Nicolas Philippe

Hossein Davarzani

Stéfan Colombano

Malorie Dierick

Pierre-Yves Klein

Manuel Marcoux

Experimental study of the temperature effect on two-phase flow properties in highly permeable porous media: Application to the remediation of dense non-aqueous phase liquids (DNAPLs) in polluted soil

Keywords: Dense non-aqueous phase liquid, two-phase flow, capillary pressure-saturation relationship, relative permeability, thermally enhanced DNAPL recovery

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

Introduction

Dense non-aqueous phase liquids (DNAPLs) are liquids denser than water and slightly soluble in water. Due to their high density, DNAPLs can deeply sink into the subsurface until reaching a less permeable layer and form a non-aqueous reservoir. Subsurface zones contaminated by DNAPLs are generally hard to delimit due to soil heterogeneity, soil dispersion, and the substantial depth of the contamination combined with gravity-driven finger flow. Thus remediating these pollutants in the aquifer is a complicated matter. DNAPL source zones are even more problematic because they may keep on dissolving toxic compounds into the groundwater. Ultimately, future health and environmental problems can arise, such as drinking water contamination or soil quality degradation.

Commonly reported DNAPL contaminants include chlorinated solvents -namely, trichloroethylene (TCE) and tetrachloroethylene (PCE) -that were previously widely used as solvents for organic materials [START_REF] Schwille | Dense chlorinated solvents in porous and fractured media -Model experiments[END_REF]. Coal tars are also a notable DNAPL found in the subsurface and are also more viscous than water. [START_REF] Brown | Comparative assessment of coal tars obtained from 10 former manufactured gas plant sites in the Eastern United States[END_REF] have measured coal tar's dynamic viscosity on multiple sites and have found that the coal tar/water viscosity ratio can vary between 10 and 10 6 [START_REF] Brown | Comparative assessment of coal tars obtained from 10 former manufactured gas plant sites in the Eastern United States[END_REF]. This range is linked to the chemical heterogeneity in coal tar. Diagnosis and risk assessment are hard because the exact composition of coal tar is generally not identifiable. Hence, a site polluted by coal tar is difficult to clean up and the choice for an applicable recovery method has to be made on a case-by-case basis.

In our study, the focus is on coal tar source zones with high pollutant concentrations. Pumping in the subsurface through production wells is a primary step used in remediation operations. However, the pumping rate is very low for DNAPLs and the remediation could take tens to hundreds of years, which is economically unsatisfying [START_REF] Usepa | Pump and Treat Ground Water Remediation A Guide for Decision Makers and Practitioners[END_REF][START_REF] Russell | Decision Analysis for Pump-and-Treat Design[END_REF][START_REF] Kavanaugh | Groundwater Management in Large River Basins[END_REF][START_REF] Navy | Plume management zone to manage chlorinated solvents[END_REF][START_REF] Newell | Contaminant Plume Classification System Based on Mass Discharge[END_REF]. The most efficient current methods for remediating DNAPL source zones are thermal treatments [START_REF] Ding | A review on the sustainability of thermal treatment for contaminated soils[END_REF]. Generally, temperature is increased to vaporize [START_REF] Mcdade | Analysis of DNAPL source-depletion costs at 36 field sites[END_REF][START_REF] Baker | In-pile thermal desorption of PAHs, PCBs and dioxins/furans in soil and sediment[END_REF][START_REF] Baston | Numerical Modeling of Thermal Conductive Heating in Fractured Bedrock[END_REF] or even burn [START_REF] Rein | Smouldering Combustion Phenomena in Science and Technology[END_REF][START_REF] Switzer | Self-Sustaining Smoldering Combustion: A Novel Remediation Process for Non-Aqueous-Phase Liquids in[END_REF][START_REF] Hasan | Self-sustaining smouldering combustion of coal tar for the remediation of contaminated sand: Two-dimensional experiments and computational simulations[END_REF][START_REF] Scholes | Smoldering Remediation of Coal-Tar-Contaminated Soil: Pilot Field Tests of STAR[END_REF] the pollutant. The vapors generated are then collected and treated with carbon activated filters. However, generating high-temperature fields can be dangerous for the nutritional and biological functions of the soil. [START_REF] Pape | Impacts of thermal and smouldering remediation on plant growth and soil ecology[END_REF] have shown that even if a contaminant can be completely treated at 773.15 K, the soil is unusable for future construction or industrial operations [START_REF] O'carroll | Exploring dynamic effects in capillary pressure in multistep outflow experiments[END_REF][START_REF] Pape | Impacts of thermal and smouldering remediation on plant growth and soil ecology[END_REF]. As a result, NAPL residual saturation is lower in the soil after a thermally enhanced process. Afterwards, the high soil temperature can increase the degradation rate of chemical treatments (oxidation, surfactant flooding) and biological methods (bio or phytoremediation) [START_REF] Mclaren | Effects of temperature on chlorophenol biodegradation kinetics in fluidized-bed reactors with different biomass carriers[END_REF].

Hot-water flooding is another technique that has shown success in treating viscous NAPLs -light or dense -contamination. The idea is to reduce the dynamic viscosity of the NAPL by increasing its temperature. During pumping, the hot water will progressively replace the NAPL and leave residual NAPL saturation in the soil. Previous authors have shown that injecting hot water increases total NAPL recovery volumes by pumping processes [START_REF] Fulton | Hot water enhanced recovery of phase separated lubricating oil[END_REF][START_REF] Davis | How heat can enhance in-situ soil and aquifer remediation: important chemical properties and guidance on choosing an appropriate technique[END_REF][START_REF] O'carroll | Hot water flushing for immiscible displacement of a viscous[END_REF]. Pumping a DNAPL phase from the subsurface can be represented by an imbibition process in porous media. The DNAPL phase is progressively pushed away from the soil and the water slowly fills the pores left vacant. Thus, it is important to analyze and better understand fluid displacements before drawing any conclusions.

To characterize the behavior of the physical system composed of DNAPL, water, and a solid matrix is still an ongoing challenge in fluid mechanics. Currently, modelling is based on the theory of immiscible multiphase flow in porous media. In our case, DNAPL and water are represented as two separate phases, and the soil is an immobile solid matrix in which both liquids may flow. For this type of engineering application, the simultaneous flow of DNAPL and water in a porous medium is commonly described using the generalization of Darcy's law to multiphase flow for each phase [START_REF] Muskat | The flow of heterogeneous fluids through porous media[END_REF].

, ! " #! $" ! 1 "
where is the Darcy velocity (m.s -1 ), $ the gravitational acceleration, the permeability tensor (m 2 ), the pressure (Pa), and respectively the dynamic viscosity (Pa.s) and density (kg.m -3 ). The subscript & designates the water or DNAPL phase. The main difference between Darcy's law and its extension to multiphase flow is the addition of relative permeability terms , . It signifies that each phase flows independently through a virtual porous medium of permeability , ! ". The DNAPL occupies a part of the total pore space, depending on its saturation, which reduces the volume of water that can flow through and vice versa. This equation generally holds true when there is no momentum transfer at the interface of both fluid phases [START_REF] Whitaker | Flow in porous media II: The governing equations for immiscible, two-phase flow[END_REF].

The presence of the two fluid phases results in an interface that is subjected to interfacial tension at the pore scale. This interface also causes a pressure discontinuity at the macroscale, with respect to capillary pressure . This is subsequently defined as the difference between the pressure of the non-wetting fluid '( and the pressure of the wetting fluid ( . Immiscible DNAPLs flowing in the aquifer can be assimilated to a two-phase flow in porous media. The dimensionless numbers controlling the actual flow pattern are respectively Bond number (Bo), capillary number (Ca), and viscosity ratio (M). In the case of the imbibition of a wetting phase inside a porous medium saturated with a non-wetting phase, these numbers can be written as:

! '( ( " ² ! 3 " ', ! 4 " ', . / ! 5 "
is a characteristic length. In unconsolidated media like glass beads, L is often chosen equal to the mean particle diameter. is the interfacial tension between both liquid phases. is a characteristic velocity representing the order of magnitude of the flow rate inside the pores. The subscripts inv and dis respectively represent the invading and displaced phase. In this work, the DNAPL and water are the invading phases during drainage and imbibition, respectively. Alternatively, the viscosity ratio may be replaced by the mobility ratio (the ratio between relative permeability and viscosity). The latter includes relative permeabilities in its calculation and can be more suitable for real porous media [START_REF] Hagoort | Displacement stability of water drives in water-wet connate-water-bearing reservoirs[END_REF]. It is possible to identify flow regimes depending on the order of magnitude of each number. The different types of two-phase flow that occur in micro-models are classified as capillary fingering, viscous fingering, and stable displacement [START_REF] Lenormand | Numerical models and experiments on immiscible displacements in porous media[END_REF]. According to the authors, the type of flow that promotes maximum coal tar recovery and minimizes residual DNAPL saturation is stable displacement, which occurs only for M << 1 and Ca > 10 -4 . In practice, coal tar is much more viscous than water, and M > 1 in the case of pumping coal tar. The main objective of thermally enhanced oil recovery is therefore to reduce this viscosity ratio in order to decrease the mobility ratio and to promote stable displacement in the porous medium. The effect of temperature on Bond and capillary number should also be estimated in order to link those to the benefits of thermal enhancement. [START_REF] Mclaren | Effects of temperature on chlorophenol biodegradation kinetics in fluidized-bed reactors with different biomass carriers[END_REF] found that, for a soil consisting of London clays, an increase from 12°C to 30°C not only reduces the kinematic viscosity of a coal tar from 10 -4 m 2 .s -1 to 10 -5 m 2 .s -1 , but also significantly reduces the residual saturation, despite a small variation in the capillary effect [START_REF] Mclaren | Effects of temperature on chlorophenol biodegradation kinetics in fluidized-bed reactors with different biomass carriers[END_REF]. [START_REF] Davis | Effect of temperature and pore size on the hydraulic properties and flow of a hydrocarbon oil in the subsurface[END_REF], experimentally obtained a decrease in residual DNAPL saturation by heating from 10°C to 30°C without any temperature effect on interfacial tension [START_REF] Davis | Effect of temperature and pore size on the hydraulic properties and flow of a hydrocarbon oil in the subsurface[END_REF]. [START_REF] Sleep | Thermal variation of organic fluid properties and impact on thermal remediation feasibility[END_REF] found only a 10% change in the interfacial tension of the PCE-water pair when heated from 20°C to 90°C [START_REF] Sleep | Thermal variation of organic fluid properties and impact on thermal remediation feasibility[END_REF]. [START_REF] Bachmann | Temperature Dependence of Water Retention Curves for Wettable and Water-Repellent Soils[END_REF] suggest that the decrease in residual saturation is also related to a decrease in the contact angle. It can decrease by 0.03 °/°C for sand, and up to 0.26 °/°C for silts [START_REF] Bachmann | Temperature Dependence of Water Retention Curves for Wettable and Water-Repellent Soils[END_REF]. More recently, [START_REF] Colombano | Thermal and chemical enhanced recovery of heavy chlorinated organic compounds in saturated porous media: 1D cell drainage-imbibition experiments[END_REF] studied the thermally enhanced recovery of heavy chlorinated solvents in a highly permeable porous medium and observed no effect of temperature on residual saturation on quasi-static drainage and imbibition conditions [START_REF] Colombano | Thermal and chemical enhanced recovery of heavy chlorinated organic compounds in saturated porous media: 1D cell drainage-imbibition experiments[END_REF].

A few similar studies have been proposed in the past for viscous oil recovery. [START_REF] Koci | Effet de la température sur les propriétés de déplacement polyphasique en milieux poreux[END_REF] studied the effect of temperature on oil-water in unconsolidated porous media (sand) between 298.15 K and 443.15 K. The main findings were that raising temperature increased the irreducible water saturation, but also decreased the residual oil saturation and the relative oil permeability [START_REF] Koci | Effet de la température sur les propriétés de déplacement polyphasique en milieux poreux[END_REF]. [START_REF] Wang | Effect of oil viscosity on heavy oil-water relative permeability curves[END_REF] investigated the effect of oil viscosity on relative permeability curves for oil-water in sand packs. The measurements were made at constant flow rate. The decrease in oil viscosity was linked to an increase in both oil and water relative permeability curves [START_REF] Wang | Effect of oil viscosity on heavy oil-water relative permeability curves[END_REF].

The impact of temperature on oil-water relative permeability, interfacial tension, and wettability has been reviewed by [START_REF] Esmaeili | Review of the effect of temperature on oil-water relative permeability in porous rocks of oil reservoirs[END_REF]. Observations on the effect of temperature on two-phase flow in porous media were mainly linked to variations in those parameters. Therefore, it is very difficult to extend the findings from literature reviews on different liquids to a complex liquid such as coal tar. Also, some authors pointed out that a lot of relative permeability available data is not exact due to the presence of experimental artifacts, e.g. viscous fingering, capillary-end effects, wettability, and pore-distribution changes due to temperature variations. Specifically, reliable relative permeability measurements require a flow regime dominated by capillary forces at the pore scale.

Equation ( 1) is only valid for low capillary numbers [START_REF] Davit | One-Phase and Two-Phase Flow in Highly Permeable Porous Media[END_REF]. Oil reservoirs generally have low permeability, less than 10 -11 m 2 . The velocity is generally low enough to validate the hypothesis behind the use of generalized Darcy's law. However, in the case of soil remediation, the NAPL phase is present due to a spill and is often found in highly permeable zones in the subsurface, above 10 -11 m 2 . For instance, in our application case, coal tar was pumped in alluvial sands with a permeability of 10 -9 m 2 .

The use of generalized Darcy's law and Eq (2) implies a quasi-static assumption. The interface between both fluid phases is described by a succession of quasi-static thermodynamic states, without any dependence on time. Capillary effects are predominant at the pore scale and are represented at macroscale using Eq [START_REF][END_REF]. Relative permeabilities represent viscous effects to generalize Darcy's law to multiphase flow. However, this assumption is not always satisfied when considering multiphase flows in a transient process. For instance, when considering pumping NAPL in the subsurface, the capillary number will be obviously very high near the production well.

The main objective of this work is to measure changes in the physical properties of coal tar caused by temperature variations and to relate those to two-phase flow properties in highly permeable porous media. We explored how temperature influences the capillary pressure-saturation and relative permeability curves under quasi-static two-phase flow conditions. Our goal was to better understand and characterize the two-phase flow in highly permeable porous media with a focus on temperature.

First, we determined the properties of coal tar sampled from a real polluted site (Section 2). Data on its density, dynamic viscosity, interfacial tension with water, and contact angle with water on a glass slide were measured between 293.15 K and 323.15 K. Then we describe an experimental setup that has been used to study two-phase flow in porous media in isothermal conditions (Section 3). We conducted experiments at two temperatures (293.15 K and 323.15 K) under quasi-static conditions. Then we carried out drainage and imbibition tests at different flow rates and temperatures to study the effect of temperature on two-phase flow under dynamic conditions. Finally, we summarize the results including the capillary pressure-saturation relationship, analysis of used dimensionless numbers, and a discussion on saturation and pressure time-profiles as well as relative permeability curves obtained under dynamic conditions (Section 4).

Fluid characterization as a function of temperature 2.1. Fluid selection and properties

The main couple of liquids for this study is coal tar and water. Coal tars are pyrolysis by-products. Polycyclic aromatic hydrocarbons (PAHs) are the main chemical compounds found in coal tars. Previous studies on coal tars indicate that their physical properties depend on their chemical origin and the geology of the source zones [START_REF] Kong | Characterization of Mineral Oil, Coal Tar and Soil Properties and Investigation of Mechanisms that Affect Coal Tar Entrapment In and Removal From Porous Media[END_REF]. In addition, the complete chemical composition of coal tars is hard to determine by analytical means [START_REF] Johansson | FerrateVI oxidation of polycyclic aromatic compounds (PAHs and polar PACs) on DNAPL-spiked sand: degradation efficiency and oxygenated by-product formation compared to conventional oxidants[END_REF]. We used a coal tar sample produced from a well situated in a former coke plant. Suspended solid particles were removed from the sample using a glass fiber membrane (GF/D, 2.7 µm pore size). Some water was also collected during coal tar sampling. The water contained inside the sample was filtered out with a vacuum pump and a 10 μm pore size hydrophobic PTFE membrane. The resulting sample, containing only coal tar, was kept in a cold room at 277.15 K to limit chemical reaction rates, evaporation, and variations in physical properties during the study. Degassed tap water was used in all coal tar and water flow in porous media experiments. Ultrapure water was also considered for the experiments but was not suitable because coal tar dissolves partially in it.

DNAPLs, like coal tar, are generally difficult to experiment with due to their high toxicity and their adhesion on laboratory glassware. As previously mentioned, coal tar has generally an unknown and very heterogeneous composition. It has also been observed that the composition of coal tar can change in the experimental setups. Therefore, we chose a model fluid pair made of two immiscible pure compounds, canola oil and ethanol. Virgin canola oil was used as the substitute for coal tar (non-wetting fluid). The choice of virgin oil was motivated by the fact that refined canola oil was found to change color when heated. Vegetable oils have however a lower density than water. Thus, we chose to work with ethanol as the wetting fluid. Sodium chloride was dissolved into the ethanol at room temperature (293.15 K) at a concentration of 0.65 g/kg. The addition of salt was required to measure soil resistivity in a parallel study [START_REF] Iravani | The influence of temperature on the dielectric permittivity and complex electrical resistivity of porous media saturated with DNAPLs: A laboratory study[END_REF]. From this point, water and salty ethanol will be referred to as the wetting phase. Coal tar and canola oil will be referred to as the nonwetting phase.

Density

We measured all the densities (coal tar, water, salty ethanol, canola oil) using a pycnometer (25 mL ± 0.04 mL) and an analytical balance (± 0.01 g). The pycnometer was first calibrated with ultrapure water at room temperature (293.15 K). Density values were then measured at the same temperature. The effect of temperature on density was determined by placing the liquid inside an insulated closet initially at 293.15 K. The temperature of the closet was increased by steps of 10 K and the density was then measured with the same pycnometer. The procedure was repeated until reaching 323.15 K in triplicate per liquid. Finally, measurements were also acquired at 277.15 K by placing the sample in a cold room.

Figure 1 presents the density measurements as a function of temperature for ethanol, canola oil, water, and coal tar. Coal tar has a higher density than water at all temperatures tested in this study. Therefore, coal tar behaves as a DNAPL in the polluted aquifer zone. The coal tar density values measured in this study were close to previously reported measurements at 295.15 K, 308.15 K, and 323.15 K [START_REF] Kong | Characterization of Mineral Oil, Coal Tar and Soil Properties and Investigation of Mechanisms that Affect Coal Tar Entrapment In and Removal From Porous Media[END_REF]. Linear regression was applied using the least-squares method. We observed that ethanol, canola oil, water, and coal tar densities decrease almost linearly when their temperature increases from 277.15 K to 323.15 K. The measurements indicate that the volumetric thermal expansion coefficient can be considered constant for these fluids. For each fluid, the estimated correlations are the following, with temperature in Kelvin:

12 ! " 1237 0.4818 ! 6 " 8 ! " 1073 0.2598 ! 7 "

: ! " 990.3 0.6224 ! 8 "

; ! " 1059 0.5033 ! 9 "

Dynamic viscosity

We measured all the dynamic viscosities (coal tar, water, salty ethanol, canola oil) using a rotational rheometer (Thermo Scientific Haake™ Mars™ III) and cone-plate geometry. The plate was connected to thermoelectric cooling (Peltier effect) in order to reach temperatures between 283.15 K and 323.15 K. The rheometer applied a constant shear rate and measured the responding shear stress of the liquid. Correct calibration of the gap between the cone and the plate is of great importance for precise measurements. In our study, this gap can change due to thermal expansion. Thus, we first heated the plate to the desired temperature then calibrated the gap prior to measurements. Finally, the liquid sample (2 mL) was injected onto the plate. The range of the shear rate measurements was comprised between 10 s -1 and 100 s -1 . For Newtonian fluids, the shear stress is proportional to the shear rate. In this case, the dynamic viscosity is defined as a constant proportionality factor between both parameters.

First, coal tar shear stress was determined at 293.15 K for shear rates varying from 10 s -1 to 100 s -1 .

The shear stress increased proportionally as the shear rate increases. Thus, the coal tar sampled in this study displays the behavior of a Newtonian fluid between 283.15 K and 323.15 K. Previous studies have found that liquid coal tar is shear thinning [START_REF] Bhatia | Rheological characteristics of coal tar and petroleum pitches with and without additives[END_REF][START_REF] Fitzer | Rheological characteristics of coal-tar pitches[END_REF]. The difference in behavior found here may be due to either the historical origin of the coal tar sample or the focus on a lower temperature (283.15 K) compared to these studies (> 373.15 K). Figure 2 presents the effect of temperature on the dynamic viscosities of ethanol, canola oil, water, and coal tar. Dynamic viscosities decreased while temperature increased in all fluids The Andrade equation (two parameters empirical exponential model) was used to fit viscosity data [START_REF] Andrade | The viscosity of liquids[END_REF]. The relative changes of dynamic viscosity due to temperature were found to be close for non-wetting liquids (canola oil and coal tar) and also wetting liquids (ethanol and water). ; ! " 1.60 × 10 =B ?

A.GF×C D 2 ! 13 "

Interfacial tension and contact angle

Interfacial tension and contact angles measurements were made with a drop shape analyzer (Krüss DSA 100). The apparatus consists of a light source, a camera, and the sample provided by a syringe. A drop of the non-wetting fluid (coal tar or canola oil) is injected into a bath filled with the wetting fluid (water or salty ethanol). The bath and the syringe are surrounded by an insulated chamber. The chamber is connected to a thermostat, which can change the measuring temperature between 285.15 K and 323.15 K. The accuracy for interfacial tension, contact angle, and temperature measurements are respectively 0.3 mN/m, 0.3°, and 0.1 K. A camera, controlled by Krüss ADVANCE software is able to detect the drop and shape contours from images for interfacial tension and contact angle measurements.

The calibration of the apparatus was checked by measuring pure water's surface tension between 285.15 K and 323.15 K. For interfacial tension measurements, a 1 mm diameter PTFE needle was used. A non-wetting drop was formed at the tip of the needle directly inside the wetting fluid. The choices of needle diameter and drop volume were motivated by a previous work [START_REF] Berry | Measurement of surface and interfacial tension using pendant drop tensiometry[END_REF]. The measurements were done in triplicate for each target temperature. The measurements were then acquired every minute and averaged over one hour. The interfacial tension was determined with Young-Laplace equation. The method used by Krüss ADVANCE software automatically detects the drop in the image and fit shape factor in order to reproduce its contour. The interfacial tension is then calculated from:

∆ E ! 14 "
where Δρ is the difference between densities of the two phases, g the standard acceleration due to gravity, R0 the drop radius at its apex, β a shape factor.

For contact angle measurements, a glass slide was introduced inside the wetting fluid (water and ethanol) bath. A non-wetting fluid (DNAPL and canola oil) drop was then injected on the glass slide.

The drop shape was reproduced by Krüss software using the previously introduced Young-Laplace equation. The contact angle was measured as the angle between the detected contour and baseline, represented by the horizontal glass slide. Pure water-air surface tension measurements were in agreement with previous ones made by [START_REF] Vargaftik | International tables of the surface tension of water[END_REF]. This validates the proposed experimental apparatus and our protocol.

Prior to temperature measurements, interfacial tension was measured against time for a coal tar/tap water system over 200 minutes at 293.15 K and 323.15 K. The interfacial tension decreased in both cases as coal tar ages in tap water. This change in interfacial tension results from a small amount of coal tar dissolving rate into the water at the interface. The measurements could not be taken for a longer time as the drop fell off afterwards. To discuss only the temperature effect, all the interfacial tension measurements that follow were taken after 2 minutes.

The interfacial tension measurements for salty ethanol/canola oil and coal tar/water are plotted in Figure 3. The reported range of interfacial tensions is generally comprised between 20-50 mN/m for TCE, PCE, and chlorinated solvents with water at 293.15 K [START_REF] Cai | Interfacial Tension Measurement in DNAPL/Aqueous Surfactant Systems[END_REF][START_REF] Dawson | Influence of Viscous, Gravitational, and Capillary Forces on DNAPL Saturation[END_REF][START_REF] Smith | Determining effective interfacial tension and predicting finger spacing for DNAPL penetration into water-saturated porous media[END_REF]). Yet, for both fluid pairs studied, we observed low interfacial tensions (< 3 mN/m). Such low values are found for liquids containing amphiphilic substances. This may be the case for the coal tar used in this study as it was a mixture sampled from a real polluted site and its exact chemical composition is unknown. Commercial oils can also contain additives that reduce the interfacial tension of water/oil [START_REF] Gaonkar | Interfacial tensions of vegetable oil/water systems: effect of oil purification[END_REF]. [START_REF] Sanaiotti | Densities, Viscosities, Interfacial Tensions, and Liquid-Liquid Equilibrium Data for Systems Composed of Soybean Oil + Commercial Linoleic Acid + Ethanol + Water at 298[END_REF] measured a range of interfacial tensions between soybean oil and water and ethanol mixtures [START_REF] Sanaiotti | Densities, Viscosities, Interfacial Tensions, and Liquid-Liquid Equilibrium Data for Systems Composed of Soybean Oil + Commercial Linoleic Acid + Ethanol + Water at 298[END_REF]. The reported values were between 1-3 mN/m, which are close to what we measured. Unknown additives inside the oil might be the cause of these differences.

Previous studies have found that the influence of temperature on interfacial tension is linked to its influence on mutual solubility. For binary mixtures composed of a polar and a non-polar liquid, solubility increases linearly with temperature. As a consequence, interfacial tension also decreases linearly [START_REF] Donahue | The boundary tension at water-organic liquid interfaces[END_REF]. The same trend was found here for coal tar and water, as well as for canola oil/ethanol in our measurements. However, the decreasing interfacial tension with temperature for canola oil/ethanol was more significant than the decrease for the coal tar/water system. The linear relationships between interfacial tension and temperatures for both fluid pairs are described by:

12/8 ! " 3.3 3.3 × 10 =A ! 15 "

;/: ! " 5.7 1.2 × 10 =E ! 16 "

The accuracy of the apparatus was 0.3 mN/m, which is around 10 % of our absolute measurement value.

Figure 4 presents contact angles for coal tar and canola oil droplets against temperature. The variations of contact angle are not significant between 283.15 K and 333.15 K and can be considered constant with temperature. The slight variations observed on coal tar sessile drops could also be due to repeatability issues with the sessile drop method. All materials were cleaned thoroughly between any two measurements and the measurement conditions (temperature, injection speed, and droplet size) were precisely respected.

The measured contact angles between both couples of liquids seem close. However, coal tar also sticks on solid surfaces while canola oil does not. Our measurements of contact angle on a glass slide are a first indication of wetting phase conditions. They do not reflect the more complex behavior happening with complex liquids, like coal tar, inside a real porous medium.

Experimental setups and procedures for capillary behavior characterizations

The parameters measured in Section 2 indicate that temperature has an effect only on the dynamic viscosities of the fluids. Here we performed the drainage and imbibition experiments for coal tar/water and canola oil/ethanol in two experimental configurations (Figure 5). First, small pressure variations were applied using a small 1D column to limit viscous fingering and measure capillary pressure-saturation curves at 293.15 K and 323.15 K. This also avoided interface instability. Thus, we focused on how parameters change with temperature in a generalized Darcy's law application case. The drainage and imbibition experiments were done using a small transparent 2D cell using the same configuration as before. Finally, the 2D cell was connected to a volumetric pump where flow rate and temperature were varied to observe their effects on saturation and pressure profiles.

Experimental setups

Experiments were carried out inside a 1D vertical column (diameter 5.2 cm, height 5.6 cm). The column (Figure 5a) was made of polyvinylidene fluoride (PVDF) to resist coal tar corrosion. The measurements were made for both fluid pairs: coal tar/water and canola oil/ethanol. Coal tar was sampled from a field mainly composed of coarse sand with d50 = 1 mm. Thus, 1 mm homogeneous glass beads were chosen for the experiments. This was to avoid inaccuracies due to adsorption effects, particle shape heterogeneity, pore-size distribution, and possible wettability changes during data acquisition. As shown in Figure 5b, the drainage-imbibition experiments for coal tar/water and canola oil/ethanol were also performed in a small 2D cell (length = 5.0 cm, height = 5.0 cm, width = 2.0 cm). This 2D cell was also made of PVDF to ensure high corrosion resistance against coal tar. The cell was made of two transparent glass plates in order to visualize the two-phase flow using imaging.

The container with both fluids was placed on a Sartorius Cubis MSE (± 0.1 g) mass scale acquiring data automatically every 6 s. Two pressure transducers (KELLER PR33X, ±300 Pa) were added to the setup as three-way junctions at the top and bottom of the cell. The tubing connected to transducers was filled with ethanol initially to avoid the presence of air in the system. Both transducers were initially calibrated to read 0±10 Pa at atmospheric pressure at their respective positions.

The homogeneity of our beads was checked with sieves with mesh sizes between 0.96 mm and 1.04 mm. The mass of glass beads inside the medium was kept constant in order to ensure similar compaction, porosity, and initial saturation between various experiments. The column was filled with glass beads and vibrated slowly during the packing process. Glass beads were held inside the setup using the stainless-steel grids (mesh size 0.9 mm).

Initially, the wetting phase was injected at 25 mL/min with a peristaltic pump inside the porous medium. The possible presence of residual air was checked by measuring porosity and permeability.

Experiments were conducted on columns with a porosity between 0.39 and 0.42. The mean permeability of the glass beads was measured to be 3.5 × 10 -10 m 2 . a) b)

Quasi-static experiment procedure

In the quasi-static experiment setup, the column was connected to two scaled reservoirs (3.5 cm internal diameter and 41.8 cm high). Details on the experimental setup in quasi-static conditions are available in [START_REF] Colombano | Thermal and chemical enhanced recovery of heavy chlorinated organic compounds in saturated porous media: 1D cell drainage-imbibition experiments[END_REF]. One reservoir was connected to the bottom of the column and filled with the non-wetting phase. The other reservoir was connected to the top and filled with the wetting-phase. In this setup, the measurements were made by successive equilibrium states. The capillary pressure pc is defined as the difference between the pressure of the non-wetting phase and that of the wetting phase. Thus, the capillary pressure can be directly defined as the difference of hydrostatic pressures between the non-wetting and wetting liquids.

'( ( ! '( ( " ! 17 "

We chose the initial conditions for the experiments so that the interface between the non-wetting and wetting phase would be at equilibrium at the bottom of the 1D column. This means that z 0 () corresponds to a level of non-wetting fluid R '( equal to ( R ( / '( . For drainage experiments, the non-wetting reservoir base level was progressively increased by R 0.02 m steps, which represents 20 Pa and 18 Pa variations at 293.15 K and 323.15 K respectively. The volume balance between the non-wetting phase level R '( and wetting phase level R ( gave the saturation change between two successive steps. This process was repeated until no difference could be observed between the nonwetting and wetting reservoir volume variations. The levels of non-wetting and wetting fluids could be determined with a precision of 1 mm. The irreducible wetting saturation Sir,w was measured as the difference between initial and produced volumes of the wetting phase reservoir during drainage. For imbibition experiments, the non-wetting phase reservoir base level was decreased by 0.02 m steps. This time, the difference between the initially injected non-wetting phase volume and the volume produced during the imbibition gave the residual non-wetting saturation Sr,nw.

The time step between each height change is an important parameter for achieving the equilibrium condition. This time step was kept constant for experimental repeatability. To choose this time step, we measured the level of the interface inside the 1D cell after a 0.02 m step. This level was stationary 3 hours after changing the non-wetting phase reservoir for coal tar/water, and 1 hour for oil/ethanol during imbibition. We saw slightly low stabilization times during drainages. This is mainly due to the influence of gravity (drainages are produced upwards, imbibitions downwards). Hence, the reservoir level was changed when the saturation in the cell was constant. These experimental conditions have been chosen in order to keep a capillary dominant process inside the cell.

The heights measurements were corrected for the evaporation rate, using a reference reservoir to quantify water level change because of evaporation. Also, we limited evaporation by closing the end of the reservoirs with a perforated plastic cap. As pointed out before, the reservoirs were made with PVDF which is hydrophobic. Therefore, the volume attached to the reservoir wall is not significant and was not considered for hydrostatic pressure measurement.

For canola oil/ethanol and coal tar/water, two sets of three cell experiments were carried out at 293.15 K and 323.15 K. The whole setup was placed in an oven when heated at 323.15 K. Each set consisted of two successive drainages and imbibitions. For the first set of experiments, the temperature was kept at 293.15 K for the whole experiment. For the second set, the primary drainage was performed at 293.15 K but the temperature was increased to 323.15 K from the first imbibition.

For each experimental case, capillary pressure versus saturation datasets were fitted on van Genuchten empirical model using a Levenberg-Marquardt Algorithm (LMA) written in Python. We found that this model gave the best least-squares residuals out of all tested for our data. Van Genuchten parameters (α,n) and residual saturations have been averaged for each experiment.

Dynamic pumping experiment procedure

In this case, the drainage and imbibition experiments were done only for the oil/ethanol system as coal tar was completely opaque and the imaging procedure was not suitable for determining coal tar saturation inside the cell.

For both drainage and imbibition, we used three constant flow rates at room temperature (6, 12, 24 mL/min at 293.15 K) in a clockwise motion. After injecting 2 pore volumes, the pump's direction of rotation was changed to recover the non-wetting phase (imbibition). Oil/ethanol imbibition was also carried out at 12 mL/min at 323.15 K to study the effect of temperature.

Figure 6 presents photographs of the cell during the dynamic experiments. The interface between salty ethanol and canola oil observed during drainages was stable for all cases. Only two pore volumes were needed to reach irreducible ethanol saturation. However, imbibitions were unstable and prone to viscous fingering. Ten pore volumes were pumped during imbibition.

In all cases, the pressure difference initially increased because of the ethanol injection until a value of 3800 Pa. The pressure was read every 6 seconds. We observed substantial oscillations on the pressure readings due to the pump's peristaltic motion. A digital low-pass filter with a cut-frequency equal to the cycle frequency of the pump was applied to the data to remove these artefacts.

The sum of the wetting and non-wetting phase outlet masses was measured continuously during the experiment. The mass was measured every 6 seconds during both drainage and imbibition.

Saturation measurements using imaging

Saturation was determined inside the 2D small cell with an imaging technique. A Nikon® D810 with NIKKOR LENS 105 (Nikon®) digital camera which has a 34 megapixel resolution was used for optical imaging. Images were acquired every 30 seconds during the injection or pumping of the non-wetting phase. The experiments were performed in a dark room with two light spots (2×300 W, Broncolor®). Camera parameters and lighting conditions were identical for all experiments. Light transmission and light reflection methods have both been used in the past to observe two-phase flow processes in porous media [START_REF] Geel | Laboratory and model simulations of a LNAPL spill in a variablysaturated sand, 1. Laboratory experiment and image analysis techniques[END_REF][START_REF] Darnault | Visualization by light transmission of oil and water contents in transient two-phase flow fields[END_REF][START_REF] Wu | Estimation of representative elementary volume for DNAPL saturation and DNAPL-water interfacial areas in 2D heterogeneous porous media[END_REF]. In this study, the light reflection method (LRM) was used. The main idea of imaging analysis is to determine a relationship between light intensity and wetting-phase saturation Sw. Previous work found an exponential relationship between light intensity and saturation [START_REF] Gerhard | Relative permeability characteristics necessary for simulating DNAPL infiltration, redistribution, and immobilization in saturated porous media[END_REF][START_REF] Colombano | Thermal and chemical enhanced recovery of heavy chlorinated organic compounds in saturated porous media: 1D cell drainage-imbibition experiments[END_REF]. The images were in RGB but were transformed in 8-bit images for image analysis. The mean gray values were obtained using Fiji freeware. The following procedure was applied for image analysis of photos from the same experiments:

-Adjust or correct lighting differences with the 8-grayscale levels. The black and white square correspond to the reflectance of 18.9% and 98.2% respectively as reference values. -Determine the pixel area to be analyzed. The pixel area has to be big enough to avoid microscale effects, but at the same time not too big to avoid shadows and non-uniform lighting. The mean gray value averaged over the pixel area was plotted before analyzing images. This plot was used to determine an area where the mean gray value was constant. -Use a calibration curve to determine the wetting phase saturation inside the cell. For the calibration, we took images of cells containing a known volume fraction of glass beads and liquids.

For each saturation endpoints (Sw=Sir,w or Sw=1-Sr,nw), the cell was uniformly saturated and the reflected light intensity was measured (Fig B .1). The optical density was defined as the ratio between this measured intensity and the intensity of a white reference (98.2%).

For oil and ethanol, we found a linear relationship ( 2.63 XY + 2.34 with a coefficient of determination R² close to 1 (R² = 0.95). This result means ethanol saturation can be determined directly from light intensity measurements. This property is useful in order for measuring saturation, especially in dynamic conditions.

For both fluid pairs, the relationship between optical density and saturation was measured at 293.15 K and 323.15 K. We saw no effect of temperature on the relationship between optical density and saturation.

Results & Discussion

4.1. Effect of temperature on quasi-static capillary pressuresaturation curves

An example of the capillary pressure-saturation curves is plotted in Figure 7 at 293.15 K for coal tar/water. The experimental data from all three experiments are presented by crosses (exp) without distinction. However, van Genuchten parameters were obtained for each experiment separately and then were averaged (solid and dashed lines). Fitted van Genuchten parameters, irreducible and residual saturations are summarized in Table 1 for all experiments. This model uses two fitting parameters: α (m -1 ) and n, which are related to the inverse of the entry pressure and the homogeneity of the porous medium, respectively. For oil/ethanol and coal tar/water experiments, α values are different. This result is coherent because their corresponding measured values of interfacial tension are not the same. The parameter n is also much higher for coal tar/water than for oil/ethanol. Wetting irreducible and non-wetting residual saturations are almost the same when comparing the same case for both fluid pairs. Following [START_REF] Dury | Dependence of hydraulic and pneumatic characteristics of soils on a dissolved organic compound[END_REF], we also calculated β coefficients (Table 1), the ratio between the inverse of α and the interfacial tension, for all experiments. This ratio is used to scale existing capillary curves to other fluid pairs. In our case, we found that the values are slightly higher for oil/ethanol than coal tar/water. Thus, using the scaling procedure, it is not possible to get coal tar/water capillary pressure-saturation curves from oil/ethanol ones. Our results indicate that the scaling laws are not suitable when using complex fluids like coal tar. We showed before that only dynamic viscosity was influenced by temperature changes. However, the dependency of the dynamic viscosity on temperature did not influence the capillary pressuresaturation curves in static conditions. The small difference between coal tar/water curves at 293.15 K and 323.15 K might be explained by either the heterogeneity of coal tar or alteration of the coal tar/water mixture at a higher temperature.

Density, dynamic viscosity, interfacial tension, and contact angle results were used to calculate the relevant dimensionless numbers related to imbibition experiments (Table 2). Bond number, Bo, and viscosity ratio, M, could be calculated directly with the measured parameters. The intrinsic velocity calculated with Darcy law was used to calculate Ca numbers. The pressure difference used in this expression is the same as the one used in our experiments. The resulting definition for Ca is thus equivalent to the one proposed in [START_REF] Chatzis | Correlation of capillary number relationships for sandstone[END_REF]. Since drainage is mostly a stable process due to a favorable viscosity ratio, dimensionless numbers will be calculated in the case of imbibition, to verify the validity of Darcy's law. 4.19×10 -1 5.24×10 -5 1.48×10 -2 4.36×10 -1 5.46×10 -5 1.32×10 -2 293.15 4.16×10 -1 5.20×10 -5 1.96×10 -2 4.66×10 -1 5.82×10 -5 1.64×10 -2 303.15 4.13×10 -1 5.16×10 -5 2.55×10 -2 4.99×10 -1 6.23×10 -5 2.01×10 -2 313.15 4.09×10 -1 5.11×10 -5 3.26×10 -2 5.35×10 -1 6.69×10 -5 2.43×10 -2 323.15 4.05×10 -1 5.07×10 -5 4.11×10 -2 5.77×10 -1 7.21×10 -5 2.91×10 -2

We found the bond number to be slightly below 1 due to the small difference in density between the two phases. The flow between coal tar/water and oil/ethanol in 1 mm glass beads can, therefore, be described by Darcy's law. Oil/ethanol Bond number increases with temperature while that of coal tar/water decreases. This can be explained by the fact that the density difference ! '( ( " increases with temperature in the case of oil/ethanol but decreases for coal tar/water. The capillary number has been found to be low enough (10 -5 ) to justify that capillary forces prevail rather than viscous forces at the pore scale. In addition, no emergence of viscous fingers has been noted during the small pressure step experiments. Thus we can conclude that measurements of capillary pressure -saturation in our case has been done with respect to hypothesis behind generalized Darcy's law, namely the fact that the interface between both liquid phases is rigid and no fingering occurs.

It has been found that viscosity ratio, during imbibition, decreases by a factor 3 for coal tar/water and a factor of 2 for oil/ethanol. Here, the influence of temperature on the viscosity ratio was not as high as expected. Those variations were not sufficient to change the order of magnitude of the dimensionless values and to change the residual saturations. Increasing the temperature has two consequences for the imbibition process: increasing the viscosity ratio and decreasing the capillary number. Also, it appears that the effect of temperature on residual non-wetting phase saturation Sr,nw was almost negligible in both cases. However, previous viscous LNAPL and DNAPL displacement experiments have shown that the injection of hot water increases the recovery rate of the contaminant and reduces the residual saturation [START_REF] Jabbour | Oil recovery by steam injection: three-phase flow effects[END_REF][START_REF] O'carroll | Hot water flushing for immiscible displacement of a viscous[END_REF]. The experiments are done here by increasing the capillary pressure with small pressure steps. Several weeks are needed to complete a drainage and imbibition cycle experiment. The capillary numbers in our quasi-static experiments are still low (Ca << 1), which corresponds to a capillary fingering regime. In this case, the displacement structure is controlled by the fluctuations in the capillary threshold pressures at the displacement front. Thus, the viscosity ratio does not have a visible effect on the results. However, for high invasion rates, the dependence on M may become relevant because of the viscous dominated displacement.

In addition, the dimensionless numbers between coal tar/water and oil/ethanol are quite close. Considering that oil and coal tar are both non-wetting fluids, the same residual saturations and capillary curves between both fluid pairs should be expected. The use of oil/ethanol as a model fluid pair is thereby justified.

Dynamic effect on two-phase flow properties

As previously noted, two pore-volumes (PV) were needed to reach ethanol irreducible saturation. The injection of oil from the bottom (drainage) into the porous column saturated with ethanol was found to be a stable process at all flow rates. During this step, only ethanol was produced from the cell outlet and as soon as oil reached the cell outlet, only oil was. The displacement front was stable due to gravity effects and because oil's dynamic viscosity is higher than ethanol's (M > 1).

We saw a completely different trend for the injection of 10 PV of ethanol from the top of the cell (imbibition). The following observations were observed for the smallest flow rate (6 mL/min): the injection of 10 PV of ethanol lasted 38 minutes. Only oil was recovered during the first 6 minutes (0 to 1.6 PV). After that, ethanol bubbles started to flow out separately from the system until 10 minutes (1.6 to 2.6 PV). Then, ethanol and oil were both recovered as separate phases at the same time. In the tubing, oil was flowing near the walls while ethanol was recovered near the center (2.6 to 4 PV). Finally, after pumping for 15 minutes (4 to 10 PV), only ethanol was produced for the rest of the injection and the oil saturation did not change. The error bars show the difference between the saturation values calculated from the imaging technique and mass balance. For all saturation points, the maximum saturation difference between both methods is 0.05 (± 1 mL), which validates the proposed imaging technique to determine liquid saturation inside the cell. As shown in Figure 9a, the irreducible saturation Sir,w has been found not to depend on the flow rate. Its value varied between 0.03 (at 6 mL/min) and 0.07 (at 24 mL/min). Also, when the pump was stopped between the injection and pumping steps, there was a small time window where a saturation change occurred. This effect was even more noticeable for a low flow rate. During this time, we noticed saturation redistribution inside the cell likely due to end effects. There are indeed local saturation gradients inside the cell at pore scale that are more important in dynamic conditions. However, we do not take it into account because we mostly focused our study on macroscopic characteristics and measure the mean saturation over the whole cell. Conversely, oil pumping heavily depends on both flow rate and temperature (Figure 9b). First, higher oil residual saturations were obtained for higher flow rates. During the experiments, more ethanol fingers swept a lower area at a high flow rate. This phenomenon had already been observed and confirmed by [START_REF] Doorwar | Fingering function for unstable immiscible flows[END_REF]. Consequently, oil saturation decreased at a slower rate even though the pumping rate was higher. Another important point is that residual saturation was obtained with lower pore volumes for low flow rates. Almost 8 PV were needed to reach Sr,o = 0.29 at 24 mL/min while only 5 PV were needed with Sr,o = 0.06 at 6 mL/min. The comparison between both profiles at 12 mL/min (one obtained at 293.15 K and the other 323.15 K) shows that temperature had two effects. The oil recovery rate was increased at high temperature. During our fluid characterization, we noted that only the viscosity ratio decreased with temperature. Thus, fewer fingers are formed during the injection of ethanol inside oil at 323.15 K, and a higher volume of oil is recovered from the cell. In addition, the residual saturation is smaller at high temperature (0.06 at 323.15 K vs. 0.15 at 293.15 K). However, as we see before, the temperature had no effect on the residual saturation in the quasi-static experiments. Thus, from a certain flow rate (capillary number) where the dynamic effect becomes important, the temperature significantly affects the residual saturation. Finally, the results also underline that with increasing temperature, which leads to a lower viscosity ratio, oil recovery is enhanced at even higher flow rates. This means that the remediation time is shortened at higher temperatures. As mentioned before, we ran the experiments with a volumetric pump. This type of pump keeps a constant flow rate, and pressure adapts itself in consequence. Thus, a higher injection flow rate leads to a higher pressure difference in the system. Conversely, if the pumping flow rate is high, the depression needed will be bigger and can go under atmospheric pressure (defined as a relative pressure equal to 0 Pa here). It is also interesting to note that the pressure required to rotate the pump at 12 mL/min was also lower for higher temperature value (323.15 K).

The cell was at zero oil saturation at the beginning of the oil injection (Figure 8a). The pressure difference inside the cell slowly increased almost linearly until irreducible wetting (ethanol) saturation. The prescribed flow rate at the pump increased the measured pressure difference inside the cell, but no difference in saturation values was observed. At irreducible ethanol saturation, the pressure still increased due to the rise of ethanol level inside the ethanol reservoir situated above the cell.

The cell was at irreducible wetting saturation at the beginning of the oil pumping (Figure 8b). The pressure difference fell almost immediately as soon as pumping began. A higher flow rate translated into a higher initial pressure drop. Then the pressure difference increased linearly until ethanol breakthrough, time at which ethanol starts to be recovered. The pressure difference stabilized at non-wetting (oil) residual saturation.

The pressure needed to pump oil from the cell at 323.15 K was lower than that at 293.15 K. This effect is mainly linked to the reduction of dynamic viscosity. It is possible to interpret this from another point of view: The recovery rate of oil would be higher at 323.15 K than at 293.15 K initially, but the pumping done with the highest flow rate (26 mL/min) is also the pumping that generated the highest residual oil saturation (0.29). A certain balance between operating pressure and temperature needs to be determined for viscous DNAPLs in permeable media to recover the largest volume from the porous media in the smallest time.

A summary of all experiments, dimensionless numbers, irreducible saturations Sir,w, and residual saturations Sr,o obtained in this work are presented in Table 3. For dynamic experiments, the characteristic velocity was considered equal to the pump flow rate (in m 3 /s) divided by the porous medium section (5.6 cm * 2.5 cm) for capillary number calculations. As seen before for quasi-static experiments, the Bond number can be considered constant. This is because the parameters contributing to capillary forces (interfacial tension, wettability, pore geometry) are invariant with temperature.

Capillary numbers during dynamic experiments were very high (Ca >> 10 -2 ) compared to quasi-static ones (Ca≈10 -5 ). Viscous forces were much more predominant than capillary forces in that case. It should be noted that the reported capillary numbers were obtained in one-dimensional flow conditions. In a real site, the velocity field will change depending on the radial distance from the well. Studies in 2D and 3D experimental setups are needed to better understand the governing conditions that may impact the dynamic effects in real sites. During injection, the flow rates tested gave almost zero irreducible saturation: the oil was able to sweep almost all the ethanol initially inside the cell.

The capillary number and mobility ratio were both important to determine the effect of temperature on residual saturation in this case. First, the flow rate increased (Ca increased), leading to a higher residual saturation due to thinner and longer viscous fingers. Then, the dimensionless numbers were almost the same at 6 mL/min at 293.15 K and 12 mL/min at 323.15 K. Thus, it is logical to obtain the same residual saturation. However, it took 10 minutes to reach this value at 323.15 K instead of 40 minutes at 6 mL/min at 293.15 K, validating the use of temperature as a way to enhance DNAPL pumping. The generalized Darcy's law with the use of relative permeability is often used in order to understand the effect of temperature. However, in the case of injection or pumping in highly permeable media, dynamic effects must be considered and cannot be represented by this law accurately. The theoretical notion of relative permeability is compromised due to the presence of viscous fingering and ethanol films around the glass beads. In this situation, it is difficult to admit the hypothesis that both phases are flowing separately in the medium. The fact that temperature reduces oil residual saturation appears to be linked to this.

Also, we noted the presence of ethanol films in all experiments under dynamic conditions. These films are generally not taken into account in numerical studies considering the effect of temperature.

Their emergence was even higher when the temperature had been increased. No experimental study has been conducted on fluid film behavior, and it is, therefore, difficult to highlight if this is an experimental artifact due to the fluids used or a phenomenon that should be taken into account. Yet, when approaching residual saturation values, the number of films decreases and becomes even less visible as the capillary number becomes high. The presence of these films has been observed in the past in trickle-bed reactors [START_REF] Reinecke | Investigation of the two-phase flow in trickle-bed reactors using capacitance tomography[END_REF]. The solid particle size used in this type of reactors is in the millimeter range, like the ones studied here. This leads us to conclude that the presence and retention of these films inside a porous medium are mostly linked to the high capillary number encountered in highly permeable porous media. [START_REF] Moura | Connectivity enhancement due to film flow in porous media[END_REF] have also observed the presence of capillary bridges during drainage experiments. Their results show that film flow can enhance the recovery of the trapped liquid [START_REF] Moura | Connectivity enhancement due to film flow in porous media[END_REF]. This is coherent with our observations: more films are present during imbibition at 323.15 K than at 293.15 K and the resulting ethanol saturation is then reduced.

Pressure difference and ethanol saturation data have been used in order to determine relative permeability curves with the JBN method [START_REF] Welge | A simplified method for computing oil recovery by gas or water drive[END_REF][START_REF] Johnson | Dynamic Capillary Pressure Curve for Water/Oil Displacement in Porous Media: Theory vs[END_REF]. [START_REF] Qin | Experimental studies on effects of temperature on oil and water relative permeability in heavy-oil reservoirs[END_REF] previously measured oil and water relative permeabilities in sand packs and determined that temperature effectively increases both fluids relative permeabilities [START_REF] Qin | Experimental studies on effects of temperature on oil and water relative permeability in heavy-oil reservoirs[END_REF]. Figure 11 represents the effect of temperature on oil and ethanol relative permeability measured in pumping conditions at 12 mL/min. Oil relative permeabilities have been found to have an exponential profile at 293.15 K and an almost linear profile at 323.15 K. Ethanol relative permeabilities have a small magnitude compared to oil relative permeability, due to the high viscosity ratio considered in our study. In addition, ethanol relative permeability increases with temperature. For all saturations, oil and ethanol relative permeabilities are higher at 323.15 K rather than at 293.15 K. This is due to the difference in saturation profiles between pumping at 293.15 K and 323.15 K. Both oil and ethanol are thus able to flow at a faster rate in pumping applications for higher temperature.

Conclusions

Changing soil temperature during DNAPL pure phase recovery involves analyses of the parameters used in generalized Darcy's law. Hypotheses behind the use of this equation were recalled and it appears that in soil remediation applications, wetting phase film and viscous fingering may appear under dynamic conditions. The influence of temperature on a coal tar sampled from a field was investigated. Density, dynamic viscosity, interfacial tension, and contact angle parameters for coal tar/water and oil/ethanol (a model two-phase liquid) have been measured between 293.15 K and 323.15 K. Dynamic viscosities of all studied liquids decrease exponentially with temperature. Liquids densities and interfacial tensions between the two immiscible coupled fluids decrease linearly with temperature. These parameters are all important in the capillary and viscous forces balance determining the flow regime. The studies on the expected flow pattern have been investigated by [START_REF] Lenormand | Numerical models and experiments on immiscible displacements in porous media[END_REF]. The further analysis of dimensionless numbers between both fluid pairs showed that it is possible to use oil and ethanol as a model fluid for studying coal tar-water twophase flow in porous media.

We performed drainage and imbibition experiments in a 1D porous cell under almost quasi-static (Ca < 10 -5 ) conditions and in the application range of generalized Darcy's law. During the experiments, no viscous fingering was noted. Nor was a temperature effect observed on two-phase flow van Genuchten parameters or residual saturations for both couples of liquids at 293.15 K and 323.15 K. It should be noted that capillary forces are relatively small in these cases. This is because of the highly permeable porous medium used and the low interfacial tension of the considered liquids.

In all our drainage/imbibition experiments, the residual saturations and observations obtained with canola oil and ethanol experiments were close to the ones observed with coal tar and water. However, it is considerably easier to obtain results with simple and almost not toxic liquids, like canola oil and ethanol, especially by image analyzing techniques. This type of simplification is done with glass beads in experiments to model a simpler and inert porous medium with the same permeability and porosity.

Finally, we further studied the effect of temperature by pumping oil in the small cell filled by the glass beads at irreducible ethanol saturation and 323.15 K. Increasing the temperature reduces the pressure difference needed to reach the same pumping rate, increases the recovery rate of oil from the porous medium (Sr,o = 16% down to 6% when heating from 293.15 K to 323.15 K), and finally increases both oil and ethanol relative permeabilities. The higher recovery rate is linked not only to reduced viscous fingering but also to the presence of interfacial films that seem to appear because of the high capillary numbers (Ca > 10 -2 ) encountered in highly permeable media. Residual saturation has also been found to decrease considerably due to decreasing the viscous fingering occurred at low flow rate. The presence and growth of viscous fingerings allow the ethanol to flush a larger part of the medium. The experiments performed under dynamic conditions have shown that dynamic effects are important when considering temperature effects during pumping operations. In complement to our study which focuses on macroscopic quantities, it would be interesting to extract local and microscopic quantities to investigate more thoroughly the impact of temperature on twophase flow in a real site. The coupling between heat transfers and two-phase flow should be also considered in order to better understand the physical mechanisms happening during thermally enhanced DNAPL pumping.

Figure 1 :

 1 Figure 1: Changes in canola oil, salty ethanol, water, and coal tar densities between 277.15 K and 323.15 K

Figure 2 :

 2 Figure 2: Dynamic viscosity of salty ethanol, canola oil, water, and coal tar as a function of temperature. Lines represent data fitting with Andrade two-parameter exponential function

Figure 3 :

 3 Figure 3: Interfacial tension of canola oil/ethanol and coal tar/water as a function of temperature

Figure 4 :

 4 Figure 4: Evolution of coal tar/water and canola oil/ethanol contact angle on a glass slide

Figure 5 :

 5 Figure 5: Drainage/Imbibition setups used for (a) capillary pressure-saturation measurements and (b) setup used for experiments in dynamic conditions

Figure 6 :

 6 Figure 6: Images taken during (a) injection at 12 mL/min and (b) pumping at 6 mL/min of oil inside 1 mm glass beads initially saturated with ethanol. As oil is being injected, a sharp horizontal interface is being pushed in. On the contrary, when oil is being pumped, viscous fingers appear and trap the oil ganglia

Figure 7 :

 7 Figure 7: Data from triplicates experiments and averaged fitted capillary pressure-water saturation curves at 293.15 K for coal tar/water (primary drainage)

Figure 8 :

 8 Figure 8: Effect of temperature on capillary pressure-saturation curves for a) oil/ethanol primary curves, b) oil/ethanol main curves, c) coal tar/water primary curves, d) coal tar/water main curves

Figure 9 :

 9 Figure 9: Evolution of ethanol saturation as a function of flow rate during a) the injection (2 PV) and b) pumping (10 PV) of oil

Figure 10 :

 10 Figure 10: Measured pressure difference during injection (a) and pumping (b) of oil

Figure 11 :

 11 Figure 11: Oil and ethanol relative permeabilities estimated during pumping at 12 mL/min at 293.15 K and 323.15 K

Table 1 :

 1 Summary of all drainage-imbibition experiments in the 1D cell

	Fluids	T (K)	Step	α (m -1 )	n	Sir,w	Sr,o	β
	Oil/Ethanol 293.15	Drainage 1	5.52±0.22 3.17±0.58 0.10±0.02	0	0.085
	Oil/Ethanol 293.15 Imbibition 1 4.90±0.30 3.57±0.45 0.10±0.02 0.14±0.02	0.096
	Oil/Ethanol 293.15	Drainage 2	4.03±0.39 4.19±0.31 0.13±0.03 0.14±0.02	0.116
	Oil/Ethanol 293.15 Imbibition 2 4.22±0.52 4.16±0.35 0.13±0.03 0.15±0.02	0.111
	Oil/Ethanol 293.15	Drainage 1	6.22±0.43 2.96±0.50 0.11±0.02	0	0.075
	Oil/Ethanol 323.15 Imbibition 1 5.34±0.45 3.09±0.38 0.11±0.02 0.13±0.03	0.104
	Oil/Ethanol 323.15	Drainage 2	4.17±0.27 3.89±0.25 0.14±0.04 0.13±0.03	0.133
	Oil/Ethanol 323.15 Imbibition 2 4.11±0.48 4.10±0.42 0.14±0.04 0.15±0.04	0;135
	Coal tar/Water	293.15	Drainage 1	7.19±0.25 5.87±0.99	0.12±0.03	0	0.058
	Coal tar/Water	293.15	Imbibition 1 6.26±0.68 10.1±1.94	0.12±0.03 0.13±0.02	0.066
	Coal tar/Water	293.15	Drainage 2	5.18±0.44 9.86±1.33	0.17±0.05 0.13±0.02	0.080
	Coal tar/Water	293.15	Imbibition 2 5.53±0.50 12.1±2.29	0.17±0.05 0.16±0.03	0.075
	Coal tar/Water	293.15	Drainage 1	8.20±1.0 6.58±0.67	0.11±0.05	0	0.051
	Coal tar/Water	323.15	Imbibition 1	8.56±1.1 8.46±0.69	0.11±0.05 0.12±0.03	0.052
	Coal tar/Water	323.15	Drainage 2	7.88±0.67 7.84±0.57	0.14±0.03 0.12±0.03	0.057
	Coal tar/Water	323.15	Imbibition 2 7.96±0.83 9.67±1.09	0.14±0.03 0.14±0.03	0.056
	For canola oil/ethanol and coal tar/water, the differences observed in capillary pressure-saturation
	curves between 293.15 K and 323.15 K are within the standard deviation and could not be attributed
	to temperature effect (Figure 8). However, for coal tar/water and the same water saturation values,
	the capillary pressure is lower at 323.15 K than at 293.15 K. The previous measurements indicate that
	temperature changes are almost negligible for density, interfacial tension and contact angle between
	293.15 K and 323.15 K.

Table 2 :

 2 Bond, capillary numbers and viscosity ratio associated with the two-phase flow of oil/ethanol and coal tar/water between293.15 K and 323.15 K

	Liquids		Coal tar -water			Canola oil -ethanol
	T (K)	Bo	Ca	M	Bo	Ca	M
	283.15

Table 3 :

 3 Summary of all conducted experiments with their associated Bond number, Capillary number, viscosity ratio, irreducible saturations Sir,w , residual saturation Sr, and image observations

	Experiment	Temperature (K)	Bo	Ca	M	Sir,w or Sr,o	Observations
							Stable flow with
	Quasi-static drainage	293.15	4.66×10 -1 5.82×10 -5 6.19×10 1 0.10±0.02	low capillary
							fingering
							Stable flow with
	Quasi-static drainage	323.15	5.77×10 -1 7.21×10 -5 3.40×10 1 0.11±0.02	low capillary fingering and low
							film
	Injection (6 mL/min)	293.15	4.66×10 -1 3.87×10 0 6.19×10 1	0.05	Stable flow
	Injection (12 mL/min)	293.15	4.66×10 -1 7.75×10 0 6.19×10 1	0.07	Stable flow
	Injection (24 mL/min)	293.15	4.66×10 -1 1.50×10 1 6.19×10 1	0.03	Stable flow
							Transition
	Quasi-static imbibition	293.15	4.66×10 -1 5.82×10 -5 1.64×10 -2 0.14±0.02	between capillary and viscous
							fingering
	Pumping (6 mL/min)	293.15	4.66×10 -1 6.50×10 -2 1.64×10 -2	0.06	High films and low viscous fingering
							Moderate films
	Pumping (12 mL/min)	293.15	4.66×10 -1 1.30×10 -1 1.64×10 -2	0.16	and viscous
							fingering
	Pumping (24 mL/min)	293.15	4.66×10 -1 2.60×10 -1 1.64×10 -2	0.29	Low films and high viscous fingering
	Pumping (12 mL/min)	323.15	5.77×10 -1 4.80×10 -2 2.91×10 -2	0.06	High films, low viscous fingering
							Transition
	Quasi-static imbibition	323.15	5.77×10 -1 7.21×10 -5 2.91×10 -2 0.13±0.03	between capillary and viscous
							fingering

Acknowledgments

This study was performed as part of the "Bioxyval" project. The authors would like to thank ADEME for co-funding the project under the "AMI" program, REMEA, and ANRT for providing the PhD grant for Nicolas Philippe. We gratefully acknowledge the financial support provided to the PIVOTS project by the "Région Centre -Val de Loire" and the European Regional Development Fund.

Canola oil w Wetting phase (ethanol or water) W Water nw Non-wetting phase (canola oil or coal tar) '( ( ! ( " ! 2 "


Louis Delaunay

Emeline Cozien

Pierre Gehannin

Nassim Mouhali

Sabrina Mace

Florence Postollec

Ivan Leguerinel

Anne-Gabrielle Mathot
email: mathot@univ-brest.fr

Occurrence and diversity of thermophilic sporeformers in French dairy powders

This study reports the prevalence of sporeforming bacteria and contamination levels of a variety of powdered dairy products from cows', ewes', goats' and mares' milk produced in France. The concentration of mesophilic spores, thermophilic spores and highly heat-resistant thermophilic spores was assessed in 61 dairy powders. Thermophilic spore concentration was highly variable between powders, likely due to the different manufacturing processes used for transforming milk into dairy powders. The different stages of processing, particularly heat treatment close to 55 °C had a strong impact on selection of thermophilic bacteria contaminating dairy powders. For some products, thermophilic spore counts were as high as 5.89 log TSC g -1 . Of the 313 thermophilic isolates selected, 93.3% belonged to the species Geobacillus stearothermophilus (43.5%), Bacillus licheniformis (30.0%) and Anoxybacillus flavithermus (19.8%). These results confirm the presence of the three majority species reported worldwide.

Introduction

The manufacture of powdered products is an important part of the dairy industry because of their long shelf life and ease of use. France is one of the leading producers and processors of milk in Europe. Although non-pathogenic, spore-forming bacteria and more specifically thermophilic aerobic species have become an important parameter for monitoring production hygiene in this type of product. Three species dominate this group of bacteria. They include Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus licheniformis [START_REF] Rückert | A RAPD-based survey of thermophilic bacilli in milk powders from different countries[END_REF]).

In the dairy industry, the provenance of bacterial spores is highly diverse as spores originate from soil [START_REF] Heyndrickx | The importance of endospore-forming bacteria originating from soil for contamination of industrial food processing[END_REF], water [START_REF] Christiansson | Bacillus cereus spores in raw milk: Factors affecting the contamination of milk during the grazing period[END_REF] or silage [START_REF] Giffel | Bacterial spores in silage and raw milk[END_REF]. The spores can be consumed by cows, resist transit and end up in the faeces, from which they can contaminate the milk, through dirty teats and thus entering milking equipment (Miller, Kent, Boor, Martin, & Wiedmann, 2015a). Contamination can also occur during the transport of milk from the farm to the dairy industry [START_REF] Huck | Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product[END_REF]. All these sources of contamination contribute to explaining the presence of bacterial spores in raw milk [START_REF] Huck | Tracking sporeforming bacterial contaminants in fluid milk-processing systems[END_REF]. Concentrations of mesophilic bacterial spores can be as high as 3.88 log mL -1 in raw milk [START_REF] Mcguiggan | Aerobic spore-forming bacteria in bulk raw milk: Factors influencing the numbers of psychrotrophic, mesophilic and thermophilic Bacillus spores[END_REF]. More specifically, thermophilic spores have been found at concentrations ranging from 0.26 to 2.41 log spore mL -1 in raw milk [START_REF] Buehner | Prevalence of thermoduric bacteria and spores on 10 Midwest dairy farms[END_REF][START_REF] Murphy | Bedding and bedding management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk[END_REF].

Those thermophilic spores are mainly represented by Bacillus licheniformis but also slightly by Geobacillus stearothermophilus or Anoxybacillus flavithermus (from not detected to minus than 2%; [START_REF] Chauhan | Rapid identification of dairy mesophilic and thermophilic sporeforming bacteria using DNA high resolution melt analysis of variable 16S rDNA regions[END_REF].

In addition to milk contamination and the concentration effect by processing, it has been shown that spore-forming (especially thermophilic) bacteria can multiply during processes and thereby persist in industrial sites as a biofilm [START_REF] Scott | The formation of thermophilic spores during the manufacture of whole milk powder[END_REF].

After pasteurisation, which has no effect on spores, the manufacturing processes for dairy powders differ according to the raw material and desired end product.

Milk powders (whole or skimmed) and whey powders are obtained by concentration in evaporators followed by atomisation, while milk protein or whey protein powders need an ultrafiltration before concentration and atomisation (see [START_REF] Mchugh | Detection and enumeration of spore-forming bacteria in powdered dairy products[END_REF] for nice scheme). In addition, other casein precipitation processes such as acid, alkali or rennet can be used. These different processes lead to different temperatures, aw, pH conditions and durations that will select and influence the development of microorganisms. Cleaning in place operations can also affect contamination levels.

Infant formulas are another category of powder products of dairy origin, which can be manufactured either by dry formulation (mixing of powders) or wet formulation (mixing of liquid ingredients), concentrated and atomised [START_REF] Happe | Infant formula[END_REF].

Studies on the prevalence of mesophilic or thermophilic spore-forming bacteria have mainly focused on whole milk powders (WMPs) and skimmed milk powders (SMPs) [START_REF] Dettling | Accurate quantification of thermophilic spores in dairy powders[END_REF]Miller et al., 2015b;[START_REF] Reginensi | RAPDbased screening for spore-forming bacterial populations in Uruguayan commercial powdered milk[END_REF][START_REF] Rückert | A RAPD-based survey of thermophilic bacilli in milk powders from different countries[END_REF][START_REF] Sadiq | A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China[END_REF]. However, few other studies have focused on whey powders, whey protein powders and even infant formula powders (Miller et al., 2015b;[START_REF] Sadiq | A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China[END_REF][START_REF] Yuan | A survey on occurrence of thermophilic bacilli in commercial milk powders in China[END_REF][START_REF] Zain | The potential source of B. licheniformis contamination during whey protein concentrate 80 manufacture[END_REF].

The objective of this study was to evaluate the contamination levels and prevalence of sporeforming bacteria in French dairy powders targeting specific products of interest for dairy powder production from cows', ewes', goats' and mares' milk.

Material and methods

Sampling and spore enumeration

A total of 61 French dairy powders was collected from 19 industrial plants on a voluntary base. Powders were derived from cows', ewes', goats' and mares' milk. Analysed samples were composed of WMP, SMP, whey powder (WP), whey protein concentrate (WPC), milk protein concentrate (MPC), casein powder (CP), caseinate powder (CtP), permeate powder (PP) and infant formula (IF).

Analyses were performed according to ISO 6887-5 (ISO, 2010) for milk solutions reconstitution; to NF V08-250 (NF, 2010), for mesophilic spore counts (MSC) and thermophilic spore counts (TSC), and according to ISO standard ISO/TS 27265:2009 (106 °C for 30 min; ISO, 2009) for highly heat-resistant thermophilic spore count (HRTSC).

All bacterial spores were enumerated on agar according to ISO/TS 27265 [START_REF] Iso | Dried milk -Enumeration of the specially thermoresistant spores of thermophilic bacteria[END_REF]. Counts were performed in duplicate for each powder. The enumeration data were processed with Statistica version 13.5.0.17.

Isolate recovery

Thermophilic spore-forming bacterial counts were used to select isolates for further identification. Based on the colony morphology diversity, a proportional number of isolates were selected and isolated twice on TSA (Biokar Diagnostics) + potato starch 0.2% (Panreac). Selected isolates were stored in a 20% sterile glycerol solution at -80 °C.

DNA extraction and isolate identification

Procedures used for DNA extraction, M13 PCR amplification, gel migration, clustering fingerprints, isolates selection for 16S rDNA sequencing, and identification, are similar to [START_REF] Ronimus | A RAPDbased comparison of thermophilic bacilli from milk powders[END_REF] and are fully explicated in Supplementary material.

Results and discussion

Milk powders analysed in this study can be classified into four major groups. This included powders processed with evaporation and atomisation (WMP; SMP and WP), which have to be redivided because of their protein composition (into WMP and SMP on the one hand and WP on the other); powders processed with filtration step (MPC and WPC); powders made of reconstitution (IF).

Finally, we arbitrarily grouped in "others" the remaining samples (CP, CtP, PP) that did not fit into these categories and for which there are few samples analysed. Enumerations and diversity between those four groups is different (Table 1).

The high concentrations of thermophilic spores in WMP or SMP (Fig. 1) from cows' milk may be explained by the temperature conditions during manufacturing processes, which were favourable for the development of thermophilic bacteria and the formation of biofilms [START_REF] Zhao | Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers[END_REF].

Indeed, in plate heat exchangers used for pasteurisation, preheaters used before evaporation, and evaporators, temperatures classically range from 45 to 70 °C [START_REF] Goff | Evaporation and dehydration[END_REF]. In WMP and SMP, G.

stearothermophilus seems to be predominant, followed by B. licheniformis and A. flavithermus.

International studies demonstrate that B. licheniformis is the main contaminant in some WMP [START_REF] Rückert | A RAPD-based survey of thermophilic bacilli in milk powders from different countries[END_REF][START_REF] Sadiq | A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China[END_REF] and SMP [START_REF] Sadiq | A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China[END_REF]. However, other studies demonstrate that A. flavithermus is also the main contaminant of some WMP [START_REF] Dettling | Accurate quantification of thermophilic spores in dairy powders[END_REF] and SMP [START_REF] Dettling | Accurate quantification of thermophilic spores in dairy powders[END_REF][START_REF] Ronimus | A RAPDbased comparison of thermophilic bacilli from milk powders[END_REF]. Results obtained in our study are in agreement with worldwide one where G. stearothermophilus reaches more than 22% of identified isolates [START_REF] Sadiq | A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China[END_REF]. The contamination level of WMP from ewes' and goats' milk seems less important (average: 1.68 log TSC g -1 ) and could be linked to the size of the industry or to the run duration, in addition, no thermophilic spores were quantified in mare milk probably because of the process used (lyophilisation).

Whey can be concentrated and evaporated directly, or fractioned by filtration, giving different types of dairy powders, protein powders or permeate powders [START_REF] Snappe | Protéines laitières[END_REF]. The high contamination of WP can be explained by evaporation steps like WMP and SMP, and filtration (for some of them), increasing the bacterial population rate. where whey protein and lactose concentrations and temperatures are favourable for its development.

For casein powder (CP), the high level of thermophilic bacteria is due to use of rennet. There was no modification of pH value and bacteria were concentrated in the protein part. Moreover, during casein power production, rennet casein is washed with hot water (45/60 °C) which may promote thermophilic bacteria growth. The low concentration of bacterial spores in CtP may be explained by the combination of acid and alkaline treatments as well as the absence of heat treatment in plate heat exchangers or evaporation before drying. To our knowledge, no comparison data for CP or CtP are available in the bibliography. Permeate powders originate from the liquid part obtained by ultrafiltration of milk or whey (bacteria and proteins are retained in the retentate). This observation explains the absence or very limited level of 1.18 log TSC g -1 in one of the four permeate powder samples analysed. No correlation between diversity and process can be done due to the lack of isolates obtained from those types of powder.

Infant powders are produced by blending various milk ingredients (SMP, lactose and WPC) from the fractionation of milk constituents to approximate the composition of human breast milk. It is, therefore, difficult to explain the variability in spore concentrations observed by the impact of the manufacturing process. This difference in composition may explain the high presence of G.

stearothermophilus, followed by A. flavithermus and B. licheniformis. These results are not in accordance with studies of [START_REF] Yuan | A survey on occurrence of thermophilic bacilli in commercial milk powders in China[END_REF] that demonstrated that A. flavithermus is the most prevalent contaminant with more than 40.4% of isolates obtained. However, [START_REF] Sadiq | A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China[END_REF] demonstrated that B. licheniformis was the most predominant isolate with more than 47.2%. Those two studies shown that G. stearothermophilus was present at a level of 21% and 17.1% respectively.

The contamination level of thermophilic spores in goat and ewe formula (average: 1.72 log TSC g -1 )

seems equal to IF from cow milk, but mesophilic concentration is higher (average: 2.51 log MSC g -1 versus 1.06 log MSC g -1 in cows' milk IF).

Regarding enumeration of TSC present in the four groups, ANOVA analysis (Table 1) can classify them in two groups (a and b) resulting in a high contamination for WMP and SMP compared with IF (group b). WP, WPC and MPC are part of groups a and b due to of their median TSC contamination.

The high concentration of HRTSC (Fig. 1; Table 1) in WMP and SMP seems to be related to the presence of G. stearothermophilus and A. flavithermus, unlike MPC and WPC that were mainly contaminated by B. licheniformis resulting of low amount of HRTSC.

Besides the three major species, other species also isolated from the dairy environment or ingredients such as Aneurinibacillus thermoaerophilus, Bacillus smithii, Brevibacillus brevis, Bacillus thermoamylovorans, Bacillus coagulans and Brevibacillus bortelensis [START_REF] Lücking | Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage[END_REF]Miller et al., 2015b;[START_REF] Ronimus | Survival of thermophilic spore-forming bacteria in a 90+ year old milk powder from Ernest Shackelton's Cape Royds Hut in Antarctica[END_REF][START_REF] Scheldeman | Incidence and diversity of potentially highly heat-resistant spores isolated at dairy farms[END_REF][START_REF] Yuan | A survey on occurrence of thermophilic bacilli in commercial milk powders in China[END_REF] or never isolated from dairy environment such as Bacillus ginsengihumi and Paenibacillus naphthalenovorans accounted for 6.71% of all the isolates in our study.

Calculated Simpson Index (1/D) [START_REF] Simpson | Measurement of diversity[END_REF] on powder type did not highlight any difference between them, but the strain diversity reflected by the M13 PCR footprint shows that diversity of A. flavithermus (1/D = 4.17) is higher (Table 1) than G. stearothermophilus (1/D = 2.22) and B. licheniformis (1/D = 1.72).

Conclusion

The presence and concentration of mesophilic and thermophilic spore-forming bacteria in dairy powders varies according to the type of powder analysed. At each stage of milk or whey processing, the environmental conditions such as temperature, pH or composition, may or may not promote their proliferation, biofilm formation and sporulation in the raw material or residual biofilms. Furthermore, concentration processes, including evaporation, filtration or precipitation, influence the concentrations of bacterial spores in dairy powders. Thermophilic spore forming bacteria are used as a hygiene indicator if the cleaning in place procedure is not well respected. Due to their resistance to processes and cleaning products, it is important to monitor these thermophilic spores to limit their proliferation and potential impact in products using the reconstituted powders.

Future experiments will be conducted on the phenotypic diversity of thermophilic spores, particularly their capacity to form biofilms and their resistance to different cleaning treatments.

Figure legend

Fig

Log cfu g -1

Table 1 Median of enumeration of mesophilic (MSC), thermophilic (TSC) and highly heat resistant thermophilic spores (HRTSC) in the four types of dairy powders studied and diversity of TSC isolated.

  The spore diversity is quite similar to that for WMP and SMP, where G. stearothermophilus is predominant

	(52%), but A. flavithermus is more present in WP (29%), which is in parallel with the study from Miller
	et al. (2015b) who found, in acid WP, that those two sporeformers represent 27.1% and 38%,
	respectively.
	Concentration of thermophilic spores of proteins obtained from whey (WPC) or milk (MPC) is
	consistent with the work of Kent, Chauhan, Boor, Wiedmann, and Martin (2016). Bacterial spores are

retained by the filtration membrane into the retentate

[START_REF] Chamberland | Biofouling of ultrafiltration membrane by dairy fluids: Characterization of pioneer colonizer bacteria using a DNA metabarcoding approach[END_REF]

. Besides predominance of B. licheniformis in this type of powder was explained by

[START_REF] Zain | The potential source of B. licheniformis contamination during whey protein concentrate 80 manufacture[END_REF]

by the development upstream of ultrafiltration processes in tanks or plate exchangers,

  . 1. Enumeration of mesophilic spores (▤, 80 °C 10 min, incubation 37 °C), thermophilic spores (▧, CtP, caseinate powder; MPC, milk protein concentrate; WP, whey powder; WPC, whey protein concentrate; PP, permeate powder; IF, infant formula.

	80 °C 10 min, incubation 55 °C) and heat resistant thermophilic spores (▨, 106 °C 30 min, incubation
	55 °C) in milk powders: □, median; box, 25 e percentiles; whisker, non-outlier min and max
	(coefficient 1); ○, outlier values (coefficient 1.5). WMP, whole milk powder; SMP, skimmed milk
	powder; CP, casein powder;

  a a Abbreviations are: WMP, whole milk powder; SMP, skimmed milk powder; WP, whey powder; WPC, whey protein concentrate; MPC, milk protein concentrate; IF, infant formula (Others are permeate powder, casein powder, caseinate powder); SE, standard error; NA, not applicable.

	Parameter	WMP / SMP WP	WPC / MPC IF	Others Total Simpson
							index (1/D)
	N samples	15	8	10	18	10	61
	Median MSC / SE	2.60 / 0.46 1.57 / 0.56 2.37 / 0.68 1.51 / 0.67 NA	NA
	Median TSC / SE	4.02 / 1.47 1.94 / 0.80 2.78 / 0.53 1.60 / 0.83 NA	NA
	Median HRTSC / SE	1.81 / 1.27 1.24 / 0.26 0 / 0.48	0.94 / 0.98 NA	NA
	ANOVA Grouping TSC	a	a/b	a/b	b	NA	NA
	Total TSC isolates	108	44	67	83	11	313
	A. flavithermus (%)	17	29	22	18	NA	19.8 4.17
	B. licheniformis (%)	25	14	63	17	NA	30.0 1.72
	G. stearothermophilus (%) 50	52	9	59	NA	43.5 2.22
	Others (%)	8	5	6	6	NA	6.7
	Simpson index (1/D)	2.94	2.78	2.27	2.50	NA

Acknowledgements

The authors acknowledge the support provided by "Quimper Bretagne Occidentale" and the regional council of Brittany (Région Bretagne, France).