*5.1. High Surface Area Electrodes*

One method of improving the rate of reaction in electrochemical bioreactors is to use electrodes with high surface areas to enable high enzyme loadings and consequently higher current densities. Electrodes can be designed with a range of three-dimensional (3D) architectures. Parameters such as the method of enzyme immobilisation, the orientation of the enzyme on the support, the porosity of the support, and diffusional limitations within the pores need to be taken into account [147].

Due to their ease of preparation, porous metal electrodes have been used for a range of applications. For example, dealloyed nanoporous gold (NPG) can be prepared on the electrode surface by electro/chemically dissolving Ag from Au/Ag alloys. The resultant pores have diameters that are sufficiently large to accommodate the enzymes and to enable substrates and products to readily diffuse into and out of the pores. Through changing parameters such as the alloy composition and the dealloying conditions, the pore diameters can vary over the range of 5–700 nm [148]. For example, dealloyed nanoporous gold (NPG) electrodes modified with osmium polymer as a mediator were used

for the determination of glucose and lactose [149]. Dealloyed nanoporous gold (NPG) electrodes have also been used for the direct electron transfer of enzymes such as laccase [69]. The immobilisation of FAD-dependent glucose dehydrogenase, bilirubin oxidases [78] and fructose dehydrogenase [150] were also investigated on porous gold electrodes. Nanoporous gold electrodes are widely used as a biofuel cell [78,151] and biosensor [152]. In the construction of biofuel cells, gold nanoparticles have been attached to the electrode surface to enhance the efficiency of the cells [153,154].

Carbon nanotubes have been widely used to modify electrodes [155–158]. Carbon nanotubes possess properties such as high conductivity and surface areas. A simple method for the modification of electrodes with carbon nanotubes and enzymes is to place a drop of a suspension of enzyme and carbon nanotubes onto the surface of the electrode surface [77]. Electrode modification with carbon nanotubes can facilitate direct electron transfer between the electrode and enzymes such as fructose dehydrogenase [159], or horseradish peroxidase [160], resulting in the preparation of efficient biofuel cells [161,162]. Jourdin et al. used multiwalled carbon nanotubes to improve the performance of a microbial system for the bioreduction of carbon dioxide. Multiwalled carbon nanotube electrodes considerably increased the rate of electron transfer between electrode and microorganisms by 1.65-fold and the acetate production rate by 2.6-fold in comparison with the graphite plate electrodes [163]. The microbial electrosynthesis of acetate from CO<sup>2</sup> was described using a vitreous carbon electrode modified with multi-walled carbon nanotubes [164]. Bulutoglu et al. immobilised fused alcohol dehydrogenase on multi-walled carbon nanotubes for the electrocatalytic oxidation of 2,3-butanediol [165]. Bucky papers are flexible, light materials prepared from carbon nanotubes [147,166]. Zhang et al. developed a bioelectrode for electroenzymatic synthesis using a bucky paper electrode on which [Cp\*Rh(bpy)Cl]<sup>+</sup> was immobilized as a mediator for the regeneration of NADH. A turnover frequency of 1.3 s−<sup>1</sup> was achieved for the regeneration of NADH and the system was used for the preparation of D-sorbitol from D-fructose using immobilized D-sorbitol dehydrogenase. −

The high porosity of 3D graphene materials can enable higher enzyme loadings, increasing the performance of the electrodes. Choi et al. used a combination of graphitic carbon nitride and reduced graphene oxide as an efficient cathode for the reduction of O2. The H2O<sup>2</sup> produced at the surface of the cathode surface was used for the peroxygenase-catalysed selective hydroxylation of ethylbenzene to (R)-1-phenylethanol [167]. Enzymes can be covalently attached on the graphene hybrid electrodes through the controlled functionalization of graphene causing high stability. Effective enzyme–graphene conjugation can pave the way for direct electron transfer on the electrode surface. Seelajaroen et al. used enzyme–graphene hybrids for the electrochemical preparation of methanol from CO<sup>2</sup> using NAD(P)-linked enzymes including formate dehydrogenase, formaldehyde dehydrogenase and alcohol dehydrogenase (Figure 11). Enzymes were covalently bound on to carboxylate-modified graphene surfaces.

**Figure 11.** The cofactor-free biosynthesis of methanol using formate dehydrogenase, formaldehyde dehydrogenase and alcohol dehydrogenase. Reprinted from [168] Copyright (2020), with permission from the American Chemical Society.

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Due to the effective rates of electron transfer from the graphene-modified electrode to the enzymes, considerable improvement was observed with a high faradaic efficiency of 12%. A higher production rate of 0.6 µmol·h <sup>−</sup><sup>1</sup> was achieved and the current was stable for 20 h. This shows the advantage of using conductive graphene carboxylic acid as support [168].
