Adaptively Evolving Bacterial Communities for ... - ACS Publications

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Environmental Science & Technology

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Adaptively evolving bacterial communities for complete and selective reduction of Cr(VI),

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Cu(II) and Cd(II) in biocathode bioelectrochemical systems

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Liping Huang1,*, Qiang Wang1, Linjie Jiang1, Peng Zhou2, Xie Quan1,*, Bruce E. Logan3

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1. Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education (MOE),

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School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024,

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China

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2. College of Chemistry, Dalian University of Technology, Dalian 116024, China

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3. Department of Civil and Environmental Engineering, The Pennsylvania State University, University

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Park, Pennsylvania, 16802

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Corresponding authors:

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(Huang L.) [email protected], Tel./Fax.: 86 411 84708546

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(Quan X.) [email protected], Tel.: 86 411 84706140

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The authors declare no competing financial interest

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ACS Paragon Plus Environment


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ABSTRACT

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Bioelectrochemical systems (BESs) have been shown to be useful in removing individual metals

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from solutions, but effective treatment of electroplating and mining wastewaters requires

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simultaneous removal of several metals in a single system. To develop multiple-reactor BESs for

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metals removal, biocathodes were first individually acclimated to three different metals using

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microbial fuel cells with Cr(VI) or Cu(II) as these metals have relatively high redox potentials,

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and microbial electrolysis cells for reducing Cd(II) as this metal has a more negative redox

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potential. The BESs were then acclimated to low concentrations of a mixture of metals, followed

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by more elevated concentrations. This procedure resulted in complete and selective metal

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reduction at rates of 1.24 ± 0.01 mg/L-h for Cr(VI), 1.07 ± 0.01 mg/L-h for Cu(II), and 0.98 ±

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0.01 mg/L-h for Cd(II). These reduction rates were larger than the no adaptive controls by

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factors of 2.5 for Cr(VI), 2.9 for Cu(II) and 3.6 for Cd(II). This adaptive procedure produced less

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diverse microbial communities and changes in the microbial communities at the phylum and

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genus levels. These results demonstrated that bacterial communities can adaptively evolve to

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utilize solutions containing mixtures of metals, providing a strategy for remediating wastewaters

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containing Cr(VI), Cu(II) and Cd(II).

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TOC art

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INTRODUCTION

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Heavy metals such as Cr(VI), Cu(II) and Cd(II) in electroplating and mining industry

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wastewaters, if not removed, can severely contaminate surface waters, groundwaters, and soils.1-2

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Electrochemical processes have high energy requirements and can require expensive catalysts to

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decrease electrode overpotentials.2-3 Conventional biological processes can be used either

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completely instead of, or coupled with traditional physical-chemical processes,4 but they have

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disadvantages such as generation of high volumes of sludge, and/or the need for a continuous

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source of organic carbon.5-6 Development of more environmentally-benign and less energy

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demanding technologies would therefore be useful for treating these wastewaters.2-4

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One new promising method for metal removal is the use of bioelectrochemical systems

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(BESs).7-8 Cr(VI) and Cu(II) can be spontaneously reduced to Cr(III) and Cu(0) on the cathodes

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of microbial fuel cells (MFCs) due to favorable half cell redox potentials of 1.14 and 0.34 V (vs.

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standard hydrogen electrode, SHE) relative to that of organic matter (ca. -0.30 V for acetate,

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standard conditions).9-10 Even under the condition of an approximately neutral pH (eg. 6.0)

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suitable bacterial survival, the pH-dependent reduction of Cr(VI) has an half cell redox potential

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of 0.64 V (vs. SHE), and therefore it can spontaneously occur in MFCs. Cd(II) has been shown

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to be adsorbed to the cathode in single-chamber, air-cathode MFCs11 but it can be chemically

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reduced in microbial electrolysis cells (MECs) at a more negative potential of -0.52 V (vs. SHE,

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standard conditions).12-13 While the newly developed MFC-MEC coupled systems show

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promising for mixed metals under extreme acidic conditions,13-14 the effluents still contain 4


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concentrations of metals that are too high to be discharged into environment. Thus, the process

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needs to be further improved or additional treatment would still be required. The use of

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biocathodes in these different BESs to reduce electrode overpotentials avoids the need for

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expensive inorganic catalysts in purely electrochemical processes, and the microorganisms are

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self-regenerating and therefore their use is sustainable.7,9,15-16 While biocathodes have been

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shown to be useful for removing individual metals such as Cr(VI), U(V) and Co(II),17-22 there

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have been no tests on removing mixtures of different metals. In addition, metal concentrations in

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BES effluents have been above drinking water limits established by the World Health

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Organization (WHO) and the US EPA, which are 0.05 mg/L for Cr, 1.0 mg/L for Cu and 0.005

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mg/L for Cd.20,23-24 The use of microorganisms to reduce a mixture of metals is not trivial as

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mixed metals can be synergetically more toxic than the additive effect of the individual

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metals.25-28

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In this study, biocathodes were first developed in MFCs individually acclimated to Cr(VI)

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(MFC-Cr) or Cu(II) (MFC-Cu), and in MECs to Cd(II) (MEC-Cd). The microbial communities

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on these biocathodes were then adaptively evolved to become acclimated to a mixture of these

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chemicals by initially using low concentrations of these metals, followed by higher

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concentrations, to achieve nearly complete removal of these metals. The mixed metal solutions

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were sequentially fed through the first two MFCs for Cr(VI) and Cu(II) removal, and then into

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the MECs for Cd removal. Species of Cr(VI), Cu(II) and Cd(II) were chosen here mainly due to

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their frequent observation in electroplating wastewaters, although the specific concentrations of 5



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these metals in electroplating wastewaters varies depending on the facility.28-29 The performance

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of this three-reactor BES system was evaluated in terms of metals and organics removal, biomass

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production, gas (hydrogen and methane) or power generation, and coulombic efficiencies (CEs).

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High-throughput 16S rRNA gene sequencing was used to characterize the microbial

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communities on the biocathodes of the BESs of the original cathodes acclimated to only one

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metal, compared to biocathodes acclimated to the mixture of metals.

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MATERIALS AND METHODS

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Reactor Setup. Duplicate tubular two-chamber reactors19 were used in all experiments and for

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each of the duplicate reactors three replicate experiments were performed. The MFCs were

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operated at a fixed external resistance of 510 Ω, while a voltage of 0.5 V was added to the circuit

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of the MECs using a power source (DC Power Supply PS-1502DD, Yihua, Guangzhou, China).

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The anodes were made of graphite brushes (PANEX33 160 K, ZOLTEK),30 and the cathodes

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were porous graphite felt (Sanye Co., Beijing, China).19 The anode (25 mL working volume) and

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cathode (40 mL) chambers were separated by a cation exchange membrane (CEM) (CMI-7000

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Membranes International, Glen Rock, NJ) with a projected surface area of 47 cm2.

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Inoculation and Operation. Anodes were inoculated using the effluent of MFCs well

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acclimated to acetate that were originally inoculated using primary clarifier effluent from the

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Lingshui Wastewater Treatment Plant (Dalian, China). Anodes were fed with a phosphate buffer

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(5 mM NaH2PO4) medium containing acetate (1.0 g/L).19 For the initial 3 - 4 fed-batch 6


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acclimation cycles with each lasting 24 – 36 h, cathodes were fed using the same phosphate

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buffer medium prepared in domestic wastewater, except acetate was replaced by NaHCO3 (1.0

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g/L) to provide a carbon source for the autotrophic microorganisms, with metals added to the

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individual reactors at a concentration of 5 mg/L [Cr(VI) or Cu(II) in MFCs, and Cd(II) in MECs].

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After the biofilm was fully acclimated, each fed batch cycle time was set at 4 h. Analytical

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reagents of K2Cr2O7, CuCl2 and CdCl2 were used to prepare these metal solutions. A total of

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96.7% of Cr2O72- was theoretically present as HCrO4- in this catholyte. Wastewater was then

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omitted from the catholyte medium in all subsequent experiments.

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After reproducible cycles of performance in terms of removal of the metals by the

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biocathodes [acclimation time reaching 25 days for both MFC-Cr and MFC-Cu, and 10 days for

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MEC-Cd], the reactor operation was switched to sequential flow of catholyte containing a

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mixture of metals through successive cathode chambers. During this acclimation stage, the

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solution contained 3.0 mg/L of Cr(VI), 1.0 mg/L of Cu(II), and 1.0 mg/L of Cd(II). This solution

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was first used in MFC-Cr(VI), and then at the end of the batch cycle it was transferred into the

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cathode of MFC-Cu(II), followed by transfer into MEC-Cd(II). After multiple cycles using this

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lower concentration metals mixture, the concentrations of each of the metals were increased to 5

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mg/L, and the acclimation cycles were continued. These biofilms were considered to be fully

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acclimated when metal removals were reproducible over 4 - 6 fed batch cycles.

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Two controls were used to evaluate biocathode performance. The first was operation of the reactors under open circuit conditions (OCC) and the second was operation with a closed circuit 7



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but without inoculation of the cathodes (CCC).31 Prior to adding the solutions into the electrode

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chambers, the anolyte and catholyte were sparged with N2 gas for 15 min. All inoculation and

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solution replacements were performed in an anaerobic glove box (YQX-II, Xinmiao, Shanghai),

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and all experiments were run at room temperature (20 ± 3 ºC). The initial pH of all catholytes

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was 5.8, and the solution conductivities in both anolyte and catholyte were adjusted to be 5.8

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mS/cm using KCl.

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Measurements and Analyses. The total chemical oxygen demand (COD), metals

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concentrations (Cr(VI), Cu(II), and Cd(II)), hydrogen and methane concentrations in the

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headspaces of cathode chambers of the MECs, and soluble COD and biomass in the catholyte

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were measured as previously described.,19,22 as summarized in the Supplementary Information

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(SI). Morphologies of the electrodes after Cr(VI), Cu(II) and Cd(II) reduction were examined

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using a scanning electronic microscopy (SEM) (QUANTA450, FEI company, USA) equipped

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with an energy dispersive spectrometer (EDS) (X-MAX 20 mm2/50 mm2, Oxford Instruments,

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UK). Samples for SEM-EDS analysis were prepared as described in SI. Cathode and anode

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potentials, cyclic voltammetry (CV) and linear sweep voltammetry (LSV) analysis were

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performed as previously described19,22 and summarized in the SI. Power density is often

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normalized by the electrode projected surface area. In the MFCs and MECs used here, both the

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graphite brush anodes and porous graphite felt cathodes had very high and much different

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surface areas, but the separator (or membrane) is known to often limit power production in

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MFCs.32 Therefore, power was normalized to the projected surface area of the separator, 8


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allowing comparison to previous studies based on power per area,32 in addition to providing

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absolute values. All the potentials reported were given in relative to a standard hydrogen

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electrode (SHE).

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Bacterial Community Analysis. Samples were collected from the biocathodes of the BESs

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at the end of experiments acclimated to single metal, and the mixture of metals at an identical 5

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mg/L. Since values of metal reduction rate, circuit current and power production in the duplicate

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reactors were highly similar, five pieces of graphite felt (1×1×1 cm3) were removed from

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multiple locations on the electrodes in each of the duplicate reactors and mixed together for

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analysis. Bacterial community results from this sample thus reasonably reflected those in the

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duplicate reactors. These electrodes were fragmented using sterile scissors. Cells attached on the

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electrodes were removed by rinsing three times with sterile water, and concentrated by

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centrifugation. DNA was extracted using Qubit2.0 DNA kit (Sangon Biotech (Shanghai) Co.

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Ltd., China) according to the manufacturer’s instructions. The quality (A260/A280) and quantity

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(A260) of the extracted genomic DNA were determined with a Nanodrop® 1000

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spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The extracted DNA from each

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sample was amplified using PCR reactions and universal 16S rRNA primers (Illumina Miseq).

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After the PCR amplification, the amplicons were recovered using a gelose extraction kit (Cat:

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SK8131, Sangon Biotech (Shanghai) Co. Ltd., China), and rinsed with Tris-HCl solution. After

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amplification and quantification, the amplicons were sequenced using an Illumina Miseq

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following standard procedures. The data were optimized through removal of low-quality 9



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sequences, unrecognized reverse primers, and any ambiguous base calls, with a length < 200 bp.

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High quality sequences were aligned, grouped into OTUs (97% similarity) using the uclust

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algorithm, and a representative sequence from each OTU was classified phylogenetically

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assigned to a taxonomic identity (phylum, class and genus level) using the RDP Naïve Bayesian

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rRNA classifier at a confidence threshold of 90%.33 Beta diversity metrics using weighted

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UniFrac distance matrix34 were calculated from the phylogenetic tree and visualized with

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principal coordinate analysis in quantitative insights into microbial ecology (QIIME software

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v1.3.0) pipeline.35 Rarefaction curves, Shannon diversity indices and species richness estimators

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of Chao1 were generated for each sample using QIIME. All analyses described were conducted

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in triplicates, and the means were reported. The sequence data will be provided upon request.

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Calculations. Cr(VI), Cu(II) and Cd(II) reduction, anodic CEan, cathodic CEca, and yields of metals, biomass, organics, hydrogen and methane were calculated as described in SI.

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RESULTS AND DISCUSSION

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Acclimation of BESs to Individual Metals. Following 25 d of acclimation (details shown in

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Table S1), biocathodes developed in the MFCs produced a maximum of 48 µW (369 µA) (Figure

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S1A) based on a current of 220 µA (510 Ω external resistance) (Figure S1B) with a Cr(VI)

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catholyte, and a maximum of 27 µW (161 µA) (Figure S1C) and 166 µA at 510 Ω (Figure S1D)

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with a Cu(II) catholyte. Each of these MFCs demonstrated efficient metals removal, with 1.21 ±

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0.02 mg/L-h for Cr(VI), and 1.18 ± 0.02 mg/L-h for Cu(II) (Figure S1E). The circuit current in 10


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the MEC used to treat Cd(II) reached 994 µA after 10 days (Figure S1F), with Cd(II) removal at

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a rate of 1.15 ± 0.01 mg/L-h (Figure S1E). This removal rate for Cd(II) was higher than the 0.78

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mg/L-h previously obtained at a similar pH of 6.0,12 but lower than the rate of 3.58 mg/L-h at a

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higher solution conductivity 8.6 mS/cm and lower pH 2.0.13 Both of the reports were at a much

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higher initial Cd(II) concentration of 50 mg/L, and used only abiotic cathodes. Rates of Cd(II)

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removal would be expected to increase in proportion to concentration and by using higher

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solution conductivities. No measurable concentrations of Cr(III), Cu(II) and/or Cd(II) were

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found in the anolyte after a fed batch cycle, excluding the possibility of metal diffusion from the

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cathode to the anode. However, this does not preclude metal retention in the ion exchange

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membrane. CEca reached 92 ± 2% in MFC-Cr and 84 ± 2% in MFC-Cu (Tab.S2), with 60% of

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this current used for Cr(VI) reduction in the MFC-Cr, and 50% for Cu(II) reduction in the

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MFC-Cu. The remaining 32% of the electrons for tests with the MFC-Cr, and 34% for the

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MFC-Cu, were likely lost primarily to oxygen reduction. Despite sparging with ultra pure N2 for

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15-20 min, there was a residual dissolved oxygen concentration of 0.7 – 1.0 mg/L in the initial

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catholyte. This dissolved oxygen could have accounted for 20 - 26% of the coulombs in the

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MFC-Cr, and 28 - 39% in MFC-Cu.36-38

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CV Analyses on Biocathodes Developed with Single Metals. There were different

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predominant reductive peak potentials in the CVs of biofilms using the three different metals,

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with 0.285 V for Cr(VI), 0.075 V for Cu(II), and -0.410 V for Cd(II) (Figures 1A, B and C). In

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each case, these reductive peak potentials were more positive than those of the abiotic controls 11



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despite not excluding the possibility of oxygen evolution from the electrode at high potentials,

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showing the biofilms catalytic activity toward metals reduction. Reductive peak potential was an

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effective parameter for assessing bacterial catalytic activities in the biocathodes.37,39-41 Reductive

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onset potentials (where current values were changed from positive to negative) on the biotic

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cathodes were also more positive than the abiotic controls for all the three different metals (0.730

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V vs 0.685 V for Cr(VI), 0.240 V vs 0.215 V for Cu(II), and -0.205 V vs -0.210 V for Cd(II))

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(inserts in Figure 1). A more positive reductive onset potential on the biotic electrode indicated a

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decrease in the overall free energy of the electron transfer reaction, mainly due to bacterial

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interactions with the electrode surface, which appeared to decrease the energy required for

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Cr(VI), Cu(II) and Cd(II) reduction.42-43 A lower reductive peak current on the biotic cathode,

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however, suggests some degree of mass transfer inhibition due to both the bacterial attachment to

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the electrode surface42 and the formation of these metal precipitants on the electrode surface or

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within the microorganisms. The speciation and location of these metal precipitants may be

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dependent on the specific electrotrophs on the cathodes. Determining the exact fate of these

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metals would require more defined conditions, such as pure culture tests. Cr(VI) would be

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expected to be reduced to insoluble Cr(OH)2+ and Cr(OH)2+, in addition to Cr(OH)3, under the

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present experimental conditions,44-46 which would explain lower biotic peak currents compared

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to abiotic controls. The positive effects of both more positive reductive onset potential and more

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positive peak potentials, together with the negative more mass transfer inhibition on the biotic

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cathode thus presumably resulted in the more efficient Cr(VI), Cu(II) and Cd(II) reductions than 12


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those in the abiotic controls. Other researchers also observed similar results, where biotic

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cathodes for hydrogen evolution and Au(III) or Co(II) recovery had lower reductive peak

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currents than abiotic cathodes despite the biotic cathodes having more positive potentials.42,47

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Current density based on only the biofilm-covered area (which can only roughly estimated by

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microscopic examination) was recently suggested as a relatively good method for assessing BES

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performance, particularly for electrodes of porous graphite felt as used here.48 However, the

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biofilm area could not be determined here as it was very sparse, and the available area could also

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be impacted by the area occupied by metal precipitates. Considering the usually sparse coverage

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of bacteria on the cathodes and the area occupied by the metal precipitates as well as the

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presence of non-electrochemically active microbes in the mixed cultures, the actual current

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density based on effective electrochemically active biofilm-covered area was presumably higher

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than that of the abiotic controls, despite the somewhat higher reduction peak currents (in terms of

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mA) in the abiotic controls (Figures 1A, B and C).

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The peak currents were examined at different scan rates (ν) (1 - 10 mV/s) to determine the

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rate limiting step.49 When a plot of the peak current versus ν is linear, the results indicate

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“thin-film behavior”, and therefore that electron transfer from the electrode to the bacteria is

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slower than intracellular reactions. If the peak is current proportional to ν1/2, this indicates a

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diffusion-controlled regime.37,50-51 Plots of peak current ν were linear for all three metals (Figures

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1D, E and F), demonstrating that current generation was not limited by diffusion for these

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biocathodes. The gradients of the scans decreased in the order of Cr>Cu>Cd, indicating different

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kinetic behavior for microorganisms using these three metals.37,51

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Morphologies of Cathode Surfaces and Product Analysis with Single Metals. The

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cathodes of BESs were sparsely covered by bacteria, with precipitates observed in association

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with groups of cells (Figures S2A, B, and C). Sparse biofilms were consistent with other reports

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of biocathode biofilms,19,40-41,52 with this morphology in contrast to the relatively thick biofilms

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that develop on bioanodes. EDS analysis confirmed the presence of Cr, Cu and Cd in these

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precipitates in the respective BESs (Figures S2D, E, and F). In addition, the presence of carbon,

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oxygen, phosphorus and sulfur may be attributed to the coating of the metal precipitates on the

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microorganisms and the microorganisms on the graphite felt, while gold can be likely traced

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back to sample pretreatment. The observations of carbon and oxygen in the absence of bacteria

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(Figures S2G, H and I) reflected that metals removal was mostly related with the bacteria. A 48 h

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abiotic control experiment demonstrated there were no changes in the concentrations of Cr(VI),

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Cu(II) and Cd(II) in the solution, excluding the formation of precipitates between metals and

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anions in the catholyte. The absence of carbon and sulfur elements in the metal precipitates-only

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samples (Figures S2J, K, and L) further precluded the formation of metal sulfide and carbonate

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metal complexes in the present system. The ratio of elemental metal and oxygen in EDS can be

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used to deduce the product composition.10,53-55 The Cr:O ratio in the precipitates was 0.30

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(Figure S2J), approaching that of 0.33 for a precipitate of Cr(OH)3. The Cu:O ratio was 10

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(Figure S2K), and the Cd:O was 7 (Figure S2L), which were much higher than those of the final 14


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products of 2 or 1 for Cu2O or CuO, and 1 for CdO. These ratios therefore show that Cu(II) and

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Cd(II) were mostly reduced to the pure metals. Based on the final pHs that ranged from 5.9 – 6.1,

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the calculated forms of the metals using the Nernst equation would be Cr(III), Cu(0), and Cd(0)

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(Figure S3). The speciation of chromium at different potentials was extensively reported to be

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reductively changed into precipitates of Cr(OH)2+, Cr(OH)2+ and Cr(OH)3 at pHs of 5.9 –

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6.1.44-46 In the case of Cr(VI) conversion in the biocathode/abiotic cathode bioelectrochemical

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systems, as summarized in Table S3, all Cr(VI) was reductively changed into Cr(III). Similarly,

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Cu(II) was mainly reduced to Cu(0) with tiny Cu2O whereas Cd(II) was transformed into Cd(0)

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(Table S3). Our results were therefore consistent with these previous reports on the speciation of

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these metals at these pHs.

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Biocathodes Adaptively Evolved to Low and Elevated Mixed Metals. Mixed metal

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solutions containing Cr(VI), Cu(II) and Cd(II), at each 5 mg/L, was sequentially fed into the

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MFCs or MECs individually acclimated only to a single metal. Reduction rates [0.49 ± 0.03

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mg/L-h for Cr(VI); 0.37 ± 0.05 mg/L-h for Cu(II); and 0.27 ± 0.03 mg/L-h for Cd(II)] were all

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much lower compared to rates when using only the individual metals (Figure S1C). It is well

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known that combinations of heavy metals are generally more toxic than single heavy metals.25-28

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The apparent decrease in metal reduction rates in response to the mixed metal influents reflects

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the detrimental impact of the other metals on the bacterial communities, in good agreement with

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previous reports.25-28

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The reactors were next acclimated to low concentrations of these metals (details in SI, and

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Figures S4 and 5), and then switched to higher concentrations of 5 mg/L of each metal for 11 –

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15 cycles. Metal reduction rates were greatly improved for Cr(VI), as there was complete

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removal to below detectable limits (< 0.04 µg/L) at a rate of 1.24 ± 0.01 mg/L-h (Figure 2A).

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This rate was 2.5 times of that without adaptive evolution (0.49 ± 0.03 mg/L-h) and slightly

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lower than the rate obtained using the lower concentration mixed metal solution (1.48 ± 0.02

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mg/L-h) (Figure S4A), but comparable to that obtained with the single metal solution (1.21 ±

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0.02 mg/L-h) (Figure S1C), demonstrating successful adaptation to the presence of elevated

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mixed metals. There was little removal of Cu(II) or Cd(II) in this reactor or in the controls

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(OCC-biotic and CCC-abiotic, 0.12 ± 0.01 - 0.22 ± 0.02 mg/L-h). Metal removals in the

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CCC-abiotic controls were mainly ascribed to chemical reduction and physical adsorption56

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whereas observation in the OCC-biotic controls included processes associated with the presence

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of bacteria, bio-adsorption in addition to physical adsorption, and biological reduction of metals.

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While adsorption may have contributed to metal removal in this study, it is not expected to be

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large here due to the sparse biofilms that typically develop on biocathodes, and there was

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relatively little metal removal in abiotic controls. Although there were decreases in the OCP and

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maximum power produced by the MFC originally acclimated to only Cr(VI) (Figures 2D and

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S4D), the high rate of Cr(VI) reduction demonstrated the successful acclimation of the

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community relative to Cr(VI) removal.

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The biocathodes in the subsequent reactors also showed good acclimation to the elevated

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mixed metal solutions. In the second MFC [originally acclimated to only Cu(II)], complete Cu(II)

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removal was observed at a rate of 1.07 ± 0.01 mg/L-h, with very little reduction of Cd(II) (Figure

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2B). This reduction rate for Cu(II) was 2.9 times of that without adaptive evolution (0.37 ± 0.05

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mg/L-h), 2.0 times of that with a low concentration of mixed metals (0.53 ± 0.02 mg/L-h)

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(Figure S4B), and a slightly lower rate than that obtained with the reactors acclimated to only Cu

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(II) (1.18 ± 0.02 mg/L-h) (Figure S1C). The OCP of 0.34 V and maximum power of 26 µW (188

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µA) (Figure 2E) were similar to those obtained with the reactor acclimated to only Cu(II) (0.33 V,

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and 28 µW) (Figure S1B).

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In the third reactor, Cd(II) was also completely removed at a rate of 0.98 ± 0.01 mg/L-h

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(Figure 2C), which was 3.6 times of that without any adaptive evolution (0.27 ± 0.03 mg/L-h),

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but slightly lower than that for the MEC acclimated to only Cd(II) (1.15 ± 0.01 mg/L-h). The

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Cd(II) reduction rates were also greater than those obtained in either of the controls (CCC-abiotic

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and OCC-biotic, 0.51 ± 0.06 – 0.89 ± 0.03 mg/L-h). The measured cathode potential in the

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MECs of -0.61 V (Figure 2F) was identical with that measured using the low concentration

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mixed metal solution (Figure S4F).

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Compared to results with a low concentration of mixed metals, an elevated mixed metal

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concentration was beneficial not only for anodic CEs in all three reactors, but also product yields

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in the MFC that removed Cu (Table S2). Metal reduction rates in the biocathode reactors could

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not be directly compared with conventional electrochemical processes due to greatly different 17



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operational conditions of much high temperatures of 70 – 90 ºC and more acidic pHs of 1.0 – 2.0,

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both of which reasonably favored metal reduction.3,57-60 However, apparently higher metal

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reduction rates in the biocathode reactors were observed than the CCC-abiotic controls,

322

indicating the higher performance of biocathodes than a conventional electrochemical process

323

without microorganisms. The energy produced by the MFCs (0.6 kWh/kg-Cr and 0.3

324

kWh/kg-Cu) was lower than the power required for the Cd removal in the MEC (10.5

325

kWh/kg-Cd). Considering the hydrogen energy of 2.8 kWh/kg-Cd concomitantly produced in

326

MEC for Cd(II) reduction, the overall power required to operate these reactors would only be 6.8

327

kWh/kg-metal. This is substantially lower than the 73 - 113 kWh/kg-metal used for purely

328

electrochemical processes.57-60

329

CV results indicated slight increases in reductive peak currents in all the reactors acclimated

330

to the elevated concentrations of mixed metals (Figures 3A, B and C), relative to those for the

331

low concentrations of mixed metals. This result illustrates the positive link between peak current

332

and substrate concentration, which always affect the electron transfer processes observed in

333

CVs.42,47-48 These changes were consistent with results showing improved metals removals

334

(Figure 2). Current was a linear function of the scan rate for all the three biofilms with these

335

elevated mixed metals (Figures 3D, E and F), demonstrating a lack of diffusional mass transfer

336

limitations in these systems.

337 338

Changes in Bacterial Communities. From six generated libraries, a total of 23254 high-quality 16S rRNA gene sequences were obtained with an average length of 440 nucleotides 18


Page 19 of 32


339

(Table S4). These sequences were assigned into 2235 OTUs with a distance limit of 0.03.

340

Although the rarefaction curves did not demonstrate a plateau (Figure S6), the Good’s Coverage

341

estimators indicated that the sizes of libraries were sufficient to cover 93 - 97% of the bacterial

342

communities (Table S4).

343

An analysis of these data show that the diversity of the microbial communities decreased,

344

and that different bacterial communities were identified on the biocathodes of the reactors

345

acclimated to the mixed metal solutions, compared to reactors acclimated only to a single metal.

346

The decrease in diversity was shown by a decrease in the Shannon indices from 4.62 to 4.22 for

347

Cr(VI), 4.60 to 4.15 for Cu(II), and 4.08 to 3.86 for Cd (II) (Table S4). These decreases in

348

diversity likely resulted from the greater toxicity of the mixed metals compared to only single

349

metals.26-28

350

Analysis of the bacterial communities based on a Weighted Fast UniFrac Principle

351

Coordinates Analysis, indicated little similarity in the communities acclimated to a single metal

352

with those acclimated to the mixtures of metals (Figure 4A). Only the MECs acclimated to Cd(II)

353

alone, or the Cd(II)-acclimated reactor subsequently acclimated to the mixed metals, grouped in

354

the same quadrant. The reactors acclimated to the mixed metals were also relatively randomly

355

dispersed. A dendrogram constructed on the basis of community phylogenetic lineages further

356

supported differences in these bacterial communities based on an absence of any close

357

alignments of the communities acclimated to single metals with those acclimated to mixtures of

358

metals (Figure 4B). These findings demonstrate that the compositions of the bacterial 19



359

communities were initially different from each other when acclimated to the individual metals,

360

and that they further diverged after acclimation to the metal mixtures.

361

The bacterial community consisted of 8 phyla, with the majority of sequences (21.3 - 72.0%)

362

belonging to Proteobacteria, which are frequently observed in BESs (Figure 5A).61-62 Compared

363

to the respective single metal acclimated evolved biofilms, the relative abundance of

364

Proteobacteria decreased in reactors acclimated to mixed metals that were originally acclimated

365

to Cu and Cd, whereas Proteobacteria and Actinobacteria increased in reactors originally

366

acclimated to only Cr. The acclimation to the mixed metals stimulated the relative abundance of

367

Firmicutes and Tenericutes in all reactors, and Bacteroidetes in the MFC originally acclimated to

368

Cu (Figure 5A). Conversely, Synergistetes was absent in all communities following acclimation

369

to the mixed metals.

370

Four classes (Alpha-, Beta-, Gamma-, and Delta-) within Proteobacteria were observed from

371

all communities except for Deltaproteobacteria in the MFCs originally acclimated to Cr (Figure

372

5B). Mixed metal acclimation led to a relative increase in Alphaproteobacteria.

373

Betaproteobacteria were the most abundant in all the communities with single metals and the

374

biofilm of MFC with the mixed metal solution that was originally acclimated to Cr.

375

Genus level characterization suggests that bacteria most similar to Geobacter were relatively

376

abundant on biocathodes acclimated only to Cr (10.7%) or Cu (10.1%) (Figure 6). In the abiotic

377

controls, no acetate was observed in the catholyte, suggesting that there was limited or no

378

diffusion of acetate from the anode to cathode at the beginning of the experiment that could have 20


Page 20 of 32

Page 21 of 32


379

sustained substantial Geobacter growth. However, the organics formed in the catholyte (Table

380

S2) could have allowed for heterotrophic growth of Geobacter, as this genus is not known to be

381

autotrophic.61-62 The predominant genus in the biocathodes of MFC acclimated to Cr was

382

Dysgonomonas (10.2%), but Azoarcus (16.7%) were relatively abundant in these reactors

383

following acclimation to the mixed metal solution. Similarly, dominant genera were shifted from

384

Geobacter (10.1%) to Myroides (10.8%) and an unclassified bacterium (10.1%) for reactors

385

switched from only Cu to mixed metals. In the case of Cd, Achromobacter (16.8%) and Brucella

386

(15.7%) were predominantly observed in biofilm with treating only Cd, compared to Alcaligenes

387

(16.5%), Tissierella (14.7%), Brevundimonas (10.0%) and Aquamicrobium (9.4%) with mixed

388

metal solutions. While ecological roles of these bacteria cannot be ascertained based on their

389

presence, these changes do show that the switch from a single metal to the metals mixtures

390

altered the composition of the microbial communities.

391

The use of biocathodes in BESs shows promise as a low energy and sustainable method for

392

remediation of toxic metals in wastewaters. These studies have shown that biocathode BESs can

393

be used to remediate Cr(VI), Cu(II) and Cd(II) individually, or together in mixtures, using

394

cathodes of MFCs and MECs. Following acclimation to a single metal, the microbial populations

395

in reactors exhibited adaptive evolution whereby the original target metals could be completely

396

removed at rates similar to that originally obtained with the reactors acclimated to a single metal.

397

In all cases, the reactors did not show acclimation for removal of the other target metals,

398

providing an effective strategy for sequential and selective recovery of chromium, copper and 21



399

cadmium from mixed wastes. Community analysis showed that the bacterial compositions were

400

significantly different after the elevated mixed metal influents, and generally they had less

401

diverse communities following exposure to the mixed metal solutions. Although there was no

402

clear identification of the specific genera responsible for the metals reduction, as the

403

communities remained relatively diverse, future identification of specific bacterial species could

404

help to improve our understanding of the operation of these systems. Such information could

405

make it possible to acclimate biocathodes in a single reactor to remove and recover the multiple

406

metals, when those metals have similar half cell potentials for reduction.

407

Compared to the biocathodes with only Cr(VI) reduction and abiotic cathodes under extreme

408

acidic conditions for Cr(VI), Cu(II) and/or Cd(II) reduction (Table S3), the present study

409

suggests it should be possible to treat neutral pH and low-strength wastewaters from

410

electroplating and mining industry processes. Practical implementation will depend on the

411

long-term operation of the biocathodes and other characteristics of the wastewaters, as well as

412

the cost of the system relative to conventional treatment processes. The costs of the materials

413

used in MFCs and MECs are decreasing,63-65 but at this point in time it is likely that this

414

technology is not yet ready for commercial applications as larger-scale studies would be required

415

for evaluating the process prior to commercial and full scale applications.

416

ACKNOWLEDGEMENTS

417

The authors gratefully acknowledge financial support from the National Natural Science

418

Foundation of China (No. 21377019), the National Basic Research Program of China (No. 22


Page 22 of 32

Page 23 of 32


419

2011CB936002), Specialized Research Fund for the Doctoral Program of Higher Education

420

“SRFDP” (No. 20120041110026), the Program for Changjiang Scholars and Innovative

421

Research Team in University (IRT_13R05) and the Programme of Introducing Talents of

422

Discipline to Universities (B13012).

423

Supporting Information Available

424

Information as mentioned in the text. This information is available free of charge via the Internet

425

at http://pubs.acs.org.

426 427

REFERENCES

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456

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Page 27 of 32


-4

10 bio-5mg/L abiotic-5mg/L bio-0mg/L abiotic-0mg/L

-10

0.65 0.70 0.75 0.80 p

0.02 0.01 0.00 -0.01 -0.02

abiotic-v1 abiotic-v1/2

y = -0.4762x - 0.468 R² = 0.9521

-2.0

y = -0.0863x - 2.7389 R² = 0.9831 y = -0.3627x - 2.3984 R² = 0.9516

0.200.220.240.26

-4.0 0.0

bio-5mg/L abiotic-5mg/L bio-0mg/L abiotic-0mg/L

bio-v1 abiotic-v1

C -1.0

0 0.04 0.02 0.00 -0.02 -0.04 -0.06 -0.22

-2

E

y = -0.1137x - 0.9128 R² = 0.9906

-3.0

-1

y = -0.2853x - 8.9798 R² = 0.9812

-1.0

Current (mA)

Current (mA)

y = -1.1977x - 7.8579 R² = 0.9469

bio-v1 bio-v1/2

1

-0.20

bio-v1/2 abiotic-v1/2

F

y = -0.3995x - 1.125 R² = 0.9787 y = -0.0941x - 1.5051 R² = 0.9915

-2.0

-3.0

y = -0.3721x - 1.5789 R² = 0.9799

y = -0.0876x - 1.9331 R² = 0.9917

-0.18

-3

-4.0 -1.5

606 607 608 609 610 611 612 613

y = -1.3104x - 5.4266 R² = 0.9592

B

0

2

y = -0.3102x - 6.6646 R² = 0.9818

-14 0.0

bio-5mg/L abiotic-5mg/L bio-0mg/L abiotic-0mg/L

-4 3

-8

-12

2

-2

D

-10

0.2 0.1 0.0 -0.1 -0.2

-15 4

Current (mA)

Current (mA)

0

-5

bio-v1/2 abiotic-v1/2

-6

Current (mA)

Current (mA)

5

bio-v1 abiotic-v1

A

-1.0

-0.5

0.0 0.5 1.0 Potential (vs. SHE, V)

1.5

-4

0

4 Scan rate (mV/s)

8

12

Figure 1 (A, B and C) CV tests and (D, E and F) peak current – scan rate on the biocathodes of (A and D) MFC-Cr at an initial Cr(VI) of 5 mg/L, (B and E) MFC-Cu at a Cu(II) of 5 mg/L, and (C and F) MEC-Cd at a Cd(II) of 5 mg/L. Inserts in subfigures (A, B and C): enlarging for comparisons of biocathodes and abiotic cathodes.

27



0.5

Metal concentrations (mg/L)

5

OCCs-Cr OCCs-Cu OCCs-Cd

abiotic-Cr abiotic-Cu abiotic-Cd

0.4

Voltage output (V)

4 3 2

30

20 0.2 10

0.1 0

0 0

1

2 Time (h)

3

0 0

4

6

0.5 bio-Cu bio-Cd


5

OCCs-Cu OCCs-Cd

0.4

Voltage output (V)

3 2

B

1

100

200 Current density (µA)

300

400 40

mixed-elevated-voltage mixed-low-voltage mixed-elevated-power mixed-low-power

abiotic-Cu abiotic-Cd

4

E 30

0.3 20 0.2 10

0.1 0.0

0 0

1

2 Time (h)

bio-Cd

OCCs-Cd

3

0 0

4

5

100

200 Current density (µA)

300

400

-0.600 abiotic-Cd

F

4 -0.605

Cathode potential (V)


D

0.3

A

1

40 mixed-elevated-voltage mixed-low-voltage mixed-elevated-power mixed-low-power

Power production (µW)

bio-Cr bio-Cu bio-Cd

Power production (µW)

6

3

-0.610

2

-0.615

1

C

0

-0.620 0

614 615 616 617 618

Page 28 of 32

1

2 Time (h)

3

4

10

12

14 16 Number of cycle

18

Figure 2 Change of Cr(VI), Cu(II) and Cd(II) in (A) MFC-Cr, (B) MFC-Cu, and (C) MEC-Cd, polarization curves in (D) MFC-Cr and (E) MFC-Cu, and (F) cathode potential of MEC-Cd with elevated mixed metals of identical Cr(VI), Cu(II) and Cd(II) at 5 mg/L.

28


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0

5

mixed-low mixed-elevated

mixed-low mixed-elevated -2

Current (mA)

Current (mA)

A 2

-1

y = -0.3835x - 2.6135 R² = 0.9819

-4

y = -0.375x - 3.0114 R² = 0.9935

-6

p

-4 1.5

-8 0 mixed-low mixed-elevated

B

-1

0.5

-3 0

-1.5 2 C



Current (mA)

-1

0

y = -0.1246 x - 0.8378 R² = 0.9803 y = -0.1425 x - 0.8570 R² = 0.9827

-2

-1

-3

-2 -1.0

620 621 622 623 624

F

C

1

Current (mA)

y = -0.1138x - 0.8915 R² = 0.9833

-2

-0.5

E

y = -0.1221x - 0.763 R² = 0.9779

Current (mA)

Current (mA)


619

D

-0.5

0.0 0.5 1.0 Potential (vs. SHE, V)

1.5

0

2

4

6 8 Scan rate (mV/s)

10

12

Figure 3 Comparison of (A, B and C) CV tests and (D, E and F) peak current – scan rate on the biocathodes of (A and D) MFC-Cr, (B and E) MFC-Cu, and (C and F) MEC-Cd under mixed metal influents of either Cr(VI), Cu(II) and Cd(II) at an identical 5 mg/L, or 3 mg/L Cr(VI), 1 mg/L Cu(II) and 1 mg/L Cd(II).

29



625 626 627 628 629 630

Figure 4 Weighted Fast UniFrac (A) principle coordinates analysis and (B) cluster of the bacterial communities on the basis of phylogenetic lineages that samples contain.

30


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120 others Synergistetes

unclassified Bacteroidetes

Actinobacteria Tenericutes

Firmicutes Proteobacteria

Deferribacteres

Relative abundance (%)

100

80

60

40

20

0 120 others Betaproteobacteria

Deltaproteobacteria Alphaproteobacteria

Gammaproteobacteria B

100

Relative abundance (%)

80

60

40

20

0 single

631 632 633 634 635 636 637 638 639 640 641

mixed MFC-Cr

single

mixed MFC-Cu

single

mixed MEC-Cd

Figure 5 Comparison of relative abundance of bacterial reads retrieved from the biocathodes adaptively evolving with single and mixed metals, and classified at (A) the phylum level and (B) the class level distribution of the most dominant phylum of Proteobacteria. Phyla and classes that represent less than 1.0% of the total bacterial community composition were classified as “others”.

31



642 643 644 645

Figure 6 Comparison of relative abundance of bacterial reads retrieved from the biocathodes adaptively evolving with single and mixed metals at the genus level. Genera that represent less than 1.0% of the total bacterial community composition were classified as “others”.

646

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Adaptively Evolving Bacterial Communities for ... - ACS Publications

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