Pushing the limits for enzyme-based membrane-less hydrogen fuel cells – achieving useful power and stability

Performance characteristics of simple enzyme-based membrane-less hydrogen fuel cells running on non-explosive H2-rich air mixtures have been established using an adjustable test bed that allows multiple unit cells to operate in series or parallel. Recent advances with ‘3D’ electrodes constructed from compacted porous carbon loaded with hydrogenase (anode) and bilirubin oxidase (cathode) have been extended in order to scale up fuel cell power to useful levels. One result is an appealing ‘classroom’ demonstration of a model house containing small electronic devices powered by H2 mixed with a small amount of air. The 3D electrodes work by greatly increasing catalyst loading (at both anode and cathode) and selectively restricting the access of O2 (relative to H2) to enzymes embedded in pores at the anode. The latter property raises the possibility of using standard hydrogenases that are not O2-tolerant: however, experiments with such an enzyme reveal good short-term performance due to restricted O2 access, but low longterm stability because the root cause of O2 sensitivity has not been addressed. Hydrogenases that are truly O2 tolerant must therefore remain the major focus of any future enzyme-based hydrogen fuel cell technology. Page 1 of 26 RSC Advances


Introduction
Isolation and characterization of O 2 -tolerant [NiFe]-hydrogenases that are able to catalyze H 2 oxidation in the presence of O 2 has stimulated new concepts in hydrogen fuel cells. [1][2][3][4][5][6][7] Not only do enzymes prove the feasibility of replacing platinum metal catalysts with ones derived from abundant elements (indeed the active sites of enzymes are more active than Pt [8][9][10][11] ) but their high selectivity lends itself to simple, membrane-less fuel cells that could be miniaturized, thus compensating for the large footprint of the catalyst. [12][13][14][15] Our focus is therefore on membrane-less cells. Although commercial development is, even optimistically, a long way ahead (depending on achieving high levels of miniaturization, longterm stability and identification of a niche use) orders-of-magnitude improvements have been made since the demonstrations, in 2005 and 2006, of an enzyme-based membrane-less H 2 fuel cell running in the presence of CO 16 or on a non-explosive H 2 -weak/air-rich mixture (3% H 2 in air). 17 The H 2weak/air-rich mixture proved problematic because even an O 2 -tolerant hydrogenase is overwhelmed by O 2 under low-load conditions, 18 but no such problem was encountered when using a non-explosive H 2rich/air-weak mixture (80% H 2 in air). Since 2010, power densities and lifetimes of enzyme-based, membrane-less H 2 fuel cells have advanced in two stages. It was demonstrated that a reasonably stable power density of 0.1 mW·cm −2 (based on the anode area) could be achieved by covalently attaching a hydrogenase and a 'blue' Cu oxidase to graphite electrodes modified with multi-walled carbon nanotubes. 19 Then, in a subsequent development, Xu and Armstrong used 3D compacted mesoporous carbon electrodes with re-proportioned anode/cathode areas to increase the power density up to 1.7 mW·cm −2 (based on geometric area of anode) with an extended half-life of approximately one week. 20 Porous 3D electrodes (we use 'CPC' as a general abbreviation for different types of compacted porous carbon) increase the number of reaction sites per unit geometric electrode area, thus substantially raising power density. [21][22][23][24][25][26][27] The enzyme molecules permeate deeply, leading to a greatly enhanced catalyst loading, while the porous channels are interconnected and open to the surface. 20 Varying the relative sizes of anode and cathode further increases power output which is otherwise limited by the low O 2 level.
Oxygen tolerance of the hydrogenase that is used as the anodic electrocatalyst is an essential requirement for all enzyme-based H 2 fuel cells, even if a membrane is used (due to unavoidable O 2 crossover). All hydrogenases characterized to date are inactivated or permanently damaged by O 2 and/or high potentials, 28 30 The term rate(inact) increases with increasing O 2 concentration whereas rate(act) increases as the potential is lowered, and both rate(inact) and rate(aact) are affected in a more complex way, by the concentration of H 2 . Hence f is increased by impeding O 2 access to the active site and increasing the electron availability for reactivation.
It is thus timely to consider further interesting implications of developing membrane-less H 2 fuel cells with CPC electrodes. We have therefore investigated the minimum configuration, using multiples of simple unit cells arranged in parallel or series, that is required to produce useful power (i.e. sufficient to run small electronic devices). A test bed has been constructed, in which parallel and series connections of sandwich-like electrode stacks can be varied. As explained below, powers are reported in mW/total cell volume as well as in mW/area (geometric electrode area), although no attempt has been made to optimize volume power density (see below) as we focused on having sufficient space to manipulate the cells easily. Secondly, we have addressed the issue of whether CPC electrodes can usefully improve the O 2 tolerance of O 2 -sensitive (standard) hydrogenases (which usually have higher activity) an idea that complements, in a timely way, a recent paper by Plumeré et al, who found that embedding an O 2 -sensitive hydrogenase into a viologen-based redox hydrogel significantly improved its O 2 tolerance. 31

Results and discussion
Test bed structure: The test bed shown in Figure 1A Figure 1A(ii)). Enzymes were applied to anode and cathode as described in Methods. To balance the anodic and cathodic currents in the non-explosive H 2 -air mixture, the area ratio of anode to cathode was re-proportioned in each unit cell: thus the cathodes (modified with bilirubin oxidase (BOD) from Myrothecium verrucaria) were 4 x 1.5 cm while the anodes (modified with Hyd-1) were smaller, at 0.8 x 1.5 cm. The additional 1 cm of exposed stainless steel plate (electrochemically inert) above the cathode was required so that the H 2 /air mixture could be bubbled into each cell without losing electrolyte. In the parallel multi-cells, 2, 3 or 4 anodic plates and 2, 3 or 4 cathodic plates were connected (denoted 2x2, 3x3, 4x4) to contain 3, 5 or 7 unit cells, respectively ( Figure 1B(i)). For a series multi-cell, a divider was used to separate the electrolyte solutions ( Figure 1B(ii)). Reductive activation of Hyd-1 was carried out electrochemically, as described previously, 19 by connecting the anodes to a potentiostat and inserting reference and counter electrodes at one side of the divider ( Figure 1A(i)). Before activating Hyd-1, the solution was raised above the central divider to connect all the cells; then after activation was complete, the level was lowered to isolate the cells series-wise. Volume power density was calculated by dividing total power by the total volume of the cells (3.75 cm 3 for each unit cell).  respectively. The volumes of those pores having diameters larger than 10 nm are 0.034 cm 3 g −1 , 0.42 cm 3 g −1 and 1.0 cm 3 g −1 , representing 65%, 75% and 85% of the total pore volumes of compacted 100% G, 60/40 G/MCNT and 100% MCNT electrodes, respectively. Based upon maximum molecular diameters of Hyd-1 (12 nm) and BOD (8 nm), 35,36 it follows that most pores of the CPC electrodes are large enough to be permeable to both enzymes. Electrodes with greater pore volumes will therefore have a higher density of electrocatalytic sites.
Assuming complete uptake of enzymes into the porous channels of CPC electrodes, the total amounts of hydrogenase (RMM approximately 100 kDa) and BOD (RMM approximately 50 kDa) deposited on respective electrodes are 1.2 nmol and 18 nmol, respectively (see Methods). A calculation indicates that an atomically flat surface occupied by a monolayer of Hyd-1 (assuming a footprint 12 x 12 nm) and BOD (assuming a footprint 8 x 8 nm) would extend to approximately 1000 cm 2 and 7000 cm 2 respectively, to be compared with geometric anode and cathode areas of 1.2 cm 2 and 6 cm 2 . The enzyme loading is therefore equivalent to that of about 10 3 monolayers in both cases, the vast excess being drawn into the pores of the electrode. An exhibit was constructed to demonstrate that the test bed with series 2[4x4] configuration, is capable of powering electronics from a non-explosive 78% H 2 -22% air mixture at 20 °C. The festive 'Hydrogen House' shown in Figure 4 contains five red LED tree lights and a miniature electronic clock. A light dependent resistor probe (LDR) 37 was used to monitor changes in LED light intensity with time, in order to establish the working stability of the fuel cell under continuous power output.
The light intensity showed no decrease over the course of 8 hours ( Figure S1).
Improving the O 2 tolerance of hydrogenases. The following experiments were carried out to determine if the H 2 oxidation activity of hydrogenases under aerobic conditions is improved if the enzyme is embedded in a 3D CPC electrode as compared to adsorption at a conventional pyrolytic graphite edge (PGE) electrode. Figure 5 compares the current-time profiles for H 2 oxidation by Hyd-1, previously fully activated, at a CPC electrode (60/40 G/MCNT) and at a PGE electrode. In each case, the electrode potential was held at +0.206 V vs. SHE (a more oxidizing value, and thus more demanding conditions, than used in earlier experiments 3,30 ) and the H 2 level in non-explosive H 2 -air mixtures was varied periodically.
The Hyd-1/CPC electrode showed no loss in H 2 oxidation current over the initial period of 1200 s under 100% H 2 . Then, upon switching the gas supply to 89% H 2 -11% air, the current dropped rapidly to 58% of its original value (f = 0.58) and remained constant at that value for a further 1200 s. Switching back to 100% H 2 resulted in a rapid and complete recovery of the original current. After a further 1200 s, during which the same stability was observed, the gas supply was switched to 78% H 2 -22% air and the current dropped rapidly to 42 % of the original value (f = 0.42), remaining stable at this value for 1200 s. Finally, the gas mixture was switched back to 100% H 2 and complete recovery of the original current was observed. Exactly the same procedures were used to measure the performance of the conventional Hyd-1/PGE electrode. The slow decrease in current measured in the 100% H 2 atmosphere is usually described as 'film loss'. After 1200 s, switching the gas mixture from 100% H 2 to 89% H 2 -11% air resulted in a rapid drop in current to approximately 7.5 % of the original value (f = 0.075). Switching the gas supply back to 100% H 2 then produced an increase in current that was much slower than observed for the Hyd-1/CPC electrode; in fact, recovery was incomplete even after 1200 s, when the gas supply was switched to 78% H 2 -22%, resulting in the current dropping rapidly to approximately 3.5% of the original value (f = 0.035). Finally, restoring the gas supply to 100% H 2 resulted again in a slow increase in activity.
These results show that the great advantage afforded by embedding a hydrogenase in a 3D carbon electrode extends to an improved ability to function when the gas supply contains O 2 . Hyd-1 is inherently O 2 -tolerant, which means that if O 2 is added to the H 2 gas supply, only the oxidized 'resting' Ni-B state is formed: 30 Ni-B is reactivated in a potential-dependent process, although the rate is slow at being offset by the far more reducing potential (which increases rate(act)). 30 Regardless of whether a porous CPC electrode improves the performance of hydrogenase or BOD electrochemistry in the absence or presence of O 2 , an obvious rationale so far is that the electrode contains so much enzyme, that losses, whatever their origin, have little effect on current or stability. The ability of Hyd-2 to operate in the presence of O 2 also varies between the different 3D CPC electrodes. Figure 6(iii) shows chronoamperograms of Hyd-2 at compacted 100% MCNT and 60/40 G/MCNT electrodes, recorded at an electrode potential of +6 mV vs. SHE. It should be noted that a more negative potential was used for the Hyd-2 experiments (compared to Hyd-1) because the characteristic potential (E switch ) at which Hyd-2 is reactivated from the Ni-B state is >0.2 V more negative than for Hyd-1. 3 Switching the gas supply from 100% H 2 to an 89% H 2 -11% air mixture results in an immediate drop in current that is significantly more pronounced for the 60/40 G/MCNT CPC electrode than for the 100% MCNT CPC electrode: furthermore, the resulting suppressed current measured with the 60/40 G/MCNT CPC electrode continues to drop at a faster rate than for the 100% MCNT CPC electrode. When, after 1200 s under 89% H 2 -11% air, the gas supply was switched back to 100% H 2 , recovery was faster and more complete for the 100% MCNT CPC electrode. A second exposure to O 2 , this time using 78% H 2 -22% air for 1200 s, revealed the same pattern but with lower currents, as expected. Finally, Figure 6(iv) shows the result of an experiment to compare the current produced by a Hyd-2−60/40 G/MCNT CPC electrode over a continuous period of 17 h under 100% H 2 with the current recorded over a 12 h period under a 78% H 2 -22% air mixture, after which the gas supply was restored to 100% H 2 . The current under 100% H 2 remains steady, whereas the switch to 78% H 2 -22% air causes an immediate drop in current that is followed by a continual slow decrease.
Upon restoring a 100% H 2 supply, only partial recovery is observed, which is barely affected by switching the electrode potential to −0.194 V for 1 minute: since this procedure should drive the activation of any remaining Ni-B, the observation shows that O 2 causes a significant degree of unrecoverable damage to Hyd-2.
The results shown in Figure 6(i and ii) 40 L is the Avogadro constant, and P is pressure. As shown in Figure 2(iii), the volumes of pores having diameters smaller than 70 nm make up >95% and 100% of the total pore volumes of compacted 100% MCNT and 60/40 G/MCNT electrodes, respectively. Here, N 2 is assumed to be inert, so only H 2 and O 2 are considered. Effusion occurs when the pore size is smaller than the mean free path of a gas molecule, 39 Figure S1 which shows 100% retention of LED intensity after 8 h.

Conclusions
The studies have shown how the power provided by a membrane-less enzyme fuel cell acting on a mixed H 2 /air feed is easily scaled up to useful levels. Stable power, readily gained by exploiting the high enzyme loading possible with '3D' porous carbon electrodes, is multiplied in terms of current or voltage using a simple stack design. There is considerable room for improvement in volume power density through miniaturization via careful attention to engineering. In support of a recent paper by

Construction of the test bed:
The fuel cell test bed consisted of one compartment constructed from polycarbonate with a partial divider also made of polycarbonate. A reference electrode (Ag/AgCl) and a Pt mesh counter electrode were positioned at one end to use for electrochemical measurements under potential control. The main part of the test bed consisted of 16 stainless-steel plates as supports for the 3D CPC electrodes. The CPC electrodes were attached to both sides of each plate using conductive silver adhesive. The exposed metal plate areas not covered by the CPC electrodes were painted to insulate against the electrolyte solution. The top edges of the plates were bent and fixed to the ceiling of the test bed by screws and nuts, which also served as electrical connectors. Such an assembly also allowed for convenient dismantling of the plates. The anodic and cathodic plates were fixed in alternating mode. Gas supply into each unit cell (up to a total of 14) was achieved through gas inlets positioned close to the bottom of the cell and positioned midway between anode and cathode. The CPC discs were then cut to size for the required geometric area and attached to the stainless steel plates as described above. After each plate had been constructed, 30 µL of Hyd-1 (or Hyd-2) (4 mg mL −1 ) was applied to the anode side and 150 µL of BOD (6 mg mL −1 ) was applied to the cathode side. The enzyme concentrations and quantities were chosen after evaluating how oxidation and reduction current densities varied with amounts of Hyd-1 and BOD respectively ( Figure S3). The enzyme-modified CPC electrodes were subsequently placed in a cold room (4 °C) for 1 h to allow the enzymes to permeate into the electrodes before starting the electrochemical characterization and fuel cell tests. The molecular quantities of Hyd-1 (RMM = 100 kDa) and BOD (RMM = 50 kDa) used to modify the CPC electrodes are therefore 1. The ratios of H 2 to air were controlled by two mass flow controllers (Sierra Instruments). A Sorptomatic 1990 instrument (CE Instruments) was used to acquire N 2 adsorption-desorption isotherms at 77 K, and BET surface areas were calculated from the linear part of BET plots. Pore size distribution plots were obtained by using the BJH model. 34 The light intensity of the red LED was monitored by a cadmium sulphide (CdS) light dependent resistor connected to a digital multimeter. Figure 1A. (i) Photograph of the test bed structure (RE = reference electrode, CE = counter electrode).

Figures
(ii) The scheme for the unit cell (A = Anode, C = Cathode).