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Biotransformation of Furanic and Phenolic Compounds with Hydrogen Gas Production in a Microbial Electrolysis Cell Xiaofei Zeng, Abhijeet P Borole, and Spyros G. Pavlostathis Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02313 • Publication Date (Web): 27 Oct 2015 Downloaded from http://pubs.acs.org on November 1, 2015

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Biotransformation of Furanic and Phenolic Compounds with Hydrogen Gas

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Production in a Microbial Electrolysis Cell

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Xiaofei Zeng1, Abhijeet P. Borole2,3, and Spyros G. Pavlostathis1,*

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School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0512, United States 2 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States 3 Bredesen Center for Interdisciplinary Research and Education, The University of Tennessee, Knoxville, TN 37996, United States

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*Corresponding author. Phone: 404-894-9367; fax: +404-894-8266; E-mail address: [email protected] (S. G. Pavlostathis)

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ABSTRACT: Furanic and phenolic compounds are problematic byproducts resulting from the

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breakdown of lignocellulosic biomass during biofuel production. The capacity of a microbial

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electrolysis cell (MEC) to produce hydrogen gas (H2) using a mixture of two furanic (furfural,

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FF; 5-hydroxymethyl furfural, HMF) and three phenolic (syringic acid, SA; vanillic acid, VA;

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and 4-hydroxybenzoic acid, HBA) compounds as the substrate in the bioanode was assessed. The

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rate and extent of biotransformation of the five compounds, efficiency of H2 production, as well

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as the structure of the anode microbial community were investigated. The five compounds were

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completely transformed within 7-day batch runs and their biotransformation rate increased with

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increasing initial concentration. At an initial concentration of 1,200 mg/L (8.7 mM) of the

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mixture of the five compounds, their biotransformation rate ranged from 0.85 to 2.34 mM/d. The

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anode coulombic efficiency was 44-69%, which is comparable to wastewater-fed MECs. The H2

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yield varied from 0.26 to 0.42 g H2-COD/g COD removed in the anode, and the bioanode

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volume-normalized H2 production rate was 0.07-0.1 L/L-d. The biotransformation of the five

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compounds took place via fermentation followed by exoelectrogenesis. The major identified

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fermentation products that did not transform further were catechol and phenol. Acetate was the

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direct substrate for exoelectrogenesis. Current and H2 production were inhibited at an initial

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substrate concentration of 1,200 mg/L, resulting in acetate accumulation at a much higher level

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than that measured in other batch runs conducted with a lower initial concentration of the five

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compounds. The anode microbial community consisted of exoelectrogens, putative degraders of

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the five compounds, and syntrophic partners of exoelectrogens. The MEC H2 production

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demonstrated in this study is an alternative to the currently used process of reforming natural gas

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to supply H2 needed to upgrade bio-oils to stable hydrocarbon fuels.

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TOC Art

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INTRODUCTION

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Lignocellulosic biomass is a promising feedstock for the production of biofuels using thermal,

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chemical or biological processes. Breakdown of lignocellulosic biomass, regardless of the

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process used, typically results in furanic and phenolic byproducts, which are inhibitory,

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problematic compounds. For instance, furanic and phenolic compounds in lignocellulosic

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hydrolysates are highly inhibitory to H2 and ethanol producing, fermentative microorganisms,

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which has been a persistent challenge in biofuel production through fermentation processes.1,2 In

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pyrolysis, a thermal process reforming biomass to bio-oil, the presence of polar and oxygen-rich

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compounds (e.g., furan aldehydes and phenolic acids) makes the bio-oil acidic, unstable,

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requiring H2 in the downstream hydrogenation process to upgrade the bio-oil to a stable fuel.3 In

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order to overcome the negative effect of furanic and phenolic compounds, physical and/or

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chemical removal, as well as microbial and enzymatic processes have been explored.4 However,

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these methods have the trade-offs of lowering biofuel yield and do not lessen the challenge of

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downstream wastewater treatment.4 Thus, a process to directly utilize furanic and phenolic

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compounds to produce biofuels would be an improvement over the existing methods.

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Microbial electrolysis cell (MEC) technology is a bioelectrochemical process, which

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produces H2. Exoelectrogenic bacteria in the anode oxidize organic substrates by transferring

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electrons to the electrode and then to the cathode. With a voltage input (> 0.3 V), protons

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transferred from the anode to the cathode via a cation exchange membrane are reduced to H2.5

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Unlike other H2 producing bioprocesses, the MEC produces H2 through an abiotic half-reaction

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in the cathode, with the supply of electrons from the microbially-assisted half-reaction in the

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anode. This feature of MEC eliminates the need for H2-producing bacteria, which are highly

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susceptible to furanic and phenolic compounds.1,6 Thus, the MEC is potentially able to convert

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these problematic compounds to H2, which in turn can be used in the hydrogenation process and

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thus minimize the external H2 supply currently generated by reforming natural gas.

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Given the advantage of the MEC technology, recent studies have investigated the

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potential of integrating MEC in biofuel production platforms. Lignocellulosic effluent, refinery

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wastewater and switchgrass-derived bio-oil aqueous phase have been treated in MECs with

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simultaneous H2 production.7,8,9 However, the fate and effect of furanic and phenolic

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compounds, which are the problematic components of the above-mentioned feedstocks, remain

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unknown. This study investigated a MEC for H2 production from two furanic and three phenolic

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compounds. This is the first attempt to understand the fate of specific inhibitory compounds and

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their potential to produce H2 in a MEC. The most commonly used substrates in MECs are acetate

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and wastewater.10-12 However, acetate is a favorable organic substrate for exoelectrogens,13,14

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whereas, furanic and phenolic compounds are less biodegradable and have not been reported as

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direct substrates for exoelectrogens. On the other hand, wastewater streams generally include

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easily biodegradable components, which may be the major contributors to H2 production. Two

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previous studies used furanic and phenolic compounds as the substrate in the anode of a

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microbial fuel cell (MFC), but had mixed results. Catal et al. reported that, with the exception of

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HMF, use of nine individual furanic and phenolic compounds (2-furaldehyde, syringaldehyde,

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vanillin, trans-cinnamic acid, trans-4-hydroxy-3-methoxy-cinnamic acid, 4-hydroxy-cinnamic

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acid, 3,5-dimethoxy-4-hydroxy-cinnamic acids, benzyl alcohol and acetophenone) did not

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generate voltage.15 In contrast, Borole et al. demonstrated that an anode microbial consortium

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was able to convert furfural, HMF, 4-hydroxybezaldehyde, hydroxyacetophenone, and vanillic

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acid to electricity.16 Thus, the question remains whether a MEC anode microbial community can

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use furanic and phenolic compounds as the sole carbon and energy source to produce H2 in the

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cathode.

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The overall goal of this study is to produce H2 through the biotransformation of specific

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furanic and phenolic compounds using MEC technology. The specific objective of the work

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presented here was to enrich a microbial community, assess its capacity to biotransform two

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furanic and three phenolic compounds, and produce H2 in a batch-fed MEC. Biotransformation

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of the selected furanic and phenolic compounds, formation of metabolites, production of current

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and H2, as well as the structure of the anode microbial community were investigated.

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MATERIALS AND METHODS

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Chemicals. Furfural (FF, 99%), 5-hydroxymethyl furfural (HMF, ≥ 99%), syringic acid

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(SA, ≥ 95%) and 4-hydroxybenzoic acid (HBA, ≥ 99%) were purchased from Sigma-Aldrich (St.

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Louis, MO). Vanillic acid (VA, ≥ 99%) was purchased from Alfa Aesar (Ward Hill, MA). The

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five compounds are soluble in water (1.5-364 g/L at 25oC), not volatile (Henry’s law constant =

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10-14 - 10-6 atm-m3/mol), and have a low hydrophobicity (log Kow = -0.09 to 1.58)(Table S1;

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Supporting Information). The standard potential at pH 7.0 (E  ) of the five compounds is from -

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0.388 to -0.303 V (Table S2) compared to the E  value of -0.414 V for proton reduction to H2.

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Inoculum. The bioanode inoculum was a piece of biofilm-attached carbon felt from a

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MFC anode developed at the Oak Ridge National Laboratory (Oak Ridge, TN), which over time

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had been fed with a mixture of furanic/phenolic compounds, corn stover hydrolysates, and

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switchgrass pyrolysis aqueous stream.9,16,17 The original inoculum of this MFC was a sample

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collected from a municipal anaerobic digester. The bioanode inoculum was further enriched in

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the present study in a different MFC anode, fed with the mixture of the two furanic and three

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phenolic compounds, prior to being transferred to a MEC anode. 5 ACS Paragon Plus Environment

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MFC Setup and Operation. An air-cathode MFC was set up to enrich the inoculum.

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The anode electrode was porous carbon felt (5 stripes, 1 cm × 1 cm × 10 cm each; Alfa Aesar,

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Ward Hill, MA) tied to a stainless steel rod. The anode chamber was a modified square glass

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bottle with an open channel on one side. The empty bed volume was 250 mL, and the liquid

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volume was 200 mL due to electrode displacement. The cathode was a membrane-electrode

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assembly with a surface area of 5.7 cm2 purchased from Fuel Cells Etc (College Station, TX),

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which was made of a cation exchange membrane and carbon cloth containing 0.5 mg/cm2 Pt.

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The cathode was clamped to the side channel extended from the anode chamber and exposed to

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air on one side.

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A piece of biofilm-attached carbon felt (approximately 1 cm ×1cm × 3 cm) was placed in

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the center of the anode carbon felt electrode. The anode medium consisted of (in g/L): NH4Cl,

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0.31; KCl, 0.13; NaH2PO4·H2O, 2.45; Na2HPO4, 4.58, along with trace metals and vitamins.18

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The pH of the medium was 7.0. The anolyte was deoxygenated by bubbling N2 through the

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liquid phase prior to use, and was continuously mixed magnetically. The MFC was maintained at

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room temperature (20-22°C). A mixture of the five compounds at equal electron equivalents

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(each at 62.5 mg COD/L) and a total concentration of 200 mg/L (312 mg COD/L) was fed to the

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MFC anode once a week (7-day fed-batch). During the first ten feeding cycles (~70 days),

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glucose (200 mg/L) was fed along with the five compounds to enhance microbial growth. A

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variable resistor was placed between the anode and cathode, and its resistance was gradually

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reduced from 500 to 250 and then to 100 Ω, in order to promote the growth of exoelectrogenic

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bacteria. The voltage was recorded by a potentiostat (Interface 1000TM, Gamry Instruments,

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Warminster, PA); culture enrichment lasted for 6 months. The MFC activity stabilized with a

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mean maximum current of 1.25 mA, soluble COD (sCOD) removal of 50-60% and coulombic

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efficiency (CE) of 40-60%, measured over the last 20 feeding cycles.

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MEC Setup and Operation. An H-type MEC was developed with two square glass

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bottles separated by a cation exchange membrane (Nafion 117, 5.7 cm2; Dupont, Wilmington,

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DE) and maintained at room temperature (20-22oC). Both chambers and the anode electrode had

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the same configuration as the above-described MFC anode. The cathode electrode was a carbon

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cloth containing 0.5 mg/cm2 of Pt (5 cm × 6 cm; Fuel Cell Etc, College Station, TX). A gas

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collection burette using displacement of an acid brine solution (10% NaCl w/v, 2% H2SO4 v/v)

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was connected to each chamber headspace for gas volume measurement (Figure S1; Supporting

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Information).

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The inoculation procedure and anolyte of the MEC were the same as for the above-

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described MFC. The MEC catholyte was a 100 mM phosphate buffer (pH 7.0), deoxygenated by

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bubbling N2 prior to use. Both the anolyte and catholyte were replaced at the beginning of each

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feeding cycle. During the startup, the anode chamber was amended with 200 mg/L of the five

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compounds mixture (same composition as the MFC feed). After the startup, which lasted for 9

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weeks, the total initial concentration of the substrate mixture was increased from 200 to 400,

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800, and then to 1,200 mg/L. A voltage of +0.6 V was set to the MEC anode relative to the

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cathode, and the current was recorded every 4 hours by the potentiostat. The duration of each

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feeding cycle was 6-7 days until the current dropped below 0.2 mA. The anolyte and catholyte

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were continuously mixed magnetically and the anode and cathode headspaces were initially

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filled with N2.

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Two controls were evaluated. Control 1 was used to investigate the stability of the five compounds in the presence of the porous carbon felt and anolyte. Four serum bottles, each

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containing 100 mL anolyte and 200 mg/L compound mixture, were kept under N2 headspaces.

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Two of the bottles contained carbon felt with equivalent quantity (v/v) as in the MEC anode. The

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concentration of the five compounds was monitored for 7 days. Control 2, setup with a biomass-

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free anode electrode in the MEC, was used to evaluate the potential contribution of the applied

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voltage on current production and transformation of the five compounds in the absence of

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microbial activity.

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Microbial Community Analysis. Microbial community analysis of the MEC anode was

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performed after 9 weeks (9 feedings) from the startup. A piece of anode electrode with attached

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biofilm (approximately 1 cm ×1cm × 3 cm) was washed several times with the anolyte and then

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cut into small pieces (< 0.5 cm). The genomic DNA was extracted with the PowerSoil DNA

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isolation Kit (MO BIO Laboratories, Carlsbad, CA), according to the manufacturer’s

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instructions. The concentration and purity of the DNA sample were determined with a ND-1000

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spectrophotometer (NanoDrop Technologies, Wilmington, DE). The 16S rRNA gene was

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sequenced using an Illumina MiSeq system (LC Science, Houston, TX). Bacterial primers 319F

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(5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3')

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were used to amplify the V3-V4 hypervariable regions of the 16S rRNA gene. The obtained

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sequences were clustered into Operational Taxonomic Units (OTUs) with 97% similarity. The

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longest read in each OTU was chosen as the representative sequence for taxonomic classification

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using the RDP classifier Version 2.7. The sequence-based phylogenetic tree of the abundant

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bacteria (>1% abundance) was constructed by applying the neighbor-joining algorithm using the

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program MEGA 6.06. The tree topology was evaluated by bootstrap resampling analysis of 1000

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data sets. The representative sequences of the abundant species have been deposited to GenBank,

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National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/) with sequence

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accession numbers from KT124613 to KT124626.

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Analytical Methods. The furanic and phenolic compounds were quantified using a high

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performance liquid chromatography (HPLC) unit equipped with a UV-Vis detector (Agilent

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1100, Santa Clara, CA). A HPX-87H column (BioRad, Hercules, CA) was used with an eluent of

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15% acetonitrile in 5 mM H2SO4 (v/v) at a flow rate of 0.6 mL/min.16 The wavelength of 280 nm

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and 210 nm was used for the furanic and phenolic compounds, respectively. Acetate and other

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volatile fatty acids were quantified by the same HPLC method at the wavelength of 210 nm,

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except that the eluent was 5 mM H2SO4 without any organic solvent. Metabolites of the five

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compounds were identified using an LC/MS/MS unit (Agilent 1260 Infinity LC system, 6410

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Triple Quad MSD) equipped with a Kinetex biphenyl column (3×150 mm, 5 µm; Phenomenex,

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Torrance, CA). The eluent consisted of (A) 5 mM ammonium acetate with 0.5% acetic acid in

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acetonitrile (v/v) and (B) 5 mM ammonium acetate in 0.5% acetic acid (v/v) at a flow rate of 0.5

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mL/min, using gradient elution as follows: eluent A was increased from 2% to 30% in 2.3 min

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and to 90% in 1.2 min, and then was maintained at 90% for 2.5 min. The MS/MS was operated

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in both positive and negative modes at 100 eV in an m/z range of 50-250. The product ions of the

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same molecular weight as hypothesized metabolites were fragmented, and the fragmentation

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patterns were compared with those of purchased pure chemicals. Soluble chemical oxygen

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demand (sCOD) and pH were measured following procedures outlined in Standard Methods.19

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Total gas production was measured by the acid brine solution displacement in the burettes,

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equilibrated to 1 atm. Headspace gas composition (i.e., H2, CO2, and CH4) was determined with

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a gas chromatography unit equipped with two columns and two thermal conductivity detectors.20

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Calculations. Coulombic efficiency and H2 yield were calculated as previously

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described.21 Current density was normalized to either the empty bed volume of the anode

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chamber (250 mL) for comparison with single-chamber MECs, or the projected surface area of

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the Nafion membrane (5.7 cm2), assuming the membrane surface area was limiting, due to the

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narrow channel of the H-type reactor.22

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RESULTS AND DISCUSSION

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MEC Startup. During the MEC startup period, fed-batch addition of 200 mg/L

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compound mixture was conducted in repetitive 7-day feeding cycles. Stable maximum current

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and H2 production was observed by day 28 (Figure S2) and the startup period continued for

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another 35 days (5 feeding cycles) to confirm stable performance. During the MEC operation,

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the anolyte and catholyte pH was in the range of 6.7-7.0 and 7.0-7.3, respectively. The maximum

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current density (Imax) was 0.16 ± 0.04 mA/cm2 or 3.6 ± 0.9 A/m3, cumulative H2 production was

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19.3 ± 1.2 mL (20°C, 1 atm), and coulombic efficiency was 44 ± 12% over the last 5 feeding

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cycles. The five compounds were completely transformed, with sCOD removal of 57 ± 10%

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during each feeding cycle. An abiotic control assay (Control 2), conducted under the same MEC

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conditions, with the exception that the anode was not inoculated, resulted in negligible current

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(1% abundance)

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detected in the MEC bioanode are shown in Figure S8. The detected species in the bioanode are

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mainly related to exoelectrogens, putative degraders of the furanic and phenolic compounds, and

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potential syntrophic partners with exoelectrogens. Desulfovibrio desulfuricans is a sulfate-

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reducing bacterium, which is able to perform exoelectrogenesis through cytochrome c.40 In

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addition, the major known degraders of furfural and 5-HMF under anaerobic conditions belong

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to Desulfovibrio genus.41 Geobacter spp. are well studied exoelectrogens, using acetate and H2 as

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primary electron donors.34,42 Eubacterium limosum is known to grow on methoxylated aromatic

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compounds, such as syringic acid and vanillic acid.43 E. limosum is also a homoacetogen, which

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could consume H2 formed during fermentation and produce acetate for exoelectrogens.44

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Pelobacter propionicus is not known to perform exoelectrogenesis or to use acetate as the

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electron donor, but is thought to be involved in syntrophic interactions with exoelectrogens

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fermenting initial substrates to acetate.14,45 Clostridium populeti and Clostridium

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aminobutyricum are known mixed-acids fermenters, and the latter has been found in acetate- and

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glucose-fed MFC anodes.46,47 Phascolarctobacterium faecium can convert succinate to

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propionate.48 Therefore, E. limosum, P. propionicus, P. faecium and Clostridium spp. could be

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syntrophic partners with exoelectrogens by converting the furanic and phenolic compounds or

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their biotransformation products to readily available substrate (e.g., acetate) for

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exoelectrogenesis. Several detected species in the present study are closely related to bacteria,

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such as Dysgonomonas spp.,17 Pleomorphomonas oryzae, 49 and the uncultured bacterium clones

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(JX462549.1 and GU083415.1)45,50 previously found in bioelectrochemical systems, but their

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functions are unknown. Other phylotypes related to the OTUs detected in the present study are

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Cloacibacillus evryensis, an amino-acid degrading bacterium found in anaerobic digesters,51 and

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a bacterial species found in cellulose and xylan-pectin enrichments of cow feces.52 These species

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may have been carried over from the original inoculum, which came from a municipal anaerobic

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digester.

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The present study demonstrated the capacity of MEC to convert furanic and phenolic

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compounds to H2, which strengthens the promise of integrating MEC technology in biorefinery

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platforms. Furanic and phenolic inhibitors, which are a great challenge in dark fermentation of

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lignocellulosic hydrolysates, were productively utilized in the MEC to produce H2, which can

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then be used for the hydrogenation of bio-oils, thus eliminating the need to reform natural gas to

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H2. Moreover, the biotransformation fate of furanic and phenolic compounds shown in the

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present study provides mechanistic insights into bioprocesses taking place in MEC and

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fermentation systems which use complex, lignocellulose-derived feedstocks. The relatively low

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H2 production rate and inhibition of exoelectrogenesis at 1,200 mg/L observed in the present

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study, need to be addressed. On-going research is investigating continuous-flow MEC operation

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to find the means to mitigate the observed inhibitory effect during the batch MEC operation, as

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well as increase the H2 production rate and yield.

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ASSOCIATED CONTENT

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Supporting Information

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Tables S1-S3, and Figures S1-S8 are available free of charge via the Internet at

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http://pubs.acs.org.

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ACKNOWLEDGEMENT

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We acknowledge funding for this work from the U.S. Department of Energy, BioEnergy

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Technologies Office under the Carbon, Hydrogen and Separations Efficiency (CHASE) in Bio-

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Oil Conversion Pathways program, DE-FOA-0000812. The manuscript has been co-authored by 20 ACS Paragon Plus Environment

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UT-Battelle, LLC, under Contract No. DEAC05-00OR22725 with the U.S. Department of

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Energy.

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significance of hydrogen scavengers. Biotechnol. Bioeng. 2010, 105 (1), 69-78.

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fuel cell in the presence and absence of a proton exchange membrane. Environ. Sci. Technol.

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2004, 38 (14), 4040-4046.

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(19) Rice, E. W.; Eaton, A. D.; Baird, R. B. Standard Methods for the Examination of Water and

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Wastewater, 22nd ed.; APHA, AWWA, WEF: Washington DC, 2012.

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(20) Okutman Tas, D.; Pavlostathis, S. G. Effect of nitrate reduction on the microbial reductive

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transformation of pentachloronitrobenzene. Environ. Sci. Technol. 2008, 42 (9), 3234-3240.

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Technol. 2006, 40 (17), 5172-5180.

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(22) Logan, B. E. Essential data and techniques for conducting microbial fuel cell and other

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types of bioelectrochemical system experiments. ChemSusChem 2012, 5 (6), 988-994.

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(23) Liu, Z.; Zhang, C.; Wang, L.; He, J.; Li, B.; Zhang, Y.; Xing, X.-H. Effects of furan

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derivatives on biohydrogen fermentation from wet steam-exploded cornstalk and its microbial

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community. Bioresour. Technol. 2015, 175, 152-159.

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(24) Lee, H.-S.; Vermaas, W. F. J.; Rittmann, B. E. Biological hydrogen production: Prospects

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and challenges. Trends Biotechnol. 2010, 28 (5), 262-271.

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(26) Cusick, R. D.; Kiely, P. D.; Logan, B. E. A monetary comparison of energy recovered from

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microbial fuel cells and microbial electrolysis cells fed winery or domestic wastewaters. Int. J.

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Hydrogen Energy 2010, 35 (17), 8855-8861.

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(27) Escapa, A.; Gil-Carrera, L.; García, V.; Morán, A. Performance of a continuous flow

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microbial electrolysis cell (MEC) fed with domestic wastewater. Bioresour. Technol. 2012, 117,

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55-62.

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bioelectrochemically assisted microbial reactor (BEAMR). Int. J. Hydrogen Energy 2007, 32

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(13), 2296-2304.

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(29) Call, D.; Logan, B.E. Hydrogen production in a single chamber microbial electrolysis cell

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lacking a membrane. Environ. Sci. Technol. 2008, 42 (9), 3401-3406.

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(30) Wang, A.; Liu, W.; Ren, N.; Cheng, H.; Lee, D.-J. Reduced internal resistance of microbial

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electrolysis cell (MEC) as factors of configuration and stuffing with granular activated carbon.

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Int. J. Hydrogen Energy 2010, 35 (24), 13488-13492.

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(31) Sander, R. Compilation of Henry's law constants, version 3.99. Atmos. Chem. Phys.

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Discuss. 2014, 14 (21), 29615-30521.

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(32) Lee, H.-S.; Torres, C. I.; Parameswaran, P.; Rittmann, B. E. Fate of H2 in an upflow single-

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chamber microbial electrolysis cell using a metal-catalyst-free cathode. Environ. Sci. Technol.

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2009, 43 (20), 7971-7976.

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(33) Rozendal, R. A.; Jeremiasse, A. W.; Hamelers, H. V. M.; Buisman, C. J. N. Hydrogen

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production with a microbial biocathode. Environ. Sci. Technol. 2007, 42 (2), 629-634.

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(34) Caccavo, F.; Lonergan, D. J.; Lovley, D. R.; Davis, M.; Stolz, J. F.; McInerney, M. J.

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Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-

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reducing microorganism. Appl. Environ. Microbiol. 1994, 60 (10), 3752-3759.

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Electrochemical investigations of the oxidation-reduction of furfural in aqueous medium:

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Application to electrosynthesis. Electrochim. Acta 2004, 49 (3), 397-403.

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597

Page 28 of 33

Table 1. Efficiency parameters of the MEC fed with the mixture of the five compounds. Initial substrate concentration (mg/L)

Parameter

200

400

800

2.9 ± 0.2 e

2.5 ± 0.2

1.7 ± 0.03

(%) b

38 ± 0.2

42 ± 7

26 ± 0.4

sCOD removal (%)

49 ± 0.1

49 ± 8

61 ± 4

58 ± 1

69 ± 8

44 ± 0.1

0.07 ± 0.0004

0.10 ± 0.002

0.10 ± 0.001

H2 yield (mol/mol) a

CE (%) c H2 production rate (L/L-d) d 598 599 600 601 602 603 604 605

a

Moles of H2 collected per mole of the compound mixture transformed

b

H2-COD per COD removed during each batch run

c

Electrons recovered as electrical current per electron equivalent of COD removal during

each batch run d

Maximum production rate, observed on day 1 during each feeding cycle (20ºC and 1

atm) normalized to the empty bed volume of the anode chamber (0.25 L) e

Mean ± standard deviation; n = 3

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606

Table 2. Possible fermentation of the identified transformation products to acetate and other

607

products (∆  ; Gibbs free energy at pH 7 and 298 K).

∆  (kJ/mol)

Compound

Reaction

Catechola

C H  + 4H O = 3CH COO + H + 3H 

-69.12

Phenola

C H O + 5H O = 3CH COO + 2H + 3H 

+8.46

FAb

9 11 2 C H O + 6 H O ⇌ HCO + CH COO + H  2 2

-763.66

HMF-OHb

C H O + 3 H O ⇌ 3CH COO + H + 3 H 

-215.76

diHBAb

2 C H O + 8 H O ⇌ HCO +

13 15 CH COO + H  2 2

608 609 610

a

! value from Yaws, 2003 53

b

! value calculated based on the group contribution method 54

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-257.66

Environmental Science & Technology

611

LIST OF FIGURES

612

Figure 1. Current and cumulative H2 production during four batch assays conducted at increased

613

initial substrate concentrations in the MEC anode (A, 200 mg/L; B, 400 mg/L; C, 800 mg/L; D,

614

1,200 mg/L). Error bars represent mean values ± one standard deviation, n = 3.

615

Figure 2. Normalized concentration profiles of the five compounds fed to the MEC anode at

616

various initial total concentrations (200 to 1,200 mg/L).

617

Figure 3. Biotransformation products detected during the four batch assays at increased initial

618

substrate concentrations in the MEC anode (200-1,200 mg/L).

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2.5

A

Current H2

2.0 1.5 1.0 0.5 0.0 2.5

B

2.0 1.5

Current (mA)

1.0 0.5 0.0 2.5

C

2.0 1.5 1.0 0.5 0.0 2.5

D

2.0 1.5 1.0 0.5 0.0 0

1

2

3

4

5

6

Time (d)

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70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0 70 60 50 40 30 20 10 0

Cumulative H2 (mL at 20oC, 1 atm)

Figure 1

Environmental Science & Technology

Page 32 of 33

Figure 2

FF

HMF

1.0

Initial concentration (mg/L):

Normalized Concentration (C/C0)

0.8

200 (biomass-free) 200 400 800 1200

0.6 0.4 0.2 0.0

SA

1.0

HBA

VA

0.8 0.6 0.4 0.2 0.0 0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Time (d)

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1

2

3

4

5

6

7

Page 33 of 33

Environmental Science & Technology

Figure 3

120

120

Phenol

Catechol 100

100

Initial concentration (mg/L):

80

80

200 (biomass-free)

60

60

400

40

40

800

20

20

0

0

Concentration (mg/L)

200

1200

6

180

diHBA

60

HMF-OH

FA

150

5

50

120

4

40

90

3

30

60

2

20

30

1

10

0

0 0

1

2

3

4

5

6

7

0 0

1

2

3

4

5

6

7

0

Time (d)

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1

2

3

4

5

6

7