Théo Guérin

Alina Ghinet

Christophe Waterlot
email: christophe.waterlot@junia.com

The phytoextraction power of Cichorium intybus L. on metal-contaminated soil: focus on time-and cultivar-depending accumulation and distribution of cadmium, lead and zinc

Keywords: Metal, Chicory, Bioavailability, Plant pigments, Remediation factor

This study is focused on the evaluation of the accumulation of Cd, Pb and Zn in five cultivars of Cichorium intybus L. (chicory) which were produced on contaminated agricultural soil. Over a growth period of 211 days, the roots and leaves were collected in four stages and then analyzed by flame atomic absorption spectrometry to measure the concentration and distribution of the target metals in these two chicory organs considering the weather and the nature of the cultivar. For all cultivars, sharp decreases of Pb (from 165 mg kg -1 to 3 mg kg -1 ), Cd (from 11 mg kg -1 to 5 mg kg -1 ) and Zn concentrations (from 157 mg kg -1 to 40 mg kg -1 ) in the roots were highlighted over time. The data collected enabled the calculation of the variation of the bioconcentration factor, the biological absorption coefficient and the translocation factor for Cd, Pb and Zn. These parameters were then correlated with the distribution of the fresh biomass of leaves and roots and several indicators such as chlorophyll content, flavonols, anthocyanin and nitrogen balance index were measured. The study concludes with the discussion on the ability of chicory to clean up contaminated agricultural soil. The current investigation has shown: i) a translocation of Cd (and Zn to a lesser extent) from the roots to the leaves; ii) an increase in the level of anthocyanins with the increase of the metal trace elements concentration in the leaf, while the content of chlorophyll and the nitrogen balance index decrease, which could be linked to the phenomenon of senescence; iii) an ability of the chicory to reduce the bioavailable pool of the three metal trace elements studied, in particular for Cd.

Introduction

Soil pollution by metal(loid)s remains a major environmental concern in the whole world. These contaminants accumulate quickly in living organisms (fauna and flora) and often reach toxic concentrations that affect the soil functioning, the ecosystem, and the human health due to their longterm persistence in the environment. Human activities (e.g. mining and smelting of metal ores) are the main sources that contributed to increase the environmental anthropogenic pollution by metal(loid)s [START_REF] Alloway | Heavy Metals in Soils[END_REF][START_REF] Alloway | Sources of Heavy Metals and Metalloids in Soils[END_REF]. The most famous contaminated area in the Hauts-de-France region (France) is located in the former coal mining and ore processing sites (Metaleurop Nord and Nyrstar). For more than a century, the main pollutants released by these two metallurgical plants were lead (Pb), cadmium (Cd), and zinc (Zn), defined as metal trace elements (MTE) in the current study. The total concentration of these MTE and their bioavailability in the surrounding agricultural soils is sometimes problematic since a high proportion of the agricultural crops for foodstuffs are not conform with the European legislation [START_REF] Douay | Assessment of potential health risk for inhabitants living near a former lead smelter. Part 1: metal concentrations in soils, agricultural crops, and homegrown vegetables[END_REF][START_REF] Pruvot | Heavy Metals in Soil, Crops and Grass as a Source of Human Exposure in the Former Mining Areas[END_REF].

Many ex-situ and in-situ methods have been developed to manage polluted soils [START_REF] Gong | An overview of field-scale studies on remediation of soil contaminated with heavy metals and metalloids: Technical progress over the last decade[END_REF]) (e.g encapsulation, soil washing, vitrification…). While these methods allow effective soil treatment, these technologies are often costly, time consuming and unsuitable for managing the soil of large polluted sites [START_REF] Liu | Remediation techniques for heavy metal-contaminated soils: Principles and applicability[END_REF]. Soil phytoremediation is a much more viable alternative because of its ease of implementation and the low costs it implies. This technology refers to the use of plants and amendments and/or microorganisms that can be added to improve the soil quality through several processes such as the degradation of organic matter, the degradation of pollutants as well as their extraction or stabilization [START_REF] Ali | Phytoremediation of heavy metals-Concepts and applications[END_REF]. Among these processes, phytoaccumulation consists in metal(loid)s accumulation in the plant organs after their extraction from the polluted soils. Some of them namely nickel (Ni), copper (Cu), manganese (Mn) and Zn are considered as essential for the growth of plants and so, they can accumulate them [START_REF] Bhargava | Approaches for enhanced phytoextraction of heavy metals[END_REF]. Some others like Cd, Pb, silver (Ag), arsenic (As), antimony (Sb) or mercury (Hg) are not essential and can cause phytotoxic effects (Finnegan and Weihua, 2012;[START_REF] Sheoran | Factors Affecting Phytoextraction: A Review[END_REF][START_REF] Shtangeeva | Bioavailability and toxicity of antimony[END_REF]. However, metallophytes are able to concentrate some of these metal(loid)s from contaminated soils within their organs without necessarily significantly alter their growth and development [START_REF] Rossini-Oliva | A review of hazardous elements tolerance in a metallophyte model species: Erica andevalensis[END_REF]. Therefore, organ of these plants can be used through many applications to develop the bio economy and so ingrate the circular economy concept [START_REF] Deyris | Woody species: a new bio-based material for dual Ca/Mg catalysis with remarkable Lewis acidity properties[END_REF][START_REF] Hazotte | A novel process to recover cadmium and zinc from the hyperaccumulator plant Noccaea caerulescens[END_REF][START_REF] Gomes | Phytoremediation for bioenergy: challenges and opportunities[END_REF]Guérin et al., 2021;[START_REF] Hechelski | From Conventional Lewis Acids to Heterogeneous Montmorillonite K10: Eco-Friendly Plant-Based Catalysts Used as Green Lewis Acids[END_REF][START_REF] Janus | Elaboration, characteristics and advantages of biochars for the management of contaminated soils with a specific overview on Miscanthus biochars[END_REF]Oustrière et al., 2019;Waterlot et al., 2019).

Cichorium intybus L. (chicory) adapts to wide temperature ranges, different types of soils and is able to grow along the roadside, in industrial sites, urban parks and wasteland, even under high level of contamination (Askoy, 2008). Several studies have shown that chicory is capable, to some extent, of accumulating MTE (Cd, Pb and Zn) when produced on moderately contaminated soil [START_REF] Mandzhieva | Plant contamination by heavy metals in the impact zone of Novocherkassk Power Station in the south of Russia[END_REF]Sekara et al., 2005;[START_REF] Zeng | Cadmium accumulation in winter crops and the assessment of paddy soil phytoremediation in southern China[END_REF]. Moreover, [START_REF] Zeng | Cadmium accumulation in winter crops and the assessment of paddy soil phytoremediation in southern China[END_REF] showed that translocation of metals to shoots depended on the nature of metals and on seedling and mature periods.

The purpose of this work is (i) to assess the dynamic of the MTE accumulation (Pb, Cd and Zn) by the roots and shoots of various cultivars of chicory produced on severely contaminated agricultural soils sampled near the former Metaleurop Nord plant, (ii) to examine the variation of several plant pigments (chlorophyll, anthocyanins, flavonols and nitrogen balance index) and (iii) to evaluate the ability of chicory in the remediation of contaminated soils by Cd, Pb and Zn.

Materials and Methods

Soil sampling, experimental set-up, and plants sampling

Contaminated soil (300 kg) was sampled from the ploughed layer (0-25 cm) of an agricultural plot (latitude: 50° 26' 15.9" N; longitude: 3° 01' 04.9" E) located not far away from the former Metaleurop Nord plant (France) in November 2018. This soil is a clay sandy loam dominated by silt and it is characterized by the following parameters: pH=7.7, Total N: 1200 mg kg -1 ; Total C: 18.2 g kg -1 ; C/N: 15.2; P2O5: 160 mg kg -1 ; Organic matter: 45 g kg -1 ; CEC: 15 cmol + kg -1 [START_REF] Hechelski | Toward a New Way for the Valorization of Miscanthus Biomass Produced on Metal-Contaminated Soils Part 1: Mesocosm and Field Experiments[END_REF][START_REF] Nsanganwimana | Metal accumulation and shoot yield of Miscanthus×giganteus growing in contaminated agricultural soils: Insights into agronomic practices[END_REF]. After homogenization, five containers (outside length: 40 cm; outside width: 30 cm; outside height: 25 cm) were filled with 20 kg of air-dried contaminated soil. The soil moisture experimental device was maintained at 40% field capacity with tap water in greenhouse at 20-25 °C. In each of containers, 7 seeds of chicory were sown in 2 rows. Five cultivars, providing by Florimond Desprez company (https://www.florimond-desprez.com/rep/en/nl/groupe-florimond-desprez-2.html), were considered (C1, C2, C3, C4 and C5) in the current study. Chicories were samples after 74, 105, 156 and 211 days after sowing, taking one plant out of every two times. Shoots and roots of chicory were then separated, crushed, and dried at 105 °C, then stored in acid-washed plastic bottles until digestion.

Digestion of plant and soil

Soil digestion and extractable-MTE in soils

300 mg of soil were digested by a mixture of 37% HCl (4.5 mL) and 70% HNO3 (1.5 mL). The suspension was heated at 120 °C for 90 min in a hot-block digester (HotBlock TM Environmental Express ® SC100, Charleston, USA). Ultra-pure water was then added until reaching 25 mL and the mixture was filtered over an acetate Millipore membrane (0.45 µm porosity). The resulting solutions were stored in acid-washed plastic bottles at 4 °C prior to analysis. Calcium chloride (0.01 M) and a mixture of Low Molecular Weight Organic Acids (LMWOAs) were used to evaluate the labile pool MTE in soils, the bioavailable MTE in short and long run [START_REF] Waterlot | Impact of a phosphate amendment on the environmental availability and phytoavailability of Cd ad Pb in moderetaly and highly carbonated kitchen garden soils[END_REF]Waterlot and Hechelski, 2019).

Plant digestion

300 mg of dry plant sample (roots or leaves) were added to 5 mL of 70% HNO3. The resulting suspension was heated at 95 °C for 90 min using the hot-block digester. After heating, 5 mL of H2O2 (30%) were slowly added in the cooled solution and the mixture was heated at 90±5 °C for 180 min.

Ultra-pure water was then added until reaching a total volume of 25 mL, the mixture was filtered over an acetate Millipore membrane (0.45 µm porosity) and stored at 4 °C prior to analysis.

Chemical and statistical analyses

The concentration of MTE (Pb, Cd and Zn) in soil and plant digests were determined by flame or electrothermal atomic absorption spectrometry (AA-6800, Shimadzu, Tokyo, Japan) following the recommendations described in the literature [START_REF] Waterlot | The problem of arsenic interference in the analysis of Cd to evaluate its extractability in soils contaminated by arsenic[END_REF][START_REF] Savio | Study of matrix effects and spectral interferences in the determination of lead in sediments, sludges and soils by SR-ETAAS using slurry sampling[END_REF]. The limit of detection was 2.1 µg Cd L -1 , 41 µg Pb L -1 , 1.5 µg Zn L -1 using flame as atomizer and 0.06 µg Pb L -1 using graphite furnace as atomizer (Waterlot et al., 2013;Waterlot and Hechelski, 2019). INCTL-OBTL-5 (Virginia tobacco leaves n=3) was used as reference material for the quality control. The digestion and analysis procedures yielded good average recovery values of 90-108 %, depending on MTE (Obtained concentrations: [Cd] = 2.36±0.12 mg kg -1 ; [Pb] = 2.11±0.21 mg kg -1 ; [Zn] = 56.2±2.2 mg kg -1 ; Certified concentrations: [Cd] = 2.64±0.14 mg kg -1 ; [Pb] = 2.01±0.31 mg kg -1 ; [Zn] = 52.4±1.8 mg kg -1 ).

Chlorophyll content (µg cm -2 ), Flavonols Index (relative units) and Anthocyanins Index (relative units) and were measured using a plant pigment content meter (Dualex® Scientific+, Force-A, Orsay, France) on green leaves at the base of the rosette. Moreover, the Dualex device provided a Nitrogen Balance Index (NBI) as reported in [START_REF] Rusinowski | New Miscanthus hybrids cultivated at a polish metal-contaminated site demonstrate high stomatal regulation and reduced shoot Pb and Cd concentrations[END_REF]. Data collected were compared with those obtained from chicories grown in the same conditions on a control unpolluted soil considered as a reference soil (R). From the data set (n1 = 120; n1 = 6n with n=20), analyses were performed using the software program XLSTAT 2021 (Addinsoft, Paris, France). Significant differences between reference and/or cultivars were determined using a one-way ANOVA. Normality and homoscedasticity of residuals were met for all tests. When significant differences occurred between reference and/or cultivars, multiple comparisons of mean values were made using post-hoc Tukey HSD tests.

Differences were considered statistically significant at p<0.05.

Indices calculation

Bioconcentration factor (BCF)

The BCF is the ability of plants to accumulate MTE with respect to their concentration in soils [START_REF] Wang | Bioconcentration and translocation of heavy metals in the soil-plants system in Machangqing copper mine, Yunnan Province, China[END_REF]. In the current study, the BCF is the ratio of MTE concentration in the roots to those in the soil substrate (Eq. 1; [START_REF] Mirzaei | Potential use of grapevine cv Askari for heavy metal phytoremediation purposes at greenhouse scale[END_REF]:

= (1)
where CMTERP is the MTE concentration in the root of plants and CMTES is the MTE concentration of in soils.

Biological absorption coefficient (BAC)

The BAC is related to the bioaccumulation of MTE in the aerial part of plants. This index is defined as the ratio between the MTE concentrations in the shoots and the soil substrate (Eq. 2, [START_REF] Zehra | Identification of high cadmium-accumulating oilseed sunflower (Helianthus annuus) cultivars for phytoremediation of an Oxisol and an Inceptisol[END_REF].

= (2)
where CMTESP is the MTE concentration in the shoot of plants and CMTES is the MTE concentration in soils.

Translocation factor (TF)

The TF indicate the ability of plants to translocate MTE from roots to leaves [START_REF] Zehra | Identification of high cadmium-accumulating oilseed sunflower (Helianthus annuus) cultivars for phytoremediation of an Oxisol and an Inceptisol[END_REF] and is defined as follows (eq 3):

= (eq 3)
where CMTEL is the concentration of MTE in leaves and CMTER is the concentration of MTE in roots.

Results and discussion

Metal concentration in soil-chicory system

The concentrations of Pb, Cd and Zn in the studied agricultural soil were 757.1 ± 35.4 mg kg -1 , 10.7 ± 0.5 mg kg -1 , 857.4 ± 74.0 mg kg -1 (n = 9), respectively. On average, these concentrations are 20-fold, 25-fold and 12-fold higher than those in the loessic reference soils [START_REF] Sterckeman | Assessment of the contamination of cultivated soils by eighteen trace elements around smelters in the north of France[END_REF].

Compared to the ploughed horizons of cultivated plots around Noyelles-Godault plants, Pb and Zn concentrations in the studied agricultural soil were the highest (45% and 33%, respectively) whereas Cd was quite similar (10.7 vs 9.8; [START_REF] Sterckeman | Assessment of the contamination of cultivated soils by eighteen trace elements around smelters in the north of France[END_REF]. The 0.01M CaCl2-extractable concentrations were 0.04 ± 0.01 mg Pb kg -1 , 0.28 ± 0.07 mg Cd kg -1 and 0.56 ± 0.11 mg Zn kg -1 (n = 9). The extractable-MTE concentrations measured in the mixture of LMWOAs were 5.34 ± 1.07 mg Pb kg -1 , 1.83 ± 0.12 mg Cd kg -1 and 172 ± 13 mg Zn kg -1 (n = 9). These results highlight the poor labile pool of these MTE. The bioavailable fraction of MTE in short run was below 0.1%. In long run, it was 0.7% for Pb, 17% for Cd and 20% for Zn. This means that a potential decrease of the total Cd and Zn will be done, but obviously it will take time.

Figure 1 shows the mean of MTE concentrations in the roots and shoot of chicory over time. Pbconcentrations in the roots of chicory at the first sampling (74 days) ranged from 101 to 165 mg.kg -1 , depending on cultivars. These concentrations were 10-fold higher than those measured in the shoots of cultivars C1, C2 and C5 and 6-fold higher for C4. From the second sampling up to the sampling at the end of the experiment, the concentration measured in the roots of chicory drastically decreased (from 60% to 85%) to reach a plateau, ranging from 3.21 to 9.07 mg Pb kg -1 for the two last samplings, which depends on cultivars. In the same time, slight modifications in the Pb concentrations measured in the shoots of chicory may be noted but without any relations with the decrease of Pb concentrations in the roots. These results are not surprising knowing that Pb is a non-phytoavailable MTE and suggests a dilution of Pb in the roots over the growing period. Indeed, the ratio of roots biomass between the second and the first samplings (105 and 74 days, respectively) is approximately 14.

However, these results are somewhat at odds with those described in [START_REF] Zeng | Cadmium accumulation in winter crops and the assessment of paddy soil phytoremediation in southern China[END_REF]. The mean Pb concentrations measured by the authors in the roots and shoots of chicory were around 50 and 25 mg kg -1 , although the Pb concentration in their soil were 10-fold lower at least than in the current study.

This result means that the labile pool of Pb is a crucial parameter to consider. The extractable-MTE concentration using the mixture of LMWOAs and 0.01 M CaCl2 represented 1% and 0.01% of the total Pb concentration, respectively, as reported in previous studies [START_REF] Hechelski | Toward a New Way for the Valorization of Miscanthus Biomass Produced on Metal-Contaminated Soils Part 1: Mesocosm and Field Experiments[END_REF][START_REF] Nsanganwimana | Metal accumulation and shoot yield of Miscanthus×giganteus growing in contaminated agricultural soils: Insights into agronomic practices[END_REF]. These data correlated well with the fact that the Pb concentrations in both studied chicory' organs are low despite the high total Pb concentrations in the soil substrate.

Variations in Cd and Zn concentrations in roots and shoots of chicory over time are presented in Figures 1B and1C. The concentrations of Cd and Zn in the roots of chicory decreased over time (from 50 to 60% and 64 to 78%, respectively) and slightly increased at the fourth sampling (211 days). In contrast, concentration of the two metals increased over the growth period, from 25 to 147% for Cd and from 5.5 to 43% for Zn. As shown on Figures 1B and1C, the Cd and Zn accumulations in plant organs at the end of the experiments were higher in shoots that in roots whatever the cultivars are. 211 days after sowing, the Cd and Zn concentrations in the shoots of chicory were up to 50 mg kg -1 DW and 172 mg kg -1 DW, respectively. These concentrations were, respectively, 7-fold and 3.4-fold higher than those in chicory' roots. These results correlated well with those obtained for Zn by Zeng et al.

(2019) but are quite different for Cd. No significant difference was highlighted by the authors between the Cd concentrations measured in shoots and in roots in seedling and mature periods. It has been reported in previous studies that the MTE accumulation capacities are affected by interspecific differences, environment conditions [START_REF] Grant | Selection and breeding of plant cultivars to minimize cadmium accumulation[END_REF]Shi and Cai 2009) and the available Cd and the total Cd concentration, which was 8-fold higher in the current study than in [START_REF] Zeng | Cadmium accumulation in winter crops and the assessment of paddy soil phytoremediation in southern China[END_REF].

Bioaccumulation factor, biological absorption coefficient and translocation factor

MTE-accumulation, MTE-translocation, and MTE-distribution in the upper and underground parts of plants were studied by means of three indices named bioconcentration factor (BCF: root-tosoil metal concentration ratio), biological adsorption coefficient (BAC: plant leaves-to-soil metal concentration ratio) and translocation factor (TF: leaves-to-root metal concentration ratio). The utilization of these indexes was recommended to define the ability of plant models in the phytoextraction and phytostabilization of MTE [START_REF] Zhou | Potential use of king grass (Pennisetum purpureum Schumach. × Pennisetum glaucum (L.) R.Br.) for phytoextraction of cadmium from fields[END_REF]. According to [START_REF] Alagić | Assessment of the quality of polluted areas based on the content of heavy metals in different organs of the grapevine (Vitis vinifera) cv Tamjanika[END_REF], a value of BCF greater than 1 (BCF > 1) indicates a good bioaccumulation of MTE in the roots of plants and if these plants have BAC values greater than 1 (BAC > 1), they are a suitable option for bioaccumulation of MTE in their shoots. Moreover, if TF > 1, the good ability of plants to translocate MTE from their roots to their aboveground parts make them as suitable for phytoextraction purpose (Vamelari et al., 2010). In contrast, for BCF > 1 and TF < 1, the potential of plants to be MTE-tolerant is great and so, they have potential for introducing as phytostabilization purpose (Vamelari et al., 2010). The three parameters were reported in Table 1 for each sample. The Pb-BCF values were low, ranging from 0.13 to 0.22 at the first sampling, and then, decreased sharply to reach 0.01 from the third sampling up to the last one. This trend was not cultivar-dependent. As shown in Table 1, the Pb-BAC values are very low over time (ranging from 0.01 to 0.03) whatever the cultivar meaning that chicories should not be suitable for Pb-phytoaccumulation. However, depending on time, the Pb-TF values were higher than 1 and were cultivar-dependent (from 1.44 to 3.53 at 156 days and from 0.86 to 1.34 at 211 days). These trends correlated well with the fact that the Pb-concentration remained quite constant from the second harvest to the end of the experiment. Caution should be taken when interpreting the results related to Pb-BAC and Pb-TF. Thus, we do not want to conclude on the capacity of chicory to phytostabilize and phytoextract Pb. However, it is worth mentioning that chicories were described in other studies as Pb-phytoextractor (Vamelari et al., 2010) and Pbaccumulator [START_REF] Baker | Accumulators and excluders -strategies in the response of plants to heavy metals[END_REF].

As reported in [START_REF] Sêkara | Cadmium and Lead Accumulation and Distribution in the Organs of Nine Crops: Implications for Phytoremediation[END_REF], [START_REF] Mandzhieva | Plant contamination by heavy metals in the impact zone of Novocherkassk Power Station in the south of Russia[END_REF] and [START_REF] Zeng | Cadmium accumulation in winter crops and the assessment of paddy soil phytoremediation in southern China[END_REF], the data for Cd distribution suggest that chicories are characteristic of good accumulators since BAC and TF were higher than 1. These two indicators increased over the growth period to reach values higher than 4 and 8, respectively corroborating the decrease of the BCF values over the same period. Unfortunately, due to the size of containers, the growth of plants was limited between 156 days and 211 days, potentially leading to a bias in the data collected. This result is even more pertinent as 60% of the total Cdconcentration was found in the acido-soluble fraction of the fourth-step sequential extraction procedure [START_REF] Pelfrêne | Effect of miscanthus cultivation bioaccessibility in metal-contaminated soils: comparison between greenhouse and field experiments[END_REF]. Thus, by removing the shoots of chicory from the field, this plant should be able to be used for the removal of the bioavailable pool of Cd from the soil. Although Cd and Zn have similar chemical properties, the Zn-BAC and Zn-BCF were lower than 1 from the first harvest to the last one. However, Zn-BAC decreases whereas Zn-BCF increases over the growth period meaning that Zn-TF increases. As shown in Table 1, this indicator increases in time up to 156 days and was higher than 3 at 211 days meaning that chicories are able to extract Zn from soil.

Recently, Wolf and Paulino (2020) pointed out the potential toxicity effects of Zn when chicories were produced on Zn-contaminated soils. In fact, the authors spiked their soils with zinc sulfate to obtain concentrations of 1200 mg Zn kg -1 and 2400 mg Zn kg -1 . They highlighted a decrease of leaf biomasses 45 days after seedling and chlorosis effects. In the current study, no visual symptoms were noticed.

Plant uptake and foresight

The total harvested-plant biomass was expressed as leaf and root fresh-biomass distribution (Figure 2). As reported on it, the absolute mass distribution of these two plant-parts depends on cultivars. Leaf and root biomasses were in the same order of magnitude for the first two cultivars whereas they were different for the last three cultivars. For those, biomasses of roots ranged from 58.6% to 59.5% and so, were higher than in leaves. Based on the total mass of sampled roots and leaves, exported metals from the soil to the chicories were calculated and presented in Table 2. The total metal uptake in leaves and roots were slightly dependent on cultivars and were highly metaldependent. The ascending order of metals mass in the leaves of chicories was Zn>Cd>Pb whereas it was Zn>Pb>Cd in roots. Whatever the results, the data highlighted that if chicories are produced on contaminated soils, collections of leaf are required to avoid the recycling of leaf-bound MTE before the root harvest. As shown in Figure 3, chlorophyll contents in the reference (R), and the cultivars C3 and C5 were statistically the same. This result corroborates with the fact that the exported MTE by these two cultivars were the lowest. On the contrary, statistical differences were highlighted between the chlorophyll contents in R and the cultivars C1, C2 and C4, for which the exported MTE were the greatest. This means that higher the exported MTE is, lower the concentration of chlorophyll in the leaves of chicory is. Indeed, among the survival mechanisms of plants are MTE-translocation to leaves and decreasing chlorophyll [START_REF] Uzu | Foliar Lead uptake by lettuce exposed to atmospheric fallouts[END_REF]. Moreover, the decrease of chlorophyll may be linked to the senescence [START_REF] Wojciechowska | Plant Organ senescence -regulation by manifold pathways[END_REF].

Although there is no statistical difference in the concentration of flavonols between the reference and the five cultivars studied, it is worth mentioning the relationship between the results obtained with chlorophyll and antocyanins. No statistical difference was highlighted between antocyanins in R and C3 and C5, whereas anthocyanin concentrations in R were statistically different from those in C1, C2 and C4. As shown in Figure 3, concentrations of anthocyanin were the highest in these three cultivars.

This corroborates with previous studies in which it was well established that one of the role of anthocyanin is the protection of plants from several abiotic and biotic stress [START_REF] Chalker-Scott | Environmental significance of anthocyanins in plant stress responses[END_REF][START_REF] Ahmed | Characterization of dihydroflavonol 4-reductase (DFR) genes and their association with cold and freezing stress in Brassica rapa[END_REF], which may provide them a better adaptation to contamination level. Acting as antioxidant, anthocyanins are able to bind with MTE reducing consequently their toxicity [START_REF] Dai | Antioxidative and chelating properties of anthocyanins in Azolla imbricata induced by cadmium[END_REF][START_REF] Akbari | Relationship of flower color parameters and metal ions of petal tissue in fully opened flowers of Gerbera[END_REF]. It is worth mentioning that the formation of anthocyanin-MTE complexes is pH-dependent since it affects the protonation/deprotonation of anthocyanins and consequently their reactivity towards heavy metals.

As for chlorophyll concentrations, the NBI indices (Figure 3) were the lowest for C1, C2 and C4

and statistically different from that of R. In addition, no statistical difference was highlighted between the reference and the cultivars C3 and C5. Defined as an indicator of C:N allocation changes [START_REF] Cerovic | A new optical leaf-clip meter for simultaneous non-destructive assessment of leaf chlorophyll andepidermalflavonoids[END_REF], the NBI could be a simple valuable tool for preventing the rate of senescence of chicory leaves in addition to the chlorophyll measure, and so the premature leaf fall of chicories produced in contaminated soils. This highlight has been suggested in several studies about miscanthus [START_REF] Mos | Impact of Miscanthus x giganteus senescence times on fast pyrolysis bio-oil quality[END_REF][START_REF] Rusinowski | New Miscanthus hybrids cultivated at a polish metal-contaminated site demonstrate high stomatal regulation and reduced shoot Pb and Cd concentrations[END_REF].

Despite the short time-experimentation (211 days) and the limited-size of the containers, the exported metal from soil ranged from 0.83 mg to 1.59 mg Pb, 1.44 to 2.03 mg Cd and 6.94 to 10.87 mg Zn. Based on the dataset, a remediation factor (RF) was calculated according to the definition described in [START_REF] Sabir | Contrasting Effects of Farmyard Manure (FYM) and Compost for Remediation of Metal Contaminated Soil[END_REF]. Briefly, this factor was defined as the ratio of the mass of metal in the set of plants cultivated in each container to soil as follows (Eq. 4):

= × ! " ## # ×" ## $ % " #
× 100 (eq 4)

It was clearly demonstrated that the mechanism uptake of MTE depended on several parameters like the root zone, the environmental conditions, the soil parameters, the plant species, the speciation of MTE [START_REF] Tangahu | A review on heavy metals (As, Pb, and Hg) uptake by plants through phytoremediation[END_REF] and so, the bioavailability of MTE. Thus, two remediation factor indexes, named RFLMWOAs and RFCaCl2 were calculated considering the extractable MTE concentrations measured in the mixture of organic acids (LMWOAs) and in the 0.01 M calcium chloride solution.

The results are summarized in Table 3. Although it was demonstrated that Pb, Cd and Zn were poorly phytoavailable using miscanthus as plant model [START_REF] Nsanganwimana | Metal accumulation and shoot yield of Miscanthus×giganteus growing in contaminated agricultural soils: Insights into agronomic practices[END_REF]2021), the RF-Cd values were in the range 0.67-0.95 %, depending on the cultivars. Even if the maturity of chicories has not been reached, this result is interesting since the RF values tend to increase with time [START_REF] Saraswat | Prospective Application of Leucaena Leucocephala for Phytoextraction of Cd and Zn and Nitrogen Fixation in Metal Polluted Soils[END_REF]. According to our knowledge, no study reported similar data using chicory cultivars. In contrast, some RF-Cd values were reported in literature using various plant models in anthropogenically or artificially contaminated soils [START_REF] Hu | Chelant-Assisted Uptake and Accumulation of Cd by Poplar from Calcareous Arable Soils Around Baiyin Nonferrous Metal Smelters, Northern China[END_REF]Chhajro et al., 2018;Moemeri and Khalaki, 2019). Chhairo et al. (2018) revealed RF-Cd values of 0.39 without any amendment and up to 1 using chelators (EDTA, NTA, citrate) and castor bean (Ricinus communis) whereas Rf-Cd values below 0.7 was highlighted whatever the treatments (EDTA, EGTA, citrate; [START_REF] Sun | Induced-phytoextraction of heavy metals from contaminated soil irrigated by industrial wastewater with Marvel of Peru (Mirabilis jalapa L.)[END_REF][START_REF] Hu | Chelant-Assisted Uptake and Accumulation of Cd by Poplar from Calcareous Arable Soils Around Baiyin Nonferrous Metal Smelters, Northern China[END_REF]. However, RF-Cd values between 1.0 and 4.0 using Secale montanum and Leucaena leucocephala were reported under a phytoextraction context (Saraswast andRai, 2009, 2011;[START_REF] Moameri | Capability of Secale montanum trusted for phytoremediation of lead and cadmium in soils amended with nano-silica and municipal solid waste compost[END_REF] As shown in Table 3, the RF-Pb values are very low, from 0.006 to 0.01. They are in the same order of magnitude as those reported in [START_REF] Moameri | Capability of Secale montanum trusted for phytoremediation of lead and cadmium in soils amended with nano-silica and municipal solid waste compost[END_REF] and [START_REF] Sun | Induced-phytoextraction of heavy metals from contaminated soil irrigated by industrial wastewater with Marvel of Peru (Mirabilis jalapa L.)[END_REF]. Nevertheless, depending on the ability of shrub and tree species to phytoextract Pb, some authors highlighted medium and high RF-Pb values (from 0.5 to 13.5; [START_REF] Aman | Assessing some shrub species for phytoremediation of soils contaminated with lead and zinc[END_REF][START_REF] Singh | Assessment of different multipurpose tree species for phytoextraction of lead from lead-contaminated soils[END_REF]. The RF-Zn values were ten-fold higher than those of RF-Pb although the total concentrations of these MTE in the soil studied were quite similar. Even if similar RF-Zn values were reported in Sun et al. (2001) as those obtained in the current study, it is worth noting that RF-Zn values are between 0.3 to 2.1, depending on plant species and soil characteristics [START_REF] Aman | Assessing some shrub species for phytoremediation of soils contaminated with lead and zinc[END_REF]Sarawat and Rai, 2011;[START_REF] Sabir | Contrasting Effects of Farmyard Manure (FYM) and Compost for Remediation of Metal Contaminated Soil[END_REF].

In the Gentle soil Remediation Options (GRO) the common goal is to decrease the labile pool of MTE because it represents the most available pool of MTE. This pool is a part of the total metal concentration and is commonly named bioavailable. In this concept, phytoextraction and phytoremediation are both strategies that can be used to achieve this objective if this plant-based soilmanagement is integrated in the bioeconomy concept. From these global considerations, we defined the RFLMWOAs and RFCaCl2 described above. Values of these two remediation factors were higher than those of RF since there were defined from the extractable-MTE concentrations. As indicated in Table 3, the RFLMWOAs-Cd is higher than 4 meaning that chicories can be used as phytoextractor for bioavailable-Cd in long run. This result correlated with the conclusion described previously from the BAC, BCF and TF values. In contrast, the results remain less evident for Pb and Zn since their respective RFLMWOAs values were generally below 1. Anyway, chicories are able to decrease the pool of the bioavailable-MTE in short time as shown by the RFCaCl2 values for the three MTE studied.

Although the growth period was limited in time due to the size of containers, this new approach and the whole of results obtained from container-experiments should provide new insights into the potential of chicories in lowering the environmental risks after a large-scale chicory production on contaminated agricultural soils. However, it is worth mentioning that further studies need to be conduct due to (i) the restriction of the root size in the current study and not for field studies and (ii) a potential lateral and vertical gradient of metal(loid)s contamination levels in real contaminated sites.

Conclusion

This study, carried out over a period of 211 days, was conducted to determine the concentration and time distribution of Cd, Pb and Zn in five cultivars of chicory which have been produced on an agricultural contaminated soil. Leaves and roots analysis at 4 time points during experimentation (at 74, 105, 156 and 211 days, respectively) revealed that the Cd and Zn concentration in plants organs at the end of the experiments were higher in shoots that in roots (up to 7-fold and 3.4-fold higher, respectively) whatever the cultivars were, while no significant differences were observed for Pb.

Indeed, Cd-BAC and Cd-TF increase during the growing period to reach values above 4 and 7, highlighting the ability of chicory to reduce the Cd content of the soil. This reasoning is also valid for

Zn, but to a lesser extent since the Zn-TF reaches values higher than 3 at 211 days. The Zn-BAC values are lower than 1 from the first harvest to the last one. In addition, MTE concentrations in shoots have been related to the content of chlorophyll, anthocyanins and nitrogen balance index. It appears that an increase of the MTE concentration in the leave induces i) an increase of the anthocyanin content, which may provide to the chicory a better adaptation to contamination level ii) a decrease of the content of chlorophyll and nitrogen balance index, which could be related to the senescence phenomenon. Finally, the phytoextracting power of chicory has been discussed regarding the bioavailable part of the total MTE concentration and has been identified as a good phytoextractor, particularly for Cd. These news results thus highlight the interest of harvesting the aerial parts of chicory thus limiting the deposition of MTE after large-scale chicory production on contaminated agricultural soils.

Fig. 3 .

 3 Fig. 3. Chlorophyll content, Flavonols index, Anthocyanins index and Nitrogen Balance Index measured in leaves of chicories (reference and

Table 1

 1 Mean values of bioaccumulation factor (BAC), bioconcentration factor (BCF) and translocation factor (TF)

	Cultivar	Time (days)	Pb	BAC Cd	Zn	Pb	BCF Cd	Zn	Pb	TF Cd	Zn
		74	0.01	2.06	0.15	0.13	1.05	0.16	0.11	1.97	0.95
	C1	105 156	0.02 0.01	2.14 2.45	0.13 0.17	0.02 0.01	0.44 0.39	0.05 0.03	0.81 1.44	4.91 6.31	2.54 5.37
		211	0.01	3.29	0.21	0.01	0.57	0.05	0.89	5.80	4.47
		74	0.02	1.93	0.15	0.17	1.09	0.20	0.10	1.77	0.75
	C2	105 156	0.03 0.02	1.77 2.89	0.11 0.15	0.07 0.01	0.44 0.33	0.04 0.04	0.42 3.36	4.06 8.70	2.62 4.22
		211	0.01	3.22	0.20	0.01	0.62	0.05	1.04	5.17	3.63
		74	-	-	-	-	-	-	-	-	-
	C3	105 156	0.02 0.02	2.03 2.28	0.12 0.14	0.05 0.01	0.60 0.31	0.09 0.03	0.45 2.48	3.38 7.45	1.40 4.10
		211	0.02	2.55	0.16	0.01	0.56	0.05	1.34	4.57	3.33
		74	0.04	1.90	0.14	0.22	1.15	0.18	0.16	1.65	0.77
	C4	105 156	0.02 0.01	2.23 2.73	0.11 0.15	0.03 0.00	0.57 0.09	0.07 0.05	0.66 3.53 28.82 3.90	1.73 2.90
		211	0.01	4.69	0.20	0.01	0.70	0.06	0.89	6.65	3.38
		74	0.01	2.06	0.14	0.13	1.14	0.18	0.10	1.81	0.78
	C5	105 156	0.01 0.02	1.57 2.26	0.11 0.13	0.03 0.01	0.50 0.30	0.06 0.03	0.44 2.02	3.12 7.49	1.90 4.05
		211	0.01	3.58	0.15	0.01	0.49	0.05	0.86	7.38	2.97

Table 2

 2 Leaf accumulation and root uptake of MTE by chicories

	cultivar	MTE accumulation in leaves	MTE uptake by roots	MTE uptake by chicories
		Cd (mg) Pb (mg) Zn (mg) Cd (mg) Pb (mg) Zn (mg) Cd (mg) Pb (mg) Zn (mg)
	C1	1.18	0.28	6.23	0.41	0.56	2.72	1.59	0.83	8.96
	C2	1.42	0.33	6.65	0.51	0.51	4.21	1.93	0.84	10.87
	C3	1.02	0.54	4.94	0.57	1.05	3.91	1.58	1.59	8.86
	C4	1.44	0.39	5.04	0.59	0.67	4.51	2.03	1.06	9.56
	C5	1.01	0.30	3.40	0.43	0.93	3.54	1.44	1.23	6.94

Table 3

 3 Remediation factors (RF) Fig. 1. Pb, Cd and Zn accumulation (A,B,C) in the roots and shoots depending on the growing time Distribution (%) of leaf and root biomass for the five cultivars studied

	cultivar	Cd	RF Pb	Zn	Cd	RFLMWOAs Pb	Zn	Cd	RFCaCl2 Pb	Zn
	C1	0.74	0.006	0.05	4.42	0.79	0.26	28.4	119.2	80.5
	C2	0.90	0.006	0.06	5.37	0.79	0.32	34.5	119.6	97.7
	C3	0.74	0.010	0.05	4.40	1.50	0.26	28.3	227.0	79.6
	C4	0.95	0.007	0.06	5.65	1.00	0.28	36.3	152.0	85.9
	C5	0.67	0.008	0.04	4.00	1.16	0.20	25.7	175.8	62.4

Acknowledgments

The authors thank the Region Hauts-de-France (FEDER funding: Fonds européen de développement regional), JUNIA and the Florimond Desprez company for financial support of this work and technical help and facilities. The authors also acknowledge B. Desprez, N. Henry and Y. Delourme for their technical advice.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Eric Pollet
email: eric.pollet@unistra.fr

Keywords: Enzymatic polymerization, Candida antarctica lipase B, renewable resources, bio-based polymers, semi-aromatic polyesters, furan-based polyesters

Enzymatic polymerization is a promising route for a greener synthesis of biobased polyesters.

However, this approach is still often limited to aliphatic polyesters, which present limited properties such as low thermal resistance. In this context and till now, introduction of aromatic monomers into polyesters very often resulted in low molar mass chains. Herein, aliphaticaromatic copolyesters based on dimethyl-2,5-furandicarboxylate were enzymatically synthesized using immobilized Candida antarctica lipase B with a particular focus on the influence of the solvent used. Two series of poly(hexylene adipate)-co-(hexylene furanoate) and poly(butylene adipate)-co-(butylene furanoate) copolyesters were successfully synthesized in diphenyl ether, displaying high molar masses (Mn up to 19 000 g.mol -1 ) for aromatic monomer contents up to 70 and 50%, respectively. High aromatic content resulted in reduced molar masses due to a loss of solubility of the growing aromatic chains. Replacing diphenyl ether by acetophenone, which had never been used before as solvent for enzymatic synthesis of polyesters, led to significantly enhanced solubility and thus an increase in the average molar masses of poly(hexylene furanoate) and poly(butylene furanoate). The resulting polyesters showed greater thermal stability and higher Tg, offering new perspectives for expanding the potential applications of such enzymatically-produced biobased aromatic copolyesters.

Lipase-catalyzed synthesis of furan-based aliphaticaromatic biobased copolyesters: impact of the solvent.

Alfred Bazin, Luc Avérous, Eric Pollet* BioTeam/ICPEES-ECPM, UMR CNRS 7515, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg Cedex 2, France

Introduction

Due to the current environmental context and the global perspectives, polymer science research is now increasingly oriented towards higher sustainability, notably by following the principles of green chemistry. Thus, the synthesis of bio-based polyesters has aroused strong interest in recent decades [START_REF] Vilela | The quest for sustainable polyesters -insights into the future[END_REF][START_REF] Zia | Recent developments and future prospects on bio-based polyesters derived from renewable resources: A review[END_REF][START_REF] Rebouillat | Recent Strategies for the Development of Biosourced-Monomers, Oligomers and Polymers-Based Materials: A Review with an Innovation and a Bigger Data Focus[END_REF], to replace fossil-based polyesters and to develop new macromolecular architectures. Numerous compounds produced from biomass, such as building blocks or monomers, have been used for the elaboration of these polyesters [START_REF] Becker | Top value platform chemicals: bio-based production of organic acids[END_REF][START_REF] Kristufek | Monomer design strategies to create natural product-based polymer materials[END_REF]. For example, the conversion of biobased succinic and adipic acids with butane-and propanediol into polymeric material has been studied [START_REF] Debuissy | Synthesis and characterization of biobased poly(butylene succinate-ran -butylene adipate). Analysis of the composition-dependent physicochemical properties[END_REF][START_REF] Debuissy | Study on the structure-properties relationship of biodegradable and biobased aliphatic copolyesters based on 1,3-propanediol, 1,4-butanediol, succinic and adipic acids[END_REF]. It is thus possible to obtain a wide range of aliphatic copolyesters but their behaviors, such as thermal and mechanical properties, are often limited. As an example, biobased poly(butylene succinate) presents a glass transition temperature (Tg) of -37°C and a Young's modulus of approximately 368 MPa [START_REF] Jin | Thermal and mechanical properties of mandelic acid-copolymerized poly(butylene succinate) and poly(ethylene adipate)[END_REF]. These values are far below the most common fossil-based polyester, namely polyethylene terephthalate (PET) with a Tg of 76°C [START_REF] Nguyen | The quest for high glass transition temperature bioplastics[END_REF] and Young's modulus above 2 600 MPa [START_REF] Bedoui | Micromechanical modeling of isotropic elastic behavior of semicrystalline polymers[END_REF]. In order to improve these properties, aromatic and semi-aromatic bio-based polyesters are now gaining a lot of attention since the aromaticity brings higher thermal resistance and rigidity linked to higher transition temperatures. Several sources of aromatic building blocks have been considered. As an example, lignocellulosic biomass is an important source of aromatic building blocks such as vanillic and ferulic acids [START_REF] Nguyen | The quest for high glass transition temperature bioplastics[END_REF][START_REF] Llevot | From Lignin-derived Aromatic Compounds to Novel Biobased Polymers[END_REF]. For instance, derivatives of ferulic acid have been copolymerized to produce high thermal transition polyesters with Tg ranging from 32 to 113 °C (Fig. 1,a) [START_REF] Nguyen | Polyethylene ferulate (PEF) and congeners: polystyrene mimics derived from biorenewable aromatics[END_REF]. Recently, vanillic-based polyester have been studied and showed thermal transitions comparable to PET with a Tg up to 77°C (Fig. 1,b) [START_REF] Xanthopoulou | Effectiveness of Esterification Catalysts in the Synthesis of Poly(Ethylene Vanillate)[END_REF].

a) [START_REF] Nguyen | Polyethylene ferulate (PEF) and congeners: polystyrene mimics derived from biorenewable aromatics[END_REF] b) [START_REF] Xanthopoulou | Effectiveness of Esterification Catalysts in the Synthesis of Poly(Ethylene Vanillate)[END_REF] Fig. 1: Examples of polyesters synthesis from biobased aromatic building blocks.

Among the aromatic monomers or building blocks that are attracting a lot of interest today, furan derivatives occupy an important place [START_REF] Poulopoulou | Biobased Engineering Thermoplastics: Poly(butylene 2,5furandicarboxylate) Blends[END_REF]. These compounds, often synthesized from fructose, can be used in various polymeric materials such as polyamides, polyethers or polyesters [START_REF] Sousa | Biobased polyesters and other polymers from 2,5-furandicarboxylic acid: a tribute to furan excellency[END_REF]. For instance, polyesters based on furan derivatives such as dimethyl 2,5-furandicarboxylate (DMFDC) have been extensively studied [START_REF] Papageorgiou | Production of biobased 2,5-furan dicarboxylate polyesters: Recent progress and critical aspects in their synthesis and thermal properties[END_REF][START_REF] Terzopoulou | Tuning the Properties of Furandicarboxylic Acid-Based Polyesters with Copolymerization: A Review[END_REF]. The aim of these studies is often to develop biobased polymers that mimic the structures and the properties of PET. Recently, it has been shown that furan-based polyesters, such as polyethylene furanoate (PEF), have properties comparable or even higher than those of PET and therefore represent a very promising alternative [START_REF] Gandini | The furan counterpart of poly(ethylene terephthalate): An alternative material based on renewable resources[END_REF][START_REF] Loos | A Perspective on PEF Synthesis, Properties, and End-Life[END_REF]. It was also claimed that PEF presented good enzymatic degradability [START_REF] Weinberger | Enzymatic surface hydrolysis of poly(ethylene furanoate) thin films of various crystallinities[END_REF]. However, the synthesis of such polyesters requires high temperatures as well as potentially toxic metal-based catalysts [START_REF] Nguyen | Polyethylene ferulate (PEF) and congeners: polystyrene mimics derived from biorenewable aromatics[END_REF][START_REF] Xanthopoulou | Effectiveness of Esterification Catalysts in the Synthesis of Poly(Ethylene Vanillate)[END_REF][START_REF] Terzopoulou | Effect of catalyst type on molecular weight increase and coloration of poly(ethylene furanoate) biobased polyester during melt polycondensation[END_REF] (such as antimony [22] and tin oxides [23]), which are not in line with a green chemistry approach.

In order to address these problems, enzyme-catalyzed synthesis of polyesters represents a very promising alternative and has been largely investigated [START_REF] Douka | A review on enzymatic polymerization to produce polycondensation polymers: The case of aliphatic polyesters, polyamides and polyesteramides[END_REF][START_REF] Kobayashi | Synthesis of Polyesters I: Hydrolase as Catalyst for Polycondensation (Condensation Polymerization)[END_REF][START_REF] Pellis | The Closure of the Cycle: Enzymatic Synthesis and Functionalization of Bio-Based Polyesters[END_REF][START_REF] Jiang | Enzymatic Synthesis of Biobased Polyesters and Polyamides[END_REF]. Enzymatic polymerization was also studied by our group to develop biobased aliphatic polyesters [START_REF] Debuissy | Lipase-catalyzed synthesis of biobased and biodegradable aliphatic copolyesters from short building blocks. Effect of the monomer length[END_REF][START_REF] Debuissy | Enzymatic synthesis of biobased poly(1,4-butylene succinate-ran -2,3-butylene succinate) copolyesters and characterization. Influence of 1,4and 2,3-butanediol contents[END_REF]. Enzymes are environmentally friendly and non-toxic catalysts that have demonstrated their efficiency in a large number of reactions and that can be immobilized on solid supports to improve their stability and to be reused [START_REF] Öztürk | Nanoclays for Lipase Immobilization: Biocatalyst Characterization and Activity in Polyester Synthesis[END_REF][START_REF] Poojari | Thermal stability of Candida antarctica lipase B immobilized on macroporous acrylic resin particles in organic media[END_REF]. The most commonly used enzymes for polyester synthesis are the esterases, such as the immobilized form of Candida antarctica Lipase B (iCALB) [START_REF] Ortiz | Novozym 435: the "perfect" lipase immobilized biocatalyst?[END_REF].

Immobilization of the enzyme allows greater stability and reusability [START_REF] Mateo | Improvement of enzyme activity, stability and selectivity via immobilization techniques[END_REF]. iCALB-catalyzed polymerization can be performed in solution or in bulk [START_REF] Douka | A review on enzymatic polymerization to produce polycondensation polymers: The case of aliphatic polyesters, polyamides and polyesteramides[END_REF]. Bulk polymerization is more relevant for industrial large scale processes but high viscosity and infusibility issues linked to the mild temperatures imposed by enzymatic catalysis severely limit this route.

Enzymatic syntheses of furan-based oligomers or polyesters catalyzed by iCALB have already been performed but always resulted in polymers with lower molar masses than their aliphatic counterparts [START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF][START_REF] Morales-Huerta | Blocky poly(ɛcaprolactoneco -butylene 2,5-furandicarboxylate) copolyesters via enzymatic ring opening polymerization[END_REF][START_REF] Flores | Synthesis of Aromatic-Aliphatic Polyesters by Enzymatic Ring Opening Polymerization of Cyclic Oligoesters and their Cyclodepolymerization for a Circular Economy[END_REF][START_REF] Comerford | Thermal Upgrade of Enzymatically Synthesized Aliphatic and Aromatic Oligoesters[END_REF]. In this case, the impact of the structure and aromaticity of the monomers on the activity of iCALB is not well understood, till now.

Thus, this study aimed at investigating the (bio)synthesis of biobased aromatic copolyesters by enzymatic catalysis. The influence of furan-based aromatic monomers on the activity of iCALB in the synthesis of several semi-aromatic copolyesters has been particularly examined. The reactivity of DMFDC combined with diethyl adipate (DEA), 1,6-hexanediol (1,6-HDO) and 1,4butanediol (1,4-BDO) in different ratio was assessed (Fig. 2). These esters were chosen to allow easier comparison with results from the literature [START_REF] Debuissy | Lipase-catalyzed synthesis of biobased and biodegradable aliphatic copolyesters from short building blocks. Effect of the monomer length[END_REF][START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF][START_REF] Flores | Synthesis of Aromatic-Aliphatic Polyesters by Enzymatic Ring Opening Polymerization of Cyclic Oligoesters and their Cyclodepolymerization for a Circular Economy[END_REF]. For that, the polymerization kinetics as well as the molar masses, compositions and chemical architectures of the synthesized copolyesters were determined by SEC and NMR analyses. Different solvents were screened in order to enhance the enzymatic synthesis of poly(hexylene furanoate) (PHF) and poly(butylene furanoate) (PBF). Finally, the influence of the aromatic monomer content on the thermal properties of the synthesized copolyesters was particularly studied and discussed.

Synthesis of the aliphatic-aromatic copolyesters

Two series of copolyesters were synthesized, one for each diol, with different ratios in the furan-based aromatic monomer. Both the Poly(hexylene adipate-co-furanoate) (PHAd-co-PHF)

and Poly(butylene adipate-co-furanoate) (PBAd-co-PBF) series were synthesized using an equimolar proportion of ester and alcohol groups.

For that, the diol (1,6-HDO or 1,4-BDO) was added to a mixture of DMFDC and DEA in various ratio (given in Table 1 and Table 2, respectively) in a 10 mL Schlenk reactor. Then, 2 ml of dry diphenyl ether (150 wt.% in comparison to the total mass of monomers) and 144 mg of pre-dried iCALB (10 wt.% in comparison to the total mass of monomers) were added.

The solution was magnetically stirred and heated at 90° C under partial vacuum. The pressure was set to 350 mbar and was then decreased to 100, 50 and 20 mbar after 4, 8 and 24 hours of reaction, respectively.

After 72h, or at precipitation of the sample, the reaction was quenched by adding 5 ml of chloroform. The mixture was filtered to remove the iCALB and the excess of chloroform in the filtrate was eliminated by rotary evaporator. The obtained polyester was precipitated in a large volume of ice-cold methanol and centrifuged at 8 000 g for 1 minute. The supernatant was eliminated. Finally, the polymer was dried in a vacuum oven at 40°C for 24h before analysis.

Poly(hexylene adipate-co-furanoate) (PHAd-co-PHF):

Influence of the solvent on the synthesis of PHF and PBF

For the synthesis of PHF and PBF in different solvents, the previous protocol was slightly modified. The proportions of diol and DMFDC were kept constant while the solvent and iCALB were doubled in order to avoid early polymer precipitation. The solution was magnetically stirred and heated at 90° C. The pressure was reduced, when possible, depending on the solvent, to values indicated in Table 3. After 72h, or at precipitation of the sample, the reaction was quenched by adding 5 ml of chloroform. The polymer was then precipitated and recovered with the same method afore described.

Kinetics monitoring

The enzymatic polymerizations were monitored by NMR spectroscopy. Aliquots of solution were withdrawn at 24, 48 and 72h from the medium. These samples were dissolved in chloroform and filtered. They were then precipitated in a large volume of ice-cold methanol. The solution was centrifuged at 8 000 g for 1 minute, the supernatant was discarded, and the polyester was dried in a vacuum oven at 40°C for 24h before analysis.

Enzymatic activity assay

Samples of 14 mg of iCALB beads were added into 5 mL vials. Before the vial sealing, 200 µl of 1-butanol, 69 µl of diethyl adipate and 380 µl of the desired solvent were added. The solution was placed at 50°C under mild magnetic agitation (100 rpm) to avoid deterioration of the fragile catalyst support. Aliquots of 10 µl were withdrawn at different times and rapidly analyzed by NMR spectroscopy to quantify the transesterification activity. In order to assess the leaching of the enzyme, samples of iCALB were incubated at 90°C for 24h in 380 µl of a given solvent. At the end of the incubation, the solvent was carefully pipetted out of the vial. 5 mL vials were filled with the solvent of incubation, 200 µl of 1-butanol and 69 µl of diethyl adipate. The vials were placed at 50°C and aliquots were withdrawn at given times and analyzed by NMR spectroscopy.

Characterizations

1 H NMR spectra were obtained on a Bruker 400 MHz spectrometer. 2D and 13 C NMR spectra were obtained on a Bruker 500 MHz spectrometer. CDCl3 was used as a solvent. For the polymers hardly soluble in CDCl3, few microliters of trifluoroacetic acid (TFA) were added to achieve proper dissolution. Calibration of the spectra was performed using the CDCl3 peak (δH = 7.26 ppm, δC = 77.16 ppm).

Size Exclusion Chromatography (SEC) was used to estimate the number average molar mass (Mn), weight average molar mass (Mw) and dispersity (Đ). The analyses were performed on a Shimadzu liquid chromatograph in chloroform at a flow rate of 0.8 mL/min. The apparatus was equipped with a PLGel Mixed-C and PLGeL 100 Å columns. A calibration curve using polystyrene (PS) standards was built for molar mass determination.

Differential scanning calorimetry (DSC) analyses have been performed on the samples to estimate their glass transition temperature (Tg), melting temperature (Tm), and crystallization temperature (Tc), if applicable. The analyses were performed using a Q2000 DSC apparatus from TA instrument (TA instrument, New Castle, Delaware, USA). Typically, a 1 mg sample in sealed aluminum pan was fast heated to 170°C. The sample was kept 5 minutes at such high temperature to erase all thermal history. The sample was then cooled a 5°C/min to -80°C followed by an isotherm of 5 minutes. Finally, a second heating was performed at 10°C/min to 170°C.

Modulated DSC (MDSC) was also performed. For that, the samples were heated 10°C above Tm, then rapidly quenched in liquid nitrogen. Samples were then analyzed by MDSC at a rate of 2°C/min, an amplitude of ± 1.2°C and a frequency of 60s.

Thermogravimetric analysis (TGA) was performed to determine the polymers thermal stability.

The measurements were conducted on a High-Res TGA Q5000 from TA instrument. Samples between 1 and 2 mg were heated at 10°C/min from room temperature to 600°C under air.

Mass measurements were carried out on an AutoflexTM MALDI-TOF mass spectrometer

Results and discussions

Semi

Core units

End-Groups Finally, the more shifted (CH2-O) protons are the ones of the diol surrounded by two furan units at 4.33 ppm (b'').

These attributions allow to formulate several assumptions about the structure of the copolymers. First, the Mn of the polyesters can be estimated thanks to the intensities of the signals corresponding to the end-groups compared to the ones of the core-units. Eq. 1 is inspired by the method given by Izunobi et al. [START_REF] Izunobi | Polymer Molecular Weight Analysis by 1 H NMR Spectroscopy[END_REF]. It is comparable to the equation given by Jiang et al.

[34] but also takes into account the adipic units as well as the ends group as part of the polymeric chain as did Debuissy et al. [START_REF] Debuissy | Lipase-catalyzed synthesis of biobased and biodegradable aliphatic copolyesters from short building blocks. Effect of the monomer length[END_REF]. It is a quick method to compare polyesters chain-lengths with each other, but long polymeric chains show low intensity (I) end-groups signals, which consequently decrease the accuracy of the calculated molar masses (Eq. 1).

Also, characteristics signals corresponding to each monomeric unit are present and confirm the synthesis of semi-aromatic polyesters. Characteristic signals of the furan and adipate moieties, the two diesters employed, can be distinguished at 7.15 and 2.32 ppm, respectively. It is clearly noticeable that the furan's signal intensity is lower when the amount of DMFDC in the feed is low and that the signal corresponding to the adipate moieties is inversely affected. The ratio of each diester inserted in the polymeric chain can therefore be estimated by calculating the intensity ratio between the furan and adipate signals (Eq. 2).

% ℎ ℎ = + 2 (2)
(1) The obtained PHAd and PHF have Mn(NMR) of 6 300 and 2 100 g.mol -1 , respectively. These values are rather low but they are in accordance with those reported in the literature. For instance, Nasr et al. [START_REF] Nasr | Enzymatic Polycondensation of 1,6-Hexanediol and Diethyl Adipate: A Statistical Approach Predicting the Key-Parameters in Solution and in Bulk[END_REF] obtained, after an extensive optimization of the reaction parameters, PHAd with an average molar mass (Mn determined by SEC) of 18 500 g.mol -1 in 48h. Concerning PHF, with a protocol similar to ours but at a temperature of 95°C, Jiang et al. [START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF] obtained Mn(NMR) of 3 500 g.mol -1 . The values determined by NMR spectroscopy and SEC are different since SEC values are given as polystyrene-equivalent molar masses (polystyrene standards were used for the calibration) which are thus overestimated compared to the true molar masses. One can also note that in all the cases, the molar masses of PHAd and PHF obtained by CALB-catalyzed polymerization are very different. However, increasing the amount of aromatic moieties surprisingly did not induce a linear decrease in the molar mass of the obtained copolyesters.

= ( + ) -- 2
Indeed, polyesters with a ratio of aromatic diester up to 70% have an identical average molar mass. Dispersity (Ɖ) values are quite high (from 1.5 to 2.1) but consistent with the transesterification activity of the lipase. The slightly lower molar mass calculated from NMR analyses for the PHAd-co-PHF containing 10 and 30% of DMFDC could be explained by remaining traces of unreacted 1,6-HDO in the final polymer. This increases the intensity of end groups in comparison to the core units, thus lowering the calculated molar mass. Indeed, these copolyesters being highly amorphous it was difficult to eliminate all traces of residual monomers.

The progress of the reaction has been monitored by NMR spectroscopy. Thanks to Eq. 1, the molar masses of the copolyesters could be calculated and plotted versus the reaction time (Fig. 4). • PHAd, ▲ 10% of DMFDC, ▬ 30% of DMFDC, ○ 50% of DMFDC, ◊ 70% of DMFDC, ♦ 90% of DMFDC, ■ PHF When the aromatic diester (DMFDC) content is lower than 70%, the kinetics of the enzymatic polymerization is not affected. However, the polyesters containing 90 % of aromatic diesters and • PBAd, ▲ 10% of DMFDC, ▬ 30% of DMFDC, ○ 50% of DMFDC, ◊ 70% of DMFDC, ♦ 90% of DMFDC, ■ PBF PBAd and PBF with Mn(NMR) of respectively 5 100 and 1 200 g.mol -1 were synthesized. Again, these results are comparable with values reported in the literature. Pellis et al. [START_REF] Pellis | Elucidating enzymatic polymerisations: Chain-length selectivity of Candida antarctica lipase B towards various aliphatic diols and dicarboxylic acid diesters[END_REF] obtained, with both enzymatic synthesis and a thermal post processing, PBF with Mn of approximatively 1 000 g.mol -1 . While Jiang et al. obtained PBF with a Mn(NMR) of 1 500 g.mol -1 [START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF]. In a previous study, our group obtained PBAd with a comparable Mn(NMR) of 5 600 g.mol -1 [START_REF] Debuissy | Lipase-catalyzed synthesis of biobased and biodegradable aliphatic copolyesters from short building blocks. Effect of the monomer length[END_REF]. Here again, the difference between Mn values determined from NMR analyses and by SEC comes from the fact that the latter are given as polystyrene-equivalent. In comparison with the aforementioned results, the average molar mass of the synthesized PBAd is significantly lower than PHAd. This phenomenon was described in several previous studies [START_REF] Debuissy | Lipase-catalyzed synthesis of biobased and biodegradable aliphatic copolyesters from short building blocks. Effect of the monomer length[END_REF][START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF][START_REF] Pellis | Elucidating enzymatic polymerisations: Chain-length selectivity of Candida antarctica lipase B towards various aliphatic diols and dicarboxylic acid diesters[END_REF][START_REF] Cruz-Izquierdo | Lipasecatalyzed synthesis of oligoesters of 2,5-furandicarboxylic acid with aliphatic diols[END_REF][START_REF] Mahapatro | Lipase-Catalyzed Polycondensations: Effect of Substrates and Solvent on Chain Formation, Dispersity, and End-Group Structure[END_REF]. In connection with its natural hydrolytic activity, CALB has a better affinity for longer monomers, especially C6 monomers, resulting in higher molar masses for the obtained polymers. Therefore, the decrease PBAd-co-PBF copolyesters with a ratio up to 50% of DMFDC could be obtained with high molar masses. However, a slight decrease in the average molar mass with the DMFDC content is noticeable. Dispersity values around 2 are obtained which is in agreement with the literature and the transesterification activity of the enzyme [START_REF] Debuissy | Lipase-catalyzed synthesis of biobased and biodegradable aliphatic copolyesters from short building blocks. Effect of the monomer length[END_REF][START_REF] Mahapatro | Lipase-Catalyzed Polycondensations: Effect of Substrates and Solvent on Chain Formation, Dispersity, and End-Group Structure[END_REF]. The molar mass distribution observed on SEC elution profiles (SI.V) revealed shouldered peaks for PBAd-co-PBF containing between 10 and 50% DMFDC. This could indicate a bimodal distribution and therefore the presence of less reactive growing chains during the polymerization, resulting from a high aromatic content. The PBAd-co-PBF containing 70% of DMFDC precipitated in the medium after 30 hours of reaction resulting in low molar mass polymers. The copolyester containing 90% of DMFDC and the PBF also precipitated and it occurred even earlier, after approximatively 8 hours. Therefore, reducing the diol length leads to the precipitation of the polymer in the medium for a lower ratio in aromatic diester. It also seems to reduce the reactivity of CALB towards highly aromatic polymeric chains.

The ratio of aromatic moieties in the polyesters depending on time could be calculated thanks to Eq. 2. These results for PHAd-co-PHF and PBAd-co-PBF, respectively plotted in Fig. 6 and7,

show similar behavior. The ratio of aromatic moieties inserted in the polymer remains equal to the feed ratio in DMFDC all along the reaction, indicating no specific preference of the enzyme toward one or another diester. These results led us to consider that the activity of the enzyme was only slightly impacted by the amount of aromatic diesters in the medium but that the solubility of the synthesized polymer chains in the medium was the main parameter responsible for the low molar masses obtained.

Maniar et al. [START_REF] Maniar | Furan-Based Copolyesters from Renewable Resources: Enzymatic Synthesis and Properties[END_REF] reported that for the CALB-catalyzed synthesis of polyester an increase in aromaticity leads to a decrease in DPn. This behavior was attributed to side reactions with the aromatic diol and to potential substrate specificity of the enzyme with intermediate structures inhibiting the polymerization. In our study, we observed that the copolyesters with a high content in aromatic monomer display a chain growth with a kinetics that does not depend on the aromatic content until they are no longer soluble in the reaction medium. At this point, the polymer precipitates and is no longer available for the enzyme, completely stopping the chain growth (see SI.XVI.1). Low molar masses polymers have a direct impact on the thermal properties of the corresponding material. It particularly leads to low Tg when the main goal of introducing aromatic moieties, when producing furan-based polymers, is to obtain high Tg materials.

In order to facilitate the solubility of the copolyester in the medium and thus avoid precipitation, different options can be considered. First, the temperature of reaction could be raised. Indeed, it has been shown that CALB can be used at temperatures higher than 90°C, without denaturation [START_REF] Poojari | Thermal stability of Candida antarctica lipase B immobilized on macroporous acrylic resin particles in organic media[END_REF][START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF][START_REF] Frampton | Synthesis of lipase-catalysed silicone-polyesters and siliconepolyamides at elevated temperatures[END_REF][START_REF] Lozano | Lipase Catalysis in Ionic Liquids and Supercritical Carbon Dioxide at 150 °C[END_REF]. However, such high temperatures increase the risk of denaturation with a rapid loss of activity of the enzyme. Besides, higher temperature conditions are against the main advantage of using enzymes, which allow polymerization under mild conditions. The second option is to find the appropriate solvent which is (i) able to support the reaction conditions (especially with a high boiling point, since the reaction is performed under partial vacuum), (ii) compatible with the enzyme and (iii) solubilizing the final polyester.

Debuissy et al. [START_REF] Debuissy | Enzymatic Synthesis of a Bio-Based Copolyester from Poly(butylene succinate) and Poly(( R )-3-hydroxybutyrate): Study of Reaction Parameters on the Transesterification Rate[END_REF][START_REF] Debuissy | Enzymatic synthesis of biobased poly(1,4-butylene succinate-ran -2,3-butylene succinate) copolyesters and characterization. Influence of 1,4and 2,3-butanediol contents[END_REF] have already screened different solvents for CALB catalyzed polymerization and reported anisole and phenetole as potential candidates. To complement these previous works, other solvents have been investigated based on their partition coefficient (log P)

and their boiling points. The log P is the logarithm of the studied product concentration in octanol divided by its concentration in water. It is an empirical method for the estimation of the lipophilicity of a given solvent. A high log P value corresponds to a highly lipophilic solvent and vice versa. Other parameters could be considered such as the dielectric properties of the solvent or the interaction of the substrates with the solvents with computational tools as did Pellis et al. [START_REF] Pellis | Safer biobased solvents to replace toluene and tetrahydrofuran for the biocatalyzed synthesis of polyesters[END_REF]. However, the log P has been used as a reference as it has already been employed in previous studies [START_REF] Juais | Isosorbide Polyesters from Enzymatic Catalysis[END_REF][START_REF] Kumar | Candida a ntartica Lipase B Catalyzed Polycaprolactone Synthesis: Effects of Organic Media and Temperature[END_REF][START_REF] Dong | Study on the enzymatic polymerization mechanism of lactone and the strategy for improving the degree of polymerization[END_REF][START_REF] Kobayashi | Lipase-catalyzed ring-opening polymerization of medium-size lactones to polyesters[END_REF].

The study of the solvents influence was focused on the synthesis of PHF and PBF, which are the polymers with the highest backbone rigidity and therefore the most difficult to get dissolved.

Solvents with a variety of log P have been investigated, taking diphenyl ether and toluene as references. Toluene having a boiling point of 111°C, the reaction has been performed at atmospheric pressure. Phenetole, anisole, acetophenone and cyclohexanone have been used at a minimal pressure of 250 mbar. Diphenyl ether having a high boiling point, it has been used as described before through a four steps method to reach 20 mbar. Early precipitation of the polymer was sometimes observed due to evaporation of the solvent. Thus the volume of solvent was doubled to avoid such issue but the amount of enzyme was also multiplied by 2 in order to keep a constant enzyme concentration. Kinetics of the reaction was proven to be similar to previous reaction conditions. The resulting polymers were analyzed by NMR spectroscopy (SI.XIV) and MALDI-TOF MS (SI.XVII) analyses. As MALDI-TOF-MS analysis of polydisperse polymers are not quantitative [START_REF] Rashidzadeh | Use of MALDI-TOF To Measure Molecular Weight Distributions of Polydisperse Poly(methyl methacrylate)[END_REF][START_REF] Mcewen | Instrumental effects in the analysis of polymers of wide polydispersity by MALDI mass spectrometry[END_REF], only rough estimates of Mn values were given in order to compare polymers with each other but they should not be taken as absolute values.

The characteristics of the solvents and molar masses of the obtained polyesters are shown in Table 3. A strong influence of the solvent on the resulting molar masses can be observed, especially for PHF. Acetophenone and anisole, solvents with log P lower than 2.5 but higher than 0.8, gave the higher molar masses for both PHF and PBF (Table 3). In these solvents, the reaction could be carried out for a longer time before observing the precipitation of the synthesized polymer.

Acetophenone was the solvent allowing the longer reaction time before precipitation (around 24 hours for PHF) and thus allowed to synthesize a semi-aromatic copolyester whose molar mass determined from NMR analyses is nearly four times greater than that obtained in diphenyl ether.

Anisole also allowed the synthesis of more than three-fold higher molar mass PHF, as calculated from NMR spectra, when compared to diphenyl ether. However, anisole has no effect on the final molar mass of PBF. One may note from the experimental part that, when synthesized in anisole and phenetole, PHF needed the addition of 5 µl of anhydrous TFA for complete solubilization before NMR analysis. This method has already been described and proved not to alter the polymer structure [START_REF] Hariharan | Useful NMR solvent mixture for polyesters: Trifluoroacetic acid-d/chloroform-d[END_REF].

In the literature, Dong et al. [START_REF] Dong | Study on the enzymatic polymerization mechanism of lactone and the strategy for improving the degree of polymerization[END_REF] studied the impact of solvent log P on the enzymatic ring opening polymerization of lactones. They reported that solvents with log P value higher than 4

were preferable whereas solvents with log P values between 4 and 2 gave unpredictable results.

Finally, solvents with log P lower than 2 resulted in low enzymatic activity. However, in their study 1,2-dichloroethane (log P = 1.52) was an exception and led to higher molar masses than expected. Kumar et al. [START_REF] Kumar | Candida a ntartica Lipase B Catalyzed Polycaprolactone Synthesis: Effects of Organic Media and Temperature[END_REF] also observed inconsistencies in the relationship between log P and CALB enzymatic activity. Our results also demonstrated that the log P value alone does not allow to predict the CALB activity. Because of precipitation issue, the solubility of the polymer in the medium is also a critical parameter to be considered. Unfortunately, this solubility in a given solvent cannot simply be predicted by log P. As depicted before, research with computational tools also showed that monomers stability in a given solvent greatly impacted the CALB reactivity and the obtained molar mass [START_REF] Pellis | Safer biobased solvents to replace toluene and tetrahydrofuran for the biocatalyzed synthesis of polyesters[END_REF]. Nevertheless, acetophenone and anisole were clearly identified here as new promising solvents for the enzymatic synthesis of PHF and PBF.

MALDI-TOF-MS analysis also allows to determine the molar mass distribution and to estimate the average molar masses of the polyesters. The obtained spectra confirm an increase in molar mass when PHF and PBF are synthesized in acetophenone or anisole in comparison to diphenyl ether. However, the average values are significantly lower than those determined from NMR analyses. Two phenomena can explain this difference. First, MALDI-TOF-MS analysis of polydisperse polymer samples is not quantitative and the largest polymer chains are underrepresented, which results in an underestimation of the average molar masses. On the other hand, the determination of molar masses by NMR spectroscopy is based on the measurement of the intensity of chain ends compared to that of the core repeating units. If the end groups are consumed by a side reaction, their intensity decreases, resulting in an overestimation of the molar masses.

However, this last hypothesis can be verified since MALDI-TOF-MS allows of the analysis of polymer chain ends. The end groups of PHF and PBF synthesized in the different solvents have been analyzed (Fig. 8 and SI.XVII). When synthesized in diphenyl ether, the PHF is mostly ended by both an ester and a hydroxyl or by two hydroxyl groups. A minority of chains with an ester group at both ends can be observed (Fig. 8a). When synthesized in anisole, the PHF was mostly ended by hydroxyl groups (SI.XVII.1). When PHF was synthesized in acetophenone, chains were ended by hydroxyl groups or by a carboxylic acid and hydroxyl group (Fig. 8b). This carboxylic acid could be produced by hydrolysis in the reaction medium even though the acetophenone used for the reaction was anhydrous. Indeed, as previously mentioned, acetophenone allows to prolong the reaction time thus leaving more time for an eventual hydrolysis of the polyester. Moreover, acetophenone is much more polar than the other solvents.

This could facilitate the hydration of the solvent under partial vacuum. Such hydrolysis of the polyester chains could be responsible for the apparent increase in molar mass calculated by NMR spectroscopy through the phenomenon impacting the chain ends depicted earlier. End groups analysis was also performed on PBF (SI.XVII.2-4). Interestingly, no hydrolysis was observed this time for the PBF chains synthesized in acetophenone. Acetophenone had already been studied as solvent or as co-solvent for the synthesis of aromatic polyesters [START_REF] Imai | Preparation and properties of aromatic polyesters and copolyesters containing phenylindane unit[END_REF][START_REF] Akutsu | Synthesis and Properties of Polyarylates Derived from 4,4″-Dihydroxy-o-terphenyl[END_REF] but had never been used in enzymatic polymerizations. The catalytic activity of iCALB was measured in the newly selected solvents, acetophenone and anisole, and compared to the one in diphenyl ether. For that, and in order to avoid the problem of polymer solubility, a simpler reaction of transesterification on model substrates was considered. The enzymatic activity has thus been assayed by studying the transesterification of diethyl adipate with 1-butanol at 50°C and atmospheric pressure (Fig. 9). The reaction being reversible, an excess of 1-butanol was used to displace the reaction equilibrium.

Fig. 9: Assessment of the activity of CALB with diethyl adipate and 1-butanol Moreover, it has been reported that in certain solvents, part of the enzyme can leak from the supporting beads [START_REF] José | Investigation of the causes of deactivation-degradation of the commercial biocatalyst Novozym® 435 in ethanol and ethanol-aqueous media[END_REF][START_REF] Ortiz | Novozym 435: the "perfect" lipase immobilized biocatalyst?[END_REF]. Presence of free enzyme could promote the reaction of polymerization.

However, this enzyme leaching reduces the reusability of the beads. The impact of the selected solvents on the CALB activity and leaching has therefore been evaluated. After incubation of the beads at 90°C in a given solvent, the catalytic activity of the solvent was determined (after removal of the immobilized enzyme) in order to detect the possible presence of free enzymes that would have leached from the support.

The progress of reaction was determined from the conversion of ester groups (Fig. 10).

Fig. 10: iCALB catalyzed conversion of diethyl adipate with 1-butanol in various solvents: ▲ Diphenyl ether, ■ Acetophenone, ♦ Anisole.

The catalytic activity of iCALB was found to be similar in all solvents. Only a slightly higher activity was observed in acetophenone. These results tend to imply that the higher molar masses obtained for the polyesters in acetophenone and anisole are not due to a higher specific activity of the enzyme in such solvents. Moreover, no activity was detected in the solvent alone after incubation with the iCALB. This indicates that among these solvents, none was found to cause significant leaching of the enzyme from the support to the medium. Therefore, no free enzyme participated to the improvement of the molar mass of PHF and PBF. The high potential of solubilization of these solvents, delaying the precipitation of the polymer in the medium, leads to longer time of reaction with a higher accessibility of the substrate to the enzyme and therefore allows to obtain higher molar masses.

Polyesters thermal properties

As depicted before, one of the main goals of increasing the aromatic content of polyesters is to improve their behaviors such as their thermal properties. Moreover, the molar masses and thus the synthesis method can also affect these properties. Consequently, the thermal behavior of the synthesized polymers has been determined to evaluate the impact of the aromatic content and synthesis method.

Thermal properties of PHAd-co-PHF and PBAd-co-PBF copolyesters

The thermal properties of PHAd-co-PHF and PBAd-co-PBF synthesized in diphenyl ether with different ratio of DMFDC have been studied by DSC, MDSC and TGA (Table 4 and SI.VI-XI). a Tg gap was too weak and in the limit of the apparatus to be precisely measured. b from Modulated DSC analyses c from classical DSC analyses For a given aromatic content, the Tg values of the hexanediol-based polyesters were systematically lower than those of the butanediol-based ones. In polyesters, the thermal properties are highly dependent on the length of the monomers. These results are in agreement with previous observations on aliphatic polyesters [START_REF] Debuissy | Lipase-catalyzed synthesis of biobased and biodegradable aliphatic copolyesters from short building blocks. Effect of the monomer length[END_REF]. Indeed, hexanediol brings more flexibility to the polymeric chains compared to a shorter diol, lowering the Tg.

The impact of the aromatic content on the Tg values of the PHAd-co-PHF and PBAd-co-PBF copolyesters have been evaluated (Fig. 11 and12). As expected, an increase in the aromatic content leads to an overall increase in the Tg value. The experimental Tg values of the copolyesters have been compared to the values calculated with the Fox law. For both polyesters, samples with an aromatic content below 70% generally followed the Fox law. However, PHAdco-PHF with an aromatic ratio above 90% and PBAd-co-PBF with an aromatic ratio above 70% endotherms of PBF50, PHF50 and PHF70 were hardly discernible. Polyesters with a DMFDC content higher than 70% showed relatively high melting temperature (above 100°C) and higher crystallinity. This is in perfect agreement with results reported in the literature [START_REF] Wu | Biobased poly(butylene 2,5furandicarboxylate) and poly(butylene adipate-co-butylene 2,5-furandicarboxylate)s: From synthesis using highly purified 2,5-furandicarboxylic acid to thermo-mechanical properties[END_REF][START_REF] Yu | Chemosynthesis and characterization of fully biomass-based copolymers of ethylene glycol, 2,5-furandicarboxylic acid, and succinic acid[END_REF][START_REF] Zhou | Synthesis, physical properties and enzymatic degradation of bio-based poly(butylene adipate-co-butylene furandicarboxylate) copolyesters[END_REF][START_REF] Papadopoulos | Synthesis and characterization of novel poly(ethylene furanoate-co-adipate) random copolyesters with enhanced biodegradability[END_REF]. Polyesters properties have been also evaluated by TGA. For both series of polyesters, increased thermal stability, measured as Td5%, is observed when the aromatic content increases, up to 70% of DMFDC (Fig. 13). Then, Td5% is dramatically reduced for higher aromatic content. This evolution is less marked on Tdmax, especially for the PBAd-co-PBF series. A similar but less pronounced phenomenon has been reported in the literature with a decrease of Td5% above 80% of aromatic content [START_REF] Wu | Biobased poly(butylene 2,5furandicarboxylate) and poly(butylene adipate-co-butylene 2,5-furandicarboxylate)s: From synthesis using highly purified 2,5-furandicarboxylic acid to thermo-mechanical properties[END_REF]. The decrease in Td5% is intensified by the low molar masses of the copolyesters [START_REF] Chrissafis | Effect of molecular weight on thermal degradation mechanism of the biodegradable polyester poly(ethylene succinate)[END_REF]. Increasing the aromatic content of these polyesters was therefore an efficient way to increase their thermal resistance until their molar masses were negatively impacted and thus led to a decrease in the thermal stability.

According to these results, reaching sufficiently high molar masses seems essential to produce polyesters with high thermal properties. Indeed, the low molar masses of the polyesters containing high aromatic contents are responsible for the decrease of the Td and Tg of the polymers.

Thermal properties of the PHF and PBF synthesized in different solvents

PHF and PBF synthesized in acetophenone and in anisole were also analyzed and compared to the polyesters synthesized in diphenyl ether to determine the impact of the higher molar masses on the thermal properties (Table 5 and SI.XII and XIII). As expected, for PHF a significant increase of at least 10°C in the Tg was observed when the polymer was synthesized in acetophenone or in anisole due to the higher molar masses evaluated from NMR analyses of 7 600 g.mol -1 and 6 700 g.mol -1 , respectively, compared to diphenyl ether (which resulted in PHF with a molar mass of 2 100 g.mol -1 ). In the case of PBF, no significant change was observed due to the lower impact of these solvents on the obtained molar masses. For both PHF and PBF, the melting temperature slightly increased for the polymers synthesized in acetophenone and anisole when compared to diphenyl ether. The thermal resistance (measured as Td5%) was improved for both polymers. PHF showed a Td5% value 6°C higher when synthesized in acetophenone. For PBF, the Td5% was increased by 23°C when synthesized in acetophenone.

For both polyesters, the improvement in thermal resistance could be linked to the higher molar masses obtained when using acetophenone. However, we do not explain the significant improvement observed for PBF synthesized in anisole since the Mn value (1 200 g.mol -1 ) was not found to be higher than the one obtained in diphenyl ether (1 400 g.mol -1 ).

Conclusion

In this work, PHAd-co-PHF and PBAd-co-PBF biobased aliphatic-aromatic copolyesters were synthesized in various solvents via the green route of enzyme-catalyzed transesterification reactions using immobilized Candida antarctica lipase B as biocatalyst.

For reactions carried out in diphenyl ether the highest molar masses were obtained for the PHAd-co-PHF series. Mw values above 30 000 g.mol -1 were obtained for aromatic diester contents ranging from 0 to 70 %. Replacing 1,6-HDO with 1,4-BDO as a shorter diol resulted in copolyesters with slightly lower molar masses at up to 50% aromatic monomer content. In this composition range, an increase in the furan-based monomer content resulted in improved thermal properties (higher Tg and degradation temperature) for both copolyesters series. The obtained molar masses are similar or even slightly higher than those reported so far in the literature for equivalent systems [START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF][START_REF] Comerford | Thermal Upgrade of Enzymatically Synthesized Aliphatic and Aromatic Oligoesters[END_REF].

As in previous published studies, the molar masses remain low and reach a plateau at high aromatic monomer content [START_REF] Jiang | A biocatalytic approach towards sustainable furanic-aliphatic polyesters[END_REF]. However, our results then showed that CALB was able to insert the furan-based aromatic monomer into the macromolecular architectures of different copolyesters chains in proportions equal to those of the feed without negative impact on the reaction kinetics or on the obtained molar masses.

In contrast, this study highlighted that it is the loss of solubility observed for polyesters with high aromatic content (above 50% and 70% in DMFDC with butanediol and hexanediol, respectively) that is stopping the polyester chain growth thus leading to lower molar masses and consequently lower thermal properties.

The screening of various alternative solvents showed that replacing diphenyl ether with acetophenone or anisole resulted in significantly improved solubility for both the highly aromatic PHF and PBF polyesters. The best results were obtained with acetophenone, which had never been reported so far as a solvent for the enzymatic synthesis of polyesters, resulting in a significant increase in molar masses for PHF up to 7 600 g.mol -1 as measured from NMR analysis and 3 500 g.mol -1 as estimated from MALDI-TOF MS. Consequently, the polymer presented a greater thermal stability and a Tg increased by 11 °C. However, a possible hydrolysis of the polyester in the reaction medium was highlighted and will be further investigated.

This study clearly demonstrated the impact of the solvent in such enzymatic systems to obtain high molar mass copolyesters based on elevated aromatic content with improved thermal behavior. To complete this study, additional investigations should be carried out (mechanical properties, biodegradation...) in connection with the potential uses of these biobased aliphaticaromatic copolyesters such as packaging or biomedical applications.

CRediT authorship contribution statement

Fig. 2 :

 2 Fig. 2: CALB-catalyzed synthesis of poly(hexylene adipate)-co-(hexylene furanoate) and poly(butylene adipate)-co-(butylene furanoate)

(

  Bruker Daltonics GmbH, Bremen, Germany) used at a maximum accelerating potential of 20 kV in positive mode and operated in linear mode. The delay extraction was fixed at 560 ns and the frequency of the laser (nitrogen 337 nm) was set at 5 Hz. The acquisition mass range was set to 1500-10000 m/z with a matrix suppression deflection (cut off) set to 1500 m/z. The equipment was calibrated with ACTH 1-17 ([M+H] + 2094.42), insulin ([M+H] + 5732.52), ubiquitin I ([M+H] + 8565.76) and myoglobin ([M+H] + 16952.31). Spectra were processed with flexAnalysis software (Bruker Daltonics). Sample preparation was performed with the dried droplet method using a mixture of 0.5 µl of sample with 0.5 µl of matrix solution dried at room temperature. The 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) matrix was prepared at 20 mg/ml in dichloromethane.

  -aromatic furan-based polyesters of different composition were synthesized by enzymatic polymerization. A first series of experiments was conducted by reacting 1,6-HDO with different ratios of DMFDC and DEA. The chemical structure of each polymer has been determined by 1 H NMR spectroscopy. The spectra of the obtained polyesters are shown in Fig. 3, and also in Fig. SI.I in Supplementary Information (SI). The structure and the attribution of the peaks were confirmed by COSY, HMBC and HSQC 2D NMR spectroscopy (Fig. SI.II).

Fig. 3 : 1 H

 31 Fig. 3: 1 H NMR spectra of the enzymatically synthesized PHAd-co-PHF

Fig. 4 :

 4 Fig. 4: Time evolution of the Mn values determined by NMR spectroscopy of the PHAd-co-PHF.

  (g/mol) Time (h) the PHF both precipitated before 24 h of reaction, which could explain the constant and low molar mass obtained after 24 h of reaction. The solubility of the polymer in the medium would therefore be a critical parameter influencing its final molar mass. In order to confirm these results, a similar study has been conducted replacing 1,6-HDO by a shorter diol, 1,4-BDO. If the precipitation of the polymer in the medium is the key parameter, a shorter diol should amplify the phenomenon at stake by inducing higher rigidity of the polymer chain backbone and thus lower solubility. The molar mass of the polymers synthesized with 1,4-BDO evaluated by SEC and NMR spectroscopy are shown in Table2and Fig.5. The NMR spectra of the resulting polymers are given in SI.III while the SEC traces and results are given in SI.V. The average molar masses were evaluated by NMR spectroscopy using an equation similar to equation 1 but using the molar mass of the corresponding constituting moieties as coefficients.

Fig. 5 :

 5 Fig. 5: Time evolution of the Mn values determined by NMR spectroscopy of the PBAd-co-PBF.

  mass between PHAd and PBAd is effective and not only due to the different molar mass of the diol. Indeed, from the NMR analysis, the DPn values of PHAd and PHF(27 and 8, respectively) are also higher than those of PBAd and PBF (25 and 5, respectively).

Fig. 6 :Fig. 7 :

 67 Fig. 6: Determined ratios of aromatic moieties in the PHAd-co-PHF depending on time for different ratio of DMFDC

Fig. 8 :

 8 Fig. 8: MALDI-TOF-MS spectra of PHF synthesized in a) diphenyl ether and b) acetophenone with c) corresponding end-groups identification.

haveFig. 11 :Fig. 12 :

 1112 Fig. 11: Evolution of Tg of the PHAd-co-PHF with different proportions of DMFDC (experimental data compared to the theoretical ones from Fox equation)

Fig. 13 :

 13 Fig. 13: TGA measurements of PHAd-co-PHF with different ration of DMFDC ▬ PHAd, ▬ 10% of DMFDC, ▬ 30% of DMFCD, ▬ 50% of DMFDC, ▬ 70% of DMFDC, ▬ 90% of DMFDC, ▬ PHF

Alfred Bazin:

  Methodology, Investigation, Formal analysis, Visualization, Writing -review & editing. Luc Avérous: Writing -review & editing. Eric Pollet: Conceptualization, Methodology, Supervision, Writing -review & editing.

  1

Table 1 :

 1 Table 1 gives the Mn values determined from NMR spectra compared to Mn, Mw and Ɖ obtained from SEC analyses (Fig. SI.IV) of the synthesized polyesters. Compositions and molar masses values of the PHAd-co-PHF, as determined by NMR spectroscopy and SEC.

	4	* " 3	% * 116.16 + 4

a Calculated from NMR spectra data.

Table 2 :

 2 Compositions and molar masses values of the PBAd-co-PBF as determined by NMR

	spectroscopy and SEC
					NMR			SEC
	Sample name	Ratio of DMFDC (%)	Ratio of DEA (%)	Yield (%) a	Measured ratio of aromatic moieties (%)	Mn (g.mol -1 )	Mn (g.mol -1 )	Mw (g.mol -1 )	Ɖ
	PBAd	0	100	98	--	5 100	16 600	34 700	2.08
	PBF10	10	90	98	9.1	4 700	16 800	33 100	1.96
	PBF30	30	70	97	30.2	4 500	11 100	19 500	1.73
	PBF50	50	50	97	48.3	4 300	10 200	17 500	1.71
	PBF70	70	30	94	68.3	2 800	6 200	10 400	1.68
	PBF90	90	10	88	85.5	1 500	2 800	4 300	1.50
	PBF	100	0	85	--	1 400	2 000	2400	1.20
	a Calculated from NMR spectra data.

Table 3 :

 3 Influence of solvents on molar masses of PHF and PBF (CALB 20 wt.%, solvent 300 wt.%, 90°C 72 h)

	PHF	PBF

a Program of pressure: 4 h at 350mbar, 4 h at 200 mbar, 16 h at 50 mbar, 48 h at 20 mbar. b Ambient pressure. c Program of pressure: 4 h at 350mbar, 68 h at 250 mbar. n.a.: not available (these polymers have not been analyzed by MALDI-TOF MS).

Table 4 :

 4 Thermal properties of PHAd-co-PHF and PBAd-co-PBF

	Polyester	Tg b	Tm c	Td5%	Tdmax
		(°C)	(°C)	(°C)	(°C)
	PHAd	--a	58	270	374
	PHF10	-52	51	305	390
	PHF30	-40	32	329	387
	PHF50	-32	83	336	384
	PHF70	-19	107	347	387
	PHF90	-6	135	334	379
	PHF	-1	140	332	381
	PBAd	--a	60	283	387
	PBF10	-58	41	302	389
	PBF30	-39	--	345	386
	PBF50	-25	102	327	387
	PBF70	-8	141	340	383
	PBF90	-3	148	307	381
	PBF	15	153	264	375

Table 5 :

 5 Thermal properties of the PHF and PBF synthesized in different solvents

	Polymer	Solvent	Tg (°C)	Tm (°C)	Td5% (°C)	Tdmax (°C)
		Diphenyl ether	-1	140	331	381
	PHF	Anisole	11	145	338	374
		Acetophenone	10	146	351	385
		Diphenyl ether	15	153	264	375
	PBF	Anisole	16	159	307	386
		Acetophenone	17	163	288	384

Acknowledgements

The authors thanks Jean-Marc Strub of LSMBO-IPHC Strasbourg for his expertise and time regarding the MALDI-TOF MS analysis.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.