Eric Dumont

Charlotte Picard

Maxime Guillerm

Emanuel Granero Fernandez

Christophe Stavrakakis

Edith Norrant

Catherine Juery

Nicolas Lesage

Franck Rouxel

Beatrice Balannec

Abdeltif Amrane
email: abdeltif.amrane@univ-rennes1.fr

Annabelle Couvert

Separation of silicone oil droplets dispersed in activated sludge

Keywords: Activated sludge, Emulsion, Liquid/liquid Separation, Silicone oil, Toluene

A process in three steps involving a gas-liquid contactor with an organic phase, a TPPB and a liquid/liquid separator to recover the organic phase for its recycling was proposed to treat hydrophobic Volatile Organic Compounds. Among the processes tested for liquid/liq uid separation (settling, hydrocyclone and centrifugation), the latter appeared to be the most efficient. The centrifugal acceleration needed for the separation of oil and water was found to be dependent on the viscosity of the oil used and the presence of microorganisms. The force required for phases' separation was found to be reduced in the presence of selected commercia l demulsifiers.

Introduction

Atmospheric pollution is one of the major issues in terms of environment. Several conferences (New York, 1997; Rio 1992; Paris Climate Conference 2015) highlighted the importance of reducing the emission of Volatile Organic Compounds (VOCs). It is possible to classify the processes of purification of gas loaded with VOCs in three groups: thermal treatment (incineration and condensation), mass transfer processes with or without chemical reactions (adsorption, absorption, membranes) and finally biological systems (biofilters, biotrick ling filters and bioscrubbers) [START_REF] Cloirec | Les composés organiques volatils dans l'environnement[END_REF]. Biological treatments are interesting to eliminate non recoverable compounds at low cost, but they are not applicable for all VOCs. These latter have to be available in the aqueous phase, biodegradable and not toxic towards biomass. Then, the performances of conventional bioprocesses are restricted to some families of compounds, and do not match with hydrophobic VOCs.

A c c e p t e d m a n u s c r i p t

Then, the addition of an organic phase, immiscible with the aqueous phase, should allow the absorption of high amounts of VOCs [START_REF] Cesário | Feasibility of using water-immisc ib le organic solvents in biological waste-gas treatment[END_REF]. VOCs dissolved in the organic phase are in equilibrium with the aqueous phase owing to mass transfer from the organic to the aqueous phase. The concentration of VOCs remains low owing to their hydrophobicity, and hence non toxic for biomass. The organic phase acts therefore as a reservoir of pollutants [START_REF] Collins | Biodegradation of phenol at high initia l concentrations in two-phase partitioning batch and fed-batch bioreactors[END_REF][START_REF] Collins | Simultaneous biodegradation of benzene, toluene, and p-xylene in a two-phase partitioning bioreactor: concept demonstration and practical application[END_REF][START_REF] Hori | Drastic change in cell surface hydrophobicity of a new bacterial strain, Pseudomonas sp. TIS1-127, induced by growth temperature and its effects on the tolueneconversion rate[END_REF]. Such multiphase reactor is named Two Phase Partitioning Bioreactor (TPPB) [START_REF] Daugulis | Two-phase partitioning bioreactors: a new technology platform for destroying xenobiotics[END_REF]. To date, silico ne oils have been identified as the best organic solvents for the biological treatment of hydrophobic VOC [START_REF] Darracq | Silicone oil: An effective absorbent for the removal of hydrophobic volatile organic compounds[END_REF]. To treat such compounds, a process in three steps is proposed (Figure 1): the first one is a gas / liquid contactor dedicated to the transfer of the VOC from the gaseous phase to the organic phase; the second step is a TPPB dedicated to the degradation of the VOC contained in the organic phase (either transferred in the aqueous phase, or at the liquid / liquid interface ); and the last step is a liquid / liquid separator aiming at recovering the organic phase and reusing it at the inlet of the whole process [START_REF] Darracq | Silicone oil: An effective absorbent for the removal of hydrophobic volatile organic compounds[END_REF][START_REF] Arriaga | Gaseous hexane biodegradation by Fusarium solani in packed-bed and stirred tank bioreactors with two liquid phases[END_REF]. Taken into account the cost of the silicone oil [START_REF] Dumont | VOC absorption in a countercurrent packed-bed column using A c c e p t e d m a n u s c r i p t water/silicone oil mixtures: Influence of silicone oil volume fraction[END_REF], the separation step between oil and water is of main importance for the viability of the process at industrial scale. The aqueous phase containing the biomass is partially recycled in the TPPB.

In the TPPB, the organic phase is dispersed in the aqueous phase into droplets which are stabilized by the biomass itself or by surfactants produced by the biomass. Indeed, biomass is able to produce high and low molecular mass surfactants which can be peptides, saccharides or lipids or their combination. Biosurfactants can be divided into low molecular mass molecules, which efficiently decrease surface and interfacial tensions, and high molecular mass polymers, which tightly bind surfaces. They are produced by a wide variety of diverse microorganis ms [START_REF] Rosenberg | High-and low-molecular-mass microbial surfactants[END_REF]. Moreover, the amount and the type of biosurfactants produced depend on the carbon and the nitrogen sources used by the biomass (i.e. the C/N ratio), the temperature, the aeration and the pH [START_REF] Desai | Microbial production of surfactants and their commercia l potential[END_REF][START_REF] Martínez-Checa | Yield production, chemical composition, and functional properties of emulsifier H28 synthesized by Halomonas eurihalina strain H-28 in media containing various hydrocarbons[END_REF][START_REF] Toledo | Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture[END_REF]. Some bacterial cells are able to stabilize oil-in-water emulsions themselves and therefore behave as fine solid particles at the interface [START_REF] Hori | Drastic change in cell surface hydrophobicity of a new bacterial strain, Pseudomonas sp. TIS1-127, induced by growth temperature and its effects on the tolueneconversion rate[END_REF][START_REF] Dorobantu | Stabilization of oil-water emulsions by hydrophobic bacteria[END_REF]. Moreover, Marin et al. [START_REF] Marin | Emulsifier production and microscopica l study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5[END_REF] A c c e p t e d m a n u s c r i p t observed bacteria clustering around oil droplets and eventually embedded in a polymer sheet forming then a complex network.

According to the process presented in Figure 1, the oil is recycled. Then, a coalescence of the oil droplets after the TPPB is necessary in order to separate the oil phase from the aqueous phase. This liquid / liquid separation can be performed according to various processes.

Evaporation (or vacuum evaporation) consists in an increase of the temperature of the emuls io n leading to the break of the emulsion, and then water vaporization. This process allows producing an aqueous effluent of excellent quality, leaving low water content in the final oily phase [START_REF] Gutiérrez | The effect of additives on the treatment of oil-in-water emulsions by vacuum evaporation[END_REF][START_REF] Gutiérrez | Treatment of a waste oilin-water emulsion from a copper-rolling process by ultrafiltration and vacuum evaporation[END_REF]. However, in the present case, the biomass present in the aqueous phase most likely stays in the organic phase (at the interface), disturbing the recirculation of the phases by fouling the gas/liquid contactor. Moreover, this process presents a high energy consumptio n compare to other techniques. Membrane technology can be a cheaper alternative method for separating emulsions. Examples of separation have been reported in the literatur e : pervaporation, reverse osmosis, ultrafiltration and microfiltration [START_REF] Scott | Application of crossflow microfiltration to emulsion separation in extraction processes[END_REF][START_REF] Koĺtuniewicz | Process factors during removal of oil-in-water emulsions with cross-flow microfiltration[END_REF][START_REF] Tirmizi | Demulsification of water/oil/so lid emulsions hollow-fiber membranes[END_REF][START_REF] Sirkar | Membrane Separation Technologies: Current Developments[END_REF]. The continuo us phase is driven through the membrane by the application of a transmembrane pressure and the dispersed phase is retained at the surface of the membrane. But the quality of the flux declines with time, which could be attributed to (i) surfactant adsorption on the pore walls; (ii) the presence of a polarized layer of concentrated emulsion at the membrane surface and (iii) pore plugging by oil droplets [START_REF] Lee | Concentration polarization, membrane fouling and cleaning in ultrafiltration of soluble oil[END_REF][START_REF] Lipp | A fundamental study of the ultrafiltration of oil-water emulsions[END_REF]. The separation of oil in water emulsion would involve the utilization of a hydrophilic membrane. However, as the biomass produces surfactants and bacteria are amphiphilic, a decrease of the hydrophobicity of the membrane by adsorption might be achieved. Moreover, the main drawback of this technology is that the membrane may retain the oil and the biomass [START_REF] Albasi | Filtration of biological sludge by immersed hollow-fiber membranes: influence of initial permeability choice of operating conditions[END_REF], while the objective is that the biomass cross the membrane with the aqueous phase.

A c c e p t e d m a n u s c r i p t

In the frequent case where the two phases present a difference of electrical conductivity, it is possible to separate them by application of an electric field. This electrochemical destabiliza tio n (electrocoagulation) does not need any chemical additive. When direct current is applied to water through a pair of metallic electrodes, the anode is dissolved to produce metal ions. Then, the electro-generated cations neutralize the surface charge on the surfactant molecules around the oil droplets, and as a result, the emulsion is destabilized and the colloidal oil particles begin to coalesce [START_REF] Yang | Electrochemical coagulation for oily water demulsification[END_REF][START_REF] Tir | Optimization of oil removal from oily wastewater by electrocoagulation using response surface method[END_REF][START_REF] Cañizares | Coagulation and electrocoagulation of oil-in-water emulsions[END_REF]. However, this process consumes lots of energy and is not well adapted to high volume.

Otherwise, liquid separation can be based on the difference of densities between phases using gravitational acceleration (decantation) or centrifugal acceleration. Settlers are known for their relative ease of design and versatility, but they require a large floor-space and often decantation is a long process. Usually, the demulsification time of surfactant-stabilized emulsions by decantation may be of order of magnitude larger than their residence time in the separator.

There is therefore a high interest in increasing the gravitational force by centrifugatio n.

Centrifugation is a compact process requiring lower time of operation compared to settling. The tangential force acting on the droplets can be hundreds of times higher than the normal gravitational acceleration of decantation. The collisions between the droplets are expected to be more efficient and to cause an increase of coalescence [START_REF] Cambiella | Centrifugal Separation Efficie nc y in the Treatment of Waste Emulsified Oils[END_REF][START_REF] Krebs | Separation kinetics of an oil-in-water emulsion under enhanced gravity[END_REF]. Although centrifuga tio n technology is an attractive method, it requires nonetheless high energy levels.

Liquid separation can also be performed using hydrocyclones. This technology is based on the energy of the fluid in order to generate a vortex effect. The oil droplets move toward the wall or the center of the vortex depending on their density, and they undergo a centrifuga l acceleration increasing coalescence [START_REF] Wolbert | Efficiency estimation of liquidliquid Hydrocyclones using trajectory analysis[END_REF].

Finally, it is possible to separate the phases using chemical agents (salts and polymers) called demulsifiers. The efficiency of the salts is linked to the valence of its cation [START_REF] Cañizares | Coagulation and electrocoagulation of oil-in-water emulsions[END_REF]. Moreover, A c c e p t e d m a n u s c r i p t their presence influences the zeta potential of the droplets and the breakup of the emulsion is due to the reduction of the surface charge of the droplets. Polymer demulsifiers can be cationic, ionic or nonionic. These molecules are able to modify the characteristics of the oil-water interface film (the interfacial rheology and the dynamic interfacial tension), allowing the coalescence of the droplets [START_REF] Kim | A study of dynamic interfacial mechanis ms for demulsification of water-in-oil emulsions[END_REF][START_REF] Sun | Influence of demulsifiers of different structures on interfacial dilational properties of an oil-water interface containing surface-active fractions from crude oil[END_REF][START_REF] Kang | Flocculation, coalescence and migration of dispersed phase droplets and oil-water separation in heavy oil emulsion[END_REF]. Besides, the demulsifier concentration influences the separation efficiency. Pincus et al. [START_REF] Pincus | Second Conference on Clinical ACTH[END_REF] suggested that increasing the concentration improves the emulsion separation and Wang et al. [START_REF] Wang | A study of interfacial dilatio na l properties of two different structure demulsifiers at oil-water interfaces[END_REF] showed the existence of an optimum value which is specific of each solution of droplets and polymers. Furthermore, Krawczyk et al. [START_REF] Krawczyk | Chemical demulsification of petroleum emulsions using oil-soluble demulsifiers[END_REF] showed that the partitioning of the demulsifier between the oil phase and the aqueous phase influe nce the efficiency of the separation. An equal partition of the demulsifier between phases seems to improve the efficiency. Moreover, the molecular size of demulsifiers is a factor influenc ing their efficiency [START_REF] Sun | Influence of demulsifiers of different structures on interfacial dilational properties of an oil-water interface containing surface-active fractions from crude oil[END_REF]. This is why, usually, commercial demulsifiers are blended mixtures of several components with various chemical structures, polymeric materials and weight distribution. As a result, their efficiency can only be measured carrying out experiments on a given emulsion.

The goal of this work was to determine the most appropriate liquid / liquid separator after stabilization of the oil droplets in the TPPB. As the most appropriate process to separate the phase uses gravity or an increase of the gravitational force, 3 technologies were tested for separation, i.e. settling, centrifugation and hydrocyclone. Addition of chemical demulsifie rs was also tested in order to determine their influence on the droplet coalescence. For each experiment, the oil in water dispersion was characterized in order to evaluate the impact of physico-chemical modifications of the dispersion.

A c c e p t e d m a n u s c r i p t 2 Experimental

Dispersion formation

The dispersion of oil in water was performed in Erlenmeyers. Silicone oils (Rhodorsil oils from Bluestar Silicones Company), whose characteristics are presented in Table 1, have been used. 100 mL of oil was introduced in a 1 L Erlenmeyer. Then, 300 mL of water containing 0.5 g L - 1 of activated sludge from the wastewater treatment plant of Rennes (France) and minera ls (Tables 2 and3) was added. The Erlenmeyers were closed with a rubber top or a cellulosic top before introducing toluene through a derivation. Erlenmeyers were then incubated at 25°C under agitation (300 rpm) for 8 days. Toluene (C7H8, Fisher Chemicals, 99%) was chosen as carbon source for biomass development because it is a highly hydrophobic and commonly used in TPPB studies as target VOC. The biomass activity was then quantified by the change in the toluene concentration in the gas phase. Toluene concentration was measured using a gas chromatography (Thermo Scientific, Focus GC, FID detector) by daily sampling of the gas phase in the Erlenmeyers. Figure 2 shows the toluene consumption by activated sludge. Based on the control, losses of about 20% could be highlighted. On day 3, 500 µL of toluene were added in order to feed the biomass with carbon and so to let time to dispersion formatio n.

According to Figure 2, the blank solution shows that the leaks of toluene are clearly smaller than its consumption confirming the use of toluene as a carbon source.

Assessment of the separation quality

As the aim of this work was to determine the most appropriate liquid / liquid separator of the emulsion, a homemade method to assess the efficiency of the separation was developed. The separation quality was evaluated using a scale developed as described in Table 4. This scale allowed to rate each parameter characterizing the separation efficiency by attributing a note from 1 to 5. The score of each parameter was weighted to give an average score and quantify A c c e p t e d m a n u s c r i p t the separation efficiency (Table 4). The higher the score was, better was the efficiency. The accuracy of the method was evaluated to be equal to ±5%.

Determination of the oil properties

The viscosity was measured using a viscometer (Brookfield, CV-II+, viscometer-Labomat) and the density was quantified using a pycnometer (Brand) and a precision balance (10 -4 g). The interfacial tension existing between oil and water was measured using a force tensiome ter (Kruss K100SF). Two kinds of oils were considered, new oil and used oil, namely oil which has been in contact with biomass several times (at least one time).

Demulsifier addition and assessment

Thirteen commercial demulsifier solutions were tested in order to perform the separation of the emulsions generated in the Erlenmeyers. For confidential reasons, the names of these products are not given, and have been replaced by letters. Each demulsifier was added to the solutio ns (from 0 to 200 mg L -1 ), which were then agitated during 30 minutes. In order to quantify the role of demulsifiers using the method presented earlier, the dispersions formed in the Erlenmeyers were carefully and faithfully split in tubes of 50 mL. Then, the oil and aqueous phases were separated using a centrifuge during 10 min (Sigma Laboratory, 6K15, centrifuga l acceleration from 500 g to 5000 g).

The demulsifier partitioning between oil and water was also evaluated. For this purpose, the dispersion of oil in water was prepared with 25% of oil and 75% of ultra-pure water. Due to the method used for partitioning assessment, experimentations were carried out without biomass in order to avoid bias in measurement. On a first approach, it was assumed that the presence of biomass has no influence in partitioning value. A known amount of demulsifier, corresponding A c c e p t e d m a n u s c r i p t to a given amount of Chemical Oxygen Demand (COD), was added to the dispersions and a dispersion without demulsifier addition was used as a control. The solutions were agitated during one day. Then, the COD was spectrophotometrically measured in the aqueous solutio n using kits (Nanocolor 500D, Marchery Nagel), and the distribution of the demulsifier in each phase was evaluated from the following equations:

𝐶𝑂𝐷 𝐷𝑊 = 𝐶𝑂𝐷 𝑊 𝑉 𝑊 -𝐶𝑂𝐷 𝑂𝑊 𝑉 𝑂 𝑉 𝑊 (1) 𝐶𝑂𝐷 𝐷𝑂 = 𝐶𝑂𝐷 𝐷 𝑉 𝑊 -𝐶𝑂𝐷 𝐷𝑊 𝑉 𝑊 𝑉 𝑂 (2)
where:

CODDW and CODDO are the chemical oxygen demands of the demulsifier dissolved in the water phase and in the oil phase respectively (mg L -1 ).

CODW is the chemical oxygen demand measured in the aqueous phase corresponding to the carbon due to the demulsifier (mg L -1 ).

CODOW is the chemical oxygen demand of the oil dissolved in water (mg L -1 ).

CODD is the total chemical oxygen demand of the demulsifier of the aqueous phase before its contact with the oil (mg L -1 ).

VW and VO are the volumes of the aqueous and oil phases, respectively (L).

The demulsifier biodegradability was evaluated from measurements of the Biological Oxygen Demand after 5 days (BOD5) carried out with Oxitop IS6 (WTW, Alès, France). Each flask used (164 mL) contained 0.034 g L -1 of MgSO4, 7H2O; 0.042 g L -1 CaCl2; 2.3 x 10 -3 g L -1 of FeCl3; 3 x 10 -3 g L -1 of NH4Cl; 0.05 g L -1 of Na2HPO4; 0.02 g L -1 of KH2PO4 and 0.006 g L -1 of dry matter of activated sludge. The sampling volume of the solution introduced in the flask was deduced from the measurement of the COD of the solution. A solution of N-allylthio urea was added as nitrification inhibitor (10 mg L -1 ). Then, the demulsifier was added. Two flasks were used for each demulsifier solution.

A c c e p t e d m a n u s c r i p t

A similar protocol was used for two control flasks containing easily biodegradable compounds (glutamic acid and glucose at 130 mg L -1 each) instead of demulsifiers. A blank test was also performed without the addition of carbon in a volume of 432 mL of saline solution in order to evaluate the endogenous respiration (DBO5 END ; mg L -1 ). The pH was initially adjusted to 7 for each flask. Biodegradability was evaluated by means of the following equation 3:

𝐵𝑖𝑜𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 𝐵𝑂𝐷 5 -𝐵𝑂𝐷 5 𝐸𝑁𝐷 𝐶𝑂𝐷 (3)
Two sets of experiments were performed in order to evaluate the demulsifier toxicity. Glass bottles of 22 mL were filled with 5 mL of aqueous solution (Table 2), 0.5 g L -1 of dry suspended matter and 1.7 mL of oil. Each demulsifier was added at a concentration of 1000 mg L -1 . One bottle was used free from demulsifier and one bottle free from both demulsifier and biomass.

The bottles were closed with PTFE-coated silicone rubber septa (Perkin, Elmer, France) and sealed with aluminum caps. Then 1 µL of liquid toluene was injected into each bottle. The bottles were incubated at 25 °C under agitation (300 rpm) and the toluene concentration in the headspace was periodically analyzed by gas chromatography.

The activated sludge distribution between the aqueous and oil phases was also quantified. Dispersions of oil in water in the presence of the biomass formed according to the protocol described in section 2.1 were used. Demulsifiers were added, and the dispersion was agitated for 1 hour at 300 rpm. Then, the oil and aqueous phases were separated by centrifuga tio n (20000 g, 20 min). The centrifugate of the aqueous phase was at the bottom of the bottle. Some sludge stayed at the oil and water interface were considered to be part of the organic phase. The centrifugate of each phase was rinsed with ultra pure water and centrifuged again. The centrifugate was then dried at 105°C for 10 hours and weighed to obtain the amount of suspended solids.

A c c e p t e d m a n u s c r i p t 3 Results and discussion

Characterization of the oil in water dispersion

The characterization was carried out using the Rhodorsil oil 47v5. The interfacial tension between oil (new and used oil) versus water containing salts was lower than that versus ultrapure water (Figure 3). Indeed, the presence of salts in water is likely to modify the surface and interfacial tension [START_REF] Tsujii | Surface Activity: Principles, Phenomena and Applications[END_REF]. The interfacial tension between new oil and water containing biomass was also lower than that observed in the presence of salts (the interfacial tension between used oil and water containing biomass was not measured). It could be due either to the biomass itself which is able to cling to the interface [START_REF] Marin | Emulsifier production and microscopica l study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5[END_REF] or to biosurfactants produced by the biomass [START_REF] Rosenberg | High-and low-molecular-mass microbial surfactants[END_REF][START_REF] Toledo | Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture[END_REF]. Figure 3 also put in evidence that the use of used oil decreased the interfac ia l tension between oil and water. Even if the interfacial tension between used oil and water containing biomass was not measured, it could be argued that the value should be slightly lower than the value obtained for new oil. This could be due to oil-soluble molecules produced by biomass. For this reason, the oil density and viscosity were measured before and after contact with the biomass. After contact with the biomass, the dynamic viscosity of oil increased from 5 to 6 mPa.s (+20%), showing an apparent effect of the biomass. Conversely, after contact with biomass, the density of used oil appeared not significantly different to that of new oil (910 kg.m - 3 ; Table 1). These results show that the biomass can impact the properties of the oil and increase the stability of the emulsion, but without influencing significantly the industrial cost (due for instance to pumping). An analytical analysis would be interesting to identify the molecules responsible of the increase of the oil viscosity; however such study appears out of the scope of the present study. The effect of the presence of salts and biomass in the aqueous media on the separation by settling and centrifugation was evaluated using the scale evaluation reported in Table 4.

A c c e p t e d m a n u s c r i p t

Separation by settling

In order to test the separation by settling, the dispersion formed was versed in a separator funne l and the settling was operated for ten days. When oil and ultra-pure water are mixed, their separation can easily be performed by settling. However, the presence of salts in water stabilizes the emulsion making the separation difficult [START_REF] Tsujii | Surface Activity: Principles, Phenomena and Applications[END_REF]. An illustration of such separation is given by the picture displayed in Figure 4. A thin (micrometer size) film of oil on the top can be observed, as well as a layer of oil in the water dispersion. After ten days, no more change in the dispersion could be noticed showing the lack of efficiency of settling in our time scale process. Settling was therefore discarded as a possible separation system. As a result, the separation has to be done through physical or chemical process.

Separation by centrifugation

Centrifugation without demulsifier

The effect of the presence of salts in the aqueous media on the separation by centrifugation was evaluated for different acceleration rates (Figure 5). This figure shows that the components of the emulsion can be efficiently separated by centrifugation. However, the presence of microorganisms in the salt solution (after 8 days of agitation) increased the stability of the dispersion and hence decreased the efficiency of the separation. This could be due to the microorganisms themselves or bio-surfactants produced by these latter [START_REF] Schmid | Effects of Biosurfactant and Emulsifica tio n on Two-Liquid Phase Pseudomonas oleovorans Cultures and Cell-Free Emuls io ns Containing n-Decane[END_REF][START_REF] Kang | Hydrophobic bacteria at the hexadecane-water interface: examination of micrometre-scale interfacial properties[END_REF][START_REF] Li | Interaction between Emuls io n Droplets and Escherichia coli Cells[END_REF].

The literature reports that the diameter of the oil droplets generated during emulsion agitatio n decreased when the agitation speed increased [START_REF] Franco | Influence of Processing Variables on the Rheological and Textural Properties of Lupin Protein-Stabilized Emulsions[END_REF][START_REF] Aronson | Highly Concentrated Water-in-Oil Emulsio ns : Influence of Electrolyte on Their Properties and Stability[END_REF]. In order to test the centrifuga tio n efficiency, oil in water dispersions (Rhodorsil oil 47v5) were prepared at three agitation speeds before separation (i.e. 200, 300 and 550 rpm). The centrifugal acceleration necessary to separate the phases are given in Table 5. Below the given range of values, the phases are not separated, while over the higher value a clear separation between both phases can be observed. In The effect of oil viscosity on the separation efficiency was investigated for the oil in water dispersions formed at 300 rpm using the three silicone oils presented in the Table 1. Table 6 shows that an increase of the viscosity from 5 to 50 mPa s implied an increase of the centrifugation rate needed for an efficient separation. For viscosity higher than 50 mPa s, centrifugation rate has not to be increased.

Centrifugation with demulsifier

The separation efficiency of centrifugation in the presence of a chemical demulsifier in the emulsion has been measured for 13 different demulsifiers. Figure 6 presents an example of the qualitative note of the separation for different concentrations of the demulsifier and for 3 centrifugation rates. According to this figure, the best separation was reached for a demulsifier concentration of 15 mg L -1 . Moreover, from 500 g to 750 g, the separation efficiency increased with the centrifugation rate, but there is no significant difference between 750 g and 1000 g. As a result, and in comparison with the results obtained without demulsifier (Figure 5), it can be concluded that demulsifier addition allows to significantly reduce the centrifugation rate needed for separation. Among the 13 commercial demulsifiers tested, 3 were retained for their efficiency to achieve separation. In order to be used at industrial scale in the presence of microorganisms, the biodegradability, the toxicity and the distribution of these 3 demulsifie rs (symbolized by letters A to C hereinafter) between oil and water phases were tested.

Biodegradability, toxicity and demulsifier distribution

For the three products A, B and C, the demulsifier distribution between the oil and water phases was determined according to the procedure described in the part 2.4. Experimental results show A c c e p t e d m a n u s c r i p t that the 3 selected demulsifiers were mainly found in the aqueous phase, for more than 90 % (Figure 7). Although the names of the demulsifiers cannot be given for confidential reasons, this finding was expected because these products are cationic polymers soluble in water.

The biodegradability of the three demulsifiers was evaluated based on the ratio COD/BOD5. A high ratio is desired, on the one hand, in order to avoid a possible consumption of the demulsifier as carbon source in competition with the VOC, and on the other hand, for economic considerations in view of their recycling into the aqueous phase in the TPPB. The calculated values of the ratio COD/BOD5 were 19, 104 and 2003 for demulsifiers A, B and C respectively.

As ratios are superior to 3, the non-biodegradability of the 3 demulsifiers is proven [START_REF] Esener | The bioenergetic correlation of COD to BOD[END_REF].

In order to assess the toxicity of the three demulsifiers towards microorganisms, the consumption of the toluene by the biomass was evaluated for 2 days. Figure 8 shows the evolution of the toluene concentration in the gas phase in the presence and the absence of demulsifier. A blank (nor microorganisms neither demulsifier) was used to evaluate toluene leaks. For each case studied, duplicate experiments were performed. In the absence of demulsifier and biomass, the toluene concentration decreased from 30 h of culture until reaching 60% of the initial concentration. In the presence of demulsifiers A and B, about 20 h lag phase was observed and toluene was completely removed after 32 h. The demulsifier C showed a different behavior since biodegradation started only after 50 h, and removal yield were in the range 50 to 80% of the initial toluene amount. From this, a clear inhibitory effect was shown for this demulsifier, which is not acceptable for possible implementation in the considered process.

The activated sludge distribution between the aqueous and oil phases was also evaluated in the absence and the presence of the demulsifiers. The objective was to minimize the amount of biomass in the oil phase. The results obtained from duplicate experiments showed that with demulsifier B, the biomass settled more than with demulsifiers A and C. An additiona l A c c e p t e d m a n u s c r i p t experiment was carried out by adding ultrapure water to the oil phase after separation of the phases. The oil and the water were then shaken and the activated sludge in each phase was evaluated again. The results showed that 40% of the sludge present in the oil phase was transferred to the aqueous phase used for washing. This part of transferred sludge represented around 6% of the total sludge initially present in the dispersion. Consequently, and according to this result, at an industrial scale, the addition of a tank for agitation and decantation before the centrifugation would allow to significantly decrease the amount of activated sludge in the oil phase.

Separation by hydrocyclone

A modeling study has been done in order to evaluate the feasibility of using a hydrocyclone to separate the phases. This study is based on the model proposed by Amini et al. [START_REF] Amini | Mathematical modelling of a hydrocyclone for the down-hole oil-water separation (DOWS)[END_REF] involving the Comsol software. The model allows to determine the trajectory of the oil droplets inside the hydrocyclone and the acceleration they undergo. The dimensions of the hydrocylone used for the study (Figure 9) are given in the Table 7. The purpose of this modeling was to determine the acceleration underwent by the fluids inside the hydrocyclone (Figure 10). Nowadays, models are not able to predict the coalescence of oil droplet but can be helpful to determine the trajectories of the oil droplets as a function of their size. The model shows that the hydrocyclo ne cannot allow to achieve a separation between the oil and water phases for droplets with a diameter smaller than 10 µm, because some of them leave the hydrocyclone in the aqueous phase. Over 10 µm diameter, all the oil droplets leave the hydrocyclone in the oil phase.

However, with the increase of the oil droplet diameter, the oil leaves the system without undergoing high acceleration, and as a result, for droplets over 500 µm of diameter, the hydraulic retention time inside the hydrocyclone was too low to achieve signific a nt acceleration. From the emulsions performed according to the procedure described in the part A c c e p t e d m a n u s c r i p t 2.1., the size of the oil droplets and the droplet size distribution were experimentally measured using a Mastersizer (Malvern Instruments 2000). Results are presented at the Figure 11. The average diameter of the oil droplets was found to be 310 µm, while the distribution was found to follow a Gaussian-type. According to the results of the model, it appears that most of the droplets should leave the hydrocyclone without achieving the acceleration needed for coalescence. As a result, hydrocyclone could not be used for the liquid / liquid separation at industrial scale.

Conclusion

In view of the use TPPB for the treatment of gas containing hydrophobic VOC, water and oil phases need to be separated with the aim of oil recycling. It was observed that the stability of the emulsion of oil droplets dispersed in the water phase was due to the presence of salts, as well as biomass, in water. Moreover, this stability increased with the stirring speed of the TPPB as well as with the viscosity of the oil phase. As a result, centrifugation appeared to be the most efficient separation process for the use of TPBB at industrial scale. The centrifugal acceleration needed for the separation of oil and water mixtures was found to be dependent on the viscosity of the oil used, as well as the presence of microorganisms which increased the stability of the emulsion. The addition of demulsifier has also to be considered in order to decrease the force required for the separation of phases. The distribution of the demulsifier between the oil and water phases, the toxicity of the demulsifiers toward biomass and the biodegradability of the demulsifiers allowed selecting 2 demulsifiers which could be subsequently tested at pilot scale for the separation of liquid phases.

A

  c c e p t e d m a n u s c r i p t agreement with the literature data, the value of the centrifugal acceleration necessary for the separation of phases increased with the agitation speed used for dispersion generation.

  Figure captions

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 12345 Figure 1: Process to treat hydrophobic VOC in three steps

Figure 6 :

 6 Figure 6: Example of separation quality according to demulsifier concentrations and centrifuga l

Figure 7 :

 7 Figure 7: Demulsifier distribution between oil and water

Figure 8 :Figure 9 :Figure 10 :

 8910 Figure 8: Demulsifier toxicity assessment: time courses of toluene consumption (the differe nt

Figure 11 :

 11 Figure 11: Oil droplet size distribution.

A

  c c e p t e d m a n u s c r i p t

Table captions Table 1 :

 captions1 Oil properties at 25 °C

Table 2 :

 2 Composition of the aqueous solution

Table 3 :

 3 Composition of the nutritive solution (adapted from[START_REF] Trinci | A Kinetic Study of the Growth of Aspergillus nidulans and Other Fungi[END_REF])

Table 4 :

 4 scale evaluation of the separation of the oil and liquid phase

Table 5 :

 5 Centrifugal acceleration rate versus the agitation rate (Rhodorsil oil 47v5).

Table 6 :

 6 Centrifugal acceleration needed versus oil viscosity

Table 7 :

 7 Dimension of the hydrocyclone used for the model

Acknowledgments

The authors would like to thank the French agency for the Environment and the control of Energy (ADEME) for their financial support through a postdoctoral fellowship for C. Picard. A c c e p t e d m a n u s c r i p t


Gilles Le Moullac

Claude Soyez

Pierre Lyonnard

Sébastien Chabrier

Léo Milhade

Yannick Gueguen

Benoit Beliaeff

Sebastien Chabrier

Leo Milhade

Non-invasive functional exploration techniques for bivalves with applications to pearl oyster Pinctada margaritifera

Keywords: bivalve, filtration, pearl rotation, respiration, valvometry

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

Introduction

The pearl oyster Pinctada margaritifera is a marine bivalve with a distribution range in the intertropical zone of the Pacific and Indian Oceans. This species is exploited, mainly in French Polynesia, for the production of cultured pearls. For many years, this species has been the subject of studies aiming to improve knowledge of its nutritional needs (Le [START_REF] Moullac | Feeding the pearl oyster Pinctada margaritifera during reproductive conditioning[END_REF][START_REF] Vahirua-Lechat | Changes in fatty acid and sterol composition during oogenesis in the pearl oyster Pinctada margaritifera[END_REF], reproduction [START_REF] Chavez-Villalba | Determination of gender in the pearl oyster Pinctada margaritifera[END_REF][START_REF] Teaniniuraitemoana | Molecular signatures discriminating the male and the female sexual pathways in the pearl oyster Pinctada margaritifera[END_REF][START_REF] Teaniniuraitemoana | Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes[END_REF] and mechanisms controlling biomineralization [START_REF] Blay | Cultured pearl surface quality profiling by the shell matrix protein gene expression in the biomineralised pearl sac tissue of Pinctada margaritifera[END_REF][START_REF] Joubert | Temperature and food influence shell growth and mantle gene expression of shell matrix proteins in the pearl oyster Pinctada margaritifera[END_REF][START_REF] Joubert | Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization[END_REF][START_REF] Luyer | Whole transcriptome sequencing and biomineralization gene architecture associated with cultured pearl quality traits in the pearl oyster, Pinctada margaritifera[END_REF]. Beyond the acquisition of purely biological knowledge, P. margaritifera is studied for the improvement of pearl production [START_REF] Gueguen | Characterization of molecular processes involved in the pearl formation in Pinctada margaritifera for a sustainable development of pearl farming industry in French Polynesia[END_REF] and its potential to adapt to environmental challenges [START_REF] Beliaeff | Perliculture en Polynesie francaise -Enjeux economiques, scientifiques et techniques[END_REF]. Ecophysiological approaches make it possible to study and model the behavioural and physiological responses of pearl oysters to their environment over their development and can be based on non-invasive exploration techniques. In contrast to invasive techniques, non-invasive functional approaches can provide information on the general functioning of organisms. For bivalves, there is a fairly wide variety of methods and instrumental means allowing online physiological assessment of intact animals. The current range of non-invasive techniques for bivalves includes systems for measuring metabolic flows (filtration and respiration) and valve activity that can be applied to the bivalve molluscs as long as the animal characteristics (e.g. size) make this technically feasible. In this paper, we present those non-invasive techniques with applications to pearl oyster and list other potentially promising approaches. We also focus on a unique method that we developed specifically for the pearl oyster to record pearl rotation characteristics.

Measuring metabolic fluxes

In living organisms, the measurement of ecophysiological parameters of nutrition and respiration makes it possible to establish an energetic balance of assimilated organic matter. In bivalves, this balance is generally expressed as a growth potential including reproduction, known as 'Scope For Growth, SFG' [START_REF] Bayne | Aspects of reproduction in bivalve molluscs[END_REF]. Based on ecophysiological functions such as filtration, consumption, absorption and respiration, which are known to vary according to the environment, the energy balance provides an 'estimate' of the physiological status of the animal at different periods of its development and according to the environmental parameters. More precisely, the calculation of an organism's energy balance consists in drawing up a balance of energy gains and losses, assuming that the amount of energy acquired through food during assimilation is used for maintenance, growth and reproduction. The energy balance calculation is more accurate when filtration and respiration measurements are carried out on the same animals, in the same enclosure.

Measurements of bivalve ingestion and respiration allow an energy balance to be established, providing indications about the relationship of these animals with their living conditions. In our ecophysiological measurement system (EMS) (Fig. 1a; [START_REF] Chavez-Villalba | Physiological responses of female and male black-lip pearl oysters (Pinctada margaritifera) to different temperatures and concentrations of food[END_REF], energy flows can be measured in an, which is composed of eleven openflow chambers. In our experiments, ten oysters are placed in ten of the chambers and the remaining eleventh chamber is left empty to serve as a control. The drains of the chambers are each equipped with a two-way electromagnetic valve activated by an automaton. When the valve of one measuring chamber is opened, the released water is analysed for 3 min using a 10AU fluorometer (Turner, San Jose, CA, USA) and an oximeter (OXI 358; WTW, Weilheim, Germany) to measure the fluorescence and dissolved oxygen. The measurements of each chamber are carried out by piloting 11 electrovans, which successively remove part of the effluent to the sensors (oximeter and fluorimeter). A National Instrument (NI) Ethernet cFP-2120 network module is controlled by a real-time LabView (NI) driver. The card transmits data and tracking graphs via Ethernet on an interface (Fig. 1b) that provides the values of incoming flows, sampling by successive opening and closing of the electrovans, real-time display of all curves (fluorescence of microalgae, dissolved oxygen and flows), and recording of average data on the computer dedicated to EMS management. Each cycle is completed within 3 min and another cycle then starts in the control chamber and lasts 3 min (sequence: chamber 1, control, chamber 2, control, etc.). The specimens remain in the chambers for 48 h and measurements can be taken every 60 min until 48 fluorescence and oxygen measurements have been recorded. The respiration rate (RR), expressed in mg O2 h1, is estimated by the difference in the oxygen concentration between the control and experimental chambers using the following formula: RR = V (O1 O2), where O1 is the oxygen concentration in the control chamber, O2 the oxygen concentration in the experimental chamber and V the water flow rate. Ten measurement series are commonly performed on groups of four pearl oysters, each composed of two grafted and two non-grafted pearl oysters. The energy expenditure is expressed as RR of individuals (total energy expenditure) and as standardized RR [START_REF] Savina | A comparative ecophysiological study of two infaunal filter-feeding bivalves: Paphia rhomboı¨des and Glycymeris glycymeris[END_REF].

Replicates from EMS thus make it possible to accurately estimate the energy status of the bivalves under various environmental pressures or according to their physiological status and/or genetics. Measurements are taken on individuals who have previously been acclimatized for at least 1 month in a contiguous room with 8500 L tanks also equipped with environmental control systems. This experimental tool allowed us to show that the oxygen requirement of female P. margaritifera is higher than that of males [START_REF] Chavez-Villalba | Physiological responses of female and male black-lip pearl oysters (Pinctada margaritifera) to different temperatures and concentrations of food[END_REF]. The energy balance of the species was also measured at different temperatures to calculate its thermal optimum and to estimate the energy risk of climate warming (Le Moullac et al. 2016a); this also demonstrated that acidification has no significant impact (Le Moullac et al. 2016b). The effect of anthropogenic environmental pressures such as microplastics has been studied, and we have thus been able to determine that the bioenergetic response is dose-dependent [START_REF] Gardon | Microplastics affect energy balance and gametogenesis in the pearl oyster Pinctada margaritifera[END_REF]. Finally, we observed that pearl oysters grafted for the production of cultured pearls did not expend significatively more energy, as estimated by oxygen consumption, than the non-grafted ones. We could therefore inferred that the energy cost associated with pearl formation is negligible (Le Moullac et al. 2018b).

Measuring pearl rotation

Cultured pearls are produced by first making a grafting operation, during which a small piece of mantle tissue from a donor oyster (the graft) is inserted in the gonad of the recipient oyster together with a nacre bead, the nucleus [START_REF] Wada | Formation and quality of pearls[END_REF]. Once positioned in the recipient oyster, the outer epithelium cells of the graft multiply and form a pearl sac around the nucleus. The pearl sac then starts to deposit nacre (aragonite) layers onto the nucleus. This is the starting point of the future pearl. A rearing period of approximately 18 months is then needed to produce a pearl with a sufficiently thick layer of nacre for the market [START_REF] Gueguen | Characterization of molecular processes involved in the pearl formation in Pinctada margaritifera for a sustainable development of pearl farming industry in French Polynesia[END_REF]. A pearl is round for at least two reasons: (i) because the nucleus is spherical and (ii) the growth front of nacre on the surface of the pearls suggests that rotation is taking place. [START_REF] Cartwright | Pearls are selforganized natural ratchets[END_REF] put forward a theory of pearl rotation, explaining how forces acting during the deposition of aragonite tablets would lead to a particular type of pearl movement. The layers of aragonite oriented in specific directions on the surface would give momentum to the pearl during the growth of its layers, thus causing movement. Once activated, a dynamic mechanism would be initiated that could lead to different rotational movements, particularly depending on whether the pearl has a defect. We were finally able to develop an unique device to demonstrate that the pearl rotates during its formation in the pearl sac of the pearl oyster P. margaritifera [START_REF] Gueguen | Yes, it turns: experimental evidence of pearl rotation during its formation[END_REF].

The magnetometer used in this study is made up of three main connected parts. The measuring part is a dome with sensors consisting of a half-sphere of acrylic glass (diameter 20 cm) on which are set 25 magnetic sensors each consisting of two components (Fig. 2), an HMC1021 compass from Honeywell (a one-axis magnetic sensor) and an offset compensation circuit. Twenty-four of the 25 sensors are spread across the convex surface of the dome in three circles of eight elements at 0°, 30° and 60° angles with the base. The last sensor is located at the top of the dome at 90°. Each sensor is glued on the dome with a cyanoacrylate paste and protected from impacts and water by an acrylic glass tube. The electrical part of the magnetometer is composed of a data acquisition board with 26 RJ45 sockets (25 used to connect the cables from the sensors and the last one to make the ethernet connection with the computer) and 25 wiring cables, each of which ends in a RJ45 plug. The human-machine interface (HMI) is a program called 'magneto', developed by VEGA Industrie (Avrainville, France), which is composed of two parts. The first part of the HMI is a microcontroller that uses internal software to collect, process and transfer data to the second. This second part is software on the computer (magneto-magnetometer interface 1.0), which collects data from the microcontroller, allows data to be collected from real-time sensors and produces visualized data, thus tracking the acquisition process. This interface is used to define communication, data acquisition parameters and to make backups. The following parameters were defined for our experiments: acquisition frequency: 50 (1/10 s); filtration rate: 5; and recording periodicity: 1 min. The export data file is a.CSV file, which the software names with references to the date and hour of start: Magneto_YYYY_MM_DD_HH_MM.csv. The data acquired by the magnetometer were processed with an R routine to convert the data acquired into 3D coordinates and perform calculations on pearl movement kinetics. Mean angular speed of rotation (min 1 ) and a graphical representation determining the type of pearl motion could then be derived from the converted data (Fig. 3).

The experiment carried out using this device showed that the pearl actually turns in the pearl sac, with rotation starting as soon as the pearl sac closes [START_REF] Gueguen | Yes, it turns: experimental evidence of pearl rotation during its formation[END_REF].

The pearl sac is the result of the development of cells of the graft that was implanted during the grafting operation, at the same time as the nucleus. The healing of the gonad and development of the pearl sac take time. It is estimated that the pearl sac becomes functionalthat is, the first aragonite deposits are observed between 30 and 45 days after the transplant operation. This was confirmed by rotational recordings using a magnetometer in pearl oysters grafted with magnetized nuclei [START_REF] Gueguen | Yes, it turns: experimental evidence of pearl rotation during its formation[END_REF].

Once the rotation of pearls was demonstrated, the question of most interest to the industry was the link between rotation and formation, and hence quality, of pearls. We first showed that rotation speed differed according to the type of movement: pearls with axial movement had a significantly higher rotation speed than those with random movement (Le Moullac et al. 2018a). When we conducted ecophysiological experiments to test the impact of the environment on pearl rotation and formation, however, we found pearl growth rate to be influenced by temperature but not by pearl rotation speed. On the basis of our results, we now consider pearl rotation to be a more complex process than formerly thought. Mechanisms involved could include strong environmental forcing in immediate proximity to the pearl. Another implication of our findings is that, in the context of ocean warming, pearl growth and quality can be expected to decrease in pearl oysters exposed to temperatures above 30°C (Le Moullac et al. 2018a).

Measuring valve activity

Bivalve valve behaviour is a potentially important tool for biological monitoring of water quality as, through these movements, bivalves can serve as environmental sentinels [START_REF] Borcherding | Ten years of practical experience with the Dreissena-Monitor, a biological early warning system for continuous water quality monitoring[END_REF]). Valve activity is also essential for understanding metabolism optimization strategies. A relatively large number of methods exist for observing and measuring valve movement, as presented below.

A strain gauge can be used to record valve movements of oysters. Composed of electrically resistant wires, it converts the stretching of these wires into variations in electrical resistance. Strain gauges are cemented onto thin, flexible strips of metal that are attached to a nylon line. This line is cemented onto the upper valve of the oyster. A system of this type (BLH Electronics, Waltham, MA, USA) was developed to measure shell-valve gape activity and periodic activity of Crassostrea virginica (Higgins 1980a,b). It was later applied to mussels following their exposure to the toxic dinoflagellate Protogonyaulax tamarensis [START_REF] Shumway | The effects of the toxic dinoflagellate Protogonyaulax tamarensis on the feeding and behaviour of bivalve molluscs[END_REF]. Subsequently, Porter and Breitburg (2016) developed a comparable system (SG13/1000-LY43 or LY41, Omega Engineering Inc., Stanford, CT, USA) directly sealed onto the shells for gape monitoring of C. virginica under diel-cycling hypoxia.

An inductance-based valvometry device was constructed to study the valve rhythm dynamics of Corbicula fluminae when exposed to copper [START_REF] Jou | Sensory determinants of valve rhythm dynamics provide in situ biodetection of copper in aquatic environments[END_REF]. This device was conceived by attaching a pair of lightweight electrical coils to the valves and using them as sensors to determine the degree of bivalve opening. This system makes it possible to record the valve behaviour of several bivalves simultaneously. An interface card (NI DAQPad-6259) controls the acquisition of the corresponding signals, and a specific LabVIEW program monitor compiles data. This way, valve activities of clams were recorded dynamically in real time.

The HFNI valvometer is a high frequency (10 Hz), noninvasive (HFNI) biosensor. The animal is equipped with two light coils (sensors), 53 mg each (unembedded), fixed on the edge of each valve. These coils are coated with a resin-sealing before fixation to the valves. One of the coils emits a high-frequency sinusoidal signal that is received by the other coil. Each animal's behaviour is thus measured every 1.6 s [START_REF] Andrade | High frequency noninvasive (HFNI) bio-sensors as a potential tool for marine monitoring and assessments[END_REF][START_REF] Sow | Water quality assessment by means of HFNI valvometry and high-frequency data modeling[END_REF].

Gaping activity is measured using magnetoresistive sensors (Honeywell). The sensor is formed by four encapsulated reeds activated by a magnet, and the supports glued onto each valve. The magnet is also encapsulated, with the reeds and slides hanging on it attached to two stainless steel rods of 1 mm diameter and 10 cm long [START_REF] Garcia-March | In situ biomonitoring shows seasonal patterns and environmentally mediated gaping activity in the bivalve, Pinna nobilis[END_REF].

In our laboratory, a type of electromagnetic valvometer was tested. This is an automatic and continuous recording device of bivalve valve movements designed according to the Hall Effect Principle (1879), which states that an electric current passing through a material bathed in a magnetic field generates a voltage perpendicular to the latter. The valvometer ('Memocollecteur' developed by Micrel, Hennebont, Morbihan), described by [START_REF] Floch | Un capteur biologique original enregistrant les mouvements valvaires des mollusques bivalves[END_REF], takes the form of cylindrical structure of approximately 300 mm in diameter, with a maximum height of 50 mm and a mass of 2 kg. Between 4 and 8 bivalves are fixed horizontally in the periphery on a PVC crown, while the centre of the device contains an autonomous miniaturized data acquisition microprocessor. The PVC blades come out from a receptor, and reach onto the top valve of the oysters, opposite the hinge. An adjustable vinyl tip adjusts the sensor position when the oyster is closed. When the oyster opens, the tip lifts the PVC blade. The gap between the stem and the central receptor thus increases. An electromagnet located in this receptor transforms this gap into an electrical signal.

When the signal is part of a time series (Fig. 4), spectral analysis can reveal the rhythms of activity. In our study, spectral analysis of the record series of the valved activity revealed a period of 23.25 h and a lower density of 2.15 h (Fig. 5a), followed by a set of repetitive high-frequency and low-amplitude micromovements (Fig. 5b). These micromovements are interesting because they were almost perpetual. These micromovements could be one of the engines 2.0 of filtration, in addition to the ciliary activity of the gills. Correspondingly, we can now hypothesize that these valve micromotions are involved in the rotation of beads in grafted pearl oysters. This hypothesis should be tested by simultaneously measuring the valve activity and the rotation of the bead at the same frequency (per second). In P. margaritifera, the records of pearl oyster valve opening variations over 15 days in the lagoon of Takapoto in 1995 (D. Buestel, unpublished data) revealed a marked diel cycle. Oysters open more, and thus filter more, at night than during the day.

The valvometers put out an electrical signal (mV) that is used in a binary manner (open/closed), together with the opening time [START_REF] Jou | Sensory determinants of valve rhythm dynamics provide in situ biodetection of copper in aquatic environments[END_REF][START_REF] Porter | Eastern oyster, Crassostrea virginica, valve gape behavior under dielcycling hypoxia[END_REF], or transformed into a spread value (mm). The spread value of the opened valves can be compared when the measurements are standardized; in this case, the opening width (mm) is transformed into the degree of aperture considering the valves as two sides of a right-angled triangle. The spread of the opening is then the measure on the opposite side of the angle, transformed into an aperture angle. Indeed, the height, unlike the angle, depends on the position of the needle on the animal and the animal's length. To eliminate this bias, by likening the oyster to a right-angled triangle (Fig. 6), it is then possible to calculate: sin (a) = H/L, and hence, a = arcsin (H/L). In a previous study (Le Moullac 2008), this transformation of valve spread values made it possible to measure responses to prolonged hypoxia in Pacific oyster C. gigas, which induced a reduction in average valve opening.

Non-invasive methods for future applications

Measuring cardiac activity could usefully complement the range of different measures described above. Heart rate was successfully used as a proxy for whole-animal stress [START_REF] Braby | Following the heart: temperature and salinity effects on heart rate in native and invasive species of blue mussels (genus Mytilus)[END_REF], in response to toxins [START_REF] Davenport | A study of the effects of copper applied continuously and discontinuously to specimens of Mytilus edulis (L.) exposed to steady and fluctuating salinity levels[END_REF][START_REF] Depledge | Automated interpulseduration assessment (AIDA): a new technique for detecting disturbances in cardiac activity in selected macroinvertebrates[END_REF][START_REF] Grace | The effects of copper on the heart rate and filtration rate of Mytilus edulis[END_REF][START_REF] Wedderburn | The field application of cellular and physiological biomarkers, in the mussel Mytilus edulis, in conjunction with early life stage bioassays and adult histopathology[END_REF], temperature [START_REF] Coleman | The effect of aerial exposure on the activity of the mussels Mytilus edulis L. and Modiolus modiolus (L.)[END_REF][START_REF] Helm | The effect of exposure on the heart rate of the mussel, Mytilus edulis L[END_REF][START_REF] Pickens | Heart rate of mussels as a function of latitude, intertidal height, and acclimation temperature[END_REF], salinity [START_REF] Nicholson | Ecophysiological aspects of cardiac activity in the subtropical mussel Perna viridis (L.) (Bivalvia: Mytilidae)[END_REF][START_REF] Stickle | Effects of salinity on the respiration and heart rate of the common mussel, Mytilus edulis L., and the black chiton, Katherina tunicata (Wood)[END_REF] and predation [START_REF] Rovero | Cardiac and behavioural responses of mussels to risk of predation by dogwhelks[END_REF]. Two main technical principles are used: impedance and infrared. Impedance, measuring an electrical resistance, can be considered as an invasive technique because it requires drilling into the bivalve shell to insert two small electrodes (copper or platinum) into the pericardial cavity. These electrodes are attached to the shell by a surgical glue or dental cement. This method produces an analogue impedance signal, which is converted to a voltage signal [START_REF] Braby | Following the heart: temperature and salinity effects on heart rate in native and invasive species of blue mussels (genus Mytilus)[END_REF][START_REF] Earll | Temporal variation in the heart activity of Scrobicularia plana (Da Costa) in constant and tidal conditions[END_REF][START_REF] Gainey | Physiological effects of Protogonyaulax tamarensis on cardiac activity in bivalve molluscs[END_REF]. The method of measuring heart rate (HR) by infrared (IR) is more recent and non-invasive because it does not require perforation of the shell. A waterproof photoplethysmograph is glued on the shell in the vicinity of the cardiac region, with optical sensors consisting of an infrared transmitter and a phototransistor. Tests of the transmission of an infrared signal through the pearl oyster shell demonstrated the a limitation of the method, however, because the shells of P. margaritifera individuals greater than 85 mm in height are too thick to allow the IR signal through.

Magnetic resonance imaging (MRI), although not greatly used, can provide a very wide range of information that is currently acquired by bivalve dissection. Application of MRI is possible in marine bivalves, especially oysters, with sufficient spatial resolution to depict anatomy without movement artefacts or magnetic susceptibilities [START_REF] Pouvreau | Investigation of marine bivalve morphology by in vivo MR imaging: first anatomical results of a promising technique[END_REF]). MRI techniques have been used to characterize gonad development and to determine the sex of live Pacific oysters through their shells [START_REF] Davenel | NMR relaxometry as a potential noninvasive routine sensor for characterization of phenotype in Crassostrea gigas[END_REF][START_REF] Davenel | Noninvasive characterization of gonad maturation and determination of the sex of Pacific oysters by MRI[END_REF]. Recent advances in MRI in real time allow us to visualize the movements of organs such as the human heart, tongue and joints [START_REF] Uecker | Real-time MRI at a resolution of 20 ms[END_REF][START_REF] Zhang | Real-time MRI at 20 millisecond resolution[END_REF]) (https://www.independent.co.uk/news/health/realtime-mrilive-video-tongue-jens-frahm-max-planckinstitute-europe an-patent-office-a8327001.html). Applying such technology to the pearl oyster would allow the observation of pearl formation and improve our understanding of the origin of pearl rotation.

Conclusion

Magnetic resonance imaging exploration can provide regular information for tracking reproduction, through maturation to spawning, and to see the first developments of the pearl sac. The latest advances in MRI, which show the dynamic capabilities of real-time MRI, should help to unravel some of the mystery about pearl rotation. Simultaneous non-invasive functional explorations can be considered, combining measures of metabolic flux, rotation of pearls and recording of valve activity, preferentially using stress gauges that would not interfere with the magnetometric system signals. This article reviews the state of the art of non-invasive functional exploration techniques that can be used in bivalves. We hope that this paper will be useful to physiologists of bivalve marine molluscs through the tools and applications presented here or others that could be conceived in the future. Overview of the magnetometer system. For data acquisition, a grafted pearl oyster Pinctada margaritifera is placed into the dome.

The colour code is that of the rainbow, the beginning of the recording is marked by the colour red.

Figure 3

Three-dimensional representation of the nucleus movement in two Pinctada margaritifera pearl oysters. The circular motion observed on the left pearl corresponds to a pearl whose shape is not perfectly spherical rotating on an axis. The random movement observed on the right pearl corresponds to a perfectly spherical pearl. The temporal dynamics of pearl movement are revealed by the coloration of the line illustrating the movement.

Figure 4

Continuous recording of the valve activity of a pearl oyster for 17 days. Schematic pearl oyster whose valve opening is likened to a right-angled where a is the opening angle at the hinge, L: the length of the shell, and H: the valve spread value opposite the hinge.

Figure 1

 1 Figure1(a) The ecophysiological measurement system for pearl oysters Pinctada margaritifera, biodeposits are harvested for analysis of organic matter assimilation; (b) software interface showing the lights corresponding to the metabolic chamber being analysed (b1), simultaneous measurements of dissolved oxygen and microalgae fluorescence (b2).

Figure 2

 2 Figure 2

  Figure 5 Spectral analysis of the continuous recording of valve activity (a) spectrogram revealing a major peak of activity at 23.25 h, (b) series enlargement showing high-frequency micromovements.

Acknowledgements

The authors express their gratitude to Belinda Hui who helped lay down the principles of the magnetometer project and Max Mastail, now retired, who led the design of the magnetometer. We also thank Dominique Buestel for providing the valvometric data. We would also like to thank the entire technical team of the Ifremer Pacific Centre (IMA team) who assembled these unique systems.


Nicolas Baghdadi

Hassan Bazzi

Mohammad El Hajj

Mehrez Zribi

ANALYSIS OF SENTINEL-1 DERIVED SOIL MOISTURE MAPS OVER OCCITANIE, SOUTH FRANCE

Keywords: Soil moisture, Sentinel-1, SAR, C-band, Neural Network. Precipitation (mm)

Monitoring the surface soil moisture (SSM) in agricultural areas at plot scale helps in many applications such as irrigation planning and crop management. Over the last decade, SAR (Synthetic Aperture Radar) data have shown great potential in estimating SSM over agriculture areas. Today, Sentinel-1 (S1) and Sentinel-2 (S2) satellites present a good opportunity for operational SSM estimates in agricultural areas because they provide free and open access data at high spatial resolution (10 m x 10 m) and high revisit time (6 days over Europe). The aim of this paper is to present an operational approach for mapping soil moisture at high spatial resolution (plot scale) in agriculture areas by coupling S1 and S2 images. The proposed approach is based on the inversion of the Water Cloud Model (WCM) using the neural network technique.

INTRODUCTION

Soil moisture plays a key role in different hydrological processes (floods, runoff, evapotranspiration, infiltration, soil erosion, and imbalances in the water and carbon cycles). The arrival of Sentinel-1 SAR data (10 m spatial resolution and a 6-day revisit period) and Sentinel-2 optical data (10 m spatial resolution and a 5-day revisit period) have encouraged the development of operational algorithm for soil moisture mapping over agricultural areas with high revisit time and high spatial resolution (up to plot scale).

Several studies tend to use the neural networks technique to invert backscattering models and estimate the soil moisture. To estimate soil moisture and roughness from the C-band polarimetric radar data, Baghdadi et al. [START_REF] Baghdadi | Estimation of soil parameters over bare agriculture areas from C-band polarimetric SAR data using neural networks[END_REF] first generated a database of backscattering coefficients for a wide range of bare soil conditions (soil moisture and surface roughness) using the Integral Equation Model IEM [START_REF] Baghdadi | Calibration of the integral equation model for SAR data in C-band and HH and VV polarizations[END_REF]. Then, the neural networks (NNs) were trained using the synthetic database. A priori information on the soil moisture and roughness were considered to improve the accuracy of neural networks. Finally, the trained neural networks were validated using a real database and show an accuracy on the soil moisture estimates of approximately 7 vol.% with the use of a priori information. El Hajj et al. [START_REF] El Hajj | Synergic use of Sentinel-1 and Sentinel-2 images for operational soil moisture mapping at high spatial resolution over agricultural areas[END_REF] inverted the Water Cloud Model (WCM) [START_REF] Attema | Vegetation modeled as a water cloud[END_REF] combined with the Integral Equation model (IEM) [START_REF] Baghdadi | Calibration of the integral equation model for SAR data in C-band and HH and VV polarizations[END_REF][START_REF] Baghdadi | Semiempirical Calibration of the Integral Equation Model for SAR Data in C-Band and Cross Polarization Using Radar Images and Field Measurements[END_REF]. In their study, they used the WCM parameterized by Baghdadi et al. [START_REF] Baghdadi | Calibration of the Water Cloud Model at C-Band for Winter Crop Fields and Grasslands[END_REF] for the C-band. The total backscattering coefficient in the WCM is considered as the sum of the direct vegetation contribution and the soil contribution multiplied by the attenuation factor. Using a vegetation descriptor derived from optical images (Sentinel-2), the direct contribution of vegetation can be calculated. El Hajj et al. [START_REF] El Hajj | Synergic use of Sentinel-1 and Sentinel-2 images for operational soil moisture mapping at high spatial resolution over agricultural areas[END_REF] validated the proposed approach for the operational mapping of soil moisture over a study site located in Occitanie, South France. They showed that the soil moisture in agricultural areas could be estimated with an accuracy of approximately 5 vol.%. Moreover, an evaluation of SMOS, SMAP, ASCAT and S1-SSM products was performed by El Hajj et al. [START_REF] El Hajj | Evaluation of SMOS, SMAP, ASCAT and Sentinel-1 Soil Moisture Products at Sites in Southwestern France[END_REF] and reveals that the S1-derived soil moisture maps provide the most accurate estimation of SSM. The higher accuracy of estimated SSM moisture is probably due to the well-calibrated IEM combined with the well parameterized WCM and the use of high spatial resolution (10 m × 10 m) land cover maps derived from S2 images to eliminate SAR scattering from forest and urban areas.

The aim of this paper is to analyze Sentinel-1 derived soil moisture maps over Occitanie region according to GPM (Global Precipitation Mission) rainfall data. The soil moisture maps were derived from the very high spatial resolution Sentinel-1 images following the algorithm developed by El Hajj et al. [START_REF] El Hajj | Synergic use of Sentinel-1 and Sentinel-2 images for operational soil moisture mapping at high spatial resolution over agricultural areas[END_REF]. The operational approach for mapping soil moisture at high spatial resolution (up to the plot scale) in agriculture areas by coupling S1 and S2 images is described in section 2. Section 3 includes a comparison between S1-SSM and precipitation derived from GPM (Global Precipitation Measurement) data. Finally, Section 4 presents the main conclusions.

S1-SSM ALGORITHM

The approach for the operational soil moisture mapping uses the neural networks (NN) technique to invert the radar backscattering signal [START_REF] El Hajj | Synergic use of Sentinel-1 and Sentinel-2 images for operational soil moisture mapping at high spatial resolution over agricultural areas[END_REF]. The development of the inversion method consists of the following steps:

 Generate a synthetic database of backscattering coefficients using a parameterized WCM [START_REF] Attema | Vegetation modeled as a water cloud[END_REF] combined with the modified IEM [START_REF] Baghdadi | Calibration of the integral equation model for SAR data in C-band and HH and VV polarizations[END_REF][START_REF] Baghdadi | Semiempirical Calibration of the Integral Equation Model for SAR Data in C-Band and Cross Polarization Using Radar Images and Field Measurements[END_REF];  Noise the synthetic SAR C-band database and NDVI values to obtain synthetic database close to the real SAR and NDVI data;  Train and validate the neural networks using the synthetic dataset;  Finally, apply the trained neural networks to S1 and S2 images to map the soil moisture.

Incidence angle, noisy radar signal at VV polarization, and noisy NDVI are the inputs of the NN. The soil roughness (root mean square surface height "Hrms") was not used as an input parameter for the neural networks training and validation. However, the synthetic database was simulated using Hrms values between 0.5 and 3.8 cm. The NN was trained using the Levenberg-Marquardt algorithm with two hidden layers. The number of neurons for each hidden layer is 20. Linear and tangent-sigmoid transfer functions were associated with the first and second hidden layers, respectively.

To improve the soil moisture estimates, a priori knowledge on the soil moisture state is introduced. Indeed, from weather forecasts (precipitation and temperature) it is easy to define if the soil is is either dry to slightly wet (no precipitation for many days before SAR acquisition) or very wet (heavy rainfall preceding SAR acquisition). The integration of a priori information constrains the range of possible soil moisture parameter values estimated by the neural networks and thus leads to a better estimation of the soil moisture:

 Case 1: A priori information is available for mv. The soil is supposed to be dry to slightly wet according to expertise based mainly on meteorological data (precipitation, temperature). Soil moisture values are assumed to range from 2 to 25 vol. %.  Case 2: A priori information is available for mv. The soil is supposed to be very wet according to the expertise of the meteorological data. The mv values are assumed to vary between 25 and 40 vol. %. An overlap of 10 vol. % on the mv is considered between the databases used for the training of the neural networks. For a priori information that soil is dry to slightly wet (case 1), the NN was developed using the training database elements with the mv between 2 and 30 vol. %. For a priori information that soil is very wet (case 2), the NN was developed using the training database elements with the mv between 20 and 40 vol. %.

The developed algorithm is currently used in operational mode on many study sites. For example, S1 soil moisture maps for the Occitanie region (South France) at high spatial resolution (up to plot scale) are available since September 2016 as open access data via the Theia French Land Data Center (http://www.theia-land.fr/en/thematic-products).

COMPARISON BETWEEN S1-SSM AND PRECIPITATION

Rainfall measurements with 30 min revisit time obtained from the GPM (Global Precipitation Measurements) sensor were considered in this study. From the GPM mission the IMERG (Integrated Multi-satellite Retrievals for GPM) data product was selected to perform our analysis. The spatial resolution of IMERG maps is 0.1 °× 0.1 °. IMERG data at different spatial and temporal resolutions are available via the NASA Precipitation Measurements Mission (PMM) website (https://pmm.nasa.gov/data-access).

For each S1-SSM map date, one cumulative rainfall map for the 6 days prior to the S1-SSM map date was computed by summing the 30 min time interval of the Late IMERG GPM product. Then, each S1-derived SSM map was overlaid with the corresponding 6 days of cumulative GPM rainfall. Starting with the initial map on 21/12/2016 (Figure 2a), departments as Tarn, Hérault, Lozère, and Aveyron of the eastern and middle parts of the region encountered high soil moisture values (more than 20 vol.%) corresponding to 10-20 mm cumulative precipitation during the past 6 days, whereas Gers and Haute-Garonne of the western part shows low soil moisture values (20 vol.% at most) corresponding to the absence of precipitation in the area for the same prior period. A map derived 6 days later on 27/12/2016 (Figure 2b) shows a decrease in soil moisture values in the Hérault and Gard due to the absence of precipitation between 21/12/2016 and 27/12/2016. Moreover, high soil moisture values are estimated for Tarn due to continuous raining episodes between 21/12/2017 and 27/12/2017, and a slight increase in soil moisture values is observed in western part due to 3 mm of precipitation recorded one day prior to the S1-SSM map according to the GPM data (Figure 2b). A general decrease in soil moisture values throughout the region is observed between 27/12/2016 (Figure 2b) and 02/01/2017 (Figure 2c) as no precipitation was recorded. A continuous lack of precipitation for 12 days from 27/12/2016 until 08/01/2017 caused soil moisture values to drop to less than 15 vol.% on 08/01/2017 in the whole region as shown in Figure 2d. Six days later on 14/01/2017 (Figure 2e), soil moisture values remained low at the east as no precipitation was recorded for an additional 6 days, whereas a moderate increase in soil moisture values is observed in the middle and western parts (Tarn, Haute-Garonne, and Gers) corresponding to precipitation between 08/01/2017 and 14/01/2017 (a cumulative rainfall increase of 5-20 mm). Twenty-four days with no precipitation recorded in the Hérault and Gard region between 27/12/2016 and 20/01/2017 caused the soil moisture values to drop more, with values less than 15 vol.% in this area on 20/01/2017 (Figure 2f).

In addition, for Gers department, low soil moisture values (less than 15 vol.%) were estimated on January 20, 2017 and high cumulative precipitation was recorded between 14 and 20 January 2017 (15 mm) (Figure 2f). This low soil moisture estimates could be linked to the presence of frozen soil in the area. In a recent study using Sentinel-1 SAR data, Baghdadi et al. [START_REF] Baghdadi | Detection of Frozen Soil Using Sentinel-1 SAR Data[END_REF] showed that a decrease of at least 3 dB in the radar backscattered signal can be observed over frozen soil conditions. Such a decrease in the SAR signal yields an underestimation of soil moisture in the S1-derived SSM maps. In our case, it seems that low soil moisture values for this area on 20/01/2017, although with the presence of rainfall, is due to frozen soil conditions. To support our assumption, the temperature curve and precipitation records for a local meteorological station in Auch city located in Gers-Occitanie were analyzed. On 19 January 2017 (S1 acquisition over Gers on January 19, 2017 17:56 UTC), the temperature varied between -8°C and 2°C with a mean value of -3°C throughout the day. Three days before (15 and 16 January) 15 mm cumulative precipitation was recorded. Thus, a precipitation event followed by a decrease in the air temperature to less than 0°C justifies the presence of frozen soil conditions as the cause of a decrease in the estimated soil moisture values in the S1-derived SSM maps.

After 29 days of dry conditions in the eastern part (Hérault and Gard between 27/12/2016 and 25/01/2017), a sudden increase in the soil moisture was observed on 26/01/2017 with the beginning of a rainfall event (Figure 2g). The mean S1-SSM mean value in this area increased from 13 vol.% in 20/01/2017 to 32 vol.% in 26/01/2017. On the other hand, the absence of precipitation in the remaining parts of the region between 20/01/2017 and 26/01/2017 caused soil moisture to attain lower values (16 vol.%) on 26/01/2017.The slight increase in soil moisture values in Gers between 20 and 26 January is probably due to the disappearance of the frozen conditions observed in the previous acquisition (20/01/2017). The temporal analysis of S1-derived SSM values over a period of time shows the direct effect of raining episodes or dry conditions on S1-SSM values.

CONCLUSION

This paper presents an analysis of the new Sentinel-1 surface soil moisture (S1-SSM) product derived at a very high spatial resolution (plot scale) and a revisit time of 6 days over Southern of France. A good coherence was observed between the temporal evolution of the soil moisture and the precipitation records derived from the GPM data. The S1-SSM values increases following rainfall event and decreases after a period without rainfall due to evaporation.

Figure 1 .

 1 Figure 1. Location of study site, Occitanie region, South France.

Figure 2 :

 2 Figure 2: S1-derived SSM map overlaid with GPM cumulative precipitation data. (a) 21/12/2016, (b) 27/12/2016, (c) 02/01/2017, (d) 08/01/2017, (e) 14/01)2017, (f) 20/01/2017, (g) 26/01/2017.

ACKNOWLEDGMENT

This research was supported by IRSTEA (National Research Institute of Science and Technology for Environment and Agriculture) and the French Space Study Center (CNES, DAR 2018 TOSCA). The authors wish to thank the ESA (European Commission and the European Space Agency) for providing the S1 images. We used Copernicus level 2A S2 data processed by the French land data service center (Theia: https://www.theia-land.fr).