Anne Marie Farnet Da Silva

G Gil

F Ruaudel

A C Chevremont

E Ferre

A M Farnet
email: m.farnet@univ-cezanne.fr

M R C N R S I R D 6116

M R C N R S 6180

Polycyclic aromatic hydrocarbon

Keywords:

HAL is

Introduction

Mediterranean forests are mainly composed of schlerophyllous species such as Quercus ilex L. because of climate conditions characterised by draught in summer. Thus particularly high amount of various phenolic compounds can be found in forest litters in this area [START_REF] Racon | Histological and chemical changes in tannic compounds of evergreen oak leaf litter[END_REF]. Under these drastic conditions, certain microbial species -mainly white-rot fungi-are able to transform phenols, which are recalcitrant molecules, via phenoloxidases such as laccases. For instance, Marasmius quercophilus is a white-rot basidiomycete which extensively colonises evergreen-oak litter and produces several laccases [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF][START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid reactivity towards natural and xenobiotic aromatic compounds[END_REF]. These blue copper oxidases are indeed polymorphic: different isoforms of laccases can be produced depending on environmental conditions and on fungal species [START_REF] Palmieri | Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus[END_REF][START_REF] Palmieri | A typical laccase isoenzyme from copper-supplemented Pleurotus ostreatus cultures[END_REF][START_REF] Baldrian | Interactions of heavy metals with white-rot fungi[END_REF]. Thus Mediterranean forest litters may be considered as a source of laccase diversity because of their specific chemical composition.

Furthermore, in the Mediterranean area, anthropogenic pressure is particularly high and do not favour the ecosystem functioning. Various organic pollutants can greatly affect organic matter turn-over in soils or litters: for instance, polycyclic aromatic hydrocarbons can drastically alter microbial activities [START_REF] Hamdi | Effects of anthracene, pyrene and benzo[a]pyrene spiking and sewage sludge compost amendment on soil ecotoxicity during a bioremediation process[END_REF][START_REF] Eom | Ecotoxicity of a polycyclic aromatic hydrocarbon (PAH)-contaminated soil[END_REF]. These widespread pollutants are recalcitrant to biotransformation because of their aromatic and condensed structure leading to a very low solubility [START_REF] Muhit | Predicting organic carbon-water partitioning of hydrophobic organic chemicals in soils and sediments based on water solubility[END_REF]. Laccases produced by white-rot fungi have been widely used in various detoxification processes since they oxidise aromatic pollutants such as chlorophenols, PAH or dyes [START_REF] Bezalel | Initial oxidation products in the metabolism of pyrene, anthracene, fluorene and dibenzothiophene by the white-rot fungus Pleurotus ostreatus[END_REF][START_REF] Potin | Biodegradation of polycyclic aromatic hydrocarbons (PAH) by Cladosporium sphaerospermum isolated from an aged PAH contaminated soil[END_REF][START_REF] Zhang | Degradation of chlorophenols catalyzed by laccase[END_REF]. Moreover, the effects of mediators on oxidation potential of laccase have also been extensively investigated: these molecules are commonly used in laccase reaction since they can enlarge laccase substrate range. Mediators are oxidised by laccases and then oxidise other compounds in a non-specific reaction. Although synthetic mediators, such as 2,2′-azino-bis-3-ehtylbenzothiazoline-6-sulfonicacid (ABTS) and hydroxybenzotriazole (HBT), are usually used as models, other studies have focused on natural phenolic compounds as mediators since it can provide information about their potential role in natural environments [START_REF] Calcaterra | Phenolic compounds as likely natural mediators of laccase: a mechanistic assessment[END_REF].

In this work we wanted to investigate the oxidation potential of laccase functional diversity towards PAH. Tools which can rapidly discriminate the various laccase isoforms are still lacking. Based on a previous work [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF], isoelectric focusing (IEF) appeared to be a promising method. Here, we used this technique to investigate laccase diversity of a M. quercophilus strain and to characterise these isoforms reactivity towards different substrates such as ABTS which can also be used as a mediator. The potential of PAH oxidation of these isoforms has indeed been investigated with or without mediators (ABTS or HBT) in order to assess the role of such diversity in natural ecosystem contaminated with these pollutants. Moreover this study has also focused on the importance of mediator pre-oxidation to test whether preoxidation by laccases favour PAH oxidation yields.

Materials and methods

Isolation of the strain

Strain 19 of M. quercophilus was isolated from a part of the site of La Gardiole de Rians, Var, France (1 ha) using the rhizomorphic form of the fungus [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white-rot fungus Marasmius quercophilus[END_REF]. A fungal cap culture was firstly grown on a malt extract agar medium (MEA), (malt extract, (Bio Mérieux, France): 20 g L -1 , agar (Bio Mérieux, France): 20 g L -1 ) supplied post-autoclaving with chloramphenicol, (Sigma, France): 50 mg L -1 . Secondly, the pure mycelial culture obtained was used to inoculate an agar medium containing whole wheat flour (Bio Mérieux, France: 20 g L -1 ) which favours rhizomorph production. Then one rhizomorph was used to inoculate a MEA plate to obtain a pure dicaryotic culture.

Culture conditions and partial purification of laccase

Precultures were performed in 200 mL Erlenmeyer flasks with 50 mL of malt extract liquid medium (MEL) containing malt extract (20 g L -1 ) and CuSO 4 (5 g L -1 ). They were inoculated with a plug (1 cm diameter) from a fungal culture grown on MEA and incubated at 25 °C in darkness for 4 days. These precultures were used to inoculate two 3 L-Erlenmeyerflasks with 400 mL of MEL. Cultures were incubated at 25 °C for 5 days in darkness under shaking at 200 rpm.

Enzyme activity was measured by following the oxidation of syringaldazine [N,N′-bis-(3,5-dimethoxy-4-hydroxybenzylidene)hydrazine] to quinone (ε M =65000 M -1 cm -1 ) at 525 nm in acetate buffer 0.1 M, pH 4.0 on a spectrophotometer Kontron Uvikon 860. One unit (U) of laccase activity is defined as the amount of enzyme that oxidises 1 µmol of the substrate/min. Sampling (two replicates) was performed from the third to the seventh day of culture. The reaction mixture contained 500 µL of the medium, 10 µL of syringaldazine (0.6% in methanol) and 2.5 mL of acetate buffer (two replicates for each sampling). Cultures were filtered on a glass microfibre filter GF/D, 2.7 µm (Whatman, England). The filtered medium was concentrated using dialysis tubes (Cellu Sep, VWR, France) rated at 10 MW (10 kDa) cut-off. A total volume of 100 mL was obtained. The concentrated filtered medium was loaded on an ion-exchange Mono Q in fast performance liquid chromatography (FPLC) equilibrated with phosphate buffer 0.1 M pH 6.0. Laccase was eluted with a step gradient (0.2 M, 0.4 M, 2 M NaCl) for 30 min at a flow rate of 1 mL min -1 . Laccase activity was measured as described above to detect fractions containing laccase. Laccase purity was checked on a PAGE (polyacrylamide gel electrophoresis) carried out according to [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] using 4% stacking gel and 7.5% separating gel at 220 V with the Mini-Protean II electrophoresis cell (Biorad) and protein was stained using the Coomassie Blue standard method.

Isoelectric focusing of the purified laccase

Isoelectric focusing was performed using the Biorad Rotofor preparative IEF Cell. The sample was prepared as follow: 10 mL of the purified enzyme solution were added to 1 mL of Bio-Lyte 3/10 ampholytes (Biorad) and distilled water q.s.p. 50 mL to obtain a pH gradient from 3 to 10. Isoelectric focusing was performed for 3 h at 200 V till the pH gradient gets stable (voltage about 360 V). 2 mL-fractions were collected under vacuum and the pH was immediately measured for each fraction. Laccase activity was also measured for each fraction using syringaldazine as a substrate as described above.

2.4. Determination of V m and K m for each laccase isoelectric isoforms ABTS and 2,6 dimethoxyphenol (DMP) were used as substrates to determine the V m and K m which were calculated using the Lineweaver-Burk transformation of Michaelis-Menten equation. The concentrations ranged from 0.5 to 50 µM and from 0.2 to 10 µM for ABTS and DMP respectively in acetate buffer 0.1 M pH 4.5.

High Performance Liquid Chromatograph (HPLC) analysis of PAH transformation

HPLC system was equipped with a C18 Reverse Phase column (Merck, 4.6×250 mm) in the following gradient system: solvent A, water/trifluoro acetic acid 0.1%/acetonitrile 90/10 v/v, solvent B water/trifluoro acetic acid 0.1%/acetonitrile 95/5 v/v, gradient =5 to 15 min, A 100% to B 100%; 15 to 25 min B 100% (flow rate 1 mL min -1 ). The PAH transformation was realised using the mixture of the six isoforms of laccase since a weak activity was obtained for some isoforms. The reaction mixture (5 mL) contained ABTS or HBT 1 mM, PAH 250 mg L -1 and 5 U of laccase in acetate buffer 0.1 M pH 4.5 and 10% acetone. The reaction mixtures were incubated at 30 °C for 6 h in the dark. A 4-hour preliminary ABTSoxidation was also performed using the same conditions and the PAH solution in acetone was added after the pre-oxidation of the mediator. An extraction with 5 mL of diethyl ether was performed for each reaction mixture prior to injection. The organic phase was evaporated and the products of extraction were dissolved in 1 mL of acetonitrile (30 µL injected). The PAH used were: naphtalene, anthracene, phenanthrene and benzo(a)pyrene. All the PAH were purchased from Sigma. The same reactions were performed without mediators. Controls were also realised using only ABTS or HBT with laccase and using ABTS or HBT with the PAH but without laccase. To test ABTS oxidation via benzo(a)pyrene oxidation products, a control with benzo(a)pyrene and ABTS in a sealed flask saturated with nitrogen was also performed. All these controls were incubated at 30 °C for 6 h. P-hydroxybenzoic acid was used as reference in each injection. 9,10-anthraquinone was also injected in order to confirm the identification of the single oxidative product obtained after anthracene oxidation. Peaks were identified based on matching retention time and UV spectra at 254 nm with those of anthracene and anthraquinone standards.

Cultures of M. quercophilus with anthracene

Cultures, previously inoculated with 10 mL of preculture of strain 19, were performed for 8 or 15 days of culture on a rotary shaker (200 rpm) at 25 °C in 1 L-Erlenmeyer flasks with 200 mL of MEL including 50 mg of anthracene. Laccase activities were measured everyday from day 4 with syringaldazine as the substrate as described above. Different control cultures without mycelium were performed: control cultures after autoclaving to assess anthracene evaporation during sterilisation and control cultures incubated in the same conditions and for both times of incubation. Each experiment was performed in three replicates. Cultures were filtered and the mycelium as well as the supernatant was extracted three times with the 200 mL of dichloromethane. The solvent was then evaporated and the solid extract solubilised in 1 mL of acetone. 30 µL of dilution 10 -3 were injected in the HPLC system. A calibration curve was also performed for anthracene concentrations ranging from 10 µM to 60 µM. Results were expressed as percentages of anthracene disappearance.

Statistical analysis

The non-parametric U-test of Mann-Whitney was used to separate significantly different means (P b 0.05) of remaining anthracene under the different experimental conditions. These analyses were performed using SPSS 11.0 statistical software (SPSS Inc., Chicago, IL).

Results and discussion

After laccase purification from M. quercophilus cultures on MEL one band on SDS-PAGE (data not shown) was obtained with a molecular weight of 60 kDa as described previously [START_REF] Farnet | Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF]. Isoelectric focusing from the partially purified laccase obtained here shows six isoforms (called isoform 1 to 6) with the following pH i : 2. 90, 3.05, 3.25, 3.55, 4 and 4.8. The diversity of the laccase isoforms highlighted here is consistent with previous studies [START_REF] Palmieri | A typical laccase isoenzyme from copper-supplemented Pleurotus ostreatus cultures[END_REF][START_REF] Stajic | Effect of different carbon and nitrogen sources on laccase and peroxidise production by selected Pleurotus species[END_REF]. A previous study on this species of basidiomycete [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF] revealed three isoforms with different pH i (4, 4.2 and 4.4) from the constitutive isoform and four others (4.75, 4.85, 4.95 and 5.1) from an isoform induced with phydroxybenzoic acid. This M. quercophilus strain (strain 17) was also isolated from an evergreen oak litter in La Gardiole de Rians (Var, France). However, the present result confirmed that isoelectric focusing can thus be considered as a simple method to describe more precisely enzymatic isoform variations at an intraspecific level. Furthermore, this methodology gives information about the amino acid composition of the protein studied: variations in pH i indicate that the amino acids of the isoforms and thus the protein sequences are different. These differences may influence the biochemical enzyme characteristics, which we partially described by calculating the V m and K m values of these different isoforms using ABTS and 2,6DMP as substrates (Table 1). For all the isoforms (except isoform 5), the K m measured with 2,6 DMP was higher than that with ABTS and V m was always higher with ABTS as the substrate. Thus the affinity of isoforms 1, 2, 3, 4 and 6 for ABTS was stronger than that for 2,6 DMP. Although certain biochemical characteristics, such as molecular weight or optimal pH of activity, are almost the same for many fungal laccases, laccase affinity for susbtrates is variable. For instance a weak laccase affinity for ABTS has been observed in previous studies: [START_REF] Pozdnyakova | Oxidative degradation of polyaromatic hydrocarbons catalyzed by blue laccase from Pleurotus ostreatus D1 in the presence of synthetic mediators[END_REF] found a higher affinity for 2,6 DMP than for ABTS with a laccase produced by Pleurotus ostreatus. On the other hand, [START_REF] Michniewics | The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physicochemical and catalytic properties[END_REF] have found that laccase isoforms from Cerrena unicolor have a higher affinity for ABTS. In further studies, a greater amount of each laccase isoform should be obtained in order to describe more precisely the respective role of each enzyme in xenobiotic oxidation. For instance, their different affinities for ABTS may favour or not PAH transformation yield and thus their respective contribution to global laccase activity may have an effect on PAH oxidation efficiency.

Because most of aromatic pollutants can be dissolved only in organic solvents, the effects of such solvents on laccase activity were assessed in a previous study [START_REF] Farnet | Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF]. The authors have shown that the laccases produced by M. quercophilus were still active in 10% acetone. Therefore, these conditions were used in the present study. Here, only anthracene and benzo(a)pyrene were oxidised by laccase (Table 2) with or without mediator (ABTS or HBT). This result may be explained by the fact that in naphthalene and phenanthrene, electronic delocalisation is observable in two or three condensed cycles respectively while anthracene or benzo(a)pyrene cycles do not exhibit this characteristic. This structural characteristic may make impossible the use of laccases for phenanthrene and naphtalene oxidation. This electronic delocalisation is indeed directly linked to the ionisation potential of a molecule. The ionisation potential of naphtalene and phenanthrene are higher than that of anthracene: respectively 8.1, 8.19 and 7.43 eV. [START_REF] Hammel | Oxidation of polycyclic aromatic hydrocarbons and dibenzo[p]-dioxins by Phanerochaete chrysosporium ligninase[END_REF] have shown that lignine peroxidase from Phanerochaete chrysosporium was able to oxidise only PAH with an ionisation potential b∼ 7.55 eV such as pyrene (IP = 7.5 eV). [START_REF] Field | The tolerance of lignin peroxidase and manganese-dependent peroxidase to miscible solvents and the in vitro oxidation of anthracene in solvent: water mixtures[END_REF] have found the same results with lignine-and manganese-peroxidases from Bjerkandera sp. Thus, when ionisation potentials are available, they can be used to forecast the ability of a laccase to oxidise a molecule. For instance, M. quercophilus laccase is probably unable to oxidise chrysene (IP = 7.8 eV). In further studies, it would be valuable to determine the IP value above which oxidation by the M. quercophilus laccase will be impossible, by testing laccase oxidation of several molecules with IP ranging from 7.5 to 8 eV.

The anthracene oxidative product obtained with M. quercophilus was 9,10-anthraquinone as revealed by the HPLC elution profile (Fig. 1). No traces of other oxidation products were detected with HPLC. 9,10-anthraquinone was also injected in order to confirm the identification of the single product obtained and both the UV scan and the elution profile corroborate our results. No benzo(a)pyrene oxidation products were detected, although this molecule was actually transformed as revealed by the strong decrease in benzo(a)pyrene amount when laccase was used with ABTS (66%, average of three experiments, standard deviation under 2%). Most studies on PAH transformation with laccase have not clearly identified oxidation products: authors have mainly depicted PAH concentration decrease [START_REF] Cho | Oxidation of polycyclic aromatic hydrocarbons by laccase of Coriolus hirsutus[END_REF][START_REF] Dodor | Oxidation of anthracene and benzo[a] pyrene by immobilized laccase from Trametes versicolor[END_REF][START_REF] Potin | Biodegradation of polycyclic aromatic hydrocarbons (PAH) by Cladosporium sphaerospermum isolated from an aged PAH contaminated soil[END_REF][START_REF] Rodríguez | Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase[END_REF][START_REF] Valentín | Biodegradation of polycyclic aromatic hydrocarbons in forest and salt marsh soils by white-rot fungi[END_REF]. [START_REF] Lau | Use of spent mushroom compost to bioremediate PAH-contaminated samples[END_REF], using spent mushroom compost, have found that PAH transformation led to an increase in phtalate concentration. However, this result was not specifically correlated to PAH transformation since phtalates were also extracted from the compost and the authors suggested that it was, in this case, associated with lignocellulose transformation. [START_REF] Hu | Biodegradation of benzo[a]pyrene with immobilized laccase: genotoxicity of the products in HaCaT and A3 Cell[END_REF] have shown the production of quinones (1,6-benzo(a)pyrene quinone, 3,6benzo(a)pyrene quinone and 6,12-benzo(a)pyrene quinone) as well as different non-identified products. Quinone production for both anthracene and benzo(a)pyrene has been linked to nucleophilic attacks and further non-enzymatic arrangements of the molecule, leading to quinones [START_REF] Collins | Oxidation of anthracene and benzo[a]pyrene by laccases from Trametes versicolor[END_REF].

In our reaction mixtures where ABTS was oxidised by laccase, absorption values increase mainly at 425 nm and to a lesser extent from 600 to 800 nm (Fig. 2). When anthracene was added to the reaction mixture, the absorption value observed at 425 nm strongly decreased as well as those from 600 to 800 nm. The reaction mixture, which was green because of the ABTS radical cation formed [START_REF] Bourbonnais | Electrochemical analysis of the interactions of laccase mediators with lignin[END_REF], turned to an almost transparent mixture. This result shows that anthracene oxidation via ABTS actually occurred simultaneously with ABTS reduction. Furthermore, the remaining anthracene percentages were significantly weakest when ABTS or HBT were previously oxidised as shown in Fig. 3 (respectively 20 and 45%). Laccases can use both mediators and anthracene as substrates although mediators are considered to be better substrates than anthracene. Thus pre-oxidation of mediators may limit competition between the two molecules and favour anthracene abiotic oxidation with the oxidised form of the mediator (in the case of ABTS, a radical cation as previously described). With benzo(a)pyrene, ABTS reacted differently: the radical cation did not disappear when benzo(a)pyrene was added to the medium. Furthermore, in the reaction control with both ABTS and benzo(a)pyrene, the ABTS radical cation was produced even though no enzyme was added to the reaction mixture. This is probably due to the products of benzo(a)pyrene oxidation with O 2 : this PAH has a particularly low IP leading to abiotic oxidation such as photooxidation or oxidation via O 2 as previously described [START_REF] Mill | Photolysis of polycyclic hydrocarbons in water[END_REF][START_REF] Miller | Photolysis of polycyclic aromatic hydrocarbons in water[END_REF]. To check this hypothesis, the same experiment (benzo(a)pyrene plus ABTS) was performed in an atmosphere saturated with nitrogen. In this case no ABTS oxidation was observable which is most probably due to the absence of benzo(a)pyrene oxidation by oxygen. These investigations using ABTS as a mediator highlight the fact that this molecule can hardly be considered as a relevant substrate for laccases under particular experiments. For instance, when laccase activities are detected in soils, many abiotic parameters are likely to cause abiotic oxidation of ABTS or reduction of ABTS radical cations since these molecules are very reactive. In soils contaminated with PAH, abiotic reactions due to PAH may occur, leading to a misinterpretation of in situ results. Thus in environmental studies, and moreover in contaminated soils, these parameters must be taken into account when measuring laccase activities. ABTS can hardly be a relevant choice as a substrate for laccases because the electrochemical characteristics make ABTS very reactive towards many molecules.

HBT is not an efficient mediator when used with the M. quercophilus laccase under study. This is supported by the fact that HBT redox potential, E 0 , is higher than that of ABTS (1080 and 475 V/NHE respectively as described by [START_REF] Baiocco | Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase-mediator systems[END_REF] and oxidation with laccase is always favoured when substrates have low E 0 since electrons are transferred from the substrate to the laccase active site. [START_REF] Pickard | Polycyclic Aromatic Hydrocarbon metabolism by white-rot fungi and oxidation by Coriolopsis gallica UAMH 8260 Laccase[END_REF] have also found that HBT was a less efficient mediator than ABTS in anthracene oxidation.

In order to quantify anthracene disappearance in M. quercophilus culture, both culture medium and mycelium were extracted (the mycelium was extracted to check whether any adsorption occurred). Furthermore, we took into account anthracene loss after sterilisation, which was 7.5% (± 0.8%), to calculate percentage of anthracene disappearance in M. quercophilus culture. These percentages were 73 and 79% (standard deviation under 1.5) after 8 and 15-day incubation time respectively. Anthracene oxidation was mainly due to laccase activity alone since neither manganese peroxidase nor lignine peroxidase activities were detected in the culture medium (data not shown). This result corroborates those found in a previous study [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid reactivity towards natural and xenobiotic aromatic compounds[END_REF], with another M. quercophilus strain, where no peroxidise activities were detected. Here, the percentage of anthracene disappearance was quite similar after 8-and 15-day incubation time (73 and 79%). This result may be explained by a peak of laccase production: laccase activity was indeed measured throughout the culture and showed that he highest activity (8 U L -1 ) was observed after 7 days of culture. Then, laccase activity progressively decreased and after 15 days of culture it was 3 U L -1 . The weak solubility of anthracene in water did not affect laccase oxidation of this molecule by decreasing its bioavailability. While acetone was added to the reaction mixture with the purified laccase to favour solubilisation, no solvent was used in fungal cultures to prevent toxic effects of the solvent on the mycelial growth.

The present study highlights the importance of fungal extracellular oxidative enzyme in pollutant degradation. [START_REF] Steffen | Enhancement of bioconversion of high molecular mass polycyclic aromatic hydrocarbon in contaminated non-sterile soils by litter-decomposing fungi[END_REF] have shown the role of various species of litter decomposing fungi in high molecular mass PAH transformation in soil. This transformation was indeed enhanced by fungi (such as Stropharia spp.) while autochthonous microorganisms had a weaker transformation potential (average transformation percentage of 80 and 20 respectively). Furthermore, our study gives evidence of infraspecies laccase diversity which was assessed using isoelectric focusing. This result corroborates those found in previous studies on laccase gene diversity in a forest soil [START_REF] Luis | Diversity of laccase genes from basidiomycete in a forest soil[END_REF] and thus, further studies should concentrate in correlating both protein and gene functional diversity. Moreover, it would be valuable to perform in situ extraction of laccases from litter in order to identify, after protein sequencing, the fungi which actually produced laccases in forest litter.

Moreover, this in vitro study has shown that measuring oxidative activities can lead to misinterpretations depending on the conditions used since PAH are responsible for abiotic reactions towards laccase substrate. In a previous study [START_REF] Farnet | Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF], we have shown that abiotic oxidation or reduction may occur on the laccase substrate with different metal ions. Thus, in complex environments such as soils or litters where many molecules can interact with the enzyme substrate, results on laccase activities and other phenoloxidases should be considered with caution. Further studies should concentrate in determining a laccase substrate which oxidation leads to a stable product to limit at least abiotic reduction.

Fig. 2 .

 2 Fig. 2. Spectral scan of the reaction mixture during anthracene oxidation with the constitutive laccase of Marasmius quercophilus: ABTS alone (---), ABTS and laccase (-), ABTS, laccase and anthracene (---). The arrows indicate the peaks corresponding to ABTS, to the oxidise form of ABTS and to anthracene.

Fig. 3 .

 3 Fig. 3. Anthracene oxidation with the constitutive laccase of Marasmius quercophilus, with or without mediators (ABTS and HBT) and with or without a preoxidation of ABTS and HBT (PREOX_ABTS, PREOX_HBT). Results are expressed as percentages of remaining anthracene detected with HPLC (average of three experiments). Stars indicate significant differences (P b 0.05) between means of percentage of remaining anthracene with or without pre-oxidation of the mediator used.

Fig. 1 .

 1 Fig. 1. Elution profile at 254 nm of the reaction mixture with anthracene and the constitutive laccase of Marasmius quercophilus after extraction and solubilisation in acetone with UV scan of 9,10-anthraquinone and anthracene respectively (a and b).

Table 1 V

 1 m and K m (using ABTS and 2,6-DMP) and specific activity of the different laccase isoforms of the constitutive laccase of Marasmius quercophilus found after isoelectric focusing

			pH i
			2.99	3.06	3.24	3.55	4	4.78
	ABTS	K m	0.78	0.667	0.755	0.521	0.378	0.136
		V m	7.61	7.593	8.025	6.94	4.82	3.017
	2,6 DMP	K m	0.911	0.988	1.13	0.813	0.12	0.285
		V m	4.766	4.372	3.462	2	0.81	1.037
	Specific activity		1.504	0.623	0.296	0.161	0.057	0.141

Table 2

 2 Ionisation potential and solubility in water of different PAH and oxidation of these compounds with Marasmius quercophilus laccase

	PAH	lonisation potential	Solubility in water c	Oxidation
		(eV)	(mg L -1 )	with M.
				quercophilus
				laccase
	Naphtalene	8.1 a	0.3	-
	Phenanthrene	8.19 b	1	-
	Chrysene	7.8 b	0.0018	ND
	Anthracene	7.43 b	0.045	+
	Benzo(a)pyrene	7.23 b	0.0016	+
	ND: Non-determined.

a Bernal-Martinez et al. (2007).

b

[START_REF] Roseen | External partial discharge resistance of polyethylene and cross linked polyethylene containing voltage stabilizers[END_REF]

. c

[START_REF] Hammel | Oxidation of polycyclic aromatic hydrocarbons and dibenzo[p]-dioxins by Phanerochaete chrysosporium ligninase[END_REF]

.

Acknowledgments

We would like to thank Mrs Marjorie Sweetko for her very helpful assistance in English language for all these years. This work was financially supported by AQUAPROX (France).


Laurent Puech

Ram Karthik

Nicolas Ramakrishnan

Stéphane Le Moigne

Pierre Corn

Anne Slangen

Hassane Le Duc

Anne Boudhani

Bergeret

Karthik Ram Ramakrishnan
email: karthik.ramakrishnan@tut.fi

Nicolas Le Moigne

Stéphane Corn
email: stephane.corn@mines-ales.fr

Pierre R Slangen

Anne Le Duc

Hassane Boudhani

Anne Bergeret

Ramakrishnan

Investigating the impact behaviour of short hemp fibres reinforced polypropylene biocomposites through high speed imaging and finite element modelling

Keywords: A. Biocomposite, B. Impact behaviour, C. Finite element analysis (FEA), D. Mechanical testing, (Additional keyword: High speed imaging)

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The use of natural fibres as reinforcements for polymer composites has gradually increased in recent years mainly because of two environmental assets: their low energy demand for production and their renewability [START_REF] Mohanty | Natural fibres, biopolymers and biocomposites: an introduction[END_REF]. Moreover, natural fibres present interesting physical properties when compared to manmade reinforcement fibres such as glass fibres: their low density leads to highly competitive specific mechanical properties in addition to good viscoelastic damping performances [START_REF] Bledzki | Composites reinforced with cellulose based fibres[END_REF][START_REF] Hepworth | The use of unretted hemp fibre in composite manufacture[END_REF][START_REF] Karus | Natural fibres in the European automotive industry[END_REF]. These properties make natural fibres good substitutes to glass fibres in many industrial applications. For instance, automotive specifications require materials with good impact properties as the parts designed are usually subjected to damages caused by bumps, crashes or falling objects. It is difficult to assess the impact resistance of composites due to the complex damage modes related to the bulk and interfacial properties of matrix and fibres that lead to matrix failure, fibre breakage and fibre pull-out. Thus the analysis of failure mechanisms in composites subjected to impact is of major interest.

Several authors compared the impact behaviour of natural fibre reinforced composites to traditional glass fibre ones [START_REF] Patel | Hemp fibre as alternative to glass fibre in sheet moulding compound. Part 2-impact properties[END_REF][START_REF] Scarponi | Impact load behaviour of resin transfer moulding (RTM) hemp fibre composite laminates[END_REF][START_REF] Santulli | Mechanical and Impact Properties of Untreated Jute Fabric Reinforced Polyester Laminates Compared With Different E-Glass Fibre Reinforced Laminates[END_REF][START_REF] Santulli | Partial replacement of E-glass fibers with flax fibers in composites and effect on falling weight impact performance[END_REF][START_REF] Santulli | A Comparative Study on Falling Weight Impact Properties of Jute / Epoxy and Hemp / Epoxy Laminates[END_REF][START_REF] Caprino | Damage and energy absorption in GFRP laminates impacted at low-velocity: Indentation model[END_REF][START_REF] Caprino | Low impact behaviour of hemp fibre reinforced epoxy composites[END_REF][START_REF] Caprino | Irreversibly absorbed energy and damage in GFRP laminates impacted at low velocity[END_REF]. Among them, Patel et al. [START_REF] Patel | Hemp fibre as alternative to glass fibre in sheet moulding compound. Part 2-impact properties[END_REF], Scarponi et al. [START_REF] Scarponi | Impact load behaviour of resin transfer moulding (RTM) hemp fibre composite laminates[END_REF], and Santulli et al. [START_REF] Santulli | Mechanical and Impact Properties of Untreated Jute Fabric Reinforced Polyester Laminates Compared With Different E-Glass Fibre Reinforced Laminates[END_REF][START_REF] Santulli | Partial replacement of E-glass fibers with flax fibers in composites and effect on falling weight impact performance[END_REF][START_REF] Santulli | A Comparative Study on Falling Weight Impact Properties of Jute / Epoxy and Hemp / Epoxy Laminates[END_REF] showed that for glass fibre reinforced epoxy and polyester resins, peak loads and absorbed energies at breakage were higher than those of hemp fibre reinforced composites, but the latter exhibited a higher strain at break. Santulli et al. [START_REF] Santulli | Mechanical and Impact Properties of Untreated Jute Fabric Reinforced Polyester Laminates Compared With Different E-Glass Fibre Reinforced Laminates[END_REF][START_REF] Santulli | Partial replacement of E-glass fibers with flax fibers in composites and effect on falling weight impact performance[END_REF][START_REF] Santulli | A Comparative Study on Falling Weight Impact Properties of Jute / Epoxy and Hemp / Epoxy Laminates[END_REF] postulated that the difference in absorbed energy between natural fibres (jute, flax and hemp) and glass reinforced epoxy composites was due to a shorter elastic phase upon impact loading in the case of biocomposites leading to lower peak loads as compared to glass fibre reinforced composites. These authors also found out that unlike the glass reinforced composites, the loading phase of the biocomposites is slightly curved which means it suffered internal damage, although it could not be seen at the surface. In those materials, failure was mainly driven by matrix cracking and the sample does not undergo penetration until a large amount of matrix cracking takes place and leads to fibre breakage and pull-out which occur at higher absorbed energy. Biocomposites could thus exhibit a good penetration resistance even after damage initiation. Besides, Patel et al. [START_REF] Patel | Hemp fibre as alternative to glass fibre in sheet moulding compound. Part 2-impact properties[END_REF] also found that for low fibre volume fraction (< 20 vol%), the differences in absorbed energy between glass and natural fibres composites were not significant. Caprino et al. [START_REF] Caprino | Damage and energy absorption in GFRP laminates impacted at low-velocity: Indentation model[END_REF][START_REF] Caprino | Low impact behaviour of hemp fibre reinforced epoxy composites[END_REF][START_REF] Caprino | Irreversibly absorbed energy and damage in GFRP laminates impacted at low velocity[END_REF] pointed out that epoxy-hemp biocomposites needed higher impact energy to initiate damage when compared to epoxy-glass composites. This result suggests that natural fibre composites can have comparable impact damage tolerance as glass fibre composites. Moreover, an interface treatment such as alkaline, silane or a combination of both could even increase the energy absorbed by the biocomposites upon impact [START_REF] Patel | Hemp fibre as alternative to glass fibre in sheet moulding compound. Part 2-impact properties[END_REF]. Most of the studies dealing with impact behaviour of natural fibres reinforced composites were mainly based on uniaxial impact tests such as Izod or Charpy tests [START_REF] Shahzad | Comparison of tensile properties and impact damage tolerance of hemp and glass fiber composites[END_REF][START_REF] Pickering | A review of recent developments in natural fibre composites and their mechanical performance[END_REF]. There is limited literature dealing with biaxial impact tests of natural fibre composites performed on drop-weight impact machine. Moreover, if low velocity impact tests were conducted on such composites [START_REF] Mandal | Short jute fiber reinforced polypropylene composites: effect of compatibiliser, impact modifier and fiber loading[END_REF][START_REF] Bledzki | Impact properties of natural fibre reinforced epoxy foams[END_REF], very few authors tried to correlate their impact behaviour to crack propagation. Nevertheless, some works attempted to correlate the impact energy to the damaged area, i.e. in the case of long fibre composite the delamination area, but the analyses were only based on post-mortem observations and not on the tracking of crack propagation during the impact test [START_REF] Scarponi | Impact load behaviour of resin transfer moulding (RTM) hemp fibre composite laminates[END_REF][START_REF] Caprino | Irreversibly absorbed energy and damage in GFRP laminates impacted at low velocity[END_REF][START_REF] Vasconcellos | Influence of low velocity impact on fatigue behaviour of woven hemp fibre reinforced epoxy composites[END_REF][START_REF] Scarponi | Low-velocity impact behaviour of hemp fibre reinforced bio-based epoxy laminates[END_REF].

The numerical simulation of the response of composite structures to impact loads is of particular interest for automotive applications related to passenger and pedestrian safety as these materials are increasingly being used in critical applications and structures [START_REF] Daiyan | Numerical simulation of low-velocity impact loading of a ductile polymeric material[END_REF]. The numerical modelling of fibre reinforced polymer composites is possible at different scales: micro, meso and macro-scale [START_REF] May | Adaptive multi-scale modeling of high velocity impact on composite panels[END_REF]. Sliseris et al. [START_REF] Sliseris | Numerical modelling of flax short fibre reinforced and flax fibre fabric reinforced polymer composites[END_REF] described the microstructural characteristics of short flax fibre and flax fabric in their numerical model and demonstrated that the simulation was able to capture the main damage mechanisms of the composite. Some researchers [START_REF] Liebold | Recent enhancements on short-fiber reinforced plastics modeling in LS-DYNA[END_REF][START_REF] Kebir | A specific finite element procedure for the analysis of elastic behaviour of short fibre reinforced composites. The Projected Fibre approach[END_REF] determined the anisotropic and locally varying effective elastic properties of short fibre reinforced plastics using the elastic properties of the fibre and the matrix and a homogenisation algorithm that mapped the orientation distribution of the fibres. Nutini and Vitali [START_REF] Nutini | Simulating anisotropy with LS-DYNA in glass-reinforced , polypropylene-based components[END_REF] recommended using an anisotropic, non-linear and strain-rate dependent material law that already exists in the library of commercial Finite Element (FE) codes and obtained useful and reliable results with limited computational cost for a range of benchmark problems. Chethan et al. [START_REF] Chethan | Experimental and Numerical Modeling of Hemp-Polyester Composites[END_REF] recommended that a more viable tool for analysing industrial components was a macro-mechanical modelling approach based on the experimental non-linear stress-strain behaviour of the composites up to breakage. Isotropic models that neglect the microstructural features and the directional dependency of the material properties and instead use preferential flow direction properties were still able to provide designers with predictive knowledge of the problem. Sandberg and Rydholm [START_REF] Sandberg | Evaluation of Material Models to Predict Material Failure in LS-DYNA[END_REF] investigated the impact response during two complex load cases, and compared the results of a simple elasto-plastic material law and failure model with a more complex crash model and showed that the elasto-plastic model gave similar material behaviour prediction and had the potential to reduce simulation time. Hosseinzadeh et al. [START_REF] Hosseinzadeh | Damage behavior of fiber reinforced composite plates subjected to drop weight impacts[END_REF] reported that a macroscopic model is capable of predicting the threshold of damage, which is a good design criterion for composites. Chethan et al. [START_REF] Chethan | Experimental and Numerical Modeling of Hemp-Polyester Composites[END_REF] used a bilinear material model defining yield strength and tangent modulus for hemp polyester composites and found good agreement between experiments and simulations for tensile tests.

The aim of this study is to develop a methodology for the characterization of the impact behaviour of short natural fibre reinforced biocomposites through the combination of experimental impact testing, high speed imaging and finite element modelling. Polypropylene (PP) -hemp fibres biocomposites and PP -glass fibres composites with identical fibre volume fraction produced by twin-screw extrusion and injection moulding were chosen as material for this study. A drop-weight impact machine was used to characterize the behaviour of the composites over a wide range of impact energies (from 3 to 126 J). Moreover, the implementation of a high speed camera during the impact test enabled the measurement of macro-cracks propagation and the tracking of the crack tip throughout the impact test. Based on these results and the tensile behaviour of the composites, a three-dimensional finite element model study was conducted so as to predict the impact behaviour of the composites.

Materials and methods

Materials and composites manufacturing

Polypropylene (PP) supplied by Faurecia Interior Systems (FIS, Meru, France) was used as the thermoplastic matrix. The mechanical and physical properties of the PP matrix are listed in Table 1.

Tensile properties of the composites

Tensile mechanical properties of the biocomposites were measured with a Zwick TH 0101 testing machine equipped with an extensometer Zwick "clip-on" for the determination of the Young's modulus. The displacement speed was set to 1 mm/min and 20 mm/min for the determination of the Young's modulus and ultimate stress respectively according to ISO 527 standard. Ten samples were tested for each composite.

Typical tensile curves of PP-H and PP-G composites are shown in Figure 1. Table 2 reports the Young's modulus, ultimate stress and strain at break values for the two composites. It should be pointed out that PP-H and PP-G composites present little and comparable standard deviations in their tensile properties.

Characterization of the impact behaviour of the composites

Impact tests were conducted on an Instron CEAST 9340 drop-weight impact machine allowing the impact force monitoring. The impact kinetic energy was varied by modifying the impactor height and weight and the resulting impact energies were 3, 6, 8, 9, 12 and 126 J. The samples were mounted, centred and clamped by a 40 mm diameter steel flanges fixture. They were then struck at their centre by a cylindrical steel striker with a hemispherical tip of 20 mm diameter. Five samples of each material were tested for the six impact energies. The force-time and force-displacement curves obtained were analysed. A visual evaluation of the broken samples was conducted in order to determine the critical energy needed for full penetration.

A high speed camera Phantom V711 (Vision Research -Ametek Materials Analysis Division) was added to the drop-weight impact set up enabling to capture 8000 fps (frames per second) videos with a for each frame as it can be seen in Figure 3. Thus, the total crack length at any time L crack (t) is given by Equation (1):

L crack (t) = OA(t) + OB(t) + OC(t) + OD(t) + Oi(t) (1)
Knowing that each video time code was synchronised to the contact onset, a correlation was established between the events occurring on the force / time / displacement curves obtained by the CEAST Data Acquisition and Analysis system (DAS) and the appearance of damages on the samples observable on the recorded images.

Failure surface observations

Failure surfaces of the broken samples obtained from the impact tests were observed in the thickness of the samples using a scanning electron microscope (SEM) (Quanta FEG 200, FEI Company). Samples were previously carbon coated and the acceleration voltage was set to 15 keV. The adhesion at the fibre/matrix interface was assessed on samples broken with impact energy of 12 J.

Three-dimensional finite element modelling

The impact response of the glass fibre and hemp fibre reinforced polypropylene plate was modelled using the Finite Element software LS-DYNA. LS-DYNA is the desired FE code for the low velocity impact analysis of composite structures as it is a general-purpose FE code for analysis of large deformation dynamic response of structures based on explicit time integration (LS-DYNA manual [START_REF] Hallquist | LS-DYNA User's Manual[END_REF]). The geometry of the FE model developed in LS-Prepost for the simulation of impact response of the composite plate is shown in The impactor hemispherical tip was modelled with 6355 tetrahedral elements, while the composite target was represented by 77768 thick shell elements with element length of 0.22 mm at the centre and 2 mm length in the periphery. The mesh on the composite plate was refined to have an adequately fine enough mesh in the central region of interest, i.e. areas of high stress gradients or large deformations. A mesh sensitivity analysis was conducted and this element size was the best compromise between calculation time and accuracy of the local material fracture representation.

The simplest models consider only the elasticity of the composite but tensile testing of the composite indicated that its behaviour is non-linear. Thus, a compromise solution is the use of Piecewise Linear Plasticity model available in the material library as MAT24. The elasto-plastic material constitutive law is based on the stress-strain curves obtained from the experimental tensile testing. The elastic Young's modulus defines the stress-strain relation up to the yield stress and above the yield stress, a curve defining effective stress and effective plastic strain coordinates is used to describe the plastic behaviour. An effective plastic strain criterion was used to define the erosion of elements, and hence the on-going damage within the plate.

If the calculated effective plastic strain for any element exceeds the predefined value, the element is removed from the model and the simulation continues with the eroded model, i.e. the damaged plate.

An initial velocity is defined to the nodes of the hemispherical impactor. The density of the steel impactor is modified to obtain the same kinetic energy as in the experiments. Four initial velocities, 1.4, 1.98, 2.2 and 2.8 m/s were simulated that correspond to initial impact energies of 3, 6, 9 and 12 J, respectively. To reduce the runtime, all simulations started with the impactor situated just 0.5 mm above the plate. An Automatic Surface to Surface contact was defined between the impactor and the composite plates.

The time-history of the kinetic energy of the impactor and the internal energy of the composite target obtained from the LS-DYNA model is shown in Figure 5. The initial energy of the impactor is 3.07 J for this case and the corresponding internal energy at t 0 is zero. As the impactor comes into contact with the target, there is energy transfer between the impactor and the composite. At 5.4 ms, the kinetic energy of the impactor reaches zero at peak displacement and there is rebound afterwards. It can be seen that the internal energy of the target does not reach the initial kinetic energy of 3.07 J as some of the energy is lost as sliding energy and hourglass energy in the model. During the rebound phase, some of the energy of the plate is returned to the impactor. It can be observed that for the 3 J impact, there is considerable energy 0.57 J that is returned to the impactor. The final internal energy of the plate of 2.4 J corresponds to the energy dissipated in the plate in plastic deformation and damage. In the case of fully perforating impact, there is no rebound and the kinetic energy of the impactor remains zero.

Results and discussions

3.1. Experimental study of impact behaviour using drop-weight impact tests

Influence of impact energy

Impact tests at increasing energy were performed in order to identify the critical impact energy at which the specimens were totally penetrated by the impactor. Figure 6 shows impacted PP-H and PP-G samples ordered by impact energy levels from 3 to 12 J. Three main tendencies can be drawn according to energy level: (i) at low impact energies, i.e. 3 to 6 J, the impacted specimen exhibit macro-cracks of moderate length that can be identified in the white zones related to crazing and cracking of the PP matrix; (ii) for intermediate energies, i.e. 9 J for PP-H and from 6 to 9 J for PP-G, macro-cracks reached their maximum lengths at the edge of the clamping steel plate; (iii) at high impact energies, i.e. 12 J and more, macro-cracks were widely opened due to the penetration of the impactor through the sample. . Tests performed at low impact energies highlight a force increase as the impactor is in contact with the samples followed by a smooth decrease when the impactor leaves contact with the surface of the plate, which can be attributed to a rebound phase. This phenomenon occurred at 3 and 6 J for PP-H and only 3 J for PP-G composite as expected from the visual observations in Figure 6. Similar results were also reported by Santulli et al. [START_REF] Santulli | Mechanical and Impact Properties of Untreated Jute Fabric Reinforced Polyester Laminates Compared With Different E-Glass Fibre Reinforced Laminates[END_REF][START_REF] Santulli | Partial replacement of E-glass fibers with flax fibers in composites and effect on falling weight impact performance[END_REF][START_REF] Santulli | A Comparative Study on Falling Weight Impact Properties of Jute / Epoxy and Hemp / Epoxy Laminates[END_REF], studying the impact behaviour of jute, hemp and flax fibres reinforced thermoset resins. Besides, the remaining whitening zone in the centre of the material corresponds to the irreversible crazing and cracking mechanisms that occurred within the PP matrix. Tests performed at intermediate impact energies, i.e. between 8 J and 9 J for PP-H and between 6 and 9 J for PP-G composite, exhibit a different behaviour as the smooth decrease in force related to rebound is not visible anymore. Indeed, a sudden drop in force is observed. At higher impact energies (9 J and above), the loading phase was followed by a drastic drop in force and a total penetration of the impactor through the samples. This drop in force is related to major damages within the material, either delamination (in the case of woven reinforced composites) or matrix cracking and fibre breaking / pull-out, as described by numerous authors [START_REF] Kontermann | Notch Support for LCF-Loading: A Fracture Mechanics Approach[END_REF][START_REF] Wu | The impact properties and damage tolerance and of bi-directionally reinforced fiber metal laminates[END_REF][START_REF] González | Simulation of drop-weight impact and compression after impact tests on composite laminates[END_REF]. Comparison of PP-H and PP-G for impact energy 6 J.

The contact force-displacement curves for the PP-H and PP-G composites subjected to 6 J and 12 J impact are presented in Figure 8. Three main loading phases can be identified: (i) a linear domain at low deformation (i.e. shorter time) which corresponds to the elastic response of the materials, (ii) a non-linear domain at higher deformation which corresponds to the plastic strain of the materials and (iii) for high impact energy (above 9 J), a final breakage phase which is characterized by a drastic drop of the force. As pointed out by Santulli et al. [START_REF] Santulli | Mechanical and Impact Properties of Untreated Jute Fabric Reinforced Polyester Laminates Compared With Different E-Glass Fibre Reinforced Laminates[END_REF], an obvious higher elasticity of PP-G composite can be noticed when considering the slope of the contact force-time and displacement curves in the linear domain. Besides, even if different impact velocities were used to obtain each level of impact energy, it appears that the curves were superimposed in the linear domain, hence demonstrating that at this loading speed the pronounced viscoelastic character of the PP matrix had no significant influence on the response in the elastic domain. In order to measure the absorbed energy upon impact, the force-displacement curves were divided into two different loading phases. In the first phase, the force increases until its maximum value (F max ) followed by either (i) a slow decrease of the force corresponding to rebound or (ii) a sudden drop of the force corresponding to the breakage of the material. The area under the curve up to the displacement at F max corresponds to the absorbed energy upon impact. Various disturbances on the curves can be observed in this first phase suggesting the development of minor damages within the material. In the second phase, the force decreases smoothly (i) with negative displacement corresponding to rebound (low impact energy), (ii) or with non-regular force oscillations and positive displacement (intermediate and high impact energies) due to the friction of the impactor passing through the broken specimens, hence dissipating residual energy. The area under the force/displacement curve of this second phase (any positive displacement after reaching F max ) is thus considered as the energy absorbed due to frictions [START_REF] Caprino | Irreversibly absorbed energy and damage in GFRP laminates impacted at low velocity[END_REF]. Although all materials exhibited the same kind of force/displacement response within the same range of impact energies, it should be noticed that PP-H composites fully broke at 9 J whereas PP-G composite broke at threshold energy of 8 J. This suggests that PP/hemp biocomposites may be submitted to higher impact energies than PP/glass composites without complete failure.

Table 3 presents the peak force (F max ) and the displacement at F max for both composites as a function of the impact energy. For a given impact energy, the maximum impact force (F max ) reached by the PP-G composite tends to be higher than those of PP-H biocomposites, e.g. 20% higher at 12 J with 2099 ± 17 N and 1753 ± 31 N for PP-G and PP-H, respectively. On the other hand, the deformations at break for PP-G composite are lower than those of PP-H biocomposites, e.g. 50% higher at 12 J with 3.52 ± 0.2 % and 5.4 ± 0.4 % for PP-G and PP-H respectively. Those results are believed to be related to differences in fibre stiffness and strain. Indeed, several authors showed a significant difference between hemp and glass fibres Young's modulus ranging from 14 to 70 GPa [START_REF] Beckermann | Engineering and evaluation of hemp fibre reinforced polypropylene composites: Fibre treatment and matrix modification[END_REF][START_REF] Duval | Influence of the sampling area of the stem on the mechanical properties of hemp fibers[END_REF][START_REF] Placet | Characterization of the thermo-mechanical behaviour of Hemp fibres intended for the manufacturing of high performance composites[END_REF][START_REF] Placet | Diameter dependence of the apparent tensile modulus of hemp fibres: A morphological, structural or ultrastructural effect? Compos Part A[END_REF] and 73 GPa [START_REF] Beukers | Lightness: The inevitable renaissance of minimum energy structures[END_REF][START_REF] Davis | Tensile testing. 2nd Editio[END_REF], respectively. Moreover, although hemp fibres have a lower elongation at break than glass fibres due to local defects and weak points (1.6-3%

[33] and 4.6-4.8% [START_REF] Guillon | Fibres de verre de renforcement[END_REF] respectively), their composites show an opposite trend with significantly higher elongation at break (see tensile curves for PP-H and PP-G in Figure 1). It can be assumed that the higher stiffness of glass fibres tends to reduce the ductility of the composite during the impact. Differences in the quality of interfacial adhesion and failure mechanisms could also be responsible for the lower elongation at break measured for glass fibres composites. In other studies, higher deformation at break and significantly lower peak load were also measured for various hemp fibre reinforced composites. Santulli et al. [START_REF] Santulli | Mechanical and Impact Properties of Untreated Jute Fabric Reinforced Polyester Laminates Compared With Different E-Glass Fibre Reinforced Laminates[END_REF][START_REF] Santulli | Partial replacement of E-glass fibers with flax fibers in composites and effect on falling weight impact performance[END_REF][START_REF] Santulli | A Comparative Study on Falling Weight Impact Properties of Jute / Epoxy and Hemp / Epoxy Laminates[END_REF] compared the impact behaviour of a flax / epoxy composite to a glass / epoxy composite with similar weight ratio reinforcement on a drop-weight impact test, and found a better impact resistance for the glass reinforced ones. Patel et al. [START_REF] Patel | Hemp fibre as alternative to glass fibre in sheet moulding compound. Part 2-impact properties[END_REF] and Scarponi et al. [START_REF] Scarponi | Impact load behaviour of resin transfer moulding (RTM) hemp fibre composite laminates[END_REF] also showed that glass fibres reinforced polyester and epoxy composites have a slightly higher energy absorbed at breakage than hemp reinforced biocomposites. However at low fibre volume fraction (< 20 vol%), the difference was much smaller. Caprino et al. [START_REF] Caprino | Low impact behaviour of hemp fibre reinforced epoxy composites[END_REF][START_REF] Caprino | Irreversibly absorbed energy and damage in GFRP laminates impacted at low velocity[END_REF] also noted a higher absorbed energy at failure for carbon or glass reinforced composites when compared to biocomposites, even if a higher impact energy was needed to initiate damages in hemp fibres reinforced epoxy composite.

Nevertheless, it should be noticed that in several studies, fibre volume fractions are different, i.e. 50 vol% for carbon fibres reinforced composites, 47 vol% for glass fibres reinforced ones and 30-35 vol% for hemp fibres reinforced ones, which does not allow to draw clear conclusions on the reinforcing effect of hemp fibres. Moreover, all the above described observations were performed on the total absorbed energy during the impact tests including the friction phase. In the present study, only the absorbed energy until F max was considered as the remaining absorbed energy is widely influenced by the friction properties of the impactor and the broken material and hence less representative of the impact resistance of the material.

Figure 9 shows the evolution of the absorbed energy at F max as a function of the impact energy. It can be easily observed that a plateau is reached above 12 J for both PP-H and PP-G composites. Considering the standard deviations in the transitory phase (between breakage and full impactor penetration: 8-12 J for PP-H composite and 6-12 J for PP-G composite), it is evident that once the maximum energy that can be absorbed by the material is reached, further increasing the initial impact energy does not produce corresponding increase in absorbed energy. This is proved by the 126 J impact case, and it can be seen that the same amount of energy is absorbed from 8 to 126 J for PP-H and PP-G composites. This result evidences the existence of three different breaking responses depending on the impact energy. At low impact energy (up to 6 J), radial macro-cracks are initiated and slightly propagated. The sample does not undergo a major failure and the absorbed energy is limited. At intermediate energy (from 6 to 9 J), cracks are fully propagated but the impact energy is not sufficient to fully penetrate the sample. Nevertheless, the material undergoes a major failure leading to a sharp force drop and the total energy that can be absorbed by the material under breakage is reached. Finally, at high impact energy, macro-cracks are fully propagated and the material exhibits the same drop in force. The higher impact energy leads to a complete penetration of the impactor within sample but the absorbed energy is similar since no additional macro-cracks propagate. Concluding, by comparing PP-H and PP-G composites at the same fibre volume fraction (12 vol%), we found that the absorbed energy until F max for PP-G composite is much lower than for PP-H composite (highest difference of 40 % being reached at 12 J). This is believed to be due to a better energy dissipation within the biocomposite microstructure related to higher plastic strain and a potential interfacial decohesion phenomenon attributed to a poor adhesion at PP / fibre interface [START_REF] Thio | Role of interfacial adhesion strength on toughening polypropylene with rigid particles[END_REF].

Failure surface observations and adhesion at the fibre /matrix interface

An analysis of literature data on the interfacial shear strength (IFSS) of PP / hemp fibres and PP / glass fibres, as measured by fibre microbond or pull-out tests, revealed that IFSS ranges from 3.3 to 5.8 MPa for PP / hemp [START_REF] Li | Determination of interfacial shear strength of white rot fungi treated hemp fibre reinforced polypropylene[END_REF][START_REF] Park | Interfacial evaluation of modified Jute and Hemp fibers/polypropylene (PP)-maleic anhydride polypropylene copolymers (PP-MAPP) composites using micromechanical technique and nondestructive acoustic emission[END_REF], whereas PP / sized glass fibres have IFSS values ranging from 8 to 10.2 MPa [START_REF] Gentles | Investigation of Interfacial Strength Between Polyethylene Terephthalate Fibre and Maleic Anhyrdide Modified Polypropylene[END_REF]. This significant difference can be primarily explained by the fact that commercial grades of glass fibres are sized to be compatible with the host matrix while it is not the case with hemp fibres. Indeed, PP / bare glass fibres has an IFSS similar to PP / hemp, i.e. ranging from 3 to 6 MPa, hence highlighting the important role of the sizing on the IFFS of PP/ glass [START_REF] Gentles | Investigation of Interfacial Strength Between Polyethylene Terephthalate Fibre and Maleic Anhyrdide Modified Polypropylene[END_REF][START_REF] Yang | Interface strength in glass fibre-polypropylene measured using the fibre pullout and microbond methods[END_REF].

Failure surfaces in the thickness of PP-H and PP-G samples broken at 12 J were observed by SEM so as to qualify the interfacial adhesion between the matrix and the two fibre types. As seen in Figure 10, no clear difference was found between the failure surfaces obtained with the untreated hemp fibres and the sized glass fibres, as they all primarily exhibit fibre pull-out phenomena and matrix failures with few fibres breakage. Moreover, no residue of polymer matrix could be found onto fibres surface even for PP-G composites, although glass fibre sizing was formulated for PP matrix. There observations conflict with the significant differences in IFSS values reported in literature for PP / glass and PP / hemp. However, it should be pointed out that micromechanical tests (microbond / pull out tests) used to determine IFSS for short fibres reinforced composites are usually conducted at very low strain rates, whereas impact testing involve very high strain rates. Our observations thus highlight that the quality of interfacial adhesion and its role on composite damage could be strain rate dependent. In this regard, Thio et al. [START_REF] Thio | Role of interfacial adhesion strength on toughening polypropylene with rigid particles[END_REF] found that the positive relation between impact toughness and weaker adhesion in PP / glass particles systems became less pronounced as strain rate was increased. Nevertheless, the authors could not conclude on the origin of this strain rate dependency, being either related to the debonding mechanism itself or to the plastic strain characteristics of the matrix polymer.

In any case, our SEM observations are characteristic of a weak interfacial adhesion which could greatly influence the absorbed energy at breakage. Indeed, Russo and its co-authors [START_REF] Simeoli | The role of interface strength on the low velocity impact behaviour of PP/glass fibre laminates[END_REF][START_REF] Sorrentino | Mechanical performance optimization through interface strength gradation in PP/glass fibre reinforced composites[END_REF][START_REF] Boccardi | Effects of interface strength gradation on impact damage mechanisms in polypropylene/woven glass fabric composites[END_REF] showed that such weak interfaces lead to adhesive interfacial failure and higher strain at break during impact promoted by fibres slipping that is known to dissipate a considerable amount of energy. George et al. [START_REF] George | Surface Modification to Improve the Impact Performance of Natural Fibre Composites[END_REF] have shown for flax fibres reinforced epoxy composites that interfacial compatibilisation can lead to lower impact strength.

For these authors, cohesive interfaces obtained by fibre treatments such as organosilanes only promoted increases in tensile stiffness and strength to the detriment of impact properties. In contrast, Patel et al. [START_REF] Patel | Hemp fibre as alternative to glass fibre in sheet moulding compound. Part 2-impact properties[END_REF] found that fibre surface treatments (silane and alkaline) promoting interfacial cohesion led to higher absorbed energy upon impact for hemp fibre reinforced polyester composites.

The differences in fibre shape factors (length over diameter) could also influence the stress transfer mechanisms upon loading and hence the impact behaviour. A microstructural analysis was performed on PP-G and PP-H composites (not shown). We found median fibre shape factors of 3.5 and 2.7 for PP-G and PP-H, respectively. Nevertheless, in both cases, fibre lengths were far from the critical fibre length for optimal stress transfer. Besides, Oksman et al. [START_REF] Oksman | The influence of fibre microstructure on fibre breakage and mechanical properties of natural fibre reinforced polypropylene[END_REF] pointed out that fibre shape factor has no critical influence when the interfacial adhesion is poor as is the case in this study. Based on SEM micrographs, the failure of PP-H and PP-G composites thus appears dominated by matrix failure and fibre pull-out, independent of the fibre type.

Impact behaviour analysis using high speed imaging

Description of impact events: image analysis versus force-displacement curves

A high speed imaging acquisition system has been implemented on the drop-weight impact machine so as to correlate the occurrence of events on the force-displacement curves and appearance and propagation of macro-cracks in the composite specimens as shown in Figure 11 in the case of a PP-H biocomposite impacted at 12 J (see also videos for PP-H and PP-G composites impacted at 3 J and 12 J).

All the force-displacement curves obtained from drop-weight impact tests exhibit several disturbances during the loading phase seen as small and short drop in force and labelled events , ,  and  in Figure 11. Synchronising the recording of the high speed imaging and the impact test, it was possible to correlate these events to the appearance of macro-cracks on the bottom face of the samples. For this particular test (PP-H at 12 J), the sampling frequency was 200 pts/ms on the impact force curves and 4 frames/ms on the recorded videos. The first drop in force occurred at 8.13 ms (event  on the curve) and the two first radial macro-cracks appeared at 7.80 ms (picture ). This phenomenon was accompanied by a whitening of the sample related to crazing phenomenon. "Drop in force" events  and  occurred at 8.60 ms and 9.70 ms, respectively, while the third and fourth macro cracks were observed at 8.30 and 9.30 ms (pictures  and ).

It should be pointed out that a constant time gap of roughly 0.30 ms was noticed between the "drop in force" events and the appearance of the macro-cracks. This time gap is assumed to be due to a synchronization fault in the recording of both signals, i.e. imaging and force-displacement data, and related to a short delay of the DAS acquisition system of the drop-weight impact machine. Nevertheless, the appearance of new macrocracks was a temporal phenomenon very close to the drop in force observed on the curves and they can thus be directly correlated. The more remarkable event on the force curve was undoubtedly the major force drop that occurred at the event . This event imputed to the total breakage of the material, was found to be related to the moment at which one of the four propagating macro-cracks reaches the edge of the clamped sample.

The drop in force occurs at 12.07 ms, while the image related to the macro-crack reaching the edge was taken at 11.80 ms. A time gap of the same order (0.3 ms) was again observed. Events ,  and  are related to the smooth decrease of the force occurring when the impactor fully penetrates the sample, spreading the four triangles formed after the complete macro-cracks propagation. This analysis was carried out for all impact energies and the two composites. Similar phenomena were observed in all cases. For low impact tests (3 and 6 J), the appearance of short macro-cracks occurred at the time of small drop in force. For higher impact tests (8 to 126 J), the major drop in force, corresponding to the total breakage of the material, always occurred when one of the macro-cracks reaches the edge of the clamped sample.

Crack length propagation during impact

Tracking the tip of each macro-crack appearing on the samples during impact enabled the quantification of the cumulated length of macro-cracks over time as seen in Figure 12. Correlating the force-time curve to the cumulated crack length, it is observed that sudden increases in crack length are related to drop in force.

Indeed, the first sharp increase in crack length of 13.68 mm measured at 7.80 ms can be correlated to the first drop in force (8.13 ms, event  on Figure 11). Accordingly, the second and third crack length increments (22.98 mm at 8.37 ms and 36.10 mm at 9.37 ms, respectively) correspond to the second and third drop in force (8.60 ms and 9.70 ms, events  and  on Figure 11, respectively). These sudden increments in crack length can be due to both the appearance of new macro-cracks and the propagation of already initiated ones.

Besides, it should be pointed out that the same time gap (about 0.30 ms) between the force curve and the crack length increments was found. The comparison of cumulated crack lengths reached at breakage for PP-H and PP-G composites is given in Figure 13 a). Despite a higher absorbed energy at break whatever the impact energy (Figure 9), PP-H exhibits the same cumulated length of macro-cracks for a given impact energy when compared to PP-G composites. Moreover, the cumulated crack length reached a plateau for impact energies higher than 8 J. A maximum cumulated crack length of roughly 50 mm was indeed reached by all composite specimens. This maximum value of cumulated crack length was not as high as expected if considering that the four macrocracks initiated in the plate would all reach the clamped edges of the sample. Indeed, in this case, a cumulated crack length of 4 times the radius of the clamped specimen, i.e. 80 mm, would be reached. This results highlights that the full breakage of the material and related large drop in force occurs as soon as one of the macro-cracks reaches the clamped edge of the sample.

Comparing the cumulated crack length versus the absorbed energy at F max of PP-H and PP-G composites (Figure 13 b), it seems obvious that for a given crack length, PP-H biocomposites absorb much more energy (p-value = 1.01x10 -5 for absorbed energy data and p-value = 0.19 for cumulated crack length data, based on the statistical non-parametric Mann & Whitney U test). This result evidences that despite having similar fracture growth patterns, the energy absorbed during crack propagation is significantly higher for PP-H biocomposite as compared to PP-G composite. This could be related to differences in failure mechanisms, adhesive interfacial failure and fibre pull-out phenomenon being predominant for the biocomposites (Figure 10). Besides, crazing phenomenon within the PP matrix might play an important role in the processes of energy absorption during impact. Unfortunately, considering the very short duration of an impact, it was not possible to collect a sufficient number of images while keeping the high resolution required to analyse crazing phenomenon in details. Cumulated crack length at F max versus absorbed energy at F max for PP-H and PP-G composites.

Numerical study of impact behaviour using three-dimensional finite element modelling

The simulated mechanical response of the plate during impact is usually recorded and analysed in terms of peak load, absorbed energy, and deflection at peak load. Figure 14 shows the simulated force-time and displacement-time curves for 3 J impact on PP-H biocomposite. The impactor displacement history shows the loading phase up to the maximum displacement of 3.8 mm. It can be seen that after peak force of 1050 N, there is a sharp drop in the force corresponding to the creation of macro-cracks. At the end of the loading phase at 5.1 ms, there is a rebound of the impactor seen as a decrease on the displacement curve. The damage of the composite plate was simulated using erosion criteria. A damage parameter D is defined to express the volume of the eroded elements. Since the mesh around the point of impact is very fine and the cracks are typically one element wide, the volume of the eroded elements as a percentage of the total volume of the plate is very small (of the order of 1.10 -3 %). Figure 16 shows the propagation of failure in the PP-H biocomposite plate for 3 J and 12 J impact. It can be seen that the initiation of cracks occurs at 2 ms and 0.98 ms for the two impact cases, respectively. There is a rapid initial crack followed by steady increase in the damage. The amount of damage is evidently higher for the 12 J impact when compared to 3 J impact.

It is also interesting to note that there is a massive increase in the eroded volume at perforation for 12 J impact at 5.1 ms. comparison of the crack growth pattern of the composite was achieved so as to determine the ability of the simulation to reproduce the phenomena observed during the experiment. Figure 17 shows the force-time history as well as bottom surface of the PP-H biocomposite for impact energy of 3 J and 12 J. A comparison of the bottom surfaces impacted at 3 J and 12 J and observed with high speed imaging at the end of the loading phase is also shown in the figure.

For the 3 J impact case, it can be seen from the comparison of the force curve that there is good overall comparison with the experiment. The force increases quasi-linearly till the first macro failure seen from the drop in the force values (Figure 17 a). In the case of the LS-DYNA model, this crack initiation force is about 1030 N while for the experiment, the critical force is 1120 N. The model also overestimates the loss of stiffness due to this failure compared to the experiment. This can be explained by the fact that the failure is modelled using an erosion criteria which instantly deleted the elements that have exceeded the failure strain. A progressive damage model would be more suitable to predict the loss of stiffness due to the accumulation of damage. The crack pattern and length at the end of the loading phase are similar to the composite plate impacted using the drop tower (Figure 17 b andc). It should be noticed that the total duration of the impact is under-estimated in the model; 8 ms compared to 9.2 ms for the experiment.

While the lower energy impact caused only crack initiation, it is evident from the drop tower experiment at 12 J that there was crack propagation and complete perforation of the plate (Figure 17 e and f).

The force-time curves shows that the beginning of the loading phase and the critical force for the first initiation of cracks at around 1000 N is well reproduced by the LS-DYNA simulation (Figure 17 d). The loss of stiffness due to these cracks is higher for the model due to the erosion of the failed elements. However, the model was able to simulate the perforation at 5.1 ms and the corresponding drop in the force. It is important to note that the material parameters input to the FE model, including the erosion parameters of maximum effective strain and number of integration points were the same for the different impact energies. The bottom surface of the plate observed from the experiments and the FE model also exhibit similar behaviour (Figure 17 e and f). In the case of the higher energy impacts, since there is no rebound phase, the image at the time of the sharp drop in force corresponding to penetration damage was chosen. The crack extends almost up to the edge of the fixture and additionally some failure can be observed in the clamped edge of the plate. One of the important outcomes of the model is its ability to correlate the crack length from the simulations with the experimental crack length measured from the high speed camera images. Table 4 shows the cumulated crack lengths measured for 3, 6, 9 and 12 J impact energy from the image analysis and from simulations. It can be seen that there is less than 12.63 % of difference between the model and the experiment. The modelling of impact damage with simple erosion criteria thus overcomes the difficulties of complex damage models such as Johnson Cook Damage model, which requires many additional experimental tests to estimate the input parameters, while still being able to predict with good agreement the impact behaviour measured experimentally, both in terms of force-time history and macro-cracks propagation. It should be pointed out that the developed model could be improved by considering the strain rate effects for example by using Cowper Symonds model [26] [28]. Obviously, the strain rate dependency of the mechanical behaviour of composites would need to be investigated by mechanical testing at increasing strain rates so as to determine the parameters to include in the model. An improved model including the strain rate effects and a Generalised Incremental Stress State dependent damage model (GISSMO) should be implemented in future works.

Conclusions

An original method combining impact tests, high speed imaging and FE modelling was developed to characterize and predict the impact behaviour of composite samples subjected to low velocity impact. PPhemp fibres (PP-H) and PP-glass fibres (PP-G) composites were manufactured with the same volume fraction (12 vol%) and their respective impact behaviour were compared. It was found that although PP-G composites could withstand higher impact loads, PP-H biocomposite exhibited a higher deformation at break leading to a significantly higher absorbed energy until breakage for any impact energy. This phenomenon is believed to be due to differences in the fibre characteristics, i.e. lower stiffness and strain at break of hemp fibres, and failure mechanisms. SEM observations revealed that damage mechanisms of PP-H and PP-G composites is dominated by matrix failure and fibre pull-out.

The implementation of a high speed camera enabled a correlation between force-displacement history and macro-cracks propagation. Independently of the fibres used, all composites seemed to break once one of the propagating macro-cracks has reached the edge of the clamped sample. Image analysis also allowed measuring the cumulated crack length until breakage. It was found that despite having similar fracture growth patterns, the energy absorbed during crack propagation is significantly higher for PP-H biocomposite as compared to PP-G composite. The origin of this phenomenon should be searched in underlying differences related to the failure mechanisms that could be approached by increasing the magnification and/or the acquisition speed to analyse fine details of the crack propagation, in particular matrix crazing.

Post-mortem analysis by CT scans could also help to analyse matrix crazing.

A numerical FE model of the impact behaviour of PP-H biocomposite was developed using LS-DYNA.

A macroscopic non-linear material model was used to model the material behaviour and a strain based erosion criteria was used to model damage. The results of the FE model were compared in terms of the forcetime histories as well as the cumulated crack lengths with the experimental data. The model was able to simulate the initiation and propagation of the macro-cracks and the results are in good agreement with the experimental drop tower data. The developed model could be used to predict the impact response of large automotive components made of natural fibre reinforced composites with minimal computational cost.

Figure 1 .

 1 Figure 1. Typical tensile stress-strain curves of PP-H and PP-G composites.

Figure 2 .

 2 Figure 2. Scheme of the drop-weight impact machine Instron CEAST 9340 and high speed imaging setup.

Figure 3 .

 3 Figure 3. Principle of macro-cracks length tracking for a PP-H sample.

Figure 4 .

 4 Figure 4. The dimensions of the square plates were 100 x 100 x 4 mm 3 . The composite was modelled as a

Figure 4 .

 4 Figure 4. Geometry and finite element mesh of the composite plate, the impactor tip and the flanges.

Figure 5 .

 5 Figure 5. Energy history of the hemp composite plate for 3.07 J impact.

Figure 6 .

 6 Figure 6. (a) PP-H and (b) PP-G samples broken on the drop-weight impact machine and ordered by

Figure 7 .

 7 Figure 7. Contact force vs time curves for (a) PP-H according to the impact energy between 3 and 12J, (b)

Figure 8 .

 8 Figure 8. Contact force vs displacement curves for PP-H (blue) and PP-G (red) samples impacted at (a) 6J

Figure 9 .

 9 Figure 9. Absorbed energy at F max according to the impact energy for PP-H and PP-G composites.

Figure 10 .

 10 Figure 10. SEM micrographs of failure surfaces of samples broken at 12 J impact for (a) PP-H and (b) PP-G

Figure 11 .

 11 Figure 11. Contact force versus time and associated time tagged cliches corresponding to the different

Figure 12 .

 12 Figure 12. Cumulated macro-crack length over time (dashed line) correlated to the force curve (solid line)

Figure 13 .

 13 Figure 13. a) Cumulated crack length at F max versus impact energy for PP-H and PP-G composites, b)

Figure 14 .

 14 Figure 14. Typical force and displacement history for 3 J impact on PP-H biocomposite.

Figure 15

 15 Figure 15 shows the bottom surface of the plate at different time intervals to show the initiation and

Figure 15 .

 15 Figure 15. Mean effective strain contours throughout propagation of impact damage in PP-H biocomposite

Figure 16 .

 16 Figure 16. Eroded volume of elements for 3 and 12 J impact case for PP-H biocomposites.

Figure 17 .

 17 Figure 17. Comparison of force-time history for (a) 3 J and (d) 12 J impact of PP-H biocomposite; Bottom

Table 1 .

 1 Tensile (ISO 527 standard) and thermal (measured by DSC with 10°C/min heating rate) properties of PP matrix ; Et Young's modulus ; σ m ultimate tensile strength ; ε b strain at break ; X c degree of crystallinity; T c crystallisation temperature ; T m melting temperature. Hemp fibres (Cannabis sativa L., variety Fedora 17) supplied by Fibres Recherche Développement (FRD, Troyes, France) were used as reinforcement. The degree of retting was adjusted by the supplier for typical use in common composite applications. Hemp fibres presented an average length of 2 mm. Their biochemical composition was characterized according to Van Soest method (standards NF EN ISO 13906

		E t (MPa)	σ m (MPa)	ε b (%)	X c (%)	T c (°C)	T m (°C)
	Mean	1242	23.9	36.1	48.2	121.7	173
	Standard deviation	30	0.1	6.5	3.5	0.1	9

Table 2 .

 2 Composites produced and tested in the study

	sample designation	PP-H	PP-G
	PP mass fraction (%)	80	70
	Fibre type	Hemp	Glass
	Fibre mass fraction (%)	20	30
	Fibre volume fraction (%)	12	12
	Young's modulus (MPa)	2572 ± 57	6138 ± 117
	Ultimate stress (MPa)	24.5 ± 0.1	64.9 ± 0.3
	Strain at break (%)	3.2 ± 0.1	1.9 ± 0.0

Table 3 .

 3 Peak force F max and displacement at F max for PP-H and PP-G according to the impact energy.

	PP-H	PP-G

Table 4 .

 4 Comparison of cumulated crack lengths obtained from drop tower experiments and LS-DYNA simulations for various impact energies.

	Impact Energy	Cumulated crack length (mm) Experiment LS-DYNA	Difference (%)
	3J	34.67	30.29	12.63
	6J	44.53	49.19	10.46
	9J	66.67	60.29	9.57
	12J	67.48	69.8	3.44

Acknowledgements

The authors gratefully thank the ANRT French Association as well as the ADEME French Organization (convention n°1682C0043) for their financial supports. KRR, NLM and SC thanks the European Union's Horizon 2020 Programme (HERMES project, grant agreement n° 636520) for its funding.


Roman Anufriev

Nicolas Chauvin
email: nicolas.chauvin@insa-lyon.fr

Jean-Baptiste Barakat

Hammadi Khmissi

Khalid Naji

Gilles Patriarche

Xavier Letartre

Michel Gendry

Catherine Bru-Chevallier

Photoluminescence polarization and piezoelectric properties of InAs/InP quantum rod-nanowires

INTRODUCTION

The growth of nanowires (NWs) using the Vapor-Liquid-Solid (VLS) method is well adapted to integrate III-V semiconductors on silicon (Si) giving to the NW technology a great potential in optoelectronics and photonics on Si substrates. One original feature of III-V semiconductor NW growth is the possibility to obtain heterostructures with a wurtzite (Wz) crystallographic phase instead of the more usual zinc-blende phase. In the present work, we report on the experimental observation of a piezoelectric field and a strong polarization anisotropy in the photoluminescence spectra of Wz InAs/InP quantum rod (QRod) NWs.

II. GROWTH CONDITIONS AND SAMPLE PREPARATION

The Wz InAs/InP QRod-NWs were grown by VLS assisted solid source MBE on a (111) oriented Si substrate [START_REF] Anufriev | Polarization properties of single and ensembles of InAs/InP quantum rod nanowires emitting in the telecom wavelengths[END_REF]. The obtained structure consists of an InP NW with an InAs insertion, called QRod due to its elongated shape (Figure 1.a). The NWs have a 80 -100 nm diameter and a 1.5 -2 μm length. The diameter and the length of the InAs QRods are measured in the 8 -11 nm range and the 20 -100 nm range, correspondingly. To study the NWs by means of low temperature micro-photoluminescence (micro-PL), the NWs were ultrasonically removed from the as-grown substrate and transferred onto a TEM copper grid, served as a host-substrate.

The sample was mounted in a He-flow cryostat and cooled to 10 K. The excitation was provided by a continuous wave He-Ne laser beam (632.8 nm) focused by a microscope objective to a spot size of ≈ 4 µm.

III. POLARIZATION RESOLVED PHOTOLUMINESCENCE

Polarized micro-PL studies have been performed on single QRod-NWs at 10K and room temperature. A strong anisotropy is observed: nearly all the PL emission is polarized parallel to the NW axis (Figure 1.b and 1.c). The degree of linear polarization (DLP) defined as

     I I I I DLP // //
is bigger than 0.9 and is temperature independent. This result is in contradiction with the fact that the ground state of the bulk Wz InAs should emit light perpendicular to the NW axis. This result could be explained either by a heavy-hole light-hole mixing [START_REF] Bleuse | Inhibition, enhancement, and control of spontaneous emission in photonic nanowires[END_REF] and/or by the photonic nature of the NW which inhibits the dipole oriented perpendicular to the NW axis when the NW diameter is small compared to the emission wavelength [3]. To investigate this last hypothesis, a second sample has been grown with the same growth conditions except a bigger InP growth time during the last step of the growth to increase the NW diameter. With this sample, we were able to investigate the polarization emission for NWs with diameters in the 100-300 nm range. With increasing NW diameter, a decrease of the QRod emission anisotropy is observed and the DLP is equal to 0.45 for a 300 nm NW diameter. A strong decrease of the DLP is also observed for the Wz InP peak: the DLP, which is close to zero for thin NWs, reaches -0.6 for NW diameters of 300 nm. This negative DLP means that the emission is mainly perpendicular to the NW axis, in agreement with the theory, and confirms that the DLP measured in thin NW is impacted by the photonic properties of the NW.

IV. PIEZOELECTRIC EFFECTS IN QUANTUM ROD-NANOWIRES

The micro-PL emission of single QRod-NW ground states has been investigated, at 10K, as a function of the excitation power and an unusual behavior has been observed. Being broad at low excitation power, the peak is blue-shifting and narrowing as the excitation power is increased up to 0.2 mW (Figure 2.a). The blueshift and narrowing of the PL emission with the excitation power increase are typically related to an electric field screening and explained in terms of quantum confinement Stark effect (QCSE) [START_REF] Lähnemann | Coexistence of quantum-confined Stark effect and localized states in an (In,Ga)N/GaN nanowire heterostructure[END_REF]. Indeed, at low excitation power, the emission energy is decreased and the linewidth is broad due to the carrier separation induced by the electric field. However, with a laser power increase, the electric field can be screened by photogenerated carriers, weakening the QCSE and leading to an emission blueshift (Figure 2.b) and a narrowing (Fig 2 .c) of the PL emission [START_REF] Liu | Modification of optical properties by strain-induced piezoelectric effects in ultrahigh-quality V-groove AlGaAs/GaAs single quantum wire[END_REF]. As the excitation power is reaching 0.2 mW, the electric field is completely screened, and the following increase of the power is leading to the more usual redshift and broadening. In the present case, due to the lattice mismatch between InAs and InP materials, the Wz InAs QRods are strained, so a strain-induced piezoelectric polarization inducing an electric field may indeed be expected.

To investigate this phenomenon, observed at 10K, the same excitation power dependence studies have been performed as a function of the temperature. A blueshift and a narrowing are observed up to 50K but their respective magnitudes decrease with the temperature increase and, starting from 70K, no clear shift or narrowing is observed. This transition, observed between 50-70K, is equivalent to a thermal energy kT = 4-6 meV, value in the order of magnitude of the blueshift observed at 10K. This means that the carriers are thermally activated and can overcome the energy of the piezoelectric field.

In order to fully understand the piezoelectric phenomenon taking place in the QRods and the role of the QRod geometry, the calculation of the expected strain and piezoelectric field inside these Wz QRod-NWs has been performed using the 3D nano device simulator "Nextnano" [START_REF] Zibold | Semiconductor based quantum information devices: Theory and simulations[END_REF]. An averaged electric field of 40 kV/cm is obtained in the case of a QRod of 40 nm in length and 8 nm in diameter. This piezoelectric field is strongly related to the diameter and length of the QRod. This could explain why the phenomena observed in Figure 2 were not observed for all the QRods of the sample.

In conclusion, we report on the evidences of the straininduced piezoelectric field in Wz InAs/InP QRod-NWs. This electric field caused by the lattice mismatch between the InAs and InP results in a QCSE and, as a consequence, affects the optical properties of the structure. Moreover, these QRods revealed a very strong polarization anisotropy (DLP>0.9) which is a consequence of the photonic nature of InP NWs.

Figure 1

 1 Figure 1. a) TEM picture of an InAs/InP QRod-NW. Polarization resolved PL of a single QRod-NW measured at 300K (b) and 10K (c).

Figure 2 .

 2 Figure 2. 10K µ-PL spectra (a), emission energy (b) and linewidth (c) of a single QRod-NW as a function of the excitation power.

ACKNOWLEDGMENT This work has been supported by the Agence Nationale pour la Recherche project INSCOOP (ANR-11-NANO-012) and by the LABEX iMUST (ANR-10-LABX-0064) of Université de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). Authors are gratefully acknowledging facilities and technological staffs from Nanolyon platform.