# Step Towards Enzymatic Bioelectrorefinery: Design of a Ligninolytic Hybrid Air-Breathing Biocathode

Vladyslav Mishyn, Juliette Floret, Brigitte Chabbert, Véronique

Aguié-béghin, David Crônier, Nunzio Giorgio G Carducci, David P Hickey,
Sofiene Abdellaoui

► To cite this version:

Vladyslav Mishyn, Juliette Floret, Brigitte Chabbert, Véronique Aguié-béghin, David Crônier, et al. "Step Towards Enzymatic Bioelectrorefinery: Design of a Ligninolytic Hybrid Air-Breathing Biocathode." ChemCatChem, 2025, 17 (3), 10.1002/cctc.202401537. hal-05066574

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Submitted on 14 May 2025

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# Step Towards Enzymatic Bioelectrorefinery: Design of a Ligninolytic Hybrid Air-Breathing Biocathode

Vladyslav Mishyn,[a] Juliette Floret,[a] Brigitte Chabbert,[a] Véronique Aguié-Béghin,[a] David Crônier,[a] Nunzio Giorgio G. Carducci,[b] David P. Hickey,[b] and Sofiene Abdellaoui*[a]

Lignins, abundant aromatic biopolymers and one of the major components of lignocellulosic biomass, remain the most underutilized renewable bioresources of aromatics and hydrocarbons on the Earth. Numerous physical and chemical processes have been developed for lignin valorization; however, they generally suffer from environmentally unfriendly, harsh conditions and lack reaction specificity. On the other hand, milder methods involving biocatalysts exist but are impeded by many limitations, such as cofactor regeneration, deleterious enzyme–lignin interactions, and low stability. In this work, we attempt to eliminate the constraints encountered in enzyme-based lignin valorization

processes through the development of a novel electrochemically assisted bioprocess. This “all-in-one” biocathode incorporates a hybrid electrocatalytic interface combining a hydrogen peroxide-generating passive air-breathing gas diffusion electrode with an immobilized hydrogen peroxide-consuming lignin peroxidase on a single surface and catalyzing the depolymerization of lignins. The ligninolytic potential of this bioelectrochemical device is demonstrated using both lignin models (veratryl alcohol and veratrylglycerol β-guaiacyl ether) and a technical lignin at room temperature in aqueous media with the reaction efficiency of 14.9% per hour.

## 1. Introduction

The limited availability of fossil fuel resources and the worsening effects of climate change have prompted the use of biomass as an alternative carbon source to produce energy, commodity chemicals, and polymer materials. As the most abundant renewable carbon source on the planet, lignocellulosic biomass is a rich source of cellulose (40–50%), hemicelluloses (20–30%), and lignins (10–25%).[1] While cellulose and hemicelluloses are already well exploited in biorefineries as feedstocks and fuel resources, about 99% of industrial lignins are generally considered as byproducts, and currently there are no green and cost-effective methods for lignin conversion due to their structural complexity.[2] Lignins have a high calorific value making them suitable for use as raw materials of biofuel. Unfortunately, the vast majority of lignins are burned and used as an energy source. As a result, lignins remain one of the most poorly valorized sources of renewable carbon on the planet, and there remains a need for strategies to depolymerize lignins

into oligomeric or monomeric aromatic compounds. The recalcitrance of lignin to valorization is a consequence of a high structural heterogeneity stemming from the final step of lignin biosynthesis, involving random radical-coupling of three phenylpropane units called monolignols: *p*-coumaryl alcohol (H unit), coniferyl alcohol (G unit), and sinapyl alcohol (S unit).[1b,3] As a result, lignins consist of many different covalent intermonomer bonds, of which carbon-oxygen bonds (C—O—C), such as *β*-ether (*β*-O-4), and various C—C bonds are the main types.[4] Therefore, the depolymerization of lignins through the specific and controlled cleavage of these bonds would provide an renewable supply of biosourced and valuable aromatics.

Existing approaches to lignin deconstruction include pyrolysis, solvolysis, and cracking.[5] These processes generally aim to cleave *β*-ether and C—C bonds under harsh conditions: *β*-O-4/*α*-O-4 ether bonds are cleaved at 200–400 °C during the pyrolysis, whereas temperatures above 400 °C are required to break C—C bonds.[6] These methods are energy-intensive and generally exhibit poor selectivity. Prompted by overcoming these drawbacks, many researchers have focused on investigating strategies for lignin deconstruction and refining that can be accomplished with catalysts operating under milder conditions, such as enzymatic and electrochemical approaches.[7] In nature, white-rot fungi and certain species of bacteria use an enzymatic arterial to degrade lignins to the point of complete mineralization to CO₂.[8] Since several decades, strategies inspired by these ligninolytic microorganisms have been intensively studied and have led to the development of new green biotechnologies aiming to depolymerize lignins under mild conditions with high selectivity.[9]

These microorganisms produce extracellular oxidoreductases, such as heme-peroxidases, laccases, and other enzymes involved in selective oxidative depolymerization of lignins. Peroxidases, especially lignin peroxidase (LiP), have received much

<footnote>[a] Dr. V. Mishyn, J. Floret, Dr. B. Chabbert, Dr. V. Aguié-Béghin, D. Crônier, Dr. S. Abdellaoui
INRAE, FARE, Chaire AFERE, Université de Reims Champagne-Ardenne, 2 esplanade Roland Garros, Reims 51100, France
E-mail: sofiene.abdellaoui@univ-reims.fr</footnote>

<footnote>[b] N. G. G. Carducci, Dr. D. P. Hickey
Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824-1226, USA</footnote>

<footnote>Supporting information for this article is available on the WWW under https://doi.org/10.1002/cctc.202401537</footnote>

<footnote>© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>
<figure><img src="image_3.png" /></figure>

<table><caption>**Table 1.** In situ electrochemical generation of H₂O₂ applied for enzymatic reactions.</caption><thead><tr><th>Application</th><th>Enzyme</th><th>Enzyme Immobilization</th><th>O₂ Source</th><th>Interface</th><th>Setup</th><th>Reference</th></tr></thead><tbody><tr><td>Halogenation</td><td>VCPO</td><td>no</td><td>Ambient air</td><td>GDE/OCNT</td><td>H-cell</td><td>[10]</td></tr><tr><td>LC pretreatment</td><td>–</td><td>no</td><td>Pure O₂</td><td>GDE/Carbon black</td><td>3-compartment</td><td>[11]</td></tr><tr><td>Hydroxylation</td><td>AaeUPO</td><td>no</td><td>Saturated O₂</td><td>CNT/Lumiflavin</td><td>–</td><td>[12]</td></tr><tr><td>Organic carbon removal</td><td>HRP</td><td>no</td><td>Saturated O₂</td><td>RVC</td><td>–</td><td>[13]</td></tr><tr><td>Organic carbon removal</td><td>LiP</td><td>no</td><td>Dissolved O₂</td><td>RVC</td><td>–</td><td>[14]</td></tr><tr><td>Lignin deconstruction</td><td>LiP</td><td>no</td><td>Dissolved O₂</td><td>AT-CNT/CoTMPP</td><td>H cell</td><td>[15]</td></tr><tr><td>Lignin deconstruction</td><td>LiP</td><td>Yes</td><td>Ambient air</td><td>GDE/OCNT/CoTMPP/Py-LPEI/LiP</td><td>Single cell</td><td>This work</td></tr></tbody></table>

AaeUPO, heme-thiolate peroxygenase; AT-CNT, acid-treated carbon nanotubes; CNT, carbon nanotubes; CoTMPP, 5,10,15,20-tetrakis(4-methoxyphenyl)-21H,23H-porphine cobalt(II); GDE, gas diffusion electrode; HRP, horseradish peroxidase; LC, lignocellulose; LiP, lignin peroxidase; OCNT, oxidized carbon nanotubes; Py-LPEI, pyrene-modified linear poly(ethyleneamine); RVC, reticulated vitreous carbon; VCPO, vanadium chloroperoxidase.

attention due to their high redox potential that gives them the ability to catalyze the oxidative cleavage of lignin by radical-based mechanisms of a wide range of phenolic and nonphenolic compounds, including methoxybenzene-containing lignin-like substrates.[16] Additionally, LiP has been utilized in numerous fields, such as catalytical removal of organic pollutants, dye decolorization, skin-lightening products, and elimination of endocrine-disrupting compounds.[17] However, LiP requires hydrogen peroxide (H₂O₂) as an electron acceptor, which must be continuously supplied in small and controlled amounts either from an external source or by using H₂O₂-generating oxidases, making their use in a bioreactor more challenging. In addition, in vitro applications of LiP-based systems are limited by their poor stability, lignin-derived inhibition, denaturing enzyme–lignin interaction, and enzyme–product separation.[18] Collectively, these limitations have hindered peroxidase-based technologies applied for lignin deconstruction and valorization.

Many H₂O₂-driven biocatalysts are found in applications involving enzymatic synthesis, such as oxidation, hydroxylation, and halogenation.[19] As mentioned above, this requires continuous addition of chemicals or enzyme with a fine control of H₂O₂ concentration. Excess peroxide concentrations can have a detrimental effect on the catalytic activity or even lead to irreversible enzyme deactivation.[20] The electrochemical oxygen reduction reaction (ORR) via a two-electron process enables the controlled generation of H₂O₂ necessary to maintain the activity of peroxidases without its external addition. In addition to being a simple, clean and safe approach for H₂O₂-dependent biocatalysis, in situ electrochemical H₂O₂ generation can be achieved under ambient and biocompatible conditions and can operate with dissolved molecular oxygen (O₂) under ambient pressures.[21] Table 1 highlights some notable works describing electroenzymatic systems operating with in situ electrogeneration of H₂O₂. However, most of these bioelectrochemical systems are limited by the poor solubility of O₂ in water, which is depleted during the electroreduction, and therefore requires O₂ to be continuously pumped into the reaction vessel. Furthermore, it has been reported that the presence of O₂ bubbles in the reaction mixture can have detrimental effect on the enzyme activity.[15] To overcome this limitation, studies have demonstrated the benefits of using gas diffusion electrodes (GDE) in which H₂O₂ is produced in situ from

the diffusion of O₂ present in a gas phase.[11] This porous three phase gas-permeable electrode is composed of a catalytic layer, a gas diffusion layer, and a metal current collector mesh. The catalytic layer faces the electrolyte solution, whereas the gas diffusion layer is in direct contact with a gas phase. Carbon-based materials (graphene, carbon nanotubes, carbon black, vitreous carbon), noble metals, transition-metals and oxides (macrocyclic compounds M-N₄, pyrolized and atomically dispersed particles), and organic molecules (anthraquinones and flavins) have been successfully utilized as electrocatalysts for oxygen reduction in bioelectrochemical systems.[22] Ko et. al. have recently demonstrated that 5,10,15,20-tetrakis(4-methoxyphenyl)-21H,23H-porphine (CoTMPP), adsorbed on oxidized carbon nanotubes (OCNT), has an outstanding electrocatalytic activity applied to peroxidase reactions.[15] The key advantages of using a OCNT/CoTMPP catalyst are that it has low overpotential of the reaction onset, high current efficiency and H₂O₂ yield, and it follows pathway close to 2-electron oxygen reduction (e.g., production H₂O₂ without formation of side reactive oxygen species (ROS) and water molecules).

Enzymes are considered as microenvironment-sensitive and long-term unstable biocatalysts, which often hinders their industrial applications. Enzyme immobilization is one of the main strategies in enzyme-based biotechnologies, which aims to simplify downstream processing in industry along with enhancing environment-tolerance and stability of enzymes during the storage and operation.[23] Moreover, this enables enzymes to be recycled/reused in a bioreactor, thus reducing the cost and quantity of biocatalysts-based technologies. Various methods have been developed to immobilize large biomolecules on surfaces: weak noncovalent physisorption, irreversible covalent surface linkage, entrapment into a polymer matrix, and cross-linking.[24] However, enzyme surface immobilization often leads to a drastic loss of catalytic activity as a result of alteration of the protein conformation and active site steric hindrance.[25] Recently, our collaborator, Hickey et. al. has described a novel material based on pyrene-modified linear poly(ethylenimine) (Py-LPEI), which is used in the design of protein-modified electrodes and facilitates bioelectrochemical redox characterization and improves residual enzyme activity compared to other common enzyme immobilization methods.[24c]
<figure><img src="image_4.png" /><figcaption>Figure 1. Schematic representation of the ligninolytic air-breathing biocathode. GDE, gas diffusion electrode; LiP, lignin peroxidase H8 enzyme from *Phanerochaete chrysosporium*.</figcaption></figure>

In summary, the application of LiP in lignin biorefinery is mainly limited by the need for controlled addition/generation of H₂O₂, the deleterious effects of the lignin on the enzyme activity (inhibition, hydrophobic interaction, etc.), and thus the necessity to use costly multi-chamber compartment reactors to protect the enzyme. In this study, we present the design and the characterization of an original ligninolytic air-breathing biocathode that offers a promising route to overcome the above-mentioned limitations. This biocathode incorporates a hybrid electrocatalytic interface devised from the assembly of two different catalyst layers acting synergistically on a single interface at the surface of a GDE, allowing the catalytic depolymerization of lignins (Figure 1). This system can generate in situ H₂O₂ from the electrochemical reduction of O₂ from air passively diffusing through the GDE whose surface is modified with the immobilization of the LiP from *Phanerochaete chrysosporium*. The ligninolytic potential of this biocathode is evaluated using a lignin dimer model (veratrylglycerol β-guaiacyl ether and VGE) and a commercial soda lignin (Protobind). In addition, it is worth noting that our proof of concept is also tested with a technical lignin (soda lignin), while the applications of LiP-based systems described in the literature are often evaluated using only simple lignin-substrate models.[15,26] To the best of our knowledge, this is the first reported single compartment bioelectrochemical reactor applied to lignin conversion, combining the benefits of enzyme surface immobilization and gas diffusion electrode.

## 2. Results and Discussion

### 2.1. Electrochemical H₂O₂ Generation

The primary objective was to select the most suitable catalyst to electrochemically generate H₂O₂ for H₂O₂-dependent

peroxidases. Among the numerous electrocatalysts capable of producing H₂O₂ in situ via electrochemical ORR, the following catalysts were evaluated: multiwalled carbon nanotubes (CNT), carboxyl functionalized multiwalled carbon nanotubes (OCNT), 2-ethylantraquinone, and cobalt(II)-porphyrin adsorbed on OCNT via π-π stacking (OCNT/EAQ and OCNT/CoTMPP, respectively). These catalysts were selected based on their high catalytic activity, potential biocompatibility, high surface area, and the possibility to confine them on carbon electrode surfaces. First, the electrochemical active surface areas obtained after catalyst immobilizations on glassy carbon (GC) electrodes were evaluated according to Randles-Sevcik equation using 1 mM ferrocene methanol (Figure S1a-b, Equation S1). The results show a significant increase in the surface area of modified interfaces compared to unmodified flat GC (Table 2). The electrochemical active surface area was improved by the factors of 2.0, 2.7, 3.2, and 3.5 for GC/OCNT, GC/OCNT/CoTMPP, GC/CNT and GC/OCNT/EAQ, respectively, due to the high surface area and surface roughness of the carbon nanotubes. The electrocatalytic ORR onset potentials obtained with the selected catalysts were measured and evaluated by linear sweep voltammetry (LSV), using a potential window between +0.2 and -1 V (versus Ag/AgCl). As shown in Figure 2, the catalyst-modified GC (GC/CNT, GC/OCNT, GC/OCNT/EAQ, and GC/OCNT/CoTMPP) exhibit higher current densities for ORR in the presence of O₂, compared with bare GC. In addition to the increase in catalytic activity of modified electrodes, as reflected by the current density, a shift of the onset potentials can be observed from -0.51 V, for unmodified GC, to -0.20, -0.18, -0.09, and +0.01 V (versus Ag/AgCl) for GC/OCNT, GC/OCNT/EAQ, GC/CNT, and GC/OCNT/CoTMPP, respectively. The same measurements were carried out in N₂-saturated electrolyte as a control experiment, showing nonsignificant catalytic currents (Figure S1c). The reduction peak at -0.48 V (versus Ag/AgCl) observed with GC/OCNT/EAQ-modified electrode under N₂-saturated electrolyte is attributed to the cathodic signal of EAQ (Figure S1d).

The ORR may undergo different reaction pathways leading to the formation of superoxide ion, H₂O₂ or H₂O, respectively (Equation (1-3)):

$$O_2 + e^- \rightarrow O_2^- \quad (1)$$

$$O_2 + 2H^+ + 2e^- \rightarrow H_2O \quad (2)$$

$$O_2 + 4H^+ + 4e^- \rightarrow 2H_2O \quad (3)$$

In the scope of our work, we aim to avoid formation of side reactive oxygen species (ROS) detrimental to the enzymes and to orient specifically the ORR towards the production of H₂O₂ involving two-electron transfer. Therefore, the number of electrons exchanged during the ORR with the different catalysts was evaluated according to the Koutecky-Levich equation (Equation S2, Figure 3a, and Figure S2). The resulted values indicate that the ORR follows different reaction pathways depending on the catalysts, with the number of electrons ranging from 1.1, for OCNT/EAQ, to 3.1 for GC (Table 2). The electrode modified with GC/OCNT/CoTMPP demonstrated n = 1.8, which is the closest value to the desired pathway leading to the H₂O₂ pro-
<figure><img src="image_5.png" /></figure>

<table><caption>**Table 2.** Electrochemical catalysts for the ORR.</caption><thead><tr><th>Interface</th><th>Surface Area (cm²)</th><th>Reaction Onset (V vs Ag/AgCl)</th><th>Number of Electrons</th><th>H₂O₂ Production (µM h⁻¹)</th><th>Current Efficiency (%)</th></tr></thead><tbody><tr><td>GC</td><td>0.06</td><td>-0.51</td><td>3.1</td><td>7.0</td><td>10.9</td></tr><tr><td>GC/CNT</td><td>0.19</td><td>-0.09</td><td>1.5</td><td>7.3</td><td>37.5</td></tr><tr><td>GC/OCNT</td><td>0.12</td><td>-0.20</td><td>1.6</td><td>16</td><td>57.1</td></tr><tr><td>GC/OCNT/EAQ</td><td>0.21</td><td>-0.18</td><td>1.1</td><td>53</td><td>82.2</td></tr><tr><td>GC/OCNT/CoTMPP</td><td>0.16</td><td>+0.01</td><td>1.8</td><td>37</td><td>70.3</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Linear sweep voltammograms obtained with different catalysts applied to H₂O₂ production at a scan rate of 50 mV s⁻¹ in O₂ gas-purged 50 mM acetate buffer/1 mM CaCl₂ pH 3.</figcaption></figure>

duction. However, the amount of electrogenerated H₂O₂ in the presence of CoTMPP (37 µM·h⁻¹) is slightly lower than the quantity obtained with EAQ (53 µM·h⁻¹). This may be due to a lower electron transfer rate with the CoTMPP-modified interface or due to interferences from side ROS produced with EAQ and affecting the H₂O₂ quantification assay.

The amount of H₂O₂ generated by the different catalyst-modified electrodes driven at a mild reduction potential of -0.5 V (versus Ag/AgCl) was monitored over time in O₂-saturated electrolyte at pH 3 (Figure 3b). The chosen pH is favorable to both H⁺-dependent ORR, which converts O₂ to H₂O₂, and the ligninolytic peroxidase activities, whose optimal pH is 2.6.[7b] The quantity of H₂O₂ produced per hour is summarized in Table 2, and the highest production rates were obtained with OCNT/CoTMPP and OCNT/EAQ, 37 and 53 µM h⁻¹, respectively. It is important to note that, regardless of the catalyst used, the reaction slows down after the first hour due to the depletion of O₂ dissolved in the electrolyte.

The current efficiency was evaluated for all selected catalysts to determine the ratio of H₂O₂ produced in solution to the theoretical mass according to Faraday's law (Equation S3). The best catalytic performances were found with electrodes modified with OCNT, OCNT/CoTMPP, and OCNT/EAQ with efficiencies of 57.1, 70.3, and 82.2%, respectively. Indeed, these catalysts use

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 3.** Quantitative characterization of H₂O₂ generation catalyzed by different catalyst using (a) Koutecky-Levich plot derived from rotating disk electrode (RDE) experiment; (b) H₂O₂ production rate in oxygen saturated conditions at the cell voltage -0.5 V (versus Ag/AgCl) in 50 mM acetate buffer/1 mM CaCl₂ pH 3.</figcaption></figure>

OCNT as a matrix where carboxylic acid functionalities play an important role in the superior catalytic activity due to the presence of the oxygen-containing groups.[27]

These characterizations led us to select GC/OCNT/CoTMPP as the catalyst for H₂O₂-producing electrode for the ligninolytic
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 4.** Electrochemical generation of H₂O₂ using a gas diffusion electrode (GDE) modified with OCNT/CoTMPP as a function of time using various gases supply (O₂, N₂, or air) in 50 mM acetate buffer/1 mM CaCl₂ buffer pH 3 at -0.5 V (versus Ag/AgCl). The inset sketch depicts the gas diffusion electrode modified with OCNT/CoTMPP.</figcaption></figure>

biocathode. Although the use of GC/OCNT/EAQ generates a greater amount of H₂O₂, GC/OCNT/CoTMPP is more suitable for driving peroxidase activity because the number of electrons used by the latter is the closest to the 2-electron ORR pathway, thus avoiding the formation of ROS deleterious to enzymes. Moreover, GC/OCNT/CoTMPP exhibits the highest onset potential obtained during the electrocatalytic reduction of O₂ (+0.01 V versus Ag/AgCl) which is thermodynamically favorable for the development of a biocathode. In addition, the rate of H₂O₂ generation and the current efficiency obtained with OCNT/CoTMPP were found to be suitable for driving peroxidase activity.

## 2.2. Electrochemical Generation of H₂O₂ Using a Gas Diffusion Electrode

To overcome the depletion of dissolved oxygen, a GDE modified with the selected OCNT/CoTMPP catalyst was used (Figure 4 inset). The oxygen permeability of the electrode was measured, and the experimental setup is depicted in Figure S3a–b. To ensure the production of H₂O₂ prior to the enzyme introduction, a constant potential of -0.5 V versus Ag/AgCl was applied, and the concentration of H₂O₂ after 1 hour of the ORR was measured as high as 1.68, 1.03, and 0.06 mM for different gases supplied to the GDE: O₂, air, or N₂, respectively (Figure 4). Indeed, the use of a triphase electrode facilitates the mass transport of O₂ to the surface of the catalytic material and helps to overcome the reaction slowdown after the prolonged period. A significant increase in the H₂O₂ concentration was observed up to 1.68 mM h⁻¹ compared to 37 µM h⁻¹ (Table 2) because of increasing the surface area (from 0.16 cm² to 0.79 cm²) and the surface porosity compared to GC-based interfaces. Importantly, O₂ and air-fed interfaces demonstrated similar results signifying that ambient air without additional pressure can be used as the oxygen source

for the ORR. Utilization of the O₂ from the air simplifies the setup and reduces cost by eliminating the need for additional reagents and equipment. The combination of the OCNT/CoTMPP catalyst and the GDE has improved and enabled a continuous generation of H₂O₂.

## 2.3. Construction and Characterization of Ligninolytic Air-breathing Biocathode

In this section, we have opted for the use of the lignin peroxidase H8 (LiP H8), a monomeric peroxidase of 38 kDa secreted by *Phanerochaete chrysosporium*, a wood-degrading fungi.[28] This fungal enzyme is characterized by a high redox potential enabling the catalytic oxidation of phenolic and nonphenolic part of lignins, making it of great interest for many applications, such as biorefineries, biotechnology, biocatalysis, and waste treatment.[15,26b,29] The oxidation of the nonphenolic part of lignins by LiP H8 leads to the cleavage of β-O-4 ether and C—C bonds in the aromatic biopolymers.[7b] The LiP H8 was a recombinant enzyme and was characterized prior to use (Figure S4–S5).

The ligninolytic air-breathing biocathode interface was prepared by cross-linking Py-LPEI with the recombinant LiP H8 onto CNT/CoTMPP-modified GDE (Figure 5a). The Py-LPEI-based immobilization strategy was selected for its ability to stabilize immobilized enzyme on carbon electrode surface as shown in the previous study.[24c] The enzyme-polymer matrix adheres to the catalytic interface via π-π stacking complexes between pyrene groups of the polymer and sp² honeycomb structure of carbon nanotubes. Surface imaging by scanning electron microscopy (SEM) has been performed and reveals the morphological differences between the surfaces of unmodified and modified GDE with OCNT/CoTMPP and LiP H8 (Figure 5b–d). The unmodified GDE surface consists mainly of amorphous carbon black particles. The interface morphology and roughness change significantly upon surface modification with OCNT/CoTMPP, showing a typical pattern of interconnected carbon nanotubes. Introduction of Py-LPEI polymer with the enzyme forms a film covering the surface of the carbon nanotubes.

The electroenzymatic activity of the immobilized LiP H8 coupled with the electrochemical generation of H₂O₂ onto the biocathode was first evaluated using veratryl alcohol (VA) as substrate. VA is a secondary metabolite produced by ligninolytic fungi and is oxidized to veratryl aldehyde (VAD) by high redox potential peroxidases, such as LiP H8 in the presence of H₂O₂ (Figure 6a inset).[30] The conversion of VA to VAD via Cα-oxidation is a conventional assay used to demonstrate a ligninolytic activity of peroxidases toward the nonphenolic part of lignins.[31] The reaction was initiated by the electrogeneration of H₂O₂ from air at -0.5 V (versus Ag/AgCl) in the presence of 1 mM VA, and the oxidation process was monitored by measuring the absorbance of VAD at 310 nm. The concentration of VAD was measured as high as 0.25 mM after 1 h of the electroenzymatic oxidation, corresponding to 25% of conversion of the initial amount of VA (Figure 6a). Aiming to ensure that the reaction was driven by the enzymatic reaction, a control experiment was per-
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 5.** (a) Assembly scheme of the ligninolytic air-breathing biocathode; (b–d) scanning electron microscope (SEM) images of the electrodes after each modification step: bare GDE and GDE covered with OCNT/CoTMPP and GDE/OCNT/CoTMPP surface with Py-LPEI immobilized LiP H8, respectively.</figcaption></figure>

formed by substituting LiP H8 enzyme by bovine serum albumin (BSA, catalytically inert protein). The oxidation rate of VA was nonsignificant in the control experiment, concluding that the electrochemically produced H₂O₂ had no capacity to oxidize VA into VAD. The specificity of the reaction was then confirmed by HPLC (Figure 6b). Prior to applying current to the electrochemical cell, a single peak was observed at 11.8 min corresponding to VA. After 1 h of bioelectrocatalysis, the chromatogram revealed peaks at 11.8 and 23.7 min attributed to nonconverted VA and newly formed VAD, respectively. The operational stability of the immobilized LiP H8 fed with electrogenerated H₂O₂ was tested over five cycles of 1 h (Figure S7). The results show that the LiP activity is stable during two cycles and decreased to 50% at the third cycle. The relative activity is 8% at the fourth cycle and becomes insignificant at the last cycle.

In a second step, the ability of the biocathode to catalyze the specific cleavage of β-O-4/C<sub>α</sub>–C<sub>β</sub> bond in lignins was validated by using 1 mM veratryl glycerol-β-guaiacyl ether (VGE), a lignin β-O-4 dimer model (Figure 6c inset). The electroenzymatic reaction was monitored by optical detection of VAD (310 nm) released after the β-O-4/C<sub>α</sub>–C<sub>β</sub> cleavage of VGE at –0.5 V (versus Ag/AgCl).

The reaction kinetics were linear during 60 min with the reaction efficiency of 14.9% per hour as concluded by measuring VAD concentration equal to 0.15 mM after the electrolysis (Figure 6c).

The control experiment with BSA (as described above) showed no oxidation of the substrate caused by the production of H₂O₂ and/or an electrochemical side reaction. The reaction products VAD and guaiacol were validated by HPLC chromatography. The initial chromatogram before the electroenzymatic reaction at *t* = 0 min showed a single peak at 30.9 min corresponding to VGE (Figure 6d). The measured chromatogram after the electroenzymatic reaction at *t* = 60 min exhibited the new peaks at 17.2 and 23.7 min attributed to guaiacol and VAD, respectively, according to the external standards (Figure S6). The assay with guaiacol glyceryl ether (GGE), a substrate similar to VGE but bearing a phenol, was also achieved (data not shown) and showed that the presence of phenolic groups hinders the oxidative bond cleavage catalyzed by LiP. The results confirm the high specificity and selectivity of the air-breathing biocathode for cleaving β-O-4/C<sub>α</sub>–C<sub>β</sub> bond in lignin-like nonphenolic substrates and support its ligninolytic potential.
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 6.** (a) Kinetics of veratryl aldehyde (VAD) formation (black) from the bioelectrocatalytic oxidation of veratryl alcohol (VA) at −0.5 V (versus Ag/AgCl) and monitored by absorbance at 310 nm (ε = 9300 M⁻¹ cm⁻¹). The control experiment was carried out substituting LiP H8 enzyme by nonactive BSA protein (grey). The inset illustrate the reaction scheme of oxidation of VA to VAD in the presence of LiP H8. (b) HPLC chromatograms of the reaction at t = 0 min (blue) and t = 60 min (red). (c) Kinetics of the formation of VAD and guaiacol from veratryl glycerol-β-guaiacyl ether cleavage (VGE, black) monitored by optical absorbance at 310 nm. Control experiment (grey): LiP H8 enzyme substituted by nonactive BSA protein. The inset shows the reaction pathway of β-O-4/Cα-Cβ bond cleavage of VGE in the presence of LiP H8 resulting in VAD and guaiacol formation. (d) HPLC chromatograms of VGE before (t = 0 min, blue) and after the electroenzymatic reaction (t = 60 min, red). The inset shows guaiacol peak.</figcaption></figure>

## 2.4. Electroenzymatic Conversion of Soda Lignin

Finally, the ability of the air-breathing biocathode to degrade a commercially available lignin, Protobind, extracted by soda pulping from wheat straw and sugarcane mixture, was investigated.[32] In addition to our results with VGE and GGE, Pham et. al. have reported that phenolic unit structures have an inactivating effect on the biodegradation of lignin by LiP.[33] Therefore, prior the electroenzymatic conversion, the samples were copiously washed with ethyl acetate to remove the soluble fraction containing the phenolic residues. Furthermore, after the washing step, the insoluble fraction was treated with the laccase from *Trametes versicolor* to oxidize the phenolic unit structures of lignin. These treatments were carried out to limit the negative effect of phenolic group on the oxidative cleavage activity of LiP.[18c] The lignin was then subjected to enzymatic electrolysis

carried out under the identical conditions as for VGE described above. The lignin conversion was evaluated after 48 h of bioelectrochemical treatment by high pressure size exclusion chromatography (HP-SEC) using a UV detector monitoring the apparent molecular weight of the substrate. Figure 7 shows HP-SEC chromatograms: before the electroenzymatic reaction (t = 0 min) different peaks were obtained at 0.46, 0.92, 3.13, 8.90, and 22.26 kDa. After 48 h of the electroenzymatic reaction, the 0.46 kDa and 0.92 kDa fractions increased by a factor of 2.5 and 1.8, respectively, confirming the release of low molecular weight fragments from lignin. The intensity of the signal of the middle molecular weight fractions of 3.13 and 8.90 kDa decreased by 18 and 22%, respectively. These postreaction signals variations suggest that the middle molecular weight fractions were degraded to form smaller mass components. In the control experiment (LiP substituted by BSA), which permits evaluation of the direct
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 7.** Electroenzymatic conversion of technical lignin using air-breathing GDE/OCNT/CoTMPP/LPEI/LiP biocathode: Size exclusion chromatography (SEC) chromatograms of soda lignin using UV detectors (280 nm). The arrows indicate the peak changes.</figcaption></figure>

effect of electrogenerated H₂O₂ on the lignin, the peak intensities at 3.13 and 8.90 kDa have also decreased slightly less as compared to the presence of LiP; however, the intensities of low molecular range peaks at 0.46 and 0.92 kDa and high molecular range peaks at 22.26 kDa have significantly decreased likely due to a restructuration rather than depolymerization of the lignin. These results demonstrate that GDE/LiP-modified biocathode can successfully degrade lignin producing low molecular weight fragments ranging from 0.46 to 0.92 kDa.

### 3. Conclusion

This work introduces the first ligninolytic biocathode developed with a hybrid catalytic interface consisting of the immobilization of an electrocatalyst and a ligninolytic peroxidase working in synergy on a single surface of a gas diffusion electrode. This biocathode can produce in situ enough amount of H₂O₂ from ambient air for the surface-confined lignin peroxidase. Assays with various catalysts for H₂O₂ generation, we have concluded that OCNT/CoTMPP is the most suitable for the electroenzymatic processes according to its electrochemical characterization. The use of GDE overcomes the depletion of the dissolved oxygen in the electrolyte by using the ambient air as costless and abundant oxygen supply. The lignin-degrading enzyme LiP H8, which is involved in the specific cleavage of the C–C and β-O-4 bonds of lignins, was then successfully immobilized in a cross-linked Py-LPEI hydrogel on the H₂O₂-generating electrode surface. The resulting biocathodes demonstrated a ligninolytic potential on a conventional high redox substrate VA and VGE, lignin dimer model, with efficiencies of 25% and 14.9% per hour, respectively, as well as on a technical lignin-producing low-molecular-weight fragments. The use of this all-in-one air-breathing biocathode provides a promising route for the development of bioelectrochemical-based technology applied to the lignin valorization in biorefineries.

### Supporting Information

The authors have cited additional references within the **Supporting Information**.[24c,28]

### Acknowledgements

This research was financially supported by the Agence Nationale de la Recherche (ANR), under grant ANR-21-CE43-0004 (Project VALBIOELEC).

### Conflict of Interests

The authors declare no conflict of interest.

### Data Availability Statement

Research data are not shared.

### Keywords: Bioelectrocatalysis · Biorefinery · Lignin

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Manuscript received: September 14, 2024

Revised manuscript received: October 20, 2024

Accepted manuscript online: October 21, 2024

Version of record online: October 28, 2024