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Arsenic oxidation by flavin-derived reactive species under oxic and anoxic conditions: Oxidant formation and pH dependence Kunfu Pi, Ekaterina Markelova, Peng Zhang, and Philippe Van Cappellen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03188 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019
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Arsenic oxidation by flavin-derived reactive species under
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oxic and anoxic conditions: Oxidant formation and pH
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dependence
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Kunfu Pi a, Ekaterina Markelova b, Peng Zhang c, Philippe Van Cappellen a,*
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a Ecohydrology
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Water Institute, University of Waterloo, Waterloo, N2L 3G1, Canada
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b Amphos21
Research Group, Department of Earth and Environmental Sciences &
Consulting S.L., C/Venecuela, 103, 08019 Barcelona, Spain
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c
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of Geosciences, 430074 Wuhan, China
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* Correspondence:
State Key Laboratory of Biogeology and Environmental Geology, China University
[email protected] 13 14
Abstract
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Flavins are ubiquitous redox-active compounds capable of producing reactive oxygen
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(O2•-, •OH, H2O2) and flavin radical species in natural environments, yet their roles in
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the redox transformations of environmental contaminants, such as arsenic (As),
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remain to be investigated. Here, we show that reduced flavins can be a source of
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effective oxidants for As(III) under both oxic and anoxic conditions. For instance, in
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the presence of 15 μM reduced riboflavin (RBFH2) 22% of 30 μM As(III) is oxidized
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in aerated solution at pH 7.0. The co-oxidation of As(III) with RBFH2 is
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pH-dependent, with a faster reaction rate under mildly acidic relative to alkaline 1
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conditions. Quencher tests with 2-propanol (for •OH) and catalase (for H2O2) indicate
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that As(III) oxidation under oxic conditions is likely controlled by flavin-derived •OH
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at pH 5.2 and 7.0, and by H2O2 at pH 9.0. Kinetic modeling further implies that
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flavin-derived reactive oxygen species are mainly responsible for As(III) oxidation
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under oxic conditions, whereas oxidation of As(III) under anoxic conditions at pH 9.0
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is attributed to riboflavin radicals (RBFH•) generated from coexisting oxidized and
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reduced riboflavin. The demonstrated ability of flavins to catalyze As(III) oxidation
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has potential implications for As redox cycling in the environment.
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1. Introduction
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Arsenic (As) is an environmental pollutant responsible for widespread groundwater
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contamination, posing severe threats to drinking water security and public health of
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over one hundred million people globally 1,2. Groundwater contamination by geogenic
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As mainly occurs under reducing conditions in coastal floodplains and
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fluvial-lacustrine basins
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mobility and bioavailability of As in near-surface environments
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redox-sensitive behavior is driven by the redox-dependent speciation of As where
1,3.
In contrast, oxidizing conditions generally decrease the
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This
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oxidized As(V) tends to have a greater affinity for various sorbents, especially Fe/Mn
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oxides
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As(III) to As(V) followed by adsorption and/or co-precipitation reactions 7.
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Under acidic to weakly-alkaline pH conditions, molecular oxygen (O2) is not an
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effective oxidant for As(III), and hence other oxygen species, such as ozone (O3) and
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hydrogen peroxide (H2O2), have been used in the remediation of As-polluted waters
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8-10.
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As(III)
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toxicity. Alternatively, Fe(III)-assisted remediation techniques are commonly used to
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promote As(III) oxidation followed by As(V) immobilization by adsorption and/or
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co-precipitation
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reactive oxygen species generated by the photolysis of Fe(III)-complexes and
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subsequent dark reactions
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decomposition of H2O2 on ferrihydrite surfaces
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Fe(II)-containing sediments 18.
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The mechanisms and kinetics of As(III) oxidation in Fe-depleted environments have
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been less studied. Nonetheless, they are becoming an increasingly important area of
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both scientific and technological investigations with respect to As transformation and
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remediation
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microorganisms and plants, as well as specific metabolites (e.g., enzymes), to help
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mitigate contamination and hazards
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immobilization of contaminants (e.g., via enzymatic detoxification or chemisorption
5-7.
Therefore, As-polluted water treatment often relies on the oxidation of
Additional industrial oxidants (e.g., active chlorines) are also able to oxidize 11,12,
but are of limited applicability in natural environments due to their
13-16.
19-21.
The presence of Fe can also enhance As(III) oxidation via
14,
the Fenton reaction system 17,
15,
the catalyzed
and the oxygenation of
In this context, bioremediation techniques rely on naturally present
22-26.
In addition to direct microbial
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onto cell walls), microorganisms may indirectly impact their environments by
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releasing reactive organic substances
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included in the extracellular signatures of Shewanella bacteria isolated from lake
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sediments 30, nitrogen-fixing bacteria
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by algae (e.g., the diatom Thalassiosira weissflogii)
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glaucus)
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nutrient depletion).
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Being highly reactive even at sub-micromolar concentrations
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electron shuttles and influence the fate of redox-sensitive contaminants in natural and
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engineered environments
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oxidized metals under anoxic conditions has been demonstrated for Fe(III), Tc(VII),
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Np(V), U(VI), Cr(VI) and Pu(IV) 26,39,41-44. Recent studies, however, have highlighted
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that microorganisms, for example, Shewanella oneidensis MR-1 and Shewanella sp.
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MR-4, produce and release flavins not only under anoxic, but also under oxic
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conditions 36,38.
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Flavins have been shown to control redox potential (Eh) measurements in artificial
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groundwater over a wide potential range, from +350 to -250 mV
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electroactivity of flavins has been attributed to their autocatalytic capacity for redox
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transformations mediated by O2 or light
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by O2, intermediate reactive species of both oxygen (e.g., H2O2 and superoxide
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radicals) and flavins (e.g., flavin radicals) are generated
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absence of O2, effective oxidants can be produced when oxidized and reduced
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27-29.
and cyanobacteria 32. They are also produced
and plants (e.g., Beta vulgaris)
26,39-41.
For example, flavin compounds are
35
33,
fungi (e.g., Aspergillus
in response to stress conditions (e.g.,
36-38,
flavins can act as
The ability of flavins to enhance the reduction of
45-47.
36.
The high
During the oxidation of reduced flavins
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Moreover, even in the
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riboflavin species co-exist in solution 46.
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The ability of flavins to yield reactive oxygen species has been demonstrated
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experimentally
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range (3-12) not only in simple solutions but also in biological systems 46. However,
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research to date has yet to investigate the fate of reduced contaminants co-existing
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with flavins under oxic conditions. Because flavins are a major class of redox-active
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microbial exudates in a variety of natural environments
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these compounds may be a natural source of efficient oxidants. Flavins could thus
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represent key actors in the oxidative transformation of inorganic and organic
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pollutants. Their potential applications in contaminant treatment technologies,
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especially under dynamic redox conditions, therefore, deserve further consideration
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46,49.
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In this study, we use riboflavin as a representative flavin compound and address its
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role in the oxidation of As(III) to As(V) in the presence and absence of O2, and over a
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range of pH between 5.2 and 9.0. Specifically, we designed a series of batch
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experiments to demonstrate that (1) the oxidation of As(III) is accelerated by the
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production of riboflavin-derived reactive oxygen species under oxic conditions, (2)
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the co-existence of oxidized and reduced riboflavin compounds may lead to As(III)
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oxidation in the absence of O2, and (3) the formation of reactive oxidant species and
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the reaction kinetics of As co-oxidation with riboflavin are pH-dependent.
45,47,49
and shown to be thermodynamically favorable over a wide pH
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we hypothesize that
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2. Materials and Methods
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2.1. Experimental series
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A stock solution of reduced riboflavin (RBFH2) was prepared from oxidized
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riboflavin (RBF, Sigma-Aldrich) following the procedure of Shi et al.
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Supporting Information (SI) for details). A typical experimental solution was prepared
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by mixing 100 mL of the designated reactants (As(III), RBFH2, RBF, quenchers) and
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100 mL of the matrix solution (10 mM NaCl plus pH buffer) in a 500-mL reactor (see
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SI for details). Six series of experiments were carried out in the dark and at three pH
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values, mildly acidic (5.2), neutral (7.0) and moderately alkaline (9.0) (Table 1).
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The first series of experiments was conducted under anoxic conditions inside a glove
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box (97-98% N2 and 2-3% H2) with initial equimolar concentrations of 7.5 µM
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RBFH2 and 7.5 µM RBF (Exp. I). Note that the corresponding total riboflavin
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concentration of 15 µM matched that used in the subsequent oxic experiments. The
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anoxic experiments were designed to derive kinetic parameters for reactions between
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As(III) and riboflavin species without the interference of reactions with O2.
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The other series of experiments were conducted under oxic conditions (Exp. II-VI,
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Table 1) with a stable level of dissolved O2 (200±15 μM). In the oxic experiments,
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As(III) oxidation was assessed in the absence of riboflavin (Exp. II) and in the
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presence of RBFH2 (Exp. III). Oxic experiments with RBFH2 were also conducted in
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the presence of 40 mM 2-propanol (•OH quencher, Exp. IV) or catalase (H2O2
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quencher, Exp. V) to evaluate the role of reactive oxygen species in As(III) oxidation. 6
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(see
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In the final oxic experimental series, RBFH2 was substituted by RBF (Exp. VI). All
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experiments were conducted in duplicate.
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Table 1. Experimental design. Experimental series Exp. I. Oxidation of As(III) by riboflavin radical (RBFH•) Exp. II. Oxidation of As(III) by O2 (control group)
pH
Initial As(III) (μM)
Initial RBFH2 (μM)
Initial RBF (μM)
Quencher (mM)
Anoxic
5.2, 7.0, 9.0
15, 20, 30
7.5
7.5
-
Oxic
5.2, 7.0, 9.0
30
-
-
-
Oxic
5.2, 7.0, 9.0
30
10, 15
-
-
Oxic
5.2, 7.0, 9.0
30
15
-
40
Oxic
5.2, 7.0, 9.0
30
15
-
40
Oxic
5.2, 7.0, 9.0
30
-
15
-
Aeration state
Exp. III. Co-oxidation of As(III) with reduced riboflavin (RBFH2) by O2 Exp. IV. Role of flavin-derived •OH
in As(III) oxidation with
added 2-propanol Exp. V. Role of flavin-derived H2O2 in As(III) oxidation with added catalase Exp. VI. Oxidation of As(III) by oxidized riboflavin (RBF) 131 132
2.2. Analytical methods
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Total riboflavin concentrations were determined by measuring the fluorescence signal
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of RBF at the excitation/emission wavelength of 450/520 nm after complete oxidation
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in the dark 36, using a Flexstation-3 Multimode Reader (Molecular Devices) equipped
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with black/clear bottom 96-well microplates. The fluorescence signals were calibrated
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using commercial RBF standard solutions (Sigma-Aldrich), with a detection limit of
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16 nM. The standard deviation of the total riboflavin concentrations was ≤5%. 7
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After sampling, riboflavin speciation was determined immediately by UV-vis
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spectrophotometry, with absorbance measured on a Flexstation-3 Multimode Reader
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(Molecular Devices). The concentration of RBF at a given time t, [RBF]t, was
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calculated as 50:
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[RBF]𝑡 =
𝐴𝑡 ― [RBFH2]0𝜀RBFH2 𝜀RBF ― 𝜀RBFH2
(1)
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where At is the total absorbance, [RBFH2]0 the initial reduced riboflavin
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concentration, εRBFH2 (1200 mol-1 cm-1) and εRBF (12200 mol-1 cm-1) the extinction
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coefficients of reduced and oxidized riboflavin at wavelength 450 nm, respectively.
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The standard deviation of the measured RBF concentrations was 6-9%.
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Concentrations of H2O2 were measured following the protocol of the commercial
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Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich). The fluorescence was
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measured at the excitation/emission wavelength of 540/590 nm, with a detection limit
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of 0.01 μM. The standard deviation of the H2O2 concentrations was ≤10%.
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Samples for As speciation analysis were pre-treated to adjust pH to 6 followed by
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separation on an anion-exchange resin cartridge (LC-SAX, Sigma-Aldrich) (See SI
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for details)
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hydrogen-generation atomic fluorescence spectrometry (HG-AFS, PS Analytical),
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with a detection limit of 0.5 μg L-1. To measure As concentrations below this limit,
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samples were analyzed by anodic stripping voltammetry using a 797 VA Computrace
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equipped with the TRACE Gold sensor (Metrohm, Application Bulletin 416/3).
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Standard deviations of As(III) and As(V) concentrations mostly fell below 8%, with
51.
The concentration of As in the cartridge outflow was measured by
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few cases over 10%. The sum of As(III) and As(V) concentrations was calculated to
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verify As mass balance against the concentration of initially added As(III).
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2.3. Kinetic modeling
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Two kinetic models were developed to simulate As(III) oxidation under various
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experimental conditions using the Kintecus software
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rate constants of As reaction with riboflavin radical (RBFH•) under anoxic conditions
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(Exp. I, Table 2); Model 2 was developed to represent the kinetics of As(III) oxidation
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by flavin-derived reactive species in the presence of O2 (Exp. III-V, Table 2). To track
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the contributions of specific pathways in the overall reaction network, the counting
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variables X1, X2, Z1, Z2, Z3, Z4 were added to the reaction stoichiometries as shown in
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Table 2. Within each experimental series, the data obtained at the three pH values
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were fitted separately. Because only minor pH fluctuations (±0.03) were observed
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during the experiments, the differences in the rate constants at different pH values
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illustrate the pH dependence of the corresponding reaction pathways (Table 2).
52.
Model 1 was used to derive
174 175
3. Results and Discussion
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3.1. As(III) oxidation: Riboflavin radical
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At acidic (5.2) and neutral pH (7.0), the As(III) concentration was stable in the
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solutions of mixed reduced and oxidized riboflavin under anoxic conditions (Exp. I).
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The lack of detectable As(V) production (
