as fuel [2]. Entire microbial cells, organelles and biological molecules have been utilized as catalyst in biofuel cells. The molecules for energy conversion in living eukaryotic cells are utilized as biocatalyst and as model reactions. The reactions are complex and involve the action of nucleotides nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). The nucleotides in the cell are reduced to NADH and FADH2 by protons coming from a chain of oxidation reactions belonging to the microbial catabolic metabolism. The cyclic oxidation and reduction of nucleotides enables the transport of charge in the mitochondria and thus in the microbial cell. The energy pathways in prokaryote cells involve a chain of transmembrane enzymatic proteins. The c-type cytochromes in the outer cell membrane enable direct contact cell-electrode and research in molecular biology shows that cytochromes are responsible for extracellular electron transfer (EET).
The reactions occurring in the living cells involve different catalytic proteins or enzymes; thus charge transfer through biological molecules has required many years of investigation. Enzymes can act in the electrolyte, or be immobilized at the electrode, and electron transfer achieved via either mediated or direct form. The contact of the enzyme with the substrate is achieved via physical or covalent adsorption. The type of contact is a function of the location of the active site in the enzyme, which can be in the periphery or in the core of the catalytic protein. The electrode material for immobilization of the bioelectrocatalyst is one of the main issues. Thus, the intrinsic properties of the electrode such as porosity and conductivity must be improved via doping, template construction or addition of nanomaterials. Another concern in bioelectrocatalysis is the lifetime of the enzymatic electrodes, which are very sensitive to environmental conditions. Plenty of strategies using polymers have been proposed, including encapsulation, cross-linking, anchoring, and self-assembly with the aim of improving the electron transfer between the enzyme and the electrode. This process can be explained by different mechanisms like percolation though immobile redox centers, collision of mobile centers, and conduction through a conjugated backbone. The direct transfer occurs via electron tunneling from the active site in the enzyme and the electrode.
In the following sections, reactions of general interest in cells catalyzed by enzymes and microorganisms are described in the first instance. The next section focuses on advances in electrode material development, as well as enzyme immobilization and bacterial biofilm preparation strategies. Finally, in the last sections the phenomena that occur in the transfer of electrons at the enzymatic and bacterial level are described, and two cases of application of bioelectrocatalysis are presented.
1.2 Reactions of Interest in Bioelectrocatalysis
1.2.1 Enzyme Catalyzed Reactions
Oxidoreductases (EC group 1†) are enzymes that are capable of catalyzing reactions in which electron transfer is involved and have been used as fuel cell components since the early 1960s. In 1962, Davis and Yarborough reported an increase in the potential of a cell in which glucose oxidase was present in one of the electrode compartments (the other being a Pt/O2 one) [3]. Two years later, Yahiro et al. reported the first polarization curves using glucose oxidase (GOx), D-amino acid oxidase and yeast alcohol dehydrogenase in bioanodes that they coupled with Pt cathodes for O2 reduction [4]. Since then, most of the enzymatic biofuel research has been centered in the oxidation of glucose and the reduction of oxygen. This has been driven by the abundance of both substances in our biosphere; while oxygen is abundant in the atmosphere, glucose is used as a source of energy by almost all living beings. Because of their predominance in the literature, this section will focus on the mechanistic description of enzymatic oxidation of glucose and reduction of oxygen by glucose oxidase and laccase, respectively. Other enzymes, like glucose dehydrogenase [5, 6] and bilirubin oxidase [5] can perform similar reactions through different mechanisms and can be useful in some situations. Furthermore, a variety of other fuels (e.g. carbohydrates [7, 8], alcohols [9, 10], lipids [11] and organic acids [12] and oxidants (mainly H2O2 [13]) have been employed in enzymatic biofuel cells. However, it is out of the scope of this work to review them all in detail. Rather, it is expected that the information presented here will allow the readers to perform similar literature search for their particular enzyme of interest.
Glucose can be oxidized by a variety of enzymes including glucose oxidase and glucose dehydrogenase. Of these, glucose oxidase has been the one massively preferred, due to its high specificity and good turnover and stability [14, 15]. Glucose oxidase (EC 1.1.3.4) is a dimeric flavoprotein that oxidizes β-D-glucose into D-glucono-δ-lactone while reducing molecular dioxygen (from here on simply referred to as oxygen) to hydrogen peroxide. In each subunit, an active site is deeply buried in a funnel-shaped pocket that contains a non-covalently bound flavin adenine dinucleotide (FAD) cofactor (Figure 1.1a). The N5 atom of this molecule is situated 13–18 Å from the surface and acts as the first electron acceptor in a so called “ping-pong” mechanism [16]. This first half reaction (enzyme reduction) takes place through simultaneous donation of a proton and a hydride from the glucose to the His516 residue and FAD, respectively. Although literature frequently states that the product of this half-reaction is FADH2, there is evidence of the resulting negative charge in the flavin moiety. Therefore, the reduced state of the cofactor is better described as the anionic form FADH− [17]. The second half-reaction is the reoxidation of FADH− to FAD, reducing an oxygen molecule to peroxide. This last process takes place in two one-electron steps that produces two intermediates, a semiquinone radical for the FAD flavine moiety and a superoxide anion radical for the O2 molecule (Figure 1.1b). Although not directly participating in the electron transfer, it is believed that His559 and Glu412 help with the pH control in the active site.
Glucose oxidase is produced by a variety of animals, plants, bacteria, algae and fungi. However, only GOx extracted from this last kingdom (mainly from Aspergillus and Penicillium genera) have gained industrial application, partly because they fall under the “generally recognized as safe” category of the U.S. Food and Drug Administration [14]. In academic fuel cell research, GOx produced by Aspergillus niger is highly preferred mainly due to its commercial availability. A few studies have been reported using GOx from Penicillium funiculosum 46.1 but the enzyme extraction and purification from the cell culture needs to be performed [13].
Although efficient, it is thought that wild-type glucose oxidase is not at its catalytic maximum, and therefore directed evolution experiments have been performed to find better versions of the enzyme. In a recent study, Petrović et al. found several GOx mutants that presented a higher rate for glucose oxidation reaction, as well as a smaller Michaelis-Menten constant (KM). Molecular dynamics simulations and X-ray crystallographic information revealed that a key mutation was the exchange of Met556 for a valine residue modifying the shape of the active site. In wild-type GOx, the His516 residue can have two conformations, a catalytic and an inactive one, in which its imidazole side chain flips into a cavity near the active site (Figure 1.2a). When Met556 was substituted for a valine, the cavity became smaller, effectively locking the His516 into the catalytic position [15]. This is reflected in the relative values of the calculated free energies (ΔG) for the catalytic and non-catalytic states. While in wild-type GOx both states have similar ΔG, the mutated enzyme clearly shows thermodynamic and kinetic preference for the catalytic state (Figure 1.2c). This example shows that deeper understanding of the mechanistic subtleties can have convenient implications in the industrial applications that employ GOx, including fuel cells.
