derivatives [63, 64]. Direct comparisons between Fc-C3-LPEI and FcMe2-C3-LPEI [51], and between FcMe2-C3-LPEI and FcMe4-C3-LPEI [63], showed an 85–90 mV decrease in the peak potentials for each pair of methyl groups added.
Mediator-less charge transport between the enzyme and the electrode surface is an attractive idea. These systems would allow the use of the whole potential difference between the cofactors on each enzyme. As well, the absence of the mediator means there is one less component that can get lost or damaged during operation. In fact, nowadays stability problems are associated more to the mediator than to the enzyme itself [94]. Direct electron transfer (DET) is, however, quite more challenging than MET in general. Let us consider the case of glucose oxidase. As mentioned earlier, it is difficult to achieve DET between GOx and the electrode because of its deeply buried active site. Attempts to covalently immobilize the FAD cofactor on the flat electrode surface and then add the apoenzyme to reconstitute the holoenzyme were futile [149]. This is most likely due to the presence of the surface that sterically hinders the proper fitting of the cofactor therefore altering the conformation of the enzyme.
A number of reports detail the preparation of electrodes which incorporate carbon nanotubes and GOx. Some of them claim that direct electron transfer takes place on the basis of the observation of the oxidation and reduction peaks of the cofactor (FAD) through cyclic voltammetry [150]. Upon addition of oxygen to the solution, an increase of the cathodic current is interpreted as proof of biocatalysis by the FAD in GOx. Furthermore, addition of glucose results in a decrease of the reductive current, which is taken as evidence of the retention of the activity of the immobilized GOx. It has been pointed out by Milton and Minteer, however, that these responses cannot be unequivocally ascribed to GOx DET [5]. A mixture of dissociated cofactor at a proper distance to transfer electrons to the electrode, and remaining active enzyme not in the right distance/conformation for DET would result in the same response. They suggest that, in order to ascertain the presence of DET, a few strategies can be employed, including the analysis of the reaction product and the evaluation of the reaction using denatured, inhibited or mutated enzymes.
Enzymes with more exposed active sites are more suitable for their use in DET. Such is the case, for example, of laccase (see Section 1.2.1). In order to have efficient DET, however, the active site must be within tunneling distance of the electrode [52]. This means that it is not enough to have an active site close to the enzyme surface. Also, the orientation of the enzymes must be such that the active site is pointing towards the electrode. Of course, most enzyme immobilization methods do not control the enzyme orientation; instead, it is random and only a few molecules are in the proper orientation. However, it is possible to use the enzyme tertiary structure itself to direct the immobilization. Certain molecules resembling the substrate (typically polyaromatic compounds) can be immobilized in the electrode surface. When laccase approaches this surface during enzyme immobilization, the substrate-binding pocket where the T1 Cu is located tends to interact (“dock”) with these groups and acquire a proper orientation for DET. These molecules have been termed DET promoters and have also been shown to work with other metalloenzymes like bilirubin oxidase [5].
Not many direct comparisons have been performed between enzymatic fuel cells in which MET or DET take place. Ishida et al. compared the performance of bioanodes containing glucose dehydrogenase, either immobilized with CNT for DET or with ferricyanide for MET [151]. Figures 1.11 c and d show that the onset for oxidation in DET is, as expected occurs at less negative potentials compared to MET (see Figure 1.10). The difference is, however, only of about 100 mV, small value compared to the difference in redox potentials between the ferricyanide and the FAD cofactor of the GDH (~300 mV). The authors attribute the high potential required for MET to the overpotential for FAD oxidation produced by the distance between the CNT and the enzyme active site. Polarization curves of complete cells sharing the same cathode (Figures 1.11e,f) are consistent with the cyclic voltammograms. A slightly higher OCV is obtained for the DET case, at the expense of current and, therefore, power. This example shows that each electron transfer mode has its advantages and disadvantages, and the most suitable strategy must be chosen in a case-by-case basis.
Figure 1.11 Comparison of direct and mediated electron transfer in electrodes containing glucose dehydrogenase. DET is achieved through the addition of carbon nanotubes while ferricyanide is used for MET. (a–d) Cyclic voltammetry of GDH anodes along with the respective negative controls. Black and red curves are in the absence and presence of glucose, respectively. (e–f) Polarization curves of fuel cells produced with the DET (e) and MET (f) anodes. Cathodes used Pt as catalyst. Reprinted with permission from (Ishida, K., Orihara, K., Muguruma, H., Iwasa, H., et al., Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenin Dinucleotide Glucose Dehydrogenase. Anal. Sci., 34, 783-787, 2018.). Copyright (2018) Japan Society for Analytical Chemistry.
1.5.2 Microorganism-Electrode Electron Transfer
Electron transfer between microbial cells and solid materials has always occurred in the nature. This phenomenon was first investigated by the Lovley’s research group in microorganism-metal interactions, particularly species with Fe(III) reducing capability [152]. Electron transfer takes place in the cytoplasm of the cell for regular metabolic pathways. However, the phenomenon at the bacteria–electrode interface was differentiated and it named extracellular electron transfer (EET). EET is defined as the process in which the electrons derived from the oxidation of substrates, are transferred to the outer surface of the cell to reduce an extracellular terminal electron acceptor [153].
Similar to the classification of electron transfer phenomena already mentioned for enzymes, microbial EET has been grouped into direct (DET) and mediated transfer (MET) and is achieved by different structural molecules or via metabolites expelled by the cell. Table 1.3 shows a classification of the EET mechanisms recognized for electroactive bacteria.
DET involves a physical contact between components of the cell membrane and the electrode; in this case the contact is maintained by an exo-polymeric matrix surrounding the cells. In the cases when the biofilm is not formed, EET is lower in comparison to bacteria in biofilm.
Outer membrane cytochrome complexes are present in different forms and depend on the electroactive species. In Geobacter various multihaem cytochromes, and multi-copper proteins have been identified. In Shewanella, redox reaction cascades from six multi-haem cytochrome complexes, mediate the EET (Table 1.4).
Another DET mechanism is performed via pilus-like structures commonly named nanowires. These structures are observed in G. sulfurreducens and S. oneidensis. The appendages favor transfer of electrons through longer distances and even at a centimeter scale. EET between species has been described for anaerobic granules but this mechanism is also associated to interspecies EET in electroactive biofilms [160]. The proteins that participate in the electron transfer via pili-like structures are still being investigated; their identification presents difficulties because the bacteria produce a variety of filaments and not all of them seem to be conductive. The conductivity in the pili is attributed to the truncated PilA monomer; packed aromatic aminoacids form a path for conduction of electrons along the pili [161]. However, this mechanism seems not be predominant because only 80% of 95 species that have Fe(III) reducing capability lack electron-conducting