→ O2 + 4H+ + 4e-
Microbial electrolysis cells (MECs) operate under a constant electrical supply, therefore this energy represents an operation cost and a decrease in the energy efficiency. To overcome these issues, the use of alternative energy technologies has been proposed [34]. Another alternative to reduce the cost of energy supply is an intermittent operation for conversion of CO2 into organic products using a biocathode [35].
The thermodynamic cell voltage for hydrogen production from acetate oxidation in a bioanode, and methane production in a biocathode from water oxidation is resumed in Table 1.1. At least theoretically, these reactions present advantages compared to totally abiotic processes for gaseous fuel production. For instance, water electrolyzers require 1.23 V for hydrogen formation while the bioelectrolysis of water requires 0.134 V.
1.3 Immobilization of Biocatalyst
1.3.1 Immobilization of Enzymes on Electrodes
Although the first proof-of-concept biofuel cells employed the enzymes freely in solution [3, 4] this approach is poorly applicable in practice. Enzymes are costly and losing them with the fuel and oxidant flow turns operation expensive. Therefore, most of the reported enzymatic biofuels include enzymes immobilized on the electrode surface.
A number of strategies have been developed to immobilize enzymes on solid supports and a significant number of reviews have been published explaining the advantages and disadvantages of each approach, often proposing a classification of the methods based on criteria that is not standardized [36–41]. As well, these reviews often focus on the immobilization of enzymes on supports that, while solid, are generally a mobile part of a bioreactor (the so-called carriers). This section, instead, focuses on the description of the different strategies in the context of the immobilization on an electrode surface, giving representative examples of their use in fuel cell research.
In general, enzyme immobilization is a balancing act between the external forces holding the enzyme on the support and the internal forces that maintain the enzyme conformation, and therefore, its function. The addition of interactions can stabilize the enzyme but, if they are too strong, they can modify the conformation of the active site or even denature the enzyme. As well, the addition of dense composite materials around the enzymes can create mass transport limitations that need to be kept in mind; else the catalytic performance can be severely affected.
Immobilized enzymes are usually evaluated measuring the current produced with different concentrations of substrate. In solution, the relationship between the enzymatic reaction rate (V) and the substrate concentration ([S]) is given by the Michaelis–Menten equation (Equation (1.1)).
where Vmax is the maximum rate at a given enzyme concentration and KM is the Michaelis–Menten constant that represents mainly the enzyme’s affinity for the substrate. The enzymatic rate can be measured by a number of techniques, spectrophotometric ones being particularly popular. Once the oxidoreductase is immobilized on the electrode, part of the exchanged electrons in the enzymatic reaction end up / come from the electrode. Therefore, the measured current does depend as well from the substrate concentration. An analogous equation has been derived which employs the apparent Michaelis–Menten constant
In this case, the
According to the forces involved, immobilization strategies can be classified as either physical or chemical. The first group includes adsorption, polymer entrapment and electrostatic binding. In adsorption, enzymes are weakly bound to the electrode surface via mainly Van der Waals forces, hydrophobic interactions and hydrogen bonds (Figure 1.4a) [40]. The main advantage of this method is the simple procedure required. Typically, the electrode is incubated in a solution of the enzyme, after which it is rinsed to remove the unbound enzymes. Laccase [42] and fructose dehydrogenase [7] have been shown to present direct electron transfer (see Section 1.5.1) when adsorbed on carbon electrodes. The main drawback of adsorption immobilization is the lability of the enzymes. Since no strong interaction is present between the enzyme and the electrode, enzyme leaching is a common limitation of the electrodes prepared in this manner, which can be aggravated if the conditions of the environment change [40].
In polymer entrapment (Figure 1.4b), enzymes are physically trapped between the network of the polymer chains. Although some interactions between the enzymes and the polymer matrix might exist, they are not the main cause for enzymes to be fixed in place. Electropolymerization [43] and photopolymerization [44] in the presence of enzyme have been used to trap glucose oxidase in the polymer layer. An interesting approach has been the use of tetrabutylammonium bromide (TBAB)-modified Nafion, to immobilize alcohol dehydrogenase, aldehyde dehydrogenase and even nanotube-bound laccase [7, 9]. The exchange of the protons in the Nafion for hydrophobic alkyl ammonium ions reduces the acidity of the polymer environment and widens the channels to allow the diffusion of relatively large enzymes substrates and cofactors [45].
Figure 1.4 Schematic of popular enzyme immobilization techniques. (a) Adsorption (b) Polymer entrapment (c) Electrostatic entrapment (d) Covalent bonding and (e) Cross-linking.
Electrostatic entrapment (Figure 1.4c) makes use of the charge in the amino acid residues of the redox enzyme. Depending on the relative values of the protein isoelectric point and environmental pH, enzymes can present a net positive or negative charge. Accordingly, they can be favorably attracted to ionic polymers bearing the opposite charge. Cationic polymers, such as poly(ethyleneimine)s (PEIs) [46], poly(allylamine) [47, 48] and chitosan [42, 49], have therefore been used to immobilize negatively charged enzymes through an anion exchange process. Conveniently, the two most commonly used enzymes, GOx and laccase, are negatively charged at their operating pH (usually 7–7.5 and 4.5–5.5, respectively [7, 50, 51]). This approach has been taken further to form a structure of alternating layers of enzyme and cationic polymer, generally known as layer-by-layer (LbL) deposition. Using this strategy on both anodic and cathodic graphite electrodes, Rengaraj et al. achieved maximum power densities of 103 μW cm−2 [50]. Although stronger than simple adsorption, the forces involved in electrostatic entrapment are relatively weak. Therefore, enzyme loss is still a problem that prevents this strategy to be reliable at making durable electrodes.
Chemical