Science of the Total Environment 466467 (2014) 490502

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Science of the Total Environment

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

Natural attenuation process via microbial oxidation of arsenic in a high

Andean watershed
Eduardo D. Leiva a, Consuelo d.P. Rmila a, Ignacio T. Vargas a, Cristian R. Escauriaza a, Carlos A. Bonilla a,
Gonzalo E. Pizarro a, John M. Regan b, Pablo A. Pasten a,
a
b

Department of Hydraulic and Environmental Engineering, Ponticia Universidad Catlica de Chile, Santiago, Chile
Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park, PA, USA

H I G H L I G H T S

Dissolved As decreases in a stream from a hydrothermal source in Chilean Altiplano.

As attenuation is governed by As(III) oxidation and a pH decrease.
The oxidation of As(III) is mediated by As(III)-oxidizing microorganisms.
Dissolved As attenuation is correlated with As immobilization on Fe-rich sediments.
Biological oxidation coupled to sorption/coprecipitation limits the ux of As from hydrothermal waters.

a r t i c l e

i n f o

Article history:
Received 15 January 2013
Received in revised form 29 June 2013
Accepted 2 July 2013
Available online xxxx
Editor: F.M. Tack
Keywords:
Fluvial arsenic
Biological arsenic oxidation
AroA-like
Chemical arsenic speciation
Altiplano

a b s t r a c t
Rivers in northern Chile have arsenic (As) concentrations at levels that are toxic for humans and other organisms. Microorganism-mediated redox reactions have a crucial role in the As cycle; the microbial oxidation of
As (As(III) to As(V)) is a critical transformation because it favors the immobilization of As in the solid phase.
We studied the role of microbial As oxidation for controlling the mobility of As in the extreme environment found
in the Chilean Altiplano (i.e., N4000 meters above sea level (masl) and b 310 mm annual rainfall), which are conditions that have rarely been studied. Our model system was the upper Azufre River sub-basin, where the natural
attenuation of As from hydrothermal discharge (pH 46) was observed. As(III) was actively oxidized by a microbial consortium, leading to a signicant decrease in the dissolved As concentrations and a corresponding increase
in the sediment's As concentration downstream of the hydrothermal source. In-situ oxidation experiments demonstrated that the As oxidation required biological activity, and microbiological molecular analysis conrmed the
presence of As(III)-oxidizing groups (aroA-like genes) in the system. In addition, the pH measurements and solid
phase analysis strongly suggested that the As removal mechanism involved adsorption or coprecipitation with
Fe-oxyhydroxides. Taken together, these results indicate that the microorganism-mediated As oxidation contributed to the attenuation of As concentrations and the stabilization of As in the solid phase, therefore controlling the
amount of As transported downstream. This study is the rst to demonstrate the microbial oxidation of As in
Altiplano basins and its relevance in the immobilization of As.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Arsenic (As) is a toxic element widely distributed in natural environments (Cullen and Reimer, 1989; Smedley and Kinniburgh, 2002).
The contamination of surface and groundwater by As is a serious
environmental concern because such contamination limits the use
of the water and has adverse effects on human health (Berg et al.,
Corresponding author at: Departamento de Ingeniera Hidrulica y Ambiental, Escuela
de Ingeniera, Ponticia Universidad Catlica de Chile, Vicua Mackenna 4860, Macul,
Santiago, Chile. Tel.: +56 2 2354 4872; fax: +56 2 2354 5876.
E-mail address: ppasten@ing.puc.cl (P.A. Pasten).
0048-9697/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2013.07.009

2001; Bhattacharya et al., 2002; Smedley and Kinniburgh, 2002).

Although the behavior of As in nature has been widely studied in recent
years (Berg et al., 2001; Manning et al., 1998; Nickson et al., 1998;
Nordstrom, 2002; Oremland and Stolz, 2003; Roussel et al., 2000;
Stollenwerk et al., 2007), the biogeochemical controls governing the
mobilization, stabilization, and release of As into river systems are still
unclear.
The mechanisms of As attenuation in natural environments include
a combination of physical, chemical, and microbial factors (Wang and
Mulligan, 2006). The sorption onto iron (Fe) oxyhydroxides is the
major natural attenuation process for the removal and sequestration
(stabilization) of As species (Drahota et al., 2012; Leblanc et al., 1996).

E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

Co-precipitation of As and Fe also contribute to the stabilization of

As in solid phase (Fuller et al., 1993). However, changes in the As speciation, particularly the oxidation from As(III) to As(V), have a crucial
role in the release and mobilization of As because As species differ in
their solubility, toxicity, transport, and bioavailability (Aposhian and
Aposhian, 2006; Casiot et al., 2005; Drahota et al., 2009; Masscheleyn
et al., 1991; Smedley and Kinniburgh, 2002). Therefore, microorganisms
can oxidize As and therefore increase its sorption onto mineral phases
because As(V) binds more strongly to Fe oxyhydroxides than As(III)
(Oremland and Stolz, 2003). The impact of As oxidation reactions catalyzed by microorganisms on As attenuation may vary according to the
climatic and geochemical conditions of a particular system (Lin and
Puls, 2003; Smedley and Kinniburgh, 2002).
The occurrence of As in arid environments is widespread due
to geogenic or anthropogenic sources (Baker et al., 1998; Del Razo
et al., 1990; Guo et al., 2003; Smedley et al., 2002). Northern Chile is
seriously affected by high concentrations (N1 mg l1) of As in rivers
(Alsina et al., in press; Caceres et al., 1992; Landrum et al., 2009;
Pizarro et al., 2010; Queirolo et al., 2000; Romero et al., 2003; Sancha,
1999), which generates serious problems for the population reliant
upon these waters in this arid region. The biogeochemical processes
that control the fate of As in river systems are quite specic because
the extreme conditions, which are high altitude (N4000 masl) and
high aridity (b 310 mm year1 of precipitation), impact the environment. In arid and semi-arid systems, metal enrichment processes
may occur via capillary transport (Dold and Fontbot, 2001). The mobilized elements (e.g., As) are transported toward the surface of the
soils and sediments where the presence of oxygen may facilitate oxidation (Bechtel et al., 2001; Dold and Fontbot, 2001; Smedley and
Kinniburgh, 2002). Therefore, the As(III) oxidation and the As stabilization (especially as As(V) species) by adsorption processes occur predominantly under oxidizing conditions (Smedley and Kinniburgh,
2002). The role of biological reactions, particularly the reactions mediated by As-oxidizing microorganisms, in the biogeochemical cycling of
As still is unknown for these types of systems.
The Chilean Altiplano is characterized by a scarcity of water and
rich mining activity. Particularly, the Azufre River sub-basin contains
a complex mixture of natural and anthropogenic contaminants from
different sources, seriously affecting the quality of the surface water
resources. We observed that in the upper section of this sub-basin,
the As concentrations are high (1.03.5 mg l1); additionally, the
hydrothermal waters and sediments exhibit naturally elevated concentrations of As (0.8 mg l1 and N4 g kg1 respectively). Interestingly, the total dissolved As (AsD) concentrations are attenuated
( 70% AsD), and a noticeable amount of As oxidation ( + 90%
As(V)/AsD) occurs in the hydrothermal springs (Leiva et al., 2011). Effective risk management and remediation efforts for such As-enriched
uvial systems as Azufre River sub-basin requires the understanding
and quantication of the changes in chemical speciation, as well as
the biogeochemical control of the mobilization/stabilization of As in
the solid phase. In this regard, the Altiplano in northern Chile is an
ideal site to study the processes that control As mobilization under
extreme conditions.
2. General setting
2.1. Climatic and environmental characteristics of the model site
The Altiplano is an area in the central Andes (1534S) covering
the western part of Bolivia, northern Chile, southern Peru, and northern Argentina, with an average altitude of 3600 masl (Allmendinger
et al., 1997). The Chilean Altiplano is an elevated plateau 4000 masl
covered by numerous andesitic stratovolcanoes that can reach up
6500 masl (Muoz and Charrier, 1996; Stern et al., 2007). Particularly,
the geological formations include conglomerates of the Upper Pliocene
Pleistocene, sandstones, shales, and rhyolitic ignimbrites (Salas et al.,

491

1966). This area has average temperatures of 0 C, concentrated rainfall

between 50 and 300 mm year1, and potential evaporation of approximately 6001200 mm year1 (DGA, 1987). The climatic conditions on
all timescales are closely related to changes in the upper-air circulation
and by the amount of near-surface water vapor, impacting wet and dry
conditions and inducing strong rainfall uctuations (Garreaud et al.,
2003).
The Azufre River sub-basin is located in the XV Region of Arica and
Parinacota, in northern Chile (Fig. 1). This sub-basin is part of the
Lluta River Watershed (LRW) and extends between 18 0018 30
S and 70 2069 22 W. The Azufre River originates in the upper
section of the LRW in the Altiplano and has an average altitude of
4250 masl (Leiva et al., 2011). This area has a semi-arid climate: rainfall rarely exceeds an average of 310 mm annually and occurs as
intense thunderstorms from late December to late February. This
area is characterized by water scarcity caused by the limited rainfall,
low humidity, strong solar radiation and high evaporation rates
(4.9 0.5 mm d1) overall. The Azufre River sub-basin is strongly
inuenced by the Tacora Volcano, which is the northernmost
volcano in Chile and located at 1743S, 6946W with an altitude
of 5980 masl. The Tacora volcano is a stratovolcano in the Central
Volcanic Zone (CVZ) of the Andes (1528S) (Stern et al., 2007;
Clavero et al., 2005). The CVZ is a chain of quaternary stratovolcanoes
and andesiticrhyodacitic domes (Allmendinger et al., 1997 and Kay
et al., 1999). The Tacora volcano has been active since at least the
Middle Pleistocene and displays permanent fumarolic (CO2 and SO2
degassing) and hydrothermal features in the southwest ank of the volcano within a drainage area contributing to the Azufre River (Capaccioni
et al., 2011; Clavero et al., 2005, 2006). Additionally, the legacy of
sulfur mining is observed on the Tacora crater (~5500 masl) and the
west ank, where tailings and waste rock deposits may be observed.
Acid mine drainage (pH b 2.0; As b 0.4 mg l1, Fe b 10 mg l1) from
uncontrolled exposure to water and atmospheric O2 is observed at
the foot of mine tailings. The mining legacy and the dry climate limit
the growth of vegetation, but in the upper section of the watershed,
there is a wetland where different biogeochemical processes can control
the environmental fate of As and other contaminants.
The study site is located in this wetland, which runs along
the middle reach of the Azufre River and receives a ow of several
hydrothermal features; these hydrothermal features nally drain into
the Azufre River as described in Fig. 1. Hydrothermal springs are characterized by elevated As content, causing the mobilization of high concentrations of dissolved As (N1 mg l1) and Fe (N1 mg l1) to the river
ow (Leiva et al., 2011).
2.2. Microbiological context
Microbial processes have a signicant impact on the mobility and
speciation of As (Oremland and Stolz, 2003). The transformations
mediated by microorganisms play an important role in their modulation of As behavior in soils, wetlands, groundwater, and surface
waters, as well as in the toxicological characteristics of environmental As (Oremland and Stolz, 2003; Mukhopadhyay et al., 2002; Tsai et
al., 2009). As(III) is more toxic and mobile than As(V) and its presence usually entails a comparatively larger environmental health
risk and concern (Mondal et al., 2006; Pierce and Moore, 1982). Bacteria capable of oxidizing As(III) have been found in sediments, soils,
mine tailings, hydrothermal sites, and natural waters (DonahoeChristiansen et al., 2004; Gihring and Baneld, 2001; Gihring et al.,
2001; Hamamura et al., 2009; Inskeep et al., 2007; Oremland et al.,
2002; Oremland and Stolz, 2003; Santini et al., 2002). Both heterotrophic and autotrophic bacteria can oxidize As. Heterotrophic bacteria oxidize As(III) through a detoxication mechanism and do not
obtain energy from the reaction (Anderson et al., 1992; Silver and
Phung, 2005). In contrast, autotrophic bacteria oxidize As(III) as a
chemolithoautotrophic growth strategy, where carbon xation is

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E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

Fig. 1. (a) Map of Chile highlighting the location of the upper section of the Azufre River sub-basin in the Lluta River watershed of northern Chile; (b) transects selected for the
study. Each transect drains into the Azufre River.*Distance after the origin of the Azufre River (panel a).

coupled with the oxidation of inorganic As(III) under aerobic, nitratereducing, or denitrifying conditions (Oremland et al., 2002; Rhine
et al., 2006; Santini et al., 2002). Microbial oxidation of As is mediated
by enzymes called arsenite oxidases, such as AroA/B, AsoA/B, and
AoxA/B (Inskeep et al., 2007; Kashyap et al., 2006; Lebrun et al., 2003;
Santini and vanden Hoven, 2004). In hydrothermal systems, the As(III)
oxidation activity correlates to the presence of arsenite-oxidizing microorganisms (Hamamura et al., 2009; Inskeep et al., 2007; Macur et al.,
2004). However, there is no information available concerning the impact of arsenite-oxidizing microorganisms on hydrothermal systems at

extreme altitude and in arid lands with high As concentrations in the

ground and surface waters.
2.3. Environmental and water resources signicance
High concentrations of AsD from the Azufre River (1.03.5 mg l1)
strongly determine the inux of As into the Lluta River Valley. The Lluta
River is a precious water resource for a population of ~213,800 located
in the city of Arica, which is the capital of the XV Region of Arica and
Parinacota, as well as in many surrounding villages. The Azufre River

E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

mixes downstream with more alkaline waters (pH increase), consequently forming Al hydroxides and Fe oxyhydroxides (Guerra et al.,
2012). These solid phases are well-known sorbents of As and occur as
suspended solids or coatings on other particles, which are transported
downstream or deposited depending on the ow velocity. Intense
storms during the wet season trigger events that mobilize As-enriched
sediments deposited along the river bed. The imminent construction
of a dam to increase water availability throughout the year for agricultural purposes causes additional concern because the new structure
will most likely become an As repository. The microbial reactions that
control the inux of As at the head waters play a signicant role in the
overall As budget and the ux of As into the Lluta River Valley water
resources. Specically, this research was performed to investigate the
inuence of the As oxidation processes mediated by arsenite oxidizers
on the changes in As speciation and attenuation within a hydrothermal
spring; these ndings will aid the development of future remediation
strategies. Furthermore, we also expect that this case will serve as a
model for other high-altitude hydrothermal systems containing elevated AsD concentrations of reduced As in the CVZ.

3. Methods
3.1. Experimental design in the study site, sampling and on site
measurements
The hydrothermal site chosen for detailed study in this work is
located on the west ank of the Tacora volcano (Fig. 1b). Two hot surface runoff channels that drain from the same hot spring were chosen
for a transect sampling, each containing four sampling points for
hydrogeochemical and microbiological analyses. The average ow
velocities were 25.4 cm s1 in transect 1 and 12.9 cm s1 in transect
2, based on suspended particle travel times within a distance of 2 m
in the surface water ow (procedure modied from Langner et al.,
2001). The transects were selected based on preliminary data analyses from July, 2010 that indicated a decrease in the AsD concentrations downstream from the discharge of the hydrothermal spring
source. In addition, the sampling points were chosen based on their
proximity to high concentrations of Fe-oxyhydroxides, allowing
the simultaneous evaluation of the effects caused by the changes
in As speciation and the natural attenuation of AsD concentrations
(adsorption onto Fe-oxyhydroxides).
As-rich waters, sediments, and microbial samples were collected
from ve eld campaigns, occurring in October 2010, February 2011,
July 2011, November 2011, and May 2012. Water samples from the
Azufre River were collected in 300 ml High Density Polyethylene
(HDPE) bottles and were immediately ltered in the eld with 0.22-m
nylon lters. Filtered subsamples were acidied with HNO3 (1:100 v/v,
Merck) for the analysis of the AsD concentration (McCleskey et al.,
2004). Unltered subsamples were also acidied to a pH of approximately 2 in the eld with HNO3 immediately after sampling (1:100 v/v,
Merck) for total As (AsT) (includes particulate As) concentration analysis
along the river.
Water samples from the hydrothermal transects were collected
in 300 ml HDPE bottles and immediately ltered in the eld with
0.22-m nylon lters. Filtered subsamples were acidied to pH 2
with HNO3 for later laboratory measurements of AsD, total dissolved
Fe (FeD) concentrations, and major dissolved cation analysis (sodium,
potassium, calcium). For the As speciation analysis (As(III) and
As(V)), the ltered subsamples were acidied in the eld to a pH of
approximately 3 with HCl (1:100 v/v, Merck) (McCleskey et al.,
2004).
Additional ltered and non-acidied water samples were collected along the Azufre River and in sampling points of the hydrothermal transects for the later determination of dissolved anions (sulfate,
nitrate, and chloride) analysis. All water samples (ltered/acidied,

493

unltered/acidied, and ltered/non-acidied) were stored in darkness at 4 C until processing and analysis occurred within 1 week of
sampling.
The on-site water analyses included temperature (PHC301, HACH),
pH (PHC301, HACH), dissolved oxygen (DO) (LDO101, HACH), and
electrical conductivity (CDC401, HACH) (Hq40d Multi, HACH, Loveland,
CO, USA). Undisturbed surface sediment samples (depth 6 cm) at each
sampling point of the hydrothermal transects were collected with
50 ml polypropylene tubes (BD Biosciences, Mountain View, CA) and
150 ml HDPE bottles, and were kept in darkness at 4 C until processing
and analysis.
3.2. Hydrogeochemical and solid-phase analyses
AsT (unltered, acid-soluble), AsD (ltered, acid-soluble), and FeD
(ltered, acid-soluble) analyses in water samples were carried out
by Total X-ray Reection Fluorescence (TXRF) (S2 Picofox, Total
X-ray reection uorescence spectrometer, Bruker AXS, Billerica,
MA, USA) (Borgese et al., 2009). The AsT, AsD, and FeD concentrations
in the water samples were conrmed by Inductively Coupled Plasma
Optical Emission Spectrometry (ICP-OES) (PerkinElmer Optima 7300
DV ICP-OES, Shelton, CT, USA) (data not shown). The dissolved inorganic As species [As(III) and As(V)] were measured in the laboratory
using ltered/HCl-acidied subsamples. The dissolved As(III) concentrations were determined by hydride generation with cysteine and
sodium borohydride reductants, as well as Inductively Coupled Plasma Mass Spectrometry (ICP-MS) detection (PerkinElmer ELAN 6100,
Shelton, CT, USA) (detection limit b 0.002 mg l1) (Garbarino et al.,
2002). The As(V) concentration was determined by subtracting the
As(III) content from the AsD concentration measured with TXRF.
The contents of the dissolved inorganic Fe species [Fe(II) and Fe(III)]
were determined by spectrophotometry. The ferrous (Fe(II)) iron concentration was determined in the eld immediately after sampling in
ltered/non-acidied subsamples with a HACH spectrophotometer
DR/2010 (110 Phenanthroline method 8146, adapted from Standard
Methods for the examination of water and wastewater 15th ed 201
(1980)) (HATCH, Loveland, CO, USA). Ferric (Fe(III)) iron concentration
was determined by subtracting the Fe(II) content from the FeD concentration measured with TXRF. Sodium, potassium, and calcium were
analyzed via ICP-OES from the ltered/acidied subsamples. Sulfate,
nitrate, and chloride were analyzed using Ion Chromatography (IC)
(882 compact IC plus, Metrohm, Herisau, Switzerland) with ltered/
non-acidied subsamples.
The sediment samples were dried at 40 C and the analysis of the
total As in the solid phase was performed with the prior microwave
digestion (Microwave Accelerated Reaction Systems, model MARS
(CEM corporation, Mattheus; North Carolina 28106)) of ~ 100 mg of
dry sample (b2 mm) in 9 ml of concentrated HNO3 and 3 ml of concentrated HCl (EPA method 3051A). The As content of the digests was
determined by TXRF. The results of the As analyses in the solid phase
were conrmed by ICP-OES (PerkinElmer Optima 7300 DV ICP-OES,
Shelton, CT, USA) (data not shown).
3.3. Analytical quality control
Continuous quality assurance/quality control (QA/QC) procedures
were performed for both the eld measurements and laboratory
analyses by TXRF, ICP-OES and IC. All eld and laboratory instruments
were calibrated using certied reference standards or analytical grade
synthetic standards. The acceptable standard deviation for results of
metals, cations, and anions was always b10%, and all analyses were
interspersed with and checked against standard reference materials.
The AsT, AsD and FeD measurements of the water samples and the
As content of acid digest samples were measured by TXRF in triplicate. The QC of the measurement was performed by inserting a synthetic standard with a known concentration of As and Fe into the

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E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

analytical run every 20 samples. Similar to the acid digest samples,

the measured concentrations of trace elements (As, Cu, Pb, Fe, Mn)
in certied reference standards (Nist 2781, Nist 2702 and SRM 2709a
San Joaquin Soil) were measured with an error range of 5 1% of
their certied values. The quantitative TXRF analysis was conducted
by adding an internal Gallium (Ga) standard of known concentration.
In addition, the metal concentrations in the blanks were always
below the TXRF lower limits of detection (LLD) calculated for the
water samples (b0.01 mg l1 for As, and b 0.03 mg l1 for Fe). The
QC of the equipment was achieved by monitoring the operational
parameters. A gain correction (adjustment of the spectroscopic amplication) was performed daily and the spectroscopic resolution, sensitivity and accuracy of quantication were checked monthly.
The measurements of metals (As and Fe) and cations (sodium,
potassium, and calcium) made via ICP-OES were performed in duplicate, and a QC sample composed of certied concentrations of metals
and cations was incorporated into the analytical run every 10 samples. The blanks for the metals (As, Fe) were always under the detection limit of the equipment (b0.006 mg l1 for As, and b0.01 mg l1
for Fe). The same was observed for the cation analysis; the values of
blanks were under the limit of detection for calcium (b 0.01 mg l1),
sodium (b 0.01 mg l1) and potassium (b0.01 mg l1). QC of equipment was accomplished by the periodic monitoring of the calibration
curve slope. Furthermore, the accuracy and precision of the measurements were evaluated by checking the results against certied standard
samples.
The anion analyses were performed by IC in duplicate and included
an internal quality control sample every 20 measurements composed of
synthetic standards of known anion concentrations to corroborate the
results. For the TXRF and ICP-OES analyses, the IC analysis also reported
blank values under the detection limit for chloride (b 0.34 mg l1), sulfate (b0.061 mg l1) and nitrate (b0.076 mg l1). The QC of equipment included a calibration curve of the anions, which was checked
monthly against synthetic analytical grade standards. After each measurement, the column was washed continuously with the mobile phase.
3.4. In-situ arsenite-oxidation assays
The biotic and abiotic rates of As(III) oxidation were evaluated in
the presence of both alive and dead microbial communities, respectively. For the abiotic oxidation assays, sediment samples (64 cm3)
upstream from the points of each transect [transect 1 (2.7 m),
transect 2 (4.2 m)] were transferred to 500 ml bottles. The sediment
samples were covered with 10 ml of 37% (v/v) formaldehyde, before
the addition of 300 ml of spring water and 2 mg l1 of As(III). The
biotic oxidation assays were performed following the same protocol,
except that formaldehyde was not added to the sediments. The
sample bottles were incubated in a neighboring hot spring of similar
temperature [transect 1 (~ 30 C), transect 2 (~ 22 C)]. Water subsamples (25 ml) were taken at 0, 3, 5, and 12 min, ltered in the
eld using 0.22-m nylon lters, and analyzed to determine the As
speciation and AsD. This protocol was modied from that of
Langner et al., 2001.
3.5. Microbiological analyses
3.5.1. Sampling and DNA/RNA extraction
The microbiological samples were collected in sterile 50 ml
polypropylene tubes (BD Biosciences, Mountain View, CA) and preserved for DNA (4 C) or RNA extraction (RNASafer Stabilizer Reagent,
MoBio, Carlsbad, CA, USA). Metagenomic DNA isolation was performed
using the Power Soil DNA Isolation Kit (MoBio, Carlsbad, CA, USA).
The integrity of the DNA was veried via 1% (w/v) agarose gel electrophoresis prepared with SYBR Green DNA binding dye (1) (Invitrogen,
Carlsbad, CA, USA). The DNA quantity and purity were measured with

spectrophotometry based on the A260 and A260/A280 ratio. The DNA

preparations were stored at 20 C until analysis.
For the RNA extraction, environmental samples were preserved
in the eld with RNASafer Stabilizer Reagent (MoBio, Carlsbad, CA,
USA), before being stored at 4 C in the laboratory. The total RNA
was extracted from 0.51.0 g of wet sample using the Power Soil
Total RNA isolation kit (MoBio, Carlsbad, CA, USA). The extracted RNA
was treated with RNase-free DNase (Promega, Madison, Wisconsin,
USA), recovered with a phenol-chloroform solution (1:1), and precipitated with ethanol. Finally, the RNA was resuspended in 50 l of
nuclease-free water. The RNA quantication was measured at A230.
RT-PCR was performed using a SuperScript RT-PCR System (Invitrogen,
Carlsbad, CA, USA). To verify the absence of DNA contamination, RT-PCR
controls were performed without the addition of reverse transcriptase.
Finally, the cDNA preparations were stored at 20 C until analysis.
3.5.2. Polymerase chain reaction (PCR) amplication
PCR amplication of the aroA-like genes was performed with degenerate primers [aroA95f (5-TGYCABTWCTGCAIYGYIGG) and aroA599r
(5-TCDGARTTGTASGCIGGICKRT T)] (Hamamura et al., 2009) from environmental DNA and cDNA. The reactions (50 l) were prepared
using similar masses of the DNA and cDNA templates, as well as 1 M
of each primer; the program used was previously described elsewhere
(Hamamura et al., 2009). The amplied products (500 bp) were visualized via agarose gel electrophoresis, and the aroA-like gene of
Agrobacterium tumefaciens 5A (aoxAB) was used as the positive control.
Subsequently, 16S rRNA gene amplication from the environmental
DNA was performed using the universal primers 8F (5-AGAGTTTG
ATCCTGGCTCAG-3) and 1392R (5-ACGGGCGGTGTGTAC-3) (Amann
et al., 1995).
3.5.3. Terminal restriction fragment length polymorphism (T-RFLP)
analysis
The T-RFLP analyses were carried out using DNA samples from
the hydrothermal source (0 m), because it presented the highest concentrations of As(III) and fed both transects. T-RFLP analysis was
performed using the previously described universal primers for the
16S rRNA gene (Amann et al., 1995); the 8F primer was labeled with
the uorophore 6-FAM and the 1392R primer was left unchanged. The
labeled products were digested separately with 20 U of HhaI and MspI
endonucleases and precipitated as described elsewhere (Moran et al.,
2008). The DNA fragments were separated via capillary electrophoresis
(ABIPrism 310, Applied Biosystems), and the fragment sizes were
estimated using the ROX 1000 internal standard (Applied Biosystems,
Foster City, CA, USA). The T-RFLP data were plotted as the area (standardized as relative abundance using the sum of all peak areas) of
each restriction fragment uorescence peak against the fragment
sizes. The terminal restriction fragments (T-RFs) with lengths b 50
and N 700 bp and those representing b 0.5% of the total area were not
included in the analyses (Schutte et al., 2008).
The in silico DNA restriction analysis of the 16S rRNA gene sequences from arsenite-oxidizing microorganisms (Hamamura et al.,
2009; Inskeep et al., 2007) was enacted to create a new database of
predicted T-RFs that included aroA-like positive microorganisms.
The database was generated using the Virtual Digest tool, which is
a component of the Microbial Community Analysis (MiCA) online
resource (Shyu et al., 2007); the new predicted T-RFs were added to
the Ribosomal Database Project (RDP) (Cole et al., 2009). Subsequently,
the phylogenetic analysis was performed by importing the generated
database into the PAT program (Kent et al., 2003) to allow a greater
tolerance range for the sizes of the T-RFs; a sizing error of 1 bp was
allowed for the smallest T-RFs (50200 bp) and a 3 bp error was
allowed with the longest T-RFs (200700 bp). The database was
matched to the T-RF lengths generated from the T-RFLP proles of
the hydrothermal spring. Two datasets were generated for the possible
assignments of the T-RFs obtained from the digestion with each

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495

4. Results

greater reduction (from 3.4 to less than 1.0 mg l1) along this same
length of the river that cannot be explained solely by this phenomenon
(the stream ow increases only 2.1 times). Based on these data, we studied the attenuation of As in hydrothermal transects in this area of the
wetland.

4.1. Arsenic concentrations in the Azufre River

4.2. Arsenic geochemistry in the transects

The AsT and AsD concentrations along the Azufre River were
observed between 3.4 and 1.0 mg l1 (Fig. 2a), widely exceeding the
maximum level for drinking water (10 g l1) (WHO, 1993). The AsT
and AsD were very high at the origin of the Azufre River (distance
0 m) (3.4 0.3 mg l1 and 3.1 0.3 mg l1, respectively), possibly
due to hydrothermal discharges with high As concentrations. There
were no signicant differences between the AsT and AsD concentrations because the pH along the river was very low b2.5, preventing
the sequestration of As by particulate solids.
Downstream of the Azufre River, in the wetland area (4001500 m)
the AsT and AsD concentrations decreased. This decrease was explained
by a dilution effect caused by owing water with lower As concentrations
and by the active natural As attenuation processes in this area. The ow in
the Azufre River is ~14 l s1 upstream of the wetland and ~30 l s1
downstream of the wetland. The dilution effect is made evident by
changes in chloride concentrations (Fig. 2b), which decreased by approximately 60% (from 536 to 205 mg l1). The AsD concentrations exhibit a

The measured AsD (Fig. 3) exhibited a marked decrease in the

sampling points farthest from the source ( 72% for transect 1
and 76% for transect 2), demonstrating a clear attenuation of
the aqueous phase As concentration. The AsD concentration at the
source was 0.8 0.2 mg l1. In addition, we also observed an increase
in the concentration of As in the solid phase at the last 3 points of each
transect (Fig. 4), which correlates with the attenuation observed in
aqueous phase. Interestingly, the source presented the highest concentration of As in the solid phase (6.4 1.7 mg kg1 soil) and XRD
analysis revealed that the lyophilized sediments of the transects area
did not contain XRD-crystalline minerals and were therefore assigned
as unidentied amorphous Fe-oxyhydroxides.
The results of the dissolved As speciation analysis demonstrated
a marked oxidation of As in both transects (Fig. 7). Similarly, the Fe
speciation analysis illustrated that Fe(III) predominated in both
transects (Fig. 8). Additionally, the Fe concentrations in the solid
phase were constant along both transects: 285.1 46.9 g kg1 soil
for transect 1 and 292.6 63.2 g kg1 for transect 2 (considering
all sampling points). There were no signicant concentrations of
suspended solids (AsD/AsT 0.910.95) or turbidity (4.0 2.2 NTU)
measured in the bulk water.
The parameters analyzed along the transects revealed that the
specic electrical conductivity was similar at the different sampling
points of each transect (~ 4 mS cm1), and the dissolved oxygen
measurements revealed the water oxygenation from the source
until the discharge of the runoff in the Azufre River (2 mg l1 to
4.5 mg l1). There was a marked decrease in temperature ( 11 C
in transect 1 and 13.5 C in transect 2), and the pH decreased
downstream from the source (Fig. 5). The decrease in pH was more
evident in transect 2 than in transect 1. While in transect 1, the pH
declined slightly, and in transect 2 the decrease was more noticeable,
falling rapidly to reach values near 4.
The sulfate, sodium, and chloride concentrations remained nearly
constant downstream from the hydrothermal source (Fig. 6) in both
transects. The same was observed for nitrate, calcium, and potassium
+

ratios
(data not shown). The AsD/SO2
4 , AsD/Na , and AsD/Cl
exhibited a marked reduction in both transects, which was likely
inuenced by the AsD attenuation along the two transects.

restriction enzyme (HhaI and MspI), and these datasets were crossed
to rene the assignment of candidate species at the sampling point.

4.3. In-situ oxidation experiments

Fig. 2. Decreases in the AsT and AsD concentrations (a); and dissolved chloride
(b) along the Azufre River in the wetland area. The graphs are presented relative to the
distance from the origin point of the Azufre River (P1). Altitude (masl) is shown for reference. The wetland area extends between 400 and 1500 m after the origin of the Azufre
River. The data are presented as the mean SEM (error bars).

As(III) oxidation was not observed when the biological activity

was eliminated via formaldehyde addition to the samples from either
transect (Fig. 9). Therefore, the As(III)/As(V) ratio remained constant
when the biological activity was eliminated. The in-situ oxidation
experiments performed in the presence or absence of sunlight revealed
no signicant differences in As speciation (data not shown).
The water residence times in both transects were relatively short at
1.04 0.12 min for transect 1 and 1.0 0.04 min for transect 2, and
the extent of the As oxidation cannot be explained by slow abiotic oxidation reactions. However, in the solidliquid interface, the residence
times may be higher. Additionally, the As(III) oxidation can proceed abiotically via Mn oxides, which assist in the oxidation of As(III) and are
consequently reduced to Mn(II) (Manning et al., 2002; Scott and
Morgan, 1995). However, the Mn concentrations in the solid phase of
the transects were low; concentrations of 0.37 0.06 g kg1 soil for
transect 1 and 0.36 0.09 g kg1 for transect 2 were observed, considering all sampling points.

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E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

Fig. 3. As concentrations in the sampling points of transect 1 (a) and transect 2 (b). The AsD concentrations are expressed as the relative concentration (Ci/C0) down gradient of the
discharge from the hydrothermal source (Ci = concentration at distance i, C0 = concentration at source). C0 in aqueous phase was 0.8 0.2 mg l1. The data are presented as the
mean SEM (error bars).

4.4. Molecular analyses

The biological As oxidation is an enzymatic reaction mediated
by arsenite oxidase (AroA). We searched for aroA-like genes in the
metagenomic DNA extracted from the sampling points. In all sampling points, the putative aroA-like gene was amplied (Fig. 10a),
indicating that there was the potential to oxidize As biologically.
Additionally, the aroA-like gene expression analysis (from cDNA)
conrmed that the aroA transcript was produced in the rst three
sampling points of both transects (Fig. 10b, upper gel) and can be
induced in the last point (two transects) via exogenous addition of
1 ppm of As(III) [as NaAsO2] (Fig. 10b, lower gel).
The in silico analysis of the T-RFLP results supported the presence
of several operational taxonomic units with species assigned to
arsenite-oxidizing bacteria (Table 1), including Hydrogenophaga sp.,
Thiobacillus sp. and Thermus thermophilus. The other microorganisms
identied were all uncultured bacteria, which were also found in
environmental samples and sites with similar characteristics to our
study site (e.g., Uncultured eubacterium env. OPS 6; YNP). Two of
these bacteria presumably correspond to members of the Chloroexi
and Alphaproteobacteria classes.

was active in this area. In particular, the reduction in the As concentrations in the surface runoff of hydrothermal waters reveals the
presence of active As stabilization processes. Additionally, there
were no signicant changes in the ionic matrix of this runoff to suggest any local dilution processes were occurring.
The stability of the conductivity (approximately 4 mS cm1) along
both transects suggests that there is no mixture of waters to explain
the results of As attenuation; although the evidence is compelling, it is
not conclusive. However, the conservative behavior of the anions and
cations observed in both transects precludes the possibility of dilution.
+

ratios
The obvious reduction in the AsD/SO2
4 , AsD/Na , and AsD/Cl
supports the hypothesis that the As attenuation has a different geo+

chemical behavior than the tracer ions, such as SO2

4 , Na , and Cl .
Therefore, the changes in the AsD concentrations along the transects
are not caused by dilution; if dilution occurred, then the As/ion ratios
would remain stable throughout both transects. While a dilution effect
with waters from the same ionic matrix but lower As concentrations
would give this same outcome, there are no known hydrothermal
water sources with lower As concentrations able to explain the observed phenomenon, and the stream ow along the two transects
exhibited no substantial change.

5. Discussion
5.2. Arsenic stabilization occurs onto Fe minerals
5.1. Arsenic natural attenuation occurs in the transects
The difference between the reduction of chloride concentrations
(~ 60%) and the reduction in the As concentrations (~ 71%) along the
Azufre River indicates than an active natural As attenuation process

The decreasing AsD and increasing amount of solid phase As

across the last three points of the two transects might be explained
by an increase in the adsorption of As onto the Fe-oxyhydroxides
present in this area. As adsorption onto Fe-oxyhydroxides and the

Fig. 4. Solid phase As concentrations in the sampling points of transect 1 (a) and transect 2 (b). The As concentrations in the solid phase are expressed as the relative concentrations
(Ci/C0) down gradient of the discharge from the hydrothermal source (Ci = concentration at distance i, C0= concentration at source). C0 in solid phase was 6.4 1.7 mg kg1 soil.
The data are presented as the mean SEM (error bars).

E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

497

Fig. 5. Geochemical parameters in the water samples from the sampling points of transect 1 (a) and transect 2 (b). DO () and temperature () reveal the expected behavior for
hydrothermal waters in contact with the atmosphere. The decrease in pH () is important for the adsorption processes of As, and stability of the conductivity () suggests that
there is no dilution. The data are presented as the mean SEM (error bars).

formation of ferric arsenate precipitates are the main natural attenuation processes for the removal of As (Drahota et al., 2012; Haffert
and Craw, 2008; Leblanc et al., 1996), supporting the hypothesis of
the relevance of these minerals in the attenuation of As in the studied
transects (Fig. 3). Incidentally, amorphous Fe-oxyhydroxides have a
high adsorption capacity for As because they have a large surface
area and therefore provide more active sorption sites (Wang and
Mulligan, 2006). Consequently, the presence of unidentied amorphous Fe-oxyhydroxides lends credence to the idea that these minerals are crucial for the attenuation of As observed in both transects.
The obvious As(III) oxidation to As(V) in both transects could
enhance the adsorption of As onto Fe-oxyhydroxides under varying pH
conditions. As(V) interacts with Fe-oxyhydroxides more strongly than
As(III) (Oremland and Stolz, 2003) because the adsorption mechanism
is strongly inuenced by pH changes. Therefore, the As(III) oxidation
to As(V) and the subsequent As(V) adsorption onto Fe-oxyhydroxides
might explain the As attenuation observed downstream from the
hydrothermal streams.
The presence of similar Fe concentrations in the solid phase of
both transects downstream of the hydrothermal source suggests
that changes in the concentrations of As in the aqueous phase were
due to an active process at the solidliquid interface and not by increasing As sorbent concentrations (Fe-oxyhydroxides). pH decrease
along the transects supports this because at low pH it does not
favor the formation of these minerals. However, the circumneutral
pH in the hydrothermal source and the presence of Fe(III) in the
aqueous phase may favor the formation of Fe-oxyhydroxides in this
location and therefore increase the availability of sediments with
free surface sites for As(III) adsorption. This phenomenon would explain the higher As concentrations in solid phase at this location
(source) (Fig. 4). Additionally, the co-precipitation of As and Fe can
also occur during the formation of Fe-oxyhydroxides (e.g., Ferrihydrite),
which may help to mitigate the As concentration in aqueous phase
(Fuller et al., 1993). Additionally, the co-precipitation of As(V) and
Fe(III) may be favored by the As(III) oxidation (Jia and Demopoulos,
2008; Richmond et al., 2004; Twidwell et al., 2005). However, the low

concentrations of suspended solids (turbidity b 6 NTU and AsD/AsT

0.910.95) invalidate the supposition that the As attenuation in aqueous phase occurred because of the As adsorbed on the suspended particulate phases. These results support the idea that the mineral phases
present in the runoff beds of both transects were responsible for the
observed attenuation of As.
5.3. pH control on arsenic immobilization
The correlation between the pH reduction and AsD attenuation in
the transects supports the hypothesis that the oxidation of As(III) to
As(V) favors As stabilization on the solid phase by increasing the
As(V) adsorption onto solid phase. As(III) and As(V) are strongly
chemisorbed on Fe-oxyhydroxides, but this process is pH dependent.
While As(V) is adsorbed onto Fe-oxyhydroxides between pH 4 and 7,
with an optimal adsorption occurring around pH 4, As(III) is more
strongly adsorbed between pH 6 and 9; and this pattern is most
noticeable with the amorphous Fe-oxyhydroxides (De Vitre et al.,
1991; Dixit and Hering, 2003; Manning et al., 1998; Pierce and
Moore, 1982).
Therefore, the decreasing pH along the transects (Fig. 5) reveals
possible changes in the differential adsorption of As(III) and As(V)
because, at circumneutral pH, the adsorption of As(III) is maximized
and favored over As(V) adsorption (Dixit and Hering, 2003; Goldberg
and Johnston, 2001). In the sampling points downstream from the
hydrothermal source, As(V) predominates in aqueous phase and the
pH becomes more acidic (~pH 4). As the As(III) is oxidized to As(V)
along the transects, and the decrease in the pH increases the As(V)
sorption; therefore, decreasing pHs partially promotes the As attenuation observed in the transects. However, the high As concentration in
the solid phase found at the source can be explained by the pH present
at this point, which is approximately 6 (Fig. 5); at this pH, the adsorption of As(III) is promoted, which has a higher concentration at this
point (Fig. 7). Additionally, this sampling point is the hydrothermal
source with naturally emerging high AsD concentrations. However,
the specic behavior of adsorption onto the Fe-oxyhydroxides is

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E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

Fig. 6. Sulfate, sodium, and chloride concentrations in the sampling points of study transect 1 (a) and transect 2 (b). AsD/SO2
4 , AsD/Na , and AsD/Cl ratios in study transect 1 (c) and
transect 2 (b). Both transects reveal the stability of ion concentrations between the different sampling points. The AsD/ion ratios indicate a marked reduction in both transects. The ratios
were calculated from the As and ion concentrations (mg l1) and were plotted by multiplying the values by 104. The data are presented as the mean SEM (error bars).

dependent on the acidbase characteristics of the As(III) and As(V)

solutions, the afnity of surface functional groups of the solid, and
the cooperative or competitive effects of the anions and cations in the
aqueous phase (Davis and Kent, 1990; Kent and Fox, 2004; Grafe
et al., 2004).

5.4. Arsenite oxidation and natural attenuation is dependent on

biological activity
The results of the in-situ experiments demonstrate that microbial
activity is necessary for the oxidation of As(III). Additionally, these

Fig. 7. Arsenite oxidation in the water samples from transect 1 (a) and transect 2 (b). Concentrations of As(III) and As(V) are presented relative to the concentration of the total AsD.
There is an obvious oxidation of As in both transects from the hydrothermal source (0 m).

E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

499

Fig. 8. Fe speciation (Fe(II) and Fe(III)) in the water samples from transect 1 (a) and transect 2 (b). Concentrations of Fe(II) and Fe(III) are presented relative to the concentration of
the total dissolved Fe. In both transects, Fe(III) predominates over Fe(II) and is stable along the transects.

results conrmed that abiotic As(III) oxidation caused by DO or

another electron acceptor was not signicant compared to the oxidation activity in the presence of microbial activity at similar time scales.
The kinetics of the oxygen-mediated As oxidation has a reported
half-life of one year (Eary and Schramke, 1990). Additionally, abiotic
As(III) oxidation may occur in association with Fe-oxyhydroxides,
where As(III) is adsorbed, oxidized, and nally desorbed through a
pH-dependent process (Belzile and Tessier, 1990; Horneman et al.,
2004; Smedley and Kinniburgh, 2002). However, the most important
abiotic pathway of As oxidation in natural systems is the transfer of
electrons to Mn oxides (Manning et al., 2002; Oscarson et al., 1981).
As(III) is adsorbed on the Mn oxide surfaces and undergoes several
redox reactions on the mineral surface before being released to the
aqueous phase (Parikh et al., 2010; Scott and Morgan, 1995). The As
oxidation rate promoted by the Mn oxides depends on the available
surface area, surface charge, and pH (the oxidation rate is slightly higher

at low pH) (Chiu and Hering, 2000; Oscarson et al., 1983). Although Mn
oxides may form on the surface of Fe-oxyhydroxides and As(III) can be
oxidized more rapidly at high temperatures (N48 C) through a photocatalytic process (Schwenzer et al., 2001), the low concentrations of Mn
in solid phase (b 0.5 g kg1) and the results of in-situ experiments
discount this mechanism. Additionally, the formation of Mn oxides is
favored at a higher pH than those observed in this system (pH 46)
(Morgan, 1967).
The photochemical oxidation of As(III) was also studied by Hug
et al. (2001), who observed a 90% removal of As(III) within 2 to 3 h
in solutions containing Fe(II, III) and citrate. At 25 C under natural
sunlight, the half-life of As(III) is 0.7 h at pH 5 in a solution of
18 M Fe(III) and 100 mg l1 DOC (Kocar and Inskeep, 2003).
Accordingly the photocatalytic As oxidation has faster kinetics
(hours) than a strictly oxygen-dependent abiotic As oxidation. However, the lack of signicant differences between the in situ oxidation

Fig. 9. In-situ As(III) oxidation assays with sediments from transect 1 (a) and transect 2 (b). As oxidation is evident when the biological activity is not inhibited by formaldehyde.

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E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

Fig. 10. Agarose gel displaying the amplication of an aroA-like gene (a) and aroA-like expression (b) in the microbial samples from the study transects. All samples were also
supplemented with 1 ppm As(III) to conrm the aroA-like gene's functional role (lower panel of (b)). The 16S rRNA gene was used as a control for DNA quality, and Agrobacterium
tumefaciens 5A was a positive control for arsenite oxidase activity.

experiments in the presence and absence of light demonstrates

that the solar radiation does not cause the observed As oxidation in
this site at the measured time scale (Fig. 7). Consequently, in other
natural systems with pHs ranging from 3 to 6, the abiotic oxidation
of As(III) is negligible relative to the biologically mediated oxidation
(Eary and Schramke, 1990; Langner et al., 2001; Taylor, 2007). Therefore, in natural environments, the available evidence indicates that
the rates of abiotic As oxidation by photochemical, photocatalytic, or
Fe or Mn oxides-mediated reactions are signicantly lower than the
oxidation mediated by microorganisms (Cherry et al., 1979; Eary and
Schramke, 1990; Ying et al., 2011). Therefore, the absence of As oxidation observed when microbial processes were neutralized (Fig. 9)
supports the idea of microbial activity performing the majority of the
As(III) oxidation.
Similarly, the microbiological analyses suggest that the functional
biological activity of the As oxidation promotes the geochemical
changes observed in the As(III), As(V), and AsD concentrations.
Among the microorganisms found by T-RFLP analyses, Hydrogenophaga

sp. and Thiobacillus sp. are chemoautotrophic arsenite oxidizers

(CAOs); both of these organisms belong to the class Betaproteobacteria,
are common in soils, and are frequently associated with metalcontaminated sites (He et al., 2007; Heinzel et al., 2009). We also
found T. thermophilus, which is a heterotrophic arsenite oxidizer
(HAO), revealing the variety of bacterial metabolisms that might impact
the As cycle in this area. Recent studies have revealed that these organisms are present in hydrothermal systems with high As concentrations
(Hamamura et al., 2009; Inskeep et al., 2007) and might contribute
to the oxidation of As(III) in natural systems (Hamamura et al., 2009).
For the other uncultured bacteria found in the study site, there is no
information available regarding their As oxidation activity. However, the phylogenetic analyses conducted by other authors have identied microorganisms belonging to both classes (Chloroexi and
Alphaproteobacteria) commonly found in arsenic-impacted soils and
geothermal environments (Hamamura et al., 2009; Inskeep et al.,
2007). The presence of As-oxidizing bacteria in this area is consistent
with the aroA-like presence and expression, suggesting that these

Table 1
Closest matching species from the 16S rRNA gene T-RFs assignment in the 16S rRNA databases. The microbial characterization corresponds to the hydrothermal source (0 m).
Closest match species

AroA-like microorganisms
Hydrogenophaga sp. str. CL3
Thiobacillus sp. str. S1
Thermus thermophilus
Other microorganisms
Uncultured eubacterium env. OPS 6
Uncultured eubacterium clone BBACe10
Uncultured Chloroexi bacterium

Uncultured bacterium
Uncultured bacterium

a
b

CAO: chemoautotrophic arsenite oxidizer.

HAO: heterotrophic arsenite oxidizer.

16S rRNA
acc. no.
(GenBank)

AroA-like
acc. no.
(GenBank)

Phylogenetic afliation

Arsenic metabolism

AroA-like gene

DQ986320
DQ986319
X07998, NC_006461

EF015462
EF015459
BAD71923

-Proteobacteria
-Proteobacteria
DeinococcusThermus

CAOa
CAOa
HAOb

aroAB
aroAB
aroAB

AF018191
GU357466
HQ397171
HQ397189
HQ397194
DA067
GQ921453
GQ921460
GQ921465

Chloroexi

-Proteobacteria

E.D. Leiva et al. / Science of the Total Environment 466467 (2014) 490502

populations are responsible for the As oxidation observed in the

studied transects. Therefore, the results of the in situ As oxidation experiments, the aroA-like gene presence/expression, and the T-RFLP analysis
strongly support the hypothesis that arsenite-oxidizing microorganisms
contribute to the As oxidation and consequently to the natural As attenuation observed in the study site.

6. Conclusions
Our results indicate that there was a natural attenuation of As in the
model system. The AsD concentrations decreased along the transects,
while the conservative ion concentrations along the transects discount
the possible effects of dilution or mixing of waters. The rapid oxidation
of As(III), which occurred within a few meters of the hydrothermal
source (As(V)/AsD increases of +95% [transect 1] and + 100% [transect 2]), suggests the possible involvement of As oxidation in the natural
attenuation process. The increase in the As concentrations in the solid
phase after the source correlates with the As oxidation in transects 1
and 2, suggesting that the stabilization is mediated by the adsorption
of As onto Fe-oxyhydroxides and controlled by the environmental pH.
The results of the in-situ oxidation experiments reveal that live microorganisms were required to catalyze the As(III) oxidation at the studied
time scale. Complementary microbiological molecular analyses support
these ndings and suggest that both the oxidation of As(III) and the attenuation of AsD is mediated directly by microorganisms via enzymatic
activity (AroA-like). The results of the aroA-like gene expression suggests that changes in the expression of this gene can depend upon
As(III) concentration. The T-RFLP analysis supported the presence of
arsenite-oxidizing bacteria in the studied area and reveals the autotrophic and heterotrophic metabolisms that are most likely to be active in
the As oxidation.
Our ndings contribute to a better understanding of biogeochemical processes of As oxidation, as well as the biogeochemical controls
of As in uvial systems impacted by As-rich hydrothermal discharges;
these As-rich hydrothermal discharges occur in upper section of the
Azufre River sub-basin. The description of the microbial As oxidation
reactions involved in these processes is crucial for improving our understanding of the As cycle in extreme, arid environments. Together,
these results illustrate the complexity of the interactions involved in
the mobilization and stabilization of As due to the interplay between
redox and sorption reactions at the mineral-liquid interface involving
biotic and abiotic reactions at different scales.
Future research should include detailed spectroscopic analysis of the
solid-phases to determine the speciation of As and Fe on the surface of
the Fe minerals and to conrm the specic mechanisms involved in the
stabilization of As at the molecular level. Additional efforts are necessary to describe the environmental conditions that might inhibit or enhance this attenuation process. This knowledge is crucial for the design
of effective risk management and remediation efforts for uvial systems
enriched with As from hydrothermal discharges.

Acknowledgments
This research was supported by a FONDECYT 1100943/2010, a
FONDECYT 1130936/2013, and a CONICYT grant 24121233/2012, as
well as Fulbright Scholar Award 9561 for J.M.R. This study was also partially supported by a CORFO 09CN14-5709 grant and a CONICYT/FONDAP
15110020 grant. Thanks are extended to the reviewers for the corrections and suggestions, which signicantly improved the manuscript.

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