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Title
Bacteria attenuation by iron electrocoagulation governed by interactions between
bacterial phosphate groups and Fe(III) precipitates
Permalink
https://escholarship.org/uc/item/27f797jr
Journal
Water Research, 103
ISSN
0043-1354
Authors
Delaire, Caroline
van Genuchten, Case M
Amrose, Susan E
et al.
Publication Date
2016-10-01
DOI
10.1016/j.watres.2016.07.020
Peer reviewed
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Title: Bacteria attenuation by iron electrocoagulation governed by
interactions between bacterial phosphate groups and Fe(III) precipitates
Article Type: Research Paper
Keywords: Iron electrocoagulation; bacteria attenuation; bacterial
surface functional groups; specific interactions; bivalent cations;
oxyanions
Corresponding Author: Mrs. Caroline Delaire,
Corresponding Author's Institution: University of California, Berkeley
First Author: Caroline Delaire
Order of Authors: Caroline Delaire; Case M van Genuchten, PhD; Susan E
Amrose, PhD; Ashok J Gadgil, PhD
Abstract: Iron electrocoagulation (Fe-EC) is a low-cost process in which
Fe(II) generated from an Fe(0) anode reacts with dissolved O2 to form (1)
Fe(III) precipitates with an affinity for bacterial cell walls and (2)
bactericidal reactive oxidants. Previous work suggests that Fe-EC is a
promising treatment option for groundwater containing arsenic and
bacterial contamination. However, the mechanisms of bacteria attenuation
and the impact of major groundwater ions are not well understood. In this
work, using the model indicator Escherichia coli (E. coli), we show that
physical removal via enmeshment in EC precipitate flocs is the primary
process of bacteria attenuation in the presence of HCO3-, which
significantly inhibits inactivation, possibly due to a reduction in the
lifetime of reactive oxidants. We demonstrate that the adhesion of EC
precipitates to cell walls, which results in bacteria encapsulation in
flocs, is driven primarily by interactions between EC precipitates and
phosphate functional groups on bacteria surfaces. In single solute
electrolytes, both P (0.4 mM) and Ca/Mg (1-13 mM) interfered with the
adhesion of EC precipitates to bacterial cell walls, whereas Si (0.4 mM)
and ionic strength (2-200 mM) did not impact E. coli attenuation.
Interestingly, P (0.4 mM) did not affect E. coli attenuation in
electrolytes containing Ca/Mg, consistent with bivalent cation bridging
between bacterial phosphate groups and inorganic P sorbed to EC
precipitates. Finally, we found that EC precipitate adhesion is largely
independent of cell wall composition, consistent with comparable
densities of phosphate functional groups on Gram-positive and Gramnegative cells. Our results are critical to predict the performance of
Fe-EC to eliminate bacterial contaminants from waters with diverse
chemical compositions.
Suggested Reviewers: Shankar Chellam
Texas A&M University
chellam@tamu.edu
Dr. Chellam is an expert on water treatment technologies, and he has
worked on iron and aluminum electrocoagulation for virus control.
Chuanyong Jing
Chinese Academy of Science
cyjing@rcees.ac.cn
Dr. Jing works on biogeochemistry and on environmental interfacial
processes. He has recently co-authored a ATR-FTIR study about bacteriagoethtite adhesion
Sharon Walker
University of California, Riverside
swalker@engr.ucr.edu
Dr. Walker is an expert on bacteria-particle adhesion in sub-surface
environments, and on the role of cell surface polymers in bacteria
adhesion and transport
Andreas Voegelin
EAWAG
andreas.voegelin@eawag.ch
Dr. Voegelin is an expert on molecular environmental geochemistry and on
the reactivity of iron(III) precipitates and interactions with cooccurring ions.
Jon Chorover
University of Arizona
Chorover@email.arizona.edu
Dr. Chorover is an expert on the investigation of the interactions
between iron oxides and bacterial cell walls using spectroscopic methods
Sanjay Mohanty
University of Pennsylvania
sanjay.mohanty@colorado.edu
Dr. Mohanty has worked on bacteria-mineral interactions in the context of
bacteria transport in biofilters
Changa Lee
Ulsan National Institute of Science and Technology
clee@unist.ac.kr
Dr. Lee has worked on pathogen inactivation and on the production of
reactive oxidants from zero-valent and ferrous iron
Cover Letter, For Editor only
Mark van Loosdrecht
Department of Biochemical Engineering
Delft University of Technology
KWR Watercycle Research
Delft
Netherlands
April 21st, 2016
Dear Water Research Editor,
On behalf of all coauthors, I am pleased to submit our manuscript “Bacteria Attenuation by Iron
Electrocoagulation Governed by Interactions between Bacterial Phosphate Groups and Fe(III)
Precipitates” enclosed for consideration as a research paper for Water Research.
In this study, we investigate a specific application –bacteria attenuation- of iron
electrocoagulation (Fe-EC), a promising technology for the treatment of arsenic-contaminated
groundwater in low-resource settings. Simultaneous arsenic and bacteria attenuation in Fe-EC
has been demonstrated in our previous study, and constitutes a significant advantage of this
technology in areas where arsenic often concurs with fecal contamination. However, the
processes leading to bacteria attenuation and the impact of groundwater composition are not well
understood. This manuscript presents new results elucidating the molecular-scale mechanisms of
bacteria attenuation in Fe-EC, and the role that major groundwater ions, such as HCO3-, P, Si, Ca
and Mg, play in such mechanisms.
Our work goes beyond presenting remediation results because we thoroughly investigate the
processes leading to bacteria inactivation and removal in Fe-EC. In addition, our findings have
significant implications for field treatment as they allow to predict the performance of Fe-EC
to attenuate various types of bacterial contamination in different groundwater matrices. We
showed that attenuation is independent of cell wall composition, which is critical to generalize
our findings to all bacterial species relevant to water quality. Finally, the molecular mechanisms
identified in this study can be used to discuss the potential of Fe-EC, and other Fe-based
coagulation processes, to treat various water sources, such as surface water, agricultural runoff
and wastewater.
The results presented here are novel, as no other study to the authors’ knowledge has
investigated the bacterial surface functional groups and the type of interaction involved in the
adhesion of Fe(III) (oxyhydr)oxides to cell walls in water matrices representative of field
conditions. Specifically, molecular-scale interactions between bacteria and Fe(III) oxides in
systems containing bivalent cations and oxyanions, which alter the surface of bacterial cells and
Fe(III) oxides, are not known.
Finally, we believe that our approach to elucidate bacteria-precipitates interactions is innovative.
In systems where bacteria are encapsulated inside flocs and in the complex groundwater-like
electrolytes, spectroscopic techniques such as ATR-FTIR cannot adequately determine bacterial
functional groups mediating bacteria-precipitate adhesion. Instead, we used a novel approach,
where macroscopic data of bacteria attenuation in systematically varied electrolytes was
combined with ζ-potential measurements to elucidate molecular-scale processes. Building on
previous spectroscopic studies in more simple controlled systems, our approach allowed us to
gain knowledge on bacteria-Fe(III) precipitate interactions in complex water matrices.
We believe that this work will be relevant to a general audience interested in mechanistic aspects
as well as in field applicability of drinking water treatment technologies.
Sincerely yours,
Caroline Delaire (corresponding author)
Department of Civil and Environmental Engineering
University of California, Berkeley
Berkeley, California, USA 94720
Phone: +1 (510) 417-9491
caroline.delaire@orange.fr
Case M. van Genuchten
Institut de Dynamiques de la Surface Terrestre
University of Lausanne
Lausanne, 1015-CH, Switzerland
cmvangenuchten@gmail.com
Susan E. Amrose
Department of Civil and Environmental Engineering
University of California, Berkeley
Berkeley, California, USA 94720
samrose@berkeley.edu
Ashok J. Gadgil
Energy Technologies Area
Lawrence Berkeley National Laboratory
Berkeley, California, USA 94720
ajgadgil@lbl.gov
*Highlights (for review)
Bacteria attenuation by iron electrocoagulation governed by interactions between bacterial
phosphate groups and Fe(III) precipitates
Caroline Delaire*,† , Case M. van Genuchten§ , Susan E. Amrose†, Ashok J. Gadgil†,‡
†
Department of Civil and Environmental Engineering, University of California, Berkeley, California
94720-1710, United States
§
Department of Earth Sciences – Geochemistry, Faculty of Geosciences, Utrecht University, Utrecht
3508TA, The Netherlands
‡
Energy Technologies Area, Lawrence Berkeley National Laboratory, Berkeley, California 94720,
United States
Highlights
In natural waters, bacteria attenuation by Fe-EC is primarily due to physical removal with flocs
Bacterial phosphate groups govern the adhesion of EC precipitates to cell walls
Ca/Mg decrease removal, Si has no effect, and P decreases removal if Ca/Mg are absent
Fe-EC is equally effective for Gram positive and negative (rough and smooth) strains
Graphical Abstract
*Manuscript
Click here to view linked References
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Bacteria attenuation by iron electrocoagulation governed by interactions between bacterial
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phosphate groups and Fe(III) precipitates
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Caroline Delaire*,† , Case M. van Genuchten§ , Susan E. Amrose†, Ashok J. Gadgil†,‡
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†
Department of Civil and Environmental Engineering, University of California, Berkeley, California
94720-1710, United States
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§
Department of Earth Sciences – Geochemistry, Faculty of Geosciences, Utrecht University, Utrecht
3508TA, The Netherlands
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‡
Energy Technologies Area, Lawrence Berkeley National Laboratory, Berkeley, California 94720,
United States
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* Corresponding author: Department of Civil and Environmental Engineering, University of California,
Berkeley, CA 94720-1710, United States. Phone: (+1) 510-417-9491; email: caroline.delaire@orange.fr
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Abstract
Iron electrocoagulation (Fe-EC) is a low-cost process in which Fe(II) generated from an Fe(0) anode
31
reacts with dissolved O2 to form (1) Fe(III) precipitates with an affinity for bacterial cell walls and (2)
32
bactericidal reactive oxidants. Previous work suggests that Fe-EC is a promising treatment option for
33
groundwater containing arsenic and bacterial contamination. However, the mechanisms of bacteria
34
attenuation and the impact of major groundwater ions are not well understood. In this work, using the
35
model indicator Escherichia coli (E. coli), we show that physical removal via enmeshment in EC
36
precipitate flocs is the primary process of bacteria attenuation in the presence of HCO3-, which
37
significantly inhibits inactivation, possibly due to a reduction in the lifetime of reactive oxidants. We
38
demonstrate that the adhesion of EC precipitates to cell walls, which results in bacteria encapsulation in
39
flocs, is driven primarily by interactions between EC precipitates and phosphate functional groups on
40
bacteria surfaces. In single solute electrolytes, both P (0.4 mM) and Ca/Mg (1-13 mM) interfered with the
41
adhesion of EC precipitates to bacterial cell walls, whereas Si (0.4 mM) and ionic strength (2-200 mM)
42
did not impact E. coli attenuation. Interestingly, P (0.4 mM) did not affect E. coli attenuation in
43
electrolytes containing Ca/Mg, consistent with bivalent cation bridging between bacterial phosphate
44
groups and inorganic P sorbed to EC precipitates. Finally, we found that EC precipitate adhesion is
45
largely independent of cell wall composition, consistent with comparable densities of phosphate
46
functional groups on Gram-positive and Gram-negative cells. Our results are critical to predict the
47
performance of Fe-EC to eliminate bacterial contaminants from waters with diverse chemical
48
compositions.
49
50
Keywords
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Iron electrocoagulation; bacteria attenuation; bacterial surface functional groups; specific interactions;
52
bivalent cations; oxyanions.
2
53
Highlights
54
In natural waters, bacteria attenuation by Fe-EC is primarily due to physical removal with flocs
55
Bacterial phosphate groups govern the adhesion of EC precipitates to cell walls
56
Ca/Mg decrease removal, Si has no effect, and P decreases removal if Ca/Mg are absent
57
Fe-EC is equally effective for Gram positive and negative (rough and smooth) strains
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Graphical abstract
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3
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1. Introduction
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Iron electrocoagulation (Fe-EC) is a process relying on the electrolytic dissolution of an Fe(0) anode
68
to generate Fe(II), which is oxidized by dissolved O2 to produce Fe(III) (oxyhydr)oxide precipitates with
69
an affinity for microbial and chemical contaminants (Delaire et al., 2015; van Genuchten et al., 2012). Fe-
70
EC can efficiently remove arsenic from contaminated groundwater (Amrose et al., 2014; Li et al., 2012),
71
and has also been shown to attenuate bacteria in a range of water matrices (Barrera-Dı́az et al., 2003;
72
Delaire et al., 2015; Ghernaout et al., 2008). In a recent study, we demonstrated that Fe-EC can attenuate
73
Escherichia coli (E. coli) from synthetic Bengal groundwater (SBGW) without detriment to arsenic
74
removal (Delaire et al., 2015), confirming that Fe-EC has promising applications for low-cost
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groundwater remediation (Amrose et al., 2014). Two processes contributed to bacteria attenuation in Fe-
76
EC: (1) physical removal, caused by bacteria enmeshment in Fe(III) flocs and subsequent settling, and (2)
77
inactivation by reactive species produced upon Fe(II) oxidation by O2. Fundamental aspects of the
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mechanisms underlying these two processes remain unknown. For example, the type of chemical
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interactions governing bacteria enmeshment in flocs is not well understood. In addition, the effect of
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major groundwater components, such as HCO3-, Ca, Mg, Si, and P, which can interfere with both
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inactivation and removal, has not been investigated. Finally, the impact of bacteria surface structure
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(Gram-positive versus Gram-negative, smooth versus rough Gram-negative) on attenuation has not been
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elucidated. By addressing these knowledge gaps, this study can improve considerably our predictions of
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Fe-EC performance in various water matrices containing different types of bacterial contamination.
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Our previous work suggests that the adhesion of EC precipitates to cell walls is a key process in
86
bacteria enmeshment in flocs (Delaire et al., 2015). Specifically, the significantly higher bacteria removal
87
by Fe-EC in comparison to coagulation with pre-synthesized ferryhydrite (for the same Fe(III)
88
concentration) shows that removal cannot be solely attributed to the mechanical sweeping of bacterial
89
cells by Fe(III) flocs (sweep flocculation). In addition, increased removal at higher Fe dosages indicates a
90
stoichiometric relationship between Fe(III) precipitates and bacterial surfaces, consistent with the primary
4
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role of precipitate adhesion to cell walls. However, important questions remain regarding the bacterial
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functional groups involved in such adhesion, the type of interaction (electrostatic versus specific
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bonding), and the effects of groundwater chemistry and cell wall structure.
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Four types of surface functional groups are present on bacterial cell walls at comparable densities:
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hydroxyl (pKa ~ 9.0), amine (pKa ~ 9.0), carboxyl (pKa ~ 4.7), and phosphate groups (pKa1 ~ 3.1, pKa2 ~
96
6.6) (Borrok et al., 2005; Ngwenya et al., 2003). Hydroxyl and amine moieties do not have a strong
97
affinity for Fe(III) oxides (McBride and Kung, 1991; Norén et al., 2008) and therefore they are not
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expected to strongly interact with EC precipitates. By contrast, carboxyl and phosphate moieties have
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strong affinities for Fe(III) oxides (Arai and Sparks, 2001; Chassé et al., 2015; Filius et al., 2000; van
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Genuchten et al., 2014a) and studies using Attenuated Total Reflectance Fourier-Transform Infrared
101
spectroscopy (ATR-FTIR) have shown direct bonding of bacterial phosphate and carboxyl groups to
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hematite and goethite (Elzinga et al., 2012; Parikh and Chorover, 2006; Parikh et al., 2014). However,
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these studies were performed in controlled laboratory systems and simple water matrices, and they cannot
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be directly extrapolated to Fe-EC in groundwater, where precipitates and bacteria interact in an agitated
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suspension and in the presence of bivalent cations (Ca and Mg) and oxyanions (P and Si), which can sorb
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to bonding sites on bacteria (Beveridge and Koval, 1981; Johnson et al., 2007) and precipitates (van
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Genuchten et al., 2014b), respectively, and may therefore interfere with adhesion.
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In addition to electrolyte composition, a number of studies have shown that the biomolecular structure
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of bacterial cell walls can affect their interactions with mineral surfaces through changes in surface
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charge, hydrophobicity and steric hindrance (Chen and Walker, 2012; Jacobson et al., 2015; Walker et al.,
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2004). Because waterborne pathogenic bacteria and indicator organisms span the range of Gram-positive,
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smooth and rough (with and without O-antigen) Gram-negative strains (WHO, 2011), understanding the
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impact of cell wall structure on bacteria attenuation with Fe-EC is essential to generalize our findings to
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all bacterial species relevant to water quality.
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Spectroscopic techniques such as ATR-FTIR, X-ray fluorescence (XRF) and X-ray absorption
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spectroscopy (XAS) have been used to study bacteria-Fe systems (Chan et al., 2009; Elzinga et al., 2012;
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Miot et al., 2009; Yan et al., 2016). However, these techniques cannot adequately determine bacteria-
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Fe(III) interactions in systems where Fe(III) is co-precipitated with bacteria in complex electrolytes
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similar to groundwater. For example, P-Fe bonds from bacteria-precipitate interactions and from aqueous
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P sorption to precipitates look very similar using ATR-FTIR (Elzinga et al., 2012) and would not be
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distinguishable with P K-edge XAS (Kelly et al., 2008). Additionally, ATR-FTIR is not suited to
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investigate interactions taking place inside large flocs due to the low penetration length of infrared beams
123
in aqueous medium (~1µm). To circumvent these limitations, the present study proposes an innovative
124
approach, where macroscopic data of bacteria attenuation in systematically varied electrolytes are
125
combined with ζ-potential measurements to elucidate the molecular interactions between bacteria and EC
126
precipitates. Although this approach can only provide indirect evidence for specific interactions between
127
bacteria and precipitates, it builds upon previous spectroscopic studies, which have identified bacteria-Fe
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oxide bonding processes in simple controlled systems (Elzinga et al., 2012; Parikh and Chorover, 2006;
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Parikh et al., 2014) and structures of Fe-EC precipitates in complex water matrices (van Genuchten et al.,
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2014a, 2014b), to gain information about bacteria removal mechanisms in groundwater-like electrolytes.
131
The goals of this study are to: (1) determine the impact of HCO3-, Ca, Mg, P and Si on bacteria
132
attenuation with Fe-EC, (2) identify the bacterial functional groups involved in the adhesion of EC
133
precipitates to cell walls and investigate the type of interaction (electrostatic versus specific), and (3) test
134
the generalizability of these conclusions to various bacteria types. To achieve these objectives, we first
135
compared Fe-EC with FeCl3 coagulation to distinguish the contributions of inactivation and removal via
136
enmeshment in flocs to overall bacteria attenuation in Fe-EC as a function of the HCO3- concentration.
137
Inactivation results were confirmed using live-dead staining. Second, we systematically investigated the
138
effect of ionic strength, Ca/Mg and P/Si on E. coli attenuation, both in single and multiple solute
139
electrolytes, to constrain the bacterial functional groups involved in precipitate adhesion to cell walls. ζ-
6
140
potential, a proxy for surface charge, was used to assess the interaction of major groundwater ions with
141
the surface of EC precipitates or E. coli cells. Third, we validated our proposed mechanism with 3
142
bacteria strains bearing different surface structures (smooth and rough Gram-negative, and Gram-
143
positive). Our results strongly suggest that Fe-EC can be used to remove various types of bacteria from a
144
wide range of water matrices representative of regions affected by arsenic and microbial contamination of
145
drinking water sources. More generally, this study can help predict the performance of Fe-EC, and other
146
Fe-based coagulation processes, to reduce bacterial contaminants from drinking water and wastewater.
147
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2. Methods
2.1. Bacteria preparation and enumeration
150
One Gram-positive and two Gram-negative bacterial strains were used: Enterococcus faecalis (ATCC
151
19433, no antibiotic resistance), Escherichia coli K12 (NCM 4236, kanamycin-resistant), and Escherichia
152
coli ECOR 10 (from STEC center, ampicillin-resistant (Mazel et al., 2000)). K12 is a rough strain (no O-
153
antigen) (Stevenson et al., 1994) whereas ECOR 10 is a smooth strain (O-antigen present, serotype O6)
154
(STEC center, 2016). After three propagations in growth media amended with appropriate antibiotics,
155
stationary-phase bacteria were rinsed 3 times and resuspended in 100 mM NaCl as detailed in the
156
Supporting Information. Bacteria were spiked in Fe-EC electrolytes to achieve initial concentrations of
157
106.1-6.7 CFU/mL (105.0-5.8 CFU/mL for E. faecalis). Bacteria concentrations were enumerated in duplicate
158
in 0.1 mL aliquots as colony forming units (CFU) using the spread plate technique on agar amended with
159
appropriate antibiotics (detection limit of 10 CFU/mL), as described in the Supporting Information.
160
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2.2. Electrolytes
The list of electrolytes used in bacteria attenuation experiments is specified in Table S1. In summary,
we first varied the concentration of HCO3- (0.1-8.0 mM) to examine its impact on bacteria inactivation.
7
164
Second, a range of ionic strengths was investigated by varying NaCl (in deionized water and in SBGW)
165
or NaClO4 (in 1 mM CaCl2). Then, concentrations of bivalent cations (Ca: 0-13.5 mM and Mg: 0-10.6
166
mM) and oxyanions (P: 0-0.4 mM and Si: 0-0.4 mM) were systematically varied, in single and composite
167
electrolytes, to elucidate their effect on bacteria removal. Finally, SBGW containing 8.2 mM HCO3-, 2.7
168
mM Ca, 2.0 mM Mg, 1.3 mM Si, 0.15 mM P, and 6.3 µM As(III), was prepared as described elsewhere
169
(Delaire et al., 2015) and used as the electrolyte in some experiments. All experiments were conducted at
170
pH 7.0 ± 0.3, except for the comparisons between the three bacterial strains, which were conducted at pH
171
7.5 ± 0.2. The pH was held constant throughout experiments by adding HCl, NaOH or NaHCO3 as
172
needed. Electrolytes were selected in part to overlap with previous work on the structure of EC
173
precipitates (van Genuchten et al., 2014a, 2014b, 2012), which we leverage in our interpretations of
174
bacteria attenuation and ζ-potential measurements.
175
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2.3. Fe-EC and FeCl3 experiments
177
The procedure used for Fe-EC experiments has been described elsewhere (Delaire et al., 2015) and is
178
detailed in the Supporting Information. Briefly, two 1 cm × 8 cm Fe(0) electrodes were submerged in 200
179
mL of electrolyte spiked with bacteria (anodic submerged area of 3 cm2). In all experiments, a current
180
density of 10 mA/cm2 was applied for 11 min, resulting in a Faradaic Fe dosage of 0.5 mM. After the
181
electrolysis stage, suspensions were stirred open to the atmosphere for 90-180 min to allow for complete
182
Fe(II) oxidation and formation of Fe(III) precipitates. Suspensions were then left to settle overnight to
183
separate individual cells from cells associated with EC precipitates. When required for floc formation and
184
settling (Table S1), 5 mg/L-Al of Al2(SO4)3 (alum) was added at the end of the mixing period, along with
185
approximately 1.5 mM NaHCO3 to avoid a pH drop. Preliminary tests confirmed that the addition of alum
186
did not significantly modify bacteria attenuation (see Supporting Information). Solution pH was not
187
controlled during the settling stage. In a subset of experiments, coagulation by FeCl3 addition was used
188
instead of Fe-EC to isolate the contribution of removal from that of inactivation. In these experiments, 1
8
189
mL of a 100 mM FeCl3 solution was added to the electrolyte and the solution pH, which dropped to ~3
190
during FeCl3 addition, was re-adjusted to 7.0±0.1 in less than 5 min.
191
Unfiltered and filtered (0.45 μm nylon filters) samples were taken before Fe-EC, and before and after
192
overnight settling, for measurements of Fe, As, Ca, Mg, P, and Si by inductively coupled plasma optical
193
emission spectrometry (ICP−OES, PerkinElmer 5300 DV, measurement error typically < 5%). All
194
samples for ICP-OES analysis were digested in 0.2 M HCl. Filtered and unfiltered samples were used to
195
measure Fe(II) and total Fe (Fe(II) + Fe(III)), respectively (Delaire et al., 2015). Across the 113 bacteria
196
attenuation experiments reported here, the total Fe concentration after Fe-EC (Fe dosage) was 96% ± 7%
197
of the value predicted by Faraday’s law (0.5 mM). Unoxidized Fe(II) (before settling) and unsettled Fe
198
(after settling) were <1.2% and <4.7% of the total Fe dosed, respectively. Because the formation of
199
calcite, magnesite or hydroxyapatite in our experiments was limited if not negligible (see Supporting
200
Information), Ca/Mg/P removal measured by ICP-OES was used as a proxy for Ca/Mg/P uptake by EC
201
precipitates. Bacteria attenuation was calculated as the difference between log CFU concentrations before
202
Fe-EC and after settling (samples taken from the supernatant, ∼ 3 cm below the surface), and therefore
203
accounts for both inactivation and removal via enmeshment in flocs. Bacteria attenuation experiments
204
were generally replicated three or more times, except for 12 experiments conducted in duplicate or less
205
(see Table S2). We report average bacteria attenuations ± one standard deviation across replicates.
206
Finally, to assess the effect of P/Si on the uptake of carboxyl moieties by EC precipitates, we
207
performed citrate removal experiments using Fe-EC in the presence and absence of oxyanions under
208
conditions identical to E. coli removal experiments, using 10 mg/L-Al of alum before settling (Table S1).
209
Citrate concentrations were measured as total C with a TOC-VCSH analyzer (Shimadzu).
210
211
2.4. ζ-potential measurements and bacterial viability tests
212
In this study, ζ-potential measurements, which are a proxy for surface charge, were used to assess the
213
interaction of major groundwater ions with the surface of EC precipitates or E. coli K12 cells. ζ-potential
9
214
was measured by dynamic light scattering (Malvern Zetasizer Nano-ZS) at 633 nm. In addition,
215
qualitative assessments of membrane permeabilization, which were used as a proxy for bacteria
216
inactivation, were performed with the BacLight LIVE-DEAD kit (Invitrogen) used in conjunction with
217
fluorescent microscopy (Zeiss AxioImager, 63× Plan-Apochromat objective, EndoGFP and mCherry
218
filters, UC Berkeley CNR Biological Imaging Facility). Sample preparation and data collection
219
procedures are described in the Supporting Information.
220
221
2.5. Model of Ca/Mg complexation by bacterial cell walls
222
Drawing on previous work (Johnson et al., 2007; Ngwenya et al., 2003), we derived a simple
223
equilibrium surface complexation model, which included three bivalent cation adsorption sites on
224
bacterial cell walls: carboxyl groups, protonated and deprotonated phosphate groups. The model predicts
225
the percentage of bacterial phosphate and carboxyl groups complexed by Ca and Mg as:
𝐾
2+
2+
𝐾𝑃1,𝐶𝑎 [𝐶𝑎2+ ]+𝐾𝑃1,𝑀𝑔 [𝑀𝑔2+ ]+ 𝐴2
+ (𝐾𝑃2,𝐶𝑎 [𝐶𝑎 ]+𝐾𝑃2,𝑀𝑔 [𝑀𝑔 ])
[𝐻 ]
226
%𝑃 𝑔𝑟𝑜𝑢𝑝𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑥𝑒𝑑 = [𝐻+]
227
%𝐶 𝑔𝑟𝑜𝑢𝑝𝑠 𝑐𝑜𝑚𝑝𝑙𝑒𝑥𝑒𝑑 = [𝐻+] 𝐶,𝐶𝑎
𝐾
𝐾
[𝐶𝑎 2+ ]+𝐾𝑃2,𝑀𝑔 [𝑀𝑔2+ ])
+ 1+ 𝐴2
+𝐾𝑃1,𝐶𝑎 [𝐶𝑎 2+ ]+𝐾𝑃1,𝑀𝑔 [𝑀𝑔2+ ]+ 𝐴2
(𝐾
𝐾𝐴1
[𝐻+ ]
[𝐻+ ] 𝑃2,𝐶𝑎
𝐾
𝐾𝐴
[𝐶𝑎2+ ]+𝐾𝐶,𝑀𝑔 [𝑀𝑔2+ ]
+ 1+𝐾𝐶,𝐶𝑎 [𝐶𝑎2+ ]+𝐾𝐶,𝑀𝑔 [𝑀𝑔2+ ]
∗ 100
∗ 100
(1)
(2)
228
Deprotonation constants of bacterial surface functional groups and Ca adsorption constants were
229
obtained directly from the literature (Johnson et al., 2007). Mg adsorption constants were derived from a
230
relationship between metal-acetate and metal-bacteria complexation constants proposed by Johnson et al
231
(Johnson et al., 2007). Additional details regarding the derivation of this model, including equilibrium
232
constants, are given in the Supporting Information and Table S3.
233
234
235
3. Results and Discussion
3.1. Effect of HCO3- on the contributions of removal and inactivation
10
236
The effect of 8 mM HCO3- on E. coli attenuation by Fe-EC and FeCl3 coagulation is shown in
237
Figure 1a. Representative images of live-dead stained E. coli are presented in Figure 1b-e. Whereas 8 mM
238
HCO3- did not significantly affect E. coli attenuation by coagulation with FeCl3, the presence of HCO3-
239
decreased attenuation by Fe-EC by ~1.2 log. Because no reactive oxidants are produced from an Fe(III)
240
salt (Hug and Leupin, 2003), minimal inactivation occurs during FeCl3 coagulation (consistent with live-
241
dead staining, Figure 1b-c), which implies that attenuation via FeCl3 addition is exclusively due to
242
physical removal (enmeshment in flocs). Any difference in precipitate-bacteria adhesion between Fe-EC
243
and FeCl3 coagulation would lead to higher removal in the latter, because the precipitates generated by
244
FeCl3 coagulation in a HCO3- electrolyte are less crystalline and thus have a higher surface area than Fe-
245
EC precipitates (Schwertmann and Cornell, 2000; van Genuchten et al., 2014b; Voegelin et al., 2010).
246
Consequently, the difference in attenuations between Fe-EC and FeCl3 coagulation can conservatively be
247
attributed to inactivation.
248
As shown in Figure 1a, HCO3- did not affect physical removal, which is consistent with ζ-potential
249
measurements showing that HCO3- does not significantly interact with the surface of E. coli cells or
250
Fe(III) precipitates (Figure S1). By contrast, 8 mM HCO3- decreased inactivation substantially by ~1.2
251
log. We found a strong correlation between bacteria inactivation in Fe-EC (Figure 1a) and membrane
252
permeabilization (Figure 1d-e). Membrane damage may be caused by reactive intermediates such as O2●-,
253
H2O2, and Fe(IV), which are generated during Fenton-type reactions (Hug and Leupin, 2003; Keenan and
254
Sedlak, 2008) and have been associated with bactericidal effects (Alt et al., 1999; Ikawa et al., 2010; Kim
255
et al., 2010). The inhibition of inactivation by HCO3- might be explained by the formation of CO3●-
256
radicals, which are produced when HCO3- or Fe(II)-carbonate complexes react with H2O2 (Hug and
257
Leupin, 2003; Medinas et al., 2007). CO3●- is much more reactive than O2●-, H2O2, and Fe(IV) (Augusto
258
and Miyamoto, 2011; Jacobsen et al., 1998; Neta et al., 1988) (see Supporting Information), and is
259
therefore a much shorter-lived and less selective oxidant. Thus, we speculate that large HCO3-
260
concentrations reduce membrane damage and inactivation by shifting the nature of reactive species
11
261
produced during Fe-EC towards a shorter-lived oxidant (CO3●-) that is more likely to die off in the bulk
262
(e.g. reacting with Fe(II), Cl-, HCO3-) than to interact with cell membranes.
263
Overall, Figure 1 shows that both inactivation and removal (via enmeshment in flocs) contribute to
264
E. coli attenuation in Fe-EC, and that the concentration of HCO3- governs the amount of inactivation. In
265
the remaining sections of our study, we will focus on removal. Interactions between EC precipitates and
266
E. coli cells are investigated by varying levels of ionic strength, Ca, Mg, P, and Si. Because these ions are
267
not expected to react with oxidants such as O2●-, H2O2, or Fe(IV) (Hug and Leupin, 2003; Li et al., 2012;
268
Roberts et al., 2004), nor to interact with lipid aliphatic chains, which are the target of oxidants on cell
269
membranes (lipid peroxidation), they are assumed to have a negligible effect on inactivation. Therefore,
270
their potential impact on E. coli attenuation will be solely attributed to changes in removal.
271
272
273
3.2. Effect of ionic strength
Increasing ionic strength over 2 orders of magnitude (2-200 mM), which results in increased charge
274
screening (Debye length decreased tenfold), did not significantly affect E. coli attenuation by Fe-EC,
275
regardless of the initial electrolyte composition (Figure S2). The negligible effect of ionic strength
276
suggests that electrostatic interactions play a secondary role compared to specific interactions in the
277
adhesion of EC precipitates to E. coli cells. In the following two sections, we investigate the bacterial
278
surface sites involved in these interactions by systematically varying the concentration of bivalent cations
279
and oxyanions in order to selectively complex adsorption sites on the surface of E. coli cells and EC
280
precipitates, respectively.
281
282
3.3. Effect of bivalent cations: Ca and Mg
283
3.3.1.
284
285
Single solute electrolytes (no oxyanions, no HCO3-)
E. coli attenuation as a function of Ca and Mg concentrations is shown in Figure 2a. Ca and Mg both
decreased E. coli attenuation, with a larger inhibitory effect observed for Mg (2.1 log decrease in
12
286
attenuation when Mg increased from 0 to 10.6 mM) than for Ca (1.3 log decrease in attenuation when Ca
287
increased from 0 to 12.9 mM). Because bivalent cations should not affect inactivation (see 3.1.), these
288
reductions in bacteria attenuation can be interpreted as reductions in E. coli removal.
289
Figure 2b shows the ζ-potential of EC precipitates and E. coli cells as a function of Ca/Mg
290
concentrations. In this single Ca/Mg solute electrolyte, EC precipitates were positively charged.
291
Increasing concentrations of Ca/Mg had a limited effect on the ζ-potential of precipitates, suggesting that
292
bivalent cations interacted minimally with their surface. This result was expected given the repulsive
293
electrostatic forces between bivalent cations and positively-charged EC precipitates, and is consistent
294
with previous work showing negligible uptake of Ca/Mg by Fe(III) (oxyhydr)oxides at circumneutral pH
295
in the absence of oxyanions (Kanematsu et al., 2013; Stachowicz et al., 2008). By contrast, Ca and Mg
296
caused a significant increase in the ζ-potential of E. coli cells, indicating a strong interaction between
297
bivalent cations and bacteria surfaces. Figure S3 shows the percentage of bacterial functional groups
298
complexed by bivalent cations, as predicted by our equilibrium surface model. According to this model,
299
raising Ca/Mg concentrations from 0 to 13 mM leads to a significant increase in the complexation of
300
carboxyl (from 0 to 70-80%) and phosphate (from 0 to 90-95%) groups, which is consistent with the
301
observed increase in E. coli ζ-potential (Figure 2b).
302
Figure 2c combines E. coli attenuation results (Figure 2a) and model outputs (Figure S3) to highlight
303
that E. coli removal decreases as the percentage of complexed bacterial carboxyl and phosphate groups
304
increases. Stronger inhibition of E. coli removal by Mg than by Ca (Figure 2a) is consistent with this
305
trend, because Mg has a higher affinity for bacterial surface functional groups (Beveridge and Koval,
306
1981) (Table S3 and Figure S3).
307
3.3.2.
Groundwater-like electrolytes (with oxyanions and HCO3-)
308
Figure 2d shows the effect of Ca (0-13.5 mM) and Mg (2.4-10.5 mM) on E. coli attenuation in a
309
groundwater-like electrolyte containing 8 mM HCO3-, 1.2 mM Si, and 0.4 mM P. Similar to the single
13
310
Ca/Mg solute system, bivalent cations reduced E. coli attenuation, with Ca/Mg concentrations above 10
311
mM leading to a 1-2 log decrease in attenuation.
312
Figure 2e shows ζ-potentials of EC precipitates and E. coli cells as a function of Ca/Mg
313
concentrations in the groundwater-like electrolyte. Bivalent cations increased the ζ-potential of E. coli
314
cells, consistent with the complexation of phosphate and carboxyl groups on cell walls, as explained in
315
section 3.3.1. In this electrolyte, EC precipitates were negatively-charged due to the sorption of P and, to
316
a lesser extent, Si (P:Fe and Si:Fe molar solids ratios of 0.7 ± 0.1 and 0.06 ± 0.04, respectively)
317
(Appenzeller et al., 2002; Hamid et al., 2011). In contrast to previous experiments in the absence of
318
oxyanions, bivalent cations significantly interacted with the surface of EC precipitates in the
319
groundwater-like electrolyte, as indicated by a substantially higher ζ-potential at larger Ca/Mg
320
concentrations. This increase in precipitate surface charge coincided with increased Ca/Mg uptake, with
321
solids ratios going from 0.5 ± 0.1 to 1.2 ± 0.7 mol Ca:mol Fe, and from 0.3 ± 0.1 to 0.5 ± 0.4 mol Mg:mol
322
Fe, respectively. EC precipitates with similar chemical compositions (i.e. Ca/Mg:P:Fe molar ratios) have
323
been documented in previous studies performed in nearly identical electrolytes, but in the absence of
324
bacteria (van Genuchten et al., 2014a, 2014b). In these studies, Ca was shown to interact with P sorbed to
325
Fe(III) precipitates, via direct Ca-O-P bonds, and to a lesser extent, electrostatically. In the present study,
326
the observed increase in precipitate ζ-potential with Ca/Mg in the groundwater-like electrolyte is
327
consistent with such interactions of Ca/Mg with P sorbed to EC precipitates.
328
Figure 2f illustrates the inverse relationship between E. coli attenuation in the groundwater-like
329
electrolyte and the percentage of bacterial functional groups complexed by Ca/Mg (derived from our
330
model). Figure 2f also includes data from our previous study of E. coli attenuation in SBGW containing
331
2.6 mM Ca and 1.9 mM Mg (Delaire et al., 2015), which are consistent with this trend. Finally, we note
332
that E. coli attenuations in groundwater-like electrolytes (Figure 2f) were overall ~1 log lower than in
333
single solute systems (Figure 2c), which is consistent with the inhibition of inactivation by 8 mM HCO3-
334
shown in section 3.1.
14
335
Taken together, Figures 2a-f show that Ca/Mg decreases E. coli removal independent of the
336
electrolyte, and more specifically, independent of the surface charge of EC precipitates: whether Ca/Mg
337
increase (Figure 2b, no oxyanions) or decrease (Figure 2e, oxyanions present) the electrostatic barrier to
338
precipitate adhesion on cell walls, bivalent cations equally inhibit E. coli removal. Combined with the
339
limited impact of ionic strength (Section 3.2 and Figure S2), this result confirms the minimal role of
340
electrostatic interactions on E. coli removal and instead points to the importance of specific interactions
341
between EC precipitates and bacterial phosphate and/or carboxyl groups. These findings are in good
342
agreement with previous ATR-FTIR studies that provided evidence for direct bonding between Fe oxides
343
and bacterial phosphate/carboxyl groups in more simple and controlled systems (Elzinga et al., 2012;
344
Parikh and Chorover, 2006; Parikh et al., 2014).
345
346
3.4. Effect of oxyanions: P and Si
347
3.4.1.
Single solute electrolytes (no bivalent cations, no HCO3-)
348
Figure 3a shows the effect of 0.4 mM Si/P on E. coli attenuation in electrolytes containing no Ca/Mg.
349
Whereas Si had no detectable effect, P reduced E. coli attenuation by 1.6 log. Because Si and P should not
350
affect inactivation, as explained in section 3.1, these effects correspond to changes in removal via
351
enmeshment in flocs. ζ-potential measurements of EC precipitates and E. coli cells as a function of P/Si
352
concentrations are presented in Figure 3b. Si and P had no detectable effect on the ζ-potential of E. coli
353
cells, reflecting the absence of interaction between these oxyanions and bacterial cell walls. By contrast,
354
Si and P significantly decreased the ζ-potential of EC precipitates, indicating oxyanion sorption
355
(Appenzeller et al., 2002; Hamid et al., 2011), which is supported by the uptake of Si and P measured by
356
ICP-OES (Si:Fe and P:Fe molar solids ratios of 0.3 and 0.6, respectively). Because electrostatic
357
interactions do not play a major role in E. coli removal, as demonstrated above, lower bacteria removal in
358
the presence of P cannot be explained by the decrease in precipitate surface charge. Rather, the results in
359
Figure 3a indicate that inorganic aqueous P competes with bacterial functional groups involved in
15
360
bonding to EC precipitates. By contrast, our results indicate that Si does not strongly compete with these
361
functional groups.
362
Because aqueous P and bacterial phosphate groups are structurally and chemically similar, they are
363
expected to compete for precipitate surfaces. However, the competition between P and carboxyl groups is
364
less straight-forward. To assess the effect of P on the adsorption of carboxyl moieties, we measured the
365
removal of citrate (a proxy for carboxyl groups) by Fe-EC in the presence and absence of P. As shown in
366
Figure 3c, P decreased citrate removal by nearly 54% (initial P:C molar ratio of 0.9). In E. coli attenuation
367
experiments, the molar ratio of aqueous P to bacterial surface carboxyl groups is ~ 2500 mol P: mol C
368
(see Supporting Information). Therefore, aqueous P is expected to strongly compete with bacterial
369
carboxyl groups in attenuation experiments.
370
Fe(III) (oxyhydr)oxides have a much higher affinity for P than for Si (Li et al., 2014; Roberts et al.,
371
2004). Therefore, Si is not expected to effectively compete with bacterial phosphate groups for precipitate
372
surfaces. However, Figure 3c shows that Si decreased citrate removal in Fe-EC by nearly 20% (initial
373
Si:C molar ratio of 0.7). In E. coli attenuation experiments, where the molar ratio of Si to bacterial surface
374
carboxyl groups is orders of magnitude higher (~ 2500, see Supporting Information), it is thus likely that
375
Si would inhibit bacteria removal if carboxyl groups played an important role in the adhesion of EC
376
precipitates. Because Si had no detectable effect on E. coli attenuation (Figure 3a), we propose that
377
phosphate groups are the primary sites for the adhesion of EC precipitates to cell walls, with negligible
378
contributions from carboxyl groups.
379
3.4.2.
Groundwater-like electrolytes (with bivalent cations, HCO3- and Si)
380
In Figure 3d, we show the effect of P (0-0.4 mM) on E. coli attenuation in the presence of Ca (2 and 9
381
mM) or Mg (8 mM) in a groundwater-like electrolyte containing 8 mM HCO3- and 1.2 mM Si. In contrast
382
to experiments in electrolytes free of bivalent cations, where P decreased E. coli removal by 1.6 log
383
(Figure 3a), 0.4 mM P had no effect on E. coli removal in the presence of Ca/Mg. We note that lower E.
16
384
coli attenuations in Figure 3d compared to Figure 3a (~ - 2 log) are due to the inhibition of inactivation by
385
8 mM HCO3- (shown in section 3.1) and to the reduction in removal caused by Ca/Mg (shown in section
386
3.3).
387
Figures 3e-f show ζ-potential measurements of EC precipitates and E. coli cells, respectively, as a
388
function of P concentration in the groundwater-like electrolyte containing bivalent cations. Figure 3e
389
shows that P did not interact significantly with bacterial cells, as expected. In contrast to single oxyanion
390
systems (Figure 3b), EC precipitates in the groundwater-like electrolyte were negatively-charged for all P
391
concentrations, due to sorbed Si/P. In addition, the ζ-potential of EC precipitates did not decrease when
392
the P concentration increased from 0 to 0.4 mM, despite substantial P uptake by precipitates (P:Fe molar
393
solids ratios of 0.6-0.8, see Table S4). This result stands in strong contrast with electrolytes containing no
394
Ca/Mg, where high concentrations of P (0.4 mM) and similar P:Fe solids ratios (0.6 mol:mol)
395
significantly decreased EC precipitate surface charge (Figure 3b). In the groundwater-like electrolytes,
396
ICP-OES measurements indicated that Ca/Mg uptake by EC precipitates increased by 20-200% –
397
depending on the initial Ca/Mg concentration– in the presence of 0.4 mM P (Table S4). This co-sorption
398
of Ca/Mg explains the negligible impact of P sorption on the surface charge of EC precipitates.
399
Based on the co-sorption of Ca/Mg and P, the behavior of precipitate and bacteria surfaces, and the
400
negligible effect of P on E. coli removal observed in our system, we propose that Ca/Mg can act as a
401
bivalent cation bridge between bacterial phosphate groups and P sorbed to EC precipitates. This Ca/Mg
402
configuration, which creates additional sites at the precipitate surface that can interact with bacterial cell
403
walls, is consistent with the Ca-P-Fe configurations documented previously in comparable systems (Senn
404
et al., 2015; van Genuchten et al., 2014a; Voegelin et al., 2010).
405
406
3.5. Attenuation of different types of bacteria
17
407
The attenuation of E. coli K12, E. coli ECOR 10 and E. faecalis in SBGW with an Fe dosage of 0.5
408
mM is shown in Figure 4. No significant difference between the log attenuations of the three different
409
bacterial strains was observed, despite their considerably different cell wall structures. For example, the
410
surface of Gram-positive E. faecalis is composed of a peptidoglycan layer topped with techoic acids,
411
whereas the surface of Gram-negative E. coli is made of phospholipids and lipopolysaccharides (LPS)
412
(Madigan et al., 2000). Furthermore, the two E. coli strains differ by the length of their LPS: ECOR 10 is
413
a smooth strain with a full-length LPS (with O-antigen), whereas K12 is a rough strain with a truncated
414
LPS (no O-antigen). Such differences in cell wall composition lead to differences in hydrophobicity,
415
surface charge, surface roughness and steric hindrance to approach mineral surfaces and nanoparticles
416
(Chen and Walker, 2012; Jacobson et al., 2015; Walker et al., 2004).
417
Previous studies have found that cell wall composition and LPS length affect the interactions of
418
bacteria with mineral surfaces (sand, iron-oxide coated sand, and gold nanoparticles) in systems governed
419
by non-specific interactions, such as electrostatic, steric, hydrophobic, and van der Waals forces (Chen
420
and Walker, 2012; Jacobson et al., 2015; Mohanty et al., 2013; Truesdail et al., 1998; Walker et al.,
421
2004). In contrast to these studies, similar attenuation of E. coli K12, E. coli ECOR 10 and E. faecalis in
422
our system is likely due to the dominant role of specific interactions in bacteria-precipitate adhesion.
423
Phosphate functional groups, which we showed are the primary binding sites for EC precipitates, are
424
present in similar abundance on Gram-negative and Gram-positive bacteria (Borrok et al., 2005) (mainly
425
on phospholipids and on techoic acids, respectively), explaining similar removal of E. coli and E. faecalis.
426
In addition, negligible steric hindrance from longer LPS on E. coli is likely due to the small size of EC
427
precipitates compared to bacterial cells (Figure S4).
428
Based on these results, we expect that Fe-EC would be similarly effective for all waterborne
429
pathogenic bacteria, both Gram-negative (e.g. Vibrio cholera, Shigella, Salmonella, pathogenic E. coli)
430
and Gram-positive (e.g. E. faecalis, Bacillus cereus, Staphylococcus aureus). Finally, similar attenuation
431
of E. coli K12 and E. coli ECOR 10 suggests that fecal pathogens, which are typically smooth strains
18
432
(Felix and Pitt, 1935), would be as effectively removed as our model indicator E. coli K12. Overall, these
433
results are promising for the application of Fe-EC to drinking water or wastewater treatment.
434
435
4. Conclusions
436
In this study, we showed that bacteria inactivation, which can be significant in the absence of oxidant
437
scavengers, is largely suppressed by HCO3- concentrations characteristic of natural waters. Therefore, we
438
expect physical removal to be the primary process of bacteria attenuation in most water treatment
439
applications. Sludge sterilization before handling and disposal (e.g. via heat treatment) may therefore be
440
necessary as flocs may contain viable pathogens.
441
We have shown that removal is driven by the interactions of EC precipitates with bacterial phosphate
442
groups, which may bind to Fe(III) surfaces directly or via a Ca/Mg bridge to P sorbed on precipitates. In
443
light of these mechanisms, the contrasted effects of P and Si observed in this study can be generalized to
444
other strongly- (e.g., arsenate) and weakly- (e.g., borate, arsenite, nitrate) sorbing oxyanions, respectively.
445
Similarly, the observed impact of Ca/Mg (hardness) can be extrapolated to metallic bivalent cations that
446
may be present in wastewater, such as Cu2+, Cd2+, Pb2+, and Zn2+.
447
Consistent with the universal presence of phosphate groups on bacteria surfaces, Fe-EC is equally
448
effective towards Gram-positive and Gram-negative bacteria, rough and smooth alike. Our results
449
strongly suggest that Fe-EC, which is a technology applicable to decentralized arsenic remediation in
450
low-resource settings (Amrose et al., 2014; Holt et al., 2005), can also effectively remove all types of
451
bacterial contamination from a wide range of groundwater sources. Field validation of these promising
452
results as well as an investigation of virus attenuation are needed to confirm the potential of Fe-EC to
453
substitute for existing disinfection methods when applied to groundwater treatment.
454
455
Acknowledgements
19
456
This work was supported by the Development Impact Lab (USAID Cooperative Agreement AID-OAA-
457
A-13-00002) and the Andrew and Virginia Rudd Family Foundation Chair for Safe Water and Sanitation
458
administered by the Blum Center for Developing Economies. Additionally, C.M.v.G. acknowledges
459
funding support from the Netherlands Organization for Scientific Research (NWO) through a Veni grant.
460
We wish to express our gratitude to Kara Nelson, who provided useful discussions regarding the
461
complexation of Ca/Mg to bacterial functional groups, and to David Sedlak, Denise Schichnes, John
462
Wertz and Aidan Cecchetti for their kind assistance along various steps of this work. ζ-potential
463
measurements were conducted at the Molecular Foundry of Lawrence Berkeley National Laboratory and
464
supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy
465
under Contract No. DE-AC02-05CH11231.
466
467
Supporting Information
468
The Supporting Information provides detailed descriptions of experimental protocols (for Fe-EC
469
experiments, ζ-potential measurements, and fluorescent microscopy), the bacteria surface complexation
470
model, and the reactivity of strong oxidants produced in Fe-EC. Supporting figures and tables referenced
471
in the text are also included.
472
20
473
474
475
476
477
478
479
480
481
Figure 1: E. coli attenuation with Fe-EC and FeCl3, with and without 8 mM HCO3-. Fe dosage was
0.5 mM in all experiments. Panel a shows E. coli log attenuations. The asterisk indicates that the detection
limit for bacteria attenuation was reached for some of the replicate experiments. Panels b-e show
fluorescent microscopy images of live (green)-dead (red) stained E. coli cells. The blue dashed line is the
average attenuation in all FeCl3 experiments (with and without HCO3-) and represents removal (blue
arrow). E. coli log attenuations are compared to this baseline to deduce approximate log inactivations (red
arrows). All experiments were conducted at pH 7.0. In 0.1 mM HCO3- experiments, 2 mM NaCl were
added for conductivity.
482
21
483
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485
486
487
488
489
490
491
492
493
494
Figure 2: Effect of Ca and Mg on E. coli attenuation in Fe-EC, in single solute electrolytes (panels a, b
and c) and in groundwater-like electrolytes containing 8 mM HCO3-, 1.2 mM Si and 0.4 mM P (panels d,
e and f). Panels a and d: effect of increasing Ca/Mg concentrations on E. coli log attenuation with an Fe
dosage of 0.5 mM. The asterisk indicates that the detection limit for bacteria attenuation was reached for
some of the replicate experiments. Panels b and e: effect of increasing Ca/Mg concentrations on the ζpotential of EC precipitates and E. coli cells (data points for 0 mM Ca and 0 mM Mg overlap on panel b).
Panels c and f: E. coli attenuation as a function of complexed bacterial surface groups (combination of
Figures 2a and S3, and 2d and S3 respectively). The dotted red lines highlight the inverse correlation
between E. coli attenuation and the complexation of bacterial functional groups. All experiments were
conducted at pH 7.0. Experiments with no Ca/Mg (panel a) were conducted in 2 mM NaCl for
conductivity.
495
22
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498
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500
501
502
503
504
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Figure 3: Effect of P and Si on E. coli attenuation by Fe-EC with an Fe dosage of 0.5 mM in single
solute electrolytes (0.4 mM P or Si in 2mM NaCl background for conductivity; panels a,b and c) and
groundwater-like electrolytes containing 8 mM HCO3-,1.2 mM Si and bivalent cations (panels, d, e and f).
a) Effect of Si and P on E. coli attenuation. Asterisks indicate that the detection limit for bacteria
attenuation was reached for some of the replicate experiments. b) Effect of Si (open symbols) and P (solid
symbols) on the ζ-potential of EC precipitates and E. coli cells. c) Effect of P and Si on the removal of
citrate (a proxy for carboxyl moieties) by Fe-EC. d) Effect of P on E. coli attenuation at different levels of
Ca/Mg. e) Effect of P on the ζ-potential of EC precipitates. f) Effect of P on the ζ-potential of E. coli
cells. All experiments were conducted at pH 7.0.
506
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Figure 4: Log attenuation of three different bacterial strains by Fe-EC, at an Fe dosage of 0.5 mM.
All experiments were conducted at pH 7.5 in SBGW (8.2 mM HCO3-, 2.7 mM Ca, 2.0 mM Mg, 1.3 mM
Si, 0.15 mM P, and 6.3 µM As(III)). The log attenuation of E. coli K12 in SBGW shown here has also
been reported elsewhere (Delaire et al., 2015).
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Electronic Supplementary Material (for online publication only)
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