Electrochemical Control of Redox Potential Arrests Methanogenesis

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Electrochemical control of redox potential arrests methanogenesis and regulates products in mixed culture electro-fermentation Yong Jiang, Lu Lu, Huan Wang, Ruixia Shen, Zheng Ge, Dianxun Hou, Xi Chen, Peng Liang, Xia Huang, and Zhiyong Jason Ren ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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ACS Sustainable Chemistry & Engineering

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Electrochemical control of redox potential arrests methanogenesis and regulates products in

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mixed culture electro-fermentation

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Yong Jiang a, b, Lu Lu b, Huan Wang b, Ruixia Shen b, Zheng Ge b, Dianxun Hou b, Xi Chen b, Peng

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Liang a*, Xia Huang a, Zhiyong Jason Ren b*

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a

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Environment, Tsinghua University, Beijing, 100084, P.R. China

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b

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Boulder, Boulder, CO 80309, USA

State Key Joint Laboratory of Environment Simulation and Pollution Control, School of

Department of Civil, Environmental, and Architectural Engineering, University of Colorado

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*Corresponding author,

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E-mail: [email protected] (P. Liang), [email protected] (Z.J. Ren).

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ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT:

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This study demonstrates the feasibility of using solid electrodes as an alternative source or sink

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of electrons to regulate the redox potential of mixed culture anaerobic reactors, so tunable

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fermentation products can be generated. The product spectrum was characterized under the working

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potentials of -1.0 V, -0.6 V, and -0.2 V (vs. Ag/AgCl), which spans the electron flow direction from

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cathodic current to anodic current. Results show that in neutral pH more negative working potential

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led to higher production of CH4, H2 and acetic acid, while increasing the potential from -1.0 V to

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-0.2 V (vs. Ag/AgCl) greatly reduced methanogenesis by 68% and acetic acid generation by 33%.

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Lowering initial pH to 6.2 reduced such effects by electrical potential. The decrease of working

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potential slightly decreased butyric acid production and showed little impacts on propionic acid

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under both pH conditions. When the reactor switched from poised conditions to open circuit

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condition, more propionic acid and acetic acid while less butyric acid production were observed.

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This redox potential based control presents a new approach to regulate the mixed culture

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fermentation and improve product tunability.

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KEYWORDS:

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Electro-fermentation, arrested methanogenesis, hydrogen, carboxylic acids, anaerobic digestion

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INTRODUCTION

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Anaerobic processes play a critical role in transforming traditional energy intensive wastewater

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treatment plants towards energy and resource recovery facilities. Anaerobic digestion (AD) is a

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model technology that breaks down and stabilizes organic waste such as wastewater sludge, food

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waste, and animal waste and generates biogas and nutrient-rich effluent and biosolids.1 The AD

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process corresponds to a cascade of oxidation and reduction reactions carried out by consortia of

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microorganisms, and CH4 is the main final product, because it has the lowest oxidation state.2 AD is

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a closed system without external inputs of electron acceptors or energy source, so it tends to reach a

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thermodynamic equilibrium with the products (CH4) having the lowest Gibbs energy change per

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electron than any other organic compounds during biological conversion.3-6 This high specificity

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provides AD an advantage over other bioenergy systems as it generates a homogeneous and easily

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separable gaseous product despite the vast heterogeneity of the substrate. However, biogas is a very

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low-value product, and it requires clean-up before use or it must go through significant upgrading to

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meet pipeline quality standards. With the abundant supply of cheap natural gas, biogas production

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from AD faces significant challenges in applications, as current economics cannot justify the

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investment in biogas.7,8 In addition, the impact on global warming has been another major barrier

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due to air and greenhouse gas regulations.9

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Newest initiatives to increase the valorization of organic waste involve the production of short

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chain volatile fatty acids (VFAs) and alcohols via anaerobic fermentation. Such products not only

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bring up the values by themselves as compared to biogas, they are also excellent chemical

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precursors for the production of even higher valued chemicals such as polyhydroxyalkanoates

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(PHAs), biofuels, medium chain fatty acids (MCFAs), and single cell protein (SCP).10 To improve

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the recovery of VFAs and alcohols, factors such as substrate, pH, temperature, and hydraulic 3



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retention time (HRT) are generally optimized to regulate the product spectrum.11,12 For example,

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lower pH values (4.5-7.0) were found favorable for mixed butyrate/acetate generation with

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methanogenesis inhibition, while higher pH (7.0-8.5) and thermophilic temperatures led to higher

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alcohol content.13-15 Similar to pH as a measure of proton activity, the extracellular

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oxidation-reduction potential (ORP) corresponds to the activity of the electrons present in the

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electrolyte and can be easily monitored with an ORP sensor, which represents important operational

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conditions that influences the NAD+/NADH ratio within cells. The NAD+/NADH ratio represents

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cells intracellular ORP because of intracellular redox homeostasis, which controls gene expression

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and enzyme synthesis for the overall cell metabolic activities.16 Therefore, it is reasonable to

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hypothesize that by controlling the redox potential, the fermentation pathways can be influenced

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and therefore the product spectrum can be regulated. Instead of using unsustainable chemical agents,

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Hirano et al., recently revealed a clear relationship between the electrochemically regulated redox

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potential provided by electrodes and pure culture methanogenesis. Under -0.8 V (vs. Ag/AgCl), CH4

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production of M. thermautotrophicus were increased by 1.6 times compared with a control, while

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methanogenesis was effectively suppressed between +0.2 and −0.2 V.17 Other studies used this

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electro-fermentation (EF) approach in pure culture systems to produce chemicals including

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ethanol,18 butanol,19 lactate,20 and lysine.21

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However, there is limited literature in EF using mixed culture of microorganisms, which is

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necessary in environmental applications because heterogeneous raw feedstocks are commonly

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used.15,22 The most related study was a combined system with a microbial fuel cell (MFC) placed in

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a mixed culture fermentation reactor, and the resulted showed H2 production was increased due to

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the timely consumption of VFAs by electroactive bacteria, which helped with pH stabilization.23,24

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Other studies reported 1,3-propandiol production from glycerol could be enhanced during 4


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electro-fermentation biorefining process.25,26 Limited information is available in understanding how

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the working potential influences mix culture EF pathways, especially beyond the surface of the

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electrode. It is not known whether controlled redox potential can be an effective approach to inhibit

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undesired methanogenesis in a mixed culture setting. Moreover, the stability of such operation is

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unknown as most studies only operated the reactors for a few days.

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In this study we investigated the feasibility of using solid electrodes as an alternative source or

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sink of electrons to regulate the redox potential of the mixed culture reactor fed with glucose, which

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therefore forces the rebalance the fermentation pathways to promote or inhibit targeted processes.

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The product spectrum was systematically evaluated under a large range of redox conditions, which

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covers the corresponding electron flow direction change from cathodic current to anodic current.

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The effects on methanogenesis under different conditions were specifically evaluated, and process

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stability was monitored for more than 3 months. System performance was also evaluated under both

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neutral and acidic conditions with different buffer capacities to understand the interactions between

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redox and pH controls.

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EXPERIMENTAL SECTION

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EF reactors construction and startup. Each EF reactor had two chambers which were

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separated with a cation exchange membrane (CMI7000, Membranes International Inc., NJ, USA).

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Each working electrode chamber had a volume of 100 mL, and each counter electrode chamber had

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a volume of 25 mL. Graphite felts (Sanye Carbon Co., Ltd., Beijing, China) were used as

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electrodes.27,28 Each electrode was in disc shape with a projected area of 28 cm2. A gas bag was

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connected to the working electrode chamber using a collection tube.29,30 Each reactor was equipped

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with an Ag/AgCl reference electrode (3 M KCl, AgCl saturated, +0.2 V versus SHE) placed near the

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working electrode. All EF reactors were run in duplicate. All electrode potentials reported in the 5



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study are relative to the Ag/AgCl reference electrode.31

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Glucose based synthetic wastewater was used as the substrate in all reactors. Every liter of

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growth medium contained 5 g glucose, 0.5 g NH4Cl, 0.5 g KCl, 0.1 g Ca2Cl, 0.1 g MgCl2.6H2O,

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and 10 mL trace minerals solution, and 200 mM PBS solution (with the initial pH of 7.0 or 6.2 as

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described in following section).32 The growth medium was purged with N2 prior to feeding to

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achieve an anaerobic condition. The same PBS solution without substrate was used as the

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electrolyte in the counter electrode chamber. Anaerobic sludge taken from a local wastewater

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treatment plant (Boulder, CO) was used as the inoculum (10% v/v in the initial 3 batches).33 The

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reactors were operated in fed-batch mode in different conditions for a total period of 93 days. From

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days 0-70, the reactors were divided in 3 groups, with each group applied a fixed potential at -0.2 V,

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-0.6 V, and -1.0 V, respectively. From days 71-93, all reactors were operated in open circuit

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condition without poised potential, so the results can be used to reveal the effects of electric current

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on product selection. Several studies have indicated that pH has effects on the product distribution

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between various acids, hydrogen and methane,12 so the effects of pH were evaluated by operating

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the reactors under 2 pH conditions. From days 0-37 the electrolyte had an initial pH of 7.0, and then

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from days 38-93 the pH was adjusted to 6.2. The operation temperature was controlled at 35 ºC, and

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the solution was constantly mixed by a magnetic stir bar. The working potentials were controlled

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using a multichannel potentiostat (CHI 1000B, Chenhua Co., Shanghai, China) in the

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three-electrode mode.34

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Analyses and calculations. The concentration of carbohydrates including formic acid, acetic

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acid propionic acid, butyric acid and lactic acid was analyzed by HPLC (Agilent 1200, USA) using

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a Bio-Rad Aminex HPX-87C column with 10 mM phosphoric acid solution as the eluent at a flow

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rate of 1 mL/min.35 The concentration and volume of hydrogen and methane were quantified by a

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gas chromatograph (Model 8610C, SRI Instruments) equipped with a thermal conductivity detector

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using nitrogen as the carrier gas.36,37 During day 60 and day 70, the electrochemical characteristics

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of the working electrode were analyzed using cyclic voltammetry (CV) from -1.0 V to +0.2 V at a 6


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scan rate of 1 mV/s.38 The working electrode was analyzed within the EF cell during the CV test.

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The carbon recovery was calculated based on the molar fraction of carbon contained in various

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products and in the substrate (glucose). The electron recovery of electron recovery from glucose

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were calculated based on the molar conversion factor of 24 e- equiv per mol glucose, 8 e- equiv per

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mol methane, 20 e- equiv per mol butyrate, 14 e- equiv per mol propionate, and 8 e- equiv per mol

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acetate, respectively.39 The uncounted carbon and electrons flows due to unmeasured products were

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classified as “others”.

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Biofilm samples on the working electrodes (namely Bl) and suspension samples (namely SP)

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were harvested on day 70. Microbial DNA was extracted using PowerSoil DNA Isolation Kit

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(MoBio Laboratories Inc.) according to the manufacturer protocol.40 The 16S rRNA genes were

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amplified by PCR using primer pair 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R

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(5′-GACTACHVGGGTATCTAATCC-3′), which target at the hypervariable V3–V4 region. The

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DNA sequence was analyzed using an Illumina MiSeq platform by Magigene Technology Co., Ltd

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(Guangzhou, China). Species abundance statistics were determined at the phylum and genus level to

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provide the microbial community information.41,42

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RESULTS AND DISCUSSION

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Anodic and cathodic current monitored for EF. The feeding frequency for the fed-batch

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operation extended from 2 days to 5 days and finally to 11 days to achieve a steady state with three

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repeatable cycles of current output, as shown with arrows in Fig. 1A. When switched to the open

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circuit mode the working potentials of all reactors were approximately -0.55 V (vs. Ag/AgCl)

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regardless of the original states (Fig. 1). Fig. 1B shows that when a working potential of -0.2 V was

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applied, the electrode potential was more positive than the open circuit potential, so anodic EF

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reactions occurred with electrons flowing toward the electrode, showing in positive current. In

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contrast, when the applied potentials were more negative than the open circuit potential (-0.6 V and

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-1.0 V), cathodic EF conditions were created, where the working electrode became an electron 7



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source, showing in negative current.43,44 The current at -1.0 V showed fluctuation at the beginning

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(days 0-10) with a maximum cathodic current of -7.9 ± 0.5 mA, while the currents in the other 2

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conditions were relatively stable. The anodic current increased with a maximum peak of 1.47 ± 0.12

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mA, while the cathodic current was slightly fluctuated within in the range from -0.14 mA to -0.04

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mA after stabilization (days 38-70). The maximum cathodic current in each batch was observed

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right after each substrate replacement, indicating possible disturbance of the redox environment. In

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mixed culture reactors, fermentative bacteria and electroactive bacteria interact synergistically to

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sustain the conversion of organic substrates to currents in an anodic EF condition,23,45-47 while in

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cathodic EF condition, the microbial consortium can also consume electrons from the electrode to

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make different products depending the redox condition.26

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Here Fig. 1.

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Applied working potentials affected hydrogen and methane production. Fig. 2 shows the

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profiles of H2 and CH4 generation during the operation in different stages. All reactors showed fast

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net H2 generation at the beginning (days 0-22), presumably due to fermentation of easily degradable

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glucose. Higher H2 was produced at -1.0 V during this stage when initial electrolyte pH was

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maintained at 7.0. When the duration of each batch was extended (days 22-38), a clear profile of

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decline in H2 was observed with minimal net H2 accumulated for each batch. When the initial pH

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was adjusted to 6.2 from day 38, higher H2 accumulation was observed with the highest buildup

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obtained at -0.6 V. When reactors were switched to open circuit, a decrease of H2 accumulation was

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observed at -0.6 V, and gradually all reactors tended to show similar net H2 production. Because H2

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was constantly consumed by hydrogenotrophic methanogens, homoacetogenic bacteria, and

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electroactive bacteria, the observed volume was a combination of generation and consumption.

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Thus, the net H2 accumulation at different stages was achieved at different working potential. It

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should be noted that the H2 production was not likely from water electrolysis, as the water 8


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electrolysis requires a large cathodic current density as the sole source of electrons for protons

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reduction, while a consistently decrease of cathodic current density was observed here (Fig. 1B).

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It is more interesting to see from Fig. 2B that CH4 generation was significantly influenced by

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the poised potential in different EF reactors. All reactors showed clear CH4 production after day 15,

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and the CH4 volume among different reactors ranged from 4.3-6.7 mL at day 38. When the initial

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pH dropped to 6.2 and the duration was extended during days 39-70, a clear increase in CH4

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generation was observed. Generally, more negative potential led to higher CH4 accumulation, while

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more positive potential showed low methanogenesis activities. Overall, an average of 12.02 ± 3.59

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mL of CH4 was generated each cycle in reactors under -1.0 V, where 7.44 ± 3.46 mL and 3.99 ±

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0.98 mL were generated each cycle from reactors under -0.6 V and -0.2 V, respectively. When

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reactors were switched to open circuit, a decrease of CH4 accumulation was observed at both -0.6 V

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and -1.0 V, and gradually these two reactors started to show similar performance in CH4 production.

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In contrast, a slightly increase of CH4 accumulation was observed when the reactor at -0.2 V was

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switched to open circuit, of which the maximum CH4 accumulation was still lower than the other

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two reactors.

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The trend of CH4 production does not necessarily align with the H2 content in each reactor,

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suggesting that the redox potential applied in each reactor played an important role in influencing

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methanogenesis. For example, a clear profile of decline of H2 accumulation was observed in all EF

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reactors during days 0-22, and only a trace amount H2 was accumulated during days 22-38. During

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day 38-71, -0.6 V working potential showed higher H2 generation, while -1.0 V working potential

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consistently showed higher CH4 generation. The findings are consistent with previous mixed culture

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studies for CO2 reduction, which showed that the activity of methanogens decreased when the

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cathode potential was increased.48,49 These results support the hypothesis that by regulating the 9



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redox potential using an electrode, methanogenesis can be effectively inhibited or improved

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depending on operational goals.50

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Here Fig. 2.

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The final pH of each cycle was monitored (Fig. S1), and a pH decrease was observed along

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with operation, presumably due to the accumulation of organic acids.14,39 The pH drop however was

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not significant due to the use of high strength PBS buffer. The final pH decreased by 0.54±0.03 in

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open circuit condition with an initial pH of 6.2. In comparison, pH drops varied among EF reactors

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poised under different potentials. The pH drop varied from 0.68 (at -1.0 V) to 0.79 (at -0.2 V) from

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the initial pH of 7.0. When the initial pH was 6.2, the drop varies from 0.28 (at -1.0 V) to 0.93 (at

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-0.2 V). The production (anodic) and consumption (cathodic) of protons were believed to contribute

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to pH changes as well.

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Applied working potentials affected carboxylic acids production. The production of

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different carboxylic acids including formic acid, acetic acid, propionic acid, butyric acid and lactic

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acid was measured under different working potentials. Fig. S2-S4 show that formic acid and lactic

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acid were produced at the beginning then disappeared after approximately 3 days, suggesting that

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they were intermediates and consumed by microbes. The concentration became stable after 5 days

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of a batch test, and the concentration of major VFAs (acetic acid, propionic acid, and butyric acid)

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were compared in this condition (Fig. 3).

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Here Fig. 3.

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Overall the total amount of VFA generation didn’t change significantly under different working

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potentials, but more negative potential did lead to slight increase in total VFA under pH 7.0 (Fig. 10


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3A). Such increase is mainly attributed to the increase in acetic acid production. Fig. 3B shows that

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when the working potential dropped from -0.2 V to -1.0 V at pH 7.0, the production of acetic acid

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increased by 55%. However, trend of acetic acid production was not obvious in more acidic

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condition (pH 6.2). The working potential didn’t seem to have big impact on propionic acid

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production, and rather higher production was observed in open circuit potential (Fig. 3C).

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Interestingly, the positive working potential led to an increase generation of butyric acid in both pH

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conditions (Fig. 3D). For example, under pH 7.0, the reactor at -0.2 V shown 21% increase of

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butyric acid production than that of open circuit potential. Under pH 7.0, the butyric acid production

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under different potential obviously showed an opposite trend compared to acetic acid. Moreover,

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neutral pH did lead to higher butyric production than that of pH 6.2 across the different working

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potentials. Overall, the decrease of working potential promoted acetic acid production by 55%

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under pH 7.0 whereas little impacts was observed under pH 6.2. More negative working potential

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slightly decreased butyric acid production and showed little impacts on propionic acid under both

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pH conditions. Plus, more propionic acids and acetic acid, while less butyric acid production were

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observed when the reactors were switched from the controlled working potentials to open circuit

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condition (Fig. 3C and 3D).

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The carbon recovery (Fig. 4A) and electron recovery (Fig. 4B) from glucose in the EF process

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was performed. A range of 61-78% in carbon recovery and 70-87% in electron recovery could be

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obtained by comparing final fermentation products and the substrate, and the spectrum of each

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product was tuned based on the difference of working potential. The contribution of current

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generation (at -0.2 V) or consumption (at -0.6 V and -1.0 V) to the electron recovery from glucose

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was in the range from 0.02% to 5% (Table. S1). The EF process is regarded as an electrochemically

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influenced spontaneous fermentation, thus the contribution of electron consumption or donation by

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the working electrode to the electron balances was limited.37 For instance, a cathodic EF study

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reported that only 0.2% of the electron inputs were used for butanol production, while 99.8% of the

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electrons were originated from the glucose substrate.19 Methane production didn’t contribute 11



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significantly to carbon and electron recoveries, because the limited molar fraction of carbon content,

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and the limited molar conversion factor of about 0.3 e- equiv per liter (@35 °C), as a gaseous

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product.39 The carbon recovery here was comparable with pervious studies.51 Generally, the carbon

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recovery was both effected by pH and the working potential, and the application of electrochemical

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control of redox potential successfully regulateed the carbon distribution of the final products.

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Here Fig. 4.

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Analysis of electroactivity of working electrodes. Fig. 5A shows that no apparent redox

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peaks were observed in any reactors at the beginning of a typical batch cycle. However, by the end

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of the batch cycle, an oxidation peak at approximately -0.26 V was observed for reactor under -0.2

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V, but no correlated reduction peak was detected (Fig. 5B). The oxidation peaks observed here were

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likely associated with redox couples possibly from flavoproteins to iron sulfur proteins.23,52 No

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obvious catalytic reactions were observed on CV curves at day 60 and day 70 in the -1.0 V reactor,

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which was consistent with the stable current observed in Fig. 1. The current ranged from -0.14 mA

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to -0.04 mA during the period. In conjunction with the metabolite profiles shown in Fig. 2-4, it is

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reasonable to hypothesize that more negative potential applied in the -1.0 V reactor influenced the

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metabolic shift toward methanogenesis. Such boost in methane production was largely due to the

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facilitated consumption of VFAs and H2 to CH4 by methanogens not direct electron update from the

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electrode. These CV results also suggest that metabolites with electroactivity were generated during

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the anodic EF process, but the electron exchanges between microbes and the electrode in cathodic

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EF might not involve mediated process.

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Microorganisms use a set of metabolic regulatory enzymes to sense the environmental redox

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state and external electron sinks. Such changes can trigger the shift of behavior and metabolisms of

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microorganisms.16 The environmental redox state could be regulated by the addition of chemical 12


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agents53 and by electrochemical methods. For example, a recent study successfully used

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ferricyanide as the mediator to carry electrons from the electrode into cells, and the electrochemical

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signals could be used to control the transcription and behavior of Escherichia coli.54 The working

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electrode with more negative potential also promoted methanogenesis of M. thermautotrophicus,

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which was hypothesized due to the stimulated activities of membrane-bound hydrogenase

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enzymes.17

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Regarding the reducing agents used for methanogenesis, there co-exist electrode and soluble

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electron donors such as H2 and acetate, whose productions are influenced by the redox potential as

285

well. Early electromethanogenesis studies showed the solid cathode served as the sole electron

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donor to produce methane from the electrochemical reduction of carbon dioxide,49 thus the

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increased methane generation was coincided with the increase of cathodic current, because more

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electrons were consumed. However, most recent studies found most microbial electrosynthesis

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reactions occurred by utilizing intermediate H25 or formate55, or other electron donors produced on

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the cathode rather than direct current uptake. Electrochemical control has been used to influence the

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methanogenesis sparging with H2 (as the main electron donor) and CO2, and the results shown that

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no obvious different of cathodic current was observed during stable stage when the working

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potential were controlled at different -0.2 V, -0.5 V, and -0.8 V (vs. Ag/AgCl), respectively.17

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In this study, the working electrode introduced by EF can be regarded as a special external

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electron sink or source depending on the potential applied, which triggered the activities of

296

regulatory enzymes. Specifically, the anodic EF is hypothesized to drive more ATP synthesis by

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creating a proton gradient, while the electron supply by cathodic EF are expected to produce more

298

reduced redox factors (NADPH)

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fermentation for ATP generation relied on substrate-level phosphorylation and have been found

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. These pathways are regarded as major routes in anaerobic

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dominant in previous studies.3,57 In summary, the electrode at more negative potentials could

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regulate the adjacent redox environment and thus stimulate the activity of methanogens. The

302

electron donors can be electrode but more likely H2 or acetate. Further studies on understanding the

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mechanisms of electron transfer in EF environment will help reveal such interactions, but it is

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beyond the scope of this study.

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Here Fig. 5.

306 307 308

Microbial community. The majority of bacterial 16S rRNA genes in all samples belonged to five

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phyla (Fig. 6A): Actinobacteria,

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Gram-negative Bacteroidetes and Gram-positive Firmicutes are known capable of polysaccharide

311

hydrolysis, and both were commonly found in mixed culture glucose fermentation39,58 and

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anaerobic digesters.59 The biofilm on electrodes were dominated by Firmicutes, while samples in

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suspension was dominated by Bacteroidetes. At the genus level (Fig. 6B), there were two genera

314

with high relative abundance, including Clostridium (35%-54% on biofilm and 7-26% in

315

suspension), and Bacteroides (1-4% in biofilm, 7-25% in suspending medium). Other genera

316

identified account for low relative abundance from 1-8.5%, which include Actinomyces,

317

Anaerofilum, Oxobacter, Bifidobacterium, Cloacibacillus,

318

Phascolarctobacterium,

319

Sporanaerobacter Genus Bacteroides is commonly found as saccharolytic bacteria, and Clostridium

320

is associated with both acetate and butyrate production from glucose.60 It should be noted that

321

Geobacter as a known electroactive bacteria were not detected with high abundance, which was in

322

agreement with the limited anodic current. Previous studies showed that when Geobacter was

323

enriched on the anode with glucose substrate, it was associated with high anodic current output.39,61

324

Bacteroidetes,

Prevotella,

Firmicutes, Proteobacteria, and Synergistetes.

Pseudoramibacter,

Desulfovibrio, Eubacterium,

Oscillospira,

Ruminococcus,

and

The archaeal community on the working electrode was dominated by Methanobacterium and 14


Page 15 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


325

Methanobrevibacter, both of which were hydrogentrophic methanogens belonging to a same family

326

of Methanobacteraceae (Fig. S5). When the working potential become more negative, the relative

327

abundance of Methanobacterium decreased, coincided with the increase of Methanobrevibacter. A

328

separate methane kinetics test was conducted to understand the roles played by hydrogenotrophic

329

and acetoclastic methanogens in the EF reactors. The results suggested that hydrogenotrophic

330

methanogen played a major role in CH4 production than acetoclastic methanogens (data not shown).

331

The change of relative abundance of archaeal community could relate to the increase of hydrogen

332

evolution on the working electrode with the potential adjusted at more negative values. For example,

333

on a methanogenic biocathode, less efficient hydrogen-forming electrode was dominated by

334

Methanobacterium, while platinum cathode with high efficient hydrogen-forming was dominated

335

by Methanobrevibacter.62

336 337

Here Fig. 6.

338 339

Glucose fermentation pathway in mixed culture electro-fermentation. This study presents

340

a new electrochemical redox control approach to achieve the valorization of organic waste by

341

regulating the fermentation pathways and promoting higher valued short chain VFAs in

342

fermentation process. The EF reactors showed various distributions of final products depending on

343

the applied electrical potential (Fig. 7). More negative working potential led to cathodic current,

344

which boosted methanogenesis.6,51,63,64 In contrast, more positive working potential led to anodic

345

current, which inhibited methanogen growth, rather promoted the growth of electroactive bacteria

346

and accumulation of butyric acid. This study demonstrates for the first time that electrodes can

347

serve as an alternative source or sink of electrons to regulate the redox potential of mixed culture

348

anaerobic reactors, so tunable fermentation products can be generated. By changing the working

349

potential poised on the electrode (-1.0 V to -0.2 V vs. Ag/AgCl), methanogenesis can be either

350

arrested or promoted, which is also associated with different amount of hydrogen and VFA 15



351

production. Our study implicated that using poised electrodes to trigger either anodic current or

352

cathodic current is a promising approach for product tuning depending on the valorization goals.

353

Here Fig. 7.

354 355 356

CONCLUSIONS

357

The results demonstrate that electrical potential had direct impacts on mixed culture

358

fermentation and influenced metabolites generation. Results show that overall the decrease of

359

working potential promoted the production of CH4, H2 and acetic acid under pH 7.0, whereas

360

smaller changes were observed on propionic acid and butyric acid production. When systems were

361

switched to open circuit condition, all reactors behaved similarly. While this study demonstrates the

362

feasibility of using electrical potential to regulate metabolite distributions in mixed culture

363

fermentation, more detailed investigations can be performed to understand the interactions among

364

different influencing factors and identify the desired pathways. The relationship between the

365

applied potential, the activities of regulatory enzymes, and microbial communities also need to be

366

further investigated.

367 368

SUPPLEMENTAL INFORMATION

369

Supplemental Information includes 4 figures can be found with this article online (PDF).

370

AUTHOR INFORMATION

371

Corresponding Author

372

*E-mail: [email protected] (P. Liang), [email protected] (Z.J. Ren)

373

Notes

374

The authors declare no competing financial interest.

375

Acknowledgments

376

This work was supported by the U.S. National Science Foundation (CBET 1510682), the Chinese 16


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377

Scholarship Council (CSC), and National Science Foundation of China (NSFC No. 51778324,

378

51422810).

379

17



380

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Fig. 1. The temporal changes of working potential (A) and electric current (B) monitored for EF

569

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570

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578

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The average of VFAs were calculated using the data at 5th day of each cycle for each scenario.

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Fig. 4. The carbon balance (A) and electron recovery (B) of the electro-fermentation reactors

581

operated under different conditions. OC: open circuit.

582

Fig. 5. Cyclic voltammetry (CV) profiles of the working electrodes under different poised potentials

583

at (A) the beginning of a batch cycle (day 60) and (B) the end of a batch cycle (day 70).

584

Fig. 6. Bacterial community distribution at the phylum level (A), and at the genus level (B).

585

Sequences less than 1% of total are grouped as “others”.

586

Fig. 7. Schematic of mixed culture glucose fermentation pathway under the influence of

587

electrochemical control of redox potential.

588 26


Page 26 of 35

Page 27 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


589

27


inoculation

0.0

pH=7.0

Page 28 of 35

open circuit A

pH=6.2

-0.5 -1.0 2

B

0

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Potential (V vs. Ag/AgCl)


-2

-0.2 V -0.6 V -1.0 V

-4 -6 -8

0

10

20

30

40

50

60

70

80

90

Time (d)

590 591

Fig. 1. The temporal changes of working potential (A) and electric current (B) monitored for EF

592

reactors during 93-day operation. The reactors were either controlled with different potentials (days

593

0-70) or in open circuit (days 71-93). The arrows show substrate replacement. The initial pH of the

594

fermentation broth was separately adjusted to 7.0 (days 0-37), and 6.2 (days 38-93).

595

28


Page 29 of 35

Hydrogen (ml)

150 inoculation

pH=7.0

open circuit

pH=6.2

A

100 50 0 15

Methane (ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


B

-0.2 V -0.6 V -1.0 V

10 5 0

0

10

20

30

40

50

60

70

80

90

Time (d)

596 597

Fig. 2. The profiles of H2 (A) and CH4 production (B) in EF reactors operated in different stages.

598

The arrows indicate substrate replacement and one batch duration. The initial pH was separately

599

adjusted to 7.0 (0-37 days), and 6.2 (38-93 days). Applied potentials are relative to the Ag/AgCl

600

reference electrode.

601

29



4

2

A

B

Acetic acid (g/L)

Total VFAs (g/L)

3

2

1

0 2

Butyric acid (g/L)

1

0

1

0 2

C

7.0-EF 6.2-EF 6.2-OC

Propionic acid (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

D

1

0

-0.2 V

-0.6 V

-1.0 V

-0.2 V

-0.6 V

-1.0 V

602 603

Fig. 3. The concentration of total VFAs (A), acetic acid (B), propionic acid (C) and butyric acid (D)

604

in EF reactors in different stages. “7.0-EF” and “6.2-EF” indicate the initial pH in closed circuit EFs

605

was 7.0 and 6.2, respectively. “6.2-OC” indicates open circuit condition with an initial pH of 6.2.

606

The average of VFAs were calculated using the data at 5th day of each cycle for each scenario.

607

30


Page 31 of 35

others

butyrate

propionate

acetate

others

B

80 60 40 20 0

608

methane

100

Electron recovery (%)

A Carbon recovery (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pH7.0 (-0.2 V)

butyrate

propionate

acetate

80 60 40 20 0

pH7 pH7 pH7 pH6.2 pH6.2 pH6.2 pH6.2 pH6.2 pH6.2 -0.2V -0.6V -1.0V -0.2V -0.6V -1.0V -0.2V -0.6V -1.0V OC OC OC

methane

100

pH7 pH7 pH7 pH6.2 pH6.2 pH6.2 pH6.2 pH6.2 pH6.2 -0.2V -0.6V -1.0V -0.2V -0.6V -1.0V -0.2V -0.6V -1.0V OC OC OC

pH7.0 (-0.2 V)

609

Fig. 4. The carbon balance (A) and electron recovery (B) of the electro-fermentation reactors

610

operated under different conditions. OC: open circuit.

611

31



2

2

A

B 1

0 -1 -0.2 V -0.6 V -1.0 V

-2 -3 -1.0

612

Current (mA)

1

Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

-0.8

-0.6

-0.4

-0.2

0.0

0.2

Electrode potentials (V vs. Ag/AgCl)

0 -1 -0.2 V -0.6 V -1.0 V

-2 -3 -1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

Electrode potentials (V vs. Ag/AgCl)

613

Fig. 5. Cyclic voltammetry (CV) profiles of the working electrodes under different poised potentials

614

at (A) the beginning of a batch cycle (day 60) and (B) the end of a batch cycle (day 70).

615

32


Page 33 of 35

A 100

-0.2V Bf

-0.6V Bf

-1.0V Bf

-0.2V SP

-0.6V SP

-1.0V SP

B 100

Relative abundance (%)

60

40

20

-0.6V Bf

-1.0V Bf

-0.2V SP

-0.6V SP

-1.0V SP

60

40

20

0

0 Others Firmicutes

616

-0.2V Bf

80

80

Relative abundance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Synergistetes Bacteroidetes

Proteobacteria Actinobacteria

Others Prevotella Desulfovibrio Bacteroides

Sporanaerobacter Phascolarctobacterium Clostridium Anaerofilum

Ruminococcus Oxobacter Cloacibacillus Actinomyces

Pseudoramibacter_Eubacterium Oscillospira Bifidobacterium

617

Fig. 6. Bacterial community distribution at the phylum level (A), and at the genus level (B).

618

Sequences less than 1% of total are grouped as “others”.

619

33



Page 34 of 35

acidogens and acetogens Glucose NAD+

Propionate

NAD+ NADH+H+

H2

NADH+H+

Pyruvate CO2 H2

Fd

Lactate

NAD+ NADH+H+

FdH2 NADH+H+

Acetyl-CoA

NAD+

accelerated by cathodic EF Butyrate

Acetate

electroactive bacteria introduced introduced by anodic anodic EF 620

hydrogenotrophic methanogen

CH4

electric current

621

Fig. 7. Schematic of mixed culture glucose fermentation pathway under the influence of

622

electrochemical control of redox potential.

623 624

34


Page 35 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

625


For Table of Contents Use Only e-

Anodic EF Glucose

e-

Cathodic EF Glucose H2O

H2O

e-

e-

e-

e-

e-

e-

e-

e-

H2

Products 626 627 628 629

O2

Products

Anode Methanogens

Anode Cathode Cathode Anode-respiring bacteria Fermenters

Synopsis: Solid electrodes can serve as a source or sink of electrons to regulate the redox potential of mixed culture fermentation and generate tunable products.

630

35


Electrochemical Control of Redox Potential Arrests Methanogenesis

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