Antonio Hernández Mañas

Léa Vilcocq

Pascal Fongarland

Laurent Djakovitch

Lignin catalytic oxidation by CuO/TiO2: role of catalyst in phenolics formation

Keywords: Catalytic oxidation, Kraft lignin, Phenols, Vanillin, Copper catalyst

Lignin, which is a by-product of pulp and paper mills, could be used as a starting material for the production of phenolic compounds for chemical industry. In this work, CuO/TiO2 catalyst prepared by incipient wetness impregnation showed a significant activity in lignin oxidative depolymerization in basic aqueous medium, producing phenolic compounds using air as oxidant, at 150°C and 20 bar pressure. The main identified products were vanillin, vanillic acid and acetovanillone. Modifications on lignin during the reaction were observed: oxidation of the residual lignin, decrease in hydroxyl moieties, decrease in β-O-4 linkages, formation of quinones and carboxylic functions. To optimize the reaction conditions, several limitations to lignin depolymerization were evaluated. These include physical limitations like formation of lignin agglomerates, diffusion limitation of lignin and vanillin inside the heterogeneous catalyst that were estimated using the Weisz-Prater criterion, and chemical limitations, particularly by over-oxidation of phenolic compounds. One of the main limitations was due to diffusion rates. When optimized, vanillin yield reached up to 5.1%.

INTRODUCTION

CO2 emissions are currently the cause of a major climate crisis. In this context, the design of chemicals from biomass is necessary to substitute fossil resource and facilitate a transition towards a diminution of energy and material consumption and the rise of a new model based on reasonable use of limited natural resource. The European Green New Deal targets 25% of biosourced materials in chemical industry in 2030. Lignocellulose, composed of cellulose, hemicellulose and lignin, became an important raw material for producing biosourced chemicals and fuels [1,2]. Different processes are used to separate its constituents, the main one in the paper mill industry being the Kraft process [3] that delivers cellulose pulp, and lignin soluble and partially degraded in alkaline aqueous medium. Ca. 630 kt lignin are produced as a by-product of Kraft process every year [START_REF] Chen | Lignocellulose Biorefinery Engineering: Principles and Applications[END_REF], but lignin remains poorly upgraded (for energy recovery through burning) due to the complex structure and the stability of this aromatic biopolymer [START_REF] Lora | [END_REF]. Nevertheless, it is a highly interesting material for producing aromatic chemicals, stimulating in the last ten years researches for its valorization. Among all reported methods, oxidative, reductive and thermochemical routes seem to be promising technologies [6]. Reductive and thermochemical routes, while delivering aromatic chemicals, are often preferred for fuel applications, whereas oxidative routes are often preferred for producing functional aromatics such as phenolics for chemical applications [7]. The work described here belongs to the latter area. Initially, most of the studies related to oxidative routes where devoted to evaluate the reactivity of model compounds. However, the last decade showed an increased number of reports related to lignin oxidative depolymerization in the presence or not of catalysts, whatever they are homogeneous or heterogeneous, and several review articles reported these studies [7][8][9].

Catalyst-free alkaline lignin oxidation under aerobic conditions was investigated by several research groups, generally under mild conditions (lignin (60 g.L -1 ), aqueous NaOH (2 N), 130-180°C, air (3-9 bar)) delivering yield in aromatic between 3% and 10% [10][11][12][13][14][15]. Noticeably, such process was commercially exploited for the production of vanillin as flavoring agent from sulphite pulping liquors by Borregaard company [16]. Actually, overoxidation of aromatic products during lignin oxidation remains one of the main difficulty to solve [17]. According Tarabanko mechanistic hypotheses, basic media help to prevent vanillin over-oxidation [18][19][20] but catalytic approach is also helpful. Cu-, Fe-, V-, Co-, Mn-, Re-based homogeneous catalyst and polyoxometalates were often reported and remain the object of intensive development [7]. On technical lignin, data generally showed that the reaction rates could be accelerated by adding a metal catalyst; however, phenolics yields were not particularly improved [21][START_REF] Bozell | Selective Catalysis for Renewable Feedstocks and Chemicals[END_REF][START_REF] Li | [END_REF][24][25][26][27]. Tarabanko et al. particularly studied the kinetic and mechanism of the oxidative lignin depolymerization in the presence of copper salts under oxygen or in the presence of water peroxide [28][29][30][31]. They showed that vanillin yield could reach up to 18 wt% when addressing directly pine wood oxidation by H2O2 in the presence of copper hydroxide as catalyst, whereas a yield of 5.4 wt% in vanillin was reached when starting from isolated lignin.

Heterogeneous catalytic systems were, as well, intensively developed [32]. Next to perovskites [33], they include metal supported catalysts based, for example, on Co [34][35][36], Mn [37], Fe [38] or noble metals (i.e. Au [39], Pd [40][41][42] or Pt [43,44]). Like for homogeneous catalysts, they were often applied to model compounds and when applied to real lignin, yields in phenolics stay low. By contrast, heterogeneous copper-based catalysts were intensively studied and applied to various lignin materials.

Kumar et al. evaluated the properties of Cu/-Al2O3 for the oxidative depolymerization of grass soda lignin (Protobind™ 1000) and alkali lignin. In water, up to 4.9% yield in vanillin was observed from alkali lignin [45,46]. Jeon et al reported the use of a Cu-Mn mixed oxide for vanillin production by alkaline wet oxidation of a commercial Kraft lignin with H2O2. Results showed that activity of the catalyst increased as Cu-content increase; however, data showed as well overoxidation of vanillin to vanillic acid under the reaction conditions [47]. Kuznetsov et al. reported several studies related to catalytic conversion of native or Organosolv lignin in the presence of Cu/TiO2 catalysts. Whatever the reaction conditions used (70-100°C, 300°C in supercritical conditions), data showed that while lignin depolymerization occurred, low yields in aromatic compounds were obtained due to over-oxidation [48][49][50][51][52]. Li et al. reported the use of Ce-Cu/MFI nanosheet catalysts for the aerobic depolymerization of an Organosolv lignin in ethanol under oxygen at 150°C. Aromatic yields are in the range of 10 to 58% [53], including alkylated derivatives as previously reported elsewhere [54,55]. Next to supported copper catalysts, massive CuO was also investigated. Villar et al. reported that CuO can produce lignin oxidation under inert atmosphere. Using 160 wt% CuO/lignin they achieved 6 wt% yield in syringaldehyde and 1.3wt% yield in vanillin [13,56]. Qu et al. showed that CuO catalysts in the presence of hydrogen peroxide under microwave irradiation can cleave lignin -O-4 dimeric model compounds to guaiacol, vanillin and vanillic acid via a dihydroxylation [57]. From softwood lignin, yields in vanillin reached ca. 4.4%; from hardwood lignin, yields in aldehydes (mainly syringaldehyde) reached up to ca. 11.4% [58]. Ninomiya et al. reported the oxidative depolymerization of eucalyptus lignin previously pretreated via enzymatic saccharification using stoichiometric CuO giving yield in aromatic aldehydes up to 39% [59]. Ouyang et al reported the use of CuO/Fe2(SO4)3 material in the presence of water peroxide in various solvent. Best results were achieved in a mixture MeOH/Water, leading to overall phenolic compounds yield of 18%. Authors suggest that the use of methanol facilitates bond cleavages and prevents recondensation reactions [60].

The mechanism of the catalytic aerobic lignin depolymerization was particularly studied by Tarabanko and co-workers. They suggested the formation of phenoxyl radicals that evolved through retro-aldol reactions [20,61]. Recently, Davaritouchaee et al. reported that pathways of aerobic lignin depolymerization depends on the nature of the initial radical oxidation sources [62]. Lately, we reported results showing that metal catalysts can influence the way in which lignin evolved during alkaline aerobic depolymerization [42]. This short literature review showed that among all evaluated catalysts, copper ones were particularly active and remained generally associated with low costs. The last is relevant for potential applications in biorefineries and lignin upgrading by oxidation processes towards phenolics, and particularly vanillin. Lignin upgrading represents a stimulating area able to meet the demand of biorefineries in improving their economical balance. Technical lignin, and particularly Kraft lignin, is particularly interesting as a starting material, as it is now only a by-product of Kraft pulp mills. However, literature pointed also out that if homogeneous copper-based catalysts led to good to excellent results, they were almost inactive when heterogenized except when used as massive CuO catalyst. This is why we reinvestigated deeply heterogeneous copper-based catalysts with the idea of developing an industrial process for lignin valorization. Thus, a home-made CuO/TiO2 catalyst was prepared and fully characterized before being implemented in aerobic oxidative lignin upgrading in basic media (i.e. aqueous sodium hydroxide). This study offers opportunities to transpose, at a later stage, the reaction conditions to Kraft black liquor upgrading [63] as an intermediate step to implementation in Kraft industries.

In this contribution, we report the results issued from studies of pinewood Kraft lignin, almost exclusively issued from the random polymerization of guaiacyl units [64], upgrading in aerobic aqueous basic conditions. Key parameters that can affect the catalytic process like lignin agglomeration and its diffusion to active catalytic centers were particularly evaluated.

Experimental

Materials and samples

General

Kraft lignin was used in this study. It has been extracted from marine pine (softwood) by a Kraft process and precipitated from black liquor by acidification with CO2 in CTP (Centre Technique du Papier, Grenoble, France) [43]. All other products are commercially available and were purchased from Sigma Aldrich or Alfa Aesar. All solvents were used as supplied without further purification. TiO2 was kindly supplied by Evonik (Aerolyst 7711 extrudates 1x4 mm).

Catalyst synthesis

CuO/TiO2 (5 wt%Cu) catalyst was used in all experiments. It was prepared by incipient wetness impregnation using an aqueous solution of Cu(NO3)2.5H2O (6.9 g.L -1 ). 32 mL of aqueous copper nitrate solution per gram of TiO2 extrudate was used for impregnation experiments. After 4 h impregnation time, the catalyst was dried for 1 night at 110°C and calcined for 5 h at 550°C (5°C/min) under air flow is a muffle furnace. After calcination, catalyst pellets were crushed and sieved. The fraction 90-200 µm was used in the reference catalytic tests.

Catalyst characterization

The elemental analysis of the catalysts was carried out by using an ICP-OES ACTIVA Jobin Yvon device. Before analysis, mineralization was carried out by a dilution with H2SO4, HNO3 and HF, a subsequent evaporation and then a heating process of the residue with HNO3.

XRD analysis was carried out in a Bruker D8-Advance with automatic sampler, using a radiation Cu-Kα (0.154184nm). Phase identifications were attributed after comparison with data provided in the crystallography database PDF-4+ from ICDD (International Center for Diffraction Data).

A Mettler Toledo TGA/DSC STAR System was used to study the thermal behavior of the prepared catalysts (5 °C/min, from 25 to 1000 °C, air as carrier gas).

A N2 adsorption-desorption isotherm was obtained on an ASAP 2020 of Micromeritics, equipped with two pressure sensors. The analysis was done using nitrogen as physisorption gas at a temperature of -196°C. Desorption pretreatment was carried out for 3 h at 300°C under vacuum.

Reaction tests

Catalytic oxidation of lignin in aqueous phase

All reactions were performed under basic condition using an aqueous 1wt% NaOH solution delivering a pH of approximately 12.7. Under these conditions, pH remains basic over all the reaction time despite the production of carboxylic acids.

Reactions were carried out in a 300 mL stainless batch reactor (Vinci Technology). 150 mL of an aqueous solution of lignin (5 g.L -1 ) and NaOH (10 g.L -1 ) was added to the reactor together with the CuO/TiO2 catalyst, if any. The reactor was purged with N2, heated up at atmospheric pressure to 150°C under stirring. Then, the reactor was pressurized at 20 bar of air and the mechanical stirring was adjusted at 1800 rpm. The completion of these operations defines the starting time of the reaction.

Reactor pressure was continuously kept constant by a regulation valve during the whole reaction time, supplying thus the necessary air. After 1 hour of reaction time, the reactor was cooled using an ice bath to 15°C and then slowly depressurized. In these operating conditions, maximum oxygen consumption during reaction was estimated to be inferior to 10% of the total amount of O2 in the reactor. The use of air as an oxidant corresponds to several objectives of green chemistry: it does not generate any by-product, it is widely available, contrarily to pure O2 which needs purification, and it increases the safety of the process.

Products fractionation

Post-reaction treatments have been developed to separate the different reaction products allowing careful qualitative and, when possible, quantitative analyses (Figure 1).

First, the aqueous reaction medium was filtered to remove the solid catalyst. 100 mL of the filtrate was acidified by adding a diluted aqueous solution of hydrochloric acid (10 wt%) until pH 1 (ca 15 mL HCl) to precipitate non-depolymerized lignin (only soluble at high pH values). The precipitate was separated by centrifugation at 4000 rpm for 20 min, washed with hydrochloric acid (10%) and water before being dried at 60°C for 9 hours under vacuum (5.10 -2 mm Hg). It was called Klason phase.

The liquid fraction recovered from the centrifugation was extracted with dichloromethane (DCM) (3x80 mL) delivering the Organic phase composed mainly by non-polar compounds (phenolic compounds and oligomers). It was dried over MgSO4 and concentrated in rotary evaporator before being dried under vacuum (5.10 -2 mm Hg) at ambient temperature for 30 minutes.

Remaining aqueous phase, containing the polar compounds, was concentrated in rotary evaporator to eliminate water and dried at 60°C under vacuum for 1 hour. The residue was taken-up with THF followed by a filtration with a 0.4 µm filter to remove insoluble inorganics, mainly sodium chloride. It was then concentrated in rotary evaporator to eliminate THF and dried under vacuum at 60°C for 1 hour. The resulting product was called "Aqueous phase".

Analyses on lignin and reaction products

Lignin and products from the oxidation reactions were characterized using different complementary analytical techniques as described below.

C, H, O, N and S content measurements were done by an external laboratory (ISA -Institut des Sciences Analytiques, Villeurbanne, France). Oxygen concentration was measured after pyrolysis and quantification of CO by an IR detector. Carbon, hydrogen, nitrogen and sulfur concentrations were measured after total combustion and quantification of CO2, H2O, N2 and SO2 by a thermal conductivity detector.

The same TGA device (Mettler Toledo TGA/DSC STAR System), as that used for catalyst characterizations, was used to study the thermal behavior of lignin and Klason phase (5 °C/min, from 25 to 1000 °C, air as carrier gas). The moisture degree was determined by the weight loss at 100-115 °C and the ashes by the remaining weight at 1000 °C.

FT-IR analyses were carried out in an absorbance mode using a Nicolet IS5 equipped with an ID7-ATR accessory with diamond crystal from Thermo Scientific. The sample was directly placed over the diamond and IR signal was recorded with a resolution of 4 cm -1 , 32 scans and a spectral range of 4000 cm -1 to 525 cm -1 . The spectra obtained with this method were qualitatively analyzed to characterize different structures in the lignin. NMR spectra were acquired on a Bruker AVANCE III 400MHz equipped with a BBFO probe (Z gradient).

For 1 H-NMR, samples were prepared by dissolving an aliquot (10-15 mg) in 600 mg of (CD3)2SO. Data were recorded over 16 scans at 50°C. Chemical shift were measured relatively to residual protic signal of (CD3)2SO at 2.5 ppm. For 13 C-NMR, data were recorded so that quantitative analysis could be applied.

Tetramethylthiourea was used as standard and Fe(acac)3 as relaxing agent. An aliquot of the sample (10-40 mg) was dissolved in (CD3)2SO (600 mg). Then, 0.1 mL of the standard solution, made from tetramethylthiourea (40 mg) and Fe(acac)3 (4 mg) in (CD3)2SO (1000 mg), was added. Data were recorded over 11000 scans at 50°C. Chemical shift were measured relatively to the signal of deuterated solvent ((CD3)2SO: 39.5 ppm). For HSQC NMR, samples were prepared by dissolving an aliquot (10-15 mg) in 600 mg of (CD3)2SO. Data were recorded over 48 scans at 50°C. For 31 P quantitative NMR analyses, data were recorded after phosphorylation using 2-chloro-4,4,5,5-tetramethyl-1,3,2dioxaphospholane (TMDP) as phosphylating agent [43]. An aliquot (10-30 mg) was dissolved in CDCl3 (200 mg) to which was added cyclohexanol (8 mg), used as standard, and TMDP (100 mg). DMF (0.25 mL) and pyridine (0.25 mL) were added to ensure full dissolution of formed salts. Data were recorded over 64 scans at 25°C. Chemical shift were measured relatively to the signal of the product of the reaction of TMDP with water (δ = 132.2 ppm) and quantitative data were measured relatively to the area to the signal provided by phosphylated cyclohexanol (144 ppm).

Size-exclusion chromatography (SEC) was performed on a Malvern OmniSEC apparatus equipped with 3 PLGel mixed C columns from Agilent Technologies. THF was used as a solvent. Online detection was performed with a viscometer and an UV detector. A calibration curve, obtained with polystyrene standards (Polymer Standards Service, Mainz, Germany) in the range of 500 to 4.2x10 6 g.mol -1 , was used to calculate the molecular weight distribution of the samples. Molecular weight was also determined from the viscometer using a correlation hydrodynamic radius-molecular weight (universal calibration). Data were acquired and processed with the OmniSEC 5.12 software. Before analysis, lignin and Klason phases were dissolved in THF.

GC-FID analyses of the organic phases were performed on a Shimadzu GC-2010 chromatograph equipped with a FID detector, a AOC-20i+ autosampler and a Phenomenex Zebron ZB-5HT column (cross-linked of 5 % Phenyl -95 % dimethylpolysiloxane, 30 m x 0.25 mm x 0.25 μm). The column temperature program was: 2 min at 60 °C, 30 °C min -1 to 150 °C, 2 °C min -1 to 200 °C, 10 °C min -1 to 260 °C, and 2 min at 260 °C. The total run time was approximately 38 min. Helium was used as carrier gas. FID was operated at a temperature of 300 °C with hydrogen and air flows of 40.0 mL min -1 and 400.0 mL min -1 , respectively. Data collection and processing were handled by a LabSystem chromatography data system. Before analyses samples were diluted in CH3CN to which naphthalene, used as internal standard, was added. GC-MS were obtained on a Shimadzu GC-MS-QP2010S equipped with an AOC-20i+ autosampler and a Sulpelco SLB-5MS column (5% Phenyl -95 % dimethylpolysiloxane, 30m x 0.25mm x 0.25μm). The conditions applied are the same that those used for the GC-FID.

Dynamic Light Scattering (DLS) analyses were carried out in a Malvern Nano-ZS. The samples were diluted in an aqueous 10 wt% NaOH solution (pH = 12.7). Results are presented as a mathematical treatment of autocorrelation data, which can be roughly assimilated to a size distribution curve.

High-performance liquid chromatography (HPLC) was carried out on a Shimadzu apparatus equipped with a photodiode array detector. To improve characterizations of aliphatics and aromatics, two columns were used: a Phenomenex Rezex ROA column with acidified water as mobile phase (0.005 N H2SO4), isocratic mode, 0.6 mL.min -1 and 70°C, and a Phenomenex Kinetex XB C18 column associated to acidified water/acetonitrile mixture (20/80) as mobile phase (0.005 N H2SO4), isocratic, 0.7 mL.min - 1 and 50°C.

RESULTS AND DISCUSSION

Catalyst preparation and characterization

The catalyst was prepared by incipient wetness impregnation. After drying and before calcination, an aliquot was crushed and studied by TGA under air. This helped us to determine the optimum calcination temperature; Figure S1 showed that full decomposition of copper nitrate occurred after 380-400°C, therefore to ensure complete calcination a temperature of 550°C was chosen. Once calcined at 550°C, the catalyst was ready to use. It was characterized by several techniques. Elemental analyses showed that the copper content reached 4.9-5 wt% as expected.

XRD of the CuO/TiO2 catalyst was measured. The specific surface area has been determined by a BET-N2 isotherm analysis. Both the TiO2 support and the CuO/TiO2 catalyst were analyzed. The BET results showed a decrease in the specific surface area from 50 ± 1 m 2 .g -1 for the TiO2 support to 32 ± 1 m 2 .g -1 for the CuO/TiO2 catalyst. Figure S2 shows the result of the adsorption-desorption isotherm analysis. The form of the hysteresis loop can be associated with a mesoporous structure. Mesoporous pores were quantified by the BJH method, obtaining a diameter size distribution of 2-50 nm and an average diameter of 25 nm.

TEM of the CuO/TiO2 catalyst was also recorded and CuO phase and particles could be visualized with the help of EDX analysis (Figure 3). Pictures showed that, as expected, CuO phase is homogeneously dispersed on the whole catalyst mass, but CuO particles are highly heterogeneous in terms of size with observation of small particles of an average size 3-4 nm and large particles/aggregates of ca.

Lignin oxidative depolymerization reaction -no catalyst

The oxidative depolymerization of lignin under basic medium was first carried out without catalyst.

Several characterizations using complementary techniques were done on the initial lignin and on the products of the reaction.

Lignin extracted from marine pine by a Kraft process was used as a starting material. It was treated in a batch reactor at 150°C, 20 bar of air and 1800 rpm stirring rate for 1 h. After cooling the reactor, reaction medium was fractionated in Klason phase (23 wt%) Organic phase (16 wt%) and Aqueous phase (44 wt%). Detailed procedure is given in experimental part.

Characterization of the initial lignin and of the Klason phase

The initial Kraft lignin was characterized after drying at 60°C for 9 hours under vacuum (5.10 -2 mm Hg) (except for wetness measurement). One can note that the Klason phase is assumed to be "unreacted lignin". Thus, detailed comparison between both materials takes sense to understand the transformation that occurred during the aerobic depolymerization of lignin.

TGA analysis (Figure S3) was performed periodically on lignin to measure the wetness degree. It was found that wetness stays relatively constant upon the time, at 4.5 wt%. Above 500°C, all the organic part of lignin was burnt, the residue observed was attributed to remaining ashes composed of inorganics present in the initial lignin and representing around ca 2 wt%.

At 500°C, the Klason phase was also fully burnt; however, mathematical derivation of TGA (Figure S4)

shows differences in the behavior of lignin and Klason phase. Lignin shows two peaks, one of them between 200°C and 390°C and the other between 390°C and 500°C. On the contrary, the Klason phase shows only the second peak between ca. 370°C and 490°C. This could be related with the presence of two fractions in the initial lignin, one would be more accessible to the oxidation, being transformed and extracted before recovering the Klason phase during the fractionation process.

In order to get better insights about this hypothesis, SEC chromatograms of initial lignin and Klason phase were recorded (Figure S5). In both cases, a molecular weight distribution between 100 and 1000 g.mol -1 was observed, with a slight decrease for the Klason phase. Data obtained from these experiments are reported in Table 1. A decrease of the average molecular weight is observed from 1505 g.mol -1 for initial lignin to 813 g.mol -1 for the Klason phase; however, the polydispersity Mw/Mn of the molecular weight is not affected by the reaction. Depolymerization is thus evidenced with an average molecular weight divided by a factor 2 after 1h of reaction. It should be noted that Klason phase only represents the acid precipitate and a part of soluble lignin (so-called "oligomers") may remain in solution after Klason phase precipitation. Elemental analysis of lignin delivers C 62.6 %; O 28.9 %; and H: 6.1 %. There is a significant presence of sulfur (2.3 %) due to sodium sulfide used during the Kraft extraction process. This ratio is similar to the theoretical ratio of guaiacyl unit (C 65%; H 6%; O 29%). It differs from the elemental composition of pine wood (i.e.: C: 47.2 %, O: 46.6 %, H: 6.1 % and N: 0.1 %) showing higher carbon and lower oxygen content that can be expected when considering the relative structure of wood constitutive biopolymers (i.e. cellulose and hemicellulose showed higher oxygen content than lignin). Elemental analysis of Klason phase delivers different values: C: 59.7 %, O: 30.5 %, H 4.3 % and S: 0.9 %. A slight decrease of the carbon content coupled to an increase in the oxygen content was observed that can be related to partial oxidation of the lignin biopolymer as suggested by Gierer mechanism [65]. This is supported also by the decrease of the hydrogen content attesting partial or total oxidation of alcohols to ketones and aldehydes. Noticeably, sulfur content decreased from 2.3 wt% in initial lignin to 0.9 wt% in the Klason phase that expresses easy oxidative cleavage of these functionalities, mainly to sulphates [START_REF] Djakovitch | Unpublished results from ANR CHEMLIVAL project[END_REF]. This is particularly interesting, since it shows that next to the possibility of producing high added value aromatics, the aerobic alkaline lignin depolymerization also allows reducing the sulfur content in products leading to a desulfuration process of Kraft lignin biopolymers.

Figure 4 shows the FTIR-ATR analyses of the initial lignin and Klason phase obtained from a noncatalytic run. FTIR-ATR of lignin shows characteristics bands corresponding to the aromatic functions at ca. 1420 cm -1 , 1504 cm -1 and 1588 cm -1 . According to general observations for softwood lignin, the band at 1504 cm -1 that is considered as "pure" band characteristic of the aromatic skeleton has higher intensity than the one at 1452 cm -1 . Additionally, bands at 1264 cm -1 , 1206 cm -1 and 1028 cm -1 characteristic of guaiacyl moieties in lignin are observed (resp. C=O stretching; C-C and C-O stretching;

Aromatic C-H deformation). Band at 1694 cm -1 corresponds to unconjugated carbonyl groups, those at 1452 cm -1 and 1420 cm -1 correspond to C-H stretching in CH2 and CH3 alkyl moieties. Bands at 2934 cm - 1 and 3360 cm -1 are assigned to, respectively, C-H stretching in CH2 and CH3 alkyl moieties and O-H phenolic stretching (Figure S6). Please note the absence of bands at 1325 cm -1 and 1118 cm -1 that would have been characteristic of syringyl units (resp. Aromatic C-H deformation S band; C=O stretching), which is consistent with the pine wood origin of Kraft lignin.

Almost all bands are observed in Klason phases; however, differences were observed in relative intensity. Particularly, a clear decrease in the intensity of the band at 1504 cm -1 , compared to the other bands, was observed attesting that "aromatic content" was lost during the aerobic alkaline depolymerization. This can be due either to effective depolymerization releasing aromatic compounds in the reaction media as observed from the analyses of the organic phases (see below), or due to oxidation of the aromatic moieties in the biopolymer. The last hypothesis is supported by the increase of the band at 1694 cm -1 (C=O non-conjugated) and 1588 cm -1 (C=O conjugated p-substituted, quinone), that overlap with vibration of aromatic moieties as well ( aromatic skeleton). These observations are coherent with the results from elemental analyses that shows an increase of the oxygen content in the Klason phases and are related with the formation of aldehydes, ketones and carboxylic acids by oxidation of hydroxyl functionalities. Full FTIR-ATR spectra of lignin and Klason phases, and associated band assignments can be found in supporting information (Figure S6 and Table S1). Initial lignin and Klason phase were further characterized using NMR techniques. 1 H and 13 C, despite allowing getting information regarding structure, were not used due to relatively severe limitations in a quantitative approach [43], we preferably performed 31 P after proper phosphitylation and 2D-HSQC experiments.

31

P NMR spectra are reported in Figure S7, and quantitative data are summarized in Figure 5. Most part of the hydroxyl contents is observed in aromatic rings (blue). As expected from a softwood lignin [3], the main part of hydroxyl groups correspond to guaiacyl units (2.1 mmol.g - Analyzing the Klason phase show significant decrease in the hydroxyl content from a total OH content of 7.2 mmol.g -1 in the initial lignin to 4.6 mmol.g -1 for the Klason phase. This decrease affects the phenolic (mainly guaiacyl units) and aliphatic hydroxyls, whereas an increase in the content of the OH linked to carboxylic acids is observed (from 0.4 mmol.g -1 in lignin to 1.4 mmol.g -1 in Klason phase).

These data agree with the results observed in FTIR-ATR. However, from elemental analyses a slight increase in the oxygen content was measured in the Klason phase. Therefore, rather than simple loss of hydroxyls due to depolymerization, it is reasonable to propose that, as well, the oxygen uptake observed in elemental analysis is also due to the oxidation of phenolic and aliphatic OH to quinones and ketone/aldehydes, that is well supported by FTIR-ATR and 31 P NMR spectra. Further structural analyses came from HSQC-NMR involving INEPT sequence. We focused mainly on the aromatic [4.9-8.5 ppm 1 H / 90-160 ppm 13 C] and oxygenated aliphatic regions [2.5-6 ppm 1 H / 50-105 ppm 13 C] (Figure 6). Beyond the qualitative analyses, HSQC-NMR spectra were analyzed in a semi-quantitative way following the procedure described by Wen et al. [START_REF] Wen | [END_REF] Results are reported in Table 2 and expressed on the basic of 100 aromatic units. Data show that the aerobic depolymerization reaction produces a significant decrease in the inter-unit linkages content. Inter-unit β-O-4 linkage content dropped by 60% during the reaction decreasing from 6.7 in initial lignin to 2.6 in the Klason phase. Other inter-unit linkages followed the same tendency; the β-β linkages decreased by 40% and the β-5 linkages by 66%. These data are consistent with those reported in the literature [43,44]. HSQC spectra after reaction did not show the appearance of new signal that could correspond to new linkages in Klason phase. Therefore, two hypotheses can be made:

i) the number of inter-unit linkages decreased drastically, during reaction, i.e. the polymerization degree of lignin decreased; ii) new inter-unit linkages were produced but they were not detected by HSQC NMR. The former hypothesis is supported by SEC analysis (Table 1) that showed a drastic decrease in molecular weight of lignin/Klason phase during reaction. However, the latter hypothesis cannot be discarded as the formation of condensed, C-C linkages such as 5-5' linkages was described in other studies; this type of linkage does not contain any H atom and thus is not detected in HSQC NMR.

To summarize the analytical data, the Klason phase presents several modifications compared to the initial Kraft lignin:

a decrease of the average molecular weight from ca. 1500 g.mol -1 to 800 g.mol -1 (SEC).

a decrease in the aromatics content (FTIR-ATR) associated to significant decrease of interunit bonds, particularly the β-O-4 linkages (HSQC NMR).

an uptake of oxygen (EA) associated to increase of carbonyl functionalities as quinone and carboxylic acids (FTIR-ATR and 31 P NMR), and to a drop in the hydroxyl content ( 31 P NMR).

Such complementary analyses help to follow lignin transformation during the reactions. Please note that this oxidized lignin polymer can present applications in the industry, for example as an anionic dispersant [68], but this is behind our project.

Characterization of organic phase

GC-MS and GC-FID analyses of the organic phase allowed identifying and quantifying the main monoaromatic compounds. Figure 7 shows a typical chromatogram obtained after an aerobic oxidative depolymerization experiment that exhibit mainly four compounds: vanillin, acetovanillone, vanillic acid and homovanillic acid. All compounds were identified via GC-MS and quantified with solutions of commercial standards. Vanillin is clearly the main mono-aromatic compound extracted in this fraction and quantifiable by GC. The sum of the areas of identified peaks was always superior to 90 % of the sum of all GC peaks (outside solvent and standard). This led us to consider that the majority of monoaromatic compounds was identified and quantified in this study. TGA analysis of organic phase (Figure S8) under air showed that around 50 % of the material was burnt at relatively low temperature (between 100 and 280°C) whereas the rest was burnt between 300 and 500°C. The first mass loss (ca. 70%) was attributed to mono-aromatic compounds (vanillin boiling point being 285°C) and it was assumed that heavy compounds (oligomers) were burnt at higher temperatures.

Finally, a 1 H-NMR analysis (Figure S9) of the organic phase allowed increasing our knowledge about the compounds not observed by GC analyses. 1 H NMR confirmed that vanillin is the main aromatic compound present in this phase; however, it also indicated the presence of oligomers and other phenolic compounds. Particularly, a significant number of protons in the aliphatic region was observed.

This could be possibly related to aliphatic chains of oligomers, or aliphatic compounds extracted together with aromatics since dichloromethane is not fully selective for aromatic compounds.

It is remarkable that mono-aromatic compounds all together (here, vanillin 3.3 wt%, acetovanillone 0.7 wt% and vanillic acid 0.5 wt%) represent, in most cases, around ca. 30 % of the total organic phase weight (here, 16 wt% from initial lignin). Clearly, other products are present, as shown by TGA and NMR (see below), but could not be identified, nor individually quantified, at this stage. Most probably, these compounds are oligomers issued from partial depolymerization of lignin whose higher molecular weight and boiling points prevented analysis by GC, together with unidentified phenolic compounds in traces.

Characterization of aqueous phase

This phase is expected to contain the aliphatic compounds obtained after oxidative depolymerization of lignin and over-oxidation of aromatic compounds. 1 H-NMR (Figure S10) confirmed this expectancy and showed that it contains a low quantity of aromatic compounds. Therefore, the main part of the aromatic compounds was extracted by dichloromethane and was found in the Organic phase.

To improve the characterization of this phase, HPLC analyses were conducted. These analyses allowed to identify several other molecules, mainly aliphatic acids like oxalic, succinic, formic, acetic acids.

These results are coherent with those obtained by 1 H NMR (Figure S11-A). This is very interesting since these acids present several applications in industry encouraging their valorization. As observed by 1 H NMR, these analyses also showed traces of phenolic compounds, like vanillin, vanillic acid and acetovanillone in Aqueous phase. (Figure S11-B)

Effect of initial lignin concentration on its depolymerisation

Before evaluating the role of the CuO/TiO2 catalyst on the aerobic depolymerization of the Kraft lignin, we first evaluated the influence of the concentration of lignin on the reaction without catalyst. The influence of the lignin concentration on its depolymerization under aerobic conditions was studied in non-catalytic conditions (150°C, 20 bar of air, 1 h and 1800 rpm). Our standard reaction conditions used a lignin concentration of 5 g.L -1 that is far away from the concentration encountered in Kraft paper industries. Several motivations encouraged us to use such concentration: keep basic pH along the reaction time, prevent foaming, limit lignin condensation reactions. For the study, the lignin concentration was evaluated from 5 g.L -1 to 20 g.L -1 .

Figure 8 reports the results obtained for both the mass balance after the different tests (Figure 5A), and associated yields in mono-aromatic products (Figure 5B). Lignin conversion decreased strongly as its concentration increased as observed with the increasing amount of Klason phase (23 wt% at a concentration of lignin of 5 g.L -1 to 52 wt% at 10 g.L -1 and 78 wt% at 20 g.L -1 ). At the same time, the amounts of the organic and aqueous phases decreased by an important degree from 16 wt% to 4 wt% and from 44 wt % to 9 wt%, respectively. Analyzing the Organic phases revealed that the vanillin yield is radically reduced from 3.2 % to 0.4 % when increasing the lignin concentration from 5 g.L -1 to 20 g.L -

1

. These results indicate that the degree of lignin depolymerization is inversely proportional to its concentration. To get deeper insight on the influence of this parameter, we underwent studies at longer reaction times. Different tests were performed at a lignin concentration of 20 g.L -1 increasing the reaction time from 1 h to 3 h (see Supporting Info Figure S12). Surprisingly, very similar results to those achieved after 1 h reaction time were obtained. In all cases, the Klason phase represent ca. 78 wt%, the Organic phase 1-4 wt% and the Aqueous phase 7-9 wt%. The vanillin yield only slightly increased from 0.4% after 1 h to 0.7% after 3 h. Since increasing the reaction time does not affect the lignin conversion, other limiting factors should be evaluated. Thus, several hypotheses are proposed:

-Limitation of the oxygen transfer from gas phase to aqueous medium preventing efficient depolymerization rate at high lignin concentrations. In order to evaluate this effect, the oxygen-transfer rate was calculated [11,69] (See Supporting Info Table S2) and compared with the oxygen consumed during the oxidation reaction. The results reported in Table 3 show a value for the oxygen transfer rate significantly higher than the value of the oxygen consumption, meaning that lignin oxidation is not limited by the oxygen transfer.

Table 3. Oxygen transfer and consumption in non-catalytic oxidation reactions.

Lignin concentration (g.L -1 )

Oxygen consumption (mmol.L -1 .s -1 ) 5

1.89 10 -3 10 2.58 10 -3 20 3.62 10 -3 Oxygen transfer (mmol.L -1 .s -1 )

9.07

-Lack of solubility of lignin material. The lignin used in this study is not soluble in neutral or acidic medium, as verified in other experiments. However, this hypothesis was ruled out as the pH of the reaction medium stay, over the reaction time, in basic values (ca. 12.75) at which Kraft lignin is still soluble.

-Recondensation reactions occurring over the reaction time. Several authors have reported such reaction during the depolymerization process, mainly during bleaching [70,71].

Generally, these recondensation reactions result in the production of 5,5' biphenolic structures, which would be unreactive under oxidative conditions. Increasing lignin concentration would favor these reactions. As a consequence of such recondensation reaction, the average molecular weight of resulting biopolymers should be affected. SEC analyses were carried out on the three Klason phases, and data are reported in Table 4. While at a lignin concentration of 5 g.L -1 the average molecular weight decreased from 1505 g.mol -1 to 813 g.mol -1 , it remained almost stable at a concentration of 10 g.L -1 (1372 g.mol -1 ) whereas it increased to 1952 g.mol -1 when using a lignin concentration of 20 g.L -1 . Last two values resulting very probably from partial lignin depolymerization followed by/together with recondensation reactions. The average molecular weight calculated by number distribution present the same tendency. Remarkably, the polydispersity of the molecular weights are very similar in all cases (Figure S13). Therefore, increasing lignin concentration improves, very probably, the rate at which the recondensation reaction occured. -Formation of lignin flocs in the reaction medium. Despite of the basic pH, the full solvation of long lignin polymeric chains is a complex phenomenon. At high concentration, refractive objects or "flocs" to solvation could be formed, limiting the access of the oxygen to the whole polymeric chain and consequently lignin conversion. Dynamic light scattering (DLS) analyses were performed for the three evaluated lignin concentrations. While performed at 25°C, due to technical limitation to operate at the reaction temperature of 150°C, DLS showed a certain tendency of lignin to form larger objects at high lignin concentrations (Table 5). The formation of such flocs supports mass transfer limitation preventing efficient lignin depolymerization. The last two hypotheses (i.e. recondensation reactions, and formation of lignin flocs) appear, at the light of our knowledge, the most reasonable to explain the results observed. In reality, very probably both phenomena occurred and combined to explain better the results observed.

Thus, in order to improve further the catalytic system, we decided to perform all other reactions at a lignin concertation of 5 g.L -1 , except mentioned.

Initial catalytic aerobic lignin depolymerization

The reactions were carried out in a batch reactor at 150°C, 20 bar of air and 1800 rpm stirring under alkaline conditions (Aqueous NaOH) in the presence of CuO/TiO2 catalyst. The reaction mixtures were treated as described above.

Initial tests aimed at evaluating the role of the air and of the catalyst in the reaction. Thus, the reaction was performed first under inert atmosphere, pressurizing the reactor with nitrogen, then under air without introducing any catalyst. Figure 9A reports the mass balance of the different tests, compared with the results from a catalytic reaction using CuO/TiO2 catalyst.

In all cases, mass balances around 80 wt% are observed. Except losses due to the fractionation protocol used, mass loss can be related to several other factors. The production of over-oxidation products evolving in gas phase (mainly CO2 or CO) from complete oxidation of the biopolymer during the reaction is one of the main factors. Next, loss of volatiles during the drying of the different phases would be another critical point that would reduce the mass balance. These support well the fact that higher mass balances are observed when the Klason phase represents the main fraction.

As expected, under inert conditions (blank) no significant amount of products (only 5 wt% of organic and aqueous phase) was obtained. In the absence of a catalyst, data obtained are close to those issued from performing the fractionation protocol on dissolved Kraft lignin without any reaction (Figure 4a, "No reaction"). Adding a catalyst slightly modified the situation, as a slight activity is observed mainly toward the formation of products evolving in the aqueous phase. This means that next to heating, an oxidant is required to observe significant lignin depolymerization, only few percent are transformed to product under nitrogen.

Under 20 bar of air, and without catalyst, the Klason phase dropped from 88 wt% to 23 wt%, attesting of a significant conversion of the substrate. The aqueous phase became the main fraction with 44 wt%.

The organic phase is produced in a quantity of 16 wt%, encouraging us in evaluating the CuO/TiO2 catalyst. While used in a relative low quantity (Cu: 5 wt%Cu/Lignin), the aromatic phase increased from 16 to 21 wt%. At the same time, the aqueous phase fraction decreased from 44 to 38 wt%, suggesting that the catalyst helps to increase the selectivity towards aromatic compounds. An alternative reaction mechanism can therefore occur in the presence of CuO/TiO2 catalyst. Noticeably, lignin conversion increased as well, as the Klason phase decreased from 23 to 18 wt%. Interestingly, using the TiO2 support alone did not modified the data observed under air in the absence of the catalyst; therefore, the active phase is CuO.

For clear analysis of the organic phase, we focused on the three main compounds derived from guaiacyl units, which are vanillin, acetovanillone and vanillic acid (Figure 9B). Vanillin is the main (mono)aromatic product obtained from the oxidative depolymerization of lignin. Under nitrogen, almost no (mono)aromatic compound was produced, and the low amount of the three main products observed when adding the CuO/TiO2 catalyst is probably due to the stoichiometric activity of CuO.

Under air, however, yields increased, particularly that of vanillin that raised from 0.5 % up to 5.1%.

Figure 9B shows that under air, the catalyst increased significantly the yield from 3.2% to 5.1%, an improvement of 40%. Interestingly, the increase of the yield of vanillin in the presence of CuO/TiO2 catalyst is higher than that of the organic phase (resp. 40% versus 2.3%) meaning that the CuO/TiO2 catalyst is active and selective towards vanillin production. The yields of acetovanillone and vanillic acid were improved in the same range than that of vanillin. Like before, the sum of the yield of identified (mono)aromatic compounds represent ca 30% of the total mass of the organic phase, meaning that other compounds were extracted by DCM but could not be analyzed by GC. Concerning the Klason phase, SEC data (see Supporting info, Table S3) showed a significant decrease of the average molecular weight from 1505 g.mol -1 for the initial lignin to 664 g.mol -1 for the Klason phase issued from the catalytic run; however, not so different from that of the non-catalytic run (i.e.: 813 g.mol -1 ). Nevertheless, the molecular weight distribution observed after the catalytic run is very different from that of the initial lignin or the Klason phase obtained after the non-catalytic run (see Supporting info, Figure S13). Like previously reported [42], our data support that the catalyst, next to improving the formation of aromatic compounds from oligomers, act as well in recondensation reactions (probably forming 5-5' interunit linkages). This probably prevents efficient depolymerization of lignin due to the resilience of condensate units. HSQC-NMR of the Klason phase showed surprisingly a strong decrease in the content of the  linkages from 5.8 in the initial lignin to 0.7 in the Klason phase obtained for the catalytic run, while other characteristic linkages like the -O-4 and-5 stay in the range form what was observed after a non-catalytic run (see Supporting info, Table S4). These data suggest that the catalyst is particularly active in modifications of the  linkages that could include bond breaking or oxidation at one of the -position.

In all cases, the catalyst was found to be stable over the reaction time as outlined by XRD analyses on spent catalyst (see Supporting info Figure S14). The only noticeable modification being related to the anatase/rutile ratio showing a transition between the two metastable phases of TiO2. Elemental analyses of the reaction mixtures after reaction revealed a copper concentration of 2.5-5 ppm, in agreement with a low leaching rate. TGA of used catalyst did not show significant amount of adsorbed organic matter.

Although the catalytic activity of the CuO/TiO2 catalyst seems to be limited to the of vanillin, it is interesting to compare these results with those reported in the literature (Table 6). Few works concerned the use of CuO. Different lignin/starting materials were used in these works and the results depends strongly on the nature of the lignin and the reaction conditions making comparison to our work speculative. When focusing on Kraft lignin from softwoods, no report concerns the use of CuO, but we found interesting to compare our data to those achieved with other catalytic systems. Best yield in vanillin (6.8 %) from Kraft lignin oxidative depolymerization was achieved with CuMn mixed oxide under oxygen atmosphere and the use of hydrogen peroxide as co-oxidant. Under our milder conditions that uses oxygen from the air as the oxidant and moderate basic conditions, the 5.1% yield in vanillin achieved is in the range of data reported in the literature. At this stage, and with these results in hand, we underwent optimizations of the reaction conditions aiming at increasing the efficiency of the catalyst toward the production of aromatic compounds, focusing on vanillin. Catalytic reaction with different sizes of particle of CuO/TiO2 (Lignin (5 g.L -1 ), 150°C, 1800 rpm, 1 h, 20 bar of (N2 or air), CuO/TiO2 (CuO/TiO2 (5 wt%Cu), 2 wt%Cu/lignin). A) Mass balance of the different products concentration (wt%), B) Organic phase yield (wt%).

The catalyst particle size had a minor impact on mass balance after reaction. An increase of lignin conversion was observed with extrudates and powder catalyst when compared to non-catalytic reaction. While both catalytic reactions showed an increase of lignin conversion, the reaction done with crushed catalyst showed a slightly higher Klason phase proportion that could be related either to a lower conversion rate, or more probably to higher rate of recondensation reactions. The organic phase shows an increase in its proportion when powder catalyst was used and no effect when extrudates catalyst was used, which can be associated with a higher catalytic activity for the powder form. At the same time, the proportion of the aqueous phase decreased slightly with the powder form. The GC analysis of the organic phase showed significant increase in vanillin yield, and its dependence to the catalyst particle size. Thus, while the use of CuO/TiO2 extrudate produced an increase by 28% of the vanillin yield (from 3.4%wt to 4.1%wt), the used of crushed CuO/TiO2 catalyst (90-200 µm) improved the vanillin yield by 59% (from 3.4%wt to 5.1%wt).

The reason of the significant difference in the catalytic activity between the extrudates and crushed catalyst can be related to internal mass transfer limitations. In a first approach, as seen in the previous part, lignin can form flocs into the reaction medium, like other polymeric chains. Although it was not possible to measure the size of these flocs at 150°C, the temperature of the reaction, we assume that they keep, or eventually, decrease in size with increasing the temperature as resulting from an increase of the system energy. A flocs size of about 44 nm was measured by DLS at a lignin concentration of 5 g.L -1 . This means that lignin could present limitations to enter into the mesopores of the catalyst, and accessing the active sites. Mesopores were measured by nitrogen physisorption with the BJH method (Figure S3) and estimated in the range of 2-50 nm with an average diameter of 25 nm. The mesoporous structure of the catalyst is not modified by crushing. However, the catalyst particle size decreased the diffusion time for lignin reaching the active site (CuO) inside the pores and for products leaving the active site to reach the catalyst surface. Diffusion is reduced in powder form since the CuO active sites attached to TiO2 surface are closer from the catalyst external surface. These phenomena could affect positively the vanillin production.

This effect was verified by calculating the Weisz-Prater (WP) parameter (Table 7). WP criterion compares values of the apparent rate of reaction and diffusion rates in catalyst grain and is used to evaluate how internal diffusion can limit the reaction rate. This parameter was calculated with the next expression [START_REF] Fogler | Elements of Chemical reaction engineering[END_REF]:

𝐶 = 𝑅𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝐷𝑖𝑓𝑓𝑢𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 = -𝑟′ (𝑜𝑏𝑠)𝜌 𝑅 𝐷 𝐶
In this equation, r'A is the apparent reaction rate per volume of catalyst, R is the catalyst particle radius and CAS the concentration of the reactant the surface of the catalyst. De is the effective diffusion, which was obtained from the bulk diffusivity DAB (Lignin [START_REF] Garver | [END_REF]: 10 -10 m 2 .s -1 , Vanillin [75]: 1,05*10 -09 m 2 .s -1 ) by the next correlation [START_REF] Fogler | Elements of Chemical reaction engineering[END_REF]:

𝐷𝑒 = 𝐷 Φ 𝜎 𝜏̃

The values for 𝜏(tortuosity) Φ (pellet porosity) and 𝜎 (constriction factor) was 3, 0.4 and 0.8 respectively, typical values used in literature for materials. Two different calculations were made, based on lignin consumption rate and on vanillin production rate. The results obtained for the Weisz-Prater criterion (Table 7) show an important limitation of the vanillin diffusion in the case of the extrudates catalyst, with a value significantly higher than 1. When the catalyst particle size decreased, the limitations are solved and the oxidation is performed in chemical regime, which is usually admitted when WP parameter presents a value inferior to 0.3. This is related with the optimal production of vanillin with a catalyst size of 90-200 µm (Figure 10B).

In the case of lignin, diffusion limitations are always present, which is related to the similar mass balance obtained with and without catalyst (Figure 10A). These limitations are clearly evidenced when the catalyst particle size is higher (Table 7).

These results are useful to elucidate the role of the catalyst. The fact that lignin presents an important internal mass transfer limitations during the catalytic reaction makes its direct conversion on the active catalytic site (CuO) unlikely under our reaction conditions. It is assumed that lignin reacts first with OH - in solution to form oligomer species small enough to diffuse in the catalyst and that vanillin and other phenolics are produced in a second step on the active catalytic sites from these oligomer intermediates. Therefore, at this point the most plausible hypothesis is that the catalyst increases the reaction rate of the vanillin formation from oligomers that present lower internal mass transfer limitation, as represented on Figure 11.

Effect of catalyst loading

Calculation of the WP criterion reported above showed that the catalyst particle size significantly affects the mass transfer rate of lignin, and, in a lower degree, that of vanillin. The catalyst loading is another factor that can affect this phenomenon, and consequently the aromatics yields. Therefore, catalytic tests at different catalyst loadings were performed under the same reaction conditions. Three catalysts loadings, corresponding to 2, 5 and 7 wt%Cu/lignin were used.

Figure 12 shows that the catalyst loading affects the proportion of the different phases. A 5 wt%Cu/lignin presents an optimum in terms of the organic phase fraction that raised from 16 wt% without catalyst to 21 wt %; however, increasing further the catalyst loading to 7 wt%Cu/lignin resulted in a drop of the amount of the organic phase to 11 wt%, probably due to over-oxidation of aromatics to aliphatic compounds. This is supported by the higher amount of aqueous phase that reached 75 wt% (compared Regarding the yields in mono-aromatics in organic phase, the results indicate also an optimum for the catalyst loading of 5 wt%Cu/lignin with a yield of vanillin that increased to this point to 5.1 wt% (Figure 12). Increasing further the catalyst loading resulted in a drop of the vanillin yield due to over-oxidation, as already mentioned. Altogether, these results evidence the difficulty in optimizing the reaction conditions to produce mainly aromatic compounds without observing their over-oxidation. This requires deeper understanding of the reaction kinetics. In order to get deeper insight on the evolution of the reaction mixture over the time, reactions were performed at different reaction times keeping the standard reaction conditions (150°C, 20 bar of air and 1800 rpm of stirring rate). Catalytic (CuO/TiO2) and non-catalytic reactions were compared.

to
13 shows the results of lignin oxidation over 120 min reaction time for both the evolution of the mass balance and the yields of the aromatics in the organic phase. Figure 13A shows that most of the depolymerization reaction occurred during the first 15 minutes with a relatively high rate, since the Klason phase dropped from 60 wt% to 40 wt% over this period of time. However, as indicated by the mass balance, this depolymerization produced mainly aliphatic compounds evolving in the aqueous phase, which raised from 3 wt% to 30 wt% over the same period. After this reaction time, lignin depolymerization rate decreased, leading to a proportion of Klason phase of 10 wt% after 2 h of reaction time. Rate of formation of products evolving in the aqueous phase shows similar behavior; a quick increase over the first 15 min, after which the rate decreased to deliver finally 50 wt% aqueous phase after 120 min for both the catalyzed and non-catalyzed reactions. For both the Klason phase and the aqueous phase, the evolutions are very similar whatever a catalyst was used or not.

The evolution of the organic phase shows different behavior, and differences exist between the catalyzed and the non-catalyzed reactions. In all cases, the organic phase is produced at a lower rate when compared to aqueous phase, which is particularly true for the non-catalyzed reaction. Thus, while the amount of the organic phase grows from 7 wt% to 14 wt% in 15 min for the CuO/TiO2 catalyzed reaction, its increase is of only 1 wt% (i.e. from 7 % to 8 %) for the non-catalyzed one over the same period of time. Additionally, on the contrary to the aqueous phase which amount increased continuously, that of the organic phase reaches a maximum at ca. 60 min reaction time before deceasing slowly. These observations can be rationalized through different factors:

-A decrease in the rate of formation of the aromatic compounds that can be then slower than that of their degradation by overoxidation to products evolving in the aqueous phase.

-Structural modifications of the lignin biopolymer preventing further bond cleavages to produce aromatics evolving in the organic phase.

-reactions between aromatics and oligomers to products evolving more probably in the Klason phase.

All hypotheses are supported by experimental facts. Analyses of the organic phase over the time (Figure 13B) showed clearly that for the catalyzed reaction the yield of the main aromatic compound, namely vanillin, decreased after 1 hour as the mass of the organic phases does. Concerning the noncatalyzed one, similar behavior is observed; however, the maximum in vanillin yield was reached after 30 min reaction time before remaining relatively constant. This suggests that the catalyst participate to the over-oxidation of produced aromatics.

The evolution of the acetovanillone yield over the reaction time follows almost the same behavior than that of vanillin. However, that of vanillic acid yield is different. While it reached a maximum between 15 and 60 min, before remaining relatively constant, for the catalyzed reaction, a maximum was observed after 30 min reaction time for the non-catalyzed reaction after which it decreased rapidly.

These differences suggest that vanillic acid formation follows different pathways than that of vanillin and acetovanillone, which can follow similar reaction pathways.

The results showed that the yields in aromatic compounds produced by lignin depolymerization are not linked to the level of the depolymerization itself as it results from various competitive reactions. These include, for example, the lignin base-catalyzed depolymerization to oligomers followed by a metal-catalyzed formation of aromatics, that is counterbalanced by (re)condensation reactions and degradation of the aromatics by overoxidation. From our results, degradation of the aromatics seems to be favored by the presence of the catalyst, like their production, pointing out the extreme complexity of these transformations.

CONCLUSIONS

This study evidenced that CuO/TiO2 catalyst helps to improve the production of aromatic aldehydes, here vanillin, by lignin oxidative depolymerization. However, this reaction remains complex and several factors affecting the efficiency were identified. Among them, two concern directly the starting lignin material:

-One of the first factor affecting the efficiency of the reaction is the lignin concentration. Its increase resulted in decreased aromatic aldehyde yields due to the formation of lignin agglomerates in solution as determined by DLS experiments. Those agglomerates could limit the mass transfer of the oxygen from the aqueous medium to the polymeric chain preventing thus deep depolymerization.

-Lignin diffusion inside the catalyst grain is another important limitation. This could be partly solved by using smaller grain size of catalyst, which decreased therefore the diffusion time and led to increase the yield of vanillin. However, our results show that the initial lignin cannot enter in the catalyst mesopores; therefore, its depolymerization relies on homogeneous reactions and on active sites available on the external surface area.

Other factors limiting lignin depolymerization efficiency are linked to the products evolving in the reaction medium and associated side reactions:

-The over-oxidation of the aromatic compounds, resulting in decreased yield in products of interest, is another important limiting factor. This was particularly demonstrated by studying the influence of the catalyst loading that showed an optimum in vanillin yield when using a charge of 5 wt%Cu/lignin. Nevertheless, deeper insights were gained from analyzing the evolution of the reaction mixture over the time. Our studies showed that lignin depolymerization occurred mainly in the first 30 min of reaction as shown by the reduction of the Klason phase amount to ca. 27 wt%. After that time, the Klason phase decreased slowly to reach a residue of ca. 15 wt% over the next 90 min. In the catalyzed reaction, the yield of vanillin production presents an optimum at 1 hour of reaction time before decreasing, indicating the degradation of vanillin, related to overoxidation in the presence of the catalyst. On the contrary, for the non-catalyzed reaction no optimum was clearly observed, the yield in vanillin reached a maximum at ca 30 min reaction time and remained almost constant.

-Finally, recondensation reaction involving lignin polymeric chain and products from its depolymerization, whatever one considers the mono-aromatic compounds or the oligomers, is another important factor that can affect efficient depolymerization. Particularly, at high lignin concentration, an increase in polymer molecular weight was observed, typical of condensation reactions. Diluting lignin helped to control this phenomenon.

To summarize, several factors affect the efficiency of catalyzed oxidative depolymerization of lignin towards aromatics. Among them, lignin agglomeration, limited diffusion in catalyst porosity, recondensation reactions and aromatic over-oxidation reactions are important parameters to take in account to optimize the reaction conditions. A balance should also be found between increasing the efficiency of catalysts for depolymerizing lignin under oxidative atmosphere and the risk of catalyzing overoxidation of aromatic compounds of interest. This requires deeper studies to develop an efficient lignin valorization process.

Methods:

X-ray diffraction:

XRD analysis was carried out in a Bruker D8-Advance with automatic sampler, using a radiation Cu-Kα (0.154184nm).

Thermogravimetric Analysis:

The same TGA device (Mettler Toledo TGA/DSC STAR System) was used to study the thermal behavior of lignin and Klason phase (5 °C/min, from 25 to 1000 °C, air as carrier gas). The moisture degree was determined by the weight loss at 100-115 °C and the ashes by the remaining weight at 1000 °C.

Fourier transform-infrared spectroscopy (FT-IR):

FT-IR analyses were carried out in an absorbance mode using a Nicolet IS5 equipped with an ID7-ATR accessory with diamond crystal from Thermo Scientific. The sample was directly placed over the diamond and IR signal was recorded with a resolution of 4 cm -1 32 scans and a spectral range of 4000 cm -1 to 525 cm -1 . The spectra obtained with this method were qualitatively analyzed to characterize different structures in the lignin.

NMR:

NMR spectra were acquired on a Bruker AVANCE III 400MHz equipped with a BBFO probe (Z gradient).

For 1 H-NMR, samples were prepared by dissolving an aliquot (10-15 mg) in 600 mg of (CD3)2SO. Data were recorded over 16 scans at 50°C. Chemical shift were measured relatively to residual protic signal of (CD3)2SO at 2.5 ppm.

For 13 C-NMR, data were recorded so that quantitative analysis could be applied. Tetramethylthiourea was used as standard and Fe(acac)3 as relaxing agent. An aliquot of the sample (10-40 mg) was dissolved in (CD3)2SO (600 mg). Then, 0.1 mL of the standard solution, made from tetramethylthiourea (40 mg) and Fe(acac)3 (4 mg) in (CD3)2SO (1000 mg), was added. Data were recorded over 11000 scans at 50°C. Chemical shift were measured relatively to the signal of deuterated solvent ((CD3)2SO: 39.5 ppm). For 31 P quantitative NMR analyses, data were recorded after phosphorylation using 2 Chloro-4,4,5,5tetramethyl-1,3,2 -dioxaphospholane (TMDP) as phosphylating agent 7 An aliquot (10-30 mg) was dissolved in CDCl3 (200 mg) to which was added cyclohexanol (8 mg), used as standard, and TMDP (100 mg). DMF (0.25 mL) and pyridine (0.25 mL) were added to ensure full dissolution of formed salts. Data were recorded over 64 scans at 25°C. Chemical shift were measured relatively to the signal of the product of the reaction of TMDP with water (δ = 132.2 ppm) and quantitative data were measured relatively to the area to the signal provided by phosphylated cyclohexanol (144 ppm). For HSQC NMR, samples were prepared by dissolving an aliquot (10-15 mg) in 600 mg of (CD3)2SO. Data were recorded over 48 scans at 50°C. Semi-quantitative analyses were calculated following the following equations: 𝐼𝐶 = 𝐼𝐺 (𝑠𝑜𝑓𝑡𝑤𝑜𝑜𝑑 𝑢𝑛𝑖𝑡𝑠) (Equation 1)

% = 𝐼𝐴 𝐼𝐶 • 100 (Equation 2)

Fig 1 .

 1 Fig 1. Oxidation test and fractionation protocol of lignin oxidation products.

Figure 2

 2 showed that the Rutile (2Θ = 27.41; 36.04) to Anatase (2Θ = 25.85; 36.43; 37.03; 38.57) ratio varies in the catalyst compared to the original support. Particularly the proportion of Rutile increased after calcination, which could be expected given the calcination temperature applied. Additionally, on the catalyst, characteristic peaks of CuO were observed at 2Θ = 35.42; 35.54; 38.71 and 38.90 angles.

Fig 2 .

 2 Fig 2. XRD analysis of CuO/TiO2 (5%Cu). TiO2 Anatase (Red), TiO2 Rutile (Blue), CuO Tenorite (Green).

Fig 3 .

 3 Fig 3. TEM pictures and EDX mapping of the CuO/TiO2 catalyst (copper detected by EDX appears in orange).

Fig 4 .

 4 Fig 4. FTIR spectra of initial Kraft lignin (red), Klason phase of non-catalyzed reaction (blue).

Fig 5 .

 5 Fig 5. 31 P-NMR analysis after phosphitylation of initial lignin and Klason phase.

Fig 6 .

 6 Fig 6. HSQC-NMR analyses of the initial lignin and Klason phase. (A): oxygenated aliphatic region, (B): aromatic region.

  OH p-hydroxyphenyl OH guaiacyl OH syringyl + condensed units OH alyphatic HSQC-NMR spectra of lignin in the oxygenated aliphatic regions (Figure 6a left) show, next to methoxy groups (H/C: 3.76/56.2 ppm) and residual carbohydrates (H/C: 3.02-3.72/68.2-78.5 ppm), all characteristics correlations corresponding to inter-unit linkages: β-O-4 linkages (A); β-β linkages (B); phenylcoumaran units (β-5 linkages)(C). Beside the methoxy groups that are predominant, the β-O-4 linkages (A/A'/A'') are the most important inter-unit bonds detected in HSQC, which is expected from a softwood lignin, with (A) (H/C: 4.79/71.7 ppm), (A) (H/C: 4.74/81.9 ppm), (A G/H) (H/C: 4.29/85.1 ppm) and (A) (H/C: 3.48/60.6 ppm), and associated (A') (H/C: 4.14/67.6 ppm) and (A'') (H/C: 3.67/63.7 ppm). β-β linkages are observed with (B) (H/C: 4.64/85.6 ppm), (B) (H/C: 3.04/54.1 ppm) and (B) (H/C: 3.75 and 4.10/71.7 ppm) and β-5 linkages gave correlations at (C) (H/C: 5.48/87.4 ppm) and (C) (H/C: 3.44/53.9 ppm). Cinnamyl alcohol signal is observed at (D) (H/C: 4.08/61.9 ppm). Aromatic region (Figure 6b, left) exhibits the characteristic correlations corresponding to guaiacyl units (G) (G2: H/C: 6.91/111.36 ppm, G5: H/C: 6.77/116.11 ppm, G6: H/C -6.77/121.74 ppm) and those of p-hydroxyphenyl units (H) (H2,6: H/C: 7.21/128.85 ppm). Correlations corresponding to ferrulic derivatives as observed as well: (FA2) (H/C: 7.41/109.69 ppm), (p-CA2.6) (H/C: 7.42/126.31 ppm) and (CH=CH) (H/C: 5.34/120.94, 5.73/122.88 ppm). Correlations corresponding to guiacyl units substituted by carbonyl moieties (G') are also observed (G'2: H/C: 7.49/112.24 ppm, G'6: H/C: 7.27/121.12 ppm). Almost all correlations subsist in HSQC-NMR spectra of the Klason phase (Figures 6a and 6b, right); however, with variation of the volume. Please note the full disappearance of the correlations corresponding to the β-5 linkages (C) and decrease of the carbohydrate content in the oxygenated aliphatic region, and disappearance of the correlations (FA2), (p-CA2.6) and (CH=CH) corresponding to ferrulic derivatives in the aromatic region.

Fig 7 .

 7 Fig 7. Typical chromatogram of the organic phase.

Fig 8 .

 8 Fig 8. Non-catalytic reaction with different lignin concentrations (150°C, 20 bar of air, 1 h and 1800 rpm). A) Mass balance of the different phases (wt%), B) Aromatic product yields (wt%).

Fig 9 .

 9 Fig 9. Aerobic depolymerization of Kraft lignin: Lignin (5 g.L -1 ), 150°C, 1800 rpm, 1 h, 20 bar of (N2 or air), CuO/TiO2 (CuO/TiO2 (5 wt%Cu), 5 wt%Cu/lignin, 90-200 µm). A) Mass balance of the different fractions (wt%), B) Aromatic product yields (wt%).

Fig 10 .

 10 Fig 10. Catalytic reaction with different sizes of particle of CuO/TiO2 (Lignin (5 g.L -1 ), 150°C, 1800 rpm,

Fig 11 .

 11 Fig 11. Diffusion of lignin and lignin oxidation products in CuO/TiO2 catalyst.

  44 wt% in the absence of catalyst) in which the products of over-oxidation are extracted. At the same time the proportion of the Klason phase, that indicate the level of lignin conversion, drop to 8 wt% (versus 23 wt% in the absence of catalyst, and 13 wt% and 18 wt% at resp. 2 wt%Cu/lignin and 5 wt%Cu/lignin) indicating enhanced catalytic activity at this loading. The slight increase of the Klason phase at 5 wt%Cu/lignin can be related to repolymerization/recondensation reactions. At 7 %Cu loading, the results can be explained by an increased catalytic conversion of lignin through its transformation to oligomers, and then aromatics that are degraded. Next to this route through oligomers, non-catalytic depolymerization of lignin could occur at higher rate, what correlates the low remaining lignin in Klason phase and the high proportion of the aqueous phase.

Fig 12 .

 12 Fig 12. Catalytic reaction with different catalyst loading (CuO/TiO2 (5 wt%Cu), 1 hour, 150°C, 20 bar of air and 1800 rpm). A) Mass balance of the different products concentration (wt%) B) Aromatic product yields (wt%).

Fig 13 .

 13 Fig 13. Catalytic (CuO/TiO2 (5 wt%Cu)-90-200µm; 5 wt%Cu/lignin) and non-catalytic reactions at different reaction times (150°C, 20 bar of air and 1800 rpm). A) Mass balance of the different products concentration (wt%). B) Product yields (wt%).

FiguresFigure S1.

  Figures

  Figure S1. TGA analysis of catalyst before calcination.

Figure S2 .

 S2 Figure S2. Isotherm analysis of TiO2 (Evonik, Aerolyst 7711 extrudates 1x4mm) (Red) and synthetized CuO/TiO2 (5%Cu) (Blue).

Figure S3 .

 S3 Figure S3. TGA analysis of Kraft lignin (Red) and Klason solid from lignin oxidation (Blue).

Figure S4 .

 S4 Figure S4. Mathematical derivation from ATG analysis of Kraft lignin (Red) and Klason solid from lignin oxidation (Blue).

Figure S5 .

 S5 Figure S5. Molar mass distribution curve obtained by the SEC analysis, Kraft lignin (Red), Klason phase (Blue) and Klason phase (CR) (Green).

Figure S2 .

 S2 Figure S2. Complete FTIR-ATR analysis of Kraft lignin (Red), Klason phase from lignin oxidation (Blue) and Klason phase from lignin catalytic oxidation (Green).

Figure S4 .

 S4 Figure S4. TGA analysis of the organic phase from lignin oxidation.

Figure S5. 1 H

 1 Figure S5. 1 H NMR analysis of vanillin (A) and aromatic phase from lignin oxidation (B).

Figure S7 .

 S7 Figure S7. HPLC chromatograms of aqueous phase using Phenomenex Rezex ROA column (A) and Phenomenex Kinetex XB-C18 (B).

Figure S8 .

 S8 Figure S8. Non-catalytic reaction with a lignin concentration of 20 g.L -1 at different reaction times (150°C, 20 bars air, 10 gNaOH.L -1 ). A) Proportion of the different phases (wt%), B) Aromatic compounds yields (GC analysis of the Organic phase) (wt%).

Figure S9 .

 S9 Figure S9. Molar mass distribution curve obtained by the SEC analysis of the Klason phases obtained by the different experiments, 5 g.L -1 (Red), 10 g.L -1 (Blue), 20 g.L -1 (Green).

Figure S14 .

 S14 Figure S14. XRD analysis of CuO/TiO2 (5%Cu) before reaction (A) and after reaction (B) (150°C, 20 bars air, 60 minutes). TiO2 Anatase (Red), TiO2 Rutile (Blue), CuO Tenorite (Green).

Table 1 .

 1 Data obtained from the SEC analysis.

		Mn (g.mol -1 )	Mw (g.mol -1 )	Mw/Mn
	Initial lignin	745	1505	2.02
	Klason phase	426	813	1.91

Calibration with polystyrene standards.

Table 2 .

 2 Semi-quantitative data obtained from HSQC-NMR analyses. a Expressed per 100 aromatic units. Calculated following the procedure reported by Wen et al.[START_REF] Wen | [END_REF](see Supporting Information)

		Initial Lignin	Klason phase
	β-O-4	6.7	2.6
	β-β	5.8	3.5
	β-5	2.4	0.8

a

Table 4 .

 4 Molecular weight obtained by size exclusion chromatography (SEC)

		Mn (g.mol -1 )	Mw (g.mol -1 )	Mw/Mn
	Initial lignin	745	1505	2.02
	Klason phase (5 g.L -1 )	426	813	1.91
	Klason phase (10 g.L -1 )	722	1372	1.90
	Klason phase (20 g.L -1 )	889	1952	2.19

Table 5 :

 5 DLS analysis of lignin basic aqueous solutions at different lignin concentrations (25°C).

	Concentration (g.L -1 )	Z average (nm)
	5	44.0
	10	61.8
	20	92.3

Table 6 .

 6 Comparing our work to the literature.

	Biomass	Catalytic System a	Conditions	Products	Reference
	Hardwood Kraft	CuO (160 wt%)	O2 (10-15 atm)	Syringaldehyde and	[13, 56]
	lignin		150°C	Vanillin: 7.0-8.0%
			NaOH (0.5-2 N)
		CuO (catalytic)	O2 (10-15 atm)	Syringaldehyde and
			150°C	Vanillin: < 3.0%, no
			NaOH (0.5-2 N)	effect of adding CuO.
	Japanese Cedar	CuO (20-60 wt%)	H2O2	Vanillin: 2.6-5.0%	[58]
	Wood Particles		µW irradiation

Table 7 .

 7 Weisz-Prater parameter calculation (Lignin (5 g.L -1 ), 150°C, 1800 rpm, 1 h, 20 bar of (N2 or air), CuO/TiO2 (CuO/TiO2 (5 wt%Cu), 2 wt%Cu/lignin)[START_REF] Fogler | Elements of Chemical reaction engineering[END_REF]

	Catalyst size	Rp (m)	rA Vanillin (mmol.L -1 .s -1 )	CWP Vanillin	rA lignin (mmol.L -1 .s -1 )	CWP Lignin
	Extrudates (1x4mm)	3.3E-4	3.8E-4	2.2	-7.9E-4	19.8
	90-200μm	2.4E-5	4.7E-4	0.3	-7.6E-4	2.0
	Rp: particle rA: apparent reaction rate; CWP: Weisz-Prater criterion.

  ). A) Proportion of the different phases (wt%), B) Aromatic compounds yields (GC analysis of the Organic phase) (wt%).

				Klason phase	Organic phase	Aquous phase
		100%
	A) Composition (wt%)	20% 40% 60% 80%		78% 9%	4%		79% 8%	1%		78% 7%	3%
		0%
			20 glignin.L^(-1)	20 glignin.L^(-1)	20 glignin.L^(-1)
				1 hour			2 hours		3 hours
				Vanillin	Acetovanillone	Vanillic acid
		1.2%
	B) Product yields (wt%)	0.4% 0.8%	0.4%	0.1%	0.4%	0.5%	0.2%	0.4%	0.7%	0.2%	0.4%
		0.0%
			20 glignin.L^(-1)	20 glignin.L^(-1)	20 glignin.L^(-1)
				1 hour			2 hours			3 hours

ACKNOWLEDGEMENTS

The authors thank Evonik for generous gift of Aerolyst catalyst support. The authors thank CNRS and the University of Lyon (UCBL) for support. IRCELYON scientific and technical services, Laurent Veyre and Stéphanie Pallier from CP2M (for respectively TEM and DLS analyses) are kindly acknowledged.

The authors thank AXELERA cluster for the chemical and environmental sectors.

STATEMENTS & DECLARATIONS

Funding

The authors gratefully acknowledge the National Agency of Research (Phenoliq N° ANR-16-CE06-0007-03) for funding. A. H.M. thanks the National Agency of Research (Phenoliq N° ANR-16-CE06-0007-03) for PhD grant.

Data availability

Effect of catalyst particle size

Initially, the CuO/TiO2 catalyst was used after preparation and calcination in pellet form. This means that the sizes of catalyst particles have those of the commercial Aerolyst 7711 extrudate support, which is ca 1 mm diameter * 3-4 mm length. In other experiments, the catalyst was used after being crushed and sieved to a size of 90-200 micrometers. The oxidation reaction was performed using the standard conditions. Figure 10 shows the mass balance and the variation in the organic phase composition for the oxidative depolymerization of lignin using different catalyst particle sizes at a catalyst concentration of 2 wt%Cu/lignin.

Conflict of interest statement

The authors have no relevant financial or non-financial interests to disclose.

Author contribution

The manuscript was written through contributions of all authors. High-performance liquid chromatography (HPLC):

These analyzes were carried in a Shimadzu apparatus equipped with two columns:

-Column Phenomenex Rezex ROA -detector DAD, acidified water as mobile phase, 0.005 N H2SO4, isocratic, 0.6 mL.min -1 and 70°C. -Column Phenomenex Kinetex XB C18 -detector DAD, acidified water as mobile phase, 0.005 N H2SO4/Acetonitrile 80/20, isocratic, 0.7 mL.min -1 and 50°C.

Physisorption (Isotherm-BET):

The mesoporosity and BET analysis were carried out on an ASAP 2020 of Micromeritics, equipped with two pressure sensors. The analysis were done using nitrogen as physisorption gas at a temperature of -196°C. Desorption pre-treatment was carried out for 3 h at 300°C.

TEM:

STEM-HAADF (Scanning Transmission Electronic Micros-copy -High Angle Annular Dark Field) images were taken by a JEOL 2100F microscope, with a 200 kV acceleration tension. These characterizations were performed at "Centre Technologique des Microstructures" (CTµ, Villeurbanne, France)

Tables

Table S1. Assignments of bands in FTIR spectra of FCBA lignin and Klason phase. 1 Wave number (cm -1 ) Assignment Table S2: Correlations used for the calculation of the oxygen transfer. KLa was calculated using a correlation already corroborated by Bellefon et al. 1 Correlation for Kla calculation.