metals include
Time (d)
bioadsorption, bioaccumulation, and metal sulfides precipitation, as
it has been described for metals such as cadmium, zinc or Fe, c
among others (Chen et al., 2000; Machemer and Wildeman, 1992; 4.0
Madejón et al., 2002). Another process that could be involved in Anodic biofilm
Electrical conductivity mS cm-1

metal removal is the formation of organo-metallic complexes and 3.5

Cathodic biofilms
Abiotic Control
bioaccumulation of heavy metals inside the anodic and cathodic
biofilms (L-J. Zhang et al., 2012). 3.0

3.3. As and Fe removal in batch reactors 2.5

2.0
In order to evaluate if the functioning of the MFC system was rele-
vant for the removal process, this was evaluated in non-MFC systems
1.5
(i.e. batch cultures) inoculated with microorganisms grown on the cath-
ode and anode. The results show the removal of Fe and As in lower mag- 1.0
nitude than the observed in MFC reactors, with a 45% and 35% removal
for Fe and As, respectively (Fig. 4). Similarly, a lower increase in pH was 0.5
also observed (from 3.7 to ~6.4) using both inocula in separate cultures
(with dissolved Fe and As) (Fig. 5b). As in MFC reactors, in the bulk 0.0
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
phase of the batch reactors, we also observed Fe precipitates, but the in-
crease in pH was lower compared with MFC reactors, which can reduce Time (d)
the amount of Fe oxyhydroxides. In addition, it has been observed that
Fig. 5. Soluble chemical oxygen demand (sCOD) consumption (a), variation of pH (b) and
Fe oxyhydroxide precipitates can be found containing significant
variation of electrical conductivity (c) in batch reactors. The sCOD consumption is
amounts of colloidal-sized material (Langmuir and Whittemore, expressed as a percentage of removal related to the initial COD concentration. The initial
1971). The sizes of Fe crystals can be from microcrystals to crystal aggre- COD concentration of all batch reactors was 807.6 ± 1.6 mg L−1. pH neutralization in
gates (Langmuir and Whittemore, 1971). This is relevant because small batch reactors inoculated with microorganisms present at electrodes was evident
particles (~100 Å) are less stable compared to well-crystallized solids. In reaching pH values between 5.5 and 7. The data are the mean of two reactors (average
of two replicates). The data are presented as the mean ± standard error of the mean
several natural and laboratory solutions, it has been observed that these (error bars).
colloidal materials can remain indefinitely in the solution (Langmuir
and Whittemore, 1971; Majzlan et al., 2003). Hence, batch conditions
 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481 477

(non-MFC) and the agitation processes can favor a disaggregation of the
Fe oxyhydroxide precipitates keeping the colloidal materials in solution.
In addition, the lowest removal of Fe and As in batch reactors
could be associated with the lower biomass:volume ratio of batch
reactors (~41–45%) with respect to MFCs. Likewise, the sCOD
consumption was between 60% and 70% (Fig. 5a) and no significant
changes were observed in the electrical conductivity (Fig. 5c) inside
the reactor. No significant differences were observed in the removal
of Fe and As between both non-MFC reactors, showing a similar
behavior with the pH increase. These results again indicate that As
removal is mediated by the precipitation of Fe, which is controlled
by the pH neutralization.
We had previously shown that the pH neutralization in this system
is controlled by the biological activity associated with the microorgan-
isms present in the cathodic and anodic biofilms and not by the MFC
electrochemical performance (Leiva et al., 2016). Our experiments
with the enriched medium in batch reactors confirm this fact and also
support the idea that removal of As and Fe is also dependent only on
the presence of the selected microbial community and not on the MFC
functioning. Also, the difference in the removal of Fe and As in both

b c

500 um 500 um

d e

500 um 200 um

Fig. 6. Photographs of minerals phases precipitated over cathodic biofilm in MFC reactor (a). Light microscope images at 4× (b, c, and d) and 10× (e) of iron phases precipitated over
cathodic biofilm covered the air-cathode. The orange solids in the biofilm attached to the air-cathode suggest the precipitation of iron-oxyhydroxides by the oxygen diffusion from the
air. Solids phases were not observed in the anode.
478 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481

systems (MFC and non-MFC) can be associated with the configuration
of the reactors. The presence of the electrodes in MFC reactors can be
decisive for the selection of the microbial community responsible for
the increase of the pH, for resistance to heavy metal stress and indirectly
for the Fe and As removal. Indeed, it has been reported that some
biofilms are more resistant to heavy metal stress than either station-
ary-phase or logarithmically growing planktonic cells. Teitzel and
Parsek (2003) observed that biofilm of Pseudomonas aeruginosa were
between 2 and 600 times more resistant to copper, lead, and zinc than
free-swimming cells. They suggest that extracellular polymeric sub-
stances (EPS) can protect the biofilm from heavy metal stress by seques-
tering heavy metals and retarding their diffusion into the biofilm
compared with planktonic cells (Teitzel and Parsek, 2003). Other stud-
ies have suggested that the mechanism of resistance to metals in
biofilms is a combined action of chemical, physical and physiological
phenomena associated with the microbial community (Harrison et al.,
2007). Biosorption in the surface of biofilms can be a mechanism of re-
sistance to the toxic concentrations of Fe and As, which can occur simul-
taneously with the precipitation of Fe oxyhydroxides on biofilms
(Harrison et al., 2007; van Hullebusch et al., 2003). In addition, it has
been observed that different biofilms can produce more EPS compared
with planktonic cells grown with soluble substrates (this has been
shown in bioleaching systems) (Vera et al., 2013), which could be deter-
minant for the resistance of heavy metal stress.
Thus, the relevance of MFC systems and the presence of electrodes in
the removal of As and Fe would be associated with the ability to select
microorganisms that are tolerant to low pH and high concentrations of
heavy metals, to select neutralizing microbial communities, and to be a
solid support for biofilm-forming bacteria. These systems could be useful
as a pre-incubation or pre-treatment system for contaminated waters.
3.4. Characterization of solids precipitated inside the MFCs
To investigate the association of As and Fe with the surface of elec-
trodes, biofilm pieces were examined by optical microscopy. Since we
observed an orangish aspect on cathodes and not anodes, it would likely
mean that Fe removal resulted in Fe minerals precipitating near the ca-
thodic zone (Fig. 6). This suggests that the precipitation of Fe
oxyhydroxides would be associated with the higher concentration of
DO near the cathode, however, in batch reactors (non-MFC) we also ob-
serve fine Fe suspended materials. As aforementioned, in these reactors
the agitation would have an opposite effect, on the one hand, it favors
the aeration and on the other hand, it can promote the disaggregation
of the Fe-flocs generating a smaller amount of precipitated solids and
a greater concentration of colloidal particles in solution.
Additionally, electrode surfaces were examined by an SEM-EDS
analysis. The carbon cloth of the abiotic control did not show the pres-
ence of microorganisms or precipitation of Fe minerals (Fig. 7d). In con-
trast, bacteria of different sizes covered a wide area of the cathode in
different zones (Fig. 7c). EDS analysis showed that Fe was the largest el-
ement present on the surface of the biofilm. Cheng et al. (2011) previ-
ously found that spherical nano-particles of Fe oxide can precipitate
from dissolved Fe present in AMD-like solutions using fuel cell
technologies.
EDS analysis also showed overlapping of the Fe and As signals in the
same areas of the biofilm, indicating the presence of Fe-As compounds

a b

2 um 2 um

10 um 10 um

c d

2 um 2 um

10 um 10 um

Fig. 7. Scanning electron micrographs of biofilms attached to the anode (a) and cathode (b) of the SC-MFC reactors. Precipitated solids were observed mainly on the surface of the cathodic
biofilm. Micrographs of abiotic control for anode (c) and cathode (d) electrodes are also displayed.
 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481 479

Fig. 8. Energy dispersive X-ray spectrum of solids precipitated on cathodic biofilms. The intensity of As, Fe, and O emission lines are shown in EDS spectrum, suggesting the precipitation of
As-rich Fe oxyhydroxides. Other elements such as Sodium (Na), Phosphorus (P) and Calcium (Ca) have also been identified.

in the precipitated solids (Fig. 8). The quantitative analysis of the iden-
tified spectrum showed that there were 71.8% of Fe and 4.2% As atoms
on the surface of the cathodic biofilm, while Fe and As were not detected
on the surface of the anodic biofilm (data not shown). These results re-
inforce the idea that the As removal is related to the precipitation of Fe
minerals through surface adsorption and/or coprecipitation.
The XRD spectrum of the mineral phases precipitated on the cath-
odes are shown in Fig. 9. XRD analysis revealed that peaks were not
identified as crystalline phases (FeS2 and As4S4) and the analysis
shows that the precipitated solids are nearly amorphous. Therefore,
the precipitated minerals would not correspond to XRD-crystalline Fe
oxides and were assigned as unidentified amorphous Fe-oxyhydroxides.
The pattern of XRD is typical of other amorphous iron oxides (HFOs) ob-
served in natural and laboratory systems, such as poorly crystalline fer-
rihydrite (Fredrickson et al., 1998; Jia et al., 2007). Although crystalline
Fe phases like Pyrite were not detected, it is not possible to completely
rule out their low-concentration presence, due to the detection
limitations of this technique and the overlapping with the strong amor-
phous line.
Freshly-formed amorphous Fe oxyhydroxides have a high affinity
for As, due to their large surface area, which favors the presence of
more active sorption sites (Wang and Mulligan, 2006). Several studies
have shown that HFOs have a higher density of sorption sites compared
to crystalline Fe oxides. Dixit and Hering (2003) observed that the max-
imum sorption density for HFO was 2100 μmol g−1, while for goethite
(a crystalline Fe-oxyhydroxide) reached only 140 μmol g−1. According
to the authors, although both have similar sorption sites per unit area,
the specific area of the HFOs is much greater than crystalline phases
such as goethite (Dixit and Hering, 2003). Furthermore, this stronger
adsorption of As species by amorphous Fe-oxyhydroxides is also related
to the fact that their structure can be freer and highly hydrated, becom-
ing permeable to hydrated ions (Bowell, 1994; Pierce and Moore, 1982).
Thus, the ions can diffuse into the amorphous mineral structure and not
be restricted only to external sorption sites.

Fig. 9. XRD pattern for solid phases precipitated on the surface of the cathode electrode. The formation of amorphous precipitates is shown as a bulking of the baseline (red line). Crystalline
solid phases were not identified (Red square = FeS2, Blue diamond = As4S4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
480 E. Leiva et al. / Science of the Total Environment 645 (2018) 471–481
4. Conclusions
Our results showed that inside MFCs Fe and As are removed. Micro-
organisms that form biofilms in the cathode and anode were tolerant to
high concentrations of Fe and As and low pH. The removal of Fe in the
system is probably associated with the increase in pH caused by micro-
bial activity inside the MFC. The pH neutralization first favors the pre-
cipitation of Fe minerals that can sorb and/or coprecipitate As,
promoting its removal from the aqueous phase. XRD analysis suggests
the presence of Amorphous Fe-oxyhydroxides. Moreover, poor crystal-
lization of the precipitated minerals may be due to early stages of pre-
cipitation that favor the formation of freshly-formed disordered Fe-
oxyhydroxides. These mineral phases have a higher affinity for As, as
they have more active sorption sites and therefore can catalyze the re-
moval of As by surface adsorption processes onto Fe minerals previously
precipitated on the biofilms. SEM-EDS analyses support this idea be-
cause Fe and As show a marked spatial association on the surface of
the cathode biofilms. Even so, this does not allow to rule out direct
precipitation of Fe-As-bearing ores. The precipitation of Fe minerals
(with As) worsened the generation of voltage and could explain
the low levels observed. In addition, the voltage generation is not a
prerequisite for the removal of Fe and As, since this removal occurs
in the same way in non-MFC reactors. However, the relevance of
MFC systems in the removal of As and Fe would be associated with
the ability to be a specific system for the selection of metal/acid-tolerant
microbial communities, and highlights the potential of these systems to
be used as biotechnological pretreatment applications for acidic waters
enriched in As and Fe.
These findings contribute to the understanding of dissolved metal
removal processes in MFC systems. Likewise, these results could expand
the use of these emerging technologies from the removal of organic
compounds to their use in scalable systems of acidic water treatment.
Interestingly, the removal of As has not been addressed in previous
studies, and due to the recalcitrance of this contaminant, the develop-
ment of new remediation methodologies could contribute to conven-
tional treatment systems.
Additional efforts are needed to understand the mechanisms in-
volved in As precipitation at the molecular level by determining the
minerals that are formed and their long-term stabilization. Likewise, ad-
vances in the characterization of the microbial communities that form
the anodic and cathodic biofilms could help to understand the specific
removal mechanisms associated with Fe and As. Finally, future research
should focus on understanding the impact of the precipitation of Fe
minerals enriched with As on cathodic and anodic biofilms, determining
their impact on electrochemical performance, Fe and As removal rates
and the divergence of microbial communities, and on understanding
the role of electrodes in the selection of microbial communities and in
the removal of Fe and As.
Acknowledgments
This research was funded by PIA-UC Eduardo Leiva/DDA-VRA,
FONDECYT project 3140515, FONDECYT project 1160917 and CEDEUS
(CONICYT/FONDAP/15110020). We thanks to Javiera Anguita and Cris-
tobal Alvear for their help in Fe-As-rich sediments sampling during field
campaign. Thanks are also extended to the reviewers for the corrections
and suggestions, which contributed significantly to improve the
manuscript.
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