(a) Structure of a subunit of glucose oxidase from Aspergillus niger elucidated by Hecht et al. [18] (PDB code 1GAL), showing the FAD cofactor buried in the protein. (b) Catalytic reaction of glucose oxidase. The semiquinone radical FAD intermediate is not shown for clarity.
Laccase (EC 1.10.3.2) is a multicopper “blue” oxidase that has a variety of organic and inorganic compounds as its natural substrates, including phenols, phosphates, acids, ketones and amines. In all cases, O2 acts as the final electron acceptor. It is formed by a single polypeptide chain that folds into three different beta barrel domains. Its active site contains four copper atoms, classified as T1, T2, T3α and T3β, according to their spectroscopic and paramagnetic properties. T1 Cu, for example, presents an intense blue color because of its coordination with nitrogen and sulfur atoms from the neighboring histidine and cysteine residues. On the other hand, T2 Cu presents similar absorption to aquo or hydroxo Cu complexes [19]. In the enzyme oxidized state, all the Cu atoms have an oxidation state of +2.
Figure 1.2 Molecular dynamics studies of the structure of the active site of wild-type (a) and mutant (b) glucose oxidase. The meshed area in the right-hand side is a pocket in which His516 can flip into. In the mutant form, the pocket is reduced in size and divided in two parts, effectively locking His516 out. (c) Comparison of the calculated free energy for the catalytic and non-catalytic states in wild-type (curve 1) and mutant (curve 2) GOx. Republished with permission, from ACS Catal., Dušan Petrović et al., 7, 2017, 6188–6197 available online at https://pubs.acs.org/doi/10.1021/acscatal.7b01575; further permissions related to the material excerpted should be directed to the ACS.
The T1 Cu is located in a substrate binding pocket close to the enzyme surface and is the initial electron acceptor in a one-electron oxidation of the substrates (Figure 1.3a). It has been proposed that the His458 and Asp206 residues can form hydrogen bonds with some of the substrates, helping to maintain them in the adequate conformation for electron transfer. Furthermore, computer simulations have shown that the H atom at ε-N in His458 is less than 5 Å away from the OH in phenolic substrates making it likely to participate in the electron transfer [20]. Once the T1 Cu2+ has been reduced to Cu1+, it transfers electrons one by one to the cluster formed by the other three Cu atoms (Figure 1.3b). This tri-nuclear cluster (TNC) is buried deeper inside the enzyme at the interface between two domains. In this cluster, oxygen is reduced to two molecules of water in a four-electron process that takes place as two sequential steps. First, the water is reduced in a two-electron process to a peroxide-level intermediate by the two T3 Cu1+ ions. Then, electrons from the T1 and T2 Cu1+ ions further reduce this intermediate to water (Figure 1.3c) [21, 22].
Different sources for laccase include insects, bacteria, fungi and plants like the Japanese lacquer tree (Toxicodendron vernicifluum), where it was first extracted from [24] and which gives the enzyme its name. Laccase varieties extracted from fungi present the highest redox potentials [21, 22], and are thus preferred for biocathode development. Laccase isolated from different species presents some variations in their amino acid sequence [20]. Laccase from Trametes versicolor is particularly popular in electrochemistry research and thus, the numbering of the residues in the previous paragraph is referred to the numbering in this species.
Figure 1.3 (a) Structure of laccase from Trametes versicolor elucidated by Bertrand et al. [23] (PDB code 1KYA), showing the surface T1 Cu and the buried trinuclear cluster (TNC). Mechanism of substrate oxidation (b) and oxygen reduction (c) by laccase.
1.2.2 Reactions Catalyzed by Microorganisms
Conventional MFCs utilize abiotic cathodes; however, limitations for the oxygen reduction reaction are present similarly to what is observed in electrochemical cells. The use of biocathodes as an alternative to metallic cathodes was proposed in 2005 [25].
The biocathodes are classified as a function of the terminal electron acceptor available. Aerobic biocathodes utilize oxygen as final acceptor; of electrons electron transfer occurs via the reduction of a mediator such as iron and manganese and then, the mediator is oxidized by the bacteria. Anaerobic biocathodes directly reduce acceptors such as nitrate and sulfate [26].
Suitable bacteria for indirect electron transfer are Leptothix discophora, which is able to reduce MnO2 to manganese ion [27], and Acidithiobacillus ferrooxidans an iron oxidizing bacterium [28], while consortia of electroactive microorganisms for direct transfer can be found in sediment and anaerobic sludge [29, 30]. An exhaustive evaluation of the electroactivity capabilities of diverse bacterial species was reported by Cournet et al. [31].
Hybrid microbial bioelectrochemical systems include MFC fueled by light energy; this is achieved by oxygenic photosynthetic organisms, such as microalgal and cyanobacteria species. Photosynthetic organisms transfer electrons to the anode, or to heterotrophic microorganisms which in turn transfer the charge to the anode.
Energy pathways in cyanobacteria occur in the thylakoid membranes containing respiratory electron transfer chain components and in the cytoplasmic membrane with a respiratory electron transfer short chain. Since the electron transfer is not adapted for extracellular electron transport, mutant strains for electron export ability are being obtained [32]. Synechocystis have three respiratory terminal oxidase complexes for the reduction of oxygen and mutants lacking respiratory terminal oxidases showed increased ferricyanide reduction rate [32]. However, the mechanism for electron excretion to the periplasmatic space and beyond remains unresolved; hypothesis on the presence of nanowires, an assimilatory metal reduction pathway, and endogenous mediators have been stated.
One additional advantage of MFCs over general bioreactors is the possibility to adapt the microbial metabolism of the inoculum as function of the set electrode potential. This procedure enables one to increase the selectivity of the reactions and the galvanic mode of operation can become electrolytic [33].
Table 1.1 Cell potential for typical reactions with microbial bioanode, and a microbial biocathode.
| Microbial bioanode | Cathode | Cell potential |
| CH3COOH + 2H2O → CO2 + 8H+ + 8e− | 2H+ + 2e− → H2 | E = 0.134 V |
| E = −0.280/NHE | E = −0.414 V/NHE | |
| Anode | Microbial biocathode | |