Josh Milgram

Anthony Herrel

Ron Shahar

Boaz Shacham

Céline Houssin

Arnaud Delapré

Raphaël Cornette

Marion Segall

Maïtena Dumont
email: maitena.dumont@gmail.com

Keywords: enamel, diet, snakes, microscopy, CT scanning, teeth

Teeth are composed of the hardest tissues in the vertebrate body and have been studied extensively to infer diet in vertebrates. The morphology and structure of enamel is thought to reflect feeding ecology. Snakes have a diversified diet, some species feed on armored lizards, others on soft invertebrates. Yet, little is known about how tooth enamel, and specifically its thickness, is impacted by diet. In this study, we first describe the different patterns of enamel distribution and thickness in snakes. Then, we investigate the link between prey hardness and enamel thickness and morphology by comparing the dentary teeth of 63 species of snakes. We observed that the enamel is deposited asymmetrically at the antero-labial side of the tooth. Both enamel coverage and thickness vary a lot in snakes, from species with thin enamel, only at the tip of the tooth to a full facet covered with enamel. There variations are related with prey hardness: snakes feeding on hard prey have a thicker enamel and a lager enamel coverage while species. Snakes feeding on softer prey have a thin enamel layer confined to the tip of the tooth

Introduction

Teeth are important tools used by many vertebrates to acquire and process food. Vertebrate teeth have evolved for millions of years and show a myriad of shapes fulfilling various functions (e.g., Teaford et al., 2007;[START_REF] Berkovitz | The Teeth of Non-Mammalian Vertebrates, 1st Editio[END_REF]. The study of teeth has provided insights into dietary adaptations in mammals given the extensive food processing done by the teeth in this group (e.g., [START_REF] Maas | Built to last: the structure, function, and evolution of primate dental enamel[END_REF][START_REF] Lucas | Dental enamel as a dietary indicator in mammals[END_REF]. Tooth shapes and sizes are highly variable, but in general their structure is highly conserved through evolution. They are made of an external layer of enamel (or enameloid), which protects the teeth as a cap and covers the dentine which, in turn, surrounds a pulp cavity (e.g., [START_REF] Schmidt | Dental tissues in man and other vertebrates[END_REF][START_REF] Berkovitz | The Teeth of Non-Mammalian Vertebrates, 1st Editio[END_REF]. The evolution of teeth and enamel (e.g., Teaford et al., 2007;[START_REF] Ungar | Mammal teeth: origins, evolution, and diversity[END_REF], their material properties [START_REF] Cuy | Nanoindentation mapping of the mechanical properties of human molar tooth enamel[END_REF][START_REF] Rensberger | Mechanical Adaptation in Enamel Balkema[END_REF]Bechtle et al., 20120, Zhao et al., 2013) and their function have been widely studied in a diversity of vertebrate lineages (e.g., Teaford et al., 2000;[START_REF] Berkovitz | The Teeth of Non-Mammalian Vertebrates, 1st Editio[END_REF]Richman & Handrigan, 2011).

The enamel comprises the outer layer of the tooth; it is directly in contact with the food or the environment and is therefore exposed to possible damage. It is the hardest and the most durable tissue found in the vertebrate body (e.g. [START_REF] Teaford | Dental microwear and microstructure in early oligocene primates from the Fayum, Egypt: implications for diet[END_REF][START_REF] Boyde | Microstructure of enamel[END_REF][START_REF] Rensberger | Mechanical Adaptation in Enamel Balkema[END_REF]. Its thickness and size are related to diet in some vertebrates. Durophagous primates, for example, develop a relatively thick enamel layer (e.g., [START_REF] Olejniczak | Three-dimensional molar enamel distribution and thickness in Australopithecus and Paranthropus[END_REF][START_REF] Scott | Dental microwear texture analysis shows within-species diet variability in fossil hominins[END_REF][START_REF] Dumont | Enamel Thickness and Dietary Adaptation among Extant Primates and Chiropterans[END_REF]. Even if enamel is highly mineralized and more brittle, thicker enamel is thought to make tooth crowns stronger and able to resist large compressive forces [START_REF] Dumont | Enamel Thickness and Dietary Adaptation among Extant Primates and Chiropterans[END_REF][START_REF] Maas | Built to last: the structure, function, and evolution of primate dental enamel[END_REF][START_REF] Lucas | Dental enamel as a dietary indicator in mammals[END_REF]. A thicker enamel coating will also be able to resist wear longer than a thinner enamel coating because more tissue will need to be removed to expose the dentine. It was also already shown mostly in mammals (e.g. [START_REF] Lucas | Dental enamel as a dietary indicator in mammals[END_REF][START_REF] Kay | The nut-crackers-a new theory of the adaptations of the ramapithecinae[END_REF]) but also in fish (e.g. [START_REF] Delaunois | Microstructural and compositional variation in pacu and piranha teeth related to diet specialization (Teleostei: Serrasalmidae)[END_REF][START_REF] Velasco-Hogan | Tooth structure, mechanical properties, and diet specialization of Piranha and Pacu (Serrasalmidae): A comparative study[END_REF]) that a thicker enamel/enameloid layer is related to harder diet Despite the importance of understanding tooth enamel, this tissue remains relatively poorly investigated in reptiles, with mostly quantitative comparisons having been performed for crocodiles and extinct species (e.g., [START_REF] Olejniczak | Assessment of the accuracy of dental enamel thickness measurements using microfocal X-ray computed tomography[END_REF]Enax et al., 2013;[START_REF] Sellers | The significance of enamel thickness in the teeth of Alligator mississippiensis and its diversity among crocodyliforms[END_REF]. Compared to mammals, reptiles replace their teeth continuously [START_REF] Edmund | Dentition[END_REF][START_REF] Zaher | Tooth Implantation and Replacement in Squamates, with Special Reference to Mosasaur Lizards and Snakes[END_REF]Richman & Handrigan. 2011), a condition called polyphyodonty. Polyphyodonty raises the question of the pertinence of lifelong durability of teeth, teeth will need to be "performant" for few weeks or months instead of decades. Continuous replacement could ensure the replacement of a broken tooth, hypothetically without the need fora tooth that resists failure (e.g. Crofts & Summers, 2013). Whether polyphyodont reptile teeth vary in enamel thickness and distribution and why remains to be investigated.

The paucity of comparative studies could be explained by the general thin nature of reptile enamel (e.g. [START_REF] Sander | The microstructure of reptilian tooth enamel: Terminology, function, and phylogeny[END_REF][START_REF] Jones | Neutron scanning reveals unexpected complexity in the enamel thickness of an herbivorous Jurassic reptile[END_REF][START_REF] Sellers | The significance of enamel thickness in the teeth of Alligator mississippiensis and its diversity among crocodyliforms[END_REF]. In extant squamates, most studies have focused on lizards and have described them as having a simple and thin enamel layer (e.g. [START_REF] Owen | Ondontography[END_REF][START_REF] Schmidt | Dental tissues in man and other vertebrates[END_REF]. There are about 4000 species of snakes with diversified diets ranging from goo-eaters to durophagous species. This dietary diversity is associated with morphological specializations of the teeth (e.g. [START_REF] Berkovitz | The Teeth of Non-Mammalian Vertebrates, 1st Editio[END_REF][START_REF] Segall | Armed to teeth: the underestimated richness of teeth shape in snakes and its relationship to feeding constraints[END_REF] but no study to date has demonstrated a link between diet and enamel distribution in snakes. [START_REF] Owen | Ondontography[END_REF], who first described snake teeth even failed to notice the presence of enamel. Some years later, [START_REF] Tomes | IX. On the structure and development of the teeth of ophidian[END_REF] studied ten genera of snakes and described a thin layer covering their teeth. [START_REF] Levy | Beiträge sur Kenntnis des Baues und der Entwicklung der Zähne bei den Reptilien[END_REF] observed in Tropidonotus (Natrix) and Pelias (Vipera) a thin layer of enamel covering the whole tooth. The most detailed description of the enamel in snake teeth can be found in [START_REF] Schmidt | Dental tissues in man and other vertebrates[END_REF]. This author observed that the enamel of the large teeth of Python is so thin that it can barely be seen under the microscope. To our knowledge there are no other studies looking at the enamel or tooth structure of snakes, especially in a phylogenetic and ecological comparative context.

The aim of this study was to investigate whether enamel thickness and coverage was related to to prey hardness in snakes. We compared the enamel coverage of 61 species using microCT and microscopy images. We then measured enamel thickness on longitudinal sections of the teeth in 63 species of snakes. We chose species to cover the phylogeny of alethinophidian snakes. We chose species with similar diets in different genera, and closely related species with different diets to highlight potential convergence.

Material and methods

Enamel distribution via CT-scan

We extracted the dentary bone of 63 species of snakes that cover both the phylogenetic and dietary diversity of the group (Table S1). Our sample is composed of specimens from museums (American Museum of Natural History, Muséum National d'Histoire Naturelle, Jerusalem University) and private collections. The dentary teeth were CT scanned using a μCT-scanner, Phoenix Nanotom S (General Electric, Fairfield, CT, USA) at the Institut de Genomique Fonctionelle at the Ecole Normale Superieure (Lyon, France) prior to any invasive techniques. The scans were run with a voltage of 100kV and a current of 70μA, for a voxel size between 0.97-7.50μm depending on the size of the sample. The 3D reconstruction was performed in the Phoenix datos|x2 software (v2.3.0, General Electric, Fairfield, CT, USA).

Segmentation of the enamel in the teeth was done using Dragonfly (Dragonfly version 3.8.6, Objects Research system, Canada). Enamel can be recognized by its higher absorption level compared to dentine (Figure 1). The microCT scans did not allow us to quantify the amount of enamel due to a number of factors, including the sample itself and scanning artefacts (e.g. beam hardening). For some species the contrast between enamel and dentine was not high enough, or the enamel was too thin and some samples had preservation artefacts which did not allow precise quantitative measurements (Figure S1,S2). For two samples it was not possible to segment the enamel because the teeth had preservation issues: Stenorrhina degenhardtii presented a lead (Pb) layer at the surface of its teeth which resulted in a density artefact (Figure S1), and Daboia russelii presented a "shrinkage" of the teeth which lead to severely fragmented enamel (Figure S2).

However, for these two samples, microscopic sections allowed us to measure enamel thickness.

The enamel coverage was qualitatively determined using the virtual longitudinal and transverse sections obtained and checked simultaneously via microscopic observations of longitudinal sections, using the methodology explained in Figure 1 and Figure S3. We categorized enamel coverage into three groups (Figure 1, S4): a) enamel only present at the tip of the tooth (Figure 1D-F), b) enamel is present on half of the tooth (Figure 1G-I), c) the enamel is present on the entire anterior face (Figure 1J-L).

Enamel thickness measurements via microscopy

Snake teeth were extracted from the dentary and embedded in epoxy resin (Epothin, Buehler, Lake Bluff, IL, USA). Longitudinal sections were prepared by sequentially grinding the samples with 800, 2500 and 4000 grit SiC films (Buehler, Lake Bluff, IL, USA), followed by polishing with a nap polishing cloth soaked in diamond suspension (3 and 1 µm; Struers Inc., Cleveland, OH, USA). Grinding and polishing were performed with a polisher (Allied High Tech Techprep Multiprep, USA). Because snake teeth are small (some teeth are below 1 mm in length) and curved, sequentially grinding was carefully checked in order to reach half longitudinal sections of the teeth. Uncoated polished sections of the snake teeth were imaged on a Phenom XL scanning electron microscope (SEM; Phenom-World BV, Eindhoven, the Netherlands) using a back-scattered electron detector (SEM-BSE) at an accelerating voltage of 15 kV, in a low-vacuum mode.

Enamel thickness was measured using Image J software (ImageJ 1.53q, National Institutes of Health, USA) on all longitudinal images obtained on the SEM (Figure 2). The measurements were made at the location where enamel is the thickest at the antero-labial side. A minimum of ten measurements were made at different locations along the enamel layer and averages and standard deviations were obtained from these measurements. Due to their small size and their curved and crooked morphology, which could lead to inaccuracy in the measurement, enamel thickness was also measured on few transverse sections. We compared the measures of the enamel thickness on the transverse sections with those on the longitudinal sections to check the accuracy of our measurements (Figure 2, Table S2). These transverse sections were prepared only for few samples at the level of the tooth where enamel was present according to preceding CTscan observations (cf. Figure 1); teeth were embedded in epoxy and a line where the enamel layer was observed on the same material in the CTscan was marked on the epoxy resin. We grind carefully from the tip to the base until reaching this mark. A minimum of 10 measurements were made on these sections (Figure 2) using Image J software (ImageJ 1.53q, National Institutes of Health, USA). Table S2 show that there is no large difference in enamel thickness average in longitudinal and transverse sections.

Tooth length measurement

Tooth length (tooth curvature length) was measured using the FIJI (v1.53q) plugin 'Kappa' (Mary & Brouhard, 2019), in a previous study by [START_REF] Segall | Armed to teeth: the underestimated richness of teeth shape in snakes and its relationship to feeding constraints[END_REF] The ratio of the enamel thickness to tooth length was used to take into account variation in tooth size.

Prey hardness

Prey hardness was divided into 3 categories: soft (e.g., gastropods, annelids, birds, soft-skinned mammals), medium (e.g., amphibians, fish, thin-scaled lizards such as anoles) and hard (insects, snakes, crustaceans, hard-scaled lizards such as skinks). For generalist species or species with several items in their diet, we considered the most consumed item. The prey hardness for each species was determined through an extensive bibliographical work (see Supplementary Table S1).

Statistics

We tested whether tooth length and enamel thickness were correlated phylogenetic generalized least squares (function gls from the 'nlme' package (Pinheiro et al., 2022). We then tested whether raw and relative enamel thickness were associated with diet and the type of enamel coverage using the function phylANOVA of the package 'phytools' using 1000 simulations and a Holm correction for the post hoc pairwise t-tests [START_REF] Revell | Phytools: An R package for phylogenetic comparative biology (and other things[END_REF]. To do so, we pruned the tree from Pyron & Burbrink (2014), if species were not present in the tree, we used the closest relative (Figure S4).

Since the raw and relative enamel thickness were not normally distributed, they were Log10transformed and normality was checked using the shapiro.test function of the 'stats' package (raw thickness: W=0.97854, P=0.36; relative thickness: W=0.97682, P=0.29). These statistics were performed in R version 4.0.5 (R Core Team 2021). Contingency tests (Fisher's exact test) were used to determine if the enamel distribution was related to prey hardness, using the JUMP software (JMP Pro 16.0.0, USA).

Results

General microstructure and enamel thickness

Snake dentary teeth are composed of dentine and a thin layer of enamel. This layer of enamel is mostly present and observed on the antero-labial facet of their teeth in all snake species examined (Figure 1, Figure 2). A very thin layer at the posterior side of the tip and the tooth (Figure 2, Figure 3A, 3B, 3E, 3G, S4) can be observed for some species. Some snake teeth present a thick and distinct enamel layer (e.g., Liodytes rigida, Acrochordus javanicus, Grayia ornata, Boiga cynodon, Malpolon insignitus) whereas others have a really thin, barely noticeable enamel layer (e.g., Anilius scytale, Boa constrictor, Corallus annulatus) (Figure 3D-G, all species represented in Figure S4). For these last species, if most of the enamel layer is rather thin, their tip is made up of a thick and pointy enamel layer. Longitudinal sections show variable enamel appearance from corrugated to smooth. A"corrugated" surface (where enamel has fluted surface, but its outer layer is parallel to the DEJ (dentino-enamel junction))can be observed for example in Acrochordus javanicus (Figure 3A),compared to a smooth surface for other species (cf. Figure 3D-F, 3I). Few snakes species (Anilius scytale, Malpolon insignitus Ophiophagus hannah, and Boiga cynodon (Figure 3B, S4) showed a worn surface, where outer enamel surface is not smooth and not parallel to the DEJ). On the other hand, Eirenis decemlineatus, shows a crenelated surface (Figure 2C,S4).

The crenellation could not be seen on the CTscan (Fig. S4) partly due to their small size (there are 5 µm in height). It is however not clear if the differences in surface, such as the corrugated surface observed in Acrochordus javanicus are responsible of the apicobasal ridges observed along the teeth (Figure 2A-B, Figure S5). The same phenomenon can be seen in Homalopsis buccata (Figure 1K). Some snake teeth show fractured enamel, but Fordonia leucobalia has a particular enamel morphology where the enamel layer appears to be fractured and detached entirely from the dentine" (Figure 3H). Atractaspis engaddensis is the only snake species in our sample which presents a cone of enamel on its teeth (Figure 3J); the enamel is not only deposited on the anterolabial side but present around the entire tooth as confirmed by the virtual transverse sections (insert image on Figure 3J). This thick enamel ring layer all around the tooth is clearly noticeable, and different from what was observed for all other snake species (cf. Figure 1, Figure 2 and Figure 3A-I, Figure S4 for microscopic observations), where the enamel is thick and distinct on the anterolabial side (on the posterior side a really thin almost indiscernible layer of enamel seems to be present). This cone of enamel covered half of the teeth.

The average enamel thickness in the snakes measured ranged from 0.8 µm (Coluber constrictor) to 12.8 µm (Liodytes rigida) (Figure 3D-G). There is no correlation between enamel thickness and tooth length (t = 0.59, P = 0.5 ;; Figure 4A). The absolute and relative enamel average thickness do not significantly differ depending on the tested ecological factors (all P > 0.1, Figure S6). However, Atractaspis engaddensis is an outlier in our sample because of its unusual enamel morphology and behavior (Figure 3J, Figure S6, Discussion). If we remove it from the sample, we obtain a statistical difference of the relative thickness depending on the hardness of the prey (F = 4.57, P = 0.05), more precisely between hard and soft prey specialists (t = -2.97, P = 0.05, Figure 4B). Snakes feeding on hard prey have a thicker relative enamel thickness (average 5.9 ± 5.2µm) compared to snakes feeding on soft prey (average 3.23 ± 3µm; Figure 4B).

We noticed that the range of the enamel thickness varied from one tooth to another suggesting a varied enamel morphology (from smooth to crenelated) as observed in Figure 3. However, there is no statistical difference of the enamel thickness standard deviation in relation to prey hardness (; F = 0.21, P = 0.85)

Enamel distribution.

The distribution of the enamel on the antero-labial part of the teeth of snakes is different between species (Figure 1, Figure S3, Figure S4). Among the 61 species analyzed, 33 species have the antero-labial facet of the tooth entirely covered by enamel, whereas enamel covering only half, or only the tip of the tooth represents less than a third (14 species for both groups) of the entire sample. There is a statistical difference in the average enamel thickness depending on enamel distribution (F = 11.23, P = 0.002), with species presenting enamel only at the tip of the tooth having a thinner enamel than species with the tooth facet entirely covered with enamel (t = -4.57, P = 0.006, Figure 4B).

Enamel distribution is also significantly related to prey hardness (Fisher's exact test P = 0.0026).

Snakes with enamel along the entire tooth eat medium or hard prey, whereas snakes with enamel only at the tip of the tooth eat soft prey (Figure 4A, Table S3).

Discussion

Most snakes have an asymmetrical distribution of enamel; an enamel layer is mostly present at the antero-labial side of the teeth and barely discernable on the posterior side. Atractaspis engaddensis which has a highly derived feeding apparatus and behavior, was the only species with a distinct enamel layer covering the entire crown of the tooth. Atractaspids, also called stiletto snakes, forage in mammal burrows. Unlike any other species, they have highly mobile maxillae that can move laterally, allowing the snake to envenomate prey without opening the mouth. In addition to this peculiar feature, atractaspids have a highly reduced number of teeth, with no pterygoid teeth, and a limited number of teeth on the palatine and dentary bones. The remaining dentary teeth are located anteriorly to the fangs and do not obstruct their lateral motion. This reduced number of teeth, along with all the peculiarities of Atractaspis teeth (short tooth, thick enamel layer and tooth capped entirely with enamel) suggest a highly derived dentition in this species that mostly feeds on small rodents.

The asymmetrical distribution of enamel in snakes is similar to what has been observed in mammals, fish, and other reptiles (e.g., [START_REF] Seidel | Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor[END_REF]Buchtova et al., 2008;[START_REF] Zahradnicek | The development of complex tooth shape in reptiles[END_REF]. This particular enamel distribution arises from differences between lingual and labial cervical loops, where only the latter contain the stem cells responsible for the deposition of the enamel (e.g., [START_REF] Seidel | Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor[END_REF]Buchtova et al., 2008;Handrigan & Richman, 2011).

Biomechanically, the asymmetrical distribution of enamel in rodents and primates (aye-aye) results in asymmetrical wear of the teeth and continual sharpening of the occlusal surface (e.g., Tatersall & Schwartz, 1974;[START_REF] Druzinsky | Mechanical properties of incisors in rodents[END_REF][START_REF] Müller | Growth and wear of incisor and cheek teeth in domestic rabbits (oryctogalus cuniculus) fed diets of different abrasiveness[END_REF]. This particular arrangement, in addition to the creation of an epithelial bulge during tooth formation, has been suggested to be a mechanism responsible for the formation of more complex tooth shapes (e.g., multicuspid teeth) or dental ornaments (like enamel ridges and crests) via an increase of the enamel thickness [START_REF] Zahradnicek | The development of complex tooth shape in reptiles[END_REF]Buchtova et al., 2008). However, previous research showed that snakes lack this epithelial bulge [START_REF] Zahradnicek | The development of complex tooth shape in reptiles[END_REF]Buchtova et al., 2008, Handrigan & Richman, 2011), possibly explaining the simple unicuspid morphology and lack of ornamentation. Most research has been done on Colubridae (corn snake E. guttata) and Pythonidae (Python regius, Python sebae, Python molurus). The teeth of Python regius are almost straight (they present a reduced curvature compared to other teeth, Table S1, previous study by [START_REF] Segall | Armed to teeth: the underestimated richness of teeth shape in snakes and its relationship to feeding constraints[END_REF] with a very thin layer of enamel at their tip, yet do not represent the variability we observed. Most snakes in our sample (33/61 species) have the antero-labial facet of the tooth entirely covered by enamel. The differences in thickness, as well as different types of ornamentation (like crenelations, carinae and apicobasal ridges), observed in different species (e.g., in fish-eaters like Acrochordus javanicus, Homolapsis buccata) suggests the possible presence of an epithelial bulge or alternatively may point to different developmental processes in snakes.

The enamel of snakes varies in thickness, distribution, and shape. In longitudinal sections of the teeth, some species appear to have a corrugated, crenelated layer, while others have a smooth enamel layer. The enamel on the teeth can be thin (under 2µm in Anilius scytale, Calabaria reinhardtiii, Tropiolaemus wagleri, Causus sp., Micrurus psyches). Yet, other small sized species show a greater enamel thickness (e.g., Eryx jaculus, Xenodermus javanicus, Gloydius halys, Cantoria violacea, Atractaspis engaddensis, Liodytes rigida). Snake species that eat slippery prey, such as fish or slugs, show apicobasal-ridges on the posterior side of their teeth (e.g., Homalopsis buccata Figure 1K, Acrochordus javanicus, Figure 2A-B, Figure S5). We suggest that the ridges may prevent the teeth from getting stuck in the prey by limiting the suction created by the layer of mucus as suggested by McCurry and colleagues (2019). Alternatively, the ridges could help rigidify the tooth, but since there is almost no enamel along the ridges, the latter hypothesis seems less likely. However, if we consider stricto sensu the definition of McCurry et al. 2019, apicobasal ridges are elevated ridges of enamel. From our preliminary observations (Figure 2A-B and Figure S5), in snakes' teeth these ridges are formed of dentine rather than by a localized thickening of enamel. Other teeth surface ornamentations such as carinae (or media-lateral ridges, [START_REF] Mccurry | The repeated evolution of dental apicobasal ridges in aquatic-feeding mammals and reptiles[END_REF] are also observed in few snake samples (cf. Figure S5) and seem to be due to a thicker enamel edge but should be further explored.

We also observed differences in the enamel distribution on the teeth of snakes ranging from teeth entirely covered by enamel, to teeth being covered only at the upper half, to teeth presenting enamel only at the tip. Enamel distribution in snakes appears to be related to prey hardness. Snakes eating soft prey have teeth with enamel at the tip only. When feeding on soft prey, the enamel at the tip is probably sufficient to allow the tooth to pierce the skin and flesh. The tip of the tooth may also undergo a higher stress for these species and need to be covered by enamel A Finite Element Analysis on fish teeth recently demonstrated that the tip of the tooth of piranha, which purpose is to cut through flesh, undergo higher stress than the tooth of pacu, a durophagous species, in which the stress is distributed along the tooth [START_REF] Velasco-Hogan | Tooth structure, mechanical properties, and diet specialization of Piranha and Pacu (Serrasalmidae): A comparative study[END_REF]. Enamel thickness is also related to the hardness of the prey, with snakes eating hard prey having a relatively thicker enamel. Enamel thickness has been well studied in mammals, especially in primates and has shown to be, with tooth size, one of the main aspects to control damage resistance according to food hardness [START_REF] Lucas | Dental enamel as a dietary indicator in mammals[END_REF][START_REF] Lee | Fracture in teeth-a diagnostic for inferring bite force and tooth function[END_REF][START_REF] Lawn | Inferring biological evolution from fracture patterns in teeth[END_REF]: an increase of enamel strengthens the teeth (e.g., [START_REF] Dumont | Enamel Thickness and Dietary Adaptation among Extant Primates and Chiropterans[END_REF][START_REF] Lucas | Dental enamel as a dietary indicator in mammals[END_REF]Mc Graw et al., 2012). Durophagous snakes present a thicker layer of enamel in both absolute and relative terms, suggesting a possible adaptation to prevent failure while feeding on hard prey. In addition to a thick enamel layer and a large enamel coverage, some durophagous snakes, such as Liodytes, have hinged teeth [START_REF] Savitzky | Hinged teeth in snakes: an adaptation for swallowing hard-bodied prey[END_REF]. These results suggest that enamel coverage is related to the functional demand on the teeth and may have been reduced in snakes eating softer prey.

The reduction of the enamel layer observed in snakes raises several questions. Enamel has always been considered as an important protective tissue, crucial for the durability of the teeth. Yet, some mammals do not have enamel on their teeth (e.g. David_Beal et al. 2009[START_REF] Kierdorf | Dental structure and tooth attachment modes in the common fangtooth Anoplogaster cornuta (Valenciennes, 1833) (Actinopterygii; Trachichthyiformes; Anoplogastridae)[END_REF]. In fish, absence of enamel or enameloid was recently described in one species of Anoplogastridae (Kierdof et al. 2022) but the authors could not find a functional explanation. On the other side, enamel reduction or loss in mammals is presumed to be due to highly specialized feeding habits [START_REF] Davit-Beal | Loss of teeth and enamel in tetrapods: fossil record, genetic data and morphological adaptations[END_REF][START_REF] Werth | Feeding in marine mammals[END_REF][START_REF] Ciancio | Dental enamel structure in long-nosed armadillos (Xenarthra: Dasypus) and its evolutionary implications[END_REF]2021) and to lead to specific morphological adaptations, such as the hypsodont/hypselodont condition in armadillos (high crown teeth, [START_REF] Davit-Beal | Loss of teeth and enamel in tetrapods: fossil record, genetic data and morphological adaptations[END_REF] and also possibly differences in the nature of the dentine [START_REF] Kalthoff | Microstructure of dental hard tissues in fossil and recent xenarthrans (Mammalia: Folivora and Cingulata)[END_REF][START_REF] Ciancio | Dental enamel structure in long-nosed armadillos (Xenarthra: Dasypus) and its evolutionary implications[END_REF]). Another explanation for the reduction of enamel in snakes is that their teeth have a different function than those other vertebrates. Snakes use their teeth to capture, manipulate, and transport their prey whole but they rarely crush or tear apart their food, with only few exceptions [START_REF] Bringsøe | Eviscerated alive: Novel and macabre feeding strategy in Oligodon fasciolatus (Günther, 1864) eating organs of Duttaphrynus melanostictus (Schneider, 1799)[END_REF][START_REF] Jayne | Snake circumvents constraints on prey size[END_REF]. Fordonia leucobalia for instance, can tear apart crustaceans, and our specimen shows a fragmented enamel which could be due to their specific diet/manipulation or can be an artefact of preservation. Thus, except during the strike, it is possible that the load on the teeth is rather low and distributed between all teeth, but this hypothesis remains to be tested.

The high-resolution laboratory CT-scans employed here, show the technical limitations due to the small sizes of the object and the thin aspect of the enamel in some snakes. Propagation phase contrast synchrotron X-ray micro-computed tomography would be helpful in order to characterize more precisely the enamel volume. Our results show that the enamel and the tooth structure of snakes is variable and more complex that what was previously expected. Future detailed investigations on the variability of enamel and tooth surface, for example, may help to better understand the microstructure of the teeth and the differential development of the enamel. Indeed, lateral carinae, and apicobasal ridges are observed to various degrees in different snake species (e.g., [START_REF] Vaeth | Observations of tooth surface morphology in snakes[END_REF][START_REF] Massare | Tooth morphology and prey preference of Mesozoic marine reptiles[END_REF][START_REF] Young | Dentitional surface features in snakes (Reptilia: Serpentes)[END_REF]Kearny, 2006;[START_REF] Mccurry | The repeated evolution of dental apicobasal ridges in aquatic-feeding mammals and reptiles[END_REF]. This structural variation is likely important during feeding and further investigations may help us to better understand the evolution and biomechanics of snake teeth.

Conclusion

Snakes present an asymmetric enamel deposition with enamel being present principally at the antero-labial side. Prey hardness is the main ecological factor associated with enamel thickness and distribution. Similar to what has been observed for mammals, snakes feeding on harder prey have a relatively thicker and wider enamel layer. Snakes feeding on soft prey have a thin enamel layer, mostly concentrated at the tip of the tooth. This is the first study showing the variability of enamel in snake teeth and the link with diet. Further work is needed to investigate potential differences in dental microstructure and to understand the biomechanical implications of variation in the enamel distribution. D-G Morelia spilota, enamel present only at the tip of the tooth D. Virtual longitudinal section of the entire tooth with the height of the tooth represented by the blue line. E. Virtual longitudinal and transverse sections at mid-height (blue line). At this level of the tooth, enamel cannot be observed. F. Virtual longitudinal and transverse sections of the tooth where the enamel can be distinguished. This corresponds to a section at roughly 10% of the tooth height. G. SEM-BSE microscopy image of a tooth from the same species confirming the enamel appearing only at the tip of the tooth.

Supplementary Figures

H-K Eirenis decemlineatus with enamel covering half of the tooth H. Virtual longitudinal sections of the entire tooth with the height of the tooth represented by the green line. I. Virtual longitudinal and transverse sections at mid-height (blue line). At this level of the tooth, enamel cannot be observed (white arrow). J. Virtual longitudinal and transverse sections of the tooth across the upper half of the tooth (less than 30% of its height) showing the distinctive layer of enamel. K. SEM-BSE microscopy image of a tooth from the same species with distinct enamel layer present. Atractaspis engaddensis appears to be the only species specializing on soft prey presenting a thick enamel layer, as thick as durophagous species.

Species

Longitudinal

FiguresaFigure 1

 1 Figures

Figure 2 :

 2 Figure 2: Enamel thickness measurements on BSE-SEM transversal and longitudinal sections images. A-B Acrochordus javanicus SEM longitudinal (A) and transversal (B) sections red lines represent examples where the enamel thickness was measured, the (cf text). C-D Gerarda prevostiana SEM longitudinal (C) and transversal (D) sections red lines represent examples where the enamel thickness was measured in both sections(cf. text).

Figure 3 :

 3 Figure 3: Variation in enamel general morphology. Differences in enamel surface

Figure 4 :

 4 Figure 4: A. Plot of the enamel thickness versus tooth length. Each dot represents one species,

Figure 5 :

 5 Figure 5: A. reconstructed tooth illustrating the three different types of enamel coverage in

Figure S1 :

 S1 Figure S1: Artefact present on the Stenorrhina degenhardtii dentary. CT scans (A, B) of the dentary of this snake display high density layer surrounding the dentary bone and the teeth (red arrows in B). The BSE-SEM microscopic images (C, D) obtained also show the same artefact at the surface of the enamel with bright spots clearly observed on the surface of the teeth (red arrow in D). EDX analysis obtained shows that the artefact is due to lead (Pb) inclusion. (EDX analyses were performed on the Phenom (cf. Material part). Area analyses were made for both dentary, dentine and artefact areas, at 15kV, square area represent 2048 points analyses).

Figure S2 :

 S2 Figure S2: Artefact present on the teeth of Daboia russelii. Tooth of Daboia russelii look unusual and seem to have suffered from shrinking as observed in the CTscan (A). Virtual sections of the CTscan shown on both longitudinal and transverse sections (B, C) some density lines are present in the teeth. One high dense one at the surface of the teeth seems to be an artefact rather than enamel (red arrow in B and C). Microscopic images (D) of the section prepared in one of the teeth of this snake show the dentine observed in the scan (which could be caused by infiltration of chemicals). The enamel is rather thin and broken and missing in a few parts of the tooth.

Figure S3 :

 S3 Figure S3: Enamel distribution obtained by CT-scan and electron microscopy. A-C Boiga dendrophila, enamel covering the entire anterior face A. Virtual longitudinal sections of the

Figure S4 :

 S4 FigureS4: Phylogenetic tree of the snake species included in the study with a 3D view of the different teeth, illustrating the distribution of their enamel (semi-transparent rendering of the teeth and enamel segmented in red) on the left. On the right are represented the different longitudinal sections obtained to confirm the enamel distribution along the teeth. Frame color represents the enamel distribution on the teeth: entire in red, half in orange, and the tip in yellow. Hard, medium, and soft represent the hardness of the dominant prey eaten by each snake species. Only the sample of Stenorrhina degenhardtii could not be segmented due to a preservation artifact (cf. FigureS1).

Figure S5 :

 S5 Figure S5: Snake teeth ornementation. A. 3D rendering of Acrochordus granulatus teeth showing carinae present in the side of their teeth (yellow arrows). Insert CT transverse section showing the carinae possibly due to a thicker enamel edge. B. 3D rendering of Anilius scytale showing a deep carinae stopping halfway on the side of one tooth. C. 3D rendering of Acrochordus javanicus showing apicobasal ridges in the posterior side of the their teeth (red arrows). Insert corresponding SEM microscopic transverse section of one tooth showing the presence of these apicobasal ridges which seem to be formed by a thicker amount of dentine. D. 3D rendering of Homalopsis buccata showing apicobasal ridges in the posterior side of the their teeth (red arrows). Insert corresponding CT transverse sections showing the presence of these apicobasal ridges, similar to Acrochordus javanicus.

Figure S6 :

 S6 Figure S6: Plot of the relative enamel thickness versus tooth length. Each dot represents one species, dot color represents prey hardness (dark purple: hard, blue green: medium, yellow: soft).Atractaspis engaddensis appears to be the only species specializing on soft prey presenting a thick enamel layer, as thick as durophagous species.

Acknowledgements

We thank the Gans Fund for funding this study. MS was also funded by the European Union H2020 -Marie Skłodowska-Curie Global Fellowship (GA101024700). Special thanks to the people and institutions who provided specimens to enrich our sample and without whom this study would not have been possible: Anthony Herrel (MNHN), David Kizirian and Lauren Vonnahme from the herpetological collection of the American Museum of Natural History (New York), Nicolas Vidal from the herpetological collection of the Museum National d'Histoire Naturelle (Paris), Marc Herbin (MNHN), Rémi Ksas and Antoine Planelles (Venomworld), Ya-Wei Li, Vincent Prémel, Ludovic Faure, Yoan Eynac and Karine Falco. We thank Mathilde Bouchet from ENS Lyon for her time helping to set the specimens and teaching MS to use the CT scanner. We thank Filipe Natalio for providing access to the SEM Phenom in the Weizmann Institute.

Availability of data and material: Microscopy images will be uploaded in an image data resource.

Authors' contributions: MD and MS conceived and led the research project. MD led the writing of the manuscript, collecting the data and performing the analyses. MS helped with the statistical analyses and figures. RC, RS, JM, AH helped in writing the grant proposal. BS provided specimens from the herpetological collection of Jerusalem. All authors MD, JM, AH, RS, BS, CH, AD, RC and MS contributed to the interpretation and discussion of the results, and to the editing of the manuscript.

Conflicts of interest/Competing interests:

The authors declare no conflict of interest. Table S1: List of specimens used in this study along with information about the collection, scans, diet, prey hardness, and tooth and enamel measurements and references for diet. + indicates the major prey item. Differences in tooth enamel in snakes with different diets d49 Table S2: Measurements of average enamel thickness (in µm) and standard deviation of both 1 longitudinal and transverse tooth sections for some species.

Supplementary Tables

2


Semsarilar

Chaimaa Gomri
email: chaimaa.gomri@umontpellier.fr

Marc Cretin
email: marc.cretin@umontpellier.fr

Mona Semsarilar
email: mona.semsarilar@umontpellier.fr

Recent progress on chemical modification of Cellulose Nanocrystal (CNC) and its application in nanocomposite films and membranes-A comprehensive review

Keywords: Cellulose nanocrystal (CNC), self-assembly, chemical modification, membrane, film, nanocomposite

Recent progress on chemical modification of cellulose nanocrystal (CNC) and its application in nanocomposite films and membranes-A comprehensive review

Introduction

According to the 2019 UN report, the world population is growing at an annual rate of 1.1%, which predicts a global population of 8.5 billion in 2030, instead of the 7.7 billion recorded in mid-2019 [START_REF] Onu | World population prospects 2019[END_REF]. Admittedly, the growth rate has been decreasing since 1950. However, it is still accompanied by strong demand for food and increased exploitation of natural resources, which contribute to sizeable environmental stress and pollution [START_REF] Okada | Chemical syntheses of biodegradable polymers[END_REF] [START_REF] Sudhakar | An introduction of Biopolymer Electrolytes[END_REF].

Thus, synthetic polymers are used extensively to meet humanity's needs and ensure a more convenient way of life.

These polymers often present many risks; they are persistent, hardly degradable, and their degradation by-products are often toxic. Hence the use of degradable polymers, especially biodegradable ones, is encouraged.

Biodegradable polymers are defined as polymers that, in the presence of microorganisms in a natural environment, degrade into carbon dioxide and water without generating toxic by-products [START_REF] Okada | Chemical syntheses of biodegradable polymers[END_REF].

Biodegradable polymers are classified into three main categories (1) polyesters produced by microorganisms, (2) natural polysaccharides and other biopolymers, and (3) synthetic polymers, in particular aliphatic polyesters.

Polysaccharide materials get a growing interest due to their abundance, non-toxicity, and biodegradability, which make them a good replacement for petroleum-based polymers [START_REF] Garcia-Valdez | Graft modification of natural polysaccharides via reversible deactivation radical polymerization[END_REF]; not only this, but they present physicochemical properties that favor their use in several other fields (Fang et al., 2019a).

Cellulose belongs to this family of polysaccharides; it is the most abundant biopolymer on earth, with an annual production estimated between 10 11 -10 12 tons [START_REF] Nechyporchuk | Production of cellulose nanofibrils : A review of recent advances[END_REF]. It is used in several fields with the following distribution: 28% in materials sciences, 17% in engineering, 16% in chemistry, 11% in chemical engineering, 8% in physics and astronomy, 5% in biochemistry, genetics, and molecular biology, 4% in environmental science, 3% in agriculture and biological sciences, 3% in medicine, 3% in energy, 1% in immunology and microbiology and 1% in pharmacology, toxicology, and pharmaceuticals [START_REF] Mohamed | An overview on cellulose-based material in tailoring bio-hybrid nanostructured photocatalysts for water treatment and renewable energy applications[END_REF].

Cellulose can originate from wood, flax, cotton, and algae in the form of microfibrils. Mechanical and chemical treatment of these microfibrils allows for obtaining cellulose nanofibrils (CNF) and cellulose nanocrystal (CNC) [START_REF] Hamad | Cellulosic Materials[END_REF]. Cellulose can also originate from bacteria and has a more crystalline structure than cellulose extracted from plants.

This review is dedicated to cellulose nanocrystal (CNC) specifically. The first part concerns the origin, structure of cellulose, and its ability to self-assemble and form films. The second part emphasizes the main chemical modification of CNC surfaces applied during the last decade. In contrast, the last part gathers the techniques applied to develop composite membrane and film based on CNC and their possible applications.

Cellulose origin, structure, and its property to form films

Natural cellulose is originated from plants through the disintegration of the microfibers that form the cell wall (Figure 1) [START_REF] Mohamed | An overview on cellulose-based material in tailoring bio-hybrid nanostructured photocatalysts for water treatment and renewable energy applications[END_REF]) [START_REF] Gopakumar | Nanocellulose-Based Membranes for Water Purification[END_REF]) [START_REF] Dong | Cellulose/carbon Composites and their Applications in Water Treatment -a Review[END_REF]; extraction can be made by chemical or mechanical treatment [14][13] and lead to two types of cellulose, cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC). This approach is known as top-down biosynthesis. CNF consists of two regions, an ordered region, in other words, a crystalline region that confers high resistance to cellulose, and an amorphous region which contributes to the flexibility of the material [START_REF] Phanthong | Nanocellulose: Extraction and application[END_REF]. CNF is characterized by a diameter between 1-100 nm and a length between 500-2000 nm. When subjected to acid hydrolysis, cellulose nanofibrils undergo transverse cleavage along amorphous regions because they have a lower density due to their random orientation, resulting in the formation of cellulose nanocrystals (CNC) [START_REF] Gopakumar | Nanocellulose-Based Membranes for Water Purification[END_REF]) [START_REF] Mohamed | An overview on cellulose-based material in tailoring bio-hybrid nanostructured photocatalysts for water treatment and renewable energy applications[END_REF]. Their dimensions depend on the starting material, their diameter varies between 2-20 nm [START_REF] Sharma | A review on cationic starch and nanocellulose as paper coating components[END_REF], and their length is between 100-500 nm [START_REF] Gopakumar | Nanocellulose-Based Membranes for Water Purification[END_REF].

Figure 1. Illustration of the origin of natural cellulose [START_REF] Han | Thermoelectric polymer-cellulose composite aerogels[END_REF].

Nanocellulose can also be obtained from bottom-up biosynthesis using bacteria [START_REF] Bwatanglang | Market analysis and commercially available cellulose and hydrogel-based composites for sustainability, clean environment, and human health[END_REF]. It is produced through the pores of bacteria membranes, so Gluconacetobacter is the species with the highest production ability [START_REF] Belaustegui | Bacterial-cellulose-derived carbonaceous electrode materials for water desalination via capacitive method: The crucial role of defect sites[END_REF]. There are other species such as Acinetobacter xylinumthrough [START_REF] Sharma | A review on cationic starch and nanocellulose as paper coating components[END_REF], Rhizobium, Agrobacterium, Sarcina and Alcaligenes [START_REF] Belaustegui | Bacterial-cellulose-derived carbonaceous electrode materials for water desalination via capacitive method: The crucial role of defect sites[END_REF]. Bacterial cellulose has the form of twisting ribbons with a diameter between 20-100 nm and length in micron order [START_REF] Phanthong | Nanocellulose: Extraction and application[END_REF].

It has the same structure as the cellulose extracted from the plant wall but presents higher purity and crystallinity [START_REF] Revin | Cost-effective production of bacterial cellulose using acidic food industry by-products[END_REF].

The formula of cellulose is (C 6 H 10 O 5 ) n , with n being the degree of polymerization that depends on the source of the cellulose and the method of isolation and purification [START_REF] Qin | Toughness improvement of epoxy thermosets with cellulose nanocrystals[END_REF]. It is composed of β-D-glucopyranose units linked together by β-(1,4) -glycosidic bonds [START_REF] Arfin | Cellulose and hydrogel matrices for environmental applications[END_REF]; each unit has 3 hydroxyl groups, a primary hydroxyl group on the C6, and two hydroxyl groups secondary on C2 and C3, presenting reactive sites that allow modification of cellulose [START_REF] Fleet | RAFT mediated polysaccharide copolymers[END_REF] (figure 2). The reactivity of these sites is governed by factors depending on the steric effects related to the supramolecular structure of cellulose and its isomerization. The C6 primary alcohol is ten times more reactive than the C2 and C3 secondary alcohols, thanks to the free rotation around the C5 and C6 bond.

( [START_REF] Garcia-Valdez | Graft modification of natural polysaccharides via reversible deactivation radical polymerization[END_REF]. These hydroxyl groups allow the formation of inter and intramolecular hydrogen bonds [START_REF] Chu | Dispersion Properties of Nanocellulose : A Review[END_REF] [START_REF] Kausar | Nanocellulose in polymer nanocomposite[END_REF]. These bonds allow the formation of a solid network giving cellulose a fibrillated structure, high resistance, and insolubility in water and most organic solvents [START_REF] Phanthong | Nanocellulose: Extraction and application[END_REF]. CNC suspension behaves as a cholesteric crystalline liquid; this phase can be preserved after evaporation of the solvent, which leads to a well-structured iridescent film [START_REF] Frka-Petesic | Controlling the Photonic Properties of Cholesteric Cellulose Nanocrystal Films with Magnets[END_REF]. Factors such as the technic of extraction and concentration of the suspension have a direct impact on the self-assembly. CNC extracted using sulfuric acid exhibits a stable suspension due to sulfate groups negatively charged attached to the surface [START_REF] Tran | Understanding the Self-Assembly of Cellulose Nanocrystals-Toward Chiral Photonic Materials[END_REF]; this stability is a prerequisite to having self-assembly for cases where CNC is isolated by using phosphoric acid or hydrochloric acid, CNC will not be enough charged and will tend to flocculate which impacts the self-assembly negatively [START_REF] Parker | The Self-Assembly of Cellulose Nanocrystals: Hierarchical Design of Visual Appearance[END_REF].

Above a particular concentration, CNC whiskers aggregate to form tactoids. While drying, they stack into layers.

Each layer has a direction orthogonal to the nematic axis, and the layers are modulated to a helicoidal form [START_REF] Dumanli | Controlled, Bio-inspired Self-Assembly of Cellulose-Based Chiral Reflectors[END_REF][START_REF] Frka-Petesic | Controlling the Photonic Properties of Cholesteric Cellulose Nanocrystal Films with Magnets[END_REF], as shown in figure 3. The origin of the helicoidal arrangement is the chirality of CNC obtained due to D-glucose units [START_REF] Tran | Understanding the Self-Assembly of Cellulose Nanocrystals-Toward Chiral Photonic Materials[END_REF]. This specific structure displays CNC film with optical functionalities that favor its use in biophotonic material [START_REF] Xiong | Self-Assembly of Emissive Nanocellulose/Quantum Dot Nanostructures for Chiral Fluorescent Materials[END_REF].

Besides the optical properties, films based on nanocellulose show good mechanical properties compared to films made from other materials (tensile strength of 100-300 MPa and a modulus of 5-30 GPa) [START_REF] Fang | Nanocellulose-based films and their emerging applications[END_REF].

Thanks to their high surface area, they can be used as templates with other materials such as TiO 2 , carbon, and organosilica [START_REF] Lagerwall | Cellulose nanocrystal-based materials: From liquid crystal self-assembly and glass formation to multifunctional thin films[END_REF]. Therefore, integrating novel functionalities in the CNC structure can extend their application as films in many different fields. [START_REF] Tran | Tactoid Annealing Improves Order in Self-Assembled Cellulose Nanocrystal Films with Chiral Nematic Structures[END_REF]. John Wiley and Sons Copyright (2020).

Chemical modification of cellulose nanocrystal

Cellulose can be chemically modified via its hydroxyl groups, mainly the primary alcohol group. This modification can broaden its application and improve its compatibility with other materials. Modification could be done following different strategies depending on the species inserted.

Graft polymerization

Graft polymerization is considered a good approach to modifying the surface and endowing it with new physical and chemical properties [START_REF] Wohlhauser | Grafting Polymers from Cellulose Nanocrystals: Synthesis, Properties, and Applications[END_REF]. It consists of attaching a sequence of monomers to the cellulose backbone. The inserted polymer will give new properties to cellulose depending on its functional group [START_REF] Roy | Cellulose modification by polymer grafting: A review[END_REF]. Two main approaches are used for graft polymerization, the "grafting to" and the "grafting from" approach (figure 4). The "grafting to" approach involves attaching a pre-formed polymer to the cellulose's functional site via the polymer's reactive end group [START_REF] Oberlintner | Hydrophobic functionalization reactions of structured cellulose nanomaterials: Mechanisms, kinetics and in silico multi-scale models[END_REF]. This approach can be limited because of the steric hindrance; larger polymer chains can intertwine and prevent the functional group from reaching the cellulose surface [START_REF] Kedzior | Recent advances and an industrial perspective of cellulose nanocrystal functionalization through polymer grafting[END_REF]. While the "grafting from "consists of the immobilization of the initiator on the cellulose surface to allow the polymer chain's growth. This approach is widely used because it provides a high grafting density with a good control length [START_REF] Wohlhauser | Grafting Polymers from Cellulose Nanocrystals: Synthesis, Properties, and Applications[END_REF] [START_REF] Kedzior | Recent advances and an industrial perspective of cellulose nanocrystal functionalization through polymer grafting[END_REF]. The two approaches can be applied by using different techniques such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT), nitroxide-mediated radical polymerization (NMP), ringopening polymerization (ROP), and free radical polymerization. polymerization is initiated from the CNC surface. [START_REF] Kedzior | Recent advances and an industrial perspective of cellulose nanocrystal functionalization through polymer grafting[END_REF]. Elsevier Copyright (2019).

Atom transfer radical polymerization (ATRP)

As its name implies, ATRP relies on the transfer of halogen atoms between a dormant species (R-X) and a transition metal catalyst M t n -X/ligand [START_REF] Mishra | Living Radical Polymerization: a Review[END_REF]. The active radical is generated due to the transfer of the halogen atoms from the dormant species to a transition metal complex by oxidative addition. The radical created will then react with the monomer to further form a polymer chain [START_REF] Roy | Cellulose modification by polymer grafting: A review[END_REF]. ATRP has been largely used to graft polymers to substrates due to its ability to form a well-controlled polymer chain with a high grafting density. A large panel of monomers was attached to cellulose nanocrystals, as summarized in table 1. The main way used to start the surface-initiated ATRP (SI-ATRP) is to first graft the initiator to the surface of CNC by esterification; the initiator usually applied are 2-Bromoisobutyryl bromide "BIBB" and α-Bromoisobutyric acid "BIBA", then the polymer chain grows from the surface. The main negative point of using ATRP is the use of toxic transition metal complexes and the need to separate them from the final product. So, to overcome these drawbacks and decrease the amount of metal, Sèbe et al. used Activator Re-Generated by Electron Transfer ATRP (ARGET-ATRP) to graft polystyrene and poly (4-vinyl pyridine). This approach uses a reducing agent in excess to regenerate the catalyst regularly from the deactivated form. By comparing ATRP and ARGET-ATRP, it has been observed that ARGET promotes the formation of a longer chain with a low grafting density; this is because the propagation step is fast, and the amount of the catalyst is of the order of ppm (Z. [START_REF] Zhang | A comparative study on grafting polymers from cellulose nanocrystals via surface-initiated atom transfer radical polymerization ( ATRP ) and activator re-generated by electron transfer ATRP[END_REF]. Another approach was applied by Cunningham et al. to reduce the amount of catalyst, based on the use of Cu(0) as a reducing agent. The Cu(0) was added in the form of wire and was easily removed at the end. [START_REF] Arredondo | Synthesis of CO2-responsive cellulose nanocrystals by surface-initiated Cu(0)-mediated polymerisation[END_REF]

Reversible addition-fragmentation chain-transfer (RAFT)

RAFT is an effective process for controlling living radical polymerization; it enables the synthesis of macromolecular with complex architecture [START_REF] Semsarilar | Green" reversible addition-fragmentation chain-transfer (RAFT) polymerization[END_REF]. It involves thiocarbonylthio compounds that serve as chain transfer agents. The effectiveness of this agent relies on the radical leaving group "R", the activating group "Z" and its compatibility with monomers [START_REF] Moad | RAFT polymerization and some of its applications[END_REF]. Table 2 regroups polymers with the corresponding RAFT agent used. The R group needs to be a good homolytic leaving group and be able to reinitiate new polymer chains. In contrast, the Z group must provide the RAFT agent with good resonance stabilization and polar effects to be more active [START_REF] Tsang | Alternative Proton Exchange Membranes by Chain-Growth Polymerization[END_REF]. Grafting polymers to CNC by RAFT polymerization surface can be applied by immobilizing the RAFT agent to the surface via either the Z approach or the R approach.

In the Z approach, the di(tri)thio group is permanently grafted to the cellulose surface while the chain grows aside.

In the R approach, the polymer chain grows from the surface [START_REF] Boujemaoui | SI-RAFT/MADIX polymerization of vinyl acetate on cellulose nanocrystals for nanocomposite applications[END_REF]. Li et al. used another method to graft "PDMAEMA-b-PGMA-b-PHFBA "to the CNC surface. The polymer was first synthesized by RAFT, and then it was grafted onto cellulose via the reaction between the epoxy ring of PGMA and the hydroxyl group of the cellulose. The hydrophobicity increased, confirming successful grafting (J. [START_REF] Zhou | V-Shaped amphiphilic polymer brushes grafted on cellulose nanocrystals: Synthesis, characterization and properties[END_REF].

Ring-opening polymerization (ROP)

ROP is a polymerization technique that involves monomers such as epoxides, cyclic esters (ex. lactones, lactides), and cyclic carbonates [START_REF] Sarazin | Discrete Cationic Complexes for Ring-Opening Polymerization Catalysis of Cyclic Esters and Epoxides[END_REF]. ROP occurs following mechanisms such as radical, cationic, anionic, and ring-opening metathesis polymerization [START_REF] Nuyken | Ring-opening polymerization-An introductory review[END_REF]. It mainly depends on the monomer, catalytic system, and the initiator used [START_REF] Carlmark | Grafting of cellulose by ring-opening polymerisation -A review[END_REF]. The hydroxyl group can be used as an initiator for ROP. Thus modification of cellulose using ROP seems interesting [START_REF] Carlsson | Surface characteristics of cellulose nanoparticles grafted by surface-initiated ring-opening polymerization of ε-caprolactone[END_REF] since no prior chemical treatment will be needed [START_REF] Carlmark | Grafting of cellulose by ring-opening polymerisation -A review[END_REF]. Grafting PLA from CNC surface was extensively studied using Tin (II) octoate as a catalyzer ( Poly(e-caprolactone) Grafting from [START_REF] Carlsson | Surface characteristics of cellulose nanoparticles grafted by surface-initiated ring-opening polymerization of ε-caprolactone[END_REF] Table 3. Summary of polymers grafted to CNC via ROP.

Nitroxide-mediated polymerization (NMP)

NMP is based on the use of nitroxide to initiate the polymerization without the implication of another radical source or the need for a metal catalyst. The main characteristic of the alkoxyamine that could be used for NMP is its ability to provide reactive radical to initiate polymerization and form a stable mediating nitroxide radical. NMP can be used to graft polymer on a natural surface such as cellulose. For the" grafting from" approach, CNC can be functionalized with nitroxide forming, thus an alkoxyamine able to induce polymerization. Roeder and co-workers first graft BlocBuilder® (BB) on CNC via a covalent bond to the vinyl group of 4-chloromethyl styrene (CMS)

that was already inserted into CNC. The polymerization was generated after adding the monomer [START_REF] Roeder | Graft modification of cellulose nanocrystals: Via nitroxide-mediated polymerisation[END_REF]. The "Grafting to" approach was applied by Glasing et al., where they first modified CNC with glycidyl methacrylate (GMA). The polymerization was generated in the presence of macroalkoxyamines based on Nhydroxysuccinimidyl BlocBuilder® and polymers such as Styrene, N, N-(diethylamino)ethyl methacrylate(DEAEMA), and N-3-(dimethylamino)ethyl methacrylamide (DMAPMAm) [START_REF] Glasing | Grafting well-defined CO2-responsive polymers to cellulose nanocrystals via nitroxide-mediated polymerisation: Effect of graft density and molecular weight on dispersion behaviour[END_REF].

Free-radical polymerization (FRP)

FRP is an uncontrolled polymerization technique, and the synthesized polymers usually have a high dispersity.

Several methods initiate FRP, such as chemical, thermal, and radiation. Jeun and co-workers successfully grafted 1H,1H,2H,2H-perfluorodecyl acrylate (PFDA) using electron beam (e-beam) irradiation as a technique to initiate the polymerization. The advantage of e-beam is that it avoids the application of harmful compounds [START_REF] Jeun | Surface Modification for the Hydrophobization of Cellulose Nanocrystals Using Radiation-Induced Grafting[END_REF]. Plasma was also applicated to graft polymers on CNC surfaces. Here, we can cite the research done by Alanis et al. where they graft different monomers (caprolactone, styrene, α-farnesene) [START_REF] Alanis | Plasma surfacemodification of cellulose nanocrystals: A green alternative towards mechanical reinforcement of ABS[END_REF]. The main advantage of this technique is the possibility of polymerizing a wide range of monomers.

Grafting small molecule

Grafting small molecules on the CNC surface is advantageous. Diverse functions can be grafted using reactions with suitable conditions and high yield. Many strategies are applied; we discussed below click chemistry, sylilation and esterification.

Click chemistry

Click chemistry was first introduced by Sharpless et al. in 2001, describing reactions that satisfy the following criteria: stereospecific, giving high yields, modular, wide in scope, implies simple experimental conditions, the product should be easily isolated and involve only benign solvent if it is necessary [START_REF] Kolb | Click Chemistry: Diverse Chemical Function from a Few Good Reactions[END_REF]. The commonly used click chemistry reactions are Diels-Alder, thiol-ene, the azide-alkyne Huisgen, and the Cu 1catalyzed azide-alkyne cycloadditions [START_REF] Filpponen | Chapter 3: Click Chemistry in Cellulose Functionalization[END_REF]. Click chemistry was used to modify CNC by either bringing two CNC chains together [START_REF] Filpponen | Chapter 3: Click Chemistry in Cellulose Functionalization[END_REF] or grafting new functionalities to its surface. CNC can be functionalized with an Azide or an alkyne functional group for the Azide-alkyne cycloaddition, and Thiol-ene click chemistry CNC should be modified with thiol function or a methacrylate (Table 4).

Modified

Silylation

CNC can be modified by inserting a Siloxane group to enhance the hydrophobicity of cellulose [START_REF] Anpilova | Methods for Cellulose Modification in the Development of Polymeric Composite Materials (Review)[END_REF] and increase the thermal stability of the modified CNC [START_REF] Khanjanzadeh | Surface chemical functionalization of cellulose nanocrystals by 3-aminopropyltriethoxysilane[END_REF]. Zanata et al. developed crosslinked aerogels based on cellulose nanocrystals using 3-isocyanotopropyltriethoxysilane (IPTS), the developed material exhibit an amphiphilic character [START_REF] Morais Zanata | Chemically crosslinked aerogels based on cellulose nanocrystals and polysilsesquioxane[END_REF]. Silylation of CNC can have another purpose: endowing nanocellulose with flame retardancy. In this sense, Kim and co-workers modified CNC with Tris(2-chloroethyl) phosphate (TCEP) to incorporate it into a polyurethane matrix, and the result showed an increase in the limiting oxygen index up to 28% [START_REF] Kim | Eco-Friendly Nanocellulose Embedded Polymer Composite Foam for Flame Retardancy Improvement[END_REF]. To enhance CNC adsorption capacity toward CO 2 , Zhu et al. modified it with 3-(2-aminoethylamino)-propyl methyl dimethoxy silane (APS); the results revealed that even after ten cycles of adsorption and desorption the adsorption capacity remained above 85% which emphasize the APS-CNC aerogel potential to adsorb CO 2 (W. [START_REF] Zhu | Preparation of an Amine-Modified Cellulose Nanocrystal Aerogel by Chemical Vapor Deposition and Its Application in CO2Capture[END_REF]. Incorporating organosilanemodified cellulose nanocrystal into natural rubber matrix proves its efficiency in improving mechanical properties of the latter; as Singh and co-workers showed in their research [START_REF] Singh | Synthesis and chemical modification of crystalline nanocellulose to reinforce natural rubber composites[END_REF].

Esterification

The modification of cellulose nanocrystals by esterification is an interesting method to modify CNC, as summarized in table 5. Esterification can be applied using a variety of catalysts such as sulphuric acid, hydrochloric acid, iodine, potassium carbonate, and triethylamine, to name a few [START_REF] Ávila Ramírez | Simple citric acid-catalyzed surface esterification of cellulose nanocrystals[END_REF]. The degree of substitution (DS), defined as the average number of substituent groups graft per base unit (anhydroglucose unit) for cellulose the DS may range from zero to three since it has three reactive hydroxyls [START_REF] Wiley | Degree of Substitution[END_REF], depending on the concentration of the reagents, the concentration of catalyst and the removal of water [START_REF] Abraham | Highly Modified Cellulose Nanocrystals and Formation of Epoxy-Nanocrystalline Cellulose (CNC) Nanocomposites[END_REF]

. Preparation of cellulose nanocrystal composite film and membrane

Cellulose nanocrystals can be used as templates with the ability to form mesoporous films (Y. [START_REF] Huang | Multifunctional cellulose nanocrystal structural colored film with good flexibility and water-resistance[END_REF].

The significant problems CNC films face are their brittleness and poor water resistance that prevent their applications. Huang et al. developed a flexible composite film using poly (ethylene glycol) diacrylate (PEGDA)

to overcome this issue. PEGDA encapsulates CNC by forming a three-dimensional crosslinking network.

PEGDA was mixed with a photoinitiator, then added to a CNC suspension and cast in a plastic Petri dish. The film was obtained by evaporation-induced self-assembly (EISA); by the end of this step, CNC and PEGDA interacts via hydrogen bonding. To stimulate the encapsulation of PEGDA to CNC, the film was exposed to UV. It was observed that the tensile strength decreased with the increasing amount of PEGDA, while the flexibility of the composite film has been improved compared to the pristine CNC film. After soaking the prepared film for 24 h, its structure was preserved, which shows its good water-resistant property. The three-dimensional structure of the developed film allows its applications as a water-content detector, anti-counterfeiting label, and photonic paper (Y. [START_REF] Huang | Multifunctional cellulose nanocrystal structural colored film with good flexibility and water-resistance[END_REF].

To avoid the coffee-ring phenomenon obtained due to the heterogeneity structure while the drying process, Zhang et al. used vacuum-assisted self-assembly (VASA) to help obtain a well-oriented and structurally homogeneous CNC film. CNC suspension was filtered using poly(tetrafluoroethene) (PTFE) as a support membrane under vacuum. After filtration and completely drying, CNC film was peeled off from PTFE. The effect of prior sonication and the volume of suspension was evaluated to emphasize their impact on the iridescent of the obtained film. It was reported that the sonication duration does not impact CNC crystallinity, but it is responsible for the coloration variation of the films obtained. A long sonication time is needed to obtain an iridescent CNC film via VASA. Beck et al. reported that sonication leads to the rejection of ions from the bound-water layer, which leads to a large pitch because of the weak chiral interaction between CNC particles [START_REF] Beck | Controlling the reflection wavelength of iridescent solid films of nanocrystalline cellulose[END_REF]. The amount of CNC also impacted the chiral nematic structure of the film. While changing the suspension volume, films with different colors were obtained, suggesting that the chiral nematic structure's helical pitch varies with the CNC film's thickness. The formation kinetics of CNC film also impact the iridescent of the latter so that variating the vacuum degree induces a color change (Q. [START_REF] Chen | Tuning the iridescence of chiral nematic cellulose nanocrystal films with a vacuumassisted self-assembly technique[END_REF].

Vacuum filtration was used by Sulaiman et al. to prepare a nanocomposite film using a layer-by-layer approach.

The group developed a bi-layer electrode using as layers polypyrrole/ graphene oxide (GO) and polypyrrole/CNC.

The bilayer was prepared by first deposing PPy/ GO layer on a membrane filter via vacuum filtering and then adding the second layer PPy/CNC by applying vacuum filtration. After each deposition, the layers were washed with deionized water [START_REF] Kulandaivalu | Rational design of layer-by-layer assembled polypyrrole-based nanocomposite film for highperformance supercapacitor[END_REF]. Wagberg et al. reported that skipping the rinsing step will gender a rougher and less-defined layer [START_REF] Wågberg | The Use of Layer-by-Layer Self-Assembly and Nanocellulose to Prepare Advanced Functional Materials[END_REF]. After drying, the free-standing film was peeled off from the membrane. Multilayer film can be assembled via electrostatic interaction, hydrogen bonding, hydrophobic, and covalent bonding [START_REF] Kulandaivalu | Recent Advances in Layer-by-Layer Assembled Conducting Polymer Based Composites for Supercapacitors[END_REF]. Herein the interaction between the two layers was insured by hydrogen bonding between the oxygen in GO and the hydroxyl group of CNC. The assembly of the two layers PPy/GO|PPy/CNC induced active sites, which attributed to the generated film's high surface area [START_REF] Kulandaivalu | Rational design of layer-by-layer assembled polypyrrole-based nanocomposite film for highperformance supercapacitor[END_REF]. In the same context, Xia et al. developed a tri-layer membrane using mid-layer polydimethylsiloxane (PDMS), surrounded by two layers of nanocrystals cellulose modified with methyltrimethoxysilane. The silylation was intended to render the CNC surface hydrophobic and increase its dispersion in PDMS. The modification did not impact the crystallinity of CNC, which endowed the PDMS matrix with rigidity, one of the fundamental properties of CNC that favored its use as a reinforcing agent. The three layers were covalently bonded due to the crosslinking curing reaction applied during the preparation of the layers that provide strong adhesion between the layers. Barrier performance of the multilayer membrane improved compared with neat PDMS material; it was noted that oxygen permeability decreased due to the presence of Si-CNC, which was explained by the fact that Si-CNC nanoparticles block O 2 from diffusing while crossing the composite layers [START_REF] Xia | Self-bonding sandwiched membranes from PDMS and cellulose nanocrystals by engineering strategy of layer-by-layer curing[END_REF].

Shojaeiarani et al. developed a nanocomposite film based on poly (lactic acid) and CNC using EISA and spin coating. A mixture of PLA-CNC was prepared in chloroform, injected into a spin coater plate, and kept for 180s under a constant spin speed of 150 rpm; the film was obtained after the evaporation of solvent at room temperature (figure 7). It was concluded that spin coating allows good dispersion of CNC due to the high drying rate, compared to EISA, which improves the degree of crystallinity and decreases the hydrophilicity [START_REF] Shojaeiarani | Spin coating method improved the performance characteristics of films obtained from poly(lactic acid) and cellulose nanocrystals[END_REF]. During the electrospinning process, it was observed that the jet was stable for the solution prepared with CNC (HCl), which led to the formation of fibers of good quality. In contrast, the fiber obtained by using CNC (H 2 SO 4 )

was non-uniform. The mechanical properties increased with the reinforcement of the matrix fiber with both types of cellulose nanocrystals [START_REF] Naseri | Porous electrospun nanocomposite mats based on chitosancellulose nanocrystals for wound dressing: effect of surface characteristics of nanocrystals[END_REF]. In another study by Nair et al., they reported a decrease in both tensile strength and Young's modulus for nanocomposite fiber mats based on CNC and Cellulose acetate (CA), and it was explained by the fact that CNC acted more defects due to the aggregation during the loading [START_REF] Naseri | Porous electrospun nanocomposite mats based on chitosancellulose nanocrystals for wound dressing: effect of surface characteristics of nanocrystals[END_REF].

Application of cellulose nanocrystal composite membrane and film

Film and membrane have promising use in electronics, sensors, solar cells, water treatment, and so on. Several studies have been carried out in this direction. They have shown the possibility of integrating cellulose into different matrices to develop films with good performance depending on their field of application.

Conductive materials

To endow cellulose with electrical properties, a conductive material such as conductive polymers (Polyaniline, Polypyrrole, etc.), metallic particles, and conductive carbon material was combined with cellulose, so the developed nanocomposite material exhibits properties of both materials [START_REF] Du | Nanocellulose-based conductive materials and their emerging applications in energy devices -A review[END_REF]. Polyaniline (PANI), one of the conductive polymers, has drawn much attention due to its high electrical conductivity. However, PANI tends to aggregate, limiting its application (S. [START_REF] Wang | Cellulose nanofiber-assisted dispersion of cellulose nanocrystals@polyaniline in water and its conductive films[END_REF] [START_REF] Nepomuceno | Evaluation of conductivity of nanostructured polyaniline/cellulose nanocrystals (PANI/CNC) obtained via in situ polymerization[END_REF]. PANI was synthesized in the presence of other materials such as CNC to overcome this issue. Nepomuceno et al. synthesized PANI via in situ polymerization in the presence of CNC; the latter played the role of a backbone to PANI deposition. The group reported that while adding aniline to CNC, the amino group of aniline interacted with the hydrogen bond between the hydroxide group of CNC, which promoted the in-situ polymerization. The negative charge on the CNC surface interacted with positively charged amine groups of polyanilines which slowed the polymerization time. In other words, increasing the amount of aniline monomer enhanced the probability of polymerization occurring, thus decreasing the polymerization time. Increasing PANI concentration led to CNC coating, facilitating electrons' mobility. For a PANI/CNC ratio of 0.4, the conductivity obtained was 8.9x10 -1 S cm -1 ; this value matches the requirement for using sensors and electromagnetic interference shielding [START_REF] Nepomuceno | Evaluation of conductivity of nanostructured polyaniline/cellulose nanocrystals (PANI/CNC) obtained via in situ polymerization[END_REF]. To increase the stability of CNC/PANI, Wang et al. used cellulose nanofibers as a dispersant. The obtained film exhibits a conductivity of 104.7 S cm -1 . The group reported that the presence of CNF avoids the agglomeration of CNC and PANI (S. [START_REF] Wang | Cellulose nanofiber-assisted dispersion of cellulose nanocrystals@polyaniline in water and its conductive films[END_REF]. Wu et al. have also used polypyrrole with CNC to develop a nanocomposite material for supercapacitor application. To obtain a well-controlled and uniform coating of PPy, poly(N-vinylpyrrolidone) PVP was first adsorbed on the CNC surface to modify the hydrophilicity of CNC and favored the coating of PPy. PPy also acts as a stabilizer that inhibits the formation of aggregates. Due to hydrogen bonds between the carbonyl groups of PVP and the N-H group of pyrroles, the PPY growth uniformly on PVP-coated CNC. The core-shell PPy/PVP/CNC material exhibit a conductivity of 36.9 S cm -1 (X. [START_REF] Wu | Conductive cellulose nanocrystals with high cycling stability for supercapacitor applications[END_REF]. nanoparticles. The formed material had unique electroconductive properties due to the support of PPy with the presence of an excess of charge carries (El-nahrawy et al., 2020).

Water treatment

Nanocomposite membranes are promising materials for mitigating fouling and compromise between selectivity and permeability. Cellulose nanocrystal presents an excellent material to be part of this nanocomposite membrane, so the last can exhibit all the properties mentioned. The choice of cellulose nanocrystal is because they can be easily functionalized with other moieties to enhance their efficiency in retaining pollutants. Moreover, their hydrophilic nature due to the presence of hydrophilic groups can help mitigate fouling (C. Xu et al., 2020). The presence of CNC contributes to the membrane's good mechanical properties by increasing the tensile strength [START_REF] Cruz-Tato | Metalized Nanocellulose Composites as a Feasible Material for Membrane Supports: Design and Applications for Water Treatment[END_REF], and it is favored over other nanoparticles because it is considered a renewable, non-toxic, and biodegradable material [START_REF] Asempour | Synthesis and characterization of novel Cellulose Nanocrystals-based Thin Film Nanocomposite membranes for reverse osmosis applications[END_REF] CNC was incorporated with different materials such as polyethersulfone (PES), polyamide as an active layer (PA), polysulfone (PSF), and polyvinylidene fluoride (PVDF), among others to remove diverse pollutants (Table 6).

Several researchers used PES to develop nanocomposite membranes due to its high thermal stability and chemical resistance in organic solvents [START_REF] Bai | Cellulose nanocrystal-blended polyethersulfone membranes for enhanced removal of natural organic matter and alleviation of membrane fouling[END_REF]. Janoobi et al. developed a nanocomposite membrane based on PES and CNC to remove color and reduce chemical oxygen demand from industrial wastewater. The group first modified CNC with (3-aminopropyl) triethoxysilane (APTES), then mixed it with polyethersulfone polymer. The membrane was then prepared based on this blend solution. Adsorption tests showed that the presence of CNC enhanced the removal of color and contributed to the reduction of chemical oxygen demand. The adsorption was ensured by hydrogen interaction between hydroxyl groups of CNC and the different groups of the colored compounds. The presence of amino group from APTES help increase the removal of color due to its interaction with anionic groups of color, so they play the role of new adsorption sites [START_REF] Jonoobi | Characterization and properties of polyethersulfone/ modified cellulose nanocrystals nanocomposite membranes[END_REF]. Another reason to prepare composite membranes using CNC is to prevent fouling. Bai et al. used CNC due to its hydrophilic property. Comparing composite membrane based on CNC and PES to neat PES membrane, they note that the composite membrane is more hydrophilic, has a higher porosity than the neat one, and exhibits good performance in terms of antifouling ability. However, the excess of CNC renders the membrane surface rough due to the poor dispersibility of high concentrated solution of CNC, which impact the porosity and the permeability of the nanocomposite membrane too (Langming [START_REF] Bai | Cellulose nanocrystal-blended polyethersulfone membranes for enhanced removal of natural organic matter and alleviation of membrane fouling[END_REF]. To endow a nanofiltration membrane with antifouling and antibacterial properties, Xu et al. prepared a composite membrane by incorporating CNC and Ag nanoparticles into an active polyamide layer, and PES was applied as the support material for this active layer. The developed membrane exhibits good antifouling and antibacterial performance and helps reduce energy consumption known for the nanofiltration process due to the enhancement in water flux and salt rejection (C. Xu et al., 2020). In the study by Jackson et al., CNC was used only as an anti-microbial agent and incorporated into the membrane; the result showed the inactivation of up to 89% of Escherichia Coli. The group relates the anti-microbial activity of CNC to its rod-like morphology that damage the cell membrane of the bacteria after contact (figure 8) [START_REF] Jackson | Sustainable Cellulose Nanocrystals for Improved Anti-microbial Properties of Thin Film Composite Membranes[END_REF]. [START_REF] Jackson | Sustainable Cellulose Nanocrystals for Improved Anti-microbial Properties of Thin Film Composite Membranes[END_REF]. American Chemical Society Copyright (2021).

Packaging

CNC has been widely used in packaging as a reinforcing material. Due to the CNC network, the mechanical properties of the reinforced material improve significantly (S. [START_REF] Liu | Polydopamine-coated cellulose nanocrystals as an active ingredient in poly(vinyl alcohol) films towards intensifying packaging application potential[END_REF]. Furthermore, this network plays the role of a barrier to water vapor and oxygen, one of the crucial criteria for selecting the packaging material.

Packaging films based on CNC blending with PLA and PVA were extensively studied and showed promising results in thermal stability, stiffness, and anti-microbial activity. [START_REF] Pal | Reduced graphene oxide and PEG-grafted TEMPO-oxidized cellulose nanocrystal reinforced poly-lactic acid nanocomposite film for biomedical application[END_REF] reinforced PLA with CNC and reduced graphene oxide (rGO). The developed film exhibited a higher crystallinity due to the strong physical interaction between CNC and PLA. Thus, the crystallinity increase reduces the water vapor permeability value. [START_REF] Salmieri | Anti-microbial nanocomposite films made of poly(lactic acid)-cellulose nanocrystals (PLA-CNC) in food applications: Part A-effect of nisin release on the inactivation of Listeria monocytogenes in ham[END_REF] developed a bioactive packaging film based on CNC-PLA coated by nisin, the results showed a promising inhibiting capacity against Listeria monocytogenes. On the other hand, [START_REF] Dey | Physical, antifungal, and biodegradable properties of cellulose nanocrystals and chitosan nanoparticles for food packaging application[END_REF] emphasize the antifungal property of CNC, incorporated into PVA film, against postharvest pathogens. The study showed the potential application of CNC for the packaging of fresh fruits. CNC was incorporated into other natural polysaccharides, such as alginate, chitosan, and carrageenan [START_REF] Yadav | Fabrication of cellulose nanocrystal/silver/alginate bionanocomposite films with enhanced mechanical and barrier properties for food packaging application[END_REF]. In general, polysaccharides are a promising material for packaging due to the considerable advantages they present as the preservation of aromas, inhibition of oxygen penetration, and water absorption, not to mention their abundance and price [START_REF] Li | Preparation and characterization of carboxymethyl cellulose-based composite films reinforced by cellulose nanocrystals derived from pea hull waste for food packaging applications[END_REF].

Biomedical

CNC has been known to be biocompatible and non-toxic for biomedical applications. It can be used for artificial blood vessels, tissue engineering [START_REF] Dugan | Directing the morphology and differentiation of skeletal muscle cells using oriented cellulose nanowhiskers[END_REF], drug delivery [START_REF] Lin | Biocompatible Double-Membrane Hydrogels from Cationic Cellulose Nanocrystals and Anionic Alginate as Complexing Drugs Codelivery[END_REF]), etc. (Zeimaran et al., 2019) reinforced urethane with CNC to develop a self-healing nanocomposite film. The developed material showed an excellent cytocompatibility toward human dermal fibroblasts, making it promising for application in material engineering. In a more concrete study, (L. [START_REF] Qin | Preparation of collagen/cellulose nanocrystals composite films and their potential applications in corneal repair[END_REF] introduced CNC into a collagen-based film for corneal repair. The film was tested in vitro on rabbit corneal epithelial cells and keratocytes. The film showed its effectiveness in promoting epithelial wound healing and inhibiting keratocyte-myofibroblast transformation, which emphasizes CNC's potential to be applied as reinforcing agent material for corneal regeneration and reconstruction. A film based only on CNC can also be applied for medical purposes. As the study by [START_REF] Prathapan | Surface engineering of transparent cellulose nanocrystal coatings for biomedical applications[END_REF] affirmed, the researchers prove the ability to use CNC film for blood typing. Despite all these advantages, the nanotoxicity of CNC needs to be more investigated since some studies reported some possible CNC toxic outcomes [START_REF] Seabra | Cellulose nanocrystals as carriers in medicine and their toxicities: A review[END_REF].

Summary

Cellulose is the most abundant biopolymer that can replace petroleum-based materials that harm health and the environment. CNC can be extracted from native cellulose and is known for its important mechanical properties (similar to Kevlar), surface reactivity, and its ability to self-assemble. Chemical modification of CNC broadens its application in different domains of material science and enhances its compatibility with other materials.

Developing film and membrane based on CNC or modified CNC is a good solution to minimize the use of nonbiodegradable material. These films and membranes could be used in various applications such as biosensors, electrodes, and membranes for water treatment, to name a few. Developing composite material from CNC could provide solutions to a broad range of technological bottlenecks in the industry and replace toxic materials currently being banned from use due to their impact on human health and environmental issues.

  1. Introduction .................................................................................................................................................... 3 2. Cellulose origin, structure, and its property to form film ............................................................................... 4 3. Chemical modification of cellulose nanocrystal ............................................................................................ 7 3.1. Graft polymerization .............................................................................................................................. 7 3.1.1. Atom transfer radical polymerization (ATRP) ............................................................................. 8 3.1.2. Reversible addition-fragmentation chain-transfer (RAFT) ........................................................... 9

Figure 2 .

 2 Figure 2. Cellobiose unit composed of two β-D-glucopyranose units.

Figure 3 .

 3 Figure 3. (A) Photograph and (B) scanning electron microscopy (SEM) image of CNC film(Tran et al.,

Figure 4 .

 4 Figure 4. Schematic representation of (a) grafting to the CNC surface and (b) grafting from where

Figure 5 .

 5 Figure 5. Modification of cellulose by grafting polymer via ATRP polymerization (Roy et al., 2009) The Royal Society of Chemistry, Copyright (2009).

Figure 6 :

 6 Figure 6: Schematic representation resuming strategies of chemical modification of CNC with some grafted species 4. Nanocomposite film and membrane-based on cellulose nanocrystal

Figure 7 .

 7 Figure 7. Formation of the film via the spin-coating method (Shojaeiarani et al., 2020). Elsevier Copyright (2019). Electrospun nanocomposite mats-based chitosan/Polyethyleneoxide /CNC was prepared by Nasri et al. using CNC isolated by hydrochloric acid or sulphuric acid to investigate the effect of surface functionalities of CNC.

  Incorporating nanoparticles into CNC matrices enhances conductivity, as reported by El-Nahrawy et al. The group developed a nanocomposite material consisting of CNC coated with PPy and Ag Nanoparticles and (Ni 2 O 3 )

Figure 8 .

 8 Figure 8. Representative scheme of membrane-based on CNC with antibacterial activity(Jackson et al.,

Table 3

 3

	Modified CNC	Polymer

); this grafting aims to enhance CNC's ability to adhesion with another polymer matrix to improve its properties.

Table 1 . Summary of polymers grafted to CNC via ATRP.

 1

	Modified CNC	Agent RAFT	Polymer

Table 2 . Summary of polymers grafted to CNC via RAFT.

 2

	Modified CNC	Polymer	Grafting Method	Reference
	CNC-g-POx	Poly(2-oxazoline)	Grafting to	(Gauche & Felisberti, 2019)
	CNC-g-PEEP	Poly (ethyl ethylene phosphate)	Grafting to	(H. Wang et al., 2015)
	CNC-g-PLA	Poly (Lactide)	Grafting from	(Jianxiang Chen et al., 2017) (Goffin et al., 2011)(Miao & Hamad, 2016)(Lizundia et al.,
				2016)(Habibi et al., 2013) (H. Wu et al., 2016)
	CNC-g-P(CL-co-LA)	Poly(D-lactide-co-	Grafting from	(Muiruri et al., 2017)
		ε,caprolactone)
	CNC-g-PLA6	Poly(caprolactam) (Polyamide 6) Grafting form	(Rahimi & Otaibge, 2016)(Kashani Rahimi & Otaigbe, 2016)
	CNC-g-PCL

Table 4 . Summary of species grafted to CNC via Click reactions

 4

	CNC	Inserted species		Click-reaction	Ref
	CNC-Azide	CNC-alkyne		Cu(I)-catalyzed Huisgen 1,3-	(Filpponen,
					dipolar	2014)
	CNC-Azide	Propargyl-terminated	poly(ethyl	Cu(I)-catalyzed azide-alkyne	(H. Wang et al.,
		ethylene	phosphate) (propargyl-	cycloaddition (CuAAC)	2015)
		PEEP)
	CNC-Azide	Polycaprolactone diol-C≡CH	(CuAAC)	(L. Zhou et al.,
						2018)
	Fe 3 O 4 /CNC-Azide	Propargyl alcohol		(CuAAC)	(Movagharnegad
						et al., 2018)
	CNC-methacrylate	1H,1H,2H,2H-perfluorodecanethiol	Thiol-ene	(Aalbers et al.,
						2019)
	CNC-SH	N-phenyl maleimide		Thiol-ene	(J. L. Huang et
						al., 2014)
	CNC-alkyne	Azide-polycaprolactone diol	(CuAAC)	(L. Zhou et al.,
						2019)

Table 5 . Summary of species grafted to CNC via esterification, with the degree of substitution obtained and their different usage.

 5 .

	Grafted species	Degree	of	Usage	References
		substitution
	Canola oil fatty acid methyl ester	0.5		Hydrophobic coating agent	(Wei et al., 2017)
	Acetic anhydride	2.18		Reinforcing nanocomposite	(Abraham et al.,
					2016)
	Lauroyl chloride	0.2-2.4		Reinforcing nanocomposite	(Trinh	&
					Mekonnen, 2018)
	Succinic anhydride	1.6-2.2		Dye removal	(Emam & Shaheen,
					2019)
	Acetic anhydride	0.18-0.34		Reinforcing nanocomposite	(Ávila Ramírez et
					al., 2017)
	Vinyl acetate-Vinyl cinnamate	-		Emulsifying agents	(Seìbe et al., 2013)
	Palmitoyl chloride	0.3-0.8		Improve dispersibility	(Fumagalli et al.,
					2013)
	Long-chain aliphatic acid chlorides	-		Reinforcing nanocomposite	(Bendahou et al.,
					2015)
	Phenylacetic acid	0.5		Composite manufacturing	(Espino-Pérez et
					al., 2014)
	Benzylacetic acid	0.3
	Rosin	-		Anti-microbial activity	(De Castro et al.,
					2016)
	Malonate	0.16		Reinforcing nanocomposite	(Spinella et al.,
	Malate	0.22			2016)
	Citrate	0.18
	Lactic acid	87.7%		Reinforcing nanocomposite	(H. Wu et al., 2018)
	Malic acid	0.035-0.20		Reinforcing nanocomposite	(Wei Wang et al.,
					2018)
	Valeric acid	0.35		Reinforcing nanocomposite	(Shojaeiarani et al.,
					2019)
	Succinic anhydride	0.12-0.32		Reinforcing nanocomposite	(Leszczyńska et al.,
					2019)
	Octenyl Succinic Anhydride	0.539-0.768		Adsorption of Cu(II) ions	(Gupta et al., 2019)
	3-thiophenetic acid	1.1		Increase planarity of the	(Silva et al., 2021)
				conjugated polymer chains

Table 6 .Summary of composite membranes based on CNC for water treatment with the targeted pollutants and the removal rate

 6

	solving thereby leaching problem related to toxic