Covalent bonding

To prevent enzyme leaching from the electrode, covalent binding has also been envisioned. An electrode based on BOD covalently bound at PQQ-modified MW-CNTs had a half-life of about 20 days compared to 5 days for a MW-CNT electrode with only adsorbed BOD.152 For Th-laccase covalently bound at PG bearing aminophenol moieties,  75% of the

initial current was retained after 10 days of continuous chronoamperometry at 0.2 V vs. Ag/AgCl, with pure O2 bubbled through the electrolyte solution.259 Stability of the covalently

bound Th-laccase at 6-mercapto-1-hexanol modified gold electrode was evaluated by continuous operation at Eapplied = 200 mV vs. Ag/AgCl, at ω = 500 rpm and under O2. 70% of

the initial current was left after 24h. Unfortunately, control experiments were not provided.249

When the same enzyme was covalently bound on AuNPs on a LDG electrode, 40% of the catalytic current was maintained after 4 days of continuous operation in chronoamperometry.393 When AuNPs were replaced by gold nanorods, the electrodes were

stable during a day and kept their stability during few days of storage at 4 °C. The stability was not tested longer in continuous operation.394

4.1.5.3. Encapsulation

It has been reported that the lifetime of an enzyme in solution could be extended to ~ one year29 when enzymes were included in micellar polymers. Similarly, at electrodes, MCOs

130 have been embedded in sol-gel matrices, sometimes with conductive nanomaterials.364,458,463

A composite film formed of tetramethyl orthosilicate sol-gel (TMOS), CNPs and laccase was deposited on an electrode and tested by chronoamperometry at 0.2 V vs. Ag/AgCl. The dioxygen reduction current decreased by ca. 60% in the first hour, but only 17% were lost over the next 30 h.364 _{Mv-BOD was adsorbed on SW-CNTs modified with pyrene sulfonic}

acid in a TMOS sol-gel. The current density, measured in air-saturated buffer at 0.2 V vs. Ag/AgCl, dropped from 20 µA.cm−2 to 16 µA.cm−2 in the first 12 hours, and was then

stable for another 60 hours.463 _{This represents an interesting stability, however the influence}

of the different elements was not tested separately. An increased stability at medium temperatures (37°C) was also recorded when Mv-BOD was embedded with CNTs in a TMOS matrix, and attributed to the stabilizing environment of the sol-gel.487

Other matrices have also been proposed to stabilize the enzyme. Mv-BOD was for example immobilized in a network of naphtylated MW-CNTs and AuNPs in a lipidic cubic phase (monoolein). The stability was evaluated by realizing daily cyclic voltammograms. 70% of the initial current density was left after 9 days, whereas only 10% were retained in the absence of the lipidic cubic phase (Fig 32).424 Mv-BOD in solution was stabilized by

131 Figure 32. Long-term stability of Mv-BOD adsorbed on a composite of naphthalene- MWCNTs and AuNPs on a GC electrode (empty circles) and Mv-BOD + AuNPs embedded in the lipidic cubic phase on naphthalene-MWCNTs on a GC electrode (full circles). The activity of the electrode in time is shown as the limiting catalytic current measured at 0.3 V in oxygen-saturated phosphate buffer (pH 7). Reproduced with permission from [424]. Copyright 2017. Wiley-VCH Verlag GmbH & Co. KGaA.

This remained true for the immobilized enzyme, but so far it has indisputably been shown only in MET.488 _{A biocathode based on Tv-laccase adsorbed at SW-CNTs was coated}

with a hydrophobic room-temperature ionic liquid.489 This protected the laccase in a buffer

neutral pH and in the presence Cl-, probably by forming a weakly acidic layer at the electrode

surface. In cyclic voltammetry, the signal was stable over 50 cycles.489 Efficient O2 reduction

by Mv-BOD adsorbed on AuNPs (15 nm) has also been reported in non-polar ionic liquids with small quantity of water, however a stabilization effect has not been described.490

Nafion® is often used as a binder or as a covering membrane for laccase electrodes. To the best of our knowledge, indisputable evidence of its stabilization effect has however not been reported so far. For example, a laccase physically adsorbed on carbon coal or carbon black and covered by Nafion®366 was more stable than in solution, retaining 55% activity

after one month of storage in buffer. However, the relative influence of Nafion® and nanoparticles has not been deciphered. A Trametes laccase was embedded with anthracene- MW-CNTs in different polymer matrixes to compare their influence on stability. Three matrixes were used: Nafion® modified with tetrabutylammonium ions (TBAB Nafion®), hydrophobically modified linear polyethyleneimine presenting surfactant like properties (C8 LPEI), and vapor deposited TMOS sol-gel.486 _{We give here the results obtained in}

potentiostatic mode, since it has been proved to be harsher than galvanostatic mode.486 The 3

different electrodes, TBAB Nafion®, C8 LPEI and TMOS, respectively lost 4.1 ± 0.4%, 9.6 ± 0.5% and 10 ± 2% of the initial current densities after 24 hours at Eapplied  0.44 V vs.

132 Ag/AgCl at pH 4.5 and 25 °C in atmospheric conditions. According to this study, Nafion®- TBAB was therefore the most suitable matrix for laccase entrapment.486 Elsewhere, authors

from the same group demonstrated that Nafion® modified with ammonium cations provided a suitable matrix for immobilization of enzymes,491 _{which could also explain the results}

obtained here.

Stabilization was also noticed in some cases when the enzyme was confined in a 3D network and closely surrounded by nanomaterials. A CNTs-enzyme composite induced by liquid shrinkage allowed a laccase to be closely surrounded by CNTs. The so-formed cathode was used in a fructose/O2 EBFC, which retained 80% of its initial power density after 24 h of

continuous operation at an external load of 35.3 kΩ.341 _{A one-year stability of an EBFC was}

also reported with enzymatic electrodes made of compressed enzyme-CNTs pellets. After one year of regular discharges, 22% of the initial power was still recorded.318 (see section 4.1.2.1)

4.1.5.4 Other strategies

Other strategies have also been proposed to extend the lifetime of a bioelectrochemical device, without necessarily stabilizing the enzyme at the electrode. The lifetime of an enzymatic cathode based on Tv-laccase and bucky paper was extended 2.5 fold by resupplying fresh enzyme in the electrolyte.492 Untreated culture supernatants containing

laccase were also used instead of purified enzymes. Here also fresh supernatant was periodically introduced in the catholyte. The potential was stable during 120 days under continuous application of 50 µA.cm-2 (i.e.  40% of the limiting current) while the half-life of

the electrode was  10 days when no fresh enzymes were supplied.493