An Ammonia

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School of Chemistry. National University of Ireland, Galway. University Road, Galway (Ireland) .... Hagen, FEBS Letters 1992, 303, 36-40. [7]. K. A. Brown, D. F. ...
COMMUNICATION Bioelectrochemical Haber-Bosch Process: An AmmoniaProducing H2/N2 Fuel Cell Ross D. Milton,[a,b] Rong Cai,[a] Sofiene Abdellaoui,[a] Dónal Leech,[b] Antonio L. De Lacey,[c] Marcos Pita[c] and Shelley D. Minteer[a]*

Abstract: Nitrogenase is the only enzyme known to reduce molecular nitrogen (N2) to ammonia (NH3). Using methylviologen (N,N’-dimethyl-4,4’-bipyridinium) to shuttle electrons to nitrogenase, N2 reduction to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode which utilizes the enzyme hydrogenase to oxidize molecular hydrogen (H2) results in an enzymatic fuel cell (EFC) that is able to produce NH3 from H2 and N2, while simultaneously producing an electrical current. To demonstrate this, 60 mC of charge was passed across H2/N2 EFCs -1 which resulted in the formation of 286 nmol NH3 mg MoFe protein, corresponding to a Faradaic efficiency of 26.4%.

The Haber-Bosch process is an industrial process that produces ammonia (NH3) from molecular hydrogen (H2) and molecular nitrogen (N2).[1] Since its advent at the beginning of the 20th century, the Haber-Bosch process has evolved into a technology that now consumes approximately 1% of the world’s energy sources[2] in the production of nearly 140 megatons of NH3 per year (2011).[3] Since the majority of NH3 (~66%) is produced by this energy-intensive process (requiring high temperature and pressures of 500 ºC and 20 MPa and contributing to around 3% of global CO2 emissions), alternative strategies to produce NH3 are of interest to scientists globally.[4] Nitrogenase is a two-component protein that primarily catalyzes the reduction 2H+ and N2, producing H2 and 2NH3.[5] All nitrogenases comprise a homodimeric reducing component protein (Fe protein) and a catalytic protein that is commonly Modependent (MoFe protein), although VFe- and FeFe-dependent proteins have been isolated.[6] In vivo, turnover occurs when reduced Fe protein (presumably reduced by a flavodoxin) binds to the MoFe protein and ATP-hydrolysis-coupled electron transfer (ET) of a single electron between the two proteins takes place by the following balanced equation:[5a]

solution. Substrate reduction by the MoFe protein (independent of the Fe protein and ATP hydrolysis) has been studied, although only a single study has been able to facilitate N2 reduction to NH3 using a photosensitizer.[7] Enzymatic fuel cells (EFCs) are devices that utilize redox enzymes as bioelectrocatalysts at anodic and cathodic electrode surfaces.[8] Such devices can operate at room temperature, ambient pressure and near-neutral pH, all of which are highly desirable for alternative NH3 production. Typically, ET between an enzyme and an electrode is sluggish and reversible electron mediators are used to improve electron transfer between enzymes and electrode surfaces. All bioelectrocatalytic studies of nitrogenase to date require electron mediators with very negative reduction potentials for turnover,[9] thus, the selection of a suitable bioanode is not trivial. Herein, we initially demonstrate the use of MV as the sole electron donor for nitrogenase facilitating the bioelectrochemical reduction of N2, H+, and acetylene (C2H2). We demonstrate the coupling of hydrogenase

16MgATP + 8e- + 8H+ + N2 à 16MgADP + 2NH3 + H2 + 16Pi In vitro, this enzymatic reaction is commonly achieved using dithionite (DT) as an electron donor and an ATP-regenerating

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[b]

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Dr. R.D. Milton, Ms. R. Cai, Dr. S. Abdellaoui, Prof. S.D. Minteer Department of Chemistry University of Utah 315 S 1400 E, Salt Lake City, UT, 84112 (USA) E-mail: [email protected] Dr. R.D. Milton, Prof. D. Leech School of Chemistry National University of Ireland, Galway University Road, Galway (Ireland) Dr. Pita, Dr. A. L. De Lacey Instituto de Catalisis y Petroleoquimica, CSIC C/ Marie Curie 2, L10, 28049, Madrid (Spain) Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))

Figure 1. Structure and turnover of (a) Mo-dependent nitrogenase from Azotobacter vinelandii and (b) hydrogenase from Desulfovibrio gigas using methylviologen as the electron acceptor or donor. PDB accession codes: 4WZA and 2FRV. For illustration purposes, only half of the Fe:MoFe complex is shown.

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Figure 2. Representative cyclic voltammogram bioelectrocatalytic N2 reduction by nitrogenase mediated by MV. Experiments were performed in an N2-purged 2+ solution of 100 µM MV in MOPS buffer (pH 7, 100 mM) containing Fe protein, MoFe protein and an ATP-regenerating solution in the absence (dashed line) and presence (solid line) of 6.7 mM MgCl2. Cyclic voltammetry -1 was performed at 2 mV s at room temperature (19-21 ºC), using Toray paper 2 as the working electrode (0.25 cm geometric surface area) under anaerobic conditions.

(anodic) with nitrogenase (cathodic) whereby MV is used as an electron mediator for the bioanode and biocathode, and NH3 is produced from H2 and N2 while simultaneously producing an electrical current. The need to supply ATP was negated by the use of an ATP-regenerating system (creatine phosphokinase/creatine phosphate). Initially, the suitability of MVŸ+ to act as the sole electron donor to nitrogenase for N2 reduction was evaluated. There are limited reports of viologens being able to reduce Azotobacter vinelandii nitrogenase while supporting catalytic turnover; C2H2 reduction has been demonstrated at relatively low rates.[10] Recently, MV was used as an intermediary electron donor to the Fe protein whereby DT is used as the primary electron donor.[11] To the best of our knowledge, N2 reduction to NH3 has not been demonstrated using only MV as the sole electron donor. Figure 1 highlights the structures and catalytic cycles of hydrogenase and nitrogenase using MV as the exclusive electron donor or acceptor, where the MV-mediated reduction of N2 by the nitrogenase Fe:MoFe complex proceeds as per the following equation:

the substrate) confirmed nitrogenase activity for MVŸ+, with 1018 ± 11 nmol C2H4 and 588 ± 76 nmol NH3 produced min-1 mg-1 MoFe protein; products were not detectable in the absence of MVŸ+ or when MV2+ was used. Counterpart N2 reduction assays conducted under the same conditions (except using 15 mM DT as the electron donor in the absence of MV) resulted in the production of 777 ± 38 nmol NH3 min-1 mg-1 MoFe protein, demonstrating that similar rates of N2 reduction can be achieved by the use of MVŸ+ as the sole electron donor. Cyclic voltammetric and amperometric i-t analyses were also performed, where a catalytic reductive current is observed when all protein components and substrates are present (Figure 2 and Supporting Information). The Faradaic efficiency was determined to be 59 ± 6 %, reflecting the high efficiency of generating NH3 from N2 and nitrogenase by bioelectrosynthesis (Supporting Information). After demonstrating that MVŸ+ generated by bulk electrolysis/bioelectrosynthesis and cyclic voltammetry is able to support substrate reduction by nitrogenase, we next evaluated the use of H2 as the electron donor in a homogeneous assay, whereby MV2+ reduction is facilitated by H2 oxidation by hydrogenase (from D. gigas). Figure 3a outlines the hypothetical reaction scheme for such a turnover event. N2 reduction was evaluated whereby 3.3 ± 0.2 µmol NH3 mg-1 MoFe protein was detected after 15 minutes; the headspace of the sealed assay vial contained 0.972 atm N2 and 0.028 atm H2 to avoid excessive competitive inhibition of N2 reduction by H2. Next, the ability to generate an electrical current while simultaneously producing NH3 from N2 was explored. Given the global energy requirement of NH3 production from N2, this

16MgATP + 8MVŸ+ + 8H+ + N2 à 16MgADP + 2NH3 + H2 + 16Pi + 8MV2+ and the MV-mediated oxidation of H2 by hydrogenase takes place by the following equation:[12] H2 + 2MV2+ à 2H+ + 2MVŸ+ To first demonstrate the ability of electrochemically-reduced MVŸ+ to support the reduction of the Fe protein and support substrate reduction by the MoFe protein as the sole electron donor (as per Figure 1), we performed C2H2 and N2 reduction assays except DT was replaced with MVŸ+.[13] Fe:MoFe protein ratios and [MVŸ+] were optimized by following the oxidation of MVŸ+ by UV-Visible spectroscopy (Supporting Information). Product analysis for C2H4 and NH3 formation (C2H2 or N2 was

Figure 3. (a) Biocatalytic oxidation of H2 by hydrogenase leads to the 2+ + reduction of MV to MVŸ , which in turn can be used to reduce N2 to NH3 by the Fe and MoFe proteins of nitrogenase (unbalanced). (b) Compartmentalization of hydrogenase and nitrogenase Fe/MoFe proteins by the use of a proton exchange membrane (PEM) leads to an EFC configuration that is able to utilize MV as the electron mediator in both chambers and simultaneously produce NH3 and electrical energy from H 2 and N2 at room temperature and ambient pressure.

COMMUNICATION approximately 2 hours) of charge had passed, resulting in the formation of 286 ± 0 nmol NH3 mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 ± 0.0 %. In summary, bioelectrocatalytic N2 reduction to NH3 by nitrogenase using MV as an electron mediator has been demonstrated. Short bioelectrosynthetic experiments (1 hour) of this MV-mediated reaction resulted in the production of 2.1 ± 0.5 µmol of NH3 mg-1 MoFe protein, at a Faradaic efficiency of 58.8 ± 6.1 %. Following the successful production of NH3, we separated both enzymes into a H2/N2 EFC with OCPs of 228 ± 28 mV, and maximum current and power densities of 48.0 ± 3.1 µA cm-2 and 1.50 ± 0.11 µW cm-2. Discharge of the EFCs

Table 1. Open circuit potentials, maximum current densities and maximum power densities of N2-reducing EFCs prepared with H2, DT, or glucose anodes (Hyd = hydrogenase). Anode

H2/Hyd/MV DT/MV Figure 4. (a) Representative evolution of the OCP of a H2/N2 EFC. The anodic 2+ compartment contained 100% H 2 and MV (oxidized) and hydrogenase was added at 60 s. The cathodic compartment contained nitrogenase, 100% N2 + and MVŸ (reduced) and MgCl2 was added at 660 s to initiate nitrogenase turnover (the addition of Mg activates ATP hydrolysis by rendering ATP biologically-active). (b) Representative polarization and power curves of a -1 H2/N2 EFC, performed linear sweep voltammetry (0.5 mV s ) from OCP until short circuit. The cathodic compartment (limiting) was stirred throughout.

represents a significant step forward in producing NH3 spontaneously (i.e., ΔG < 0). To date, MV as an electron donor to nitrogenase remains among the most positive (in terms of potential) of electron donors and the most negative mediator for enzymatic bioanodes, thus, we turned to the use of MV at the bioanode (H2 oxidation by hydrogenase) and the biocathode (N2 reduction by nitrogenase) of the EFC (Figure 3b). Even though the reduction potentials of the bioanode and biocathode are theoretically equal when at equilibrium (therefore not possessing a potential difference across the EFC), the potential of the MV2+/MVŸ+ redox couple at each bioelectrode changes upon a shift of equilibrium between MV2+ and MVŸ+, as per the Nernst equation (simulated within the Supporting Information), due to their respective enzymatic activities. EFCs were evaluated whereby the N2-reducing biocathodes were coupled with 3 anodic systems to demonstrate the ability to generate NH3 and electrical energy. Nitrogenase biocathodes were coupled with DT/MV2+, glucose/MV2+ or H2/hydrogenase/MV2+ anodes, where DT spontaneously reduces MV2+ and glucose is oxidized by MV2+ in carbonate buffer (pH 11);[14] Table 1 reports the OCPs, current densities and power densities for the 3 EFC configurations. After allowing the OCP to evolve over 10 minutes (Figure 4a), the performance of the EFC was evaluated by slow linear polarization (0.5 mV s-1) from OCP until short-circuit (Figure 4b). Next, a low potential difference (10 mV) was applied to afford high current flow from the anode to the cathode and NH3 was quantified. NH3 produced in the cathodic compartment was quantified after 60 millicoulombs (mC,

2+

2+

Glucose/MV (pH 11)

2+

OCP / mV

Maximum current -2 density / µA cm

Maximum power -2 density / µW cm

228 ± 28

48.0 ± 3.1

1.50 ± 0.11

254 ± 14

41.7 ± 2.8

0.82 ± 0.06

238 ± 30

12.4 ± 1.6

0.76 ± 0.04

resulting in the passage of 60 mC across the EFC yielded 286 ± 0 nmol NH3 mg-1 MoFe protein, operating at a Faradaic efficiency of 26.4 ± 0.0 %, demonstrating the ability to simultaneously produce NH3 and electrical energy from H2 and N2. We highly anticipate that these results will spur significant interest into N2based fuel cells, where NH3 and electrical energy can be simultaneously produced from N2 and a fuel. Future work to overcome limitations associated with ATP hydrolysis and the anaerobic requirements of nitrogenase is required. Due to the required transient association of the Fe and MoFe proteins, bioelectrochemistry was performed for non-immobilized nitrogenase proteins; future work will evaluated the immobilization of both proteins at the surface of an electrode.

Acknowledgements This work is supported by a Marie Curie-Skłodowska Individual Fellowship (Global) under the European Commission’s Horizon 2020 Framework (project 654836 “Bioelectroammonia”), the Army Research Office and Spanish MINECO project CTQ201571290-R. Keywords: Haber Bosch process • nitrogenase • ammonia • molecular nitrogen reduction • fuel cell [1]

F. Haber, R. Le Rossignol, Z. Elektrochem. Angew. Phys. Chem. 1913, 19, 53.

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B. E. Smith, Science 2002, 297, 1654-1655. J. D. Ayotte, J. M. Gronberg, L. E. Apodaca, Trace elements and radon in groundwater across the United States, United States Geological Survey Report, 2011. T. Kandemir, M. E. Schuster, A. Senyshyn, M. Behrens, R. Schlögl, Angew. Chem. Int. Ed. 2013, 52, 12723-12726. a) B. K. Burgess, D. J. Lowe, Chem. Rev. 1996, 96, 2983-3012; b) I. Dance, Chem. Commun. 2013, 49, 10893-10907. a) B. J. Hales, D. J. Langosch, E. E. Case, J. Biol. Chem. 1986, 261, 15301-15306; b) A. Müller, K. Schneider, K. Knüttel, W. R. Hagen, FEBS Letters 1992, 303, 36-40. K. A. Brown, D. F. Harris, M. B. Wilker, A. Rasmussen, N. Khadka, H. Hamby, S. Keable, G. Dukovic, J. W. Peters, L. C. Seefeldt, P. W. King, Science 2016, 352, 448-450. a) S. C. Barton, J. Gallaway, P. Atanassov, Chem. Rev. 2004, 104, 4867-4886; b) A. Heller, Phys. Chem. Chem. Phys. 2004, 6, 209216. a) R. D. Milton, S. Abdellaoui, N. Khadka, D. R. Dean, D. Leech, L. C. Seefeldt, S. D. Minteer, Energy Environ. Sci. 2016, 9, 25502554; b) P. Paengnakorn, P. A. Ash, S. Shaw, K. Danyal, T. Chen, D. R. Dean, L. C. Seefeldt, K. A. Vincent, Chem. Sci. 2017. a) R. V. Klucas, H. J. Evans, Plant Physiol. 1968, 43, 1458-1460; b) R. R. Eady, B. E. Smith, K. A. Cook, J. R. Postgate, Biochem. J. 1972, 128, 655-675; c) D. A. Ware, 1972 1972, 130, 301-302; d) S. Lough, A. Burns, G. D. Watt, Biochemistry 1983, 22, 4062-4066.

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Z.-Y. Yang, R. Ledbetter, S. Shaw, N. Pence, M. TokminaLukaszewska, B. Eilers, Q. Guo, N. Pokhrel, V. L. Cash, D. R. Dean, E. Antony, B. Bothner, J. W. Peters, L. C. Seefeldt, Biochemistry 2016, 55, 3625-3635. A. L. De Lacey, J. Moiroux, C. Bourdillon, Eur. J. Biochem. 2000, 267, 6560-6570. Acetylene reduction to ethylene by nitrogenase is commonly used to evaluate activity, due to the ease of ethylene quantification by gas chromatography. D. R. Wheeler, J. Nichols, D. Hansen, M. Andrus, S. Choi, G. D. Watt, J. Electrochem. Soc. 2009, 156, B1201-B1207.

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COMMUNICATION Electrifying the Haber-Bosch Process: The bioelectrochemical coupling of nitrogenase and hydrogenase yields an enzymatic fuel cell that produces electrical energy from H2 and N2 while simultaneously making NH3 as a useful chemical commodity.

Ross D. Milton, Rong Cai, Sofiene Abdellaoui, Dónal Leech, Antonio L. De Lacey, Marcos Pita and Shelley D. Minteer* Page No. – Page No. Bioelectrochemical Haber-Bosch Process: An Ammonia-Producing H2/N2 Fuel Cell