Linda De Bont

Elissa Naim

Delphine Arbelet-Bonnin

Qiong Xia

Emily Palm

Patrice Meimoun

Stefano Mancuso

Hayat El-Maarouf-Bouteau

François Bouteau
email: francois.bouteau@univ-paris-diderot.fr

Activation of plasma membrane H+-ATPases participates in dormancy alleviation in sunflower seeds

Keywords: Dormancy, H + -ATPase, Hormones, Reactive oxygen species, Seeds, Sunflower

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

1. introduction

Although successful germination of seeds is a critical step in agriculture, some of the underlying molecular mechanisms of seed germination and dormancy regulation are still unknown. Seed dormancy allows some species to persist, blocking the germination of mature seed until conditions become favorable for seedling establishment. Seeds become capable of germination after a dry storage period called after-ripening, a process which reduces dormancy. The duration of this period depends on environmental factors such as temperature, light, oxygen and moisture. Germination begins with the absorption of water by the seed and ends when the embryonic axis begins to lengthen.

Reactive oxygen species (ROS) and hormones have emerged as key regulatory actors in the regulation of seed germination and dormancy [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF][START_REF] Finkelstein | Molecular aspects of seed dormancy[END_REF][START_REF] North | Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research[END_REF]. Abscisic acid (ABA) is a positive regulator of dormancy and a negative regulator of seed germination while other hormones such as gibberellins (GA) or ethylene (ET) promote germination completion by antagonising ABA effects [START_REF] Holdsworth | Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination[END_REF][START_REF] Finkelstein | Molecular aspects of seed dormancy[END_REF][START_REF] Corbineau | Ethylene, a key factor in the regulation of seed dormancy[END_REF][START_REF] Vishal | Regulation of Seed Germination and Abiotic Stresses by Gibberellins and Abscisic Acid[END_REF]. In sunflower, ET is the major hormone acting as an antagonist of ABA and a positive regulator of seed germination [START_REF] Corbineau | Breakage of Pseudotsuga menziesii seed dormancy by cold treatment as related to changes in seed ABA sensitivity and ABA levels[END_REF][START_REF] Oracz | Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway[END_REF][START_REF] Xia | One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds[END_REF]. In seeds, ROS and ABA have antagonistic effects [START_REF] Barba-Espín | Understanding the role of H(2)O(2) during pea seed germination: a combined proteomic and hormone profiling approach[END_REF][START_REF] Oracz | Phytohormones Signaling Pathways and ROS Involvement in Seed Germination[END_REF], and ROS produced during soybean seed imbibition could promote ET production [START_REF] Ishibashi | A role for Reactive oxygen species produced by NADPH oxidases in the embryo and aleurone cells in barley seed germination[END_REF] and conversely, ET induced ROS accumulation in sunflower seeds [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF]. Interaction of ROS with either hormone metabolism or signaling pathways seems important in this process [START_REF] Petrov | Hydrogen peroxide-a central hub for information flow in plant cells[END_REF][START_REF] Wrzaczek | ROS signaling loops -production, perception, regulation[END_REF] as it has been documented in other plant process such as stomatal opening regulation [START_REF] Wang | Guard-cell signalling for hydrogen peroxide and abscisic acid[END_REF][START_REF] Agurla | Convergence and Divergence of Signaling Events in Guard Cells during Stomatal Closure by Plant Hormones or Microbial Elicitors[END_REF], plant microbe interactions [START_REF] Mersmann | Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity[END_REF][START_REF] Wi | Synergistic biosynthesis of biphasic ethylene and reactive oxygen species in response to hemibiotrophic Phytophthora parasitica in tobacco plants[END_REF] and cell death [START_REF] Kadono | Increased anion channel activity is an unavoidable event in ozoneinduced programmed cell death[END_REF]. It is also noteworthy that ion transport systems are systematically involved and cross linked with ROS and hormones in these processes [START_REF] Kadono | Increased Anion Channel Activity Is an Unavoidable Event in Ozone-Induced Programmed Cell Death[END_REF][START_REF] Munemasa | Mechanisms of abscisic acid-mediated control of stomatal aperture[END_REF]. Although ABA is known to play a critical role in plant stress responses through transcriptional induction of defense genes in different organs, ABA does have nontranscriptional mechanisms modulating ion homeostasis resulting in plasma membrane (PM) H + -ATPase inhibition during stomatal closure [START_REF] Joshi-Saha | A brand new START: abscisic acid perception and transduction in the guard cell[END_REF]. The effect of ABA on the root PM H + -ATPase is sometimes controversial [START_REF] Planes | A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H + -ATPase and decreased cytosolic pH, K + , and anions[END_REF], but these varied responses could also be linked to ROS generation as observed in cultured cells of Arabidopsis thaliana where ROS are a central messenger of ABA in the signaling pathway leading to PM H + -ATPase inhibition [START_REF] Trouverie | Anion channel activation and proton pumping inhibition involved in the plasma membrane depolarization induced by ABA in Arabidopsis thaliana suspension cells are both ROS dependent[END_REF]. On the contrary, stimulation of PM H + -ATPases by ET precursors have been suggested in various plant and cell types [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF][START_REF] Waters | Ethylene involvement in the regulation of the H(+)-ATPase CsHA1 gene and of the new isolated ferric reductase CsFRO1 and iron transporter CsIRT1 genes in cucumber plants[END_REF][START_REF] Wang | Determining the Ester and Linoleic Acid Methyl Ester Content to Comply with EN14103 Application Authors[END_REF].

The PM H + -ATPase is an important ion pump in the plant cell plasma membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM [START_REF] Sze | Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis[END_REF], a prerequisite to the activity of nutrient transporters (K + , sugars, amino-acids) coupled to the electrochemical proton gradient and important for growth [START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF]. Therefore, a mechanism related to cell polarization through PM H + -ATPase activity regulation could be operating in seeds to allow for the participation of secondary ion transport in the energization of cells [START_REF] Sze | Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis[END_REF] and thus, dormancy release.

Previous studies could not validate this hypothesis in the small seeds of A. thaliana and instead used roots because biochemical and electrophysiological measurements in roots correlated with growth phenotypes in germinating seeds. In the present work, we took advantage of the large size of sunflower seeds and used pharmacological agents to investigate in vivo the effects of hormones, namely ABA and ET, and ROS on the seed cell polarization as a marker of PM H + -ATPase activity.

We found that these hormones and ROS pharmacology act on seed cell polarization, suggesting that dormancy could be based on ABA dependent inhibition of PM H + -ATPase and that ET-and ROSdependent PM H + -ATPase stimulation could be involved in dormancy release in sunflower. and optically controlled with a microscope (Leitz, Germany). A pellet (E205, Phymep, France) was used as a reference electrode in the recording chamber. Impalements of sunflower embryo axis cells (Fig. 1A) were carried out in 5 cm Petri dishes filled with 5 ml of bathing medium (BM: 5 mM MES, 1 mM CaCl2 and 1 mM KCl buffered at pH 5.8 with 5 mM Tris) at room temperature (20-22°C) under light. Due to the thickness of the embryonic axis of the sunflower seeds, only blind impalement could be performed. We systematically check that the insertion of the microelectrode was accompanied by a rapid and steep rise in electrical resistance representing a time constant compatible with those of a plasma membrane (PM), indicating that the electrode has passed through the PM. Upon microelectrode entry into the sunflower embryo axis cells, the recorded potential drops within a few ms to an initial value (Vp) that could be maintained (Fig. 1B) or decays to a depolarized potential in few seconds (Fig. 1C). When an initial fast peak transient was measured upon microelectrode entry, it indicates that the microelectrode probably introduces a leakage into the PM, causing a change in PM potential (Vm) from its real value before impalement to a depolarized value. In this last case, the membrane could thereafter develop a slow hyperpolarization reaching a sustained membrane potential close to the recorded Vp (Fig. 1C), as has already been observed in various animal and plant cells [START_REF] Lew | Osmotic effects on the electrical properties of Arabidopsis root hair vacuoles in situ[END_REF][START_REF] Lassen | The membrane potential of Ehrlich ascites tumor cells microelectrode measurements and their critical evaluation[END_REF][START_REF] Ince | Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry[END_REF][START_REF] Ince | Small Cells Evaluated With the Patch Clamp[END_REF]. The initial fast peak transient Vp is considered as a good estimate of the true Vm and that the slow hyperpolarizations after impalement should be regarded as transient repolarizations back to the original Vm [START_REF] Lassen | The membrane potential of Ehrlich ascites tumor cells microelectrode measurements and their critical evaluation[END_REF][START_REF] Ince | Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry[END_REF][START_REF] Ince | Small Cells Evaluated With the Patch Clamp[END_REF]. Thus, analysis of the initial fast impalement voltage (Vp) is considered as a valuable aid in the estimation of Vm of cells by microelectrodes. Pharmacological studies were performed by direct application of the pharmacological agents in the Petri dishes after stabilization of the running potential or by pretreating the embryos in the BM with the pharmacological agent 30 min before voltage recordings.

Material and methods

Sunflower seeds and germination assays

ATP measurements

Adenosine-triphosphate (ATP) contents were determined using the bioluminescent method and following the protocol provided with an ATP Detection Kit (FLAA, Sigma-Aldrich, St. Louis, USA).

Hulled seeds were placed on water-soaked cotton for imbibition at 10˚C for 3, 15 and 24 hours. Dry and imbibed axes of D and ND seeds were collected and ground immediately on ice with 1 mL of acid extract from the ATP Detection Kit. Samples were centrifuged at 8000× g for 10 min at 4 °C. The supernatant was transferred to a new 1.5 mL tube and the same volume of alkaline extract was added.

After mixing and again centrifuging for 10 min at 4 °C and 8000× g, the supernatant was placed in a new tube on ice for the ATP assay. The luminescence was assayed from a 30 µL sample using a luminometer (FB12, Berthold, Germany) with 100 μL of the ATP detection buffer provided in the ATP detection Kit. All of the experiments were repeated in triplicate and the mean values were determined based on the three replicates ± standard deviation.

Seed water content determination

For water content measurements, hulled seeds were placed on water-soaked cotton for imbibition at 10˚C for 3, 15 and 24 hours. Dry and imbibed sunflower seed axes (embryos without cotyledons) were put in coded, pre-weighed eppendorf tubes and weighed again. Tubes with open caps were put in a drying oven at 105˚C for 24 hours. They were then placed in a desiccator for 10 min and weighed again. Values are means of three technical and three biological replicates ± standard deviation. WC (%) was determined using the following formula: (FW-DW)/DW*100); FW= weight of axes before drying; DW weight of axes after drying.

Statistical analysis

Significant differences between treatments were determined by the Mann-Whitney test, and P-values <0.05 were considered significant.

Results

Due to their hardness, dry seeds could not be impaled with glass microelectrodes, and thus impalements were achieved only 3h after imbibition of the seeds in the bathing medium (Fig. 1A). Upon impalement (i.e. microelectrode entry into the sunflower embryo axis) the recorded potential drops within a few ms to an initial value (Vp) that could either be maintained (Fig. 1B) or decays to a depolarized potential in a few seconds (Fig. 1C). The same behaviors were observed in both D seeds and ND seeds but the initial values of Vp were generally more hyperpolarized for ND when compared to D seeds (Fig. 1B,C). by microelectrodes [START_REF] Lassen | The membrane potential of Ehrlich ascites tumor cells microelectrode measurements and their critical evaluation[END_REF][START_REF] Ince | Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry[END_REF][START_REF] Ince | Small Cells Evaluated With the Patch Clamp[END_REF]; Vp were thereafter considered as Vm.

Role of H + -ATPases in the polarization of cells from D and ND seed axes.

Since the PM H + -ATPase is an important ion pump in the plant cell membrane that participates in PM polarization by extruding protons from the cell [START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF], we checked if H + -ATPase activity could be involved in polarization of cells from D and ND seed axes. Figure 3A (left) illustrates one of the profiles recorded in a cell from a ND seed axis upon addition of 400 µM vanadate (VAN), an inhibitor of H + -ATPases [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF][START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF]. Vanadate rapidly depolarizes the PM of this cell while a cell of a D seed axis was not depolarized upon addition of vanadate (Fig. 3A, right). On the contrary, the addition of 5 µM fusicoccin (FC), an activator of H + -ATPase [START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF], to a cell of a D seed axis induces a hyperpolarization of the PM (Fig. 3A On the contrary, 5 µM FC hyperpolarized D seed axis cells to a value close to that of ND seed axis cells (Fig. 3B, right). Addition of 5 µM FC to ND seeds only slightly hyperpolarized the axis cells (Fig. 3B, left). These data suggest that H + -ATPases are not active in cells from a D seed axis though they can be stimulated by FC, and that they are responsible for a part of the polarization of cells from an ND seed axis. We further checked for the impact of VAN on the rate of germination of ND seeds. As shown in Figure 4A (left), imbibition in presence of 400 µM VAN slows the germination of ND seeds, with the T50 being delayed for more than 8 hours. On the contrary, imbibition in the presence of 5 µM FC accelerates the germination of D seeds (Fig. 4A, right). We further checked the availability of ATP, which is necessary for PM H + -ATPase functioning, in D and ND seed axes. No difference in ATP contents could be detected between D and ND seeds (Fig. 4B). The increase in ATP contents follows precisely the seed imbibition curve recorded for D and ND seeds (Fig. 4C), strongly suggesting that ATP is not the limiting factor for the activation of PM H + -ATPases in D seeds.

The roles of hormones in the polarization of cells from D and ND seed axes.

Hormones, especially ABA and ET, are important actors in seed dormancy [START_REF] Arc | ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination[END_REF][START_REF] Corbineau | Ethylene, a key factor in the regulation of seed dormancy[END_REF][START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF]. They are also known to regulate ion transport systems, notably PM H + -ATPase [START_REF] Planes | A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H + -ATPase and decreased cytosolic pH, K + , and anions[END_REF][START_REF] Munemasa | Mechanisms of abscisic acid-mediated control of stomatal aperture[END_REF], thus impacting cell polarization. We checked if ABA, known to maintain the dormant state of seeds, and 1aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene known to alleviate dormancy [START_REF] Linkies | Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana[END_REF], could regulate the polarization of D and ND seed axis cells. The average values of Vm recorded on cells from ND seed axes pretreated 30 min before voltage recordings with 40 µM ABA (Fig. 5A)

show that ABA depolarizes the cells of ND seed axes to a value close that cells of D seed axes.

Addition of 40 µM ABA to D seeds did not modified the axis cell polarization (Fig. 5B). The average values of Vm recorded on cells from D seed axes pretreated 30 min before voltage recordings with 1 mM ACC show only a weak effect on the polarization of cells, but overnight pretreatments with 1 mM ACC allow for the hyperpolarization of these cells (Fig. 5A). These data show that ABA could maintain a low cell polarization, while in contrast, ET could participate in the hyperpolarization of these cells, further suggesting that hormones could participate in the dormancy process by regulating the polarization of seed axis cells. We further confirmed the known impact of ABA on the rate of germination of ND seeds. As shown in Figure 4B (left), imbibition in presence of 20 µM ABA slows the germination of ND seeds, the T50 being delayed for more than 14 hours. On the contrary, as expected from previous data [START_REF] Linkies | Ethylene interacts with abscisic acid to regulate endosperm rupture during germination: a comparative approach using Lepidium sativum and Arabidopsis thaliana[END_REF], imbibition in presence of 1 mM ACC accelerates the germination of D seeds (Fig. 5B, right).

Roles of reactive oxygen species (ROS) in the polarization of D and ND seed axis cells.

Reactive oxygen species have also been described as important actors in seed dormancy [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF][START_REF] Oracz | ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation[END_REF], possibly through the activation of NADPH-oxidases or peroxidases [START_REF] Singh | Superoxide and its metabolism during germination and axis growth of Vigna radiata (L.) wilczek seeds[END_REF][START_REF] Singh | Role of peroxidase activity and Ca2+ in axis growth during seed germination[END_REF]. They are also known to regulate ion transport systems and interact with hormone pathways [START_REF] Pottosin | Cross-talk between reactive oxygen species and polyamines in regulation of ion transport across the plasma membrane: implications for plant adaptive responses[END_REF][START_REF] Singh | Reactive oxygen species signaling and stomatal movement: Current updates and future perspectives[END_REF]. axis to a value close to that of cells from D seed axes (Fig. 6A). We also checked for the putative role of methylviologen (MV), known to produce ROS and alleviate dormancy [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF][START_REF] Oracz | ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation[END_REF], on the polarization of cells from D seed axes. The average values of Vm recorded on these cells pretreated 30 min before voltage recordings with 100 µM MV indicate the hyperpolarization of these cells (Fig. 6A). These data show that down regulation of ROS levels could maintain a low cell polarization while inversely, ROS generation could participate in the hyperpolarization of these cells. We further confirmed the known impact of ROS treatments on the rate of germination of ND seeds. As shown in Figure 5B, imbibition in presence of 5 mM TIRON, 50 µM DPI or 10 µM SHAM slow the germination of ND seeds, the T50 being delayed for more than 8, 9 or 15 hours, respectively. In contrast, as expected from previous data, imbibition in presence of 100 µM MV accelerates the germination of D seeds (Fig. 6C). This further suggests that the regulation of seed axis cell polarization by ROS levels could participate in the dormancy process.

A role for seed axis cell polarization in sugar influx?

The alleviation of dormancy is thought to require an active metabolism fueled by sugar. The PM H + -ATPase energizes the PM by extruding protons from the cell and generating a membrane potential [START_REF] Sze | Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis[END_REF][START_REF] Haruta | The Effect of a Genetically Reduced Plasma Membrane Protonmotive Force on Vegetative Growth of Arabidopsis[END_REF], which is a prerequisite for secondary transport of various solutes comprising hexoses [START_REF] Chen | Transport of sugars[END_REF][START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF]. Hexoses are thus able to cross the PM through H + /hexose symporters due to the H + gradient, the entry of H + with sugar inducing a depolarization of the PM [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF][START_REF] Chen | Transport of sugars[END_REF]. We recently showed in a metabolomic study [START_REF] Xia | One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds[END_REF] that the amount of glucose and fructose was increase in ND as compared to D sunflower seed axis. We thus checked for the effect of addition of glucose on polarization of cells from D and ND seed axes. Figure 7A illustrates a profile recorded in a cell from a ND seed axis upon addition of 500 µM glucose (Glc) which rapidly depolarizes the PM. The averages values of Vm recorded on cells of ND seed axis pretreated 30 min before voltage recordings with 500 µM or 1 mM Glc (Fig. 7B) show that Glc could depolarize the cells from ND seed axis in a dose dependent manner.

The addition of 1 mM phlorizin, a competitive inhibitor of Na + /Glc cotransporter in animal cells [START_REF] Choi | Sodium-Glucose Cotransporter 2 (SGLT2) Inhibitors from Natural Products: Discovery of Next-Generation Antihyperglycemic Agents[END_REF] and H + /Glc symporter in plants [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF], during the pretreatment with 1mM Glc prevents the depolarization of the cells from ND seed axis (Fig. 7B). D seed axis cells pretreated with 500 µM Glc were not depolarized (Fig. 7B). These data strongly suggest that in cells from ND seed axis Glc could be loaded through H + /Glc symporter. Thus we checked for the impact of phlorizin on the rate of germination of ND seeds. As shown in Figure 7C (left), 1 mM phlorizin slows the germination of ND seeds, the T50 being delayed for more than 10 hours. On the contrary, imbibition in the presence of 500 µM Glc accelerates the germination of D seeds (Fig. 7C, right).

Discussion

In the present work, we took advantage of the relatively large size of sunflower seeds to perform electrophysiological recordings with microelectrodes and investigate cell polarization of D and ND seeds as an in vivo marker of H + -ATPase activity, a well-known enzyme responsible for cell energization [START_REF] Sze | Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis[END_REF][START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF]. In accordance with our hypothesis that the energization of the PM could be required in dormancy alleviation and subsequent germination we found that the embryonic axis cell polarizations were different between D and ND seeds, with ND seeds showing more hyperpolarized cells. Using an inhibitor of PM H + -ATPase (VAN), we could further show that ND seed cell polarization was reduced as previously observed in other plant cells [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF][START_REF] Sze | Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis[END_REF][START_REF] Brault | Plasma membrane depolarization induced by abscisic acid in Arabidopsis suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca 2+ dependent[END_REF]. Vandate was ineffective in D seeds, supporting the argument for an absence of active PM H + -ATPases. The addition of FC, a toxin known to activate the PM H + -ATPase by promoting its binding with 14-3-3 protein [START_REF] Sze | Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis[END_REF][START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF], allowed for observations of D seed cell hyperpolarization. This toxin is known to activate PM H + -ATPase at the post-translational level [START_REF] Olsson | A phosphothreonine residue at the C-terminal end of the plasma membrane H+-ATPase is protected by fusicoccin-induced 14-3-3 binding[END_REF] suggesting that PM H + -ATPase proteins were present in the PM of D seeds but that their activity was down regulated. ATP availability was not responsible for the lack of PM H + -ATPase activity in D seeds since no difference in ATP levels was found between D and ND seed axes. As a whole, these data suggest that the regulation of PM H + -ATPase activity could be responsible for the ND cell polarization in accordance with the known role of PM H + -ATPase in hyperpolarization of the PM through the extrusion of protons from the cell leading to their energization [START_REF] Sze | Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis[END_REF][START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF][START_REF] Haruta | The Effect of a Genetically Reduced Plasma Membrane Protonmotive Force on Vegetative Growth of Arabidopsis[END_REF]. The fact that VAN could delay the germination of ND seeds and that FC activates the germination of D seeds further argues for the involvement of PM H + -ATPase activity in dormancy alleviation. These data correlate with the decrease in H + -ATPase protein concentration in PM during seed aging, which is related to slow germination in maize [START_REF] Sveinsdóttir | Seed ageing-induced inhibition of germination and post-germination root growth is related to lower activity of plasma membrane H+-ATPase in maize roots[END_REF].

We also showed that the energization of the PM likely allows the influx of hexoses through the functioning of symporters using the H + gradient (proton motive force). Effectively, the Glc-induced depolarization of ND seed cells could not be due to the uncharged sugar influx but could be caused by the coupled influx of H + with Glc through a symporter [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF][START_REF] Chen | Transport of sugars[END_REF]. This data is supported by the absence of depolarization in the presence of phlorizin, a compound known to block H + /Glc symporters [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF] and the absence of depolarization in D seed cells upon addition of Glc, with no favorable H + gradient being available for the symporter functioning due to the inactivity of PM H + -ATPase in D seeds. Phlorizin could delay the germination of ND seeds suggesting a role for sugar uptake in dormancy alleviation.

These data are also in line with the increase in Glc and fructose contents that we have recently reported for sunflower ND embryonic axes relative to D axes [START_REF] Xia | One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds[END_REF]. It's worth noting that among the 86 metabolites detected in sunflower seeds, only the amounts of Glc, fructose and xylose were increased in ND axes [START_REF] Xia | One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds[END_REF]. The proton motive force generated by PM H + -ATPase activity could allow influx not only of sugars but also of various solutes such as K + that are necessary for the cell especially during growth processes such as germination. We have previously shown that putative sugar and K + transporters transcripts are present in D and ND seeds [START_REF] Xia | One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds[END_REF].

The rapid stimulation of PM H + -ATPases by FC on D seed cells suggests that the difference of polarization of the D and ND seed cells could be due to post-transcriptional or post-translational regulation of PM H + -ATPase rather than transcriptional regulation. These data are in accordance with our recent transcriptomic study [START_REF] Xia | One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds[END_REF] that showed, the same transcript levels for AHA5-like gene, a homolog of A. thaliana AHA genes coding PM H + -ATPases [START_REF] Falhof | Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology[END_REF], and the absence of effect of ABA on AHA5-like transcript level in both ND and D sunflower seed axes. However, the delayed effect of ACC that was observed in D seed cell polarization suggests an additional transcriptional regulation of the gene coding PM H + -ATPases in accordance with the ET-induced increase in AHA5-like gene transcription by >4 fold in D sunflower seeds axis [START_REF] Xia | One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds[END_REF]. ABA, known to favor dormancy, demonstrates a non-transcriptional mechanism resulting in inhibition of PM H + -ATPase in the guard cell model [START_REF] Joshi-Saha | A brand new START: abscisic acid perception and transduction in the guard cell[END_REF].

Interestingly, in ND sunflower seed cells we observed a depolarization after pretreatment with ABA that suggests that ABA could participates in dormancy by blocking PM H + -ATPases in accordance with what was described by Planes et al. [START_REF] Planes | A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H + -ATPase and decreased cytosolic pH, K + , and anions[END_REF] during root cell growth inhibition. The opposite effect of ET that allows alleviation of dormancy and hyperpolarization of D seed cells, probably through the stimulation of PM H + -ATPase, as observed in other plant cells [START_REF] Bouteau | Electrogenic laticiferous cells proton pump Hevea brasiliensis. Its in activating sucrose / H + and glucose / H + symports at the plasma membrane[END_REF][START_REF] Wang | Ethylene and nitric oxide are involved in maintaining ion homeostasis in Arabidopsis callus under salt stress[END_REF], further suggests that the control of PM H + -ATPase could have a central role in dormancy regulation. This hypothesis is reinforced by our data on ROS, also known to participate in dormancy regulation. Effectively, MV allows D seed cell hyperpolarization suggesting a stimulation of PM H + -ATPase as observed with FC. Inversely, the decrease of ROS levels by superoxide scavengers like TIRON, or by inhibitors of NADPH-oxidases and peroxidases known to generate superoxides, such as DPI or SHAM, depolarized the ND seed cells possibly through a decrease in PM H + -ATPase activity. The effect of ROS on PM H + -ATPases is not well known but may not be transcriptional in our model,as no change in the level of AHA5-like gene transcript was observed in MV treated D sunflower seeds [START_REF] El-Maarouf-Bouteau | Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination[END_REF]. However, the H + transport activity of AHAs could be regulated by the cytoplasmic redox state which is linked to ROS [START_REF] Mangano | How Does pH Fit in with Oscillating Polar Growth?[END_REF]. The effects of the ROS pharmacology on dormancy demonstrated MV-stimulated dormancy alleviation and germination delay by inhibitors of superoxide generation, confirming the link between the seed cell polarization and its ability to germinate reinforcing the hypothesis of a role in regulation of PM H + -ATPase during dormancy alleviation.

The exact pathways involved in the regulation of the PM H + -ATPases in seeds remain to be determined and none of the inhibitors used in our physiological studies could be claimed to be highly specific for PM H + -ATPases. However, our in vivo electrophysiological data and germination tests on D and ND sunflower seeds with PM H + -ATPases pharmacology are consistent with known roles of ABA, ET and ROS in dormancy control. They further highlight that a mechanism related to cell polarization though PM H + -ATPase activity regulation could be operative and participate in energization of the cells, and thus lead to dormancy release in sunflower seeds.

  Sunflower seeds (Helianthus annuus L.) of the cultivar LG5665 were grown and harvested by the agricultural cooperative Valgrain in an open field in the South of France in 2015. Mature seeds were stored at -20°C to preserve dormancy, or at 20 °C for 2 months to release dormancy [30][31]. Germination assays were carried out by placing in the dark four replicates of 25 sunflower embryos (i.e. seeds without pericarp) on a layer of cotton wool moistened with water, with or without various pharmacological agents (abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid (ACC), sodium orthovanadate (VAN), fusicoccin (FC), 1,4-diazabicyclo[2.2.2]octane (DABCO), 4,5-dihydroxy-1,3benzenedisulfonic acid (Tiron), diphenyleneiodonium chloride (DPI), salicylhydroxamic acid (SHAM), glucose (Glc), l-[β8-D-glucopyranoxyloxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-l-propanone (phoridzin)), in 9 cm Petri dishes in the darkness at 10 °C. Methyl viologen (MV) treatment was carried out by placing embryos on cotton wool moistened with a solution of 100 µM MV for 3 h in darkness at 10 °C, rinsed with water, and then placed on cotton wool moistened with water only for the remainder of the germination trial. Germination was counted daily, and an embryo was considered as having germinated when its radicle elongated by 2 mm. The final germination percentage (%) was expressed as G (%) = (A/B) × 100, where A is the total number of seeds germinated at the end of experiment and B is the total number of seeds tested.

  Figure 2A presents the typical rate of germination for dormant (D) and nondormant (ND) seeds after imbibition with deionized water. Half of the ND seeds germinated after 39.7 hours. The distribution of Vp values recorded for cells impaled in the axis of D and ND seeds from 3 to 28 h after the beginning of seed imbibition are shown in Figure 2B. For that entire period, D seed cells presented more depolarized values with a mean Vp of -42 ± 15 mV (n=71) when non-dormant seed cells display more negative Vp with a mean of -75 ± 16 mV (n=82). The effects of various pharmacological agents were evaluated (see below), using Vp as a valuable estimation of Vm of cells

  right). The average values of Vm recorded on cells from ND and D seed axes pretreated 30 min before voltage recordings with 400 µM VAN (Fig. 3B, left) show that VAN depolarized the cells from ND seed axes to a value close to the Vm recorded from D seed axis cells.

  Thus we checked if ROS scavengers namely TIRON, a scavenger of anion superoxide, and DABCO, a scavenger of singlet oxygen, and inhibitors of NADPH-oxidases and peroxidases, diphenyleneiodonium (DPI) and salicylhydroxamic acid (SHAM), respectively, could regulate the polarization of D and ND seed axis cells. The average values of Vm recorded on cells from ND seed axes pretreated 30 min before voltage recordings with 5 mM DABCO show that it does not depolarize these cells, while TIRON (5 mM), DPI (50 µM) and SHAM (10 µM) depolarize the cells from ND seed

Figure 1 :

 1 Figure 1: (A) From left to right: impalements of sunflower embryo axis cells were carried out in 5 cm Petri dishes filled with 5 ml of bathing medium (BM: 5 mM MES, 1 mM CaCl2 and 1 mM KCl buffered at pH 5.8 with 5 mM Tris); sunflower embryo showing axe (a) and cotyledon (c) and the apex of the axe with an example of putative position of the microlelectrode tip into an epidermal cells after blind impalement. (B) Examples of maintained running potentials for cells of D and ND seeds recorded after blind impalements of the seed axis. (C) Examples of decaying running potentials for cells of D and ND seeds recorded after blind impalements of the seed axis (in = impalement of the axis with microelectrode, out = exit of the axis).

Figure 2 :

 2 Figure 2: (A) Typical rate of germination for dormant (D) and non-dormant (ND) seeds after imbibition. Half of the ND seeds are germinated after 39. 7 hours (B) Values of cell polarizations after impalement for dormant (D) and non-dormant (ND) seed axes bathed in BM from 3 to 30 h after imbibition of the seeds. (C) Mean values of Vp from ND and D cells for different time spans 3 to 6h, 15 to 20h and 22 to 28h. Dashed lines with points and without points refer to the means values for Vp of D cells and ND cells respectively. *Significantly different from ND.

Figure 3 :

 3 Figure 3: Role of H + -ATPases in polarization of D and ND seed cells. (A) Hyperpolarized ND seed cells could be depolarized upon addition of 400 µM vanadate (VAN), an inhibitor of H + -ATPases while depolarized D seed cells could be hyperpolarized by addition of 5 µM fusicoccin (FC) an activator of H + -ATPase, but not depolarized by vanadate. (B) When imbibed in the presence of 400 µM vanadate, the mean value of ND seed cell polarization was increased whereas the mean values of D seed cell polarization imbibed in the presence of 5 µM fusicoccin was decreased. Data represent mean values of at least ten cells and bars represent the standard error. Dashed lines with points and without points refer to the means values from Fig 1B for instantaneous polarization for D cells and ND cells, respectively. *Significantly different from non-treated seeds.

Figure 4 :

 4 Figure 4: (A) Imbibition in the presence of 400 µM vanadate slows the germination of ND seeds, while imbibition in the presence of 5 µM fusiccocin accelerates the germination of D seeds. Dashed lines refer to ND seed germination rate for the left panel and D seed germination rate for the right panel (cf

Figure 5 :

 5 Figure 5: Role of hormones in the polarization of D and ND seed cells. (A) The ND seed cells appeared depolarized when imbibed in the presence of 20 µM µM abscisic acid (ABA), a plant hormone known to maintain dormant state, while D seed cells are hyperpolarized as compared to nontreated D seed cells, especially after overnight treatment with 1 mM 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene known to alleviate dormancy. Mean values are of at least ten cells, and bars represent the standard error. Dashed lines with points and without points refer to the means values from Fig 1B for instantaneous polarization for D cells and ND cells respectively. (B) Imbibition in the presence of ABA slows the germination of ND seeds, whereas imbibition in the presence of ACC accelerates the germination of D seeds. Dashed lines refer to ND seed germination rate for the left panel and D seed germination rate for the right panel (cf Fig.1A). All these data were recorded in the same set of experiments. *Significantly different from non-treated seeds.

Figure 6 :

 6 Figure 6: Role of reactive oxygen species (ROS) in the polarization of D and ND seed cells. (A) The ND seed cells appeared depolarized when imbibed in the presence of 5 mM Tiron, a scavenger of anion superoxide, but also in the presence of 50 µM diphenyleneiodonium (DPI) and 10 µM salicylhydroxamic acid (SHAM), inhibitors of NADPH-oxidase and peroxidase, respectively, while 5 mM DABCO (a scavenger of singlet oxygen) did not depolarize these cells. D seed cells were hyperpolarized when compared to non-treated D seed cells after overnight treatment with 100 µM methylviologen (MV), known to produce ROS and alleviate dormancy. Mean values are of at least ten cells and bars represent the standard error. Dashed lines with points and without points refer to the means values from Fig 1B for instantaneous polarization for D cells and ND cells respectively. (B) Imbibition in the presence of Tiron, DPI and SHAM slow the germination of ND seeds, while imbibition in the presence of MV (C) accelerates the germination of D seeds. Dashed lines refer to ND seed germination rate for the left panel and D seed germination rate for the right panel (cf Fig.1A). All these data were recorded in the same set of experiments. *Significantly different from non-treated seeds.

Figure 7 :

 7 Figure 7: ND cell hyperpolarization allows for sugar influx through H + /sugar symporter. (A) Addition of 500 µM glucose (Glc) induced the depolarization of ND seed cells. (B) Imbibition in presence of 500 µM or 1 mM glucose allowed the depolarization of ND seed cells when compared to untreated ND seed cells, while co-imbibition with glucose and phlorizin, a sugar H + /Glc inhibitor, prevented sugar-induced depolarization. Imbibition in the presence of 500 µM glucose did not permit the depolarization of D seed cells when compared to untreated D seed cells. Mean values are of at least ten cells and bars represent the standard errors. Dashed lines with points and without points refer to the means values from Fig 1D for instantaneous polarization for D cells and ND cells, respectively. (C) Imbibition in the presence of 1 mM phlorizin slows the germination of ND seeds, while imbibition in the presence of 500 µM glucose accelerates the germination of D seeds. Dashed lines refer to ND seed germination rate for the left panel and D seed germination rate for the right panel (cf Fig.1A). All these data were recorded in the same set of experiments. *Significantly different from nontreated seeds.

Figure 1

 1 Figure 1:

  The time to reach 50% germination (T50) was calculated according to the formula of Coolbear et al.[START_REF] Coolbear | The Effect of Low Temperature Pre-Sowing Treatment on the Germination Performance and Membrane Integrity of Artificially Aged Tomato SeedsNo Title[END_REF] modified by Farooq et al.[START_REF] Farooq | Thermal Hardening: A New Seed Vigor Enhancement Tool in Rice[END_REF]: T50 = ti + [(N / 2 -ni) (titj )] / ni -nj (N is the final number of emergence, and ni and nj, the cumulative number of seeds germinated by adjacent counts at times ti and tj, respectively when ni < N / 2 < nj).

	2.2.	Electrophysiology
	Intracellular voltage measurements were carried out on a vibration-stabilized bench in a Faraday
	cage. Microelectrodes were made from borosilicate capillary glass (Clark GC 150F, Clark
	Electromedical, Pangbourne Reading, UK) pulled on a vertical puller (Narishige PEII, Japan), filled
	with 600 mM KCl and connected by a reversible Ag/AgCl half-cell to the measuring set-up consisting
	of an electrometer (Axoclamp 2A, Molecular Devices, USA) and an oscilloscope (Gould 1425, Gould
	Instruments Ltd, UK). Microelectrodes were placed with micromanipulators (WR6-1, Narishige, Japan)

Acknowledgements

This work was supported by grants from MESRI to LIED.

Membrane potential (mV)


Tien Cuong Nguyen

Dominique Anne-Archard

Xavier Cameleyre

Eric Lombard

Kim Anh To

Luc Fillaudeau

Tien Cuong

Bio-catalytic hydrolysis of paper pulp using in-and ex-situ multi-physical approaches: Focus on semidilute conditions to progress towards concentrated suspensions

Keywords: biorefinery, enzymatic hydrolysis, kinetics modelling, paper pulp, rheometry, yield stress

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

The latest global agreement, the Paris agreement, was established in 2015 to reduce the scale of the fossil-fuel-based economy. Greater use of renewable resources with low carbon footprints is being advocated to substitute the usage of fossil fuels and to achieve the goal of a decarbonised economy. Biofuel stands as one potential alternative, and is being scrutinised in many countries [START_REF] Chang | Potential of bioenergy production from biomass wastes of rice paddies and forest sectors in Taiwan[END_REF]. This development is not only justified by economic reasons, but also by societal demand and environmental constraints that necessitate the move towards renewable energies. Cellulosic biomass provides a lowcost, renewable, and abundant resource that has the potential to support large-scale production of fuels and chemicals via biotechnological routes [START_REF] Gibbons | Integrated biorefineries with engineered microbes and high-value co-products for profitable biofuels production[END_REF]. Among the major users of lignocellulose resources generated through forestry and agricultural practice, the pulp and paper industry holds a strategic position. Currently, the promotion of biorefineries producing multiple products, including higher-value chemicals as well as fuels and power, is a major objective of numerous consolidated programs in the world.

In order to achieve economic viability, the biorefinement of lignocellulosic resources must be operated at very high feedstock dry matter content. This strict prerequisite imposes a considerable constraint, particularly on the physicochemical and biocatalytic steps, which, overall, aim to produce high-quality, fermentable sugar syrups. Moreover, industrial criteria regarding maximum reactor volumes, energy and water consumption, and wastewater management must also be respected. The pulp and paper industry is able to provide a tried and tested industrial model for processing lignocellulose biomass into pre-treated cellulose pulps. The pulp product of this industry is appropriate for modern biorefining because it displays low lignin content, is free of inhibitory compounds that can perturb fermentations, and is devoid of microbial contaminants [START_REF] Vallette | Le bois, la pêtes, le papier[END_REF]. The process of biofuel production can thus be coupled with the pulp and paper industry. Two main advantages are highlighted here. The first is the perfect control of paper pulp quality due to the efficiency of woody substrate pre-treatment and to the elimination of the lignin fraction in the initial biomass. The second is that the pulp and paper industry produces energy by valorising residues and co-products, thus reducing final biofuel costs [START_REF] Moshkelani | The forest biorefinery and its implementation in the pulp and paper industry: Energy overview[END_REF].

In order to produce 2 nd -generation biofuels, pre-treated paper pulp would be hydrolysed and then fermented to convert the simple sugars (hexoses and also pentoses) into molecules of interest. Conventionally, this process involves two separate steps:

hydrolysis and fermentation (SHF). In addition, it can also use simultaneous saccharification and fermentation processes or, more recently, consolidated bioprocessing, which integrates enzyme production, saccharification, and fermentation into a single process. In the SHF process, cellulose is enzymatically hydrolysed by cellulases during the first step to form simple sugars (e.g. glucose), which are consumed in the second step by Saccharomyces, Zymomonas, or other microorganisms to obtain the desired product [START_REF] Fan | Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor[END_REF][START_REF] Herrera | Production of Xylose from Sorghum Straw Using Hydrochloric Acid[END_REF][START_REF] Zhao | Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis[END_REF]. The main advantage of SHF is the ability to carry out each step in its optimum conditions: temperature, pH, etc.

The key steps are always the pre-treatment techniques and the conversion into fermentable sugars [START_REF] Eggeman | Process and economic analysis of pretreatment technologies[END_REF]. A better scientific understanding, and ultimately exact technical control, of these critical biocatalytic reactions, which involve complex matrices at high solid content, currently pose a major challenge that must be overcome to facilitate the intensification of biorefining operations. Among the main parameters to be studied, the rheological behaviour of the hydrolysis suspension and the fibre particle size stand out as major determinants of process efficiency, and are responsible for the choice of equipment to be used and the strategies to be applied [START_REF] Wiman | Rheological characterization of dilute acid pretreated softwood[END_REF]. Nevertheless, only a limited number of studies cover the rheological behaviour of lignocellulose matrices in highly concentrated suspensions during biocatalytic degradation [START_REF] Nguyen | Rheology of Lignocellulose Suspensions and Impact of Hydrolysis: A Review[END_REF].

The purpose of this study was to investigate the destructuring of fibre during enzyme attack under a multi-scale approach using different analytical techniques. Semidilute conditions were chosen, as they introduce the complexity of particle-particle interactions, which are strongly involved in the transfer limitations observed in high dry matter content, without inhibiting the bioreactions. Figure 1 illustrates the three blocks corresponding to the three levels of observation: macro-scale with viscometry and rheometry, micro-scale with chord length distribution (CLD) and molecular scale with biochemistry (chemical analyses of soluble fraction and solid fraction). This threemembered framework permits the analysis and comparison of in-and ex-situ methods (excluding biochemical analysis). Experiments were conducted using three substrate concentrations in a semidilute regime and two enzyme concentrations to explore the impact of substrate properties and enzyme ratio on fibre destructuring kinetics (bioconversion rate). Then, phenomenological models can be established and considered as a whole to provide a full overview of the mechanism. In this regard, the phenomenological models should fulfill certain criteria, such as reliability, simplicity, and congruity with the experimental information. It is hoped that a global result from these three sub-blocks will provide a "knowledge block" to explain certain scientific limitations and lead to the implementation and intensification of considered bioprocesses.

Materials and Methods

Experimental set-up

An experimental set-up was specifically developed which consisted of a bioreactor and a home-designed impeller system associated with several in-situ sensors (temperature, pH, rotation speed, torque, FBRM sensor). The bioreactor was a homemade glass tank (diameter: 130 mm, Hmax: 244 mm, V: 2.0 L) with a water jacket for thermal regulation.

A specific agitator included a double impeller to minimise the difficulty in substrate mixing and ensure suspension homogeneity (Figure 2).

Physical analysis

Lignocellulose suspensions demonstrate complex rheological behaviour, and there is no standard method for studying their flow behaviour. To characterise their rheological properties as finely as possible, two measurement strategies were combined: (i) ex-situ rheometry (oscillation mode), which provided yield stress and elasticity information, and (ii) in-situ viscometry, which followed suspension viscosity in real time during enzyme attack. Particle size was analysed by in-situ chord length measurement (FBRM).

Ex-situ rheometry

Classic rheometry based on continuous permanent shear rate appears irrelevant for lignocellulosic substrates because of the rapid aggregation of fibres [START_REF] Nguyen | Rheology of Lignocellulose Suspensions and Impact of Hydrolysis: A Review[END_REF]. Thus, oscillatory measurements (including strain sweeps and frequency sweeps) were performed. These measurements offer several advantages: firstly, they prevent fibre aggregation caused by constant unidirectional shear flow; secondly, they provide additional information on the rheological behaviour of the suspensions (yield stress in this case). The storage modulus G' and the loss modulus G'' were measured using a Mars III rheometer (Thermo Scientific). Dynamic measurements were performed with serrated plates (60 mm, roughness 400 µm, gap size: 1.5 mm) on samples taken during hydrolysis. First, an oscillatory shear flow was set up with increasing shear stress amplitude from 0.1 to 20 Pa and at a fixed frequency (1 Hz). This first measurement was used to determine the linear domain. Then a scan was carried out in the linear domain for frequencies from 0.5 Hz to 20 Hz and fixed shear stress amplitude. The analysis was performed at 20 °C. Several methods can be used for yield stress determination. It can be interpreted as the stress amplitude at which the elastic modulus G' becomes smaller than the shear modulus G", or as the stress amplitude at which the loss modulus G" reaches a maximum. It can also be identified as the maximum elastic stress verifying a linear relation = . where γ is the strain amplitude [START_REF] Damani | Viscoelastic characterization of medium consistency pulp suspensions[END_REF][START_REF] Walls | Yield stress and wall slip phenomena in colloidal silica gels[END_REF].

Especially for lignocellulose substrates, which cannot stand long measurement times, it was defined as the first departure from the linear viscoelastic region [START_REF] Wiman | Rheological characterization of dilute acid pretreated softwood[END_REF][START_REF] Stickel | Rheology measurements of a biomass slurry: an inter-laboratory study[END_REF]. In the present study, a 20% reduction in G' was chosen.

In-situ rheometry

Ex-situ measurement was limited by the number of samples and the substrate properties, predominately decantation and flocculation of material. To overcome these difficulties, in-situ viscometry was conducted throughout hydrolysis. It was based on the determination of power consumption (or power number

C N P N d P Np ⋅ ⋅ = ⋅ ⋅ = π ρ 2 ; 3 5
) versus the Reynolds number (

µ d N 2 Re ⋅ ⋅ = ρ ) during
suspension mixing (see [START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF] for details). The viscosity was calculated from the power consumption curve of the mixing system under consideration using a semi-empirical model including laminar and transition regions for the reference curve with a one-to-one relationship between Np and Re:

782 . 0 ; 128 . 0 : Re 0 1 0 = =         +       = q N with N Kp Np p q q p q (Eq. 1)
The Kp constant for the mixing system was 97.9, while the Metzner-Otto constant used to estimate an equivalent shear rate was Ks = 32. All the geometric constants were determined from measurements using Newtonian (water, Marcol oil, and glycerol) and non-Newtonian fluids (xanthan-sucrose solutions). Once the experimental set-up was characterised by its power consumption curve Np(Re) and its Ks value, in-situ viscometry of the suspension was performed before the addition of enzymes and then throughout the biocatalytic reaction.

In-situ particle size analysis

Focus beam reflectance measurement (FBRM) enabled in-situ quantification and characterisation of chord length distribution (CLD). The FBRM sensor (FBRM G400, Mettler-Toledo, range: 0.1 to 1000 µm) was set up in the bioreactor to detect and monitor the changes of particle dimensions during enzymatic hydrolysis in real time.

FBRM measurement is a laser-based technique. A solid-state laser light source (λ = 795 nm) provides a continuous beam of monochromatic light that is sent down the FBRM probe. A precision motor-pneumatic or electric-is used to rotate the precision optics at a constant speed. The scan speed is fixed at 2 m.s -1 . As the scanning-focused beam sweeps across the face of the probe window, individual particles or particle structures (agglomerated or floc) backscatter the laser light towards the probe. Particles and droplets closest to the probe window are located in the scanning focused spot and backscatter distinct pulses of reflected light, which are detected by the probe and translated into chord lengths based on the simple calculation of the scan speed (velocity) multiplied by the pulse width (time). A chord length is simply defined as the straightline distance from one edge of a particle or particle structure to another edge. Typically, thousands of individual chord lengths are measured each second to produce the chord length distribution, which is the fundamental measurement provided by FBRM.

Biochemical analysis

Dry matter content

The water content of substrates and hydrolysed suspensions was determined by drying at low temperature and pressure. Volumes of samples (≈1 mL) were put in Eppendorf tubes (known mass, mepp). These Eppendorfs (mini) were then placed in an oven at 60 °C, 200 mbar for 5 days, and afterwards, weighed (mfin). Water content (W) and dry matter content (DM) were calculated using Eq. 2 (accuracy ±0.5%):

W DM m m m m W epp ini fin ini - = ⋅ - - = 100 (%) ; 100 (%) (Eq. 2)

Monomers and oligomers

Samples were centrifuged at 13000 rpm for 5 min. Glucose and soluble cellooligosaccharides, (i.e. with a degree of polymerization lower than 6) in the supernatants were quantified by an AMINEX HPX-87P carbohydrate analysis column (Bio-Rad Laboratories, Richmond, CA) using a high-performance liquid chromatography system (separations module: Waters Alliance 2690; refractometer detector: Waters 2414, Milford, MA). Conditions were optimised to detect and quantify the different cellosaccharides. Analysis was performed at 60 °C with deionised water and 0.1M Pb(NO2)2 as mobile-phase (ratio 80/20 v/v) at a flow rate of 0.5 mL/min for 30 min. Glucose and cello-oligosaccharide standards with a DP ranging from 2 to 5 were used (Sigma Chemical Co., St. Louis, MO).

Experimental strategy

Enzyme hydrolysis was carried out at 40 °C due to enzyme heat stability (activity reduction at high temperature), energy conservation, and taking into account the optimal conditions for the cell cultivation step. The pH of the medium was adjusted to 4.8. To prevent microbial contamination, 0.2 mL of a solution of chloramphenicol (5 g.L -

Results and Discussions

Bioconversion rate

The production of monomers (C6, C5) and water-soluble oligosaccharides is crucial for the development of new intensified bioprocesses. First of all, the bioconversion rate was calculated from the glucose produced using Eq. 3.

[

] [ ] [ ]         +       ⋅ ⋅ ⋅ = = s s w s theory theory measured m V Cellu DM m Glc Glc Glc ion Bioconvers ρ 9 . 0 100 100 (%) (Eq. 3)
The theoretical conversion coefficient from cellulose to glucose is 1/0.9.

Under enzyme activity, the polymeric cellulose chain is broken down to produce monomers. Oligomers (DP > 2) were not detected for any of the experiments. Xylose was quantifiable only in the case of 3% w/v -0.5mL enzyme/g cellulose.

As expected, the concentration of monomers (glucose, xylose) increases with hydrolysis time. For example, in the 3% w/v cases, the monomer concentration increased regularly during hydrolysis, so that enzyme loading had an impact on the quantity of the glucose released: 5.1 to 19.1 g.L -1 , which corresponds to 19 and 73% bioconversion for 0.1 and 0.5 mL/g cellulose respectively (Table 1). Few articles have examined the evolution of water-soluble cello-oligosaccharides. Sun and Cheng [START_REF] Sun | Hydrolysis of lignocellulosic materials for ethanol production: a review[END_REF] analysed the hydrolysis of microcrystalline cellulose (10 g.L -1 ) by cellulase produced by Cellulomonas fimi. They did not detect soluble oligomers with DP≥4, but the cellotriose concentration varied between 0.2 and 0.6 g.L -1 depending on the enzyme used-endo-glucanase or cellobiohydrolase. Solubilisation reached 61% and 50% respectively for each enzyme separately. In contrast, the intermediates of cellulose hydrolysis were not found because the Accellerase 1500 cocktail contains all the types of activity required to rapidly degrade these intermediates during hydrolysis.

The bioconversion yields are comparable to data reported in the literature. For a 2% (w/w) suspension of oven-dried corncob, after 24 h hydrolysis the glucose conversion varied between 30 and 82% for 6 and 30 FPU/g [START_REF] Carvalheiro | Hemicellulose biorefineries: a review on biomass pretreatments[END_REF]. With the hydrolysis of a pretreated wheat straw suspension at 1% (w/v), a doubled bioconversion (from 45.9 to 87%) after 18 h with enzyme loading between 9.6 and 57.6 FPU/g was reported [START_REF] Pierre | Highperformance hydrolysis of wheat straw using cellulase and thermomechanical pretreatment[END_REF].

Changes in dry matter content were monitored by determining the water content in the sample at different hydrolysis times. This technique involves the error inherent to sampling heterogeneous suspensions. Beside this technique, the dry matter content was also determined from dissolved and undissolved substrate measurements. According to the hypothesis of the conservation of substrate mass before and after hydrolysis, the dry matter in suspension can be deduced from the initial quantity and the hydrolysed quantity (which released soluble components-monomers and cello-oligosaccharides).

As previously reported, the water-soluble cello-oligosaccharides with DP > 2 were not quantifiable, so only glucose, xylose, and cellobiose were used to calculate the quantity of hydrolysed substrate. Mass balance was conserved around 95% (Table 1).

Rheological behaviour during hydrolysis

Viscosity and yield stress

In-situ viscometry showed that substrate suspensions were non-Newtonian shearthinning fluids. The viscosity of suspensions at 100 rpm (corresponding to an equivalent shear rate of 50 s -1 ) as a function of hydrolysis progress is illustrated in Figure 3-A. As expected, slurry viscosity decreased during hydrolysis. Excluding the very beginning of the reaction (t < 0.2 h), viscosity changed drastically at the beginning of hydrolysis. This sharp reduction in viscosity was observed whatever the concentration or enzyme/substrate ratio. For example, at 3% dm (w/v), 0.5 mL E/g cellulose, the viscosity decreased from 68 to 3 mPa.s. This change in the physical appearance of the slurry is associated with the biochemical changes and particle size changes occurring in the fibres [START_REF] Kaar | Benefits from Tween during enzymic hydrolysis of corn stover[END_REF][START_REF] Roche | Particle concentration and yield stress of biomass slurries during enzymatic hydrolysis at high-solids loadings[END_REF]. The drop in viscosity is a combination of the decrease of solid concentration (solubilisation) and of the fragmentation of cellulose fibres [START_REF] Rosgaard | Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw[END_REF][START_REF] Vázquez | Hydrolysis of sorghum straw using phosphoric acid: Evaluation of furfural production[END_REF].

Under the effects of enzymes, the cellulose chains are broken up to produce smaller particles and non-dissolved cellulose is converted into soluble compounds such as monomers and water-soluble oligomers. In addition, the viscosity drop is suggested to be strongly connected with the degradation and decrease in water binding capacity of the lignocellulose matrices during enzyme-based hydrolysis [START_REF] Chang | Structure, pretreatment and hydrolysis of cellulose[END_REF].

The initial viscosities of the 3% (w/v) suspensions were 14-fold higher than those of the 1% (w/v) suspension (~70 and ~5 mPa.s, respectively). The nonlinear dependence of viscosity on the concentration is expected and can be explained by an increase in particle interactions, less free water, and hydrogen bonding between cellulose chains.

As the solid concentration increases, the average distance between particles in the slurry decreases, leading to enhanced contact between particles, especially if there is an entanglement of amorphous fibres between particles [START_REF] Dasari | A scraped surface bioreactor for enzymatic saccharification of pretreated corn stover slurries[END_REF]. The high increase in initial viscosity of between 1% (w/v) and 3% (w/v) signifies that the increase of substrate concentration becomes the major stress for enzymatic hydrolysis at high (and very high) dry matter content levels. That is why a special strategy to reach the condition of high substrate concentration is absolutely necessary. During hydrolysis, a significant drop in slurry viscosity is observed within the first 10 h of the hydrolysis reaction. These results are supported by the literature for a wide range of matrices, particle sizes, and enzyme/cellulose ratios [START_REF] Geddes | Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse[END_REF][START_REF] Pereira | Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection[END_REF][START_REF] Um | Optimization of Ethanol Production from Concentrated Substrate[END_REF], although a large heterogeneity of viscosity data is reported. This can be explained by different substrates, pre-treatments, morphogranulometry and enzymes, and operating conditions.

The effect of enzyme concentration on viscosity is clearly demonstrated even for the lowest concentration in Figure 3-A. For the same substrate concentration, the higher the enzyme/g cellulose ratio (E/C), the faster the viscosities decrease. Within 5 h, the viscosity of a 3% (w/v) suspension was reduced by 85% for 0.5 mL E/g cellulose; whereas its reduction was limited to 30% for 0.1 mL E/g cellulose. This observation is in agreement with other authors [START_REF] Rosgaard | Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw[END_REF][START_REF] Geddes | Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse[END_REF][START_REF] Du | Enzymatic liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a horizontal rotating bioreactor[END_REF]. Furthermore, two tendencies in viscosity evolution were found for suspensions containing 0.1 mL/g cellulose. Firstly, the suspension viscosity increased during the first hour consecutive to swelling and unbinding effects (clearly observed for 2% w/v and 3% w/v, not significant for 1% w/v); after that, the viscosity decreased because of depolymerisation by the enzymes.

Shear stress sweep and frequency sweep experiments were performed in oscillation mode (see §2.3.1). The frequency sweep revealed that the elastic modulus G' and the viscous modulus G'' do not depend on frequency (Figure 3-B). In addition, G' is always greater than G'', with a ratio G'/G'' which can be identified in Figure 3-B. This is characteristic of viscoplastic behaviour, and shear stress sweeps were then used to evaluate yield stress. All suspensions showed viscoplastic shear-thinning behaviour both initially and throughout hydrolysis. Shear-thinning has already been mentioned by other authors [START_REF] Wiman | Rheological characterization of dilute acid pretreated softwood[END_REF][START_REF] Pereira | Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection[END_REF][START_REF] Du | Enzymatic liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a horizontal rotating bioreactor[END_REF]. Before the introduction of enzyme, the rheological behaviour for small deformations is mainly elastic, with a high value of G' and a ratio G'/G'' close to 5. For a 3% (w/v) suspension at t = 0 h and in the linear domain, G' and G" reached about 1000 and 200 Pa. This is supported by the literature for different matrices. For corn stover, at 12% dm, G' and G" were reported as 2000 and 300 Pa [START_REF] Stickel | Rheology measurements of a biomass slurry: an inter-laboratory study[END_REF]; for acid pretreated softwood, these values were 100 and 50 Pa respectively [START_REF] Wiman | Rheological characterization of dilute acid pretreated softwood[END_REF]. When increasing the shear stress, all the samples exhibited two zones: a first where G' and G" did not depend on shear stress (linear domain) and a second where both moduli decreased. In this second zone, G' deviated sharply from a stable curve and crossed (or tended to cross) G". During hydrolysis, a regular decrease in both moduli was observed. For example, in the 3%-0.5 case, a 1000-fold reduction was observed for both G' and G" after 24 h of enzyme attack. Moreover, the elastic character is noticeably preserved. One also notes that the higher the enzyme activity, the faster the decrease in elasticity (in agreement with the viscosity results above). This discussion highlights, yet again, the strong impact of enzyme concentration on how rheological behaviour varies during hydrolysis.

As viscometric yield stress measurements were not possible for these suspensions, this yield stress τ0 was deduced from the elastic modulus (G') (see Figure 3-B). Yield stress can be regarded as the stress required to initiate flow. Yield stress values between 1 and 20 Pa were determined at 1% and 3% dm (w/v) before hydrolysis. With an increase in substrate concentration from 1% to 3% dm (w/v), yield stress increased 20-fold (Figure 4). This confirmed the critical substrate concentration (≈3% dm (w/v) determined by insitu measurements) beyond which the viscosity increased exponentially [START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF]. The results presented were slightly higher than those obtained from pre-treated softwood: 0-28 Pa for 4-12% substrate concentration [START_REF] Wiman | Rheological characterization of dilute acid pretreated softwood[END_REF] and from pre-treated corn stover: 0.26-22.9

Pa for 5-17% dm [START_REF] Pimenova | Effect of corn stover concentration on rheological characteristics[END_REF]. This difference could be due to the nature and the physical characteristics of the matrices and to the method used to determine yield stress.

During hydrolysis, yield stress decreased sharply as enzyme attack progressed (Figure .    4). This can be explained by the drop in solid content in suspension and by a reduction in fibre-fibre interactions due to hydrolysis progress. The decrease of yield stress was greater for experiments with higher enzyme concentrations. The final yield stress (at 24 h hydrolysis) of 3%-0.1 was 10 times higher than that of 3%-0.5. This observation is directly correlated to the impact of enzyme activities on fibre degradation. In addition, the yield stress of the hydrolysed samples (3%-0.5 at 24 h, DM = 1.14%) exhibited lower values for similar DM content compared to the original material (yield stress at 0 h of 1% -0.5, DM = 0.97%). This can be explained by the modification of fibre structure, diameter, and shape. Decreasing yield stress during enzyme hydrolysis was previously reported for corn stover [START_REF] Roche | Particle concentration and yield stress of biomass slurries during enzymatic hydrolysis at high-solids loadings[END_REF] and pre-treated softwood [START_REF] Wiman | Rheological characterization of dilute acid pretreated softwood[END_REF]. These two studies also reported a lower yield stress value for hydrolysed slurries when compared to nonhydrolysed slurries at the same solid content.

Figure 4 shows the dependence of yield stress during hydrolysis presented in terms of dry matter content. Taking the criterion that the fluid behaves as a pourable liquid at a yield stress below 1 Pa (the values of 1% are negligible), interestingly, results for 3% show that the two yield stress curves collapse onto a single curve when plotted against dry matter content. It decreased exponentially with the decrease in dry matter content during hydrolysis. Yield stress becomes negligible when solid matter < 2%. This has real significance for the choice of substrate flow rate for the cumulative feed strategy or if the slurry has to be pumped into another bioreactor.

Uniqueness of viscosity-time curve

In order to explore the change in viscosity during enzyme hydrolysis and to compare its kinetics between different experimental conditions, a critical time is estimated through a normalised viscosity defined as follows:

fin fin t µ µ µ µ µ - - = 0 * (Eq. 4)
µ* is a dimensionless viscosity; and µ0, µt, µfin are the viscosities at t = 0 h, ti and the final viscosity (the viscosity of a suspension containing the whole dry matter as soluble fractions -hypothesizing total conversion).

µ* describes the reduction of viscosity during enzyme hydrolysis. It starts from 1 (t = 0 h) and tends to 0 (t = 24 h). From this quantity, a critical time noted t(µ* = x) can be defined as the time at which the viscosity was x% of the initial suspension viscosity.

The relationship between these critical times t(µ* = x) and dimensionless viscosity, µ* only depends on the enzyme concentration used regardless of the substrate concentration. These critical times decreased exponentially with the dimensionless viscosity. The ratio of these critical times between both conditions was around 4-fold.

Thus the hydrolysis time, t, can be normalised using a selected critical time t(µ* = x0) (Eq. 5):

) * ( * 0 x µ t t t = = (Eq. 5)
The dimensionless time-viscosity curves are plotted in Figure 5 (µ* = 0.25).

Interestingly, a single curve is exhibited for t* > 0.05, i.e. when the possible swelling and unbinding effects can be neglected. This uniqueness of dimensionless timeviscosity curves was observed whatever the hydrolysis conditions in the studied cases (substrate concentrations, enzyme ratio). These results suggest that during the period and for a 75% reduction of initial viscosity, a similar degradation mechanism could be assumed in semidilute regime (≤3% dm w/v), although rheological behaviour strongly differs between 1% and 3% w/v suspensions. The uniqueness of viscosity-time curves at macroscopic observation suggests a similar mechanism of fibre degradation. Considering these in-situ and ex-situ rheological results, macro scales will be explored with chord length distribution analysis, and then kinetics will be modelled.

Particle size evolution

The time evolution of the mean chord length and chord number for the four different conditions is shown in Figure 6. Considering chord length distribution (CLD) for the initial suspension, we define four classes (I: < 60 µm, II: 60 to 90, III: 90 to 160 µm, IV: 160 to 600 µm) which correspond to 25% of this population. Figure 7 illustrates the evolution of each class during hydrolysis for the case 1% w/w -0.5 mL enzymes/g cellulose.

A sharp decrease in mean chord length from 120 to 60 µm (Figure 6) is observed: within two hours for E/S = 0.5 mL/g and six hours for 0.1 mL/g. Considering chord number, initial values are almost proportional to concentration and during hydrolysis, two successive trends are observed. During the first step, the total chord number increased for all experiments, although the durations of this step differed strongly: around 8 to 10 hours for E/S = 0.1 mL/g, and 1 to 3 hours for E/S = 0.5 mL/g. Meantime, the relative increase of chord number is lower for the largest E/S ratio. Beyond that, enzyme activity occurs and the finest particles are converted into dissolved compounds, generating a reduction in chord number. As demonstrated in Figure 6, the fragmentation mechanisms appear to be the dominant effect in the strong viscosity reduction observed during the first 5 h, while solubilisation increased to 31%. In contrast, after 5 h, fragmentation is negligible (class IV almost constant) while solubilisation increased to 76% (at 24 h). Correlatively, the viscosity evolution was limited and the finest populations still increased from 38% to 50%.

Considering now

Modelling kinetics at macro-, micro-, and biochemical scales

Hydrolysis induces a reduction in particle size, dry matter content, and viscosity, which promotes mixing and fibre accessibility. The kinetics of viscosity (macro), chord length (micro) and substrate and product concentrations (biochemical) stand as key indicators for understanding and controlling bioprocess performance. The kinetics of biochemical enzymatic reactions have been extensively reported in the literature [START_REF] Frémaux | Eléments de cinétique et de catalyse[END_REF], contrary to physical parameters such as viscosity and granulometry. A lot of enzyme reactions (e.g. hydrolysis, oxidation, and reduction or cofactor mechanisms) are second-or higher order reversible reactions. Many are limited by diffusion and physical accessibility. Adsorption (reaction) in high molecular weight structures (for example, proteins, polynucleotides, polysaccharides, or heterogeneous protein-phospholipid, proteinnucleotide, and protein-polysaccharide structures) is more complex. Among the numerous models reported in the literature, one of the best known is the Michealis-Menten equation [START_REF] Michaelis | Die Kinetik der Invertinwirkung[END_REF] but a first-order model was also reported by Chrastil [START_REF] Chrastil | Enzymatic product formation curves with the normal or diffusion limited reaction mechanism and in the presence of substrate receptors[END_REF].

The time evolution of viscosity µ, mean chord length lcm, and residual substrate concentration in dry matter S (%) are modelled by the general equation Eq. 6:

α X k dt dX ⋅ = - (Eq. 6)
where X is the variable being modelled (µ, lcm and S); k is the kinetic constant, and α the model/reaction order (/). As boundary conditions, X varies from X0 to X∞, corresponding to initial and final values respectively.

A second-order model accurately describes the time dependence of the physicochemical parameters-viscosity, chord length, and dry matter content. For the lowest enzyme ratio, an increase of the mean chord length was observed during the first hours.

In these cases, the proposed model was restricted to the time interval corresponding to the maximal value of lcm until the end of hydrolysis (experiments with 0.1 mL enzyme/g cellulose). This lcm increase step could be explained by fibre swelling and unwinding due to limited enzyme activity. To sum up, the most suitable kinetic models are second order and written as Eq. 7 to 9:

+

(Eq. 9)

The model was adjusted via the least squares method for each of the physicobiochemical parameters. The coefficients and the correlation coefficients are presented in Table 2. The correlation coefficients indicated the fine agreement between the models and experimental data. The enzyme cocktail (Accellerase 1500) contained only cellulases and hemicellulases, so the lignin fraction can be considered as contributing to the non-hydrolysable fraction ∞ S . For our substrate, lignin represents less than 2%, and thus can be neglected. The final viscosity µ∞ corresponds to the viscosity of a suspension containing all soluble fractions (total conversion). In the present case, this value would be close to the supernatant viscosity (0.7 mPa.s) and µ∞ is negligible compared to the initial suspension viscosity. In ideal conditions, the solid fractions should be converted into soluble fractions and the ultimate chord length lcm∞ would then be null. However, experimentally, there is always some part that remains nonhydrolysable substrate, so lcm∞ cannot be neglected compared to lcm0, and is assumed equal to lcm24h.

Effects of enzyme and substrate concentrations are clearly observed for all constants.

With kS, the increase of the E/S ratio from 0.1 to 0.5 mL/g cellulose led to a 7-to 9-fold increase. When the initial substrate concentration increased from 1 to 3% w/v, a reduction of 2-3 times was observed. The same tendency was found with kµ, which demonstrated the strong impact of enzyme and substrate concentrations on the variation of suspension viscosity during hydrolysis. Similar impacts were observed for enzyme concentration and substrate concentration: 8-fold and 4-fold respectively. The final value of the mean chord length, lcm∞, seemed identical in magnitude; however, the impact of enzyme ratio can be clearly distinguished by the absolute values of klc. An increase of 3.5-5.5 -fold of klc was observed when the enzyme concentration passed from 0.1 to 0.5 mL/g cellulose.

Using equations 7 to 9, Figure 8 illustrates the successive phenomenological limitations observed during hydrolysis. Rheological behaviour appears to be the primary limiting factor. Transfer limitation due to high viscosity and yield stress is, however, temporary (during the first 5 h) but constitutes an important phenomenon. Viscosity collapse and particle fragmentation are concomitant. Beyond this, the limited reduction in size indicates a threshold for biocatalytic reaction. This could be due to recalcitrant fractions or inefficient enzyme activities. It is therefore necessary to study the physical and biochemical structures of this recalcitrant fraction in order to identify how it could be degraded.

The major challenge in 2 nd -generation biofuels is to reduce costs so as to compete with 1 st -generation ones. Therefore, processing at high solid content is mandatory. However, the rheological behaviour of the hydrolysis suspension stands out as the first and major determinant of process efficiency, and has to be considered a key criterion in proposing a rational strategy for reaching high dry matter content.

To increase transfers and bioreaction efficiency, a strategy can be built from the identified parameters in a semidilute regime: the critical substrate concentration C* (meaning a drastic increase of initial suspension viscosity beyond C*) and a targeted hydrolysis time, t(µ* = x0) (corresponding to a chosen relative reduction of initial viscosity), can be used to define a reference feed rate Qc (Eq. 10): ! = " * .$ * %& (. ℎ (Eq. 10)

A cumulative feeding strategy can be defined, based on Qc, which would allow working in a favourable regime so as to reach high hydrolysis yield by avoiding instantaneous high substrate concentrations.

Conclusion

This study aimed to delve deeply into the biophysical and transfer limitations occurring during enzymatic hydrolysis in the cellulosic biofuels context. Considering paper pulp substrates, semidilute conditions were chosen, as they introduce the complexity of particle-particle interactions, which are strongly involved in transfer limitations, without inhibiting the bioreaction. Biochemical and physical phenomena were explored through a specific experimental set-up associating in-situ and ex-situ analyses.

Non-Newtonian behaviour associated with non-negligible yield stress stand as the major factors limiting process efficiency and progress towards high solid loading. The uniqueness of dimensionless time-viscosity curves was observed whatever the operating conditions, and suggests a similar mechanism for fibre degradation. The evolution of biophysical parameters during hydrolysis was observed as the results of a combination of fibre fragmentation, which dominated during the first step, and solid solubilisation.

Kinetics modelling enabled demonstration of the successive phenomenological limitations and their magnitude. All these elements lead to the proposal of a reference feed rate which would be used in a cumulative feeding strategy for reaching high solid loading and balancing energy consumption and process efficiency.

Figure 7 ,

 7 coarse population (class IV) decreased regularly while the finest population (Class I) increased. The initial augmentation of chord number can be explained by the fragmentation of cellulose fibres. Coarse particles are attacked and divided into several fine particles.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Experimental methodology and strategy

Figure 4 :Figure 5 :

 45 Figure 4: Yield stress during hydrolysis versus dry matter content (1%-0.5 stands for: 1% dm

Figure 6 :

 6 Figure 6: Overview of in-situ (FBRM) particle size analysis: A: Chord number and B: Mean

Figure 7 :

 7 Figure 7: Population balance (classes defined from initial chord length distribution), in-situ

Figure 8 :

 8 Figure 8: Reduction of physico-biochemical parameters during hydrolysis using kinetic models

Table 1 :

 1 Bioconversion rate (%) during enzyme hydrolysis

	Substrate concentration	Enzyme/Substrate		Mass balance
			0h 0.25h 2h 5h 10h 24h
	(%gdm.L -1 )	(mL/g cellulose)		(24h,%)
	1	0.1	0 1.0 4.7 9.6 11.6 13.2	93.7
	1	0.5	0 6.0 19.6 31.1 52.4 76.6	94.1
	3	0.1	0 1.3 6.4 11.3 14.5 19.1	94.6
	3	0.5	0 6.0 20.6 32.7 54.1 73.5	95.2

Table 2 :

 2 Kinetic coefficients of rheological, granulometric (lcm∞ in µm) and biochemical parameters, and associated correlation coefficients R 2 .

	Coefficient	1%w/v		3%w/v
		0.1mL/g	0.5mL/g	0.1mL/g	0.5mL/g
		cellulose	cellulose	cellulose	cellulose
	kµ (Pa -1 .s -2 )	-	61.5	2.1	15.3
	R²	-	0.936	0.979	0.988
	klc (µm -1 .s -1 )	8.1x10 -3	44.7x10 -3	9.0x10 -3	32.1x10 -3
		(65 µm)	(66 µm)	(60 µm)	(59 µm)
	R²	0.875	0.876	0.950	0.938
	kS ((gdm/L) -1 .s -1 )	7.6x10 -3	71.7x10 -3	3.7x10 -3	26.9x10 -3
	R²	0.822	0.987	0.885	0.995

Acknowledgements

This work was realised within the ProBio3 project ANR-11-BTBR-0003, selected in the Investissements d'Avenir Programme, with financial support from the French government, managed by the National Research Agency. The authors are grateful to the "Programme de Bourses d'Excellence 2011" of the French Embassy in Vietnam.