Bioelectrochemical reduction of volatile fatty acids in anaerobic digestion effluent for the production of biofuels

Accepted Manuscript Bioelectrochemical reduction of volatile fatty acids in anaerobic digestion effluent for the production of biofuels Sanath Kondaveeti, Booki Min PII:

S0043-1354(15)30223-2

DOI:

10.1016/j.watres.2015.09.011

Reference:

WR 11519

To appear in:

Water Research

Received Date: 8 June 2015 Revised Date:

30 August 2015

Accepted Date: 5 September 2015

Please cite this article as: Kondaveeti, S., Min, B., Bioelectrochemical reduction of volatile fatty acids in anaerobic digestion effluent for the production of biofuels, Water Research (2015), doi: 10.1016/ j.watres.2015.09.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Bioelectrochemical reduction of volatile fatty acids in anaerobic

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digestion effluent for the production of biofuels

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Sanath Kondaveeti and Booki Min*

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Department of Environmental Science and Engineering, Kyung Hee University,

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1 Seocheon-dong, Yongin-si, Gyeonggi-do 446-701, Republic of Korea

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Corresponding author; Tel.: +8231-201-2463 (office); E-mail address: [email protected] (Booki Min)

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Abstract

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This study proves for the first time the feasibility of biofuel production from anaerobic digestion

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effluent via bioelectrochemical cell operation at various applied cell voltages (1.0, 1.5 and 2.0 V).

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An increase in cell voltage from 1 to 2 V resulted in more reduction current generation (-0.48 to -

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0.78 mA) at a lowered cathode potential (-0.45 to -0.84 mV vs Ag/AgCl). Various alcohols were

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produced depending on applied cell voltages, and the main products were butanol, ethanol, and

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propanol. Hydrogen and methane production were also observed in the headspace of the cell. A

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large amount of lactic acid was unexpectedly formed at all conditions, which might be the

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primary cause of the limited biofuel production. The addition of neutral red (NR) to the system

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could increase the cathodic reduction current, and thus more biofuels were produced with an

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enhanced alcohol formation compared to without a mediator.

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Keywords: Bioelectrochemical cell operation, Biofuel, Biocathode, Mediators, Anaerobic

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digestion effluent

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1 1. Introduction

The substantial decrease in fossil fuels has resulted in the search for alternative and low-

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cost methods of energy production (Kakarla and Min 2014). In this respect, bioelectrochemcial

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systems (BESs) have gained immense attention for generating electricity and biofuels with the

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simultaneous treatment of organic waste and wastewater(Kondaveeti et al. 2014). The

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potentiality of BES application for biofuel production has been evaluated for hydrogen,

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methane(Cheng et al. 2009, Logan et al. 2008), and other value-added products (Rabaey and

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Rozendal 2010)using a bioelectrocatalyzed cathode electrode. In the process of biofuel

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generation using BES, an external power supply serves as the energy source that drives the

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microbial electrochemical reduction of proton ions, carbon dioxide (CO2), and volatile fatty acids

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(Sharma et al. 2013). However, biofuel production in BESs is still in the beginning stages, and

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requires improvement in several areas to make it commercially available (Srikanth et al. 2014).

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In general, biofuels production from organic sources are limited to complex substrates

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such as lignocelluloses and wheat straw hydrolysate, which are further reduced to simple

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substrates (Acetic and butyric acid) (Srikanth et al. 2014). In this aspect, anaerobic digestion

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(AD) effluent with volatile fatty acids such as acetic and butyric acid can serve as organic

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compounds for biofuel production (Spirito et al. 2014).In typical wastewater treatment plants

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(WWTP), the effluent from AD are subjected to activated sludge systems or recycled for further

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treatment processes (Appels et al. 2008).These biofuel generations from AD effluent can be

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enhanced using exocellular mediators to modify energy metabolism. Mediator compounds cycle

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between oxidized and reduced states and play a vital role in bacterial metabolism by creating an

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additional electron flow and thereby enhancing biofuel production. Mediators such as neutral red

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(NR), methyl viologen (MV), methylene blue (MB), and anthraquinone disulfonate (AQDS) are

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used in ABE (Acetone-Butanol-Ethanol) fermentation systems and in BES to shift fermentative

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metabolism to biofuel (bioalcohol and biogas) synthesis and to limit organic acid synthesis

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(Steinbusch et al. 2009, Yarlagadda et al. 2012). Although the exact mechanism of how

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mediators regulate fermentative pathways is not fully understood, the supplementation of

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mediators is an attractive method for enhancing the electron flow from organic acids to biofuels.

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The primary objective of the present study is to evaluate the feasibility of using anaerobic

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digestion effluent to generate biofuels (bioalcohol and biogas) in bioelectochemical systems

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(BES) with the help of an external power supply. A bioelectrochemical cell (BES) operation was

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performed using an abiotic anode electrode and biocatalyzed cathode electrode for the formation

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of biofuel in a cathode chamber. The production of biofuel from anaerobic digestion effluent was

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tested at various external power inputs (1.0, 1.5 and 2 V), and the effect of mediators (Neutral

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red; NR) was also evaluated. The presence of NR has been known to influence the bacterial

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exocellular electron transfer metabolism with elevated current generations. Electrochemical

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techniques such as cyclic voltammetry (CV) were pursued for a better understanding of BES

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operation with AD effluent and in the presence of mediators.

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2. Materials and Methods

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2.1Microbial inoculum and electrolyte

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Inoculum for the microbial consortium and anaerobic digestion (AD) effluent for the cathode

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compartment were obtained from Yongin Respia Wastewater Treatment Plant (Yongin-si,

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Korea). The initial concentrations of VFAs in AD effluent were 0.5±0.04 (Acetic acid) and

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1.84±0.19 g/l(Butyric acid), respectively. The electrolyte for BES operation in the absence of 4

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planktonic cells (suspended culture) was pursued using growth media (GM). The GM consisted

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of a vitamin and mineral solution and was prepared as described in previous studies (Kakarla and

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Min 2014).

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2.2 Mediator for microbial redox reactions

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The external supplementation of mediators can provide an alternative electron flow by

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enhancing the production of biofuel, and also can accelerate power generations due to variations

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in the physiology of bacteria.NR was selected based on certain characteristics such as high

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solubility, complete reversibility, stability and lack of combination with other molecules(Fultz

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and Durst 1982). Moreover, one of the primary biocomponents involved in microbial redox

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reactions is NADH, with a redox potential of -320 mV, as similar to NR (-325 mV). NR was

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demonstrated to be biologically active in anaerobic conditions; mediator was used in

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concentrations (1 mM) similar to those used in other research studies (Steinbusch et al. 2009).

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2.3 Bioelectrochemical system set-up and operation A double chamber “H” type BES cell with two compartments was chosen and operated in

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fed-batch mode by varying the external cell voltages. Each compartment cell consisted of 200 ml

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as a working volume with a total volume of 250 ml. Platinum-coated carbon paper (0.5 mg/cm2)

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with a surface area of 40 cm2 was used as an anode, and a carbon brush (3 × 2.5 cm) was used as

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cathode. The carbon brush cathode was prepared by twisting the carbon fibers with stainless steel

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wire. The electrical connections on the electrode were established using copper wire and by

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sealing the contact area with non-conductive epoxy resin. The anode and cathode compartments

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were separated using pretreated Nafion-117 (PEM; Dupont Co., USA). The membrane and

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cathode electrode (carbon brush) pretreatment were pursued as described in previous literatures

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(Feng et al. 2010, Moon et al. 2014). To avoid membrane swelling when placed in BES,

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pretreated membranes were stored in deionized water and taken out prior to being used. Ag/AgCl

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(sat’d NaCl) from bio analytical instruments (USA) was used as a reference electrode in the

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cathode chamber during the electrochemical analysis and for noting cathode potentials. The

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anode and cathode electrodes were connected to a DC power supply and various external energy

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inputs (1.0, 1.5 and 2 V) were supplemented(Huang et al. 2013).

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Catholyte consists of either AD effluent or GM, as per requirements. Prior to

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supplementation, the cathode influent pH was adjusted to 6 using either 1 M HCl or NaOH. In

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both cases, the anode chamber of the reactor consisted of GM (pH 6). The cathode and anode

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solutions were continuously stirred for effective mixing and operated in a temperature-controlled

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incubator at 30±2oC.BES reactors were sealed using silicone gel to ensure strict anaerobic

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conditions. Prior to sealing of the BES reactors, headspace and solutions were purged with

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nitrogen gas for 15 to 20 min, minimizing the effect of dissolved oxygen. The anolyte and

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catholyte were replaced when a decrease in cell voltage was noticed. After 94 days of operation

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with AD effluent, the electrically-active biocathode electrode was transferred to fresh growth

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medium and operated at 1.5 V with similar concentrations of acetic and butyric acid.

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Along with BES, three additional sets of reactors were operated as a control. The first

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control reactor was operated similar to the BES with the exception of sterilization (autoclaving)

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of the cathode solutions (Anaerobic digestion effluent). The second control reactor was operated

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by maintaining abiotic cathode (no inoculums) conditions with an external supplement of acetic 6

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and butyric acids. The third control reactor was operated without supplementation of external

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energy (open circuit conditions).These control experiments were pursued to illustrate the

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importance of biological catalyst (biofilm), as well as the influence of external cell voltage to

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crossover thermodynamic barriers for product formation. All experiments and analysis were

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pursued in replicated (duplicate or triplicate) cycles to maintain consistency.

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2.4 Analytical Methods

For liquid analysis, 10 ml of samples were collected at the end of operation (4 to 6 days)

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using a sterile and nitrogen-purged syringe. The collected samples were filtered using an

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acrodisc (0.2 µm) syringe filter.VFA concentrations in the liquid phase were measured using an

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ion chromatogram (IC-861, Metrohm, USA) equipped with an organic acid column (model:

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Metrosep 250/7.8, Metrohm, USA). Head-space biogas analyses were pursued using a gas

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chromatograph (Younglin Instruments, Korea) equipped with a thermal conductivity detector

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(TCD) and carboxen column (Supelco, USA). The oven temperature was maintained at 150oC,

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whereas the injector and detector temperatures were 100 and 200 oC, respectively. Nitrogen gas

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was used as a carrier at a flow rate of 6 ml/min. Sampling was performed with a sterile gas-tight

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syringe (Hamilton, USA) and analyzed immediately. Bioalcohols such as ethanol, methanol,

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proponol, acetone, glycerol, butanol etc. were analyzed using gas chromatography (GC; Agilent,

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USA) equipped with a flame ionization detector (FID) and fitted with an HP innowax column

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(Agilent, USA). The injector, oven, and detector temperatures were 150, 220, and 230oC,

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respectively. Helium was used as a carrier gas with a flow rate of 5ml/min. All analytical

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systems (GC and IC) were calibrated with standard chemicals in a range of sample

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concentrations. The GC and IC sample preparations were pursued as described in previous

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studies (Merzougui et al. 2011).

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2.5 Electrochemical measurements and calculations

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External energy inputs of 1.0, 1.5, and 2.0 volts were applied to test the effect of voltage on

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product formation. The desired external voltage was applied to the circuit using a DC power

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supply. The negative lead of the power source was connected to the cathode with an external

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resistance of 15 Ω and the positive lead of the circuit was connected directly to the anode. The

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voltage was measured across an external resistor with a 5 min time interval using a data

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acquisition system in a personal computer. Cyclic voltammetry (CV) analyses were pursued to

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analyze the effect of mediators in BES using a potentiostat(Versa Stat 3, Princeton Applied

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Research, USA).CV measurements were evaluated within a range of -0.9 to 0.9 V (vs. Ag/AgCl)

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at a scan rate of 5 mV/s.

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The Coulombic efficiency (CE) of BES, using a bioelectrocatalyzed cathode for the

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reduction of VFAs (Acetic and Butyric acid), was calculated by dividing the Coulombs of the

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product (Ca) by the total coulombs (CI). Total Coulombs were obtained by integrating the area

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under the current vs. time (I-t curve). The amount of Coulombs (Ca) distributed in the moles of

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individual products (ni) by BES in the process of the bioelectrocatalyzed reduction of VFAs was

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calculated by following Equations included in the body of the text should be punctuated

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accordingly. (Marshall et al. 2012):

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Ca= Fbini,Eq. (1)

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where bi is the number of electrons in the product; F is Faraday constant (96,485 C/mol); and n is the number of moles of product.

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Capacitance and energy calculations were performed by analyzing the CV data (Raghavulu et al.

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2012) using the following equation:


=

 

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where C is the capacitance (F), Q0 is the charge obtained (C), and V0 is the maximum applied

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voltage (V).

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3. Results and discussion

3.1 Current generation in the bioelectrochemical reduction process

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Reductive biocatalytic current generations were observed in BES at various applied voltages

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(1.0, 1.5 and 2.0 V) by measuring the cell voltage across the external resistor (Fig.1, Table 2).

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Based on other studies (Sharma et al. 2013, Srikanth et al. 2014), it is presumed that the applied

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voltages used in this study were sufficient to reach activation energy. Initial current generations

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without mediator were observed after 18 days of operation with an applied voltage of 1 V, and

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the applied voltage was further increased to 1.5 and 2V. At 1 V, the current generation and

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cathode potentials (vs Ag/AgCl) were -0.48±0.03 mA and -0.45±0.02 V, respectively. The

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current generations and cathode potentials were further enhanced to -0.64±0.02 mA and -

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0.68±0.018 V, with operation at 1.5 V. Reductive current appeared to increase with increases in

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applied voltages; at 2 V the BES current generation and cathode potentials were-0.78 ± 0.01 mA

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and -0.84 ± 0.032V, respectively.

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After 63 days of operation, 1mM of neutral red was added to the cathode chamber as a

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mediator for the bioelectochemical reduction. Increased current generation and cathode

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potentials were noticed in the presence of the mediator. The variation of external cell voltage (1,

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1.5, and 2 V) with neutral red mediator resulted in enhanced cell voltages of -0.52±0.016,-

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0.7±0.021, -1.1±0.018 mA and cathode potentials of -0.51±0.014, -0.69±0.016, and -0.89 ±0.018

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V, respectively. Elevated current generations were observed in the presence of mediator due to

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the enhanced extra cellular electron transfer metabolism of biocatalytic activity. Steinbush et al.,

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demonstrated similar results for increased current generation in the presence of mediators

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(Steinbusch et al. 2009). In their study, initial current generation of 7.8 mA was amplified to18.5

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mA using methyl viologen (MV) as a mediator.

The bioelectochemical cell operation at 1.5 V with synthetic growth media and

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externally-supplemented acetic and butyric acid and in the absence of planktonic cells exhibited

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an immediate (0.8 d) reduction in current of -0.78 ± 0.032 mA (Fig S1). This result suggests that

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the bioelectrochemical reduction was obtained via the biocatalytic activity of microorganisms

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attached to the cathode electrode. No current generation and biofuels formation were observed in

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the BES control reactors (Fig. S2).

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3.2Bioalcohol and biogas production from the bioelectrochemcial reduction of anaerobic

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digestion effluent

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The product profiles represented here were drawn after examination in replicated cycles, and the

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results obtained are within the border of experimental errors. The characteristics of AD effluent

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used in the BES reactor were analyzed using standard methods (APHA 2005), as presented in 10

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Table 1. The initial concentrations of acetic and butyric acids in anaerobic digestion effluent

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were 0.5±0.04 and 1.84±0.19 g/l, respectively (Fig. 2). Bioelectrochemical reduction at 1volt

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revealed no bioalcohol generations (Fig. 2a), which might be due to a smaller cathodic potential

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(-0.45 V) (Rabaey and Rozendal 2010). By increasing the applied voltages (1.5 and 2 V), the

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bioelectrocatalyzed synthesis of bioalcohols was observed as represented in Fig 2a.At 1.5V, the

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concentration of ethanol and butanol were 31 and 57 mg/l, respectively. The ethanol

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concentration (31 mg/l) at 1.5V in this study was smaller than the value (50 mg/l) from the other

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BES operation (Sharma et al., 2013). This difference in ethanol production may be due to

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different initial concentration of acetic acid (100 mM vs 8.3 mM in this study) and butyric acid

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(100 mM vs 20 mM in this study). By increasing the cell voltage to 2 V, amplifications in

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product profiles were observed. Butanol (91 mg/l) and proponol (60 mg/l) were the main

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products observed, and other alcohols were in range of 50 mg/l. Although ethanol production

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was observed at 1.5 V, it was not observed at 2 V, possibly due to the further reduction of

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ethanol to other products or the usage of ethanol as a bacterial substrate (Müller 2001, Sharma et

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al. 2013). Several researchers reported that oxidation of ethanol in ABE fermentation favored the

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synthesizing of butyrate and propionate (Agler et al. 2011). The product profiles observed in this

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study at 2 V with a cathode potential of -840 mV (vs Ag/AgCl) were similar to other studies

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operating with a working electrode (cathode) potential of -850mV (Sharma et al. 2013). In their

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study, the concentrations of butanol and proponol were 100 and 87 mg/l, respectively.

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BES operation at various external cell voltages of 1, 1.5, and 2 V resulted in hydrogen

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(H2) and methane (CH4) as predominant headspace biogas products (Fig 2b). No methane

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generation was noticed with applied voltages of 1 V and 1.5 V; whereas at 2 V, methane

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generation was observed at 11±2.1%. An increase in hydrogen generation was noticed with 11

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increase in applied voltages. The percentages of hydrogen in the headspace at 1, 1.5, and 2 V

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were 3.7±0.5, 7.2±0.8 and 25.4±1.3% respectively. No bioalcohol and current generation were

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detected in the BES control reactor, suggesting that product formation was solely due to

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bioelectrocatalytic activity on the cathode electrode. The hydrogen gas concentrations in the

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control at various voltages (1, 1.5, and 2 V) were 1.6±0.4 2.0±0.1, and 5.9±1.1%, respectively.

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3.3 Cyclic voltammogram of bioelectochemical reduction of anaerobic digestion effluent

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both with and without mediator

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Cyclic voltammetry (CV) techniques help to determine the ability of biocatalysts on an electrode

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surface to drive reductive reactions. In the present study, CV was used to pinpoint the influence

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of extra cellular mediators on the biocatalyst to show a charge transfer phenomenon. The CV

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profiles indicated variations in electrochemical behavior in the presence of mediators (Fig. 3).

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The catalytic currents were similar with variation in the supplementation of mediators; however

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a significant variation in peak current was noticed with increases in mediator concentration (0 to

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1 mM). This might be due to the unidirectional pass of electrons from electrode to NR and

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NADH (Park and Zeikus 2000). The reductive peak potential with various concentrations (0, 0.5,

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0.7 and 1 mM) of an exocellular mediator was noticed at around -410 to -450 mV (vs. Ag/ AgCl)

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with variations in peak current. The bioelectrochemical reduction of fumarate to succinate in the

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other study exhibited a redox potential of about -520 mV (vs. Ag/AgCl) in the presence of NR

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mediator (Park and Zeikus 1999). BES operation without mediators and with various

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concentrations of mediators (0.5, 0.75, and 1mM) resulted in peak currents of -0.11, -0.14, -0.19,

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and -0.28 mA, respectively.

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This redox-coupled reaction of NR with NADH might increase the synthesis of ethanol

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and butanol, which was also indicated by enhanced alcohol formation with mediator, as shown in

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Fig.4. The charge and capacitance were calculated based on the voltammetric profiles. Charge is

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an indicator of the number of electrons available at an instant during an electrochemical reaction

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(Kondaveeti and Min 2013). The bioelectrochemical reduction processes both without and with

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NR mediator (1 mM) exhibited 3.1× 10-3and 7 × 10-3 C, respectively. This result suggests that

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the presence of mediator in BES for bioelectrochemical reduction processes can increase the

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availability of electrons for favorable reductive reactions. Capacitance is a measurement for the

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amount of electrical charge stored at a maximum applied potential. These charges stored in

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capacitive electrodes are directly proportional to the migration of electrons towards the electrode.

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Similar to charge, higher capacitance values were noticed in BES with the presence of mediator

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(7.7 mF) in comparison to without mediator (3.5 mF). These values indicate that the enhanced

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holding capacity of charge with mediator can increase biofuel synthesis as shown in previous

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studies (Srikanth et al. 2014).

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The effect of mediator on biofuel (bioalcohol and biogas) production

In the presence of mediator (NR), the formation of biofuel production was detected with the

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consecutive initiation of electron utilization (Fig.4). Accordingly, it is assumed that product

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synthesis was the consequence of electro-catalyzed biofilm reductive reactions. At 1 V (Cathode

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potential: -0.51 V), ethanol, proponol, and butanol appear in concentrations of 19.8 mg/l, 30

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mg/l, and 31 mg/l, respectively (Fig 4a). These concentrations were lower in comparison to

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other studies (Steinbusch et al. 2009) carried out by Steinbusch et al., where acetic acid was

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used as an electron acceptor (Cathode potential: -0.55 V). In their study, ethanol concentrations

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of 83 mg/l were noticed using upflow anaerobic sludge blanket (UASB) reactor inoculum; with

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methyl viologen as an external cellular mediator. The lower concentration of ethanol generation

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might be due to variations in reactor configuration (flat plate vs. double chamber), type of

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mediator (methyl viologen vs. neutral red), concentration of substrate, and biological activity on

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the cathode electrode. These concentrations of ethanol were higher with increases in applied cell

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voltage. At 1.5 V; ethanol (50 mg/l) and butanol (70 mg/l) were noticed as predominant

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bioalcohols, where as the propanol concentration was 40 mg/l.

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Bioelectrochemical cell operation at 2 V (cathode potential: -0.9 V) exhibited butanoland

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ethanol formations in concentrations of 130 mg/l and 49.8 mg/l, respectively. The concentration

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profile observed at 2 V (Cathode potential: -0.9 V) was higher in comparison to recent studies

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pursued by Sharma et al., where mixed cultures of sulfate reducers were used as the predominant

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bacteria on a working electrode (Cathode potential: -0.85 V). In their study, they produced up

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to50 mg/l of ethanol and 100 mg/l of butanol as bioalcohols. The amplified production of butanol

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in comparison to other studies might be due to the usage of redox mediators (Girbal et al. 1995,

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Yarlagadda et al. 2012).Proponol (41 mg/l) and butanol (47 mg/l) were prevalent bioalcohols

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recorded in BES by transferring the electrocatalyzed biofilm electrode into growth media with

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similar concentrations of acetic and butyric acids as in anaerobic digestion effluent (Fig S1,

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Table 2).

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The presence of neutral red (mediator) enhanced the generation of biogas (hydrogen and

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methane)(Fig 2b). The hydrogen production of 15.1±1.7, 26.3±2.1, and 31.8±2.4 % was

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observed at cell voltages of 1, 1.5, and 2V, respectively. The overall concentration of methane

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was less than 9.4±1.6% in the headspace. The absence of bioalcohol formations and current 14

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generations was noticed in the BES control reactors. The hydrogen gas concentrations at external

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supplemented voltages of 1, 1.5, and 2 V were 5.9±0.7, 8.7±1.4, and 10.9±1.4%, respectively.

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During BES operation with AD effluent and in the presence of NR at various cell voltages; the

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initial catholyte pH of 6.0 was decreased to 4.9 ± 0.2.The decrease in catholyte pH might be due

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to an increased concentration of biofuels (Nevin et al. 2010, Rabaey and Rozendal 2010). No

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variation in pH was observed in BES control reactors at various applied voltages.

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3.5Coulombs distribution in synthesized products from anaerobic digestion effluent The Columbic efficiency (CE) is defined as the percentage of supplied electrons to final products

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in a cathode. The CE was calculated at both operational conditions (the presence and absence of

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mediator) with various applied voltages (1, 1.5 and 2 V) using anaerobic digestion effluent as

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catholyte. The distributions of Coulomb into biofuels and end products were calculated and are

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depicted in Fig. 5. BES operation at various cell voltages (1, 1.5 and 2 V) with anaerobic

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digestive effluent (Fig. 5a) exhibited total Coulombs (supplemented electrons) of 77165, 277055,

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175525 C and total CE of20, 4.9, and 9.8%, respectively. The total Coulombs from current

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generation were higher in the BES operation with the supplementation of mediators, due to

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variations in bacterial extra cellular activity. The presence of mediator (Fig. 5b) and operation

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with various external cell voltages (1, 1.5 and 2 V) resulted in total coulombs (supplemented

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electrons) of 80051, 177870, and 243493 C, and CE of 15, 8, and 7.8 % respectively. Total

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Coulombs were distributed to diversified products of biofuels (bioalcohols and biogas) in both

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operational conditions. The BES operation with anaerobic digestion effluent at various external

21

cell voltages (1, 1.5 and 2 V) for biofuel production resulted in Coulombs of 22.3, 4064, 7426 C,

22

and CE of0.029, 1.5, and 4.2%, respectively. With the supplementation of mediator, the

23

Coulombs of biofuels at voltages of 1, 1.5, and 2 V were increased to 2811, 5226, and 8279 C,

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respectively, and the CEs were 3.5, 2.9, and 3.5 %. BES operation at 1.5 V with GM containing

2

acetic and butyric acids exhibited a total Coulombs (current)of 89116, 4130, and a CE for

3

biofuels of4.6 %.The CE observed in this study were slightly smaller (11~24 %), in comparison

4

to other studies of BES operation for VFAs reduction at -0.85V (Sharma et al. 2013, Sharma et

5

al. 2014).The product recovery (conversion) percentage based on the Coulombs of VFAs

6

supplemented (45923 C) in the BES operation using AD effluent at various external cell voltages

7

(1, 1.5, and 2 V) are 28, 29, and 34 %, respectively. These values are amplified with the

8

supplementation of mediators, due to increased biofuel concentrations. During BES operation in

9

the presence of mediators, the recovery (conversion) percentages based on Coulombs of VFAs

10

supplemented (46698 C) at various external cell voltages (1, 1.5 and 2 V,) are 30, 31, and 40 %,

11

respectively. By changing the catholyte solution from AD effluent to GM media, the recovery

12

(conversion) percentage based on Coulombs VFAs supplemented (43688 C) was decreased to 13

13

%, due to decremented biofuels concentration. The recovery percentages noticed in this study

14

were similar in comparison to other BES (30 %)studies using CO2to generate value-added

15

products(Annie Modestra et al. 2015).

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Lactic and formic acids were observed as major byproducts of BES operation with

17

anaerobic digestion effluent and in the presence of mediator. The coulombic efficiency of lactic

18

acid as a byproduct at various cell voltages (1, 1.5, and 2 V) and with anaerobic digestion

19

effluent were 6.6, 2.9, and 4.6 %, respectively. In the presence of mediator, the coulombic

20

efficiencies of lactic acid were 8.2 (1 V), 4.1 (1.5 V), and 3.5 %( 2 V). The generation of lactic

21

acid in BES from reductive substrates such as acetic and butyric acids can be detrimental to

22

biofuels formation. Although the production of large amounts of lactic acid compared to the

23

other alcohols could not be clearly understood in this study, some operational conditions such as

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pH, dissolved solid, and temperature may be used as the controlling factor to enhance biofuel

2

production by eliminating lactic acid–producing bacteria (Narendranath and Power 2005).

3

Alternatively, lactic acid can be used for the synthesis of bioplastics, and also used as acidulant,

4

favoring and preservative in food industry (Abdel-Rahman et al. 2013, Venkateswar Reddy et al.

5

2014). Recent studies carried out by Sharma et al., using VFAs (acetic and butyric acids) as

6

electron acceptor, also observed lactic acid (1.5 g/l) as a predominant byproduct(Sharma et al.

7

2013).The major interference noticed in the conversion of electrical energy into organic

8

chemicals using BES were selective transformations of product at consistent kinetic rates

9

inbiocathodes.BES operation was pursued with a simple combination of acetic and butyric acids

10

from anaerobic digestion effluent; however, the biofuels (bioalcohols and biogas) were

11

diversified. Moreover the presence of mediator did not alter the biofuel formation, but enhanced

12

the end product concentration. This was possibly due to the presence of various microorganisms

13

in the cathode biofilm, or to the various metabolic pathways of bacteria(Wu et al. 2012).

14

Therefore, further research in biocathodes using advanced cell configuration is needed to achieve

15

higher conversion rates and specificity of product formation at low cost. This is also essential in

16

the separating and recovery of fermentation products. Recently, Lu and his co-workers (Lu and

17

Li 2014)developed a gas-stripping process for the extraction of solvents from fermentation

18

processes. This is of special interest because products synthesized in the BES process can be

19

easily coupled to their system, with the simultaneous synthesis and recovery of products.

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Conclusion

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The bioelectrochemcial reduction of anaerobic digestion effluent at various applied cell voltages

23

was evaluated for biofuel production. Increases in current (-0.48 to -0.78 mA) generation and

17

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biofuels concentration were noticed with increased applied voltages (1, 1.5, and 2 V). Ethanol,

2

butanol, and proponol were determined to be predominant bioalcohols. Hydrogen and methane

3

were present in the headspace biogas. The concentrations of biofuels were amplified with the

4

supplementation of mediators due to increases in current generation and capacitance. Enhanced

5

biofuels and specificity in biofuels can be resolved in further optimized bioelectrochemcial

6

reduction processes.

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Acknowledgments:

9

The authors would like to thank Professor Hong Liu for providing valuable comments on the

10

manuscript. This study was funded by a National Research Foundation of Korea Grant

11

(2012R1A1A2042031) and BK plus 21 (20140260).

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Figure legends

20

Figure 1: Current generation observed in the bioelectrochemical reduction of anaerobic

21

digestion effluent at various external cell voltages (in the absence and presence of mediator)

22

Figure 2: Bioalcohol (a) generation and biogas (b) concentration after the bioelectrochemical

23

reduction of anaerobic digestion effluent at various applied cell voltages (1.0, 1.5, and 2.0 V)

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Figure 3: Cyclic voltammetry profile at various concentration of externally-supplemented

2

neutral red mediator

3

Figure 4: Bioalcohol (a) generation and biogas (b) concentration after the bioelectrochemical

4

reduction of anaerobic digestion effluent at various applied cell voltages (1.0, 1.5, and 2.0 V) and

5

in the presence of mediator (neutral red at 1 mM)

6

Figure 5: Distribution of Coulombs in synthesized products after bioelectrochemical reduction

7

in the absence (a) and presence (b) of neutral mediator (1mM)

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Tables:

Table 1: Characteristics of anaerobic digestion (AD) effluent supplemented to the BES reactor in

11

present study.

12

Table 2: Product profiles, current generation and cathode potentials observed in BES operation

13

at various operational conditions

14

Supplementary Data:

15

Figure S1: Current generation (a) and concentration of biofuels (b) observed in the BES reactor

16

with growth media at 1.5 V. (Externally supplemented acetic and butyric acid with similar

17

concentration of AD effluent)

18

Figure S2: Current generations observed in the BES reactor with sterilized wastewater (a) and

19

abiotic control (b).

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Concentration 6.8 ~7.2 0.91 9.2 7.8 86 8.1 6.9 4.9 ±0.21 0.8 ±0.01

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Unit mS/cm g/L g/L % g/L g/L g/L g/L

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Parameter pH Conductivity Total solids (TS) Volatile solids (VS) VS/ TS TSS VSS TCOD SCOD

Table 1: Characteristics of anaerobic digestive (AD) effluent supplemented to BES reactor in

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present study.

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Products (g/l) EtOH

MeOH

BuOH

-0.45 -0.68 -0.84 -0.51 -0.69

N/A 0.03 N/A 0.02 0.05

N/A 0.02 0.05 0.02 0.02

N/A 0.05 0.09 0.03 0.07

-0.89

0.05

0.03

-0.72

0.03

0.03

PrOH

Gly

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Cathode Potential (V, Ag/AgCl)

Ac

Lactic Acid

Formic Acid

Propionic Acid

N/A 0.03 0.06 0.03 0.04

N/A N/A 0.05 N/A N/A

N/A N/A 0.04 N/A N/A

0.39 0.62 0.68 0.59 0.57

0.47 0.34 0.38 0.37 0.38

0.47 N/A N/A N/A N/A

0.13

0.04

0.02

0.03

0.67

0.35

N/A

0.04

0.04

N/A

N/A

-

-

-

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Applied Current voltage (mA) (V) BES operation 1.0 -0.48 with anaerobic 1.5 -0.64 digestive effluent 2.0 -0.78 1.0 -0.52 BES operation 1.5 -0.7 with anaerobic digestive effluent 2.0 -1.1 and mediator Growth Media with Acetic & Butyric 1.5 -0.78 Acid

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N/A –Not available; EtOH-Ethanol; MeOH-Methanol; BuOH-Butanol; PrOH-Proponal; Gly- Glycerol; Ac- Acetone

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Table 2: Product profiles, current generation and cathode potentials observed in BES operation at various operational conditions

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Graphical Abstract

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Fig. 1

2.0 V

1.5 V

1.0V

1.5 V

2.0 V

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Without Mediator

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1.0 V

With Neutral red

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Fig. 1: Current generation observed in bioelectrochemical reduction of anaerobic digestion effluent at various external cell voltages.

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Fig. 2

Fig. 2: Bioalcohol (a) and biogas (b) concentration after bioelectrochemical reduction of anaerobic digestion effluent at different applied cell voltages (1.0, 1.5, and 2.0V)

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Fig. 3

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Fig. 3: Cyclic voltammetry profile at various concentration of externally supplemented neutral red mediator

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Fig. 4

Fig. 4: Bioalcohol (a) and biogas (b) concentration after bioelectrochemical reduction of anaerobic digestion effluent at different applied cell voltages (1.0, 1.5, and 2.0V) and in the presence of mediator (neutral red at 1 mM)

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Fig. 5

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With Neutral red

B

Total Coulombs(C)

Coulombs distributed in products(C)

A Total Coulombs(C)

Coulombs distributed in products(C)

Without mediator

Fig. 5: Distribution of Coulombs in synthesized products after bioelectrochemical reduction in absence (a) and presence(b) of neutral red mediator (1 mM)

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Highlights: •

Anaerobic digestion effluent were used for biofuel production via bioelectrochemical reduction Various forms of bioalcohols and biogases were recovered from anaerobic digestion

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•

effluent •

The main bioalcohols in bioelectrochemical reduction were butanol, ethanol, and

Supplementation of mediator to the system enhanced reductive current and biofuels

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production

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proponal. .

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Fig.S1

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Bioalcohol Concentration (g/l)

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A

Fig. S1: Current generation (a) and concentration of biofuels (b) observed in BES reactor with growth media at 1.5 V. (Externally supplemented acetic and butyric acid with similar concentration of AD effluent)

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Fig.S2

1.0 Volt

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1.5 Volt

1.5 Volt

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1.0 Volt

Fig. S2: Current generations observed in bioelectrochemcal cell at various applied voltage with sterilized wastewater (a) and abiotic control (b).