*5.1. Enzymatic Biofuel Cells (EBCs)*

One of the most studied energy conversion devices is the enzymatic biofuel cell (EBC) which uses oxidoreductase enzymes as biocatalysts to convert chemical energy into electrical energy [157–159]. A typical EBC consists of a two-electrode cell in which the biofuels (such as H2, formate) are oxidised at the bioanode and the oxidants are reduced at the biocathode (usually O<sup>2</sup> is reduced to water) [160]. Among these, H2/O<sup>2</sup> biofuel cells are one of the most investigated enzymatic systems [161]. Hydrogenases are promising biocatalysts to fabricate high performance H2-oxidation bioanodes. However, the extreme oxygen sensitivity of highly active hydrogenases is one of the main limitations of hydrogenase based H<sup>2</sup> bioanodes [162]. The O<sup>2</sup> sensitive hydrogenase can be protected by a low-potential viologen redox polymer matrix [163] or enzymatic O<sup>2</sup> scavenger [164]. However, these protection mechanisms either consume the electron from H<sup>2</sup> oxidation or add chemicals for protection. O2-tolerant MBH therefore shows great advantage for H2/O<sup>2</sup> biofuel cell. The first membrane-less H2/O<sup>2</sup> cell was assembled by Armstrong group. Two pyrolytic graphite electrodes, coated respectively with MBH from *R. eutropha* and laccase from *Trametes versicolor*(*Tv*), were immersed in a H2/air flushed solution. The system reached an OCV of ~970 mV and a maximum power output of ~5 µW [165]. High OCVs were achieved because MBH directly exchanged electrons with the electrode and no mediators were required. The *Re* MBH was solubilised from the membrane extracts by 2% Triton-X 114 and isolated via a *Strep*-tag sequence on the small subunit. It is possible that the MBH was devoid of the transmembrane cytochrome *b* anchor and this could explain why there was no need to use mediators. The *Re* MBH also showed CO-tolerance [165]. The same group further improved the performance of the H2/O<sup>2</sup> cell by replacing the *Re* MBH with MBH (similarly *Strep*-tagged on the small subunit) from *R. metallidurans*, which is more active and stable to O<sup>2</sup> exposure (Figure 3a). The authors emphasised that such a system would have the advantage to perform H<sup>2</sup> conversion even in H2-poor mixtures. They also showed that three cells in series provided a total OCV of 2.7 V which was sufficient to power a wristwatch for 24 h [166].

As described earlier, we have presented a strategy for using the full heterotrimeric MBH as a biocatalyst (including the cytochrome *b* anchor) and have used multi-layer membrane stacks on gold electrode to increase MBH loading [86]. In our approach, we used cytoplasmic membrane extracts of *R. eutropha* and created interconnected layers of membranes with each layer containing anchored MBHs. Lipophilic quinones were used as mediator, shuttling electron between electrode and protein. However, the irreversible electrochemical behaviour of the quinone redox reaction increases the required overpotential for H<sup>2</sup> oxidation, which will limit the power output of the devices and more research is needed to resolve this.

To optimise EBCs, gas diffusion electrodes have been investigated and enzyme coverage optimised. The porous structure of the gas diffusion electrode can increase the mass loading of the biocatalyst and overcome the mass transport limitation of gases. In 2016, Kano group used an O2-tolerant MBH from *Hydrogenovibrio marinus* and an O2-sensitive [NiFe]-hydrogenase from *Desulfovibrio vulgaris* "Miyazaki F" to create DET-type gas diffusion electrodes [167]. The authors did not specifically comment on whether their enzyme purification methods might have affected the presence of the cytochrome *b* anchor subunits of MBH. The MBHs were isolated through two different procedures. The large and small subunits of the O2-tolerant MBH were isolated through detergent solubilisation and maintained in 0.025% Triton-X. The O2-sensitive MBH went through a trypsinisation process which could lead to the separation of the transmembrane cytochrome *b* anchor. These H<sup>2</sup> oxidation electrodes generated a current density of 10 mA·cm−<sup>2</sup> in the half-cell configuration. Contrary to the O2-tolerant MBH, the O2-sensitive MBH did not show overpotential for H<sup>2</sup> oxidation and, on this basis, was selected

by the authors as bioanode of a H2/O<sup>2</sup> EBC for a further study. Coupling this O<sup>2</sup> sensitive MBH anode with bilirubin oxidase (BOD) from *Myrothecium verrucaria* immobilised on Ketjen black-modified waterproof carbon papers (KB/WPCC) electrode, a dual gas-diffusion membrane- and mediator-less H2/air-breathing biofuel cell was constructed (Figure 3b) which showed maximum power density in the range of 6.1 mW·cm−<sup>2</sup> at 0.72 V [168].

**Figure 3.** Schematic representations of enzymatic biofuel cells (EBC). (**a**) EBC comprises of graphite electrodes modified with O<sup>2</sup> -tolerant MBH of *R. metallidurans* CH34 (anode) and fungal laccase (cathode) in aqueous electrolyte exposed to 3% H<sup>2</sup> in air. Reprinted with the permission from ref [166]. Copyright (2006), The Royal Society of Chemistry. (**b**) A dual gas diffusion membrane-free H<sup>2</sup> /air powered EBC comprises of a [NiFe]-MBH (anode) and a bilirubin oxidase (cathode). Reprinted with the permission from ref [168]. Copyright (2016), Elsevier B.V.

The O2-reduction biocathodes for EBC are usually based on multi-copper oxidases like bilirubin oxidase or laccase which can reduce O<sup>2</sup> almost without overpotential [169]. Among the membrane proteins, cytochrome *c* oxidase has been studied as O<sup>2</sup> reduction catalyst. Katz and Willner assembled a membrane-less glucose/O<sup>2</sup> biofuel cell with cytochrome *c*/cytochrome *c* oxidase as O<sup>2</sup> reducing cathode [170]. The cytochrome *c* was assembled on a maleimide modified gold electrode through a cysteine residue to link cytochrome *c* oxidase. In a follow-up work, the authors developed an electroswitchable and tunable biofuel cell. In this case, the cathode was modified with poly(acrylic acid) loaded with Cu2<sup>+</sup> to covalently attach cytochrome *c* and link the latter to cytochrome *c* oxidase [171]. Although cytochrome *c* was able mediated electron transfer to cytochrome *c* oxidase for O<sup>2</sup> reduction, only an OCV of 0.12 V was obtained, likely limited by the redox potential of cytochrome *c* (~0.25 V vs. SHE), which is much lower than that of H2O/O<sup>2</sup> (0.82 V vs. SHE at pH 7). Although cytochrome *c* oxidase is not commonly used for O<sup>2</sup> reducing cathode in biofuel cell, the recent work with gold NPs by the group of Hellwig has shown renewed possibilities for cytochrome *c* oxidase as catalyst (see Section 3.3). Recently, Wang et al. [172] reported that cytochrome *c* oxidase from acidophilic bacterium *Acidithiobacillus ferrooxidans* can reduce O<sup>2</sup> at exceptionally high electrode potentials (+700 to +540 mV vs. NHE). The low overpotential for O<sup>2</sup> reduction of this cytochrome *c* oxidase makes it an attractive biocatalyst as cathode of biofuel cells in the future.

Besides the use of H<sup>2</sup> as fuel for EBC, formate is also a valuable feedstock for biofuel cells because its redox potential is similar to H2/H+. The Kano group reported a mediated electron transfer type formate/O<sup>2</sup> biofuel cell by coupling formate dehydrogenase modified bioanode with BOD modified biocathode [173]. In nature, some formate dehydrogenases can catalyse the inverse reaction to reduce CO<sup>2</sup> to formate [174]. However, to the best of our knowledge, the research in this field has been conducted only on soluble formate dehydrogenases. The ability of CO<sup>2</sup> reduction and the applications of membrane-bound formate dehydrogenase need to be further explored.

− − − − − − − Respiratory nitrate reductase (Nar) catalyses NO<sup>3</sup> <sup>−</sup> reduction to NO<sup>2</sup> <sup>−</sup>, this is an essential step to produce NH3. Today there is no enzyme known to reduce NO<sup>3</sup> <sup>−</sup> to NH<sup>3</sup> directly [175]. DET with nitrate reductase has been shown to support the electrochemical reduction of NO<sup>3</sup> <sup>−</sup> to NO<sup>2</sup> <sup>−</sup> [34,35]. A full reduction of NO<sup>3</sup> <sup>−</sup> to NH<sup>3</sup> was demonstrated by a cascade electrocatalysis process which combined nitrate reductase and noble metal catalyst to reduce NO<sup>2</sup> <sup>−</sup> to NH<sup>3</sup> [58].
