Morgane Desmau
email: morgane.desmau@northwestern.edu

Clément Levard

Vladimir Vidal

Georges Ona Nguema

Gaëlle Charron

Marc F Benedetti

Alexandre Gélabert
email: gelabert@ipgp.fr

How microbial biofilms impact the interactions of Quantum Dots with mineral surfaces?

Keywords: Biofilm, Dissolution, Quantum Dots, Mineral, Interactions QD/QDs= Quantum Dots, LP-XSW-FY= Long Period -X-ray Standing Wave -Fluorescence Yield, GI-XAS= Grazing Incidence -X-ray Absorption Spectroscopy, NP/NPs= Nanoparticles, EPS= exopolymeric substances, TGA= thioglygolic acid, ICP-QMS= inductively coupled plasma-quadrupole mass spectrometry, SEM= Scanning electron microscopy, LCF= linear combination fitting, ROS= reactive oxygen species, EPM= electrophoretic mobility, DLS= Dynamic Light Scattering

The increasing use of Quantum Dots (QDs) -nanoparticles exhibiting unique optical propertiesand their incorporation in multiple engineering products is likely to result in the release of this new class of contaminants into natural systems. In soils, bacterial biofilms and mineral surfaces form highly reactive interfaces, which may control QDs' environmental fate. However, little is known regarding QDs' stability in, and modes of interactions with, biofilm/mineral interfaces. This study examines the interactions, distributions and stability of thioglycolic acid-capped CdSe/ZnS QDs at the corundum (-Al 2 O 3 )/Shewanella oneidensis MR-1 interface, for exposure times ranging between 1h to 24h. Long Period -X-ray Standing Wave -Fluorescence Yield spectroscopy and Grazing Incidence -X-ray Absorption Spectroscopy were used. Results indicate increases in Zn and Se concentrations within the biofilm/crystal system with time, demonstrating its high accumulation capacity over 24h. In addition, dissolution of a part of the ZnS shell occurs within 1h, highlighting the potential degradation of QDs when exposed to the biofilm/crystal compartment. Once released, Zn(II) migrates toward the biofilm-crystal interface and interacts preferentially with the crystal surface. In contrast, the remaining CdSe core is mostly preserved, and stays within the biofilm thickness. However, at 24h, Se and Zn present similar distribution profiles indicating a general reduction in ZnS shell dissolution at this longer exposure time.

I. Introduction

Quantum Dots (QDs) are fluorescent semiconductor nanocrystals which present unique optical and electronic size-dependent properties, such as electroluminescence. QDs exhibit sizes between 2 and 10 nm, which place them in the most reactive class of NPs (Auffan et al., 2009a).They are incorporated into solar cells to increase efficiency [START_REF] Lin | CdS quantum dots sensitized ZnO spheres via ZnS overlayer to improve efficiency for quantum dots sensitized solar cells[END_REF], are used in medicine for in-vivo and in-vitro diagnosis [START_REF] Aldeek | Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms[END_REF][START_REF] Liu | Bacteriamediated in vivo delivery of quantum dots into solid tumor[END_REF], and are at the center of intensive research for innovative low-energy applications in imaging technologies and for designing novel solution-processed functional optoelectronic materials [START_REF] Kagan | Building devices from colloidal quantum dots[END_REF]. These nanoparticles (NPs) usually have a core-shell structure, with the core composed of CdSe, InP, PbSe, or ZnSe, surrounded by a shell of wider band-gap material such as ZnS or CdS [START_REF] Chen | The stability and removal of water-dispersed CdSe/CdS core-shell quantum dots from water[END_REF]. For potential industrial uses [START_REF] Hardman | A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors[END_REF], to allow QDs dispersion in aqueous phases and to enhance their biological compatibility or their stability, they are capped by organic or inorganic ligands [START_REF] Breus | The effect of surface charge on nonspecific uptake and cytotoxicity of CdSe/ZnS core/shell quantum dots[END_REF]. Nevertheless, given their high reactivity, these engineered materials are sometimes considered as potential contaminants, and substantial evidence of NP and QD toxicity to microorganisms have been reported [START_REF] Brayner | Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium[END_REF][START_REF] Fabrega | Impact of silver nanoparticles on natural marine biofilm bacteria[END_REF][START_REF] Mahendra | Quantum Dot Weathering Results in Microbial Toxicity[END_REF].

The recent global increase in NP production volumes has raised societal and environmental concerns. During their life-cycles, NP-containing materials can experience abrasion or leaching that could result in significant release to the environment [START_REF] Mueller | Exposure Modeling of Engineered Nanoparticles in the Environment[END_REF] where NPs are expected to accumulate in soils, sediments and landfills, according to probabilistic modeling results [START_REF] Keller | Global life cycle releases of engineered nanomaterials[END_REF][START_REF] Wang | Dynamic probabilistic material flow analysis of nano-SiO2, nano iron oxides, nano-CeO2, nano-Al2O3, and quantum dots in seven European regions[END_REF]. In those compartments, QDs are likely to undergo various physico-chemical processes such as homo-or hetero-aggregation, complexation with organic matter or biomass, chemical transformations, and partial or complete dissolution, all of which affect the reactivity, toxicity, transport and fate of QDs in natural systems [START_REF] Lowry | Transformations of nanomaterials in the environment[END_REF]. Some of these processes have already been investigated under environmentally relevant conditions, highlighting the importance of pH [START_REF] Kaur | Size tuning of MAA capped CdSe and CdSe/CdS quantum dots and their stability in different pH environments[END_REF], ionic strength [START_REF] Slaveykova | Effect of natural organic matter and green microalga on carboxyl-polyethylene glycol coated CdSe/ZnS quantum dots stability and transformations under freshwater conditions[END_REF][START_REF] Zhang | Stability and Removal of Water Soluble CdTe Quantum Dots in Water[END_REF] and the presence of natural organic matter [START_REF] Navarro | Natural Organic Matter-Mediated Phase Transfer of Quantum Dots in the Aquatic Environment[END_REF] on QDs' stability in different types of aqueous conditions [START_REF] Chen | The stability and removal of water-dispersed CdSe/CdS core-shell quantum dots from water[END_REF][START_REF] Slaveykova | Effect of natural organic matter and green microalga on carboxyl-polyethylene glycol coated CdSe/ZnS quantum dots stability and transformations under freshwater conditions[END_REF]. Nevertheless, given the complexity of natural systems and the multiplicity of the associated physico-chemical processes, many questions remain open regarding the fate of released QDs, especially with regard to interactions with microorganisms.

In soils and sediments, one of the most reactive compartments is composed of microbial biofilms growing at the surfaces of minerals [START_REF] Costerton | Bacterial biofilms in nature and disease[END_REF]. By far the main microbial organization modes are biofilms [START_REF] Flemming | Bacteria and archaea on Earth and their abundance in biofilms[END_REF], structures composed of cells encased in a complex three-dimensional organic matrix of exopolymeric substances (EPS), and found in virtually all subsurface environments on Earth [START_REF] Ménez | Life in the hydrated suboceanic mantle[END_REF]. These structures exhibit heterogeneity in compositions, hydrophobic microdomains [START_REF] Aldeek | Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms[END_REF], pH [START_REF] Hidalgo | Functional tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica nanoparticle sensors[END_REF], redox conditions [START_REF] Babauta | pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer[END_REF], thickness, spatial organization, etc. depending on parameters such as microbial strains or nutrient availability [START_REF] Allison | The biofilm matrix[END_REF][START_REF] Sutherland | The biofilm matrixan immobilized but dynamic microbial environment[END_REF]. Thus, biofilms are highly reactive dynamic systems [START_REF] Sutherland | The biofilm matrixan immobilized but dynamic microbial environment[END_REF], exhibiting elevated specific surface areas, high site densities [START_REF] Borrok | A universal surface complexation framework for modeling proton binding onto bacterial surfaces in geologic settings[END_REF] and reactive microenvironments [START_REF] Stewart | Diffusion in biofilms[END_REF]. Bacterial cells generally present an overall negative surface charge at neutral pH due to the presence of carboxyl (pK a : 3-4.5) or phosphoryl (pK a : 7-8) groups [START_REF] Ha | Role of extracellular polymeric substances in metal ion complexation on Shewanella oneidensis: Batch uptake, thermodynamic modeling, ATR-FTIR, and EXAFS study[END_REF][START_REF] Palmer | Bacterial cell attachment, the beginning of a biofilm[END_REF].

In addition, despite their small concentrations at the surface of S. oneidensis, sulfhydryl groups seem to play an important role on metal sorption [START_REF] Yu | The effect of metal loading on Cd adsorption onto Shewanella oneidensis bacterial cell envelopes: the role of sulfhydryl sites[END_REF]. The negative surface charge of bacteria at neutral pH indicates that biofilms can be viewed as negatively charged entities, since EPS add supplementary functional sites that are also negatively charged [START_REF] Tourney | The role of bacterial extracellular polymeric substances in geomicrobiology[END_REF]. Functional groups and surface charge can partly control the interactions and speciation of metals (Wang et al., 2016b), metalloids [START_REF] Templeton | Selenium speciation and partitioning within Burkholderia cepacia biofilms formed on α-Al2O3 surfaces[END_REF] and NPs [START_REF] Golmohamadi | The role of charge on the diffusion of solutes and nanoparticles (silicon nanocrystals, nTiO2, nAu) in a biofilm[END_REF] within biofilm thicknesses. However, studies suggest that attractive forces (hydrophobic or van der Waals forces) may overwhelmed the electrostatic forces when the NPs have penetrated the diffuse layer. For example, we showed in our previous work that hydrophobic interactions controlled the transport of silver NPs coated with polyvinylpyrrolidone at the biofilm/mineral interface [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF]. Similarly, [START_REF] Lerner | The effects of biofilm on the transport of stabilized zerovalent iron nanoparticles in saturated porous media[END_REF] demonstrated that the increase in coating hydrophobicity favor the retention of NPs within biofilm. Thus, the attractive forces need to be considered to explain the interactions between NPs and biofilm [START_REF] Fulaz | Nanoparticle-biofilm interactions: the role of the EPS matrix[END_REF][START_REF] Mitzel | Hydrophobicity of biofilm coatings influences the transport dynamics of polystyrene nanoparticles in biofilmcoated sand[END_REF]. Besides, the transport of solutes, antibacterial agents, metal(loid)s and NPs within biofilms are also controlled by the density, the organization and the overall specific characteristics of the matrix such as the size of the water channels and fluid voids or the development of chemical gradients and microenvironments within biofilms [START_REF] Allison | The biofilm matrix[END_REF][START_REF] Choi | Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures[END_REF][START_REF] Couasnon | Experimental assessment of occurrences and stability of lead-bearing minerals in bacterial biofilms[END_REF]Dranguet et al., 2017;[START_REF] Peulen | Diffusion of Nanoparticles in a Biofilm[END_REF][START_REF] Stewart | Antibiotic resistance of bacteria in biofilms[END_REF]. In addition to the biofilm's reactivity, the mineral surfaces where they developed are often also highly reactive and drive numerous processes in soil [START_REF] Brown | How Minerals React with Water[END_REF][START_REF] Brown | Metal Oxide Surfaces and Their Interactions with Aqueous Solutions and Microbial Organisms[END_REF] such as the sorption of metals or surface precipitation.

Nevertheless, to the best of our knowledge, the role of the biofilm/mineral interface on the transport and transformation of NPs has been under-investigated, despite its elevated reactivity and known impact on metal speciation and mobility (Wang et al., 2016a).

To predict QD fluxes, fate and potential impacts to ecosystems, it is critical to understand the behavior and physico-chemical transformations undergone by QDs when exposed to the biofilm/mineral compartment, as highlighted by [START_REF] Saleh | Mechanistic lessons learned from studies of planktonic bacteria with metallic nanomaterials: implications for interactions between nanomaterials and biofilm bacteria[END_REF]. However, the associated mechanisms remain poorly constrained and need to be investigated. For instance, the ecotoxicological potential of some QDs, whose deleterious impact toward bacteria has been demonstrated, is intimately related to their stabilities, with aged-QDs being more toxic than fresh ones [START_REF] Mahendra | Quantum Dot Weathering Results in Microbial Toxicity[END_REF]. Due to the high reactivity of the biofilm/mineral interface, the high site densities and the presence of microenvironments within the biofilm, the stability of the QDs and the speciation of the constituting elements are likely to evolve. [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF], Zn(II) and Pb(II) [START_REF] Templeton | Pb(II) distributions at biofilm-metal oxide interfaces[END_REF]Wang et al., 2016b) in biofilm/crystal systems. In addition, Grazing Incidence-X-Ray Absorption Spectrocopy (GI-XAS) measurements provide information on speciation of elements, giving an overall view of QDs' physico-chemical transformations at the biofilm/crystal interface.

II. Materials and Methods

Quantum Dots

QDs were obtained in a single-step synthesis procedure described by [START_REF] Bae | Single-Step Synthesis of Quantum Dots with Chemical Composition Gradients[END_REF]. The QDs present a chemical composition gradient with a CdS transition between the CdSe core and the ZnS shell (see Fig. 1 for a schematic representation of the QD). To allow their transfer to aqueous solution, they were functionalized with a layer of thioglycolic acid (TGA) ligand, following a protocol previously established by [START_REF] Supiandi | Isotopically Labeled Nanoparticles at Relevant Concentrations: How Low Can We Go? The Case of CdSe/ZnS QDs in Surface Waters[END_REF]. To isolate and purify QDs from the reaction medium, several cycles of concentration-dispersion steps in borate buffer and milli-Q water were performed. QDs at the end of synthesis were placed in milli-Q water at pH=10 to ensure their chemical stability during storage. This solution was green fluorescent under 312 nm UV light, characteristic of QDs with a size around 7 nm [START_REF] Bae | Single-Step Synthesis of Quantum Dots with Chemical Composition Gradients[END_REF], and a core size of 3.4 nm as determined by absorbance measurements between 400 and 650nm, and according to the core diameter to wavelength relationship [START_REF] Jasieniak | Reexamination of the Size-Dependent Absorption Properties of CdSe Quantum Dots[END_REF]. According to [START_REF] Faucher | Characterization of polymer-coated CdSe/ZnS quantum dots and investigation of their behaviour in soil solution at relevant concentration by asymmetric flow field-flow fractionation -multi angle light scattering -inductively coupled plasma -mass spectrometry[END_REF], the CdSe core measures 3 nm, and the total diameter, with the ZnS shell, is 6-7 nm based on STEM Electron Energy Loss Spectroscopy performed on the same QDs. Finally, the ZnS shell is not homogeneous and presents occasional holes in its structure (Fig. 1) [START_REF] Faucher | Characterization of polymer-coated CdSe/ZnS quantum dots and investigation of their behaviour in soil solution at relevant concentration by asymmetric flow field-flow fractionation -multi angle light scattering -inductively coupled plasma -mass spectrometry[END_REF].

In QD stock solutions, the initial concentrations of Cd, Se and Zn were measured by inductively coupled plasma atomic emission spectroscopy. The average concentrations of four syntheses are 38.54.9 M, 8.10.8 M and 4.60.5 M for Zn, Cd and Se, respectively with an average molar ratio between elements of 0.20.1, 1.91.3 and 9.06.1, for Cd/Zn, Cd/Se and Zn/Se, respectively. The calculated molar ratio of Cd/(Cd+Zn) is around 25%, in agreement with [START_REF] Bae | Single-Step Synthesis of Quantum Dots with Chemical Composition Gradients[END_REF]. The electrophoretic mobility (EPM) and zeta potential (ZetaSizer, Malvern) of QDs functionalized with TGA were measured in 5mM NaNO 3 at pH ranging from 2 to 10. The hydrodynamic diameter was also assessed by Dynamic Light Scattering (DLS) at neutral pH (Dynapro Nanostar, Wyatt Technology, California, USA).

Sample preparation

The biofilm/crystal system and the biofilm growth protocol were previously described in [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF]. Briefly, the system is composed of a highly polished, cleaned and oriented single crystal substrate of -Al 2 O 3 (1-102) with a surface roughness of 3Å (commercial Pi-KEM Ltd.) adequate for LP-XSW-FY spectroscopy. S. oneidensis MR-1 biofilms were grown for 10 days on substrates placed in a sterilized flow-through bottle, following the protocol established by Wang et al. (2016b). First, a tripticase soy broth (BioMérieux, 51019) suspension inoculated with S. oneidensis MR-1, in a reproducible way [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF], was left to settle for 1 hour in the bottle. Then, sterile synthetic growth medium (table S1) at pH 7.0 was pumped continuously through the bottle for 10 days at ambient temperature. After 10 days, the biofilm-coated surfaces were then gently placed in 10 mL Falcon centrifuge tubes containing the same growth medium, and were stored at 4°C

for 1 week before measurement. The number of colony-forming units was similar before and after 1 week at 4°C (data not shown). Prior to measurement, the biofilm/crystal systems were rinsed to remove excess nutrients, then exposed to a QD suspension in 5 mM NaNO 3 solution at ambient temperature. This background electrolyte can be considered an ideal soil solution which minimizes particle aggregation [START_REF] Chen | The stability and removal of water-dispersed CdSe/CdS core-shell quantum dots from water[END_REF]. The experiments were performed with a Cd concentration of 1372 nM and a Zn concentration of 74860 nM.

Solution pH was adjusted to 7.00.1, if necessary, in the course of experiments to ensure acceptable living conditions for S. oneidensis MR-1, using 0.1 M HNO 3 or 0.1 M NaOH.

A new biofilm/crystal system was used for each exposure time (1h, 3h, 10h and 24h) and technique, with measurements conducted in either duplicate or triplicate as detailed below. The 24h maximum exposure time was chosen to minimize biofilm structure alteration due to QD toxicity [START_REF] Dumas | Interfacial Charge Transfer between CdTe Quantum Dots and Gram Negative Vs Gram Positive Bacteria[END_REF].

Samples were gently immersed in 21 mL of fresh QD solutions, with the biofilm side facing down to ensure the study of QD transport. All tubes were shielded from light using aluminum foil to avoid QD degradation [START_REF] Li | Oxidative dissolution of polymer-coated CdSe/ZnS quantum dots under UV irradiation: mechanisms and kinetics[END_REF], and were gently shaken at 20 rpm. For LP-XSW-FY measurements, samples were placed in Kapton-covered sample holders, mounted vertically, and purged with humid He gas. For GI-XAS measurements, the fluorescence detector was mounted perpendicular to the sample surface. In order to obtain a detailed characterization of QD fate in the system, control experiments, mass balance measurements and SEM imaging were performed. Cd and Zn uptake were estimated by biofilm digestion in 2% HNO 3 , after 1h, 3h and 24h of exposure, in triplicate. To obtain concentration in mg per gram of biofilm, four biofilms were weighed just after growth, and after 12h oven drying at 450°C. The average biofilm dry weight was 0.300.02 mg per sample. As Cd and Zn are naturally present within biofilms, originating from impurities present in nutrient solutions during growth, their concentrations in biofilms that were not exposed to QDs were also measured in triplicate by the same method. The amount of Cd and Zn in solution, and sorbed onto the Falcon tube walls, were also measured using an Agilent 7900 ICP-QMS (see (2018) and is detailed in appendix 3 (see SI). More information on LP-XSW-FY principles and applications can be found in [START_REF] Trainor | Structure and reactivity of environmental interfaces: Application of grazing angle X-ray spectroscopy and longperiod X-ray standing waves[END_REF]. The limitations of using this technique in such systems (biofilm/crystal interface + NPs) are presented in [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF] and in appendix 3 (SI). Briefly, the technique is highly dependent on the roughness and the thickness of the sample. The high heterogeneity of the biofilm associated with the presence of dense nano-metric objects are likely to impact the formation of the standing waves. Thus, the modeling can be quite complicated. These parameters have been considered in our models, allowing the semi-quantification of the distribution of Zn and Se, although the interpretation has to be performed carefully. The monochromatic 13.3 keV X-ray beam was collimated using a pair of 1 meter, Rh coated Si mirrors in Kirkpatrick-Baez geometry. The final beam profile of 1000 m vertical by 10 m horizontal was defined by slits. X-ray reflectivity measurements were performed by scanning X-ray incidence angle between 0.0° and 0.5° while monitoring the intensities of the incident (I 0 ) and reflected (I 1 ) X-ray beams using N 2 -filled gas ionization chambers. Zn K and Se K fluorescence yield data were collected using a 4-element silicon drift detector (SII NanoTechnology, Vortex-ME4) in two or more locations for each sample to verify reproducibility.

The reflectivity and the fluorescence yield are modeled to obtain a semi-quantitative distribution of the elements of interest (here Zn and Se). The full description of the physical model is presented in the SI (appendix 3). In this study, the biofilm/crystal system was divided into three compartments: the crystal surface-biofilm interface (labeled C 1 ), the biofilm thickness (C 2 ) and the biofilm-gas interface (C 3 ) (Fig. S4). The model (see appendix 3, SI) enables estimation of the distribution of the elements across the compartments. Goodness of the fit were estimated by performing a 2 test and confidence interval were estimated using a Student's test, as we did it in our previous work [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF] (see appendix 3, SI).

4. GI-XAS measurements and data analysis GI-XAS (Grazing Incidence -X-ray Absorption Spectroscopy) allows the determination of elemental speciation at different depths within the biofilm/crystal system. We estimate a minimum detection limit of 15% by weight. Thus, local phenomena resulting in small quantities of certain species would not be detected. EXAFS spectra were collected at Zn Kedge (9.6 keV) and XANES spectra were collected at Se K-edge (12.6 keV) at the crystal surface-biofilm interface (C 1 ), and within the biofilm thickness (C 2 ). Measurements were performed at beamline 11-2 at the Stanford Synchrotron Radiation Lightsource (SSRL) using a grazing-incidence spectrometer in vertical scattering geometry. GI-XAS spectra were collected at room temperature in fluorescence mode using a Canberra 100-pixel Ge solidstate monolith pixel detector. The incident beam energies were selected using a LN 2 cooled Si (220) monochromator and collimated using a pair of 1 m-long Rh-coated Si mirrors. Zn and Se metal foils were used during experiments for energy calibration. In order to specifically probe the two compartments of interest, Zn K-edge and Se K-edge fluorescence data were collected at incidence angles of 0.30° and 0.25° to interrogate the crystal surfacebiofilm interface (C 1 ), and at 0.18° and 0.10° for the biofilm thickness (C 2 ), respectively. Note that for the crystal surface-biofilm interface, it is not possible to only probe the speciation of Zn and Se at the surface of the crystal. Indeed, part of the signal originated also from the first nanometers of the biofilm, meaning that the signal from the surface could be partially "contaminated" by the signal from the biofilm. For XAS data analysis, three or four scans were averaged, background subtracted, and fitted using the SIXPack interface [START_REF] Webb | SIXpack: a graphical user interface for XAS analysis using IFEFFIT[END_REF] and the IFEFFIT XAFS analysis package [START_REF] Ravel | HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT[END_REF]. Linear combination fitting (LCF) was used to quantify the presence of several possible species. Additional species were considered only when they improved the goodness-of-fit by at least 15%.

III. Results

Characterization of QDs

The surface charge of the QDs is negative over the entire measured pH range, with an increase in the negativity of the surface charge for pH>8 (Fig. S5). At pH=6.800.05 and 8.010.05, the EPMs values are -1.30.3 and -0.80.7 m cm/Vsec, respectively. The corresponding zeta potentials are -16.63.1 mV and -9.80.7 mV, respectively. The EPM measured at pH 8 present an uncertainty higher than for the other pH studied (Fig. S5), which could indicate that those points are outliers. When pH decreases, pH 8 could also correspond to the pH where the carboxylates could start slowly to protonate (carboxylate pK a on surfaces can be higher than in solution [START_REF] Chen | Determination of pKa Values of Carboxyl Groups in the N-Terminal Domain of Rat CD2: Anomalous pKa of a Glutamate on the Ligand-Binding Surface[END_REF]), favoring the presence of different particles (individual particles, dimmers, trimers…). In addition, at pH lower than 8, surface charge is lower which would probably favor aggregation of the particles due to the increase of the protonation, and the EPM, or zeta potential, would be estimated for aggregates and not for a unique QD. The partial homo-aggregation of QDs at neutral pH seems to be confirmed by the average hydrodynamic diameter measured by DLS (41.7 nm).

In addition, more than 60% of the particles present a diameter less than 30 nm (with size ranging from 5 to 200 nm, DLS results), validating the hypothesis of different types of particles in solution. This has been considered for the discussion of results.

Nevertheless, the surface charge of the QDs, or their agglomerates, at pH 7 is regarded as negative, considering the whole analysis.

Zn and Se distributions

All LP-XSW-FY profiles collected from different locations on a given sample were similar, and these profiles were thus averaged. The measured critical angle is located at 0.1670.004° (Fig. 2, marked with black arrows), in agreement with the theoretical value of 0.17° at 13.3 keV. All profiles are presented with their fit depicted as a continuous line (Fig. 2). In general, for each exposure time, Zn and Se FY data present a first peak located between 0.03°-0.07° corresponding to the biofilm-gas interface, and a second one around 0.17° corresponding to the crystal surface-biofilm interface (Fig. 2). At 1h and 3h of exposure, the most intense Se FY peaks are located at small incidence angle, while Zn FY peaks are located around 0.04° and 0.17° (Fig. 2). However, at 10h and 24h, the FY profiles are quite similar between Zn and Se, with a broad distribution between 0.05° and 0.17° (Fig. 2). Normalized FY intensities increase with time of exposure, from 1h to 24h (Fig. 3).

The Zn and Se distributions determined by LP-XSW-FY profile modeling are presented in Fig. 4. After 1h of exposure, most of the Zn (803.5%) and Se (604.5%) are located in the biofilm (C 2 ). The remaining Zn is mostly located at the crystal surface-biofilm interface (C 1 , 173.5%), whereas the remaining Se is detected at the biofilm-gas interface (C 3 , 404.5%), indicating a difference in Zn and Se distribution. This distribution is further pronounced at 3h, Se distribution is similar to the one obtained at 1h, whereas 725.5% of Zn is now located at the crystal surface-biofilm interface (C 1 ). At 10h and 24h, identical distributions are observed for Se and Zn, with 25% at the biofilm-gas interface (C 3 ), 70% in the biofilm (C 2 ), and 5%

at the crystal surface-biofilm interface (C 1 ) (see Fig. 4 for estimated confidence interval).

One could note here the high percentage of Zn located at the mineral surface (C 1 ) at 3h compared to the percentage of Zn at 1h, even if the shape the shape of the curves describing normalized fluorescence intensity appears to be relatively similar. The higher percentage of Zn obtained at 3h could be explained by the larger peak of the modeled data at 3h, at high incident angle, that could increase the percentage of Zn at the mineral surface, at the expense of the percentage of Zn present in the biofilm thickness (C 2 ). In addition, compared to the others samples, the confidence interval of the Zn-3h sample is a little bit larger (see appendix 3, SI), and the value of the 2 test is smaller for the Zn-sample at 1h compared to the one at 3h, which could indicate a better fit for the data at 1h. Thus, the percentage of Zn at the mineral surface could be overestimated and this spectrum could be considered as an outlier. Nevertheless, we considered that, even if the percentage of Zn at the mineral surface is overestimated, the observed tendency seems to be similar to the one at 1h with Zn present at the mineral surface, while Se is absent.

Zn and Se speciation in the different compartments

Zn-EXAFS and Se-XANES spectra for reference compounds and samples exposed to QDs are presented along with their fits in Fig. 5 and S6, respectively. LCF and  2 results are reported in Fig. S6 for Se, and in Table S2 for Zn. The Zn reference compounds used to perform LCF are aqueous Zn(II) (from dissolved Zn(NO 3 ) 2 ) exposed to a S. oneidensis biofilm for 3h, and native Zn-QDs. Other reference compounds were tested to improve the fit quality, such as Zn-acetate (Zn associated with carboxyl functional groups), Zn-phosphate (Zn complexation with phosphoryl groups), and Zn-cysteine (Zn complexation with thiol groups). None of those references improved the fit quality by at least 15%. Wang et al. (2016b) identified the first neighbor of Zn(II) exposed to S. oneidensis biofilm as an oxygen atom at a distance of 1.980.01 Å (fourfold oxygen coordination). In our Zn-QDs reference compound, the first neighbor is sulfur with a distance of 2.340.02 Å consistent with the Zn-S distance in Wurtzite [START_REF] Chukavin | Study of the ZnSxSe1-x@Al2O3 nanostructures by X-ray diffraction and EXAFS spectroscopy[END_REF]. For our samples, only Zn linked to S as first neighbor is detected within the biofilm thickness (C 2 ) for all exposure times. At the crystal surface (C 1 ), all Zn is linked to S at 24h of exposure, while for shorter times (1h and 3h) 79 to 85% Zn is linked to S and the remaining fraction is associated with O in the first shell. For these last two conditions, inclusion of a Zn-O bond in LCFs improves the goodness-of-fit values ( 2 reduced from 3.2 to 2.3 and from 1.9 to 0.9).

As Se is supposed to be associated with Cd in QDs, its speciation is expected to be Se(-II) with a K-edge energy of 12658 eV [START_REF] Ryser | Micro-spectroscopic investigation of selenium-bearing minerals from the Western US Phosphate Resource Area[END_REF]. However, the K-edge energy of the Se-QD reference compound is measured at a slightly higher energy, 12659.9 eV, which is between that of Se(-II) and Se(IV) (i.e. 12662 eV [START_REF] Ryser | Micro-spectroscopic investigation of selenium-bearing minerals from the Western US Phosphate Resource Area[END_REF]). Thus, in these QDs, Se is not only present as Se(-II) but some of it is oxidized. The study of [START_REF] Faucher | Characterization of polymer-coated CdSe/ZnS quantum dots and investigation of their behaviour in soil solution at relevant concentration by asymmetric flow field-flow fractionation -multi angle light scattering -inductively coupled plasma -mass spectrometry[END_REF] by STEM Electron Energy Loss Spectroscopy on QDs using the same synthesis showed that the ZnS shell thickness is not homogeneous, and parts of the CdSe core could be directly exposed to the solution and therefore undergo oxidation. After 1h and 3h of exposure, within both the biofilm thickness (C 2 ) and at the crystal surface (C 1 ), the Se redox state is similar to the QDs reference compound. However, at 24h, a small fraction of Se appears to be more oxidized since a Se(IV) reference compound is now required for the fitting procedure ( 2 reduced from 0.33 to 0.15 and from 0.41 to 0.13; Fig. S6).

Elemental sequestration in the biofilm/crystal system

The amounts of Cd and Zn remaining in the supernatant, trapped in the biofilm, and sorbed on tube walls were measured by ICP-QMS. In solution, the total concentrations of Zn and Cd decreased (from 74860 nM to 28059 nM and from 1372 nM to 4112 nM, respectively), whereas they increased within the biofilm (from 2.21.4 to 10.31.8 mg/g biofilm for Zn and from 75 g/g biofilm to 2.60.7 mg/g biofilm for Cd) and on tube walls (less than 1 nM at the beginning of the experiment to 2876 nM for Zn and 1123 nM for Cd). Regarding Zn and Cd distribution in the whole experimental system at 24h of exposure, 46.5% and 26% of total Zn and Cd, respectively, remained in solution while a much larger fraction was sorbed onto tube walls (47.5% of Zn and 70% of Cd). Within the biofilm thickness, 6% of total Zn and 4% of total Cd are trapped (Fig. S7).

The percentage of dissolved Zn, compared to total Zn in the experiment, in the supernatant as a function of time of exposure remains constant (around 10%), corresponding to an average concentration of 8125 nM (Fig. S8a). The fraction of dissolved Cd is closer to zero (around 1% in average), corresponding to a concentration of 2.52.9 nM (Fig. S8b). As dissolved concentrations of Zn and Cd in supernatant remain constant over time (Fig. S8), the presence of those dissolved species is likely to result from an initial presence of dissolved Zn and Cd in the experiments. Indeed, ZnS is known to be stable in water [START_REF] Priadi | X-ray Absorption Fine Structure Evidence for Amorphous Zinc Sulfide as a Major Zinc Species in Suspended Matter from the Seine River Downstream of Paris, Ile-de-France, France[END_REF] so no dissolution in solution is expected during the course of experiments. Note that the mass balance is conserved in all experiments (Fig. S9).

Control experiments performed with Cd(II) and Zn(II) ions at similar concentrations show that most Zn and Cd remain in solution, 96.5% and 96% respectively, while only 5% Zn and 1.5% Cd are sorbed onto tube walls. In addition, 2.5% of the Cd is found in the biofilm while no additional Zn is detected in this compartment (Fig. S7).

IV. Discussion

1. QD sequestration at the biofilm/crystal interface When the samples are exposed to QDs, most of the Zn and Cd is found associated with the tube walls, highlighting a high affinity of QDs for this type of plastic. Despite exhibiting a lower surface area compared to tube walls (estimated at 2.4 cm 2 for the biofilm/crystal system vs. 50 cm 2 for the tube), the biofilm/crystal system traps QDs in appreciable amounts, with 13% of Zn and 15% of Cd being present in the biofilm relative to the solution after 24h.

In addition, the increase in Zn, Se and Cd in this compartment with time, as measured by ICP-QMS and fluorescence intensity (Fig. 3), indicates continuous accumulation in the biofilm/crystal system over time (Fig. 6-1). As described in the introduction, the overall surface charge of S. oneidensis at pH=7 is negative [START_REF] Ha | Role of extracellular polymeric substances in metal ion complexation on Shewanella oneidensis: Batch uptake, thermodynamic modeling, ATR-FTIR, and EXAFS study[END_REF] meaning that the interactions with negatively charged QDs would not be favored. Nevertheless, the interaction between negatively charged NPs and S. oneidensis MR-1 biofilm has been previously observed with 60 nm silver NPs coated with polyvinylpyrrolidone [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF]. In this previous study, we highlighted the role of other parameters, such as NP size and hydrophobicity, on the interactions between NPs and biofilms, when both of them are negatively charged. For example, smaller particles are able to diffuse into all parts of the biofilm. In the present study, the relatively small size of the QDs (60% have a hydrodynamic diameter less than 30 nm) could explain their ability to strongly accumulate in the biofilm. In addition, the strong interactions between negatively charged QDs and S. oneidensis biofilm have also been explained in the past by the presence of hydrophobic microdomains within the biofilm thickness [START_REF] Aldeek | Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms[END_REF][START_REF] Aldeek | Patterned Hydrophobic Domains in the Exopolymer Matrix of Shewanella oneidensis MR-1 Biofilms[END_REF], validating the role of hydrophobic interactions in NP-biofilm interactions.

QD fast dissolution within the biofilm

A semi-quantitative distribution of Zn and Se in the biofilm/crystal system can be obtained by modelling LP-XSW-FY data, using the 3 compartments previously described (Fig. 4).

Interestingly, at short time of exposure (i.e. 1h and to a certain extend at 3h), an absence of co-localization between Zn and Se at the interfaces (C 1 and C 3 ) is observed. This lack of co-localization can be explained by the presence of dissolved Zn(II) that interacts differently with the biofilm and crystal surface than QDs (Fig. 6-2). Zn in solution could originate either from (i) dissolved Zn(II) present in the supernatant (10%), or (ii) from dissolution of QDs in the biofilm matrix. Regarding (i), when exposed to free Zn(II) at 899 nM, the amount of Zn trapped in the biofilm/crystal system is minimal compared to QD exposure experiments (0 vs. 6 %, see III.3 and Fig. S7). As a result, the 10% of Zn(II), ~96 nM, present in the supernatant could not fully explain the Zn detected at the crystal surface in QD experiments by LP-XSW-FY at 1 and 3h. In addition, Wang et al. (2016b) studied the distribution of dissolved Zn(II) in the S. oneidensis MR-1 biofilm/-Al 2 O 3 (1-102) crystal system and showed that for all concentrations (10 -4 to 10 -7 M) and exposure times investigated, Zn(II) was mostly associated with the biofilm and did not interact with the crystal surface. Dissolved Zn(II) present in the supernatant in our study is expected to behave the same and would partition mostly to the biofilm. Since this is not observed, the involvement of dissolved Zn from the supernatant can thus be discarded.

Therefore, Zn at the crystal surface is likely to originate from a partial QD dissolution in the biofilm thickness (ii). The fast dissolution of ZnS nanoparticles [START_REF] Dehner | Roles of siderophores, oxalate, and ascorbate in mobilization of iron from hematite by the aerobic bacterium Pseudomonas mendocina[END_REF], in contact with bacteria has been observed before, as has their dissolution in other systems such as organic wastes (Le [START_REF] Bars | Drastic change in zinc speciation during anaerobic digestion and composting: Instability of nanosized zinc sulfide[END_REF]. In our study, the Zn(II) from the ZnS shell would be released much closer to the crystal surface, and interact more easily with the highly reactive functional sites from the crystal (Wang et al., 2016b). The local dissolution of the ZnS shell in the biofilm is further supported by the GI-XAS results at 1h and 3h of exposure (Fig. 5, Table S2). Within the biofilm (C 2 ), Zn remains associated with QDs, as indicated by the presence of S in the first coordination shell. However, at the crystal surface-biofilm interface (C 1 ), a fraction of Zn (15-21%) shows O in the first-coordination shell. This indicates the partial dissolution of the ZnS shell and the interaction of the released Zn(II) with Obearing reactive sites located at the bottom of the biofilm and the crystal surface.

Unfortunately, to the best of our knowledge, no accurate binding constant is available for Zn(II) adsorbed onto -Al 2 O 3 (1-102). Nevertheless, studies conducted on Zn(II) sorption onto Al-oxide surface coatings on aquifer sand [START_REF] Coston | Pb2+ and Zn2+ adsorption by a natural aluminum-and iron-bearing surface coating on an aquifer sand[END_REF] and at the alginate/alumina interface [START_REF] Wang | Competitive sorption of Pb (II) and Zn (II) on polyacrylic acid-coated hydrated aluminum-oxide surfaces[END_REF], point out the relatively high affinity of Zn for the alumina surface. We infer that in our system, the partial dissolution of the ZnS layer of the QDs occurs first in the biofilm microenvironments (C 2 ), then the released Zn(II) migrates toward the crystal surface (C 1 ) where it is partially sorbed. Unlike the results of Wang et al. (2016b) , in which Zn was found mostly located in the biofilm, QD dissolution occurs close enough to the surface (Fig. 6-2) to allow Zn to reach the crystal without being trapped or inhibited by interactions with biofilm functional groups during its transport.

Dissolution of the ZnS shell exposes the QD core, and could favor ionic Cd and Se release. However, according to our GI-XANES measurements (Fig. S6), no modification of Se speciation is detected after 1 and 3h of exposure, indicating a higher stability of CdSe cores compared to ZnS shells. A more stable CdSe core has already been observed in oxidative environments [START_REF] Metz | Engineered nanomaterial transformation under oxidative environmental conditions: Development of an in vitro biomimetic assay[END_REF], and when QDs are in contact with algae [START_REF] Slaveykova | Effect of natural organic matter and green microalga on carboxyl-polyethylene glycol coated CdSe/ZnS quantum dots stability and transformations under freshwater conditions[END_REF]. At 1h and 3h of exposure, Se is present at the surface of the biofilm-gas interface (C 3 , 40-50%) and within the biofilm (C 2 , 50-60%), indicating that CdSe cores, stable over time, have a preferential interaction with the biofilm compartment for short term exposure.

NP dissolution when in contact with bacteria has been observed before (Auffan et al., 2009b), specifically in toxicity studies, and can occur from three different processes: it can be ligand-mediated [START_REF] Wirth | Natural organic matter alters biofilm tolerance to silver nanoparticles and dissolved silver[END_REF], redox-mediated [START_REF] Kroll | Extracellular polymeric substances (EPS) of freshwater biofilms stabilize and modify CeO2 and Ag nanoparticles[END_REF] or due to change in the local physico-chemical conditions [START_REF] Dehner | Roles of siderophores, oxalate, and ascorbate in mobilization of iron from hematite by the aerobic bacterium Pseudomonas mendocina[END_REF]. The high site density of functional groups at the bacteria surfaces and in the EPS matrix, along with the presence of extracellular organic ligands in the biofilm pores [START_REF] Ha | Role of extracellular polymeric substances in metal ion complexation on Shewanella oneidensis: Batch uptake, thermodynamic modeling, ATR-FTIR, and EXAFS study[END_REF][START_REF] Morel | The Biogeochemical Cycles of Trace Metals in the Oceans[END_REF]Wang et al., 2016b) favor ligand-mediated dissolution. The complexation of metal(loid)s in the system can limit the concentration of free species (a change in local physico-chemical conditions) and thus, trigger dissolution: when the ion activity product is less than the solubility product, dissolution is enhanced. Finally, the production of reactive oxygen species (ROS), by bacteria such as S.oneidensis [START_REF] Diaz | Influence of chemical speciation and biofilm composition on mercury accumulation by freshwater biofilms[END_REF], is a well-known phenomenon that could be amplified in the biofilm [START_REF] Wan | Loss of OxyR reduces efficacy of oxygen respiration in Shewanella oneidensis[END_REF] and in the presence of NPs [START_REF] Lu | Mechanism of Antimicrobial Activity of CdTe Quantum Dots[END_REF]. Production of ROS would enhance the redox-mediated dissolution mechanism, as has been proposed by [START_REF] Zhang | Aggregation, Dissolution, and Stability of Quantum Dots in Marine Environments: Importance of Extracellular Polymeric Substances[END_REF] for QDs exposed to EPS.

3. Crystal surface as a driver of QD accumulation with time At longer exposure times (i.e. after 10h), FY profiles of Zn and Se become similar (Fig. 2). This is either indicative of ZnS shells that are not dissolving anymore, or that crystal sites are saturated and unable to attract any additional free Zn while the biofilm/crystal system continues to accumulate QDs.

The relative increase in Zn and Se FY over time (Fig. 3) indicate that the total uptake of the QDs and their products of dissolution continue over 24 hours, which means that the whole interface does not reach saturation [START_REF] Templeton | Pb(II) distributions at biofilm-metal oxide interfaces[END_REF]. Nevertheless, over time the relative amount of Zn in the crystal surface compartment (C 1 ) compared with the biofilm compartments (C 2 and C 3 ) decreases (Fig. 4), which could indicate that some fraction of the crystal sites reach saturation within the 24 hours [START_REF] Templeton | Pb(II) distributions at biofilm-metal oxide interfaces[END_REF]. Thus, it is likely that partial saturation of the crystal surface with respect to Zn(II) occurs in the present system. On the other hand, the relative amount of Se increases a little bit at the crystal surface (C 1 ), from 0% at 1h to 62.5% at 24h which could indicate the presence of sites still available. This migration toward the biofilm/crystal interface (C 1 ) could indicate that the transport of QDs or CdSe cores is partially driven by their interaction with the crystal surface, with the biofilm slowing their progression. Indeed, the accumulation of QDs over time would favor the partial saturation of the functional sites of the biofilm, advancing from the top (where the QDs enter the system) to the bottom. The attraction of negatively charged NPs by -Al 2 O 3 surfaces has been previously reported for silver NPs coated with polyvinylpirrolidone [START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF]. For QDs, their small size, and the chemical interactions they establish with the biofilm's functional groups and microdomains, explain their relatively slow transport rate toward the crystal, while this surface remains attractive to NPs at longer exposures times (Fig. 6-3).

In addition to the partial saturation of the surface sites, slowing of ZnS dissolution after 10h of exposure could explain the observed results. The fact that, at 24h, Zn is mostly linked to S as first-shell neighbor, even at the crystal surface-biofilm interface (C 1 ) (Table S2), in contrast with the Zn speciation observed at 1h and 3h, where 15-21% of Zn was associated with O as first-shell neighbor, suggests a decrease in dissolution rate of the ZnS shell. This decrease could be the result of changes in physico-chemical conditions in the biofilm microenvironments, with fast consumption of extracellular organic chelatants during the first hours of exposure leading to a slowing of ligand-mediated dissolution processes.

The saturation of the biofilm's functional sites over time would also impact this type of dissolution.

Finally, minor oxidation of CdSe was observed at 24h in both the biofilm thickness (C 2 ) and at the crystal surface-biofilm interface (C 1 ), shown by a slight increase in Se oxidation state from Se(-II) to Se(IV) (Fig. S6). This late oxidation originates from the exposure of Cd and Se from the core to oxidative environments [START_REF] Derfus | Probing the cytotoxicity of semiconductor quantum dots[END_REF], and suggests that oxidative conditions are presented within the biofilm. Thus, the oxidation of the CdSe cores would be favored by the presence of holes in the ZnS shell as observed after QD synthesis [START_REF] Faucher | Characterization of polymer-coated CdSe/ZnS quantum dots and investigation of their behaviour in soil solution at relevant concentration by asymmetric flow field-flow fractionation -multi angle light scattering -inductively coupled plasma -mass spectrometry[END_REF], or because of partial ZnS dissolution in the biofilm/crystal system, occuring in the first hours of exposure increasing the ZnS shell porosity.

Similarities among Zn and Se distributions in the whole biofilm/crystal system indicate first the accumulation of QDs, and later, the control exerted by the mineral surfaces over QD, CdSe core and Zn(II) distributions. The accumulation of elements within the biofilm/mineral system at long exposure times masks the details of processes such as dissolution and oxidation that we were able to observe at shorter exposure times.

V. Conclusion

The present study provides an overall view of the fate of QDs when these NPs are exposed to the biofilm/crystal interface (Fig. 6), a widespread environmental compartment.

Even in a system that minimizes the biofilm to solution volume ratio, the high accumulation potential of this interface is manifest. Most importantly, the fast dissolution of the ZnS shell shortly after QD exposure constitutes a potential key process when regarding the fate of QDs in the environment, by promoting the fast degradation of these NPs and thus by limiting their persistence. This process is likely to be promoted at lower concentration, as in natural environments, and thus has to be taken into account when studying the environmental fate of QDs. Here, ZnS dissolution is likely to occur in the biofilm thickness as a consequence of high functional site densities as well as local oxidative conditions, and is followed by the migration of dissolved Zn(II) toward the crystal surface. The CdSe cores, however, remain mostly intact in the biofilm thickness. At longer exposure times, a general partitioning closer to the crystal surface is observed, highlighting the importance of -Al 2 O 3 in the whole system reactivity. ZnS shell dissolution is not discernible, partly masked by the accumulation of QDs, but also likely occurs as a result of biofilm functional site saturation. Oxidative conditions in the biofilm thickness seem to be partly preserved with evidence of slight Se oxidation.

Further studies are needed, particularly in order to investigate the involvement of bacterial metabolic activity in ZnS dissolution processes for potential use of bacterial biofilms as remediation tools in QD polluted environments.

  Zeiss Ultra Device SEM with field emission gun at 15 keV, using the protocol presented by[START_REF] Desmau | Dynamics of silver nanoparticles at the solution/biofilm/mineral interface[END_REF].

	Please refer to appendix 2 and Fig. S1 and S2 (see SI) to see protocols and results.
	3. LP-XSW-FY measurements and data analysis
	LP-XSW-FY (Long Period -X-ray Standing Wave -Fluorescence Yield) spectroscopy
	enables the measurement of elemental depth profiles at nanoscale resolution. Compared to
	other techniques used to study the transport of NPs in biofilms, this technique enables the
	study of the distribution of NPs within the whole interfacial region (biofilm+mineral), without
	modification of the system (Desmau et al., 2018), and thus allow us to obtain an overall
	understanding of the NPs' fate. However, LP-XSW-FY does not permit the monitoring of
	specific mechanisms that could occur in some parts of the biofilm due to the local presence
	of microenvironments, for example. Indeed, a surface area of 1 mm x 1 cm was probed for
	each location.
	LP-XSW-FY	measurements	were	conducted	at	beamline	13-ID-C	at
						2% HNO 3 after the
	experiments to assess the quantity of QDs sorbed onto the tube walls. The amounts of
	dissolved species in the supernatant were estimated by QD removal by centrifugal
	ultrafiltration (3 kDa, Amicon, Millipore) and measured by ICP-QMS. Control experiments

appendix 1, SI), in triplicate. The Falcon tubes were rinsed with were performed with biofilm/crystal systems exposed to Cd(II) (36.10.2 nM) and Zn(II) (3246 nM) ions for 24h in triplicate. Samples were imaged using a GeoSoilEnvironCARS (GSECARS) at the Advanced Photon Source (APS). The LP-XSW-FY set-up, data analysis and modeling is similar to the protocol described in

Desmau et al.

Acknowledgements

This work was supported by the ANR QUADOS project, grant ANR-14-CE01-0013-01 of the French Agence Nationale de la Recherche and project EC2CO-PRONTO financed by the Centre National de Recherche Scientifique (CNRS). Portions of this work were performed at GeoSoilEnviroCARS (The University of Chicago, Sector 13), Advanced Photon Source, Argonne National Laboratory. GeoSoilEnviroCARS is supported by the National Science Foundation -Earth Sciences (EAR -1634415) and Department of Energy-GeoSciences (DE-FG02-94ER14466). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The authors acknowledge Joanne Stubbs and Peter Eng for their help during X-ray Standing Waves measurement at the beamline. Parts of this work were performed at Stanford Synchrotron Radiation Lightsource

(SSRL) on beamline 11-2, and the authors acknowledge Gordon E. Brown for his help in proposal writing and Ryan Davis for his help during the experiment.

The authors acknowledge Jessica Brest, at the Environmental Chemistry Platform (IMPMC), and Fériel Skouri-Planet, at the Biology Platform (IMPMC), for their help during experiments. The authors thank the reviewers for their constructive comments and help during the reviewing process.

Conflicts of interest

There are no conflicts to declare.


Antoine Lucas

Adrien Tauleigne

Fernande Da Cruz-Boisson

Agnès Crepet

Aurélie Bergeron-Vanhille

Grégory Martin

Nicolas Garois

Philippe Cassagnau

Véronique Bounor-Legaré

F Da

Cruz Boisson

A Crépet

A Vanhille Bergeron

Mechanical Properties Enhancement while Decreasing the Viscosity of Copolyether-Ester from In Situ Formation of Star-Based Structures by Reactive Extrusion

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

Introduction

Decreasing the melt viscosity of the polymers while keeping high levels of mechanical properties is an essential axis of research. Indeed, a low melt viscosity allows a reduction of the energy used to process and inject the polymer. Furthermore, it can bring new application fields to the polymer such as the impregnation of fiber tissues for example. A decrease in viscosity can be reached by simply reducing the molar mass of the polymer, for instance by chain cleavage reactions. However, the side effect of such action is usually also a decrease of the mechanical properties. In order to decrease the melt viscosity while keeping suitable mechanical properties, very interesting results have been reached by using supramolecular polymers.

Actually, this notion was first really introduced by Lehn 1,2 in 1989 with the synthesis of a liquid crystalline supramolecular polymer by self-assembly of the two complementary monomers uracil and 2,6-diacylamino-pyridine which formed triply hydrogen-bonded pairs. Then, quadruple hydrogen bonding between the end-groups of the polymers were obtained by T. Long et al. [START_REF] Yamauchi | Journal of the American Chemical Society Combinations of Microphase Separation and Terminal Multiple Hydrogen Bonding in Novel[END_REF][START_REF] Yamauchi | Macromolecules Synthesis and Characterization of Novel Complementary Multiple-Hydrogen Bonded (CMHB) Macromolecules via a Michael Addition[END_REF][START_REF] Yamauchi | Macromolecules Thermoreversible Poly(alkyl acrylates) Consisting of Self-Complementary Multiple Hydrogen Bonding[END_REF][START_REF] Yamauchi | Macromolecules Thermoreversible Polyesters Consisting of Multiple Hydrogen Bonding[END_REF][START_REF] Elkins | Macromolecules Synthesis and Characterization of Star-Shaped Poly(ethylene-co-propylene) Polymers Bearing Terminal Self-Complementary Multiple Hydrogen-Bonding Sites[END_REF][START_REF] Mather | Macromolecular Rapid Communications Morphological analysis of telechelic ureidopyrimidone functional hydrogen bonding linear and star-shaped poly(ethylene-co-propylene)s[END_REF] by functionalizing the end-groups of different synthesized polymers of small molar masses with ureidopyrimidinone UPy. This addition was successfully tested onto poly(styrene) [START_REF] Yamauchi | Journal of the American Chemical Society Combinations of Microphase Separation and Terminal Multiple Hydrogen Bonding in Novel[END_REF][START_REF] Yamauchi | Macromolecules Synthesis and Characterization of Novel Complementary Multiple-Hydrogen Bonded (CMHB) Macromolecules via a Michael Addition[END_REF] , poly(isoprene) [START_REF] Yamauchi | Journal of the American Chemical Society Combinations of Microphase Separation and Terminal Multiple Hydrogen Bonding in Novel[END_REF] , poly(butyl acrylate) [START_REF] Yamauchi | Macromolecules Thermoreversible Poly(alkyl acrylates) Consisting of Self-Complementary Multiple Hydrogen Bonding[END_REF] , poly(butylene terephthalate) [START_REF] Yamauchi | Macromolecules Thermoreversible Polyesters Consisting of Multiple Hydrogen Bonding[END_REF] , poly(butylene isophthalate) [START_REF] Yamauchi | Macromolecules Thermoreversible Polyesters Consisting of Multiple Hydrogen Bonding[END_REF] and star-shaped poly(ethylene-co-propylene) [START_REF] Elkins | Macromolecules Synthesis and Characterization of Star-Shaped Poly(ethylene-co-propylene) Polymers Bearing Terminal Self-Complementary Multiple Hydrogen-Bonding Sites[END_REF][START_REF] Mather | Macromolecular Rapid Communications Morphological analysis of telechelic ureidopyrimidone functional hydrogen bonding linear and star-shaped poly(ethylene-co-propylene)s[END_REF] . This resulted in an increase of the mechanical properties of the polymers at room temperature. For example, these modified poly(butylene terephthalate) (PBT) with multiple hydrogen bonding (MHB) units exhibits a 30% higher impact strength and 400% higher elongation at break than a PBT with the same molar mass.. This improvement of mechanical properties was coupled with an excellent thermal control of the viscosity. Indeed the H-bonds dissociated for a temperature above 80°C and the different polymers recovered the low viscosities of their nonfunctionalized equivalents. More recently, Leibler et al. [START_REF] Agnaou | Journal of the American Chemical Society Supramolecular Thermoplastic with 0.5 Pa•s[END_REF] were able to synthesize by polycondensation supramolecular semicrystalline telechelic polymers with terminal Hbonding groups and presenting an impressive viscosity of less than 1 Pa.s above their melting point. At room temperature, their ability to crystallize allowed them to present interesting mechanical properties with, for example, stress at break values between 5 and 12 MPa. These works certainly showed impressive results on the thermo-reversibility of H-bonding and the low melt viscosity it allows at high temperature. However, excepting for a few articles, the mechanical properties of the created polymers were not extensively studied.

Moreover, the synthesis of these oligomers can be complex, and the scale-up of the process can quickly become very complicated and expensive.

Another way to obtain a significant decrease in viscosity coupled with a relative conservation of the mechanical properties is to blend a matrix with dendrimers, star-shaped or hyperbranched polymers (HBP). Unlike dendrimers, HBP can be produced in industrial quantities at low cost and still inherit the interesting properties of dendrimers. Such polymers have been first used as additives in 1992 by Kim and Webster 10 who observed the decrease in viscosity induced when HBP were mixed with linear polymers. Highly branched polyphenylenes were blended with linear polystyrene, and despites the poor compatibility, the viscosity of the blend with 5%wt of polyphenylene was lower to about 50% at 180 °C and 80% at 220 °C, while keeping good tensile modulus. Hyperbranched polyesters have been widely used as processing aids for linear polymers. Hong et al. [START_REF] Hong | Journal of Rheology A novel processing aid for polymer extrusion: Rheology and processing of polyethylene and hyperbranched polymer blends[END_REF] , Jang et al. [START_REF] Jang | Macromolecules Crystallization behavior of poly(ethylene terephthalate) blended with hyperbranched polymers: The effect of terminal groups and composition of hyperbranched polymers[END_REF] and Mulkern et al. [START_REF] Mulkern | Polymer Processing and characterization of reactive polystyrene/hyperbranched polyester blends[END_REF] all developed polymer/hyperbranched polyesters systems and witnessed a decrease of the polymer viscosity. This decrease is due to the compact structure of the HBP which adopts a sphere-like hydrodynamic volume in solution or in the melt. This results in a decrease of the chain entanglement which allows polymers to flow past each other more easily under applied stress. Moreover, the high density of functional terminal groups on the HBP can improve the miscibility of the additive and the polymer. Many other studies have been made on various HBP/polymer blends [START_REF] Run | Materials Chemistry and Physics Influences of hyperbranched poly(amide-ester) on the properties of poly(butylene succinate)[END_REF][START_REF] Huber | Macromolecular Materials and Engineering Blends of hyperbranched poly(ether amide)s and polyamide-6[END_REF][START_REF] Lin | Macromolecules Study of Hydrogen-Bonded Blend of Polylactide with Biodegradable Hyperbranched Poly(ester amide)[END_REF] , but once again, in all these works, mechanical properties of the final blends have been poorly studied. Moreover, in the majority of these studies, the branched polymers are used as rheology modifiers and processing aids, where H-bonding is the only chemical interaction witnessed when a decrease of viscosity takes place. In the case where covalent bonds are created between the branched additive and the matrix, a chain-extension mechanism occurs and the viscosity increases drastically [START_REF] Kil | Polymer Engineering and Science Rheological properties of hyperbranched polymer/poly(ethylene terephthalate) reactive blends[END_REF] . A last cited example of blend is the one from Leibler et al. [START_REF] Chabert | International Journal of Adhesion and Adhesives Supramolecular polymer for enhancement of adhesion and processability of hot melt polyamides[END_REF] who were able to lower the viscosity of an amorphous biosourced polyamide (PA) resin to less than 10 Pa.s while increasing its elastic modulus from 4MPa to 30MPa by mixing it with 10-50%wt of a small supramolecular polymer. This additive was able to partially crystallize and thus reinforce the amorphous PA at room temperature, while acting as a plasticizer in the molten state and reducing the viscosity of the PA. Besides, all these examples represented a two-steps approach with first the synthesis of the additive and then the blending with the main matrix.

To our knowledge, a significant decrease in viscosity coupled with an enhancement of the mechanical properties has never been reached for any polymer by a one-step reactional based approach. Thus, in the present study, we propose a new method to obtain a decrease of viscosity coupled with an increase of mechanical properties on a copolyether-ester matrix (COPE) by reactive extrusion with an amine-terminated polyether. These COPE have been widely studied industrially and in the literature because of their excellent flex fatigue resistance and broad temperature usage range. They resist tearing, flex-cut growth, creep, abrasion, and exhibit a very good toughness while resisting to hydrocarbons and many other fluids. In a first part we will present the rheological and mechanical results obtained for reactive blends of COPE and polyetheramines of different molar masses and functionalities.

In a second part, the more promising systems regarding their properties were chemically characterized by different specific NMR techniques, MALDI-TOF mass spectrometry and SEC-MALLS.

Materials and blends preparation

Polymer and additives

The Keyflex BT-1055D matrix (COPE) from LG Chem was kindly supplied by Hutchinson Research Center. It is a copolyether-ester thermoplastic elastomer with a Mn = 30000 g.mol -1 determined by size exclusion chromatography (SEC). Its chemical structure was characterized by NMR (Figure S1) and is presented in Figure 1. This polymer is a copolyether-ester composed of semi-crystalline poly(butylene terephthalate) (PBT) and amorphous poly(tetramethylene oxide) (PTMO) blocks. The average number of oxybutylene units in a PTMO block is 11.7 and the molar ratio between the two blocks PBT/PTMO is 7.2:1. COPE presents a melting point of 206 °C. 1. 2). The mean residence time of the polymer in the extruder was measured at around 90s using colored pellets. PBT/T-403 reference blends were mixed with an internal Haake mixer Rheomix OS (Thermo Fisher Scientific) at 220 °C with a rotor speed of 50 rpm for 20 min, time for which we observe a good stabilization of the measured torque.

Methods of characterization

NMR spectroscopy

1D-1 H, 2D COSY, HMBC and HSQC and DOSY spectra were recorded at T = 300 K using standard sequences on a Bruker Avance-III spectrometer equipped with a 5 mm BBFO+ probe operating at 400.1 MHz for 1 H. 1D- [START_REF] Mulkern | Polymer Processing and characterization of reactive polystyrene/hyperbranched polyester blends[END_REF] C NMR spectra were recorded in the same conditions on a Bruker Avance-II spectrometer equipped with a 10 mm 13 C selective probe operating at 100.6 MHz for [START_REF] Mulkern | Polymer Processing and characterization of reactive polystyrene/hyperbranched polyester blends[END_REF] C. The different amine compounds were analyzed in CDCl3 while all the COPE/amine blends were analyzed in a mixture of hexafluoroisopropanol (HFIP)/CDCl3 (80:20 v/v) with typical concentrations of 20 mg.mL -1 for 1D-1 H analysis and 70 mg.mL -1 for 1D- [START_REF] Mulkern | Polymer Processing and characterization of reactive polystyrene/hyperbranched polyester blends[END_REF] C and 2D COSY, HMBC and HSQC analysis. Chemical shifts were referenced to tetramethylsilane (TMS, δ = 0 ppm). For 2D 1 H-DOSY experiments, a double stimulated echo sequence with bipolar gradient pulses (dgste-bp) was employed for selfdiffusion coefficients measurement, and the samples were analyzed in a mixture of HFIP-d2/CDCl3 (80:20 v/v) with a concentration of 20 mg.mL -1 . Gradients were incremented from 0.96 G.cm -1 to 47,19 G.cm -1 in 40 steps using an exponential ramp, diffusion time and gradient duration were adjusted for each. NMR results were processed on Topspin 3.5 software (Bruker).

Size Exclusion Chromatography (SEC) with a conventional calibration or a Multi-Angle Light Scattering detector (MALLS)

Molar For both SEC, with a conventional calibration or MALLS detector, solutions with a concentration of 1 mg.mL -1 were prepared in a mixture of CHCl3/HFIP (98:2 v/v), filtrated with polytetrafluoroethylene (PTFE) 0.45 μm filters and eluted in the columns with CHCl3 at a flow of 1 ml.mn -1 . The positive ions were detected in all cases.

MALDI-TOF mass spectrometry

Rheological characterization

Dynamic frequency tests were conducted on a strain-controlled ARES Rheometer (TA Instruments) using a parallel plate geometry (25 mm diameter, 0.95 mm gap). Cylindrical samples (25 mm diameter, 1 mm thickness) were prepared with a bench type press Polystat 200 T (Servitec) and measurements were performed at 220 °C to determine the complex shear modulus (storage modulus (G') and loss modulus (G'')) and the absolute complex viscosity (|η*|) by varying the frequencies from 100 to 0.01 rad.s -1 .

Mechanical characterization

Uniaxial traction measurements were performed at room temperature on bone shaped H2 tensile samples on a MTS Criterion Model 43 machine (MTS Systems Corporation) equipped with a 500 N force sensor. After being dried 15 h at 80 °C under vacuum, extruded pellets of the different formulations were injected into bone shaped H2 samples on a hydraulic injection molding machine Babyplast 6/10P equipped with a piston diameter of 14 mm and presenting a clamping force of 62 kN. Chamber temperature was 220 °C, mold temperature40 °C, and the injection pressure was adapted to each type of sample in order to provide the optimal filling of the mold. The deformation speed was 2 mm.min -1 for the measurement of Young's modulus, and 30 mm.min -1 for the measurement of yield strength, breaking strength and elongation at break. At least 6 different samples were tested for each formulation, with a standard deviation under 10%.

Results and discussion

The impact of the reaction between mono-, di-and tri-amine and the COPE through reactive extrusion on the rheological behavior and mechanical properties at room temperature was first evaluated and then in a second part related to the specific created structures during the extrusion process. The mechanical properties of these different blends are reported in Table 3. Note that the melting temperature and the melting enthalpy were measured and that no specific change has been observed. Despite the decrease of the molar mass, the addition of M-600 to the COPE led to a conservation of the mechanical properties of the COPE when 2 and 4%wt were added. Above this concentration however, the Young's modulus and the breaking strength underwent a clear decrease due to a drastic chain cleavage.

Rheological and mechanical properties of the modified COPE

The reactive blends of COPE with D-400 exhibit an increase of the Young's modulus of approximately 10% for the smallest D-400 concentration while the COPE + 4.5%wt blend was too brittle to be injected into stable traction samples.

For the reactive blends of COPE with T-403, the values in Table 3 clearly indicate a change in mechanical behavior between the blends obtained with the smallest amounts of added T-403 (1 and 2%wt) and the most concentrated ones (3 to 5%wt, the blend with 10%wt of T-403 was too brittle to form stable traction samples). Indeed the Young's modulus undergoes a 30% increase for the 1%wt T-403 based blend and a 25% increase on the 2%wt T-403 based blend while maintaining a good elongation at break and a similar breaking strength. The introduction of 3%wt or more of T-403 led to a higher Young's modulus but the elongation at break dramatically decreased and the resulting materials were very brittle.

These data shows that the improvement of the Young's modulus was significantly better for the COPE/T-403 systems than for the COPE/M-600 and COPE/D-400 blends. An optimal concentration around 2wt% of T-403 comes out for which the Young's modulus of the COPE matrix is increased while the elongation at break is maintained. Above this concentration, the benefice allowed by the decrease of viscosity of the matrix is counterbalanced by an increase of the chain cleavage phenomenon and the material becomes too brittle. This obvious increase of mechanical properties evolves in the opposite direction with the measured complex viscosities and molar masses. This result indicates a difference between the room temperature behavior and the one observed in the molten state and cannot be due only to the PBT chain cleavage.

COPE/T-403 blends structure

In the molten state, the chain cleavage evidenced by SEC involves a decrease of the complex viscosity because the flow of the polymer is facilitated with smaller chains. The increase of Young's modulus with the addition of T-403 at room temperature was antagonist and indicated that other structures should have been created for specific concentrations. In order to investigate such structures, NMR, SEC-MALLS and MALDI-TOF mass spectrometry analyses were conducted on the COPE/T-403 blends. technique correlates 1 H NMR chemical shifts to self-diffusion coefficients of molecules in solution [START_REF] Johnson | Progress in Nuclear Magnetic Resonance Spectroscopy Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications[END_REF][START_REF] Walderhaug | Progress in Nuclear Magnetic Resonance Spectroscopy Self-diffusion in polymer systems studied by magnetic field-gradient spin-echo[END_REF] and allows performing a selection on compounds according to their size, which is mainly governed by their molar mass. A double stimulated echo sequence with bipolar gradient pulses was used in order to compensate convection and eddy-current effects [START_REF] Jerschow | Journal of Magnetic Resonance Suppression of Convection Artifacts in Stimulated-Echo Diffusion Experiments[END_REF] .

NMR spectroscopy

The resulting 2D DOSY map of the non-reactive solution of neat COPE and 10%wt of T-403 is shown in Figure 6. Diffusion coefficients are determined by using the Stejskal-Tanner equation [START_REF] Stejskal | Journal of Chemical Physics Spin Diffusion Measurements: Spin Echoes in the Presence of a Time-Dependent Field Gradient[END_REF][START_REF] Sinnaeve | Concepts in Magnetic Resonance Part A The Stejskal-Tanner Equation Generalized for Any Gradient Shape-An Overview of Most Pulse Sequences Measuring Free Diffusion[END_REF] :

𝐼 = 𝐼 0 • 𝑒𝑥𝑝(-𝐷(𝛾𝛿𝑔) 2 • (𝛥 - 𝛿 3 - 𝜏 2
)

)
Where I is the attenuated intensity of a signal at a define frequency, I0 is the reference intensity of the same signal with 0% gradient strength, D is the diffusion coefficient, γ is the gyromagnetic constant of the hydrogen nucleus, δ is the gradient pulse length, g is the gradient strength, Δ is the diffusion delay and τ is the time between bipolar gradients. However, those analysis were unable to bring any information on the number of T-403 amino groups that reacted, and thus on the actual architecture of the formed structures.

SEC-MALLS analysis

To We observe that for COPE and COPE + 8%wt M-600 samples a similar plot shape with a linear curve, typical of linear polymer structures, was obtained. However, a non-linear shape was observed for the COPE + 3%wt T-403 sample. This kind of plot was already witnessed in the literature and shows that linear chains are not the only structures present in this sample.

Different explanations have been advanced to explain this phenomenon: presence of microgels [START_REF] Gores | Abstracts of Papers of the American Chemical Society Copolymer Characterization using conventional SEC and molar mass sensitive detectors[END_REF] , adsorption on the column, diffusion coefficients of the larger molecules too high to give a correct measure [START_REF] Frater | Journal of Polymer Science Part B-Polymer Physics Synthesis and dilute solution properties of divinylbenzene-linked polystyrene stars with mixed arm lengths: Evidence for coupled stars[END_REF] , or an entanglement of branched molecules in the column [START_REF] Frater | Journal of Polymer Science Part B-Polymer Physics Synthesis and dilute solution properties of divinylbenzene-linked polystyrene stars with mixed arm lengths: Evidence for coupled stars[END_REF][START_REF] Podzimek | Journal of Applied Polymer Science Characterization of branched polymers by size exclusion chromatography coupled with multiangle light scattering detector. I. Size exclusion chromatography elution behavior of branched polymers[END_REF][START_REF] Gerle | Macromolecules Main Chain Conformation and Anomalous Elution Behavior of Cylindrical Brushes As Revealed by GPC/MALLS, Light Scattering[END_REF][START_REF] Percec | Journal of the American Chemical Society Visualizable cylindrical macromolecules with controlled stiffness from backbones containing libraries of self-assembling dendritic side groups[END_REF] . The last hypothesis, known as the "Argentinean bolas effect", is the one that seems to correspond to our system. The entanglement of one chain from a star or double star-shaped molecule in the column packing would lead to a delay in the elution time of the polymer molecules presenting this kind of structure. So, after the normal decrease of the molar mass or RMS radius with increasing elution time, both curves curved up. This effect is more pronounced in the RMS radius versus elution time plot and results in a curvature of RMS radius versus molar mass as seen in Figure 11.

However, if this typical curvature was observed many times, the explanation of this phenomenon is not fully understood and is still debated. Moreover, it was mainly witnessed on synthesized and well-defined branched polymers with high molar masses. Here, during the reactive extrusion process, the reaction of the amine on the copolyester blocks led to the formation of different polymer populations in the final material. Therefore, even if precautions must be taken on the interpretation of the SEC-MALLS measurements, they highlight once again structural differences between COPE/T-403 blends and linear COPE or COPE/M-600 blends.

MALDI-TOF mass spectrometry

In 4, and one of the detected molecules is presented as an example in Figure 12. The mass detection limit of the MALDI-TOF mass spectrometry technique did not allow us to observe hypothetical star-shape polymers with molar mass of several thousand g.mol -1 .

However, the observation of such small star-shaped molecules is once again a solid contribution to the hypothesis that larger star-shaped structures are formed during the reactive extrusion of the COPE with T-403, leading to both a decrease of the viscosity and an enhancement of the mechanical properties. All these data are also in good agreement with our previous study dedicated to a polyamide-6 structuration induced by its chemical reaction with a polyether triamine in the molten state [START_REF] Auclerc | Polymer Polyamide-6 structuration induced by a chemical reaction with a polyether triamine in the molten state[END_REF] .

Conclusion

An increase of the Young's modulus up to 30% coupled with a decrease of the viscosity (from Such results are very promising as it demonstrates that with a single reactive extrusion step, by a judicious selection of the triamine structure additive, a copolyether-ester based material presenting an enhancement of the Young's modulus and flowing properties can be developed.

Figure 1 :

 1 Figure 1: Chemical structure of the COPE matrix (x=0.9, y=0.1, n=9.7)

Table 1 : 2 . 2 Figure 2 .

 1222 Chemical structures and characteristics of the different amine compounds Values calculated from 1 H NMR spectra recorded in CDCl3 at room temperature Preparation of the reactive blends COPE pellets were dried for 15h at 80 °C under vacuum in order to remove residual water. The different formulations were prepared by melt blending in a co-rotating twin-screw extruder (Leistriz LSM model, diameter 34 mm, L/D 34) at 220 °C. The twin-screw profile is presented in It particularly contains two reverse conveying elements which promote an important shearing during the extrusion process.

Figure 2 :

 2 Figure 2: Twin-screw profile with two left-handed conveying elements

  masses of the different formulations were determined by SEC on a device composed of one PLgel 20 μm Guard 50x7.5 mm pre-column (Agilent), two PLgel 5 μm Mixed-C 300x7.5 mm columns (polystyrene/divinylbenzene) (Agilent), a Perkin Elmer Series 200 pump injector, a LC-20AD pump (Shimadzu) and a RID-10A differential refractive index detector (Shimadzu). Polystyrene conventional calibration was used to determine the average molar masses of the compounds.SEC was also coupled with a multi-angle light scattering (MALLS) detector (TREOS 3 angles, Wyatt) and a refractometer RID 10A (Shimadzu) to determine the average molar masses. Differential index of refraction dn/dc of 0.13 ml.g -1 used in the data processing was measured with the Wyatt Optilab T-rex (λ = 658 nm) refractometer.

T

  -403 and COPE + 10%wt T-403 samples were solubilized in CHCl3 at 10 g.L -1 . The dithranol matrix was also solubilized in CHCl3 at 10 g.L -1 . A volume of 9 μL matrix solution was then mixed with 1 μL of sample solution. An aliquot of 1 μL of each resulting solution was spotted onto the MALDI sample plate and air-dried at room temperature. All mass spectra were acquired with a Voyager-DE Pro (AB Sciex, Framingham, MA) with a final tension of 20 kV, operating in the linear or reflectron mode. An external mass calibration of mass analyzer was used (mixture of peptides from Sequazyme TM standards kit, AB Sciex).

First,

  Figure 3a-c shows the variation of the absolute complex viscosities versus frequency in the molten state (T=220°C) for different blends obtained by reaction between the COPE with M-600, D-400 and T-403 respectively.

Figure 3 :

 3 Figure 3: Variation of the absolute complex viscosity versus frequency (T=220°C) a) COPE/M-600 blends, b) COPE/D-400 blends, c) COPE/T-403 blends

Figure 4 :

 4 Figure 4: Variation of the average molar masses versus amine concentration for COPE/M-600, COPE/D-400 and COPE/T-403 blends

13 C

 13 NMR spectra of concentrated COPE/T-403 blend (10%wt T-403) is shown in Figure 5 and compared to the one of the neat COPE. It appeared clearly that the signal of the carboxylic acid end-groups of the COPE at 171.1 ppm was no more present in the reactive blend while new signals were detected in the carbonyl area at 178.2 ppm and in the aromatic area at 141.8, 133.7, 131.5 and 130.3 ppm. Those signals were attributed to amide bonds 19 coming from a reaction of the amines with the COPE, either by transamidification of the ester groups or by condensation of the amines on the carboxylic acid end-groups of the COPE. 2D-HMBC experiments correlate our interpretation (spectra shown in Figure S1).

Figure 5 :

 5 Figure 5: 13 C NMR Spectra [115-185 ppm] (a) COPE (b) COPE + 10%wt T-403 in HFIP/CDCl3 (80:20 v/v)

Figure 6 :

 6 Figure 6: 2D DOSY spectrum of non-reacted COPE + 10%wt T-403solution in HFIP-d2/CDCl3 (80:20 v/v).

For a single component

  in a diluted solution, when plotting for each characteristic signal the logarithm of the normalized signal attenuation (ln(I/I0)) versus the square root of the gradient strength, a straight line which slope is proportional to (-D) should be obtained. To plot those curves for the non-reacted blend, we selected only the resonances of both COPE and T-403 that were not overlapping to follow the signal attenuation: signals at 1.27, 1.38, and 1.44 ppm were integrated together to characterize the triamine T-403 and the signal at 2.04 ppm was chosen for the COPE. The resulting curves are presented in Figure 7.

Figure 7 :

 7 Figure 7: Logarithm of the normalized signal attenuation as a function of the gradient strength for the signals of interest of the non-reacted COPE + 10%wt T-403 solution

Figure 8 :

 8 Figure 8: 2D DOSY spectrum of reacted COPE + 10%wt T-403 blend in HFIP-d 2 /CDCl3 (80:20 v/v).

Figure 9 :

 9 Figure 9: Logarithm of normalized signal attenuation as a function of the gradient strength for the signals of T-403 in the reacted and non-reacted COPE + 10%wt T-403 blends

Figure 10 :

 10 Figure 10: Transamidification/condensation reactions between COPE and T-403 leading to star-shaped structures These 2D DOSY experiments allowed us to prove the covalent bond between T-403 and COPE and 13 C NMR showed that the reaction involved is the formation of amide functions.

  go further on the structure analysis of the COPE/T-403 reactive blends, 3 different samples were analyzed by SEC-MALLS: neat COPE, COPE + 3%wt T-403 blend, and COPE + 8%wt M-600. SEC analysis of the COPE + M-600 blends showed that this additive causes a chain cleavage of the COPE, but its linear structure prevents any eventual branching to occur. COPE + 3%wt T-403 and COPE + 8%wt M-600 blends were specifically chosen because they present molar masses of the same orders, meaning that a chain cleavage of the same importance took place during the extrusion process for the two blends. Plots of the root mean square (RMS) radius versus the molar mass for these 3 samples are presented in Figure 11.

Figure 11 :

 11 Figure 11: SEC-MALLS analysis of the COPE, the COPE + 8%wt M-600 blend and the COPE + 10%wt T-403 blend

  order to bring last complementary information about the structures created during the reaction between the COPE and the T-403, the soluble part of the COPE + 10%wt T-403 blend obtained through a Soxlhet extraction was analyzed by MALDI-TOF mass spectrometry. The extractions were performed with refluxing ethanol. Drying of the extracted fraction was performed first on a rotary evaporator RC600 (KNF), and the last traces of solvent were removed from the samples with a Schlenk line. A wide variety of peaks was detected. Especially, different compounds resulting from the reaction of the three branches of the T-403 with PBT monomers or terephthalic acid were highlighted. The list of these compounds is presented in Table

Figure 12 :

 12 Figure 12: Structure of a T-403 macromolecule (n = 6) grafted by two PBT units and one terephthalic acid

300

  Pa.s to 20 Pa.s) of the copolyether-ester COPE matrix was achieved through reactive extrusion with 2%wt of Jeffamine T-403. The unique character of the T-403 on the properties of the COPE was highlighted by a comparative study with amine compounds having similar chemical functions but presenting different functionality and molar mass. Similar decrease of viscosity was obtained but no enhancement of the mechanical properties was witnessed for additives of the same molar mass with weaker functionality. Even for additives with higher molar mass, no remarkable effect neither on mechanical properties nor complex viscosity of the COPE were observed. Different mechanisms occurring during the reactive extrusion have been studied. A chain scission caused by the reaction of the amino groups on the ester groups of the COPE was observed by SEC measurements. In the same time, grafting of the T-403 on the COPE was proved by 2D DOSY NMR analysis of the COPE/T-403 blends. MALLS and MALDI-TOF analysis of the blends as well as a comparison with COPE/monoamine and COPE/diamine blends allowed us to advance the hypothesis that this increase of mainly the Young's modulus properties coupled with an enhancement of the processability of the COPE is due to the creation of star-shaped structures with a T-403 core. Such structures will facilitate the flowability of the polymer in the molten state by avoiding chain entanglement while reinforce it in the solid state at room temperature. Such structures are not created with others mono or diamine and are in good agreement with behavior encountered in the literature for hyperbranched polymers.

Table 2 :

 2 Molar concentration of reactive functions for the different COPE/Jeffamine blends (mol.kg -1 of COPE)

	Reactive blend	Additive composition (%wt) [COOH] COPE	[NH2] added	[NH2]/[COOH]
	COPE	0	0.033	0	0
		2	0.033	0.035	1.06
		4	0.032	0.070	2.19
	COPE + M-600
		6	0.031	0.105	3.39
		8	0.031	0.140	4.52
		1.5	0.033	0.081	2.45
	COPE + D-400	3	0.032	0.162	5.06
		4.5	0.032	0.243	7.59
		1	0.033	0.070	2.12
		2	0.033	0.140	4.24
	COPE + T-403	3	0.032	0.210	6.56
		5	0.032	0.350	10.94
		10	0.030	0.699	23.30

Table 3 :

 3 Mechanical properties of the COPE/M-600, COPE/D-400 and COPE/T-403 reactive blends

	Added Jeffamine (%wt)	Young's modulus (MPa)	Yield strength (MPa)	Breaking strength (MPa)	Elongation at break (%)
	COPE		140 ± 4	16 ± 1	35 ± 2	800 ± 20
		2	150 ± 6	16 ± 1	33 ± 1	780 ± 10
	M-600	4 6	140 ± 3 125 ± 3	15 ± 1 14 ± 1	29 ± 1 26 ± 1	850 ± 50 890 ± 60
		8	115 ± 3	13 ± 1	23 ± 2	860 ± 80
	D-400	1.5 3	155 ± 4 155 ± 2	16 ± 1 16 ± 1	31 ± 2 25 ± 3	810 ± 60 780 ± 100
		1	185 ± 5	17 ± 1	20 ± 3	590 ± 100
	T-403	2 3	175 ± 2 200 ± 4	18 ± 1 -	24 ± 3 14 ± 2	760 ± 100 14 ± 4
		5	195 ± 8	-	10 ± 1	7 ± 1

Table 4 :

 4 PBT/Terephthalic acid grafted T-403 compounds detected in MALDI-TOF mass spectrometry in

	reflectron mode (dithranol matrix)
	n (T

-403) Number of grafted PBT monomer unit Number of grafted terephthalic acid Chemical formula [M+H] + Monoisotopic mass (m/z) Observed [M+H] + (m/z)

			1	C56H82N3O17	1067.5	1068.5
		3	0	C60H90N3O18	1139.6	1140.6
		1	2	C43H56N3O13	821.4	822.4
	3	2	1	C47H64N3O14	893.4	894.4
		3	0	C51H72N3O15	965.5	966.5
		1	2	C46H62N3O14	879.4	880.4
	4	2	1	C50H70N3O15	951.5	952.5
		3	0	C54H78N3O16	1023.5	1024.5
		1	2	C49H68N3O15	937.5	938.5
	5	2	1	C53H76N3O16	1009.5	1010.5
		3	0	C57H84N3O17	1081.6	1082.6
	6	1	2	C52H74N3O16	995.5	996.5

Acknowledgments

The authors are grateful to the joint laboratory IMP/ HUTCHINSON for the financial support.

The authors thank Flavien Mélis for his help for the extrusion and injection of the different blends, Catherine Ladavière for her help in MALDI-TOF mass spectrometry and her involvement in the interpretation of our results. This work has also benefited from the facilities and expertise of the Liquid Chromatography Platform (Institut de Chimie de Lyon) for the characterization of polymers.

Supporting Information.