Célia Chamignon
email: celia.chamignon@inrae.fr

Virgile Guéneau
email: virgile.gueneau@inrae.fr

Sonia Medina

Julien Deschamps
email: julien.deschamps@inrae.fr

Angel Gil-Izquierdo
email: angelgil@cebas.csic.es

Romain Briandet
email: romain.briandet@inrae.fr

Pierre-Yves Mousset
email: py.mousset@gynov.com

Philippe Langella
email: philippe.langella@inrae.fr

Sophie Lafay
email: s.lafay@jenwin.fr

Luis G Bermúdez-Humarán

Evaluation of the Probiotic Properties and the Capacity to Form Biofilms of Various Lactobacillus Strains

Keywords: Lactobacillus, probiotics, permeability, biofilm, stress tolerance

Over the last 20 years, Lactobacillus species inhabiting the gastrointestinal tract (GIT) have received much attention, and their health-promoting properties are now well-described. Probiotic effects cannot be generalized, and their uses cover a wide range of applications. It is thus important to proceed to an accurate selection and evaluation of probiotic candidates. We evaluate the probiotic potential of six strains of Lactobacillus in different in vitro models representing critical factors of either survival, efficacy, or both. We characterized the strains for their ability to (i) modulate intestinal permeability using transepithelial electrical resistance (TEER), (ii) form biofilms and resist stressful conditions, and (iii) produce beneficial host and/or bacteria metabolites. Our data reveal the specificity of Lactobacillus strains to modulate intestinal permeability depending on the cell type. The six isolates were able to form spatially organized biofilms, and we provide evidence that the biofilm form is beneficial in a strongly acidic environment. Finally, we demonstrated the ability of the strains to produce γ-aminobutyric acid (GABA) that is involved in the gut-brain axis and beneficial enzymes that promote the bacterial tolerance to bile salts. Overall, our study highlights the specific properties of Lactobacillus strains and their possible applications as biofilms.

Introduction

For centuries, lactic-acid bacteria (LAB) have been widely used as starters cultures in food fermentation and dairy products. Lactobacillus species have been largely studied as part of a healthy gut microbiota, and investigators have proposed their uses as probiotics: live microorganisms that confer a health benefit to the host when administered in appropriate amounts [START_REF] Hill | The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic[END_REF]. Although the underlying interactions with the host are strain-dependent, the general mechanisms of action by which probiotics exert their beneficial effects include enhancement of the intestinal epithelial barrier, the ability to adhere to the intestinal mucosa, the competitive exclusion of pathogens, the production of antimicrobial substances, and modulation of the immune system [START_REF] Bermudez-Brito | Probiotic Mechanisms of Action[END_REF].

In their natural habitats, the microorganisms are found in a spatially organized form called biofilms, "a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other and are embedded in a matrix of extracellular polymeric substances that they have produced" [START_REF] Donlan | Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms[END_REF]. The biofilm form provides the bacteria with several properties, such as tolerance to the action of antimicrobial peptides and other stressful conditions of the body [START_REF] Flemming | Biofilms: An emergent form of bacterial life[END_REF], as well as improving the health of the host in the case of Lactobacillus spp. through their beneficial effects. Several studies have shown that the biofilm formation can boost the probiotic activity by enhancing immunomodulatory properties and intestinal permeability, among other actions [START_REF] Olson | An Enhanced Lactobacillus Reuteri Biofilm Formulation that Increases 2 Protection Against Experimental Necrotizing Enterocolitis[END_REF][START_REF] Rieu | The biofilm mode of life boosts the anti-inflammatoryproperties of Lactobacillus[END_REF].

Dysbiosis of the gut microbiota has been clearly linked to diverse pathological conditions, such as irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD), for which a direct connection has been established, as well as in less obvious disorders, such as metabolic syndrome or behavioral disorders (depression and autism spectrum disorders) [START_REF] Gilbert | Microbiome-wide association studies link dynamic microbial consortia to disease[END_REF]. Several studies, from in vitro experimentation to clinical trials, have demonstrated a key role of intestinal permeability in these diseases. Moreover, a beneficial effect of Lactobacillus species on intestinal permeability has been demonstrated in several conditions, such as obesity [START_REF] Kawano | Lactobacillus gasseri SBT2055 inhibits adipose tissue inflammation and intestinal permeability in mice fed a high-fat diet[END_REF], IBS [START_REF] Han | Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids[END_REF], alcoholic liver disease [START_REF] Zhao | Inhibition of miR122a by Lactobacillus rhamnosus GG culture 2 supernatant increases intestinal occludin expression and protects mice 3 from alcoholic liver disease[END_REF], and stress-mediated alterations and associated visceral sensitivity [START_REF] Ait-Belgnaoui | Lactobacillus farciminis treatment suppresses stress induced visceral hypersensitivity: A possible action through interaction with epithelial cell cytoskeleton contraction[END_REF]. The manipulation of intestinal permeability can thus be proposed as a target not only for the prevention but, also, the therapy of human diseases [START_REF] Camilleri | Leaky gut: Mechanisms, measurement and clinical implications in humans[END_REF].

Due to the wide range of applications of probiotics and their various mechanisms of action, the FAO/WHO has proposed a "Guidelines for Evaluation of Probiotics in Food" [START_REF] Fao | Guidelines for the Evaluation of Probiotics in Food[END_REF], suggesting safety and effectiveness criteria for their evaluation. These criteria include a resistance to the stressful conditions encountered in the human body, the ability to adhere to the mucosa, the secretion of antimicrobial molecules, a safety assessment, and evidence of a benefit for human health [START_REF] De Melo Pereira | How to select a probiotic? A review and update of methods and criteria[END_REF].

We aimed to determine the potential probiotic properties of six different Lactobacillus strains. We selected the Lactobacillus strains on their ability to enhance intestinal permeability using the transepithelial electrical resistance (TEER) of Caco-2 and T84 intestinal epithelial cells. We then evaluated some characteristics of the probiotic candidates, such as their capacity to adhere on the intestinal epithelial cells Caco-2 and HT-29 MTX and to produce beneficial host/bacteria metabolites. We further characterized the strain by evaluating their capacity to form biofilms and the benefits associated with this microbial lifestyle versus a planktonic lifestyle.

Materials and Methods

Bacterial Strains and Growth Conditions

The origin, identity, and growth conditions of the strains that were used are presented in Table 1.

Strains were stored at -80 • C in PBS X-1 + 16% (v/v) glycerol. Before experiments, strains were grown overnight, under their respective growth conditions, from frozen stocks and passaged one time before use. In all experiments, the strain of Lactobacillus rhamnosus GG was used as a positive control.

Cell Cultures

The human colorectal adenocarcinoma cell lines Caco-2 and T84 and the mucus-producing cell line HT-29 MTX were used. The Caco-2 cell line was originally obtained from the American Type Tissue Collection (ATCC ® HTB-37™), T84 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC, 88021101), and HT-29 MTX cells were obtained from the Sloan Kettering Memorial Cancer Center (SKMCC). Caco-2 and HT-29 MTX cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids (except for HT-29 MTX), and 1% streptomycin/penicillin. T84 cells were maintained in Nutrient Mixture Food F12 (DMEM/F12), supplemented with 6% heat-inactivated fetal bovine serum, 1% nonessential amino acids, and 1% streptomycin/penicillin. The cell lines were maintained at 37 • C in a humidified atmosphere of 10% CO 2 and passed at 80% confluence.

Study of Intestinal Permeability by the Measurement of Transepithelial Electrical Resistance (TEER)

The bacterial effect on different epithelial barriers was evaluated by measuring TEER through Caco-2 and T84 cells using a REMS Autosampler (World Precision Instrument, Sarasota, FL, USA). Each cell line was seeded onto cell culture inserts (Transwell, 0.4-µm pore size, 6.5-mm diameter, polycarbonate, (Corning, New York, NK, USA) at 1.45 × 10 5 cells/membrane. The membrane was maintained with a volume of 200 µL in the apical compartment and 800 µL in the basal compartment. The cell media were changed every two days. For this experiment, cells were grown in their respective complemented growth mediums and maintained in a humidified atmosphere (as described in Section 2.2.), until they reached their highest polarity. Two days before the experiment, the medium of the cells was changed to their respective mediums without antibiotic supplementation. TEER values were measured before the addition of bacteria (t = 0 h) and after 24 h of incubation. Bacterial suspension was added at a ratio of bacteria per cell of 40:1. The respective media were used as a negative control. The cells were challenged 3 h after the beginning of the coincubation with proinflammatory cytokines. Caco-2 cells were challenged with 100 ng/mL of tumor necrosis factor-α (TNF-α) (Peprotech, London, UK) on the basal side of the cells, and T84 cells were challenged with 50 ng/mL of interferon-γ (IFN-γ) (Peprotech, London, UK) on the basal side of the cells, and 1 ng/mL TNF-α was added on both sides. The TEER ratio was calculated as follows, and the bacterial effect of the strain was investigated by comparison with the controls.

TEER ratio = (

TEER treatment t24 TEER treatment t0 / TEER control t24 TEER control t0 )

Capacity of the Strains to Adhere to Intestinal Epithelial Cells

Quantitative analysis of bacterial adherence to differentiated Caco-2 cells and mucus-producing HT-29 MTX cells was performed. Each cell line was seeded into cell culture wells at 5 × 10 5 cells/well. For this experiment, the cells were grown in their respective complemented growth mediums and maintained in a humidified atmosphere (as described in Section 2.2) until 21 days of the culture. Bacterial strains were grown overnight in their respective growth conditions (as described in Section 2.1), added to each well at a concentration of approximately 10 8 CFU/well, and the plates were incubated at 37 • C in 10% CO 2 for 1 h. After 1 h of incubation, adherent bacteria were detached from the cells using 0.5% Triton X-100 (Merck, Kenilworth, IL, USA). Each inoculum was serial diluted with de Man, Rogosa and Sharpe (MRS) medium and enumerated on MRS agar plates after 48 h of incubation at 37 • C.

Biofilm Structural Dynamics

Biofilm structural dynamics by the selected strains were observed, according to the protocol initially described by Bridier et al. [START_REF] Bridier | The biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput CLSM method[END_REF] and using a LEICA SP8 high-content screening confocal laser scanning microscope (HCS-CLSM) at the INRAE MIMA2 microscopic platform [START_REF]Microscopy and Imaging Facility for Microbes, Animals and Foods (MIMA2)[END_REF]. For the kinetics, overnight bacterial cultures were diluted in order to obtain a concentration of 1 × 10 6 CFU/mL and 200 µL of the resulting bacterial solution deposited into micro-clear microscopic-grade 96-well microplates (Greiner, Kremsmünster, Germany). After 1 h of incubation at 37 • C, bacteria were washed with MRS medium to keep only the adherent bacteria. Bacterial cultures were incubated for 0, 4 and 24 h at 37 • C. At each timepoint, bacteria were washed with saline. They were labeled with solutions of 1.66 µM SYTO9, a cell-permanent green dye that label all bacteria, and 10-µM propidium iodide (PI), a cell-impermeant nucleic acid dye that contrasts dead bacteria in red (Invitrogen, Carlsbad, CA, USA). SYTO9 and PI were excited with an argon laser set at 488 nm, and the emitted fluorescence was collected in the range 500 to 550 nm for the green signal and 650 to 70 nm for the red signal. For each biofilm analyzed, a series of 512 × 512 pixel images was acquired every micrometer on the whole biofilm thickness. Three-dimensional projections of the biofilm were reconstituted using IMARIS software. Quantitative biovolumes of the biofilms were extracted from HCS-CLSM images using ImageJ [17].

Comparison of Resistance to Stressful Conditions Depending on the Mode of Growth of the Lactobacillus Strains

Planktonic Form

For this experiment, selected bacterial strains were grown overnight in their respective growth conditions (as described in Bacterial Strains and Growth Conditions). One milliliter of 1 × 10 6 CFU of each bacterial culture was centrifuged, and the pellet was resuspended in 1 mL culture medium (control) or 1-mL culture medium at pH 2 (VWR) or 1 mL culture media containing 1% bile salts (Sigma Aldrich, Saint Louis, MO, USA) or 10-mM H 2 O 2 (Sigma Aldrich, Saint Louis, MO USA). The cultures were incubated for 1 h at 37 • C. After a 48-h incubation (37 • C), bacterial cultures were enumerated on MRS agar plates by successive dilutions. After enumeration, the log reduction was calculated and compared to the control group (MRS).

Biofilm Form

After overnight incubation, bacteria were centrifuged and diluted to a concentration of 1 × 10 6 CFU/mL. Bacteria (200 µL) were deposited in 96-well plates (Corning, New York, NY, USA) and incubated for 1 h at 37 • C (adhesion step). The culture was washed with MRS medium to keep only the adherent bacteria and further incubated with fresh MRS medium at 37 • C for 24 h. After biofilm formation, the culture supernatants were removed and replaced with the medium (control) or medium at pH 2 (VWR) or media containing 1% bile salts (Sigma Aldrich, Saint Louis, MO, USA) or 10-mM H 2 O 2 (Sigma Aldrich, Saint Louis, MO, USA) . The cultures were incubated for 1 h at 37 • C. After incubation, biofilms were resuspended in MRS medium by gentle pipetting and enumerated on MRS agar plates by successive dilutions. The log reductions were calculated and compared to those of the planktonic form.

Bile Salt Hydrolase Assays

The six Lactobacillus strains were tested for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) hydrolase activities on MRS agar plates supplemented with either 0.5% TDCA (Cayman Chemical Company, Ann Arbor, MI, USA) or 0.5% GDCA (Merck Millipore, Burlington, CO, USA), following the protocol described by Moser and Savage [START_REF] Moser | Bile Salt Hydrolase Activity and Resistance to Toxicity of Conjugated Bile Salts Are Unrelated Properties in Lactobacilli[END_REF]. Strains were grown for 16 h at 37 • C in MRS broth, streaked on MRS agar plates supplemented with 0.5% TCDA or 0.5% GDCA, and incubated at 37 • C for 48 to 72 h. Bile salt hydrolase (BSH) activity was detected by the observation of an iridescent halo below the precipitation of deoxycholic acid.

Analysis of Gamma-Aminobutyric Acid (GABA) in Culture Supernatants

Supernatant samples (500 µL) were extracted twice with 500 µL MeOH/H 2 O (50:50 v/v) in a sonicator bath for 10 min and then centrifuged for 10 min. Derivatization of the amino acid, GABA, was performed as previously described by Salazar et al. [START_REF] Salazar | Combination of an AccQ•Tag-Ultra Performance Liquid Chromatographic Method with Tandem Mass Spectrometry for the Analysis of Amino Acids[END_REF] and Collado-Gonzalez et al. [START_REF] Collado-González | Effects of water deficit during maturation on amino acids and jujube fruit eating quality[END_REF]. Briefly, 350 µL borate derivatization buffer, 50-µL amino acid standard (or 20 µL supernatant samples), and 100 µL reconstituted ACQ (10-mM ACQ dry powder in acetonitrile) were combined. This solution was vortexed and then heated in a heating block for 10 min at 55 • C. Then, the samples were injected into an ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) device. The chromatographic separation was achieved using a ACCQ-TAG TM ULTRA C18 (2.1 × 100 mm, 1.7 µm) column [START_REF] Riga | Rootstock effect on serotonin and nutritional quality of tomatoes produced under low temperature and light conditions[END_REF][START_REF] Riga | Diffuse light affects the contents of vitamin C, phenolic compounds and free amino acids in lettuce plants[END_REF] through a chromatographic gradient developed by applying a varying mixture of solvents A and B. Solvent A consisted of 50 mL of an aqueous solution (acetonitrile, formic acid, and 5 mM ammonium acetate in water) (10:6:84 v/v/v) diluted with 950 mL Milli-q water and the mobile phase; solvent B consisted of a mixture of acetonitrile and formic acids (99.9:0.1 v/v). The injection volume was 20 µL, and the elution was performed at a flow rate of 0.5 mL/min [START_REF] Riga | Rootstock effect on serotonin and nutritional quality of tomatoes produced under low temperature and light conditions[END_REF]. The MS analysis was performed in the multiple reaction monitoring mode (MRM) using the positive ionization mode. GABA was quantified based on a freshly prepared calibration curve (0.5-0.0039 µM) of the corresponding authentic standard (Sigma Aldrich, Saint Louis, MO, USA). The GABA concentration in the culture supernatant is expressed as nanogram per mL (ng/mL) [START_REF] Cerrillo | Effect of Fermentation and Subsequent Pasteurization Processes on Amino Acids Composition of Orange Juice[END_REF].

Statistics

Results are expressed as the mean ± the standard error of the mean (SEM) of three experiments with duplicate determinations. The statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). One-way ANOVA was carried out, followed by Dunnet's or Tukey's multiple comparisons test. For the three-factor analysis, a two-way ANOVA followed by Sidak's multiple comparison test was used. p-values < 0.05 were considered significant.

Results

Modulation of Intestinal Permeability in Cellular Models

We initially screened a panel of 50 strains for their impact on the transepithelial electrical resistance (TEER) of Caco-2 and T84 intestinal epithelial cells (Figure S1) based on evidence reported in the literature. The six Lactobacillus investigated in this study (Table 1) displayed the greatest effect on in vitro permeability, and the well-documented LGG was used as a positive control. The selected strains did not display the same level of beneficial effect on epithelial alterations induced by either TNF-α or IFN-γ in the two cell types (Figure 1). LC03 and CNCM had a strongly beneficial effect on Caco-2 permeability, similar to that observed with the positive control LGG (Figure 1a). The effect observed between the control and the strains on the T84 cell lines was less-marked, as the cells did not react to the same level to the mix of IFN-γ and TNF-α as did the Caco-2 cells with TNF-α. However, there was a clearly different response with the LGG and LC03 strains, and CIP and ROQB1 showed more promising effects than in the Caco-2 model (Figure 1b).

Capacity of the Strains to Adhere to Intestinal Epithelial Cells

We next characterized the capacity of our collection of Lactobacillus strains to adhere to intestinal mucosa by investigating their capacity to adhere to Caco-2 and mucus-secreting HT-29 MTX cells (originally isolated from a colorectal adenocarcinoma of human intestinal mucosa). The adhesive capacity of the strains varied considerably between the two cell lines, with a percentage of the adhesion of all strains up to five times higher to Caco-2 cells (Figure 2). There were also considerable differences between the strains, with CIP showing even higher levels of adhesion than the positive control LGG (p < 0.05).

Capacity of the Strains to Adhere to Intestinal Epithelial Cells

We next characterized the capacity of our collection of Lactobacillus strains to adhere to intestinal mucosa by investigating their capacity to adhere to Caco-2 and mucus-secreting HT-29 MTX cells (originally isolated from a colorectal adenocarcinoma of human intestinal mucosa). The adhesive capacity of the strains varied considerably between the two cell lines, with a percentage of the adhesion of all strains up to five times higher to Caco-2 cells (Figure 2). There were also considerable differences between the strains, with CIP showing even higher levels of adhesion than the positive control LGG (p < 0.05).

Capacity of the Strains to Adhere to Intestinal Epithelial Cells

We next characterized the capacity of our collection of Lactobacillus strains to adhere to intestinal mucosa by investigating their capacity to adhere to Caco-2 and mucus-secreting HT-29 MTX cells (originally isolated from a colorectal adenocarcinoma of human intestinal mucosa). The adhesive capacity of the strains varied considerably between the two cell lines, with a percentage of the adhesion of all strains up to five times higher to Caco-2 cells (Figure 2). There were also considerable differences between the strains, with CIP showing even higher levels of adhesion than the positive control LGG (p < 0.05).

Analysis of Biofilm Formation by the Selected Lactobacilli Using Confocal Laser Scanning Microscopy (CLSM)

We analyzed the biofilm formation by the selected strains, in microscopic-grade 96-well plates, at 0, 4, and 24 h by using HCS-CLSM. The representative images were treated with IMARIS dedicated software (Figure 3a). Virtual shadow projections of the biofilm are represented on the right side of the images. The three isolates, LC03, LR04, and LGG, formed short chains that were visible after the adhesion (0 h). The chains formed by the LC03 strain were over-winded. For these three strains, the morphotypes observed were not specific to the adherent populations, as they were also observed for the planktonic bacteria in the stationary phase. After a short period of incubation (4 h), the adherent chains of the three isolates elongated to form an aerial structure. The LGG isolate showed a particular phenotype after 4 h with 3D aerial organization, which is likely associated with chain elongations and cohesion factors. The existence of sparse cells and aggregates labeled with PI indicate the presence of dead or damaged bacteria in those 24-h communities. The six Lactobacillus strains were able to form biofilms with different biovolumes of between 2 and 9 µm 3 /µm 2 after 24 h of biofilm formation (Figure 3b, p < 0.05).

Microorganisms 2020, 8, x FOR PEER REVIEW 7 of 16

Analysis of Biofilm Formation by the Selected Lactobacilli Using Confocal Laser Scanning Microscopy (CLSM)

We analyzed the biofilm formation by the selected strains, in microscopic-grade 96-well plates, at 0, 4, and 24 h by using HCS-CLSM. The representative images were treated with IMARIS dedicated software (Figure 3a). Virtual shadow projections of the biofilm are represented on the right side of the images. The three isolates, LC03, LR04, and LGG, formed short chains that were visible after the adhesion (0 h). The chains formed by the LC03 strain were over-winded. For these three strains, the morphotypes observed were not specific to the adherent populations, as they were also observed for the planktonic bacteria in the stationary phase. After a short period of incubation (4 h), the adherent chains of the three isolates elongated to form an aerial structure. The LGG isolate showed a particular phenotype after 4 h with 3D aerial organization, which is likely associated with chain elongations and cohesion factors. The existence of sparse cells and aggregates labeled with PI indicate the presence of dead or damaged bacteria in those 24-h communities. The six Lactobacillus strains were able to form biofilms with different biovolumes of between 2 and 9 µm 3 /µm 2 after 24 h of biofilm formation (Figure 3b, p < 0.05).

(a)

Comparison of Tolerance to Stressful Conditions Depending on Lifestyle

We compared the tolerance of the six Lactobacillus strains to stressful conditions as a function of their lifestyle (planktonic versus biofilm). From an overnight culture in the stationary phase, the bacterial strains were cultured for 1 h under hostile conditions using either oxidative stress (H2O2), low pH, or bile salts (Figure 4). Bacteria exhibiting either lifestyles were not statistically affected by the medium complemented with bile salts, suggesting their ability to tolerate high transitory concentrations of bile salts. In contrast, the growth rate of all strains was reduced at pH 2 (p < 0.05), with the exception of the LC03 strain. A protective effect of the biofilm lifestyle was especially marked at a low pH with LR04, LGG, and CIP strains for which there was a significant delta log (∆log), ranging from 8 to 5 (p < 0.05). Of note, the average of the ∆log for the ROQB1 strain was similar to those previously observed; however, the results were not significant because of the large variations that were observed between the replicates. Despite the fact that our isolates appeared less-affected by the oxidative medium than the acidic, we observed a benefit of the biofilm lifestyle for our control strain LGG with the H2O2 medium. Finally, these results also highlight that the LC03 strain possesses the highest tolerance to stressful conditions, either in the planktonic or biofilm lifestyles.

Comparison of Tolerance to Stressful Conditions Depending on Lifestyle

We compared the tolerance of the six Lactobacillus strains to stressful conditions as a function of their lifestyle (planktonic versus biofilm). From an overnight culture in the stationary phase, the bacterial strains were cultured for 1 h under hostile conditions using either oxidative stress (H 2 O 2 ), low pH, or bile salts (Figure 4). Bacteria exhibiting either lifestyles were not statistically affected by the medium complemented with bile salts, suggesting their ability to tolerate high transitory concentrations of bile salts. In contrast, the growth rate of all strains was reduced at pH 2 (p < 0.05), with the exception of the LC03 strain. A protective effect of the biofilm lifestyle was especially marked at a low pH with LR04, LGG, and CIP strains for which there was a significant delta log (∆log), ranging from 8 to 5 (p < 0.05). Of note, the average of the ∆log for the ROQB1 strain was similar to those previously observed; however, the results were not significant because of the large variations that were observed between the replicates. Despite the fact that our isolates appeared less-affected by the oxidative medium than the acidic, we observed a benefit of the biofilm lifestyle for our control strain LGG with the H 2 O 2 medium. Finally, these results also highlight that the LC03 strain possesses the highest tolerance to stressful conditions, either in the planktonic or biofilm lifestyles.

at a low pH with LR04, LGG, and CIP strains for which there was a significant delta log (Δlog), ranging from 8 to 5 (p < 0.05). Of note, the average of the Δlog for the ROQB1 strain was similar to those previously observed; however, the results were not significant because of the large variations that were observed between the replicates. Despite the fact that our isolates appeared less-affected by the oxidative medium than the acidic, we observed a benefit of the biofilm lifestyle for our control strain LGG with the H2O2 medium. Finally, these results also highlight that the LC03 strain possesses the highest tolerance to stressful conditions, either in the planktonic or biofilm lifestyles.

Bile Salt Hydrolase Assays

We directly assessed the ability of the six selected strains to produce BSH in a colorimetric test on agar plates supplemented with either TDCA or GDCA. A large iridescent halo was observed with both the ROQB1 and CNCM strains (Figure 5), suggesting that they have the capacity to hydrolyze glyco-conjugated salts. None of the other four strains grew on 0.5% GDCA agar medium. Moreover, none of the strains showed TDCA activity, but, in contrast to the GDCA assay, they were all able to grow on 0.5% TDCA agar medium (Table 2). 2.

Table 2. Bile salts activities and tolerance. The absence or presence of bile salt hydrolase (BSH) activity and tolerance to 0.5% taurodeoxycholic acid (TDCA) or glycodeoxycholic acid (GDCA) on agar plates were evaluated.

Lactobacillus Species Strains TDCA Hydrolase Activity/Growth GDCA Hydrolase Activity/Growth L. rhamnosus

LGG

-/+ -/- L. rhamnosus LR04 -/+ -/- L. plantarum CNCM -/+ +/+ L. casei LC03 -/+ -/- L. plantarum CIP -/+ -/- L. paracasei ROQB1 -/+ +/+
3.6. Secretion of the Neurotransmitter γ-Aminobutyric Acid (GABA)

We tested the six Lactobacillus strains for their ability to secrete the neurotransmitter GABA. Culture supernatants were collected after overnight growth, and GABA was quantified by UHPLC-QqQ-MS. All strains were able to produce GABA, ranging from 7 to 130 ng/mL (Figure 6). Strikingly, ROQB1 and CIP exhibited the highest level, reaching 130.2 ± 23.86-ng/mL and 123.9 ± 23.29-ng/mL GABA. 2.

Table 2. Bile salts activities and tolerance. The absence or presence of bile salt hydrolase (BSH) activity and tolerance to 0.5% taurodeoxycholic acid (TDCA) or glycodeoxycholic acid (GDCA) on agar plates were evaluated.

Lactobacillus Species

Strains TDCA Hydrolase Activity/Growth GDCA Hydrolase Activity/Growth

L. rhamnosus LGG -/+ -/- L. rhamnosus LR04 -/+ -/- L. plantarum CNCM -/+ +/+ L. casei LC03 -/+ -/- L. plantarum CIP -/+ -/- L. paracasei ROQB1 -/+ +/+
3.6. Secretion of the Neurotransmitter γ-Aminobutyric Acid (GABA)

We tested the six Lactobacillus strains for their ability to secrete the neurotransmitter GABA. Culture supernatants were collected after overnight growth, and GABA was quantified by UHPLC-QqQ-MS. All strains were able to produce GABA, ranging from 7 to 130 ng/mL (Figure 6). Strikingly, ROQB1 and CIP exhibited the highest level, reaching 130.2 ± 23.86-ng/mL and 123.9 ± 23.29-ng/mL GABA.

Discussion

It is now well-accepted that alterations of the intestinal barrier are associated with chronic intestinal disorders, such as IBS; IBD [START_REF] Salvo Romero | The intestinal barrier function and its involvement in digestive disease[END_REF]; and other conditions, such as obesity [START_REF] Cheru | Gastrointestinal Barrier Breakdown and Adipose Tissue Inflammation[END_REF], liver diseases

Discussion

It is now well-accepted that alterations of the intestinal barrier are associated with chronic intestinal disorders, such as IBS; IBD [START_REF] Salvo Romero | The intestinal barrier function and its involvement in digestive disease[END_REF]; and other conditions, such as obesity [START_REF] Cheru | Gastrointestinal Barrier Breakdown and Adipose Tissue Inflammation[END_REF], liver diseases [START_REF] Cui | Intestinal Barrier Function-Non-alcoholic Fatty Liver Disease Interactions and Possible Role of Gut Microbiota[END_REF], and behavioral disorders [START_REF] Julio-Pieper | Intestinal Barrier and Behavior[END_REF]. Interactions between the various layers that constitute the intestinal barrier preserve normal permeability. However, several factors can disturb normal permeability, enabling the passage of toxins or pathogens from the lumen to the immune layer, leading to low-grade inflammation [START_REF] Bischoff | Intestinal permeability-A new target for disease prevention and therapy[END_REF].

Various strategies have been explored for the treatment of these conditions, such as the use of probiotics. Indeed, numerous studies have demonstrated the potential benefit of Lactobacillus species for several conditions [START_REF] Di Cerbo | Mechanisms and therapeutic effectiveness of lactobacilli[END_REF]. However, the positive effects of probiotics are often strain-and disease-dependent; thus, it is important to identify the strains that display the best and strongest beneficial effects [START_REF] Sniffen | Choosing an appropriate probiotic product for your patient: An evidence-based practical guide[END_REF].

Here, we evaluated six Lactobacillus strains for their ability to protect the epithelial layer from increased permeability (induced by proinflammatory cytokines) using TEER assays (Figure 1). We tested the potential probiotic effects of these strains on two types of cell lines: Caco-2 and T84 cells, to reflect different specific areas of the intestines. Caco-2 cells have been used for decades as an intestinal cellular model, as they have the ability to spontaneously differentiate, expressing morphological and biochemical characteristics of small intestine enterocytes [START_REF] Sambuy | The Caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics[END_REF]. In contrast, although T84 cells (less used in permeability assays) can also spontaneously differentiate, they differ from Caco-2 cells in that they express native intestinal characteristics, such as those found in undifferentiated crypt cells [START_REF] Devriese | T84 monolayers are superior to Caco-2 as a model system of colonocytes[END_REF]. Our results confirmed the beneficial effects of the six Lactobacillus strains on Caco-2, with a significant effect of the CNCM and LC03 strains. In contrast, the CIP and ROQB1 strains showed the highest beneficial effect on T84 cells, whereas LC03, LR04, and the positive control LGG showed only slight effects. These results suggest that the enhancement of the permeability provided by the strains depends on the cellular localization.

The ability of bacteria to adhere to intestinal epithelial cells is a common and well-known criterion for probiotic selection. It has been proposed that the ability to adhere confers longer residence inside the gut, increasing the transit time, thus allowing them to exert their beneficial effects and interact with the host [START_REF] Monteagudo-Mera | Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health[END_REF]. Of note, we recently described the role of a gene involved in the adhesive properties of lactobacilli (through pili) and the effects conferred to the intestinal barrier by the L. rhamnosus CNCM I-3690 strain [START_REF] Martín | The potential probiotic Lactobacillus rhamnosus CNCM I-3690 strain protects the intestinal barrier by stimulating both mucus production and cytoprotective response[END_REF]. The most common intestinal cell lines used for the determination of the adhesive capacity of bacteria are Caco-2 and mucus-producing HT-29 MTX [START_REF] Leteurtre | Differential mucin expression in colon carcinoma HT-29 clones with variable resistance to 5-fluorouracil and methotrexate[END_REF]. The panel of the six tested strains displayed the ability to adhere to both cell lines (Figure 2). However, we observed a significant difference in the ability of the bacteria to adhere to the two cell lines, suggesting that mucus production may block adhesion-binding sites. Moreover, it is possible that the major determinants of bacterial adhesion, for the tested strains, are more highly associated with components of mature enterocytes than interactions with mucus glycoproteins [START_REF] Laparra | Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium[END_REF].

Numerous studies have reported the beneficial properties of biofilm formation by Lactobacillus species by promoting longer survival of the bacteria on the host intestinal epithelium [START_REF] Salas-Jara | Biofilm Forming Lactobacillus: New Challenges for the Development of Probiotics[END_REF]. Moreover, given this advantage, recent studies have focused on biofilm-encapsulated bacteria based on two synthetic coatings, a new concept called "fourth-generation probiotics". Bacteria exhibiting this particular status are better protected from the hostile environment and better at targeting specific localizations of the gut [START_REF] Cheow | Controlled release of Lactobacillus rhamnosus biofilm probiotics from alginate-locust bean gum microcapsules[END_REF]. In this context, we tested the six Lactobacillus strains for their capacity to form biofilms on microtiter plates and to resist oxidative stress, low pH, and bile salts. All were able to form biofilms but of varying final biovolumes at 24 h, and they showed differing morphological changes during their growths (Figure 3). We compared the biofilm cells to their planktonic counterparts and found that the biofilm lifestyle clearly protected three of the tested bacteria (LR04, LGG, and CIP) better from low pH, confirming the advantage of this mode of growth in an acidic environment, such as that encountered in the gastrointestinal tract (GIT) (Figure 4). The formation of biofilms by microorganisms is strongly dependent on the bacterial species/strains; culture mediums; and conditions (temperature, surface properties, and pH) of the intestinal tract environment [START_REF] Donlan | Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms[END_REF][START_REF] Lebeer | Impact of Environmental and Genetic Factors on Biofilm Formation by the Probiotic Strain Lactobacillus rhamnosus GG[END_REF]. Moreover, the biofilm matrix represents 90% of the total biofilm and is responsible for its internal cohesion [START_REF] Flemming | The biofilm matrix[END_REF]. The matrix is composed of extracellular polymeric substances (EPS), of which some of their functions have been confirmed to be advantageous for the biofilm lifestyle and the enhancement of probiotic properties [START_REF] Lebeer | Exopolysaccharides of Lactobacillus rhamnosus GG form a protective shield against innate immune factors in the intestine[END_REF]. Moreover, EPS can be an important factor for the survival of bacteria in the microenvironment of the GIT. Indeed, the EPS content proportion has been correlated to bacterial tolerance to acidic environments and bile salts [START_REF] Caggianiello | Exopolysaccharides produced by lactic acid bacteria:from health-promoting benefits to stress tolerance mechanisms[END_REF][START_REF] Dertli | Impact of the exopolysaccharide layer on biofilms, adhesion and resistance to stress in Lactobacillus johnsonii FI9785[END_REF]. It is therefore important to better characterize biofilms under specific physiological conditions to achieve the optimal release and effects.

Primary bile acids are synthetized from cholesterol in the liver and become conjugated bile salts after their assembly with taurine or glycine amino acids. BSH enzymes, components of various species of the gut microbiota, hydrolyze and deconjugate glycine or taurine and generate secondary bile salts, which are more easily reabsorbed by the host [START_REF] Foley | Bile salt hydrolases: Gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract[END_REF]. BSH activity is now considered to be a suitable property of probiotics, as it may promote a tolerance to bile and survival of the bacteria in a hostile environment (such as that found in the intestinal tract) [START_REF] Bustos | New insights into bacterial bile resistance mechanisms: The role of bile salt hydrolase and its impact on human health[END_REF]. In addition, the beneficial impacts of certain BSH-producing bacteria have been reported for dietary lipid absorption, cholesterol lowering, and energy harvest [START_REF] Begley | Bile Salt Hydrolase Activity in Probiotics[END_REF][START_REF] Chand | Molecular features of bile salt hydrolases and relevance in human health[END_REF]. Here, we aimed to compare the BSH activity towards either TDCA or GDCA-conjugated bile salts (Figure 5 and Table 2). We observed strong BSH activity on GDCA agar medium for two Lactobacillus strains, ROQB1 and CNCM, indicating their specificity for glyco-conjugated bile salts under these conditions. Of note, some of the strains were unable to grow in the presence of glyco-conjugated salt in the medium, demonstrating the low resistance of the strains to GDCA. It is worthwhile comparing these results with those obtained with the survival test (discussed above), as most of the strains were tolerant to higher concentrations of primary and secondary bile salts for a short period (Figure 3). These results suggest that the presence of glyco-conjugated bile acids, specifically, might be more toxic for the four Lactobacillus strains that lack BSH activity, with respect to long-term growth (48 h).

Probiotic strains are able to produce molecules that can influence the functioning of the central nervous system (CNS), behavior, and nociception [START_REF] Yunes | A Multi-strain Potential Probiotic Formulation of GABA-Producing Lactobacillus plantarum 90sk and Bifidobacterium adolescentis 150 with Antidepressant Effects[END_REF]. GABA is a major neurotransmitter of the CNS, inhibiting the action potentials of neurons. Pérez-Berezo et al. [START_REF] Pérez-Berezo | Identification of an analgesic lipopeptide produced by the probiotic Escherichia coli strain Nissle 1917[END_REF] has shown that the inhibition of neuron activation by GABA is mediated by the formation of a lipopeptide, allowing diffusion of the neurotransmitter through the intestinal barrier. Furthermore, Bravo et al. highlighted the role of bacteria in gut-brain axis interactions and demonstrated a direct impact of L. rhamnosus (JB-1) on the GABAergic system through the vagus nerve [START_REF] Bravo | Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve[END_REF]. Various studies focused on GABA-enriched functional foods with, for most, the use of an optimized-growth medium [START_REF] Rayavarapu | Statistical optimization of γ-aminobutyric acid production by response surface methodology and artificial neural network models using Lactobacillus fermentum isolated from palm wine[END_REF][START_REF] Gharehyakheh | Production of GABA-enriched honey syrup using Lactobacillus bacteria isolated from honey bee stomach[END_REF]. In this study, we confirmed that the selected Lactobacillus strains were able to produce GABA, but we were not able to state on the production of low or high amounts. However, normal concentrations of GABA in body fluids are found to be approximately 10 ng/mL in human plasma and 100 ng/mL in cerebrospinal fluid [START_REF] Song | A capillary liquid chromatographic/tandem mass spectrometric method for the quantification of γ-aminobutyric acid in human plasma and cerebrospinal fluid[END_REF][START_REF] Lu | Determination of glycine, glutamine, glutamate, and γ-aminobutyric acid in cerebrospinal fluids by capillary electrophoresis with light-emitting, diode-induced fluorescence detection[END_REF]. In addition, various studies reported a decrease of the GABA concentration in a variety of psychiatric and neurological disorders [START_REF] Cortes-Saladelafont | Gamma-aminobutyric acid levels in cerebrospinal fluid in neuropaediatric disorders[END_REF][START_REF] Zheng | Body fluid levels of neuroactive amino acids in autism spectrum disorders: A review of the literature[END_REF][START_REF] Wu | A sensitive and practical RP-HPLC-FLD for determination of the low neuroactive amino acid levels in body fluids and its application in depression[END_REF]. The imbalance between GABA and its glutamate precursor, an excitatory neurotransmitter, seems to be strongly associated to these conditions [START_REF] He | Glutamate Transporters, and Circadian Rhythm Sleep[END_REF]. Overall, our findings provide new insights for the use of the Lactobacillus strains for the regulation of the GABAergic system and abdominal pain, commonly associated with intestinal barrier dysfunctions.

Conclusions

In conclusion, we demonstrated a significant protective effect of CNCM on both in vitro models of intestinal permeability. The LC03 and LR04 strains were better able to protect mature enterocytes of the small intestine, whereas ROQB1 and CIP showed a strong protective effect on crypt-like cells. We also compared the biofilm and planktonic forms of the bacteria and provided evidence of the advantages of the biofilm lifestyle in a low pH environment. Our data and others suggest that biofilm phenotypic assays should be systematically integrated in probiotic screening campaigns and provide new strategies for probiotic administration. Moreover, we found that the Lactobacillus strains were able to produce

Figure 1 .

 1 Figure 1. Screening of Lactobacillus species for their protective effects on intestinal epithelial cells permeability. TEER was assayed using (a) Caco-2 and (b) T84 cells grown on 24-well Transwell plates. TEER was measured before adding 6.10 6 CFU of bacterial onto the apical surface for 3 h prior to treatment of the cells with a solution of TNF-α for Caco-2 cells and a mix of IFN-γ and TNF-α for T84 cells for 21 h at 37 °C and 10% CO2. TEER was measured a second time at the end of the 24 h of coincubation. Statistical analysis consisted of one-way ANOVA followed by Dunnet's multiple comparison test. *p < 0.0332, **p < 0.0021, ***p < 0.0002, and **** p < 0.0001.

Figure 2 .

 2 Figure 2. Adhesion of the six Lactobacillus strains on intestinal epithelial cells Caco-2 and HT-29 MTX.A bacterial suspension of a concentration of 10 8 CFU/mL was applied to the intestinal cells. After 1 h of incubation, cells were washed to remove nonadherent bacteria and detached using 0.5% Triton X-100. Harvested solutions were then diluted in PBS X-1 and the bacteria enumerated. Results are expressed as the percentage of bacteria adhering to the intestinal cells. Statistical analysis consisted of

Figure 1 .

 1 Figure 1. Screening of Lactobacillus species for their protective effects on intestinal epithelial cells permeability. TEER was assayed using (a) Caco-2 and (b) T84 cells grown on 24-well Transwell plates. TEER was measured before adding 6.10 6 CFU of bacterial onto the apical surface for 3 h prior to treatment of the cells with a solution of TNF-α for Caco-2 cells and a mix of IFN-γ and TNF-α for T84 cells for 21 h at 37 • C and 10% CO 2 . TEER was measured a second time at the end of the 24 h of coincubation. Statistical analysis consisted of one-way ANOVA followed by Dunnet's multiple comparison test. * p < 0.0332, ** p < 0.0021, ***p < 0.0002, and **** p < 0.0001.

Figure 1 .

 1 Figure 1. Screening of Lactobacillus species for their protective effects on intestinal epithelial cells permeability. TEER was assayed using (a) Caco-2 and (b) T84 cells grown on 24-well Transwell plates. TEER was measured before adding 6.10 6 CFU of bacterial onto the apical surface for 3 h prior to treatment of the cells with a solution of TNF-α for Caco-2 cells and a mix of IFN-γ and TNF-α for T84 cells for 21 h at 37 °C and 10% CO2. TEER was measured a second time at the end of the 24 h of coincubation. Statistical analysis consisted of one-way ANOVA followed by Dunnet's multiple comparison test. *p < 0.0332, **p < 0.0021, ***p < 0.0002, and **** p < 0.0001.

Figure 2 .

 2 Figure 2. Adhesion of the six Lactobacillus strains on intestinal epithelial cells Caco-2 and HT-29 MTX.A bacterial suspension of a concentration of 10 8 CFU/mL was applied to the intestinal cells. After 1 h of incubation, cells were washed to remove nonadherent bacteria and detached using 0.5% Triton X-100. Harvested solutions were then diluted in PBS X-1 and the bacteria enumerated. Results are expressed as the percentage of bacteria adhering to the intestinal cells. Statistical analysis consisted of

Figure 2 .

 2 Figure 2. Adhesion of the six Lactobacillus strains on intestinal epithelial cells Caco-2 and HT-29 MTX.A bacterial suspension of a concentration of 10 8 CFU/mL was applied to the intestinal cells. After 1 h of incubation, cells were washed to remove nonadherent bacteria and detached using 0.5% Triton X-100. Harvested solutions were then diluted in PBS X-1 and the bacteria enumerated. Results are expressed as the percentage of bacteria adhering to the intestinal cells. Statistical analysis consisted of two-way ANOVA followed by Sidak's multiple comparison test. **** p < 0.0001, *** p < 0.0002, ** p < 0.0021, and * p < 0.0332.

Figure 3 .Figure 3 .

 33 Figure 3. Cont.

Figure 3 .

 3 Figure 3. Observation of biofilm formation. (a) High-content screening confocal laser scanning microscope (HCS-CLSM) images at 0, 4, and 24 h after adhesion step. All bacteria are labeled in green (SYTO 9) and dead bacteria in red (PI). Images were treated using IMARIS software. The grey shadow to the right represents the thickness and relief of the biofilms. (b) Quantitative biovolumes of biofilms, at 24 h, were extracted by HCS-CLSM images using ImageJ. Statistical analysis consisted of one-way ANOVA followed by Tukey's multiple comparison test. **** p < 0.0001 and ** p < 0.0021.

Figure 4 .

 4 Figure 4. Survival test of selected bacteria to stressful conditions. Bacterial strains were grown under conditions to result in one of two lifestyles: planktonic or biofilm. The cells were then incubated in

Figure 4 .

 4 Figure 4. Survival test of selected bacteria to stressful conditions. Bacterial strains were grown under conditions to result in one of two lifestyles: planktonic or biofilm. The cells were then incubated in medium at pH 2 or in medium containing 1% bile salts or 10-mM H 2 O 2 for 1 h. (a) Images represent the serial dilution plates that compare the different bacteria tested after the treatments with de Man, Rogosa and Sharpe (MRS) (control), pH 2, H 2 O 2 , and bile salts. (b) The log reduction after treatment was calculated by subtracting the bacterial concentration before incubation in the media at t = 0 h (MRS) to that at t = 24 h. Statistical analysis consisted of two-way ANOVA followed by Sidak's multiple comparison test. **** p < 0.0001, *** p < 0.0002, and * p < 0.0332.

Figure 5 .

 5 Figure 5. Bile salt hydrolase (BSH) activities. The six strains were tested for their (a) glycodeoxycholic acid (GDCA) and (b) taurodeoxycholic acid (TDCA) hydrolase activities. BSH activity was detected by the observation of an iridescent halo demonstrating the precipitation of deoxycholic acid. The presence or absence of the activity and growth capacity for each strain are summarized in Table2.

Figure 6 .

 6 Figure 6. Production of γ-aminobutyric acid (GABA) after 16 h of incubation with separately inoculated Lactobacillus strains. Analysis was performed by UHPLC-QqQ-MS/MS, and the chromatographic separation was achieved by using an ACCQ-TAG TM ULTRA C18 column. GABA was quantified based on the calibration curve (0.5-0.0039 µM). Results are expressed as nanogram per mL (ng/mL). Statistical analysis consisted of one-way ANOVA followed by Tukey's multiple comparison test. *** p < 0.0002.

Figure 5 .

 5 Figure 5. Bile salt hydrolase (BSH) activities. The six strains were tested for their (a) glycodeoxycholic acid (GDCA) and (b) taurodeoxycholic acid (TDCA) hydrolase activities. BSH activity was detected by the observation of an iridescent halo demonstrating the precipitation of deoxycholic acid. The presence or absence of the activity and growth capacity for each strain are summarized in Table2.

  Microorganisms 2020, 8, x FOR PEER REVIEW 11 of 16

Figure 6 .

 6 Figure 6. Production of γ-aminobutyric acid (GABA) after 16 h of incubation with separately inoculated Lactobacillus strains. Analysis was performed by UHPLC-QqQ-MS/MS, and the chromatographic separation was achieved by using an ACCQ-TAG TM ULTRA C18 column. GABA was quantified based on the calibration curve (0.5-0.0039 µM). Results are expressed as nanogram per mL (ng/mL). Statistical analysis consisted of one-way ANOVA followed by Tukey's multiple comparison test. *** p < 0.0002.

Figure 6 .

 6 Figure 6. Production of γ-aminobutyric acid (GABA) after 16 h of incubation with separately inoculated Lactobacillus strains. Analysis was performed by UHPLC-QqQ-MS/MS, and the chromatographic separation was achieved by using an ACCQ-TAG TM ULTRA C18 column. GABA was quantified based on the calibration curve (0.5-0.0039 µM). Results are expressed as nanogram per mL (ng/mL). Statistical analysis consisted of one-way ANOVA followed by Tukey's multiple comparison test. *** p < 0.0002.

Table 1 .

 1 Species, strain identifier, origin, and growth conditions.

	Lactobacillus Species	Strains	Abbreviation	Origin	Growth Conditions
	L. rhamnosus	LGG	LGG	Lesaffre
	L. casei	LC03 (DSM27537)	LC03	Probiotical	Facultative
	L. rhamnosus	LR04 (DSM16605)	LR04	Probiotical	anerobia
	L. plantarum	CNCM I-4459 (LP-Onlly)	CNCM	Novanat	MRS
	L. plantarum	CIP 104450	CIP	Institut Pasteur	37 • C
	L. paracasei	ROQB1	ROQB1	Roquette Frères

Microorganisms 2020, 8, 1053; doi:10.3390/microorganisms8071053 www.mdpi.com/journal/microorganisms

Microorganisms 2020, 8, x FOR PEER REVIEW

Funding: This research was funded by INDIGO Therapeutics and Association Nationale de la Recherche et de la Technologie (2016/0709).

beneficial host/strain metabolites, and our findings on GABA production open new perspectives of characterization for their use in the management of gut-brain interactions. This step-by-step characterization provided interesting information of the Lactobacillus profile and highlighted the most promising strains for further evaluation in animal models of intestinal hyperpermeability.

Supplementary Materials:

The following are available online at http://www.mdpi.com/2076-2607/8/7/1053/s1: Figure S1: Original screening of various bacterial species for their protective effect on intestinal cells' permeability.

Conflicts of Interest:

The authors declare that C.C. received a salary from INDIGO Therapeutics, as part of a CIFRE contract, a society that researches and produces probiotic/symbiotic formulations where S.L. and P-Y.M. were employees.


Rabia Badreddine
email: rabia.badreddine@ineris.fr

D François

Assessment of the PCDD/F fate from MSWI residues used in road construction in France

Keywords: incineration, ash, road, dioxin, furan, particle

MSWI fly ash is susceptible to contain high amount of dioxins (PCDD) and furans (PCDF). However, the use of MSWI residues for road construction started in France at period when the mixture of MSWI Bottom Ash with MSWI fly ash was used. From four old road sites, MSWI residues, road soils, reference soils and geo-textiles were sampled and their PCDD/F contents were analyzed. MSWI residues show a great heterogeneity but also high amounts of PCDD/F between 14 and 2960 ng I-TEQ.kg -1 DM. Road soils show less heterogeneity and contents between 0.57 et 7.23 ng I-TEQ.kg -1 DM lower than ordinary soils. Moreover, the specific analysis of the 17 toxic PCDD/F congeners (notably the 2,3,7,8-TetraCDD) indicates the very low noxiousness of road soils. The study also allows to assert the relation between the MSWI residue particle size and the PCDD/F content.

Introduction

In many areas across the world where the high demand of construction materials compared to the availability of natural materials, as well as the lack of available space for waste disposal, are a problem, the use of by-products and wastes for road construction has been seen for a long time as an appropriate solution to reduce the amount of disposed materials and to provide at the same time alternative materials for construction. A typical case of these alternative materials is that of the Municipal Solid Waste Incinerator (MSWI) residue which is produced from the household wastes combustion and used for road and car-park construction.

In France, the use of MSWI residue in road construction is supervised since the 90's through an order (1991) and a memorandum (1994) both from the ministry of Environment. The environmental assessment of MSWI bottom ash is based on the measurement of its unburned fraction and the leaching potential of some heavy metals, arsenic, sulfate and total organic carbon. Yet, MSWI residues may contain persistent organic pollutants (POP) such as polychlorinated dibenzo-dioxins (PCDD) and polychlorinated dibenzo-furans (PCDF). PCDD/F molecules are poorly water soluble and leaching tests are known for not being relevant toward them, nonetheless, the affinity of PCDD/F molecules for particles is also known, and such affinity may be higher toward the finest particles. As a consequence, one wonders if under the effect of rainwater infiltration into the road body, the washing of the MSWI residue layer may induce the transfer, downward, of PCDD/F molecules bound to the finest particles of the material. In such a case, on road sites, compared to some reference soils, an increase of the PCDD/F content in the soil underlying the road (called the road soil), may be observed.

In order to answer to this question, PCDD/F contents of MSWI residues sampled into old road structures, were analyzed (including an assessment of their noxiousness).

The relation between the dimension of MSWI residue particles and the PCDD/F content was studied. Then MSWI residue PCDD/F contents were compared to those measured in the road soil and in neighbouring soils (local references).

As before the enforcement of the 1991 orderthus PCDD/F compounds -MSWI residues were susceptible to contain higher amounts of fly ash, the present study was focused on pre-1991 constructions. At the same time, this allows to assess the medium-term state of road soils.

Context of the study

Production of PCDD/F during household waste incineration

As a result of incomplete combustion, incineration of household waste produces several organic compounds such as chlorinated species (polychlorinated biphenyls (PCB), polychlorinated dibenzodioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs)). Two temperature ranges are responsible for the production of persistent organic pollutant (POP). The first one (200 to 400°C) which results from a catalysed reaction taking places on the ash particles present in combustion systems. The second one (500 to 800°C) is the result of a rearrangement of chlorinated precursors such as chlorophenols and chlorobenzenes in the gas phase [START_REF] Stanmore | The formation of dioxins in combustion systems[END_REF].

Dioxin air emissions from incinerators have decreased in the last decade due to improvements in the pollution control technology and to the regulation implementation and enforcement. Two orders of 20 September, one for hazardous waste and another one for non hazardous waste, both limit the level of PCDD/F emission to 0.1 ng.m -3 .

Use of MSWI residues for road construction in France

The use of MSWI residue started in France during the 1950's in the area of Paris and spread all over the country during the 1980' -1990's, a period during which many incinerators were built [START_REF] Aghtm | Politique de gestion des déchets en France: Etat de l'art de l'incinération[END_REF].

As for some other alternative materials (coal fly ash, blast furnace slag…), the use of MSWI residue was codified by the ministry for public works and transports, allowing its assimilation to one of the various kinds of natural materials considered for the classical road structure design (MELT, 1997;SETRA and LCPC, 2000). The analogy is based on geo-technical responses of the MSWI residue sample to a set of usual standard mechanical tests (resistance to fragmentation and wear, sand equivalent, surface cleanliness, compactibility…).

Until the 1990's the environmental question about the use of MSWI residue was not seen as a major concern. In 1991, an order required from the 1 st of December 1992, the separating of MSWI residue into bottom ash (the fraction of the incineration residue which is collected from the incinerator grate) and fly ash (the fraction made of fine particles carried away by the flux of combustion gas which is later captured by dust collectors). While the production of MSWI bottom ash is around 250 kg per ton of incinerated residue, the production of fly ash is 20 kg per ton [START_REF] Aghtm | Politique de gestion des déchets en France: Etat de l'art de l'incinération[END_REF][START_REF] Autret | Incineration of municipal and assimilated wastes in France : Assessment of latest energy and material recovery performances[END_REF]. Due to its high pollutant potential (notably high content in chlorides, arsenic, lead, zinc, mercury, cadmium and organic compounds), MSWI fly ash was classified as hazardous waste and has to be directed toward specific landfills for hazardous waste. Usually, the MSWI bottom ash fraction is classified as non-hazardous waste and is authorized in non-hazardous waste landfill, or also potentially authorized for road construction under specific conditions. Indeed, in 1994, a memorandum from the Ministry in charge of the Environment (French Environment Ministry, 1994) provided recommendations for the use of MSWI bottom ash in road construction (embankment, capping layers, subgrades….). The use of MSWI bottom ash depends on its unburned fraction and on its leaching potential controlled through a standard batch leaching test (NF X 31-210, 1992). The 1994 memorandum sets maximum limit values related to the leachate: its global soluble fraction (total dissolved solids), plus specific limit values for seven chemical parameters: arsenic, cadmium, chromium VI, lead, mercury, sulfate and total organic carbon.

Knowledge about the PCDD/F fate in the road environment

The 1994 memorandum did not set any particular recommendation regarding dioxins and furans. Since the 1990's, MSWI residue (and more specifically MSWI bottom ash) has been extensively studied in France and worldwide from the environmental point of view. This was essentially carried out with relation to its heavy metal leaching potential and poorly with relation to its organic pollutants, notably regarding PCDD/F content and release risk [START_REF] Chandler | Municipal Solid Waste Incinerator Residues[END_REF][START_REF] Bartet | Dioxine dans les MIOM : Teneurs observées et étude préliminaire de leur potentiel de transfert vers l'environnement[END_REF]. Consequently, as opposite to heavy metals for which laboratory, lysimeter and some field studies have provided leaching data [START_REF] Silvestre | Valorisation en structure routière du mâchefer d'incinération d'ordures ménagères de l'usine de Lyon-Sud[END_REF][START_REF] Adam | Valorisation des mâchefers d'incinération en technique routière : Evaluation de leur comportement en condition réelle d'utilisation[END_REF]Drouadaine et al., 1997;[START_REF] Paris | Etude du comportement de mâchefers de DIS utilisés en technique routière -Comparaison avec des mâchefers d'OM[END_REF]Drouadaine and Badreddine, 2003), very little knowledge exists today regarding the fate of PCDD/F, notably possible transfer of PCDD/F to the open environment from the use of MSWI residues in road infrastructures.

In the French context, this issue is essentially related to the effect of pre-1991 MSWI residues, as at that time bottom and fly ashes were not separated. Considering on the one hand the long term potential effect of PCDD/F, and on the other hand the infiltration of rainfall through road surface (van Ganse, 1978) and the permeability of the MSWI residue layers (i.e. 10 -5 to 10 -4 m.s -1 ) [START_REF] Badreddine | Impact sur les sols des dioxines de MIOM utilisés en technique routière[END_REF], understanding the fate of PCDD/F hold into old MSWI residues used in road structures has became a necessity. Indeed, due to its relatively poor mechanical properties, MSWI residue has primarily been used by road engineers mainly in the deeper layers of the road structure (SETRA and LCPC, 2000), where mechanical constraints are the lowest, immediately above the underlying natural soil. The latter (called road soil), due to its direct contact with the MSWI layer and to its ability to retain pollutants represents a major target to be considered in the context of alternative material use in road construction (Jullien and François, 2006).

Materials

Road site identification

In order to take advantage of the longest possible period of contact between the MSWI residue layer and the road soil, road sites for study were chosen as old as possible. However, due to of the lack of written records for the oldest roads, it was only possible to go back in time as early as 20 years ago, thanks to the memory of the people who took part to the construction. For more recent sites (around 10 years old), more data was available.

The road body is a multi-layer structure. The pavement layers (surface course, base course, sub-base) are built on the pavement foundation, consisting of the natural ground after earthworks (scrapped and compacted) called the subgrade, generally topped with a capping layer in the French design technique (SETRA and LCPC, 2000). Figure 1a presents the different road layers of a complete structure. In practice, depending on the physical stress expected on the road body during its use (load due to traffic, climatic agents) and on the properties of the road materials, the number of layers in the structure and their thickness can vary.

When the natural soil on which the road structure is built contains too many fine particles, this may induce a transfer of fine particles from the road soil to the upper layer. This phenomenon can induce an increase of the upper layer sensitivity to moisture variation, detrimental to its bearing capacity. This risk is usually avoided using a geo-textile; such a geo-textile was found in two of the investigated sites.

Four sites (noted A, B, C and D) were investigated from various regions in France.

Sites A and B were built in the second half of the 1970's, while sites C and D were built between 1991 and 1994. Their structure is described in Figure 1b. In both site, MSWI residues were used as 0-31.5 mm unbound graded aggregates.

Site A is the private road of a MSW incinerator plant, which essentially bears a heavy lorry traffic. The structure corresponds to a flexible pavement in which the MSWI residue was used in a thick sub-base layer (70 cm). The road soil is sandy. Built in 1978, site A was sampled at the age of 20.

Site B is an urban pavement which only undergoes light vehicle traffic. The structure is that of a flexible pavement in which the MSWI residue was used in the sub-base layer (25 cm). The underlying soil is silty. Built in 1976, Site B was sampled at the age of 22.

Site C is another urban pavement which undergoes light traffic. The structure corresponds to a semi-rigid pavement in which the MSWI residue was used in a quite thick sub-base layer (40 cm), topped with a bound layer made of blast furnace slag and coal fly ash. Due to the silt nature of the road soil, a geo-textile was laid by the road constructor below the MSWI residue layer in order to prevent fine particles lifting towards the sub-base layer. Built in 1992, site C was sampled at the age of 9.

Site D is a public car park platform. The uncovered structure (a simple 1 cm-thick gravelling) is unusual for traditional car parks but is sometime chosen for occasionally used ones. The MSWI ash is used in the base layer (30 cm). Due to the silty nature of the road soil, a geo-textile was also laid by the road constructor below the MSWI residue layer in order to prevent fine particles lifting towards the base layer. Built in 1994, site D was sampled at the age of 9.

Material sampling

Sampling

On each site, the sampling operation starts by digging a trench in the pavement, down to the top of the MSWI residue layer. For safety reasons, on circulated roads, the trench is dug on the road edge side. No such consideration had to be taken into account regarding the car park. In all case, beforehand, information is collected regarding the location of buried networks (water, gas, electricity…).

Usually, the thickness of the MSWI residue layer is not known exactly; on one site it can vary by few centimeters from one point to another, which makes preferable, when possible, to dig more than one trench. A core sampler is used to collect at once the MSWI residue layer and the road soil. Then, the real thickness of the MSWI residue layer at the sampling point is measured on the core sample. The thickness of the sampled underlying soil is also measured. The core sample can then be cut in different sub-layers in order to assess the contaminant content at different depths.

The upper sub-sample of the road soil, just below the MSWI residue layer, is always rather thin (usually 5 cm thick). Samples for analysis are collected in the middle of the core section. This is a precaution avoiding peripheral contamination from the upper levels to the lower ones, as while the core sampler is pushed in, particles can move along its internal wall.

When the road site makes it easy to obtain a reference situation of the soil away from the MSWI residue road layer influence, reference samples are collected with the core sampler for analysis and comparison with the road soil. Providing data on the surrounding situation regarding PCDD/F influence on the site, the so-call reference soil makes it possible to have a view of the net impact on the road soil. For all materials, before analysis, all samples were stored in plastic bags and drums, hermetically closed.

Samples

Table 1 provides an overview of the set of samples that were analyzed, together with their codification, the numbering is for the trench (1, 2 or 3). Soil samples were collected at different depths in order to detect any vertical content variation. Depth of sampling was conditioned by the soil own-thickness.

For sites A, B and C it was possible to identify a reference soil, but not for site D. In site A, four A R samples were collected for PCDD/F content analysis (0-20 cm, 20-40 cm, 40-60 cm and 80-95 cm). In sites B, two samples were collected: 0-20 cm and 60-80 for B R ,. In site C, the reference soil (C R ) was not sampled as deep as the two others: 0-5 cm and 5-15 cm.

Regarding MSWI residues, in site A, two trenches were dug into the road structure.

Only one sample was collected in the first trench (A M1 ) because at that point the MSWI residue layer was thin (12 cm). In the second trench (A M2 ), the layer thickness was greater (65 cm) and four samples were collected in the MSWI residue (A M2/14-29 ; A M2/35-45 ; A M2/45-65 and A M2/65-85 ). In site B, one trench was dug, providing one sample (B M1 ) of MSWI residue (35-60 cm). Similarly for site C (C M1 ), only one sample was collected (50-88 cm). Lastly, in site D three trenches were dug providing each one a sample (D M1 , D M2 and D M3 ) with the same geometrical characteristics (10-30 cm).

Regarding road soils, in site A, three samples were collected in the sandy road soil of trench T1 (A S1 ) (32-37 cm, 37-42 cm and 42-52 cm). Three road soil samples were also collected at trench T2 (A S2 ) ( 29 In sites C and D, during construction geo-textiles were laid between the MSWI residue layer and the road soil. One sample of geo-textile was collected in the trench of site C (C G1 ) and two geo-textile samples were collected at site D, one from trench T1 (D G1 ), the second from trench T2 (D G2 ).

Methods

Grading of materials

The total sample, dried at 105°C, is sieved at 2 mm. The fraction below 2 mm is sieved in a dried phase in a stainless steel sieve to separate the fractions below 1 mm and 0.5 mm. The particle size distribution of the fraction below 0.5 mm is achieved using the laser diffraction technique (Mastersizer, Malvern Instrument).

PCDD/F analysis

Determination of dry matter content was achieved by drying subsamples at 105°C following the standard EN ISO 11465.

The samples were beforehand dried at room temperature. The dried samples were treated and digested with chlorydric acid. Materials (MSWI residues and soil samples) were prepared using an accredited method, based on the standard EN 1948-2 andEN 1948-3, which consists in extracting the analyzed components with mixture of toluene and acetone. This step is followed with several stages of clean-up by chromatography on columns filled with absorbents using solvents or various elution strength. After filtration, the dioxin measurement requires a solid/liquid extraction followed with a cleaning up step. The purified extracts are reduced to a minimum volume and then mixed in a solvent compatible with the final analysis by gas chromatography.

The total eluate was prepared following the same methodology as the one used for MSWI residues.

Analyses of PCDD/F were carried out by means of a High Resolution Mass Spectrometry coupled with a High Resolution Gas Chromatography (HRMS/HRGC) VG/AutoSpec. The analysis consisted in measuring 17 toxic congeners (7 congeners for PCDD and 10 congeners for PCDF). The seven dioxin congeners are 2,3,7,8-TetraCDD; 1,2,3,7,1,2,3,4,7,1,2,3,6,7,1,2,3,7,8,1,2,3,4,6,7,8-HeptaCDD and OctaCDD. The ten furan congeners are 2,3,7,8-TetraCDF; 1,2,3,7,8-/1,2, 3,4,2,3,4,7,1,2,3,4,7,2,3,4,7,1,2,3,6,7,1,2,3,7,8,1,2,3,4,6,7,1,2,3,4,7,8,9

Leaching

Regarding the geo-textiles found on two sites, before chemical analyses, the recovery of particles was realized by means of leaching, intended to wash the geotextile. The operation was renewed until the complete extraction.

Regarding MSWI residues, as PCDD/F are not water soluble compounds (Sakai et al., 2000a), the purpose of the test was not to assess their solubility but their possible residual release associated to very fine particle extraction (namely below 0.45 µm in the condition of the test due to the filter size) under the effect of water flushing. The leaching test was performed for MSWI residues from sites B and D following the EN 12457-2 standard protocol (2002). Determination of the dry matter content was made after drying of test portion at 105°C, according to the international standard ISO 11465 (1994). The liquid/solid separation is performed by means of filtration on a 0.45 µm membrane filter using a pressure filtration device. The eluate obtained after filtration was analyzed to determine PCDD/F compounds' nature and quantity. The PCDD/F congener with the greatest toxic potential, and for which the greatest amount of toxicological data is available, is 2,3,7,8-TetraCDD (Mac Kay, 2002). Its content in all material samples is provided (in ng.kg -1 DM).

Results

Results

Additionally, in order to assess the distribution of the different PCDD/F compounds in the different materials the sum of the seven PCDD congeners and that of the ten PCDF congeners was calculated (in ng.kg -1 DM).

MSWI residues' characteristics

PCDD/F contents in MSWI residues

Results are presented in Table 2. In site A, the only one showing values below 100 ng I-TEQ.kg -1 DM, the PCDD/F content for the MSWI residue in trench T1 (A M1 ) is 14.0 ng I-TEQ.kg -1 DM. In trench T2 (A M2 ), it increases from the upper sample to the third one (from 35.7 to 227 ng I-TEQ.kg -1 DM) and decreases in the lower sample (63.2 ng I-TEQ.kg -1 DM).

In site B (B M1 ), the PCDD/F content for the MSWI residue is 721 ng I-TEQ.kg -1 DM, three time higher than the maximum value measured in site A.

In site C (C M1 ), the PCDD/F content is similar to the maximum value found in site A (235 ng I-TEQ.kg -1 DM).

The three trenches of site D (D M1 , D M2 and D M3 ) show, by far, the highest PCDD/F contents. These contents are respectively 1960, 2160 and 1640 ng I-TEQ.kg -1 DM.

Relation between PCDD/F amount and particle size

A sample with a high PCDD/F content (D M1 ), was chosen in order to assess the possible relation between the particle size distribution of MSWI residues and their PCDD/F load. PCDD/F contents related to fractions < 0.1 mm; 0.1-0.5 mm; 0.5-1 mm; 1-2 mm; 2-10 mm; 10-31.5 mm of the material are presented in Figure 2.

The PCDD/F contents of fractions between 2 and 31.5 mm are similar, comprised between 733 ng I-TEQ.kg -1 and 888 ng I-TEQ.kg -1 . The finest fractions and particularly the one below 0.1 mm is 7 times higher, reaching 6590 ng I-TEQ.kg -1 .

Leaching of MSWI residues

The leaching test was carried out in order to detect the proportion of PCDD/F particles inferior to 0.45 µm. For both sites (B and D), leaching results (Table 3) show that leachates contain a small amount of PCDD/F: 2.19 pg I-TEQ.l -1 for sample B M1/35-60 ; and 4.4.1 pg I-TEQ.l -1 for sample D M1/10-30 . Brought to the mass of dry material involved in the leaching test, such releases are respectively equal to 0.019 ng I-TEQ. kg -1 DM for the first sample, and 0.041 ng I-TEQ. kg -1 DM for the second.

Geo-textiles' load of PCDD/F

The load of PCDD/F on the geo-textile from site C (C G1 ) is 175 ng I-TEQ.kg -1 DM (Table 4). Site D geo-textile samples show far higher values : 754 ng I-TEQ.kg -1 DM in trench T1 (D G1 ), and 1600 ng I-TEQ.kg -1 DM in trench T2 (D G2 ). The latter value is close to the content of the MSWI residue layer above.

PCDD/F contents in road soils

For site B (B S1 ), it varies from 7.23 to 0.57 ng I-TEQ.kg -1 DM from the upper to the lower sample. In site C (C S1/88-93 ), the single sample value is 2.87 ng I-TEQ.kg -1 DM.

In site D, from the top to the bottom of the investigated thickness in trench T1 (D S1 ), the three values are respectively 0.86, 0.79 and 1.99 ng I-TEQ.kg -1 DM. In trench T2 (D S2 ), the values are of the same order (2.22 and 0.64 ng I-TEQ.kg -1 DM). Lastly, trench T3 sample (D (D S3 ) PCDD/F content is 2.04 ng I-TEQ.kg -1 DM (Table 5).

Site A presents a contrasted situation regarding its two trenches. In trench T2 (A S2 ), the PCDD/F contents of the three samples are in the range of the previous cases B, C and D, with values evolving from 2.98 to 2.24 and 1.31 ng I-TEQ.kg -1 DM, from the upper to the lower sample. On the other hand, in trench T1 (A S1 ), whereas the two first samples present low values (1.35 and 0.74 ng I-TEQ.kg -1 DM), the deeper sample shows a content of 24.8 ng I-TEQ.kg -1 DM, which is relatively high in comparison with the content of the MSWI residue layer located just above (i.e. 14.0 ng I-TEQ.kg -1 DM). As a consequence, this values (A S1/42-52 ) is very uncertain and will not be considered in the interpretation.

PCDD/F contents in reference soils

Results are presented in the Table 6. In site A (A R ), the PCDD/F content of the reference soil decreases from 9.4 to 0.30 ng I-TEQ.kg -1 DM, from the surface to the deeper sample. The top sample value can be related to the atmospheric contamination in the vicinity of the incineration plant, from the chimney, at a time (the 1970's) when fume de-pollution by means of old dust separators was not as efficient as the nowadays air pollution control systems [START_REF] Chandler | Municipal Solid Waste Incinerator Residues[END_REF] In site B (B R ), contents are homogeneous and low (1.05 and 0.79 ng I-TEQ.kg -1 DM). Contrary to site A, it seems that no particular source of pollution has an effect on the vertical profile of content.

Site C (C R ) shows intermediate values between the two previous ones i.e. 5.40 and 6.49 ng I-TEQ.kg -1 DM for the upper and the lower samples respectively. Site C is not located in the vicinity of a particular source of PCDD/F, but it is however located in a large urban area with several potential sources.

Distribution of PCDD/F congeners in the road structure

Results related to the distribution of the different PCDD/F congeners (Tetra, Penta, Hexa, Hepta and Octa) in the MSWI residues, on geo-textiles samples, and in the road soils, for all sites, are illustrated by the example of site A trench T2 (Figure 3) and site C (Figure 4). For MSWI residues, the distribution between dioxin and furan congeners is provided in Table 2. The proportion of dioxin congeners is of 50% for C M1 but it is higher for other samples: 73% for B M1 ; 74 to 79% for D M and 57 to 97% for A M .

The distribution between dioxin and furan congeners is provided in Table 4 for geotextiles. In the geo-textile from site D, the proportion of dioxin congeners (74-75%) is equivalent to that of the above MSWI residue. For site C, it is higher (69%) to that of the MSWI residue.

The distribution between dioxin and furan congeners is provided in Table 5 for road soils. For road soils, the proportion of dioxin congeners for all sites, ranges from 54 to 89%. For sites A, B and D it is comparable to the distribution observed for the MSWI residue. For site C, it is higher.

The distribution between dioxin and furan congeners is provided in Table 6 for reference soils. It is comparable to that of the respective road soils, 85% for B R ; 72% for C R ; 69 to 90 for A R .

The toxicity of PCDD/F varies substantially depending on the different congeners. It is generally agreed that only 17 out of the 210 dioxin and furans congeners are toxic.

The examination of is 2,3,7,8-TetraCDD content in the different samples of MSWI residue samples, geo-textiles, road soils and reference soils, reveals that its amount in road soils is very low.

For road soils (Table 5), contents range from less than 0.04 ng.kg -1 (samples A S2/85-90 and D S2/35-45 ) to 2.35 ng.kg -1 (sample A S1/42-52 ), not very different from those of reference soils (Table 6), ranging from less than 0.01 ng.kg -1 (samples B R/0-20 and C R/0-5 ) to less than 0.9 ng.kg -1 (sample A R/0-20 ).

As a matter of comparison, for MSWI residues (Table 2), the 2,3,7,8-TetraCDD content is below 1.5 ng.kg -1 for site A, and ≤ 50 ng.kg -1 for sites B, C and samples D M1 and D M3 . Sample D M2 content is 190 ng.kg -1 . For geo-textiles (Table 4), contents are from 6.8 to 57.5 ng.kg -1 .

Discussion

Characterization of MSWI residues

PCDD/F contents

A great heterogeneity of MSWI residue PCDD/F contents, i.e. a factor 154 from 14.0 (A M1 ) to 2160 ng I-TEQ.kg -1 DM (D M2 ) is observed between sites. A great heterogeneity can also be observed in a site such as A (a factor 16 between 14.0 and 227 ng I-TEQ.kg -1 DM).

The lowest recorded value (14.0 ng I-TEQ.kg -1 DM for A M1-12-24 ) is in the middle of the range of values reported by Damien (1997) for bottom ash produced by recent incinerators in France in the 1990's (4.0 to 20.6 ng I-TEQ.kg -1 DM). All other contents are two times to 100 times above the maximum value recorded by Damien Such results are also well above the contents reported by [START_REF] Badreddine | Evaluation du transfert des composés organiques des MIOM utilisés en sous-couche routière dans des ouvrages de construction récente[END_REF] on MSWI bottom ash from recent incineration and treatment facilities (i.e. around 10 ng I-TEQ.kg -1 DM) (Figure 5). Highest values (M D ) are even above the highest value (i.e. 1500 10 ng I-TEQ.kg -1 DM) reported by Sakai et al. (2000b) for Japan. A comparison showing difference between MSWI residue from the old and from recent site was proposed in figure 5.

Importance of the fine fraction

The analyses of size distribution of MSWI residues have shown an important proportion of the fine fraction.

The possible relation between the particle size and the PCDD/F content was postulated in an earlier study [START_REF] Badreddine | Impact sur les sols des dioxines de MIOM utilisés en technique routière[END_REF]. The good relation between the PCDD/F content and the particle size demonstrates that the fine fraction is enriched with PCDD/F compounds. The sub-sample with the finest fraction (< 0.1 mm) shows by far a higher PCDD/F content (6590 ng I-TEQ.kg -1 ), 7 times above the other particle fractions. As the fraction below 0.1 mm represents less than 10% of the all graded aggregate (MSWI residue), this means that at least 659 ng I-TEQ.kg -1 (i.e. 33%) of the 1960 ng I-TEQ.kg -1 of the whole material (sample D M1/30-60 ), are included in the finest fraction.

These results confirm the assumption of the relation between the high value of the PCDD/F and the presence of fly ash mixed with bottom ash before 1991 in France.

Indeed, the PCDD/F levels in fly ash are generally much higher than in bottom ash (Mac Kay, 2002). [START_REF] Chang | Dioxin contents in fly ashes of MSW Incineration[END_REF] reported values between 41 ng.g -1 and 703 ng.g -1 DM for MSWI fly ash).

Considering on one hand the range figures provided by Damien (1997) for MSWI bottom ash (39 to 648 ng I-TEQ.kg -1 ) and for MSWI fly ash (765 to 4815 ng I-TEQ.kg - 1 ), and on the other hand the ratio of production between bottom ash (250 kg.ton -1 ) and fly ash (20 kg.ton -1 ) from incineration, one reaches the range of 67 to 814 ng I-TEQ.kg -1 for a theoretical mixture of bottom and fly ash. PCDD/F contents observed for MSWI residues from site A trench T2 (A M2 ), from site B (B M1 ) and site C (C M1 ) are in this range. The sample from site A trench T1 is below, but those from site D are two times above the highest value of the theoretical range.

Mobility of PCDD/F

Very low PCDD/F releases were assert (0.0219 and 0.0441 ng I-TEQ.kg -1 ).

Compared to the total content of PCDD/F compounds in the respective MSWI residue samples (i.e. 721 ng I-TEQ.kg -1 DM for B M1/35-60 and 1960 ng I-TEQ.kg -1 DM D M1/10-30 ), the released fractions are respectively 0.003 and 0.002% of the total PCDD/F amount.

The low value recorded can be linked to the low solubility of the PCDD/F and the presence of the PCDD/F in the particles superior to 0.45 µm [START_REF] Badreddine | Impact sur les sols des dioxines de MIOM utilisés en technique routière[END_REF]. Depending on congeners, solubility values are comprised between 0.74 10 -7 and 3.75 10 -3 for PCDD (Inserm, 2000) and between 4.19 10 -4 and 1.16 10 -6 for PCDF (OMS, 1997).

Assessment of PCDD/Ffate

PCDD/F contents of road soil

The heterogeneity among all road soil PCDD/F contents, i.e. a factor 9 between 0.74 (A S1 ) and 7.23 ng I-TEQ.kg -1 DM (B S1 ) is far lower than among MSWI residues. Such values are low in comparison to those reported by [START_REF] Nominé | Méthodologie pour l'évaluation de la contamination par les dioxines au voisinage d'une source fixe[END_REF] for soils of urban areas in the absence of neighbouring sources of pollution (from <1 to more than 30 ng.kg -1 I-TEQ.kg -1 DM), for soils nearby MSW incineration plants (more than 1000 ng.kg -1 I-TEQ.kg -1 DM), and even to those of soils of pasture in Europe (from <1 to 43 ng.kg -1 I-TEQ.kg -1 DM).

Comparison to reference soils' contents

In order to assess the state of road soils, comparison can be made with their respective reference soils. Depending on the context (more or less potential sources of pollution in the area), the difference between them is negative (site B), negligible (site A), or even positive (site C where pollution sources affect the reference soil). Indeed, regarding Site B, the ratio between the average contents in road soil (3.9 ng I-TEQ.kg -1 DM) and the reference soil (0.92 ng I-TEQ.kg -1 DM) is around 4.

Regarding Site A, depending on the consideration of sample A R/0-20 or not in the comparison, one can consider that the PCDD/F content in the road soil is lower (9 times) or slightly higher (1.04 ng I-TEQ.kg -1 DM in average for road soil vs 0.44 ng I-TEQ.kg -1 DM in average for reference soil) in trench T1. Considering trench T2, the conclusion is the same, i.e. respectively 4 times lower, or slightly higher (2.17 ng I-TEQ.kg -1 DM in average for road soil vs 0.44 ng I-TEQ.kg -1 DM).

In the case of Site C, the content in the road soil sample (C S1/88-93 ) is 2 times lower than in the reference soil (5.94 ng I-TEQ.kg -1 DM in average). Located in a large urban area, references soil of Site C can have been affected for years by several sources, which can lead to a situation similar as for Site A.

Comparison to MSWI residues' contents

Some vertical profiles of PCDD/F contents in road soils (sites A and B) show decreasing values from the upper sample, downward, indication of a possible contact effect of the MSWI residue layer. Contents in the road soil are however low and the transition with the MSWI residue layer with high contents, is very well marked.

For sites A and B, where the influence of the geo-textile is not susceptible to interfere, the ratio between the road soil upper sample content and the MSWI residue layer content can serve as an indicator. Regarding Site A trench T1, this ratio (1.35 vs 14.0 ng I-TEQ.kg -1 DM) is 9.6%, and for trench T2 (2.98 vs 101 ng I-TEQ.kg -1 DM in average) it is 2.9% (the MSWI residue content is 101 ng I-TEQ.kg -1 DM in average). Regarding Site B, the ratio (7.23 vs 721 ng I-TEQ.kg -1 DM) is only 1.0%.

For sites with geo-textiles, such ratios are 1.2% for site C (2.87 vs 235 ng I-TEQ.kg -1 DM), or lower for site D, i.e. 0.04% for trench T1 (0.86 vs 1960 ng I-TEQ.kg -1 DM) and 0.1% for trench T2 (2.22 vs 2160 ng I-TEQ.kg -1 DM).

Role of geo-textiles

The PCDD/F load of geo-textiles compared to the content of the MSWI residue located just above is equal to 74% of the latter in the case of site C (C G1 /C M1 ). For site D, such ratios are equal to 86% in the case of trench T1 (D G1 /D M1 ) but only 35% in the case of trench T2 (D G2 /D M2 ).

In order to assess the role of geo-textiles toward the possible PCDD/F transfer, their load can also be compared to the content of the road soil upper sample. In the case of site C, Such ratio is 61 (C G1 /C S1 ), and it is higher in the case of site D, 1860 for trench T1 (D G1 /D S1 ), and 339 for trench T2 (D G2 /D S2 ).

The role of the geo-textile inserted between the MSWI residue layer and the road soil should be clarified : may be it acts as a filter toward the transfer of fine particles downward. But may be the porosity of the road soil in itself is low enough to act as a filter, which would explain the slightly higher PCDD/F contents in some road soil upper samples.

Conclusion

This study shows that some MSWI residues used for road construction before the enforcement of the 1991 order can contain very high amount of PCDD/F compared to the MSWI bottom ash produced and used afterward. The heterogeneity between MSWI residues can be great from one road site to another but also on a single site.

The important contribution of the fraction below 0.1 mm to the MSWI residue total contents of PCDD/F was asserted. Crossed with leaching test results, this indicates that the fraction between 0.45 and 100 m should be investigated in more detail in order to specify the most loaded particle sizes.

As a whole, road soils PCDD/F contents are below the contents recorded for ordinary urban or rural soils, and consequently, no significant difference is observed with local reference soils. This indicate the absence of transfer of MSWI residue to the road soil.

In addition, thanks to the very low proportion of the 2,3,7,8-TetraCDD congener, the noxiousness of road soils is reduced. In the presence of geo-textile or not, the PCDD/F content reduction between the MSWI residue and the road soil is great. The role of the geo-textile inserted between the MSWI residue layer and the road soil should be clarified. Table 1 : Overview of the set of samples

List of Tables

  -35 cm, 85-90 cm and 95-105 cm). Sample A S2/29- 35 taken from trench T2 is a thin sand layer which accidentally slid on the MSWI residue during the road work; it was not removed and has remained inserted in the MSWI residue layer since then. In site B (B S1 ), two samples were collected in the silty road soil (B S1/60-70 and B S1/70-80 ). In site C, a single road soil sample (silty) was collected (C S1/88-93 ). In site D, three silty road soil samples were collected in trench T1 (D S1/30-35 ; D S1/35-45 and D S1/93-103 ); two in trench T2 (D S2/30-35 and D S2/35-45 ); and a single sample in trench T3 (D S3/30-35 ).

  -HeptaCDF and OctaCDF. Interferences with the other 193 non toxic congeners and some other components such as Polychlorobiphenyls (PCBs), Polychloroterphenyls (PCTs), or Polychloronaphtalens (PCNs) are eliminated. The determination of congeners was realized by the isotopic dilution method using isotope as interne markers for congeners identification and quantification. The concentration of one PCDD or PCDF congener can be converted into an International Toxic Equivalent Quantity (I-TEQ), with a Detection Limit (DL) of 1.22 nanogram I-TEQ.kg -1 of dry material (noted DM) and an accuracy of 4.7% on the Toxic Equivalent value. The model used for determining the I-TEQ is NATO (1998).

  concerning old MSWI residues are first presented. Then are those of geotextiles, road soils and reference soils. All material samples are defined in relation to their depth from the surface (expressed in centimeters) and all PCDD/F contents are expressed in nanogram of I-TEQ per kilogram of dry matter (noted ng I-TEQ.kg -1 DM).

  For site A, regarding the MSWI residue (A M2/14-29 ; A M2/35-45 ; A M2/45-65 ; A M2/65-85 ) they show the predominance of OctaCDD, and the secondary importance of HeptaCDD and HeptaCDF. For site C, they show the predominance of OctaCDD and OctaCDF in the MSWI residue and the secondary importance of HeptaCDD. The geo-textile of site C also show the predominance of OctaCDD and the secondary importance of HeptaCDD.

  Figure 1a: The typical road structure in France

Figure 2 :Figure

 2 Figure 2 : relation between particle size and PCDD/F content

Table 2 :

 2 PCDD/F contents of MSWI residues

Table 3 :

 3 PCDD/F contents from eluates

Table 4 :

 4 PCDD/F loads of geo-textiles

Table 5 :

 5 PCDD/F contents of road soils

Table 6 :

 6 PCDD/F contents of reference soils

Table 1 :

 1 Overview of the set of samples

	Site	A	B	C	D
	Material (M)	A M1 , A M2	B M1	C M1	D M1 , D M2 , D M3
	Geo-textile (G)	-	-	C G1	D G1 , D G2
	Road soil (S)	A S1 , A S2	B S1	C S1	D S1 , D S2 , D S3
	Reference soil	A R	B R	C R	-
	(R)

Table 2 :

 2 PCDD/F contents of MSWI residues

	Site

Table 4 :

 4 PCDD/F loads of geo-textiles

	Site	C	D
	Sample	C G1	D G1	D G2
	ITE (ng I-TEQ.kg -1 DM)	175	1600	754
	2,3,7,8-TetraCDD	6.84	34.9	57.5
	(ng.kg -1 )
	7 Dioxin congeners	5346	74545	28437
	(ng.kg -1 )
	10 Furan congeners	2407	26384	9317
	(ng.kg -1 )
	Dioxin (%)	69	74	75

Table 5 :

 5 PCDD/F contents of road soils

	Site

Site