Ludger Johannes
email: ludger.johannes@curie.fr

Massiullah Shafaq-Zadah
email: massiullah.shafaq-zadah@curie.fr

Estelle Dransart

CLATHRIN-INDEPENDENT ENDOCYTOSIS, RETROGRADE TRAFFICKING, AND CELL POLARITY

Keywords: endocytosis, retrograde trafficking, and cell polarity Endocytosis, intracellular trafficking, retrograde transport, polarity, glycobiology, galectin, Shiga toxin

Introduction

The plasma membrane delimitates eukaryotic cells from their environment. Due to its capacity to internalize cargoes from the extracellular space or the cell surface, endocytosis controls a multitude of cellular functions, ranging from nutrient uptake and signaling to cell migration and neurotransmission [START_REF] Doherty | Mechanisms of endocytosis[END_REF].

The plasma membrane also protects cells from extracellular insults. Yet, pathogens (e.g. viruses) and pathogenic agents (e.g. protein toxins) have found ways to breach this barrier to gain access to the intracellular space to exert their harmful action, and/or to replicate.

In this review, we will first summarize endocytic mechanisms with an emphasis on thoseoften still poorly characterized -that do not depend on the conventional clathrin machinery.

We will then discuss how these relate to the polarized distribution of molecules within cells.

We will notably focus on retrograde trafficking from endosomes to the Golgi apparatus, which allows cargo proteins that follow this pathway to have access to the polarized secretion capacity of the Golgi for targeted delivery to specialized areas of the plasma membrane, such as the leading edge in migratory cells, or the immunological synapse in lymphocytes or specialized antigen presenting cells.

Endocytic mechanisms -focus on non-clathrin uptake processes

Clathrin-dependent endocytosis remains the best characterized endocytosis pathway [START_REF] Doherty | Mechanisms of endocytosis[END_REF]. The clathrin triskelion is recruited to the plasma membrane via adaptor proteins, which directly bind consensus signals found in the cytosolic tails of cargo proteins. Based on its selfassembly properties and the recruitment of curvature inducers such as epsins and BARdomain proteins, the clathrin coat drives the formation of clathrin-coated pits from which clathrin-coated vesicles detach in a process that depends of the pinchase dynamin [START_REF] Antonny | Membrane fission by dynamin: what we know and what we need to know[END_REF].

Several endocytic processes continue to operate efficiently even when the clathrin pathway is blocked [START_REF] Ferreira | Mechanisms of Carrier Formation during Clathrin-Independent Endocytosis[END_REF][START_REF] Johannes | Glycolipids and Lectins in Endocytic Uptake Processes[END_REF][START_REF] Mayor | Clathrin-independent pathways of endocytosis[END_REF]. Since their discovery in the early 1980s, it has been a conundrum to know how cargo proteins are recruited and membranes bent in the absence of the clathrin coat. In the following, we discuss particularly well explored examples for which elements of response to these key questions have been proposed. Due to length limitations of the current review, we unfortunately cannot be fully exhaustive.

FEME

A first model for clathrin-independent endocytosis, termed fast endophilin-mediated endocytosis (FEME), relies on the membrane curvature-active BAR-domain protein family member endophilin [START_REF] Boucrot | Endophilin marks and controls a clathrinindependent endocytic pathway[END_REF] (Figure 1,(a)). FEME occurs preferentially in the leading edge of migrating cells, is triggered by ligands, and used by a number of receptors, including heterotrimeric G-protein, growth factor, and IL-2 receptor. Recently, it was described that the small GTPase Cdc42 brings 2 BAR-domain proteins to the plasma membrane, FBP17 and CIP4, which then recruit the phosphatase SHIP2 and lamellipodin to drive the local production of PIP2 and thereby, the enrichment of endophilin [START_REF] Wah Hak | FBP17 and CIP4 recruit SHIP2 and lamellipodin to prime the plasma membrane for fast endophilin-mediated endocytosis[END_REF].

CLIC/GEEC

Another model for clathrin-independent endocytosis involves short sometimes crescentshaped tubular clathrin-independent carriers (CLICs) [START_REF] Kirkham | Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles[END_REF] that then mature into glycosylphosphatidylinositol (GPI)-anchored protein-enriched early endocytic compartments (GEEC) [START_REF] Mayor | Clathrin-independent pathways of endocytosis[END_REF] (Figure 1,(b)). This CLIC/GEEC process has initially been described for the ganglioside-binding B-subunit of cholera toxin, GPI-anchored proteins (such as CD59 and Thy-1), the transmembrane protein CD44, and a major fraction of internalized fluid phase.

CLIC/GEEC endocytosis is regulated by the small GTPases Arf1 and CDC42 [START_REF] Kumari | ARF1 is directly involved in dynamin-independent endocytosis[END_REF][START_REF] Sabharanjak | GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway[END_REF], the GTPase activating factor GRAF1 [START_REF] Lundmark | The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway[END_REF], the actin nucleation factor ARP2/3 [START_REF] Sathe | Study on endocytic carrier formation in clathrin-and dynaminindependent CLIC/GEEC endocytosis. Concomitant CDC42-mediated recruitment of IRSp53 and ARF1-dependent inhibition of PICK1 are both required to induce branched actin nucleation, leading to the formation of clathrin-independent carriers. Exciting findings on how BAR domain proteins and small GTPases function together to regulate actin polymerization during CLIC/GEEC endocytosis[END_REF], and the BAR domain protein IRSp53 [START_REF] Kast | IRSp53 coordinates AMPK and 14-3-3 signaling to regulate filopodia dynamics and directed cell migration[END_REF]. CLIC/GEEC endocytosis is dynamin-independent for the endogenous cargoes that have been analyzed [START_REF] Mayor | Clathrin-independent pathways of endocytosis[END_REF], and not strictly dynamin-dependent for exogenous cargoes such as cholera [START_REF] Kirkham | Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles[END_REF] and Shiga toxins (Ref. [START_REF] Renard | Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis[END_REF], see below).

GL-Lect

How membrane bending might be operated in at least some processes of CLIC/GEEC endocytosis has been addressed at the examples of Shiga and cholera toxins, and the cellular CD44 and α5β1 integrin, which all are found in CLICs (Refs. [START_REF] Kirkham | Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles[END_REF][START_REF] Howes | Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells[END_REF] and unpublished). The glycosphingolipid (GSL)-binding homo-pentameric B-subunits of Shiga and cholera toxin (termed STxB and CTxB, respectively) induce tubular endocytic pits on cells and model membranes as a first step of their internalization [START_REF] Romer | Shiga toxin induces tubular membrane invaginations for its uptake into cells[END_REF]. This relies on curvature active properties of the B-subunit-GSL complexes [START_REF] Watkins | Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature[END_REF] and the capacity to undergo membranemediated clustering [START_REF] Pezeshkian | Mechanism of Shiga Toxin Clustering on Membranes[END_REF]. The scission process is not strictly dynamin-dependent [START_REF] Kirkham | Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles[END_REF][START_REF] Renard | Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis[END_REF], and involves other scission modalities [START_REF] Renard | Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis[END_REF][START_REF] Simunovic | Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins[END_REF].

The capacity to drive narrow membrane bending in interaction with GSLs leading to the formation of tubular endocytic pits and CLICs has also been observed for cellular lectins of the galectin family [START_REF] Lakshminarayan | Galectin-3 drives glycosphingolipiddependent biogenesis of clathrin-independent carriers[END_REF] (Figure 1,(c)). In the specific case of galectin-3 (Gal3), the lectin binds as a monomer to carbohydrates on cargo proteins such as CD44 or α5β1 integrin. Gal3 then oligomerizes and thereby gains to capacity to also interact with GSLs to drive membrane bending and the biogenesis of tubular endocytic pits from which CLICs detach for the cellular uptake of the cargoes. A similar sequence of events has recently also been described for galectin-8 and GSL-dependent cellular endocytosis of CD166 [START_REF] Renard | -lectin (GL-Lect) hypothesis. It is quite unexpected that endocytosis according to the GL-Lect hypothesis is tuned with such precision according to the isoform of a given BAR domain protein family member[END_REF]. This mechanism has been termed glycolipid-lectin (GL-Lect) hypothesis [START_REF] Johannes | Glycolipids and Lectins in Endocytic Uptake Processes[END_REF].

All these clathrin-independent processes -i.e. FEME, CLIC/GEEC, and GL-Lect -have in common that they are particularly sensitive to interference with the activity of the actin cytoskeleton, and the organization of the membrane into raft-type nanodomains. It also appears noteworthy that some cargoes and trafficking factors are overlapping between these endocytic modalities. We tentatively favor a view according to which clathrin-independent endocytic processes are driven by elements of molecular machinery that are recruited in an interchangeable way according to physiological needs [START_REF] Johannes | Bending "on the rocks"--a cocktail of biophysical modules to build endocytic pathways[END_REF].

Another emerging theme from these studies is that different forms of endocytic uptake couple to different intracellular distribution schemes, sometimes for the same receptor in the same cells. The molecular mechanisms (ligand concentrations, post-translational modifications, conformational changes…) underlying this complexity often still remain to be elucidated. In the following sections, we will address specifically one aspect of this riddle: a possible link between clathrin-independent endocytosis, retrograde trafficking, and cell polarity.

Clathrin-independent endocytosis and cell polarity

Several lines of evidence indicate that clathrin-independent endocytosis is linked to cell polarity. Well established cargoes of non-clathrin uptake processes are localized and internalized in a polarized manner: The leading edge of migratory cells for i) CD44 leading to extracellular matrix interaction and persistent cell migration, ii) the GPI-anchored protein Thy-1 for cell-cell interaction, and iii) β1 integrin for cell adhesion [START_REF] Howes | Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells[END_REF]; the apical membrane of epithelial MDCK cells for Thy-1 [START_REF] Wilson | Polarity of endogenous and exogenous glycosylphosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells[END_REF]; the basolateral membrane in hepatic epithelial cells for the GPI-anchored protein CD59 for its transcytosis to the apical side [START_REF] Ait-Slimane | Basolateral internalization of GPIanchored proteins occurs via a clathrin-independent flotillin-dependent pathway in polarized hepatic cells[END_REF] (Figure 2,(g)).

The apico-basal polarization of the colorectal cancer cell line Caco-2 is an important feature for the proper binding of the plant toxin ricin, a non-clathrin cargo, to the basolateral membrane [START_REF] Jackman | Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance[END_REF]. In MDCK cells, clathrin-independent endocytosis of ricin occurs equally efficiently at both, the apical and basolateral surfaces [START_REF] Eker | Selective regulation of apical endocytosis in polarized Madin-Darby canine kidney cells by mastoparan and cAMP[END_REF]. Upon cAMP stimulation, however, binding and uptake now preferentially operate at the apical membrane, indicating that both processes are subjected to regulation. Key regulators of the non-clathrin uptake machinery are also localized in a polarized manner.

Once activated, FEME asymmetrically operates at the leading edge of migrating cells, while clathrin-coated pits exhibit a nonpolarized distribution [START_REF] Boucrot | Endophilin marks and controls a clathrinindependent endocytic pathway[END_REF]. FEME component Endo-A2 mediates endothelial cell migration to ensure sprout angiogenesis by regulating the clathrinindependent endocytosis of ligand-activated VEGFR2 at the leading edge [START_REF] Genet | link between clathrin-independent but endophilin-dependent endocytosis and polarized cell migration in the context of sprouting angiogenesis[END_REF]. Endo-A2 silencing strongly alters the front-rear asymmetry, notably by repositioning the Golgi behind the nucleus, away from the leading-edge, suggesting a major function for FEME during persistent cell migration [START_REF] Genet | link between clathrin-independent but endophilin-dependent endocytosis and polarized cell migration in the context of sprouting angiogenesis[END_REF].

Similarly, CLIC structures were shown to be preferentially distributed at the leading edge of migrating mouse embryonic fibroblast, where the CLIC/GEEC regulator CDC42 is located [START_REF] Howes | Clathrin-independent carriers form a high capacity endocytic sorting system at the leading edge of migrating cells[END_REF]. Interestingly, CDC42 is an essential contributor in the initiation and maintenance of cell polarity, by acting as an epithelial polarity determinant that notably regulates the proper apical localization of podocalyxin and E-cadherin [START_REF] Bryant | A molecular network for de novo generation of the apical surface and lumen[END_REF][START_REF] Shafaq-Zadah | AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine[END_REF], and polarized cell migration in the context of wound-healing [START_REF] Etienne-Manneville | Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta[END_REF]. The CLIC/GEEC regulator GRAF1 is also implicated in cell polarity, regulating the orientation of cell spreading in migrating cells, and apico-basal polarity during lumen formation [START_REF] Vidal-Quadras | Endocytic turnover of Rab8 controls cell polarization[END_REF].

Lipid-rafts play critical roles in cell polarity, especially in migrating cells where they preferentially localize at the leading edge [START_REF] Guirland | Lipid rafts mediate chemotropic guidance of nerve growth cones[END_REF], such as during chemoattractant-induced neutrophil polarization to recruit calcium-dependent calpain 2 [START_REF] Nuzzi | Asymmetric localization of calpain 2 during neutrophil chemotaxis[END_REF]. GSLs also exhibit a polarized distribution. In migrating mouse embryonic fibroblasts, the polarized localization of the GM1 ganglioside at the leading edge [START_REF] Bisel | Decoupling polarization of the Golgi apparatus and GM1 in the plasma membrane[END_REF] is maintained through Rho/mDia-mediated microtubule stabilization, and further regulated by integrin-activated FAK [START_REF] Palazzo | Localized stabilization of microtubules by integrin-and FAK-facilitated Rho signaling[END_REF]. In contrast, GM1 is enriched at the uropod at the rear of migrating T lymphocytes, together with CD44 and β1 integrin, while the leading-edge is enriched in GM3 [START_REF] Gomez-Mouton | Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization[END_REF]. This differential ganglioside distribution is key for T-cell polarization, and is further regulated by cholesterol and the actin cytoskeleton [START_REF] Palazzo | Localized stabilization of microtubules by integrin-and FAK-facilitated Rho signaling[END_REF][START_REF] Gomez-Mouton | Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization[END_REF]. The GD3 ganglioside shares a similar leading-edge localization in melanoma cells where it controls the polarized recruitment of the lipid raft-associated neogenin, which is essential for cell migration and invasion [START_REF] Kaneko | Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells[END_REF].

Interestingly, GSLs are also crucial in the establishment and the maintenance of apico-basal polarization. In C. elegans, GSLs act as determinants to initiate apical domain identity [START_REF] Zhang | Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis[END_REF]. In mice, their loss alters the identity of apical brush border membrane, and GSL-depleted animals fail to take nutrients up from the intestinal lumen, which is likely due to deficiency in clathrin-independent endocytosis [START_REF] Jennemann | Glycosphingolipids are essential for intestinal endocytic function[END_REF].

As suggested by the GL-Lect hypothesis, GSLs function together with galectins for the clathrin-independent construction of endocytic pits [START_REF] Johannes | Glycolipids and Lectins in Endocytic Uptake Processes[END_REF]. The latter also appear to be important for cell polarization. In kidney, Gal8 binds in a carbohydrate-dependent manner to podocalyxin (gp135) for its post-Golgi delivery to the apical surface such as to specify lumen identity [START_REF] Lim | Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells[END_REF] (Figure 2 (c)). In kidney tubules, Gal9 preferentially binds sialylated glycoproteins for apical delivery [START_REF] Mo | Sialylation of N-linked glycans mediates apical delivery of endolyn in MDCK cells via a galectin-9-dependent mechanism[END_REF]. Similarly, Gal3 enables the polarized transport of key apical markers, i.e. the DPPIV and LPH enzymes [START_REF] Delacour | Apical sorting by galectin-3-dependent glycoprotein clustering[END_REF], and Gal4 was found in post-Golgi flotillin-containing membrane fractions that are responsible of apical delivery [START_REF] Delacour | Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells[END_REF] (Figure 2, (e)). Of note, Gal3 [START_REF] Lakshminarayan | Galectin-3 drives glycosphingolipiddependent biogenesis of clathrin-independent carriers[END_REF], Gal4 [START_REF] Delacour | Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells[END_REF], and Gal9 [START_REF] Mishra | Galectin-9 trafficking regulates apical-basal polarity in Madin-Darby canine kidney epithelial cells[END_REF] all interact with GSLs in the establishment/maintenance of cell polarity, which highlights the possibility that the GL-Lect process plays a specific role in this context (Figure 2 (d-e)).

In the following section, we discuss evidence that links clathrin-independent endocytosis to retrograde transport in the context of cell polarity.

Retrograde trafficking and cell polarity

The Golgi apparatus faces the leading edge in migrating cells [START_REF] Bisel | Decoupling polarization of the Golgi apparatus and GM1 in the plasma membrane[END_REF], and the apical membrane in epithelial cells [START_REF] Bacallao | The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium[END_REF]. The Golgi is thereby strategically positioned in a way such that it facilitates polarized secretion to these specialized areas of the plasma membrane. Retrograde transport from the plasma membrane to the Golgi apparatus allows in some cases for cell surface proteins to be subjected again to this polarized secretion program such as to keep them dynamically localized to corresponding plasmalemmal subdomains [START_REF] Carpier | Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation[END_REF][START_REF] Shafaq-Zadah | Persistent cell migration and adhesion rely on retrograde transport of beta(1) integrin[END_REF]. In the following, we will discuss links between clathrin-independent endocytosis, retrograde transport, and cell polarity.

A possible link between clathrin-independent endocytosis and retrograde trafficking has been noticed for protein toxins, i.e. bacterial Shiga and cholera toxins, and the plant toxin ricin [START_REF] Johannes | Tracing the retrograde route in protein trafficking[END_REF], and viruses or viral products, i.e. AAV [START_REF] Nonnenmacher | Syntaxin 5-dependent retrograde transport to the trans-Golgi network is required for adeno-associated virus transduction[END_REF], human papillomavirus [START_REF] Schelhaas | Entry of human papillomavirus type 16 by actin-dependent, clathrin-and lipid raft-independent endocytosis[END_REF], and HIV [START_REF] Blot | Targeting of the human immunodeficiency virus type 1 envelope to the trans-Golgi network through binding to TIP47 is required for env incorporation into virions and infectivity[END_REF].

The transferrin receptor was amongst the first cellular proteins for which it was shown that they undergo retrograde transport [START_REF] Snider | Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells[END_REF]. Only a small fraction of transferrin receptor is transported to the Golgi, however, which is mirrored by a small fraction that binds to Gal3 [START_REF] Carlsson | Galectin-3 guides intracellular trafficking of some human serotransferrin glycoforms[END_REF] and that enters cells by clathrin-independent endocytosis [START_REF] Renard | -lectin (GL-Lect) hypothesis. It is quite unexpected that endocytosis according to the GL-Lect hypothesis is tuned with such precision according to the isoform of a given BAR domain protein family member[END_REF]. Whether these fractions correspond to each other remains to be established.

Apico-basal polarity

In association with MIG-14/Wntless (WIs), Wnt morphogen undergoes retrograde transport to the Golgi apparatus [START_REF] Zhang | A novel requirement for ubiquitinconjugating enzyme UBC-13 in retrograde recycling of MIG-14/Wntless and Wnt signaling[END_REF], which is regulated by the polarity determinants Cdc42, the Cdc42associated PAR-3/PAR-6/aPKC complex, as well as the Cdc42-dependent actin assembly F-BAR domain proteins TOCA-1 and TOCA-2 [START_REF] Bai | A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling[END_REF]. Different endocytic processes seem to be operating here: Clathrin for uptake at the basolateral membrane of MDCK cells [START_REF] Yamamoto | The apical and basolateral secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by different mechanisms[END_REF]; clathrinindependent for the canonical Wnt3a; clathrin-dependent for the non-canonical basolateral Wnt5 [START_REF] Bandmann | Membrane capacitance recordings resolve dynamics and complexity of receptormediated endocytosis in Wnt signalling[END_REF]; non-clathrin and Gal3-dependent for the apical secretion/localization of Wnt11 [START_REF] Johannes | Glycolipids and Lectins in Endocytic Uptake Processes[END_REF][START_REF] Yamamoto | The apical and basolateral secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by different mechanisms[END_REF], putatively via the GL-Lect mechanism [START_REF] Johannes | Glycolipids and Lectins in Endocytic Uptake Processes[END_REF] (Figure 2,(f)).

In Drosophila, the retromer complex strongly contributes to apical polarity by regulating the retrograde transport/recycling of the apical membrane determinant Crumbs [START_REF] Pocha | Retromer controls epithelial cell polarity by trafficking the apical determinant Crumbs[END_REF] (Figure 2,(a)). In MDCK cells, Crumbs together with Scribbles stabilizes the epithelial junction protein E-cadherin. Upon Scribble (SCRIB) silencing, internalized E-cadherin accumulates in a retromer-dependent manner in the Golgi [START_REF] Lohia | The Scribble polarity protein stabilizes E-cadherin/p120catenin binding and blocks retrieval of E-cadherin to the Golgi[END_REF] (Figure 2,(b)). This interaction is likely conserved in C. elegans, contributing to Wnt signaling during polarized neuronal migration [START_REF] Wassmer | The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network[END_REF].

Front-rear polarity

The retrograde pathway plays an essential role in the polarized localization of β1 integrin to the leading edge of migrating cells [START_REF] Shafaq-Zadah | Persistent cell migration and adhesion rely on retrograde transport of beta(1) integrin[END_REF]. Interfering with retrograde transport leads to the redistribution of β1 integrin and the inhibition of persistent cell migration, while imposing high front-rear polarization stimulates retrograde transport of β1 integrin [START_REF] Shafaq-Zadah | Persistent cell migration and adhesion rely on retrograde transport of beta(1) integrin[END_REF] (Figure 3,(a)).

Both clathrin-dependent and independent uptake processes have previously been documented for β1 integrin [START_REF] Palamidessi | Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration[END_REF][START_REF] Vassilieva | Lipid rafts mediate internalization of beta1-integrin in migrating intestinal epithelial cells[END_REF][START_REF] Furtak | Galectin-3 mediates the endocytosis of beta-1 integrins by breast carcinoma cells[END_REF], the latter involving the GL-Lect mechanism based on Gal3 and GSLs [START_REF] Lakshminarayan | Galectin-3 drives glycosphingolipiddependent biogenesis of clathrin-independent carriers[END_REF][START_REF] Furtak | Galectin-3 mediates the endocytosis of beta-1 integrins by breast carcinoma cells[END_REF]. The relationship between these entry modes and retrograde transport has not yet been assessed.

The matrix metalloproteinase MT1-MMP undergoes retrograde transport prior to be secreted back to the plasma membrane [START_REF] Wang | Co-recycling of MT1-MMP and MT3-MMP through the trans-Golgi network. Identification of DKV582 as a recycling signal[END_REF] (Figure 3,(b)). The clathrin-independent cargo protein CD147 [START_REF] Eyster | Discovery of new cargo proteins that enter cells through clathrinindependent endocytosis[END_REF] stimulates MT1-MMP expression within lipid-raft enriched invadopodia [START_REF] Grass | Regulation of invadopodia formation and activity by CD147[END_REF],

suggesting that this may occur as the result of polarized secretion from the Golgi to this specialized area of the plasma membrane, following prior retrograde transport.

Immunological and neuronal synapse

Upon T cell receptor (TCR) activation, T-lymphocytes build a highly polarized structure: the immunological synapse (Figure 4). The adaptor molecule LAT, a key organizer of signalosome formation, undergoes retrograde transport to the Golgi to be secreted in a polarized manner to the immune synapse [START_REF] Carpier | Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation[END_REF] (Figure 4,(a)). LAT and T-cell signaling components such as the TCR are associated with lipid rafts [START_REF] Varshney | Lipid rafts in immune signalling: current progress and future perspective[END_REF], and it was recently reported that the TCR is internalized in a clathrin-independent manner [START_REF] Compeer | A mobile endocytic network connects clathrin-independent receptor endocytosis to recycling and promotes T[END_REF] (Figure 4,(b)). These findings again reinforce the hypothesis of a tight relationship between clathrin-independent endocytosis and retrograde transport-dependent polarized secretion as a common theme to several biological processes involving specialized areas of the plasma membrane.

In C. elegans, glutamate receptor is localized in a polarized manner towards the dendrites, and retrograde trafficking is required to ensure efficient post-synaptic activity [69]. Both clathrindependent and independent endocytosis have been implicated in glutamate receptor turnover [70]. Which of these internalization modes couples to the retrograde route has yet to be addressed.

Concepts and perspectives

In this review, we have pointed to a possible link between the clathrin-independent construction of endocytic sites at the plasma membrane and specific types of intracellular distribution, notably via the retrograde route. Further work is required to further establish this concept, and to identify molecular mechanisms that can explain how such coupling might be operated.

We have also addressed the role of retrograde trafficking in the establishment and maintenance of cell polarity, by enabling the polarized secretion of reinternalized cargoes to specialized areas of the plasma membrane within different cellular contexts, such as the leading edge of migratory cells, and the immunological synapse of activated T cells. We expect that this concept will apply more widely in the realm of cell polarity, including apicobasal polarity of epithelial cells.

Further investigation of all these aspects is likely to benefit domains of molecular cell biology research that are still relatively poorly explored, such as the role of carbohydrates and raft lipids in the dynamic compartmentalization of biological functions. cell activation. Nat Commun 2018, 9:1597. * The authors have highlighted the existence of a tight relation at the immunological synapse of T cells between clathrinindependent endocytosis and polarized recycling. Remarkably, the T cell receptor (TCR) is internalized via clathrin-independent processes, and incorporated into mobile long-lived endocytic tubules containing the lipid-membrane organizer flotillin, which is necessary for T cell activation. This thorough study provides further evidence on a tight relationship between clathrin-independent endocytosis and polarized recycling within activated T lymphocytes, which might mirror the finding on LAT [START_REF] Nonnenmacher | Syntaxin 5-dependent retrograde transport to the trans-Golgi network is required for adeno-associated virus transduction[END_REF]. Molecular hypothesis according to which sugar-binding proteins of pathogenic (e.g. the GSLbinding B-subunits of Shiga and cholera toxin, STxB and CTxB, respectively, or the GSLbinding VP1 capsid protein of SV40) or cellular origin (e.g. galectins) reorganize glycolipids to which they bind in a way such as to drive the biogenesis of tubular endocytic pits from which CLICs are generated for the cellular uptake of pathogens (e.g. SV40 virus), pathogenic products (e.g. Shiga and cholera toxins), or cellular proteins (e.g. CD44, integrins, CD59) that are recruited by the galectins.

(f)-

Wnt11 is specifically secreted to the apical surface in a process that involves Gal3. As part of its functional cycle, the Wnt receptor wntless (Wls) undergoes retrograde transport to bind newly synthetized Wnt in the Golgi for subsequent polarized secretion. Whether Gal3 also contributes to the retrograde transport of Wls still needs to be investigated. (g)-The GPIanchored protein CD59 is first transported to the basolateral membrane before being transcytosed to the apical side in a clathrin-independent but dynamin and flotillin-dependent manner. A link between this transcytotic route and the retrograde pathway presents an interesting possibility.

Figure 3 Retrograde trafficking in migratory cells

During migration (directionality indicated by thick black arrow), the cell exhibits two distinct domains: the retracting rear, and the migration front, also known as leading edge. Here again, the Golgi apparatus is localized in a polarized manner such that it directly faces the leading edge. (a)-The non-ligand-bound integrin α5β1 uses the retrograde route to the Golgi prior to being re-secreted in a polarized manner to the leading edge. Of note, α5β1 integrin uses both clathrin-dependent and -independent endocytosis processes. The possible link between retrograde transport and the modality of endocytosis remains unexplored. (b)-The matrix metalloproteinase MT1-MMP also follows a similar retrograde transport and polarized secretion cycle. Of note, MT1-MMP expression is regulated by the clathrin-independent cargo CD147, and both are recruited to the same lipid raft nanodomains. Other metalloproteinases such as MMP9 are also found at the leading edge and are positively regulated by Gal3. Altogether, this may suggest a general scheme where adhesion molecules and metalloproteinases cooperate and share similar endocytic and intracellular delivery pathways, further regulated by galectins to facilitate efficient cell migration. During this event, T lymphocytes build specialized membrane domains, termed immunological synapses (IS) to which the linker for activation of T-cell (LAT) localizes in a polarized and dynamic manner to organize the signalosome. Here again, the Golgi apparatus is localized in a polarized manner such that it directly faces the IS. (a)-LAT efficiently undergoes Rab6, retromer, and Synt16-dependent retrograde trafficking to the Golgi compartment for subsequent polarized secretion to the IS, a trafficking loop that is required for efficient T cell activation. (b)-LAT and other T cell components such as TCR as well as APC cargoes like CD166 have been reported to be internalized in a clathrin-independent manner. Whether similar to LAT the other cargoes also undergo retrograde transport for their dynamic localization to the IS remains to be studied directly.

Figure 1

 1 Figures Legends

Figure 2

 2 Figure 2 Retrograde trafficking in the polarized epithelium

Figure 4

 4 Figure 4 Retrograde trafficking in immune cells

Acknowledgements

We acknowledge support by grants from European Research Council (advanced grant 340485), Human Frontier Science Program (RGP0029-2014), Swedish Research Council (K2015-99X-22877-01-6), Mizutani Foundation for Glycosciences (reference n° 200014), Agence Nationale pour la Recherche (ANR-16-CE23-0005-02, ANR-19-CE13-0001-01), Institut National du Cancer (n°2018-1-PLBIO-01-ICR-1, n°2019-1-PL BIO-05-CEA-1), Plan Cancer program LipoCanPredict. The Johannes team is member of Labex CelTisPhyBio (ANR-11-LBX-0038) and Idex Paris Sciences et Lettres (ANR-10-IDEX-0001-02 PSL).


B Santraine

A Berne

G Caulier

J Colnel

N Ravenel

F Leroux

West Team

From Tore Supra to WEST: Evolution of the CODAC infrastructure

In 2013 Tore Supra limiter tokamak at CEA Cadarache went through major changes to become the WEST tokamak (Tungsten [W] Environment in Steady state Tokamak). The tokamak was upgraded into an X point divertor device. Taking advantage of its long discharge capability, WEST's goal is to minimize technological and operational risks of the actively cooled tungsten divertor for ITER.

At the same time, Control Data Access and Communication (CODAC) system has been redesigned in order to satisfy the performance and evolutions needed by researchers, and to facilitate maintenance and evolution for the CODAC team. This paper describes the upgrade of the CODAC system from Tore Supra to WEST.

Introduction

Build between 1984 and 1988, the Tore Supra limiter Tokamak has evolved during 30 years of operation and different technologies have been used. The last major update started in 2014 when the WEST project was started [START_REF] Bucalossi | Measurements and controls implementation for WEST[END_REF].

In parallel to the physical and mechanical transformation of the tokamak to integrate an ITER like divertor, the infrastructure of the CODAC system was also updated for several reasons: -Obsolescence: some components were not supported or even manufactured anymore. -Performance: physics diagnostics required more acquisition channels and higher sampling frequency.

-Maintenance: distributed and heterogeneous acquisition units are difficult to maintain (lot of knowledge required and time consuming).

-Cost: minimize the cost for procurement and maintenance.

Several drivers were taken into account when updating the infrastructure: cost, durability, performance, backward compatibility, maintenance … At the end, the chosen solution had to be a trade-off between the three constraints: cost, time (development and maintenance) and quality (performance and durability).

Acquisition unit

In the previous Tore Supra infrastructure, two types of acquisition units were used: -VME: CPU(s) and acquisition boards inside VME crate, a central server distributes the operating system (LynxOS).

-PC: independent system running a local Windows XP. Both types have advantages and drawbacks. The VME is easy to maintain due to its centralized architecture but is aging and costly. The PC has low cost components but is more difficult to maintain (replacement, upgrade, end of support for Windows XP). The solution to be chosen had to benefit from the experience of the previous architecture. It was decided to keep the centralized architecture and select generic components that offer high availability with effective cost.

The PXE (Preboot eXecution Environment) technology was selected. This solution is composed of two elements: -Server: it contains the operating system for the clients, its configuration and its user space. -Client: it recovers from the server all the software elements and runs it from internal RAM. This solution however introduce a single point of failure in the system, if the server fails all the clients fail. The solution in place is a redundant server which can supersede the primary server (with a nightly replication).

This architecture was called "diskless" as hard drive is not necessary. Presence of high magnetic fields and drive mechanical fatigue were also arguments to avoid having any hard drive (not true anymore with SSD).

For units generating high data throughput, a local drive (or disk array) is installed as a temporary storage (to avoid network saturation, for NAS or NFS).

Acquisition board

New acquisition boards had to be chosen due to the increase of performance (more channels, higher sampling rate), the new format of acquisition units (PCIe, PXIe, Ethernet) and new specification (real time, embedded processing, reconfigurable …). For some diagnostics, a partial upgrade has been done. The VME card has been kept and a PCIe/VME bridge is used to communicate with the PC acquisition units.

The Real Time Network is a shared memory between the diagnostics, it is used for plasma control and machine safety (actuator controlredundant with wired security). The previous network used on Tore Supra could not be maintained anymore: obsolescence, end of support, no PCIe interface.

As for the acquisition boards, a supplier selection was performed and "Dolphin Interconnect Solutions" was selected [START_REF]Dolphin express reflective memory[END_REF]. In addition to the performance increase (up to 20/40Gbps vs. 150Mbps) and the PCIe format supported, the network has a star topology, which allows a great flexibility (compared to the previous ring topology) for adding/removing nodes, for extending the network and for troubleshooting. The communication is data-driven based on master/slaves communication, the master being the PCS and the slaves being the acquisition units and the actuators (coils and heating systems). A global mapping is shared among all nodes, each node has a dedicated data area (read/write) and all nodes can access all the data (read only). The network is currently composed of 25 nodes, 5 switches and more than 1km of fibre optics. The system does not guarantee natively the integrity and the delivery of large packets.

Additional software overheads were added (CRC computation, timestamping and acknowledgement) that affects the performance, best data throughput achieved is around 110MBytes/s [880 Mbps]. Transactions are generally triggered by the acquisition/control loop of the units (typically 1 or 2 ms).

Timing System Network

This network [START_REF] Moulin | Upgrade of the timing system for Tore Supra long pulses[END_REF] is used to distribute timing information to all the acquisitions and control units: -1 MHz clock source for data timestamping -Triggers for acquisition -Safety events -Actuator controls (heating, fuelling, coils) -Shot scheduling (plasma start/stop, start/stop acquisition …) It is a star topology and is natively a simplex communication (workaround exist for providing feedback from specific system).

For WEST, no update was required in terms of performance, functionality and architecture, but new formats were needed to fit the acquisition unit upgrade. Three new boards were developed (commercial products with customized firmware): -PXIe and PCIe: FPGA boards programmed in VHDL. -PCIe: FPGA board programmed with LabView The board is either in the computer or in the acquisition crate, depending on needs. This network is running 24/7 to provide information for continuous data acquisition (between shots, during night).

Infrastructure status

Over the last 4 years, the Linux "diskless" solution for acquisition units has been deployed and represents more than 50% of all the units. Maintenance operations done so far proved that the centralized architecture is time-effective: -Unit replacement takes about 15 minutes (configuration file update, PC replacement and restart).

-New OS deployment on all units can be done in less than 20 minutes (configuration file update and reboot).

A wide variety of acquisition unit configuration can be done using a single architecture: any timing system interface (VME, PCIe, PXIe), various acquisition board format (VME, PCIe, PXIe, MXI, Ethernet, USB, RS232/485), real time network (not mandatory), various hardware (motherboard, single/multiple processor).

This architecture is the "preferred" solution for WEST but the infrastructure is still open for other stand-alone system (especially for collaborators coming with their equipment).

The operation of the CODAC also relies on various pieces of software which are in charge of the data acquisition and processing [START_REF] Leroux | New developments on Tore Supra data acquisition units[END_REF][START_REF] Guillerminet | The acquisition system for Tore Supra 1000 s discharges[END_REF][START_REF] Caulier | WestBox: an object-oriented software component for WEST CODAC[END_REF]. As for the infrastructure, an effort was made to keep it generic and easily maintainable.

Regarding the data archiving and retrieval, the database architecture is used for both the legacy system and new system and manages the experience data for Tore Supra and WEST. Different data converters can be used for converting data from/to other formats such as MDSplus or IMAS.

Experiments and results

More than 4500 experiments have been done with the WEST CODAC infrastructure, ensuring operation safety and providing data to the physicists. Several milestones has been achieved (X-point configuration, >30 seconds discharge) and the machine is ramping up.

Conclusion and plan forward

The upgrade of the CODAC infrastructure for WEST was started in 2013 and was completed at the end of 2016. Since then, the experimental operations are on-going and the system is intensively used. The return on experience showed that the infrastructure is cost-and time-effective and that the expected performances are reached.

Maintenance operation are performed regularly and several improvements are planned (or on-going): real time performances, parallel computing on GPU, time synchronization, inter-process communication … Efforts continue to upgrade old units (VME and Windows XP) and to maximize the reusability of the standard system.

Figure 1 :

 1 Figure 1: Constraints for the infrastructure update

  Configuration files are stored on the server and used to associate client MAC address / IP address / OS version / User space. Modifications are taken into account at client reboot. Several OS version are available (debug, production, evaluation) and one client can be switched from one to another easily. Replacement of a unit is done easily as well by changing the MAC address in the configuration file. The current OS image (July 2019) have the following specifications: -CentOS 7.5 -Linux kernel 3.10 -64 bits -Include drivers for acquisition boards: NI, Dolphin, Marvel, Spectrum, CAEN … -Support wide variety of hardware: single/multi processors, Intel/AMD processor, desktop/industrial single board computer … -Include software tools: gcc, cmake, openmp, cppcheck, clang … No specific hardware is required: software only at server side, all motherboards support now PXE on client side. Therefore, Commercial Off-The-Shelf (COTS) components can be used.

Figure 2 :

 2 Figure 2: Architecture for the acquisition units

Figure 3 :

 3 Figure 3: Increase of performances for WEST

Figure 3 :

 3 Figure 3: Topology of the real time network.

Figure 4 :

 4 Figure 4: X-point configuration and 30s plasma discharge.

Acknowledgments

The CODAC infrastructure upgrade was made possible thanks to the support from CEA staff and collaborators. This work has been carried out within the framework of the EUROfusion Consortium and has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement number 633053. The views and opinions expressed herein do not necessarily reflect those of the European Commission.

Acronyms


Bhawna Sharma

Christian Larroche

Claude-Gilles Dussap
email: c-gilles.dussap@uca.fr

Comprehensive assessment of 2G bioethanol production

Keywords: 2G (second generation) bioethanol, lignocellulosic biomass, fermentation, process simulation, environmental impact Highlights -

The advancements in second-generation bioethanol produced from lignocellulosic biomass, such as crops residues, woody crops or energy grasses are gaining momentum. Though, they are still representing less than 3% of total bioethanol production, the GHG reduction potential is higher than for 1G-bioethanol. The environmental impacts of bioethanol production are totally dependent on feedstock availability and conversion technology. The biochemical conversion route must overcome several technological and economical challenges such as pre-treatment, fermentation, hydrolysis process and separation. A completely mature technology is still to be developed and must adapted to the nature of the feedstock.

Nevertheless, using process simulation software, Life Cycle Assessment and integrating the different steps of bioresource harvesting and treatment processes, including the energy balances and the water requirements, it is shown that 2G bioethanol production will reduce environmental impacts provided the evaluation addresses a long-time perspective, including all conversion steps and the regeneration of the bioresource.

Introduction

Due to the increasing consumption pattern of fossil fuels, the demand for alternate energy is continuously growing. Almost 100 GWy are issued from biomass. This represents almost one third of wind-power energy and 10 % of hydropower [START_REF] Bertrand | Economic and Environmental Aspects of Biofuels[END_REF]). The transportation sector with about 15 % of the emission of greenhouse gases mainly depends on liquid biofuels, due to their high caloric volumetric value: 35.7 MJ/L for petrol, 23.4 MJ/L for ethanol. Consequently there is a lifelong interest for improving ethanol production pathways.

The world production market of bioethanol was 110 BL (billion litres) in 2018 and is expected to reach 140 BL in 2022. This increasing market reflects an intensified demand for the development of techno-economically feasible and sustainable processes based on the recycling of a fraction of carbon biomass. Considering the different raw materials and technology used for their production, both liquid biofuels (ethanol, butanol, biodiesel…etc.) and gas biofuels (methane, hydrogen and syngas) are classified into four generations [START_REF] Demirel | 22 Biofuels[END_REF] depending on the way they are produced and the nature of the feedstock.

When biofuels are produced using specific cultivation areas from seeds, grains and starchbased feedstock such as sugarcane, corn, potato, cassava, they are referred to as firstgeneration biofuels. Using food-grade crops for energy production requires cultivable land and generates conflicts with food/feed use of feedstock [START_REF] Min | Gas Fermentation for Commercial Biofuels Production[END_REF]. Therefore, even if they represent more than 96% of total worldwide production of biofuels in 2020, they cannot successfully become a long-term viable fuel source. The advancements in second-generation biofuels produced from lignocellulosic biomass, such as crops residues, woody crops or energy grasses are gaining momentum. Lignocellulosic biomass (rice straw, corn cob, wheat straw, sugarcane bagasse, cotton stalk) are the best alternatives as they are abundant, renewable and relatively cheap [START_REF] Cardona | Fuel ethanol production: Process design trends and integration opportunities[END_REF][START_REF] Hahn-Hägerdal | Bio-ethanol -the fuel of tomorrow from the residues of today[END_REF][START_REF] Zaldivar | Fuel ethanol production from lignocellulose: A challenge for metabolic engineering and process integration[END_REF]. Algal biomass (macroalgae and microalgae) is considered as a third-generation biofuel. Microalgae are known as emerging feedstock for the biodiesel production as they contain high lipid concentration up to 50% of dry biomass [START_REF] Chen | Determination of Microalgal Lipid Content and Fatty Acid for Biofuel Production[END_REF]. This permits economically feasible scale-up and development of large scale cultures plants [START_REF] Gouveia | Biodiesel from microalgae, in: Microalgae-Based Biofuels and Bioproducts: From Feedstock Cultivation to End-Products[END_REF]. It has several advantages: eco-friendly, non-toxic, less water consumption, high photosynthetic growth rate in autotrophy and therefore a high capacity for CO2 capture [START_REF] Simas-Rodrigues | Microalgae for economic applications: Advantages and perspectives for bioethanol[END_REF]. Some algal strains (Chlamydomonas reinhardtii, Porphyra, Chlorella vulgaris) are rich in carbohydrates (starch, glycogen, and cellulose) that are be directly used in the production of ethanol [START_REF] Khan | The promising future of microalgae: Current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products[END_REF]. However, the commercial production of third generation biofuels by microalgae cultivation has still several technical bottlenecks that need to be solved, including important investment costs, energy input for harvesting, risks of contamination in open pond systems, extensive downstream processing [START_REF] Griffiths | Advantages and Challenges of Microalgae as a Source of Oil for Biodiesel[END_REF]. Fourth-generation biofuels is basically an extension of third-generation biofuels in which algae are modified by the use of genetic engineering to improve the yield and lipid content of the cells. Genetically modified algae (GM algae) increase the ability of CO2 capture and rate of production of specific compounds, e.g. triacylglycerol. With genetic modifications, they are upgraded in terms of properties and cellular metabolism [START_REF] Abdullah | Fourth generation biofuel: A review on risks and mitigation strategies[END_REF]. Despite these advantages, high investment cost is necessary for using GM algae at industrial scale in order to limit and control the risk of contamination for the environment and the ecosystems. These high-tech requirements are the principal limitation for the developments of fourth-generation biofuels. It is still in its infant stage or in under research on a lab scale.

The market of bioethanol fuel has shown a dramatic increase and growing at a compound annual growth rate (CAGR) of 7.6% from 2016 to 2022. Several countries, like the U.S.A., Brazil, European Union, China, India has made an effort in order to diversify the use of fossil fuels by adding ethanol in different proportions to gasoline. The bioethanol fuel market is distributed on the basis of feedstock, blend (E5, E10, and E85) type of vehicle and geographical area. Global bioethanol production has been increased from the last few years and the U.S.A. is one of the global powerhouses in the ethanol industry followed by Brazil, the European Union, China, and Canada ("Annual Ethanol Production," 2019).Two countries answer for 84 % of the bioethanol produced worldwide: the USA with 56 % a production capacity of 62.5 BL plus 1.3 BL in construction, Brazil with 28 % (30 BL) followed by the European union with 5%, (5.5 BL), China 4% and Canada 2% (Figure 1). [START_REF] Cooper | Ethanol Industry Outlook[END_REF][START_REF] Mohanty | Bioethanol Production From Corn and Wheat: Food, Fuels and Future[END_REF] More than 94% of US bioethanol is obtained from corn starch, the remaining part coming cellulosic biomass. A major part of bioethanol produced in Brazil is issued from sugar-rich feedstock sugarcane and to a less extend from starchy crops (corn and wheat) [START_REF] Mohanty | Bioethanol Production From Corn and Wheat: Food, Fuels and Future[END_REF]. According to the EU regulatory framework 2009, a 10% contribution to the use of renewable energy in road transport fuels was targeted by 2020 (Al-Riffai et al., 2010; European Union, 2009). The total European production and consumption of bioethanol have reached 5.5 BL in 2018. Only a small fraction (less than 1%) was 2G bioethanol (IEA Bioenergy Task 39, 2020). This globally meets the global objective of 20 % for renewable energy but does not meet the 10 % renewable energy target for the transport sector (EU-28, 2018). Around 81% of the renewable ethanol produced in Europe is used for fuel. European Union is the fourth largest ethanol market in the world. The production capacity of bioethanol is expected to increase as increasing in the commercialization of cellulosic ethanol (second generation ethanol) (Phillips et al., 2018). The consumption of bioethanol is increasing with more blending as according to taxation policy, reduction in tax on E-10 fuel while increase for pure gasoline.

Figure1. World Ethanol production in 2018

The fast growth of the bioethanol production market in the transport sector has also generated some challenges in regards to sustainability and impact on biodiversity, direct and indirect land-use change and feedstock and water availability. There are also some challenges to the feedstock supply system. The main hurdle is the transportation of a massive volume of feedstock in an energy-saving manner to the biorefineries. The sustainability of a biorefinery will depend on the type of feedstock and its growing conditions and its transportation. The total ethanol production cost depends on various factors, such as feedstock production including the use of fertilizers, labour for farming, harvesting, collecting, transporting the biomass and the conversion process [START_REF] Hess | Cellulosic biomass feedstock and logistics for ethanol production[END_REF]. Among all of these, lignocellulosic biomass valorisation is a potential resource for production of second-generation ethanol, which can significantly reduce greenhouse gas emissions and offer positive environmental impacts. In general, second generation liquid biofuels (ethanol, biodiesel and liquid alkanes) are considered as important renewable fuels able to at least partly replace petroleum-based ones. The production of ethanol has gained industrial maturity and is considered as a future alternative to gasoline for transport, as it has the appropriate characteristics such as high octane number, the high heat of vaporization and low cetane number. It is also less toxic to the environment considering the reduction in GHG emissions and other pollutant emissions [START_REF] Nigam | Production of liquid biofuels from renewable resources[END_REF][START_REF] Ojeda | Exergy analysis and process integration of bioethanol production from acid pre-treated biomass: Comparison of SHF, SSF and SSCF pathways[END_REF].

There are different criteria to measure the sustainability of biofuels [START_REF] Baudry | The challenge of measuring biofuel sustainability: A stakeholder-driven approach applied to the French case[END_REF]. Life cycle assessment (LCA) is one of the assessment methods to analyse the efficiency of the production process in terms of environmental sustainability. It is mandatory to determine which conversion process and feedstock can qualify in terms of environmental sustainability. This review examines 2G-ethanol production from the biochemical conversion process and its environmental impacts by recent LCA studies with the help of process simulation.

Feedstock for second-generation ethanol production

Second-generation ethanol production is derived from non-food crops or crop residues with the main component that is lignocellulosic biomass. Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin including also water in small amount and some trace amounts of protein, minerals, and other components that are also present in the raw material.

The general structure of lignocellulosic material shows that the cellulose microfibrils (at least 1000 -5000 glucose units connected by β-1,4-glucosidic linkages) are bonded by van der Waals forces and hydrogen bonds. Hemicellulose is an amorphous heterogeneously branched polymer of pentoses and hexoses (at least 500-3000 sugar monomers) located at the primary cell wall. Lignin differs from cellulose and hemicellulose as it contains aromatic rings rather than long molecular chains with diverse chemical structures and monomer composition. The location of lignin is between the cellulose and the hemicellulose to bind them together (Bamdad et al., 2018).

Lignocellulosic biomass includes wood and woody biomass (poplar, sawdust, willow, wood residue, etc.), energy crops such as perennial grass (miscanthus grass, switchgrass, etc.), agricultural biomass (corn straw, rice straw, wheat straw, etc.) and municipal solid biomass.

Each group of biomass has persuasive characteristics that show potential, promising and abundant feedstock for the bio-refineries [START_REF] Zabed | Bioethanol production from renewable sources: Current perspectives and technological progress[END_REF]. Some chemical compositions of different biomass feedstock are reported in Table 1.

Cellulose is a major component (makes up about 45% dry weight of wood) and polymers of d-glucopyranose (C6 sugars) linked with the β 1-4 glycosidic bond between the glucose monomer forming cellobiose molecules [START_REF] Chen | Chemical Composition and Structure of Natural Lignocellulose[END_REF][START_REF] Kumar | Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production[END_REF]. These chain polymers linked together by hydrogen bond and van der Waals force, in which the glucose unit is compactly tie-up with each other and form elemental fibrils. These united or grouped microfibrils are called cellulose fibrils which are coated with hemicellulose [START_REF] Chundawat | Multi-scale visualization and characterization of lignocellulosic plant cell wall deconstruction during thermochemical pretreatment[END_REF][START_REF] Lee | Conversion of Lignocellulosic Biomass to Nanocellulose: Structure and Chemical Process[END_REF][START_REF] Pereira Ramos | The chemistry involved in the steam treatment of lignocellulosic materials[END_REF]. The crystallinity of cellulose depends on this organized structure of cellulose fibrils (Cellulose Chemistry and Properties: Fibers, Nanocelluloses and Advanced Materials, 2016; [START_REF] Saini | Lignocellulosic agriculture wastes as biomass feedstock for second-generation bioethanol production: concepts and recent developments[END_REF]. Due to the strong interaction of hydrogen bonding between the cellulose fibrils, it is insoluble in the water but soluble in dilute acid solution at high temperature (Chen and Chen, 2014).

Hemicellulose is a heteropolymer composed of different pentose, hexose sugars and uronic acids. The commons sugars found in the hemicellulose are xylose, arabinose, mannose, galactose with 50-200 units [START_REF] Saha | Hemicellulose bioconversion[END_REF]. The structure and composition of hemicellulose are different in different raw materials/biomass and their producing areas (Chen and Chen, 2014) 1,4 β-D-xylopyranose linked with each other to form a linear chain of xylan (Chen and Chen, 2014). It also contains the branch chain of glucuronic acid that are easily degraded in acid and alkaline solutions (Bajpai, 2016a;[START_REF] Saha | Hemicellulose bioconversion[END_REF]. Mannose sugar residues are linked together by a β 1-4 glycosidic bond to form a backbone chain of mannan. The linear chain of mannan is linked to galactose with α 1-6 glycosidic bond to form galactomannan and if it is linked with glucose by β 1-4 bond then the linear chain of glucomannan is formed (Bajpai, 2016b; Chen and Chen, 2014). The main content of agricultural biomass and hardwood hemicellulose is xylan whereas glucomannan is the main content in softwood (Bajpai, 2016b). Due to the presence of pentose sugars, hemicellulose has an affinity to water and at high concentration aqueous solutions become viscous (Chen and Chen, 2014).

Lignin is a heteropolymer of phenyl propionic alcohol units i.e. coumaryl alcohol, sinapyl alcohol and coniferyl alcohol, which are linked with each other by carbon-carbon (C-C) and ether (C-O) linkages. This type of linkage makes the stability of plant cell walls and resistance to pathogen infections [START_REF] Mooney | The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods[END_REF]. It acts as a physical barrier for enzymatic hydrolysis and microbial degradation as it is tightly bound to the cellulose fibrils [START_REF] Avgerinos | Selective solvent delignification for fermentation enhancement[END_REF]Bajpai, 2016b). Enzymatic or microbial delignification is difficult as the derivatives from lignin act as toxic compounds for microorganisms and reduces the activity of hydrolytic enzymes (Bajpai, 2016b).

The physical structure of feedstock and its carbohydrate composition directly impact the selection of an efficient and sustainable pre-treatment process. There are several pre-treatment methods used for the removal of lignin from the other components and make cellulose and hemicellulose accessible for enzymes of degradation. The final efficiencies of the different treatment processes, including pre-treatment pathways are very similar in terms of final ethanol yields for a given feedstock. It almost depends on the composition of raw materials.

For woody biomass, containing 25 -30 % lignin the yield is at average 300 -350 L / ton, provided both cellulose and hemicellulose fractions are converted into ethanol. On the contrary, the greenhouse gases (GHG) reduction potential versus gasoline depends on the feedstock, the cultivation methods and harvesting and treatment pathway. Table 1 summarizes the key values for different feedstock including GHG reduction potential obtained with current processes. Clearly, the robustness of the physical structure of the vegetal biomass has consequences on the efficiency of the treatment and finally on the GHG reduction potential.

The total production capacity of 2G bioethanol, including pilot plant facilities under development has reached 3 BL in 2018 with a rapid increase. These data are calling for a deeper investigation of what are the bottlenecks to overcome for reaching a good technical degree of maturity and the key descriptors that must be considered in overall conversion a given feedstock into bioethanol.

Table 1: Chemical composition of different feedstock and estimated GHG reduction potential versus gasoline for 2G-ethanol production (Bertrand et al, 2016)

Conversion technologies for 2G bioethanol production

There are mainly two conversion technologies for the production of bioethanol: biochemical and thermochemical. The biochemical conversion comprises pre-treatment, hydrolysis, and fermentation [START_REF] Soccol | Chapter 5 -Lignocellulosic Bioethanol: Current Status and Future Perspectives[END_REF] while in the thermochemical conversion gasification of the lignocellulosic biomass leads to syngas and then ethanol is produced from FT (Fischer-Tropsch) conversion [START_REF] Damartzis | Thermochemical conversion of biomass to second generation biofuels through integrated process design-A review[END_REF].

The biochemical conversion process is a common and effective technique for producing bioethanol (Achinas and Euverink, 2016; Robak and Balcerek, 2018). The general steps in bioethanol production via the biochemical route starts with pre-treatment in which lignocellulosic biomass is processed (separation of lignin, cellulose, and hemicellulose) and makes cellulose more accessible for the next step, i.e. hydrolysis. There are various pretreatment methods such as physical, chemical, physicochemical and biological. The purpose of pre-treatment is to decrease the crystallinity of cellulose and make it more susceptible to the hydrolysis in which sugar monomers are produced either by the use of acids or enzymes. The next step after hydrolysis is fermentation; it involves the conversion of sugars into ethanol during microbial fermentation mainly performed by bacteria and/or yeast [START_REF] Soccol | Chapter 5 -Lignocellulosic Bioethanol: Current Status and Future Perspectives[END_REF] .

a. Pre-treatment

Pre-treatment is the first step and very crucial, complicated and costly step in order to make bioethanol production from the lignocellulosic biomass. The goal of any pre-treatment technology is delignification to alter the structure of cellulose to make it more accessible to hydrolysis and increase the yield of sugars from cellulose and hemicellulose which can be fermented into ethanol [START_REF] Zabed | Bioethanol production from renewable sources: Current perspectives and technological progress[END_REF]. The main goal is to separate into independent fractions cellulose, hemicellulose and lignin fibres in order to make accessible for further conversion cellulose and hemicellulose, lignin remaining to be separated from the soluble media and valorised independently, at least for its calorific value.

There are different pre-treatment processes that have been developed to fractionate, solubilize and separate cellulose, hemicellulose and lignin components by using physical, chemical, physicochemical and biological treatment methods. As shown in Figure 2 each method has some pros and cons (Tomás-Pejó et al., 2011).

Figure 2: General scheme of pre-treatment processes

Physical methods such as grinding, milling, sonication are used to reduce the size of the raw material and crystallinity of biomass but they are considered as an energy-demanding process.

Chemical pre-treatment includes ionic liquids/green solvents, acid treatment (strong and dilute) and Organosolv treatment, the latter using an aqueous organic solvent (acetone, methanol, ethanol, etc.) at temperatures ranging from 140 to 220 °C. The main drawback of chemical pre-treatment is the inhibitors formation for downstream processes and pH adjustment before hydrolysis when acid treatment is used.

Biological pre-treatment uses microbial degradation of feedstock. It is considered environmentally safe because of various reasons, such as no chemicals required for this process, low energy demand; in turns, it doesn't produce toxic products. However, the low conversion rates and required long residence times make this process inefficient and laborious.

Physicochemical pre-treatment involves steam-explosion, Ammonia fibre explosion (AFEX), Co-solvent Enhanced Lignocellulosic Fractionation (CELF). Steam explosion treatment is a hydrothermal method involves both mechanical and chemical effects under high temperature and high pressure in the presence of steam where chipped biomass is treated to high pressure, high-temperature steam (160 -270 °C, 20 -50 bar) for few minutes. Then the pressure is released and due to explosive decompression, fibres get separated with minimal loss of other components. In this pre-treatment method, hemicellulose and lignin hydrolysis improves the accessibility of biomass to enzymes [START_REF] Alvira | Steam Explosion for Wheat Straw Pretreatment for Sugars Production[END_REF].

There are some parameters that affect the efficiency of this method such as residence time, particle size and temperature. It requires a strong catalyst like H2SO4 to increase the high sugar yield but it also leads to the formation of inhibitory compounds. Thus, it requires washing with water after pre-treatment for the removal of inhibitors [START_REF] Pielhop | Steam explosion pretreatment of softwood: the effect of the explosive decompression on enzymatic digestibility[END_REF][START_REF] Sassner | Steam pretreatment of H2SO4-impregnated Salix for the production of bioethanol[END_REF]. It has also many disadvantages like the production of inhibitory components such as furfural, HMF, which acts as inhibitors for the fermentation downstream process and the requirement of costly equipment increases the cost of the overall process.

b. Hydrolysis

The next step is hydrolysis, in which polymeric carbohydrates are converted into sugar monomers. Acid-catalyzed and enzyme-catalyzed are the two ways to perform saccharification [START_REF] Jahnavi | Status of availability of lignocellulosic feed stocks in India: Biotechnological strategies involved in the production of Bioethanol[END_REF]. Acid-catalyzed hydrolysis mostly occurs in the high temperature (100 -240 °C) and a high probability of inhibitor formation because of the high temperature and acidic condition. In fact, it is a combination of pre-treatment and hydrolysis processes. Enzymatic hydrolysis has several advantages over acid-catalyzed hydrolysis as it can operate at mild conditions (the temperature at 50 -60 °C with pH 4.8 -5.0) and high sugar recovery with no inhibitor formation [START_REF] Balat | Production of bioethanol from lignocellulosic materials via the biochemical pathway: A review[END_REF]. There are various factors such as temperature, pH, enzyme loading, time that affects the productivity of enzymatic saccharification. Cellulases are the group of enzymes that are used to degrade the cellulose by cleaving the β 1-4 glycosidic bond (Kuhad et al., 2011a). The demand for cellulases is continuously increasing as it has broad applications in several industries like pulp and paper industry, textile industry, food processing industry, etc. The cost of enzyme is the biggest hurdle in bioethanol refineries because of its huge consumption in the saccharification [START_REF] Wang | Bioprocessing technologies in biorefinery for sustainable production of fuels, chemicals, and polymers[END_REF]. Nowadays, microbial enzymes are widely used in various industries to make the process economically feasible. There are a number of microorganisms that are naturally secreting cellulolytic enzymes. Clostridium, Thermomonospora, Trichoderma, Aspergillus are the common bacterial and fungal strains for the production of cellulases (Kuhad et al., 2011a). Among all, Trichoderma species are the most desirable and potential candidate for cellulase production [START_REF] Imran | Cellulase Production from Species of Fungi and Bacteria from Agricultural Wastes and Its Utilization in Industry: A Review[END_REF]. There are three main types of cellulase that are involved in the enzymatic saccharification, i.e. exoglucanases (EC 3. This yield is a maximum conversion yield, knowing that associated biomass and by-products production decrease the actual yields at least by a factor of 10 to 20% [START_REF] Barnett | Beginnings of microbiology and biochemistry: The contribution of yeast research[END_REF][START_REF] Fugelsang | Fermentation and Post-fermentation Processing[END_REF]. In terms of kinetics of production, there are several factors that inhibit the efficiency of Saccharomyces cerevisiae yeast to produce ethanol like high ethanol concentration, high temperature, osmotic stress, bacterial contamination and of course presence of inhibitors.

There are some fermentation technologies, i.e. separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and cofermentation (SSCF) and consolidated bioprocessing (CBP) used in biorefineries [START_REF] Jahnavi | Status of availability of lignocellulosic feed stocks in India: Biotechnological strategies involved in the production of Bioethanol[END_REF].

In Separate Hydrolysis and Fermentation (SHF), hydrolysis and fermentation are carried out separately in different units. After pre-treatment, biomass slurry is subjected to the hydrolysis reactor where it is degraded into simple sugars and thereafter a fermentation process occurs in the fermentation unit to convert sugars into ethanol [START_REF] Taherzadeh | Enzyme-based hydrolysis processes for ethanol from lignocellulosic materials: A review[END_REF]. Endproduct inhibition is the main drawback of this process. By example, cellobiose produced in hydrolysis acts as an inhibitor for cellulase itself [START_REF] Axelsson | Separate Hydrolysis and Fermentation of Pretreated Spruce[END_REF]. It is also a timeconsuming process considering the residence time is long in hydrolysis and expensive due to high enzyme loading and material cost for two separate units.

In Simultaneous Saccharification and Fermentation (SSF), hydrolysis and fermentation are carried out in the same unit [START_REF] Kádár | Simultaneous saccharification and fermentation (SSF) of industrial wastes for the production of ethanol[END_REF]. It is considered as a better option for industries as compared to SHF as it has many advantages with high ethanol yield [START_REF] Wingren | Techno-economic evaluation of producing ethanol from softwood: comparison of SSF and SHF and identification of bottlenecks[END_REF]. The main advantage of SSF is low investment cost and no end product inhibition by glucose as it is consumed immediately by ethanol-producing microorganism resulting in an increase of the rate of hydrolysis and ethanol yield. Ethanol inhibition for enzymes, microorganisms and temperature in the reactor are the only drawback of this process. The optimal temperature for yeast is 35 °C and hydrolysis occurs at 50 °C [START_REF] Axelsson | Separate Hydrolysis and Fermentation of Pretreated Spruce[END_REF].

In Simultaneous Saccharification and Co-Fermentation (SSCF), hydrolysis and fermentation are carried out in the same unit with co-fermentation of pentose sugars (Bondesson and Galbe, 2016). Knowing that Saccharomyces cerevisiae cannot ferment the pentose sugars, genetically modified Saccharomyces cerevisiae strains that are xylose-fermenting are used for the co-fermentation process. An ethanol yield of 0.32g / g of sugars was observed for a genetically modified Saccharomyces cerevisiae strain (Bondesson and Galbe, 2016). It is a suitable process for xylose-rich lignocellulosic although the ethanol yield is 35% lower than the maximum yield. It is a suitable process for feedstock like hardwood and agricultural residues [START_REF] Olofsson | Improving simultaneous saccharification and co-fermentation of pretreated wheat straw using both enzyme and substrate feeding[END_REF]. Consolidated BioProcessing (CBP) is a single-step process in which all the processes including enzyme production, hydrolysis and fermentation occur in the same unit.

Clostridium thermocellum is the mainly used microorganism in this process with a natural synthesis of cellulases to degrade the biomass into fermentable sugars and produce ethanol [START_REF] Hasunuma | Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains[END_REF]. Though it is at an infant stage, this process has several advantages like low investment and enzyme cost, less chance of contamination.

d. Distillation

The main challenge of bioethanol biorefinery is the separation of ethanol from the fermentation broth to produce pure bioethanol. Due to the presence of an azeotrope, the separation of ethanol from the ethanol-water mixture requires special attention. Three conventional methods exist: azeotropic distillation, Liquid-Liquid extraction, and extractive distillation [START_REF] Nitsche | Extractive and Azeotropic Distillation[END_REF]. There are other techniques that are raising in the future like pervaporation and salt distillation [START_REF] Nagy | Analysis of energy saving by combination of distillation and pervaporation for biofuel production[END_REF][START_REF] Soares | Dehydration of ethanol with different salts in a packed distillation column[END_REF] considering they are less energy consuming. The most commonly used technique is extractive distillation, which is used for large scale operations [START_REF] Ravagnani | Anhydrous ethanol production by extractive distillation: A solvent case study[END_REF]. A separating solvent is used. It has specific properties: non-volatile (high boiling point) and miscible to the mixture. The separating agent is added to the azeotropic mixture to alter the relative volatility of the key component without additional azeotrope formation. When these solvents are added to the mixture, it causes the change in the volatilities of the components [START_REF] Pfenning | Kirk-Othmer Encyclopedia of Chemical Technology[END_REF]). In the extractive distillation technique, a dissolved salt can be used as a separating agent for the ethanol-water mixture, e.g. Calcium Chloride salt. Extractive distillation works on two variables, the solubility of the solvent and the difference in the boiling point of the components whereas in Liquid-Liquid distillation the process is based on the solubilities of ethanol in an organic phase, permitting to significantly increase the ethanol concentration resulting to decrease the energy consumption.

Process Simulation

Process simulation is basically a representation of chemical, physical, biological, other technical processes and unit operations in a simulation software (Luyben, 1989). The simulation models also allow the interpolation and extrapolation within certain limits. The process simulation software delineates the whole process in a flowsheet where all unit operations are connected with the streams in order to find the optimal parameters or conditions. The basic knowledge of chemical kinetics, chemical reactions, and the thermodynamic properties are required for calculation of process parameters of any unit operation. The main purpose of the process simulation is to generate mass and energy balances. It also gives details about the performances of the various elements of the process, i.e. reactors, distillation columns and analyses them in terms of balances in the context of interconnected unit operations leading to economical evaluation and sustainability.

Numerous chemical simulators are available, which are used industrially or on the academic scale. Aspen plus, Chemcad, Prosimplus, Hysys are some examples of chemical engineering simulators that are widely used in the industries. Aspen plus is widely used process simulation software in the field of biorefinery. In the case of bioethanol production from biomass feedstock, there are several unit operations and complex process streams. Pre-treatment, hydrolysis, fermentation and distillation are interconnected with each other. The pre-treatment is the most crucial and complex step to incorporate in simulation software when addressing ethanol production from lignocellulosic biomass. The main reason is that pre-treatment is related to a Solid Liquid Gas unit operation and requires a consistent knowledge of physicochemical properties of the compounds, including specific heats, heat of dissolution and thermophysical properties of complex materials at relatively high temperature (100 -250°C). Moreover, the different pre-treatment techniques that are mainly classified into four categories physical, chemical, physiochemical and biological involve acid base pre-treatment requiring being able to simulate complex concentrated acid base solutions and highly concentrated aqueous salt solutions.

In order to give a baseline value, the global ethanol yield obtained with Aspen plus for a liquid hot water pre-treatment process is in the order of 350 L bioethanol / ton dry matter and with an energy consumption at least reaching 87 MJ / L bioethanol (Quintero and Cardona, 2011) that is consistent with values obtained at pilot and industrial plant level. These values give departure orders of magnitude for final results that can be obtained with different feedstock.

Life cycle assessment a. General framework

The concept of life cycle assessment (LCA) has been introduced in the 1970s and several The goal and scope stage examines the inputs and outputs of materials, including technical details like the functional unit, system boundaries, and allocation methods. The functional unit is based on volume (L), mass (kg) of the product and the land area (ha), energy (MJ) and distance (km) are also used. It gives the reference unit to which the inputs and outputs can be related. The Life Cycle Inventory Analysis stage (LCIA) involves the compilation and quantification of inputs and outputs for a given product system. It gives a description of flow model of input (water, raw material) and output (products and emissions such as CO2, SO2, NOX, CO, etc.) for a given product system. The impact Assessment stage converts the inventory results into related environmental impacts including global warming potential, acidification, eutrophication, human eco-toxicity, etc. In the characterization steps, the impact potential of each emission is estimated by using scientific factors using LCIA methodologies

b. LCA studies on bioethanol produced from biochemical conversion pathway

The life cycle of lignocellulosic ethanol has been evaluated by the methodology of LCA to measure the economic and environmental impact of the production system (Kim and Dale, 2005a).

. In several studies, it has been observed that lignocellulosic bioethanol leads to a remarkable reduction in GHG emission ( [START_REF] Wyman | Ethanol from lignocellulosic biomass: Technology, economics, and opportunities[END_REF]. However, crop residues are the leftover in the field after harvest. It contributes to improve the structure of soil and also provide protection from the direct impacts of rain, sunlight, and wind. Excessive use of crop residues leads to soil erosion and decreases the quality and fertility of the soil. Although N-based fertilizer during cultivation can be used, it leads to eutrophication and acidification because nitrogen and phosphorus are released from the soil. The overall result must be evaluated considering that synthetic fertilizers may also contribute to global warming (Kim and Dale, 2005a).

(i)

LCA studies on ethanol produced from agro-residues Wheat straw is widely used as a feedstock in the production of lignocellulosic ethanol. In corn stover ethanol is used as compared to gasoline, which is independent of any allocation method. However, GWP (Global Warming Potential) increases in economic allocation while it decreases when mass/energy allocation method is applied [START_REF] Luo | An energy analysis of ethanol from cellulosic feedstock-Corn stover[END_REF]. Some researchers reported that the fuel from straw doesn't show a positive effect on the environment because of water eutrophication and land use [START_REF] Uihlein | Environmental impacts of a lignocellulose feedstock biorefinery system: An assessment[END_REF].

Excessive removal of straw (wheat straw) from the field shows the effect on environmental emissions [START_REF] Gabrielle | Life-cycle assessment of straw use in bio-ethanol production: A case study based on biophysical modelling[END_REF]. It is also observed that some definite portion of crop residues can be removed without affecting the quality of soil, conditional to the soil types, season and location [START_REF] Nelson | Resource assessment and removal analysis for corn stover and wheat straw in the Eastern and Midwestern United states -Rainfall and wind-induced soil erosion methodology[END_REF][START_REF] Reijnders | Ethanol production from crop residues and soil organic carbon[END_REF]. Some researchers suggest that 60% of corn stover should be harvested and collected for the biorefineries (Sheehan et al., 2003b), while some stated that only 25% would be harvested, higher proportions affecting the soil fertility (Blanco-Canqui and Lal, 2009). In a recent agricultural implementation, the study shows that only 28% of corn stover might be considered to be harvested and collected without degrading the soil quality [START_REF] Graham | Current and Potential U[END_REF]. The carbon proportion of ethanol from corn stover would have to be in the range 10 -44% of gasoline (Kaufman et al., 2010), knowing that when higher proportions are used the GHGs emission during the storage of resource plays an important role,, except if performing equipment available on the commercial scale is used (Sheehan et al., 2003b).

Nevertheless, the lignocellulosic biomass is considered as most favourable feedstock regarding its present high availability and low cost. In any case the production cost of feedstock will have to be incorporated in the global analysis when intensifying and concentrating the production in specific areas, the economic viability being to consider with this perspective. Technological issues need to be solved for the large-scale commercial an environmental advantage over liquid hot water considering higher enzyme load is required for liquid hot water pre-treatment. The enzyme used in the hydrolysis process also contributes to 20-40% of environmental burdens in toxicity and eutrophication. E85 shows a similar environmental profile to E100 [START_REF] Guo | The environmental profile of bioethanol produced from current and potential future poplar feedstock in the EU[END_REF]. In another study, the net process energy efficiency is estimated for the production of bioethanol from poplar (35% to 37%) with the ethanol yield approximately ranging between 303 to 316 L / t dry biomass. Modeling was done by the process simulation tool Aspen Plus to understand the production process from poplar and help in the design and optimization of the production process [START_REF] Porzio | Modelling lignocellulosic bioethanol from poplar: Estimation of the level of process integration, yield and potential for co-products[END_REF].

Black locust is also used as a potential woody biomass for the production of bioethanol and also has a lower impact on environment because of low levels of agricultural inputs used during cultivation whereas poplar has higher impact in acidification and eutrophication due to emission of N2O in environment from the N-based fertilizers required in the poplar farming.

Eucalyptus also shows higher impact on environment due to use of heavy machinery requirements during harvesting (González-García et al., 2012). In their study González-García et al., (2012) also compare bioethanol production from poplar, black locust and eucalyptus with E-85 blend with gasoline. Black locust is the best candidate in terms of minimal contributions from the field activities with an estimated reduction by 97% in GWP100 (Global Warming Potential over 100 years). In a similar way, sugar beet, genetically modified for herbicide-free crop shows less toxic impact to the environment and human health in comparison to conventional growing crops in terms of lower emissions from herbicide, transportation and field operations [START_REF] Bennett | Environmental and human health impacts of growing genetically modified herbicide-tolerant sugar beet: A life-cycle assessment[END_REF].

Conclusion

This overview shows that environmental impacts of 2G bioethanol production depend on the availability and the regeneration of feedstock and reactants and on the distribution of the products. Bioethanol produced from lignocellulosic biomass via biochemical route must overcome several difficulties like fermentation of both pentose and hexose carbohydrates, configuration of hydrolysis process in such a way that inhibitors are eliminated, and recycling of byproducts and waste treatment. The evaluation of economical viability and environmental sustainability must include: i) simulation for designing optimal process design; ii) analysis of soil quality and agriculture techniques to prevent productivity loss and soil exhaustion.

  2.1.74 and 3.2.1.91), endoglucanases (EC 3.2.1.4), and β-glucosidases (EC 3.2.1.21). Exoglucanase (E.C. 3.2.1.91) acts on the reducing and non-reducing ends of cellulose and releases cellobiose units; endoglucanase (E.C. 3.2.1.4) cleaves randomly β 1-4 glycosidic bonds resulting in the production of new ends, i.e. cellobiose and cellotriose units; βglucosidase (E.C. 3.2.1.21) attacks the cellobiose units to form simple sugar (glucose) units (Kuhad et al., 2011b; Wang, 2013). 1,4-β-D endoxylanases (E.C. 3.2.1.8) and β-xylosidases (E.C. 3.2.1.37) are the most common enzyme used for the degradation of the xylan present in the hemicellulose (Walia et al., 2017). Enzyme stability, substrate/product inhibition and catalytic efficiency are the main challenges in the production of microbial enzymes. Several recent advances in genetic modifications, recombinant DNA techniques and adopting different strategies for strain improvement lead to increase the production and make the enzyme more robust and economically feasible (Adrio and Demain, 2014). c. Fermentation Fermentation is the biological process in which microorganisms, mainly yeast and bacteria, convert monomeric sugars into acids, gases, and ethanol. Saccharomyces cerevisiae (baker's yeast) is the commonly used organism in the alcohol production process because of its high productivity and ethanol yield from different feedstock (Mohd Azhar et al., 2017). Other than Saccharomyces cerevisiae, Pichia stipites, Kluyveromyces fagilis, and Candida shehatae were also reported yeast strains for the production of ethanol from different sugars (Gonçalves et al., 2016; Mohd Azhar et al., 2017; Tian et al., 2009). The master stoichiometric equations characterizing the fermentation of hexose (C6H12O6 equivalent) and pentose (C5H10O5 equivalent) to ethanol lead to the same mass yields (51.1 %) the complementary part being CO2.

  efforts have been done to develop the methodology of LCA. Many international and national organizations promoted, supported and developed the concept of LCA (SETAC-Society of Environmental Toxicology and Chemistry, USEPA-the United States Environmental Protection Agency, ISO-International Organisation of Standardization, ILCAJ-Institute of Life Cycle Assessment, Japan). As a result, a Concord has been established on the LCA framework and inventory methodology (The International Standards Organisation, 2006). The Global Energy Partnership Task Force (GBEP) has also been developed the LCA methodology for the bioenergy system and compare it from the lifecycle of the fossil-fuel energy system (Hayashi et al., 2014). The LCA methodology comprises mainly 4 stages: Definition of goal and scope, Life Cycle Inventory Analysis (LCIA), Impact assessment, and Interpretation of the results.

(

  The International Standards Organisation, 2006). There are various LCIA methodologies depending on the different impact categories and selection of indicators such as CML 2002, Eco-indicator 99, ReCiPe, LIME, Lucas, TRACI, etc. (Owsianiak et al., 2014; Rosenbaum et al., 2017). The interpretation of the results provides a conclusion for decision-making. It is a technique to identify and quantify the results of the inventory analysis and life cycle impact assessment.

Figure1

  Figure1. World Ethanol production in 2018[START_REF] Cooper | Ethanol Industry Outlook[END_REF][START_REF] Mohanty | Bioethanol Production From Corn and Wheat: Food, Fuels and Future[END_REF]

Table 1 )

 1 (IEA Bioenergy Task39, 2019; Fleming et al., 2006; González-García et al., 2012; Mabee et al., 2006; Spatari et al., 2005; van Vliet et al., 2009;

  [START_REF] Borrion | Environmental life cycle assessment of bioethanol production from wheat straw[END_REF], study, there is an evaluation and comparison of two ethanol blend fuel i.e. E15 and E85 from wheat straw. The highest the blending the better are the results, i.e. E85 reduces by 73% GHG while E15 reduces only by 13% for GHG emission compared to conventional gasoline. However, in acidification, eutrophication and water depletion environmental issues, the ethanol blend fuel doesn't show any benefit compared with conventional fuel. The assessment of global environmental performances included global warming, ozone depletion, acidification, eco-toxicity, etc. It was characterized by the ReCiPe methodology and SIMAPRO was used to build the inventory and impact assessment analysis[START_REF] Borrion | Environmental life cycle assessment of bioethanol production from wheat straw[END_REF]. In the case of rice straw, high energy is consumed, i.e. 72% of the total for the preparation of rice straw from paddy production. CML 2001 method is used to assess the acidification, climate change, eutrophication, and, in turns, the impact on environment.

	Borrion et al. (2012) concluded that bailing, straw transportation, methane, and N2O in flue
	gas and high-pressure construction are the main sources of GHG emission that has been
	confirmed by Shafie et al. (2014). Perennial grasses can increase carbon sequestration in soils.
	Process residues and co-products should be considered for energy production in order to save
	fossil energy (Cherubini et al., 2009).
	E-85 blend lignocellulosic ethanol from corn stover is capable of reducing GHG emission by
	86 -113% as compared to conventional fuels (Davis et al., 2009; Sheehan et al., 2003a). Corn
	stover ethanol lowers the GHG emission as compared to corn-grain ethanol (Kim and Dale,
	2005b; Williams et al., 2009). Ozone layer depletion and abiotic resources are decreased if

bioethanol produced from forest residues and woody biomass

  Presently, the contribution of bioethanol produced from forest residues and woody biomass is not really significant compared to energy crops in terms volume of produced bioethanol but it may become active and will be developed in the future[START_REF] Sims | Energy crops: current status and future prospects[END_REF] considering the resource is mostly composed of forest industry waste and residues. This entails reduced production costs and availability but on the contrary high transportation costs for concentrating the production to large-scale industrial plants. The majority of LCA studies on energy crops report that reduction in GHG emission and fossil energy consumption must be evaluated globally (Cherubini and Ulgiati, 2010; Schmer et al., 2008; Sims et al., 2006; von Blottnitz and Curran, 2007). It is mandatory to consider all environmental factors both related to conversion process of feedstock and to the production of feedstock itself, such as acidification and eutrophication increase (Cherubini and Ulgiati, 2010). Guo et al. (2014) have compared two pre-treatment methods on poplar and with different blends, i.e. E-10, E-85, and E-100. Pre-treatments of poplar are done by dilute acid or by liquid hot water and followed by SHCF by Zymomanas mobilis to produce bioethanol. LCA modeling is done by SIMAPRO7.3.3 and simulation by ASPEN PLUS. Dilute acid showed

	(ii) LCA studies on By example, the lignocellulosic ethanol pathway from switch-grass avoids 65% of GHG
	emissions if E85 is used. E85 reduces GHG emission but is less effective for other impacts
	such as photochemical oxidation and eutrophication (Bai et al., 2010). Bioethanol produced
	from switch-grass leads to a reduction in GHG emission of 94% compared to gasoline but it
	will be necessary to further intensify the biofuel yield and energy sustainability of switch-
	grass by genetic and agronomical improvement (Schmer et al., 2008). It is reported that
	switch-grass is effective at storing soil organic carbon (SOC) not only at soil surface but at
	depth beneath 30 cm where carbon is less vulnerable to mineralization and loss (Liebig et al.,
	production of lignocellulosic bioethanol (Hatti-Kaul et al., 2007; Tan et al., 2008). For 2005; Schmer et al., 2008). Studies show that the soil organic carbon (SOC) and water-
	instance, the use of various chemicals such as sulphuric acid, lime in the biochemical extractable organic carbon (WEOC) are higher in the perennial grass systems than the annual
	conversion cannot be neglected as it contributes to GHG emission and water consumption corn system and switch-grass had lower SOC and WEOC in among all perennial grass
	(Mu et al., 2010). systems (Hahn-Hägerdal et al., 2006).

Table 1 :

 1 . World Ethanol production in 2018[START_REF] Cooper | Ethanol Industry Outlook[END_REF][START_REF] Mohanty | Bioethanol Production From Corn and Wheat: Food, Fuels and Future[END_REF]) Chemical composition of different feedstock (weight % fresh matter) and estimated GHG reduction potential versus gasoline for 2G-ethanol production[START_REF] Bertrand | Economic and Environmental Aspects of Biofuels[END_REF]

	China; 4%	Rest of World; 2%
	European Union; 5%		Brazil
	Canada; 2%		India
	Argentina; 1%		Thailand
		Brazil; 28%
			US
			Argentina
		India; 1%	Canada
	US; 56%	Thailand; 1%	European Union
			China
			Rest of World

Figure 2: General scheme of pre-treatment processes

Acknowledgement

The authors acknowledge the support received from the French Government Research Program "Investissements d'avenir" through the IMobS3 Laboratory of Excellence (ANR-10-LABX-16-01).