Nicolas Mano
email: mano@crpp-bordeaux.cnrs.fr

Anne De Poulpiquet

O2 Reduction in Enzymatic Biofuel cells

Keywords: ABTS, 2,2'-azinobis(3-ethylbenzothiazolin-6-sulfonate) AFM, Atomic force microscopy AMT, 4-aminothiophenol AR, Alternative resting AuNPs, Gold nanoparticles BDD, Boron doped diamond BET, Specific surface area determined Carbon nanoparticles CNTs, Carbon nanotubes Laccase from Cerrena unicolor Cyclic voltammetry CVD, Chemical vapor deposition CMF, Carbon microfiber Density functional theory Dual polarization interferometry EBFCs, Enzymatic biofuel cells 2008

Catalytic four-electron reduction of O2 to water is one of the most extensively studied electrochemical reactions due to O2 exceptional availability and high O2/H2O redox potential, which may in particular allow highly energetic reactions in fuel cells. To circumvent the use of expensive and inEfficient Pt catalysts, multicopper oxidases (MCOs) have been envisioned because they provide Efficient O2 reduction with almost no overpotential. MCOs have been used to elaborate enzymatic biofuel cells (EBFCs), a sub-class of fuel cells in which enzymes replace the conventional catalysts. A glucose/O2 EBFC, with a glucose oxidizing anode and a O2 reducing MCO cathode, could become the in vivo source of electricity that would power sometimes in the future integrated medical devices. This review covers the challenges and advances in the electrochemistry of MCOs and their use in EBFCs with a particular emphasis in the last 6 years. First basic features of MCOs and EBFCs are presented. Clues provided by electrochemistry to understand these enzymes, and how they behave once connected at electrodes, are described. Progresses realized in the development of Efficient biocathodes for O2 reduction relying both on direct and mediated electron transfer mechanism are then discussed. Some implementations in EBFCs are finally presented.

Introduction

About 2.5 billion years ago, oxygen became an important component of the earth atmosphere [START_REF] Farquhar | Atmospheric Influence of Earth's Earliest Sulfur Cycle[END_REF] due to the emergence of cyanobacteria, organisms that produce oxygen during photosynthesis. One of the consequences of this event is the biological utilization of copper, a transition metal now omnipresent in biology, while only iron used to be predominant in the first living forms. In the oceans, oxygenation led to a shift from high-iron to high-copper contents, because oxygen favors the oxidation state +III of insoluble Fe 3+ , while Cu is preferentially in the Cu 2+ form which is soluble and therefore bioavailable. [START_REF] Festa | Copper: an Essential Metal in biology[END_REF] In living organisms, copper is still linked with oxygen metabolism : a recent study examining copper utilization by 450 bacteria demonstrated that around 80% of anaerobes did not depend on copper, while more than 90% of aerobes were copper dependent. [START_REF] Ridge | Comparative Genomic Analyses of Copper Transporters and Cuproproteomes Reveal Evolutionary Dynamics of Copper Utilization and Its Link to Oxygen[END_REF] Copper can be found in numerous metallo-proteins, in which the metal is coordinated by amino-acids. Copper-containing proteins are present in bacterial, fungal, plant and mammalian organisms. [START_REF] Muttkowski | Copper: Its Occurrence and Role in Insects and Other Animals[END_REF] In some of the proteins, copper is frozen in the reduced or oxidized state. Of particular importance in the frame of electrochemistry are the proteins in which copper centers are able to undergo the redox Cu 2+ /Cu + reaction. These metallo-proteins contain either one or several copper centers, and the latter are either mononuclear (type T1, T2 or CuB sites) or binuclear (type T3 or CuA sites). [START_REF] Solomon | Copper Active Sites in Biology[END_REF] Copper ligands and protein environment modulate the potentials of the +II/+I transition that varies accordingly from 85 mV to >1000 mV, [START_REF] Liu | Metalloproteins Containing Cytochrome, Iron-Sulfur, or Copper Redox Centers[END_REF] while in water the apparent standard potential E°' of the Cu 2+ /Cu + couple is 150 mV vs. the normal hydrogen reference electrode (NHE). [START_REF] Solomon | Electronic Structures of Metal Sites in Proteins and Models: Contributions to Function in Blue Copper Proteins[END_REF] Copper-containing proteins cover different functions ranging from electron transfer to catalysis, and are involved in a wide variety of mechanisms among which the most important are energy conversions in respiration or metabolism. It must be noted however that copper is also toxic at high concentrations so that many proteins are linked to copper regulation and homeostasis. [START_REF] Festa | Copper: an Essential Metal in biology[END_REF] Copper based enzymes are associated with the metabolism of N2O (nitrous oxide reductase), NO2 -(nitrite reductases), O2 .-(superoxide dismutase), or O2. [START_REF] Solomon | Copper Active Sites in Biology[END_REF] More relevant are the two classes of enzymes involved in oxygen reduction: terminal oxidases, and multicopper oxidases (MCOs). Terminal oxidases can be divided in cytochrome or quinol oxidases, and contain both copper centers and hemes. Oxygen reduction occurs with cytochrome or reduced quinone as a primary electron donor. The reaction is coupled to proton transfer and the enzyme-catalyzed reactions create the proton gradient necessary for ATP synthesis. [START_REF] Yoshikawa | Reaction Mechanism of Cytochrome c Oxidase[END_REF] MCOs catalyze the 4-e -oxygen reduction coupled to oxidation of various organic or metallic substrates. [START_REF] Solomon | Copper Active Sites in Biology[END_REF] In MCOs, inner sphere electron transfers are involved in binding, activation and reduction of O2.

Among the desperately sought, but rarely well-engineered devices envisioned for sustainable energy conversion, fuel cells could harvest energy from fuels like dihydrogen, alcohols, etc. [START_REF] Debe | Electrocatalyst Approaches and Challenges for Automotive Fuel Cells[END_REF][START_REF] Tiwari | Recent Progress in the Development of Anode and Cathode Catalysts for Direct Methanol Fuel Cells[END_REF] Their advantages stem from direct conversion of chemical energy into electricity, and long-term operation enabled by continuous fueling. The reaction involved in fuel cells requires a combustive, and O2 is the most widely used because of its availability and high O2/H2O redox potential. [START_REF] Shleev | Oxygen Electroreduction versus Bioelectroreduction: Direct Electron Transfer Approach[END_REF] Designing catalysts for O2 reduction in conventional fuel cells (CFCs) is challenging. They generally require high overpotentials, [START_REF] Kjaergaard | Enzymatic versus Inorganic Oxygen Reduction Catalysts: Comparison of the Energy Levels in a Free-Energy Scheme[END_REF] or only catalyze the incomplete O2 reduction to H2O2. [START_REF] Zagal | A Mechanistic Study of O2 Reduction of Water Soluble Phtalocyanines Adsorbed on Graphite Electrodes[END_REF][START_REF] Shi | Catalytic Pathways for the Electroreduction of O2 by Iron Tetrakis(4-N-methylpyridyl)porphyrin or Iron Tetraphenlyporphyrin Adsorbed on Edge Plane Pyrolytic Graphite Electrodes[END_REF] Ideally, the platinum-based catalysts widely used in CFCs would be replaced by less expensive biological oxidation catalysts. Enzymatic biofuel cells (EBFCs) are a sub-class of fuel cells in which enzymes replace the conventional catalysts used in CFCs. They offer numerous advantages such as selectivity and specificity, allowing in particular operating without the separation membrane otherwise required to avoid cross-reactions at the electrodes. Moreover, they can operate in mild environments, and are also renewable and biodegradable.

The glucose/O2 biofuel cell, with a glucose oxidizing anode and a O2 reducing cathodes could become the in vivo source of electricity that would power sometimes in the future integrated medicals devices. [START_REF] Heller | Integrated Medical Feedback Systems for Drug Delivery[END_REF] In this context, the enzymatic MCO catalysts are especially interesting because they provide Efficient 4-e -O2 reduction [START_REF] Solomon | Copper Active Sites in Biology[END_REF] with almost no overpotential. [START_REF] Mano | A Laccase-Wiring Redox Hydrogel for Efficient Catalysis of O2 Electroreduction[END_REF] In the last decades, electrochemical research of redox proteins showed ways to connect MCOs at electrodes, and to use them as Efficient and specific electrocatalysts for O2 reduction, [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF][START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF] making some the enzymes of choice in EBFCs.

In this review we cover the challenges and advances in the electrochemistry of MCOs and their use in EBFCs since their first connection at an electrode 40 years ago, with a particular emphasis in the last 6 years. The interested reader will find in Table 1 additional reviews on this topic, with a particular focus on electrode materials for EBFCs. Here, we first briefly present basic features of MCOs and EBFCs, before focusing on the electrochemical approach of MCO study and applications. We describe what electrochemistry teaches us about these enzymes, and how they behave once connected at electrodes. We then discuss the progresses realized in the development of Efficient biocathodes for O2 reduction relying both on direct and mediated electron transfer mechanism (DET and MET respectively). Finally, we present some implementation in EBFCs.

Table 1: Reviews related to O2 reduction by MCOs and in Biofuel cells 2004 cuprous oxidase that oxidize metal substrates. The last category includes ascorbate oxidase (EC 1.10.32) or bilirubin oxidase (EC 1.3.3.5) which can be specific towards single substrate (i.e ascorbate or bilirubin) while also being able to oxidize other organic substrates. 100 A general trait of MCOs is the presence of at least four copper ions which permit oxidation of substrates while reducing O2 to water without production of any toxic intermediates. Each copper is classified according to its optical and magnetic properties. Type I Cu displays an intense absorption band near 600 nm resulting from a charge transfer transition from Cys to Cu (II) and is responsible for the name, blue copper oxidases, given to these enzymes (Fig 1A). It also exhibits a small Cu(II) A|| hyperfine coupling constant in EPR (Fig 1B).

Society of Chemistry

The exact nature of the electron transfer has been the subject of numerous investigations and is summarized in Figure 3. [START_REF] Solomon | Copper Active Sites in Biology[END_REF][START_REF] Jones | Electron Transfer and Reaction Mechanism of Laccases[END_REF][START_REF] Solomon | Electronic Structures of Metal Sites in Proteins and Models: Contributions to Function in Blue Copper Proteins[END_REF][START_REF] Wherland | Multicopper Oxidases: Intramolecular Electron Transfer and O2 Reduction[END_REF][START_REF] Komori | Structural Insights into the O2 Reduction Mechanism of Multicopper Oxidase[END_REF][102][103][104][105][106] Reproduced with permission from ref [START_REF] Solomon | O2 Reduction to H2O by the Multicopper Oxidases[END_REF]. Copyright 2012 Royal Society of Chemistry Briefly, after oxidation of the substrate at the T1, electrons are shuttled to the TNC where the two two-electron steps of O2 reduction to H2O occurs. [START_REF] Solomon | O2 Reduction to H2O by the Multicopper Oxidases[END_REF]107,108 The formation of the peroxide intermediate (PI) results from the first two-electron step with a fast reaction rate ( 10 6 M -1 .s -1 ). In the second two-electron step, the O-O bond is cleaved with a fast rate constant leading to the formation of the Native Intermediate (NI). In presence of substrate, NI is reduced to the fully reduced form of the enzyme and is ready for another catalytic cycle. In absence of substrate, NI slowly decays to the resting oxidized (RO) form and only reintegrates the catalytic cycle upon reduction with a substrate.

Laccases

Laccases (Fig 4A) are the most studied MCOs because of their use in different biotechnological applications; ranging from food applications, pulp bleaching, hair-coloring to organic synthesis. [START_REF] Mate | Laccase: A Multi-Purpose Biocatalyst At The Forefront Of Biotechnology[END_REF][109][110][111] More than 220 species have been identified in insects, fungi or bacteria. All Coppers are displayed in blue Among them, fungal laccases have been the most studied. [START_REF] Solomon | Copper Active Sites in Biology[END_REF][112][113][114] Laccases are known to oxidize a variety of polyphenolic compounds concomitant with the reduction of O2. Due to this latter property, Tarasevich et al. have pioneered the use of laccases for O2 reduction. In 1979, they reported the immobilization of monolayers of laccases on vitreous carbon electrodes, generating O2 reduction currents of 175 A.cm -2 at pH 5. 115 The geometry/coordination of the T1 copper deserves attention since this is the entry point of electrons coming from the electrode or from the redox mediator. It has been shown that its redox potential was modulated by the nature of its coordination. A T1 with a weakly coordinating Met in the axial position exhibits generally a lower redox potential than MCOs where Met has been replaced by non-coordinating Phe or Leu. 116 However, many exceptions exist and the type of coordination cannot explain alone the difference in the observed electrochemical potentials. Other factors such as electrostatic interactions and hydrogen bonds must be taken into account, making therefore difficult to predict the redox potential of MCOs. 117 We list here some of the most commonly studied laccases in bioelectrochemistry.

Fungal laccases have been identified in basidiomycetes. Among them we use the abbreviation Th-laccase to call without differentiation the two isoenzymes from Trametes hirsutus, also known as Coriolus hirsutus 118 , and Tv-laccase to call the laccase from Trametes versicolor, also known as Coriolus versicolor or Polyporous versicolor. Other laccases are extracted from trees, among which Rv-laccase the laccase from the Japanese lacquer tree Rhus vernicifera.

Bilirubin oxidases

BODs (Fig 4B) have been discovered in 1981 by Tanaka and Murao. 119 Because of their ability to oxidize bilirubin, they were classified as BODs and not laccases, even though BODs can also oxidize a variety of phenolic compounds. [START_REF] Mano | Features and Applications of Bilirubin Oxidases[END_REF] BODs have been primarily used in the medical field to elaborate bilirubin biosensors. [START_REF] Mano | Features and Applications of Bilirubin Oxidases[END_REF][START_REF] Mano | Bilirubin Oxidases in Bioelectrochemistry: Features and Recent Findings[END_REF] Only 7 BODs have been identified/characterized so far, among which BOD from Myrothecium verrucaria (Mv-BOD), the most studied BOD, BOD from Trachyderma tsunodae 120 (Tt-BOD), BOD from Magnaporthae orizae 121 (Mo-BOD) and BOD from Bacillus pumilus (Bp-BOD). 101 Unlike Tt-BOD or Mv-BOD, the two newly identified Mo-BOD and Bp-BOD display good thermal stability, a high activity at pH 7 and high temperature, a low sensitivity towards NaCl and no sensitivity to urate. Bilirubin is not a substrate that has been systematically tested with laccases or other MCOs. Therefore, it is not unlikely that MCOs already identified and characterized also display BOD-like activity. The use of Mv-BOD for the electrocatalytic O2 reduction at neutral pH has been reported for the first time in 2001 by Tsujimura et al. 122 Ever since, the number of studies relying on BODs has been flourishing for one main reason: unlike laccases, BODs display a high activity and stability at neutral pH and in presence of chloride. Two points deserve attention. The first concerns the denomination of the enzyme. Reiss et al. recently identified a CotA enzyme from Bacillus pumilus 123 which was classified as a laccase while Durand et al. classified an enzyme sharing 98% identity as a BOD. 101 BOD-like activity has also been observed in a CotA from Bacillus subtilis but the enzyme was classified as a laccase. 124 Reiss et al. have suggested that the denomination of BOD was improper since these enzymes could oxidize a variety of substrates. Therefore, they suggested to introduce the term of laccase-like MCOs (LMCOs). 125 On the other hand, not all laccases oxidize bilirubin. Therefore, the denomination of BOD would permit to differentiate between those MCOs that oxidize bilirubin or those that do not. The question of classification remains open. The second point deserving attention, which is linked to the first, is the core structural difference between laccases and BODs. Detailed analysis of amino acid sequences does not permit to differentiate BODs and laccases. 126 Combining different analytical tools such as molecular modeling or X-rays analysis would definitely help, but only two X-rays structures of BODs are available so far, for Mo-BOD 127 and Mv-BOD. 128,129

Cuprous oxidase and tyrosinase

The two enzymes have been rarely used in O2 biocathodes. Cuprous oxidase, or copper efflux oxidase (CueO) (Fig 4C) is involved in copper efflux in E.coli. 130,131 CueO possesses the same catalytic site as other MCOs but exhibits a strong activity towards cuprous ion and, unlike other MCOs, no activity towards phenolic compounds. Tyrosinase, or polyphenol oxidase (Fig 4D), only carries a couple of T3 copper atoms and is involved in the ortho-hydroxylation of monophenols and two-electron oxidation of diphenols. [START_REF] Solomon | Multicopper Oxidases and Oxygenases[END_REF] In the following, we will not consider cerulosplasmin and ascorbate oxidase (EC 1.10.3.3) which have not yet proven their utility in O2 reduction. [START_REF] Shleev | Oxygen Electroreduction versus Bioelectroreduction: Direct Electron Transfer Approach[END_REF] It has been shown that direct electron transfer (DET) between ceruloplasmin and an electrode surface was feasible under non turnover conditions, but in most cases electrocatalytic reduction of O2 was not achieved. [START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF][132][133][134][135] The only example of O2 reduction with ceruloplasmin on gold modified with ferromagnetic particles encapsulated in carbon was reported in 2015 by Matysiak et al. 136 However, the current density is too low to envision, so far, application in EBFCs. The DET of ascorbate oxidase has been reviewed by Shleev [START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF] and also investigated by Atanassov et al. 137

Basic feature of enzymatic biofuel cells

Detailed thermodynamic and kinetic analyses of enzymatic biofuel cells (EBFCs) have been provided in earlier reviews. [START_REF] Barton | Enzymatic Biofuel Cells for Implantable and Microscale Devices[END_REF][START_REF] Bullen | Biofuel Cells and Their Development[END_REF][START_REF] Davis | Biofuel Cells -Recent Advances and Applications[END_REF][START_REF] Luz | Enzyme Biofuel Cells: Thermodynamics, Kinetics and Challenges in Applicability[END_REF] Therefore, in the following, we only briefly summarize key points and advantages of EBFCs.

Despite the fact that electrochemical reactions are catalyzed by enzymes at one or both electrodes of the EBFCs, there is no conceptual difference between the latter and conventional fuel cells (CFCs). [START_REF] Debe | Electrocatalyst Approaches and Challenges for Automotive Fuel Cells[END_REF] The fuel is oxidized at the anode while the oxidant is reduced at the cathode, and electrons are exchanged between the two electrodes in an external electrical circuit. The driving force of the reactions is the difference in electrochemical potential between the two electrodes. The transport of charges in the electrolyte solution (or polymer) is ensured by ions. However, unlike CFCs, EBFCs operate at near-neutral pH and ambient temperature. Enzymes also have advantages as electrocatalysts: specificity towards their substrates, renewability, biodegradability, high turnover frequencies, and low intrinsic overvoltages. Moreover, the great diversity of enzymes allows envisioning EBFCs working with diverse fuels and oxidants. In addition, if the bioelectrodes are properly designed, no membrane is needed in EBFCs to separate anodic and cathodic compartments, allowing thereby miniaturization of the devices.

An EBFC is characterized by its current intensity, I, its voltage Ecell, and its power, defined as the product of these latter:

P [W] = Ecell [V] x I [A] Eq. 1
The electrical current intensity I is the charge flux entering the anode or leaving the cathode.

In lots of studies the current density, defined as the ratio of current I by surface area A and denoted j, is preferred. Similarly, the power is often expressed in terms of power density. This makes actually hard to compare the efficiency of different devices because in some (rare) cases the real electroactive surface area of the electrode is considered for the calculation, whereas it is more common to use the projected geometric area. This point deserves a particular attention. While the normalization of the current is critical for comparing the results of the numerous publications, normalization is difficult.

Other difficulties for comparison arise from the fact that experimental conditions (pH, temperature, scan rate, electrode rotation…) differ and in some studies, experimental details (enzymes activities and purities, loading…) are not provided. Natural convection which is often underestimated may also significantly affect current densities. 138 Comparisons are even more difficult when porous materials are used and particularly when detailed characterization of the materials are not provided (BET, thickness, porosity, mass,..). In addition, the different electrochemical techniques used to evaluate the power profile of EBFCs may also be problematic in some cases. For example, using linear sweep voltammetry at a quite fast scan rate (>5 mV.s -1 ) to evaluate the power profile of EBFCs based on highly porous electrode materials may induce errors. Since porous materials usually have a high capacitance, it is more than likely that the measured currents are composed by both catalytic and capacitive current, which artificially enhances the biofuel cell performances. In order to evaluate the actual power, the effective catalytic currents have to be determined using chronoamperometry (for individual bioelectrodes) and under continuous discharge (for the biofuel cell setup) at different imposed potentials, or using linear sweep voltammetry at very low scan rates so that the steady-state can be reached. For all the above considerations, it would undoubtedly be useful to develop a protocol for comparison of data, as was already suggested by Minteer and others, [139][140][141] but was not favorably received. The authors of this review believe that such normalization is much needed, after answering the question of the best way to normalize performances, taking into account the variety of electrode materials, EBFC designs and enzymes used.

While in CFCs currents can be measured in A, they are mostly in the range of few tenths of µA to few hundreds of µA (resp. few µA.cm -2 to few mA.cm -2 ) in EBFCs, or more generally at enzymatic electrodes. Similarly, power (resp. power densities) are in the range of kW to MW in CFCs whereas they are mostly between few µW and few mW in EBFCs. Some important features of enzymatic electrodes and EBFCs are described in Figure 5. (red) and power curve (blue) of the complete EBFC. Adapted from [27]. Copyright 2008 American Chemical Society.

The potential adopted by the electrode at j = 0 is the open circuit potential (OCP). It is close to the onset potential, the starting potential of catalysis. It must be underlined that the definition of "onset potential" is quite fuzzy since it is difficult to define the exact point at which catalysis actually "starts". The difference between the OCP and the apparent standard redox potential, the overvoltage (), characterizes the deviation to ideality of a catalyst and is a significant measure of its efficiency. Enzymes and MCOs in particular, are potentially powerful catalysts since for most of them electrochemical overvoltages are lower than for conventional catalysts. The difference between the OCP of the biocathode and the bioanode determines the maximal electrochemical driving force or open circuit voltage (OCV, also Ecell for j = 0) of the EBFC. The OCV of biofuel cells lies below the thermodynamic limit of 1.23 V for H2/O2 EBFCs, but sometimes very close to it, 142,143 and below the maximal OCV of 1.18 V for glucose/O2 EBFCs, where glucose is oxidized into gluconolactone at the anode.

EBFC performances can be characterized in terms of OCV, polarization curve and power curve. These two curves show respectively the evolution of the cell voltage and of the power density with the current density (Fig 5B). Particular data are the maximum power density Pmax, and the maximum current density jmax which corresponds very often to the shortcircuit current (i.e. current density for Ecell = 0). As seen in Figure 5B, the polarization curve can be divided into three regions. In Segment I, the steep decrease of the cell voltage corresponds to kinetic losses, which are related to the slow rate of the electrode reaction due to e.g. slow electron-transfer kinetics. After this first decrease of the cell voltage, the polarization curve continues decreasing more linearly with a smaller slope over a wide range of current density values. This decrease, which can be seen in Segment II, corresponds to the ohmic voltage drop originating from the system's intrinsic resistance as well as from the resistance to the flow of ions within the electrolyte. Finally, (concentration polarization) losses related to mass transport limitations result in the final decrease of the potential until it reaches zero (Segment III). At that point, the current reaches its maximal value and the system behaves as if the electrodes were in a short circuit. Each of the mentioned potential losses can be described with corresponding overpotentials (Eq. 2). This allows calculating the cell voltage as: 𝐸 𝑐𝑒𝑙𝑙 = 𝐸 𝑒𝑞 -𝜂 𝑎𝑐𝑡 -𝜂 𝑑𝑖𝑓𝑓 -𝐼 ∑ 𝑅 𝐸𝑞. 2

where: 𝐸 𝑐𝑒𝑙𝑙 and 𝐸 𝑒𝑞 are respectively measured and theoretical (thermodynamic) voltages, 𝜂 𝑎𝑐𝑡 and 𝜂 𝑑𝑖𝑓𝑓 are respectively kinetic (activation) and mass transport based overpotentials, 𝐼 is the current that flows through the cell and ∑ 𝑅 is the sum of all resistances that are present in the system. The challenge is to minimize all kinetic losses by optimizing each ingredient of the biofuel cell; the electrode material, enzymes, and redox mediators if needed; and by Efficiently integrating them together. It could be done for example by optimizing the cell design and the immobilization of the enzymes.

Electronic connection of immobilized enzymes

One of the major challenges of using an enzyme as an electrocatalyst is to establish an Efficient electronic connection between the enzyme and the electrode, particularly because of the important size of the protein molecule, and the anisotropy of its electronic properties. A poor electrical connection will induce consequent kinetic losses. Two mechanisms allow electron transfer between an enzyme and an electrode. On one hand, in direct electron transfer (DET), electrons tunnel directly between an enzyme and an electrode. This mechanism relies on the ability of the enzyme to accept electrons not only from its natural substrate but also from artificial electron donors like the electrode. On the other hand, in mediated electron transfer (MET), electrons are shuttled via a redox molecule, called mediator, which exchanges electrons with the enzyme and is reversibly oxidized or reduced at the electrode surface (Fig 6). DET can only occur if the enzyme active site or an electron relay is close enough to the electrode surface. The rate of interfacial electron transfer kET, described by Marcus semiclassical theory 144,145 (Equation 3), depends on the driving force of the reaction (∆G°), the nuclear reorganization energy (λ), and the electronic coupling (HDA) between donor D and acceptor A.

𝑘 𝐸𝑇 = 1 ℎ 𝐻 𝐷𝐴 2 √4𝜋𝜆𝑅𝑇 𝑒 -(∆𝐺°+𝜆) 2 4𝜆𝑅𝑇
𝐸𝑞. 3

The simplest model to describe the electronic coupling in case of proteins is the « square-barrier model », according to which 𝐻 𝐷𝐴 2 decreases exponentially with increasing distance between donor and acceptor (rDA):

𝐻 𝐷𝐴 2 = (𝐻 𝐷𝐴 0 ) 2 𝑒 -𝛽(𝑟 𝐷𝐴-𝑟 0 ) 𝐸𝑞. 4
where HDA 0 is the electronic coupling at Van-der-Waals distance (r0). The tunnel parameter β reflects the protein efficiency for electron transfer and depends on the protein structure. 146,147 In DET, the catalytic wave is centered on a potential that is only affected by the properties of the enzyme molecule. This is discussed further in section 3. Therefore, a performant DET can only be obtained for a monolayer of enzymes properly oriented. Experimentally, it has been verified for redox proteins including MCOs immobilized on SAMs with different alkyl lengths that the efficiency of electron transfer decreases exponentially with increasing numbers of methyl groups. [149][150][151] In proteins, electrons tunnel through a maximum distance of  14 Å. 146 MET is possible if the affinity between the enzyme and the mediator is high enough, and if the difference in their redox potentials induces a driving force for the electron transfer. Both the oxidized and the reduced forms of the mediator must be stable chemically, and of course none of them must inhibit the bioelectrochemical reaction. The mediator can be for example another protein like cytochrome c, 152 diffusive species like ABTS or transition metal complexes, or a metal complex bound to a polymer, like osmium complexes. 153 The corresponding polymers are used in the form of a hydrogel, whose reticulated structure allows permeation of solvent and diffusion of ions and non-charged species. Unlike most hydrogels, osmium based redox polymers are also capable of conducting electrons via self-collision between neighboring oxidized and reduced osmium redox moieties. The "diffusivity" of electrons in the hydrogel depends on the length of the tether that links the osmium complex to the polymer backbone, the characteristic electron-hoping distance between neighboring redox sites, the self-exchange rate constant, and the concentration of redox sites. 154 In MET configuration, the catalytic wave is centered on the mediator potential (Fig 7B ).

The two approaches have advantages and drawbacks. DET is reasonably simple and cheap, and allows working with overvoltages close to zero thanks to intrinsic properties of enzymes. However, it requires a unique orientation and only allows the simultaneous connection of a maximum of one monolayer of enzymes. In contrast, MET allows to wire electronically lots of enzymes regardless of their orientation and even in multilayers. The hydrogel especially is a convenient matrix for enzyme immobilization that even protects enzymes from inhibitors. However, electron-hopping in the redox polymer introduces additional steps that can lower the catalytic efficiency of the bioelectrode. A drop of cell voltage is also induced because catalysis is recorded at the potential of the mediator which must be lower than that of the enzyme. Finally, in case of diffusive mediators, a cell membrane is required to avoid cross reactions.

Mechanistic studies of MCOs

The catalytic mechanism of MCOs in homogeneous solution has been largely investigated by different spectroscopic techniques, crystallography and density functional theory (DFT) calculations and only partly elucidated. [START_REF] Jones | Electron Transfer and Reaction Mechanism of Laccases[END_REF][START_REF] Solomon | O2 Reduction to H2O by the Multicopper Oxidases[END_REF][START_REF] Solomon | Multicopper Oxidases and Oxygenases[END_REF] For example, the mechanisms of MCO inhibition at high pH or high concentrations of halides, or the differences between BODs and laccases are still unclear. Step 5: hypothesized electron transfer from the electrode to the TNC; Step 6: Electron transfer from the electrode to the redox mediator; Step 7: electron transfer within the redox mediator;

Step 8 electron transfer from the redox mediator to the T1. Adapted with permission from

[155] Copyright 2017 Elsevier During homogeneous catalysis, oxidation of organic substrates occurs via a ping-pong type mechanism. T1 is the primary center at which electrons from the reducing substrates are accepted, [START_REF] Jones | Electron Transfer and Reaction Mechanism of Laccases[END_REF][START_REF] Solomon | O2 Reduction to H2O by the Multicopper Oxidases[END_REF][START_REF] Solomon | Multicopper Oxidases and Oxygenases[END_REF]155,156 and O2 is reduced at the TNC (Fig 8). One of the major questions, however, is whether the mechanism is the same for an enzyme immobilized at the electrode.

Intuitively, we may for example expect different rate limiting steps, depending whether the MCOs are connected at the electrode in DET or in MET.

The influences of pH and chloride on the MCOs activity are of particular importance, especially if MCO-based biocathodes are envisioned for BFCs operating in physiological media. In this section, we first present commonly adopted opinion about how pH and halide inhibitors impact on MCO mechanism in homogeneous solutions. Due to the general similarity between mechanism in homogeneous solution and in electrochemistry in MET connection, we simultaneously develop investigations realized in electrochemistry in MET.

The reader can find more information about mechanism in homogeneous solution in different specialized reviews. [START_REF] Solomon | O2 Reduction to H2O by the Multicopper Oxidases[END_REF][157][158][159][160][161] The following part presents a comprehensive view of DET connected immobilized enzymes. We also consider two less-known inhibitors, hydrogen peroxide and methanol, without differentiating DET and MET, because a priori their effect is similar in both modes.

Effect of pH on MCOs

The effect of pH on the MCO catalytic mechanism has been the subject of intense research and has only recently been partly elucidated. The first studies were reported in homogenous solution in 1997 by Xu. 162 They demonstrated that both high-and low-potential laccases from Trametes villosa, Rhizoctonia solani and Myceliopthora thermophila were inhibited at neutral pH and above. It was hypothesized that laccases were inhibited upon increasing the pH due to the binding of OH -to the T2 site, therefore interrupting intramolecular electron transfer between the copper sites. 157,158 Solomon et al. investigated in details how pH influences laccases reaction mechanism. [START_REF] Solomon | O2 Reduction to H2O by the Multicopper Oxidases[END_REF] They particularly tried to decipher which step of the reaction cycle was inhibited at high pHs. Since the rates of formation of the PI and NI states are pHindependent 163 , these steps cannot be associated to the pH inhibition (Fig. 3). The remaining possibilities are therefore the formation of the fully reduced form, either from NI or from the resting oxidized form, or an alternative mechanism. More recently, by combining protein film volammetry, 164 computational modeling and numerical analysis, Blanford et al. 165 investigated the pH dependence of O2 reduction of two high and two low-potential MCOs immobilized in DET mode. They attributed the pH inhibition to another redox state of the enzymes, unknown so far, called "X state". This point is detailed further in section 3.4.8.

However, since there are no direct spectroscopic or crystallographic features of the "X state", it cannot be totally excluded that the inhibition effect may come from the NI state. If the formation of NI has been studied extensively, this is not the case of the protons involved during the reduction of NI. 105 While the residual activity of laccases is almost nil at neutral pH, irrespective of the redox mediator used, this is not so for BODs. [START_REF] Mano | Features and Applications of Bilirubin Oxidases[END_REF] Since the specific activity of BODs is declining with the pH (when ABTS is used as a substrate), one can hypothesize that OH -also inhibits BODs. Nevertheless, detailed kinetics studies on BODs still have to be performed.

Another question remains open: why are BODs less sensitive to pH than laccases? Different strategies have been explored to prevent laccases inhibition at neutral pH, or to get around it. For example, by switching from a fungal Tv-laccase to the small bacterial laccase from Steptomyces coelicolor, which was reported to be highly active at pH 7, Calabrese-Barton et al. reached 1.5 mA.cm -2 at 40°C and 900 rpm while they only reached 0.2 mA.cm -2 with the fungal one. 166 The exact reason for the higher activity of Sc-laccase at neutral pH still has to be identified. Enzyme engineering and molecular biology are also powerful tools to evolve laccases. 167 For example in 2009, Cusano et al. 168 succeeded in shifting the pH profile of chimeric laccases and in 2013, Alcalde et al. widened the pH profile of a fungal laccase. 169 The same authors later used directed molecular evolution to evolve a basydiomycete laccase. 170 After several rounds of evolution, they succeeded in getting a high redox potential laccase with an optimum activity shifted towards neutral pH and more resistant to chloride. Surprisingly, all mutations were located in the second coordination sphere of the T1. All these engineered enzymes have yet to be tested in electrochemistry. Pita,De Lacey et al. 171 proposed a different approach. By locally acidifying the pH at pH 5 near a modified electrode with Th-laccase, the authors succeeded in keeping the enzyme active while the pH of the bulk solution was 6.5. To create an acidic local pH around the Th-laccase electrode, they used magnetic particles modified with glucose oxidase.

Halides inhibition

The first detailed studies in homogeneous solution on the effect of halides (Cl -, F -, Br -) on MCOs activity have been reported in 1996 by Xu using ABTS as a substrate. 172 In the study, the author tested 5 different laccases and Mv-BOD. He concluded that the inhibition order was following the general trend F -> Cl -> Br -and depended on the accessibility of ions to the TNC. The author also noticed that the magnitude of the inhibition was dependent on the species of laccases, particularly because the latter influences the size of the channel leading to the TNC. All MCOs were inhibited by F -, and Mv-BOD was 1000 times more tolerant to Cl - compared to laccases. In 2001, Xu reviewed the effect of pH and anions on the inhibition on 5 laccases from different species. 157 In 1998, Hirose et al. 173 specifically studied the effect of halides inhibition on bilirubin ditaurate oxidation by Tt-BOD. The inhibition order was SCN - (thiocyanate) > F ->> Cl -> Br -, and the inhibition was non-competitive.

Mechanistically, the affinity between fluoride and the TNC is a general trait of MCOs and has been the subject of numerous investigations particularly by combining EPR, X-ray crystallography and modeling. 158-161 In 2005, Quintanar et al. 174 proposed a model in which the binding of F -only occurs in presence of the fully oxidized TNC. It is explained by strong electrostatic interactions between the negatively charged fluoride and the positively charged fully oxidized TNC. On the contrary, the exact mechanism of chloride inhibition of MCOs in homogeneous solution is not yet elucidated. It is not excluded that Cl -may interact with the T1 or with the TNC but spectroscopic and crystallographic evidence is still missing.

Because of the envisioned applications of MCOs, the effect of halides on the catalytic reduction of O2 by MCOs immobilized at electrodes has been studied. The current density of the "wired" Mv-BOD and Tt-BOD electrodes with osmium redox polymer only declined respectively by 6% and 3% in presence of 140 mM NaCl at pH 7.4. 120,175 Moreover, even though fluoride has a very high affinity with the MCOs, the presence of 100 mM F -did not significantly affect the current density. It can be hypothesized that the electrostatic adduct formed by the enzyme and the redox polymer acts as a shield against Cl -and to some extent F - . A similar effect has been seen by Calvo et al. with a self-assembled layer of laccase and osmium bound to poly(allylamine), when the concentration of Cl -in solution was below the concentration of chloride bound to positively charged groups in the polycationic film. 176 When the same redox polymer used to wire BODs was quaternized, 177 the effect of fluoride was much more pronounced, the current declining already by 40% in presence of 20 mM F -.

Quaternization introduces positive charges within the redox polymer and may therefore affect the interaction between enzymes and polymer, exposing the TNC to fluoride. Although BODs already share a relative tolerance to chloride, to prevent even more the inhibition by Cl -, a different strategy consists in identifying new BODs with increased resistance to it. Durand et al. have for example reported a new BOD from Bacillus pumilus which only loses 20% of its activity towards SGZ oxidation in presence of 500 mM NaCl in a 100 mM citrate-phosphate buffer pH 7. 101 Once immobilized within an osmium redox polymer, this new enzyme showed a high tolerance to chloride.

In contrast to BOD electrodes, the effect of Cl -on laccase-modified electrodes is pronounced. The activity of an electrode modified with osmium polymers and Th-laccase declined by 60% at pH 5 when the chloride concentration was raised from nil to 100 mM. 178 It has been hypothesized that Cl -was a competitive inhibitor towards redox mediators, blocking their access to the T1 site. 179 Even though this hypothesis still has to be clearly proven, strong evidence has already been reported. For example, Bey et al. designed a new osmium redox polymer with the objective to improve the interaction between the T1 and the osmium, therefore restricting the access of anions to the catalytic site. It resulted in the formation of a chloride-resistant cathode based on Trametes hirsuta. 180 The authors also applied the same strategy to shield CotA from Bacillus subtilis from chloride. 181 With the same objective but a different strategy, Leech et al. used a laccase from Melanocarpus albomyces, which displays an optimal activity near neutral pH, 182 co-immobilized with (polyvinylimidazole)10[Os(2,2'bipyridine)2 Cl ] +/2+ . They could reach 3.8 mA cm -2 at 0.2 V in a physiological buffer solution containing 100 mM NaCl, 183 the highest current density reported for laccase at neutral pH, and later used it in a glucose/O2 BFC. 184 The inhibition effect of Cl -on laccase modified electrodes is also observed in DET, to some extent. For example, when Cerrena maxima laccase was immobilized on graphite particles, the current declined by 30% upon addition of 0.1 M NaCl. 185 Salaj-Kosla et al. showed that the activity of nanoporous gold electrodes modified with a laccase from Trametes hirsuta was strongly dependent on halides in a citratephosphate buffer pH 4. 186 Addition of fluoride completely inhibited the enzyme because, as discussed above, F -blocks the electron transfer between the T1 and the TNC. The addition of 200 mM Cl -decreased the current by 50%. In contrast, when the same enzyme was immobilized on graphite carbon electrodes through phenyl derivatives, De Lacey et al. did not observe any inhibition by Cl -. 187 Interestingly, the addition of ABTS as a diffusing redox mediator made the modified electrodes sensitive again to Cl -. This observation tends to confirm that Cl -may act as a competitive inhibitor towards redox mediators.

Model of mediated electron transfer

Model

Mathematical models predict and describe the observed steady-state electrochemical currents and concentration profiles of enzymes, substrates and redox mediators. The models consider mass and charge transport, enzymatic reactions and electrode reactions. Modeling coupled reaction/diffusion problems leads to a set of non-linear differential equations that cannot always be solved analytically. Therefore, either analytical solutions are derived under certain hypothesis or simplifications (i.e describing limiting cases), or alternatively numerical full solutions can be simulated. We give below a brief introduction of the models of an MCO cathode based on mediated electron transfer (MET) mechanism. Further details and last achievements can be found in a recent review. [START_REF] Rajendran | Mathematical Modeling of Nonlinear Reaction-Diffusion Processes in Enzymatic Biofuel Cells[END_REF] MET mechanism includes two substrates, O2 and the redox mediator. The mechanism is described by a non-sequential, or "ping-pong" mechanism, where all steps of enzyme reaction with O2 are included in a global oxygen reduction reaction (ORR) mechanism ( Fig 9). The redox mediator transfers one electron to the electrode. The reaction mechanism for MCO can therefore be described by the following equations, derived from a general model of ping-pong mechanism [188][189][190][191] 𝑂 2 + 4𝐻 + + 𝑀𝐶𝑂 𝑟𝑒𝑑 → 𝑀𝐶𝑂 𝑜𝑥 + 2𝐻 2 𝑂 𝐸𝑞. 5

4𝑀𝑒𝑑 𝑟𝑒𝑑 + 𝑀𝐶𝑂 𝑜𝑥 → 4𝑀𝑒𝑑 𝑜𝑥 + 𝑀𝐶𝑂 𝑟𝑒𝑑 𝐸𝑞. 6
and the mediator reduction at the electrode can be expressed as:

𝑀𝑒𝑑 𝑜𝑥 + 𝑒 -→ ⃖ 𝑀𝑒𝑑 𝑟𝑒𝑑 𝐸𝑞. 7
Typically only the case where both the enzyme and the redox mediator are confined by a membrane or a polymer in the reaction layer at the electrode surface was considered. [188][189][190][191][192][193][194] In this case, the enzyme is immobile while the mediator is diffusing with an apparent diffusion coEfficient DMed, app to/from the electrode surface where it is reduced. Alternatively, in case of a linked redox mediator, charges are propagating by electron-hopping between the neighboring redox sites across the hydrogel layer and the model is formally unchanged.

Oxygen is diffusing in the film with an apparent DO2,app. The bulk solution is supposed to be ideally mixed (i.e. [O2]x=l = constant) and the convection in the catalytic layer can be neglected. Assuming a one-dimensional diffusion, the following differential equations can then be derived (for 0 < x < l): 188,189,193 𝜕

[𝑀𝑒𝑑 𝑟𝑒𝑑 ] 𝜕𝑡 = 𝐷 𝑀𝑒𝑑,𝑎𝑝𝑝 𝜕 2 [𝑀𝑒𝑑 𝑟𝑒𝑑 ] 𝜕𝑥 2 -4𝑣 𝐸𝑛𝑧 𝐸𝑞. 8 𝜕[𝑂 2 ] 𝜕𝑡 = 𝐷 𝑂2,𝑎𝑝𝑝 𝜕 2 [𝑂 2 ] 𝜕𝑥 2 -𝑣 𝐸𝑛𝑧 𝐸𝑞. 9
where 𝑣 𝐸𝑛𝑧 is the rate of the enzymatic O2 reduction. In some studies, only the substrate consumption rate was supposed to exhibit a Michaelis-Menten kinetics, 188 while in other cases the catalytic rate constants of the enzymatic reaction were supposed to be independent on both O2 and mediator concentrations. 192 The pH-dependency of enzymatic activity was not taken into account.

It may reasonably be proposed that the model assuming a Michaelis-Menten kinetics both for O2 and the redox mediator, first derived for a stoichiometric coEfficient for electrons, can be extended to the case of MCOs for which 4 electrons are necessary to reduce 1 molecule of O2. In these conditions, 𝑣 𝐸𝑛𝑧 at steady-state is therefore defined as: 189,191,193 𝑣 𝐸𝑛𝑧 = 𝑘 𝑐𝑎𝑡 [𝑀𝐶𝑂 𝑇𝑜𝑡 ] The kinetics of equation 7 defines the boundary conditions of [Mred] at x = 0. It is reasonable to consider that the electrochemical reaction is reversible and that its kinetics follows a

Butler-Volmer law. In most cases, diffusion is considered to be the only mass transport mechanism. In some recent models however, an additional term has been introduced to take into account the mass transport by migration (i.e. the influence of electric field), 194 or convection. 192

Mathematical modeling

Both analytical and numerical methods have been used to derive the equations describing the behavior of an enzymatic electrode [188][189][190][191][192][193]195,196 or more rarely of complete EBFCs. [START_REF] Rajendran | Mathematical Modeling of Nonlinear Reaction-Diffusion Processes in Enzymatic Biofuel Cells[END_REF]194

Analytical solutions

In a first fundamental study Bartlett et al. 188 built a case diagram based on complete analytical solutions derived for a set of limiting cases. In their study, they only considered a Michaelis-Menten kinetics for the substrate conversion (i.e. O2 reduction in our case). The two dimensionless coordinates of the resulting case diagram are , which describes the relative thicknesses of the film and diffusion layer; and , the relative concentrations of enzyme in its reduced or oxidized forms. In the first case (I), the limiting step is the enzyme-mediator reaction (i.e. mass and charge transport of substrates and redox mediators within the film are faster than the enzymatic reaction). The second case (II) corresponds to limitation by the mass and charge transport of the redox mediator. In case III, the reaction kinetics are much faster than substrate and mediator diffusion, so that the enzymatic reaction is limited by the substrate in one part of the film, while it is limited by the mediator in the other part of the film. This situation results in the reaction occurring only in a very thin region inside the film. The fourth case (IV) corresponds to limitation by the mass and charge transport of the substrate. Finally, in case V the limiting step is the enzymesubstrate reaction (i.e. mass and charge transport of substrate and redox mediators within the film are faster than the reaction).

More recently, analytical solutions were derived using the homotopy perturbation method (HPM) combined with the inversion conjuncture in the Laplace plane. 192,195,196 While limiting cases mostly describe steady-state electrochemical responses, this method allowed describing current densities in chronoamperometry at different potentials both in the transient and steady states. It was applied to an electrode based on MET of laccase in an osmium redox hydrogel. The time to reach steady-state was determined, and proved to depend on the potential of the redox mediator. Interestingly this approach could show how much the steady state current densities were influenced by the enzyme characteristics (catalytic and Michaelis-Menten constants), concentration of substrate in the bulk, and film thickness. 192 In a further study, the same approach showed its significance for the derivation of analytical expressions of steady-state current densities and O2 and mediator concentrations, valid in the potential step methods. Moreover, two graphical procedures were proposed for the estimation of Michaelis-Menten constants. In both cases, the model was validated by fitting with experimental data of oxygen-reducing laccase cathodes. 192,196

Full numerical derivation

In the first study of Bartlett mentioned above, numerical simulations of the one dimensional model were performed based on the relaxation method, and were in good agreement with the analytical study. 188 This 1D model was then applied to an enzymatic O2 reducing porous cathode. 193 The composite electrode was modelled as cylindrical conducting fibers around which the reaction layer film was coated. The influence of morphology (in terms of film thickness, composite porosity and fiber diameter) was evaluated. The authors also studied how to improve mass transport of the oxygen substrate, and predicted that the maximum current densities could be close to 100 mA.cm -2 for a pure O2-breathing electrode, and 60 mA.cm -2 for an air-breathing electrode. 193 While this study mainly focused on thick films, another proposed to consider high-surface area electrodes grafted with thin polymer layers. 189 In that case also it was proposed that currents around 100 mA.cm -2 could be achieved with the porous electrode.

Another important contribution 190 used modeling to determine the effect of ∆Eet (difference between the T1 and the redox mediator potentials) on the bimolecular rate constants of the enzymatic reaction; kcat/Km and kcat/KS towards mediator and oxygen respectively (corresponding to kcat/KM,Med and kcat/KM,O2 with our notations). Using a finite difference method, expressions of substrate and redox mediator concentrations were determined, from which the expression of current density was derived. This expression was used to extract kinetic parameters from experimental CVs recorded with T. hirsuta laccase immobilized in redox hydrogels with osmium based redox mediators covering a wide range of potentials. 190 The bimolecular rate constants were found to be slightly lower in the film than those determined in free solution. 156 In addition, the performance of the cathode was found to vary linearly with ∆Eet for ∆Eet < 300 mV, and to be independent on ∆Eet for ∆Eet > 300 mV (Fig 11). 190 and constant mediation rate at lower E 0 values. Reproduced with permission from [190].

Copyright 2008 American Chemical Society.

A model of a complete glucose/O2 EBFC composed of a cascade of enzymes and a redox mediator at the anode, and an air-breathing cathode based on BOD with ferricyanide has also been reported. 194 This model was used to describe an EBFC formerly reported by Sakai et al. 197 where both enzymatic systems were immobilized on porous carbon-fiber electrodes. The simulated data agree with the experimental results. Clues could be obtained concerning the evolution of concentrations and currents over time at a given cell voltage, but also concerning the evolution of several parameters in space. For example, pH was shown to vary in the cell, being maximal at the anode. When a voltage was applied across the cell for a long time, the overpotential at the carbon-fiber cathode was found to drop drastically with the distance from the electron collector/carbon-fiber interface. 194

Studies of the mechanism of direct electron transfer

In this part, we discuss the electrochemical behavior of enzymes at the electrode interface in DET and explain general mechanistic clues provided by electrochemical studies.

In the DET mode, electrons could theoretically be transferred directly to the TNC, provided that this latter is accessible and close enough to the electrode surface, thus bypassing the internal electron transfer. Therefore, one of the major questions is whether the O2-reduction mechanism remains unchanged for an enzyme directly connected at the electrode (DET mode).

Redox potentials of the Cu sites

Redox potentials characterizing the different Cu sites of various MCOs have been determined by redox titration in homogeneous solutions. T1 Cu, whose oxidation state can only vary between +I and +II, is well characterized by a single redox transition at a determined apparent standard potential E°'. Slightly different values for the same enzyme can however be found in literature. MCOs can be classified according to the redox potential of their T1 site in low (340-490 mV vs. NHE), middle (470-710 mV vs. NHE) and high (730-780 mV vs. NHE) redox potential MCOs. [START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF]198 Tree laccases belong to the first group and fungal laccases to the third one, [START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF]199,200 while BODs are part of the 2 nd with for example T1 potentials of 660 mV and 670 mV vs. NHE at pH 7 for Tt-BOD and Mv-BOD. [201][202][203][204] Moreover, the T1 titration curve is indicative of a one electron process since the potential varies by 49-90 mV per unit of log(Abs). 118,202 The molecular origin of these potential differences is discussed in section 2.1. It is more difficult to attribute redox potentials to the TNC because this cluster can be characterized by several redox transitions. In very early studies, the redox potentials of the T3 sites of R. vernicifera and T. versicolor laccases have been determined at 480 and 780 mV vs. NHE resp., 199,200 while a redox potential of 390 mV vs. NHE was attributed to the T2 Cu of the low-potential Rv-laccase. 199,200 In this section, unless stated otherwise, we quote redox potentials against the NHE reference electrode for the sake of consistency with the literature.

Determination of the redox potential of T1 in MCOs has also been realized by nonmediated spectroelectrochemistry following the vanishing of the blue band at 600 nm upon application of reductive potentials and its re-apparition upon re-oxidation. 118 Direct electron transfer between a gold electrode and a high-potential laccase was also observed for the first time with this technique, with Trametes hirsuta in a gold capillary. 118 Non-mediated spectroelectrochemistry furthermore confirmed the possibility to establish DET with the T1

Cu for several fungal laccases from different basidiomycetes (Trametes hirsuta, Trametes ochracea, Cerrena maxima). 205 Simultaneous detection of T1 and T3 Cu (at resp. 600 and 300 nm) also proved that enzymes could be fully reduced via DET from an electrode. 205 Here again, analysis of the titration curves gave information about the electron transfer mechanism.

In case of high potential Th-laccase and Mv-BOD, DET to the T1 was very slow, and the curve showed a hysteresis and 2 noticeable electron transfer (ET) processes. This was attributed to ET to the T1 (resp. at 860 mV for Th-laccase and 805 vs. NHE for Mv-BOD) mediated by another redox site in the MCO (resp. at 290-370 and 460 mV vs. NHE for Thlaccase and Mv-BOD). 118,202 Since only ascorbate can fully reduce the laccase, implying that the redox potential of the T2 might be considerably more negative than the T1, it was

proposed that the T2 redox potential for high-potential laccases was about 400 mV vs. NHE at pH 6.5. Therefore, although in these cases electron transfer from the electrode to the T1 was realized without the help of an external redox mediator, it was not, strictly speaking, "direct electron transfer" to the T1. It proceeded via another enzymatic redox site of lower potential and most likely the T2 Cu. 118

Non-catalytic faradaic processes under anaerobic conditions

In several cases, direct electron transfer (DET) between MCOs and electrodes has been observed under anaerobic conditions and characterized by the appearance of reversible or quasi reversible redox peaks in cyclic voltammetry (CV), linear sweep voltammetry (LSV)

or square wave voltammetry (SWV). For high-potential laccases, different redox peaks have been observed corresponding to either a high or a low-potential process, or both. The midpoint redox potentials (given by 𝐸 𝑚 =

𝐸 𝑎 +𝐸 𝑐 2
where Ea is the potential of the anodic peak and Ec is the potential of the cathodic peak) were usually observed in the 300-450 mV vs.

NHE range for the low-potential processes, and between 600 and 900 mV vs. NHE for the high-potential processes respectively. This low-potential process, mostly observed with a gold working electrode, [START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF]118,[205][206][207][208] can hardly be assigned to the T1. It was hypothesized that it could be related to the T2 redox transition. To confirm this hypothesis, experiments were realized with T2-depleted enzymes. 118 While a 50% T2-depleted Th-laccase gave 50% peak current, no low-potential redox peak was observed for the apo-enzyme. In addition, in presence of F -, whose affinity for the TNC has been shown in homogeneous solution, the midpoint potential increased by about 35 mV. All these experiments tended to confirm that T2 potential is around 400 mV vs. NHE, and that in all the above mentioned cases, at least a fraction of laccases were oriented with the T2 Cu facing the surface. 118 Experiments performed with a recombinant laccase bearing a Cys-tag, inducing different orientations of the enzyme on gold via the S-Au bond, also supported the attribution of the peak at lower potential to a TNC intermediate. When upon immobilization the T1 was repelled far from the electrode surface, and the T2/T3 approached close to it, a redox process at Em = 377 mV vs. NHE was observed and attributed to the T2.

On the contrary when T1 and T2/T3 were at the same distance from the electrode surface, a more irreversible pair of redox peaks with Em = 470 mV vs. NHE was observed and attributed to simultaneous effect of both T1 and TNC Cu clusters. 209

High redox potential

For high-potential laccases, a high-potential redox process, more certainly corresponding to the T1 has also been identified (Fig 12). A reversible redox process on a graphite electrode at 645-700 mV vs. NHE at pH 5 has been observed for Tv-laccase, but only in the presence of a promotor (2,6-dimethoxyphenol (DMP) or 4,4'-bipyridine). 211 Later, Tv-laccase adsorbed on edge-plane pyrolytic graphite exhibited a reversible redox process at 790 mV vs. NHE. 212 Similarly, redox peaks between 780 and 800 mV vs. NHE at pH 6.5 were recorded by SWV for T. hirsuta, T. ochracea, and C. maxima laccases adsorbed on HOPG. 210 The same enzymes adsorbed on gold electrodes exhibited high redox processes (between 857 and 903 mV vs. NHE), which lowered to approx. 800 mV when the enzymes were covalently bound on AMT. 205 Similarly, the high potential redox peak of laccases from T. versicolor and T. hirsuta entrapped in a cationic polymer matrix on a gold electrode were respectively 784 ± 3 and 778 ± 4 mV vs. NHE, in good agreement with the T1 potential. 208

Low-potential laccases

For low potential laccases, electrochemical studies described only one pair of redox peaks coinciding with the T1. A non-catalytic process with semi reversible behavior was observed for Rv-laccase on a gold electrode modified with an anionic polymeric film at 410 mV (pH 5.5) or 400 mV vs. NHE (pH 7), 213 and at 410 mV vs. NHE at pH 7 for the same laccase covalently bound on mercapto-propionic acid (MPA) on gold. 214 This anaerobic process did not change upon addition of F -, 214 and T2-depleted enzyme still exhibited the same redox process while the apo-protein was electrochemically silent. 213 Melanocarpus albomyces and Rv-laccases entrapped in a cationic polymer matrix on a gold electrode also exhibited only one redox process at resp. 422 ± 4 and 449 ± 5 mV vs. NHE at pH 6.5, in good agreement with T1 potential. 208

Bilirubin oxidases

BODs behave approximately like laccases with respect to their electrochemical response in absence of O2. For Mv-BOD adsorbed on SPG, a unique and very small quasi reversible process at Em = 515 mV vs. NHE at pH 7.4 with a large peak separation was first recorded. 215 Later, two pairs of peaks were simultaneously identified at Em = 690 mV (resp. Furthermore, a quasi-reversible redox process at Em = 360 mV vs. NHE at pH 7, observed for Tt-BOD covalently bound on MPA on gold, was attributed to a resting form of the enzyme because it was almost not influenced by F -. 203 This led to the suggestion that an endergonic tunneling from the T1 to T2/T3 could be involved in MCO mechanism. 203 A noncatalytic low-potential redox signal of Mv-BOD (360 mV vs. NHE at pH 7), attributed to a resting form of the enzyme, also appeared when the enzyme was oriented with the TNC facing the electrode surface. 216 3 non-catalytic cathodic peaks (at resp.  693, 574 and 403 mV vs. NHE), and only one anodic (at the lowest potential) were visible on a CV at pH 6 for Mv-BOD entrapped in a matrix of Ketjen black (KB), carbon nanotubes (CNTs) and chitosan.

The highest was assigned to the T1, consistently with previous studies, and it was proposed that the 2 others might be attributed to different redox states of the TNC, containing or not bridging oxygen species. 217 It is noteworthy however that a low potential redox peak at  420 mV had previously been assigned to a surface group of the enzyme. 218,219 The simultaneous presence of two pairs of peaks suggested that for different fractions of BOD only the T1 or the T2 was in contact with the electrode. 208 The appearance of the low-potential redox process was attributed to either the T2 or to a coincidental electrochemical response of the 4 Cu ions, [START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF] while the high potential peak was attributed to the T1. Since different redox processes could be observed for the same enzyme immobilized at different materials, it was proposed that different protein sites were communicating with the electrode, depending on the electrode material. 203

Two-versus four-electron reduction of dioxygen

The redox processes observed in anaerobic conditions, especially those at low potential, could not always be correlated to a catalytic current in aerobic conditions. This means that some enzymes were immobilized in a configuration that permitted direct electron exchange with the electrode, without however enabling the catalytic mechanism. This remark suggested that in these cases the enzymes were not connected via the proper redox center. 118,207 This hypothesis was confirmed in some cases by the addition of a redox mediator that enabled MET. 203,220 The onset of catalysis was observed in some rare cases in the low-potential range for high-or middle-potential MCOs. Onset at  300 mV vs. NHE at pH 5 was for example reported for the fungal Th-laccase on bare gold 221 or AMT self-assembled monolayer (SAMs) on gold. 222 Similarly, oxygen reduction by Mv-BOD in solution started at  450 mV vs. NHE, when a gold wire modified with a mixed mercapto-undecanol/mercapto-undecanoic acid SAM was used as the working electrode. 223 Only low catalytic currents were observed with these systems, while non-catalytic signals were not detected. [221][222][223] The influence of F -, which is known to inhibit the IET in homogeneous catalysis, has been also studied. For example, Th-laccase covalently bound on 4-AMT-modified gold electrode exhibited a first reduction current starting at 510 mV vs. NHE at pH 5, which was followed by a second more steeper increase at 240 mV. F -inhibited only the high-potential (E > 240 mV) reduction, while the current at lower potentials remained unchanged (Fig 13).

Similar observation was done for Cm-laccase on a bare gold disc: O2 reduction started at a quite low potential, 320 mV vs. NHE, and was not influenced by F -. This led to the supposition that in both cases no IET was occurring. 205 Moreover, the catalysis starting at low potential has been correlated to the incomplete O2 reduction to H2O2 in several cases. For example, when a fungal laccase was adsorbed on bare gold electrodes, H2O2 production in the low potential range (E < 240 mV vs. NHE) was 3 times higher than for naked gold alone. It was lower both in presence or absence of enzyme at higher potentials (240-440 mV vs. NHE). 205 Similarly, for an engineered laccase adopting different orientations on a gold electrode, oxygen reduction was observed either at 495 mV vs.

NHE or at 340 mV vs. NHE and assigned respectively to DET by the T1-TNC or by the TNC alone. It was associated with H2O2 production of resp. 2.75 and 7.53 µM. 209 It seems therefore that DET from the electrode to the T2/T3 is possible but leads either to no catalysis or in the best cases to the 2-e -reduction of O2 to H2O2. It clearly appears that the orientation of the enzyme at the electrode influences both the electron transfer pathway and the electron efficiency of O2 reduction. 209 Contrary to the catalysis observed at low potential and assigned to the reduction of O2 to H2O2, no significant formation of hydrogen peroxide has been reported for catalysis starting at the T1 potential. H2O2 evolution was for example estimated with a porphyrincoated electrode in a solution in which O2 had been reduced by a Tv-laccase electrode. Direct ORR started at  0.84 V vs. NHE at pH 5.5 with the fungal Tv-laccase adsorbed on graphite.

The experiment confirmed that no more than 5% O2 could have been reduced to H2O2. 211 A similar experiment was performed with Mv-BOD attached to multi-wall carbon nanotubes (MW-CNTs) on the disc of a rotating ring disc electrode (RRDE), for which the ORR onset was  0.7 V vs. NHE at pH 7.5. During the CV, the platinum ring was polarized to measure the reduction of H2O2. Zero hydrogen peroxide was detected on the disc, and according to the mass and charge balance of the RRDE no more than 7.5% O2 in total could have been reduced to H2O2. 224 In both cases, Koutecky-Levich analysis of the rotating electrode matched a 4electron reduction to water and not H2O2. 211,224 It is therefore of interest to determine the number of electrons exchanged by an enzyme respectively in high-versus low-potential anaerobic redox processes. For a peak at high potential, the ratio of anodic to cathodic currents were shown to vary from 1:1 (which actually can be converted to 4:4 by normalizing the peak intensity ip by the scan rate v) at very low scan rates (below 1 mV.s -1 ) to 4:1 at fast scan rates (above 200 mV.s -1 ). 208,214 Further analysis of the peak separation ∆Ep as a function of ∆log(f) in SWV indicated that 3.65 electrons were transferred for Tv-laccase and 3.85 for Th-laccase. 208 These results support a 4

e -transfer to/from the T1 site, which would correspond to a possible full reduction/oxidation of the enzyme via the T1, with the full reduction being much slower than the oxidation. For a low-potential peak of high-potential laccases, the width of the peak (given by the difference between the half-wave potential EHW and the peak potential EP) was consistent with a 1 e - transfer. 118 The anodic to cathodic Qa/Qc charge ratio increased from 1:1 to 3:1 with increasing v; and SWV analysis gave numbers of electrons of 2.78 for Tv and 2.9 for Thlaccase 208 which suggests that full reduction of the enzyme cannot occur via the low-potential intermediate whereas it is possible via the T1 Cu. For the unique redox peak of low-potential Ma and Rv-laccases, Qa/Qc varied from 1:1 for v < 100 mV.s -1 to 4:1 for scan rates above 800 mV.s -1 , and SWV gave n values close to 4 (n = 3.9 for Rv and 3.8 for Ma-laccases). 208 The general observation that the reduction peak is lower than the oxidation peak 118,205 or that the rate of oxidation is faster than the reduction in CV or SWV 208,214 is consistent with the hysteresis noticed in spectroelectrochemistry, which also shows that the oxidation process is significantly faster than the reduction process. 205 All this suggests that IET might be the ratelimiting step.

Causes of the low catalytic efficiency and stability on gold electrodes

Different behaviors were observed for BODs and laccases on gold electrodes. On bare gold, besides the H2O2 related mechanism described above, no catalysis at high potential or no catalysis at all was recorded for Th-laccase, 205,225 Cm-laccase, 205 or small laccase from Fusarium proliferatum. 226,227 On the other hand, a clear DET catalysis was observed for Mv-BOD on bare monocrystalline gold [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF]225 or platinum electrodes, [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF] with an onset above 0.7 V

vs. NHE at pH 5.8 consistent with the T1 redox potential. With this latter enzyme, the observed electrocatalytic responses vanished however relatively quickly [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF] with for example half-lives of resp. 15 minutes and 2.5 hours at pH 4 and 7.4, rather due to gradual deactivation than to desorption of the enzyme. 225 The efficiency and stability of catalysis at gold electrodes was investigated by coupling electrochemistry to other techniques like quartz crystal microbalance (QCM), 151,228 QCM with dissipation monitoring, [229][230][231] surface plasmon resonance (SPR), 220 ellipsometry, 225 atomic force microscopy (AFM) [225][226][227] or dual polarization interferometry (DPI). 231 No desorption of either BOD or laccase immobilized on bare or on thiol-modified gold electrodes was measured by ellipsometry, SPR or QCM, confirming the irreversible character of the adsorption of the enzymes on these surfaces. 220,225,228,230,231 Therefore, the decrease in cathodic current observed in numerous studies cannot be related to enzyme loss but most probably to enzyme denaturation or structural rearrangement. 220,228,229 The enhancement of stability observed with amide bonding of MCOs on the electrode surface was more likely due to a stiffening of the immobilized layer preventing conformational changes, rather than due to a better binding of enzymes to the electrode. 220,229

Denaturation by enzyme-metal interaction

For MCOs adsorbed on bare metal surfaces, a certain loss of activity towards oxidation of organic substrates was demonstrated. DMP oxidation was 7.5 times less Efficient

for Fp-laccase adsorbed on bare gold than on HOPG. 227 Enzymatic assay with 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) showed that Th-laccase was completely deactivated on bare polycrystalline gold, while some activity was still recorded for Mv-BOD. 225 Denaturation of MCOs adsorbed on bare metal has already been suggested. 232 Very fast adsorption of Fp-laccase on bare gold was observed by atomic force microscopy (AFM), and the formation of aggregates with a wide variety of sizes suggested an intense interaction with bare gold. 227 For Mv-BOD and Th-laccase on bare gold, the size of globular features ( 20 nm) was considered normal, taking into account the tip dilation effect of the AFM. 225 Similarly, the low heights of the molecules, ranging between 2.5 and 3 nm, was attributed to a tip compression effect that makes soft biological molecules appear always smaller with AFM, 225,227 although it could also correspond to a flattening of enzymes at the surface. 231 Mv-BOD was also more stable on MPA-modified gold than on bare gold. 225 BOD faster deactivation on single crystal might be due to tighter holding. [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF] Under O2, a stronger STM contrast was observed and the process was less reversible than under air. This study therefore highlights a structural change of the protein, that enables current tunneling through the enzyme only in its oxidized form. 233 Other studies suggested either a conformational change or reorientation under applied potential. 220,229,234 For Mv-BOD adsorbed or covalently bound to thiol-modified gold electrodes, currents decay did not coincide with the mass decay 228,229 but with the energy dissipation change, 229,230 indicating a non-desorptive degradation and most probably structural changes. Since no activity was recovered upon addition of a diffusing redox mediator; the current loss could not be explained by reorientation of some enzymes. 220 The current was even less stable when the potential was applied continuously, compared to punctual measurements (Fig 14B ). 220,229 At the same time, the dissipation losses were much higher, meaning that the BOD layer stiffened under application of a potential, and supporting a potential-induced enzyme denaturation. Moreover, under potential cycling, a mass drop was also recorded, therefore suggesting protein dehydration. 229 This could be an important cause of activity loss, since different studies have shown that drying the BOD electrode in a desiccator led to complete loss of the enzymatic activity, which was attributed to denaturation of the enzyme by losing water. 235,236 To rationalize the direct influence of potential on the active site, the behavior of the fraction of enzymes that were not directly connected at the electrode was investigated in MET upon addition of a redox mediator. A more stable current was observed for Mv-BOD adsorbed or covalently bound on NH2 terminated SAM on gold electrode in MET configuration, i.e.

when the enzyme was not directly submitted to potential cycling. 220 However, on carboxylate SAMs, the fraction of enzymes oriented in MET also lost its activity. These results suggest that direct influence of the potential on the active site is not the only origin of denaturation. 220

The electric field created at the electrode interface could also be deleterious for the enzyme structure, but to the best of our knowledge, no direct proof of this hypothesis has been reported. Since in the case discussed above the systems essentially differ by the chemistry of the electrode surface, it also indicates that the electrode surface charges influence on the enzyme stability. 220 This effect is discussed further in section 4.1.4.

Enzyme concentration

Catalysis constants kcat much lower than values for homogeneous catalysis (250 s -1 ) could be calculated for a surface coverage close to one monolayer of Mv-BOD adsorbed on bare gold (ca 4 s -1 ) 228 or on 3-MPA-modified flat polished gold surface of a QCM sensor ( 35s -1 ). 229 The decrease in activity with time was higher for immobilized enzymes than for enzymes in solution. 220 This suggested that BOD was poorly oriented at the electrode, and that some denaturation occurred upon immobilization. Furthermore, at pH 4 more enzyme was adsorbed but the measured current was even smaller than at pH 7. 228 Similarly, a lower efficiency was recorded for a monolayer of Mv-BOD on mercapto-hexanoic acid (MHA)modified gold (5 pmol.cm -2 ) than for a sub-monolayer (0.6 pmol.cm -2 ) with global kcat of respectively 7 and 48 s -1 . 220 It was therefore likely that higher BOD concentrations were detrimental, due to the increased interactions between neighboring BOD molecules. However, the opposite effect has also been observed. The behaviors of Mv-BOD and Th-laccase upon immobilization on bare gold electrodes were compared. Mv-BOD-always led to higher surface coverages, and at the same time to the highest enzyme specific activity. It was consequently proposed that steric effect or electrostatic interactions between neighboring molecules, induced by higher enzyme coverage, could prevent the enzyme from flattening, thereby lowering inactivation due to conformational changes. 225,231 The influence of the initial Mv-BOD concentration on the adsorption was investigated by DPI: intermediate concentrations (10-25 mg.mL -1 ) were shown to lead to the most Efficient (in terms of kcat) and persistent (in terms of kcat stability) layers, with an optimized kcat of 60 s -1 . 231 Adsorption was shown to occur in 2 stages: first an irreversible binding of a protein layer attributed to the spreading of the molecules at the surface, and then the formation of a less rigid layer of proteins. Higher concentrations of the enzyme solution produced less rigid layers in the first phase, because faster adsorption prevented enzyme from relaxing and flattening at the electrode surface. This correlated well with higher enzymatic specific activity. 231 However too dense layers also led to decreased efficiency. 231

Catalytic mechanism on carbon electrodes

When BOD and laccase are immobilized on bare polished GC, catalytic currents have not always been observed. 235,237 Therefore unless stated otherwise, this paragraph is only focused on studies of MCOs adsorbed on graphite electrodes. A well-defined catalytic wave for O2 reduction was recorded for most enzymes (Fig 15). To determine the primary electron acceptor in heterogeneous electron transfer (HET), characteristic potentials of the ORR under aerobic conditions as well as their variation with pH have been first considered. This follows the general assumption that catalysis is controlled by the potential of the redox center at which electrons enter/leave the enzyme, and not by the potential of the actual catalytic center. 238,239 Ecat, the potential corresponding to the maximum acceleration of the catalysis (i.e. E for maximal di/dE) is a convenient tool to describe values of characteristic potentials in catalysis, because it contains both thermodynamic information and clues about kinetics of the transformation. 165,240 However the steady-state potential of the electrode (OCP), onset potential or half-wave (EHW) potential of the catalytic ORR wave are more widely described in the literature. , 204,215,217,241 which coincided well with the high-potential quasi-reversible redox transformations observed under anaerobic conditions, and 810 mV at pH 4, i.e. 120 mV above E°'T1, for Tt-BOD. 204 All these values of onset potential are slightly higher than the redox potential of the T1 Cu center, indicating that the T1 might be the electrochemical control center of catalysis.

The onset pH-dependence was precisely 60 mV/pH unit in the first report for laccase, 242,243 which was consistent with a 1 proton-1 e -exchange, but slightly higher than 52 mV/pH unit reported later for Mv-BOD on SW-CNTs. 244 Furthermore it was more than twice higher than slopes of 20-25 mV/pH unit described for Mv and Tt-BODs on graphite. 204 These latter values were consistent with 23 mV/pH unit observed for the non-catalytic signal of both BODs, and close to the pH dependence described for laccase T1 potential. 200 This is a further

indication that T1 controls the potential of catalysis.

In the absence of diffusion limitation, it is also consistent to consider the half-wave potential EHW, which coincides with the redox potential of the electrochemical control center in catalysis, under kinetic limitation. For Mv-BOD, EHW was 670 mV vs. NHE at pH 7.4 in one study, 241 while a lower value of 600-605 mV vs. NHE at pH 7.4 can be found elsewhere. 215 This latter value shifted to 700 mV vs. NHE at pH 4.0, consistent with a change of 30 mV/pH unit. 215,217 Potentials lower than expected were ascribed to a slow HET. On the other hand, EHW for Tt-BOD coincided well with Em of the high-potential redox process (690 mV). 203 EHW was between 740 and 800 vs. NHE and changed by 5-30 mV/pH unit in the pH For the different MCOs adsorbed on various graphite electrodes, reproducible values were obtained for characteristic potentials of catalysis (OCP, onset, EHW and E°') and all correlated with values of the T1 Cu potential. Although one must be careful that CV curves are not in all cases centered on the potential of the first electron relay when enzymes present several redox centers, 246 it is clear that the T1 Cu strongly influences the potential of catalytic O2 reduction. Moreover, the catalytic potential correlates in numerous cases with a noncatalytic signal centered on the T1 potential, indicating that this latter must be directly facing the electrode surface. This strongly supports that DET at graphite electrodes under turnover conditions occurs via the T1 Cu, similarly with the O2-reduction mechanism in homogeneous catalysis. [START_REF] Shleev | Direct Electron Transfer between Copper-containing Proteins and Electrodes[END_REF]198,202 This conclusion is furthermore supported by the electrochemical behavior of a mutant of Mv-BOD, Met467Gln in which the axial methionine ligand of T1 Cu was replaced by a glycine residue, and its comparison to that of the recombinant wild type BOD. 247 The onset potentials for O2 reduction by both enzymes were consistent with the previously-determined T1 potentials, with a negative shift for the mutant at 230 mV vs. Ag/AgCl instead of 460 mV vs. Ag/AgCl at pH 7. 248 A further indication that a similar reaction mechanism is adopted is the consistency of the enzyme activity with that observed in homogeneous solution. pH dependence of maximal currents under kinetic control display a bell shape typical of reaction rate of MCOs vs.

pH. [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF]215,221,242 KM for O2 of resp. 0.11 mM and 0.27 mM at 500 and 50 mV vs. NHE at pH 7, 204 and 0.7 mM at 342 mV vs. NHE at pH 5 were determined for graphite-adsorbed Mv-BOD. [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF] These values are close to  0.2 mM at pH 7 measured in homogeneous system, which suggests that Mv-BOD preserves its native state after adsorption at the carbon material.

Even though the same tendency has been reported in the literature concerning the characteristic potentials at steady state and catalysis, the pH dependence of activity, KM constants…, slight variations have been observed. They are probably due to different surface states of the electrode, which result in varying affinities with MCO molecules (see section 4.1.4). However, no considerable deviation from homogeneous catalysis has been observed.

The same general conclusion can therefore be drawn for both laccases and BODs: since the properties of heterogeneous electron transfer at graphite electrode are similar to those in homogeneous reaction, the T1 copper must be the first electron acceptor of DET between

MCOs and carbon electrodes. This is furthermore consistent with T1 being situated not further than 8 Å from the enzyme surface. Electrons accepted from the T1 are then transferred to the TNC where the 4e --O2 reduction occurs.

Similarly, at modified gold electrodes, DET occurring at the T1 was also observed for both high and low-potential laccases. 233,249 Rv-laccase covalently bound on MPA-modified gold showed an onset at 500 mV vs. NHE, and a half-wave potential EHW = 400 mV vs.

NHE at pH 7. 214 For Rv-and Ma-laccases in a cationic polymer on gold, the onset was 450 mV vs. NHE at pH 6.5 and EHW was close to the non-catalytic process at 350 mV. 208 Addition of fluoride inhibited the catalysis both for BOD and laccase, [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF]214 while no change of the anaerobic voltammogram was observed, 214 which pointed to the inhibition of the internal electron transfer. All these data showed that a similar mechanism accounts for homogeneous and heterogeneous O2 reduction by MCOs, provided that DET to the T1 can be established.

The effect of surface modification on DET bioelectrocatalysis is discussed further in section 4.1.4

Possibility of endergonic electron-transfer

The general mechanism at carbon (or conveniently-modified gold) electrodes has been described in previous paragraph. However, a more intriguing phenomenon has sometimes been observed. Two consecutive catalytic waves in the cyclic voltammogram of O2 reduction were detected in following cases. When Th-and Tv-laccases were immobilized within a polymer on a gold electrode, an onset at 730 mV vs. NHE at pH 6.5 followed by a more drastic increase at 620 mV was recorded. 208 For both Mv-and Tt-BODs at graphite electrodes, two consecutive reduction waves were observed in the absence of diffusion limitation (Fig 15). 203,204 These processes coincided well with 2 quasi-reversible redox transformations at low and high potential observed under anaerobic conditions (Fig 15). 203,204,208 This curious phenomenon was particularly investigated for BOD electrodes. 203,204 Several hypotheses had to be examined before considering that it was catalytically relevant.

First, a damaged and a native active fraction of enzyme could have been simultaneously immobilized onto electrodes. Second, two different catalytically active BOD fractions could coexist. The catalytic currents decreased over time but did not result in change of the voltammogram shape, thus ruling out the first possibility. Moreover, since BODs had been purified the second hypothesis was very unlikely. 204 It was therefore proposed that a single enzyme form could adopt 2 different orientations at the electrode interface. Several redox centers of the oxidoreductase could therefore be addressed simultaneously. 204,208 This proposition is not contradictory with published BOD structures, in which both Cu clusters are situated within tunneling distance from the enzyme surface. 129,146 The low-potential wave was attributed to a catalytically active intermediate of the TNC 118,203,204 with a potential close to 400 mV vs. NHE. The redox transformation of this intermediate was shown to be O2-independent and pH-dependent. As a consequence, it was attributed to the redox transformation between the first and second partly reduced TNC intermediates, i.e. to the proton-coupled redox transformation from the peroxide intermediate to another state. [START_REF] Solomon | Multicopper Oxidases and Oxygenases[END_REF]250 This latter enzyme state would be comprised between peroxide and native intermediates. It is noteworthy that, later, catalytic oxygen reduction by Mv-BOD starting exclusively at low potential could be observed when the controlled immobilization of the enzyme at CNTs specifically approached the TNC close to the electrode surface. In that case, the low potential was attributed to a resting form of the enzyme. 216 Despite the disputable opinion concerning the redox intermediate involved in the above described catalytic mechanism, it is not unreasonable to consider that at least one IET O2 reduction by MCOs can therefore be decomposed in two parts, heterogeneous electron transfer between enzyme and electrode, and catalysis. If the second step is considered irreversible, two rate constants, k°, the standard surface rate constant, and kc, the catalytic rate constant, are enough to describe the kinetics of O2 reduction. It is noteworthy that kc normally follows a Michaelis-Menten dependence on O2 partial pressure. Moreover, only HET rates are supposed to be dependent on the electrode potential, and this dependency can be described in a first approximation by Butler-Volmer kinetics (equations 13 and 14).

𝑘 𝑓 = 𝑘°𝑒 -𝛼 𝑛𝐹 𝑅𝑇 (𝐸-𝐸°′) 𝐸𝑞. 13 𝑘 𝑏 = 𝑘°𝑒 (1-𝛼) 𝑛𝐹 𝑅𝑇 (𝐸-𝐸°′) 𝐸𝑞. 14
where kf and kb are forward and reverse interfacial electron-transfer rate constants, resp. for the reduction and oxidation, and k° is the standard heterogeneous electron-transfer rate constant at the apparent standard potential E°' of catalysis. α is the charge transfer coEfficient (usually considered equal to 0.5), and R is the universal gas constant. Missing parameters can be determined by fitting the experimental CV curves with the expression of steady-state current density given by:

𝑗 = 𝑛 𝐸 𝐹𝑘 𝑐 𝛤 𝑡 1+ 𝑘 𝑐 𝑘 𝑓 ⁄ + 𝑘 𝑏 𝑘 𝑓 ⁄ 𝐸𝑞. 15
where 𝑛 𝐸 is the number of electrons exchanged (a single electron exchange at the T1 copper is considered), F is the Faraday constant, and Γt is the total surface concentration of the enzyme (in mol.cm -2 ). This model was also extended for enzymes diffusing in a reaction layer, just replacing Γt by µ[E] in eq. 15, where [E] is the total concentration of enzyme in the reaction layer, and µ is the thickness of this latter. 252 µ is given by µ = √ 𝐷 𝐸 𝑘 𝑐 ⁄ , with DE being the diffusion coEfficient of the enzyme in the layer.

This model considers a very fast internal electron transfer (IET), therefore the enzyme behaves as if only one redox center able to take 2 different redox states (reduced or oxidized)

were present. Although this is a strong simplification, the resulting equation is able to generate curves that closely fit to the experimental voltammetry data. However it is quite disputable if E°' can account for a real thermodynamic potential of one of the Cu centers.

A refined, yet still simplified, model is described in Figure 16B. 204,233 The internal electron transfers between T1 and TNC are separated from the catalytic O2 reduction at the TNC and considered reversible, while all steps of O2 reduction at the TNC are still included in a global catalytic mechanism considered irreversible. 5 kinetic rate constants are therefore needed to account for the forward and reverse HET (k1 and k-1), forward and reverse IET (k2 and k-2) and irreversible catalysis (k3). Heterogeneous electron-transfer can still be described by Butler-Volmer type equations (equations 13 and 14, with kf = k1 and kb = k2), while catalysis is best described by Michaelis-Menten kinetics (equation 16). 233,253

𝑘 3 = 𝑘 3,𝑚𝑎𝑥 [𝑂 2 ] 𝐾 𝑀 + [𝑂 2 ] 𝐸𝑞. 16
In this approximation, IET is considered to occur as a bimolecular reaction of electron exchange between T1 and TNC. If forward and reverse IET rates are considered equal (i.e. k2

= k-2), the total current can be expressed as: 233

𝑗 = -𝑘 1 𝑘 2 𝑘 3 𝑛 𝐸 𝐹𝛤 𝑡 𝑘 1 (𝑘 2 + 𝑘 3 ) + 𝑘 -1 (𝑘 2 + 𝑘 3 ) + 𝑘 2 𝑘 3
𝐸𝑞. 17

It is interesting to note that by then considering fast IET, i.e. when catalysis and HET are the limiting steps, equation 17 can be further simplified to give back equation 15, with kc = k3, kf = k1 and kb = k2. 233,253 On the contrary, when IET is the rate limiting case, i.e. when catalysis is very fast (k3 >> k2), eq. 17 becomes :

𝑗 = 𝑛 𝐸 𝐹𝑘 2 𝛤 𝑡 1 + 𝑘 -1 𝑘 1 ⁄ + 𝑘 2 𝑘 1 ⁄ 𝐸𝑞. 18
Importantly, as evidenced by the absence of k3 in the equation, no dependence of the catalytic current on O2 concentration is expected in this latter case. 233 This catalytic scheme is however still incomplete as it does not consider all electron transfer steps and catalytic intermediates of Copyright 2014 American Chemical Society.

However, contrary to Blanford et al., they considered that the resting oxidized form was formed irreversibly from the native intermediate, and did not consider the formerly introduced X state. Moreover, as in the previous models, they considered reversible HETs.

Based on the assumption that the limiting step is O2 binding, they could derive an expression of the cathodic current that contains 8 rate constants (Fig 18 ):

𝑗 = 𝑛 𝐸 𝐹𝛤 𝑡 𝑘 1 ′ (𝑘 2 [𝑂 2 ] -𝑘 -2 𝑓) 𝑔(1 + 𝑘 1 𝑔 (1 + 𝑓 + 𝑘 3 𝑓 + 𝑘 -1 𝑘 -3 + 𝑘 1 ) 𝐸𝑞. 19
Where f and g are respectively given by:

𝑓 = 𝑘 2 [𝑂 2 ](𝑘 -3 + 𝑘 1 ) (𝑘 -3 + 𝑘 1 )(𝑘 -2 + 𝑘 3 ) -𝑘 3 + 𝑘 -3 𝐸𝑞. 20 𝑔 = 𝑘 -1 + 𝑘 -1 ′ + 𝑘 2 [𝑂 2 ] -𝑘 -2 𝑓 -𝑘 1 𝑘 3 𝑓 + 𝑘 -1 𝑘 -3 + 𝑘 1 𝐸𝑞. 21
The potential-dependent HET rate constants k1, k-1, k'1 and k'-1 can be described by Butler-Volmer kinetics, where k°, the exchange rate at 0 overpotential, can be further expressed by the semi-classical Marcus equation (equations 3 and 4 in part 2.3). This allowed to evaluate both the electronic coupling and the mean distance between enzyme and electrode. This theoretical approach was validated with experiments performed with Thermus termophilus laccase.

Resting forms of MCOs

Electrochemical experiments did not only allow to determine all rate constants of O2 reduction on MCOs modified electrodes, but were also useful to evidence and study different resting forms. 127,165,255,256 The resting oxidized (RO) form, an inactive fully oxidized form of MCOs, already characterized by spectroscopic, crystallographic and DFT techniques, is known to re-enter the catalytic cycle only upon reduction. Similarly, activation of a fraction of enzymes at reducing potentials was evidenced in electrochemistry both in CV or in chronoamperometry (CA) (Fig 17B), but only for high-potential MCOs (Tv and Coriolopsis gallica laccases, and Mv-BOD) and not for low potential (Bs-BOD or Rv-laccase). 165 The inactive state, formed outside the catalysis, was identified as the RO form. By simple electrochemical experiments, the formation rate of this RO form was proved to increase with decreasing pH. In addition, while the formation of RO was usually considered to be reversible, the fraction of inactive form did not tend to 1, thus supporting an equilibrium between RO and an active state (indicated by the red arrow for the reverse reaction added in the catalytic cycle on Fig 17A). The existence of another inactive "X-state", to the best of our knowledge not described elsewhere, was also evidenced, based on the observation that under certain conditions of pH and potential a fraction of enzymes seems to inactivate during catalysis and to reactivate outside of catalysis. 165 Different scenarios were envisioned to elucidate the origin of the "X-state": it could either be formed from the PI and then evolve to give the NI; or be the result of reversible transformation of NI or PI. Experimental data permitted to eliminate the first hypothesis since the PI to NI transition is pH-independent. The second hypothesis was also ruled out by a modeling approach. The "X state" was therefore assumed to be in a "dead-end" equilibrium with PI. (Fig 17)

Pronounced hysteresis with shift of O2-reduction onset towards a more negative potential, characteristic of an inactivation, followed by reactivation at reductive potentials, has also been observed for fungal BOD from Magnaporthe oryzae 127,255 and in the presence of Cl - for bacterial BODs from Bacillus subtilis and Bacillus pumilus (Fig 19)

. 256 Complementary UV-Vis and spectroscopic studies allowed to assign this feature to the "alternative" resting form (AR), a partially reduced resting form of MCOs. 127,255 Its formation and reactivation were then fully characterized by electrochemistry, showing that its rate of formation increased upon decreasing pH and increasing Cl -concentrations. AR formation did not require the presence of O2 which is therefore not involved as bridging species in this form, and sufficiently oxidizing potentials were needed to partially oxidize the Cu centers. Contrary to the case of RO formation, all enzymes were converted to the AR form, supporting the hypothesis that this transformation is irreversible. Enzymes in the AR form only reenter the catalytic cycle upon full reduction.

Halides affecting the direct electron transfer

As discussed above, in homogeneous solution, by using combined spectroscopic and DFT studies, Cl -has been shown to bind to T1, and F -to the TNC. In terms of inhibition mechanism, Cl -and I -are competitive inhibitors while F -is non-competitive with respect to electron donor. In DET mode, MCOs are also to some extent inhibited by halides. 234,257,258 Inhibition of high potential catalysis by F -for BOD or laccase both in DET and MET 186,203,204,220,221,257,259 is in agreement with F -binding at the TNC and/or inhibition of the IET. Inhibition by Cl -has been also observed. For example, the current delivered by Cerrena maxima laccase immobilized on graphite particles declined by 30% upon addition of 100 mM NaCl. 258 Addition of 200 mM Cl -decreased the current by 50% for laccase from Trametes hirsuta adsorbed in nanoporous gold electrodes at pH 4. 186 However, higher resistance to Cl - even for laccases has been sometimes observed in DET. It suggests that the electrode, in particular when it is nanostructured, prevents Cl -access to the T1. 257,259 For example, when

Th-laccase was covalently bound on LDG electrodes, DET was stable in presence of 150 mM NaCl, while MET with a diffusing mediator was completely inhibited for the same chloride concentration. 259 Potential shifts similar to those described above, have also been observed in DET upon addition of halides. EHW of Cerrena maxima laccase shifted 94 ± 3 mV in the negative direction in presence of 150 mM Cl -at pH 5. This effect disappeared as soon as the electrode was transferred in a chloride-free buffer. 258 The EHW shift observed with Trametes laccase immobilized on SW-CNTs increased with increasing Cl -concentration, being respectively 60 mV at 10 mM and 150 mV at 100 mM Cl -. 257 For the same enzyme, similar potential shifts were observed upon addition of I -. 257 This potential shift can be explained by Cl -or I -binding at the T1. In presence of F -, the low-potential anaerobic redox process observed for Mv-BOD decreased, consistently with F -binding to TNC. 204 More surprisingly, a negative shift of the high potential catalysis has also been observed upon addition of F -. 234,256,257 The slight 30 mVdecrease in conjunction with 90% current loss was attributed to F -binding both at the T1 and the TNC, thus shifting the T1 potential and blocking IET at the same time. 257 A higher 100 mV-decrease for Didymocrea laccase at pH 4, with lesser-pronounced inhibition, was attributed exclusively to F -binding to the TNC. In this case, surface-enhanced Raman spectroscopy (SERS) studies of the enzyme orientation suggested that the T1 was relatively far from electrode surface, so that DET was more likely occurring through the TNC. 234 Interestingly this study is the first to propose a DET via TNC occurring at a higher potential than T1. 234 However, this is not a direct demonstration that DET to TNC is feasible and additional experiments with T2-depleted MCOs would be required. A redox titration showed that the T1 potential, ca. 100 mV lower than the half-wave potential correlated to TNC, was little affected by F -addition. Exactly the same shift was observed for Bp-BOD, while the primary electron acceptor in that case was supposed to be the T1 Cu. 256

Rate limiting steps of O2 reduction

Different studies have shown that the rate limiting step during O2-reduction catalysis by MCOs connected in DET mode varied depending on the experimental conditions and applied potentials. The enzyme affinity for O2 was shown to depend on electrode potential, which means that "apparent" Michaelis constants are measured at a given potential. This led to the conclusion that the rate determining steps of the catalytic mechanism also depend on the electrode potential. 233 In the case where an endergonic IET was considered, the calculated IET rates were influenced by pH, due to the different pH dependencies of the high and lowpotential intermediates. The thermodynamic driving force decreased at basic pH, so that IET started to be the rate limiting step in alkaline solution. 204 The same conclusion was drawn from analysis of the O2-reduction CV shape in the absence of O2 diffusion limitation. At low pH, the CV exhibited a trailing edge well described by the model introduced by Léger et al.,

whereas it was almost sigmoidal at high pH. [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF] This was attributed to fast IET and catalysis at low pH. In this case, both current and turnover rate are determined by the rate of HET, which increases at high overvoltage. On the contrary, at high pH, slow IET and catalysis are determined by the turnover rate. T1 is therefore the electrochemical control center, and defines the potential of electro-catalysis. The authors proposed that the process at high pH is limited because the deprotonated carboxylate D105 close to TNC cannot supply the required protons. 17

Inhibition by H2O2

While studies describing the effect of pH and halides on MCOs activity are widespread in the literature, the impact of hydrogen peroxide on laccases and bilirubin oxidases has only more recently attracted attention. In the context of a glucose/O2 biofuel cell where glucose oxidase is used at the anode, it is hypothesized that H2O2 generated during the oxidative half reaction of glucose may affect BOD or laccase. The first paper addressing this issue was reported in 2010 by Calvo et al. 260 Their cathode was composed of a self-assembled layer of laccase from Trametes versicolor or Trametes trogii and osmium complex bound to poly(allylamine). They evidenced the inhibition of biocathodes by H2O2 during oxygen reduction. In 2015, they extended their work and demonstrated the reversible non-competitive inhibition of laccase by H2O2 both in MET 261 and DET. 253 The authors attributed the inhibition of the laccase to the oxidation of the T3 Cu by H2O2, earlier hypothesized by Solomon et al.,262,263 which therefore interferes with the mechanism of oxygen reduction. [START_REF] Solomon | Multicopper Oxidases and Oxygenases[END_REF] Minteer and co-workers also demonstrated the reversible non-competitive inhibition of laccase by H2O2 whether the laccase operated in DET or MET (when ABTS was used as a redox mediator). 264 They furthermore studied the effect of H2O2 on bilirubin oxidases. [265][266][267] They demonstrated that, unlike laccases, Mv-BOD was irreversibly inhibited by H2O2 (Fig 20). However, the exact mechanism of inhibition still has to be understood. Fortunately, it is less complicated to overcome the problem of H2O2 than the pH or Cl -effect. Different strategies have been developed to protect the MCO cathode. To prevent the formation of the inhibiting H2O2, O2-independant glucose-oxidizing enzymes such as glucose dehydrogenase can be used. Alternatively, H2O2 can be degraded via the formation of bi-enzymatic systems such as glucose oxidase/horseradish peroxidase, glucose oxidase/catalase or BOD/catalase. In this latter case, H2O2 is dismutated to water by the second enzyme, thus protecting the MCO.

Although these studies on H2O2 inhibition are of interest from a mechanistic point of view, they are not necessarily relevant for the field of biofuel cells since highly concentrated solutions of H2O2 have been used (mM range and up to 20 mM). For example, a carbon fiber coated with 35 wt% of an engineered glucose oxidase 268 and an adequate redox polymer for a total loading of 2000 µg.cm -², would generate 20 mM H2O2 after 4.2 months of continuous operation in presence of 5 mM glucose in a saturated O2 buffer pH 7.2.

Effect of methanol

Palmore et al. envisioned the use of enzymatic electrodes in direct methanol fuel cells, which typically operate with concentration of MeOH comprised between 1 and 5 M. 269 The authors showed that the activity of laccases either in solution or entrapped in polypyrrole film was decreasing upon increasing MeOH concentrations. 270 However, the decline was slower for the immobilized enzymes. They attributed the loss of activity to the replacement of water molecules by MeOH within the enzyme. 271 In a series of three papers, Calabrese-Barton et al.

also investigated the impact of methanol on the oxygen reduction current delivered by an electrode modified with an osmium polymer and Tv-laccase. When the bioelectrocatalysts were immobilized on a carbon-fiber paper support incorporated in a membrane-electrode assembly, the authors demonstrated the tolerance of laccase to methanol. A 6% increase in current density at Ecell = 0.2 V was found for 10 M methanol as compared to 1 M methanol. 272 They later demonstrated that in presence of 1 M methanol, the performance of a biocathode based on Th-laccase on carbon cloth was only reduced by 4.5%. This decrease was explained by a de-swelling of the redox polymer rather than by the enzyme deactivation. 273 In 2011, 274 they showed that the sensitivity of O2 reduction to methanol depended not only on the concentration of MeOH, but also on the osmium polymer used and particularly on its redox potential. The loss in current was nil below 2.5 M methanol, it was 30% between 2.5 and 5 M, while 100% current was lost above 7.5 M. For a 7.5 M methanol concentration, the authors showed that Tv-laccase inactivation was irreversible. 235 In the first studies of DET catalysis by MCOs, the main efforts were directed towards elucidation of O2 reduction mechanism by enzymes directly connected at the electrode (see section 3.4), and less importance was attributed to electrocatalytic performances. In this section, we describe works performed to achieve Efficient enzyme connection in DET, with the purpose to use MCOs as cathodic enzymes in EBFCs. Important parameters are therefore high onset potential, high current densities and stabilities. To obtain high onset potentials the enzyme must be connected to the electrode via the T1. Lots of efforts have been devoted to reach large current densities at sufficiently high potentials. This involves immobilization of high amounts of enzymes, and high apparent catalytic rates. We consider first random orientation of the enzyme at the electrode, and describe current densities reached at different planar electrodes. Since current densities remained limited in that configuration, two main strategies have then been developed. On one hand, current magnitudes could be increased by immobilizing higher number of catalysts at a given geometric surface. Nano-or micro-structuration of the electrode surface has been envisioned in this context, to multiply the real electroactive surface area of an electrode. We develop this in part 4.1.2 and 4.1.3. On the other hand, the enzyme electronic connection had to be optimized, which mostly relied on achieving functional orientation of the enzyme at the electrode surface. We describe strategies developed to ensure optimal orientation of MCOs at both planar and structured electrodes in part 4.14. Then, we discuss the stability issue in part 4.1.5.

Contrary to previous section describing the mechanistic study of MCOs, and still for the sake of consistency with the literature, we will here quote the potentials versus the Ag/AgCl reference electrode, unless stated otherwise. The potentials are therefore typically  200 mV lower than when quoted vs. the NHE reference electrode.

Planar electrodes

"Planar" Carbon electrodes

In the first studies, MCOs have been immobilized on various kinds of carbon electrodes: glassy carbon (GC), 235 various types of graphite (low density graphite (LDG), spectroscopic graphite (SPG), pyrolytic graphite (PG), basal plane or edge plane highly oriented pyrolytic graphite (HOPG)…), 203,210,215,221,235 and screen printed carbon. 279 Except glassy carbon, carbon surfaces are however rarely strictly planar. While it is clear that DET catalysis has been observed with different MCOs simply adsorbed on most of these materials, it is difficult to compare their efficiency as enzyme support. Indeed, it is well-known that carbon electrodes differ from one another as far as microstructure, roughness, and chemical functions are concerned.

To give an idea of the magnitude of currents or current densities recorded with MCOs adsorbed on carbon electrodes, we list below some values obtained at 0 V vs. Ag/AgCl. Tvlaccase adsorbed on edge plane graphite gave currents of  40 µA. In that case, the maximum rate at which O2 could be reduced, deduced from the intercept of a Koutecky Levich plot, indicated a turnover rate of ca. In the very first study of DET by a BOD, Mv-BOD was also adsorbed on different electrode materials: GC, HOPG and PFC (plastic formed carbon). PFC and edge-plane HOPG gave the best results (200 µA.cm -2 with HOPG at pH 7), even exceeding the highest current densities obtained at that time with laccase. It was proposed that surface functional groups and/or nanostructure governed the surface ET kinetics and adsorption property of BOD, but no more details were provided. 235 In the following reports, the current densities diverged. The differences cannot be assigned merely to different operating conditions; they may be explained by the fact that, as already mentioned, the electrode surface was not well characterized. For example  25 µA.cm -2 at pH 7 were recorded with Mv-BOD adsorbed on HOPG, 245 and  30 µA.cm -2 for the same enzyme adsorbed on SPG. 215 Tt-BOD adsorbed on SPG led to 20 µA.cm -2 at pH 7 203 and  0.5 mA.cm -2 under O2 at pH 4, 204 i.e. the results differed by more than one order of magnitude. Although it is not possible to identify the exact origin of this difference, we can note that it is much higher than those observed in studies of pH influence performed in homogeneous solution.

DET on carbon electrodes was also routinely used to characterize less widespread or newly discovered MCOs. 166,181,280,281 Interestingly, the proof of concept of some EBFCs was established with enzymatic planar carbon cathodes. Tv-laccase adsorbed on graphite provided the cathode of the first totally enzymatic H2/O2 BFC. 282,283 Similarly, the first mediator-and cofactor-free EBFC based on cellulose dehydrogenase (CDH) operating in neutral chloridecontaining buffers as well as in serum relied on Mv-BOD adsorbed on SPG electrode as a cathode. 241 A cathode based on Mv-BOD adsorbed on a LDG electrode was also combined to a bi-enzymatic anode for the 6 e --oxidation of glucose in a membrane-less EBFC operating in a buffer mimicking human physiological fluid. 284 This is detailed further in section 6.

Other planar electrodes

DET was also achieved for different MCOs on bare or modified gold surfaces. Since this is explored in details in section 3.4.4. and 4.1.4., we only remind here some current densities obtained when MCOS were randomly immobilized. 221 For most laccases, no DET current was recorded when they were adsorbed on bare gold. Rv-laccase on MPA-modified gold gave 84 µA.cm -2 at pH 7. 214 With Mv-BOD, very different values ranging between 5 and more than 100 µA.cm -2 were recorded on bare gold electrodes. [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF]225,228,231 Some less conventional materials have been proposed, like boron doped diamond (BDD) or semiconductors. BDD has a wide working potential window, low background current, high stability, tunable hydrophilicity and optical transparency. Direct O2 reduction by

Cerrena unicolor laccase was demonstrated on BDD 285 with the enzyme either in solution or immobilized in a liquid crystalline cubic phase at the electrode. However, DET currents were very low (< 1 µA.cm -2 ) while much higher currents were achieved upon addition of a redox mediator (25 µA.cm -2 ). This proved that the enzyme orientation was not optimal. 285 Tvlaccase was immobilized on p-type nanostructured silicon (111) as a model semiconductor. 286 Under illumination with visible light, the material was theoretically able to supply electrons for the enzyme activation. O2 reduction started about 200 mV more positive when laccase was present, but still at a very low potential (-0.24 V vs. SCE). The plateau current was 30 µA.cm - 2 . 286

Strategies for increasing the enzyme loading

To overcome the limitation in current densities, and reach values usable in EBFC, the surface concentration of enzymes on electrode surfaces had to be improved. A study by Ikeda et al. showed that multiple layers of enzymes could be connected in DET configuration on a planar electrode. 252 Multilayers of Mv-BOD were entrapped electrostatically in poly-L-lysine (PLL) on a PFC electrode. In O2 saturated buffer, the process was diffusion controlled. A plateau at 845 µA.cm -2 was reached upon stirring at 1400 rpm, while only 300 µA.cm -2 were recorded when BOD was simply adsorbed on this PFC electrode. Calculations according to the model presented in section 3 indicated that the reaction layer was 0.2 µm-thick, i.e. much smaller than the real thickness of the BOD-PLL film (50 µm). HET standard rate constant was 19 s -1 , i.e. about 10 times smaller than for adsorbed BOD (178 s -1 ). These two calculations highlight that this design was not optimized, although it allowed to connect more enzymes and to reach higher currents. 252 Another option for immobilizing more enzymes is to increase the roughness of the In both cases, currents increased compared to the planar electrode: 0.27 mA.cm -2 were reached at 0 V in the first case, supposedly due to higher number of electrochemically active laccases 287 , and 102 µA.cm -2 in the second thanks to the nanostructure. 288 The importance of the roughness has also been highlighted with Mv-BOD immobilized on a planar electrode together with platinum group metal-free catalysts. Current densities did not only increase because of the inherent properties of the catalysts, but the contribution of the increased roughness was also important, providing better results when pores within the ratio 3-5 nm where formed. No significant increase occurred with larger pores (12-20 and 40-80 nm). 289 Alternatively, a large variety of materials can be used to enlarge the electroactive specific surface area (ESSA) of the electrode. Matrices based on nanomaterials with a high surface/volume ratio have been developed. Typical nanomaterials are carbon nanotubes, nanoparticles or nanofibers (resp. CNTs, CNPs or CNFs), and gold nanoparticles (AuNPs).

Various metallic nanoparticles (Fe, Pt...) have also been described. Ideally, it would be beneficial to find materials with hierarchical porosity allowing Efficiently immobilizing enzymes and preventing mass transport limitations. Therefore, highly porous 3D materials like mesoporous gold or carbon with the adequate and tailored porosity have also been engineered. In the following of this section we will focus on studies where the nanostructuration of the electrode has been evaluated, first with carbon and then with gold-based materials.

Carbon nanostructures

Carbon based materials are especially available, cheap, biocompatible, and can be produced in a variety of structures. Moreover, they are chemically and electrochemically inert.

Carbon nanotubes

Discovered in 1991, carbon nanotubes (CNTs) 290 are rolled layers of sp 2 carbon atoms.

Single-wall (SW) CNTs consist of one single graphene layer, while multi-wall (MW) CNTs are composed of several layers. Their typical diameters range from 1 to few nm (SW-CNTs)

or 20-50 nm (MW-CNTs), and their lengths are in the range of few µm to few tens of µm.

Their nanometer-sized dimensions impart them a high aspect ratio. Moreover, they possess a high electronic conductivity, and a huge mechanical strength. 291 Their surface chemistry can be divided into two types. Their walls, made of sp 2 graphene carbon atoms, are hydrophobic pristine, while the defects on the side walls and edge sites at the open ends are full of hydrophilic carboxy groups. As for their electrochemical properties, it is widely accepted that atoms at the ends are like edge planes of HOPG and favor electrochemical activation; and atoms at the sidewalls are like basal plane of HOPG and show very slow electron transfer kinetics. 292,293 However, recent SECM studies have demonstrated that electrochemical properties are more likely to be similar at the ends and at the walls. 294 It is often stated that DET is favored by the appropriate dimensions of CNTs. It is true that DET has been often reported on CNTs, whether it was a catalytic [295][296][297] or a non-catalytic signal, [295][296][297][298] while the same enzyme on a planar electrode gave no DET. It must however be noted that in the case where no DET was observed on the planar electrode, this latter was most of the time made of bare gold or GC. 295,296,298 The properties of these surfaces towards DET have already been discussed in previous section. Therefore, the reasons why DET is appearing are most of the time unclear. It could be due to an increase in ESSA, and thus an increase in enzyme loadings, that makes visible a signal non detectable with fewer enzymes.

Alternatively, it could come from chemical functions inducing appropriate immobilization of the enzyme, to the increase in roughness, or to the peculiar properties associated to nano-size of CNTs. The very few fundamental studies will be discussed herein.

Preparation of the biocathode

Both SW-CNTs 142,296,297,[299][300][301][302][303][304] and MW-CNTs 218,244,295,298,303,[305][306][307][308] have been used to immobilize the MCOs. No clear trend could be established if one of these two kinds of CNTs induces more Efficient O2 reduction. With the purpose to allow for Efficient DET, CNT-based 3D-structures for MCO immobilization have been built following various methods. In many studies, a CNT suspension in water, buffer, or organic solvent was deposited on a planar electrode and left to dry to allow for the formation of a 3D network. 142,244,299,309 However, CNTs tend to aggregate due to Van-der-Waals interactions, so that their high surface area is not entirely exploited for the electrochemical reaction. Since it is desirable to form electrodes with a high degree of CNT dispersion, a specific care has to be used for homogenization of the suspension in different solvents. Surfactants, or biopolymers like cellulose, can for example help the dispersion and improve compatibility with biomolecules. 295,310,311 Another issue arising from this method is the effective electrical connectivity of CNTs with the Reproduced with permission from [312]. Copyrigth 2016 Elsevier

Another possibility is to fabricate ordered arrays of CNTs grown by chemical vapor deposition on the electrode, 257,313,314 or by electrophoretic deposition. 313 Covalent binding of CNTs on Au 218 has also been performed. Free standing CNT-based electrodes have been designed, either in the form of buckypaper (BP), 315 fibers 310 or 3D pellets. [316][317][318] These structures have ensured DET for different MCOs: Mv-BOD, 142,296,302,303,305,308,311,319,320 Mo-BOD, 310 Th-laccase, Rv-laccase, 320 Tv-laccase, 295,299,321 Cerrena unicolor laccase, 301,322 Agaricus bisporus laccase, 313 or even tyrosinase. 317 Similar protocols have been used for the immobilization of MCO, whatever their origin, and their peculiar surface properties have been little considered. This latter point will be discussed further in section 4.1.4.

CNTs were often treated with HNO3 299,300,323 or H2SO4 before use, which eliminates impurities but also introduces defects and oxygenated functions. 142,313,320 Enzymes have been directly adsorbed on the CNTs network, 299,320 mixed with CNTs prior to the formation of the network, 297 covalently bound using EDC-NHS on CNT carboxy groups, 142,244 or cross-linked with glutaraldehyde. 295,296,323 Another possibility has taken benefit of the π-conjugated systems of CNT walls to induce functionalization, using a bi-functional molecule that bears a defined aromatic functionality to bind by π-π stacking to the CNT walls, and a functional tether group for attachment of the enzyme. 324,325 This method has been used for example to introduce pyrene-butyric succinimidyl ester (PBSE) 306,307,321,326,327 or pyrene butyric acid. 308 The impact of the chemical functionalization is discussed further in section 4.1.4. The strong interaction between concavalin A immobilized on SW-CNTs and the glycosylated laccase has been exploited. 328 The laccase presented a higher activity towards oxidation of organic substrates than the enzyme directly adsorbed, however no electrochemical catalysis was proved in this case.

CNT properties affecting direct electron transfer to MCOs

Very few studies have been performed to establish a clear link between the different kinds of CNTs, or the different protocols used to immobilize the MCOs, and the efficiency of the DET. The electrocatalytic activity of cross-linked Tv-laccase 329 and Mv-BOD 309 on two types of CNTs has been compared. The first kind had a uniform inner diameter (10-30 nm), carboxylic surface groups, a higher graphitization degree, a better conductivity and a higher electroactive surface area. The second kind had tapered inner tube diameter and mostly quinone surface groups. Without much surprise both enzymes exhibited a nicer behavior on the first kind. For Tv-laccase, a faster electron transfer, a clearer non catalytic signal, and 6 times higher current densities in catalysis (reaching 25 µA.cm -2 ) were observed. 329 For Mv-BOD, the non-catalytic signal was observed only on the first kind that led also to 5 times higher current densities (90 µA.cm -2 ). However, currents did not reach a plateau for any of these CNT-types, meaning that the interfacial electron transfer was a quite slow process. 309 In another report, the authors tried to compare electrophoretic deposition and chemical vapor deposition (CVD) of SW-CNTs on porous silicon. 313 Better DET for a covalently linked laccase was obtained with electrophoretically-deposited CNTs, but these results have to be considered carefully since porous silicon was covered with a conductive gold layer, while an insulating layer was formed during CVD growth. Therefore, higher cathodic currents could be assigned to a better conductivity of the electrode/nanomaterial interface rather than to better inherent properties of the material. Highly crystalline SW-CNTs (grown by CVD on a gold wire) were also compared to Toray carbon paper treated with UV-ozone. 257 Adsorbed laccase from Trametes sp. led to catalysis on both materials but kET constants were respectively 1200 and 800 s -1 , reflecting higher degree of appropriate orientations on SW-CNTs. The coverage of electrochemically active enzymes was also much higher on these latter (3.6.10 -12 against 0.31.10 -12 mol.cm -2 ), translating a higher affinity.

Recently, the effect of the length of MW-CNTs has also been evaluated 330 The spatial homogeneity of the mediator-less bioelectrocatalysis in a CNT composite was also evaluated by SECM. Cerrena unicolor laccase was encapsulated in a sol-gel silicate matrix with SW-CNTs. ORR was recorded in the presence of the laccase at Eapplied = 0.3 V and compared to a control experiment with the laccase at OCP. The activity was more evenly distributed than in a silicate film without CNT where proteins were shown to aggregate. 332

Increasing the current densities through CNT networks

In evaluating the benefits of CNTs, performance improvements are reported most of the time in terms of higher current densities. Importantly, very little change in onset potentials has been reported. Although CNTs were shown to increase DET efficiency, currents were small when MCOs were first immobilized in CNT-networks: 5 µA at the plateau with Mv-BOD and Tv-laccase cross-linked with glutaraldehyde on MW-CNTs for the first demonstration; 295 28 µA.cm -2 under air and 102 µA.cm -2 under O2 with Mv-BOD immobilized on SW-CNTs. 300 Later the increase in current was mostly assigned to the higher electroactive surface area rather than to a better electrical connection. We give below some examples of MCOs immobilized in 3D networks constructed by drop casting of a CNT-solution on the electrode.

A 300-fold current increase was reported for Mv-BOD covalently linked on SW-CNTs compared to bare PG under 100% O2 at 25°C, giving almost 1 mA.cm -2 . 142 Laccase embedded in a bioceramic (Hydroxyapatite)-CNTs self-assembled nanocomposite led to around 100 µA.cm -2 under air with an OCP of 0.56 V vs. Ag/AgCl at pH 6, and the currents were diffusion limited. 323 When Mv-BOD was immobilized in a MW-CNT/cellulose network, 800

µA.cm -2 were reached under air at pH 5, and 60% were still recorded after 45 h at the constant potential 0.2 V vs. Ag/AgCl. 311 Cerrena unicolor laccase incorporated in SW-CNTs covered with a transparent and highly viscous cubic phase of monoolein gave 200 µA.cm -2 . This was however not optimal since the addition of covalently bound ABTS raised the current to 400 µA.cm -2 , indicating that some enzymes could not participate in DET currents. 301 In another study, when Mv-BOD was covalently attached at MW-CNTs, themselves covalently attached on a gold electrode, currents increased up to 500 µA.cm -2 in an unstirred solution. 218 Although these results already represented a considerable increase compared to most bare carbon electrodes, the current densities remained limited. Therefore, specific and oriented attachment of the enzymes has been the main concern in most of the following studies (section 4.1.4.).

Some of the developed cathodes were successfully used in EBFCs, for example with MW-CNTs oxidizing ascorbate at the anode, 295 or with glucose dehydrogenase (GDH) on SW-CNTs at the anode. 296,299,300,302 The first Glucose/O2 EBFC based on SW-CNTs showed OCV = 0.8 V and Pmax = 9.5 µW.cm -2 at Ecell = 0.52 V at pH 6. 299 Other devices included a H2/O2 EBFC with a hyperthermophilic hydrogenase at the anode leading to OCV = 1.1 V, more than 300 µA.cm -2 at Ecell = 0.65 V, and less than 40% loss after 24 h at an applied Ecell = 0.65 V. 142 A CNT-based cathode was also used as a proof of concept in a photosynthetic electrochemical cell for the light-to-electricity conversion with water as the only input. Water was oxidized by thylakoid membranes while the laccase reduced O2, regenerating water in the system. The maximal generated power was 5 µW.cm -2 . 306 The hydroxyapathite-CNT cathode combined with glucose oxidase (GOx) also gave an OCV of 0.58 V and Pmax = 15.8 µW.cm -2 at 0.28 V. 323 In a fructose/air one chamber EBFC, OCV = 663 mV and Pmax = 126 µW.cm -2 at pH 5 were obtained. 90% of the initial power remain after 12 h, and 52% after 87 hours at

Ecell = 0.35 V. 311
To obtain a hierarchical porosity, CNTs were also deposited in an already porous material. 3D hierarchically structured composites were built by growing MW-CNTs by CVD on CMF with diameters ranging from 6 to 9 µm, 314,333,334 or by depositing CNTs on a porous carbon fabric to form a gas diffusion layer (GDL), 305 or by coating CNTs-Anthracene or CNTs-COOH in a highly porous carbon felt. 335,336 In the latter case, after deposition of CNTs-COOH by LBL, ESSA increased 5 times, and cathodic currents reached almost 1.5 mA.cm -2 at 0 V. This allowed the development of a H2/O2 EBFC with an OCV of 1.12 V and net currents of 765 µA at short circuit. Absolute Pmax was 410 ± 5 µW which represented a successful scale-up compared to a previously reported device, 142 indicating that the diffusion limitation of O2 was overcome by the porosity of the electrode. 335

Free-standing CNT-based 3D layers

Besides porous random networks, CNTs can be used as electrospun fibers 310,337 or buckypaper. [319][320][321]326,327,338,339 BP is a flexible self-supporting membrane-type film made of densely packed CNTs maintaining close contact by Van der Waals interactions. It is obtained by filtration of CNTs dispersion. This nanoporous material has attractive properties such as very high surface area, high porosity, chemical and electrochemical stability, high mechanical strength and high conductivity (a value of 51.2 S.cm -1 has been reported 326 ). Importantly for the construction of potentially implantable biodevices, it has also a low toxicity both in vivo and in vitro. 340 To quote some performances: BP enabled an enhancement in current densities for Th and Rv-laccases as well as Mv-BOD. Currents reached 120-220 µA.cm -2 compared to 12-35 µA.cm -2 for spectrographic graphite, leading to a mass-transport limited regime. 320 An EBFC based on BP had an OCV of 710 mV and Pmax > 100 µW.cm -2 319 and this device was furthermore able to work in urine and saliva. 338 BP was also compared to "conventional" MW-CNTs electrodes where CNTs formed aggregates of 500 µm in diameter. Tv-laccase was adsorbed on a 30 µm thick BP made of 0.9 mg.cm -2 MW-CNTs homogeneously dispersed, and presenting a median pore diameter of 48 nm. 315 Reporting the current to the mass of material showed that properties of MW-CNTs were better exploited in BP: at  0.54

V vs. Ag/AgCl, the current was 471 ± 64 µA.mg -1 , which represented a 68-fold increase. In terms of current densities, they were an order of magnitude higher than previously reported: 487 ± 117µA.cm -2 against only 50 µA.cm -2 . 315,326 Miyake Such homogeneous entrapment was also achieved by trapping Magnaporthe oryzae BOD and SW-CNTs in wet-spun fibers. 310 Homogeneous distribution of enzymes in the resulting material was confirmed by fluorescence imaging and energy dispersive X-Ray analysis (EDX). Amounts of immobilized BOD were higher than when the enzyme had to diffuse in a previously made CNT-fiber. The currents increased 3 times, and no further increase was recorded upon addition of ABTS, indicating a proper orientation of the enzymes. In the previous system, 50% increase in current was recorded after ABTS addition.

Cosnier et al. developed free standing pellets obtained by mechanical compression of

CNTs mixed with enzymes. They showed that a 3D porous network was formed with 10 nm thick CNTs, and that pores inter CNTs were ranging between 50 and 100 nm. 317 The CNT matrix had a high surface area and high porosity. With 250 mg MW-CNTs and 50 mg enzyme, the electroactive surface area was 52 cm 2 , the conductivity 3300 S.m -1 , and the percentage of void 43%. 316 This technique allowed in particular to record the first DET-based O2 reduction by a tyrosinase 317 , and to develop highly powerful cathodes and EBFCs.

Cathodes exhibited for example 5.8 mA.cm -2 at 0 V under O2 with the quantities mentioned above, 316 or a current plateau of 0.17 mA at pH 7 with a smaller electrode made of 35 mg MW-CNTs and 15 mg enzyme. 342 The cathode OCP was shown to increase from 740 to 820 mV vs. NHE with increasing enzyme amounts. 316 The first glucose/O2 BFC using these electrodes had an OCV = 0.95 V and Pmax = 1.3 mW.cm -2 at Ecell = 0.6 V in a pH 7 buffer containing 50 mM glucose. 316 Upon improvement of the bioanode, Pmax was 1.54 mW.cm -2 in an air-saturated buffer pH 7 containing 50 mM glucose. 342 Impressive stabilities were recorded. [316][317][318] An EBFC based on 35 mg MW-CNTs and 15 mg laccase at the cathode and both GOx and catalase at the anode had an initial Pmax of 160 µW.cm -2 and OCV of 0.75 V at pH 7, with a buffer containing 5 mmol.L -1 glucose under air. It was operated regularly during one year with 1 hour constant-load discharges, and kept in a pH 5 buffer when not used. 22%

of the initial power was still recorded after 1 year. 318 However, this corresponds to the "apparent stability" described by Armstrong et al. [START_REF] Cracknell | Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis[END_REF] With a high amount of enzymes, the current is indeed limited by the substrate diffusion, therefore enzymes can be lost from the electrode surface or inactivated without altering the current magnitude.

Graphene

Discovered in 2004, graphene is a honeycomb crystalline lattice of sp 2 carbon atoms with high in-plane electron conductivity, mechanical strength, and transparency. 343 It can be obtained via mechanical exfoliation of graphite, chemical synthesis like chemical vapor deposition, or by chemical or electrochemical reduction of graphene oxide (GO). 237,[344][345][346] This latter method restores partially the sp 2 structure and electron conductivity of graphene, while conserving oxygen functionalities. Despite its promising properties, graphene has not yet been widely used in EBFCs. [START_REF] Karimi | Graphene Based Enzymatic Bioelectrodes and Biofuel Cells[END_REF]347 Graphene has been used as a building block for the construction of 3D networks, either alone or in combination with CNTs or AuNPs. [348][349][350] Unless otherwise stated, in the cases described here ORR started at a potential close to T1 of the enzyme, proving that currents were due to DET between the MCO and the electrode. Moreover, like in most studies, the DET performances will be described in terms of current densities.

In earlier studies, it was not obvious that graphene could be of any interest for DET of MCOs, and that DET was obtained at all. An ORR onset at potentials below 0 V vs. Ag/AgCl was for example reported for Mv-BOD immobilized on graphene, while no control without the enzyme was presented, suggesting that direct electroreduction of O2 on graphene was more likely recorded. 351 In another case, with a graphene-modified electrode, Tv-laccase in solution reduced O2 at 0.25 V vs. Ag/AgCl. This potential was attributed to a redox process of hydroxyle, which suggests a MET mechanism with hydroxyle acting as a redox mediator. 345 A combination of GO and cobalt hydroxide with chitosan was described for a glucose/O2

BFC employing laccase at the cathode. An OCV of 0.6 V and a Pmax of 517 µW.cm -2 were reported, but from the article it is not clear if a redox mediator has been used or not. 352 In case of assessed DET mechanism, the current densities were very low at the beginning of the use of graphene. Tv-laccase adsorbed on electroreduced GO gave currents inferior to 5 µA.cm -2 , probably due to aggregation of the enzymes. 345 With BOD adsorbed on graphene, reduction current 74 times higher than at GC were obtained, but they still only reached 1.5 µA. 353 BOD covalently attached to a block copolymer bearing carboxylate moieties modified with graphene was employed in an EBFC using GOx at the anode. In an oxygenated solution containing 5 mM glucose at pH 7.4, power and current densities reached 0.115 µW.cm -2 and 0.16 µA.cm -2 , and 3.7 µW.cm -2 and 5.1 µA.cm -2 , respectively without and with graphene. Not many details about the electrodes were however given. 354 However, DET currents could be increased by simply optimizing the electrode preparation protocol. Different protocols were evaluated for Mv-BOD: 1/ layer-by-layer deposition of electrochemically reduced GO and BOD; 2/ electrochemical reduction of BOD mixed with GO flakes presenting a wide size distribution of 0,5-5 µm; 3/ the same protocol as

Elsevier

The total pore volume of a network of graphene nanoplatelets could also be increased by adding in the graphene suspension NaH2PO4 that crystallized in the carbon thin film during the drying process, thereby acting as a mold. 356 The adsorbed Trametes sp. laccase gave catalytic currents of 1.6 mA.cm -2 at pH 3, a 2-fold increase compared to the network without Na2HPO4. This enhancement corresponded to an increase of enzyme coverage thanks to a more appropriate porosity. Competitive ORR currents of respectively 2.1 mA.cm -2 at pH 4 and 0.4 mA.cm -2 at pH 7.4 were obtained with Mv-BOD and Th-laccase covalently bound on graphene after simultaneous electrodeposition and electroreduction of GO. A rotation rate of 1500 rpm had to be used to overcome mass transport limitation. 237 Finally, BOD adsorbed on graphene and coated on a carbon fiber cloth rendered hydrophilic by UV-ozone treatment led to a plateau current of 0.2 mA.cm -2 . 357

Very recently, parameters influencing adsorption and electrocatalytic properties of BOD integrated in an electrochemically reduced GO matrix were investigated. 358 GO flakes with different negative charge densities were studied. It was found that the current density (j), the heterogeneous electron transfer rate (kET) and the coverage of electrochemically active enzyme (Γa) depended on the negative surface charge density of graphene, and that j was more influenced by Γa than kET (i.e. the electron transfer was fast enough not to be limiting).

The maximum current density was 597 ± 25 µA.cm -2 .

Hierarchical 3D graphene-based structures have also been designed. A graphenelaccase composite was electrodeposited and covalently bound on carbon micropillars (50 µm in diameter, 200 µm in height) of a 3D array. The cathode was combined to a GOx-based anode developed following the same procedure. The power density of this EBFC was 7 times higher than that of the graphene-less EBFC. However, the stability was lower, which could be related to enzyme denaturation within the graphene matrix. 359 Tv-laccase was also immobilized on carbon fiber electrodes decorated with graphene nano-sheets produced in situ by an electrochemical procedure. This cathode was coupled to a PQQ-dependent GDH in a glucose/O2 EBFC leading to jmax = 70 µA.cm -2 and Pmax = 5.5 µW.cm -2 at an external load of 6 kΩ. 360 More generally, CNPs seem to promote DET but in many cases poor results were obtained. The protocols mostly involved drop-casting of CNP-suspensions with Nafion® or PVDF as a binder. [361][362][363] When Mv-BOD was adsorbed or cross-linked with glutaraldehyde, less than 5 and 10 µA.cm -2 were respectively obtained under air and O2, while no DET at all was observed with a GC electrode. This corresponds likely to an increase of ESSA. 361,362 A biocomposite of Cerrena maxima laccase, glutaraldehyde and CNPs delivered 43 µA.cm -2 , while only 17 µA.cm -2 were obtained on graphite. 258 For Mv-BOD adsorbed on Carbon Vulcan 0.27 mA.cm -2 were obtained in O2 at 600 rpm without a mediator, and 1.6 mA.cm -2 upon ABTS addition. This underlines that despite the structuration of the electrode not all enzymes were favorably connected for DET. 363 Different polymers have also been introduced

for the dispersion of CNPs, like chitosan 346 or polylactic acid (PLA), 377 leading respectively to 83 ± 19 µA.cm -2 and 255 ± 42 µA.cm -2 and underlying the influence of the charge of the polymer. Interestingly, the example of Trametes trogii laccase adsorbed on large surface area graphite and Vulcan carbon electrodes showed that current densities were in the nA.cm -2 range when referred to the real electroactive surface area, while they reached 600 µA.cm -2 when calculated with the geometric surface. 253 It is therefore questionable if DET was really promoted or if a "zoom factor" was more likely observed.

Enzymatic BFCs based on carbon nanoparticles

Despite generally relatively low performances, the described mesoporous

Importance of porosity

What seems nevertheless important for entrapping an enzyme is the adequacy between the size of the enzyme and the size of the meso-pores. The increased current densities observed with pores of appropriate sizes were attributed not only to the ESSA enlargement but also to a "curvature effect" promoting DET. 371 Contrary to low current densities reported previously, currents of 4 mA.cm -2 were obtained under stirring for Trametes sp laccase adsorbed on carbon aerogels with an average pore size of 22 nm, 374 reaching even 10 mA.cm -2 with a RDE at 8000 rpm. 375 Stabilization in the meso-pores was observed with stable currents recorded continuously during 10 days. 375 Mv-BOD immobilized in the same carbon aerogel gave 6 mA.cm -2 at 3000 rpm and pH 7 375 while Bp-BOD adsorbed in a mesoporous matrix of carbon cryogel with average radius of 20 nm gave 3 mA.cm -2 at -0.1 V vs. Ag/AgCl, 37°C and pH 7.2. 376 The same enzyme Bp-BOD adsorbed on carbon nanofibers made of stacked graphene cones presenting a large volume of mesopores gave 4 mA.cm -2 under O2 at 60°C and pH 4. 372 Similarly, around 1 mA.cm -2 was obtained when Mv-BOD was adsorbed at KB with mesopores in the range 2-100 nm, while only 3.5 µA.cm -2 were recorded at a planar electrode. 371 On the contrary, with too small pores of 6.3 nm, currents delivered with immobilized BOD were only 20 µA.cm -2 in DET and twice more in MET, showing that both enzyme immobilization and orientation were not optimized. 373 A mathematical model of random orientation of enzymes on planar and mesoporous electrodes was derived from Laviron's model 378 for the non-catalytic signal 379 and from

Léger's model 148 for the catalytic signal. 371 It estimated the possibility to establish DET between enzyme and electrode. BOD was chosen as a model protein, among others. It was assimilated to a sphere with a radius r = 3 nm, and a distance between enzyme center and active site rAS = 2 nm (a unique redox center was considered), while electrode pores were considered to be spherical with a radius Rp. Computation showed that no clear non-catalytic redox process in the absence of substrate was expected with the BOD model on planar or mesoporous electrode with Rp >> r, but a clear redox peak when Rp was close to r. 379 Catalytic currents on the contrary were predicted both for planar and mesoporous electrodes, but they were much higher when pore and enzyme sizes were close to each other. 371 Although the model showed that an adequate porosity should promote DET, it did not take into account the problems related to adsorption of enzymes in the pores, that may be sterically restricted in case of small pores radii (typically adsorption is not expected for pores smaller or of the same size than enzymes).

Besides the pores needed for enzyme immobilization, a network for Efficient transport of substrate is also important. Mv-BOD was for example adsorbed in macro/mesoporous carbon with different ratios of meso/macropores, obtained by carbonization of magnesium oxide hard template (MgOC). This technique ensures the control of morphology and pore size, and the material was composed of interconnected macropores (150 nm) with an additional morphology of 40 nm mesopores. The highest current density was obtained with the material containing 33% macropores, with which around 12 mA.cm -2 was reached in O2 saturated pH 5 buffer at 25°C and 10000 rpm, i.e. approx. twice more than when either only meso or only macropores were present. Mesopores are important for enzyme immobilization, and ensure better stabilization. For the materials containing only mesopores, only macropores or the 33% mixture, respectively 80%, 66% and 75% of the current density was retained after 100 CV cycles. However, additional macropores are necessary to ensure O2 mass transport, as demonstrated by the high increase in current densities recorded with increasing macropores contents. 380

Free-standing carbonaceous films

Free-standing carbonaceous materials have been developed, either with micro/macroporous carbonaceous monoliths made from hard macrocellular silica templating 141 or cylinders of compacted nanomaterials: CNTs (see above), graphitized mesoporous carbon 143 or mixtures of graphite and MW-CNTs 381 . The first presented excellent mechanical properties, and tunable size, geometry and porosity. Small pores of diameters slightly bigger than enzymes were present for effective immobilization of enzymes, micropores of 5.4 Å representing a total microporous area of 600 m 2 .g -1 were supposed to help the enzyme attachment, and larger pores (> 10 µm) were introduced to allow mass transport of fuels. In the second case, mixing different nanoparticles in various proportions influenced the ratio of pores with diameters larger than 10 nm. With a ratio of 60/40 graphite/MW-CNTs, these latter pores represented 0.42 cm 3 .g -1 , i.e. 75% of the total pore volume. Currents delivered with adsorbed Tt-and Mv-BOD were respectively 2.1 mA.cm -2 at 1000 rpm, 25°C and pH 7.2, i.e. a 500-fold increase compared to GC, 141 and 1.23 mA.cm -2 at 25°C and pH 6. 381 Interestingly, catalytic currents increased linearly with electrode thicknesses and volumetric current densities were constant, reflecting that the same intrinsic efficiency was obtained. It is also interesting to note that when currents were normalized by the specific area, values of 1.58 µA.cm -2 in mesoporous carbon and 2 µA.cm -2 at GC were calculated (resp. at 7500 and 1000 rpm). 141 Similarly, with mixtures of graphite and MW-CNTs, currents were divided by 2 when electrodes were cut in half. For higher thicknesses, a linear scale up of the current was however not observed. 381 Despite the tuned microstructure in carbon monoliths, systems were still limited by random orientation of enzymes, as shown by the wide distribution in ET interfacial rate constants reflected by the CV shape, and mass transport was not fully optimized. 141 In the second system, designed for H2/O2 fuel cells, intrinsic limitations were induced because non-explosive gas mixtures were used to design a membrane-less EBFC. A low level of O2 (4.6%) was therefore present. This, combined with a poor affinity of Mv-BOD towards O2, explains why cathodic currents were much lower than anodic currents where a hydrogenase was oxidizing H2. The electrodes were scaled to compensate this cathodic limitation, and ultimately permitted to get an EBFC delivering 1.67 ± 0.24 mW.cm -2 (relative to the anode area). 143 Power densities could be further multiplied by a simple stack design. For example, assembling in series two composed cells, themselves made by parallel assembling of 4 individual cells, gave: OCV = 2.1 V and Pmax= 7.5 mW (0.18 mW.cm -3 ) with the same gas mixture at pH 6 and 20°C. 381 Enzyme stability inside the pores was comparable to that in solution. The current kept a constant value of 0.7 mA.cm -2 during 18 hours at 0 V vs. Ag/AgCl and 25°C, while the current at a GC electrode was nil after 2 h in the same conditions. 141 In an EBFC configuration, 90% of the initial current densities were retained after 1 day, and 54% after 1 week at an applied voltage of Ecell = 0.8

V. 76% were retained after 24 h when the voltage was Ecell = 0.55 V. 381 This last point underlines that estimated stability is not only an intrinsic characteristic of a system, but also depends on the conditions chosen for the test (See section 4.1.5.).

Comparison of carbon nano-structures

To conclude, it is interesting to quote a study comparing the efficiency of different carbon-based nanomaterials or porous 3D materials as DET promoters for MCOs. Catalysis was described both in terms of volume and surface current densities for the various materials.

To calculate surface current densities, the real surface (BET) was taken into account. Tvlaccase was adsorbed on graphite felt, CNFs, SW and MW-CNTs or porous carbon tubes, and

ORR was recorded at  0.45 V vs. Ag/AgCl. In terms of volumetric current densities, the least Efficient was graphite felt with less than 10 µA.cm -3 , and the most Efficient were MWCNTs and CNFs with resp. 77 µA.cm -3 and 70 µA.cm -3 . SW-CNTs and porous carbon tubes gave intermediate results (resp. 40 µA.cm -3 and 37 µA.cm -3 ). The rankings were totally different when surface current densities were compared. CNFs and graphite felt gave the highest current densities (resp. 129 and 125 µA.cm -2 ), SW-CNTs gave the lowest (2 µA.cm -2 ), while intermediate results were recorded with porous carbon tubes and MW-CNTs (resp. 14 and 10 µA.cm -2 , i.e. 1 order of magnitude lower than the CNFs or graphite felt). When the current was normalized by the surface, the results highlighted a possible limitation: CNTs may form aggregates so that their surface is not entirely available for enzyme adsorption. 382 This study also demonstrated that nanomaterials have to be selected carefully depending on the envisioned application.

Gold nanostructures

Besides widespread carbon, metallic 3D electrodes have also been designed for MCOs immobilization and DET bioelectrocatalysis. The most described metal for this purpose is gold, either as gold nanoparticles (AuNPs) for the nanostructuration of planar electrodes, or by direct synthesis of porous gold materials. Gold has a good electronic conductivity, a high chemical stability, and superior surface tunability by chemisorbed thiols, used to form selfassembled monolayers (SAMs), or by other techniques like electrochemical reduction of diazonium salts. 383,384 AuNPs in particular present a high surface area, a controllable size and are easily synthesized. Immobilization of already synthesized AuNPs, or direct synthesis of AuNPs at the electrode surface allowed the development of a 3D network. The first method involved deposition by drop casting, [385][386][387][388][389][390] layer-by-layer, 391,392 or covalent bonding. 393,394 The second method relied on electrochemical or chemical reduction of gold salts, [395][396][397] or on dealloying of Au alloys. 398 Porous gold electrodes could also be formed by covering with gold a 3D silicon material, 399 by electrodepositing a porous gold film on a gold electrode, 400 or by bottom-up templating approach. [401][402][403] Pores of different sizes, depending on the Au/Ag ratio, 398 or a nanoporous gold structure displaying 3D networks of interconnected pores with typical pore diameters of ca. 30-40 nm, were also obtained via de-alloying of Au alloys. 404 Calculation of the real electroactive surface area of 3D gold electrodes is then possible by integrating the peak associated to the reduction of gold oxide, which corresponds to a theoretical charge density of 390 ± 10 µC.cm -2 . 405

The use of other metallic nanoparticles has also been reported. Thioaniline modified BOD and Pt nanoparticles (Ø  2 nm) were simultaneously electro polymerized on a gold electrode. The presence of BOD increased the onset potential, and higher current densities (ca 90 µA.cm -2 ) were recorded, as a synergic effect of Pt and BOD. 406 Nano-structuring the electrode surface with nano-crystalline hematite semiconductor (αFe2O3) allowed DET currents to be multiplied by 20 compared to a flat surface. However, the onset was 50 mV vs. Ag/AgCl at pH 6.4, which is quite low for Mv-BOD. 407 No DET was recorded for the same enzyme with other metal oxides like TiO2 or WO3. 407 Alloys were also used. For example, a BFC was built with a cathode based on Mv-BOD encapsulated in a mesoporous

Pd-Pt aerogel. The electrode delivered a maximum current density of  200 µA.cm -2 , with combined contributions of the enzyme and the alloy to catalysis. 408 DET was observed when Tv-laccase was immobilized at TiO2 nanoparticles, but the catalysis was more Efficient when ABTS was added. 409 For a laccase covalently bound on ordered arrays of polyaniline-coated polycrystalline copper nanotubes (200 nm in diameter and 30 µm in height) the plateau current density was 1.2 mA.cm -2 (which corresponded to an absolute current of 220 µA). 410 To the best of our knowledge, no DET current was observed for a laccase covalently bound on 40-nm magnetite Fe3O4 nanoparticles, 411 nor with platinum nanoparticles. 412 As already stated for carbonaceous nanostructures, there is a discussion whether AuNPs increase DET efficiency by enhancing the electroactive area and thus by connecting more enzyme molecules, or favor a better relative orientation of the enzyme towards the conducting material. Actually one can imagine that when the size of the AuNP is comparable or smaller than the enzyme, the nanoparticles should approach closer to the active site and therefore drive a better electronic communication while allowing more freedom of orientations. This must be carefully evaluated in the case of MCOs. There is sometimes a clear evidence that DET appears on nanostructured gold, while not on planar gold (all other parameters being kept constant), 413,414 or that catalytic currents are enhanced. Nonetheless, precise evaluation of electron transfer rate is rarely performed. Contradictory results can even be found in the literature. We will first discuss this point in the following, then address the increase in current densities via surface increase and finally discuss the actual usefulness and limitations of gold nanostructured electrodes.

Effect of the size of AuNPs on DET

First studies that compared the influence of different sizes of AuNPs showed quite contradictory results. 5 and 16-nm spherical AuNPs were used for oriented attachment of Thlaccase (see section 4.1.5.). The recorded efficiency of DET was attributed not only to an appropriate orientation but also to an adequate size of the AuNPs. Results were indeed better when the particle dimensions were close to those of Th-Laccase (5 nm), leading to catalytic O2 reduction in DET, and only 6% increase in currents after addition of the diffusing redox mediator ABTS. 393 Mv-BOD was also covalently bound to small nanoparticles (5 ± 3 nm), giving 375 µA.cm -2 under pure O2, with only 5% increase upon ABTS addition. 415 The catalytic efficiency decreased when nanorods (average size 31 ± 6 nm) were used instead of nanoparticles, supporting the assumption that when the nano-elements are bigger than the immobilized MCO, the improvement effect in the electronic coupling does not take place. 394 Higher values of βd0 (16.6) than on AuNPs (11.0), representing higher dispersion of orientations, were also attributed to the presence of different crystalline facets of the nanorods. 394 Yet, on the other hand, another study suggested that better electronic coupling was favored with bigger AuNPs. Diameters of 7, 15 and 70 nm, leading to the same electrode roughness, were used as a support for Mv-BOD. The highest current density was obtained with the biggest AuNPs, bringing the conclusion that the most important parameter is the size of the spaces between the AuNPs where the enzyme must penetrate. 416 Moreover, the presence of AuNPs is not enough by itself to allow for Efficient DET, as shown by the following examples. Rv-laccase covalently bound on small nanoparticles (1.7 ± 0.4 nm) encapsulated in polyamidoamine (PAMAM) dendrimers exhibited a non-catalytic redox peak which increased with AuNPs compared to dendrimers only, but no O2 reduction catalysis was recorded. 417 In another study Tv-laccase was cross-linked with glutaraldehyde on AuNPs forming uniform aggregates of 200-500 nm. While AuNPs increased the conductivity of the ITO electrode, only MET catalysis could be observed. 396 Polypyrrole nanotubes were decorated with AuNPs whose sizes ranged between 40 and 1000 nm. When attached on them, neither Tv-laccase nor Mv-BOD exhibited DET but only MET, probably due to the high abundance of repulsive amino groups on polypyrrole. 418 Calculations suggested also that even at AuNPs not all enzymes were in DET configuration. 413,414 The number of Th-laccases per AuNP (22 nm) was evaluated at 44 by QCM-D coupled to electrochemistry. 413,414 On the other hand, the maximum current density, obtained for a monolayer of AuNPs, was 31.10 -18 A at a single nanoparticle. The low value of kcat (13 s -1 ) was explained by unfavorable orientation of some enzymes. 413,414 Interestingly, another laccase from Trichaptum abietinum (Ta-laccase) showed the same electronic coupling with AuNPs, but a better catalytic activity was recorded. 419 This latter result was attributed to the smaller size of Ta-laccase reducing conformational changes at the AuNPs surface.

Shleev et al. proposed to rationalize the influence of the size of the nanoparticles by taking into account not the geometric area but the real area of the electrode. 386,387,419 The study was performed with Mv-BOD immobilized on sub-monolayers of AuNPs with uniform sizes significantly bigger than that of enzymes (20, 40, 60, 80 nm). imax clearly increased with AuNPs size, but when the current was reported to the real area of the electrode, the same value of 15 ± 3 µA.cm -2 was obtained for all particle sizes (Fig 26). kcat, app and kET, resp 15.0 ± 1.5s -1 and 10.4 ± 0.4s -1 , were also not influenced by the AuNPs size. These results demonstrated that kinetic constants were not influenced by the addition of AuNPs nor by their size. The increase in DET on AuNPs was in general more likely due to an increase in electroactive surface than to an accelerated heterogeneous electron transfer or biocatalytic rate. However, as the authors stated it, these conclusions are not necessarily true for all enzymes and for particles smaller than enzymes. Recently, Bilewicz's group designed a fructose/O2 EBFC with Cerrena unicolor laccase immobilized on macroporous reticulated vitreous carbon (RVC) covered with a gold thin layer and decorated with very small AuNPs (1.8 nm) at the cathode. 420 The maximal current density, calculated with the BET surface of bare RVC, was 301 ± 26 µA.cm -2 , i.e. approx. 1.5 times more than when bigger AuNPs (14 nm) were used instead. It is nevertheless hard to conclude on the effect of the size of AuNPs, since the calculation does not take into account the developed surface of AuNPs.

Increasing the current density by enlarging the electroactive surface

We list in this section some examples of increase in current density attributed to a greater electroactive surface area. A 70-fold increase in surface area with AuNPs (Ø  50 nm) provided currents of 5.2 mA.cm -2 with Mv-BOD at 4000 rpm. 390 Catalytic O2 reduction with Th-laccase on 5-nm AuNPs reached 1.3 mA.cm -2 at 200 mV vs. Ag/AgCl, 393 and 0.6 mA.cm -2 on gold nanorods at the same voltage. 394 Mv-BOD directly adsorbed on AuNPs (Ø  20 nm) on a gold disk gave 110 µA.cm -2 (= 2.2 µA) in an air-saturated quiescent solution. 385 When the 3D structure was built with 17-nm AuNPs on a gold wire (3 mm in length, 100 µm in diameter), the ESSA increased around 100 times and Mv-BOD O2 reduction reached 1.5 mA.cm -2 in an O2-saturated buffer at pH 7. 391,[421][422][423] For a surface ratio of AuNPs (22 ± 3 nm) to planar electrode of 40, Mv-BOD adsorbed on carboxylate-bearing SAMs gave 0.4 mA.cm - 2 . 392 In all the above mentioned studies, ESSA increases more than the current densities, which means that the surface is not entirely exploited.

Hierarchical porosity

To obtain a hierarchical porosity, besides mixing AuNPs with other nanomaterials, 424 different structures have been developed. A nano/microstructured porous silicon was covered with a gold thin layer, further chemically modified for covalent binding of Trametes sp.

laccase. The structure presented micrometer-size channels ( 1-2 µm in diameter) and an additional nano-morphology (50-300 nm). However, DET was limited and currents of 70 µA.cm -2 were only obtained in MET configuration. 399 In another study, Mv-BOD 236 and Thlaccase 186 were adsorbed in nano-porous gold electrodes of roughness factor 26. Currents of 0.3 mA.cm -2 and 2 µA.cm -2 were obtained with BOD and laccase respectively, which further increased to 0.8 mA.cm -2 and 28 µA.cm -2 when the enzymes were covered with an epoxy polymer. For both enzymes the current was nil on planar gold. Currents increased even further with addition of an Os polymer, proving that not all enzymes were in DET configuration. 186 Another article reports the use of porous gold consisting of large micropores connected with nanopores, with a wide pore size distribution (10 nm-30 µm), at both electrodes of a glucose/O2 EBFC. Pmax was 6 µW.cm -2 at Ecell = 0.2 V, but no details were given on the electrochemistry of the cathode. 400 Tv-laccase was also covalently bond in an orientation favoring DET in a 3D macroporous sponge-like network based on gold "supraspheres", themselves made of 20-nm AuNPs. The current density reached 0.61 mA.cm -2 compared to 0.13 mA.cm -2 on a planar electrode, while the ESSA was multiplied by 45 (Fig 27). Mv-BOD was also adsorbed on nanostructured gold electrodes obtained by nanoimprint lithography, presenting cavities of 300 nm in lateral dimension and 100-200 nm in depth. Currents reached 186 µA.cm -2 compared to 48 µA.cm -2 at the planar electrode, and the increase was attributed to surface increase, the electrode roughness being multiplied from 1.06 to 1.7. 425 Recently a more rational approach of the effect of the pore size has been proposed.

Mv-BOD was adsorbed under vacuum in nanostructured gold, consisting in a 3D network of channels with evenly distributed pores, and obtained by de-alloying Ag/Au alloys. 398,426 The maximal currents of 65 µA.cm -2 were obtained with pores of 8 ± 2 nm (i.e close the enzyme size), while increasing the pore size to 24, 42 and 62 nm decreased the current densities. 398

Mass transport issues and stabilization in pores

In some cases, O2 mass transport limitation was also reported and clearly evidenced by the shape of the cyclic voltammogram (a peak shape characteristic of diffusion-limitation was observed in some cases), or by using a rotating electrode. 385,390 In other cases, even with elevated current densities, this limitation was absent. 399 This can be related to the size of the channels of the porous network: nanometer-sized in the case of mass transport limitation and micrometer in its absence, but other parameters like the length of the diffusing distance for the substrate, could also intervene. A stabilization effect has also been noticed. The stability was enhanced compared to a flat gold electrode and attributed to the confinement of enzymes or their retention in the mesopores. 390,426,427 The confinement could not only prevent their leaching but reduce their flattening at the electrode surface, thus reducing the activity loss. 425 Some of the gold nanostructures described above have been implemented successfully in EBFCs. 385,388,[390][391][392]400,421,426 This point is detailed later in section 6.

Tailored orientation of MCOs at the electrode interface

We have already discussed that increasing the amount of immobilized enzymes allowed reaching higher catalytic currents. However, the magnitude of these latter was still limited, and most of the time not all immobilized enzymes could take part in the catalysis.

Another requirement is therefore to ameliorate the enzyme electronic connection.

Whatever the material of the electrode, there is a huge interest in controlling and optimizing the heterogeneous electron transfer at the enzyme/electrode interface. The major parameter involved, the distance between the active site of the enzyme and the electrode surface, has to be minimized. A first possibility, previously developed, is to use nanomaterials with adequate porosity/dimensions, while random orientation is allowed. However, the limitations encountered in terms of current densities and percentage of favorable connection underline that this approach is rarely completely successful. On the other hand, particularly when the dimensions of the electrode surface structure are big compared to the enzyme, this distance mainly depends on the relative orientation of the protein towards the electrode surface. Enormous efforts have been accomplished to understand the parameters that drive Surfaces can be chemically functionalized via different methodologies. To quote only a few used here, monolayers can be formed by chemisorption of thiols (SAMs) on gold or reduction of diazonium salts on carbon, gold or platinum. 216,249,348,428 This theoretically allows grafting of any desired chemical function. The reduction of diazonium salts is supposed to be more stable than SAMs that can be easily oxidatively or reductively desorbed, but can lead to more than one monolayer. 429 It can then possibly increase the distance between enzyme and the conductive surface, or even lead to the formation of insulating layers. Oxygen groups can be created by oxidative chemical or plasma treatments 430 or by UV/ozone treatment. 431 A fast and easy way to functionalize CNTs is to use the non-covalent π-π interactions with aromatic molecules, which is stronger than simple electrostatic interactions or physical adsorption but conserves electronic properties of CNTs contrary to covalent grafting. 325,337,[432][433][434][435][436] This even allowed wrapping CNTs in DNA, enabled by the presence of π-electrons in stacked base pairs of DNA. 437 To evaluate the efficiency of the adopted strategy towards enzymes orientation, it is possible to evaluate the increase in DET, which however reflects also the increase in enzyme loadings. The comparison DET/MET, that is a reciprocal function of the amount of properly oriented enzymes, is therefore more rigorous. Kinetic models that extract information from voltammograms give useful and precise information. Several models have been developed by Tsujimura et al. 235 ,Léger et al. 148 ,Hexter et al. 239 The first one, already described in section 3.4.7, assumes that all electrocatalytically active enzymes have the same orientation.

Therefore, heterogeneous electron transfer can be described by a unique average rate constant, k 0 . 235 In the second model, it is assumed that enzymes adopt a distribution of slightly different orientations. 148,238,239 The distance between the electrode surface and the enzyme redox center is evenly distributed between a minimal distance dmin, and a distance dmin+d0. The product βd0 reflects the scattering of orientations (β is the decay constant in the Marcus theory), and determines the wave shape at high driving force where the slope of i vs. E is given by:

𝑑𝑖 𝑑𝐸 = 𝑖 𝑙𝑖𝑚 𝛽𝑑 0 * 𝐹 2𝑅𝑇
𝐸𝑞. 22

Laccases immobilized by supramolecular/hydrophobic interactions

This approach is quite simple and cheap since the enzyme is directly adsorbed on the chemically modified electrode. The idea to orientate the laccase via a "lock and key" or "plug-in-the socket" interaction with a substrate-like function at the electrode surface was first introduced by the group of Armstrong. 438 They considered that the T1 Cu in the structure of Tv-laccase (PDB ID 1KYA) was surrounded by a hydrophobic and electron-rich region, the "substrate-binding pocket", where the oxidation of the substrate takes place. In their first studies, a wide variety of polycyclic aromatic molecules 275,438 was covalently grafted on a "planar" PG electrodes. This led to more uniform and higher enzyme coverage of the electrode, as evidenced by epifluorescence microscopy. Importantly, the wave shape and the onset of O2 electro-reduction were not affected by the chemical functions, thus attesting no mediation nor change of the enzymatic mechanism. This result also underlines that the grafted chemical functions do not have to possess redox properties. However, the chemical modification, especially with anthracene and chrysene moieties, proved to be very good both for increasing the current density and improving the stability ( In the following, most studies were performed with laccases from Pycnoporus cinnabarinus, 275,438 Trametes versicolor, 275,435,439,440 Trametes hirsuta, 438 , Cerrena unicolor, 441,442 but also with other enzymes with considerably different structure like the lowpotential laccase from Streptomyces coelicolor. 443 The use of very smooth planar surfaces like monolayers obtained by protecting the electrode during the electrochemical grafting on polished glassy carbon electrodes 444 or closely packed SAMs on gold surface, 445,446 raised the question whether a good orientation is only due to chemical modification or also to the roughness of the electrode. Very low DET was observed in these cases, although the electrode surface was decorated with anthracene or anthraquinone moieties. This result was explained by the fact that on a smooth surface, or on a well-organized monolayer, the chemical functions are less accessible. On the contrary, when a mixed (antracene-methanethiol/ethanethiol) SAM was formed on an electrochemically roughened gold surface, the grafted moieties became accessible to the laccase and the current doubled. 445 A direct proof that isolated accessible molecules are needed for an Efficient immobilization of laccase via supramolecular interaction was given by a SECM study of mixed SAMs on monocrystalline gold (111). 446 Mixed thiolated veratric acid (tVA) / mercaptopropionic acid (MPA) SAMs were built with different water contents, leading either to separated pure tVA or MPA phases or to a unique well-mixed phase. In the case of the pure phase, neighboring tVA moieties were closely interacting, thus forming clusters bigger than the enzyme hydrophobic substrate-binding pocket. This was directly linked to quasi-inexistent DET for adsorbed Tv-laccase on this SAM. On the contrary, tVA molecules were widely separated by MPA molecules in the well-mixed phase. Adsorbed Tv-laccase exhibited in this case a clear cathodic wave with j = 0.58 µA.cm -2 at 0.4 V vs. Ag/AgCl. 446 This approach was further extended to different electrode materials and nanostructured architectures: carbon cloth, 275 carbon nanotubes covalently modified with anthracene, anthraquinone, naphthalene or related compounds at their ends or walls, 424,439,441,442,[447][448][449][450] CNTs covalently modified with bisphenyl groups, 451 or CNTs non-covalently modified with pyrene molecules bearing the desired chemical functions. 440,452 Even on nanostructures, a considerable increase in catalytic currents was observed.

A major problem with CNTs is that the terminal « substrate-like » moieties are rich in aromatic electrons and can also bind to CNTs by π-stacking, thus reducing the amount of available anchoring groups for the attachment of the enzyme. Better availability of anchoring groups at the electrode surface can be induced by bi-functionality where at least one functional linker is not bound to CNT walls due to steric hindrance, 436,452 as it has been done

with pyrene-bis anthraquinone, 452 pyrene-bis naphtoquinone, 436 or by functionalization of graphene oxide further immobilized by π-π interactions on MW-CNTs. 348 Another possibility is to use another hydrophobic moiety unable of π-stacking like adamantane. 433 Interestingly, although the binding energy of anthraquinone (evaluated by theoretical calculations) is almost twice higher than that of adamantane, catalytic currents for the reduction of O2 were much higher with adamantane, reaching 2.2 mA.cm -2 . This result, combined with QCM-D experiments showed that more enzyme was immobilized, supporting the idea that more groups were accessible and allowed for enzyme binding and orientation.

The hydrophobic interactions between electrode and substrate-binding pocket was also exploited for SW-CNTs modified with a steroid biosurfactant bearing a large, rigid and planar hydrophobic moiety. 453,454 This allowed very fast heterogeneous electron transfer rates between T1 Cu site and SW-CNTs (3000 s -1 ). Hydrophobic polymers were also able to act as a conductive wire approached to the T1 via π-π stacking. 455,456 However, a possible limitation of this approach is the lack of specificity of the hydrophobic interaction, since several hydrophobic regions co-exist at the surface of the enzyme, and not only close to the T1. This was evidenced in several cases by different experimental observations. Either a trailing edge was observed in the cyclic voltammograms 275 or currents were consistently enhanced upon addition of a soluble mediator, whatever the surface modification, 444 consistent with the assumption that a unique molecule orientation was probably not achieved. In addition, the immobilization of Tv-and Sc-laccases on SW-CNT modified with the molecular tether PBSE was investigated via molecular dynamic. SW-CNTs (2.71 nm in diameter) were positioned near the hydrophobic substrate binding pocket of the laccases (position 1) and close to 2 other regions where adsorption was also likely (positions 2 and 3). In case of Tv-laccase, T1 Cu was situated resp.

.2.1 Laccases

Although in most studies laccase orientation has been performed via the supramolecular orientation strategy, another approach relies on electrostatic interaction. This approach was proposed for Cerrena unicolor laccase (pI = 3.95) immobilized on CNPs 364 or SW-CNTs 458 bearing sulfonate functionalities in a silicate sol-gel matrix. This surface chemistry induced better reproducibility and at least twice higher currents. However, the increased performances observed in this case are more likely favored by the hydrophilicity of the structured matrix, rather than due to better orientation on negative charges, since for the same laccase a better orientation on positive charges was confirmed by PM-IRRAS studies. 428 Electrostatic interactions could also drive an enhanced immobilization for an engineered enzyme, as demonstrated with an unusual homo-trimeric laccase bearing a His-affinity tag at the C-ter close to the T1. 459 In contrast, QCM studies performed with different laccases showed that the nature of the charges did not influence the enzyme coverage. 428,459 Studies performed with various laccases highlighted that electrode surface chemistry has to be customized according to the specific structure of each enzyme. Ulstrup et al.

compared the influence of electrode surface charge for 3 different laccases: Coprineus cinereus (Cc-laccase), Streptomyces coelicolor (Sc-laccase), Myceliophtora thermophila (Mtlaccase), and Mv-BOD. 233 The differences in electrocatalytic behavior were notable, and could be easily correlated with the residues surrounding the T1 Cu atom. Mv-BOD and Cclaccase, who share a positive charge area around the T1 center, displayed the highest activity on carboxylate-terminated SAMs. On the contrary, Sc-laccase showed the best activity on alkyl-terminated thiols, consistently with a hydrophobic pocket around the T1 Cu. Mt-laccase, the only one bearing a negative patch around the active site, was also the only one not to display a catalytic current. 233

Bilirubin oxidases

A considerably different behavior between most laccases and Mv-BOD was observed: contrary to laccases, adsorbing Mv-BOD on molecules with a single aromatic ring or presenting a hydrophobic end lowered catalytic currents. [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF]233 When a complete structure of Mv-BOD was obtained, 129 this could be explained by the presence of a narrow hydrophilic substrate binding site close to T1, with basic amino acid residues positively charged at acidic and neutral pH. The differences in terms of amino-acid sequence between bacterial BODs and fungal BODs also resulted in different behaviors. 434 Due to the difficulty to rationalize how DET with Mv-BOD could be obtained on carbon surfaces, which generally bear lots of functionalities and have a porous structure, 460 studies were performed on well-ordered and atomically flat single gold crystal 151 and then extended to carbon or more complex architectures: KB, 461 GO, ECR-sputtered CNFs 431 and CNTs. 152,216,338,437,462,463 Here also DET was proved because the catalysis started at a potential that could be attributed to the T1 whatever the chemical functions at the electrode surface, 151 even when the surface was modified with redox active groups. 152,338,458 Studies globally showed the importance of the electrode charges, 151,437,463 catalytic currents being low for positive charges, and higher for negative charges (most studies were performed at neutral or near-neutral pH). This proved that amino-acids near the T1 are more important than the global charge of the BOD whose isoelectric point is 4.1. Especially the adequacy of carboxylic functions [START_REF] Dos Santos | Mechanistic Studies of the 'Blue' Cu Enzyme, Bilirubin Oxidase, as a Highly Efficient Electrocatalyst for the Oxygen Reduction Reaction[END_REF]151,220,432 has been highlighted. On carbon presenting carboxylate functions on aromatic protrusion, 17 kinetic analysis 148 evidenced that the enzyme adopted a narrow cone of orientations. This can be partly attributed to an increase of hydrophilicity, or a better wettability of the electrode 431,437,462 rather than to enhanced enzyme loadings. As a matter of fact, similar enzyme loadings have been calculated for different electrode surface charges. 216 However, the interactions favoring a functional immobilization could be more complex than simply electrostatic. Indeed, some studies have suggested that for optimal immobilization of Mv-BOD, the anchoring molecules need to possess both aromatic properties and negative charges. 432,461 This is consistent with previous studies where Mv-BOD adsorbed on MW-CNTs modified with different compounds, all bearing carboxylic functions, showed no special difference with unmodified CNTs, except when the compound was Pyrroloquinoline quinone (PQQ). For this latter the catalytic currents almost doubled. 152,319,338 Consistently also, Mv-BOD was perfectly oriented on MW-CNTs bearing carboxynaphthalene moieties. DET currents reached 4 ± 0.4 mA.cm -2 in a stirred oxygenated solution, and no increase was observed upon ABTS addition. 216 On the contrary, when Mv-BOD was immobilized on CNTs bearing positively charged amino groups, O2 reduction was observed at much lower potential (EHW = 0.19 V vs. Ag/AgCl at pH 7). This was attributed to DET to the TNC. 216 In that case, addition of ABTS led to high MET currents ( 2.5 mA.cm -2 ). This is also consistent with DET at T1 requiring negative surface charges at the electrode.

Quite contradictory results were however recorded, with Mv-BOD showing a better orientation than Tv-laccase on anthracene-modified MW-CNTs. Upon addition of ABTS only 11% increase in current densities were observed, while 59% increase was recorded for Tvlaccase. 266,267,464,465 Mv-BOD immobilized on PG modified with naphthyl-2-carboxylate moieties was compared with platinum. The limiting current density, jlim, was 10 mA.cm -2 for the platinized PG electrode, with an estimated platinum concentration of 2150 pmol.cm -2 . For Mv-BOD, jlim was 0.7 mA.cm -2 , which represents a much better efficiency since the enzyme concentration was supposed to be lower than 10 pmol.cm -2 . In addition, the reaction overpotential for oxygen reduction was much smaller at the Mv-BOD naphthyl-2-carboxylate modified PG electrode than at Pt under all experimental conditions. 17

Although electrostatic interactions have enabled Efficient immobilization of different MCOs, the specificity of the electrode modification towards immobilization of each particular enzyme is of crucial importance. A few examples underline it. The presence of COO -on the electrode surface did not improve the bioelectrocatalytic activity for a CueO, whose T1 is surrounded by acidic residues, while an improvement was recorded with positively charged modification. 461 Two different BODs, the bacterial Bp-and the fungal Mv-BODs, sharing only 36% sequence homology had also very different behaviors on CNTs with different surface charges. 434 The comparison of DET/MET currents, and modeling with Armstrong's model, 148,239 showed that Mv-was correctly oriented on negative surfaces (as already demonstrated), while Bp-was much better on positive. This result was consistent with their respective positive and negative charges surrounding the T1 in the considered pH range. This difference was also observed for naphtoate-CNTs, which were favorable for Mv-BOD but detrimental for Bp-BOD. 466

Orientation of bilirubin oxidase with substrates

The supramolecular arrangement involved in orientation of BOD with its substrate is also consistent with the fact that both aromatic and negative functions are needed. 129,461 The onset of catalysis did not change 467 but currents increased when Mv-BOD was adsorbed on PG electrode or MW-CNTs modified with bilirubin. 129,468,469 A shift in half wave potential towards more oxidizing potentials was ascribed to a better control of the orientation when BOD was adsorbed on Ketjen black modified with bilirubin or other bilirubin related compounds. 470 Both the coverage of electrochemically active enzyme (λΓ) and standard surface electron-transfer rate constant (k°, determined according to Tsujimura's model) increased with bilirubin. 470 This latter induces rearrangements in the binding pocket that favor the interaction of BOD with the electrode, as was recently demonstrated by molecular docking and DFT. 471 The idea of binding the BOD through its substrate was extended to other compounds; syringaldazine on CNTs, 472 or CNTs with different porphyrins (by considering that bilirubin is a decomposition product of protoporphyrin IX), 432 leading to current densities up to 4.66 mA.cm -2 . This furthermore showed that the aromatic moiety (here the porphyrin skeleton) influences the enzyme coverage, while carboxylate groups help orientating the proteins.

Covalent attachment

Covalent binding has also been introduced to force the desired MCO orientation by attaching a specific part of the enzyme, mostly a determined amino-acid residue or a carbohydrate of the glycosylation shell, at the electrode surface. Linking is ideally performed at a site that does not affect the catalytic activity of the immobilized enzyme, and is also enhanced when the enzyme is linked via several points 307 due to a more constraint orientation.

It is of course necessary to identify the appropriate site. In a first approach, this was often done by trial and errors rather than by extensive modelling analysis. Even if a good position is identified, this does not guarantee the success of the immobilization.

Different chemistries for the covalent binding have been compared in terms of DET/MET ratios for Th-laccase on planar gold 249 , gold nanoparticles 393 , gold nanorods 394 or on a composite of carbon microfibers and carbon nanotubes (CNT/CMF). 333 The Schiff's base or imino-bond formation involving glycosylated residues close to the T1 binding pocket led consistently to a preferential orientation, contrary to amide bond formations involving carboxylic functions located at the opposite of T1. 222,249,333,394,[473][474][475] Therefore, chemical modification can be used to tune the electrode surface for the enzyme: for example, amino aryl functions were introduced on graphene for amide bonding with oxidated sugar residues of Th-laccase, and naphtoic acid was grafted to enable amide bond formation with Lys residues of Mv-BOD. 237 An Efficient DET cannot however always be attributed to optimal orientation, as shown by the covalent anchoring of Mv-BOD on a CNT/CMF material. The best results

were obtained when a lysine of the enzyme was linked to the CNTs via a phenylbutanoic acid or glutaraldehyde spacer, i.e. a very long chain (23 atoms in the case of glutaraldehyde). 314 An explanation is that the flexibility of the long chain allowed repositioning of the enzyme at the CNT surface for an Efficient electron transfer. 314 Covalent binding often leads to random immobilization because the involved residues are in most cases distributed over the protein surface. For example, C. gallica laccase bears 7

reactive lysines that can be involved in a covalent bond, but none of them is situated close to T1. 476 In case of laccase covalently bond on NHS-SAMs, DET was evidenced but no electrocatalytic activity was recorded, suggesting that the copper ion undergoing electron transfer was not the primary electron acceptor in electrocatalysis. 207 In another case, addition of ABTS improved the current density by respectively 20% (laccase) and 24% (BOD)

indicating that not all molecules were correctly oriented for DET. 237 In case of Mv-BOD, reactive dye labelling was performed to identify lysine involved in covalent attachment: all of these groups would position the metal center at least 2 nm from a planar electrode surface. 229

Engineered enzymes

To be even more specific, the orientation of MCOs could be driven via supramolecular chemistry as described above, and followed by covalent binding for example via carbodiimide chemistry. 337,476 But another solution was to artificially introduce amino acids at a desired location via site-directed mutagenesis. This approach exploited the well-known ternary metalchelate complex formed between His-tagged proteins and nitrilo-triacetic acid, which is easily grafted on a planar electrode. 477 With His-tagged laccases, 478,479 although the specific binding of the protein was proved, this approach did not allow a DET probably because of a too long distance between T1 and electrode, even in case of a proper orientation. But importantly the activity towards mediators in solution was not affected neither by the tag nor by the immobilization. With a His-Cys tag at the N-or C-terminus, a laccase from Trametes sp.

could also be directly attached to a gold surface. A DET communication could be established with the electrode, but the quite low onset potential was attributed to either the TNC alone, or a mixed T1-TNC signal, and the incomplete O2 reduction to H2O2 was recorded. Copyright 2016 American Chemical Society.

Other strategies

Some more anecdotic studies rely on oriented immobilization of the MCO under the influence of an external magnetic field. 482,483 Tv-laccase was immobilized on a GC electrode modified with nanomagnets, made of carbon nanocapsules (Ø  30-70 nm) with an iron core.

O2 reduction currents were doubled under application of an external magnetic field 𝐵 , 482,483 reaching 5 mA.cm -2 when PPY and GA were added. 483 𝐵 was supposed to influence on the migration of paramagnetic O2, but also on the enzyme orientation via attraction of the paramagnetic T1 Cu(II).

A very recent study proposed MCO orientation assisted by organic solvents. 484 Tvlaccase was mixed with SW-CNTs in the presence or absence of 20% organic solvent. Results

were particularly detailed in case of ethanol. When the mixtures were immobilized on an electrode surface, currents were increased by 70% in presence of ethanol compared to controls made in absence of ethanol, leading to 711 µA.cm -2 at 0.4 V vs. Ag/AgCl at pH 6 and 500 rpm. The enzyme structure was studied by circular dichroism (CD) and attenuated total reflectance infrared (ATR-IR) spectroscopy in presence of CNTs and ethanol, either in solution or dried at the electrode surface. Surprisingly, no drastic influence of ethanol on enzyme denaturation/unfolding was observed. An orientation change was recorded instead, with β sheets of the enzyme aligned perpendicularly to CNT sidewalls, indicating that the substrate binding pocket was facing the sidewalls. 484

Stability issues

Bioelectrochemical devices, either single electrodes, complete EBFCs or various biosensors, generally share a relatively poor operational stability. Enzymes in solution are typically active for a few hours to a few days 485 , and most enzymatic systems are rarely stable for more than few hours under continuous operations. [START_REF] Cracknell | Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis[END_REF] However, it is important to be aware that the stability is not only an inherent property of the device, but that numerous other parameters such as the experimental conditions, the method to evaluate the stability or the storage conditions have a huge influence on it. The problems encountered in assessing, and therefore comparing, the stability of various devices are comparable to those encountered for the normalization of current, since in both cases there is no specific guidelines or established

protocol.

An interesting study published by Minteer and coworkers underlined for example the impact of the measurement method. They compared galvanostatic and potentiostatic measurements, and different experimental conditions for both techniques. 486 For a laccase from Trametes sp. immobilized on anthracene-modified MW-CNTs, at pH 4.5, 25°C and in atmospheric conditions, galvanostatic measurement (at 30% of the electrode maximal current) led to a 3% decrease over 24 hours, while 15% were lost in potentiostatic measurement (at  0.44 V vs. Ag/AgCl, i.e. 200 mV more negative that the onset potential) ( Fig 30). Stability in potentiostatic conditions also decreased with increasing overvoltage, 21 ± 6% being lost after 24 hours at  0.54 V vs. Ag/AgCl, and 42 ± 12 % at  0.24 V vs. Ag/AgCl. In galvanostatic conditions, the stability also decreased with increasing applied currents, only 3.0 ± 0.9% being lost after 24 hours at 10% of the electrode limiting current, and 100% at 90% of the limiting current. 486 Many mechanisms could account for the relative instability of an enzymatic electrode: lack of intrinsic stability of the enzyme itself; 27 denaturation, change of conformation or configuration, reorientation, and chemical inactivation upon immobilization; [START_REF] Cracknell | Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis[END_REF]229,320 or just enzyme leaching from the electrode surface due to a weak enzyme/electrode interaction.

Electrochemistry by itself does not permit to link the catalytic stability with the structure/conformation of proteins, so combined approaches have been put forward. Efforts to elucidate the nature of the damage on gold electrodes involved the coupling of electrochemistry to other analytical techniques (QCM, PM-IRRAS, ellipsometry, AFM, SPR: see section 3.4). The problem is more complex with carbon electrodes due to the lack of Efficient analytical tools. Therefore, it is still unclear which factor is mainly responsible for instability.

In this context, different techniques have been envisioned to improve the stability of enzymatic electrodes. Most are directed towards retaining the enzyme molecule at the electrode surface, or ensuring a stabilizing environment for the enzyme at the electrode.

Reducing damaging surface-interactions with a promoter

Promoters used to favor enzyme orientation have also shown to stabilize the electroenzymatical O2 reduction current. When Pycnoporus cinnabarinus laccase was adsorbed on a 2-anthracene modified PG electrode, 57% of the initial current was retained after 8 weeks. On the contrary, on an unmodified electrode, more than 95% were lost after 4

weeks. The stability was measured by daily cycling in O2-saturated solution. 438 The stability of Tv-laccase on carbon cloth modified by reduction of anthracene-2-diazonium (A2D) was measured continually over 2000 hours. Without A2D the current dropped from 75 to 10 µA.cm -2 in 72 hours; with A2D a rapid decrease was observed, followed by a stabilization at 75% of the initial current density (50 µA.cm -2 ). 275 An electrode modified with Th-laccase coated on monolayers of anthracene or anthraquinone exhibited still half of the initial current after 30 days of storage in the buffer. However, no comparison was reported with the promoter-free electrode. 444 For Tv-laccase adsorbed on CNTs bearing bis-anthraquinone moieties, 48% of the initial signal were retained after 20 days with one hour discharge every day, while all the current was lost for the same experiment without the promoter. 452 Similarly,

Tv-laccase was more stable when adsorbed at MW-CNTs bearing adamantane moieties than immobilized on pristine MW-CNTs. The stability was evaluated over one month with a daily 1 h discharge at 0.24 V vs. Ag/AgCl. On bare MW-CNTs, currents were below 5% of their initial value after 20 days, while 66% was conserved after one month on adamantane-MW-CNTs. 433 Similar observations were made when Mv-BOD was immobilized at an interface covered with bilirubin or related compounds. Lifetime of the BOD-PG electrode assessed by chronoamperometry or cyclic voltammetry was increased by 50% when the surface was coated with bilirubin. 129 An increase in the stability was also recorded when KB CNPs were modified with bilirubin. With daily measurements, 60% of initial current densities were left after 7 days at bilirubin-coated KB, while only 20% were left after 7 days (or 40% after 3 days) when Mv-BOD was adsorbed at bare KB (Fig 31)

. 470 With phenylcarboxylate-modified KB, 60% of initial current densities were retained after 4 days. 461 In all these cases, the stability increase could be due to a better retention of the enzyme at the electrode surface, or to the promoter reducing conformational change or reorientation. Published by the PCCP Owner Societies

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

Encapsulation

It has been reported that the lifetime of an enzyme in solution could be extended to ~ one year [START_REF] Moehlenbrock | Extended Lifetime Biofuel Cells[END_REF] when enzymes were included in micellar polymers. Similarly, at electrodes, MCOs 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 polyammonium cations. 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.

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)

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

Mediated electron transfer

Direct electrical communication between MCOs and electrodes has been largely

exploited in the recent past, and nowadays 60% of the published papers concern DET-based MCO cathodes, and 40% MET-based electrodes. Although DET efficiency has been greatly enhanced over the last decade, MET still possesses numerous advantages, as described above.

An obvious asset of MET, beside its versatility, is that multilayers of enzymes can be simultaneously connected at an electrode surface whatever their orientation. This might be an explanation why between 1982 and 2004, most of the published papers on MCOs for O2 reduction were based on mediated electron transfer connection. This ability to electronically connect multilayers of enzymes is a precious tool to enhance the current densities. This is the major focus of this section. We will willingly not compare the stabilities obtained for such

MCOs electrode (except in some particular cases) because all the necessary information for a fair evaluation are, most of the times, not provided.

MET connection is often optimized on planar rotating or non-rotating electrodes, made of glassy carbon, platinum or gold, and then usually scaled to 3 dimensional electrodes to enhance specific surface and enzyme loading. Diffusive redox mediators are commonly used to probe enzymatic kinetics while immobilized redox mediators are rather used to elaborate devices. For the latter, 80% are osmium based redox polymers, and 20% other redox mediators including conducting polymers, proteins or organic/inorganic compounds. In this section, we will first discuss MET on various types of bare electrodes with diffusive or immobilized redox mediators. In a second time, we will describe the use of 3D materials in MET based cathodes.

Bare electrodes

Diffusing redox mediators

Diffusing redox mediator are used to probe electrochemically the kinetics between enzymes and redox mediator, 201,494 to probe the effect of mutations on MCOs 247,488,495 or to evaluate the potentiality of an enzyme as a bioelectrocatalyst. 496,497 For this purpose, the most common diffusive redox mediator is 2,2'-azinobis (3-ethylbenzothiazolin-6-sulfonate) (ABTS), 216,363,[498][499][500][501][502] first introduced in 2001 by Tsujimura et al. 122 with Mv-BOD. In an airsaturated pH 7.0 buffer containing 0.5 mM ABTS 2-and 0.11 M of enzyme, they could reach 2 mA on a carbon felt electrode. ABTS is also used routinely as a control to verify the efficiency of enzymes connected in DET. [503][504][505][506][507] However, at neutral pH, ABTS has the disadvantage to undergo gradual decomposition 122,494,508 and other redox mediator should be From an engineering point of view, the use of diffusive redox mediator is not the "ideal" strategy when designing electrochemical devices. In the case of EBFCs, a membrane separating anodic and cathodic compartments is required to avoid cross over of the redox mediator. Diffusive redox mediators therefore complexify the system and introduce new limitations, kinetic losses and additional costs. The use of osmium based redox polymers has been pioneered by Adam Heller in the 90's. [514][515][516][517][518][519] These hydrogels have been first used with glucose oxidase to make glucose sensors. 520,521 Due to its success, this system was then extended to numerous other enzymatic devices. [522][523][524][525] Os-based polymers constitute the only hydrated electron conducting system which allows for rapid diffusion of ions, substrates (glucose, O2,…) and products. In addition, these hydrogels are particularly versatile. Their redox potential can be tuned at will to be close to the redox potential of the enzymes, by choosing the adequate ligands around the osmium atom. The concentration of osmium, the nature and composition of the polymer backbone, 528-532 but also the spacer that connect osmium complexes to the backbone, 527,533 can be designed depending on the enzymes and the future applications.

Furthermore, quaternization of the pyridine rings permits to introduce positive charges in the osmium redox polymer, enhancing both the rate of electron transport and the strength of the electrostatic complex formed between the polycationic redox polymer and the polyanionic MCO. 534,535 The electrochemical properties of the modified electrodes can be for example optimized by tuning the ratio between enzymes, redox polymer and cross linker 536 , by

A B

choosing an adequate film thickness 537 and/or by optimizing the temperature and drying time. 538 In 2004, Leech et al. 539 reported the co-immobilisation of Tv-laccase with an osmium based redox polymer (E°' = +0.4 V vs. Ag/AgCl) on GC electrodes which reached a current density of 240 A.cm -2 in pH 5.0 buffer, at +0.3 V vs. Ag/AgCl. At pH 7.5, the current was only 7% of the catalytic current observed at pH 5. The authors attributed this loss in current to the low activity of laccases at neutral pH, but also to a change in electron transport upon hydrogel swelling. 540 The same authors later optimized the cathodic redox polymer and increased its redox potential by 90 mV. Their objective was to improve the OCP of an EBFC, and to demonstrate it was possible to yield a significant power density in a 0.05 M phosphate buffer pH 7.4 containing 0.1 M NaCl, even though laccases are sensitive to chloride and less active at neutral pH. 541 The cathodic current was increased by 20%, and a maximum power density of 16 W.cm -2 was reached at a cell voltage of 0.25 V when a glucose oxidizing anode was used. To improve the stability of their modified electrodes, they specifically tailored Os redox polymers to three enzymes Mv-BOD, Ma-laccase and Th-laccase and evaluated their performance and stability. 542 They showed that the magnitude of the O2 reduction current densities depended on the enzyme selected, the thermodynamic driving force induced by the difference between the enzyme and the polymer redox potentials, and also on the chemical structure of the osmium complex. The same authors pre-treated graphite electrode by reduction of diazonium salts, or gold electrodes with cysteamine, to introduce anchoring amine groups 543 to further improve the stability, and particularly improve the interface between the osmium polymer and the electrode surface. After functionalization and modification with osmium polymer and enzymes, the authors showed for example that a pretreated graphite electrode retained 90% of the original redox signal after 48 hours. By replacing GC electrodes with their newly developed pretreated graphite electrodes, they showed a four-fold increase of the power density of their EBFC. 544 The wide variety of developed protocols is another proof of the versatility of osmium More recently, Le Goff et al. 559 reported the synthesis of original osmium complexes bearing chelating N-heterocyclic and bipyridine ligands to wire Mv-BOD. These complexes were easily grafted at the surface of both planar and nanostructured CNT electrodes, either by electrogeneration of a redox polymer, or by supramolecular π-π interactions. The immobilization of diazonium derivatives of an osmium bipyridine complex has also been reported, but has not been used with an enzyme so far. 560 stabilized by an electrodeposited film of PPY. 565 In their later system, they also added catalase to benefit from an extra boost at their EBFC by dismuting H2O2, which is naturally present in bodily fluids. 566 PPY has also been used by Tingry et al. 567,568 In a series of papers, they immobilized Mv-BOD or laccase and ABTS in a PPY film 569 and for example used it as a cathode in a concentric EBFC generating Pmax = 42 µW cm -2 at Ecell = 0.3 V. 570 To improve the stability of the biocathode, functional groups were also introduced on PPY to covalently attach BOD. 571 Redox mediators such as porphyrin, ferrocene, osmium, and ruthenium complexes were also entrapped in electropolymerized PPY. 572

Proteins as redox mediators

The use of Cytochrome c (Cyt c) as a redox mediator in homogeneous solution has been reported by Sakurai in 1992. 575 The authors studied the kinetic parameters of the electron

Carbon particles

Carbon particles of various shapes and sizes (graphite, graphene, carbon black, ketjen black, carbon vulcan,..) have been used to make carbon electrodes. 376 The common point of these electrodes is the preparation method which comprises the mixture of the particles with a binder to form a paste, further deposited onto electrode surfaces. It is a very easy way to increase the specific surface, however without controlling or maximizing the porosity. For example, Kondo et al. mixed Mv-BOD, ABTS and carbon paste to elaborate a biocathode, further used in a starchy biomass EBFC delivering 99 W.cm -2 at 0.55 V. 583 The second modified with ABTS 593 on glassy carbon electrodes, and further cover with a mixture of laccase/Nafion®. Covalent linking of ABTS to SW-CNTs prevented its leaking from the electrode surface and allowed a better interaction with laccase molecules. The presence of Nafion® also prevented the leaching of the enzyme without impeding the diffusion of O2. The combination of CNTs and graphene has also been reported by Chen et al. 594 After growing 3D graphene networks, the electrode was first modified with SW-CNTs, and then laccase was covalently immobilized on the composite electrode. Poor electron transfer was observed on the graphene modified electrode compared to the composite electrode. However, even in presence of SW-CNTs, ABTS was needed to improve the electron transfer.

Carbon fibers

Even if carbon fibers usually do not offer a large volumetric area, their geometry allows them to reduce mass transport limitations. 595 Radial diffusion with electrode diameters smaller than the diffusion length of the reactants allows for faster mass transport compared with semi-infinite planar electrodes. In addition, the use of carbon fibers allows for miniaturization of EBFCs. 7 m diameter-2 cm long commercial carbon fiber electrodes have been used by 545,[596][597][598][599][600][601] Tvlaccase 533 and Bp-BOD. 602 Current densities 1mA.cm -2 were reached in PBS buffer. Homemade carbon fibers were also designed by Tingry et al. 603 by electrospinning polyacrylonitrile followed by carbonization. The carbon fiber electrodes were then modified with PPY, ABTS and laccase. 1 mA.cm -2 at +0.1 V vs. Ag/AgCl in a 0.1 M pH 3 phosphate buffer was reached.

3D materials

Although on planar electrodes MET permits to significantly enhance O2 current densities compared to DET, the performances are still not sufficient to envision the elaboration of high power EBFCs. In such a context, the most intuitive way to enhance the power densities is to increase the specific surface of the electrode, to allow for higher enzyme loading. It is especially crucial to develop a microstructure suitable both for enzyme immobilization and mass transfer of substrates. Regarding that, different carbonaceous or metallic matrices based on conductive nanomaterials, as well as free-standing 3D structures, have been proposed for MET electrodes and will be discussed in the following. Of particular interest is the recent paper from Eychmuller et al. [START_REF] Wen | Enzymatic Biofuel Cells on Porous Nanostructures[END_REF] and Zhao et al. [START_REF] Zhao | Nanostructured Material-Based Biofuel Cells: Recent Advances and Future Prospects[END_REF] reviewing the use of porous nanostructures for enzymatic EBFCs.

Porous carbon electrodes

Carbon cloth

Carbon cloth is usually made with carbon fibers of different diameters, which can be further functionalized with CNTs to enhance even more the specific surface. 604 It is usually fixed on a planar electrode with a conductive carbon paint, and further modified with polymer and enzymes (Fig 39). Calabrese- Barton et al. 605,606 were the first to report the use of a 375 m thick carbon cloth with a porosity of 78% and made with 10 m diameter carbon fiber.

Their electrode was composed of PVI-Os(tpy)(dme-bpy) 2+/3+ (with 1/5 th of the imidazoles complexed with [Os(tpy)(dme-bpy)] 2+/3+ (tpy = terpyridine; dme-bpy = 4,4'-dimethyl-2,2'bipyridine)]) and Th-laccase. They reached a current density of 5 mA.cm -2 at 0.57 V vs.

Ag/AgCl in a pH 5 chloride-free citrate buffer at 37.5ºC (Fig 39B). In this pioneering work, the authors demonstrated that a 50-fold increase in surface area lead to a multiplication of the O2 reduction current by a factor of 5. A glucose/O2 EBFC built with this cathode had a five-fold higher power density than the earlier cells. 606 At neutral pH and in presence of 0.14 M chloride, the current density of this cathode was almost nil, due to the inactivity/inhibition of this laccase species in physiological conditions. However, when using laccase from Pleurotus ostreatus, the same authors could obtain ~100 µA.cm -2 at +0.4 V vs. Ag/AgCl at pH 7 in a phosphate buffer containing NaCl. 178 In 2008, Gallaway et al. reached 6.25 mA.cm -2 at +0.3 V vs. Ag/AgCl in an oxygenated pH 7 buffer when Sc-laccase was used in a porous composite electrode. 166 To build a faster O2 cathode operating at pH 5, a spacer has been introduced between the backbone and the osmium polymer to enhance the B A rate of collisions between neighboring osmium complexes and therefore the apparent electron diffusion from (6.2  0.8)×10 -9 cm 2 .s -1 to (7.8  0.4) × 10 -7 cm 2 .s -1 . 533,607 As seen in Fig 33B ,   the presence of the tether permitted to significantly increase the current. 500 A.cm -2 was reached at only +0.63 V vs. Ag/AgCl for the polymer with the tether while it was at +0.54 V vs. Ag/AgCl for the polymer without spacer. A glucose/O2 biofuel cell built with this new cathode operated at Ecell = 0.88 V while producing Pmax = 350 W.cm -2 in a stagnant pH 5 buffer at 37°C. 533 At the cathode, a current density of 3 mA.cm -2 at +0.54 V vs. Ag/AgCl at pH 5 was reached, which is at only -140 mV vs. the potential of the reversible O2/H2O electrode. In 2002, Mano and Heller were the first to report the reduction of O2 under physiological conditions (pH 7.4, 0.15 M NaCl, 37°C) with immobilized redox mediators. 175 The electrode consisted in the crosslinked adduct of Mv-BOD and PAA-PVI-[Os (4,4'dichloro-2,2'-bipyridine)2Cl] +/2+ with a redox potential of +340 mV vs. Ag/AgCl. The modified electrode reached 5 mA cm -2 when poised at +0.3 V vs. Ag/AgCl. The same year, to upshift the operating voltage of the EBFC, a new cathode was elaborated with the same redox polymer but with a new BOD from Trachyderma tsunodae. 120 Cardoso et al. have also reported the use of carbon cloth modified with polyamidoamine dendrimers for the immobilization of Tv-laccase using ABTS entrapped into polypyrrole films. 278 Minteer et al. used a different approach. 510 Their carbon cloth electrode was first coated with a suspension of Nafion® modified with quaternary ammonium salt and containing bilirubin and Mv-BOD, and further soaked in 1 mM Ru(bpy)3 2+ to pre-concentrate the redox mediator in the membrane. In addition to be ethanol tolerant, the advantage of this cathode was its extended active lifetime provided by the treated porous Nafion® membrane. 491 The authors postulated that the modified structure of Nafion® with ammonium salts could help stabilizing and increasing the enzyme activity, by providing a protective outer shell and an ideal chemical environment resisting to a decrease in pH within the pore structure. This cathode was used in an ethanol/oxygen biofuel cell which displayed an active lifetime of 30 days and power densities of up to 0.46 mW.cm -2 . With the same idea of improving the active lifetime of Mv-BOD, Heller et al. adopted a different strategy. 608 After modification of their carbon cloth electrode with osmium polymer, crosslinker and BOD, they coated their electrode with lyotropic liquid crystals. The authors demonstrated that such coating permitted to prevent mechanical stripping of the bioelectrocatalyst upon shearing.

Moreover, it reduced the damage due to urate electro-oxidation without affecting the reduction of O2. A similar strategy has been recently developed by Jian et al. with the same system, but using a supramolecular coating made with glycosylated nucleoside fluorocarbon amphiphile instead of liquid crystals. 609 Carbon nanotubes were also controllably grown on carbon cloth as reported by Schuhmann et al. 610 With such electrode modified with osmium polymer and Th-Laccase, the authors could increase 125 fold the power density of their EBFC compared to an EBFC made with a bare carbon cloth electrode and identical bioelectrocatalysts.

Buckypapers

Buckypaper (BP)-based nanostructured electrodes have been widely used due to their simplicity of fabrication. BPs are usually obtained by classical filtration technique of a CNT suspension. Redox mediators may be added during the filtration or a posteriori to functionalize the electrode surface. However, with this filtration process, it is not possible to control neither the porosity, nor the orientation of the nanotubes, and non-homogenous papers are usually obtained.

Cosnier et al. developed free standing buckypaper electrodes by combining multiwalled carbon nanotubes (MW-CNTs) and bis-pyrene modified with ABTS. The latter acted both as a cross linker between the nanotubes and as a redox mediator for the Tt-laccase. The authors could reach 1.6 mA at 0 V vs. Ag/AgCl in a 0.1 M pH 5 buffer with an operational stability evaluated at two weeks. 611 Hussein et al. 612,613 also described the use of buckypapers.

Novel BPs fabricated from commercially available multi-walled carbon nanotubes (MW-CNTs) were coated with Mv-BOD, ABTS and Nafion®. 613 The authors demonstrated that they could obtain a higher electrocatalytic activity of BOD for the ORR than at a commercially available carbon black (Vulcan XC-72R). 613 In their last report, 614 BP was modified with Mv-BOD and ABTS and further coated with Nafion®. When this electrode was implemented in an EBFC with a glucose oxidizing anode, a power density of 26 W.cm -2 at Ecell = 0.2 V was attained at 37°C in a quiescent O2-saturated physiological buffer containing 5 mM glucose.

Carbon-nanotube fibers

3D-structured carbon fiber is the ideal candidate to develop miniaturized EBFCs. It is the ideal combination for achieving large surface area and fast mass transport while maintaining the geometric surface as small as possible. In 2010, Gao et al. developed a new porous cylindrical electrode combining radial transport and large specific surface area allowing for fast mass transport. It was made of assembled and oriented nanotubes further coated with osmium redox polymer and Mv-BOD. 615 In a PBS buffer and under air, the current density was 10 folds higher on the newly engineered porous microwire electrode compared to a conventional modified carbon fiber. It also allowed smaller overpotentials for O2 reduction. In addition, the mechanical stability was considerably enhanced due to the presence of nanotubes. Such strengthening effect has also been reported for numerous electrodes made with CNTs. For example, Kang et al. 551,616 Copyright 21014, Nature Publishing Group After twisting to obtain a yarn, the electrodes were modified with osmium polymer and Mv-BOD. The authors could reach 6 mA.cm -2 at +0.2 V vs. Ag/AgCl in an O2-saturated PBS buffer at 37°C. The high current density resulted from the presence of the hydrophilic PEDOT layer which permitted to enhance the connectivity, conductivity and wettability of CNTs while providing electrolyte accessibility within the entire yarn. A different strategy has been developed by Cosnier et al. 337 The authors produced electrospun carbon nanofibers containing CNTs. After annealing, the fibers were first functionalized via the reduction of 4carboxyphenyldiazonium to generate carboxy functions. Then, Tt-laccase was immobilized on the modified fiber by amide coupling. To further enhance the amount of wired enzymes, a bispyrene modified with ABTS was added on the fibers. Such modified fibers permitted to reach 100 A.cm -2 at +0.2 V vs. Ag/AgCl in a 0.1 M phosphate buffer pH5.

3D carbonaceous electrodes

In 2014, Tsujimura et al. 618 reported the formation of Magnesium oxide-templated mesoporous carbon (MgOC, mean pore diameter 38 nm). The advantage of magnesium oxide templating is the possibility to tune at will the pore size. In 2015, they used this technique to elaborate a cathode modified with Mv-BOD and ABTS. 508 The adsorption of ABTS on the electrode surface improved both the BOD loading and the electron transfer rate from the electrode to the electro-active site of BOD. No significant leaching of the redox mediator was observed. Willner et al. 373 described the design of highly porous electrodes based on glassy carbon first modified with mesoporous carbon particles (500 nm diameter, with pore dimensions 6.3 nm) perfused with ABTS after drying. The modified electrodes were then coated with Mv-BOD and Nafion®. According to the author, the advantage of this method was the combination of the high surface area, the close proximity between the redox mediator and the BOD, and the possibility to entrap the redox mediator within the matrix (Fig 41 ). Mv-BOD and osmium polymer, 175 A.cm -2 at +0.1 V vs. Ag/AgCl in a PBS buffer was obtained with or without rotation. 619 Additional examples can also be found in the review of Bellino et al. [START_REF] Catalano | Wired Enzymes in Mesoporous Materials: a Benchmark for Fabricating Biofuel Cells[END_REF] who described in details the use of mesoporous materials for wired enzymes.

Gold electrodes

Magner et al. 620 fabricated and characterized nanoporous gold substrates, with different morphologies and roughnesses, which were attached to GC electrodes. After modification with an osmium polymer and Mv-BOD, the authors reached 500 µA. cm -2 at neutral pH in unstirred conditions. Interestingly, the authors also demonstrated that the diffusion layer was exceeding the film thickness, resulting in the overlapping of the diffusion zone between adjacent nanopores, and therefore yielding to similar currents than a planar

Other enzymatic systems

If state-of-the art biocathodes mainly rely on the use of laccases or BODs, alternative approaches have been developed to enzymatically reduce O2 to water. This includes the use of bienzymatic systems, or of different enzymes such as copper efflux oxidase (CueO) or tyrosinase (also called polyphenol oxidase, abbreviated PPO). Even if the reduction of oxygen has been achieved with these systems, these approaches have not been widespread for two mains reasons: on one hand, CueO and PPO display an overpotential for O2 reduction higher than BOD and laccase; and on the other hand the design of bienzymatic systems is often time consuming and more complicated to implement. Ag/AgCl. The onset potential was 0.6 V vs. Ag/AgCl, close to the voltage that could be obtained with a high potential laccase. They also demonstrated that in presence of 6 mM glucose, the addition of 100 mM Cl -did not affect the current density. Cosnier et al. elaborated two types of electrodes relying on the same concept. In the first, they used doublewalled carbon nanotubes modified with HRP and GOx. 625 The authors reached 200 A.cm -2 at +0.3 V vs. Ag/AgCl in an oxygenated 5 mM glucose solution. For their second biocathode, they developed a more complex system (Fig 43B ). 624 First, they coated buckypaper with pyrene modified with ABTS, and then incubated it in a HRP solution. The modified electrodes were functionalized with pyrrole-concanavalin A and incubated in a GOx solution.

Their freestanding electrode generated 11 mA.cm -2 at +0.1 V vs. Ag/AgCl during 1 hour of continuous discharge in an oxygenated 5 mM glucose buffer, and was insensitive to Cl -. Ramanivicius et al. 626 used graphite rod electrode modified with GOx and HRP, and generated 150 A.cm -2 in an aerated 100 mM glucose solution at 25°C. The resulting multilayers were then further cross-linked to provide stability. The authors used this kind of biocathodes for a variety of biocatalytic systems. [START_REF] Willner | Integrated Enzyme-Based Biofuel Cells -A Review[END_REF][START_REF] Katz | Biofuel Cells with Switchable Power Output[END_REF][629][630][631] For example, in 2001, they reported the first self-powered biosensors with such bienzymatic cathode. 632 In 2003, 633 they developed an EBFC with an electrochemically switchable power output. Their cathode was composed of poly(acrylic acid) loaded with Cu 2+ and further functionalized with Cyt c and COx. Depending of the applied potential, i.e -0.5 V or +0.5 V vs. Ag/AgCl, the authors could respectively tune the biocathode into a conductive or nonconductive state. In 2005, 634 they also developed a magnetically controlled EBFC still based on this biocathode and demonstrated that a 3-fold increase in power output could be obtained when a magnetic field of 0.92 T was applied.

Cytochrome c and cytochrome oxidase

Copper efflux oxidase

The use of CueO has been proposed in 2007 by Kano et al. as an alternative to laccase or BOD, and has been mainly developed by this group. 635 After immobilization of the enzyme on highly oriented pyrolytic graphite electrodes by simple drop coating, the authors could reach 4 mA.cm -2 at pH 5, 25°C, with the electrode rotating at 1500 rpm. The authors also evaluated the redox potential of the T1 Cu at +0.3 V vs. Ag/AgCl by redox titration, which was later confirmed when CueO was immobilized in carbon aerogel. 636 In these conditions, they reached 8 mA.cm -2 at 0 V vs. Ag/AgCl in a pH 5 buffer and at 4000 rpm. They further improved their biocathode by using mesoporous carbon electrodes, and reached 12 mA.cm -2 at -0.4 V vs. Ag/AgCl in a pH 5 buffer and at 10 000 rpm (Fig 45) They also showed that the limiting current densities were pH independent in the 2-8 pH range. The exact kinetic mechanism of this enzyme, and particularly the role of the fifth copper, has yet to be elucidated. While it has been shown that the fifth copper of the enzyme is required to oxidize ABTS, 638 its presence is not necessary to achieve DET with an electrode surface. Kerzenmacher et al. have also reported the use of crude supernatants containing CueO. 639 The implementation of CueO in EBFCs using dissolved O2 has however not been done so far.

Polyphenol oxidase

Because it was shown by Kang et al. that some BOD species were sensitive to urate, 640,641 which would prevent their use in blood, Cosnier et al. proposed to use instead the urate-tolerant tyrosinase as a cathodic enzyme. In 2010, they designed an EBFC implanted in rats with a biocathode composed of PPO, graphite and ubiquinone. 642 PPO could reduce O2 to water in complex media, and the implanted EBFC generated Pmax = 24.4 W.mL -1 . Since a dialysis bag was needed to prevent the leaching of ubiquinone, the authors replaced ubiquinone with tetrathiafulvalene. 643 An original electrode has also been developed by Wang et al. A mixture of banana pulp, rich in PPO, and graphite particles, was used to elaborate a biocathode. In presence of hydroquinone, as diffusing redox mediator, the authors were able to reach 0.27 mA.cm -2 in 0.1 M PBS pH 7.4. 644 Non catalytic DET between PPO and graphite electrodes has been first reported in 1996 by Gorton et al., but the catalytic reduction of O2 to water was not observed. 134 PPO has also been immobilized on nano titanium dioxide particles, 645 on silver electrodes, 646 on singlewalled carbon nanotubes, 647 or onto glassy carbon electrodes, 648 but again catalytic electroreduction of O2 was never achieved. The only success has been reported by Cosnier et al. in 2012. 649 The authors made an electrode by compressing MW-CNTs and PPO together.

With the resulting electrode, a current density of 0.55 mA.cm -2 was recorded at 0 V vs.

Ag/AgCl in a 0.2 M PBS buffer pH 7 at 25°C (Fig 46A). The authors concluded that they succeeded in connecting the T3 copper to the electrode surface. Mechanistically, the authors proposed a kinetic model previously discussed for tyrosinase. 102,650 The oxy form might undergo an O-O cleavage via the DET, or via the formation of an intermediate which is further reduced at the electrode surface. After addition of 4 protons and 4 electrons, the generated deoxy form further reacts with O2 which completes the catalytic cycle (Fig. 46B).

MCOS based biocathodes in enzymatic biofuel cells

In this paragraph, we only give some examples of the implementation of MCO-based biocathodes in EBFCs. The interested reader will find in

Operated in buffer

As mentioned above, the first membrane-less glucose/O2 EBFC was reported in 1999 by Katz,Willner et al. 627 This paper is important for the field as it was the first paper that demonstrated that by properly choosing and/or designing bioelectrocatalysts, a membrane separating anodic and cathodic compartments was not necessary. It also opened the way to miniaturized systems. However, both power density and operating voltage were quite low at that time. The next important step has been taken by Chen et al. two years later. 652 Their EBFCs consisted in two 7-m diameter, 2-cm long carbon fibers respectively coated with glucose oxidase and an osmium redox polymer at the anode, and Tv-laccase and a second osmium redox polymer at the cathode. At 37°C, in a pH 5 aqueous solution containing 15 mM glucose, and under quiescent conditions, the EBFC reached Pmax = 137 W.cm -2 at Ecell = 0.4

V. Ever since, the numbers of MET and DET based glucose/O2 EBFCs have been growing

exponentially due to the envisioned applications in bodily fluids. [START_REF] Barton | Enzymatic Biofuel Cells for Implantable and Microscale Devices[END_REF][START_REF] Bullen | Biofuel Cells and Their Development[END_REF][START_REF] Davis | Biofuel Cells -Recent Advances and Applications[END_REF][START_REF] Minteer | Enzyme-Based Biofuel Cells[END_REF][START_REF] Willner | Integrated Enzyme-Based Biofuel Cells -A Review[END_REF][START_REF] Leech | Enzymatic Fuel Cells: Recent Progress Electrochim[END_REF][START_REF] Luz | Enzyme Biofuel Cells: Thermodynamics, Kinetics and Challenges in Applicability[END_REF][START_REF] Shleev | Quo Vadis[END_REF] Laccases and BODs have been mainly used at the cathodic side in combination with glucose oxidase, 596,601,653,654 pyranose dehydrogenase, 655 PQQ-dependent glucose dehydrogenase, 426,590,[656][657][658] FAD-dependent glucose dehydrogenase, 591,659 or cellobiose dehydrogenase 388,660-663 at the anode. It is worth mentioning that bionanodes based on enzyme cascades have also been developed [664][665][666] to improve the coulombic efficiency of EBFCs. One of the highest power densities was reported by Sakai et al. in 2009. 197 Their EBFC generated a maximum power density of 1.45 mW.cm -2 at 0.3 V under quiescent conditions, with a cathode based on Mv-BOD immobilized within a matrix composed of polylysine and K3[Fe(CN)6].

The work of Xiao et al. deserves a particular attention because they reported the first and only example of EBFC operating in organic solvents containing 5 mM glucose. 276 Their nanoporous gold EBFC was respectively modified with glucose oxidase and osmium redox polymer on the anodic side, and bilirubin oxidase and osmium redox polymer on the cathodic side. The EBFC could operate in acetonitrile, acetone and alcohols. In this latter case, the power output of the cell decreased with increasing hydrophobicity of the alcohols; methanol  ethanol  1-propanol  1-butanol 1-pentanol. More importantly, even though the power density was lower in organic solvents than in aqueous solvents, the anode and the cathode recovered their initial activities when transferred in aqueous buffer after operation in organic solutions. This inspiring work may lead to new applications of EBFCs, such as for example to their use in bioelectrosyntheses.

In fruits/vegetables

As a proof of concept of possible implantation in media containing glucose and O2,

EBFCs have been inserted in different fruits, vegetables or juices containing glucose and O2 (Fig 47)

. [667][668][669] The first has been reported by Mano et al. in EBFCs. Of interest is the experiment made by Southcott et al. in 2013. 679 To mimic the human blood circulating in a 8 m capillary at 1 mm.s -1 , the authors elaborated a biofuel flow cell. When a human serum solution with 6.4 mM glucose concentration was used, a current density of 0.83 mAcm -2 was obtained with a laccase-based biocathode.

In serum and blood

Numerous examples of EBFCs operating in bovine or human serum can be found in the literature. To the best of our knowledge, the first EBFC operating in human serum has been reported by Gao et al. in 2007. 680 There are two major explanations for the low number of experiments in human blood.

First, beyond respecting strict ethical guidelines for experimental practice, governmental authorizations are needed, which may be cumbersome in some countries. Then, blood contains numerous proteins, ions or molecules that may interfere, inhibit or passivate enzymes, redox mediators and electrodes. This is also the reason why power densities attained in whole human blood are lower than in serum, which is 90% water, and relatively interferentfree. In addition, the venous blood-dissolved O2 concentration is lower than in aqueous buffers, making the cathode even more limiting than in buffer. The proof of concept that EBFCs can harvest energy for whole human blood has been, however, proven unequivocally.

The first real in vivo experiment has yet to be done. To take this next step towards human implantation, other considerations will have to be taken into account such as biocompatibility and cytotoxicity. We note in this context that MCOs based cathodes have also been operated in other human fluids such as saliva, tears or urine. 339,391,421,685,687 2005 of the proof of concept of this kind of devices, that displayed a maximum power density of 5 μW cm -2 at a cell voltage above 0.8 V at pH 5, 282,283 was followed by further developments and fundamental studies. Particular care is indeed required due to the specificities of Hase as an EBFC catalyst, and the inherent problems of the H2 fuel that, combined with O2, leads to explosive mixtures under certain conditions. 142,282,308 Power densities have overcome 100 µW.cm -2 in 2012 thanks to the structuration of electrodes with CNTs. 142,308 The same kinds of performances were also obtained when the enzymes were immobilized at AuNP-modified electrodes. 392 H2/O2 EBFCs with power densities above 1 mW.cm -2 have been developed in the groups of Armstrong 143 and Lojou. 372 In particular, the second was able to operate over a wide range of temperatures thanks to the combination of a hyperthermophilic Hase and the thermostable Bp-BOD, both immobilized in DET configuration at CNF-modified electrodes. In 2014 the first H2/O2 EBFC using an O2sensitive Hase was reported by Plumeré et al. 688 No membrane separated the anodic and cathodic compartments, and the Hase, operating via a MET mechanism, was protected from O2 denaturation by a redox hydrogel. 689 The ability of these EBFCs to operate small devices was also recently demonstrated. 335,381 A membrane-less H2/O2 EBFC using an air-breathing Mv-BOD cathode was reported in 2015 466 , followed by the development of an EBFC using gas-diffusion electrodes both at the anode and the cathode by Kano et al. in 2016 467,690 . In this configuration, a maximal power density of 6.1 mW.cm -2 at Ecell = 0.72 V was reached at room temperature and under quiescent conditions. 467 Besides glucose or H2, other fuels have been studied in combination with MCO-based biocathodes 666 : to name a few, methanol, 277,278,691 ethanol, 692-694 fructose, 470,695 ascorbate, 391 or sulfite 423 have also been reported in the literature. The interested reader will find additional information in the reviews listed in Table 1.

Microfluidic biofuel cells

MCOs based electrodes have also been used to elaborate microfluidic EBFCs even if, so far, their use for such applications is less widespread. This field has been reviewed by Kjeang et al.,[START_REF] Lee | A Perspective on Microfluidic Biofuel Cells[END_REF] Sinton et al. [START_REF] Kjeang | Microfluidic Fuel Cells: A Review[END_REF] and more recently by Sanchez et al. [START_REF] Safdar | Microfluidic Fuel Cells for Energy Generation[END_REF] Microfluidic biofuel cells are usually composed of two channels, of various designs and geometries, into which circulate the respective anodic and cathodic substrates. Redox mediators and enzymes can also be added in this stream if they are not immobilized. The lack of convective mixing at low Reynolds numbers permits to eliminate the need for a membrane between the anode and cathode, therefore minimizing ohmic losses. Protons diffuse through the liquid-liquid interface between the two substrate streams. Despite the conceptual simplicity, numerous difficulties can be encountered such as the cross-diffusional mixing of substrates at the interface between the two streams. The fabrication of the microchannel and the lengths/diameters of the electrodes require particular care. One of the first use of a MCObased biocathode in a microfluidic BFC has been reported by Palmore et al. 696 The anolyte consisted of 10 mM ABTS and the catholyte of 10 mM ABTS and 0.5 mg.mL -1 of laccase.

Different strategies have been later proposed to immobilize the enzymes. 697 While PDMS has mainly been used to create microfluidic EBFCs, paper based microfluidic EBFCs are now emerging. For example, a compact and low volume carbon paper-based enzymatic microfluidic glucose/O2 biofuel cell has been reported recently. 698 Over the 2.5-30 mM glucose concentration range, the power density was a linear function of the glucose concentration, and ranged from 20 to 90 µW.cm -2 .

Paper based EBFCs

Despite the low price of paper, its availability and ease of use, the first example of Japanese paper has been chosen because it has a high water absorbency. To be made conductive, it was first coated with a carbon ink. On the anodic side, the electrode was further modified with a mixture of Ketjen black, glucose oxidase and tetrathiafulvalene. At the cathode, a mixture of KB and polytetrafluoroethylene was coated on the conductive paper before Mv-BOD addition. The power density of the fabricated EBFC reached 0.12 mW.cm -2 at Ecell = 0.4 V in presence of 100 mM glucose in a 1 M phosphate buffer pH 7 at room temperature, which was the highest power reported in 2013. Paper based electrodes have also been used in microfludidic EBFCs, 700 in self-powered systems, 701 or to elaborate a selfpowered hybrid micro-supercapacitor integrated system. 702 All used MCO-based cathodes. A recent and excellent review by Desmet et al. [START_REF] Desmet | Paper Electrodes for Bioelectrochemistry: Biosensors and Biofuel Cells[END_REF] summarizes the use of paper electrodes in biofuel cells.

MCOs with air-breathing electrodes

Most of the EBFCs are limited by the cathode because of the low diffusion coEfficient (< 5.10 -5 cm -2 .s -1 ) and concentration of dissolved O2 (1.2 mM maximum at neutral pH) in aqueous solution. Increasing the low current densities and enhancing mass transport on the cathodic side are key challenges in EBFCs. These limitations could be circumvented by using an air-breathing cathode, where O2 would diffuse directly from the air to the cathode. In 2005, Calabrese-Barton 703 reported a particularly interesting numerical simulation of a MCO-based cathode. The authors predicted that in an O2 saturated pH 5 buffer at 37°C, a maximum current density of 9.2 mA.cm -2 could be attained with dissolved oxygen. By introducing an air-breathing cathode, the model predicted that jmax = 60 mA.cm -2 in air or jmax = 100 mA.cm -2 in pure O2 could be reached. However, designing a gas-diffusion cathode is not trivial. The gas-diffusion cathode usually consists of two layers: a gas-diffusion layer made of hydrophobized carbon black 704,705 or Ketjen black 690,706,707 , which has to be permeable to gas; a hydrophilic catalytic layer made with carbon black 708 , carbon cloth 709 , CNTs 710-712 or carbon felt 713 and containing the MCO. These first two layers may also be added on top of a third layer providing for mechanical stability. A delicate balance between hydrophobicity and hydrophilicity of the carbon/air and carbon/electrolyte interfaces has to be achieved (Fig 51). , 368,466,712,715 laccases 708,709,716 and CueO. 706 For example, Atanassov et al. reported a DET-based laccase-modified gas-diffusion electrode with a current density 0.5 mA.cm -2 . 307 20 mA.cm -2 have been reached for a DET-based CueO biocathode. 706 An interesting approach has been reported recently by Atanassov et al. 717 who combined BOD and non-platinum based catalysts. jmax = 1.2 mA.cm -2 was reached in a 0.1 M phosphate buffer pH 7.5. MCO-based gas-diffusion electrode have been used in H2/O2, 466,467,690 methanol/O2 718 or in fructose/O2 biofuel cells. 305,368

Biofuel cells for self-powered devices

Self-powered sensing devices do not need any external power source, and usually consist of only two electrodes. One electrode acts as the sensing part and both electrodes allowed electron transfer. Two Boolean logic gates (AND/OR) were assembled using this concept. MCO-based biocathodes have also been used for example for self-powered immune sensors, 725 self-powered biosensors for arsenite and arsenate, 726 lactate biosensors, 336 antiobitic residue determination, 727,728 glucose measurement with amperometric 602 or optical readout, 729 neurochemical monitoring in rat brain, 730 self-powered fluorescence switch systems, 731 or for wireless signal transmission. 732 This field has been reviewed by Zhou et al. [START_REF] Zhou | Bioelectrochemical Interface Engineering: toward the Fabrication of Electrochemical Biosensors, Biofuel Cells and Self Powered Logic Biosensors[END_REF][START_REF] Zhou | Biofuel Cells for Self-Powered Electrochemical Biosensing and Logic Biosensing: a Review[END_REF] and by Arechederra et al. The authors reported also the same year a self-charging ascorbate/O2 electrochemical capacitor. 734 In 2014 also, Cosnier et al. 735 published a similar concept using CNT matrixes modified with Tv-laccase. The authors demonstrated that their hybrid system was stable for at least 40 000 pulses of 2 mW. In 2016, the first self-charging Nernstian bio-supercapacitor operating with an identical redox mediator at the anode and cathode was reported. 736 In 2017, Xiao et al. 737 reported a hybrid device using Mv-BOD immobilized on de-alloyed nanoporous gold. Good operational stability was observed for 50 charge/discharge cycles at j = 0.2 mA cm -2 . A power density of 608.8 μW.cm -2 was obtained when discharged at 2 mA cm -2 , i.e.

468 higher than the power output of the BFC itself. The work of Bilewicz et al. using a laccase-based cathode deserves also attention in this field. [738][739][740][741] For example, they used a biocathode covered with laccase adsorbed on naphthylated multiwalled carbon nanotubes to elaborate an hybrid devices generating Pmax = 1.6 mW.cm -2 at Ecell = 0.76 V. Hybrid devices may also rely on solar power, which allows simultaneous solar energy conversion and storage.

Nanostructured indium tin oxide electrodes were therefore used. 742,743 This new promising field, which may circumvent low power densities and substrates depletion arising in EBFCs, has been reviewed in 2014 by Pankratov et al. [START_REF] Pankratov | Hybrid Electric Power Biodevices[END_REF] and in 2016 by Sode et al. [START_REF] Sode | Biocapacitor: a Novel Principle for Biosensors[END_REF]

Wearable biofuel cells

Wearable electronics, and particularly wearable electrochemical sensors, have attracted recently a lot of attention. 744 Since they are likely to be in contact with the human skin or human tears [START_REF] Pankratov | Tear Based Bioelectronics[END_REF]339 , they should be flexible, light, and biocompatible. The detection of a wide variety of substrates, ranging from glucose to uric acid has been realized. Even more recently, the use of wearable biofuel cells has been envisioned as a power source for wearable applications. Bandodkar has written two reviews on this subject. 745,746 Surprisingly, Pt has been preferred so far to MCOs as the cathodic catalyst. For example, an epidermal EBFC able to harvest chemical energy from human perspiration and particularly from lactate, 747 or the fabrication of a wearable biofuel cell printed directly onto textile substrates have been reported. 748 In both cases, Pt was the catalyst of O2-reduction. In 2014 however, Mv-BOD has been used by Ogawa et al. 749 The authors developed an organic transdermal iontophoresis patch with an integrated EBFC (Fig 54). The anodic electrode was made with fructose dehydrogenase coated on a CNT-modified carbon strip, and an O2 diffusion cathode was based on Mv-BOD. Once immobilized on the skin, the OCP was 0.75 V and the maximal current density 300 A.cm -2 .

Conclusions and Outlook

The major cause of inefficiency in common EBFCs, envisioned as energy-converting devices for more than half a century, is the four-electron electroreduction of oxygen to water, particularly because of the low concentration and slow diffusion of O2 in aqueous media.

Studies in the recent decades aimed to overcome this limitation. We have witnessed an exponential growth in publications on optimizing MCO-based biocathodes. The different strategies that have been put forward are listed in Table 1. Two major research axes have developed: 1) design of increasingly sophisticated nanostructured porous electrodes, allowing increased enzyme loadings without impeding the diffusion of O2 91 and 2) improvement of the electrical connection between MCOs and the electrode surfaces, whether in MET or DET. 750 The continuous increase in OCP, current densities or power densities reflect particularly well the significant progress made along the two lines of research.

The studies have led to a fundamental milestone with the first implantation in a vertebrate and to a second milestone with the first operation in human blood. The latter now raises new questions and exciting challenges, such as immunogenicity, bioinertness and cytotoxicity issues. Solutions have to be found to minimize the reaction with the foreign body. It is likely that a protective/bioinert coating or membrane will be required to minimize the foreign body reaction. It must not be, however, detrimental to O2 diffusion.

Because the enzymatic reduction of O2 is becoming increasingly Efficient, new applications have emerged e.g. in wearable enzymatic biofuel cells and new promising field such has the development of hybrid supercapacitors, which may circumvent low power densities and substrates depletion arising in EBFCs.

In their 2004 pioneering review on EBFCs Calabrese-Barton and Atanassov [START_REF] Barton | Enzymatic Biofuel Cells for Implantable and Microscale Devices[END_REF] said that the development of successful biofuel cells is driven by demand arising from specific applications. The low current densities prevented the use of EBFCs in most of applications.

However, high current densities are not a prerequisite in some, as a current of a few pA suffices, when the voltage is high enough, to power ACSICs. 751 For high power applications, EBCs/supercapacitor systems are of particular interest due to their ability to deliver high power densities within a very short burst.

Despite enormous advances and encouraging achievements, some limitations remain.

One of the most underappreciated problem is the stability. In addition to catalyzing reactions in biofuel cells, enzymes also inspire the design of analogous inorganic and metal-organic catalysts. 752-754 "Bio-inspired" catalysts aim to reproduce the metal coordination observed in biological molecules to attain similar efficiency in a less complex structure. Further development is expected also along this axis.

Acknowledgments

The

  Quartz crystal microbalance with dissipation monitoring QM/MM: Quantum mechanical and molecular mechanics RO: Resting oxidized RRDE: Rotating ring disc electrode RVC: Reticulated vitreous carbon Rv-laccase: Laccase from Rhus vernicifera SAMs: Self-assembled monolayer Sc-laccase: Small laccase from Steptomyces coelicolor SECM: Scanning electrochemical microscopy SERS: Surface enhanced Raman spectroscopy SGZ: Syringaldazine SPG: Spectroscopic graphite SPR: Surface plasmon resonance STM: Scanning tunneling microscopy SW-CNTs: Single-wall carbon nanotubes SWV: Square wave voltammetry Ta-laccase: Laccase from Trichaptum abietinum TBAB: Tetrabutylammonium ions TNC: Tri-nuclear copper cluster To-laccase: Laccase from Trametes ochracea TMOS: Tetramethyl orthosilicate Tt-BOD: Bilirubin oxidase from Trachyderma tsunodae Tt-laccase: Laccase from Trametes troggii Th-laccase: Laccase from Trametes hirsuta tVA: Thiolated veratric acid Tv-laccase: Laccase from Trametes versicolor PPY: Polypyrole

Figure 1 .

 1 Figure 1. UV-visible (A), X-band EPR (B) of purified BOD from Bacillus pumilus highlighting the spectral feature in MCOs. Reprint with permission from ref [101] Copyright 2010 Elsevier.

Figure 2 .

 2 Figure 2. The structure of the MCO active site with arrows marking the flow of substrates, electrons (e -), and O2. Reproduced with permission from ref [ 93 ]. Copyright 2012 Royal

Figure 3 .

 3 Figure 3. Mechanism of O2 reduction to water by the MCOs. Red arrows indicate the steps that take place in the catalytic cycle of the MCOs. Black arrows indicate steps that can be experimentally observed but are not part of the catalytic cycle. The dashed arrows at the right indicate the transfer of an electron from the T1 Cu to the T2 Cu to create PI + e-, that occurs in going from PI to NI but is not experimentally observed in the wild type enzyme.

Figure 4 .

 4 Figure 4. X-rays structure of different MCOs. A) Native fungus laccase from Trametes hirsuta (PDB: 3FPX); B) BOD from Myrothecium verrucaria (PDB: 2XLL); C) Copper

Figure 5 .

 5 Figure 5. Electrochemical characterization of an EBFC. A: Current/Voltage response of the bioanode (blue curve) and of the biocathode (red curve). The purple arrows indicate the current density and the cell voltage giving the maximum power density. B: Polarization curve

Figure 6 .

 6 Figure 6. Two different mechanisms of electron exchange between an enzyme (here a laccase) and an electrode. Left: Direct Electron Transfer (DET) and Right: Mediated Electron Transfer (MET), here with a diffusive mediator.

  4.5 and 3.4.7. The distribution of orientations allowing DET results in distribution of electronic transfer rates 148 reflected by the trailing edge in the catalytic cyclic voltammogram (Fig 7A).

Figure 7 .

 7 Figure 7. Representative cyclic voltammograms of a MCO in DET (A) and MET (B). Curves 1 and 1' are recorded in non-catalytic conditions in the absence of O2. Curve 1' clearly shows the redox process of the mediator. Curves 2 and 2' are recorded in catalytic conditions under O2. The dotted vertical bars indicate the apparent standard potentials of the enzyme (E°'enzyme) and of the mediator (E°'mediator).

Figure 8 .

 8 Figure 8. Comparison of different steps for electron transfer for enzymes in solution (A) and immobilized (B) with Direct electron transfer (DET) or Mediated electron transfer (MET). A) Step 1: Substrate oxidation at the T1; Step 2: IET; Step 3: O2 reduction. B) Step 1: Electron transfer from the electrode to the T1; Step 2: IET; Step 3: O2 reduction; Step 4: O2 diffusion;

Figure 9 .

 9 Figure 9. Schematic illustration of a MCO-based oxygen-reducing cathode. The processes described in the mathematical model occur throughout the film from x = 0 (electrode surface) to x = l (film thickness). Partition of oxygen between the film and solution is described by the partition coEfficient KP,O2. The motion of charges is described by the apparent diffusion coEfficient DMed,app of the redox mediator. Oxygen diffusion occurs within the film with a diffusion coEfficient DO2,app. O2 is reduced by the reduced MCO to H2O, the oxidized MCO is reduced by the mediator, and the mediator is re-reduced at the electrode surface. Adapted with permission from [188]. Copyright 1995 Elsevier.

Figure 10 shows

 10 the 2D case diagram corresponding to the further assumption that diffusion/reaction of redox mediators are well balanced with diffusion/reaction of substrate in the reaction film (in our case it would correspond to 𝐷 𝑂2the saturation condition in the film (KP,O2[O2] << KM,O2).

Figure 10 . 1 (

 101 Figure 10. Two-dimensional case diagram showing the five limiting cases predicted analytically. The concentration profiles surrounding the case diagram are from simulations at the points marked () on the case diagram: case I,  = 0.1,  = 0.01; case II,  = 10,  = 0.001; case III,  = 10,  = 1; case IV,  = 10,  = 1000; case V,  = 0.l,  = 100. The continuous lines indicate dimensionless mediator concentration profiles ([Medred]/[MedTot], assuming that the mediator is confined in the film); and the dotted lines the dimensionless substrate concentration profiles ([O2]/KP,O2[O2]). Horizontal scale is from x = 0 (electrode) to x = 1 (bulk solution). Vertical scale is dimensionless concentration. Note the compressed vertical scale for profiles I and V. Reprinted with permission from [188]. Copyright 1995 Elsevier.

Figure 11 .

 11 Figure 11. Effect of mediator redox potential, E 0 , on laccase-mediator bimolecular rate constant, kcat/Km, and laccase-oxygen rate constant, kcat/Ks. Also shown are redox potentials of laccase and O2/H2O at pH 4. A linear free energy region is observed for high E 0 (small ΔEet)

Figure 12 .

 12 Figure 12. Voltammograms recorded with HOPG electrodes (1) without and (2) with T. hirsuta laccase (30 mg.mL -1 laccase solution) entrapped under a dialysis membrane at the electrode surface. (A) Cyclic voltammograms recorded in air-saturated 50 mM citratephosphate buffer pH 3.5 containing 100 mM NaClO4; scan rate: 10 mV.s -1 . (B) Osteryoung square wave voltammograms recorded under anaerobic conditions in 100 mM phosphate buffer pH 6.5; amplitude: 10 mV, frequency: 10 Hz. Reprinted with permission from [210]. Copyright 2005 Elsevier.

Figure 13 .

 13 Figure 13. Representative cyclic voltammograms of (A) Cerrena maxima and (B) Trametes hirsuta laccases immobilized on (A) bare and (B) 4-aminothiophenol self-assembled monolayer (SAM) modified gold disc electrodes (0.1 M phosphate buffer pH 5.0; scan rate: 10 mV.s -1 ; second scan). (A) CVs of Cerrena maxima laccase under aerobic and anaerobic conditions, and in the absence or presence of sodium fluoride starting at 1040 mV vs. NHE. (B) CVs of Trametes hirsuta laccase under aerobic or anaerobic conditions, and in the absence or presence of sodium fluoride starting at 1040 mV vs. NHE. Reprinted with permission from [205] Copyright 2006 from Elsevier.

3 . 4 . 4 . 2

 3442 These observations can be correlated with the faster denaturation caused by stronger electrostatic interactions observed when MCOs are adsorbed on N-doped carbon nanotubes. (see section 4.1.2 for further details) Applied electrode potential A potential-induced structural change of the enzyme was evidenced by in situ STM studies of Streptomyces coelicolor laccase adsorbed on butanethiol modified Au (111) surface. 233 This technique allowed at the same time the first single-molecule resolution in aqueous buffer. Interestingly the oxidized enzyme (at 0.89 V vs. NHE) appeared transparent, while bright spots were observed for the enzyme under turnover (at 0.44 V vs. NHE under air) (Fig 14A).

Figure 14 .

 14 Figure 14. Influence of applied electrode potential on enzyme. (A): structural change induced by potential, evidenced by in situ STM of Streptomyces coelicolor laccase on a butanethiolmodified monocrystalline gold under pure O2 atmosphere. A1: Initial image at 0.84 V vs. NHE. Bias: 0.70 V. A2: Image at 0.44 V vs. NHE. Bias: 0.30 V. A3: Image after returning at 0.84 V vs. NHE. Bias: 0.70 V. Tunneling current in all cases: 0.035 nA. Scan area: 100 × 100 nm 2 . A4 and A5 are histograms showing the height distribution of the bright spots in A2 and A3, respectively. Height normalization was done against the maximum height measured in A2. Lines are best fits to a normal distribution. Adapted with permission from [233], copyright 2012 American Chemical Society. (B): Comparison of the catalytic current loss measured in cyclic voltammetry after the Mv-BOD electrode has been left at OCV (red columns) or submitted to an applied potential E = +0.2 V vs. Ag/AgCl (black columns). The error bars are represented in green. For better accuracy with respects to the original figure, the timescale is not linear. Adapted with permission from [220]. Copyright 2016 American Chemical Society.

Figure 15 .

 15 Figure 15. (A) Cyclic voltammogram of BOD adsorbed on SPGE in the absence of O2. pH 7.0, 0.1 M phosphate buffer; scan rate: 100 mV.s -1 ; starting-scan potential: 1000 mV vs. NHE. Curve 1: uncorrected for the background current; Curve 2: circles, background current subtracted. The broken line curve represents the calculated theoretical voltammograms. (B) Effect of the scan rate on the background-corrected voltammograms under O2. Curve 1: 10.mV s -1 ; Curve 2: 100 mV.s -1 . Reprinted with permission from [203] Copyright 2008 Elsevier.

range 3 - 5 for

 35  An approximation of the standard apparent potential of the redox center involved in catalysis, E°', was determined for Mv-BOD by fitting the curves with the model developed by Ikeda. 235 E°' was found quasi constant in the pH range 2-5.5 ( 670 mV vs NHE) and a shift to less positive values with an average variation of 45 mV/pH unit was recorded between pH 6 and 8.245

3 . 4 . 7

 347 is uphill.Page et al. 146 and other studies 251 have indeed calculated for a series of redox enzymes that endergonic ET can occur, as long as neighboring redox centers are not separated by more than 14 Å. Taking into account this maximal distance, reorganization energy λ calculated from quantum mechanical and molecular mechanics (QM/MM) evaluations, and ∆G° calculated from the measured potential difference between the two detectable sites, pHdependent internal electron transfer rate constants between T1 and the TNC low redox intermediate were calculated. They ranged from 3 s -1 up to 138 s -1 , in agreement with experimental kcat values. 204 Mathematical modeling of the catalytic current Different DET models can account for the mechanism of the O2 reduction by MCOs electronically connected to the electrode via the T1. Importantly, all models are derived considering non-limiting O2 diffusion. In the first general model proposed by Tsujimura et al., 235 the internal electron transfer (IET) and the different steps of O2 reduction at the TNC are included in a general "catalytic" mechanism (Fig 16 A).

Figure 16 .

 16 Figure 16. Schematic representation of DET mechanism for O2 reduction by MCOs. (A): model proposed by Tsujimura et al. (adapted from [235]). kf and kb are the rate constants of the reversible heterogeneous electron transfer , and kcat is the global catalytic constant for O2 reduction by the BOD including intramolecular electron transfer and all steps of O2 reduction at the TNC. The second step is considered irreversible in the model. (B): refined model. The heterogeneous electron transfer is considered reversible and characterized by the rates for forward and reverse electron transfers k1 and k-1. Similarly, the internal electron transfer is considered reversible and characterized by the rates of forward and reverse electron transfers k2 and k-2. The only irreversible step is the O2 reduction at the TNC characterized by the rate constant k3. Adapted with permission from [204,233]. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA, and American Chemical Society.

  the general mechanism of O2 reduction by MCOs. Blanford et al. proposed a complete general mechanism for O2 reduction by immobilized MCOs, based on that developed by Solomon et al. 92,93 for homogeneous conditions (Fig 17A, see sections 3.1 and 3.4.8 for more details).

Figure 17 .

 17 Figure 17. A: A mechanism for MCO-catalyzed O2 reduction based on that of Solomon and co-workers, with additions from Blanford et al. shown in red. B: Evidence for the formation of the Resting Oxidized (B1) and X state (B2) in response to a series of applied potentials. Eapplied is the experimental potential used, typically 0.4 V vs. NHE at pH 4; when the electrode is at OCP no potential is applied. B1 indicates how the fraction of enzymes in the RO state is calculated. B2 is observed in some particular combinations of pH and time at OCP. Adapted with permission from [165] Copyright 2012 Royal Society of Chemistry.

Figure 18 .

 18 Figure 18. Mechanism for MCO-catalyzed O2 reduction used in the analytical model developed by Agbo et al. The steps including HET are indicated by orange arrows, the catalytically relevant intermediates and internal steps are indicated in blue, and the resting form outside of the catalytic cycle is indicated in green. For the n th step, the forward rate constant is indicated by kn and the reverse by k-n. Adapted with permission from [254].

Figure 19 .

 19 Figure 19. Electrochemical evidence of the alternative resting form in Bp-BOD in presence of NaCl at pH 4 and 37°C, observed in cyclic voltammograms of O2 reduction at 5 mV.s -1 in 200-100 mM phosphate-citrate buffer. Blue curve: usual catalytic response in the absence of NaCl. Red curve: hysteresis observed after 15 mM NaCl addition. The arrows indicate the direction of the potential sweep. Bp-BOD was adsorbed on a pyrolytic graphite electrode first modified with carbon nanofibers. Reproduced with permission from [256]. Copyright 2017 American Chemical Society.

Figure 20 .

 20 Figure 20. Chronoamperometric trace for a (A) BOD/Anthracene-MWCNTs (pH 6.5) and (B) laccase/Anthracene-MWCNTs (pH 4.5) Toray-paper electrode in a hydrodynamic citrate/phosphate buffer solution (0.2 M), poised at 0.2 V (vs. Ag/ AgCl). NB: air and argon

Figure 21 .

 21 Figure 21. (A) AFM topographical images and height line profiles at points indicated by the arrows. Top: before UV/ozone irradiation. Down: after irradiation for three hours. The thorn height distribution is centered at 2,25 nm. (B) Voltammograms of BOD physically adsorbed on original and nanostructured carbon films with different surface wettabilities. 50 mM PB pH 7.0, scan rate 20 mV.s -1 and electrode area 0,0314 cm 2 . Top: real scale and down: enlargement of the dotted square in Top. Adapted with permission from [288]. Copyright 2017 American Chemical Society.

Figure 22 .

 22 Figure 22. (A) Percentage of CNTs connected to the electrode in a 125-mm thick film for various volumes. % CNT fill, ΦC: 0.25% (o); 1.6% (); 3% (∆); 4.5% (▲). (B) MATLAB image displaying only those CNTs connected to the electrode, shown as black lines.

  for Mv-BOD in a kinetically controlled regime. The intermediate length(1-4 µm) led to the highest current densities (6.1 mA.cm -2 at 0.1 V) while performances decreased for longer (10 µm) and shorter (<1 µm) CNTs sharing the same diameter(10-15 nm) (Fig 23). This quite complex behavior was explained by considering determinant the ratio of hydrophobicity to density of carboxy groups. Since the walls are mostly hydrophobic, while carboxy groups are mostly at the ends, this ratio is driven quasi exclusively by the length of the CNTs.

Figure 23 .

 23 Figure 23. Influence of the length of MW-CNTs on ORR by Mv BOD. LSVs in O2-saturated 100 mM phosphate buffer (pH 7) at 41 C, ω = 4000 rpm, and v = 5 mV.s -1 . Mv BOD is adsorbed at MW-CNTs of different lengths (L) and outer diameter (OD), or at CNPs. Black solid line: L = 1-4 µm and OD = 10-15 nm ; black broken line: L = 1-4 µm and OD = 10-15 nm and a surfactant is added ; black dotted line : L < 1 µm and OD = 8-10 nm ; and gray solid line : L = 10 µm and OD = 10-15 nm. The gray broken line represents CNPs. Adapted with permission from [330]. Copyrigth 2016 Elsevier

  et al. constructed an innovative free-standing CNT-based enzymatic film. 341 After introducing Trametes sp. laccase in a forest made of 1 mm-long CNTs separated by 16 nm, further drying induced shrinkage to a close packed structure, whose total thickness was 20 µm (Fig 24). Current densities of 2.1 ± 0.3 mA.cm -2 were reached with stirring, and further increased to 4 mA.cm -2 when 2 films were joined (Fig 24).

Figure 24 .

 24 Figure 24. (A) Schematic diagram of laccase entrapment in a CNT forest by "liquid-induced shrinkage". The photograph shows a free-standing enzyme-CNT film that can be manipulated with tweezers. (B) Corresponding CV of the so-formed single-piece electrode in N2 (black curve) and O2-saturated stirred Mc Illvaine buffer pH 5 (red curve). The green curve shows the currents obtained with a double-piece electrode, v=10 mV.s -1 . The electrode films were prepared using a 20 µm thick CNT film and 0.25 mg.mL -1 laccase solution. Adapted with permission from [341]. Copyright 2011 American Chemical Society.

in 2 Figure 25 .

 225 Figure 25. Comparison of different protocols for Mv-BOD immobilization on GO flakes. Small GO flakes are labelled GO-S2. (A): The LBL biocathodes with BOD adsorbed on layers of GO (black), electrochemically reduced GO (red), GO-S2 (blue) and electrochemically reduced (GO-S2) (green). (B): The biocathodes prepared by in-situ mixing of BOD on the GCE with GO (red) and GO-S2 (blue) with their subsequent reduction. The biocathode prepared by "ex situ" incubation of BOD with GO-S2 and consequent electrochemical reduction is shown in black and control electrode with only electrochemically reduced GO layer is shown in green. Adapted with permission from [355]. Copyrigth 2014

Figure 26 .

 26 Figure 26. AuNPs: (A) SEM images of bare electrode (1) and electrode modified with 20 (2), 40 (3), 60 (4) and 80nm (5) AuNPs. (B) Experimental (points) vs. modelled (lines) voltamogramms of electrodes modified with Mv-BOD in O2 saturated buffer. In 1 currents are reported to the geometric area and in 2 they are reported to the real area. Black: bare gold; Green: 20 nm AuNPs; Red: 40 nm; Purple: 60 nm and Blue 80 nm. Adapted with permission from [386]. Copyright 2014 The Royal Society of Chemistry

  401

Figure 27 .

 27 Figure 27. (A) SEM images of the building blocks, gold supraspheres. (B) top view of the resulting porous gold surface. (C) Polarization curves of the biocathode for O2 reduction in 50 mM acetate buffer pH 5.5 under saturated N2, ambient air and saturated O2 conditions. Scan rate 1 mV.s -1 . Adapted with permission from [401]. Copyright 2014 American Chemical Society.

  this orientation, and to engineer adequately the chemistry of the electrode surface to obtain the desired positioning of the enzyme molecule, both on planar and on structured electrodes. It is easy to understand that rationalization and improvement of the orientation necessitate a precise knowledge of the enzyme structure. Both the positioning of the active site in the protein molecule, and the chemical properties of the enzyme surface are important parameters. Several examples showed that different enzymes, even originated from the same class and sharing very close amino-acid sequences, did not display the same behavior with respect to their orientation at a given interface. Most chemical modifications intend to approach the T1 Cu closer to the electrode surface, although there is still some controversy about which Cu atom is the electron entry point during bioelectrocatalysis (see above). In the following we classify the different developed strategies according to the nature of the main interaction involved: hydrophobic interactions, supramolecular arrangement, electrostatic interactions, and covalent bonding.

  Fig 28); leading to up to 4-fold increase in the current density. These results were attributed to the presence of specific hydrophobic and π-electron conductor recognition sites with the right length, angle and rigidity.

Figure 28 .

 28 Figure 28. Catalytic cyclic voltammogramms of a laccase at a modified or unmodified PG electrode. Inserts show the grafted surface groups. (A) Pc-laccase at an electrode modified with anthracene groups. The black curves are the catalytic waves immediately after spotting on laccase solution and the red curves are the catalytic wave after cell solution was changed for fresh buffer. (B) Tv-laccase at an electrode modified with chrysene groups. Adapted with permission from [438] and [275]. Copyright 2007 and 2008, Royal Society of Chemistry.

  at 0.5, 2.1 and 3.0 nm from the surface in the considered positions 1, 2 and 3. Calculations demonstrated that adsorption was favored in region 1 over region 2 by 20-40 kcal.mol -1 , but by only 11-15 kcal.mol -1 in region 1 over region 3. For Sc-laccase, the distance between T1 Cu and the protein surface was resp. 0.7, 3.5 and 1.6 nm in the regions 1, 2 and 3. No preferential adsorption was shown between regions 1 and 3. This modeling study is a further indication that several positions of the enzyme at the electrode surface have approximately the same probability

  209 An unnatural amino acid, 4-azido-L-phenylalanine, was also genetically introduced at different positions of Sc-laccase to enable specific attachment at the electrode surface via clickchemistry. Surprisingly however, the best results in terms of O2 reduction were obtained with positions that were supposed to keep the Cu sites away from the electrode surface.480 More recently, a recombinant laccase from Trametes sp. was engineered to present a unique surface lysine residue close to the T1 copper atom.481 A pyrene was grafted at this lysine enabling immobilization on β-cyclodextrine decorated AuNPs (3 nm) on MW-CNTs. O2 reduction currents reached 2.75 mA.cm-2 , and this ten-fold increase compared to random adsorption was attributed both to a better coverage, and to an excellent DET proved by a pure Nernstian behavior (Fig29).

Figure 29 .

 29 Figure 29. Site-specific pyrene modified laccase immobilized either at β-cyclodextrin-AuNPs/MW-CNTs (1) or directly via π-π stacking of the pyrene on MW-CNTs (2) and the corresponding cyclic voltammogramms under O2. Reproduced with permission from [481].

Figure 30 .

 30 Figure 30. Comparison of operational stability of the anthracene-MW-CNTs/laccase cathode in potentiostatic (solid line) and galvanostatic (dashed line) mode. Potentiostatic: cathode was poised at 0.4 V vs. SCE in 50 mM citrate buffer, pH 4.5 at 25°C for 24 h in atmospheric conditions and the current was measured as a function of time. Galvanostatic: Under the same conditions, 30% of the individual electrode current was passed through the electrode for 24 h, and the voltage measured as a function of time. Reproduced with permission from [486]. Copyright 2014. The Electrochemical Society.

Figure 31 .

 31 Figure 31. Long-term stability of the BOD adsorbed at a KB-GC electrode modified with bilirubin (circles) and at an unmodified KB-GC electrode (triangles). The activity was measured from the steady-state catalytic current in CA at 0 V vs. Ag/AgCl and 60 s after the potential step in an O2-saturated phosphate buffer (pH 7) at ω= 4000 rpm. The current is plotted as the relative value. Reproduced with permission from [470]. Copyright 2014.

Figure 32 .

 32 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.

  preferred. For example, Cavaco-Pualo et al.509 used 6 redox mediators (ABTS, promazine, hydroquinone, syringaldazine, Ferrocyanide, hydroquinone) with redox potentials ranging from +150 mV to +800 mV vs. Ag/AgCl to mediate the reduction of O2 by a glassy carbon electrode modified with the fungal laccase T. villosa in a pH 5 buffer. Leech et al. were the first to report the use of an osmium complex, [Os(2,2'-bipyridine)2(N-methylimidazole)Cl] + , as a diffusing redox mediator for the reduction of O2 to water. 135 They determined the homogeneous second order rate constant for tyrosinase from Agaricus bisporus in a 0.1 M phosphate buffer pH 7.4 (3×10 4 M -1 s -1 ) and for laccase from Trametes versicolor in a 0.1 M acetate buffer pH 4.7 (3.3×10 6 M -1 s -1 ). Other cyano-metal complexes such as [W(CN)8] 3-/4-, [Os(CN)6] 3-/4-, [Mo(CN)8] 3-/4-and Ru(bpy)3 2+ have also been used in this context. 497,510 In presence of 0.25 mM [W(CN)8] 3-/4-in an O2 saturated pH 7 buffer at 4000 rpm, Tsujimura et al. reached 17 mA.cm -2 at +0.25 V vs. Ag/AgCl with a carbon felt electrode modified with Mv-BOD. 511 Later Ikeda et al. used [Fe(CN)6] 3-/4-as a redox mediator to study the effect of poly[oxyethylene(dimethylimino)propyl-(dimethylimino)ethylene] 495 and of polyammonium cations as enzyme stabilizers for Mv-BOD. 488

  154 Such systems permit to immobilize simultaneously enzymes and redox mediators on the electrode surface. Phase separation is avoided by creating an electrostatic adduct between the polycationic osmium redox polymer and enzymes, which are polyanionic at neutral pH (for example, the pI of most MCOs lies between 3 and 6). In addition, this adduct can be further cross-linked to enhance its stability. Because the enzymes are surrounded by the redox hydrogel, they are electrically wired irrespective of their orientation. This allows the electrical connection of multiple layers of enzymes in 3 dimensions. Conduction within the hydrogel results from the collisions between neighboring oxidized and reduced osmium moieties tethered to the polymer backbone(Fig 33A).514

Figure 33 .

 33 Figure 33. (A) Schematic representation of electron conduction in an Os-based redox polymer. Electron transfer in between reduced and oxidized osmium centers is based on a self-collision phenomenon. Introducing a long tether increases electron "diffusivity" along the polymer "wire" (Adapted from [ 526 ] with permission from the PCCP Owner Societies); (B) Chemical structure of a tris(4,4'-dimethyl-2,2'-bipyridil)osmium complex tethered to a partially quaternized polymer (poly(4-vinilpyridine)) backbone through an eight atoms long link. Reprinted with permission from [527]. Copyright 2003 American Chemical Society

  polymers. Apart from the original method developed by Heller, different authors have proposed alternative synthesis and immobilization strategies.Gao et al. 550 developed a cross linking technique taking advantage of the labile Cl -, which is substituted upon electrodeposition by nitrogen-containing amino acid residues (histidine, lysine and arginine) naturally present in enzyme. This new immobilization technique was used to successfully wire bilirubin oxidases551,552 and laccases.553,554 Photoinitiated polymerization using poly(ethyleneglycol)diacrylate and 2-hydroxy-2-methyl propiophenone has also been shown to be an Efficient method to co-immobilize Mv-BOD and osmium based redox polymers.555 This new protocol permitted to reduce the preparation time of electrodes from 18 h to 1 h. The possibility to modify electrodes by self-assembling alternating layers of polycationic redox polymers and polyanionic enzymes has also been exploited. Calvo et al. designed a biocathode made by self-assembling laccase from Trametes troggii and an osmium complex bound to polly(allylamine).176 For three enzymes layers, the authors reached 80 A.cm -2 at +0.3 V vs. Ag/AgCl in a citrate buffer.Leech et al. used the same approach to elaborate a supramolecular hydrogel(Fig 36). This hydrogel was composed of two building blocks. The first one consisted in two -helical leucine zipper domains separated by a randomly coiled domain rendered conductive by the addition of an osmium complex. The second building block was composed of a -helical leucine zipper domain attached to a random coiled domain genetically fused to Sc-laccase. Upon mixture, the helical domains assemble into tetrameric coiled coils, thus forming an ordered and conductive supramolecular hydrogel. As seen in Fig36B, after drop casting such hydrogel on a GC electrode, the authors could reach 15 A.cm -2 at +0.2 V vs. Ag/AgCl in an oxygenated 100 mM phosphate buffer pH 7 at 900 rpm and 25 °C.

Figure 36 .

 36 Figure 36. Bioelectrocatalytic hydrogel. (A) Schematic diagram of the supramolecular hydrogel. (B) Bioelectrocatalysis of a mixed hydrogel of 7.5 wt% OsHSH-1 and 2.5 wt% HS-SLAC on a 3-mm glassy carbon rotating disk electrode. Buffered to pH 7.0 with 100 mM phosphate buffer, 900 rpm, 25°C, 1 mM O2. Adapted with permission from [558]. Copyright (2008) National Academy of Sciences, U.S.A.

  exchange between horse heart Cyt C and Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. Cyt C was subsequently used as a redox shuttle between immobilized enzymes and gold electrode surfaces. This has been reviewed in deeper details by Siveria et al.576 Of particular interest is the work ofLisdat et al. 577 In 2007, 223 they demonstrated that Mv-BOD could accept electrons from Cyt C, whether this latter was used as a diffusive redox mediator or electrostatically immobilized with the enzyme. It was then possible to achieve a direct inter-protein electron transfer. In 2008, 578 they built an electrode by alternating layers of poly(aniline sulfonic acid) and a mixture of Mv-BOD and Cyt C, and demonstrated the possibility to electrically connect multilayers of both proteins. The electrochemical properties of such electrode depended on the number of deposited layers of proteins. Later, they extended this concept to other enzymes such as laccases,579,580 and in

Figure 38 .

 38 Figure 38. Chimeric Rubredoxin (Rd) as a redox mediator for the bioelectrocatalytic reduction of oxygen by laccase. A) Graphical depiction of the electron transfer from the electrode towards laccase through Rd-HET bionanowires. B) CV of electrocatalytic-mediated oxygen reduction of Rd-HET/laccase in sodium phosphate buffer (pH 5) at 37 °C. Both the redox couple of Chimeric-Rd/laccase in deoxygenated sodium phosphate buffer (dashed black line) and the catalytic oxygen reduction of Rd-HET/laccase in the presence of oxygen (blue line) are shown. Reproduced with permission from [582]. Copyright 2016, Nature Publishing Group

Figure 39 .

 39 Figure 39. (A) SEM (electron backscattering mode) of the composite 350 m thick carbon cloth cathode loaded with 0.78 mg.cm -2 "wired" laccase (45 wt % laccase, 49 wt % redox polymer, 6 wt % PEGDGE) The lighter areas are coated with more heavy atoms (osmium). (B) (a) Cyclic voltammograms (50 mV.s -1 sweep rate, 500 rpm) of the vitreous carbon and the composite carbon cloth electrodes under argon. (b) Potential-dependence of the O2 electroreduction current density at 1 mV.s -1 sweep rate at 1 atm O2 pressure. Current densities were based on the projected area of the 4-mm carbon cloth disk (0.13 cm 2 ). Reprinted with permission from [606]. Copyright 2001 American Chemical Society.

  observed a similar effect on glassy carbon electrode modified with oxidatively treated carbon nanotubes on top of which was deposited an adduct of Mv-BOD and osmium polymers. Imbrication of BOD and CNTs in the core of the fiber was later made possible by developing a one-step fabrication of microfibers. 310 Baughman et al. 617 reported the fabrication of biscrolled yarn electrodes produced from MW-CNTs sheet and coated by PEDOT (Fig 40).

Figure 40 .

 40 Figure 40. The fabrication and structure of biscrolled yarn electrodes for BFCs. (a) Schematic illustration of the fabrication method for biscrolled yarn electrodes. (b,c) SEM micrographs showing the surface of a 50-mm diameter biscrolled yarn cathode based on PEDOT/MW-CNT host. The scale bars were 20 mm and 500 nm, respectively. The yarn bias angle (the angle between the MW-CNT fiber direction on the yarn surface and the yarn direction) is 37°. (d,e) SEM cross-section images at low and high magnifications of the fracture surface of a biscrolled yarn cathode, which shows yarn corridors between scrolled PEDOT/MW-CNT sheets that contain the yarn guests. The scale bars were 5 mm and 500 nm, respectively. (f) Schematic illustration of the structure and function of BFC anode and cathode, which indicate associated electron transfer processes and chemical transformations. The enzyme and redox mediator polymer used were glucose oxidase (GOx) and (poly(N-vinylimidazole)-[Os(4,4'-

Figure 41 .

 41 Figure 41. Schematic illustration of the modified cathode with BOD and ABTS 2-. Adapted with permission from [373]. Copyright (2013) American Chemical Society

  electrode. Leech et al. developed highly-ordered macroporous electrodes with 500 nm diameter pores (Fig 42). 621

Figure 42 .

 42 Figure 42. SEM images, at different magnification of a macroporous gold scaffold obtained upon electro-depositing though templates formed by vertical deposition of 500 nm diameter polystyrene spheres to yield 2½ deposited layers of gold. Cyclic voltammograms at planar (black), 1/2 sphere (red), 1½ sphere (green) and 2½ sphere (blue) gold layer electrodes modified with [Os(2,2-bipyridine)2(PVI)10Cl] + and Ma-laccase recorded in 0.05 M phosphate buffer, pH 7.4, 0.15 M NaCl, 10 mM glucose. Scan rate 5 mVs -1 and current is normalised to projected geometric area of underlying gold substrate (0.1964 cm 2 ). Adapted with permission from [621]. Copyright 2012 The Royal Society of Chemistry

4. 2 . 5 . 1 AFigure 43 .

 25143 Figure 43. (A) Representation of CMF/CNT composite carbon structure onto Graphite rod and involved enzymatic/heterogeneous reactions responsible for reductive current at cathode. Reproduced from [ 623 ] with permission from John Wiley and Sons. (B) Sketch of the biocathode functioning based on bis-pyrene-ABTS BP after adsorption of HRP and subsequent electropolymerization of pyrrole-concanavalin A for the immobilization of GOx. Reproduced with permission from [624]. Copyright 2015 The Royal Society of Chemistry

  In 1999, Katz and Willner reported the first membrane-less glucose/O2 EBFC and paved the way for the development of such systems. They reached 4 W for an external load of 0.6 k.627 Their biocathode was composed of an assembly of Cyt c and cytochrome oxidase (COx)(Fig 44).

Figure 44 .

 44 Figure 44. Schematic configuration of a biofuel cell employing glucose and O2 as a fuel and an oxidizer, respectively, and PQQ-FAD/GOx and Cyt C/COx-functionalized electrodes as biocatalytic anode and cathode, respectively. Reproduced with permission from [627]. Copyright 1995 Elsevier

  .

  637

Figure 45 .

 45 Figure 45. Cyclic voltammograms of CueO-KB-GCE in pH 5.0 McIlvaine buffer at 500 rpm (top curve), and 1000, 2000, 4000, 6000, 8000, and 10 000 rpm (bottom curve) at 1 atm O2 and 25°C. The data were taken at the scan rate of 20 mV.s -1 . The dotted and dashed lines represent the CVs obtained in an Ar-saturated solution and in a quiescent O2-saturated solution, respectively. Reproduced with permission from [637]. Copyright 2009 Elsevier

Figure 46 .

 46 Figure 46. (A) Cyclic voltammograms recorded at MW-CNT/tyrosinase bioelectrodes in argon-deaerated solution (dotted line) or oxygen saturated solution (straight line). The dashed line corresponds to a MW-CNT electrode without tyrosinase in oxygen saturated solution (v = 0.2 mV.s -1 , 0.2 M PBS, pH 7, 25 °C). Adapted with permission from ref [649]. Copyright 2011 Elsevier. (B) Proposed kinetic mechanism of O2 reduction.

Figure 47 .

 47 Figure 47. Different examples of EBFCs implanted in fruits and cactus. (A) in a grape 598 (Reprinted with permission from ref [598] Copyright 2003 American Chemical Society.); (B) in oranges and apples; 374 Published by the PCCP Owner Societies; (C) in grape 670 (Reproduced with permission from [670] Copyright 2011 The Royal Society of Chemistry); (D) in Cactus 671 (Reprinted with permission from [671]. Copyright 2010 American Chemical Society.

Figure 48 .

 48 Figure 48. EBFCs implanted in animals. In rats in 2010[ 642 ] and 2013[ 674,675 ]; in rabbit in 2011[ 670 ]; in cockroaches in 2012[ 548 ] and 2015[ 549 ]; in snail [ 676 ] and clams[ 327 ] in 2012 and in lobsters in 2013 [ 677 ]. Adapted with permission from [678]. Copyright 2015, University of Bordeaux.

  With a cathode based on Mv-BOD immobilized on carbon nanotubes, they obtained Pmax = 5 µW.cm -2 at Ecell = 0.4 V. After further optimization of their electrodes with SW-CNTs, they reached Pmax = 35 µW.cm -2 at Ecell = 0.39 V.302 In 2010, Pmax = 3 µW.cm -2 at Ecell = 0.37 V was reported for the first EBFC operating in DET with Mv-BOD coated on graphite electrodes.241 In 2014,Scherbahn et al. 319 reached Pmax = 45 µW.cm -2 at Ecell = 0.39 V in an unmodified serum for an EBFC cathode made with Mv-BOD immobilized on vertically aligned carbon nanotubes. With the same enzyme at the cathode, Pmax = 57.5 µW.cm -2 at 37°C in human serum containing 5 mM glucose was obtained by Milton et al.464 Ammam et al. reported Pmax = 160 µW.cm -2 at Ecell = 0.21 V on glassy carbon electrodes 564 and Pmax = 69 µW.cm -2 at Ecell = 0.151 V on multi-walled carbon nanotubes when laccase was used.681 The EBFC developed by Ó Conghaile et al.591 generated Pmax = 37 µW.cm -2 at Ecell = 0.26 V with a Sc-laccase immobilized within an osmium redox polymer, and Pmax = 60 µW.cm -2 at Ecell = 0.3 V in artificial plasma when Myceliophthora thermophila laccase was used.682 The highest power density obtained so far for an EBFC operating in human serum has been reported in 2015 by Kwon et al. using the device described in Figure40. They first reached Pmax = 1.02 mW.cm -2 at Ecell = 0.40 V in 2014 617 and then Pmax = 1,1 mW.cm -2 at Ecell = 0.50 V in 2015.683 Experiments in human blood have been rarer, and only 5 can be found in the literature.In 2010, an EBFC made with a laccase entrapped within a matrix of Nafion® and poly(vinyl pyrrolidone) generated Pmax = 5.56 µW.cm -2 at Ecell = 0.07 V.684 However, neither the concentration of glucose nor the concentration of O2 were provided in this work. Pmax = 2.8 µW.cm -2 at Ecell = 0.47 V in presence of 4.6 mM glucose was reached byWang et al. 663 with Mv-BOD immobilized in three-dimensional gold electrodes. More recently, Pmax = 73 µW.cm - 2 at Ecell = 0.3 V has been obtained byÓ Conghaile et al. 685 in whole blood containing 5.4 mM glucose. Their cathode was made with adsorbed Mv-BOD on a gold electrode modified with gold nanoparticles. The highest power density in whole and undiluted human blood so far has been reported byCadet et al. 602 It was Pmax = 129 µW.cm -2 at Ecell = 0.38 V with a glycaemia of 8.2 mM. The cathode was made with a combination of Bp-BOD and an osmium redox polymer(Fig 49A). The first ex vivo experiment in human blood under homeostatic conditions has been reported in 2016 in a vein replica.686 The power generated by this EBFC made with a Mv-BOD cathode was enough to power a flexible low-voltage display(Fig 49B).

Figure 49 .

 49 Figure 49. EBFCs operating in human blood: (A) ex vivo conditions. Reprinted from [602] with permission from Elsevier. (B) ex vivo conditions in a vein replica. Reproduced with permission from [686]. Copyright 2016 The Royal Society of Chemistry.

  EBFCs have been reviewed in details byLojou et al. in 2014 [START_REF] De Poulpiquet | Biohydrogen for a New Generation of H2/O2 Biofuel Cells: a Sustainable Energy Perspective[END_REF] and some more recent developments have been described by Cosnier et al.[START_REF] Cosnier | Recent Advances on Enzymatic Glucose/Oxygen and Hydrogen/Oxygen Biofuel Cells: Achievements and Limitations[END_REF] Except of the first device combining microbial H2 oxidation and enzymatic mediated O2 reduction by Mv-BOD and ABTS 496 , these devices mainly rely on a hydrogenase (Hase) at the anode, and a MCO at the cathode, both operating in DET conditions (unless otherwise specified below). Even if high interests arise from the high OCV of H2/O2 EBFCs, and the high turnover frequencies and ability to establish DET of Hases, their story is rather short compared to that of glucose/O2 EBFCs due to the problematic high sensitivity of most Hases towards O2. The discovery of O2-tolerant Hases allowed recently envisioning H2/O2 enzymatic BFCs. The publication in

  paper based EBFCs has only been reported in 2012 by Jenkins et al. with Whatman filter paper. 699 Its versatility allowed its use for various types of EBFCs. For example, Japanese paper has been used by Tsujimura et al. 369 to construct a flexible and high performance EBFC with an O2 breathing cathode (Fig 50).

Figure 50 .

 50 Figure 50. Illustration of (A) a screen-printed paper-based porous carbon electrode for bioanode or biocathode, (B) a paper-based biofuel cell. Reproduced with permission from [369]. Copyright 2013 The Royal Society of Chemistry.

Figure 51 .

 51 Figure 51. Schematic of the three-phase interphase of a gas-diffusion electrode. Reproduced with permission from [307] Copyright 2012 John Wiley and Sons.

45

 45

5. 7

 7 Hybrid bio-devices Hybrid biodevices exhibit dual function and are a combination of an enzymatic biofuel cell and an electrochemical capacitor. Once charged by the internal EBFC, the device can be discharged as a supercapacitor. The concept and denomination of "self-charging electrochemical biocapacitor" have been first published in 2014 by Pankratov et al. 733 Their 2-electrodes device is illustrated in Figure 53.

Figure 53 .

 53 Figure 53. Schematic representation of a self-charging biological supercapacitor. The electrochemical capacitor is built using a graphite foil modified with a polyaniline/CNTs composite on one side (the capacitive side/face), and the enzymatic fuel cell is designed from three-dimensional gold NPs based nanobiostructures on the other (the charging side/face).Gold NPs: pink-orange spheres; thiol layer: black sticks. The structure of bilirubin oxidase was taken from the know crystal structure (PDB: 2XLL) and cellobiose dehydrogenase was

Figure 54 .

 54 Figure 54. (A) Schematic of the transdermal iontophoresis patch that is an assembly of enzyme-modified CFs that serve as the electrodes, hydrogel films containing a biofuel and a chemical to be delivered, a conducting polymer-based stretchable resistor, a poly(dimethylsiloxane) (PDMS) frame, and O2 permeable medical tape. (B) Photograph of the patch mounted on a human arm, and a schematic of the iontophoresis-assisted drug delivery mechanism. The fructose/O2 biofuel cell generates a transdermal ionic current that is accompanied by an osmotic flow from the anode to the cathode, thereby assisting the electrophoretic movement of small molecules into the skin. Reprinted with permission from [749] Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA

  Relatively few studies are dedicated to the study of stability of immobilized MCOs 231 , and even fewer deal with their thermostability. Stability studies are often only corollaries of other optimizations. If one envisions the use of EBFCs in medical or other long-term applications, this problem has to be tackled. As discussed in this review, the causes of the poor stability of MCOs immobilized on electrode surfaces are many and most are interrelated. Fundamental and mechanistic studies are needed to resolve these issues. Because electrochemistry by itself does not permit to link the catalytic stability with the structure/conformation of proteins, the electrochemical studies must be combined with others. Enzyme engineering can be a valuable tool to improve the stability/thermostability of MCOs in homogeneous solutions. However, solution phase stability does not predict the stability of immobilized enzymes. This is particularly well illustrated by their inhibition by halides. Inhibition by Cl -varies between the MCOs, and differs for different MCOs immobilized on different electrodes, as well as for MET, depending on the redox mediator used. Again the combination of different analytical techniques in homogeneous and heterogeneous conditions is required to enhance our understanding of the MCOs and further improve cathodes for EBFCs applications.

  authors thank Prof Adam Heller (The university of Texas-Austin-USA) for critical reviewing/editing this MS. NM thanks his past PhD/Post Doctoral students and collaborators as indicated in the references cited for their outstanding contributions to this field. AP thanks her collaborators at the Laboratory of Bioenergetics and Protein Engineering, and especially Drs. V. Fourmond, M. Ilbert, E. Lojou, I. Mazurenko and M. Roger for fruitful discussions. NM also acknowledges support from the Région Aquitaine, the Agence Nationale de la Recherche (12-BS08-0011-01 and 16-CE19-0001-02), the European project BIOENERGY (FP7-PEOPLE-2013-ITN607793) and the Labex AMADEus (ANR-10-LABX-0042-AMADEus) belonging to the program Initiative d'Excellence IdEx Bordeaux (ANR-10-IDEX-003-02). Biographies: Nicolas Mano is senior researcher at the CNRS. He received his Ph.D. from the University of Bordeaux (France) under the supervision of Prof Alexander Kuhn in 2001. He then carried out postdoctoral studies at the University of Texas at Austin (USA) with Prof Adam Heller, working towards the development of glucose sensors and miniature membraneless glucose/O2 biofuel cells. In 2006, he integrated the CNRS and created his team at the Centre de Recherche Paul Pascal located in Pessac (France) working on different fields of bioelectrochemistry with a special emphasis on biofuel cells. He also developed an activity in enzymes engineering on various oxidases including bilirubin oxidases. His contribution to the field of bioelectrochemistry has been recognized by numerous National and International Prizes Anne de Poulpiquet got interested in electroenzymatic O2 reduction during her PhD dedicated to the development of enzymatic H2/O2 biofuel cells. She obtained her degree under the direction of Dr. Elisabeth Lojou at Aix-Marseille University (France). After a postdoctoral stay at the Institute of Molecular Sciences in Bordeaux (France), she joined the Bioenergetic and Protein Engineering Laboratory (Marseille, France) as an assistant professor. Her research interests now focus on coupling electrochemistry to optical techniques to study the electroenzymatic reactivity.

  𝑘 𝑂 2 , the maximal catalytic rate constant at saturating mediator concentration and 𝑘 𝑀𝑒𝑑 , the maximal catalytic rate constant at saturating O2 concentration. With the notations used above, theseconstants are respectively defined as:

	𝑘 𝑂 2 =	𝑘 𝑐𝑎𝑡 𝐾 𝑀,𝑂 2 + [𝑂 2 ]	𝐸𝑞. 11
	𝑘 𝑀𝑒𝑑 =	𝑘 𝑐𝑎𝑡 𝐾 𝑀,𝑀𝑒𝑑 + [𝑀𝑒𝑑 𝑟𝑒𝑑 ]	𝐸𝑞. 12
	1 +	𝐾 𝑀,𝑀𝑒𝑑 ⁄ [𝑀𝑒𝑑 𝑟𝑒𝑑 ]	+	[𝑂 2 ] ⁄ 𝐾 𝑀,𝑂 2	𝐸𝑞. 10

kcat is the effective rate constant and 𝐾 𝑀,𝑂 2 and 𝐾 𝑀,𝑀𝑒𝑑 are the effective Michaelis-Menten constants for the ping-pong mechanism. This allows introducing two additional kinetic constants,

3.4.2.1 Strongly oxidizing laccases 3.4.2.1.1 Low redox potential

	range (resp. Em = 394 ± 3 mV vs. NHE for Tv-laccase and Em = 408 ± 4 mV vs. NHE for Th-
	laccase at pH 6.5). 208
	In 1999, a low redox quasi reversible process at Em = 411 mV vs. NHE was observed
	at pH 6 for a recombinant truncated Tv-laccase immobilized on a thiol-modified gold
	electrode. 206 Numerous other examples of this low-potential redox process can be found in the
	literature: a pair of peaks with a midpoint at Em = 337 mV vs. NHE at neutral pH was
	recorded for Tv-laccaseat thiol modified monocrystalline gold (111) surfaces; 207 a single quasi
	reversible redox process at Em = 405 mV vs. NHE was observed for Th-laccase in a gold
	capillary. 118 Th-laccase adsorbed on a bare gold disc, and laccases from Cerrena maxima,
	Trametes hirsuta and Trametes versicolor covalently bound on 4-aminothiophenol (AMT) on
	gold exhibited two pairs of redox peaks. One of them had a low-potential resp. at Em = 486
	mV vs. NHE for Th-laccase on bare gold, and approx. 400 mV for the three laccases on
	aminothiophenol. 205 T. versicolor and T. hirsuta laccases entrapped in a cationic polymer
	matrix on a gold electrode also exhibited two pairs of peaks, with one in the low-potential

  A compartmentless fructose/O2 EBFC with FDH immobilized on KB at the anode gave a 790 mV OCV, jmax = 2.8 mA.cm -2 and Pmax = 850 µW.cm -2 at Ecell = 410 mV at pH 5, room temperature, and in presence of 200 mM fructose under stirring. In a quiet solution,

	performances were lower: jmax was 1.1 mA.cm -2 , while Pmax was 390 µW.cm -2 at Ecell = 410
	mV. 63% of power was lost after 12 h of continuous operation under stirring. But the
	delivered power was already usable: 4 EBFCs connected in series lighted a red LED (1.8 V)
	during 60 days. 374 CNFs also allowed the development of a H2/O2 EBFC delivering Pmax=
	1.2 ± 0.2 mW.cm -2 at Ecell = 0.75 V while the OCV was 1.1 V at pH 6 and 50°C. The
	biocathode was quite stable, with 90, 70 and 50% current remaining after 30 minutes cycling
	at 50, 60 and 70°C. 372
	carbonaceous cathodes were used for EBFC development. A glucose/air EBFC with GOx at
	the anode had a 0.93 V OCV, and Pmax = 40.8 µW.cm -2 at Ecell = 0.41 V with a pH 7.2 air-
	saturated buffer containing 4 mM glucose. Connected 24 h at an external load of 1 MΩ, the
	EBFC lost 7.1% power density in 24 h and 37.4% in 7 days while the OCV remained
	constant. 361 A compartmentless lactate/O2 BFC had a 0.6 V OCV and Pmax = 3.13 µW.cm -2 at

Ecell = 0.4 V. Connected 24 h at 1 MΩ, in a pH 7 buffer containing 40 mM lactate and 20 mM NAD + under ambient air, the power losses were 6% in 24 h and 41% in 7 days.

362

  496,512 For example, Palmore et al. reported one of the first membrane EBFC using ABTS and laccase from Pyricularia oryzae generating

	Pmax = 42 W.cm -2 at Ecell = 0.61 V in a 0.2 M acetate pH 4 buffer. However, Nafion® had to
	be used as a separating membrane. 513
	4.2.1.2 Immobilized redox mediators
	4.2.1.2.1 Osmium based redox polymers

  In 2003, Mano et al. demonstrated that the overpotential of an enzymatic cathode was lower than platinum for the reduction of oxygen at any current density(Fig 34). 545 O2 biofuel cells in combination with a thermophilic hydrogenase.372,466 With the high redox potential Mo-BOD, it was possible to reach 1.37 mA.cm -2 for the enzymatic O2 reduction at +0.2 V vs. Ag/AgCl under physiological conditions by designing a new Os redox polymer with an increased density of catalytic sites.531 The onset for O2 reduction was +0.46V vs. Ag/AgCl at pH 7. By combining Tt-BOD and PVI-Os(4,4-dimethyl-bpy)2Cl on carbon rod electrodes, Scherson et al. reached 40 A.cm -2 at neutral pH. They further combined this cathode with a bi-enzymatic anode composed of trehalase and glucose oxidase to build an EBFC able to operate in a living insect.548,549

	temperatures up to 70 °C376,547 making it particularly suitable for the design of high-
	temperatures H2/
		0		2
	I / mA.cm -2	-4 -2	I/ mA.cm -2	-6 -2
		-6		-10
		-0.8 -0.6 -0.4 -0.2		-0.4	-0.2	0
		 / V			 / V
	Figure 34. Dependence of the current density on the overvoltage for electrodes rotating at
	500 rpm (top curve) 1000 rpm, 2000 rpm, and 4000 rpm (bottom curve) under 1 atm O2 at
	37C. Scan rate. 5 mV.s -1 Left. Polished Pt in 0.5 M H2SO4. Right. "Wired"-BOD on polished
	vitreous carbon in a pH 7.2 saline (0.15 M NaCl) phosphate (0.02 M) buffer. 32.3 wt% BOD,
	60.2 wt% polymer, 7.5 wt% PEGDGE, at 0.17 mg.cm -2 loading. Reprinted with permission
	from [545]. Copyright 2003 American Chemical Society.
	For example, under 1 atm O2 pressure, 9.5 mA.cm -2 at an overpotential of -0.31 V was
	reached on the enzymatic electrode and only 0.6 mA.cm -2 (16-fold lower) on a polished
	platinum cathode in 0.5 M H2SO4. The electrode consisted in the cross-linked adduct of Mv-
	BOD and a copolymer of polyacrylamide and poly(N-vinylimidazole) (PAA-PVI) complexed
	with [Os (4,4'-dichloro-2,2'-bipyridine)2Cl] +/2+ whose redox potential is +340 mV vs.
	Ag/AgCl. The modified electrode reached 5 mA cm -2 when poised at -0.18 V vs. the
	reversible O2/H2O electrode potential in a phosphate buffer saline. With the same redox
	polymer, the same authors reported the wiring of Bp-BOD which led to an Efficient
	biocathode that could operate in physiological conditions with a high tolerance to chloride, 101
	in serum with less than 9% of current loss after 3.3 h of continuous experiments 546 and at

  Different examples of the use of conductive polymers and particularly polypyrole (PPY) can be found in the literature. PPY is a versatile polymer which can be easily functionalized to introduce functional groups for the covalent binding of enzymes.561 However, since PPY is a poor electronic conductor, redox mediators are preferably added to ensure a better electronic conduction. The use of PPY with laccase was first described in 2007 byPalmore et al. 270 Films of PPY doped with ABTS and laccase were grown on glassy carbon electrodes. Under 1 atm O2, a current density of 3 mA.cm -2 was reached at +0.33 V in a pH 5 buffer at 500 rpm. The same system has also been grafted by Yoo et al. on a gold electrode.562 With the same idea, PPY was used by Mousty et al. to entrap laccase and modified anionic clays with ABTS.563 Fransaer et al.564 reported the electrodeposition of a solution containing PPY, a derivative of ruthenium as a redox mediator, 4-sulfonyldiphenol and Tv-laccase, yielding 50 A.mm -2 at 0 V in a pH 7.4 phosphate buffer. The same author also reported a biocathode made by adsorbing laccase and ABTS on MW-CNTs, further

	4.2.1.2.2 Conducting polymers

  Shimomura et al. 573 used different conductive polymers, polythiophene derivatives, but also had to dope them with ABTS. Upon immobilization with Mv-BOD on a modified gold electrode, 0.4 mA.cm -2 was reached at +0.2 V vs. Ag/AgCl in a 0.1 M pH 7 phosphate buffer. The use of conductive polymers such as polyaniline has also been reported more recently. Shleev et al. elaborated a composite electrode comprising Mv-BOD, polyaniline and CNTs.574 In a pH 7.4 phosphate buffer complemented with active blood components, a current density of 0.48 mA.cm -2 was reached at 37°C. This demonstrated the ability of such system to operate in a blood-like medium.

Table 1 a

 1 series of reviews published since 2004 that provide countless information on the history, applications and challenges of EBFCs. Of particular interest are the most up-to-date reviews byRasmussen et al. 651 and

	Shleev. 89

membrane-less EBFC with a cathode made by layer-by-layer self-assembling of Th-laccase and osmium polymers. 556 A new synthesis route for osmium based redox polymers has been elaborated by Schuhmann et al. (Fig 35). 557 Instead of using the classical "ligand exchange method", they proposed a covalent binding approach with an osmium complex already bearing the suitable ligands. Surprisingly, the authors considered their system as a DET. Even though organic/inorganic redox mediators are not used, it may not be considered as a DET per se since electrons are shuttled from one protein to another one, i.e. from Cyt C to BOD, and not directly transferred between enzymes and electrode. With the same idea of using proteins as redox mediators, a paper by Altamura et al. is of particular interest (Fig 38). 582 The authors designed an electroconductive bionanowire which resulted from the attachment of a prion domain with a rubredoxin. The newly formed bionanowire was then used as a redox mediator for the reduction of O2 by Tv-Laccase in a sodium phosphate buffer pH 5. The current density obtained for such system was higher than those obtained with Cyt C. This technique may provide a new strategy to elaborate enzymatic electrodes. common point of this preparation method is the frequent need to add a membrane to prevent the leaching of the redox mediator. For example, Kokoh et al. used Carbon Vulcan XC72 to elaborate an electrode further modified by adsorbing Mv-BOD and ABTS. Nafion® was added on top of the modified layer, or in the mix of enzyme and ABTS, to prevent the leaching of the bioelectrocatalyst. 363,584 Nafion® was also used by Cosnier et al. in their compressed electrode made of graphite, laccase and ABTS. 585 Sol-gel 586 , poly-L-lysine 497 or lipid liquid crystalline cubic phases 587 were also used to prevent the leaking of redox mediators.

Carbon nanotubes

The general properties and advantages of CNTs have been presented in section 4.1.2.1. These outstanding nanomaterials have also been implemented in MET-based electrodes. They were generally used to formulate carbon ink. Such carbon ink was developed by Viikari et al. 588 by mixing carbon nanotubes, ABTS and Trametes hirsuta laccase. After coating on different printing substrates, they could reach current densities up to 39.1 µA.cm -2 at 0.25 V vs. Ag/AgCl on Whatman filter paper. Such methodology has also been reported later by Leech et al. 589 CNTs can also be incorporated to the enzyme solution prior to casting on an electrode. For example, in 2013, Leech et al. used osmium redox polymers to elaborate three different biocathodes with two laccases and Mv-BOD. 590 To enhance the electroactive surface of their electrodes, they added MW-CNTs in the mixture containing enzymes and redox polymer. In presence of MW-CNTs, the current density was increased by a factor of 2.5 compared to the corresponding planar electrode without MW-CNTs. Such increase was attributed to higher loading of enzymes enabled by the addition of carbon nanotubes. The authors exploited this advantage to elaborate various EBFCs. 591,592 In 2010, Sadowska et al. described the first deposition of single-walled carbon nanotubes (SW-CNTs) covalently combined together provide the power for the sensing device. The power output is usually proportional to the concentration of the analyzed substrate. The first self-powered system for the measurement of glucose using an EBFC, made with a glucose oxidase anode and an O2reducing cathode combining Cyt C and COx, has been reported by Willner and Katz in 2001. 632 Katz et al. have also pioneered the use of MCO-based cathodes to develop EBFCs with switchable power output and their use in enzymatic logic systems. [START_REF] Katz | Biofuel Cells with Switchable Power Output[END_REF]633,[719][720][721][722][723] The principle of such systems has been described in their first report in 2009. 724 The authors synthesized a polymer brush covalently functionalized with osmium redox centers. For pH > 5.5, mediated electron transfer was prohibited because of the shrinkage of the hydrogel; while for pH < 4.5, the fully swollen and hydrated hydrogel