Facultative Nitrate Reduction by Electrode-Respiring Geobacter

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Facultative Nitrate Reduction by Electrode-Respiring Geobacter metallireducens Biofilms as a Competitive Reaction to Electrode Reduction in a Bioelectrochemical System Hiroyuki Kashima, and John Michael Regan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504882f • Publication Date (Web): 27 Jan 2015 Downloaded from http://pubs.acs.org on February 9, 2015

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Environmental Science & Technology

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Facultative Nitrate Reduction by Electrode-Respiring Geobacter metallireducens

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Biofilms as a Competitive Reaction to Electrode Reduction in a Bioelectrochemical

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System

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Hiroyuki Kashima and John M. Regan*

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Department of Civil and Environmental Engineering, The Pennsylvania State University, University Park,

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PA, 16802, United States

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*corresponding author:

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212 Sackett Building

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University Park, PA 16802 U.S.A.

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[email protected]

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Tel: +1-814-865-9436, Fax: +1-814-863-7304

1 ACS Paragon Plus Environment


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ABSTRACT

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Alternative metabolic options of exoelectrogenic biofilms in bioelectrochemical systems (BESs) are

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important not only to explain the fundamental ecology and performance of these systems but also to

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develop reliable integrated nutrient removal strategies in BESs, which potentially involve substrates or

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intermediates that support/induce those alternative metabolisms. This research focused on dissimilatory

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nitrate reduction as an alternative metabolism to dissimilatory anode reduction. Using the exoelectrogenic

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nitrate reducer Geobacter metallireducens, the critical conditions controlling those alternative

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metabolisms were investigated in two-chamber, potentiostatically controlled BESs at various anode

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potentials and biofilm thicknesses and challenged over a range of nitrate concentrations. Results showed

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that anode-reducing biofilms facultatively reduced nitrate at all tested anode potentials (-150 to + 900 mV

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vs Standard Hydrogen Electrode) with a rapid metabolic shift. The critical nitrate concentration that

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triggered a significant decrease in BES performance was a function of anode biofilm thickness but not

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anode potential. This indicates that these alternative metabolisms were controlled by the availability of

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nitrate, which is a function of nitrate concentration in bulk solution and its diffusion into an anode-

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reducing biofilm. Coulombic recovery decreased as a function of nitrate dose due to electron-acceptor

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substrate competition, and nitrate-induced suspended biomass growth decreased the effluent quality.


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TOC Art

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INTRODUCTION

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Electrode reduction by exoelectrogenic biofilms is central to anodic bioelectrical system (BES)

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performance 1. These systems have potential applications in mixed-culture bioprocesses, such as

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wastewater treatment or bioremediation, that often harbor undesirable alternative microbial activities with

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the potential to compete with the desired bioelectrochemical process. For example, aerobic organic

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degradation supported by oxygen crossover at the air cathode decreases the coulombic efficiency of

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single-chamber microbial fuel cells (MFCs)2-5, and methanogenic activity decreases hydrogen yields in

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microbial electrolysis cells (MECs) 6-8. While these alternative metabolisms involve non-electrode

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respiring microbes residing in a system, some exoelectrogens have alternative metabolisms to electrode

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reduction, such as ferric, nitrate, or sulfate reduction. BESs offer a unique experimental platform for

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studying these alternative electron-acceptor metabolisms relative to electrode reduction to help us better

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understand dissimilatory electrode reduction. In addition, this understanding would be useful for the

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development and operation of BESs in applications that include these alternative electron acceptors, such

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as integrated nutrient removal from wastewater 9 and groundwater treatment 10. Several studies have

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reported the negative effects of these alternative electron acceptors on mixed-culture BES anode

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performance 11, 12, but fundamental research is needed to understand these alternative metabolisms

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specifically on the exoelectrogenic populations in the biofilms.



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The effect of nitrate on electrode reduction is of particular interest because some exoelectrogens

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such as Geobacter metallireducens 13, Shewanella oneidensis 14, Comamonas denitrificans 15,

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Geoalkalibacter subterraneus 16, 17, and Calditerrivibrio nitroreducens 18 conduct this energetically

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favorable dissimilatory metabolism in anoxic natural and engineered systems, and it is a key factor for the

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development of nitrogen removal technologies in BESs from wastewater 19-23 and groundwater 10, 24.

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Recent designs involving simultaneous nitrification and denitrification with single-chamber air-cathode

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MFCs 23, 25, denitrifying biocathode MFCs with an anion exchange membrane to avoid ammonia

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crossover 22, and BES-driven in-situ groundwater nitrate removal systems 10, 24 have brought us new anode

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systems under the influence of nitrate. Thus it is critical to understand the effects of nitrate on

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exoelectrogens to develop effective and reliable nitrogen-removal strategies in BESs. Sukkasem et al.

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examined the effects of nitrate on performance of mixed-culture single-chamber MFCs 12. The study

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reported decreased coulombic recovery and maximum voltage output in the presence of nitrate under high

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current conditions, and attributed these negative effects to competition for electrons between anode

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reduction and denitrification 12. Also, other studies reported heterotrophic nitrate reduction activity in the

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anode compartments of nitrogen-removal BESs 22, 24. Given the mixed-culture conditions in these studies,

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it is unknown whether such negative nitrate effects were just a minor coulombic loss by non-

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exoelectrogenic denitrifiers, or nitrate led to a striking deterioration in BES performance due to a

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metabolic shift of anode reducers to nitrate reduction.

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To address the above questions, it is important to understand the critical conditions that control

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the facultative metabolisms of anode and nitrate reduction in dissimilatory nitrate-reducing

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exoelectrogens. As an analogous system, the facultative metabolic shift between aerobic respiration and

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nitrate respiration by facultative denitrifiers is controlled by dissolved oxygen (D.O.), with D.O. above a

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critical level inhibiting nitrate reductase activity 26 and also its gene expression 27. This understanding of

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critical D.O. conditions has been used as a fundamental engineering parameter to design anoxic

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denitrification processes. Unlike the well-studied case of D.O. effects on the facultative metabolic shift


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between aerobic respiration and nitrate respiration, the conditions that determine anode versus nitrate

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reduction are unknown. With respect to the anode conditions, while its availability cannot be described in

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terms of concentration, it can be described with electrode potential according to the Nernst equation. An

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anode with a higher electrode potential is a thermodynamically better electron acceptor due to its larger

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possible metabolic energy gain determined by the redox couple of electron donor and electron acceptor

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(anode) 28.

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This research is focused on understanding the dynamics of nitrate reduction and anode reduction

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for the exoelectrogenic nitrate-reducing bacterium G. metallireducens to determine the critical conditions

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controlling these possibly competitive metabolisms and the effects on anode performance. Geobacter

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species are often the dominant taxon in various mixed-culture bioanode systems 29-32, and 16S rRNA gene

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clones closely related to G. metallureducens were found in different mixed-culture BESs 29, 33. This

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bacterium is also known to reduce nitrate coupled with the oxidation of organics 13, ferrous 34, and

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graphite electrode 35 as an electron donor. We hypothesized that graphite anode-reducing G.

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metallireducens biofilms facultatively reduce nitrate and its negative effect on current production would

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be a function of nitrate concentration, anode potential, and biofilm thickness.

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MATERIALS AND METHODS

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Microbial source and culture condition

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G. metallireducens GS-15 (ATCC 53774) stored at – 80 ᴼC was cultured in the dark at 30°C

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using sterile, anaerobic ATCC medium 1768 with modified concentrations of the following components:

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ferric citrate (40 mM), acetate (20 mM), and NaH2PO4 (1.67 mM). Stationary phase culture was

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transferred (10 % in volume) into the same medium and cultured again to obtain more robust growth. This

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second stationary phase culture was anaerobically centrifuged (3500 ×g, 20 min, 4ᴼC) and cell pellets

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were resuspended in the anaerobic medium described above but omitting ferric citrate and acetate. A 25 ACS Paragon Plus Environment


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mL aliquot of these cell suspensions (O.D.600 = 0.23 - 0.28) was transferred into the working electrode

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chamber of each electrochemical cell as inoculum.

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Electrochemical cell configuration and biofilm acclimatization on graphite anode Dual chamber, H-type electrochemical cells (Glasstron Inc., Vineland, NJ) with Nafion 117

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cation exchange membrane separating the working electrode chamber and the counter electrode chamber

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were used for the tests. Graphite plate blocks (Isomolded Graphite Plate, GM-10 grade, Graphitestore) cut

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into 1.0 cm × 3.0 cm × 0.3 cm were sanded with 400 and 1500 grit sandpaper, sonicated to remove

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graphite particles, and then polished with aluminum oxide as described elsewhere 36. Graphite plates were

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then soaked in 1 M HCl overnight for three times to remove metal contaminants. Cleaned graphite plates

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were connected to titanium wire and used as working electrodes. The same graphite plate blocks cut into

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1.0 cm × 6.0 cm × 0.3 cm and prepared in the same way were used as counter electrodes. Reference

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electrodes (Ag/AgCl, 3 M NaCl, +0.211 V vs Standard Hydrogen Electrode (SHE)) were inserted into the

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working electrode chambers. The electrode chambers (approximately 180 ml of working volume) were

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filled with 120 mL of growth media described above but without ferric citrate and vitamins solution in the

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working electrode and without acetate in the counter electrode. All electrochemical cells and media were

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sterilized and sparged with CO2:N2 (20 %: 80 %) gas to keep the system anaerobic prior to inoculation.

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Working electrodes were poised at potentials of -150 mV, 0 mV, +400 mV, +650 mV, or +900

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mV vs. SHE with a potentiostat (Uniscan model PG580, Buxton, United Kingdom) to acclimatize

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biofilms. The BESs were operated in batch mode in a 30 ᴼC temperature-controlled chamber without

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stirring. Abiotic controls were run under the same conditions but without inoculation. An aluminum gas

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collection bag was connected to each chamber through sterile 0.2-μm mesh filters to release any pressure

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imbalance between the two chambers during operation. To acclimatize anode biofilms of different

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thicknesses, acclimatization schemes included one acclimatization batch cycle with 0.5 mM acetate, one

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acclimatization cycle with 10 mM acetate, or two sequential acclimatization cycles with 10 mM acetate.


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Each acclimatization batch cycle was ended once current production decreased below 100 μA.

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Acclimatized biofilms were then tested in a subsequent nitrate spike batch cycle.

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Nitrate spike tests

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After biofilm acclimatization, media in the electrochemical cell chambers were replaced

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anaerobically with new media containing 10 mM acetate for the working electrode chamber and without

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acetate for the counter electrode chamber. The working electrode was poised at the same potential as the

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acclimatization cycle by a potentiostat (VMP3, Bio-Logic USA, TN), and working electrode chambers

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were stirred at ~180 rpm. After observing stable current (approximately 2.5 hours), a defined amount of

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nitrate was spiked every 30 minutes into the working electrode chamber to gradually increase the nitrate

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concentration. Current production was recorded by the potentiostat during the tests, and the working

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chamber medium was sampled at each spike. The nitrate concentration at which the current decreased

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without recovery within 30 minutes after a nitrate spike was defined as the critical nitrate concentration

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(i.e., significantly hampers current production) at that working electrode potential and biofilm thickness.

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In each experimental condition with a specific anode potential and biofilm thickness, duplicate anodes

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were tested with gradual nitrate spikes, as well as a control anode with gradual sodium chloride spikes of

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equivalent concentration, and duplicate anodes for biofilm protein concentration measurement that were

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sacrificed at the time of the first nitrate spike. Gradual nitrate spikes were stopped after passing the

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critical nitrate concentration, and then the test batch cycle was continued until current production

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increased once again.

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In addition to the gradual nitrate spike tests, biofilms acclimatized at 0 mV were subjected to a

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single nitrate spike to see the effects of nitrate at different levels. Two sets of biofilms were acclimatized,

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one with a single acclimatization cycle in 0.5 mM acetate medium to form thin biofilms, and the other

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with two sequential acclimatization cycles in 10 mM acetate medium to form thick biofilms.

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Analytical methods 7 ACS Paragon Plus Environment


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Nitrate, nitrite, and acetate concentrations in anode media were measured with ion

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chromatography (ICS-1100 with AS-25 anion separation column, Dionex, CA). Ammonia concentration

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in anode and cathode media was measured with the Salicylate method (Nitrogen-Ammonia Reagent Set,

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TNT, AmVer (Salicylate), Hach, CO). Cells in biofilms and suspension were solubilized by sodium

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hydroxide 37, and the resultant solubilized protein was measured by the modified Lowry method with DC

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Protein Assay Kit (Bio-Rad, CA). Bovine serum albumin fraction V was used to prepare protein standards.

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RESULTS AND DISCUSSION

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Anode-reducing G. metallireducens biofilms preferentially reduced nitrate

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G. metallireducens biofilms reducing graphite anodes poised over a potential range of -150 to

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+900 mV facultatively reduced nitrate as an alternative electron acceptor. For thin biofilms (acclimated

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with 0.5 mM acetate as electron donor), current production fluctuated in response to each incremental

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nitrate spike and then continuously decreased once the nitrate concentration in the chamber exceeded a

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critical level. Thick biofilms (acclimatized with one or two consecutive cycles of 10 mM acetate) showed

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little perturbation in current with initial incremental nitrate additions until an abrupt drop in current once

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reaching a critical nitrate concentration. For example, thin biofilms acclimatized on graphite electrodes

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poised at 0 mV (0.09 mg-protein/cm2) showed a critical nitrate concentration of approximately 0.82 mM

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(Figure 1). Nitrite accumulation in bulk solution was observed in most nitrate-spiked anode biofilms

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during significant current decrease, but nitrite did not accumulate for some thick biofilms even though the

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current showed an appreciable drop (Figure S-1A). Nitrate was sequentially reduced to nitrite and

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ammonia via dissimilatory nitrate reduction to ammonia (DNRA) 26, which G. metallireducens has been

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shown to conduct in suspended culture coupled with acetate oxidation 13, 38. Current production recovered

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once both the nitrate and nitrite concentrations in the chamber decreased. Current production did not

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recover even after the nitrate concentration dropped below the critical level, suggesting that nitrite formed 8 ACS Paragon Plus Environment

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with nitrate reduction was preferentially reduced over the anode and/or nitrite inhibited the anode

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reduction reaction. Stable anodic performance of sodium chloride-spiked biofilms showed that the

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impaired current production observed in nitrate-spiked biofilms was not due to multiple spikes and

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samplings that might introduce trace amounts of oxygen into an anode chamber and inhibit activity of G.

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metallireducens, an obligate anaerobe (Figure 1). In addition, the solution redox potential remained

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constant throughout the nitrate spikes (Figure S-2) and an abiotic nitrate-spike test showed no current,

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indicating that the nitrate effect was not indirect or abiotic. Furthermore, there were no measurable

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negative effects of nitrate on current production by G. sulfurreducens, an anode reducer that cannot use

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nitrate as an electron acceptor (Figure S-3), providing further evidence that the negative nitrate effects on

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anode reduction by G. metallireducens were attributed to its role as a competitive electron acceptor to the

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anode.

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The availability of nitrate controlled facultative anode and nitrate metabolisms

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The critical nitrate concentration that triggered a significant current decrease in anode-reducing G.

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metallireducens biofilms was a function of the biofilm protein concentration (Figure 2). Assuming the

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biofilms had uniform protein concentrations throughout their thickness, the critical concentration

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increased with biofilm thickness, which correlates with the diffusion barrier that controls nitrate

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penetration into the biofilm matrix 39. Consistent with the findings reported for G. sulfurreducens biofilms

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that peak current increased with biofilm thickness up to 40 µm, above which the peak current was

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generally lower 40, the maximum current produced by chloride-spiked control anode biofilms in this study

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were lower for thick biofilms (Figure S-1) than thin biofilms (Figure 1). This inverse relationship between

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peak current and protein concentration suggests the presence of mass transport limitations in the thicker

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biofilm systems. (There was no correlation between critical nitrate concentration and peak current.)

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Because the current measurements represent the composite biofilm activity, the contributions of each part

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of the anode biofilm (i.e., the outer layers near the bulk solution or inner layer immediately next to the

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anode surface) to current production are not known. Some studies showed that G. sulfurreducens anode 9 ACS Paragon Plus Environment


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biofilm had relatively low differences in transcript abundance throughout a biofilm 41 with similar activity

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42

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anode-reducing G. sulfurreducens biofilms 43, 44. There is a discrepancy in the reported nature of this

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heterogenous activity, with a suggestion of higher activity near the anode 43 versus higher activity at the

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outside of the biofilm due to acetate depletion within highly conductive biofilms 44. However, it is

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possible in our experiments with surplus electron donor that nitrate reduction by cells in the outer layer of

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a thick anode biofilm did not appreciably decrease current production because these cells that were most

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distant from the anode surface may have had a slight contribution to the current. This inference is further

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supported by the current responses in an experiment in which mixing was briefly paused; current

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production resumed in nitrate-spiked systems, showing an increased nitrate diffusion barrier relative to

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the high acetate diffusion rate promoted by excess acetate concentrations (Figure S-4).

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, while other studies reported spatially heterogeneous metabolic activity throughout thick (> 100 µm)

Anode potential had no significant impact on critical nitrate concentration (Figure 2). These

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facultative reactions were strictly determined by the availability of nitrate as the preferred electron

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acceptor and not the availability of the anode, similar to the facultative preference of oxygen over nitrate

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for facultative denitrifiers 26. Different anode potentials resulted in different current production in the

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absence of nitrate (Figure S-5), especially the -150 mV anodes versus the 0 and +400 mV anodes, which

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is expected based on a thermodynamic perspective and previous reports. However, this variable anode

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availability did not result in different critical nitrate concentrations. This was even the case at the positive

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extreme of +900 mV, which is essentially the highest technically feasible electrode potential due to the

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emergence of significant abiotic anode reduction from anode corrosion or water splitting for graphite

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anodes at higher potentials (Figure S-6), and represents the most thermodynamically favorable anode as

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an electron acceptor.

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The preferential use of nitrate over an anode cannot be explained by a simple thermodynamic

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perspective of available energy. Biofilms respiring anodes poised at +900 mV, which provide greater

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available energy than nitrate respiration (Figure S-7), still facultatively reduced nitrate with a critical 10 ACS Paragon Plus Environment

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nitrate concentration comparable to anodes with lower potentials. This is likely because the real biological

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energy gain of each biofilm was less than the available energy due to the inability of the respiratory

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machinery to capture all of the available energy. For example, G. sulfurreducens reportedly had higher

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biomass yield with the reduction of an anode poised at 0 mV vs SHE over an anode at -160 mV, but the

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biomass yield at +400 mV was not higher than 0 mV, illustrating that G. sulfurreducens obtained larger

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energy to support biomass growth from a more positive anode between -160 mV and 0 mV but they could

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not obtain additional energy from 0 to +400 mV 28. In anode-reducing G. sulfurreducens cells, outer

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membrane cytochromes (OMCs) have been proposed as terminal electron donors (TEDs) that donate

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electrons to an anode and potentially constrain actual energy gain if the anode potential is higher than the

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TED working in the system. Electrochemical analyses such as cyclic voltammetry (CV) and differential

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pulse voltammetry have identified redox-active components in BES anodes 36, 45, 46 with similar potential

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ranges and midpoint potentials of purified OMCs such as OmcB (-190 mV) 47, OmcS (-360~-40 mV, -212

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mV vs SHE) 48, and OmcZ (-420~-60 mV, -220 mV vs SHE) 49. Although the electron transfer

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components of G. metallireducens for electrode reduction have not yet been fully determined, no visible

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oxidation peak that was unique to biofilms acclimatized at high anode potential, +650 and 900 mV, was

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found in CVs (data not shown), suggesting G. metallireducens cannot capture thermodynamic energy

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from such high anode potentials.

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In fact, the anodes with high potentials of +650 mV or +900 mV seemed to be less favorable

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electron acceptors for G. metallireducens cells. It was difficult to acclimatize stable biofilms at 900 mV

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and it required a medium with higher acetate dose (1.7 mM) than the lower anode potentials, which

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showed stable current and biomass production with 0.5 mM acetate. Also, the +650 mV and +900 mV

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anodes showed lower peak current relative to 0 mV and +400 mV in the second batch (Figure S-5),

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perhaps indicating decreased biological activity due to oxidative stress with these high anode potentials

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that may cause unfavorable effects such as degradation of electron-transfer proteins at the electrode

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surface 50. Another study reported a similar decrease in anode reduction rate with G. sulfurreducens-



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dominated mixed culture biofilms grown at 0.81 V vs SHE relative to anodes with lower potentials (-0.09,

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0.21, and 0.51 V vs SHE) in single-chamber MECs 32.

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It is also possible that G. metallireducens preferentially used nitrate as electron acceptor based on

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criteria other than actual energy gain, as G. sulfurreducens preferentially reduces Fe(III) over fumarate

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even though it provides significantly less energy to support cell growth 51. For example, perhaps soluble

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electron acceptors are preferentially used over insoluble electrodes. Further research is needed to

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understand the mechanisms that govern these facultative metabolisms.

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Nitrate above critical level resulted in significant BES performance decrease

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To further investigate abrupt nitrate influences on anode-reducing biofilms, a set of thick anode-

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reducing biofilms was subjected to nitrate spikes at different levels, higher and lower than the estimated

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critical nitrate level. The critical nitrate concentration for these biofilms was estimated to be 3.87 mM

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based on the protein concentrations of two sacrificed biofilms (0.49 ± 0.03 mg-protein/cm2) and the linear

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regression of critical nitrate concentrations (Figure 2). With this estimate, four different levels of nitrate

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were spiked into duplicate anodes: 1.5 mM (low), 4.0 mM (critical), 10.0 mM (high), and 0 mM control

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with a 1.5 mM sodium chloride spike. Current production showed that bulk nitrate concentrations above

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the critical level resulted in a significant decrease of current down to the abiotic control level (Figure S-6),

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whereas anode biofilms subjected to nitrate below the critical level did not show significant current

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decrease (Figure 3). Another set of anodes acclimatized with thin biofilms (0.08 mg-protein/cm2) at 0 mV

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and subjected to low or high levels of nitrate showed similar results (Figure S-8). As seen in the gradual

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nitrate spike tests, nitrate was facultatively reduced to ammonia and the anode biofilms returned to anode

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reduction once nitrate and nitrite were used up except under high nitrate concentration (10 mM), which

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resulted in substantial intermediate nitrite accumulation in the system. These high nitrite concentrations

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seemed to result in inhibition of anode biofilm cells by accumulated nitrite, or free nitrous acid (FNA)


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that may have accumulated at the base of the biofilms near the electrode-biofilm interface, at which the

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pH would be expected to be lower than bulk solution 52 (Figure 3).

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Time-series electron distribution profiles also show preferential nitrate reduction over anode

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reduction by biofilms (Figure 4). In the presence of nitrate below the critical level, anodes and nitrate

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were simultaneously reduced in concert with acetate oxidation, while sequential nitrate reduction

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followed by anode reduction was observed in biofilms subjected to nitrate over the critical level. Greater

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acetate oxidation and a slight increase of circuit charge recovery was observed in 1.5 mM nitrate-spiked

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anodes relative to the no-nitrate control anodes over the course of a batch (Figure 4). This is possibly due

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to the DNRA reaction (Equation 1) mitigating acidification associated with anode reduction 53 (Equation

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2), since the final averaged pHs in bulk anode media were 5.63 in the 1.5 mM nitrate anodes and 5.30 in

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the no nitrate anodes.

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‫ܪܥ‬ଷ ‫ ିܱܱܥ‬+ ܱܰଷି + 2‫ ܪ‬ା = ܰ‫ܪ‬ସା + ‫ܱܥ‬ଶ + ‫ܱܥܪ‬ଷି

(Eq. 1)

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‫ܪܥ‬ଷ ‫ ିܱܱܥ‬+ 3‫ܪ‬ଶ ܱ = ‫ܱܥ‬ଶ + ‫ܱܥܪ‬ଷି + 8݁ ି + 8‫ ܪ‬ା

(Eq. 2)

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Intermediate nitrite was detected only in anodes spiked with nitrate higher than the critical level (Figure

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3) (Figure S-8). This is possibly because G. metallireducens cells preferentially conducted nitrate

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reduction to nitrite over nitrite reduction to ammonia. DNRA is a sequential two-step reduction of nitrate

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to ammonia with nitrite as intermediate, and it is thought that energy generation is mostly from the first

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step, whereas the second step is used to dump excess electrons and degrade toxic nitrite 26.

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Previous mixed-culture studies demonstrated decreased BES kinetics performance and cell

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voltage due to nitrate addition to the anode, and attributed such performance decreases to electron donor

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competition between strict anode reducers, facultative anode reducers, and non-anode reducing nitrate

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reducers 9, 12. By using pure-culture anode-reducing biofilms to exclude the activities of non-anode

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reducing nitrate reducers, at least in the thin-biofilm systems that would not have had inactive cells at the

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outer biofilm surface, this study showed that significant BES performance impairment can be caused by a 13 ACS Paragon Plus Environment


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metabolic shift of exoelectrogens from anode reduction to nitrate reduction. The BES performance

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decrease due to the presence of nitrate concentrations higher than a critical level can occur in mixed-

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culture BES anodes composed of exoelectrogenic nitrate reducers such as G. metallireducens and C.

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denitrificans, and it is important to keep the nitrate level below this biofilm thickness-dependent critical

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level to secure stable BES performance. While the findings of this study cannot be directly compared to

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previous mixed-culture reports due to significant differences in systems and conditions, we expect that the

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critical nitrate levels for mixed-culture anode biofilms would be higher than those reported here for two

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reasons: non-exoelectrogenic nitrate reducers could contribute to lowering the nitrate concentration in the

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biofilm, and the anode-reducing activity of non-nitrate reducing exoelectrogens would not decrease due to

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nitrate as long as there is surplus electron donor. The anode-reducing activity of non-nitrate reducing

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exoelectrogens can also be decreased due to inhibition by nitrite/FNA that might be formed by nitrate

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reduction, and critical nitrite concentration rather than critical nitrate concentration would be a better

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measure to describe nitrite/FNA inhibition on non-nitrate reducing exoelectrogens.

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Decreased anode reduction with metabolic shift from anode to nitrate and nitrite/FNA inhibition

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The observed bulk shift from current production to nitrate reduction may be a composite of

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several different nitrate-induced effects such as a metabolic shift of anode-reducing cells to nitrate/nitrite

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reduction, nitrite/FNA inhibition of cell metabolisms, and a contribution from previously inactive cells to

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nitrate reduction. The extent of contribution from these different alternatives depended on the biofilm

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thickness and the nitrate concentration. For thin biofilms (protein concentration ca. 0.08-0.09 mg/cm2),

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which were estimated to be only a few cell layers thick based on an areal protein density of 0.02-0.03

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mg/cm2 for monolayers of G. sulfurreducens 54, the biofilm was presumed to be composed almost entirely

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of active anode-reducing cells without stratification of activities or biochemical conditions across the

314

biofilm cross section. Therefore, the shift from current production to nitrate reduction in these thin-

315

biofilm systems was due to a change in metabolism from anode to nitrate reduction.


Page 14 of 28

Page 15 of 28


The interpretation of the systems with thick biofilms is more complicated due to the possibility of

316 317

stratification in activity and conditions. As mentioned above, the thick biofilms had lower peak currents

318

than the thin biofilms, suggesting there were mass transport constraints through the thick biofilms. Prior

319

to nitrate addition, these biofilms may have had cells in the outer layers that were inactive due to electron

320

acceptor limitation. The sudden current drop coupled with nitrate reduction by thick biofilms may have

321

involved nitrate reduction at the outer surface by these previously inactive cells that had not been

322

contributing to current production. This would not be expected in the thin biofilms, in which there would

323

not be an accumulation of inactive cells. A second possible contributing factor in thick-biofilm systems

324

was inhibition by nitrite and/or FNA. Biofilms subjected to nitrate concentrations significantly higher

325

than the critical concentration resulted in a high level of nitrite accumulation, and did not resume anode

326

reduction or reduce the accumulated nitrite even in the presence of excess electron donor (ca. 2.60 mM of

327

acetate). Nitrite inhibition may have contributed to the response of these systems. Moreover, in these

328

thick-biofilm cases where pH gradients would be expected to establish 55, FNA inhibition may also

329

explain the decreased current since the pH at the anode-biofilm interface might be low enough to form

330

FNA at a level sufficient to inhibit cell metabolism 52. During the current drop coinciding with nitrate

331

addition, active anode-reducing cells around the low-pH anode-biofilm interface could be selectively

332

inhibited by FNA, while the outer part of the biofilm with neutral pH could conduct nitrate reduction

333

without FNA inhibition. However, this FNA inhibition was unlikely in thin biofilms, which would lack a

334

significant pH gradient. Thin biofilms may have been inhibited by accumulated nitrite, but since nitrate

335

reduction was always observed, if nitrite inhibition occurred it was only partial inhibition of microbial

336

activity.

337

Concurrent reduction of anode and nitrate, and quick metabolic shift from anode to nitrate

338

The concurrent reduction of both the anode and nitrate was observed in several tests. Anode

339

biofilms exposed to nitrate concentrations less than the critical level reduced nitrate while also producing

340

current (Figure 3). Electron distribution analysis showed that almost 50 % of the electrons extracted from 15 ACS Paragon Plus Environment


341

acetate oxidation were used for nitrate reduction while the remaining 50 % was used for anode reduction

342

in the initial 5 hours after the nitrate spike, and concurrent electron flow to these two metabolisms was

343

observed until the nitrate in the system was used up (Figure 4). This concurrent reduction of two electron

344

acceptors may have been due to a functionally stratified biofilm. The outer parts of the biofilm would be

345

exposed to higher nitrate concentrations and might have switched to nitrate reduction, while the inner

346

biofilm may have continued anode reduction because of lower nitrate concentrations in the interior of the

347

biofilm. The facultative metabolic shift between anode reduction and nitrate reduction at the bulk biofilm

348 349

level occurred within a short time, on the order of minutes to an hour. For thin biofilms, the anode

350

reduction rate decreased immediately (in less than a minute) in response to nitrate spikes, and reduction of

351

nitrate started less than an hour after the spike (Figure S-8). This bulk metabolic shift was very rapid, and

352

distinct from previously reported results for this bacterium under different conditions. Specifically,

353

Finneran et al. (2002) reported that soluble Fe (III) citrate grown G. metallireducens suspended cells did

354

not reduce nitrate, and nitrate grown cells continued to reduce nitrate following Fe(III) citrate addition 34.

355

The metabolic shift from anode reduction to nitrate reduction as bulk biofilm reaction within a short

356

amount of time may be due to the insoluble anode-reducing growth condition or due to heterogeneous

357

metabolic status such as stratified biofilm composed of active anode-reducing cells and inactive cells

358

which had potential to quickly initiate nitrate reduction. Further study is needed to address this question.

359

Nitrate in the anode chamber lowered coulombic recovery and increased suspended biomass

360

growth

361

An electron balance analysis showed that electron recovery to the circuit decreased with

362

increased nitrate concentration in the system (Figure 5). Almost all of the nitrate spiked in the anode

363

chamber was reduced, resulting in fewer available electrons for anode reduction. The addition of nitrate

364

induced suspended biomass growth (Figure 5), as previously reported in mixed-culture MFCs 12. Cells


Page 16 of 28

Page 17 of 28


365

with an anode as the sole electron acceptor cannot respire independently from the anode, and thus

366

suspended biomass production in an anode chamber without nitrate was due solely to biofilm detachment.

367

However, once nitrate was introduced into the anode chamber, detached cells can grow independently

368

from the anode by using nitrate as the electron acceptor. Detached cells and cells growing in suspension

369

can reduce nitrate and result in lower coulombic recovery, though they do not decrease the anode

370

reduction rate under non-electron donor limited conditions. However, once the biofilm cells actively

371

reducing the anode shift their metabolism to nitrate reduction, there was a significant performance drop

372

due to decreased anode reduction rate. From a wastewater treatment perspective, suspended growth

373

induced by nitrate would yield high suspended solids in the effluent, and additional processes such as

374

clarification or coagulation might be required to remove suspended solids from the treated water body.

375

Acknowledgement

376

This study was supported by U.S. Army Research Office (W911NF-11-1-0531) and graduate fellowship

377

program from Japan Student Services Organization (L10126050001).

378

Supporting Information Available

379

Figures mentioned within the text are provided. This information is available free of charge via the

380

Internet at http://pubs.acs.org/.

381

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electrical current generation by anode‐respiring bacteria. Biotechnol. Bioeng. 2008, 100, (5), 872-881.

519

54.

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sulfurreducens Biofilms as a Function of Growth Stage and Imposed Electrode Potential. Electroanalysis

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2010, 22, (7-8), 865-874.

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55.

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for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory

524

effects of proton accumulation within the anode biofilm. Energy Environ. Sci. 2008, 2, (1), 113-119.

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525



526

Figure 1. Current, nitrate, and nitrite profiles in a gradual nitrate spike test of G. metallireducens biofilms

527

acclimatized on anodes poised at 0 mV with 0.5 mM acetate. Nitrate and nitrite data points are means of

528

duplicate. Error bars designate one standard deviation.

529 530 531 532 533 534 535 536 537 538 539


Page 24 of 28

Page 25 of 28


540

Figure 2. Critical nitrate concentrations of anode biofilms with different biofilm protein concentrations at

541

different anode potentials. For each data point, biofilm protein concentration was obtained from two

542

anodes sacrificed for protein analysis at the first nitrate spike (mean, ±S.D.), and critical nitrate

543

concentration was obtained by two anodes (mean, ±S.D.) in a gradual nitrate spike test. Linear regression

544

equation of all averaged data points was as follows: Critical NO3- concentration (mM) = 7.901 × biofilm

545

protein concentration (mg/cm2) - 0.002 (R2 = 0.974)

546 547 548 549 550 551 552 553



Page 26 of 28

554

Figure 3. Current, nitrate and nitrite profiles of anode reducing biofilms in response to exposure to various

555

levels of nitrate. Biofilms acclimatized on anodes poised at 0 mV (protein concentration: 0.49 (mg-

556

protein/cm2), estimated critical nitrate concentration: 3.87 mM) were subjected to various levels of

557

nitrate: High condition (10 mM, more than two times higher than the critical level), Critical condition

558

(target 4 mM, approximately the same as the critical level), Low condition (1.5 mM), and No nitrate

559

control (target 1.5 mM sodium chloride). Each condition was tested in duplicate.

560 561 562 563 564 565


Page 27 of 28


566

Figure 4. Time-series electron distribution profile of an anode biofilms under the presence of various

567

levels of nitrate. Panel A, B, and C shows time-series electron distribution profiles of duplicate anodes of

568

Control, Low, and Critical nitrate conditions shown in Figure 3. Error bars designate one standard

569

deviation. Electrons donated by acetate oxidation, electrons used for anode reduction and electrons used

570

for nitrate reduction were calculated based on decrease of acetate, decrease of nitrate and presence of

571

nitrite, and circuit recovered charge. Electrons consumed by nitrate reduction was calculated by assuming

572

dissimilatory nitrate reduction to ammonia as the electron sink for the loss of nitrate as well as nitrate

573

reduction to nitrite for the accumulated nitrite.

574 575 576 577 578 579 580 581



582

Figure 5. Electron balance of anode chambers in the presence of nitrate in different levels. This figure

583

describes anodes with 4 different levels of nitrate described in Figure 3. Each data point shows mean of

584

duplicate and bars designate one standard deviation. Electrons consumed by nitrate reduction was

585

calculated by assuming dissimilatory nitrate reduction to ammonia as the electron sink for the loss of

586

nitrate as well as nitrate reduction to nitrite for the accumulated nitrite. Electrons used for biomass

587

synthesis was calculated from protein concentration data by assuming proteins account for 50 % in mass

588

of the biomass composed of C5H7O2N.

589


Page 28 of 28

Facultative Nitrate Reduction by Electrode-Respiring Geobacter

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