Changes in Glucose Fermentation Pathways as a Response to the

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Changes in glucose fermentation pathways as a response to the free ammonia concentration in microbial electrolysis cells Mohamed Mahmoud, César Iván Torres, and Bruce E. Rittmann Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05620 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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Changes in glucose fermentation pathways as a response to the free ammonia concentration in microbial electrolysis cells Mohamed Mahmoud § ¥ £ *, César I. Torres § Φ, and Bruce E. Rittmann § ¥

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§

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Tyler Road, Tempe, Arizona, USA.

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¥

Biodesign Swette Center for Environmental Biotechnology, Arizona State University, 727

School of Sustainable Engineering and the Built Environment, Arizona State University,

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Tempe, Arizona, USA.

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Φ

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Tempe, Arizona, USA.

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£

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Cairo 12311, Egypt

School for Engineering of Matter, Transport and Energy, Arizona State University,

Water Pollution Research Department, National Research Centre, 33 El-Buhouth St., Dokki,

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* Corresponding author: Mohamed Mahmoud

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Email: [email protected] & [email protected]; tel.: +1 480 727 7596; fax: +1 480 727

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0889.

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Abstract

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When a mixed-culture microbial electrolysis cell (MEC) is fed with a fermentable substrate, such

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as glucose, a significant fraction of the substrate’s electrons ends up as methane (CH4) through

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hydrogenotrophic methanogenesis, an outcome that is undesired. Here, we show that free

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ammonia-nitrogen (FAN, which is NH3) altered the glucose fermentation pathways in batch

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MECs, minimizing the production of H2, the “fuel” for hydrogenotrophic methanogens.

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Consequently, the Coulombic efficiency (CE) increased: 57% for 0.02 g FAN/L fed-MEC,

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compared to 76% for 0.18 fed-MECs and 62% for 0.37 g FAN/L fed-MECs. Increasing FAN

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concentration was associated with the accumulation of higher organic acids (e.g., lactate, iso-

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butyrate, and propionate), which was accompanied by increasing relative abundances of

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phylotypes that are most closely related to anode respiration (Geobacteraceae), lactic-acid

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production (Lactobacillales), and syntrophic acetate oxidation (Clostridiaceae). Thus, the

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microbial community established syntrophic relationships among glucose fermenters, acetogens,

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and anode-respiring bacteria (ARB). The archaeal population of the MEC fed 0.02 g FAN/L was

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dominated by Methanobacterium, but 0.18 and 0.37 g FAN/L led to Methanobrevibacter

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becoming the most abundant species. Our results provide insight into a way to decrease CH4

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production and increase CE using FAN to control the fermentation step, instead of inhibiting

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methanogens using expensive or toxic chemical inhibitors, such as 2-bromoethane sulfonic acid.

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Keywords: Microbial electrolysis Cells, Free ammonia-nitrogen, Syntrophy, Anode-respiring

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bacteria, Fermenters.

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Introduction

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Microbial electrolysis cells (MECs) represent one of the newest environmental

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biotechnologies for wastewater treatment coupled to the production of renewable energy in the

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form of electrical power, hydrogen gas (H2), or valuable chemicals.1 Since the organic

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compounds – the “fuel” for the MECs – are complex in nature, their biodegradation requires

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cooperation among different trophic groups: fermenters, homoacetogens, and anode-respiring

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bacteria (ARB), as well as competition with methanogens.2,3 Generally, an MEC’s microbial

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community has a high complexity in terms of community structure and diversity, but key

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microbial populations have been identified.4,5

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The defining group in an MEC is the ARB, which perform a unique type of respiration.

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Most respiring anaerobes transfer electrons intracellularly to a terminal electron acceptor, such as

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sulfate, nitrate, or carbon dioxide.6,7 ARB oxidize organic matter internally, but transfer the

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resulting electrons outside their membrane to a solid electron acceptor, the anode.2,5 Another

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feature of majority of ARB in mixed community is that their electron donors are limited to only a

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few simple substrates, such as acetate and H2.8–10 Thus, fermentation is necessary to generate the

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mixture of simple fermentation products that the ARB can utilize directly.

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In environments lacking electron acceptors, fermentable organic compounds, such as

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glucose, are transformed into a variety of organic acids and H2. Acetate is the most prevalent

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organic acid, and Table 1 presents a number of reactions in which acetate and H2 are formed or

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consumed. H2-consumers include hydrogenotrophic methanogens, which use H2 as the main

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electron donor and produce methane (reaction 3 in Table 1).7,11–13 Homoacetogens also scavenge

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H2 to yield acetate. Homoacetogenesis and hydrogenotrophic methanogenesis have very low

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energy yields.14 H2 must be kept at low level to allow fermentation to be thermodynamically

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possible.3,11,15 When H2 builds up, the fermentation stoichiometry changes such that higher

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organic acids, such as lactate, butyrate, propionate, and ethanol, are produced rather than acetate

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and H2.3,15

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When the MEC’s anode is the only respiratory electron acceptor, ARB can out-compete

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methanogens for acetate, due to their thermodynamic and kinetic advantages over the acetate-

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consuming methanogens (reaction 5 in Table 1); hence, acetoclastic methanogens cannot

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compete with acetate-oxidizing ARB, although H2-oxidizing methanogens compete well with

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H2-oxidizing ARB.8 However, ARB do not have similar advantages for H2 consumption, and the

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electrons in H2 often are channeled to methane (CH4) via hydrogenotrophic methanogens, which

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are able to grow in suspension, as well as in the anode’s biofilm.9,16 Thus, one of the challenges

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for MEC scale-up and commercialization is to minimize production of CH4 from the H2

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generated via fermentation in the presence of hydrogenotrophic methanogens.

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Table 1.

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Previous researchers proposed different strategies to inhibit methanogenesis in

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laboratory-scale MECs, including thermal treatment, periodic exposure to oxygen (O2), pH

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excursions, alamethicin exposure, and use of chemical inhibitors such as 2-bromoethanesulfonate

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(BES).8,17, 18 Among the proposed strategies, chemical inhibitors seem to be the most effective

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approach, due to their selectivity for inhibiting the activity of methyl coenzyme M reductase A

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(mcrA) gene in acetoclastic and hydrogenotrophic methanogens. For example, Parameswaran et

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al.8 inhibited methanogens in an ethanol-fed MEC with 50 mM BES, which boosted the

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Coulombic efficiency (CE) by ~40%, as electrons from H2 were rerouted to anode respiration

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instead of to CH4 production. Although BES is an effective way to inhibit methanogens,8,9 it is

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not practical for field applications due to the continuous need to supply the inhibitor, which is

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environmentally toxic and expensive.

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We recently revealed that ARB are tolerant to relatively-high free-ammonia nitrogen

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(FAN) concentrations, up to at least 200 mg/L; however, high total-ammonia nitrogen (TAN)

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concentrations (i.e., > 2.2 g/L) might cause inhibition (or lowering) of the anode respiration

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rate.19 The relatively high threshold for FAN inhibition by ARB may be a tool for managing

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microbial communities to favor ARB and high electron recovery in MECs fed with fermentable

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substrates, as long as TAN is not too high. Therefore, we hypothesize that high-enough FAN

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can promote the desired syntrophy in MECs fed with fermentable substrate by inhibiting

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hydrogenotrophic methanogens. Suppressing H2-oxidizing methanogens provides ideal

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conditions for homoacetogens to oxidize H2 and grow, leading to the generation of acetate, the

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ideal substrate for ARB. 8,9 This hypothesis requires that a FAN concentration high enough to

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suppress methanogens not have a strong inhibitory effect on ARB and fermenters.14,20 This

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strategy is highly relevant to MECs treating real wastewater that are characterized by high FAN

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concentrations, including landfill leachate and animal wastewater.21,22

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A high FAN concentration also might promote a new pathway for acetate consumption to

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produce CH4 at very low H2 partial pressure: syntrophic acetate oxidation (SAO) (alternatively

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known as reverse acetogenesis), which has been explored in anaerobic digesters.23,25 SAO is a

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two-step process in which acetate is utilized by syntrophic acetate-oxidizing bacteria (SAOB)

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(reaction 7 in Table 1) with the generation of reducing equivalents, often in the form of H2. This

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step is a highly energy-demanding reaction that requires syntrophy with H2-consuming bacteria

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(e.g., hydrogenotrophic methanogens or hydrogenotrophic ARB) to maintain a very low level of

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H2 (reaction 3 or reaction 6 in Table 1, respectively) in order to make the overall reaction

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thermodynamically favorable.11,24 In the absence of other electron acceptors, such as nitrate and

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sulfate, the produced H2 is most likely routed to CH4 through hydrogenotrophic methanogenesis,

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since ARB are known of being relatively poor H2-consumers.8,25 So far, nothing is known about

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the SAO process in MECs fed with high FAN concentration.

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Our overarching goal is to investigate the effect of different FAN levels on the

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interactions among ARB and other members of the communities, particularly the fermenters and

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methanogens. We document the microbial interactions through a combination of chemical,

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electrochemical, and genomic tools. In order to study electron-flow and synergies in an MEC’s

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anode, we use a fixed concentration of glucose (5 mM) as the sole electron-donor substrate, and

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we vary the influent FAN concentration (i.e., 0.02, 0.18, and 0.37 g FAN/L) going into the anode

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during batch and semi-continuous (hydraulic retention time of 2 days) MECs. Key is that we

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establish electron-equivalent mass balances. We also characterize the relative abundance and

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composition of bacteria and Archaea by Illumina sequencing, and we track homoacetogens and

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methanogens by targeting the formyltetrahydrofolate synthetase (FTHFS) gene – a conserved

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gene involved in their CO2 fixation pathway – and mcrA gene, respectively, by quantitative real-

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time PCR (qPCR).

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Materials and methods

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MEC design and operation For our experiments to understand the fundamentals of how the microbial community

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changes in response to high FAN concentration, we used half-cell MECs with its anode potential

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controlled using a potentiostat. Each MEC held ~ 320 mL in each chamber and had a square

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graphite anode having a total surface area of ~ 11.6 cm2, and a 0.8-cm OD graphite rod as

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cathode. This setup excluded any interference from potential losses at the cathode or due to a

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large distance between the anode and the cathode; fixing the anode potential allowed us to focus

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only on the anodic reactions. In order to ensure that the ARB were not limited by a low anode

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potential, we poised the anode at a fixed potential of – 0.3 V vs. Ag/AgCl (or ~ – 0.04 V vs.

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standard hydrogen electrode (SHE) in our media) using a VMP3 digital potentiostat (Bio-Logic

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USA, Knoxville, TN) by placing an Ag/AgCl reference electrode (BASI Electrochemistry, west

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Lafayette, IN) about 0.5 cm away from the anode. We separated the anode chamber from the

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cathode chamber with an anion exchange membrane (AMI 7001, Membranes International, Glen

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Rock, NJ). The pH of the cathode chamber was adjusted to 12 by addition of 10 N NaOH to

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facilitate the transport of hydroxide ions (OH–) from the cathode compartment to the anode

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compartment through the anion-exchange membrane.

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We inoculated the MEC’s anode chamber with 2 mL of biofilm inoculum from a

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previously operated MEC fed with 5 mM glucose as the sole electron donor. After sparging the

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MEC with ultra-high purity N2 gas (≥99.9%) for ~ 45 min, we fed the MEC with autoclaved (for

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90 min at 121°C) glucose medium (initial pH of 8.1) containing: 5 mM glucose (or 38.4 me–eq),

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100 mM phosphate buffer (KH2PO4/Na2HPO4), 2.1 g NaHCO3, and 10 mL of trace minerals as

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outlined in Parameswaran et al.8 We added different amounts of ammonium chloride as the sole

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N-source, giving FAN (and TAN) concentrations of 0.02 g FAN/L (0.2 g TAN/L), 0.18 g FAN/L

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(2 g TAN/L), and 0.37 g FAN/L (4 g TAN/L). We eliminated the possibility of ARB biofilm

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needing to fix atmospheric N2 by using an ammonium concentration more than sufficient for

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growth in the control experiments (0.2 g N/L or 14 mM-N). Prior to the MEC’s electron balance

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experiments, we acclimated the inoculum by continuously feeding the developed biofilm with 5

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mM glucose medium as the sole electron donor at relatively short hydraulic retention time (i.e.,

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24 h), followed by performing 2 consecutive batch cycles to select for a microbial community

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that was efficient at consuming glucose and producing electrical current. We controlled the

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temperature at 30°C in a temperature-controlled room, and the liquid in both chambers was

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mixed using a magnetic stirrer at 220 rpm. We performed all batch MEC experiments in

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duplicate.

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In order to confirm and expand the results from the batch MEC experiments, we

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evaluated MEC performance with semi-continuous operation. We operated two independent

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MECs in parallel and fed in a semi-continuous mode once every 2 days. The MECs were

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operated at a fixed temperature of 30°C and fed with a 5-mM glucose medium having the same

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composition as mentioned in the previous paragraph. The liquid in both chambers was mixed

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using a magnetic stirrer at 220 rpm. We tested the MECs with different FAN concentrations

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achieved with different combinations of TAN concentration and pH: (1) 0.003 g FAN/L (pH = 7

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and TAN = 0.2 g/L), (2) 0.02 g FAN/L (pH = 8.1 and TAN = 0.2 g/L), 0.18 g FAN/L (pH = 8.1

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and 2 g/L), and (4) 0.37 g FAN/L (pH = 8.1 and TAN = 4 g/L).

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Chemical analyses We measured TAN, in duplicate, using HACH kits (HACH, Ames, IA) after filtration

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through a 0.22-µm membrane filter (PVDF GD/X, Whatman, GE Healthcare, Ann Arbor, MI).

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We quantified liquid samples for organic fermentation products and glucose using high-

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performance liquid chromatography (HPLC; Model LC-20AT, Shimadzu, Columbia, MD) with

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an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) after filtration through a

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0.22-µm membrane filter according the method described in Mahmoud et al.21 Briefly, we used

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2.5 mM sulfuric acid and 18-MΩ reverse-osmosis water as eluents for determining organic

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fermentation products and glucose, respectively, at a constant flow rate of 0.6 mL/min. We

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developed a five-point calibration curve for every set of analyses, performed duplicate assays,

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and report the average concentrations.

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We measured the volume of gas produced with a friction-free glass syringe of 10- or 25-

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mL volume (Popper & Sons, Inc., New Hyde Park, NY, USA). We estimated gas percentages of

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H2, CH4, and CO2 in samples taken with a gas-tight syringe (SGE 500 µL, Switzerland) using a

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gas chromatograph (GC 2010, Shimadzu Corporation, Columbia, MD) equipped with a thermal

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conductivity detector and a packed column (CarboxenTM 1010 PLOT Capillary Column,

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Supleco, Inc.). Helium was the carrier gas at a constant flow rate of 10 mL/min and pressure of

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42.3 kPa. Temperature conditions for column, injection, and detector were 80, 150, and 220 °C,

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respectively. We employed analytical grade H2, CH4, and CO2 gases for standard curves, carried

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out gas analyses in duplicate, and averaged the two values.

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Microbial community analyses DNA extraction. At the end of each batch experiment, we harvested the entire biofilm

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biomass from each MEC anode by scraping it off with a sterilized pipette tip and suspending the

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biomass sample in a sterile centrifuge tube containing DNA-free water. We then centrifuged the

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contents at 10,000g (Eppendorf Centrifuge 5414 D, USA) for 10 min to concentrate the biomass,

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which we stored at −20 °C prior to DNA extraction. We also centrifuged the entire liquid of the

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MEC chamber to concentrate the suspended phase for extraction. We extracted the total

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genomic DNA using the MOBIO Powersoil DNA extraction kit according to manufacturer’s

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instructions, and determined the quality and quantity of the extracted DNA using a nanodrop

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spectrophotometer (ND 1000, Thermo Scientific) by measuring absorbance at 260 and 280 nm.

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Quantitative real-time PCR (qPCR). We evaluated the presence and abundance of

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methanogenic Archaea and homoacetogens by targeting the mcrA and FTHFS genes,

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respectively, using qPCR. We carried out all PCR reactions in optically clear tubes with caps in

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an Eppendorf Realplex 4S realcycler with a 20 µL total reaction volume. We performed all

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qPCR reactions in triplicate along with a six-point standard curve by following modified assays

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for FTHFS gene9 and mcrA gene.26 We performed negative-control assays by using DNA-free

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water instead of DNA templates. We reported the results as the number of gene copies per

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reactor, after calculating the number of 16S rRNA genes per the entire biofilm or suspended

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phase of each MEC.

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Illumina sequencing. We amplified the extracted DNA using 16S rRNA gene-targeting

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forward and reverse fusion primers at MR DNA laboratory (www.mrdnalab.com, Shallowater,

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TX, USA). We performed bacterial and archaeal sequence using the bar-coded primer set 515F

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(5′–GTGCCAGCMGCCGCGGTAA–3′)/806R (5′–GGACTACHVGGGTWTCTAAT–3′)27 and

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349F (5′–GYGCASCAGKCGMGAAW–3′)/806R (5′–GGACTACVSGGGTATCTAAT–3′),28

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respectively, in a single-step 28 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen,

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USA) with the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s,

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53°C for 40 s, and 72°C for 1 min, after which a final elongation step at 72°C was for 5 min. In

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order to determine the amplification success and the relative intensity of bands, PCR products

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were checked in 2% agarose gel.29 Then, the amplified products were pooled in equal

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proportions based on their molecular weight and DNA concentrations, purified using calibrated

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Ampure XP beads, and used to prepare DNA libraries following the Illumina TruSeq DNA

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library preparation protocol. Sequencing was performed on a MiSeq Illumina sequencer

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(Illumina Inc., USA) following the manufacturer’s guidelines.

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Bioinformatics analysis. We analyzed the sequences data using QIIME software package

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version 1.9.130 after trimming off low-quality bases and discarding sequences shorter than 25 bp,

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longer than 450 bp, or labeled as chimeric sequences. We performed taxonomic classification at

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3% sequence divergence (97% sequence similarity) and assigned taxonomy to operational

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taxonomic units (OTUs) by using the Ribosomal Database Project (RDP) classifier with a 50%

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confidence threshold.31 We performed rarefaction on the OTU table at a depth of 100 sequences

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in 10 replicates, and we analyzed the rarefaction measures with the same sequence numbers per

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sample (i.e., 223,000 sequences per sample for bacteria population) with a python script in

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QIIME software.31 We performed alpha- and beta-diversity calculations, including richness of

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each samples with Chao1 index,32 the diversity with Shannon and Phylogenetic Distance Whole

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Tree metrics,33 the evenness with the equitability coefficient on a normalized scale from zero, in

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which community is perfectly even, to 1, in which community has one dominant OTU and many

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singlets,29 and principal-coordinates analysis (PCoA) using python script in QIIME software.

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Sequence data sets are available at NCBI/Sequence Read Archive (SRA) under study with

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BioProject accession number PRJNA343831.

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Electron balance and electron flow into various sinks We established electron balances by estimating the electron equivalents of experimentally

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measured glucose and fermentation products with the following equivalences:12 24 me– eq per

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mmole glucose, 8 me– eq per mmole acetate, 14 me– eq per mmole propionate, 20 me– eq per

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mmole butyrate, 12 me– eq per mmole lactate, 25 me– eq per mmole valerate, 0.32 me– eq per mL

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CH4 (@30°C), and 0.08 me– eq per mL H2 (@30°C).

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First, we established electron flow from glucose when hydrogenotrophic methanogens

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are not suppressed or are completely suppressed. We assumed stoichiometric fermentation of

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glucose (24 me– eq) into 2 moles of acetate (16 me– eq) and 4 moles of H2 (8 me– eq). Acetate is

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efficiently consumed by acetate-consuming ARB to generate electric current, while H2 is

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consumed by hydrogenotrophic methanogens that have thermodynamic, kinetic, and metabolic

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advantages over H2-consuimng ARB and homoacetogens.8 In parallel, a fraction of electrons is

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utilized by different microbial groups in the MEC’s anode to synthesize new microbial cells. We

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quantify the fraction of electrons used for biomass synthesis based on fs° (me– eq biomass per

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me– eq substrate) values of 0.18 for fermenters,12 0.08 for hydrogen-consuming methanogens,12

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~0.02 for homoacetogens, 34,35 and ~ 0.09 for ARB.19 Supplementary Figure S1 shows the

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different paths for electron flow from glucose when hydrogenotrophic methanogens are not

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suppressed (Figure S1A) or are completely suppressed (Figure S1B). The resulting CE for the

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electron flows in Figure S1A (when methanogenesis is not suppressed) is 50%, with 25% of the

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electrons routed to CH4 gas and 25% to biomass. CE increases to 76% when methanogenesis is

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completely inhibited, with comparable electrons routed to biomass (24%) (Figure S1B).

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Results and discussion

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Framework for explaining electrons distribution for batch MECs

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Figure 1 presents the electron distribution from glucose into the possible electron sinks

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over the course of the experiments with different FAN concentrations. The inoculum had been

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pre-acclimated to glucose, and then we performed an electron balance on batch experiments with

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glucose as the sole electron donor. With the lowest initial FAN (0.02 g FAN/L), current

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production increased significantly during the first 2 days (Figure 1A). Simultaneously, acetate

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production increased to ~ 32% of the total electrons, and glucose decreased below the detection

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limit. Methane slowly accumulated within 2 days of operation and reached a plateau after day 6,

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when methane generation accounted for ~ 15% of the total electrons supplied from glucose.

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15% methane is less than the estimate in Figure 1A (25%), probably due to either H2

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consumption by either ARB or homo-acetogenesis. The total electrons contained in other

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fermentation products were small: ~ 3.3 % for propionate and ~ 3.1% for butyrate within the

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first 4 days of operation. Propionate and butyrate were completely consumed at day 17 and day

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8, respectively.

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We detected no H2 in the headspace gas of the MEC’s anode. Thus, H2 produced during

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glucose fermentation was quickly channeled to methane by H2-consuming methanogens, to

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current by ARB, or to acetate and then current by homoacetogens and ARB.13,24 The Coulombic

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recovery at the end of batch cycle (~ 57%) was slightly greater than the estimated value in Figure

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S1, which suggests that a modest flow of electron where routed through H2 to the anode.

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Figure 1B shows the distribution of electrons from glucose during 20 days of batch operation with 0.18 g FAN/L, where methane generation was partly inhibited. Compared to the

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MEC fed with 0.02 g FAN/L (Figure 1A), current slowly increased during the first 2 days and

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was associated with complete glucose fermentation. However, lactate started to increase,

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becoming the largest electron sink (~ 30%) at day 2, followed by acetate (~ 13%), propionate (~

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5%), and iso-butyrate (~ 2%). Since lactate fermentation is more likely than direct anode

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respiration, it has to be fermented first before it can be utilized by ARB. Lactate fermentation

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proceeded rapidly, and the current increased to become the largest electron sink after day 6, at

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which time methane accounted for ~ 9% of the total electrons supplied from glucose. Although

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we did not detect any H2 in the headspace gas, formate was detected in low concentration (0.67

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mM), accounting for nearly 2% of glucose’s total electrons. These results support that that 0.18

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g FAN/L delayed lactate fermentation, rather than glucose fermentation, and significantly

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inhibited methanogenesis. A key finding is that CE was ~ 76%, which illustrates the benefit to

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inhibiting methanogenesis.

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Further increasing the initial FAN concentration to 0.37 g FAN/L (Figure 1C) had a more

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pronounced effect on fermentation, although the impact on methanogenesis was not much

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greater than 0.18 g FAN/L. Similar to the lower FAN concentration, the rate of glucose

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consumption did not change; however, 0.37 g FAN-N/L altered the fermentation kinetics and

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pathway. During the first 4 days, lactate, iso-butyrate, propionate, butyrate, and acetate

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accumulated, collectively accounting for ~ 25% of glucose’s electrons. After 4 days,

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fermentation proceeded rapidly with concurrent increases in electric current, which became the

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largest electron sink by the end of the batch cycle (~ 62%), suggesting that this high FAN

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concentration might partially inhibit ARB. However, methanogenesis was not completely

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inhibited, as it accounted for ~ 6% of glucose’s electrons.

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As a result of organic-acids accumulation, the pH at the end of all batch cycles decreased

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from their initial pH value (8.1) to a narrow range of 6.85−7.10. Since the pH values were not

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much different from each other, variations in pH should have had a minimal effect on

317

fermentation pathway and kinetics.36

318 319

Figure 1.

320 321 322

Electron balances for batch MECs Figure S2 presents the electron balances at the end of batch-cycle operation: 57±2.9%,

323

76±3.5%, and 62±2.7% of the total electrons from glucose ended up as electric current for MECs

324

fed with 0.02, 0.18, and 0.37 g FAN/L, respectively. Theoretically, ~ 25% (or 9.7 me– eq) of

325

glucose’s electrons (i.e., 38.4 me– eq) should have been ended up as methane, if all the generated

326

H2 were utilized by H2-consuming methanogens (Figure S1A). However, we detected as

327

methane only about 15%, 9%, and 6%, respectively, of the original electrons.

328

Electrons ending up as organic acids at the end of the experiment were by far the highest

329

for the MEC fed with 0.37 g FAN/L: ~ 16% of the influent electrons, versus ~ 0.2% and ~ 2%

330

for MECs fed 0.02 and 0.18 g FAN-N/L, respectively. Approximately 28%, 13%, and 16% of

331

the electron from glucose were unaccounted by directly measured components. Unidentified

332

components include biomass, soluble microbial products, and fermentation products not

333

measured by HPLC. Figure S1 shows an estimate of 25 and 24% of electrons ending up in

334

biomass when methanogenesis is active versus completely inhibited.

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335 336

Semi-continuous glucose fermentation in MECs To examine whether high FAN concentration affected fermentation, methanogenesis, or

337

both, we performed MEC experiments using semi-continuous operation by replacing the feed

338

medium (i.e., 5 mM glucose) once every two days. Figure 2 summarizes the results for both

339

MECs, which showed similar trends. The current density was stable over the influent FAN

340

range of 0.003 to 0.18 g FAN-N/L (Figure 2A), but a further increase of influent FAN to 0.37 g

341

N/L led to a gradual decrease in the current density after 2 semi-continuous cycles. This pattern

342

suggests that higher FAN slowed acetate oxidation by ARB, since acetate accumulated at the end

343

of semi-continuous cycle (Figure 2B).

344

Similar to the batch MECs, glucose was completely fermented for all FAN conditions,

345

confirming that high FAN concentration did not affect the first fermentation step. As the FAN

346

concentration increased, more electrons ended up in organic acids, and this was associated with

347

17 – 29% decreases in methane yield. Perhaps high FAN altered the fermenters’ intracellular pH

348

homeostasis, leading them to divert electrons away from H2 production and toward formation of

349

more-reduced organic products.37 A shift from H2 to organic acids is consistent with high FAN

350

maintaining a low H2 concentration in the MEC, a change that should be beneficial for

351

syntrophic interactions not involving hydrogenotrophic methanogens.

352 353

Figure 2.

354

355

Distribution of bacterial population in batch MECs

356

Figure 3 and Figure S3 report the sequence analyses of the V4 region in the bacterial 16S

357

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358

belonged to 3 phyla: Proteobacteria, Firmicutes, and Bacteroidetes (Figure 3A), which is

359

consistent with previous studies.9,38,39 Several members of phyla Bacteroidetes and Firmicutes

360

are known to ferment sugar, whereas many members of Proteobacteria are known to perform

361

anode respiration.40,41 The lower abundance of Proteobacteria in the suspended-phase (SP)

362

samples agrees with the fact that most of ARB can only use the anode surface as terminal

363

electron acceptor.

364

Among the Proteobacteria, Deltaproteobacteria were the largest sub-group. The

365

predominant genus among Deltaproteobacteria was Geobacter, and their relative abundance

366

increased with higher FAN. Whereas biofilm (Bf) samples from 0.02 g FAN/L had ~ 2.6% of

367

the total genus sequences in Geobacter, samples from 0.18 and 0.37 g FAN/L had 34.5% and

368

62% of the total genus sequences, respectively (Figure 3B). The high abundance of

369

Geobacteraceae in 0.18 and 0.37 g FAN/L Bf samples agrees with their higher CEs compared to

370

the control MEC, and it confirms that the relatively high FAN concentration gave the ARB an

371

ecological advantage that enhance electron flow towards anode respiration versus

372

methanogenesis. Although we observed high relative abundance of Geobacteraceae for the MEC

373

fed with 0.37 g FAN/L, its CE was lower when compared to MEC fed with 0.18 g FAN/L (p-

374

value < 0.05; pairwise t-test). This probably was due to partial inhibition to the biofilm’s ARB at

375

higher FAN. Partial inhibition of acetate uptake by ARB in the biofilm anode made the

376

microbial community less functionally effective for anode respiration, even though the relative

377

abundance of Geobacteraceae in anode’s biofilm remained high.

378 379

Figure 3

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380

The relative abundance of Firmicutes was similar in all samples except for 0.37 g FAN/L

381

Bf, but their compositions varied significantly. Figure S4 shows community breakdown at the

382

family level within the phylum Firmicutes. An increase in FAN concentration led to the

383

emergence of Clostridiaceae and Ruminococcaceae families, which belong to the order

384

Clostridiales. Compensating losses occurred for the family Tissierellaceae. Recently, Müller et

385

al. 20 revealed that several members of the Clostridia class, mainly belonging to the orders

386

Clostridiales, can perform syntrophic acetate oxidation (SAO) (See Table 1), which was the

387

predominant pathway for methane production with elevated ammonia levels.20,42

388

We also detected an increase in the relative abundance of Lactobacillales, which include

389

genera Lactobacillus and Enterococcus, at the highest FAN conditions (0.18 and 0.37 g FAN/L).

390

Lactobacillales are reported to play a major role in lactic-acid production in fermentation,43 and

391

this is consistent with the observation of lactate accumulation during glucose fermentation in

392

MECs fed with 0.18 and 0.37 g FAN/L (Figure 1).

393

Supplementary Table S1 and Figure S5A show all the values for the bacterial community

394

diversity metrics for Bf and SP samples. The Chao1, Shannon, and PD values reflect that the Bf

395

samples developed more diverse bacterial community than the corresponding SP samples. Also,

396

the bacterial diversity of Bf and SP samples from 0.18 g FAN/L-fed MEC, which achieved the

397

highest CE (Figure 1), was greater than from the other two MECs. Consistent with the diversity

398

results and based on the equitability coefficient, the evenness increased as FAN concentration

399

increased from 0.02 to 0.18 g FAN/L, while the 0.37 g FAN/L-fed MEC was least even (Figure

400

S5B). Previous studies revealed that microbial communities with greater evenness are often

401

associated with more robustness and functional stability compared to less even microbial

402

communities.29,44

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The results of weighted PCoA analysis, Figure 4, show that principal components (PC) 1

403 404

and 2 explained 33.7% and 29.8% of the total bacterial community variations, respectively.

405

Increasing FAN concentration correlated with the PC2 vector, whereas the type of sample (Bf

406

versus SP) correlated with the PC1 vector. The trend along PC2 was associated with the emergence of the orders

407 408

Desulfuromonadales, Lactobacillales, and Synergistales at elevated FAN concentration; to

409

compensate, the relative abundances of other orders, such as Clostridiales and Bacteroidales,

410

decreased in response in change in FAN concentration (Figure 4). Several members of

411

Desulfuromonadales, a sub-group of Deltaproteobacteria, and Lactobacillales are well-known

412

ARB and lactic acid producers, respectively. Although Synergistales, a sub-group of phylum

413

Synergistetes, have been detected in anaerobic digesters treating different wastewater, their

414

functions are still mysterious.45,46 One study suggested that members of this order are potential

415

propionate consumers,47 but another study suggested that they might be SAOB.48 The

416

emergence of those orders, coupled with the decreases of Clostridiales and Bacteroidales, well-

417

known acetic acid-producing fermenters, supports that higher FAN altered glucose fermentation

418

towards the production of higher organic acids (e.g., lactate) and away from H2, rather than the

419

formation of 2 moles of acetate and 4 moles of H2 per mole of glucose, as illustrated in Figure

420

S1.

421

The trend along PC1 was associated with the emergence of the orders Bacteroidales and

422

Lactobacillales, which play an important role in fermentation of complex substrates.

423

Compensating for these increases, the relative abundance of Desulfuromonadales decreased

424

along PC1 vector. These results confirm that most of fermentation occurred in the suspension

425

phase, since we operated MECs in batch mode; ARB dominated the MECs’ biofilm.

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426

Figure 4.

427 428

429

Distribution of Archaea population in batch MECs Figure 5 summarizes the sequencing results for Archaea. Hydrogenotrophic

430

methanogens dominated all Bf and SP Archaea communities, regardless the FAN concentration,

431

which is consistent with previous work.9,40 The percentage of Archaea relative to bacteria, based

432

on the prokaryotic library results, decreased as FAN increased, supporting that the methanogens

433

were partially inhibited by higher FAN (Figure S6). For example, the percentage of Archaea

434

relative to Bacteria in Bf samples was about 10.8%, 3.8%, and 4.0% for 0.02 g FAN/L, 0.18 g

435

FAN/L, and 0.37 g FAN/L, respectively. The percentage of Archaea to bacteria was higher for

436

all SP samples than for Bf samples, confirming that the methanogens were predominantly in the

437

suspended phase of MECs operated in batch mode. This trend is reinforced by the qPCR results

438

targeting the mcrA gene (Figure 5A) and by the decrease in CH4 production in the MECs’

439

headspace fed with 0.18 g FAN/L and 0.37 g FAN/L (i.e., 9% and 6% of the original electrons,

440

respectively) compared to control MEC (i.e., 15% of the original electrons) (Figure 1). The

441

mcrA gene copies in the MECs’ anodes fed with high FAN concentrations (0.18 g FAN/L, and

442

0.37 g FAN/L) were statically significant lower (p-value < 0.05; pairwise t-test) compared to

443

control experiment (0.02 g FAN/L). The most likely reason for increasing the mcrA gene copies

444

per reactor volume in the SP sample is either biomass detachment from the biofilm into the

445

supernatant or DNA residual from the inoculum.

446

For all experimental runs, we did not detect any acetoclastic methanogens in all Bf and

447

SP samples, which were dominated by hydrogenotrophic methanogens (i.e., Methanobacterium

448

and Methanobrevibacter) (Figure 5B). For the 0.02 g FAN/L bf, Methanobacterium was

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449

dominant (~83% of sequence), while Methanobrevibacter (74% for 0.18 g FAN/L biofilm and

450

91% for 0.37 g FAN/L biofilm) and Methanobacterium (16% for 0.18 g FAN/L biofilm and 11%

451

for 0.37 g FAN/L biofilm) shared the archaeal community for higher FAN concentrations.

452

Several members of both genera are reported to be resistant to ammonia inhibition up to 400 mM

453

(i.e., 5.6 g TAN/L or ~ 0.04 g FAN/L at pH 6.8).49

454

The higher relative abundance of Methanobrevibacter with higher FAN suggests that

455

they contained the strains more resistant to FAN inhibition. Following the FAN increase,

456

Methanobacterium started to outcompete Methanobrevibacter (Figure 5B). A new uncultured

457

Archaea, classified as a candidate genus vadinCA11 (Thermoplasmata sp.), emerged as FAN

458

decreased. This genus was detected in an anaerobic fluidized bed fed with wine-distillation

459

waste,51 and it is potentially halophilic.52 The results suggest that the community shifted toward

460

methanogens tolerant to FAN.

461 462

Figure 5.

463 464 465

High FAN did not affect homo-acetogens Previous work revealed that suppressing methanogenesis was the main reason that high

466

CEs were achieved in MECs.9 Suppressing H2-oxidizing methanogens provides ideal conditions

467

for homoacetogens to oxidize H2 and grow, leading to the generation of acetate, the ideal

468

substrate for ARB. 8,9 If this scenario were true, we should have observed a larger population of

469

homoacetogens with higher FAN concentration. To test this hypothesis, we tracked the relative

470

abundance of homoacetogens by targeting the FTHFS gene using qPCR; the results are in Figure

471

S7. FTHFS gene copies for all Bf and SP samples were comparable, regardless of the FAN

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472

concentration. The qPCR data also are consistent with bacterial 16S rRNA gene sequence data,

473

where we detected genus Treponema, a well-known homoacetogenic Spirochaetes (Figure 3B),

474

but at a very low relative abundance (< 1% of total sequences) for all samples. Thus,

475

homoacetogens were consistently present, and changes in their numbers likely were not

476

responsible for enhancing CE.

477 478 479

New insights into metabolic flexibility in MECs Our results provide insight for possible way to decrease CH4 production and increase CE

480

by controlling the fermentation step using FAN, instead of inhibiting methanogens using

481

expensive or toxic chemical inhibitors. Altering fermentation led to less production of H2, which

482

is the “fuel” for methanogens, resulting in less CH4 production and higher CE, even though

483

methanogenesis was not completely suppressed. The lowering of H2 generation was

484

accompanied by more production of short-chain organic acids. However, relatively high FAN

485

(0.37 g FAN/L in my study) might promote SAOB, which are competitors for acetoclastic ARB,

486

introducing the possibility of a new pathway for acetate consumption in MECs fed with

487

fermentable substrates; it is a pathway to be avoided.

488

SAO might be the reason for the lower CE we observed with the MEC fed with 0.37 g

489

FAN/L, compared to MEC fed with 0.18 g FAN/L. Although acetate-based anode respiration is

490

thermodynamically favorable compared to SAO (See Table 1), the half-saturation constant (Ks)

491

for SAOB ranges from 4.7 to 13 mM,48,53 values ~2.5- to 7-fold higher than the maximum Ks

492

reported for mixed-culture ARB (1.86 mM).54 Despite the higher Ks value for acetate, the SAOB

493

may be more tolerant to high FAN, which would give them a kinetic advantage compared to

494

ARB, since ARB often are susceptible to a high FAN concentration. This observation coincides

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495

with emergence of Clostridiaceae and Ruminococcaceae families, which are potential SAOB.

496

Further investigation of SAOB in MECs is warranted.

497

An important implication of this study is that methanogenesis does not need to be

498

completely suppressed to achieve high current production and CE from MECs fed with

499

fermentable substrates. Using FAN to suppress methanogenesis is a realistic option for scaling-

500

up MECs during the biodegradation of fermentable substrates, particularly when the feed stream

501

has a high nitrogen content, such as animal manures and landfill leachate.

502 503

Acknowledgements

504 505

We thank Dr. Juan Maldonado, Dr. Zehra Esra Ilhan, and Dr. Prathap Parameswaran for helpful

506

discussion and guidance. This study was partially supported by the endowment fund from Mr.

507

Brian Swette to the Swette Center for Environmental Biotechnology and Graduate and

508

Professional Student Association at Arizona State University. Mohamed Mahmoud was

509

supported by an Egyptian government fellowship (GM 0931).

510

511

Conflicts of interest

512 513

The authors declare that there is no conflict of interest regarding the publication of this paper.

514 515 516 517

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518

Supporting Information

519 520

Available in the Supporting Information are: Figure S1-S8: Electron flows from glucose into

521

different electron sinks in an MEC’s anode, Electron balance of MECs at the end of batch-cycle

522

operation, bacterial community distribution at the class level, composition of phylum Firmicutes

523

at the family level, bacterial community diversity and richness results, archaea-to-bacteria ratio,

524

FTHFS gene copies per reactor determined by qPCR at the end of batch cycles; and Table S1:

525

Chao1, Shannon, and PD indices for bacterial sequences.

526

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Table 1. Overview of reactions involving acetate and H2

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Table 1

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Process (1) Glucose fermentation: C6H12O6 + 2H2O → 2 CH3COO− + 4H2 + 2CO2 + 2H+

∆G°rxn (kJ/e– eq) a – 8.59

(2) Acetoclastic methanogenesis: CH3COO− + H2O → CH4 + HCO3−

– 3.88

(3) Hydrogenotrophic methanogenesis: HCO3− + 4H2 + H+ → CH4 + 3H2O

−16.38

(4) Homoacetogenesis: 2 CO2 + 4H2 → CH3COO− + H+ + 2H2O

− 11.88

(5) Acetoclastic anode respiration b: CH3COO− + 2H2O → 2CO2 + 7H+ + 8e−

− 19.30

(6) Hydrogenotrophic anode respiration b: 2H2 → 2H+ + 2e−

− 9.65

(7) Acetate oxidation to H2: CH3COO− + 4H2O → 2HCO3− + 4H2 + H+

+13.10

730

a

We calculated ∆G°rxn at standard conditions (i.e., 298 K, 1 atm for gases, pH 7, and 1 M for

731

soluble reactants) based on the thermodynamic data provided in Thauer et al.11; b Near-optimum

732

anode potential = +0.2 V versus SHE.15

733

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734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756

Figures caption Figure 1. Electron distributions in MECs fed with 5-mM glucose at an initial pH of 8.1: (A) 0.02 g FAN/L or 0.2 g TAN/L (final pH = 6.85), (B) 0.18 g FAN/L or 2 g TAN/L (final pH = 7.10), and (C) 0.37 g FAN-N/L or 4 g TAN/L (final pH = 6.89). Figure 2. Performance of semi-continuous MECs fed with different FAN concentrations: (A) Current generation profile of duplicate MECs versus time. (B) Electron balance of MECs at the end of semi-continuous cycle operation in panel A. Figure 3. Bacterial community sequencing results. (A) bacterial community distribution at the phylum level. Phyla with less than 1% of total sequences are grouped as “others”. (B) bacterial community distribution at the genus level. Genera with less than 1% of total sequences are grouped as “others”. Figure 4. Weighted Unifrac analysis shows that the relative abundance of order-level phylotypes on the Principal Coordinates. FAN concentration determined the main phylotypes that drove the community structures in MECs. Figure 5. Archaeal community analyses. (A) mcrA gene copies per reactor determined by qPCR at the end of batch cycles. All error bars show standard deviations of triplicate measurements. (B) Archaeal community distribution at the genus level. Genera with less than 1% of total sequences are grouped as “others”.

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Figure 2

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Figure 4

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Figure 5

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