*4.3. Electrochemical Reactors*

The coenzyme nicotinamide adenine dinucleotide is required as a co-substrate for over 300 dehydrogenases [141]. A number of studies have been carried out to use electrochemical methods to regenerate the cofactor. Yoon et al. prepared an electrochemical laminar flow microreactor to regenerate NADH in the synthesis of chiral L-lactate from the achiral substrate pyruvate (Figure 4). For this purpose, gold electrodes were deposited on the inside of a Y-shaped reactor, with two separate streams, one with buffer and the second with reagents (FAD, NAD+, enzymes and substrate), with the flow directed to the cathode. The reduced cofactor, FADH<sup>2</sup> was produced at the cathode and used for the reduction of NAD<sup>+</sup> to NADH, resulting in a 41% yield of L-lactate (theoretical yield of 50%) [142].

**Figure 4.** Schematic diagram of a laminar Y-shaped microreactor used for the electrochemical regeneration of NAD<sup>+</sup> using formate dehydrogenase (FDH) [142].

A filter-press microreactor with semi-cylindrical channels on the electrode surface resulting in high surface areas was used to electrochemically regenerate FADH<sup>2</sup> (Figure 5) [143].

**Figure 5.** A schematic of a filter-press microreactor. Reprinted from [143] Copyright (2020), with permission from John Wiley and Sons.

− − − As electrochemical cofactor regeneration has, by definition, to occur at the surface of the electrode, scaling up reactions is likely to cause diffusion limitations resulting in reduced rates of reaction [23]. This challenge can be addressed by using three-dimensional electrodes in continuous flow reactors. Kochius et al. designed a system for the efficient electrochemical regeneration of NAD+, based on 3D electrodes with a high working surface area 24 m<sup>2</sup> . The anode, comprised of a packed bed of glassy carbon particles, was surrounded by two cathodes (titanium net) (Figure 6). The oxidation of NADH was carried out using ABTS as a mediator, with turnover numbers of 1860 and 93 h−<sup>1</sup> for the mediator and the cofactor, respectively, rates that were significantly higher than previously reported. A space time yield 1.4 g L−<sup>1</sup> h <sup>−</sup><sup>1</sup> was achieved for the three-dimensional electrochemical reactor, a value higher than that at a two-dimensional cell [48]. − − −

**Figure 6.** Schematic diagram of the three-dimensional electrode [48].

− Ruinatscha et al. fabricated a reactor with three-dimensional porous carbon electrodes with a surface area to volume ratio of 19,685 m2m−<sup>3</sup> (Figure 7).

− The reactor was used to produce FADH2, which was then coupled with styrene monooxygenases for the synthesis of styrene oxide. The rate of mass transfer increased in this system and FAD was reduced at a rate of 93 mM h−<sup>1</sup> producing styrene oxide at a rate of 1.3 mM h−<sup>1</sup> [144]. Due to the benefits of using microreactors for cofactor regeneration, the direct regeneration of NAD<sup>+</sup> was examined. Rodríguez-Hinestroza et al. designed an electrochemical filter press microreactor for the direct anodic regeneration of NAD+, via the horse-liver alcohol dehydrogenase-catalysed synthesis of β-alanine (Figure 8). The platinum and gold electrodes used had 150 microchannels, with a high surface area

−

of 250 cm−<sup>2</sup> . NADH was directly oxidized at the gold electrode, with a 92% conversion of NADH to NAD+. The produced NAD<sup>+</sup> was used for the enzymatic oxidation of carboxybenzyl-β-amino propanol [145]. −

− −

**Figure 7.** Schematic diagram of system for a reactor for the regeneration of FAD. Reprinted from [144] Copyright (2020), with permission from Elsevier.

β **Figure 8.** Schematic diagram of an electrochemical microreactor system and the electroenzymatic oxidation of carboxybenzyl-β-amino propanol. Reprinted from [145]. Copyright (2020), with permission from Elsevier.

Fisher et al. prepared a multichannel segmented flow bioreactor in which the oxidoreductase pentaerythritol tetranitrate reductase (PETNR) was regenerated electrochemically with the use of methyl viologen as a mediator (Figure 9), replacing NADPH as cofactor. The use of methyl viologen resulted in rates of substrate reduction that were 15–70% of those observed with NADPH [146].

Mazurenko et al. carried out mediated regeneration NAD<sup>+</sup> via poly(methylene green) in a flow reactor for the synthesis of D-fructose from D-sorbitol using D-sorbitol dehydrogenase [142]. Electrochemical methods can also be used to immobilise enzymes on the surface of electrodes for subsequent use in a flow reactor. For example; Xiao et al. designed a continuous flow bioreactor based on lipase immobilised by the electrochemical generation of a silica film nanoporous gold-modified glassy carbon electrodes (Figure 10a). In addition, −1.1 V vs. Ag/AgCl was applied on the electrode surface immersed in tetraethoxysilane and lipase solution. This triggered hydroxyl ion production that caused tetraethoxysilane condensation on the electrode surface and lipase was entrapped into silica structure.

β

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− −

β

β

**Figure 9.** (**a**) Schematic diagram of a segmented flow bioreactor system, (**b**) 3D representation of the bioreactor. Reprinted from [146] Copyright (2020), with permission from The Royal Society of Chemistry.

**Figure 10.** (**a**) Schematic diagram of the electrodeposition of a silica layer with entrapped enzyme on an electrode and (**b**) an image of the continuous flow reactor [107].

The modified electrodes were inserted into the flow reactor (Figure 10b) and used to prepare p-nitrophenol from 4-nitrophenyl butyrate (4-NPB), with the full conversion of the substrate (0.075 mM in 2 mL) after eight cycles [107].
