Clémentine Mansas

Loubna Atfane-Karfane

Eddy Petit

Julie Mendret

Stéphan Brosillon

André Ayral

Functionalized ceramic nanofilter for wastewater treatment by coupling membrane separation and catalytic ozonation

Keywords: ceramic nanofilters, catalytic ozonation, hybrid process, Fe2O3 catalyst, micropollutant elimination

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

Introduction

Water treatment and reuse are key topics with respect to the scarcity of drinking water on earth [START_REF]Human Development Report 2006, beyond scarcity: power, poverty and the global water crisis[END_REF], [START_REF]European Water policies and human health -Combining reported environmental information[END_REF]. A global concern is associated with the elimination of organic micropollutants before a possible reuse or a discharge in the environment [START_REF] Benotti | Pharmaceuticals and Endocrine Disrupting Compounds in U.S. Drinking Water[END_REF], [START_REF] Ratola | Occurrence of organic microcontaminants in the wastewater treatment process. A mini review[END_REF]. Different technologies are implemented for tertiary water treatment including filtration processes (using fixed beds or membranes) and advanced oxidation processes like ozonation [START_REF] Matilainen | Natural organic matter removal by coagulation during drinking water treatment: A review[END_REF]- [START_REF] Van Der Sloot | Characteristics, treatment and utilization of residues from municipal waste incineration[END_REF]. Combining membrane separation and catalytic ozonation is a promising technology for micropollutant removal [START_REF] Mansas | Coupling catalytic ozonation and membrane separation: A review[END_REF]. This should especially be the case when the separative surface of a nanofiltration (NF) membrane is functionalized in order to act as catalytic contactor. Oxidation by ozone can indeed be enhanced by using a suitable catalyst. Catalytic ozonation is mainly based on heterogeneous catalysis; the active solids are usually metals or metal oxides [START_REF] Mansas | Coupling catalytic ozonation and membrane separation: A review[END_REF]- [START_REF] Von Gunten | Ozonation of drinking water: Part I. Oxidation kinetics and product formation[END_REF]. Two types of NF membranes are available, organic and inorganic membranes. With organic membranes it is difficult to deposit a continuous and stable catalytic layer because of different factors, especially the very limited thermal stability and the very high mechanical flexibility of the organic polymers in comparison with inorganic materials [START_REF] Hora | Inorganic Thin Film Deposition and Application on Organic Polymer Substrates[END_REF]. Moreover such membranes exhibit a limited durability in the presence of ozone [START_REF] Mori | Ozone-microfiltration system[END_REF]- [START_REF] Flyborg | Can treated municipal wastewater be reused after ozonation and nanofiltration? Results from a pilot study of pharmaceutical removal in Henriksdal WWTP, Sweden[END_REF]. The use of ceramic nanofilters permits to avoid these difficulties thanks to their good thermal, mechanical and chemical stability [START_REF] Ayral | Chap.25 -Ceramic membrane processing; new approaches in their design and applications[END_REF]. The successful deposition of very thin nanoporous layers of the targeted catalytic materials on nanoporous ceramic membranes has already been demonstrated [START_REF] Gwak | Porous ceramic membranes exhibiting ferri/ferromagnetic properties for separation[END_REF]. The objective of the present work was to prepare a catalytic ceramic nanofilter with very low molecular weight cut-off (MWCO) and to demonstrate its operational performances. To do that, iron oxides have been chosen for several reasons: (i) they are identified in literature as ones of the most active catalysts [START_REF] Lv | Catalytic ozonation of toxic pollutants over magnetic cobaltdoped Fe3O4 suspensions[END_REF]- [START_REF] Yuan | Enhanced photocatalytic degradation of humic acids using Al and Fe co-doped TiO2 nanotubes under UV/ozonation for drinking water purification[END_REF], (ii) robust and low-cost sol-gel routes exist leading to stable colloidal suspensions [START_REF] Bee | Synthesis of very fine maghemite particles[END_REF], (iii) their toxicity is rather limited, (iv) they have been successfully used for the functionalization of ultrafiltration ceramic membranes [START_REF] Karnik | Fabrication of Catalytic Membranes for the Treatment of Drinking Water Using Combined Ozonation and Ultrafiltration[END_REF]- [START_REF] Byun | Mn oxide coated catalytic membranes for a hybrid ozonation-membrane filtration: Comparison of Ti, Fe and Mn oxide coated membranes for water quality[END_REF]. A commercial nanofilter with a very 2 low MWCO of 200 Da was here functionalized by sol-gel deposition of a mesoporous maghemite γ-Fe2O3 thin layer. Preliminary experiments enabled to determine the maximum temperature usable for the thermal strengthening of the catalytic layer without significant permeance change. The catalytic activity of the maghemite equivalent powder was tested in batch reactor with ozone and para-chlorobenzoic acid (pCBA; MW = 156.6 Daltons), which quickly reacts with hydroxyl radicals formed with ozone at the catalyst surface, and only very slowly with ozone itself [START_REF] Park | Characterization of natural organic matter treated by iron oxide nanoparticle incorporated ceramic membrane-ozonation process[END_REF], [START_REF] Ibn Abdul | Ozone combined with ceramic membranes for water treatment: Impact on HO radical formation and mitigation of bromate[END_REF]- [START_REF] Park | Kinetic decomposition of ozone and para-chlorobenzoic acid (pCBA) during catalytic ozonation[END_REF]. The operational performance of the functionalized ceramic membrane was finally evaluated with a dedicated pilot. The obtained results unequivocally showed the catalytic activity of this functionalized membrane.

Experimental 2.1. Materials

Single channel tubular membranes used in this study were supplied by IKTS (Germany). They are 250 mm in length with internal and external diameters of 7 and 10 mm, respectively, and glass sealing of 13 mm on both ends. The effective filtration area (usable length x internal diameter x π) was equal to 4.9 10 -3 m², with the separative layer on the inner side of the tube. Four intermediate layers are deposited between the titania-based microporous top layer and the α-Al2O3 macroporous support. The maghemite (γ-Fe2O3) colloidal sol was synthetized according to the Massart's synthesis [START_REF] Bee | Synthesis of very fine maghemite particles[END_REF]. Synthesis details are given in supplementary information (SI). The catalytic deposit was done by filling the membrane tube with the maghemite sol with a contact time of 4 min and a withdrawal rate of 10 cm min -1 . After drying at room temperature, the functionalized membranes were heated to 300 °C (maximum acceptable temperature considering the thermal stability of the selected ceramic nanofilter; see later) for one hour with a heating rate of 1 °C min -1 . In order to obtained equivalent powder, the hydrosol was poured in a large beaker, dried at 60 °C and then heated to 300 °C for one hour with a heating rate of 1 °C min -1 .

Physicochemical characterization

The equivalent powder was analyzed by X-ray diffraction (XRD) and N2 gas adsorption. Cross-section observations of pristine and functionalized membranes were done by scanning electron microscopy (SEM). The N2 molar permeance, Pe , across the membranes was determined at room temperature by measuring the molar flux density, J, as a function of transmembrane pressure, ∆P, in a dead-end module:

= ∆ (1)
With: J, the molar flux density (mol m -2 s -1 ); Pe, the molar permeance (mol m -2 s -1 Pa -1 ); ∆P, the transmembrane pressure (Pa). More details about these different experiments are given in supplementary information (SI).

Batch experiment with equivalent powder

In order to test the catalytic activity, pCBA aqueous solutions ([pCBA] = 2 or 3 µmol L -1 depending on the amount of catalyst used) were prepared. The pCBA concentration in water was analyzed by highperformance liquid chromatography (see SI for more details). The ozone concentration in solution was determined by an amperometric O3 micro-sensor (AMT Analysenmesstechnik GmbH) and by using the Indigo method [START_REF] Bader | Determination of ozone in water by the indigo method[END_REF] (see SI for more details). For testing the catalytic activity of the equivalent powder, experiments were done in batch conditions. First, adsorption of pCBA on powder was investigated by mixing a pCBA solution ([pCBA]= 3 µmol L -1 ) and powder ([γ-Fe2O3] = 3 g L -1 ). After a contact time of one hour, the suspensions were filtered using PTFE syringe filters with a pore size of 0.2 µm and the filtrate solution was analyzed. Ozone produced by an ozone generator (Ozonia LAB2B, Suez, France) was continuously bubbled in a Dreschel bottle containing a pCBA solution ([pCBA]= 3 µmol L -1 ) and γ-Fe2O3 powder ([Fe2O3] = 3 g L -1 ) under stirring. The ozone flux was equal to 20 L h -1 and the resulting gas concentration was [O3]g = 11 g.Nm -3 . The concentration of dissolved ozone was about 4 mg O3.L -1 . After one hour bubbling, the suspensions were filtered using PTFE syringe filters with a pore size of 0.2 µm and the filtrate solutions were analyzed.

Assessment of operational performance with a dedicated pilot

The schematic diagram of the home-made pilot and the protocol are given in SI. Experiments were carried out with or without pCBA ([pCBA] = 2 µmol.L -1 ) in the feed tank. The concentration of the injected gaseous ozone was ~ 28 g.Nm -3 . The dissolved concentration of ozone in the transfer reactor and in all the experimental setup was ~3.5 mg O3.L -1 . A phosphate buffer (NaH2PO4/Na2HPO4) was used to maintain the pH of the feed solution equal to 6. Permeation measurements (pure water or pCBA solution) were done both on pristine and functionalized membranes, with a transmembrane pressure equal to 10 bars, and continuous weighting of the collected permeate during one hour. After each experiment, the membrane was rinsed with pure water and cleaned by filtration of ozonized water during 20 min. The pCBA retention, Rf(pCBA) was determined with the two types of membranes, with or without dissolved O3, by measuring the pCBA concentration in the permeate and in the loop:

= - , , (2)
with: Rf(pCBA), the retention factor of pCBA by the membrane; C(pCBA, loop), the concentration of pCBA in the filtration loop; C(pCBA, permeate), the concentration of pCBA in the permeate.

As proposed by Elovitz and von Gunten [START_REF] Elovitz | Hydroxyl Radical/Ozone Ratios During Ozonation Processes. I. The Rct Concept[END_REF], the Rct concept was here employed:

= • ! " ! (3)
It is defined as the ratio of OH • exposure to ozone exposure and quantifies the production of OH • radicals More details are given in SI.

Results and discussion

Thermal stability of the selected ceramic nanofilter

To the best of our knowledge, the ceramic nanofiltration membrane selected for this study is the unique one commercially available with a very low MWCO of 200 Daltons. Such a low MWCO is required for the retention of the most current organic micropollutants exhibiting molecular weights of few hundred Daltons. Preliminary analyses showed that the titania-based microporous (pore size less than 2 nm) separative layer is amorphous. It can be thus suspected that its thermal stability in terms of microstructure and crystallinity is rather limited. Because the mechanical strengthening of the additional mesoporous catalytic layer requires a thermal treatment after deposition, it was decided to first investigate the thermal stability of the pristine membrane. A same membrane was successively treated for 30 min at different temperatures (from 200 °C to 800 °C). N2 permeation measurements were then performed. Fig. 1 shows the variation of the N2 permeance (determined from Equation (1)) as a function of the temperature of thermal treatment. The N2 permeance significantly increases between 300 and 350 °C. Assuming a viscous flow for nitrogen through the separative layer, a pressure drop across the overall membrane mainly due to its microporous separative layer, and considering the Darcy law (Equation ( 4)), it can be deduced that the increase of the membrane permeance is directly related to an increase of the intrinsic permeablity D of the microporous separative layer.

= # ∆ % & ⁄ (4)
with: J, the flux density; D, the permeability of the microporous separative layer; ∆P, the transmembrane pressure; ηf, the dynamic viscosity of the fluid; L, the thickness of the separative layer.

From Carman-Kozeny relation, it appears that the permeability of a porous medium increases when its porosity or its pore size increases [START_REF] Brinker | Sol-Gel Science The Physics and Chemistry of Sol-Gel Processing[END_REF]. Considering the expected microstructural rearrangement for such a microporous layer with temperature [START_REF] Sekulic | Synthesis and Characterization of Microporous Titania Membranes[END_REF], it can be deduced that the permeability increase is mainly due to an increase in the pore size and by way of consequence of the MWCO of the membrane. In order to prevent a change of the nanofilter performance, it was decided to limit to 300 °C the temperature of thermal strengthening of the deposited catalytic layer.

Characterization of the equivalent iron oxide powder

The physicochemical properties determined for the equivalent powder thermally treated at 300 °C are summarized in Table 1.

Table 1 Physicochemical properties of the equivalent powder thermally treated at 300 °C.

Colour brick-red

Crystalline phase γ-Fe2O3

Crystallite size (nm) 10.4 ± 0.1 BET specific area (m² g -1 )

99 ± 1

As expected from the literature, its XRD pattern matches with that of maghemite, γ-Fe2O3 (Fig. 2).

From the Full-Width Half-Maximum of the diffraction peaks and the Scherrer equation, the crystallite size can be estimated as equal to 10.4 ± 0.1 nm. Assuming a set of identical spherical crystallites with such a diameter and considering that the maghemite bulk density is equal to 4.9 g cm -3 , the developed specific surface area is equal to 118 m 2 g -1 . This calculated value which neglects the area loss due to the contacts between nanoparticles is in rather good agreement with specific surface area experimentally measuring by the BET method, 99 ± 1 m 2 g -1 . Considering a random close packing arrangement of the nanocrystallites, the expected porosity is around 40 % and the average pore size equal to around one third of the size of the crystallites, i.e. ~ 3.5 nm (mesopores). (peaks with Miller indexes hkl written in black: common peaks between maghemite γ-Fe2O3 and magnetite Fe3O4; peaks with Miller indexes hkl written in red: specific peaks of maghemite).

The maghemite powder was then tested in presence of an adapted concentration of pCBA and ozone in order to quantify its catalytic activity. pCBA retention by adsorption and elimination by ozonation with and without catalyst are presented in Fig. 3. As previously mentioned, the pCBA elimination is directly related to the production of OH • thanks to the presence of the catalyst. As expected without catalyst, the pCBA degradation is very low because very few OH • were formed. Fig. 3 highlights i) a very low adsorption of pCBA on the powder, ii) an important generation of hydroxyl radicals when ozone was injected in presence of the selected catalyst iii) pCBA displays slow reaction rates with ozone. These results confirm that this material is a good candidate for the functionalization of the ceramic nanofilter. (c) pCBA elimination by ozonation

([Fe2O3]=0 g L -1 , [pCBA]0= 5 µmol L -1 , QgasO3=20 L h -1 , [O3]=10 g Nm -3 )

Microstructural observation of the modified membranes

Operational performance of the modified membranes

The permeance values for pure water and a pCBA aqueous solution were measured using the hybrid pilot, with a pristine membrane or a functionalized membrane, and with or without ozone injection (Fig. 5). The permeance values were quite stable during all the experiments (one hour long). Using pure water without ozone, the permeance was not significantly modified by the membrane functionalization (~ 10 L (h m² bar) -1 . Injection of ozone induces a measurable decrease of permeance only for the pristine membrane. This unexpected result is difficult to explain. It could possibly be assigned to a weak change of pH due to ozone injection, knowing that complex electrokinetic phenomena occur during the forced flow of ionic solutions inside nanopores [START_REF] Mazzoni | Role of electrolyte type on TiO2-ZrO2 nanofiltration membranes performances[END_REF]. This effect would

not be observed with the functionalized membrane due to a decomposition of ozone in hydroxyl radicals during the crossing the catalytic layer. Another, but less substantiated, explanation would be the possible presence of ozone nanobubbles inside the micropores of the titania layer inducing a partial pore blocking effect and consequently the observed permeance reduction. In fact, nucleation of such ozone nanobubbles would be explained by the confinement of the ozone-saturated water in these very small pores [START_REF] Bories | 11 -Isothermal Nucleation and Bubble Growth in Porous Media at Low Supersaturations[END_REF]. The permeance values for the pristine and functionalized membranes with the pCBA solution are quite the same, around 9 L (h m² bar) -1 . This decrease compared to pure water is usually observed. With ozone injection, as for pure water, the permeance is higher for the functionalized membrane than for the pristine one (~ 8.0 versus ~ 6.5 L (h m² bar) -1 ). In absence of ozone, the retention factor Rf (pCBA) is larger for the functionalized membrane (~ 65 %) than for the pristine one (~ 50 %). Considering that the pH of the treated solution was maintained close to 6 and that the pKa of pCBA is equal to 4, it means than this compound was mainly present in its deprotonated negative form. On the other hand, the zero point of charge (ZPC) of titania depends on its crystalline nature. It is located between 4 and 5 for rutile form and between 6 and 7 for anatase form [START_REF] Parks | THE ZERO POINT OF CHARGE OF OXIDES1[END_REF], [START_REF] Kosmulski | Isoelectric points and points of zero charge of metal (hydr)oxides: 50years after Parks' review[END_REF]. For maghemite (γ-Fe2O3), the ZPC is in the range from 6 to 9 [START_REF] Kosmulski | Isoelectric points and points of zero charge of metal (hydr)oxides: 50years after Parks' review[END_REF]. Based on all these data, it can be concluded that a pH equal to 6 is more favourable to the development of positively charged surfaces in the case of γ-Fe2O3 and by way of consequence to an electrostatic attraction of the negatively-charged molecules of pCBA. The pCBA degradation by ozone using pristine or functionalized membranes is illustrated in Fig. 6 and the calculation details are given in SI. It is clearly enhanced with the catalytic membrane which favours the production of OH • . In order to quantify the catalytic performance of the functionalized membrane, the Rct value was determined by plotting the pCBA elimination as a function of O3 exposure (Fig. 7). As reference a configuration without membrane was also implemented. In such a case, Rct is quite low (2.58 10 -10 ).

With the pristine membrane, Rct increases to 4.26 10 -10 . The pristine membrane thus exhibits a weak catalytic activity. The larger value of Rct, 1.49 10 -9 , is obtained for the functionalized membrane. These results undoubtedly demonstrate the significant enhancement of the catalytic ozonation due the presence of the added mesoporous maghemite thin layer.

Conclusions

Direct coupling between nanofiltration and catalytic ozonation is a very promising approach for solving the emerging issue of micropollutant elimination in water. The associated key parameters are the development of performing nanofilters exhibiting a low MWCO, an excellent durability in presence of ozone and a high catalytic activity. In this context, a commercial ceramic nanofilter with a MWCO equal to 200 Daltons has been selected and was successfully functionalized using a simple a robust method consisting in the deposition of mesoporous maghemite thin layer on the microporous top-layer of the nanofilter. The preliminary results show that it is possible to functionalize this ceramic nanofilter without disturbing its filtration features. They also demonstrate the good catalytic activity of the deposited iron oxide. Despite a non-optimized design of the used pilot, a significant enhancement of the catalytic activity has been evidenced for the functionalized nanofilter.

The future experiments will deal with the implementation of this functionalized nanofilter for the treatment of aqueous solutions containing one or several micropollutants. Both micropollutant elimination, and also membrane fouling, will be considered for the assessment of its operational performance.

Fig. 1 .

 1 Fig. 1. N2 permeance versus temperature of thermal treatment.

Fig. 2 .

 2 Fig. 2. XRD pattern of the equivalent powder thermally treated at 300 °C.

Fig. 3 .

 3 Fig. 3. pCBA removal by the equivalent powder and without after one hour of contact time. (a) pCBA retention by adsorption ([Fe2O3]=3 g L -1 , [pCBA]0= 3 µmol L -1 ); (b) pCBA elimination by OH • resulting from catalytic ozonation ([Fe2O3]=3 g L -1 , [pCBA]0= 3 µmol L -1 , QgasO3=20 L h -1 , [O3]=11 g Nm -3 ). (c) pCBA elimination by ozonation ([Fe2O3]=0 g L -1 , [pCBA]0= 5 µmol L -1 , QgasO3=20 L h -1 , [O3]=10 g Nm -3 )

Fig. 4

 4 shows a SEM image of the functionalized membrane cross-section. It is focused close to the inner side of the single channel tube. It is possible to identify one part of the third intermediate layer, the fourth intermediate layer, the microporous titania-based top layer (thickness of ~ 110 nm) and the additional mesoporous maghemite layer exhibiting a uniform thickness of ~ 80 nm.

Fig. 4 .

 4 Fig. 4. SEM cross-section image of the functionalized ceramic nanofilter close to the inner side.

Fig. 5 .

 5 Fig. 5. Permeance values after 1h for pure water and a pCBA aqueous solution ([pCBA]0= 2 µmol L -1 )with a pristine membrane and a functionalized membrane, with or without ozone injection.

Fig. 6 .

 6 Fig.6. Percentage of the pCBA elimination for the pristine and catalytic membrane in presence of ozone in permeate and retentate after 2.5h.

Fig. 7 .

 7 Fig. 7. Quantification of the elimination of the pCBA versus O3 exposure in the pilot loop for different configurations in order to determine Rct values.

Acknowledgements

The authors warmly thank the French National Agency for Research (ANR) for the financial support of this study through the ANR JCJC 2016 project Saware (ANR-16-CE-04-0002-01).


Charlène Guillaumot

Thomas Saucède

Simon A Morley

Starrlight Augustine

Bruno Danis

Sebastiaan Kooijman

Guillaumot Charlène

Saucède Thomas

Augustine Starrlight

Danis Bruno

Kooijman Sebastiaan

Can DEB models infer metabolic differences between intertidal and subtidal morphotypes of the Antarctic limpet Nacella concinna (Strebel, 1908)?

Keywords: ecological modelling, Southern Ocean, marine benthic species, model relevance, model accuracy

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

so far estimated for more than 2,000 animal species and collected in the 'Add-my-Pet' (AmP) collection (http://www.bio.vu.nl/thb/deb/deblab/add_my_pet/). It constitutes one of the most powerful approaches to characterize individual metabolic performances [START_REF] Nisbet | Integrating dynamic energy budget (DEB) theory with traditional bioenergetic models[END_REF][START_REF] Kearney | Dynamic energy budget theory: an efficient and general theory for ecology[END_REF][START_REF] Jusup | Physics of metabolic organization[END_REF] and can be calibrated for data-poor animals [START_REF] Mariño | Dynamic Energy Budget theory predicts smaller energy reserves in thyasirid bivalves that harbour symbionts[END_REF]. DEB models rely on thermodynamic concepts [START_REF] Jusup | Physics of metabolic organization[END_REF] and study how energy flows are driven within individuals during their entire life cycle [START_REF] Kooijman | Dynamic energy budget theory for metabolic organisation[END_REF]. Each individual is divided into four energetic compartments, reserve E, structure V, maturation EH and reproduction ER from which the energy is allocated in contrasting proportions according to the different life stages and two forcing environmental factors (i.e. food resources and temperature).

DEB models can be built with data coming from experiments and/or literature, to quantify age, length, weight of the different life stages and provide information on reproduction, growth and metabolic rates to calibrate the model [START_REF] Van Der Meer | An introduction to Dynamic Energy Budget (DEB) models with special emphasis on parameter estimation[END_REF][START_REF] Marques | Can we reach consensus between marine ecological models and DEB theory? A look at primary producers[END_REF].

Application of DEB models to Antarctic species is increasing. They can be easily extracted from the AmP collection, using the software AmPtool. The Matlab command "select_eco('ecozone', {'MS'})" presently gives a list of 37 species, where MS stands for "Marine, Southern Ocean". Command "select_eco('ecozone', {'TS'})" gives another 3 species for the terrestrial Antarctic environment, among which the mite Alaskozetes antarcticus. Among the most common and well studied Southern Ocean benthic invertebrates are the sea star Odontaster validus [START_REF] Agüera | Parameter estimations of dynamic energy budget (DEB) model over the life history of a key Antarctic species: the Antarctic sea star Odontaster validus Koehler[END_REF], the bivalve Laternula elliptica (Agüera et al. 2018), the bivalve Adamussium colbecki (Guillaumot 2019a) and the sea urchins Sterechinus neumayeri [START_REF] Stainthorp | AmP Sterechinus neumayeri[END_REF] and Abatus cordatus (Arnould-Pétré et al. this issue). DEB models have also been developed for some pelagic species such as the Antarctic krill Euphausia superba, the salp Salpa thompsoni (Jager andRavagnan 2015, Henschke et al. 2018) and are under development for marine mammals such as the elephant seal Mirounga leonina [START_REF] Goedegebuure | Modelling southern elephant seals Mirounga leonina using an individual-based model coupled with a dynamic energy budget[END_REF]. Antarctic species have a range of notable physiological traits when compared to their temperate counterparts. Among others, they are physiologically adapted to constant cold temperatures [START_REF] Peck | Animal temperature limits and ecological relevance: effects of size, activity and rates of change[END_REF], Morley et al. 2009, 2014), shifting day length also imposes a marked seasonal feeding behaviour [START_REF] Mcclintock | Trophic biology of Antarctic shallow-water echinoderms. Marine ecology progress series[END_REF][START_REF] Clarke | Seasonal and interannual variability in temperature, chlorophyll and macronutrients in northern Marguerite Bay, Antarctica[END_REF][START_REF] Halanych | Challenging Dogma Concerning Biogeographic Patterns of Antarctica and the Southern Ocean[END_REF], and they exhibit slow metabolic and growth rates, explaining their longer lifespans and higher longevities compared to species in other regions (Peck andBrey 1996, Peck 2002).

The limpet Nacella concinna [START_REF] Strebel | Dei Gastropoden[END_REF] (Mollusca: Patellogastropoda) is a common and abundant gastropod of shallow marine benthic communities. Distributed all along the Western Antarctic Peninsula [START_REF] González-Wevar | Phylogeography and demographic inference in Nacella (Patinigera) concinna (Strebel, 1908) in the western Antarctic Peninsula[END_REF], phylogeny recently reviewed in [START_REF] González-Wevar | Systematic revision of Nacella (Patellogastropoda: Nacellidae) based on a complete phylogeny of the genus, with the description of a new species from the southern tip of South America[END_REF], it has widely been studied for decades [START_REF] Shabica | General ecology of Antarctic limpet Patinigera-Polaris[END_REF][START_REF] Shabica | The natural history of the Antarctic limpet Patinigera polaris (Hombron and Jacquinot)[END_REF][START_REF] Walker | Introduction to the ecology of the antarctic limpet Patinigera polaris (Hombron and Jacquinot) at Signy Island, South Orkney Islands[END_REF][START_REF] Hargens | Protection against lethal freezing temperatures by mucus in an Antarctic limpet[END_REF][START_REF] Houlihan | Oxygen consumption of some Antarctic and British gastropods: an evaluation of cold adaptation[END_REF][START_REF] Peck | Temperature and basal metabolism in two Antarctic marine herbivores[END_REF][START_REF] Clarke | Faecal production and an estimate of food intake in the wild of the Antarctic limpet Nacella concinna (Strebel)[END_REF][START_REF] Cadée | Shell damage and shell repair in the Antarctic limpet Nacella concinna from King George Island[END_REF][START_REF] Ansaldo | Does starvation influence the antioxidant status of the digestive gland of Nacella concinna in experimental conditions?[END_REF][START_REF] Fraser | Growth in the slow lane: protein metabolism in the Antarctic limpet Nacella concinna (Strebel 1908)[END_REF][START_REF] Markowska | Encounters between Antarctic limpets, Nacella concinna, and predatory sea stars, Lysasterias sp., in laboratory and field experiments[END_REF], Morley et al. 2011, 2014[START_REF] Suda | The biology and ecology of the Antarctic limpet Nacella concinna[END_REF][START_REF] Souster | Seasonality of oxygen consumption in five common Antarctic benthic marine invertebrates[END_REF]). The limpet is found from intertidal rocky shores down to over 100 meters depth [START_REF] Powell | Antarctic and subantarctic mollusca: Pelecypoda and Gastropoda[END_REF][START_REF] Walker | Introduction to the ecology of the antarctic limpet Patinigera polaris (Hombron and Jacquinot) at Signy Island, South Orkney Islands[END_REF]. It has a 2-5 cm long shell (Fig. 1), that grows only a few millimeters a year with a seasonal pattern. It is sexually mature after four to six years and has a life span of up to 10 years [START_REF] Shabica | The natural history of the Antarctic limpet Patinigera polaris (Hombron and Jacquinot)[END_REF][START_REF] Picken | The distribution, growth, and reproduction of the Antarctic limpet Nacella (Patinigera) concinna (Strebel, 1908)[END_REF][START_REF] Brêthes | Distribution, growth and reproduction of the limpet Nacella (Patinigera) concinna (Strebel 1908) in relation to potential food availability, in Esperanza Bay (Antarctic Peninsula)[END_REF]. The limpet mainly feeds on microphytobenthos and microalgae [START_REF] Shabica | The natural history of the Antarctic limpet Patinigera polaris (Hombron and Jacquinot)[END_REF][START_REF] Brêthes | Distribution, growth and reproduction of the limpet Nacella (Patinigera) concinna (Strebel 1908) in relation to potential food availability, in Esperanza Bay (Antarctic Peninsula)[END_REF]. It spawns free-swimming planktonic larvae once a year, when water temperature rises in the austral summer [START_REF] Shabica | General ecology of Antarctic limpet Patinigera-Polaris[END_REF][START_REF] Picken | The distribution, growth, and reproduction of the Antarctic limpet Nacella (Patinigera) concinna (Strebel, 1908)[END_REF][START_REF] Picken | Unique spawning behaviour by the Antarctic limpet Nacella (Patinigera) concinna (Strebel, 1908)[END_REF]. Larvae drift in the water column and metamorphose after more than two months (Stanwell-Smith and Clarke 1998).

N. concinna does not have a homing behaviour (Stanwell-Smith and [START_REF] Stanwell-Smith | The Timing of reproduction in the Antarctic limpet Nacella concinna (srebel, 1908)(Patellidae) at Signy Island, in relation to environmental variables[END_REF][START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets Nacella concinna to aerial exposure[END_REF][START_REF] Suda | The biology and ecology of the Antarctic limpet Nacella concinna[END_REF] and intertidal individuals can either migrate to subtidal areas in winter to escape freezing air temperatures that may drop below -20°C [START_REF] Walker | Introduction to the ecology of the antarctic limpet Patinigera polaris (Hombron and Jacquinot) at Signy Island, South Orkney Islands[END_REF][START_REF] Branch | The ecology of limpets: physical factors, energy flow, and ecological interactions[END_REF][START_REF] Brêthes | Distribution, growth and reproduction of the limpet Nacella (Patinigera) concinna (Strebel 1908) in relation to potential food availability, in Esperanza Bay (Antarctic Peninsula)[END_REF] or shelter in rock cracks and crevices in the intertidal area. In the latter case, they do not become dormant but have a limited access to microphytobenthos, as recently observed around Adelaide Island [START_REF] Obermüller | Antarctic intertidal limpet ecophysiology: A winter-summer comparison[END_REF]. Two morphotypes of N. concinna have been distinguished, an intertidal and a subtidal type, with the intertidal type having a taller, heavier and thicker shell compared to the subtidal one that is characterized by a lighter and flatter shell [START_REF] Beaumont | Morphological and genetic variation in the Antarctic limpet Nacella concinna (Strebel, 1908)[END_REF]Wei 1991, Hoffman et al. 2010). Initially, [START_REF] Strebel | Dei Gastropoden[END_REF] and [START_REF] Powell | Antarctic and subantarctic mollusca: Pelecypoda and Gastropoda[END_REF] referred to these two morphotypes as the 'polaris' (intertidal) and 'concinna' types (below 4m depth). From that point, the potential genetic differentiation between the two morphotypes has been investigated, some of the studies concluding an absence of genetic distinction [START_REF] Wei | Morphological and genetic variation in natural populations of Antarctic limpet Nacella concinna[END_REF][START_REF] Beaumont | Morphological and genetic variation in the Antarctic limpet Nacella concinna (Strebel, 1908)[END_REF][START_REF] Nolan | Size, shape and shell morphology in the Antarctic limpet Nacella concinna at Signy Island, South Orkney Islands[END_REF]) while contrarily, de Aranzamendi et al. (2008) reported significant differences based on inter-simple sequence repeat (ISSR) markers. More recently, this last method was questioned [START_REF] Hoffman | No evidence for genetic differentiation between Antarctic limpet Nacella concinna morphotypes[END_REF]) and several studies using different markers and populations [START_REF] Chwedorzewska | Low genetic differentiation between two morphotypes of the gastropod Nacella concinna from Admiralty Bay, Antarctica[END_REF][START_REF] Hoffman | No evidence for genetic differentiation between Antarctic limpet Nacella concinna morphotypes[END_REF], Gonzalez-Wevar et al. 2011) have concluded an absence of genetic differentiation between the two morphotypes.

Apart from the absence of genetic differences, intertidal and subtidal populations strongly contrast in morphology and physiology, which has been explained by the prevalence of habitat heterogeneity and strong environmental gradients along rocky shore habitats, a common feature also observed in other gastropods [START_REF] Johannesson | Evolution in Littorina: ecology matters[END_REF][START_REF] Butlin | Sympatric, parapatric or allopatric: the most important way to classify speciation?[END_REF][START_REF] Hoffman | No evidence for genetic differentiation between Antarctic limpet Nacella concinna morphotypes[END_REF]. For instance, in N. concinna, the higher shell thickness observed in the shallow morphotype was hypothesised to play a role in resistance against crushing pack ice [START_REF] Shabica | General ecology of Antarctic limpet Patinigera-Polaris[END_REF][START_REF] Morley | Depth gradients in shell morphology correlate with thermal limits for activity and ice disturbance in Antarctic limpets[END_REF]. Intertidal morphotypes are further resistant to air exposure thanks to higher shells, bigger inner volumes relative to their shell circumference, a combination that makes them more efficient than subtidal individuals, able to store more water and oxygen, reducing desiccation risks and delaying the metabolic switch to anaerobic fermentation (Nolan 1991, Weihe and[START_REF] Weihe | Differences in the physiological response of inter-and subtidal Antarctic limpets Nacella concinna to aerial exposure[END_REF]. The subtidal morphotype has also proved to be less resistant to cold than the intertidal population [START_REF] Waller | Ecophysiological strategies of Antarctic intertidal invertebrates faced with freezing stress[END_REF], due to extra production of mucus and stress proteins in intertidal morphotypes [START_REF] Clark | Antarctic marine molluscs do have an HSP70 heat shock response[END_REF], Clark and Peck 2009, Obermüller et al. 2011) and due to diverse metabolic processes that contrasts between both populations (reviewed in [START_REF] Suda | The biology and ecology of the Antarctic limpet Nacella concinna[END_REF].

The development of ecological models enables precise models to be built, that highlight subtle differences in parameters between ecologically similar or closely related species [START_REF] Freitas | Temperature tolerance and energetics: a dynamic energy budget-based comparison of North Atlantic marine species[END_REF][START_REF] Holsman | A comparison of fisheries biological reference points estimated from temperature-specific multi-species and single-species climate-enhanced stock assessment models[END_REF][START_REF] Marn | Comparative physiological energetics of Mediterranean and North Atlantic loggerhead turtles[END_REF], Lika et al. 2020 this issue). The idea of building individual-specific models for understanding of physiological processes is not new [START_REF] Bevelhimer | Assessing significance of physiological differences among three esocids with a bioenergefics model[END_REF][START_REF] Deangelis | Individual-oriented approaches to modeling ecological populations and communities[END_REF]) and grew from the development of computational ecology that resulted in the possibility of generating "individual-oriented" models (IOM's) [START_REF] Hogeweg | Individual-oriented modelling in ecology[END_REF]Hesper 1990, DeAngelis et al. 1994). The IOM theory relies on the principle that "no two biological organisms are exactly alike, even when they have identical genes". A group of organisms within a population can have contrasting size or physiological performances according to, for example, food conditions or competition. Modelling each individual, separately, therefore constitutes a powerful approach to enhance the understanding of the entire community [START_REF] Deangelis | Individual-oriented approaches to modeling ecological populations and communities[END_REF].

In this study, due to the known morphological and physiological differences between the morphotypes, we first separately built independent DEB models for the intertidal and subtidal morphotypes of the limpet N. concinna, based on field experiment and literature data, to assess the potential differences between the models.

Secondly, we analyse whether the two model outputs suggest contrasting physiologies between the morphotypes, using a method recently developed in DEB theory, that tries to reduce differences in parameter values that are still consistent with the data (Lika et al. 2020 this issue). Using this method, the augmented loss function, tries to merge the information of the two species models into a single one. If DEB parameters of the two species can be merged, it means that the physiological differences between these two species are not strongly different. These results will finally help assess DEB model accuracy giving the amount of data available to build the models in the context of Antarctic case studies and help evaluate which type of information is necessary to gather in order to fill model gaps. Finally, the study evaluates if such models are valuable for studying Southern Ocean organisms in the context of altered environments.

2.Material and methods

DEB Model description

DEB models are based on an ensemble of rules that allocate energy flows to four main compartments (reserve E, structure V, maturity EH, reproduction ER) according to a set of priorities and the level of complexity (i.e. maturity) gained by the organism (Fig. 2, [START_REF] Kooijman | Dynamic energy budget theory for metabolic organisation[END_REF]. Maturity is treated as information, having mass nor energy. Food is first of all ingested and assimilated ( A) and energetically stored into a reserve compartment (E). A fraction of the energy that is mobilised from reserve, C, is divided into two branches according to the 'kappa-rule': a part of the energy contained in the reserve compartment (κ. C) is allocated to somatic maintenance and structure growth, whereas the second part (1κ). C contributes to maturity (before the 'puberty' threshold) or reproduction (after the 'puberty' threshold).

The energy is allocated within and in between these branches by the establishment of some priorities, where somatic maintenance ( M) has priority over growth and maturity maintenance ( J) has priority over maturity and reproduction. During its lifetime, the organism allocates energy to maturity which symbolizes its complexity and reaches some life stages at some defined thresholds ( , birth, when the organism is capable to feed; , metamorphosis; , puberty, when it can reproduce). After reaching sexual maturity, the energy that was formerly allocated to maturity is attributed to the reproduction buffer and the available energy is allocated to the development of gametes.

Different types of DEB models have been developed and coded for parameter estimation, see frequently updated https://github.com/add-my-pet/DEBtool_M page [START_REF] Marques | The AmP project: comparing species on the basis of dynamic energy budget parameters[END_REF][START_REF] Marques | Fitting Multiple Models to Multiple Data Sets[END_REF]. Here, the abj model was used for N. concinna. This model considers that growth acceleration occurs between birth and metamorphosis [START_REF] Kooijman | Dynamic energy budget theory for metabolic organisation[END_REF][START_REF] Mariño | Dynamic Energy Budget theory predicts smaller energy reserves in thyasirid bivalves that harbour symbionts[END_REF]).

The DEB model is forced by food availability and temperature. Temperature acts on metabolic rates following the Arrhenius principle (see [START_REF] Kooijman | Dynamic energy budget theory for metabolic organisation[END_REF], Jusup et al. 2017 for details). A temperature correction factor is applied to each rate that takes into account the lower and higher optimal boundaries of the individual tolerance range.

Food available for ingestion is represented by the functional response f comprised between [0,1], where 0 is starvation condition and 1 very abundant food. (mature individuals). κ. C is the proportion of the mobilized energy diverted to M and G, while the remaining part (1κ). C is used for J and R.

The parameters of the DEB model can be estimated from multiple data on the eco-physiology of a species.

The ones studied in this work are presented in Table 1. Table 1. List of the main DEB parameters, definition and units

Parameter Description Unit

Primary DEB parameters

{ Am} surface-area-specific maximum assimilation rate J.cm -2 d -1 energy conductance (velocity) cm.d -1
κ fraction of mobilised reserve allocated to soma - [Em]

[Em]= { Am}/ ; ratio of specific assimilation over energy conductance J.cm -3

Data collection and DEB calibration

DEB models were calibrated using zero-variate data (single data points at defined life stages, such as length or weight at sexual maturity, number of eggs produced per female) and uni-variate data (relationships between two variables such as oxygen consumption and temperature, length~weight relationship, weight or size~time relationships)(van der Meer 2006, Guillaumot 2019b). Data that were collected from the literature (Table 2), paying attention to the different taxonomic names adopted for the species through time (see http://www.marinespecies.org/aphia.php?p=taxdetails&id=197296, accessed December 2018); to the sampling area to enable the two morphotypes to be distinguished (intertidal/subtidal) and to the environmental conditions under which each dataset was recorded (available food resources and temperature).

Data from the literature were supplemented by experiments led by S. Morley at Rothera Station (Adelaïde Island, Western Antarctic Peninsula) in January-February 2018 (details in Appendix A). Individual shells were brought back to Europe and processed with imagery to collect growth ring data (Appendix B).

Some data are shared between the intertidal and subtidal morphotypes due to a lack of information on the morphotypes physiology in the literature (Table 2). The characteristics of the first developmental life stage, when the larvae become able to feed (i.e. age, length and weight at birth) and the pace of development (i.e. age at puberty, maximal observed age) are assumed to be identical.

Each data set was characterised by the corresponding temperature and food resources present in the field.

Food resources were represented in the model by a scaled functional response f constrained between 0 and 1, with 0 meaning no food availability and 1 maximal food abundance. f parameters were differentiated between the different stations along with temperatures. Food is very abundant in the field for the limpet and f parameters were therefore kept fixed with values ≥ 0.9. Food availability from the Rothera Station was described by pictures taken in the field and was estimated at f=1. Signy and Anvers Islands f was set at 0.9 because physiological traits (growth rate, maximal size) are very close (but slightly lower) than Rothera's observations, but no precise information is available for food conditions in the different publications for these stations.

DEB parameter estimation and goodness of fit

Sets of zero and uni-variate data, supplemented by pseudo-data were used to estimate the DEB primary parameters. Pseudo-data are extra data coming from different taxa that help calibrate the model estimation similarly to a prior element (Lika et al. 2011a). This procedure has similarities with Bayesian estimation, but are not embedded in a maximum likelihood context, since the stochastic component is not modelled. Before parameter estimation, each data set can be subjectively linked by a weight coefficient to quantify the realism of reducing variation in parameter values. Selected weight coefficients are always selected small enough in order to hardly affect parameter estimation if the information contained in the real data set is sufficient.

The DEB parameters estimation is done by simultaneously estimating each parameter using these empirical and pseudo-data by minimizing a loss function (eq. 1), using the Nelder-Mead simplex method, updated and explained in [START_REF] Marques | The AmP project: comparing species on the basis of dynamic energy budget parameters[END_REF][START_REF] Marques | Fitting Multiple Models to Multiple Data Sets[END_REF]. The loss function that is minimized is

∑ ∑ ! eq.1
where i scans datasets and j points in this dataset. dij and pij are respectively the data and the predictions and "i and i their average values in set i. wij are the attributed coefficients, is the number of data sets, ni denotes the data in a dataset, nj the data in data-points.

The value of the loss function is evaluated for each parameter trial. The goodness of fit of each prediction was quantified by the relative error (RE). The mean relative error (MRE) quantifies the overall model performance.

RE corresponds to the sum of the absolute differences between observed and predicted values, divided by the predicted values. Contrarily to the loss function, the MRE does not take into consideration the weights of the different data [START_REF] Marques | The AmP project: comparing species on the basis of dynamic energy budget parameters[END_REF]. MRE values can have values form 0 to infinity, with 0 value meaning that predictions match data exactly.

Merging parameters

The augmented loss function approach developed by (Lika et al. 2020

Fsb = ∑ =1 ∑ =1 ! + ∑ & ' ()*(, ' ) ./)0(, ' ) 1 2 '=3
eq.2 where w's are weights, d's data, p's predictions, 4's parameters, j scans data-points with a data-set of ni points (ni = 1 is allowed), i scans the data-sets and k the parameters.

In this second term, when wk=0, the parameter 4k between species are different, but when increasing wk, the parameter 4k tends to be similar between species. Therefore, the augmented loss function method uses this mathematical principle to spot potential differences between parameters of different species. First, the set of DEB parameters are separately estimated for each species and weight coefficients are set to zero. Then, for each parameter, the weight coefficient will be step-wise increased, making the loss function shift as a result. If a maximal weight value is reached without sharp changes in the loss function value along the weight increase, it means that the parameter value has a minimum variance between species. Contrarily, if the loss function value presents a sharp increase due to the change in weight coefficient, it means that the studied parameter should present contrasting values between the related species.

By applying this method to the case study of an intertidal and subtidal morphotype of the limpet N. concinna, we aim to evaluate whether there are any differences between both morphotypes caused by differences in parameters, or whether these differences are explained only by differences in environmental conditions (i.e.

food resources and temperature). Initially, the sets of parameters have been estimated separately for both morphotypes and all weight coefficients are set to zero. By step-wise increasing the weight coefficient for a particular shared parameter, the overall loss function may increase and a common merged DEB parameter is reached. If a common value of the DEB parameter can be found without important increase in MRE or loss function values, it means that the intertidal and subtidal morphotypes do not significantly differ for this parameter. A similar procedure is applied for each DEB parameter separately and iteratively. In order to have a quick idea of replicability in the results, the procedure was replicated five times, contrasting in different orders of DEB parameters merging (Appendix C). The order of permutation of merged parameters of these five replicates was chosen randomly among the 11! possible solutions. Changes in MRE and loss function values at each weight modification were reported and the predictions of the intertidal, subtidal and merged models were compared.

3.RESULTS

Parameters of the DEB models

DEB predictions for the separate intertidal and subtidal models are accurate, with MRE values lower than 0.2 (Table 3). Average MRE value of the AmP collection is close to 0.06. Relative Errors are quite low, with the highest values obtained for length~GSI data (RE= 0.6089 and 0.8702 for intertidal and subtidal models respectively) and time~length relationships, obtained from the sclerochronology measurements, that are highly variable between each measured shell (respectively RE= 0.3645 and 0.5924 for intertidal and subtidal models) (Fig. 3, Table 3, Appendix B).

In view of the substantial morphological difference between the populations, we expected to see a clear difference in the shape coefficient δM. We found a slightly larger value of δM for the intertidal morphotype, meaning that for the same shell length, it has slightly more structure, compared to the subtidal one.

Subtidal morphs have a lower energy conductance as well as double the value of maximum surface area specific assimilation rate { Am} with respect to the intertidal morphs. The ratio of specific assimilation over energy conductance [Em]= { Am}/ , determines the maximum reserve capacity of a species.

The fraction of mobilised reserve allocated to soma κ is also bigger (0.9368 for subtidal vs. 0.9084 for intertidal type), and the intertidal individuals also present a lower value for somatic maintenance rate [ M] compared to the subtidal ones. This highlights contrasts between the morphotypes in energy allocated to maturation along the first life stages ( , ) and more available energy for growth for the intertidal morphotype that has lower values of somatic maintenance. Intertidal morphotypes seem to accelerate metabolism a two-fold difference in acceleration factor sM between intertidal and subtidal types (respectively 7.862 and 4.049). The maturity threshold to reach puberty, is also lower for the intertidal morphotype than the subtidal.

The MRE values of the merged models stay below 0.25 and the value of the loss function for the merged situation is only a little larger than the sum of both populations, reflecting the fact a substantial reduction in the total number of parameters by almost a factor 2, hardly affects the goodness of fit (Table 3, Appendix C).

DEB parameters of the merged models are quite close to the values of the intertidal and subtidal models, with

[ M], δM, , , [EG] and merged values being almost exactly in between the values of the intertidal and the subtidal morphotypes. κ, 5, , { Am} and ℎ a are closer to the intertidal predictions.

Univariate predictions are also extremely close between the two models and the merged model (Fig. 3), with only a small difference for the subtidal model for which the GSI~length predictions are higher than the intertidal and merged predictions, mainly due to errors in predictions and scatter in the data. This higher potential of energy allocation to reproduction can, however, be linked to the higher values estimated for the subtidal type (Table 3).

Merging process

DISCUSSION

DEB models relevance

DEB models are powerful tools enabling predictions of the individuals energetic scope for survival, growth and reproduction, given the considered environmental conditions [START_REF] Kooijman | Dynamic energy budget theory for metabolic organisation[END_REF][START_REF] Jusup | Physics of metabolic organization[END_REF]). These mechanistic approaches have been of interest for several years to the marine Antarctic community [START_REF] Gutt | Correlative and dynamic species distribution modelling for ecological predictions in the Antarctic: a cross-disciplinary concept[END_REF][START_REF] Constable | Chapter 9.3. Assessing status and change in Southern Ocean Ecosystems[END_REF][START_REF] Gutt | Cross-disciplinarity in the advance of Antarctic ecosystem research[END_REF], and have been increasingly developed during recent years (e.g. [START_REF] Agüera | Parameter estimations of dynamic energy budget (DEB) model over the life history of a key Antarctic species: the Antarctic sea star Odontaster validus Koehler[END_REF], 2018[START_REF] Goedegebuure | Modelling southern elephant seals Mirounga leonina using an individual-based model coupled with a dynamic energy budget[END_REF][START_REF] Henschke | Modelling the life cycle of Salpa thompsoni[END_REF]).

This study is based on the example of the limpet Nacella concinna and uses data from literature supplemented by experiments conducted in Antarctica in February 2018, to build the DEB models of the intertidal and subtidal morphotypes of the species. The separately produced models were accurate, with a reduced error between observations and model predictions, except for some scatter among data such as Length~GSI relationship.

Such accuracy was mainly possible thanks to the important amount of uni-variate data that were provided by the complementary experiments conducted in Rothera, which filled knowledge gaps about reproduction, collected more precise length weight relationships to observe the morphological contrasts between intertidal and subtidal individuals and collected more precise information on the limpet's metabolic performance through its development.

Rates (and ages) depend on temperature. Here we correct for differences in temperature using an Arrhenius relationship. However, in order to meaningfully compare differences in parameters between species living in different habitats, it is useful to standardize all parameters to a common reference temperature: Tref= 20°C. This is the standard for presenting and comparing DEB parameters across the 2,000 different species in AmP.

When comparing DEB parameters estimations of N. concinna to those of their temperate counterpart Patella vulgata [START_REF] Kooijman | AmP Patella vulgata, version 2017/01/30[END_REF] at Tref= 20°C, we notice clear differences between the species in term of metabolic strategies, although the limpets morphology and therefore size and volume are close between these species (close length and predicted shape coefficient δM). For N. concinna, predicted κ is much higher and close to 1 (0.9256 vs 0.617 for P. vulgata), meaning that almost all the energy available in the reserve compartment is allocated to somatic maintenance and growth, and only a small amount is available for reproduction. This is clearly visible with the ultimate rate of reproduction more than 40 times lower for the Antarctic limpet compared to the temperate one. The capacity to assimilate resources { Am} was estimated to be 10 times higher for P. vulgata, explaining the 2.5-fold lower growth rate for N. concinna. The two metabolisms also contrast by the fact that P. vulgata is predicted to store more reserves than N. concinna in similarly abundant food conditions. These results are consistent with published experiments, where it was shown that rasping rates (i.e. feeding potential) were higher for temperate and tropical species than for N.

concinna [START_REF] Morley | Limpet feeding rate and the consistency of physiological response to temperature[END_REF]) and that development rates of Antarctic marine molluscs are much slower than at higher temperatures [START_REF] Peck | Very slow development in two Antarctic bivalve molluscs, the infaunal clam Laternula elliptica and the scallop Adamussium colbecki[END_REF][START_REF] Peck | Very slow embryonic and larval development in the Antarctic limpet Nacella polaris[END_REF], which could be partially due to the increased costs of protein production in the cold [START_REF] Marsh | High macromolecular synthesis with low metabolic cost in Antarctic sea urchin embryos[END_REF][START_REF] Robertson | The effects of temperature on metabolic rate and protein synthesis following a meal in the isopod Glyptonotus antarcticus Eights (1852)[END_REF][START_REF] Pörtner | Thermal limits and adaptation in marine Antarctic ectotherms: an integrative view[END_REF].

Such examples of comparison of energetic performance between these two species highlight the performance of DEB models to be efficiently applied for Antarctic case studies and powerful and accurate enough to enhance physiological contrasts even between closely related species; as previously discussed in other works [START_REF] Van Der Veer | The estimation of DEB parameters for various Northeast Atlantic bivalve species[END_REF][START_REF] Gatti | Comparing biological traits of anchovy and sardine in the Bay of Biscay: A modelling approach with the Dynamic Energy Budget[END_REF][START_REF] Marques | The AmP project: comparing species on the basis of dynamic energy budget parameters[END_REF][START_REF] Marn | Comparative physiological energetics of Mediterranean and North Atlantic loggerhead turtles[END_REF].

Comparison between morphotypes 20

In a second step, we evaluated if known contrasts in physiological traits between the morphotypes could be highlighted by the modelling approach. By simply comparing the two single models, we observed minimal energetic contrasts between the intertidal and subtidal morphotypes (small differences in assimilation rate and ability to store reserves, Table 3, Fig. 3). By using the augmented-loss-function method, we tried to merge the models into a single one, parameter by parameter, to evaluate the contrasts in parameters between the types (Lika et al. 2020, this issue). Results show that models were merged without generating significant changes in MRE and loss function values (Fig. 4, Appendix D). Predictions of the uni-variate data are really similar between the three models (Fig. 3), with only minor differences in temperature-corrected parameter values between both populations, meaning that the observed differences are best explained by differences in environmental conditions (temperature and food availability).

Despite the known physiological contrasts in the field, the available data did not allow the models to capture these physiological differences between the morphotypes, using only the available data. Scatter distribution of the data used to calibrate the model (Fig. 3) can hide metabolic differences, which calls for more experiments to describe the physiology of the different morphotypes. Using more complete datasets, for which all parameters are independent between intertidal and subtidal morphotypes, may also help to further constrain the differences. In our case study, several zero-variate data are shared between the intertidal and subtidal models, among which age, length and weight at birth, that control the very beginning of the development. The observed results of a twofold difference in metabolism acceleration of intertidal morphotypes compared to subtidal ones (sM ≈ 8 and 4 for intertidal and subtidal) is in fact an artefact caused by common parameters related to birth and puberty stages (age, length, weight). Indeed, specific assimilation at birth for the subtidal is two times larger than that for the intertidal, which indicates that subtidal individuals develop faster. However, according to available data provided in the model, puberty is reached at the same time for both types.

consequently needs to be smaller for the intertidal type to reach puberty at the same age ap and length Lp, explaining the observed contrasts between the intertidal and subtidal groups.

Improving the completeness of these models would therefore be necessary to enable further detailed conclusions.

A common approach in biology is to focus on differences between individuals, populations and species. Here we adopt a contrasting strategy in which we force models to determine in what manner the populations are similar in order to quantify in what manner they differ. This work is a first step to compare the energetics of both populations, and we discovered how (given the data) they seem more metabolically similar than what their appearance would suggest as first. We also highlight some artefacts that come from the quality of the data and the scatter therein. New data (so new knowledge) that fill current knowledge gaps will yield further insight into how the metabolisms of these populations have diverged to adapt to differences in environment. The current work is a contribution to understanding the relationship between observations (data) and metabolism for these two populations.

Models drawbacks and improvements

Apart from data availability, a drawback of our model construction is the lack of information about environmental properties that makes comparisons between estimations of the two morphotypes quite difficult to perform. In the models, we just considered an average temperature for intertidal or subtidal habitats from where the limpets come from, but do not add any supplementary detail on environmental contrasts between these habitats not in the difference of food availability between the morphotypes. However, contrasting environmental pressures (desiccation, salinity, hydrodynanism) and habitat characteristics (immersion time, substratum type, and surrounding physico-chemical factors) contribute to contrasting adaptative strategies among which morphological adaptation is really important for limpets, but have not been integrated into our DEB models (because it requires more data we don't have) [START_REF] Vermeij | Morphological patterns in high-intertidal gastropods: adaptive strategies and their limitations[END_REF][START_REF] Branch | The ecology of limpets: physical factors, energy flow, and ecological interactions[END_REF][START_REF] Denny | Hydrodynamics, shell shape, behavior and survivorship in the owl limpet Lottia gigantea[END_REF][START_REF] Sa Pinto | Patterns of colonization, evolution and gene flow in species of the genus Patella in the Macaronesian Islands[END_REF][START_REF] Bouzaza | Etude systématique, phylogénétique et phylogéographique de quelques espèces de Patelles[END_REF][START_REF] Grandfils | Contribución al conocimiento de Patella ferruginea (Gmelin, 1789)[END_REF][START_REF] Gray | Growth and reproduction in the high-shore South African limpet Helcion pectunculus (Mollusca: Patellogastropoda)[END_REF][START_REF] Espinosa | Effect of human pressure on population size structures of the endangered ferruginean limpet: toward future management measures[END_REF]). Desiccation is one of the strongest hypothesis to explain the morphological differences between the intertidal and subtidal morphotypes [START_REF] Mauro | Morphological and molecular tools in identifying the Mediterranean limpets Patella caerulea, Patella aspera and Patella rustica[END_REF][START_REF] Bouzaza | Discriminant-based study of the shell morphometric relationships of Patella caerulea (Gastropoda: Prosobranchia) of the western Mediterranean Sea[END_REF]. The presence of high upstream shifted apex form for the intertidal morphotypes, more exposed to desiccation, could help to store more water and absorb more oxygen, as described for Patella ferruginea [START_REF] Branch | Limpets: evolution and adaptation[END_REF][START_REF] Paracuellos | Conservational status and demographic characteristics of Patella ferruginea Gmelin, 1791 (Mollusca, gastropoda) on the Alboran Island (Western Mediterranean)[END_REF]. Similarly, shell volumes are bigger for the intertidal type and help reduce water loss [START_REF] Vermeij | Morphological patterns in high-intertidal gastropods: adaptive strategies and their limitations[END_REF][START_REF] Wolcott | Physiological ecology and intertidal zonation in limpets (Acmaea): a critical look at" limiting factors[END_REF][START_REF] Branch | Ecology of Patella species from the Cape Peninsula, South Africa. IV. Desiccation[END_REF][START_REF] Branch | The ecology of limpets: physical factors, energy flow, and ecological interactions[END_REF]) but also infer resistance to the effects of ice damage [START_REF] Morley | Depth gradients in shell morphology correlate with thermal limits for activity and ice disturbance in Antarctic limpets[END_REF]. Differences in the energetic responses of the two morphotypes of N. concinna to the difference in mean intertidal (0.45°C) and subtidal (-0.1°C), or the much greater difference in maximum (12.3 versus 1.7°C respectively; [START_REF] Morley | Thermal reaction norms and the scale of temperature variation: latitudinal vulnerability of intertidal nacellid limpets to climate change[END_REF], could be a proximate cause of the morphological differences.Taking into consideration differences between the environments is therefore important but strongly lacking in the analysis presented here.

In our study, field data show a slight difference in shell length of +5% and a small difference also in the predicted shape coefficient of 0.45 against 0.39 (Table 3) for respectively the intertidal and subtidal individuals.

This indicates very small differences in inner volumes between the studied populations as calculated by the DEB, meaning that the DEB model does not adequately reflect the difference in morphology between the intertidal and subtidal morphotypes. In the raw data, shell heights present a 33% difference between intertidal and subtidal individuals (Appendix A) but shell length was used, rather than shell height, in the model to characterize the growth structure of the species. Fine tuning the models with extra shape information could have helped to bring further contrasts between the two models, but also requires much more information on shell growth.

Moreover, the difference in food availability and quality was hypothetized between the orphotypes when calibrating the model, despite food abundance and quality knowledge being responsible for strong contrasts in DEB model outputs [START_REF] Kooijman | Dynamic energy budget theory for metabolic organisation[END_REF][START_REF] Thomas | Modelling spatio-temporal variability of Mytilus edulis (L.) growth by forcing a dynamic energy budget model with satellite-derived environmental data[END_REF][START_REF] Saraiva | Validation of a Dynamic Energy Budget (DEB) model for the blue mussel Mytilus edulis[END_REF][START_REF] Sarà | Predicting biological invasions in marine habitats through eco-physiological mechanistic models: a case study with the bivalve Brachidontes pharaonis[END_REF]. During winter time, the intertidal type seems to have supplementary access to ice-algae and microphytobenthos in rock crevices, whereas the subtidal type mainly grazes on the diatoms films growing on encrusing red algae (Appendix A, [START_REF] Obermüller | Antarctic intertidal limpet ecophysiology: A winter-summer comparison[END_REF]. But food abundance and quality were assumed for the construction of the models, as no data accurate enough were available to characterize the feeding behaviour of the limpets.

Moreover, in the case of intertidal type, no clear hypothesis is available for their behaviour during winter period, as several authors hypothesize either a migration into the subtidal or a dormance period hidden into crevices during the period where ice is covering their habitat [START_REF] Brêthes | Distribution, growth and reproduction of the limpet Nacella (Patinigera) concinna (Strebel 1908) in relation to potential food availability, in Esperanza Bay (Antarctic Peninsula)[END_REF], Obermüller et al. 2011). However, this information would be essential to explain how these individuals energetically behave during this period.

Potential of the approach

This study showed that it is feasible to build a DEB model for a marine Antarctic species, with the few available data. Adding extra information from sampling and experiments during a single expedition in the field considerably increased the accuracy of the model and highlighted some small differences in energy allocation priorities, maintenance costs and reproductive potential between the intertidal and subtidal morphotypes. But the method is then limited by model calibration and data availability since it could not prove that these contrasts are explained by anything else but environmental conditions.

Such DEB models would already be sufficient to (1) describe the performance of the species physiological traits in spatially or temporally contrasting environmental conditions [START_REF] Kearney | Biomechanics meets the ecological niche: the importance of temporal data resolution[END_REF], Teal et al. 2012), (2) to be upscaled to the population level to assess population structure and density dynamics [START_REF] Klanjscek | Integrating dynamic energy budgets into matrix population models[END_REF], Arnould-Pétré et al. 2020 this issue), or (3) to be integrated into a dynamic network by adding knowledge about interaction with other species [START_REF] Ren | An ecosystem model for estimating potential shellfish culture production in sheltered coastal waters[END_REF][START_REF] Ren | An ecosystem model for optimising production in integrated multitrophic aquaculture systems[END_REF]. Adding some data from extra experiments would easily enable further development of these models for ecophysiological or ecotoxicological applications [START_REF] Muller | Modeling the effect of toxicants on the parameters of dynamic energy budget models[END_REF][START_REF] Pouvreau | Application of a dynamic energy budget model to the Pacific oyster, Crassostrea gigas, reared under various environmental conditions[END_REF][START_REF] Peeters | Influence of low and decreasing food levels on Daphnia-algal interactions: Numerical experiments with a new dynamic energy budget model[END_REF][START_REF] Sarà | Combining heat-transfer and energy budget models to predict thermal stress in Mediterranean intertidal mussels[END_REF], or to improve knowledge about development stages, behaviour or reproduction [START_REF] Pecquerie | Modeling fish growth and reproduction in the context of the Dynamic Energy Budget theory to predict environmental impact on anchovy spawning duration[END_REF][START_REF] Rico-Villa | A Dynamic Energy Budget (DEB) growth model for Pacific oyster larvae, Crassostrea gigas[END_REF], Kooijman et al. 2011).

In this study, we wanted to explore whether the amount of data that was available to build these models were sufficient to see the known physiological and morphological differences between the two morphotypes, and results show that more data are necessary.

To conclude, we advise the use of DEB approach for ecological modelling for Antarctic case studies but modellers should be aware of the necessity to calibrate models with accurate data to fine tune results. Among these data, the description of the species habitat is complex information to be integrated into a model and most of the time only partial information is available. Working in narrow scale areas where habitat is known and described and where experiments can be run might be a good option.

Our study also hightlights the interest of DEB models to reuse data from published experiments from historical published works from Antarctic campaigns and highlights the importance of precisely documenting the associated metadata (notably the description of the environment and the conditions in which the limpets are living), data that is not always available.

Figure 1 .

 1 Figure 1. Nacella concinna in apical view (a) and lateral view (b). Scale bar: 1cm. Source: Q. Jossart, B121 expedition.

Figure 2 :

 2 Figure 2: Schematic representation of the standard DEB model, with energy fluxes (arrows, in J.d -1) that connect the four compartments (boxes). Energy enters the organism as food (X), is assimilated at a rate of A into the reserve compartment (E). The mobilization rate ( C), regulates the energy leaving the reserve to cover somatic maintenance ( M),

  costs of structure; better replaced by [EV]= κG, where κG is the fraction of growth energy fixed in structure: [EV]=[EG] compare body sizes inter-specifically; z = 1 for Lm = 1 cm -sM Acceleration factor at f =1, it is equal to the ratio of structural length at -metamorphosis and birth.

Figure 3 .

 3 Figure 3. Comparison of model predictions (uni-variate data). Blue dots joined by lines: subtidal model predictions, blue stars: subtidal data (observations); orange dots joined by lines: intertidal model predictions, orange stars: intertidal data (observations); black triangle joined by lines: merged model predictions. Prediction points may overlap (D).

  Along the merging procedure, the loss function and MRE values of the model at each step of the merging procedure are observed, one 'step' corresponding to the interative increase of the weight coefficient of the studied parameter (i.e. merging step, Fig.4) . Changes in loss function and MRE values are not that important between the initial step and the final step of the merging procedure (Fig.4, Appendix D) (respectively from 0.170 to 0.196 and from 0.192 to 0.227 for the MRE intertidal and MRE subtidal values), meaning that merging parameters is possible.and δM seem to be the parameters that are the most influencing the model during the merging procedure for both the intertidal and subtidal models and [ M] seems to further influence the intertidal model.

Figure 4 .

 4 Figure 4. Evolution of Mean Relative Error (MRE) values along the merging of the different parameters. MRE intertidal in solid blue line, MRE subtidal in dashed purple line. Example of Trial #5 (merging of z, [ M], δM_larvae, κ, ℎ a, , EH b , [EG], EH j , EH p , δM).

Table 2 .

 2 Zero and uni-variate data used to build the intertidal and subtidal models.

	INTERTIDAL GROUP	SUBTIDAL GROUP

Uni-variate data, (unit) Reference Reference

	LJO (cm, μmol/h)
	Temperature ~ Oxygen consumption	Peck 1989	Peck 1989
	TJO (K, μL/h)
	Time ~ Length	S. Morley experiment 2018****	S. Morley experiment 2018****
	tL (d, cm)
	* Max sized collected individual on the field during Belgica121 expedition (Danis et al. 2019)
	** Shabica 1976 indicates an observed value of 5.8cm and S. Morley measurements indicate a ratio between
	intertidal/subtidal lengths of the morphotypes of 1.05. The unknown subtidal Li value was calculated as
	5.8/1.05= 5.52cm.
	Length ~ AFDW	Nolan 1991, Signy Island	Nolan 1991, Signy Island
	LWd_signy (cm, g)
	Length ~ AFDW	S. Morley experiment 2018	S. Morley experiment 2018
	LWd (cm, g)
	Length ~ Gonado somatic index	S. Morley experiment 2018	S. Morley experiment 2018
	LGSI(cm, -)
	Length ~ Oxygen consumption	S. Morley experiment 2018	S. Morley experiment 2018

*** based on egg diameter of 221μm **** imagery and growth ring measurements, see Appendix B

  this issue) is a new extension that enables to compare small variation in parameter values between (close) species. The second term (in bold) of the following equation (eq. 2) is the new extension of the 'symmetric bounded (sb)' loss function:

Table 3 .

 3 Summary of goodness of fit, DEB model estimates at a reference temperature of Tref= 20°C. RE:

		INTERTIDAL	SUBTIDAL		MERGED
	MRE	0.166	0.192	0.196	0.227
	Loss function	0.2441	0.2345	0.7936
	z (-)	0.3055	0.4317	0.2579
	{6 Am} (J/d. cm -2 )	8.361	19.07	8.859
	( (cm/d)	0.0501	0.0426	0.0499
	Κ (-)	0.9084	0.9368	0.9256
	[6 M] (J/d. cm -3 )	19.62	31.68	24.62

Observed and predicted values for zero-variate data, relative error (RE) for the uni-variate data. See Fig.

3

for comparisons for uni-variate predictions between the models. MRE= Mean Relative Error. For the merged model, the MRE values respectively correspond to the mean relative error of model prediction for data of both intertidal and subtidal populations. All DEB parameters indicated were allowed to vary during covariance estimation. The abj parameters that are not mentioned in that table were kept constant with the standard initial values.

Acknowledgements

We are thankful to Philippe Pernet for N. concinna pictures, and Jonathan Flye-Sainte-Marie, Jean-François Cudennec for sclerochronology protocol advices, Eric Dabas, Rémi Laffont for sclerochronology lab trials. This work was supported by a "Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture" (FRIA) and "Bourse fondation de la mer" grants to C. Guillaumot. SMOR was supported by Natural Environment Research Council core funding to the British Antarctic Survey. This is contribution no. 40 to the vERSO project (www.versoproject.be), funded by the Belgian Science Policy Office (BELSPO, contract n°BR/132/A1/vERSO). Research was also financed by the "Refugia and Ecosystem Tolerance in the Southern Ocean" project (RECTO; BR/154/A1/RECTO) funded by the Belgian Science Policy Office (BELSPO), this study being contribution number 14.

Authors' contribution


Ting Su

Deyang Zhao

Mohamad Khodadadi

Christophe Len
email: christophe.len@chimieparistech.psl.eu

Lignocellulosic biomass for bioethanol: Recent advances, technology trends, and barriers to industrial development

Keywords: lignocellulose, bioethanol, pretreatment, hydrolysis, fermentation

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

Introduction

Our current way of life and the constant increase in population make energy one of the main necessities of the modern world [START_REF] Mortensen | A review of catalytic upgrading of bio-oil to engine fuels[END_REF]. Fossil resources are being depleted and will be economically unviable soon, so there is an urgent need to develop renewable energy to replace fossil fuels derived from oil. Biomass, the only renewable resource of organic carbon in nature, is a response to petroleum substitution [START_REF] Ragauskas | The path forward for biofuels and biomaterials[END_REF][START_REF] Hoffert | Advanced technology paths to global climate stability : Energy for a greenhouse planet[END_REF]. Among the components of biomass, lignocellulose is the most abundant with a production of ~170 billion metric tons per year [START_REF] Li | Catalytic transformation of lignin for the production of chemicals and fuels[END_REF][START_REF] Chang | Fundamental factors affecting biomass enzymatic reactivity[END_REF]. The different sources of lignocellulosic biomass are agricultural residues (leaves, stovers, straws…), agro-wastes (solid cattle manure…), forest biomass (cedar, spruce, willow…), forest wastes (sawdust, wood chips…), industrial wastes (chemical pulps …) and municipal solid wastes (food waste, kraft paper…). Lignocellulosic biomass consists of three major components: cellulose (40-60%) [START_REF] Lee | Biological conversion of lignocellulosic biomass to ethanol[END_REF], hemicellulose (20-40%) [START_REF] Ren | Hemicelluloses. 1st ed. Cereal Straw as a Resource for Sustainable Biomaterials and Biofuels[END_REF] and lignin (10-25%) [START_REF] Buranov | Lignin in straw of herbaceous crops[END_REF], which are intertwined together to form lignin-carbohydrate complexes (LCCs). To date, most commercial biorefinery processes focus on the exploitation of cellulose and hemicellulose, while the lignin fraction is still treated as a waste product [START_REF] Guan | High catalytic efficientcy of solid acid catalysts for lignin valorization[END_REF][START_REF] Schutyser | Chemicals from lignin : an interplay of lignocellulose fractionation, depolymerisation, and upgrading[END_REF].

Lignocellulosic biomass, particularly agricultural residues, is not associated with food and is available in abundance worldwide. Therefore, the consumption of lignocellulosic biomass on a large scale for bioethanol production does not divert the use of arable land as is the case for first generation bioethanol [START_REF] Agbor | Biomass pretreatment: Fundamentals toward application[END_REF].

Second-generation bioethanol production

The production of second-generation bioethanol starts with the collection of lignocellulosic feedstock and then its transportation to the plant site. Depending on the feedstock, an appropriate preprocessing step (e.g., grinding, milling) is realized to obtain the adequate the feedstock particle size. After this preprocessing, second-generation bioethanol is produced using a process involving four primary steps: (i) pretreatment, (ii) hydrolysis to sugars, (iii) fermentation, and (iv) product/product upgrading [START_REF] Chundawat | Deconstruction of lignocellulosic biomass to fuels and chemicals[END_REF][START_REF] Robak | Review of second generation bioethanol production from residual biomass[END_REF][START_REF] Abo | Lignocellulosic biomass for bioethanol : an overview on pretreatment, hydrolysis and fermentation processes[END_REF] (Figure 1). This bioethanol production includes a separate hydrolysis and fermentation (SHF) ( [START_REF] Roca | Increasing ethanol productivity during xylose fermentation by cell recycling of recombinant Saccharomyces cerevisiae[END_REF]. Using SHF process, the temperature for the enzymatic hydrolysis and the fermentation temperature can be optimized independently. Moreover, SHF process permits to recycle yeast fermentation which is not the case with other alternatives. CLCs are recalcitrant to degradation, so pretreatment is required for their destruction to separate the cellulose component from hemicelluloses and lignin so that enzymatic hydrolysis can be performed to release D-glucose/D-xylose for ethanol fermentation [START_REF] Kumar | Recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review[END_REF]. Different methods for the LCC pretreatments exist: physical pretreatment, chemical pretreatment, physico-chemical pretreatment and biological pretreatment [START_REF] Tu | Recent advances in the pretreatment of lignocellulosic biomass[END_REF][START_REF] Mahmood | Recent advances in the pretreatment of lignocellulosic biomass for biofuels and value-added products[END_REF][START_REF] Costa | Lignocellulosic to biofuels: an overview of recent and relevant advances[END_REF][START_REF] Kucharska | Pretreatment of lignocellulosic materials as substrates for fermentation processes[END_REF]. Conventional pretreatments of lignocellulose are quite costly, time-consuming and also require substantial downstream processing. Physical pretreatment such as fragmentation (hacking, grinding, milling, rolling) [START_REF] Arenas-Cardenas | Waste and biomass valorization[END_REF], microwave radiation [START_REF] Aguilar-Reynosa | Microwave heating processing as alternative of pretreatment in second generation biorefinery: an overview[END_REF], sonication [START_REF] Subhedar | Intensification of enzymatic hydrolysis of lignocellulose using ultrasound for efficient bioethanol production : a review[END_REF], spray drying with gamma radiation [START_REF] Yang | Effect and aftereffect of gamma radiation pretreatment on enzymatic hydrolysis of wheat straw[END_REF], pyrolysis [START_REF] Dickerson | Catalytic fast pyrolysis: A review[END_REF] permits to reduce cellulose crystallinity, increase surface area, reduce the degree of polymerization. Physico-chemical pretreatment such as a steam explosion [START_REF] Kumari | Pretreatment of lignocellulosic wastes for biofuel production: A critical review[END_REF], carbon dioxide explosion [START_REF] Carneiro | Cpt 17 -Biomass Pretreatment With Carbon Dioxide[END_REF], ammonia fiber explosion [START_REF] Alizadeh | Pretreatment of switchgrass by ammonia fiber explosion (AFEX)[END_REF] and ammonia recovery process [START_REF] Jung | Effect of sodium hydroxide treatment of bacterial cellulose on cellulase activity[END_REF] permits to increase pore volume, remove hemicellulose, improve enzyme accessibility, reduce the particle size. Chemical pretreatment such as acid hydrolysis [START_REF] Silverstein | A comparison of chemical pretreatment methods for improving saccharification of cotton stalks[END_REF], alkaline pretreatment [START_REF] Mosier | Optimization of pH controlled liquid hot water pretreatment of corn stover[END_REF], oxidation [START_REF] Den | Lignocellulosic biomass transformations via greener oxidative pretreatment processes : access to energy and value-added chemicals[END_REF] and ozonation [START_REF] Travaini | Ozonolysis : an advantageous pretreatment for lignocellulosic biomass revisited[END_REF], treatment with ionic liquids [START_REF] Klein-Marcuschamer | Techno-economic analysis of a lignocellulosic ethanol biorefinery with ionic liquid pre-treatment[END_REF], treatment with organic solvents [START_REF] Zhang | Organic solvent pretreatment of lignocellulosic biomass for biofuels and biochemicals : a review[END_REF] permits to increase surface area, have high reaction rate and remove hemicellulose. Biological pretreatment such as bacterial treatment [START_REF] Sharma | Biological pretreatment of lignocellulosic biomass for biofuels and bioproducts : an overview[END_REF], enzymatic treatment [START_REF] Sindhu | Biological pretreatment of lignocellulosic biomass -an overview[END_REF] permits lignin degradation and reduces the degree of polymerization of cellulose and hemicellulose as well. Following the pretreatment, enzymatic or chemical hydrolysis of the cellulose and hemicellulose releases D-glucose and hexose and pentose, respectively, as simple fermentable carbohydrates [START_REF] Sun | Hydrolysis of lignocellulosic materials for ethanol production: a review[END_REF]. It is noteworthy that hydrolysis of cellulose is more difficult than that of hemicellulose. These C-6 and C-5 carbohydrate monomers can then be fermented to ethanol by bacteria and yeasts that have been modified to ferment both hexose and pentose sugars and adapted to treat the inhibitors that are produced during pretreatment and inevitably associated with the hexose and pentose sugars. Among the large number of bacteria and yeasts that are able to ferment these sugars, Saccharomyces cerevisiae and Zymomonas mobilis are the two species currently used on an industrial scale for ethanol production [START_REF] Lau | Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production[END_REF]. The last steps of the bioethanol production is the distillation and dehydration of the aqueous ethanol solution producing ethanol with a purity of 99.9%.

Initially, hydrolysis and fermentation were performed in two separate two-stroke reactors (Separate Hydrolysis and Fermentation -SHF). Nevertheless, the two processes can be realized in the same reactor. In this way, simultaneous saccharification and fermentation (SSF) [START_REF] Olofsson | A short review on SSF -an interesting process option for ethanol production from lignocellulosic feedstocks[END_REF], pre-hydrolysis and simultaneous saccharification and fermentation (PSSF) [START_REF] Mcintosh | Pilot-scale cellulosic ethanol production using eucalyptus biomass pre-treated by dilute acid and steam explosion[END_REF], simultaneous saccharification and co-fermentation (SSCF) [START_REF] Liu | Dry biorefining maximizes the potentials of simultaneous saccharification and co-fermentation for cellulosic ethanol production[END_REF] and direct conversion (consolidated bioprocessing) (CBP) [START_REF] Hasunuma | Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains[END_REF]. SSF is the most used process in industry. This process performs the two stages in the same reactor and the same time permitting to reduce the investment cost and to limit the risk of inhibition of the enzyme by D-glucose and cellobiose. The main drawback of SSF is the temperature used (<37 °C) that is not the optimized temperature, but that permits to have less degradation of the yeasts. PSSF is developed to solve the problem encountered during the SSF process of hydrolysis at a lower temperature than the optimum temperature. In this process, enzymatic pre-hydrolysis is performed at 50 °C and then after reducing the temperature to 35 °C, the yeast is added in the same reactor. In contrast with SHF, SSF and PSSF, SSCF permits to have the hydrolysis and fermentation stages of D-glucose and D-xylose coming from cellulose and hemicellulose, respectively.

CBP is an attractive process incorporating enzyme production, saccharification and fermentation in a single reactor without the step of pretreatment. The reduction of the number of reactors and facile operation increase the competitiveness of lignocellulosic bioethanol but the design of an appropriate microbial microorganism/consortium is the main drawback.

Technology Trends and Barriers to industrial development

To date, the industrial production of lignocellulosic ethanol is perfectible. Although all stages of the process can be improved, pretreatment, pentose fermentation, simultaneous saccharification and fermentation (SSF), downstream and the reduction of by-products remain an important source of study. Despite the means developed in research and development (pretreatment, hydrolysis to carbohydrates, fermentation, distillation and purification), pretreatment remains one of the most expensive steps in the process. The pre-processing stages account for 11 to 27% of the production costs of lignocellulosic ethanol. This variation depends on the pre-processing method used. For optimal efficiency of the pretreatment stage, it must guarantee high yields of sugar required for the next fermentation stage, a very low content of inhibitors in the fermentation stages, the separation of lignin and incidentally hemicellulose which will then be transformed into molecules of interest. These steps will also reduce the cost of lignocellulosic ethanol, a low impact of the extraction, washing, neutralisation stages and of course a low cost of energy, reagents and catalysts and water.

To date, there are no methods for pretreating lignocellulosic biomass which is preponderant over another. The choice of pretreatment technology depends on various factors, including the type of biomass used. Nevertheless, steam explosion is recognized as one of the most efficient and cost-effective technologies for pretreating agricultural residues, particularly in processes scaled up for commercial production.

During the hydrolysis step, the presence of hemicellulose leads to the formation of pentoses, which are less fermentable derivatives than glucose from cellulose. Therefore, an efficient fermentation of pentoses reduces the cost of ethanol production, when we know that pentose derivatives can reach 26% by weight in the example of sugar cane bagasse and 75% by weight in sugar cane. Microorganisms capable of producing ethanol with high yields at high concentrations of substrate, inhibitors and products are therefore an avenue of research to be developed.

To overcome the problems of the SHF process where the accumulation of carbohydrates such as cellobiose causes cellulase inhibition, the SSF type processes reduce investment costs by limiting the number of reactors. Moreover, the inhibition problem is limited since D-glucose molecules are immediately fermented. However, the major problem to be solved is related to the temperature of the process: the temperature required (50-55 °C) for saccharification is higher than the cell required for fermentation. Downstream processing of lignocellulosic ethanol is also an important source of research. Indeed, the separation of ethanol from the water used in large quantities is one of the challenges for an industrial optimization of the process. Technological progress on materials (membranes, adsorbents) to obtain high-performance membranes is necessary. Similarly, a large quantity of water is used to date. This is necessary in order to reduce the viscosity of the medium during fermentation and to limit the inhibition of bacteria. In this context, waste water must be limited to prevent contamination of the environment. Research on a possible fermentation in a more concentrated medium is therefore a new avenue of research. Organic solvents that are highly flammable, volatile, and potentially toxic, requires additional safety and storage considerations which all add to the total cost. An efficient process for separating biomass constituents after pretreatment and the valorization of lignin and hemicellulose are also lines of research to be developed. To date, lignin and hemicellulose can be converted into value-added products. Similarly, the fermentation broth as well as the solid residues (including bacterial/yeast cell mass) are rich in nutrients and can be recycled in the process, used as animal feed or added to biogas plants.

Conclusion

Lignocellulosic ethanol is not yet sufficiently competitive and several parameters need to be improved. Among these, the toxicity of the reagents used, waste disposal and cost are the main ones. Indeed, many of these processes require water and organic solvents in at least part of the process. Wastewater must be sanitized to avoid environmental contamination, while many organic solvents are highly flammable, volatile and potentially toxic, requiring additional safety and storage considerations, all of which add to the total cost. In addition to toxicity and disposal, certain process steps depending on the pretreatment method used, additional equipment and energy requirements must also be considered in assessing the cost and feasibility of the whole process. In addition, physical/chemical pretreatments use acid or alkaline solutions or a solvent for catalysis, which causes equipment corrosion. The majority of the steps in the ethanol production process is carried out in a batch reactor. The discontinuous reactor-continuous reactor transfer is the subject of much work for the transformation of biomass and high value-added molecules [START_REF] Garcia-Olmo | Activity of continuous flow synthesized Pd-based nanocatalysts in the flow hydroconversion of furfural[END_REF][START_REF] Galy | Glycerol oligomerization in continuous flow reactor[END_REF][START_REF] Len | Continuous flow conversion of glycerol into chemicals: an overview[END_REF][START_REF] Bruniaux | Continuous flow reductive alkylation: synthesis of biobased symmetrical and dissymmetrical ethers[END_REF][START_REF] Wang | Batch vs continuous flow performance of supported mono-and bimetallic nickel catalysts for catalytic transfer hydrogenation of furfural in isopropanol[END_REF][START_REF] Wang | Comparative study of supported monometallic catalysts in the liquidphase hydrogenation of furfural : batch and continuous flow[END_REF][START_REF] Ouyang | Continuous flow conversion of biomass-derived methyl levulinate into gamma valerolactone using functional metal organic framework[END_REF], and the near future of ethanol production will involve a continuous process. This route will still require the destructuring of lignocellulosic material, the elimination of lignin, the reduction of the crystallinity of cellulose ... but will bring all the advantages of continuous flow chemistry: improved safety, increased productivity and selectivity, improved quality, space savings…

Figure 1 .

 1 Figure 1. Schematic representation of lignocellulosic ethanol process.

Competing interest statement

The authors declare having no competing interest.