Naila Bouayed

Manon Montaner

Claude Le Men

Johanne Teychené

Christine Lafforgue

Nicolas Dietrich
email: nicolas.dietrich@insa-toulouse.fr

Chung-Hak Lee

Christelle Guigui

Johanne Teychene

Experimental Solid-Liquid Mass Transfer around Free-Moving Particles in an Air-Lift Membrane Bioreactor with Optical Techniques

Keywords: Membrane Bioreactors (MBRs), Air-Lift Reactors (ALRs), Mass Transfer

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

Introduction

A membrane bioreactor (MBR) is a wastewater treatment that combines a membrane process (microfiltration, ultrafiltration, etc.) with a biological treatment. Over the last few decades, MBRs have proved to be highly efficient for advanced wastewater treatment and reuse, and have provided high standards of effluent quality, biomass retention, biomass concentrations, organic removal efficiency, and organic loading rate, together with a low production of sludge and a small environmental footprint [START_REF] Gander | Aerobic MBRs for Domestic Wastewater Treatment: A Review with Cost Considerations[END_REF][START_REF] Melin | Membrane Bioreactor Technology for Wastewater Treatment and Reuse[END_REF]. Yet the extensive development of MBRs for wastewater treatment is still restricted by membrane fouling, which results from the formation of a fouling layer on the membrane surface [START_REF] Xie | Particle-Scale Modelling of Rapid Granular Filtration in a Dual-Media Filter[END_REF](deposition and accumulation of solids, biofilm, pore clogging, adsorption of products, etc.). Membrane fouling gives rise to an overall reduction in performance by causing a loss of permeability, thus increasing the energy consumption and the operating costs. Several cleaning methods (physical, chemical and biological) have been developed in attempts to mitigate this phenomenon. One of the most commonly applied is air-sparging, which consists in the injection of bubbles to induce shear stress between two flat membranes, creating an Air-Lift Reactor (ALR) [START_REF] Liu | Study on Hydraulic Characteristics in a Submerged Membrane Bioreactor Process[END_REF][START_REF] Liu | Hydrodynamic Effect on Sludge Accumulation over Membrane Surfaces in a Submerged Membrane Bioreactor[END_REF][START_REF] Prieske | Prediction of the Circulation Velocity in a Membrane Bioreactor[END_REF][START_REF] Yan | Hydrodynamic Optimization of Membrane Bioreactor by Horizontal Geometry Modification Using Computational Fluid Dynamics[END_REF][START_REF] Yan | Hydraulic Optimization of Membrane Bioreactor via Baffle Modification Using Computational Fluid Dynamics[END_REF]. The addition of solid particles, such as synthetic micro-particles , granular activated carbon [START_REF] Wu | Effect of Mechanical Scouring by Granular Activated Carbon (GAC) on Membrane Fouling Mitigation[END_REF], or other granular scouring agents (reviewed by [START_REF] Aslam | Membrane Bioreactors for Wastewater Treatment: A Review of Mechanical Cleaning by Scouring Agents to Control Membrane Fouling[END_REF]) has also demonstrated an effective reduction of fouling in MBRs due to a mechanical washing effect. Recently, a novel biological method has been developed for fouling mitigation, related to the perturbation of cell-to-cell communication between bacteria [START_REF] Kim | Macroencapsulation of Quorum Quenching Bacteria by Polymeric Membrane Layer and Its Application to MBR for Biofouling Control[END_REF][START_REF] Kim | Biofouling Control with Bead-Entrapped Quorum Quenching Bacteria in Membrane Bioreactors: Physical and Biological Effects[END_REF][START_REF] Köse-Mutlu | Quorum Quenching MBR Operations for Biofouling Control under Different Operation Conditions and Using Different Immobilization Media[END_REF][START_REF] Lee | Crossing the Border between Laboratory and Field: Bacterial Quorum Quenching for Anti-Biofouling Strategy in an MBR[END_REF][START_REF] Maqbool | Membrane Biofouling Retardation and Improved Sludge Characteristics Using Quorum Quenching Bacteria in Submerged Membrane Bioreactor[END_REF]. The technique, named "Quorum Quenching", QQ [START_REF] Yu | In Situ versus Pre-Quorum Quenching of Microbial Signaling for Enhanced Biofouling Control in Membrane Bioreactors[END_REF] is based on an enzymatic degradation of the signal molecules (AHLs), that are produced for bacterial communication (Quorum Sensing [START_REF] Li | Longer Persistence of Quorum Quenching Bacteria over Quorum Sensing Bacteria in Aerobic Granules[END_REF], by excreted or intracellular enzymes produced by specific QQ bacteria. Recent works have shown that, when QQ bacteria are immobilized in small capsules and implemented in MBRs, the biofouling phenomenon is considerably inhibited [START_REF] Oh | Control of Membrane Biofouling in MBR for Wastewater Treatment by Quorum Quenching Bacteria Encapsulated in Microporous Membrane[END_REF]. For more information on the QQ mechanism and its application to MBRs, the reader is invited to consult the reviews by [START_REF] Siddiqui | Quorum Sensing Based Membrane Biofouling Control for Water Treatment: A Review[END_REF][START_REF] Siddiqui | Targeting N-Acyl-Homoserine-Lactones to Mitigate Membrane Biofouling Based on Quorum Sensing Using a Biofouling Reducer[END_REF][START_REF] Bouayed | Process-Oriented Review of Bacterial Quorum Quenching for Membrane Biofouling Mitigation in Membrane Bioreactors (MBRs)[END_REF][START_REF] Börte | Quorum Quenching for Effective Control of Biofouling in Membrane Bioreactor: A Comprehensive Review of Approaches, Applications, and Challenges[END_REF]. With the development of QQ entrapping methods [START_REF] Bouayed | Process-Oriented Review of Bacterial Quorum Quenching for Membrane Biofouling Mitigation in Membrane Bioreactors (MBRs)[END_REF], the mass transfer appeared as one question that it was essential to address, for two main reasons: firstly, as in every (bio)chemical reaction, the mass transfer of the reagents is a key phenomenon as it can be a limiting step. Secondly, the importance of studying the mass transfer involved in QQ is also linked to the different entrapping methods [START_REF] Börte | Quorum Quenching for Effective Control of Biofouling in Membrane Bioreactor: A Comprehensive Review of Approaches, Applications, and Challenges[END_REF][START_REF] Yu | Core-Shell Structured Quorum Quenching Beads for More Sustainable Anti-Biofouling in Membrane Bioreactors[END_REF][START_REF] Iqbal | Impact of Encapsulated Quorum-Quenching Bacterial Dose and Feed Type on Biofouling Control in Membrane Bioreactors[END_REF] that have been developed (introduction of solid beads, hollow cylinders or sheets into the reactors [START_REF] Bouayed | Hydrodynamics Characterization of the Impact of Free-Moving Particles in an Air-Lift Membrane Bioreactor[END_REF] and to the existing kinds of QQ bacteria producing endo-enzyme-or exo-enzyme [START_REF] Bouayed | Hydrodynamics Characterization of the Impact of Free-Moving Particles in an Air-Lift Membrane Bioreactor[END_REF][START_REF] Choo | 12 -Quorum Sensing and Quorum Quenching in Membrane Bioreactors[END_REF][START_REF] Liu | Quorum Quenching in Anaerobic Membrane Bioreactor for Fouling Control[END_REF][START_REF] Jiang | Quorum Quenching Bacteria Bioaugmented GO/PPy Modified Membrane in EMBR for Membrane Antifouling[END_REF]. From these specificities, two cases of mass transfer can be distinguished: from the mixed liquor towards the inner part of solid media, in the case of endo-enzyme degradation, and from the solid media to the mixed liquor in the case of exo-enzyme-mediated QQ. Even though some attempts have been reported in the literature previously cited, the mass transfer phenomena involved in QQ are not fully understood yet and the present study aims to improve this situation. These studies mostly focused on the observation of diffusion (with no liquid flow) at the scale of one single QQ medium.

The experimental approach described here was based on the use of a dye as model molecule in order to quantify the mass transfer at the reactor scale (not only at the QQ media scale). More specifically, the objective was to provide a quantitative characterization of the mass transfer of the molecules involved in QQ, from the liquid to the QQ media and vice versa for different MBR operating parameters (aeration flow rate, kind of QQ media…). In this context, this article offers the determination of mass transfer parameters such as the transferred flux, the mass transfer coefficient, and the Sherwood number [START_REF] Dietrich | Optical Techniques to Evaluate Hydrodynamics and Mass Transfer in Biorectors[END_REF][START_REF] Dietrich | A New Direct Technique for Visualizing and Measuring Gas-Liquid Mass Transfer around Bubbles Moving in a Straight Millimetric Square Channel[END_REF][START_REF] Xu | Comparison of Three Different Techniques for Gas-Liquid Mass Transfer Visualization[END_REF]. On the basis of these quantitative parameters, the effect of the operating conditions on the mass transfer phenomena will be discussed with reference to the effect of the shape of QQ media and will provide valuable suggestions for future research in this area.

Materials and Methods

Physical properties of QQ media

The QQ media used in this study were made of sodium alginate. Three different shapes of media were used in this study: QQ beads, QQ hollow cylinders and QQ sheets (Figure 1).

The physical properties of the QQ media are summarized in Table 1.

Mass transfer experiments

The mass transfer of real signal molecules (such N-acyl-l-homoserine lactones, acyl-HSLs or AHLs) was initially considered in this study. However depending on the considered bacteria, these molecules can have various composition and the analysis of the transfer would require the detection and the quantification of several AHLs. Among the proposed methods, a biological one relying on the use of the reporter strain A.tumefaciens is often proposed to detect the AHLs [START_REF] Oh | Biofouling Inhibition in MBR by Rhodococcus Sp. BH4 Isolated from Real MBR Plant[END_REF][START_REF] Wang | Purification and Identification of Bacterial Signal Molecules Based on N-Acyl Homoserine Lactones[END_REF]. However this method requires perfectly sterile conditions to be maintained, which could not be achieved at the reactor scale. In addition, the amount of AHLs in the mixed liquor is very small, given that these signal molecules are usually produced at very low concentrations (in the range of picograms to nanograms per liter) and that they are present as a complex mixture with different compounds, an extraction procedure is necessary before their quantification. Several methods to measure the AHL concentration after their extraction have been reported to date and are summarized by [START_REF] Siddiqui | Quorum Sensing Based Membrane Biofouling Control for Water Treatment: A Review[END_REF]. After extraction, AHL's detection by mass-spectrometry (HPLC-MS, GC-MS, LC-MS-MS, UPLC6FTIR-MS, CZE-MS), NMR or IR can be performed. However, the choice was made to study mass transfer at the global scale, rather than at the media scale. Thus, the selection of a mimic molecule (tracer) was preferred. Rose Bengal Lactone is light red to pink powder used as a dye, the main properties of which are presented in Table 2. Like the AHLs, Rose Bengal Lactone contains a lactone group, reason why it was chosen as a model molecule to study the mass transfer. The Rose Bengal Lactone was purchased from Sigma-Aldrich. Solutions at a concentration of 0.4 g.L -1 were prepared in tap water. In order to help the dissolution of the powder, the solutions were sonicated 3 to 4 times for 15 min and shaken between two sonication cycles, until a clear dark-pink solution was obtained.

The concentration of the dye was determined using optical methods. The absorbance of dye solutions was measured using a spectrophotometer (Jasco-V630, Germany). The measurements were done at 548 nm, wavelength corresponding to the maximum of absorbance in the visible range.

Mass transfer from the liquid to the media

The study of the mass transfer from the liquid phase into the solid medium was performed in jar-tests in order to control hydrodynamic conditions in the different 1L-beaker reactors (Floculateur, Bioblock Scientific). QQ media in a solution of Rose Bengal Lactone (around 0.4 g.L -1 ) at a solid:liquid volume ratio of 1:9 and under stirring (90 rpm) at room temperature. Two controls without QQ media (one with water and one with the saturated solution of Rose Bengal Lactone) were done to ensure that the observed decrease of concentration in the liquid phase could only be attributed to a transfer into the QQ media, and not to additional phenomena such as natural degradation, precipitation, etc. Liquid samples were collected from the jars every 30 min at first and then spaced out after the first 7 hours. The experiments lasted 24 h, time for the concentration to reach a constant value, corresponding to the stabilization of the system. After 24 h, the stained QQ media were collected and drained to be used for the subsequent experiments.

Mass transfer from the media to the liquid

The mass transfer from the media to the liquid phase was studied in the lab-scale Air-Lift-Membrane-Bio-Reactor (ALMBR) previously described. The QQ media saturated (stained) collected at the end of the jar-test experiments were introduced in a volume fraction of 0.45 % v/v with respected to the total volume of reactor (13 L). The air-lift was set to a riser width of 15 mm. Different operating conditions were investigated in order to highlight the effect of the QQ media shape, as well as the effect of the hydrodynamics (three air flowrates) on the transfer from the QQ media to the liquid phase. Three air flowrates were injected at the bottom of the reactor, corresponding to SAD (Specific Aeration Demand) of 0.75, 0.9 and 1 Nm 3 .h -1 .m -2 . In order to visualize the transfer phenomena in the reactor, a camera technique was developed to measure the absorbance (and thus the concentration of Rose Bengal Lactone in the liquid phase) and consisted in using a 12-bit (4096 gray level) CMOS camera (Basler-Ace Aca1920-155 um) equipped with a green filter (495 to 505 nm). A backlight panel (Phlox-LedW-BL, 400 x 200 mm², 24 V, 2A, Phlox) was set behind the reactor as presented in Figure 2. The observation window was 97.5 mm wide and 150 mm long and was located at the bottom of the reactor (under the membrane) so that the whole depth of the tank could be observed (thanks to the clear baffles). The camera technique was preferred to a simple absorbance measurement via a light sensor because of the coexistence of three phases in the reactor: gas, liquid and solid, which can be taken into account more accurately thanks to image processing. An image was recorded every 10 s (acquisition frequency of 0.1 Hz) and the total acquisition time was 1 day or more, depending on the time required to reach equilibrium. The size of the images was 1000 x 650 pixels ² (1 pixel = 150 µm). Pylon software (Basler, Germany) was used to control the camera and to configure all of its settings.

The filter was used to narrow down the range of wavelengths passing through the liquid in the reactor to approach a monochromatic beam (~ 500 nm, for which the absorbance is high enough for measurement accuracy) and to link the intensity of light to the concentration of Rose Bengal Lactone, according to the Beer-Lambert law (Equation 1), where 𝐴 is the instantaneous absorbance, 𝐼 is the instantaneous light intensity, 𝐼 ! is the light intensity through the blank (water), 𝐶 is the concentration of Rose Bengal Lactone, 𝑙 is the optical path length and is the molar attenuation coefficient. The latter coefficient was obtained from the calibration.

(

) 1
Images of standard solutions of Rose Bengal Lactone with known concentrations (Table 2) were recorded using the camera setup and the absorbance of these solutions was also measured using the spectrophotometer at 500 nm (Table 3). The absorbances per unit of optical path length are plotted on the same graph for comparison in Figure 3. It can be observed that the ratios of the absorbance to the optical path length are very close for both measurement techniques. Also, the attenuation coefficients were found to be very close (Table 2), with less than 2 % deviation. From this comparison, it is possible to conclude that the camera technique that was specifically developed for the study of the mass transfer in the ALMBR, indeed allowed the measurement of an absorbance which followed the Beer-Lambert law. Given these results, the camera was selected as the only measurement technique to study the dye transfer from the QQ media to the liquid phase in the ALMBR. The images recorded during the experiments were processed using a Matlab (MathWorks, USA) program. Prior to each experiment (before the addition of the stained solid medium), around 60 images of the blank (tap water) in the reactor were recorded to determine the intensity 𝐼 ! , and 100 images were recorded of the dark (turning the light panel off) for further calibration of the sensitivity of the measurement. After defining the total number of images to be processed, the acquisition period was selected and a threshold factor of 60 % (used to remove the bubbles or solid media from the image) was applied.

The matrices corresponding to the dark images were first averaged. This average dark image was subtracted from the images to be processed in order to attenuate the signal noise (especially at low intensities). The resulting matrices of the blank gave "flat" matrices that were averaged in a single flat matrix. In the same way, the raw images of the experiment were processed by subtracting the matrix of the average dark image. In the following step, the threshold factor was applied in order to remove the objects (bubbles and solid particles) and only keep the pixels corresponding to the liquid phase (intensity > 60 % of maximum intensity). The mean intensity 𝐼 of the processed image was then determined by averaging the remaining pixels and the intensity 𝐼 -was obtained by averaging the intensities of the corresponding pixels (in the same location) on the flat matrix. Finally, the corresponding absorbance 𝐴 was determined according to Equation 1.

First of all, the stability of the Rose Bengal Lactone over time was evaluated by introducing a solution of known concentration into the reactor and using the camera to measure the light intensity of the light panel through this solution over time. The dye was found to be sensitive to strong, long-term exposure to the light panel, as the intensity recorded increased (concentration decrease) over time. Thus, the strength of the light panel was adjusted with a view to avoiding the degradation of Rose Bengal Lactone during the experiment so as to be able to draw reliable conclusions on the mass transfer.

Liquid-solid mass transfer from the liquid to the solid medium

Analysis of the adsorption kinetics

The main mathematical models proposed in the literature to describe the overall adsorption phenomenon are based on the quantity 𝑞(𝑡) of solute adsorbed per unit mass of solid (in mg.g -1 ). The pseudo-first-order model was proposed by Lagergren in 1898 [START_REF] Yuh-Shan | Citation Review of Lagergren Kinetic Rate Equation on Adsorption Reactions[END_REF] and is expressed according to Equation 1, where 𝑞 . is the adsorbed quantity reached per unit of mass at equilibrium, and 𝑘 / is the constant of the pseudo-first-order kinetics (in s -1 ). Determining this constant requires the linearization of Equation 2, which results in Equation 3, where 𝑘 / is the slope of the curve ln(𝑞 . -𝑞(𝑡)) versus time.

𝑑𝑞(𝑡) 𝑑𝑡 = 𝑘 / 4𝑞 . -𝑞(𝑡)5 (2)
ln(𝑞 . -𝑞(𝑡)) = 𝑙𝑛(𝑞 . ) -𝑘 / 𝑡 (3)
Another model, the pseudo-second-order model [START_REF] Wu | Characteristics of Pseudo-Second-Order Kinetic Model for Liquid-Phase Adsorption: A Mini-Review[END_REF] , assumes the existence of strong chemical bonds between the solute molecule and the solid. This model is described by Equation 4, where 𝑘 0 is the constant of the pseudo-second-order kinetics (in g.mg -1 .s -1 ). The linear form presented in Equation 5 allows the constant 𝑘 0 as well as the adsorbed quantity at equilibrium, 𝑞 . to be determined.

𝑑𝑞(𝑡) 𝑑𝑡 = 𝑘 0 4𝑞 . -𝑞(𝑡)5 0 (4) 𝑡 𝑞(𝑡) = 𝑡 𝑞 . + 1 𝑘 0 𝑞 . 0 (5)
In the present case, the adsorbed quantity at time t, 𝑞(𝑡), can be deduced from Equation 6, where MS is the mass of solid particles, and 𝑉 1 are the wet density (in kg.m -3 ) and the total volume (in m 3 ), respectively, of the solid particles, 𝐶 2 (𝑡) is the concentration in the bulk, and 𝑉 2 is the total liquid volume.

(

) 6
The jar-test experiments were repeated three times. The results are presented in Figure 4, in terms of the total amount of Rose Bengal Lactone per unit of mass of medium (𝑞(𝑡)) (defined in Equation 6) for the different shape of QQ media. The curves in Figure 4 describe a very rapid increase in the first hour and then an overall slowdown until equilibrium is reached after 24 h. This kind of trend is typical of a mass transfer phenomenon in which the gradient of concentrations tends to decrease over time, thus slowing down the increase of the total amount transferred. A comparison of the three experiments performed for each media reveals that the jar-test experiment was repeatable, with less than 10 % standard deviation between the three final values of the amount of Rose Bengal Lactone transferred and a similar time evolution for the experiments done with a same QQ medium.

The comparison of the three curves obtained for three media shapes in Figure 5 suggests that the global adsorption dynamics depends on the shapes of QQ media. In addition, the time to reach 96 % of the final value was 0.5 h and 2 h for sheets and hollow cylinders, respectively, whereas, for beads, this time was much longer -about 8.5 h. According to the adsorption theory previously presented, the pseudo-first-and pseudo-second-order models were considered to describe the kinetics of the phenomenon under study. The variations of and 4. The parameters of the pseudo-second order adsorption model (𝑘 0 and 𝑞 . ) are deduced from the linear correlation parameters 𝑎 and 𝑏 (from Equation 5), according to Equations ( 7) and [START_REF] Yan | Hydraulic Optimization of Membrane Bioreactor via Baffle Modification Using Computational Fluid Dynamics[END_REF]. It appears first that the calculated maximal amounts 𝑞 . of Rose Bengal Lactone adsorbed at equilibrium (also representing the adsorption capacities of the media), are very close to the experimental values reached after 24 h (Figure 4), with discrepancies of less than 5 %, which confirms that the phenomenon studied has reached the stable state after 24 h, even for the beads. When comparing the final amounts, 𝑞 . , reached for the three shapes in each experiment, the differences were not significant, since the three (experimental) values differ by less than 5 %. This finding indicates that the shape of the solid media has no effect on their adsorption capacities, which is consistent since the media are made from the same material. However, significant differences appear in the pseudo-second-order rate constant of adsorption 𝑘 0 (representing the adsorbed flux per unit of mass of solid media), since it was found to be, respectively, 12 and 24 times greater for the hollow cylinders and sheets, than for the beads (Table 4). This trend is confirmed by observation of the adsorption rate index (𝑞 . 𝑘 0 ) defined by [START_REF] Wu | Characteristics of Pseudo-Second-Order Kinetic Model for Liquid-Phase Adsorption: A Mini-Review[END_REF], which purely reflects the kinetic performance of the system. Thus, considering the same volumes of solid media, the adsorption kinetics performance for sheets is considerably greater than for the hollow cylinders and beads.

𝑞 . (𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑) = 1 𝑎 (7) 𝑘 0 = 𝑎 0 𝑏 (8)
Knowing that the adsorption phenomenon involves a succession of mechanisms (external mass transfer, internal mass transfer, pore diffusion, etc.), and that the constant 𝑘 0 englobes all of these mechanisms, these results prove that the differences between the three shapes of solid particles necessarily originate in at least one of the steps.

Analysis of the external liquid-solid mass transfer mechanism

According to the method proposed by Furusawa and Smith [START_REF] Furusawa | Fluid-Particle and Intraparticle Mass Transport Rates in Slurries[END_REF], the external mass transfer step is related to the changes of concentrations over time by the mass balance expressed in Equation 9, where is the liquid-solid mass transfer coefficient (in m.s -1 ), is the total area of the liquid-solid interface (in m 2 ) is the total liquid volume, and ) are the concentration in the bulk and the concentration at the surface of the solid particles respectively. A simple method was proposed to deduce , and consists in substituting Equation 9with the corresponding values at the initial conditions (when ), which results in Equation 10. Therefore, the liquid-solid external mass transfer coefficient can be deduced from the initial slope [ ] of the curve versus time. This approach has been adopted

in the literature to study different systems involving liquid-solid mass transfer, some examples of which are the removal of food dyes using chitosan particles [START_REF] Dotto | Application of Chitosan Films for the Removal of Food Dyes from Aqueous Solutions by Adsorption[END_REF][START_REF] Dotto | Diffusional Mass Transfer Model for the Adsorption of Food Dyes on Chitosan Films[END_REF] or the transfer of ions using ion-exchange resin [START_REF] Sänger | Liquid-Solid Mass Transfer in Aerated Suspensions[END_REF].

𝑉 2 𝑑𝐶 2 (𝑡) 𝑑𝑡 = -𝑘 21 𝑆 1 4𝐶 2 (𝑡) -𝐶 1 (𝑡)5 (9)
( 67 * (3) 63 ) 3:-= ; = *+ > + ? * 𝐶 2 (𝑡 = 0) ( 10
)
This approach is based on the assumption that the concentration in the liquid, 𝐶 2 (𝑡), is uniform. The evolution of the concentration in the liquid phase 𝐶 2 (𝑡), is presented in Figure 5. In order to evaluate the external mass transfer coefficient, the initial slope ( 67 * (3)

63

) 3:-of each curve was determined (Figure 5) and used to calculate the external mass transfer 𝑘 21 via Equation 10, which resulted in Equation 11. The results are presented in Table 5. The product 𝑘 21 𝑆 1 , representing the transferred flux, describes the same trend as the adsorption kinetics parameters (greater for sheets than for hollow cylinders and beads). Despite the uncertainty in the product 𝑘 21 𝑆 1 , for the different QQ shapes that can reach 30 %, the differences between the three shapes are significant (on average 50 % between beads and cylinders, and 33 % between cylinders and sheets). This result confirms the assumptions made in the adsorption analysis that the shapes of media induce significant differences in the external mass transfer step of the whole phenomenon. In particular, the external mass transfer is governed by the external surface area of the solid medium, which explains the greater flux obtained for the sheets. It should be pointed out that in the case of cylinder, the considered surface is the total one (sum of external, internal and sides surfaces) and it is obvious that the liquid renewal inside the cylinder can be weak. Therefore, the measurement of the global transfer at the begining of the experiement could be affected by a concentration of dye inside the QQ media lower than that measured in the liquid bulk. The external mass transfer coefficient 𝑘 21 was determined for the three shapes (Table 5). On average, an 18 % difference was found between the mass transfer coefficients of the beads and the hollow cylinders, and a 30 % difference between hollow cylinders and sheets, which can be viewed as insignificant (< 30 %). Therefore, the external mass transfer coefficient for the three shapes of media can be considered as similar, which is consistent with the fact that the hydrodynamic conditions created in the jar-tests were similar. Knowing the external liquid-solid mass transfer coefficient as well as the diffusion coefficient of Rose Bengal in water, the Sherwood number could be determined for each condition according to Equation 12.

𝑘 21 = - 𝑉 2 𝑆 1 𝐶 2 (𝑡 -) ( 𝑑𝐶 2 (𝑡) 𝑑𝑡 ) 3:- (11)
𝑆ℎ = 𝑘 @> 𝐿 𝐷 (12)
In order to take account of the fact that the transfer occurs from all around a solid particle to its center, the characteristic length 𝐿 in Equation 12was taken to be the radius for the beads, and half the thickness for both the hollow cylinders and the sheets. According to the literature [START_REF] Dotto | Diffusional Mass Transfer Model for the Adsorption of Food Dyes on Chitosan Films[END_REF] , the external convective mass transfer step is completely prevalent for 𝑆ℎ < 0.5, whereas the diffusion is significant for 𝑆ℎ > 10. In the present case (Table 5), the Sherwood numbers roughly ranged between 0.5 and 5, and thus were between 0.5 and 10, indicating that there was no strong predominance of one mechanism over another under the conditions investigated. This result shows that the internal diffusion and the external convection can be considered as two mechanisms of equivalent importance, both governing the overall transport of molecules from the bulk to the solid media. In addition, when the three shapes of solid media particles were compared, the greatest Sherwood number was obtained for the solid beads, which means that the internal diffusion in this shape is more significant than for the other two shapes.

To conclude, with the perspective of applying quorum quenching for biofouling mitigation by using endo-enzyme-producing bacteria (such as Rhodococcus sp. BH4), the (external) mass transfer of AHLs from the mixed liquor towards the core of the media could be favored, in terms of transferred flux (𝑘 21 𝑆 1 ), by the presence of sheets rather than the other two shapes for the same volume of medium. The beads seem to be the least favorable shape for mass transfer: not only does their small surface area give rise to the lowest transferred flux (𝑘 21 𝑆 1 ) (in comparison to the other shapes) but their great diameter also slows down the internal transfer step. Considering that quorum quenching is based on an enzymatic reaction, further investigation is needed to know whether the transferred flux or the transfer kinetics of AHLs (substrate) towards enzymes is the key parameter to promote when the aim is to reduce biofilm formation effectively.

Solid-liquid mass transfer from the solid media to the liquid

In the case of the experiments conducted in the aerated lab-scale ALMBR, the transfer of the Rose Bengal Lactone took place from the solid media to the liquid. Different operating conditions were investigated to highlight the effect of the solid medium shape (beads, hollow cylinders and flat sheets), as well as the effect of the hydrodynamics (three air flowrates, Specific Aeration Demand, called SAD, of 0.75, 0.9 and 1 Nm 3 .h -1 .m -2 ) on the transfer from the solid media to the liquid phase. This kind of configuration (solid-liquid) has been much less investigated than the previous one (liquidsolid), especially under similar hydrodynamic conditions. Although it can be assumed that transfer from the solid to the liquid is a combination of several mechanisms, as illustrated in Figure 6, to the best of our knowledge, no complete model of the whole phenomenon, taking all the steps involved into account, is reported in the literature. Some studies have focused on characterizing the solid-liquid mass transfer involved in the dissolution of solid substances in their own solution, using the mass balance presented in Equation 13 [40]. This approach, similarly, to the one developed for the liquid-solid mass transfer (the opposite phenomenon) (Equation 9), allows a coefficient 𝑘 12 to be determined for the solid-liquid external mass transfer. The solid-liquid mass transfer coefficient can be deduced from Equation 14, which corresponds to the very early stage of the operation (when ) at which the concentration in the liquid is equal to zero and the initial concentration at the surface of the solid 𝐶 1 (𝑡 = 0) can be considered as homogeneous in the solid media, and equals the ratio of the initial mass of dye (𝑚 -) adsorbed on the QQ media to the total volume of solid media 𝑉 1 . In practice, 𝑘 12 is deduced from the initial slope of the curve 𝐶 2 (𝑡) versus time.

𝑉 2 𝑑𝐶 2 (𝑡) 𝑑𝑡 = 𝑘 12 𝑆 1 4𝐶 1 (𝑡) -𝐶 2 (𝑡)5 (13)
( 67 * (3) 63 ) 3:-= = +* > + ? * 𝐶 1 (𝑡 = 0) = = +* > + A , ? * ? + (14)
Mass transfer from the media to the liquid phase was studied in the ALMBR under different aeration conditions in order to evaluate how the hydrodynamics and the QQ media shape could influence the transfer. However, before analyzing the results, it is worth mentioning that, in the case of this study, it can be assumed that the hydrodynamics is involved at several levels in the mass transfer phenomenon: by promoting turbulence in the liquid phase, it can reduce the concentration gradient between the surface of the media and the bulk and, thus, accelerate the mass transfer phenomenon; at the local scale, it can enhance the motion of the particles, and therefore favor the fast renewal of the liquid boundary layer around them; in addition air flowrate affects the proportion of fluidized QQ media in the reactor, and thus control the exchange surface between the solid media and the liquid phase. Therefore, it can be assumed that the effect of hydrodynamics on the global mass transfer is complex because of the coupling of all these phenomena. For each air flowrate, the total surface corresponding to the added QQ media S, the surface corresponding to the moving particles (fluidized ones) 𝑆 BC , and the volume of these suspended QQ media 𝑉 BC are reported in Table 6. Since the used QQ media are those obtained at the end of the Jar tests, their initial dye concentration is known.

Effect of hydrodynamics on solid-liquid mass transfer

Using the camera and the image processing, the concentration of Rose Bengal Lactone over time was monitored when the stained QQ media were introduced into the ALMBR, under the conditions presented in Table 6. The results are presented in Figure 7, in terms of normalized amount of dye (

7 * (3)? * A ,
) released to the liquid overtime. It is first possible to observe that the overall aspect of the curves includes a rapid increase at the beginning and then tends to a stable value. This kind of trend was to be expected since, in this case, the mass transfer is driven by the difference of concentrations between the solid and the liquid, which tends to be attenuated over time, inducing a slowdown of the transfer. The overall mass transfer phenomenon appears to be relatively slow, since all the experiments lasted more than 24 h. The time required to approach the equilibrium (the moment t F at which the normalized concentration reached 96 % of its final value) was measured for each condition. The times t F for the three different air flowrates were quite similar for a given shape of solid media. It could then be assumed that the global transfer dynamics was not significantly affected by the aeration in the range of air flowrates investigated. In addition, the solid-liquid mass transfer pforùatarameters (𝑘 12 and 𝑘 12 𝑆 .GHIJKL. , where 𝑆 .GHIJKL. is the exchange surface between the solid medium and the liquid phase) were determined considering the initial conditions (

) and according to the approach explained previously (Equations 13 and14). The initial slopes of the curves 𝐶 2 (𝑡) were determined over the first 10 points (the first 90 s of the operation) for each condition, and one example is presented in Figure 8 for the mass transfer from the beads to the liquid under the three air flowrates investigated. The correlation parameters and the mass transfer parameters are grouped in Table 7 as the mass transfer parameters obtained for the different shapes. The observation of the transferred flux 𝑘 12 𝑆 .GHIJKL. reveals no clear effect of the aeration, in the range investigated, since different trends are described for the three shapes of solid media. The determination of the mass transfer coefficient 𝑘 12 requires the transferred flux that was measured to be divided by the exchange surface area 𝑆 .GHIJKL. to compare the transfer kinetics. However, in the present case, the exact exchange surface area is hard to evaluate, since it depends on the fluidization of the solid media, as previously mentioned. Therefore, two possible limit-cases could be considered for discussion: in the first case, the exchange surface area corresponds to the total surface 𝑆 -of solid medium introduced in the reactor, whether the particles are fluidized or settled. In the second case, the exchange surface corresponds to the surface area 𝑆 ./ of the fluidized particles only. The mass transfer coefficients were calculated in both cases (Table 7) and are also represented as a function of the aeration for the three shapes of solid media in Figure 9. Significant differences can be observed between the two cases considered. In the first case, where the totality of solid media introduced (with surface area 𝑆 -) in the reactor is assumed to participate in the global transfer, the mass transfer coefficients range roughly between 0.5 10 -7 and 2.0 10 -7 m.s -1 .

In the second case, where the assumption is that only the fluidized particles (with surface area 𝑆 ./ ) participate in the global transfer, the mass transfer coefficients range between 0.2 10 -6 and 2.0 10 -6 m.s -1 . These observations are consistent and actually indicate that, in the second case, where the number of particles transferring (only the fluidized ones) is lower, the mass transfer would have to be much faster to reach the same transferred flux as in the first case. However, the effect of the air flowrate on the mass transfer coefficient is still hardly identifiable in the investigated range, whichever the case considered. These results show that the effect of the air flowrate on the solid-liquid mass transfer is not clearly identifiable in the conditions investigated, because of the complexity of the mechanisms, which depend on the aeration, and the difficulty of assessing the exchange surface area involved in the transfer with certainty. The two cases considered here are "extreme" cases, which are most likely not representative of the reality since (i) the settled media can also take part in the global transfer and (ii) because there is an alternation phenomenon in the fluidization (the media in suspension are not necessarily the same during the whole operation). Thus, the effective surface area of exchange 𝑆 .GHIJKL. between the solid medium and the liquid phase should be somewhere between the surface area of the fluidized particles and the total surface area of the medium (𝑆 BC < 𝑆 .GHIJKL. < 𝑆 1 ).

Effect of solid particle shapes on the mass transfer

Because the initial conditions were different from one experiment to another and the levels reached at equilibrium were different for the three shapes of solid particles (Figure 7), the concentration curves were normalized with respected to the final value (

) and the curves presented in Figure 10 take only the dynamics of the transfer phenomenon into account (independently of the amounts transferred). The effect of the media shapes can be determined first by visualizing the curves, since it appears clearly that the overall dynamics is faster for the sheets than for the hollow cylinders or the beads. This can be confirmed by the comparison of the times t F necessary to reach 96 % of the equilibrium. It transfer phenomenon into account (independently of the amounts transferred).

appears that the time to reach equilibrium for the beads is approximately 3 times greater than that for the hollow cylinders and 9 times that for the sheets, in the range of air flowrates investigated. In the case of beads, the curves presented in Figure 10 reveal that the equilibrium is not completely reached and that the mass transfer continues to evolve very slowly, possibly because of their larger diameter and thus slower internal mass transfer. The mass transfer parameters determined in Table 7 show that significant differences are obtained in the transferred flux 𝑘 12 𝑆 .GHIJKL. for the three shapes. For example, for a SADm of 1.00 Nm 3 .h -1 .m -2 (corresponding to the highest fluidization rates), 𝑘 12 𝑆 .GHIJKL. in presence of sheets is up to 3 or 4 times greater than that of beads or hollow cylinders, respectively. In terms of mass transfer coefficient 𝑘 12 , the differences between the three particle shapes depend on which of the two cases mentioned above is considered. It appears that, for the same volume of solid medim introduced into the reactor, the mass transfer phenomenon is considerably enhanced in the presence of sheets compared to the other shapes, in terms of both time to reach equilibrium and flux transferred. In addition, the time 𝑡 . needed to reach equilibrium for sheets is only around 4 h, which represents one third of the residence time (12 h) of the ALMBR, whereas the time needed for beads (about 30 h) is at least 2.5 times greater than the residence time. These results suggest that, among the three shapes of media, sheets would be the most favorable to enhance the mass transfer phenomenon from the medium to the liquid phase, and thus to quickly mitigate fouling via the biological activity. The Sherwood numbers were determined according to Equation 12 for each medium shape and are shown in Table 7. Again, two cases were considered, corresponding to the two exchange surface areas. When the total surface 𝑆 1 is taken into account, the Sherwood numbers range from 0.2 to 1.6 for the different particle shapes under the different air flowrates. According to the literature, the fact that the Sherwood numbers are lower than 0.5 (in this case for hollow cylinders and sheets) indicates that the diffusion mechanism is very fast compared to the external mass transfer step in the investigated conditions, and that the diffusion time can be considered negligible. However, for the beads (0.6 < 𝑆ℎ < 1.6), the internal diffusion is not negligible, which is consistent with the fact that equilibrium was never completely reached in the investigated conditions. For Sh <1, it has been possible to propose a scaling law between the Sherwood number and the media Reynolds number ( 𝑅𝑒 = Q -*./0 ×S */1./0 /3 456 7/+67 6 860/9 T -*./0

) with a good precision (22% of standard deviation):

𝑆ℎ = 0.1 × 𝑅𝑒 -.0 (15)
This correlation shows a strong effect of the local hydrodynamics impacted by the particle on the global mass transfer rate of the medium for all the shapes studied. It is worth noting that this correlation could be improved with more data to include other dimensionless number such as the Schmidt number. When the fluidized surface area 𝑆 BC is considered, the Sherwood numbers are much greater and range from 0.4 to 17. In most cases, the Sherwood numbers are between 0.5 and 10 (except for the beads under 0.75 Nm 3 .h -1 .m -2 ) which, according to the literature, indicates that there is no clear prevalence of one mechanism over another. However, in the particular case of the beads under a SADm of 0.75 Nm 3 .h -1 .m -2 , the Sherwood number is 16.80 (> 10) which refers to a slow diffusion step. In all cases (whatever the exchange surface considered), the Sherwood numbers obtained for the beads are much higher than for the other two shapes, which undeniably shows that the diffusion into the beads is slower because of their larger diameter.

A comparison of the investigated air flowrates led to the conclusion that no notable effect on the solid-liquid mass transfer coefficient was observed in the conditions of the study. This result could be explained by the fluctuations in the fluidization of media during the experiment and the difficulty of evaluating the effective exchange surface with precision, which made the precise determination of a mass transfer coefficient difficult. Concerning the effect of the media shapes, it was found that the sheets gave rise to greater mass transfer in the shortest time to reach equilibrium and the greatest transferred flux. With a view to applying quorum quenching to reduce membrane biofouling, sheets may therefore be the most appropriate shape in the case of exoenzyme-producing bacteria (Pseudomonas sp. 1A1, for example) as the exoenzyme produced in the core of a sheets could rapidly transfer out of it to reach and degrade the AHLs present in the mixed liquor.

Finally, the determination of the Sherwood number in the conditions investigated demonstrated that the overall phenomenon is clearly limited more by the external mass transfer step than by the internal diffusion in the solid media. Nevertheless, comparing the three shapes of media tended to show that the diffusion could again be a little more important in the case of beads, as was the case for the liquid-solid mass transfer, because of their greater diameter.

In order to evaluate the impact of the presence of solid media on a possible membrane clogging, the permeability of the membrane has been measured for several operating conditions. In the case of a constant flow filtration, clogging is characterized by the increase in transmembrane pressure (TMP) over time. A pressure sensor on the permeate side allows to follow the pressure Pp over time. The initial pressure Pi in the reactor is measured at the beginning of the experiment when the pump on the permeate side is stopped. The transmembrane pressure is calculated according to the equation below:

TMP=𝑃𝑖-𝑃𝑝 (16)
The permeability measurement is performed by maintaining a constant volume of water in the reactor. The operation consists of setting the permeate flow rate and following the pressure evolution to determine the permeate flux 𝐽 and the permeability Lp, and is determined according to the Darcy's law with the following expression:

𝐽 0-°7 = 𝐿𝑝 × 𝑇𝑀𝑃 = 𝑃𝑇𝑀 𝜇 0-°7 × 𝑅 A (17)
Figure 11 represents the evolution of the water permeability at 20 °C as a function of time. It is obtained for a SADm of 0.75 Nm 3 /h/m². A first experiment was carried out with demineralized water without addition of media, it will be used as reference. Two other experiments were performed with the addition of inert media (beads and hollow cylinders) for a volume fraction of 0.45% v/v. The results highlight the establishment of an operating regime that stabilizes after one hour of filtration. If we consider only the measurements from one hour of filtration, in which the permeability measurement is stable. For the three experiments carried out (in the presence or absence of media) under similar operating conditions, it can be seen that the results of water filtration are practically identical (considering an error on the permeability measurement of ± 15%). It can therefore be stated that the addition of inert media does not lead to membrane clogging since the permeability is similar with or without QQ media.

Conclusion

In this article, an attempt has been made to quantitatively characterize the mass transfer phenomena that can be involved in the application of a bacterial antifouling technique (Quorum Quenching) using different shapes of alginate supports for bacteria for the first time in literature. Considering that the Quorum Quenching can be based on an endo-enzyme-producing bacterium or an exoenzyme-producing one, two kinds of mass transfer were distinguished for the study: liquid-solid mass transfer to mimic the transport of signal molecules from the mixed liquor to the inner part of solid media; and solid-liquid mass transfer to mimic the transport of exoenzymes from the solid media to the mixed liquor. Using a model (dye) molecule under different hydrodynamic conditions, the mass transfer kinetics characterization enabled the following main conclusions to be drawn. First, the tools selected for the characterization of the mass transfer proved to be efficient for the purpose. The experimental setups and the theoretical approaches provided relevant information about both kinds of mass transfer: the study of the liquidsolid mass transfer in jar-tests determined mass transfer coefficients in the order of magnitude of 10 -6 m.s -1 . The results brought to light the fact that both sheets and the hollow cylinders can be appropriate shapes for an efficient mass transfer of AHLs from the mixed liquor to the entrapped endo-enzyme producing bacteria, in terms of transferred flux under similar hydrodynamic conditions. For the beads, the liquid-solid mass transfer was found to be less efficient because their specific shape gave the internal diffusion step more weight in the overall transfer phenomenon. The study of the solid-liquid mass transfer in the aerated ALMBR was performed under different air flowrates for the three shapes of media. The effect of the air flowrate on the mass transfer coefficient was hardly quantifiable because of the difficulty of assessing the effective exchange surface area. However, the effect of shape could be determined and the most favorable shape in terms of transferred flux appears to be sheets. In the case of exoenzyme-producing bacteria entrapped in the solid media, the transfer of exoenzymes could be more efficient for sheets than for hollow cylinders or beads. The investigation of the two kinds of mass transfer gave interesting insights for the quorum quenching application. Actually, the mass transfer coefficient for liquid-solid transfer (in the jar-test) was found to be of the order of magnitude of 10 -6 m.s -1 whereas the solid-liquid transfer (in the aerated ALMBR) was 10 -7 m.s -1 . The two mass transfer coefficients are not directly comparable because they were obtained under different hydrodynamic conditions, but this indicates that mechanical stirring can provide better hydrodynamic conditions to foster mass transfer. In all the cases, the beads were found to give rise to the smallest transferred flux compared to the other two shapes. However, when the fluidized surface area 𝑆 BC is considered, the mass transfer coefficient for beads is greater, which means that this shape could be valuable in that case, by increasing the surface (decreasing the diameter) and increasing their fluidization (increasing the air flowrate, decreasing their diameter and/or density). These findings are of great importance because they open the way to an optimized application of quorum quenching. Finally, the development of the optical technique was revealed to be relevant to study the solid-liquid mass transfer from the solid media to the liquid phase of the aerated part of the ALMBR.

Figure 1 .

 1 Figure 1. Photographs of media used in the study: (a) beads, (b) hollow cylinders, and (c) sheets.

Figure 2 .

 2 Figure 2. Experimental setup for the visualization of the mass transfer in the MBR.

Figure 3 .

 3 Figure 3. Comparison of the ratios of the absorbance to the optical path length obtained by the spectrophotometer and the camera technique.

Figure 4 .

 4 Figure 4. (Left) Amount of Rose Bengal Lactone transferred in the solid media over time. (Middle) and (Right) Identification of the adsorption model parameters for the experimental data. Jar-test experiments.

3 4

 3 over time were thus deduced from the experimental data and are presented in Figure 4. The variations of over time do not show a linear trend, unlike the variations of 3 4 , which suggests that the pseudo-second-order model could be more appropriate (R² > 0.99) to describe the adsorption phenomenon. The parameters of the linear correlations (𝑦 = 𝑎𝑥 + 𝑏) are presented in Table

Figure 5 .

 5 Figure 5. Concentration of the Rose Bengal Lactone in the liquid phase over time. Jartest experiments.

  𝒕)𝒅𝒕)𝒕=𝟎 (mg.L -1 .h -1 ) the three experiments ± standard deviation 𝒌 𝒍𝒔 𝑺 𝒔 (10 -7 m 3 .s

Figure 6 .

 6 Figure 6. Illustrative representation of the transfer of Rose Bengal Lactone from the solid medium to the liquid, in the aerobic tank of the lab-scale MBR.

Figure 7 .

 7 Figure 7. Effect of the hydrodynamics on the normalized amount of Rose Bengal Lactone released into the liquid phase for the three different shapes of solid media. ALMBR experiments.

Figure 8 .

 8 Figure 8. Initial evolution (over the first 90 s) of the concentration of Rose Bengal Lactone in the liquid phase using beads with three air flowrates. ALMBR experiments.

Table 7 .𝒌 1 ) 15 𝒌

 7115 Parameters of the mass model and Sherwood numbers for the solid-liquid mass transfer of Rose Bengal Lactone from the medium to the liquid under different air flowrates (the 𝑆ℎ numbers were calculated with the diffusion coefficient 𝐷 = 3.92 10 -10 m 2 .s -1 ). ALMBR experiments. SADm (Nm 3 .h -1 .m mass transfer parameters for beads Tot. exchange surf. area 𝑺 𝒔 (m 2 ) 𝒌 𝒔𝒍 𝑺 𝒆𝒙𝒄𝒉𝒂𝒏𝒈𝒆 (10 -8 m 3 .s -1 ) 𝒔𝒍 (considering 𝑺 𝒔 ) (10 -7 m.s -𝒔𝒍 (considering 𝑺 𝑭𝑴 ) (10 -7 m.s -1 ) mass transfer parameters for hollow cylinders Tot. exchange surf. area 𝑺 𝒔 (m 2 ) 𝒌 𝒔𝒍 𝑺 𝒆𝒙𝒄𝒉𝒂𝒏𝒈𝒆 (10 -8 m 3 .s -1 ) 𝒔𝒍 (considering 𝑺 𝑭𝑴 ) (10 -7 m.s -1 ) mass transfer parameters for sheets Tot. exchange surf. area 𝑺 𝒔 (m 2 ) 𝒌 𝒔𝒍 𝑺 𝒆𝒙𝒄𝒉𝒂𝒏𝒈𝒆 (10 -8 m 3 .s -1 ) 𝒔𝒍 (considering 𝑺 𝑭𝑴 ) (10 -7 m.s -1 )

Figure 9 .

 9 Figure 9. Mass transfer coefficients under different air flowrates and for the three shapes of media particles. ALMBR experiments.

Figure 10 .

 10 Figure 10. Effect of the shapes of the solid media on the normalized concentrations of Rose Bengal Lactone in the liquid phase for the three different air flowrates. ALMBR experiments.

Figure 11 .

 11 Figure 11. Evolution of the permeability of the membrane for water at 20°C with and without solid media.

Table 1 .

 1 Properties of the solid media.

	Solid medium	Beads	Hollow cylinders	Sheets
	Dimensions (mm)	Diameter: 3.5	Inner diameter: 1.7	Length: 20
			Outer diameter: 3.5	Width: 10
			Length: 27	Thickness: 0.5
	Volume of a particle (mm 3 )	22.5	198.5	100
	Surface area of a particle (mm²)	8.5	455.8	400

Table 2 .

 2 Physicochemical properties of Rose Bengal Lactone.

Chemical formula/Molecular structure C20H4Cl4I4O5 Molecular weight (g.mol -1 ) 973.67 Appearance Pink powder Purity 95 %

Table 3 .

 3 Spectrophotometer and camera-deduced optical parameters for standard solutions of Rose Bengal Lactone.

Concentration of dye Lactone solution 𝑪 (g.L -1 ) 0.0002 0.0006 0.0012 0.0023 0.0044 Spectrophotometer Absorbance at 500 nm 𝑨 𝒔𝒑𝒆𝒄𝒕𝒓𝒐 (-)

	0.0037	0.0088	0.0188	0.0346	0.0673

Optical path length 𝒍 𝒔𝒑𝒆𝒄𝒕𝒓𝒐 (cm) 1.0 𝑨 𝒔𝒑𝒆𝒄𝒕𝒓𝒐 𝒍 𝒔𝒑𝒆𝒄𝒕𝒓𝒐 (cm -1 )

	0.0037	0.0088	0.0188	0.0346	0.0673

(L.g -1 .cm -1 ) 15.136 Camera Absorbance at 500 nm 𝑨 𝒄𝒂𝒎𝒆𝒓𝒂 (-)

	0.0490	0.1000	0.2050	0.4180	0.7960

Optical path length 𝒍 𝒄𝒂𝒎𝒆𝒓𝒂 (cm) 11.6 𝑨 𝒄𝒂𝒎𝒆𝒓𝒂 𝒍 𝒄𝒂𝒎𝒆𝒓𝒂 (cm -1 )

	0.0042	0.0086	0.0177	0.0360	0.0686

(L.g -1 .cm -1 ) 15.425

Table 4 .

 4 Linear correlation and parameters of the pseudo-second-order kinetics model for the adsorption of Rose Bengal Lactone onto solid media. Jar-test experiments.

		beads	hollow cylinders	sheets
	Linear correlation parameters (𝒚 = 𝒂𝒙 + 𝒃)
	𝒂 (g.mg -1 )	0.3187	0.3157	0.3260
	𝒃 (h.mg -1 .g)	0.3015	0.0712	0.0376
		0.9996	0.9995	0.9996
	Model parameters
	𝒒 𝒆 (calculated) (mg.g -1 )	3.14		3.17		3.07
	𝒒 𝒆 (experimental)	3.0	0.2	3.1	0.2	3.0	0.2
	(mg.g -1 ) 𝒌 0.34		1.40		2.83
	𝒌 𝟐 (

𝟐 (g.mg -1 .h -1 )

10 -5 g.mg -1 .s -1 )

	9.36	38.89	78.51
	𝒌

𝟐 𝒒 𝒆 (10 -4 s -1 ) 2.94 12.33 24.10

Table 5 .

 5 External liquid-solid mass transfer parameters for the three media shapes and the three experiments repeated for each shape. Jar-test experiments (under 90 rpm and solid:liquid ratio of 1:9).

		beads	hollow cylinders	sheets
	Experiment 1
	𝑺𝒔 (m 2 )	0.100	0.135	0.234
	𝑪𝒍(𝒕=𝟎) (mg.L -1 )	484	499	495
	(𝒅𝑪𝒍(𝒕)𝒅𝒕)𝒕=𝟎 (mg.L -1 .h -1 )	-316	-514	-668
	𝒌𝒍𝒔𝑺𝒔 (10 -7 m 3 .s --1 )	0.95	1.51	1.98
	𝒌𝒍𝒔 (10 -6 m.s -1 )	0.95	1.12	0.85
	Experiment 2
	𝑺𝒔 (m 2 )	0.086	0.115	0.200
	𝑪𝒍(𝒕=𝟎) (mg.L -1 )	424	409	436
	(𝒅𝑪𝒍(𝒕)𝒅𝒕)𝒕=𝟎 (mg.L -1 .h -1 )	-320	-455	-612
		1.12	1.21	0.88
	Experiment 3

𝒌𝒍𝒔𝑺𝒔 (10 -7 m 3 .s --1 ) 0.94 1.39 1.75

𝒌𝒍𝒔 (10 -6 m.s -1 )

𝑺𝒔 (m 2 )

Table 6 .

 6 Solid media properties in the ALMBR under different air flowrates.

	SADm		0.75			0.90			1.00
	(Nm 3 .h -1 .m -2 )
	Solid medium	Beads	Hol-	Sheets	Beads	Hol-	Sheets	Beads	Hol-	Sheets
			low			low			low
			cylin-			cylin-			cylin-
			ders			ders			ders
	Tot. exchange	0.086	0.115	0.200	0.086	0.115	0.200	0.086	0.135	0.234
	surf. area 𝑺 𝒔 (m 2 )
	Fluidization rate	6.2	26.2	30.1	10.9	53.2	47.8	16.1	63.3	55.9
	(%)
	Fluidized surface	0.0062 0.0354	0.0704	0.0109	0.0718	0.1118	0.0161	0.0845	0.1308
	area 𝑺 𝑭𝑴 (m 2 )
	Fluidized volume	0.31	1.53	1.76	0.64	3.11	2.79	0.94	3.70	3.27
	𝑽 𝑭𝑴 (10 -5 m 3 )


domain: chim sdv

text:
Muhammad Haseeb Iqbal

Jeanne Rosine Faratiana

Emeline Pradel

Varvara Gribova

Kamel Mamchaoui

Catherine Coirault

Florent Meyer

Fouzia Boulmedais
email: fouzia.boulmedais@ics-cnrs.unistra.fr

Revana

Kamel Mamchaoui #

Catherine Coirault #

Brush-Induced Orientation of Collagen Fibers in Layer-by-Layer Nanofilms: A Simple Method for the Development of Human Muscle Fibers

Keywords: Orientation, nano-patterning, multilayers, polyelectrolyte, myogenesis

come   L'archive ouverte pluridisciplinaire

Introduction

Musculoskeletal tissues are one of the most important components of the human body representing around 40% of adult body mass. Containing thousands of muscle fibers bundled together into an aligned parallel organization, their pivotal role is to perform routine voluntary locomotion by creating forces that are then transmitted by tendons towards the skeleton. [START_REF] Beldjilali-Labro | Biomaterials in Tendon and Skeletal Muscle Tissue Engineering: Current Trends and Challenges[END_REF] Muscle injuries are commonly caused by contusions during sports, accidental trauma, age-related wear etc. [START_REF] Järvinen | The Effects of Early Mobilisation and Immobilisation on the Healing Process Following Muscle Injuries[END_REF] Since the development of collagen-coated substrate to culture avian myotubes, [START_REF] Vandenburgh | Maintenance of highly contractile tissue-cultured avian skeletal myotubes in collagen gel[END_REF] extensive research has been carried out to regenerate skeletal muscle tissue in vitro for the treatment of muscle injuries or diseases, such as muscular dystrophies or paralysis. Muscle development involves differentiation of myoblasts into myotubes with parallel orientation, to ultimately form aligned myofibers to compensate for the muscle loss. [START_REF] Meyer | Elucidation of extracellular matrix mechanics from muscle fibers and fiber bundles[END_REF] For skeletal muscle, myotube alignment is difficult to achieve on flat surfaces, like tissue culture polystyrene (TCPS) which gives randomly organized myotubes. [START_REF] Stern-Straeter | Advances in skeletal muscle tissue engineering[END_REF] The alignment and differentiation of myoblasts were controlled by microstructure topography [START_REF] Lam | The effect of continuous wavy micropatterns on silicone substrates on the alignment of skeletal muscle myoblasts and myotubes[END_REF][START_REF] Charest | Myoblast alignment and differentiation on cell culture substrates with microscale topography and model chemistries[END_REF] and ECM proteins-based micropatterned surfaces in vitro. [START_REF] Bajaj | Patterning the differentiation of C2C12 skeletal myoblasts[END_REF] Since the early use of collagen (COL) to functionalize biomaterial surfaces, [START_REF] Vandenburgh | Maintenance of highly contractile tissue-cultured avian skeletal myotubes in collagen gel[END_REF][START_REF] Morra | Surface engineering of titanium by collagen immobilization. Surface characterization and in vitro and in vivo studies[END_REF][START_REF] Grant | Layer-By-Layer Assembly of Collagen Thin Films: Controlled Thickness and Biocompatibility[END_REF] various approaches were developed to align COL with nanometric control [START_REF] Pastorino | Oriented collagen nanocoatings for tissue engineering[END_REF] to guide fibroblasts, [START_REF] Guido | A methodology for the systematic and quantitative study of cell contact guidance in oriented collagen gels. Correlation of fibroblast orientation and gel birefringence[END_REF] and/or differentiate myoblasts. [START_REF] Bailey | Identification and change of collagen types in differentiating myoblasts and developing chick muscle[END_REF] For instance, alignment of COL fibers was achieved via lithography, [START_REF] Xia | Unconventional Methods for Fabricating and Patterning Nanostructures[END_REF] micro-grooved substrates, [START_REF] Vernon | Microgrooved fibrillar collagen membranes as scaffolds for cell support and alignment[END_REF] microfluidics, [START_REF] Lee | Microfluidic alignment of collagen fibers for in vitro cell culture[END_REF] electrospinning, [START_REF] Zhong | An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture[END_REF] magnetic fields, [START_REF] Dubey | Guided Neurite Elongation and Schwann Cell Invasion into Magnetically Aligned Collagen in Simulated Peripheral Nerve Regeneration[END_REF][START_REF] Guo | Flow and magnetic field induced collagen alignment[END_REF] flow, [START_REF] Lanfer | Aligned fibrillar collagen matrices obtained by shear flow deposition[END_REF] stretching, [START_REF] Chaubaroux | Cell Alignment Driven by Mechanically Induced Collagen Fiber Alignment in Collagen/Alginate Coatings[END_REF] and by rubbing. [START_REF] Uto | Orientation of Cultured Myotube on Rubbed Collagen[END_REF][START_REF] Chai | Rubbing-assisted approach for highlyoriented collagen fibril arrays involving calcium phosphate precipitation[END_REF] However, these techniques possess certain limitations such as the requirement for specialized instrumentations [START_REF] Vernon | Microgrooved fibrillar collagen membranes as scaffolds for cell support and alignment[END_REF] or microfluidic equipment, [START_REF] Lee | Microfluidic alignment of collagen fibers for in vitro cell culture[END_REF] mechanical assemblies, and strong electric or magnetic fields, which could damage the COL structure. [START_REF] Zhong | An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture[END_REF][START_REF] Guo | Flow and magnetic field induced collagen alignment[END_REF] Aside, the use of the nanostructured surface for cell culture are implemented with soluble factors, such as insulin, [START_REF] Beldjilali-Labro | Biomaterials in Tendon and Skeletal Muscle Tissue Engineering: Current Trends and Challenges[END_REF][START_REF] Ewton | Effects of the somatomedins and insulin on myoblast differentiation in vitro[END_REF] dexamethasone, 26- or differentiation processes. Polyphenols consumption is known to contribute to muscle health due to their antioxidant and anti-inflammatory properties. It was shown in vitro that polyphenol-rich plum extract treatment enhanced the size of C2C12 mouse myoblasts and accelerated their differentiation. [START_REF] Alsolmei | Polyphenol-Enriched Plum Extract Enhances Myotubule Formation and Anabolism while Attenuating Colon Cancer-induced Cellular Damage in C2C12 Cells[END_REF] Previously, we reported the use of tannic acid (TA) to develop hydrogen-bonded layer-by-layer (LbL) nanocoatings of COL type I using the dipping method. [START_REF] Reitzer | Use of Gelatin as Tannic Acid Carrier for Its Sustained Local Delivery[END_REF][START_REF] Iqbal | Effect of the Buffer on the Buildup and Stability of Tannic Acid/Collagen Multilayer Films Applied as Antibacterial Coatings[END_REF] Hydrogen-bonded TA LbL with neutral polymers offer tunable stability over wide pH range, thanks to the pKa of TA. [START_REF] Erel-Unal | Hydrogen-Bonded Multilayers of a Neutral Polymer and a Polyphenol[END_REF] In 2018, Park et al. introduced the brushing method, using a paintbrush, to functionalize a surface with chitosan/alginate LbL films for drug delivery, however no alignment of macromolecules was investigated. [START_REF] Park | Nanostructured Polymer Thin Films Fabricated with Brush-based Layer-by-Layer Self-assembly for Site-selective Construction and Drug release[END_REF] The brushing process was then used to align a monolayer of clay nanotubes allowing the orientation of human fibroblasts and mesenchymal stem cells along the axis of nanotubes. [START_REF] Zhao | Clay Nanotubes Aligned with Shear Forces for Mesenchymal Stem Cell Patterning[END_REF] Mimicking natural plywood, the brushing method was used to develop hierarchically structured hydroxyapatite micro-to nano-fibers/alginate films. [START_REF] Chen | Biomimetic twisted plywood structural materials[END_REF] Herein, we report a potent and simple brushing LbL method to develop oriented COL type I based nanocoating, i.e., TA/COL LbL film (Figure 1). Unlike dip-coating, this method is fast, requiring only a few seconds of deposition time for one layer. Moreover, it does not require specific or expensive instrumentation. Built at acidic pH, the oriented TA/COL LbL nanofilms released TA in solution upon contact with a physiological medium with a minor loss of thickness. After characterization of COL fibers orientation, human myoblasts (C25CL48) were seeded in a differentiation medium on the oriented TA/COL film, ended by COL. The alignment and differentiation of the cells were studied after 12 days of culture in comparison to nonoriented TA/COL films, obtained by the dipping method, uncoated glass, and TCPS (polystyrene) substrates.

Results and discussion

The brushing method aligns collagen fibrils in TA/COL LbL films. The buildup of brushed TA/COL films was performed manually on silicon or glass substrate using a nylon paintbrush at each deposition of TA and COL, alternating with a rinsing step and a drying step (Figure 1 and Supplementary video 1). Based on previous work on dipped TA/COL LbL films, an anchoring layer of a poly (ethylene imine) was deposited to allow a homogeneous distribution of COL on the substrate. [START_REF] Iqbal | Effect of the Buffer on the Buildup and Stability of Tannic Acid/Collagen Multilayer Films Applied as Antibacterial Coatings[END_REF] The brushed film buildup was optimized using different concentrations of TA, two types of paintbrush (Figure S1 in the SI), two angles of the paintbrush to the substrate and two different rinsing solutions (Figure S2-6 in the SI). The rinsing and drying procedures were chosen to sustain COL orientation and according to Park et al, [START_REF] Park | Nanostructured Polymer Thin Films Fabricated with Brush-based Layer-by-Layer Self-assembly for Site-selective Construction and Drug release[END_REF] i.e. by pipetting and drying in a vertical direction along the brushing direction. Nonoriented TA/COL LbL films were obtained using the conventional dipping method. Figure 2a shows the buildup of brushed and dipped TA/COL films. After 8 deposited bilayers, the thickness of the brushed TA/COL film was around 50 ± 1.5 nm in comparison to 167 ± 19 nm for the dipped film. The buildup by the brushing method follows strictly a linear growth (R² = 0.999) with COL and TA thickness increments of 4.6 ± 1 and 1.3 ± 0.7 nm, respectively. The greater thickness increment of COL is probably due to its higher molecular weight (300 000 g/mol) compared to TA with only 1 702 g/mol. Indeed, isolated COL deposited on mica substrate has a thickness of around 2.8 ± 0.2 nm. In citrate buffer, COL tends to form large bundles of around 5.7 nm in thickness which can justify the higher thickness of COL adlayers (Figure S7 Evolution of the thickness, followed by ellipsometry in dry state, as a function of the last deposited layer (mean and standard deviation from three different samples). The standard deviation of the brushed films is not visible due to the scale of the y-axis (the values are gathered in Table S1 in the SI, Figure S5b surface. Fibrillary structures were observed for the brushed and dipped TA/COL films (Figure 2b).

In the case of the brushed TA/COL film, the alignment of COL fibers was visible (zoom-in Figure 2b) and quantified on the AFM topography image using "OrientationJ" (Figure S4 in the SI). The diameter of the COL fibers was 60 ± 8 nm. The orientation distribution graph of the brushed TA/COL films shows a broad peak at 0° indicating that some COL fibers are oriented between -45° and +45° (Figure 2b) unlike the dipped TA/COL films showing randomly distributed COL fibers with a flat distribution graph. Figure S9 in the SI shows different AFM images from four different brushed TA/COL films at different scales, with even sharper peak at 0° (the brushing direction) in the orientation distribution graph at higher magnification. The degree of COL orientation of the brushed TA/COL films decreases by increasing the number of layer pairs with a complete loss at 16 bilayers (Figure S10 in the SI). This might be due to the loss of effective shear of the brushing by increasing the thickness of underlying LbL film. The secondary structure of COL was then determined by circular dichroism (CD). In solution, COL usually displays characteristic optical activity in the far UV spectrum with a small positive peak around 220-225 nm and a large negative peak in the range 197-200 nm. The CD spectrum of both the brushed and dipped TA/COL films shows a broad negative peak around 197 nm (Figure 2c). The intensity of the negative peak of brushed TA/COL films is lower than the dipped film, which could be explained by the difference in thickness of both films. No significant positive peak at 225 nm was observed in both cases. Generally, the absence of the positive peak at 225 nm, associated with the triple helix, is an indication of partial to full denaturation of COL. [START_REF] Velmurugan | Investigation on interaction of tannic acid with type I collagen and its effect on thermal, enzymatic, and conformational stability for tissue engineering applications[END_REF] However, COL hydrogels crosslinked by TA were reported to sustain COL native conformation with no positive peak (225 nm). [START_REF] Brazdaru | Structural and rheological properties of collagen hydrogels containing tannic acid and chlorhexidine digluconate intended for topical applications[END_REF] The absence of the positive peak of COL solutions was also observed at around 30°C, at which the triple helix structures assemble through fibrillogenesis into highly organized fibrils. [START_REF] Drzewiecki | Circular Dichroism Spectroscopy of Collagen Fibrillogenesis: A New Use for an Old Technique[END_REF] In the following, the brushed and dipped (TA/COL)8 films will be named the oriented and the nonoriented TA/COL films, respectively.

The tannic acid in solution triggers myoblasts' elongation. Before seeding human myoblasts on TA/COL films to study their differentiation and orientation, we first analyzed the cytotoxicity of TA in solution. Figure S11 in the SI shows that TA is toxic to human myoblasts with less than 70% of viability after 24 h of contact at concentrations above 33 µg/mL. For instance, the cell viability was close to zero (0.3±0.1 %) above 375 µg/mL of TA. The treatment of myoblasts with TA shows an important change in the cell area and morphology for concentrations below 33 µg/mL (Figure 3). Such an effect was quantitatively evaluated in terms of cell area (Figure 3a) and aspect ratio (Figure 3b), using ImageJ. The incubation with TA induced the elongation of cells resulting in roughly 20% reduced cell area and 4 times higher aspect ratio compared to the cells without TA treatment.

In the absence of TA, cells were well spread with no significant elongation (Figure 3c). At 10 and 25 µg/mL after 24 h of incubation, myoblasts showed elongated morphology, also known as a prodifferentiation phenotype, which is an essential precursor for cell fusion (Figure 3d and Figure 3e). [START_REF] Bruyère | Actomyosin contractility scales with myoblast elongation and enhances differentiation through YAP nuclear export[END_REF] Until now, no studies on the effect of TA on the differentiation of myoblasts was performed, only the uptake of TA by myoblasts was reported so far. [START_REF] Shin | Targeting protein and peptide therapeutics to the heart via tannic acid modification[END_REF] The observed elongation in the presence of TA is consistent with the studies related to the effect of polyphenols on muscle pathology and myotubule formation. [START_REF] Alsolmei | Polyphenol-Enriched Plum Extract Enhances Myotubule Formation and Anabolism while Attenuating Colon Cancer-induced Cellular Damage in C2C12 Cells[END_REF][START_REF] Evans | Green tea extract decreases muscle pathology and NF-κB immunostaining in regenerating muscle fibers of mdx mice[END_REF] Human myoblasts align and differentiate on the oriented TA/COL films. The orientation and differentiation of human myoblasts were investigated on TA/COL LbL films via immunocytochemistry to stain heavy chain myosin (MHC) sarcomere protein, typically expressed by mature myotubes. Myoblasts were seeded on LbL samples and uncoated glass for 24 h in complete growth medium followed by 12 days in differentiation medium, without any other supplements. We checked that the nanostructure of the oriented TA/COL film was preserved after 3 days of incubation in the DMEM medium (Figure S12 in the SI). Confocal microscopy images were recorded on day 12 and treated by OrientationJ, as shown in Figure 4. On the oriented TA/COL nanofilms, the immunostaining showed that the cells aligned and differentiated into multinucleated long myotubes with the oriented arrangement of myonuclei (Figure 4a). Myofibers' actin filaments appeared aligned which was expressed as a broad peak at 0°, i.e., the brushing direction (Figure 4b). Such myotubes formed thicker myofibers of diameter around 100 µm, i.e., approximately equivalent to the diameter of human muscle fibers (Figure 4a and Figure S13 in the SI). At this stage, some myofibers were also observed to neglect the LbL orientation (data not shown). On the nonoriented TA/COL nanofilms, randomly distributed and elongated myoblasts can be observed as in the case of the glass control substrate with almost no myosin staining (Figure 4a). In comparison, randomly distributed myoblasts and shorter myotubes (fewer fused nuclei) were observed on TCPS (Figure S14 in the SI). Regarding the cell morphology, a strong difference can be observed after 12 days of culture (Figure S15 in the SI). Of critical importance in muscle regeneration, the differentiation of myoblasts follows a contact-dependent mechanism, where movement or migration to come closer, fuse, and form myotubes is important. [START_REF] Beldjilali-Labro | Biomaterials in Tendon and Skeletal Muscle Tissue Engineering: Current Trends and Challenges[END_REF][START_REF] Vandenburgh | Maintenance of highly contractile tissue-cultured avian skeletal myotubes in collagen gel[END_REF] The myoblasts movement was recorded by time-lapse video for the first 48 h in contact with the oriented TA/COL films in the growth medium (Supplementary video 2). The oriented COL LbL guides undifferentiated myoblasts to align in the direction of the brushing and form denser cell regions favoring later the fusion and differentiation into myotubes in the differentiation medium. On the nonoriented TA/COL films and the uncoated glass surface, a random movement of cells was observed on 48 h time-lapse video (Supplementary videos 3 and 4, respectively). We then assessed the cell density on the different coatings after 12 days of differentiation. Based on the mean values of total nuclei, the oriented TA/COL film presented the same cell density as the uncoated glass substrate. In the case of nonoriented TA/COL film, the cell density is one-third compared to the glass control (Figure 5a). TA/COL films switch significantly from biocompatible to cytotoxic towards human myoblasts depending on the buildup method. TA/COL LbL films were shown to be stable in the physiological medium with a part of TA released. [START_REF] Reitzer | Use of Gelatin as Tannic Acid Carrier for Its Sustained Local Delivery[END_REF][START_REF] Iqbal | Effect of the Buffer on the Buildup and Stability of Tannic Acid/Collagen Multilayer Films Applied as Antibacterial Coatings[END_REF][START_REF] Erel-Unal | Hydrogen-Bonded Multilayers of a Neutral Polymer and a Polyphenol[END_REF] We then monitored the TA release of both TA/COL films. Upon incubation in PBS at room temperature, the release of TA followed an initial linear kinetic with roughly 86% of release within the first 4 h, which then stabilized over 1-4 days. The nonoriented TA/COL films, i.e. (TA/COL)8, released approximately 9 times more TA than the oriented film with 37 ± 3.6 µg/mL vs 4.2 ± 0.5 µg/mL, respectively (Figure 6). The difference in the amount of released TA can be explained by the thickness of the films (Figure 2a). The quantity of TA released by the nonoriented film (37 ± 3.6 µg/mL) is close to the cytotoxicity threshold towards myoblasts leading to a lower cell density in comparison to the glass control.

This effect can even be strengthened by local release of TA at the surface of the nanocoating as found for the antibacterial effect on the same system. 31 Lastly, we evaluated the final myotube formation after 12 days of differentiation by the quantification of the fusion index (defined as a ratio of nuclei contained in myotubes, with ≥2 nuclei, to the total nuclei), the aspect ratio of nuclei, the area, dimension and prevalence of myotubes (defined as the number of nuclei per myotube represented as % of total nuclei) [START_REF] Coletti | Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment[END_REF] (Figure 5b-f). The oriented TA/COL films showed an ability for the development of aligned myotubes from human myoblasts. Indeed, the fusion index calculated on the oriented TA/COL films is around 90 ± 9 % in comparison to 22 ± 14 % for the nonoriented films and 5% for glass control (Figure 5b). This indicates that approximately all the attached and proliferated myoblasts differentiated into myotubes after 12 days of culture on the oriented TA/COL films. Interestingly, the nanotopography of TA/COL films not only impacted the myoblast differentiation, but also the morphology of the nuclei (Figure S16 in the SI). Elongated nuclei with an aspect ratio of around 2.8 were observed on the oriented TA/COL films, whereas a broad shape morphology with the aspect ratio of approximately 1.5-1.6 is observed on the nonoriented TA/COL film and the glass control (Figure 5c). Myoblasts were reported to exhibit deformed nuclei morphologies depending on surface nanostructures. [START_REF] Bruyère | Actomyosin contractility scales with myoblast elongation and enhances differentiation through YAP nuclear export[END_REF] Besides the fusion index, we observed significant differences in the area and dimensions of myotubes formed on the different samples. Myotube area on the oriented TA/COL films is around 3.6 times higher than the one on the nonoriented film, the glass control (Figure 5d). Consistent with the myotube area, the myotubes on the oriented TA/COL films are 698 ± 28 µm in length and 230 ± 21 µm in width i.e., approximately 4 times longer and 2 times thicker than the ones on the nonoriented films and glass (Figure 5e). We can notice the presence of myotubes with more than 30 to 100 nuclei only on the oriented TA/COL films (Figure 5f). To investigate the impact of TA release on the myoblasts' differentiation, TA was washed out from the oriented TA/COL films, by 24 h incubation in PBS medium, prior to the cell seeding. After 12 days of culture, myoblasts appeared partially elongated. Actin cytoskeleton of cells were randomly oriented as shown by Image J treatment (Figure S17 in the SI) with the maxima of the orientation distribution not centered at 0° (the brushing direction). In contrast to the oriented TA/COL films (not washed by PBS), a significantly lower number of cells showed myosin labeling and the obtained myotubes were not aligned along the oriented COL fibers of PBS-incubated films (Figure S17 in the SI). In addition to COL-induced orientation of myoblasts, TA release appears to trigger efficient cell differentiation, as shown in Figure 4. Indeed, on PBS-incubated oriented TA/COL films, the fusion index of myoblasts was of 69 ± 22 % vs 91 ± 9 % on the untreated films with a similar nuclei aspect ratio, myotube area, dimensions, and prevalence (Figure S18 in the SI). The comparison with TCPS as control substrate is also shown in Figure S18 in the SI, presenting comparable results as the glass substrate. To confirm the synergistic role of TA release and COL orientation, we performed experiments on nonoriented TA/COL films having similar thickness (54 ± 4 nm) and amount of TA released (5.9 ± 1.2 µg/mL) as the oriented TA/COL films, i.e. dipped (TA/COL)3 films (Figure S19 in the SI). After 12 days of incubation on the nonoriented (TA/COL)3 film in the differentiation medium, elongated and randomly distributed myoblasts were observed with no myosin expression (no green staining), similar to the nonoriented film (TA/COL)8 (Figure S20a in the revised SI) with a higher cell density due to the lower amount of TA released in the supernatant (Figure S20c in the SI). The glass control was also performed for comparison (Figure S20b

Conclusion

We demonstrated that the brushed LbL films appear to be a potent and simple way to produce surfaces with aligned topography using macromolecules of high aspect ratio, such as COL. In comparison to the other methods developed so far, there is no need for sophisticated apparatus.

The aligned TA/COL nanofilms influenced the cells behavior, by improving their adhesion to a substrate, influencing their morphology, alignment, and differentiation. The differentiation of human myoblasts was obtained in 12 days when seeded on the oriented TA/COL films in differentiation medium without any other supplement thanks to two distinct properties: (i) COL orientation which helps to align myoblasts favoring their close contact, and (ii) TA release which favors the differentiation. On the TA/COL films, the topography cues and the strong links between cells and the collagen can be further used to mimic the complexity of in vivo conditions and design model tissues to regenerate anisotropic tissues, the treatment of injuries or diseases or for pharmacology research.

Experimental Section

Materials. Collagen (COL, purified type I from calf, Symatese, France), tannic acid (TA, Mw = 1701.2 g/mol, Sigma-Aldrich, France), poly(ethylene imine) (PEI, branched, 30% w/w aq.

solution, Mw = 50 000-100 000 g/mol, Alfa Aesar, Germany), citric acid monohydrate (Mw = 210.14 g/mol, Sigma-Aldrich), sodium citrate tribasic dihydrate (Mw = 294.10 g/mol, Sigma- prepared in deionized water. Prior to thin film buildup, all the solutions were filtered using a 0.20 µm filter (Sarstedt, no. 831826001) to remove any impurity or undissolved substances. The citrate buffer used in this study was prepared at pH 4 by mixing 41 mL of 0.1 M sodium citrate tribasic dihydrate solution and 59 mL of 0.1M citric acid monohydrate solution. PEI solution was prepared at 0.1 mg/mL in 0.15 M sodium chloride (NaCl) aqueous solution (no pH adjustment). TA solution was prepared at 0.5 mg/mL in citrate buffer at pH 4 (otherwise stated) and kept in dark to avoid its photooxidation. COL solution was prepared at 1 mg/mL in citrate buffer at pH 4 (otherwise stated) by overnight agitation using a magnetic stirrer at 4 °C.

TA/COL LbL buildup. Silicon wafers or microscopic glass slides were cut using a sharp diamond pen to obtain 1 × 6 cm² rectangular substrates. Silicon wafers were cleaned with ethanol/water (50% v/v) solution for 15 min prior to their use. Glass substrates were cleaned by immersing in the solutions of 10 mM SDS and 0.1 M HCl at 70°C for 10 min each, followed by extensive rinsing with deionized water. Prior to the film buildup, the substrates were plasma etched for 2.5 min with air using Harrick PDC-002-HP at high RF level to remove particles and to render the surface negatively charged and hydrophilic. PEI, a branched polycation, was used as precursor layer on each substrate before LbL buildup. Immediately after plasma cleaning, the substrate was dipped in PEI solution for 10 s, rinsed twice with ultrapure water for 5 s and dried with filtered compressed air. The substrate is then a positively charged surface ensuring a good anchoring of the films.

Oriented TA/COL based films were obtained by the brushing method. The PEI-functionalized substrate was clipped with a support in a horizontal direction facing upwards to brush the solutions always in the same direction with nylon paintbrush with hard bristles (Søstrene Grenes, Aarhus, Danemark). Horsehair paintbrush with soft bristles (Søstrene Grenes, Aarhus, Danemark) was also used at the optimization of the buildup. Regarding the optimized buildup of oriented TA/COL films, TA and COL solutions were brushed manually with the nylon paintbrush tilted at an angle of 30° to the substrate and left to dry for 10 s, followed by two rinsing steps with ultrapure water using a disposable pipette and dried with filtered compressed air at 2.5 bar, both in a vertical direction along the brushing direction. With n the number of bilayer deposition, the following brushed PEI-(TA/COL)n films were studied and named oriented TA/COL film with n = 8.

Nonoriented TA/COL films were obtained by the dipping method. The PEI-functionalized substrate was immersed in TA solution for 10 s, followed by two rinsing steps by dipping in ultrapure water for 5 s and dried by filtered compressed air. The same process was followed to deposit COL adlayer, to obtain one bilayer. The process was repeated n times to obtain brushed PEI-(TA/COL)n named nonoriented TA/COL film with n = 8. Once the desired film architecture is achieved, the homogeneous part of the sample (in the center of the substrate) was selected and cut to obtain 1×1 cm2 area samples to characterize the film properties.

Ellipsometry. The film thickness was measured, after rinsing with water and drying by compressed air, using an Ellipsometer (SD2300, PLASMOS) with an incident laser beam (632.8nm) and a constant angle of 45°. Refractive index was assumed to be constant and equal to 1.465 for all the measurements. The dry thickness values reported here are the average values with standard deviation of three individual LbL buildup experiments. Ten measurements were performed at random areas on each one of the multilayer sample surfaces. The routine ellipsometry measurements might show insignificant differences in absolute dry thickness and a more advanced ellipsometry equipment would be required for accurate measurements. However, the thickness readings are satisfactorily accurate to compare the buildup of different LbL films reported here.

Atomic Force Microscopy (AFM). COL/TA films, in dry state using nitride coated silicon tips on a nitride cantilever (Model ScanAsyst-Air, with force constant k = 0.4 N/m and frequency fn =70 kHz) and Peak Force Tapping (ScanAsyst) mode. The images were obtained at a scan rate of 1 Hz with a resolution of 512 × 512 pixels. The data analysis was performed using NanoScope Analysis software version 1.7. Random areas of the samples were scanned to obtain typical topography images. Open-source software Image J was used to determine the orientation of COL fibres using plugin "Orientation J" developed at the École Polytechnique Fédérale de Lausanne (EPFL). [START_REF] Püspöki | Transforms and Operators for Directional Bioimage Analysis: A Survey[END_REF] Firstly, AFM images were converted into grey scale (8-bit) and then the program computes the local orientation properties. From this software, a visual directional analysis is obtained in which the orientation is encoded in color following the color-coding reference image.

bovine serum, and 1% penicillin and streptomycin, named as "growth medium (GM)". The cells were pre-cultured to reach around 70% confluence.

Human myoblasts' viability. The cell (3000/well) were seeded in a TCPS 96-well plate and allowed to proliferate for 24 h. The cells were rinsed with PBS, and 100 µL of TA solutions at different concentrations prepared in the growth medium was dispensed in the wells. After 24 h of contact with the TA, followed by a PBS rinsing step, 100 µL of the CellTiter-Glo 2.0 assay reagent (Promega, G9242) was added and incubated in a shaker for 15 min at room temperature. Finally, luminescence was recorded using a spectrofluorometer (SAFAS Xenius XC). The cell area was calculated using Freehand tool in the ImageJ software and presented as a mean value of 40 cells on five individual images (200 cells in total).

Myoblasts differentiation.

The freshly prepared TA/COL film samples were UV sterilized in dry state for 20 min before the cell seeding. 20,000 cells/mL were seeded on each tested sample and allowed to proliferate for 24 h. Then, the growth medium was switched to differentiation medium (DM) (DMEM high glucose supplemented with 2% horse serum and 1% penicillin and streptomycin). Cell differentiation was followed for 12 days. DM was replaced with fresh DM after every 3-4 days. After 12 days of differentiation, cells were fixed with 500 µL of 4% PFA for 15 min at room temperature, followed by two rinsing steps using 1mL PBS, 5 min each. The cells were permeabilized by incubating in 1 ml of 0.5% Triton X-100 in PBS for 4 min, followed by three rinsing steps using 1mL PBS, 5 min each. The cells were incubated in 1% BSA solution in PBS for 1h at room temperature to block unnecessary sites to minimize background noise. To stain heavy chain myosin, 500 µL of the primary antibody (MF-20, AB_2147781, 0.039 µg/mL, 1/564 dilution in 0.1% BSA in PBS) was added for 2h at room temperature, in dark. After rinsing with PBS, cells were incubated in 500 µL of Abcam 150113 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) 1/500 diluted in 0.1 % BSA in PBS for 1 h at room temperature, in dark followed by two rinsing steps with PBS (2 x 1000 µL). Then, the cells were incubated in 500 µL of phalloidin-TRITC solution (dilution 1/800) in PBS for 30 min at room temperature, in dark followed by PBS (1000 µL) rinsing step. Finally, 500 µL of Hoechst 33342 (Invitrogen, B2261, dilution 1/1000) at 5 μg/ml in PBS were added at room temperature for 10 min to stain nuclei. Finally, the samples were mounted in transparent glass slides to observe under Zeiss LSM 710 confocal microscopy.

Table of contents

Figure 1 .

 1 Figure 1. (a) Manual brushing experimental set-up using nylon paintbrush, (b) Schematic

  in the SI). The size of the TA molecule was determined at 1.3 ± 0.5 nm by dynamic light scattering (Figure S8 in the SI).

Figure 2 .

 2 Figure 2. Physico-chemical characterization of the brushed and the dipped TA/COL films. (a)

  in the SI). (b) Typical AFM topography images of the (TA/COL)8 films and the orientation distribution graphs calculated by Image J and as an inset the zoom of the topography. The brushing direction, visible on the video camera, is indicated by the white arrow. (c) Circular dichroism spectra of the TA/COL films. The surface topography of both TA/COL films was characterized by Atomic Force Microscopy (AFM) in dry state by scanning random areas of various sizes, from 5×5 to 1×1 µm², on the sample

Figure 3 .

 3 Figure 3. Effect of TA in solution on human myoblasts (C25 CL48) morphology after 24 h of

Figure 4 .

 4 Figure 4. Orientation and differentiation of human myoblasts after 12 days of culture on the

Figure 5 .

 5 Figure 5. Quantitative information from immunofluorescence images (surface image area 691 456

Figure 6 :

 6 Figure 6: Cumulative release profiles of TA from the oriented and the nonoriented TA/COL films

  in the SI). On the nonoriented (TA/COL)3 and (TA/COL)8 films, the fusion index was similar at 17 ± 7% and 22 ± 14%, respectively, and the aspect ratio of the nuclei was approximately 1.5 (FigureS20d-e in revised SI). The dimensions of the myotubes were different, with cells being more elongated in the nonoriented (TA/COL)3 than (TA/COL)8 film with a length/width of 185 ± 61 µm/24 ± 8 µm vs 173 ± 31 µm/129 ± 13 µm, respectively (FigureS20f-h in the SI). Regarding the prevalence of myotubes, no myotubes with more than 10 nuclei were observed on the nonoriented films (TA/COL)3. In summary, the absence of COL orientation prevents the orientation of myoblasts as well as their differentiation into myofibers and the lower amount of TA released allows for better cytocompatibility, the presence of more elongated myotubes but with fewer nuclei on the nonoriented films. The orientation of the myoblasts is due to the orientation of the COL chains and their elongation to the TA released into the supernatant by the oriented film which leads to their differentiation into myofibers. Myofibers of length around 1.7 mm reconstructed from confocal images were observed on the oriented TA/COL films, showing the distribution of the nuclei (blue), myosin heavy chain (in green) and actin filament (in red). The myotube dimensions obtained might range from hundreds of micrometers to millimeters, depending on the size of the sample.

Figure 7 .

 7 Figure 7. Confocal microscopy images of a human myotube, obtained from myoblasts after 12

  Aldrich), Dulbecco's phosphate buffer saline (PBS, L0615, Dominique Dutscher, France), Dulbecco's Modified Eagle Medium (DMEM, L0103, Dominique Dutscher, France), sodium dodecyl sulfate (Prolabo, Poland), NaCl (VWR), HCl, and NaOH (Sigma-Aldrich) were used as received. Collagen and tannic acid solutions. All solutions were prepared freshly prior to use except COL. Deionized water (Milli-Q, 18.2 MΩ•cm at 25 °C) supplied by Advantage A10 (MERCK) was used in the entire study. The pH values were adjusted by using 0.1 M HCl or 0.1 M NaOH solution

For cell viability in

  contact with different concentrations of TA solutions, only actin filament (phalloidin-rhodamine) and nuclei (Hoechst 33342) were stained following a similar protocol as above. The quantitative information calculated from immunofluorescence images of human myoblast differentiation after 12 days of culture on the oriented and the nonoriented TA/COL films, uncoated glass and TCPS were calculated on a given surface image of area 691 456 µm². Three different images were measured for each condition and the experiments were conducted in triplicate. Statistical analysis. The two-tail student t-test was performed on all cell biology experimental data to determine the statistical significance of differences between the oriented and the nonoriented multilayer films with uncoated glass substrates (control). The obtained p-values are indicated on the corresponding graphs with symbols such as *, **, ***, and **** for p ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. ASSOCIATED CONTENT Supporting Information. Optimization of TA/COL brushing method, AFM topography/crosssection of isolated COL fibers on mica, hydrodynamic size of TA, "OrientationJ" color coded analysis of the AFM topography images of brushed and dipped TA/COL films, AFM topography and OrientationJ analysis of oriented (TA/COL)4, (TA/COL)8, and (TA/COL)16, in vitro cytotoxicity of TA on human myoblasts, AFM topography/orientation of oriented TA/COL after 3 days incubation in DMEM medium, myoblasts after 12 days of differentiation on oriented TA/COL and TCPS, morphology of myoblasts after 12 days of differentiation, TA release from the TA/COL films, morphology of isolated nuclei on differentiation day 12, differentiation/orientation of myoblasts oriented TA/COL_PBS films, quantitative information for oriented TA/COL_PBS films and controls on differentiation day 12, Physico-chemical characterization of the nonoriented (TA/COL)3 and myoblasts after 12 days of differentiation the nonoriented (TA/COL)3. The video 1, video 2, video 3 and video 4 are available as separate files (.avi). Supporting Information is available free of charge. Institut Carnot MICA DIAART project, Fonds Régional de Coopération pour la Recherche of Région Grand Est ERMES project and International Centre for Frontier Research in Chemistry (icFRC) are acknowledged for financial support. ACKNOWLEDGMENT Sarah Benmalek-Kehili and Christine Affolter-Zbaraszczuk are acknowledged for their technical help. M.H.I. thanks the Higher Education Commission (HEC) Pakistan for his Ph.D. scholarship. Institut Carnot MICA DIAART project, Fonds Régional de Coopération pour la Recherche of Région Grand Est ERMES project and International Centre for Frontier Research in Chemistry (icFRC) are acknowledged for financial support.

and insulin-like growth factor (IGF-1),

to trigger, support or even accelerate the regeneration

An orientation histogram can be calculated by taking into consideration few parameters such as a

Gaussian window of 1 pixel to account for the smallest possible nanostructure, and a minimum coherency and energy of 1% to eliminate the effect of isotropic areas. The distribution of orientation for TA/COL multilayers was adjusted to 0°. The same method was applied on fluorescence microscopy images to determine the orientation of cells seeded the different substrates.

TA release in physiological conditions. TA release from the oriented and the nonoriented TA/COL films in contact with PBS buffer at room temperature was followed by measuring the absorbance intensity at 277 nm using a spectrofluorometer (SAFAS Xenius XC). COL used in the study showed no absorption at this wavelength. First, a calibration curve of TA was obtained by measuring the absorbance intensity at 277 nm for different concentrations of TA in PBS buffer.

The samples were prepared on glass slides with final dimensions of 1×1 cm². Five hundred microliters of PBS buffer at pH 7.4 at room temperature was put in contact with the films. One hundred microliters of the supernatant were withdrawn and put in a UV-Star 96-well plate (Greiner bio-one) to measure the absorbance at 277 nm. To compensate 100 μL of supernatant loss, 100 µL of fresh PBS was added.

Human

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources