Tuan Le
email: tuan.le@insa-toulouse.fr

Dominique Anne-Archard
email: dominique.anne-archard@imft.fr

Véronique Coma
email: veronique.coma@u-bordeaux.fr

Xavier Cameleyre
email: xavier.cameleyre@insa-toulouse.fr

Eric Lombard
email: eric.lombard@insa-toulouse.fr

Kim Anh To

Tuan Anh Pham

Tien-Cuong Cuong Nguyen

Luc Fillaudeau
email: luc.fillaudeau@insa-toulouse.fr

Using in-situ viscosimetry and morpho-granulometry to explore hydrolysis mechanisms of filter paper and pretreated Sugarcane bagasse under semi-dilute suspensions

Keywords: In-situ viscosity Particle size distribution Filter paper Sugar cane bagasse Enzyme Hydrolysis CLD, chord length distribution, DLS, diffraction light scattering, d SE , diameter of circle equivalent (m), FBRM, focused beam reflectance measurement, FP, filter paper, FPU, filter paper unit, lc , chord length particle size distribution, Re, Reynolds number (/), SCB, sugarcane bagasse

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Introduction

Lignocellulosic feedstock is certainly the most abundant and the cheapest resource in the world [START_REF] Ogier | Production d'éthanol à partir de biomasse lignocellulosique[END_REF]. It can consist of several agriculture and industry by-products such as sugarcane bagasse, rice straw, corn stove, and wood chips. Sugarcane bagasse is the lignocellulosic residue accumulated during the production of sugar. Chemically, bagasse contains about 50% cellulose, 30% pentosans, and 2.4% ash [START_REF] Pandey | Biotechnological potential of agro-industrial residues. I: sugarcane bagasse[END_REF]. Because of the low ash content in chemical composition compared to other lignocellulosic biomasses such as rice straw (17.5% ash content) and wheat straw (11% ash content), sugarcane bagasse is a very promising feedstock for use in bioprocesses. According to Kim and Dale [START_REF] Kim | Global potential bioethanol production from wasted crops and crop residues[END_REF], the world global production of sugarcane is 328.10 9 kg, which corresponds to around 180.10 9 kg of dry sugarcane bagasse. This resource is an abundant source of raw material for biotransformation. Thus, the conversion of sugarcane bagasse to fuel ethanol has long been under consideration [START_REF] Carvalho | Kinetic study of the enzymatic hydrolysis of sugarcane bagasse[END_REF][START_REF] Gottschalk | Cellulases, xylanases, ␤-glucosidase and ferulic acid esterase produced by Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane bagasse[END_REF][START_REF] Zhao | Batch and multi-step fed-batch enzymatic saccharification of Formiline-pretreated sugarcane bagasse at high solid loadings for high sugar and ethanol titers[END_REF]. However, the processes proposed in these publications remain in laboratory scale and need to be optimized for industrial application.

In the bioconversion of lignocellulosic biomass, enzymatic hydrolysis is crucial because it transforms cellulose/hemicellulose fibers into fermentable substrate. In order to achieve a viable economy, the biotransformation of lignocellulose into monomer sugar has to be performed at high concentrations of raw material [START_REF] Modenbach | Enzymatic hydrolysis of biomass at high-solids loadings-a review[END_REF]. Working at high dry matter concentration, the challenges to solve are not only in the field of biochemistry but also extend to physical aspects. An increase in suspension viscosity leads to a rise in the energy needed for mixing. It also causes poor mixing capacity and limits mass transfer. Consequently, low enzyme distribution and localized hydrolysis can possibly occur. In order to overcome these difficulties, an investigation by physical approach is necessary to understand the mechanism of hydrolysis, as well as to identify key factors limiting conversion yield. To get in-depth knowledge, the biochemical and physical phenomena should be investigated simultaneously because of their strong coupling.

The physical research on lignocellulosic slurries started more than a decade ago [START_REF] Palmqvist | Torque measurements reveal large process differences between materials during high solid enzymatic hydrolysis of pretreated lignocellulose[END_REF][START_REF] Pimenova | Effect of corn stover concentration on rheological characteristics[END_REF][START_REF] Knutsen | Rheology of high-solids biomass slurries for biorefinery applications[END_REF][START_REF] Viamajala | Rheology of corn stover slurries at high solids concentrations-effects of saccharification and particle size[END_REF][START_REF] Um | A comparison of simple rheological parameters and simulation data for Zymomonas mobilis fermentation broths with high substrate loading in a 3-L bioreactor[END_REF]. It includes several analyses, such as suspension viscosity, yield stress, particle morphology characterization, crystallinity, porosity, surface properties, and polymerization degree. Properties of mass and heat transfer, rheological behavior of suspensions, particle morphology and fiber degradation mechanisms appeared as determinant factors of the effectiveness of the process. Lignocellulosic suspensions exhibit non-Newtonian behavior (shear-thinning, viscoplasticity). Suspension viscosity and yield stress strongly depend on substrate concentration and particle size and shape distributions. It is reported that reducing particle size may improve enzymatic hydrolysis efficiency and reduces the viscosity of biomass slurries [START_REF] Dasari | The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries[END_REF][START_REF] Rezania | Enzymatic saccharification and viscosity of sawdust slurries following ultrasonic particle size reduction[END_REF]. The literature reports on the physical properties (yield stress, viscosity, particle size distribution (PSD)) of fiber suspensions at the initial stage [START_REF] Knutsen | Rheology modification and enzyme kinetics of high solids cellulosic slurries[END_REF][START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF][START_REF] Dibble | Particle morphology characterization and manipulation in biomass slurries and the effect on rheological properties and enzymatic conversion[END_REF] and during enzymatic hydrolysis [START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF][START_REF] Pereira | Sugarcane bagasse enzymatic hydrolysis: rheological data as criteria for impeller selection[END_REF][START_REF] Dunaway | Characterization of changes in viscosity and insoluble solids content during enzymatic saccharification of pretreated corn stover slurries[END_REF][START_REF] Geddes | Optimizing cellulase usage for improved mixing and rheological properties of acid-pretreated sugarcane bagasse[END_REF]. A decreasing trend in overall conversion yield with increasing solids loading has been reported for several lignocellulosic substrates [START_REF] Jørgensen | Liquefaction of lignocellulose at high-solids concentrations[END_REF][START_REF] Cara | Influence of solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive tree biomass[END_REF][START_REF] Roche | Laboratory-scale method for enzymatic saccharification of lignocellulosic biomass at high-solids loadings[END_REF]. At high solid concentration, the physical limitations become a sticking point and need to be scrutinized.

A decreasing trend in overall conversion yield with increasing solids loading has been reported for several lignocellulosic substrates [START_REF] Jørgensen | Liquefaction of lignocellulose at high-solids concentrations[END_REF][START_REF] Cara | Influence of solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive tree biomass[END_REF][START_REF] Roche | Laboratory-scale method for enzymatic saccharification of lignocellulosic biomass at high-solids loadings[END_REF]. At high solid concentration, the physical limitations become a sticking point and need to be scrutinized. The rheological understanding of lignocellulosic suspensions is fundamental to the optimization of process efficiency [START_REF] Knutsen | Rheology of high-solids biomass slurries for biorefinery applications[END_REF][START_REF] Stickel | Rheology measurements of a biomass slurry: an inter-laboratory study[END_REF]. Through the bibliography analysis, there is only 3 publications that investigates in parallel the biochemical, morphological and in-situ [START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF] and ex-situ [START_REF] Liu | Optimization of high solids fed-batch saccharification of sugarcane bagasse based on system viscosity changes[END_REF][START_REF] Sánchez | Rheology of pulp suspensions of bleached sugarcane bagasse: effect of consistency and temperature[END_REF] rheological approaches. Aiming to complete this knowledge, and for further research on high dry matter conditions, our strategy was to investigate enzymatic hydrolysis at semi-dilute conditions to understand the fragmentation and solubilization mechanisms. The relationship between in-situ viscosimetry and biochemistry was specifically scrutinized during hydrolysis of two substrates -pretreated sugarcane bagasse (SCB 3% w/v) and milled filter paper (FP 1.5% w/v) -by a commercial cocktail (Ctec2-Novozyme). These concentrations were 1.5-2 times higher than critical concentrations (previously identified [START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF]), which led to non-Newtonian suspension behaviors. Consequently limited particle-particle interactions occurred contrary to dilute conditions, but without the highest complexity of high dry matter contents. This controlled complexity enabled further exploration of the physical limitations during hydrolysis of cellulosic substrates. Various enzyme-to-substrate (E/S) ratios (0.3-25 FPU/g cellulose) were studied with a multi-instrumented experimental set-up. Semi-dilute conditions and low activities (0.3-3 FPU/g cellulose) highlighted specific phenomena such as viscosity overtaking and magnitude of particle fragmentation versus solubilization. These operating conditions provided slow hydrolysis kinetics that facilitated observation and characterization of the phenomena at macroscopic (viscosimetry), microscopic (granulometry) and biochemical (sugar concentration) levels. Moreover, these conditions allow negating end-product inhibition, minimizing the transfer limitations, homogenizing enzyme and suspension distributions.

First, biochemical and physicochemical properties of initial suspensions are compared. Second, hydrolysis rates and viscosity are discussed as a function of the E/S ratio. Then, the final step focuses on PSD and chord length distribution (CLD) analysis, with exploration of the particle size evolution under enzymatic reaction in relation to glucose conversion yield and suspension viscosity.

Materials and methods

Substrates and enzymes

Pretreated sugarcane bagasse (SCB) obtained from the Center of Research and Development in Biotechnology, SBFT-HUST, Hanoi, Vietnam (CRDB), was used as real lignocellulose substrate. Raw bagasse was collected from the Lam Son Sugar factory (Thanh Hoa, Vietnam), then pretreated using the organosolv process (thermochemical pretreatment using formic acid). It was deformylated by diluted NaOH and washed until neutral pH. Pretreated SCB was extruded to homogenize the particle size (EuroLab 16 Extruder, 400 mm failure, extrusion line: 25 L/D 18/25 conveying, 7/25 shear stress). Extruded SCB was stored at 4 • C until use (dry matter content ∼25%). Its chemical composition consisted of 4.5% hemicellulose, 79.9% cellulose, 1.4% lignin. The analysis was performed using FibertecTM following the Van-Soest method [START_REF] Van-Soest | Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin[END_REF].

Dried and milled (Bosch MKM6003 mill) filter paper (Whatman International Ltd., Maidstone, England, Cat No. 1001 090) was selected as reference for cellulosic substrate. The composition of filter paper (FP) was 99.68% cellulose. It did not contain hemicellulose.

Surface tension analysis showed interfacial interaction free energy ( G SES ) of 34.2 mJ/m 2 and 6.44 mJ/m 2 , and hydration free energy ( G SE ) of -146.3 mJ/m 2 and -130.1 mJ/m 2 respectively for FP and SCB. Analysis was carried out using a Drop Shape Analyzer DSA-100 (KRUSS) following tangent and Young-Laplace methods. These results proved the similar hydrophilic properties of both substrates.

With increasing concentration, both FP and SCB suspensions behaved in an increasingly non-Newtonian way. A critical concentration specific to each substrate can be observed [START_REF] Nguyen | In-situ and Ex-situ Multi-scale Physical Metrologies to Investigate the Destructuration Mechanisms of Lignocellulosic Matrices and Release Kinetics of Fermentable Cellulosic Carbon, Ingenieries Microbiennes Et Enzymatiques[END_REF]; this concentration is defined as the passage from dilute behavior (substrate concentration has minor effect on suspension viscosity) to semi-dilute and concentrated behavior (substrate concentration greatly influences suspension viscosity and non-Newtonian behavior). Rheological behavior of suspensions was investigated at 40 • C, with substrate concentration varying up to 15 g/L for FP and 53 g/L for SCB. The mixing rate was in the range of 20-250 min -1 . The critical concentration was identified as 7 g/L for FP and 23.9 g/L for SCB.

The enzymatic cocktail used was Cellic Ctec2 from Novozymes (Serial 0139 batch VCN10002), which is a commercial lignocellulase cocktail. Protein concentration was 64.8 ± 4.7 mg/mL following Bradford assay and Bovine Serum Albumin (BSA) as standard. Enzy-matic activity and thermo-stability were characterized in specific conditions (T = 40 • C, pH = 4.8) following the IUPAC method [START_REF] Simha | A treatment of the viscosity of concentrated suspensions[END_REF].

One unit of filter paper (FPU), endoglucanase (CMCU) and exoglucanase (AVCU) activities [START_REF] Riedel | Synergistic interaction of the Clostridium stercorarium cellulases Avicelase I (CelZ) and Avicelase II (CelY) in the degradation of microcrystalline cellulose[END_REF] equal to the amount of enzyme to release 1 mol/min of glucose as reducing sugar from respectively 50 mg filter paper, 2% CMC (carboxyl methyl cellulose, degree of substitution 7 M) and 2% Avicel suspension (micro-crystalline cellulose 90 m). One unit of cellobiase activity (CBU) is equivalent to the amount of enzyme to release 2 mol/min of glucose from 15 mM cellobiose.

Activities per mL of cocktail are equal to 103 FPU, 104 AVCU, 831 CMCU and 3796 CBU. Enzyme was stored at 4 • C until used. Enzyme dosage in hydrolysis experiments was based on FPU per gram of cellulose on dry weight basis.

Experimental setup

The experimental setup includes a double jacket glass bioreactor (d = 130 mm, H = 244 mm, V = 2.0 L) equipped with a homedesigned impeller system associated with several in-situ sensors (temperature, pH, rotation speed, torque, FBRM). The impeller is an assemblage of two classical ones, mounted on the same shaft. The first one is a three inclined blades (diameter: 73.5 mm, angle: 45 • , h = 38 mm) located at 75 mm height from the bottom to ensure mixing in the central region of reactor as well as suspension homogeneity. The second is a close bottom mixer including 2 large blades (diameter: 120 mm, h = 22 mm) to avoid substrate decantation. A Haake VT550 viscometer (Thermo Fisher Scientific ref: 002-7026) was used to ensure mixing at specific rotation speed as well as in-situ torque measurements. The temperature was controlled by water circulation (combined cryostat Haake DC30-K20, Thermo Fisher Scientific) through the water jacket of the bioreactor. The viscometer and the cryostat were controlled by original software from Haake (RheoWin Job Manager) that also ensured real-time data recording (temperature, torque, mixing rate). The pH of the suspension was controlled and auto-adjusted by a Biostat-B (Sartorius Stedim Biotech) via home-designed software created in the LabVIEW environment. Finally, a focused beam reflectance sensor (FBRM-G400-Mettler Toledo) was located inside the reactor in order to measure the distribution of particle chords. A global description of the whole system is illustrated in Fig. 1.

Chemical and physical analysis 2.3.1. Mono-and disaccharide concentrations

Mono-and disaccharides were analyzed using highperformance liquid chromatography (HPLC, Agilent Technologies, 1200 series). Measurements were performed on an Aminex HPX-87P column #1250098 with micro-guard Carbo-P refill cartridges #1250119 (both from Bio-Rad). Separation was carried at flow rate 0.5 mL/min, column temperature 60 • C using HPLC-grade water (LiChrosolv, Merck) as mobile phase and refractive index detector. All samples were centrifuged at 10,000 min -1 for 10 min, then filtered through 0.22 m filters before injection into the column. Calibration curves were established with standard solutions of cellobiose, glucose, xylose, arabinose, galactose, and mannose (concentration ranging between 0 to 1 g/L). The lower and upper LOD are equal to 0.01 and 1 g/L respectively and the LOQ is equal to 0.036 g/L.

Dry matter content and volume fraction

The water content of substrates and hydrolyzed suspensions was determined by drying at high temperature and ambient pressure. A known mass of sample (m 1 equivalent to 1-3 mL) was filtered through filter paper (Whatman paper N • 1, known mass, m 0 ), then placed in the oven at 105 • C, for 12 h and weighed up to constant mass (m 2 ). Water content (W) and dry matter (DM) were then calculated from these measurements with an accuracy of ±2%. The volume fraction ( V ) is defined as the volume of particles (V p ) divided by the volume of whole suspension (V sus = V p + V water ) prior to mixing. In the present work, this volume fraction is calculated from mass balance by considering the initial concentration, the hydrolysis yield and particle density. DM and x DM are respectively particle density (g/L) and water-insoluble fraction (g).

v = V p V sus = x DM / DM V sus (1)
2.3.3. In-situ viscosimetry In-situ viscosimetry was conducted throughout hydrolysis, based on the real-time monitoring of torque and mixing rate and using the Metzner-Otto concept when fluids are non Newtonian ones [START_REF] Metzner | Agitation of non-Newtonian fluids[END_REF].

In laminar flows and for Newtonian fluids, the product of the Reynolds number (Re) by the Power number (Np) is constant and usually written as:

Np = Kp • 1 Re (2)
The power consumption curve, Np = f (Re) was characterized using Newtonian reference fluids-distilled water, glycerol, and Marcol 52 oil (Exxon Mobil). Eq. ( 2) for laminar regime can be extended to turbulent ones (until a critical Re value) and described by a unique equation: Eq. ( 3).

Np = Kp Re ˛+ Np 0 1/˛( 3)
Kp and ␣ are two constants that only depend on the mixing system geometry. Np 0 is the power number for turbulent flows. Experimental results on our system gave Np 0 = 0.017, ␣ = 0.75, Kp = 115.2 and shown that a laminar regime prevailed up to Re = 41.

When fluids are non-Newtonian, Eq. ( 3) is still valid as long as a generalized Reynolds number is used. This last one is calculated using the Metzner-Otto (MO) concept which allows the determination of an equivalent viscosity from the rotation frequency. The MO concept introduces a constant Ks which only depends on geometrical characteristics of the mixing system. Ks was determined experimentally using 0.04%-0.1% shear-thinning xanthan solutions prepared in a saturated solution of glucose and saccharose as reference fluids. The Ks value of our system is equal to 32. The concept can be extended up to transition flow [START_REF] Jahangiri | Investigation of the viscoelastic effect on the metzner and otto coefficient through LDA velocity measurements[END_REF]. In this study, the application of a power consumption curve to calculate suspension viscosity and establish an in-situ rheogram was extended to transitional flow, which is equivalent to Re < 1000. In turbulent flow, the power consumption curve is limited to viscosity determination, but limited up to Re = 30000 (beyond this value, Np is almost constant). Detailed methodology was previously described [START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF].

Ex-situ diffraction light scattering (DLS)

The volume-weighted particle size distribution (PSD) was determined by diffraction light scattering (DLS, Mastersizer 2000, Malvern Inst., range from 0.02 to 2000 m, red = 632.8 nm and blue = 470.0 nm light) using Mie scattering theory. A known volume of suspension (0.5-3 mL) was added to a water circulation loop (20 • C ± 2) in order to obtain laser obscuration between 5% and 40%. The whole suspension was mixed by a Heidolph magnetic stirrer at 200 min -1 while the circulation loop was maintained by a Masterflex L/S model 7553-79 at pump speed 240 min -1 . For each sample, measurements were performed in triplicate for 3 dilution rates and the average result was taken.

Laser diffraction analysis converts the detected scattered light into a PSD. This method involves measurement based on volume and identifies a diameter of equivalent sphere, d SE . This interpretation of DLS measurements is based on strong assumptions, namely spherical particle shape and a known refractive index. For our substrates (fibrous shape and multimodal distribution), it suffers from a significant deviation compared to the ideal case. Nevertheless, the equivalent sphere diameter was used and will be discussed under these restrictions. Volume-weighted particle size distribution (E v ) was calculated by the Mastersizer software and then weighted by volume fraction ( v ) in order to integrate the impact of solubilization on suspended population and to allow comparison between distributions at different hydrolysis times.

In-situ focus beam reflectance measurement (FBRM)

Focus beam reflectance measurements enable in-situ quantification and characterization of chord length distribution (CLD). In-situ CLD of particles was analyzed using an FBRM ® G400 probe (Mettler Toledo, range: 0.1-1000 m, laser light source = 795 nm, laser source rotation: 2 m/s). This probe was placed in the reactor and allowed for real-time tracking of chord length and particle count during enzymatic hydrolysis. However, operating conditions (mixing rate, suspension viscosity) that affect flow pattern may also influence the count number. Thus for N c , discussion and comparison will be restricted to almost constant viscosity phases. Thousands of individual chord lengths are typically measured each second to produce the number-weighted CLD, which is the fundamental measurement provided by FBRM ® . The conversion from number-weighted CLD into volume-weighted PSD is possible under severe assumptions [START_REF] Nguyen | Rheology of lignocellulose suspensions and impact of hydrolysis: a review[END_REF], but they are inappropriate for FP and SCB substrates because they contain particles of various shapes and dispersed sizes. Consequently, the number-weighted CLD, E n (lc), and the average number of chords counted per second, N c , will be used as indicators of population evolution. In order to facilitate compari-son between experiments, normalized parameters were defined as the ratio between instantaneous value and initial value at t = 0 h Normalized mean chord length:

l * c (t) = l c (t) l c (t = 0h) (4)
Normalized total chord count:

N * c (t) = N c (t) N c (t = 0h) (5)
Furthermore, as native measurements are number-based, the underrepresented classes in widespread population may become non-significant. This will be the case for the largest particles with FP and SCB substrates.

Operating conditions and protocols

Experiments were performed at 1.5% w/v for FP, and at 3% w/v for SCB. These concentrations are 1.5-2 times higher than critical concentration, in order to work with non-Newtonian behaviors. These conditions generate particle-particle interactions, which give rise to the complexity emanating from high dry matter contents.

The enzymatic reactions were carried out over 24 h at 100 min -1 , 40 • C, pH 4.8 ± 0.2 with a total volume 1.3 L. Enzyme-tosubstrate ratios (E/S) were adjusted from 0.3 to 25 FPU/g cellulose. An antibiotic (1.3 mL of chloramphenicol 50 g L -1 ) was added to prevent microbial contamination. Experiments were divided into two steps: i) homogenization of the suspension, and ii) enzymatic hydrolysis. In the first step, the material was mixed with water at 100 min -1 for a given duration (2-3 h) in order to achieve a homogeneous suspension and constant torque. In the second step, enzymes were added (at time t = 0) and in-situ measurements (torque, pH, T, CLD) made. In the results presentation, knowing the non-Newtonian behavior of suspensions, one specific value of viscosity was chosen to track the time evolution of hydrolysis, namely the viscosity at 100 min -1 . Moreover, the non-Newtonian behavior was investigated every 30 min by repeating the cycle: (i) mixing at 100 min -1 for 28 min, (ii) shifting at 125 min -1 for 1 min, and (iii) linear slowdown to 100 min -1 in 1 min. Data acquisition period was adjusted to 1 min at 100 min -1 and decreased to 10 s in other conditions. Mean data were recorded in each period. During the step at 125 min -1 (1 min), mean values were calculated for stabilized torque. Samples (20 mL) were taken at 0 h, 1 h, 2 h, 3 h, 6 h, 12 h, 18 h and 24 h for biochemical and granulometric analysis.

Results and discussion

Comparison of initial suspensions

To evaluate the reproducibility of experimental conditions and the differences between FP and SCB, morphogranulometry and viscosity were specifically scrutinized. The morphogranulometric properties were investigated via different analyses: (i) microscopic observation provided qualitative information about particle shape and size, (ii) DLS measurements were well adapted to coarse particle fraction (volume distribution), and (iii) focused beam reflectance measurements highlighted the fine particle population (number distribution).

Microscopic observations of FP and SCB suspensions (Fig. 2A,B) show complex mixtures of coarse and fine particles with different morphologies and ratios. For FP suspensions, Fig. 2-A indicates a majority of long cellulosic fibers that are crossed or separated, with a large fiber length-to-diameter ratio. For SCB suspensions, the particle size appears generally smaller than for FP. The proportion of fine particles seems to be higher for SCB suspensions.

Volume-weighted distributions of equivalent sphere diameter are reported in Fig. 3-A In volume-weighted distributions, few coarse fibers may account for a large volume fraction, whereas they will be negligible when considering a number-weighted distribution. This is exacerbated with multimodal distributions with high size differences between fine and coarse subpopulations.

Considering the initial suspension viscosity measured at 100 min -1 , large differences were observed between FP and SCB that cannot be explained by surface tension and hydrophobic/hydrophilic properties. Particle size dispersion needs to be carefully considered, as it strongly affects suspension rheology. Strongly different behaviors may be exhibited between mixtures of hard sphere populations (ideal case with monodisperse particles) [START_REF] Nguyen | In-situ and Ex-situ Multi-scale Physical Metrologies to Investigate the Destructuration Mechanisms of Lignocellulosic Matrices and Release Kinetics of Fermentable Cellulosic Carbon, Ingenieries Microbiennes Et Enzymatiques[END_REF][START_REF] Giesekus | Disperse systems: dependence of rheological properties on the type of flow with implications for food rheology[END_REF] and complex shape populations (real case with broad size distribution -spreading -and complex mechanical properties) [START_REF] Viamajala | Rheology of corn stover slurries at high solids concentrations-effects of saccharification and particle size[END_REF][START_REF] Dasari | The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries[END_REF][START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF]. For mixtures of hard spheres (D = 33-236 m for glass beads and D = 84-141 nm for latex beads) with narrow distribution at a given volume fraction, monomodal suspensions have a higher viscosity than bimodal suspensions. This relationship was demonstrated on both Brownian and non-Brownian hard spheres for various diameter ratios from 2.7 to 22.1. The minimal viscosity is attributed to the presence of small particles between the larger ones in bimodal suspensions. In that way, at a given concentration, if the fiber length grows, the interaction probability between particles increases, especially due to entanglement, and this generates an increase in viscosity [START_REF] Nguyen | Rheology of lignocellulose suspensions and impact of hydrolysis: a review[END_REF]. Suspension viscosity is controlled by particle concentration, but also by parameters such as morphological aspects (aspect ratio, shape, size, etc.), agglomerates and particle entanglement, surface properties, swelling propensity, and deformation capability [START_REF] Nguyen | Rheology of lignocellulose suspensions and impact of hydrolysis: a review[END_REF][START_REF] Roberts | Using the microsoft excel solver tool to perform non-linear curve fitting, using a range of non-Newtonian flow curves as examples[END_REF][START_REF] Correa | Power consumption evaluation of different fed-batch strategies for enzymatic hydrolysis of sugarcane bagasse[END_REF][START_REF] Samaniuk | Effects of process variables on the yield stress of rheologically modified biomass[END_REF][START_REF] Radeva | Kinetic model of enzymatic hydrolysis of steam-exploded wheat straw[END_REF] These numerous parameters lead to an ambiguous and hardly quantifiable definition of particle volume fraction. In this context, results reported by Dasari and Berson [START_REF] Dasari | The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries[END_REF] indicated correlation between particle size and suspension viscosity with sawdust without considering this complexity. The same observations were stated by Viamajala et al. [START_REF] Viamajala | Rheology of corn stover slurries at high solids concentrations-effects of saccharification and particle size[END_REF] with pretreated corn stover slurries, and Nguyen et al. [START_REF] Nguyen | In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis[END_REF] for concentrated cellulose fiber suspensions. Viscosities and PSD of FP and SCB suspensions were in agreement with this last assumption considering the relation between suspension viscosity and PSD.

Investigation of suspension viscosity as a function of mixing rate confirms non-Newtonian shear-thinning behavior. The mean viscosities of FP suspension decreased from 0.249 Pa s at 100 min -1 to 0.183 Pa s at 125 min -1 . This behavior is largely reported in literature [START_REF] Nguyen | Rheology of lignocellulose suspensions and impact of hydrolysis: a review[END_REF][START_REF] Derakhshandeh | Rheology of pulp fibre suspensions: a critical review[END_REF].

Fig. 4 reports initial values of size and viscosity for the different experiences with FP and SCB suspensions. It illustrates the reproducibility of these measurements. For FP, the deviations are equal to 5.4% and 3.5% for count numbers and mean chord length respectively. These same numbers for SCB are 1.9% and 2.6%, indicating good reproducibility between experiments. The same accuracy is observed for the viscosity of initial suspensions, with deviation of 2.8% and 6.2% for FP and SCB respectively.

Hydrolysis rates

Experiments were conducted for enzyme-to-substrate ratios ranging from 0.3 to 25 FPU/g cellulose. The bioconversion yields from cellulose into glucose after 24 h hydrolysis varied to a large extent as illustrated in Fig. 5. Our research strategy was not to reach the highest yields but to observe physical phenomena under limited and controlled hydrolysis conditions. Thus, the characterization of rheological and morphogranulometric properties during enzymatic reaction at different levels of substrate solubilization was considered. At 0.3 FPU/g cellulose, the bioconversion rates were negligible and equal to 3.5% and 3.1% for FP and SCB respectively. As the conversion rate stayed very low, the variation of substrate concentration during hydrolysis at 0.3 FPU/g cellulose can be neglected. Up to 3 FPU/g cellulose, the hydrolysis yields were almost similar between the two substrates. However, at the highest dosage (25 FPU/g cellulose), it is reported that FP was less accessible to enzymes than SCB, and the glucose yields after 24 h reached respectively 50.1% and 82.1% for their suspensions. These results are in good agreement with the morphogranulometric properties of the initial suspension ( §3.1), as SCB suspensions contain a majority of short fragments enhancing specific surface.

In-situ viscosimetry

The time-viscosity curves of FP and SCB suspensions during hydrolysis for various Ctec2 ratios are reported in Fig. 6A andB. These curves demonstrate the ability to finely quantify in-situ viscosity during bio-catalysis, even for low values (SCB). The reference curves (no activity) demonstrate the absence of significant change in viscosity over 24 h. With enzyme activities, the curves fulfill a dose-effect response, but in a non-linear mode. However, the evolution and the curve shapes strongly differ between FP and SCB. These trends and differences are exacerbated with the lowest E/S ratios.

With FP suspensions, a monotone decrease of viscosity is observed, and this decrease is all the more rapid because enzyme loading is high. At 25 FPU/g cellulose, the FP suspension was liquefied in a short time: after 1 h hydrolysis, viscosity was only 5.8% of its initial value. At 3 FPU/g cellulose, it took 3 h to observe a 90% reduction in viscosity. In both cases, when hydrolysis was carried out, the suspension viscosity quickly reached the supernatant (water) viscosity. Turbulent flow regime was then rapidly established, and the estimation of in-situ viscosity was poorly reliable. The same limitation during ex-situ rheometry was encountered by Rosgaard et al. [START_REF] Rosgaard | Effects of substrate loading on enzymatic hydrolysis and viscosity of pretreated barley straw[END_REF] with steam-pretreated barley straw, due to a shift in turbulent flow at high shear rates. At 0.3 FPU/g cellulose, the viscosity decline was slower and longer, even after 12 h of hydrolysis. A final viscosity of 0.09 Pa s was observed, equivalent to a decrease of 64% compared to the initial value. During enzymatic hydrolysis at 3 FPU/g cellulose, the FP suspension preserved its shear-thinning properties. The ratio of viscosity between 100 min -1 and 125 min -1 fluctuated around 1.35-1.41 from the beginning up to 10 h. The shear-thinning behavior of the reaction medium was then roughly constant during this period and did not seem to be drastically modified. Beyond this point, viscosity calculation was forbidden due to the appearance of the turbulent regime. For SCB, similar analysis was not performed because of the low magnitude of viscosity at 100 and 125 min -1 , which did not ensure accurate calculation.

It is noticeable that the liquefaction (viscosity reduction) is not directly related to the hydrolysis (glucose) yield. At 25 FPU/g cellulose, the glucose yield was only 14.8% after 1 h hydrolysis, while viscosity was reduced by 94.2%. From 1 h until 24 h, no evolution in suspension viscosity was observed, whereas glucose yields increased gradually up to 53.0%. At 3 FPU/g cellulose, a similar assessment was established with 6.8% glucose yield after 3 h, which corresponded to a 90% reduction in viscosity. Beyond 3 h, the glucose yield increased up to 24.8%. Liquefaction kinetics were higher than solubilization ones, whatever the operating conditions. The hydrolysis of cellulosic fiber consists of three actions associated with enzyme activities present in the cocktail. The random breakage of high-DP molecules into smaller ones by endoglucanase facilitates exoglucanase activity (attacking fibers from their ends and liberating cellobiose). Then ␤-glucosidase finally works on the conversion of cellobiose into glucose and has a minor effect on suspension viscosity but greatly increases hydrolysis yield. Obtained results prove that for semi-dilute conditions, liquefaction is a preliminary step for glucose production. At higher solid loading, when physical limitation becomes predominant, the role of liquefaction might be exacerbated.

With the SCB suspension (Fig. 6A), the time evolution of viscosity strongly differs and exhibits an initial overtaking before decrease. With the reference experiment (no enzyme added), a slight and linear increase in suspension viscosity by less than 15% after 24 h was observed. At enzyme loading ranged between 0.3 and 10 FPU/g cellulose, a significant increase in suspension viscosity at almost the same magnitude was witnessed during the first stage of hydrolysis. This was then followed by a stable or decreasing trend depending on the E/S ratio. Compared to the reference experiment (no activity), the viscosity overtaking then results from enzyme activities on SCB. At 0.3, 3, and 10 FPU/g cellulose, the glucose conversion yield at excess points was insignificant and equal to 1.2%, 2.9%, and 1.9%, respectively. Therefore, the substrate concentration cannot explain the evolution of suspension viscosity. A swelling effect of paper pulp, which is gradually hydrated, was suspected to explain this viscosity overtaking, but no conclusion can be stated on the basis of the results that have been presented. Basically, the enzymatic hydrolysis of lignocellulosic materials causes viscosity decrease via two mechanisms, including the fragmentation of coarse particles and the solubilization of fine particles. The viscosity excess under enzymatic action appears to be incomprehensible following a biochemical approach. Its interpretation requires further investigation of physical parameters, such as particle size and/or shape, in order to propose coherent mechanisms.

Granulometric analysis

As expected, a shift to smaller dimensions was observed during hydrolysis. Considering the influence of different physical factors on suspension rheological behavior, the volume-weighted PSD as well as the particle volume fraction ( v ) were specifically scrutinized and discussed. Meantime, the chord number N c and the number-weighted CLD of particles, E n (lc), will provide specific information about the evolution and contribution of the finest population. DLS ( §2.3.4) and FBRM ( §2.3.5) have specificities and limitations in relation to the volume-and number-weighted distributions that constitute the raw measurements. Their complementarity will be used to investigate changes in coarse and fine populations.

Particle size distribution during enzymatic hydrolysis

In order to allow comparison of particle size, PSD was weighted by the particle volumetric fraction ( v ), taking into account the reduction of suspended matter by solubilization during the process. For FP suspensions, Fig. 7 reports two E/S ratios at 3 and 25 FPU/g cellulose in order to examine slow and rapid viscosity evolutions. For SCB suspensions, Fig. 8 reports 0.3 and 3 FPU/g cellulose, considering a focus on viscosity overtaking.

For FP, as previously reported in Fig. 3A, the initial PSD exhibits a large, bimodal spreading distribution of 54% fine (d SE < 200 m) and 46% coarse (d SE > 200 m) populations. Results from DLS analysis (Fig. 7A andB) highlight the fragmentation mechanism on the coarse population at all tested dosages. This suggests a transition into fine population at a magnitude depending on the amount of enzyme loaded. However, the increase in volume-weighted distribution of the fine population was not as strong as expected. It may be due to the fact that DLS measurements based on volume-weighted distribution are probably less sensitive to fine particles. On the other hand, concomitantly with the increase of the fine population by fragmentation of the coarse population, the solubilization mechanism possibly affects and balances the fine population. The evolution of the fine population will be scrutinized deeply following number-weighted distributions using FBRM (see

§3.4.2).

At 25 FPU/g cellulose (Fig. 7B) and after 1 h, E v (d SE ) × v indicates a transition from coarse to fine population. The total volume-weighted of coarse population, v-coarse decreases by 46.3% from t = 0 h to t = 1 h. Simultaneously, the volume fraction of the fine population v-fine increases by 9.4%. These size and vol- ume evolutions correspond to a viscosity collapse of 94.2% (Fig. 6). Beyond 1 h, in parallel with the decrease in coarse population, a significant reduction of fine particle fraction by 45.3% was observed when comparing 1 h and 24 h. However, nearly no change in suspension viscosity is reported during this period. The reduction of total particle volume fraction is correlated to substrate solubilization, as indicated by the increase of glucose yield from 14.8% (t = 1 h) to 53.0% (t = 24 h).

At 3 FPU/g cellulose, similar trends were observed with slower kinetics. Between 0 h and 3 h, 38.2% viscosity reduction was observed, which corresponds to a 26.5% decrease in coarse population (from 0.0053 to 0.0043 for v-coarse ), whereas a 12.5% increase in fine population (from 0.0071 to 0.0079 for v-fine ) . At 3 h, the conversion yield of cellulose into glucose remains inferior to 6%, meaning that no significant change in total particle fraction (from 0.0124 to 0.0116 for V ) was observed. Beyond 3 h, the reduction of coarse and fine populations was demonstrated association with viscosity reduction up to 75.9% at 24 h. Meanwhile, the glucose conversion yield increased from 5% (t = 3 h) to 28.3% (t = 24 h). At 0.3 FPU/g cellulose (data not shown), similar phenomena were observed, with the coarse population decreasing in volume fraction, whereas negligible changes were reported for the fine population.

Szijarto et al. [START_REF] Szijártó | Liquefaction of hydrothermally pretreated wheat straw at high-solids content by purified Trichoderma enzymes[END_REF] reported the dominant role of purified endoglucanase in the liquefaction of pretreated wheat straw slurries. This activity randomly breaks the high DP chains into shorter fragments and causes a quick decrease in suspension viscosity. In our conditions, and assuming that the initial hydrolysis step is controlled by endoglucanase activity on the coarse population, the evolution of viscosity and PSD weighted by particle volume fraction ( V ) appears consistent with this assumption in increasing the number of ending chains. However, granulometric and biochemical mechanisms result from a complex balance between coarse and fine populations taking into account microscopic properties up to biochemical structure. Afterwards, the action of exoglucanase and ␤-glucosidase become more and more significant, leading to an increase in glucose conversion yield and a decrease in the fraction of fine particles.

By hydrolyzing substrate at low E/S loadings (0.3 and 3 FPU/g cellulose), the fraction of coarse particles (d SE > 200 m) was proven to act as the key contributor to viscosity change for FP suspensions. Meanwhile, the fine particle population (d SE < 200 m) was demonstrated as weakly impacting the suspension viscosity. During enzymatic hydrolysis at dilute conditions, the viscosity decreased significantly, mainly due to reductions of particle size, although morphological aspects could also have an effect. At higher solid loading conditions, the role of biomass concentration may become more pronounced. Working on pretreated spruce chips, Wiman et al. [START_REF] Wiman | Rheological characterization of dilute acid pretreted softwood[END_REF] also stated the changes in suspension viscosity during enzymatic hydrolysis are due to both factors: fiber properties and water insoluble solid content.

For SCB, the initial population exhibited monomodal distribution of finer size with small spreading compared to FP (Fig. 3A). Considering the curves of Fig. 8, the evolution of E v (d SE ) × v during enzymatic hydrolysis is less pronounced than for FP, but a slight change in particle size was observed despite a reduction of v . At 3 FPU/g cellulose, a slight increase in size is observed at 1 h and 3 h, although the distribution function was weighted by the volume fraction. This change just follows the viscosity overtaking which was observed at t = 30 min (Fig. 8B). From this point, the evolution of E v (d SE ) × v indicated a reduction in particle volume fraction associated with a shift to lower size. At 0.3 FPU/g cellulose, a slight increase of particle size was observed up to 3 h which then stabilized until t = 12 h (Fig. 8A). This distribution was nearly identical with that of 3 FPU/g cellulose at 1 h. Beyond 3 h, roughly corresponding to the peak in suspension viscosity, the population mainly consisted of coarse particles. The viscosity overtaking was assumed to be related to E v (d SE ) × v evolution. For aggregates corresponding to the biggest particles (Fig. 2B), the release of fibers from aggregates may contribute to a slight shift in PSD, with a moderate increase in particle number (discussed with CLD) and then Many apparent contradictory results can be found in the literature concerning the evolution of viscosity with particle size (for a given concentration or volume fraction). This illustrates the fact that parameters such as particle morphology and/or particle interactions greatly influence rheological behavior [START_REF] Nguyen | Rheology of lignocellulose suspensions and impact of hydrolysis: a review[END_REF][START_REF] Roberts | Using the microsoft excel solver tool to perform non-linear curve fitting, using a range of non-Newtonian flow curves as examples[END_REF][START_REF] Correa | Power consumption evaluation of different fed-batch strategies for enzymatic hydrolysis of sugarcane bagasse[END_REF][START_REF] Samaniuk | Effects of process variables on the yield stress of rheologically modified biomass[END_REF][START_REF] Radeva | Kinetic model of enzymatic hydrolysis of steam-exploded wheat straw[END_REF]. Moreover, the reason for E v (d SE ) × v evolution needs to be examined and assumptions may be postulated. It is evident that cellulase enzymes are able to break down cellulosic fibers and solubilize them in order to produce glucose. Basically, this reaction leads to a decrease in particle size. With respect to the granulometry and morphology of pretreated SCB, a considerable number of agglomerates are present. Once enzymes are added to the substrate suspension, both agglomerates and individual particles are attacked at the same time. Biocatalyst actions on agglomerates probably open the structure and extract/separate the fibers from the agglomerates. In this case, an increase in particle size distribution is realistic. In addition, it would lead to a moderate increase in particle number, as well as particle surfaces, in other words, the average distance between particles would be reduced and interactions increased. Consequently, particle-to-particle interactions are enhanced and the suspension viscosity increases. In contrast, the attack of enzymes on individual particles results in a decrease in viscosity due to the solubilization mechanism. In the first stage of hydrolysis, the effect on agglomerates is stronger than the solubilization of small individual particles, causing a growth in suspension viscosity. As concerns the amount of E/S loading, once the de-structuration of agglomerates reaches a threshold where all agglomerates are separated, the suspension viscosity will gradually decrease. An additional phenomenon to explain this overshot in viscosity is linked to particle shape. Giesekus [START_REF] Giesekus | Disperse systems: dependence of rheological properties on the type of flow with implications for food rheology[END_REF] studied the dependence of glass fiber suspension viscosity versus length/diameter (L/D) ratios. This research revealed that the relative viscosity rose as the L/D proportion increased, or in other words, a suspension of longer fibers will possess higher viscosity than a suspension of shorter ones. For SCB suspensions, the dispersion of agglomerates into individual fibers can be considered an evolution of particle morphology, from low to high L/D proportion.

In-situ chord length distribution

Focusing on the evolution of fine particles, in-situ CLD measurements were performed, and normalized quantities were defined as the ratio to their initial values. The evolutions of normalized total chord count, N * c and l * c are reported in Figs. 9 and 10 respectively, for FP (Fig. A) and SCB (Fig. B).

For FP, both N * c (Fig. 9A) and l * c (Fig. 10A) indicate growing trends, whereas they remain almost constant in the reference case (no activity, deviation < 5%). Small peaks may be explained by the mixing rate changes, from 100 to 125 min -1 every 30 min. Considering N c analysis, the interpretations will be limited to regions of stable viscosity, i.e., times beyond the markers in Fig. 9A, which correspond to a maximum 25% deviation based on final viscosity. Accounting for DLS volume-weighted distribution (Fig. 3A), the initial FP suspension contained 46% coarse particles and 54% fine ones. A few large fibers possibly account for a considerable volume fraction equivalent to a huge number of fine particles. For number-weighted distribution obtained by FBRM analysis, the fraction of large particles is almost invisible, whereas the finest are well described. During enzymatic attack, different phenomena leading to opposite evolutions of N * c and l * c are encountered and associated with different mechanisms. The fragmentation of large particles generates a significant growth in number of medium-sized particles while the solubilization of the finest particles leads to a decrease of l * c . For SCB, most of the time N * c (Fig. 9B) and l * c (Fig. 10B) exhibit opposite trends when compared to FP. Note that viscosity variations are almost negligible, allowing N * c interpretation throughout the whole hydrolysis. Biochemical analysis (Fig. 5) confirms the existence of a solubilization mechanism at enzyme loading from 3 FPU/g cellulose. It is suggested that there is a balance between four mechanisms: agglomerate separation, fiber fragmentation, solvation and solubilization. At 0.3 FPU/g cellulose, with only 3.1% glucose yield at 24 h, the effect of solubilization is almost negligible and the fragmentation is not enough to reduce particle size. A swelling effect of substrate by solvation may be responsible for the slight increase in l * c . At higher enzyme loading, the separation of agglomerates and the fragmentation of coarse fibers may explain the rise in chord number as well as the fall in mean chord length in the first stage. Then, depending on enzyme dosage, a solubilization mechanism becomes significant and predominant (illustrated by an increase in glucose yield), and both N * c and l * c exhibit decreasing trends. The coexistence of four mechanisms-separation of agglomerates, fragmentation of coarse fibers, solvation and solubilization of fine ones-is well established and consistent with the enzyme/substrate ratio.

For a better understanding of the enzymatic attack on the finest population, CLD weighted by N c are reported in Fig. 11A andB for FP (1.5% w/v), and in Fig. 12A andB for SCB (3% w/v) at 3 and 25 FPU/g cellulose.

The interpretation for FP was restricted to the region of stable viscosity (t ≥ 1 h for 25 FPU/g cellulose, t ≥ 4 h for 3 FPU/g cellulose and t ≥ 9 h for 0.3 FPU/g cellulose). As reported in Fig. 11A and B, a significant evolution in particle number was observed for 25 FPU/g cellulose, while almost no change was witnessed for 3 FPU/g cellulose. With number-weighted distribution, the FBRM analysis provides information about the population of fine particles. The trend at low enzyme loading might assume that there was an equilibrium between fragmentation of coarse and solubilization of fine particles, leading to a balance in total fine particles. Thus, by increasing the enzyme loading ratio to 25 FPU/g cellulose, this balance was broken and the evolution was clearly observed, with an increase of 25% in chord number for the fraction of 10 <l c < 100 m between 1 h and 3 h. The increasing trend continued until 12 h, and finally reached 140% compared to t = 1 h. During this time, the fragmentation mechanism seemed to be stronger than solubilization one. Beyond 12 h, the trend was reversed and a fair reduction in number-weighted distribution was observed for the fraction of 10 < l c < 100 m.

For SCB, the fraction of 10 <l c < 100 m was scrutinized in order to be compared to FP. The phenomenon strongly differs, with almost no evolution at 3 FPU/g cellulose. At the highest loading (25 FPU/g cellulose), distribution curves exhibit a sharp evolution. From 0 h to 3 h, no significant variation was quantified. Nevertheless, a decrease of 30.4% and 64.2% were witnessed after 12 h and 24 h respectively. Below 8 m (the size of the finest particle), a constant shoulder is identified.

With both substrates, evolution in chord population is linked to the balance and relative magnitude of two mechanisms: (i) the fragmentation of coarse particles into smaller insoluble particles that cause a rise in total number of chords, and (ii) the solubilization of fine particles, which leads to a drop in total chord population. The different phenomena observed between FP and SCB are consistent with their initial morphogranulometric properties. Biochemical results (Fig. 5) confirmed the presence of a solubilization mechanism and sustained the fragmentation mechanism illustrated by PSD and CLD. At 25 FPU/g cellulose, the fragmentation mechanism played a predominant role in FP suspensions that contain considerable amounts of coarse fibers (46% by volume for d SE > 200 m). For SCB suspensions with a lower size spread (96% of the fine particles are inferior to 200 m), the two mechanisms, fragmentation and solubilization, appear to be balanced at 3 FPU/g cellulose. When enzyme loading is increased up to 25 FPU/g cellulose, this equilibrium is maintained during the first 3 h, and then solubilization becomes the dominant mechanism. This assumption is supported by biochemical results, with glucose yield reaching 53% and 82% for FP and SCB respectively at 25 FPU/g cellulose and 24 h. For FP and SCB, the obtained results suggest that smaller particles are more accessible to enzymatic hydrolysis than larger ones.

Conclusions

The relationships between in-situ viscosimetry, in-and ex-situ morphogranulometry (PSD, CLD) and biochemistry were investigated during enzymatic hydrolysis of lignocellulose substrates under semi-dilute conditions (1.5-2 times higher than critical concentrations) using a dedicated, multi-instrumented set-up. These concentrations generate non-Newtonian behaviors and avoid neglecting particle-particle interactions, contrary to dilute conditions. In light of their industrial potentials, two specific substrates (pretreated SCB 3% w/v) and milled FP 1.5% w/v) were scrutinized during hydrolysis by a commercial cocktail (Ctec2, Novozyme) with ratios ranging between 0.3 and 25 FPU/g cellulose. The appropriate and specific mechanisms (e.g., viscosity overtaking) corresponding to different phenomena were revealed and explained via biochemical analysis, ex-situ PSD, and in-situ CLD analysis.

Firstly, the obtained results highlight the strong influence of PSD and dispersion as well as morphology on suspension viscosity. In semi-dilute conditions, suspension viscosity was found to be closely linked to size distribution spreading, and to depend mainly on the fraction of coarse particles. The population of fine particles had almost no effect on viscosity.

Secondly, during enzymatic hydrolysis, four mechanisms were identified: solvation, particle fragmentation, particle solubilization, and agglomerate separation (especially for SCB). Change in suspension viscosity was related to three of them, excluding solubilization, which mainly occurred with the finest population fraction. The increase in suspension viscosity by solvation may be attributed to the swelling effect of lignocellulose fibers, which increases the effective volume fraction. In contrast, the fragmentation mechanism resulted in a collapse of viscosity. The mechanism was explained by the shift in volume fraction of the coarse population, which was known as the determinant factor of viscosity, into fine population. The solubilization mechanism showed almost negligible effect, as the suspension can be totally liquefied at very low bioconversion yield.

Interestingly for suspension that contains agglomerates at low enzyme loading ratios, the separation of agglomerates by both mechanical mixing and enzymatic actions caused a significant viscosity overtaking, which is an unfavorable phenomenon for bioprocess intensification. The mechanism was explained by two assumptions: (i) the separation of agglomerates into individual particles strengthens the interactions between them by increasing the total number of particles in suspension and (ii) the separation induces an evolution of population shape from sphere-like (agglomerate) into fiber-like (individual fragments) that affects the suspension viscosity. At high enzyme loading, the viscosity overtaking was almost non-observable.

From an academic standpoint, the kinetics associated with viscosity, morphology and biochemistry need to be modeled. Bottleneck and balance between these phenomena will be scrutinized by local metrology (e.g., tomography to measure void fraction, specific surface, and enzyme accessibility).

From an industrial standpoint, enzymatic hydrolysis at high dry matter content stands as a major challenge. Thus the activity profile of the cocktail and the physical properties of the substrates are worth considering beyond the merely biochemical yield. In order to minimize transfer limitations (mass, heat, momentum) and ensure adequate mixing and energetic efficiency, for FP and SCB, a high proportion of endoglucanase activity is required to quickly liquefy the substrate via a fragmentation mechanism. Monodisperse lignocellulose material has the additional advantage of low suspension viscosity for the bioconversion process.

Fig. 1 .

 1 Fig. 1. Process and instrumentation diagram of experimental setup.

Fig. 2 .

 2 Fig. 2. Microscopic observation of FP suspension, 1.5% w/v (A), and SCB suspension, 3% w/v (B), both captures represent an area of 0.5 cm × 0.5 cm, 10× magnification, dark field mode (Morphologi G3S, Malvern Inst.).

  for FP and SCB suspensions. FP suspensions show a multimodal population of coarse particles (46% with d SE > 200 m) and fine particles (54% with d SE < 200 m), whereas fine particles with d SE < 200 m represent 94% of the population for SCB. The volume-weighted diameters, d(v,0.1), d(v,0.5), d(v,0.9), corresponding to 10%, 50%, and 90% of the population, are 9.8 ± 0.3, 43.5 ± 1.8, 161.3 ± 3 m for SCB and 20.9 ± 0.8, 169.4 ± 18.8, 1153.7 ± 71.9 m for FP. Volume-weighted mean diameter D(4,3) of FP (412.4 ± 34.4 m) is approximately 6 folds higher than for SCB (71.4 ± 1.3 m). Number-weighted CLD are illustrated in Fig. 3-B. The difference of size range between FP and SCB is noticeable with an expected shift to smaller dimensions. Both populations range between 1 to 200 m. Mean chord lengths of 25.1 ± 0.47 m and 16.4 ± 0.43 m were reported for FP and SCB respectively. A slight shoulder is observed for FP.

Fig. 3 .

 3 Fig. 3. Volume distributions of sphere equivalent diameter by DLS measurements (A) and number distribution of chord length by FBRM (B) for FP and SCB (dotted lines: average deviation issued from at least 6 experiments).

Fig. 4 .

 4 Fig. 4. Initial count number (Nc), mean chord length (lc), and suspension viscosity () for FP at 1.5% w/v and SCB at 3% w/v. Error bar represents the average of the absolute deviations from their mean.

Fig. 5 .

 5 Fig. 5. Glucose conversion yield of all experiments.

Fig. 6 .

 6 Fig. 6. In-situ viscosity during the first 5 h of hydrolysis of FP suspensions 1.5% w/v (A) and SCB suspensions 3%w/v (B) using Ctec2 cocktail at different dosages (0-25 FPU/g cellulose).

Fig. 7 .

 7 Fig. 7. Particle size distribution weighted by particle volume fraction during hydrolysis of FP suspensions for two enzyme loadings (A: 3 FPU/g cellulose, B: 25 FPU/g cellulose).

Fig. 8 .

 8 Fig. 8. Particle size distribution weighted by particle volume fraction during hydrolysis of SCB suspensions for two enzyme loadings (A: 0.3 FPU/g cellulose, B: 3 FPU/g cellulose).

Fig. 9 .

 9 Fig. 9. Evolution in N * c during enzymatic hydrolysis of FP 1.5% w/v (A) and SCB 3% w/v (B) for E/S loading ranging from 0.3 up to 25 FPU/g cellulose (•: points beyond which viscosity variations are negligible).

Fig. 10 .

 10 Fig. 10. Evolution in l * c during enzymatic hydrolysis of FP 1.5% w/v (A) and SCB 3% w/v (B) for E/S loading ranging from 0.3 up to 25 FPU/g cellulose.

Fig. 11 .

 11 Fig. 11. Evolution of number-weighted chord length distribution multiplied by Nc (FP suspension, 1.5% w/v) during hydrolysis with Ctec2 ratios 3 FPU/g cellulose (A) and 25 FPU/g cellulose (B).

Fig. 12 .

 12 Fig. 12. Evolution of number-weighted chord length distribution multiplied by Nc (SCB suspension, 3% w/v) during hydrolysis with Ctec2 ratios 3 FPU/g cellulose (A) and 25 FPU/g cellulose (B).

Acknowledgements

This work was supported by TECHNO 1 & 2-Erasmus Mundus (European Commission, project managed by UPS -Toulouse) and HTMS BioAsie (Ministère des Affaires Étrangères et du Développement International, MAEDI, programme BIO-Asie, N • 34082NF). It participated in the ANR Grant ProBio3-Investissement d'Avenir under the contract number ANR-11-BTBR-0003.

Authors are gratefully to the Centre de Traduction -Université Toulouse III-Paul Sabatier for English language review.

Conflict of interest

None.


Charlotte La Fay

Celia Hoebeke

Marine Juzaud

Anne Spraul

Pauline Heux

Jean-Christophe Dubus

Alice Hadchouel

Alexandre Fabre

Deep phenotyping of MARS1 (interstitial lung and liver disease) and LARS1 (infantile liver failure syndrome 1) recessive multisystemic disease using Human Phenotype Ontology annotation: overlap and differences. Case report and review of literature

Keywords: tRNA synthetase deficiency, Infantile liver failure syndrome type 1, Pulmonary alveolar proteinosis, LARS, MARS, MARS1, LARS1

come

Deep phenotyping of MARS1 (interstitial lung and liver disease) and LARS1 (infantile liver failure syndrome 1) recessive multisystemic disease using Human Phenotype

Ontology annotation: Overlap and differences.

INTRODUCTION

Aminoacyl transfer RNA synthetases (ARSs) are a highly conserved ubiquitously expressed class of enzymes that covalently attach amino acids onto transfer RNA (tRNA) as part of the protein synthesis process. [START_REF] Fuchs | Aminoacyl-tRNA synthetase deficiencies in search of common themes[END_REF][START_REF] Van Meel | Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype[END_REF] MARS1 and LARS1 respectively encode cytoplasmic methionyl and cytoplasmic leucine-tRNA synthetase.

Homozygous or compound heterozygous mutations in the MARS1 gene cause interstitial lung and liver disease (ILLD; OMIM #615486 ) including a specific form of pediatric pulmonary alveolar proteinosis (PAP) [START_REF] Sun | Mutations in methionyl-tRNA synthetase gene in a Chinese family with interstitial lung and liver disease, postnatal growth failure and anemia[END_REF][START_REF] Enaud | Pulmonary alveolar proteinosis in children on La Réunion Island: a new inherited disorder?[END_REF], while recessive mutations in the LARS1 gene have been identified as the cause of a multisystem disorder referred to as infantile liver failure syndrome 1 (ILFS1; OMIM # 615438) [START_REF] Casey | Identification of a mutation in LARS as a novel cause of infantile hepatopathy[END_REF].

An outstanding question raised by Fuchs et al. for LARS1 and MARS1 is whether tRNA synthetase deficiencies have a common phenotype [START_REF] Fuchs | Aminoacyl-tRNA synthetase deficiencies in search of common themes[END_REF]. The aim of this study was to address this question by comparing the phenotypes of three patients diagnosed in our hospital: two children respectively 8 months and 12 years of age with homozygous pathogenic variations in MARS1 and a 10 month-old infant with compound heterozygous variants in LARS1.

METHODS

Three cases of recessive LARS1 or MARS1 disease are described and these are analyzed with previous case reports of recessive LARS1 or MARS1 recessive disease in the literature using Human Phenotype Ontology (HPO) codes [START_REF] Köhler | The Human Phenotype Ontology in 2017[END_REF]. HPO provides a standardized vocabulary of phenotypic abnormalities encountered in human diseases, with each term describing a phenotypic abnormality. HPO coding reveals phenotypic similarities between diseases. Venn diagrams were drawn using Van de Peer et al's online tool (http://bioinformatics.psb.ugent.be/webtools/Venn/. ) Cases in the literature were only considered if they had been described in peer-reviewed articles (i.e. excluding theses and congress abstracts) with a confirmed genetic diagnosis. Some previously described cases of PAP were therefore excluded [START_REF] Enaud | Pulmonary alveolar proteinosis in children on La Réunion Island: a new inherited disorder?[END_REF]. One of our patients with MARS1 mutations (Patient 3) was also included in Hadchouel et al.'s study (ID: P6). Here, we analized his phenotype in more detail and provide updated information.

All three patients provided written consent for publication of their case details. For Patient 1, western blot analysis using LARS1 (D7Q4Q) Rabbit mAb 35509 Cell Signaling Technology is shown in Supplementary Figure (S3)

PATIENT DESCRIPTION Patient 1

The first patient is a boy born at 37 +6 weeks of gestation with a birth weight of 1995 g (1 st percentile) after labor was induced because of intrauterine growth retardation. He is the third child of a non-consanguineous couple with an unremarkable medical history. Routine clinical examination at two months of age revealed icteric skin and sclera and failure to thrive (10 g per day) with normal stool color. Blood tests showed acute hepatic failure with severe coagulopathy: prothrombin rate, 11%; cofactors II and X < 0.05 IU/L (normal range (NR), > 0.7 IU/L); Factor V, 66% and unresponsive to vitamin K; elevated transaminase levels (AST, 122 IU/L (NR, 5-87 IU/L); ALT, 74 IU/L (IU/L 5-60)); hyperbilirubinemia (total bilirubin, 82 µmol/L (NR, 2-17 µmol/L), direct bilirubin, 62 µmol/L (NR, 2-5 µmol/L)); hypoalbuminemia (serum albumin, 34 g/L (NR, 38-54 g/L)) and hypochromic microcytic anemia (hemoglobin, 8.8 g/dl; unresponsive to iron therapy). The patient was hospitalized and had lactic acidosis (pH, 7.25; lactates, 2.8 mmol/L) on admission. The plasma ammonia level was normal. Abdominal ultrasound findings were normal and there was no sign of hepatomegaly.

Neurological clinical examination was normal. Metabolic disorders were ruled out and all tests were normal except urine amino acid chromatography, which showed elevated levels of all amino acids, suggesting a tubulopathy, and urine organic acid analysis, which showed isolated elevation of 3-hydroxyisovaleric acid. Viral hepatitis was excluded by serological testing. Further evaluations excluding other diagnoses are listed in Table S1.

Sequencing of genes associated with cholestasis (MiSeq, Illumina) revealed two heterozygous variants in LARS1 (NM_020117), respectively in exon 19 (c.1818dup, p.(Ala607Cysfs*8,) and exon 6 (c.463A>G, p.(Lys155Glu). The patient's parents were asymptomatic heterozygous carriers (c.463A>G, p.Lys155Glu in father and c.1818dup, p.Ala607Cysfs*8 in mother). The missense variant is predicted as pathogenic by Mutation Taster (www.mutationtaster.org) and not reported in gnomAD. The other is a nonsense variant not reported in gnomAD Western blot analysis using LARS1 (D7Q4Q) Rabbit mAb 35509 Cell Signaling Technology showed decreased LARS1 signal intensity in parents and in proband versus control (29.1% of signal in proband, 48.5% in mother and 97.3% in father) S3.

At 6 months of age, growth was improving (weight, -1 SD). The patient had moderate axial hypotonia between 3 and 5 months of age but his head circumference and neurodevelopment were subsequently normal with no seizure or encephalopathy and normal muscle tone. Concentrations of bilirubin, albumin and coagulation factors normalized, and transaminase levels decreased(< 2×NR). He still had mild tubulopathy and anemia, but transfusion had not been considered necessary. Hepatomegaly had developed. Treatment consisted of vitamin (A, C, D, E, K), ursodesoxycholic acid, sodium bicarbonate, MCT oil and iron supplementation.

Patient 2

The second patient, a girl, was born prematurely at 34 +5 weeks of gestation to a non-consanguineous couple from Mayotte with an unremarkable medical history. There was no intrauterine growth retardation. In the first months of life, the patient had malnutrition due to feeding difficulties, recurrent vomiting and failure to thrive, leading to the placement of a gastrostomy tube for enteral nutrition. She showed neurodevelopmental delay at two months of age (poor head control). MRI showed hypoplasia of the corpus callosum and bilateral subependymal pseudocysts. At 6 weeks of age, the patient developed respiratory distress with tachypnea (80 /min), cough, chest retraction, crackles, and hypoxia requiring oxygen supplementation for over a month. Chest CT showed interstitial lung disease. Pulmonary alveolar proteinosis was considered the most likely diagnosis given the patient's ethnic origin, CT images (extensive ground-glass opacities, and peribronchovascular thickening with a crazy paving pattern), and respiratory condition.

Blood tests showed hypoalbuminemia, thrombocytosis and microcytic hypochromic nonregenerative anemia, which required three blood transfusions then resolved with iron supplementation.

Liver enzymes suggested transitory cholestasis (maximum GGT, 182 IU/L (NR, 0-40)) and abdominal ultrasonography showed hepatomegaly with steatosis and persistent hydronephrosis. She had sinus tachycardia but echocardiography and Holter results were normal.

At 2 months of age, the patient had CMV infection (PCR positive, up to 72000 copies/ml), treated with valganciclovir, having tested negative at birth. Sequencing of genes implicated in interstitial lung disease (MiSeq, Illumina) revealed a homozygous missense mutation c.1700C>T, p.(Ser567Leu) and a heterozygous variant c.1177G>A, (p.Ala393Thr) of the MARS1 gene, both of which are associated with pulmonary alveolar proteinosis and have already been described as pathogenic by Hadchouel et al. [START_REF] Hadchouel | Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island[END_REF]. Bronchoalveolar lavage confirmed the diagnosis of PAP. The patient is currently 10 months old, her respiratory symptoms have improved and she no longer needs oxygen supplementation. She still has feeding and eating disorders, with enteral nutrition through a gastrostomy tube and moderate undernutrition. Her neurological development is normal.

Patient 3

The third patient is a boy referred to the hospital at 2 months of age for failure to thrive, hepatomegaly with hepatic cytolysis and respiratory distress. He was born at term with a normal birth weight and is the first child of a non-consanguineous couple; his father is of Comorian descent and his mother, Malagasy.

He had tachypnea without need for oxygen supplementation. CT showed alveolar-interstitial syndrome in the lower lobes and bronchoalveolar lavage findings were consistent with pulmonary alveolar proteinosis. Digital clubbing developed over the following years.

He had slight splenomegaly and significant hepatomegaly with cytolysis and intermittent cholestasis without jaundice. Liver biopsy showed severe macrovesicular steatosis.

He had several episodes of cholecystitis without gallstones and major feeding difficulties with recurrent vomiting, abdominal flatulence and no weight gain leading to parenteral nutrition then enteral nutrition through a gastrostomy tube.

Blood tests showed normocytic normochromic nonregenerative anemia, hypoalbuminemia and hypergammaglobulinemia. Bone marrow biopsy showed erythroblastopenia and granulocytic lineage maturation disorder. The patient also had fasting intolerance and recurrent hypoglycemia and required continuous enteral feeding. All endocrine and metabolic tests were normal however.

A primary CMV infection resolved without antiviral treatment and the patient also had several bacterial infections with septicemia. Between 1 and 6 months of age, he developed severe eczema that disappeared with topical steroid treatment. He also had moderate psychomotor delay in infancy (he could not stand alone at 14 months of age) that resolved over time. The patient is currently 12 years old, with steady growth (height -1.5 SD, weight between the 3rd and 10th percentile) and has enteral nutrition through a gastrostomy tube at night. His neurological status is normal and he has no learning delay.

As described by Hadchouel et al. ( 6) sequencing of the MARS1 gene in this patient revealed two homozygous missense variants (c.1177G>A, (p.Ala393Thr) and c.1700C>T).

RESULTS

Our review of the literature identified 14 articles, 7 on LARS1 mutations and 7 on MARS1 mutations, such that including our 3 patients, 65 patients with LARS1 or MARS1 mutations (respectively 27 and 38) have so far been described in the literature. [START_REF] Fuchs | Aminoacyl-tRNA synthetase deficiencies in search of common themes[END_REF][START_REF] Van Meel | Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype[END_REF][START_REF] Sun | Mutations in methionyl-tRNA synthetase gene in a Chinese family with interstitial lung and liver disease, postnatal growth failure and anemia[END_REF][START_REF] Casey | Identification of a mutation in LARS as a novel cause of infantile hepatopathy[END_REF][START_REF] Casey | Clinical and genetic characterisation of infantile liver failure syndrome type 1, due to recessive mutations in LARS[END_REF][START_REF] Hadchouel | Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island[END_REF][START_REF] Lin | Clinical feature and molecular diagnostic analysis of the first noncaucasian child with infantile liver failure syndrome type 1[END_REF][START_REF] Lenz | Genotypic diversity and phenotypic spectrum of infantile liver failure syndrome type 1 due to variants in LARS1[END_REF][START_REF] Rips | MARS variant associated with both recessive interstitial lung and liver disease and dominant Charcot-Marie-Tooth disease[END_REF][START_REF] Alzaid | Methionyl-tRNA synthetase novel mutation causes pulmonary alveolar proteinosis[END_REF][START_REF] Abuduxikuer | Novel methionyl-tRNA synthetase gene variants/phenotypes in interstitial lung and liver disease: A case report and review of literature[END_REF][START_REF] Lenz | Rescue of respiratory failure in pulmonary alveolar proteinosis due to pathogenic MARS1 variants[END_REF][START_REF] Peroutka | Severe Neonatal Manifestations of Infantile Liver Failure Syndrome Type 1 Caused by Cytosolic Leucine-tRNA Synthetase Deficiency[END_REF][START_REF] Hirata | Severe course with letal hepatocellular injury and skeletal muscular dysgenesis in a neonate with infantile liver failure syndrome type 1 caused by novel LARS1 mutations[END_REF] Twenty-one of these patients (32.3%) patients died: 6/27 of the patients with LARS1 mutations (4 boys and 2 girls) and 15/38 of the patients with MARS1 mutations (11 boys and 4 girls). One of the patients with a LARS1 mutation underwent liver transplantation. Mean age of death was 4.5 year (range 51 days-25.2 years).

Using HPO terms, the mean number of phenotypic abnormalities identified per article was 26.1 for LARS1 mutations and 20.4 for MARS1 mutations (range, 11-45). The number of phenotypic abnormalities described only exceeded 30 in articles describing more than one patient. In single case reports, the mean number of phenotypic abnormalities described was 18 for LARS1 and 20.5 for MARS1 mutations. We described 23 clinical phenotypic abnormalities for our patient with LARS1 mutation, and 24 phenotypic abnormalities for each of our patients with MARS1 mutations.

In total, 117 HPO phenotypic abnormalities have been described at least once in patients with MARS1 or LARS1 mutations (Table S2), among which 41 (35%) have only been described in patients with LARS1 mutations, 27 (23.1%) only in patients with MARS1 mutations, and the remaining 49 (41.9%) for both MARS1 and LARS1 mutations.

Table 1 lists all the phenotypic abnormalities associated with both LARS1 and MARS1 mutations and the most frequent phenotypic abnormalities that differ between the two populations. Among the phenotypic abnormalities described for both LARS1 and MARS1 mutations, 4/49 were observed in more than 50% of cases, 8/49 in 30-50% of cases, 17/49 were observed in 10-30% of cases, and 20/49 in less than 10% of cases.

Fig. 1 shows a Venn diagram of the different phenotypic abnormalities described in the literature for MARS1 and LARS1 mutations. The proportion of phenotypic abnormalities shared between these two groups of patients was 34% and 39% after including our three patients. Only 9 (24.3%) of the phenotypic abnormalities described in the first two single case reports of patients with MARS1 and LARS1 mutations were common to both, whereas now 44 (37.9%) of the phenotypic abnormalities described in the literature have been reported for mutations in both genes. Our patient with LARS mutations had 7 phenotypic abnormalities in common with the first of our patients with MARS1 mutations, and 5 with the second, among which 4 were observed in all three patients. Interestingly, our two patients with MARS1 mutations only had 9 phenotypic abnormalities (23%) in common.

Classifying the abnormalities in terms of the affected organ (Fig. 1e) shows that while there is no overlap between phenotypes in the two groups for the kidney and lungs, for other organs (e.g. the liver and gastrointestinal tract) most of the phenotypic abnormalities overlap.

DISCUSSION

Aminoacyl-tRNA synthetase deficiencies are very complex and polymorphic pathologies. This work is an attempt to clarify the phenotypes of LARS1 and MARS1 mutations using HPO terms [START_REF] Köhler | The Human Phenotype Ontology in 2017[END_REF], which are a means to describe and compare disease phenotypes more precisely.

Among the three patients treated in our hospital whose clinical phenotypes we describe, one had compound heterozygous LARS1 variants while the two others had homozygous mutations in MARS1. Decompensation (hepatic, neurological or pulmonary) triggered by an infection, mainly in infancy, is described for most of the cases in the literature and was observed here in all three of our patients. Both our patients with MARS1 mutation had CMV infection in the first months of life. This had not been described until a recent report of a boy with MARS1 mutation who presented CMV colitis at 4 months of age. [START_REF] Lenz | Rescue of respiratory failure in pulmonary alveolar proteinosis due to pathogenic MARS1 variants[END_REF] Regarding phenotypes, all three of our patients have 12-15 HPO terms specific to their condition, and 5-9 terms in common with the other two. In these three patients, LARS1 and MARS1 phenotypes largely overlap. This is generally the case for LARS1 and MARS1 mutations. Indeed, blood, metabolism, gastrointestinal and growth abnormalities are described in more than half of patients with these mutations and are frequent in both phenotypes. However, certain characteristics seem to be more specific to one or other of the phenotypes. For instance, nervous system and muscle abnormalities (global or motor developmental delay, hypotonia, encephalopathy, MRI findings) are described in less than 10% of patients with MARS1 mutations but in more than half of those with LARS1 mutations. Conversely, LARS1 mutations are rarely associated with respiratory abnormalities but these are a characteristic feature of MARS1 mutations. For the kidney and lung finally, none of the phenotypic abnormalities overlap.

The number of common phenotypic abnormalities has increased with the number of cases and phenotypes described in the literature. One would indeed expect the number of specific abnormalities to either decrease as more cases are described, or stay constant as they are confirmed as characteristic of a specific mutation.

The relative frequency of shared phenotypic abnormalities may also differ in the two groups of patients.

Respiratory abnormalities are indeed far more frequent in patients with MARS1 mutations and precise diagnoses as alveolar proteinosis or interstitial pulmonary disease are well described. However, most recent reports of LARS1 mutations [START_REF] Fuchs | Aminoacyl-tRNA synthetase deficiencies in search of common themes[END_REF][START_REF] Lenz | Genotypic diversity and phenotypic spectrum of infantile liver failure syndrome type 1 due to variants in LARS1[END_REF][START_REF] Hirata | Severe course with letal hepatocellular injury and skeletal muscular dysgenesis in a neonate with infantile liver failure syndrome type 1 caused by novel LARS1 mutations[END_REF] also describe associated respiratory abnormalities.

It therefore seems as if no condition is specifically associated with mutations in either of the two genes. Many phenotypic abnormalities have been described in patients with either LARS1 or MARS1 mutations but based on the deep phenotyping of our patients, it appears that all these abnormalities may in fact be seen in both populations.

These complex and rare diseases having only recently been described, their phenotypic description still needs to be completed. The description of our three cases increases the number of phenotypic abnormalities associated with both LARS1 and MARS1 mutations. The number of phenotypic abnormalities described and the proportion of common phenotypic abnormalities have both increased (from 37 to 117 and from a quarter to more than a third, respectively) since the publication of the first case reports of LARS1 and MARS1 mutations. This suggests that LARS1 and MARS1 mutations have a mainly common phenotypic expression. Continued description of these patients is important for a more precise understanding of these phenotypes, one limitation being that in most cases, only identified abnormalities are reported while negative tests for other abnormalities are rarely mentioned. Aminoacyl transfer RNA synthetases attach amino acids to tRNA. Leucine and methionine have different sidechains (aliphatic for leucine and sulfur-containing for methionine) but both are hydrophobic and uncharged at neutral pH. This raises the question whether phenotyping studies of other recessive aminoacyl transfer RNA synthetase diseases would return similar results to ours or if some phenotypes stand apart.

CONCLUSION

Precise descriptions of the clinical phenotypes of LARS1 and MARS1 mutations patients are in our view essential. A few of the phenotypic abnormalities described here have only been reported in one or two patients and while they may therefore not be actually part of the phenotype of these mutations, they may also be found in future patients.

In complex diseases such as those due to LARS1 and MARS1 mutations, the more phenotypic abnormalities are described, the more the proportion found to be common to the two pathologies increases. LARS1 and MARS1 phenotypes seem to fit on a continuum, but with certain specificities.

Since the first descriptions of patients with MARS1 and LARS1 mutations appear to have been only partial, further studies and case reports are needed to clarify the common and specific features of aminoacyl-tRNA synthetase deficiency phenotypes.

Table 1a : Phenotypic abnormalities associated with both LARS and MARS mutations

 1a

		Patient	Literature	Patients	Literature
		LARS	LARS	MARS (N/2)	mars
	HPO		(N/26)		(N/37)
	HP:0003073	1	24	2	7	Hypoalbuminemia
	HP:0002240	1	7	2	31	Hepatomegaly
	HP:0002910	1	23	1	22	Elevated hepatic transaminases
	HP:0001508	1	22	2	30	Failure to thrive
	HP:0004840	1	18	1		Hypochromic microcytic anemia
	HP:0001622		16	1	3	Premature birth
	HP:0001396	1	1	2	24	Cholestasis
	HP:0001392	1	14		6	Abnormality of the liver
	HP:0012758		21	2	5	Neurodevelopmental delay
	HP:0001270		16	1	4	Motor delay
	HP:0001511	1	21		1	Intrauterine growth retardation
	HP:0001518		13

ACKNOWLEDGEMENTS:

This article was edited by Paul Guerry (Green Grow Scientific).

Conflicts of interest: none


Xavier Dallaire

Claire Mérot
email: claire.merot@gmail.com

A setback into a success: what can batch effects tell us about best practices in genomics?

The increasing access to high-throughput sequencing is certainly one of the major changes that molecular The reduced costs of next-generation sequencing have translated into a rapidly growing number of genomic studies, which are providing new insights into fundamental and applied questions in evolutionary biology and ecology. For comparative genomics and population genomics, increasing data availability brings the opportunity to increase sample size and to broaden the spatial or temporal scale of studies by reusing and merging datasets.

However, uncorrected bias, missing data, filtering parameters, sample heterogeneity, and some analytical choices are known to generate artefactual signals [START_REF] Goh | Why batch effects matter in omics data, and how to avoid them[END_REF]Tom et al., 2017). Moreover, the need to analyze large genomic datasets turned most of us into apprentices at bioinformatics. Along such a tortuous road, who hasn't wondered what the best practices are? How can we be sure that we are using the best tools or the best parameters to analyze our specific dataset? Is there uncorrected bias that may explain the observed patterns more than biological reasons? The matter can complicate further when reusing and/or combining previously generated datasets: how to deal with uncontrolled study design, variable qualities and coverage, differences in sequencing technologies, etc? The study by [START_REF] Lou | Batch effects in population genomic studies with low-coverage whole genome sequencing data : Causes, detection, and mitigation[END_REF] provides helpful answers to such questions as they explore the consequences of different technical artefacts that emerged in their dataset.

The first issue faced by [START_REF] Lou | Batch effects in population genomic studies with low-coverage whole genome sequencing data : Causes, detection, and mitigation[END_REF] is batch effect, which is a pattern of variation due to the merging of datasets produced separately that can confound or mask biological patterns. While usually mitigated by randomization or consistency in methods (e.g., library preparation and sequencing procedure), batch effect represents a major concern for studies carried over several years or across multiple labs, as well as for re-analysis of public datasets. In particular, with the rapid evolution of sequencing technology, whole-genome sequences may have been produced by different methods and may differ in read type and length. For instance, the switch from a 4-colour technology (Illumina HiSeq) to a 2-colour technology (NextSeq) as well as a different assessment of quality score between technologies led to spurious SNPs (single-nucleotide polymorphisms) and an overestimate of heterozygosity differences [START_REF] Lou | Batch effects in population genomic studies with low-coverage whole genome sequencing data : Causes, detection, and mitigation[END_REF]. Moreover, the switch from single-end to paired-end, and from 125 to 150 base pairs, affected read mapping and lead to artifactual high differentiation between the two batches. Fortunately, while such artefacts are worrying, they can be detected by simple bioinformatic procedures and mitigated by filtering choices accounting for technology differences.

Besides batch effects, the paper also draws attention to usually overlooked biases that may arise from heterogeneity in DNA quality and sequencing depth, even within the best-controlled single batch design. For example, the authors observed higher heterozygosity in samples with degraded DNA, a pattern they link to the identification of false polymorphism through deamination of cytosines. This effect might be particularly strong in long-term studies if samples vary significantly in age and were preserved in various conditions. DNA quality, as well as fragment size of the libraries, imperfectly balanced pooling, and sequencing choices, may also lead to heterogeneity in sequencing depth between samples and between batches. Lou & Therkildsen explored this issue with simulated data and showed that the higher proportion of missing data in shallow-covered samples might dampen population structure signals, although this can be controlled with appropriate tools even for low (<4X) to very low coverage (<1X).

More generally, some emerging artefacts reported by [START_REF] Lou | Batch effects in population genomic studies with low-coverage whole genome sequencing data : Causes, detection, and mitigation[END_REF] are worth considering in most sequencing datasets. For instance, this study confirms the warning from De-Kayne et al. ( 2021) about the enrichment of guanine (G) at the end of NextSeq reads that might cause artifactual SNPs and an overestimation of genetic diversity, suggesting that the trimming procedure may need adjustment. Moreover, the current trend is reducing sequencing depth to allow the analysis of large numbers of biological samples in a cost-effective way [START_REF] Lou | A beginner's guide to low-coverage whole genome sequencing for population genomics[END_REF]. While this shift has merits based on the fact that many population genomics analyses produce more robust results with more samples at low coverage than with fewer samples at high coverage (Alex [START_REF] Buerkle | Population genomics based on low coverage sequencing: how low should we go?[END_REF], [START_REF] Lou | A beginner's guide to low-coverage whole genome sequencing for population genomics[END_REF] remind us that low-coverage whole-genome sequencing is more sensitive to artefacts due to DNA degradation, depth heterogeneity or DNA quality. That being said, some artefacts such as reference bias and alignment errors are equally problematic with high-coverage data [START_REF] Gage | Multiple maize reference genomes impact the identification of variants by genome-wide association study in a diverse inbred panel[END_REF][START_REF] Lloret-Villas | Investigating the impact of reference assembly choice on genomic analyses in a cattle breed[END_REF], and more importantly, Lou & Therkildsen show that appropriate bioinformatic procedures are key to control and correct for the impact of multiple factors. Most of those mitigation methods include more stringent filtering that may reduce the fraction of genome actually analyzed or the number of polymorphic markers. This strategy remains nevertheless viable considering that whole genome sequencing produce amounts of data far superior to what is needed for many population genomics applications.

Beyond warning and practical recommendations to deal with heterogeneous sequencing datasets, Lou & Therkildsen (2022) also foster good practices for both designing and analyzing next-generation sequencing studies.

The step-by-step approach leads to a collection of tests and tools that molecular ecologists can adapt to the peculiarities of their own datasets in a modular way. Moreover, even though the list of studied technical artefacts is inevitably incomplete, the approach undertaken by Lou & Therkildsen provides an inspiring example of how to explore and deal with future problems. For example, testing the effects of different levels of filtering on key statistics and using simulations to validate some methods appears as a sensible framework applicable to other datasets. Altogether, this paves the way towards a more accurate use of different kind of genomic datasets and draws a promising picture of future research taking advantage of the incredible amount of sequence data already available. This will encourage ambitious research in molecular ecology based on larger datasets, more temporal or geographical replicates, and more non-model species.

Last but not least, it should be noted that [START_REF] Lou | Batch effects in population genomic studies with low-coverage whole genome sequencing data : Causes, detection, and mitigation[END_REF] clearly reported a problem in their data, describing not only what did work but also what didn't, and which steps of analyses and filtering succeeded or failed. In an academic world heavily focused on high-impact positive results, there is a tendency to shorten methods section in peer-reviewed papers. We believe that method-orientated work is equally important and highlights the relevance of methodological robustness in genomics.

Identifying and mitigating batch effects in whole genome sequencing data. BMC bioinformatics, 18(1), 1-12.

  ecology has gone through over the last decade. With the positive trend towards more open science, most sequencing datasets are now available on public databases, which holds amazing potential, but also risks of introducing batch effects in studies combining datasets. In this issue of Molecular Ecology Resources, Lou & Therkildsen (2022) offer a timely discussion on the matter by analyzing an imperfect low-coverage Whole Genome Sequencing dataset, in which they test the effects of differences in sequencing choices, DNA degradation, and read depth on routine population genomics analyses. Through a series of diagnostic tools, they uncover multiple factors producing technical artefacts that can bias estimates of genetic diversity, inference of population structure, and selection scans. For each confounding factor, they demonstrate the effectiveness of mitigation approaches and suggest other avenues to deal with the issue. In this perspective, we highlight considerations regarding (1) effects that arise from differences between batches of sequencing, (2) unavoidable heterogeneity within datasets, and (3) more general concerns around the use of next-generation sequencing in population genomics. Altogether, by exploring what may have appeared at first glimpse as a "failed" sequencing project, Lou & Therkildsen (2022) end up setting a standard of best practices to make the most of heterogeneous whole-genome sequences, opening a promising avenue towards efficient reuse of published datasets.

Figure 1 :

 1 Figure 1: Schematic overview of the technical artefacts explored and discussed in Lou & Therkildsen (2022).


Study of carbon nanotubes network modification in polyethylene under temperature variation and deformation

Anatole Collet

To cite this version:

Université Claude Bernard Lyon 1 Ecole Doctorale N° 34 Matériaux Spécialité de doctorat

Anatole Collet. Study of carbon nanotubes network modification in polyethylene under temperature variation and deformation. Materials. Université de Lyon, 2021. English. NNT : 2021LYSE1100. tel-03464723v2 N°d'ordre NNT : THESE de DOCTORAT DE L'UNIVERSITE DE LYON opérée au sein de l': Polymères et matériaux composites Discipline : Physique des matériaux Soutenue publiquement le 15/06/2021, par :

Anatole Collet Etude des mécanismes de dispersion et de structuration multi-échelle de nanotubes de carbone dans une matrice polyéthylène sous sollicitation thermique et mécanique

	Devant le jury composé de :
	Dr. Edith Peuvrel-Disdier	Mines ParisTech CNRS Nice	Rapporteure
	Prof. Thierry Aubry	Université de Bretagne Occidentale Rapporteur
	Dr. Jean-Paul Chapel	CRPP CNRS bordeaux	Examinateur
	Prof. Catherine Journet	Université Lyon 1	Examinatrice
	Prof. René Fulchiron	Université Lyon 1	Directeur de thèse
	Prof. Philippe Cassagnau	Université Lyon 1	Directeur de thèse
	Dr. Olivier Lhost	Total Research & Technology Feluy	Invité
	M. Yves Trolez	Total Research & Technology Feluy	Invité

  Anatole Collet 1 , Anatoli Serghei 1 , Olivier Lhost 2 , Yves Trolez 2 , Philippe Cassagnau 1 , René Fulchiron 1 *

	Table of contents
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