Véronique Solé-Jamault
email: veronique.sole@inrae.fr

Joëlle Davy
email: joelle.davy@inrae.fr

Rémy Cochereau
email: remy.cochereau@inrae.fr

Adeline Boire
email: adeline.boire@inrae.fr

Colette Larré
email: colette.larre@inrae.fr

Sandra Denery-Papini

⁻ Adeline

Optimization of large-scale purification of omega gliadins and other wheat gliadins

Keywords: Wheat protein fractionation, gluten proteins, omega-gliadins, cation exchange chromatography

Among wheat storage proteins, omega-gliadins display a singular amino acid sequence only composed by repetitive sequences. They have been described as a major wheat allergen and also contain T-cell epitopes implicated in coeliac disease. To study the structural and biophysical properties of omega-gliadins and other gliadins (e.g., gluten network formation, allergic response), highly purified and concentrated fractions are needed. In the present work, we used chromatography media screening to improve their fractionation. A S Ceramic Hyper D (Pall) resin was selected for its ability to concentrate omega-gliadins in the first elution peaks.

Gamma-and beta-gliadins were then separated by hydrophobic interaction chromatography and eluted with an ethanol gradient. Alpha-gliadins were purified by gel filtration. Each fraction was characterized by electrophoresis and reverse phase HPLC. The developed preparative protocol enabled the purification of several grams of highly purified gliadins to the detriment of the yield.

INTRODUCTION

Wheat is overwhelmingly present in the diets of Western civilizations. Unfortunately, wheatderived products are also responsible for adverse reactions such as coeliac disease and food allergies. The largest number of allergenic plant proteins is found among the prolamin superfamily, which notably contains cereal storage proteins such as gliadins [START_REF] Tatham | Allergens to wheat and related cereals[END_REF]. Gliadins are monomeric proteins characterized by their solubility in alcohol-water media, with molecular weights (MW) ranging from 30 to 45 kDa (major fraction) and up to 75 kDa (minor fraction) and isolelectric points (pI) from 5 to 8 [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. Gliadins consist of highly polymorphic polypeptides divided into four groups: alpha, beta, gamma, and omega, according to their decreasing electrophoretic mobility at low pH. Because of their molecular weight, their low sulfur content and their particular amino acid sequence based on only repetitive motives, omega-gliadin differ from other gliadins [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. According to their N-terminal and repetitive sequences, to their elution order in reverse phase high performance liquid chromatography (RP-HPLC) and to their electrophoretic mobility, they are separated into 2 subgroups: omega 5-gliadin, also called fast omega-gliadins (eluted first in RP-HPLC), and omega 1,2-gliadins, slow omega-gliadins (eluted second in RP-HPLC) [START_REF] Schalk | Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials[END_REF][START_REF] Van Eckert | Towards a new gliadin reference material-isolation and characterisation[END_REF]. Omega 5 gliadins have been reported as a major allergen in Wheat-Dependent Exercise-Induced Anaphylaxis (WDEIA) [START_REF] Morita | Fast ω-gliadin is a major allergen in wheat-dependent exercise-induced anaphylaxis[END_REF], while omega 1,2 gliadins have been identified as one of the dominant allergens in allergic reactions to deamidated gluten [START_REF] Denery-Papini | Allergy to deamidated gluten in patients tolerant to wheat: specific epitopes linked to deamidation[END_REF]. Gliadins are the main triggers of coeliac disease. In particular, T-cells from coeliac patients recognize epitopes containing the repetitive sequences present in omega-gliadins [START_REF] Sollid | Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules[END_REF]). Yet, omega-gliadins are only a minor group among prolamins, amount-wise, and have been until recently less well characterized than other prolamin classes for their polymorphism and contribution to gluten properties [START_REF] Altenbach | Towards reducing the immunogenic potential of wheat flour: Omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA[END_REF].

The storage proteins of wheat grains have the unique property of forming a viscoelastic network called gluten, which is essential for the preparation of a wide diversity of wheat-based foods.

Gliadins are essential in the formation of the gluten network in combination with glutenins: in dough, hydrated gliadins are commonly thought to contribute to the viscosity and extensibility of the network [START_REF] Cornec | Characterisation of Gluten Subfractions by SE-HPLC and Dynamic Rheological Analysis in Shear[END_REF][START_REF] Shewry | What is gluten-Why is it special?[END_REF]. Baking quality has been correlated positively with the contents in glutenin polymeric proteins and omega-gliadin [START_REF] Malalgoda | Association between gluten protein composition and breadmaking quality characteristics in historical and modern spring wheat[END_REF]. The repeated sequences present in omega-gliadins and glutenins are believed to form inter-protein hydrogen bonds resulting in the particular viscoelastic properties of the gluten network [START_REF] Belton | Mini Review: On the Elasticity of Wheat Gluten[END_REF]. Besides, omega-gliadins were recently found to form supramolecular assemblies with glutenin through non-covalent interactions [START_REF] Morel | Insight into gluten structure in a mild chaotropic solvent by asymmetrical flow field-flow fractionation (AsFlFFF) and evidence of non-covalent assemblies between glutenin and ω-gliadin[END_REF].

To study the structural, biophysical or immunochemical properties of omega-gliadins, highly purified and concentrated fractions are needed. Gliadins comprise several groups of proteins encoded by multigene families responsible for heterogeneity within each group. This complexity and the variable amount of each gliadin within wheat varieties makes their fractionation and study difficult. For example, the proportion of omega-gliadin may vary from 10 to 20 % of the total gliadins as recently reviewed by Shewry [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. Their proportion is increased under increased nitrogen availability or sulfur deficiency in the cultivation conditions [START_REF] Wan | Effects of nitrogen nutrition on the synthesis and deposition of the ω-gliadins of wheat[END_REF]. Several methods have been described to obtain more or less purified gliadins but, generally, they yielded fractions comprising several components or only small quantities of individual proteins.

Recombinant proteins or peptides have been produced to study the role of the immunedominant part of gliadins [START_REF] Denery-Papini | Anti-Peptide Antibodies Directed Against Omega-Gliadins for the Detection of Sequences from Bread and Durum Wheats[END_REF][START_REF] Mameri | Structural Basis of IgE Binding to α-and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains[END_REF][START_REF] Matsuo | Molecular cloning, recombinant expression and IgE-binding epitope of omega-5 gliadin, a major allergen in wheat-dependent exerciseinduced anaphylaxis[END_REF][START_REF] Tamás | Heterologous expression and protein engineering of wheat gluten proteins[END_REF]. Such a strategy ensures the authenticity of the expressed proteins but masks the heterogeneity of the group and yields only low amounts of proteins. Moreover, differences may be observed between purified and recombinant gliadin fractions in their secondary structure contents and antigenic activities [START_REF] Mameri | Structural Basis of IgE Binding to α-and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains[END_REF].

Classically, purification of gliadins starts with obtaining gluten by extensive washing of hydrated flour with water followed by successive extraction according to Osborne fractionation [START_REF] Marsh | Extraction, Separation, and Purification of Wheat Gluten Proteins and Related Proteins of Barley, Rye, and Oats[END_REF][START_REF] Schalk | Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials[END_REF]. These conditions of extraction separate gliadins from glutenins, even though gliadin fractions may still contain traces of glutenins [START_REF] Shewry | What is gluten-Why is it special?[END_REF].

Usually, omega-gliadins were fractionated with a combination of cation exchange chromatography at acidic pH [START_REF] Charbonnier | Isolation and characterization of ω-gliadin fractions[END_REF]. All cation exchange chromatography resins are effective in separating omega-gliadins from other groups of gliadins [START_REF] Charbonier | Large Scale Isolation of Gliadin Fractions[END_REF][START_REF] Larre | Fractionation of gliadins from common wheat by cation exchange FPLC[END_REF][START_REF] Patey | Large-Scale preparation of gliadin proteins[END_REF][START_REF] Popineau | Purification and characterisation of ωgliadin components from common wheat[END_REF]. A low salt concentration makes it possible to selectively elute omega-gliadins. They are thus obtained in the first eluted fractions, but many authors noted that some of these omega-gliadins were eluted later, thus scattering the omega fraction and contaminating the other fractions [START_REF] Charbonier | Large Scale Isolation of Gliadin Fractions[END_REF]Larré et al., 1991). The yield is often not specified in literature but is generally around 60 % for all gliadins groups corresponding to a few hundreds mg for omega-gliadins [START_REF] Patey | Large-Scale preparation of gliadin proteins[END_REF][START_REF] Schalk | Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials[END_REF]. To produce more than one gram of pure omegagliadins, the fractionation should run over several hours up to several days when large gliadin amounts are treated [START_REF] Patey | Large-Scale preparation of gliadin proteins[END_REF]. A regeneration step in harsh conditions is necessary to eliminate proteins clogged onto the column to keep a good yield and reproducibility [START_REF] Larre | Fractionation of gliadins from common wheat by cation exchange FPLC[END_REF][START_REF] Popineau | Purification and characterisation of ωgliadin components from common wheat[END_REF]. This work aims to identify a cation exchange chromatography medium able to separate omegagliadins from other gliadins, with a high yield. Different chromatography media were compared: strong (sulfoethyl, S and sulfopropyl, SP) and weak cation (carboxymethyl, CM) exchanger resins and the following parameters were explored: resolution, yield and duration.

Several chemical features for the support matrix were tested to evaluate possible interactions with the resin. A resin was selected and applied at a preparative scale to obtain highly purified fractions. This procedure helped us to purify omega-gliadins and all other gliadin groups for future physico-chemical investigations.

MATERIALS AND METHODS

2.1. Materials Bread wheat grains (7 kg) were obtained from INRAE, Le Rheu, France (cv. Récital harvested in 2018) and were grounded into flour (5 kg) by Livrac (SA) (France). Protein content of wheat flour were 13.6 % on a dry basis.

Gliadins extraction

The flour was hydrated, kneaded into a dough and washed with water using a Martin-type process on a preparative scale [START_REF] Popineau | Fractionation and characterisation of γ-Gliadins from bread wheat[END_REF]. The recovered gluten was freezedried, ground with a Waring blender and defatted with dichloromethane during 2 hours at room temperature (gluten/solvent ratio (m/v) : 1/4). After filtration (Whatman filter paper n°3), two rinsing steps with the same gluten/solvent ratio were performed. Then, the solvent was evaporated under vacuum and defatted gluten was freeze-dried.

Total gliadins were extracted from dried gluten powder (514 g) with ethanol 70% (v/v) (gluten/solvent ratio (m/v): 1/6) during 3 hours with a propeller mixer at 850 rpm. The suspension was centrifuged at 10 000 g during for 30 min (Avanti J-26 XP, Beckman Coulter) to pellet glutenins not soluble in alcohol. The pellet was discarded and the supernatant was extensively dialyzed against water and then acetic acid 0.01 M and freeze-dried. It contained total gliadins and glutenins in a small amount.

Chromatography media screening

Different ion exchange chromatography resins were tested under fixed conditions: Toyopearl CM 650 M (Tosoh), Cellufine CM C500 (Amicon), CM Ceramic hyper D, S Ceramic Hyper D and SP Trisacryl M (Pall). Each resin (30 mL) was packed in XK 16/15 support following the manufacturer's instructions. Gliadins (1 g) were dispersed in 50 mL buffer (Na lactate 0.01 M pH 3.6, urea 2 M) overnight on a rotary shaker. The extract was centrifuged 30 min at 20 000 g (SR 12.22, Jouan Centrifuge, France) and 5 mL supernatant were injected onto the column at 1 mL/min. First, the column was equilibrated with the extraction buffer and washed with 60 mL (corresponding to twice the column volume). Proteins were eluted in a 420ml gradient from 0 to 30% of the same buffer pH 3.6 with NaCl 0.5 M using a total volume corresponding to 14 times the column volume. Protein absorbance was recorded at 280 nm. Fractions of 5 mL were collected and analyzed with polyacrylamide gel electrophoresis.

Preparative fractionation

Total gliadins (10 g) per run were dissolved in buffer (Na lactate 0.01 M, pH 3.6, urea 2 M) and stirred for 2 hours at room temperature on a rotary shaker. They were then centrifuged for 30 min at 17 000 g (Beckam, Avanti JSP26, rotor JSP F500). The supernatant was injected on an S Ceramic Hyper D column (5 x 15 cm) using a preparative purification system (Pharmacia biotech, Sweden). The column was first equilibrated with buffer (Na lactate 0.01 M, pH 3.6, urea 2 M) and gliadins were eluted with a step gradient of NaCl 0.5 M. The absorbance of proteins was recorded at 280 nm. This chromatography step allowed for separating the different groups of gliadins. Omega-gliadins were eluted in the first peaks. They were dialyzed first in water, then in acetic acid 0.01 M and freeze-dried.

Eluted fractions containing alpha gliadins (2 g) were purified on a Sephacryl S100 HR column (5 x 100 cm) equilibrated in buffer (Na lactate 0.01 M, pH 3.6, urea 0.5 M). Different fractions were pooled, dialyzed first in water, then in acetic acid 0.01 M and freeze-dried.

The other eluted fractions containing beta and gamma gliadins (10 g) were loaded on SP Sepharose FF (5 x 15cm) in buffer (Na lactate 0.01 M, pH 3.6, urea 2 M). Proteins were eluted using a gradient from 0 to 25 % of the same buffer with NaCl 0.5 M. Different peaks were pooled, dialyzed first in water, then in acetic acid 0.01 M and freeze-dried. This chromatography step separated gamma-44 and 46 gliadins forms, and removed traces of alpha/beta gliadins.

Gamma gliadins were last purified on a phenyl sepharose FF column (5 x 15cm). The column was equilibrated in buffer (Na lactate 0.01 M, pH 3.6, urea 2 M), then the sample (1.5 g) dissolved in the same buffer was injected. The column was washed with buffer (ammonia 0.02 M pH 11), and proteins were eluted in a gradient from 0 to 100 % of the same buffer with ethanol 70 % (v/v). Different fractions were pooled, dialyzed first in water, then in acetic acid 0.01 M and freeze-dried.

Beta gliadins were purified from the flowthrough peak of phenyl sepharose step. Different fractions were pooled, dialyzed first in water, then in acetic acid 0.01 M and freeze-dried.

Analytical characterization

Determination of water, ash, and protein contents

Prior to analysis, protein powders were stored at 20 °C in a desiccator containing K2CO3 saturated salt to ensure constant moisture content. The water content was measured on equilibrated powders using thermogravimetric analysis under nitrogen atmosphere (TGA 2050, TA-Instrument). About 10 mg of powder were heated from 20 to 105 °C at 3 K/min and at 105 °C for at least 80 min until constant weight was achieved.

Protein content was determined according to the Dumas method with an elemental analyzer for nitrogen (Vario Micro Cube, Elementar, Frankfurt, Germany) with nitrogen-to-protein conversion factor of 5.4 as recommended for wheat proteins [START_REF] Mariotti | Converting nitrogen into protein -Beyond 6.25 and Jones' factors[END_REF].

Polyacrylamide gel electrophoresis at acid pH (A-PAGE)

Gliadins were separated onto acid polyacrylamide gel according to the method described by [START_REF] Morel | Acid-Polyacrylamide gel-electrophoresis of wheat glutenins -a new tool for the separation of high and low-molecular-weight subunits[END_REF]. Briefly protein solutions were loaded onto a 12 % polyacrylamide gel composed of urea 2 M, ascorbic acid 0.1 %, ferrous sulfate 7 H2O 0.0014% (w/v) and acetic acid 1 N 13 %, pH 3.1 polymerized by 60 µL H2O2 0.6 % (v/v). Migration in acetic acid buffer pH 3.1 was run during 1 h 40 under 250 V. Then gels were fixed in trichloroacetic acid 10 % and stained with Coomassie Brilliant blue R250 0.4 % (w/v).

Polyacrylamide gel electrophoresis in sodium dodecylsulfate (SDS-PAGE)

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions. Proteins were dissolved at 1 mg/mL in a pH 6.8-buffer constituted of Tris 0.5 M, SDS 10%, glycerol 30%, β-mercaptoethanol 8% and bromophenol blue buffer. Samples were incubated at room temperature overnight and heated at 95 °C for 5 min before being loaded onto 4-12% Bis-Tris precast gels Novex Nupage™ (Invitrogen, Thermo Fisher Scientific) with MES SDS running buffer (Invitrogen, Thermo Fisher Scientific) and Recombinant Precision Plus unstained protein standards (Biorad). Electrophoresis was performed at constant voltage (150 V) and the gels were stained with a colloidal Coomassie blue solution. Gels were washed with water and scanned.

RP-HPLC

Purity of gliadin fractions was probed by RP-HPLC [START_REF] Bietz | Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data[END_REF]. Briefly, 120 µg of proteins were dissolved in acetonitrile 25 % TFA 0.1 % and injected onto a C18 analytical column (Nucleosil C18, 4 x 250 mm, 5µm, 300 Å). They were separated at 50 °C using a gradient running from acetonitrile 25 % TFA 0.1 % to acetonitrile 55 % TFA 0.1 % during 70 min at 0.8 mL/min. Monitoring the protein was carried out using the absorbance at 214 nm.

RESULTS AND DISCUSSION

Total gliadins extraction

First, 514 g of gluten were obtained from the wheat dough prepared from 5 kg of flour extensively washed with water to remove starch and water-soluble constituants. This process concentrated proteins: the resulting dry gluten contained 73 % proteins of the flour and corresponds to 10.2 % of the flour and 79% of the flour proteins. This quantitative value was in good agreement with the isolation protocol from [START_REF] Popineau | Fractionation and characterisation of γ-Gliadins from bread wheat[END_REF] wich yielded routinely 10.8 % of gluten from flour. Then, gliadins were separated from glutenins following Osborne fractionation by solubilisation in 70% v/v ethanol. 84 g of total gliadins from 5 kg of flour were produced with 86 % protein content. The extracted gliadins represent 1.7 % of the initial flour. In comparison with the yield of 2.8% for the preparation of Prolamin Working Group (PWG) gliadin reference [START_REF] Van Eckert | Towards a new gliadin reference material-isolation and characterisation[END_REF], a large amount of gliadins may be lost in the pellet during alcohol extraction or the initial gliadin content of our flour was lower.

The total gliadins (TG) extract was composed by the four types of gliadins : alpha-, beta-, gamma-, omega-gliadins and small amount of glutenins as revealed by electrophoresis at low pH (Figure 1 A, B, C, D bottom, lines TG).

Chromatography media screening

The different groups of gliadins were fractionated by cation exchange chromatography according to their charge at acidic pH (pH 3.6) under denaturing conditions (urea 2 M). Five different cation exchanger chromatography resins varying in their electrostatic and chemical characteristics were tested under the same conditions to separate the different groups of wheat gliadins. The fractionation of omega-gliadin and the loading capacity were compared for each resin.

Peak area is used to evaluate the protein amount bound to the resin. The largest area under the peaks eluted between between 0 and 600 min was obtained with with Toyopearl CM 650 M, S Ceramic Hyper D and Cellufine C-500 resin, respectively 36, 34 and 34 AU*min. CM Ceramic Hyper D and SP Trisacryl M had the smallest peak area (respectively 27 and 30 AU*min), indicating that they bind fewer proteins than the other resins. Moreover, CM Ceramic Hyper D did not lead to any gliadin separation (Figure 1A). All gliadins were eluted in the first unbound peak during the initial washing step and few proteins were eluted by the NaCl gradient. This resin was therefore eliminated. SP Trisacryl M (Figure S1) having the smallest peaks area under curve was not retained.

Omega-gliadins were eluted in the first peaks of the chromatograms. They were either unbound or eluted at the very beginning of the gradient, which is in line with other studies [START_REF] Patey | Large-Scale preparation of gliadin proteins[END_REF][START_REF] Popineau | Purification and characterisation of ωgliadin components from common wheat[END_REF]. Then, the salt gradient induced the joint elution of gamma and beta-gliadins, followed by alpha-gliadins (Figure 1B,C,D). Only the resin S Ceramic Hyper D (Figure 1B, fractions 2 -40) allowed for a clear split of the omega gliadins from other gliadins, all of them were eluted before 7 mS/cm conductivity in three separated peaks. The well-resolved peaks containing omega-gliadins for Cellufine C500 (Figure 1C fractions 4 -6) and for Toyopearl CM 650 M (Figure 1D fractions 4 -6) were smaller than for S Ceramic Hyper D. Furthermore, it can be noticed that omega-gliadins were not separated (Figure 1C, fractions 25 -29) and partly co-eluted with gamma gliadins with Cellufine C500 (Figure 1C, fractions 33 -35). The same observation applies to Toyopearl CM 650 M (Figure 1D, fractions 29 -35).

Since S Ceramic Hyper D (Figure 1 B) had a good loading capacity (34 AU*min) and concentrated omega-gliadins in the first peaks of elution, this resin was selected.

Omega-gliadins are mainly composed of glutamine (40 -50 % mol) and proline (20 -30% mol) [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. Therefore, they have few charges on their surface, and their binding capacity to cation exchanger resin is expected to be low. S Ceramic Hyper D has particular properties: it is a hybrid resin called "gel-in-a-shell". The functionalized hydrogel is entrapped in a rigid ceramic bead [START_REF] Boschetti | Advanced sorbents for preparative protein separation purposes[END_REF]. Large pores at the surface of the beads allow proteins to diffuse rapidly and bind to the hydrogel throughout its volume, not just on the surface [START_REF] De | Mass transfer mechanisms in Hyper D media for chromatographic protein separation[END_REF]. The pore size of S Ceramic Hyper D is the largest among the tested resins (Table 1). Its binding sites would be more accessible than in the other resins, thus improving the sorption capacity. Moreover, omega-gliadins could have a compact structure even in 8 M urea while the other gliadins are unfolded in 2 M urea [START_REF] Paananen | Nanomechanical force measurements of gliadin protein interactions[END_REF], it could be speculated that omega-gliadins could diffuse faster into the ceramic bead. Altogether, this could explain the better performance of S Ceramic Hyper D resin.

Preparative fractionation

Following the analytical screening, the S Ceramic Hyper D column was retained for the first step of gliadin fractionation to reach a good separation of omega-gliadins. The process scaledup involved the same extraction and purification buffers as at analytical scale. The general purification process is presented in Figure 2 A. S Ceramic Hyper D could fractionate roughly the four gliadins groups, and concentrate omega-gliadins in the first peaks of elution with the same efficiency as in the optimization step. At preparative scale, a stepwise elution of increasing NaCl concentration was preferred to speed up the separation time and reduce buffer consumption while retaining the required purity level. An extension of the washing step at 5% of elution buffer corresponding to 25 mM NaCl was necessary to avoid contamination of gliadin peaks by omega-gliadins. Two fractions of omega-gliadins were produced according to the salt elution gradient. They contained both a mixture of omega 5-and omega 1,2-gliadins. The fraction of omega-gliadins eluted with 25 mM NaCl was further called "Omega NaCl25" and the fraction eluted with 50 mM NaCl "Omega NaCl50". Then gamma and beta gliadins were eluted with 100 mM NaCl and finally alpha-gliadins and traces of glutenins with 500 mM NaCl (Figure 2 B). The preparative fractionation allowed to produce very concentrated protein fractions with a protein content greater then 92% for each gliadin fraction (Table 2).

Eighty four grams of total gliadins were applied to the column in nine runs. A wash with NaOH 1 M was necessary after five runs to avoid loss of capacity. Omega-gliadin recovery including chromatography, dialysis and freeze-drying was 1.5 g of Omega NaCl25 and 3 g of Omega NaCl50 (Table 2). This recovery was in accordance with large scale fractionation of gliadins which produced from 4 g to 6 g of omega-gliadins from a greater amount of total gliadins [START_REF] Patey | Large-Scale preparation of gliadin proteins[END_REF][START_REF] Popineau | Purification and characterisation of ωgliadin components from common wheat[END_REF]. It was noticed that omega gliadin powders were highly sensitive to water content. A change in relative humidity induced a solid-to-liquid transition at ambient temperature in a few hours after removal from the freeze dryer. To avoid such a transition, powders were stored in a desiccator containing K2CO3 saturated salt to avoid an increase of moisture content during storage. Such a transition was not observed with other gliadin powders.

The next separation step was another cation exchanger chromatography (Figure 2B). A SP sepharose FF resin had previously been shown to fractionate different isoforms of gamma gliadins according to charge [START_REF] Popineau | Fractionation and characterisation of γ-Gliadins from bread wheat[END_REF]. The fraction obtained from the S Ceramic Hyper D column, containing gamma and beta gliadins, was injected onto SP FF with the same buffers as the first step in four runs. A NaCl gradient from 25 mM to 125 mM was used to separate gamma 46 and gamma 44 (Figure 2B). These isoforms could be detected in A-PAGE and correspond to the two major bands at the level of gamma-gliadin migration in the total gliadin lane (Figure 1B). They were not well separated with S Ceramic Hyper D column because of step elution. Gamma and beta gliadins were separated by hydrophobic interaction chromatography (HIC) with a Phenyl Sepharose FF column and eluted with an ethanol gradient [START_REF] Popineau | Fractionation and characterisation of γ-Gliadins from bread wheat[END_REF]. Briefly, the whole gamma fraction solubilized in urea lactate buffer was injected on the Phenyl sepharose FF column, then was eluted by a linear gradient of ethanol in ammonia buffer pH 11. Beta-gliadins were eluted by 10 % (v/v) ethanol and gamma 46 and 44 were eluted respectively with 39% and 46% (v/v) ethanol (Figure 2B). They were separated according to their surface hydrophobicity. A similar elution pattern was observed in literature, confirming that separation by HIC is highly reproducible [START_REF] Popineau | Fractionation and characterisation of γ-Gliadins from bread wheat[END_REF]. The gamma gliadin recovery was 1.6 g of gamma 46, 0.6 g of gamma 44 and 0.2 g of gamma 44 and gamma 46 mixture (Table 2). Three grams of beta-gliadin were purified (Table 2). The overall recovery for gamma and beta was 3 and 4 % repectively. Such very low recovery, which are encountered when targeting fractions with high purity (Figure 3 -4) were due to the numerous steps and a technical problem with one batch at the freeze-drying step which led to losses. From batch to batch, the mean recovery for HIC was around 65% which gave a final yield of 7 %.

Alpha-gliadins were last purified by gel filtration. They represent the major protein in total gliadins. Whole alpha gliadins were collected in the first chromatographic step and corresponded to the last peak eluted with 500 mM NaCl. This fraction contained traces of glutenins eliminated by size exclusion chromatography [START_REF] Charbonier | Large Scale Isolation of Gliadin Fractions[END_REF][START_REF] Patey | Large-Scale preparation of gliadin proteins[END_REF]. Glutenins are composed of sub-units stabilized by disulphide bonds. Their apparent molecular weight is much larger than that of gliadins in non-reducing conditions [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. The sephacryl S100-HR column had a separation range adapted to the size of the targeted proteins, and was equilibrated with the same buffers as in the first step. Glutenins were excluded from the gel (27 % peak area at 280 nm) and alpha-gliadins were eluted at elution volume of 1000 mL in the main peak (62% peak area at 280 nm) (Figure 2B). More than 4 g of alpha gliadin were purified (Table 2) with a final recovery of 11 %. Only 11.8 g over 22 g of fraction was purified by Sephacryl S100-HR. This purification step had a 40 % yield. If the entire fraction containing alpha-gliadins had been processed, 9 g of alpha could have been purified in total.

The overall procedure was rather long but highly purified fractions (over 85% for each gliadin) were recovered in the gram-range. Due to the purification step series, the final recovery was relatively low (< 25%). Protein losses may also be due to protein bonding onto the columns. Indeed, regular cleaning of the columns with sodium hydroxide was necessary to keep reproducible results.

Analysis of the purified fractions

The purity of each gliadin fraction was evaluated by electrophoresis and analytical reversed phase HPLC. In SDS-PAGE, proteins are only separated according to their size, as negatively charged SDS masks the protein charges. The characteristic bands for each gliadin are expected at the corresponding MW ranges of 60 000-80 000 Da for omega-gliadins, and 32 000 to 45 000 Da for alpha, beta and gamma-gliadins [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. In the present work, purified gliadins showed a typical electrophoretic profile with only one band for highly pure proteins.

Omega-gliadins showed one band at 70 kDa in SDS-PAGE (Figure 3A). Omega-gliadins showed a weak Coomassie staining, probably due to the repetitive pattern of their sequence and lack of basic amino acids as reviewed by Shewry [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. Beta-gliadins (Figure 3A) and Gamma (Figure 3A) migrated around 40 kDa. Alpha-gliadins (Figure 3A) had an intense band at 37 kDa with minor bands at lower MW. These bands, around 10 -15 kDa, could correspond to alpha-amylase/trypsin inhibitors (ATIs) [START_REF] Shewry | What is gluten-Why is it special?[END_REF].

Gliadins could be subclassified as omega, gamma, beta and alpha based on increasing mobility in electrophoresis at acidic pH [START_REF] Shewry | What is gluten-Why is it special?[END_REF]. In A-PAGE, proteins are separated according to their molecular weight and their apparent charge at pH below 3 [START_REF] Bietz | Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data[END_REF].

Migration profiles showed characteristic bands for each gliadin according to the expected profiles, with only traces of contaminants (Figure 3B) [START_REF] Bietz | Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data[END_REF]. Omega NaCl25 fraction showed two bands with low electrophoretic mobility in A-PAGE corresponding to omega1,2-gliadins, whereas Omega NaCl50 fraction presented three bands corresponding to a mixture of omega1,2 and omega5-gliadins (Figure 3B). Gamma-gliadins (Figure 3B) had a similar profile. They migrated to the middle of the gel with an intense band and another weak band just above, meaning that these fractions had homogeneous charges. The beta-gliadin fraction (Figure 3B) migrated between gamma-and alpha-gliadins. Alpha-gliadins (Figure 3B) migrated to the migration front with several bands. It could be different isoforms of proteins [START_REF] Mameri | Structural Basis of IgE Binding to α-and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains[END_REF]. Analytical C18 columns have also been widely used to characterize gluten proteins since 1980's [START_REF] Bietz | Electrophoresis and chromatography of wheat proteins: available methods, and procedures for statistical evaluation of the data[END_REF]. Proteins are separated by surface hydrophobicity and are eluted with an acetonitrile gradient. Figure 4 presents the elution pattern of purified gliadins on C18 column. Each fraction was eluted in a single major peak which highlights a high protein purity rarely obtained because of gliadin polymorphism. Omega5-gliadins were eluted between 30 min and 35 min, omega1,2-gliadins around 38 min (Figure 4). Both omega-gliadin fractions are composed of a mixture of omega5 and omega1,2. Based on the respective peak areas, Omega NaCl25 fraction contained 71% of omega1,2 and 29% of omega5-gliadin, while Omega NaCl50 fraction contained 69% of omega1,2 and 31% of omega5-gliadin . When both types of omega 1,2 and omega 5 need to be separated from the omega-gliadin fraction, preparative RP-HPLC is the most appropriate technique. Beta gliadins were eluted around 42 min (Figure 4) in a major peak, which confirms the purity (88 % of total peak area). Only a slight peak was visible at 45 min, which corresponds to gamma gliadins and represents 7 % of the fraction. Alpha-gliadins were eluted between 44 min and 49 min (Figure 4). Two main peaks were eluted at 46 min and 47.5 min (76% peak area) meaning that alpha-gliadins do not have a homogeneous composition in terms of polarity. Gamma44-gliadins (Figure 4) were eluted at 50 min (93 % peak area).

Gamma46-gliadins (Figure 4) were eluted at 51 min (92 % peak area) and a more polar fraction is eluted at 59 min (8 % peak area). These gamma-gliadin fractions are highly pure.

CONCLUSION

The developed preparative protocol enabled the purification of several grams of highly purified gliadins. Analytical techniques confirmed that each gliadin fraction was purified near electrophoretic and HPLC homogeneity. Gliadins with high purity at the level of grams are rarely obtained because of the considerable polymorphism of gliadins. We choose a S Ceramic Hyper D (Pall) column for its ability to concentrate omega-gliadins in the first elution peaks and to separate different groups of gliadins. Its specific chemistry "gel in a-shell" could explain the improved resolution of this resin compared to the other tested. The mass transfer mechanism in Ceramic hyper D resin is described as intraparticle convection rather than diffusion which allows larger proteins as omega gliadins to enter the pores of the gel.

Table 1 List of tested cation exchanger chromatography resins

  Figure 4 RP-HPLC chromatograms of purified gliadins fractions.Each fraction (120 µg) is injected on analytical C18 column. Proteins are eluted in a gradient from 25 to 55% acetonitrile-TFA 0.1% in 58 min at 0.8 mL/min. The absorbance of proteins recorded at 214 nm is represented in light blue for omega NaCl25, dark blue for omega NaCl50, red for beta-gliadins, yellow for alpha-gliadins, light green for gamma44-gliadins and dark green for gamma46-gliadins. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

Figure 3

 3 FIGURE 1

FIGURE 4 Figure 4

 44 FIGURE 4

ACKNOWLEDGMENTS

The authors thank J-C. Helleisen and B. Rolland from INRAE UMR1349 IGEPP for providing wheat seeds, C. Blassel for its significant contribution to purification experiments, and F.Pineau for her technical help. We also thank C. Berton-Carabin for critical reading of the manuscript. A special thank to J-P Compoing who initiated this work.

FUNDING

The authors acknowledge the French National Research Institute for Agriculture, Food and the Environment (INRAE) and the region Pays de la Loire for the financial support. AUTHORS CONTRIBUTIONS Véronique Solé-Jamault: conceptualization, formal analysis, original draft, review & editing; Joëlle Davy: investigation; Rémy Cochereau: investigation review & editing; Adeline Boire: supervision, funding acquisition review & editing; Colette Larré: supervision, review & editing; Sandra Denery-Papini: supervision, review & editing

DECLARATION OF COMPETING INTEREST

The authors declare to have no conflicts of interest. B.

LIST OF FIGURE CAPTIONS


Experimental measurements of the bacterial oxidation of HT in soils: impact over a zone influenced by an industrial release of tritium in HT

Keywords: HT, bacterial oxydation in soil, soil texture, reprocessing plant

Tritium is a radionuclide released to the atmosphere by nuclear industries in various forms, mainly HTO and to a lesser extent HT. However, some nuclear sites may emit predominantly HT in the atmosphere. The HT is oxidized to HTO essentially in the top cm of soils, and that the formed HTO is then possibly released into the atmosphere. HTO is an assimilable form by plants. Therefore, it is important to understand the environmental behaviour of HT.

 by laboratory experiments on soils typical of western France, and we have in particular adapted the frequency factor A and the Michaelis-Menten enzymatic reaction parameter (Km) on the basis of an Arrhenius equation in function of the porosity of the soil.

We then applied this model to the environment near the reprocessing plant of Orano la Hague (France), which emits a significant amount of HT. Based on the adapted model, and knowing the atmospheric variations of HTO and HT over the period 2013-2016, we estimated that the mean HTO activity in soil due to atmospheric HT reached 0.6 Bq.L -1 (with a peak value of 5 Bq.L -1 ) while the mean value with all sources taken into account is 6.2 Bq.L -1 . Then, in an environment such as that surrounding the Orano La Hague plant, where near-field atmospheric HT activity is very high, the bacterial oxydation contribution to produce HTO in the soil can be considered as approximately 10%. The flux to the atmosphere from these source representing approximately. 1.5 Bq.m -2 .d -1 . If we consider an area of 2 km around the plant (i.e. 13 km 2 ), we estimate 218 Bq.s -1 of HTO was released by the soil, representing less than 0.1% of the direct atmospheric release of HTO around the site. From this work, it appears clear that this secondary source term from the soil is insignificant at this specific site.

Introduction

Tritium ( 3 H) is a low energy β-emitting radionuclide with a half-life of 12.32 years. Tritium is produced by naturally-occurring processes through the interaction of gases and cosmic radiation. Natural abundance is estimated at about 4.5 kg of tritium, produced at an average rate of 0.32 atom cm -2. s -1 of earth surface [START_REF] Masarik | An updated simulation of particle fluxes and cosmogenic nuclide production in the Earth's atmosphere[END_REF]Beer, 2009, UNSCEAR, 2016), corresponding to ≈258 g y -1 [START_REF] Oms | Inventory and distribution of tritium in the oceans in 2016[END_REF]. Atmospheric tests of nuclear weapons between 1945 and 1980 led to an estimated release of 668 kg, corresponding to about 242x10 6 TBq during that period [START_REF] Okada | Overview of tritum : characteristics, sources, and problems[END_REF][START_REF] Unscear | Sources: United Nations Scientific Committee on the effects of Atomic Radiation[END_REF]; in 2016, the tritium remaining from the fallout from atmospheric nuclear weapons testing is estimated to be 22-23.5 kg [START_REF] Oms | Inventory and distribution of tritium in the oceans in 2016[END_REF]. Today, 3 H is mainly released into the environment by nuclear power plants, military nuclear facilities and nuclear reprocessing plants [START_REF] Cnsc | Rejet de tritium et conséquences sur les doses au Canada en[END_REF]ASN, 2010). Atmospheric releases of tritium to the world's nuclear power plants range from 500 to 600 TBq.y -1 (1.4-1.7 g.y -1 ). At country scale, for example in France, the 58 reactors (PWR Pressurized Water Reactor) from nuclear power plants release approximately 34 TBq.y -1 (~0.1 g) of tritium gas and 1000 TBq.y -1 (~3 g) of tritium liquid [START_REF] Oms | Inventory and distribution of tritium in the oceans in 2016[END_REF].

Spent fuel reprocessing plants (Orano La Hague in France, Sellafield in UK, Tokai-Mura in Japan) released approximately 200 to 300 TBq.y -1 in gaseous form between [START_REF] Ichimasa | In vitro determination of HT oxidation activity and tritium concentration in soil and vegetation during the chronic HT release experiment at Chalk river[END_REF][START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF][START_REF] Irsn | Le tritium dans l'environnement : synthèse critique des connaissances et perspectives de recherches[END_REF]. These three plants in the world contribute proportionally strongly to the release of tritium to the atmosphere if we compare with the large number of Nuclear Power plants.

Since 2010, Orano La Hague (France) has released 64 TBq.y -1 of gaseous tritium annually on average, and a major part of this release is in HT form (85%, [START_REF] Connan | Atmospheric tritium concentrations under influence of AREVA NC La Hague reprocessing plant (France) and background levels[END_REF]. Many facilities other than nuclear power plants and fuel reprocessing plants may release tritium in gaseous form, such as military weapons manufacturing plants, or tritium plants in the luminescent product industry (mainly in Canada). For the luminescent produce industry, in 2006, gaseous releases were estimated at 300 TBq.y -1 (ASN, 2010). Military weapons plants can also release gaseous tritium. Thus in France, the Valduc and Marcoule factories releases about 200 TBq.y -1 (IRSN, 2018). In the future, the construction of new nuclear facilities (e.g. European Pressurized Reactor, International Thermonuclear Experimental Reactor ITER) as well as changes in nuclear fuel management may lead to an increase in 3 H released from the nuclear industry. For example, ITER in the south of France could release between 220 and 900 TBq.y -1 [START_REF] Cortes | Rejets liquides et gazeux de l'installation experimentale ITER en fonctionnement de routine[END_REF] and we cannot exclude, on a global scale, the possibility of HT releases related to tritium production and underground tests by countries that are actively developing nuclear weapons programs.

While the HT form is not the dominant content of these releases, it is important to understand the behaviour of this HT form when oxided in the top cm of soils, because this tritium can then be released into the atmosphere in assimilable HTO form. The HT form is not directly bio-available in the ecosystem for plants, but becomes bio-available after oxidation into HTO [START_REF] Sweet | Oxidation of molecular tritium by intact soils[END_REF][START_REF] Conrad | Kinetics and electron transport of soil hydrogenases catalyzing the oxidation of atmospheric hydrogen[END_REF][START_REF] Dunstall | Elemental tritium deposition and conversion in the terrestrial environment[END_REF][START_REF] Bunnenberg | Differences in the Behaviour of HTO and H2O in Soil After Condensation from the Atmosphere and Conversion of HT to HTO and OBT in Soil Relative to Moisture Content and Pore Volume[END_REF][START_REF] Spencer | HTO Concentration Relationships Between Air, Soil and Vegetation and Seasonal Dependence of HT Deposition to Soil[END_REF][START_REF] Golubev | Studies of HTO transport and oxidation in unsaturated soil[END_REF][START_REF] Peterson | Tritium doses from chronic atmospheric releases: a new approach proposed for regulatory compliance[END_REF][START_REF] Kim | HT to HTO conversion and field experiments near Darlington Nuclear Power Generating Station (DNPGS) site[END_REF]. In HTO form, tritium can be readily exchanged with hydrogen atoms in the environment, thus entering the hydrological cycle, and then can enter the human/biological body through ingestion and inhalation (Belot, 1986).

HT can oxidize into HTO in the environment at atmospheric level [START_REF] Clerc | The experimental determination of the oxidation rate of tritium in the atmosphere, Radiation protection practice[END_REF][START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF], Ehalt and Rohrer, 2009) and on the surface of a plant leaf [START_REF] Cline | Absorption and metabolism of tritium oxide and tritium gas by bean plants[END_REF][START_REF] Dunstall | Elemental tritium deposition and conversion in the terrestrial environment[END_REF][START_REF] Ichimasa | In vivo fixation of atmospheric tritium gas in pine and Zelkova trees and their surroundings[END_REF][START_REF] Ichimasa | In vitro determination of HT oxidation activity and tritium concentration in soil and vegetation during the chronic HT release experiment at Chalk river[END_REF]. However, HT mainly oxidizes in the top centimeters of soil due to bacterial action [START_REF] Mcfarlane | Environmental tritium oxidation in surface soil[END_REF][START_REF] Schuler | Soils contain two different activities for oxidation of hydrogen[END_REF], Spencer et Dunsdall, 1986[START_REF] Kim | HT to HTO conversion and field experiments near Darlington Nuclear Power Generating Station (DNPGS) site[END_REF] and, to a lesser extent, under the effect of abiotic enzymes in the soil [START_REF] Conrad | Decomposition of atmospheric hydrogen by soil microorganisms and soil enzymes[END_REF][START_REF] Schuler | Soils contain two different activities for oxidation of hydrogen[END_REF].

HT bacterial oxidation can be anaerobic or aerobic [START_REF] Popelier | Soil H2-uptake in relation to soil properties and rhizobial H2-production[END_REF][START_REF] Schuler | Soils contain two different activities for oxidation of hydrogen[END_REF][START_REF] Libert | An original methodology to study the transfer of tritium as HT and HTO in grassland ecosystem[END_REF][START_REF] Piché-Choquette | H2-saturation of high affinity H2oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups[END_REF], but it has been shown that, in soil, aerobic bacteria are the most active type [START_REF] Schuler | Soils contain two different activities for oxidation of hydrogen[END_REF]. [START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF] proposed an empirical equation to quantify the oxidation of HT into HTO in soil. This reference equation for the scientific community is based on deuterium oxidation works carried out in controlled laboratory conditions for a given soil type. However, it has been shown that the oxidation rate of HT varies depending to the soil type [START_REF] Conrad | Kinetics and electron transport of soil hydrogenases catalyzing the oxidation of atmospheric hydrogen[END_REF][START_REF] Golubev | Studies of HTO transport and oxidation in unsaturated soil[END_REF][START_REF] Kim | HT to HTO conversion and field experiments near Darlington Nuclear Power Generating Station (DNPGS) site[END_REF] and it is important to adapt the work of [START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF] to soils corresponding to all existing environment.

In this study, to begin with, we started with laboratory experiments focusing on the oxidation of HT to HTO for several soil types typical of north-west France, and corresponding to the soils we found around the Orano La Hague spent fuel reprocessing plant. We particularly attempted to better integrate the influence of the soil texture on the rate of HTO production from atmospheric HT. The aim was to improve the precision of the current bacterial oxidation equation used by Ota et al. (2017) and, in addition, to generally apply this equation to any soil type depending on porosity estimated from soil fractions (silt, loam, clay).

Our subsidiary and more operational aim was to improve forecasts for HTO stock potentially "generated" in the soils in the immediate environment of the Orano La Hague plant, which could be available for atmospheric release based on precise available information on variation in HT activity. The idea behind the work is, as the Orano la Hague plant releases 85% of tritium in HT form, to estimate the ability of soils to convert this HT form into HTO, and to compare this "new stock" of HTO from bacterial oxidation with usual direct atmospheric releases of HTO.

Modelling HT oxidation in soil and expressing the frequency factor A as a function of soil porosity

HT oxidation in soil is mainly driven by the hydrogen oxidation enzymes (hydrogenesis) contained in the streptomyces [START_REF] Komuro | HT oxidation in soils in Ibaraki and isolation, identification of HT oxidizing soil bacteria[END_REF]. The generation of HTO by HT oxidation is considered as an enzymatic reaction [START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF], and the kinetics are described by the Michaelis-Menten equation (Quinlan, 1981):

= . + (1)
The terms of the equations in this section are described in Table 1. It has been proven that the bacterial oxidation of HT depends on temperature, water content, porosity and the vertical distribution of oxidizing micro-organisms (bacterial concentration gradient) in the soil profile [START_REF] Sweet | Oxidation of molecular tritium by intact soils[END_REF][START_REF] Conrad | Kinetics and electron transport of soil hydrogenases catalyzing the oxidation of atmospheric hydrogen[END_REF][START_REF] Dunstall | Elemental tritium deposition and conversion in the terrestrial environment[END_REF][START_REF] Bunnenberg | Differences in the Behaviour of HTO and H2O in Soil After Condensation from the Atmosphere and Conversion of HT to HTO and OBT in Soil Relative to Moisture Content and Pore Volume[END_REF][START_REF] Spencer | HTO Concentration Relationships Between Air, Soil and Vegetation and Seasonal Dependence of HT Deposition to Soil[END_REF][START_REF] Peterson | Tritium doses from chronic atmospheric releases: a new approach proposed for regulatory compliance[END_REF][START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF]. These different factors affect the rate of an enzymatic reaction and were defined by [START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF] using the equation (2). The parameters . . are defined in Table 1 and readers should refer to [START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF] for a detailed parametrization of these factors.

= . . . (2)
The Michaelis-Menten enzymatic reaction parameter (Km) for equation 1 is expressed as a function of the temperature on the basis of an Arrhenius equation [START_REF] Quinlan | The thermal sensitivity of Michaelis-Menten kinetics as a function of substrate concentration[END_REF][START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF] according to equation (3):

= . ! " (3)
In the model of [START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF], the parameters A and E are determined by adjusting according to experiments for a given soil type. The activation energy of the bacterial reaction of the HT (noted E) exclusively depends on the type of catalysing enzyme and the substrate [START_REF] Triebwasser-Freese | Catalytic kinetics and activation energy of soil peroxidases across ecosystems of differing lignin chemistries[END_REF][START_REF] Bock | Peptide translocation by the lysosomal ABC transporter TAPL is regulated by coupling efficiency and activation energy[END_REF]. We postulate that E is identical for all pedological soil types as, for our study, hydrogenesis is assumed as the type of catalysing enzyme and HT is assumed for the substrate, for all soil types [START_REF] Conrad | Kinetics and electron transport of soil hydrogenases catalyzing the oxidation of atmospheric hydrogen[END_REF]. A is defined as a frequency factor, integrating steric effects and the probability of collision between an oxidizing enzyme and HT [START_REF] Hupp | The frequency factor for outer-sphere electrochemical reactions[END_REF][START_REF] Weilandt | Particle-based simulation reveals macromolecular crowding effects on the Michaelis-Menten mechanism[END_REF]. On this basis, parameter A will depend on soil porosity and type and is likely to differ depending on the pedological characteristics of the soil: this parameter A must be adjusted to refine the oxidation model.

The process applied in this context, to express frequency factor A for any soil type, includes several stages:

-Measuring particle distribution and expressing soil porosity;

-Performing bacterial oxidation laboratory experiments with HT for different soil types, using soils from our local environment and air enriched in HT sampled from the Orano La Hague plant stack plume;

-Calculating the value of A according to equation (3) by adjusting the outputs from the model (equation 1) and the HTO activity produced based on experimental measurements;

-Expressing A as a function of soil porosity.

Experimental design and methodology

Soil samples

The soils used for laboratory experiments were taken in situ in zones outside of the influence of the sites releasing tritium in the north-west region of France (Table 2) in order to obtain initial HTO activity values less than 1 Bq.L -1 . Samples were taken using PVC tubes with a diameter of 10 cm, and a depth varying between 5 and 15 cm. Each soil sample (see characteristics in Tables 2 and3) corresponds to a meadow type farming soil, planted with ray-grass. The granulometric characteristics of the 5 soil types studied can be found in Table 2. Analyses were performed according to standards NF ISO 11464 and NF X 31 107 by an external laboratory (National Institute of Agronomical Research, INRA, France).

During this study, three types of soil cores were systematically sampled at each site:

-One core intended to check the absence of HTO in the sample;

-One core containing grass intended to determine the bacterial oxidation of HT in the top centimetres of soil, in laboratory conditions;

-One core without grass intended to study the bacterial oxidation of HT for bare soil (soil in which the top 4 cm, corresponding to grass roots, have been removed).

The cores are isolated from the environment by attaching a sleeve (PVC) to the "root" end of the core sampling tube and a parafilm to the "surface" end of the core sampling tube. Once the core has been isolated from contact with ambient air, it is stored in a chiller during transport, and then in a freezer prior to use in laboratory experiments.

Calculating soil porosity

The soil porosity index for our 5 experiments was calculated in several stages. The apparent bulk density of the soil, noted # $%&'( )*+, , and expressed in kg.m -3 , corresponding to the mass of soil contained in a given volume, including the volume of interstitial air, is determined using equation 4.

# $%&'( )*+, = -./'&0$ 1+ 2 . # $%&'( 1+ 2 + -./'&0$ 3 +4 . # $%&'( 3 +4 + -./'&0$ 3 56 . # $%&'( 3 56 (4)
Where the term fraction corresponds to the share of a type of particle i (i = clay, silt, sand) in kgi.kg soil -1 and densityi is the apparent density of a particle i. The different types of soil particle (Clay, Silt, Sand) and their apparent densities can be found in the website (Density of Sand, dry in 285 units and reference information (aqua-calc.com) ; Table A1).

The density of a soil particle (densityparticle) corresponding to the mean mass of a soil particle per unit of volume is then calculated. Schjønning et al. (2017) propose equation 5 to determine densityparticle from the fraction of clay in the soil (fractionclay).

# $%&'( 7 4 1*+ = -./'&0$ 1+ 2 . 2.86 + ;1 --./'&0$ 1+ 2 >. 2.65 (5)

Finally, porosity (equal to the volume of porous space per volume of soil) stated in m 3 space m -3 soil) is determined from the two terms calculated earlier, # $%&'( )*+, and # $%&'( 7 4 1*+ by equation 6 [START_REF] Chaudhari | Soil Bulk Density as related to Soil Texture, Organic Matter Content and available total Nutrients of Coimbatore Soil[END_REF].

@0-0%&'( = 1 - # $%&'( )*+, # $%&'( 7 4 1*+ (6)

Sampling of air enriched with HT

The aim in this context is to circulate air enriched with tritium in HT form in the laboratory in order to observe the incorporation of HT in the soil and oxidation into HTO. The volumes of air containing high concentrations of atmospheric HT are sampled in the environment near to the Orano La Hague (France) spent fuel reprocessing plant, which releases tritium in HT and HTO forms [START_REF] Connan | Atmospheric tritium concentrations under influence of AREVA NC La Hague reprocessing plant (France) and background levels[END_REF] during its industrial processes. These tritium releases are concomitant with the 85 Kr releases. This gas can be measured in real time, which allows samples to be specifically taken during periods with high HT activity. Air samples containing HT were therefore taken downwind from the Orano la Hague plant, making sure that the air sampled was enriched in tritium thanks to the 85 Kr measurements, using an air compressor (Bauer compressor Junior). Several B20 type (4 m 3 ) gas cylinders were prepared with a vacuum prior to sampling for the purposes of these experiments; the air sampled for our experiments contained HT levels between 1 and 23 Bq.m -3 . Due to the compression sampling technique, ambient humidity (water vapour containing tritium in HTO form) is eliminated and the air only contains HT.

In order to determine the exact HT content of the air used for the experiments, the protocol described by [START_REF] Connan | Atmospheric tritium concentrations under influence of AREVA NC La Hague reprocessing plant (France) and background levels[END_REF] was applied; For HT measurements, the volume of air collected in the pressurized container was transferred to gas treatment lines in order to obtain the HT concentration after transformation to HTO. The air passed successively at a rate of 20 L.h -1 through an activated charcoal trap made of two carbon canisters (ACR, Agilent technologies), which trap volatile organic compounds; two first bubblers (Bubbler N°1 and N°2) filled with 150 cm 3 of tritium-free water (Abatilles spring water) are used to eliminate any residual tritiated water vapor after compressing while sampling. A heated 200°C catalysis oven (palladium pellets SDEC, France) was then used to oxidize the HT to HTO (Bubblers N°3 and N°4). After the passage of a volume of about 500 L of air (depending on the expected level), air without 3 H was injected (flow: 30 L.h -1 ) to purge the pipes and ovens of all traces of 3 H in order to prevent any cross-contamination between the different samples.

During this step, which lasts 60 minutes, both ovens were heated to a temperature of 450°C.

Then, after these different steps, a volume of water was collected from the bubbler for measuring purposes. After storage in darkness at 4 °C prior to analysis, water (10 mL) was mixed with a liquid scintillator (10 mL) to allow for measurements by liquid scintillation counting.

HT bacterial oxidation chamber

The experimental set-up includes a leaktight bag containing soil cores, and HT is injected into this bag at a constant rate (0.2 L.min -1 ). The oxidation kinetics of HT driven by soil bacteria is quantified by measuring the HTO produced in the soil core after a given period. The experimental set-up is described in Figure 1. The oxidation chamber consists of a 300x400x500 mm 3 airlock in leaktight Tedlar in which the soil cores taken from the environment are introduced. The full length of the bag is open so that cores can be added or removed, and a clip system is used to ensure leaktightness. The HT is injected into the experimental set-up using a B20 (4m 3 ) cylinder with known HT activity (see table 6 for HT activities in the cylinder). The injection flow is controlled by a pressure gauge to maintain a constant pressure at equilibrium. To avoid any contamination of the bag by traces of HTO, an upstream bubbler-type system containing Abatilles spring water is inserted between the gas cylinder and the bag. This ensures that only the HT hydrogen form is injected into the bag.

The same set-up is placed downstream of the bag to prevent external air backflow and to evacuation any extra air inside the oxidation chamber. During the experiments, temperature and humidity sensors (WATCHDOG, SDEC France) are placed in the soil cores and in the oxidation chamber to track temperature and water-content changes. A beta radioactivity meter (LB123, Berthold Technology Germany) is also placed against the bag to track 85 Kr activity (present in the air injected containing the HT) in real time. The real time 85 Kr measurements can be used, by analogy, to monitor and avoid any sudden change in HT activity in the bag.

Processing soil after exposure to HT:

After a few days, when the HT soil contamination experiments have been completed, the core(s) are removed from the bag (cores can be removed at different times as part of the same experiment). The soil sample is then wrapped in parafilm and temporarily stored in a cold chamber at 4°C. Each core is then opened and placed on an aluminium tray with a diameter of 20 cm, and then vacuum packed to avoid any contamination, and frozen for 48h at -80 °C.

This freezing phase optimises the upcoming lyophilization process. Samples are then lyophilized (ThermoElectron HETO PowerDry PL3000) in order to remove the HTO from the soil produced during HT oxidation. After 24 to 72 h of lyophilization, the water from the soil is recovered and the HTO will be measured by liquid scintillation counting of 10 mL of water (Maro et al., 2017).

Tritium measurements

Tritium activity (HTO) was measured by liquid scintillation counting (TRI-CARB 3180 TRSL, PerkinElmer) with a counting time of 16 hours per sample. For HTO measurements, a volume of 10 cm 3 of collected water was placed in a vial with 10 cm 3 of liquid scintillation cocktail (Ultima gold LTT). Measurement uncertainty depends on the counting time and level of activity. Typically, for the levels to be measured, uncertainty was 3-10%. The background count rate and counting efficiency were 2.09 cpm and 14.9%. Our measurement practices are regularly validated by comparison exercises between French and international laboratories.

Limit of detection are 1 Bq.L -1 .

Application of the methodology to the Orano La Hague reprocessing plant site

The Orano La Hague reprocessing plant (49.677 °N, -1.883°W) is located in North-West

France, in the Normandy region. Reprocessing releases 3 H into the environment, mainly through two main discharge stacks at the UP2-800 and UP3 plants. For example, in 2016, 66

TBq of tritium was released into the atmosphere, with 85% in HT form and 15% in HTO form [START_REF] Connan | Atmospheric tritium concentrations under influence of AREVA NC La Hague reprocessing plant (France) and background levels[END_REF]. As part of earlier works, for an experimental study site around the Orano site, located at 2 km from the plant (Plateforme Technique Instrumenté de La Hague (La Hague Instrument-fitted Technical Platform), PTILH, 49.694°N, -1.873°W), we developed tools which can be used to reconstruct atmospheric variation in HT and HTO activity, over specific time periods, by measuring 85 Kr, HTO and HT activity at minute, weekly and monthly intervals respectively, as these elements are discharged simultaneously at the main stacks (Maro et al, 2017[START_REF] Dizès | The VATO project: Development and validation of a dynamic transfer model of tritium in grassland ecosystem[END_REF][START_REF] Renard | Modélisation à pas de temps horaire des transferts de tritium au sein d'un écosystèlme prairial[END_REF]. On this basis, we can access highly precise timelines for some past periods. Further to the adjustments made, based on these experiments, to the relationship between soil porosity and factor A used to complete the oxidation model proposed by Ota et al. (2017), we attempted to calculate the variation in HTO activity induced by bacterial oxidation in soil in the immediate environment around the Orano plant, when ambient HT activity is known. We used the TOCCATA-χ transfer model for 3 H (and 14 C) in terrestrial ecosystems, developed by the IRSN (Radiation Protection and Nuclear Safety Institute) (Le [START_REF] Dizès | The VATO project: Development and validation of a dynamic transfer model of tritium in grassland ecosystem[END_REF][START_REF] Renard | Modélisation à pas de temps horaire des transferts de tritium au sein d'un écosystèlme prairial[END_REF], for this purpose. This compartmented model covers specific time periods and integrates the day/night cycle inherent in the physiological behavior of plants based on a simplified grassland model [START_REF] Riedo | A pasture simulation model for dry matter production and fluxes of carbon, nitrogen, water and energy[END_REF][START_REF] Dizès | The VATO project: Development and validation of a dynamic transfer model of tritium in grassland ecosystem[END_REF]. The pool of tritium in soil is considered to exclusively comprise tritiated water dissolved in the unsaturated soil compartment. Soil HTO activity concentration is expressed as a function of (1) a (net) exchange process with an exchange velocity rate at the soil/air interface, (2) percolation (downward migration) of rainwater in soil, (3) root uptake of soil HTO and (4) soil HT oxidation. Similarly, soil water content results from the integration of precipitation over time minus water losses by percolation and root uptake, which is estimated using the Penman-Monteith model [START_REF] Allen | Crop Evapotranspiration: Guidelines for Computing Crop Water Requirements[END_REF] and a soil water stress factor that also possibly affects stomatal conductance (Le [START_REF] Dizès | The VATO project: Development and validation of a dynamic transfer model of tritium in grassland ecosystem[END_REF][START_REF] Renard | Modélisation à pas de temps horaire des transferts de tritium au sein d'un écosystèlme prairial[END_REF].

Results and discussion

Soil granulometry

The different proportions of clay, total loam and total sand can be used to characterize the soil type with reference to the texture triangle proposed by USDA (Table 4, United States Department of Agriculture, USDA). The soils we focused on were loam or silt loam type, with clay content of less than 19%, sand content varying between 25 and 51% and loam content varying between 40 and 57%.

Porosity of the soil types studied

The porosities of the soil types covered by our study have been estimated using equations 4, 5 and 6 and the respective proportions of clay/loam/sand measured (Table 5); we also ran the same calculations for various works found in the literature, as mentioned in table 5, and which allow us to complete this project by extending the soil types considered. Porosity varies from 0.43 to 0.44 for the soil types covered by our experiments. For the studies in the literature mentioned in this paper for the purpose of comparison, porosities vary from 0.39 to 0.46.

Determining the frequency factor A

Frequency factor A (defined by equation 3) was calculated for our soil types based on the rate at which HTO is generated by soil bacteria ( ) calculated using equations 1, 2 and 3, and which depends, in situ, on the HTO activity measured at the end of the HT exposure experiments. The oxidation rate is initially determined by measuring the HTO activity in the water in the soil produced by bacterial HT oxidation (Table 6) at the end of the experiment, with a known period of exposure given in hours (Table 3 and6). It has been demonstrated that the rate of bacterial HT oxidation varies linearly over time [START_REF] Piché-Choquette | H2-saturation of high affinity H2oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups[END_REF].

During our experiments with soils with initial HTO values < 1 Bq.L -1 , the activity measured at the end of the experiments reached values between 6.6 and 32 Bq.L -1 , directly influenced by HT activity (from 1.1 to 23.0 Bq.m -3 ) and exposure time (224 to 1224 h). The rate of production of HTO in soil in the experimental system of HTO varied from 8 to 64 mBq.L -1 .h - 1 , which is equivalent to 0.2-1.54 Bq.L -1 .d -1 .

The frequency factor A is then calculated by minimizing errors between the model (equation 1) and the measurements (Table 6) using an Excel solver. The frequency factors for our experiments vary between 6.97x10 4 and 3.06x10 5 ppm.mol.m -3 .

For soil types from the same sampling site (Experiments 1, 4 and 5), the frequency factors A obtained are similar, despite different core coverage (Table 3). The minor variation in A between these 3 soil types sampled at the same location indicate that the plant cover does not play a significant role in the rate of bacterial oxidation of HT.

This methodology was applied to data taken from the literature if sufficient experimental details were available in the original publications. Table 7 combines different values for frequency factor A calculated using the associated soil types.

According to Table 7, the highest A values are found in sandy soils and the lowest A values are found in loamy to silt-loam soils. According to equations 1 and 3, this means that the rate of HT oxidation by soil bacteria is faster in loamy soils than in sandy soils in equal experimental conditions (temperature, water content, HT concentration, soil depth, plant cover). This pedological variability in the frequency factor reflects the impact of soil porosity on A:

-For a soil with low porosity levels (e.g. sandy soil): steric repulsion and the probability of collision between an oxidizing enzyme and HT will be high;

-For a soil with high porosity levels (e.g. very fine loamy/silty soil): steric repulsion and the probability of collision between an oxidizing enzyme and HT will be low.

Relationship between frequency factor A and soil porosity

The frequency factor was plotted as a function of soil porosity (Figure 2) using the data in tables 5 and 7 (porosity calculated for all soil types in our study and in the literature and A calculations). By using data from the literature and our experimental data, we cover a soil porosity range of 0.39 -0.46, and a second degree polynomial type equation is proposed (Figure 2). This equation can also be written in canonical form as equation 7): on this basis, the minimum theoretical value of A is 8.19x10 D ppm.mol.m -3 (calculated as the minimum of the regression function in Figure 2), which is reached when soil porosity is equal to 0.44.

= 4.43x10 G . @0-0%&'( -0.44 H + 8.19x10 D (7) By using this equation, which depends on the soil porosity calculated according to Eq. 6 with standard particle density values for each soil type listed according to USDA (Table 5), we can estimate the frequency factor A for each potential existing soil type (Table 4). The frequency factor will vary by a factor of 46 between the sand, which has the highest factor A (1.00x10 7 ppm.mol.m -3 ), and the Silty Clay, which has the lowest factor A (2.19x10 5 ppm.mol.m -3 ).

Operational application in the Orano La Hague environment

Our operational aim behind this experiment was to determine the ability of the soil in the immediate environment around the Orano La Hague reprocessing plant to "generate" HTO from release of HT. As previously mentioned, tritium is released into the atmosphere during the process as gaseous HTO (15%) but mostly HT form (85%) representing about 60 -70

TBq.y -1 [START_REF] Connan | Atmospheric tritium concentrations under influence of AREVA NC La Hague reprocessing plant (France) and background levels[END_REF].

Theoretical simulation for air HT activity = 1 Bq m -3 , for all soil types

In this environment with a high ambient HT activity, it is worth preparing a report and estimating if the HTO stock generated by bacteria in soil is significant on an annual scale, and must be considered (as this stock could be re-released to the atmosphere) or if, on the contrary, it can be ignored versus the HTO already present in the soil (from a different origin).

We calculated the formation of HTO in soil by the bacterial oxidation of HT for this purpose, based on equations ( 1), ( 2) and ( 3) and the data from Table 4.

To begin, the calculation was run assuming a constant atmospheric HT activity of 1 Bq.m -3 (Table 8), for all potential soil types (including types which are not present in the immediate environment around the plant, Figure 3), using mean water density and temperatures for 4 seasons (Table 8) obtained in previous works (Maro et al., 2017). On this basis, equation 8 can be used -after calculating the oxidation rate -to convert a mole of HT in the soil (mol HT.kg -1 dry soil) into a mole of HT per surface area depending on soil type (mol HT.m -2 ).

_3* 1 = . # $%&'( )*+, . J

Where z is the depth of soil (we use a value of z = 0.05 m to represent the top cm of soil, corresponding to the soil thicknesses on which we based our experiments, and because this slice contains nearly 70% of oxidizing soil bacteria, [START_REF] Komuro | HT oxidation in soils in Ibaraki and isolation, identification of HT oxidizing soil bacteria[END_REF] and

_3* 1 is
the oxidation rate for superficial HT (mol.m -2 ). # $%&'( )*+, is determined using equation ( 5).

The HTO formation values obtained vary from 0.14 to 5.86 Bq.m -2 .d -1 during the winter and from 0.15 to 6.86 Bq.m -2 .d -1 during the summer (Table 8), i.e. the differences between the seasons are minor, however significant deviations are recorded depending on soil types.

Figure 3 shows HTO activities obtained by bacterial oxidation (for 1 Bq.m -3 of HT) for a temperature of 12°C and a soil water content of 22.5% by volume, to illustrate the clear differences between soil types. SiC (Silty Clay) type soil is the most productive in terms of HTO formation by bacterial oxidation, followed by SiCL (Silty Clay Loam) and L (Loam) soils. The most common soil types are Loam and Silt Loam in our focus area.

Simulation for real ambient HT activities over the 2013-2016 period

For the 2013-2016 period, hourly atmospheric HT content varied from 0.01 to 28.7 Bq.m -3 around the industrial site covered (Maro et al., 2017). We also considered a Loam soil type with porosity of 0.428 and a frequency factor of 3.92x10 5 . Soil temperatures and water content per volume are taken from data measured for each time period (Maro et al., 2017).

Bacterial oxidation calculations (improved by this work) using the TOCCATA-χ model were initially performed exclusively using this source of atmospheric HT, and after we carried out an identical simulation, but added to the atmospheric HT the contents for atmospheric HTO and HTO in rainwater (the atmospheric concentrations of the various tritated forms present on the La Hague site from 2013 to 2016 are present Appendix 2). The results can be found in Figure 4. If we exclusively consider HT and therefore bacterial production, it appears that a significant percentage of HTO is produced in this way (blue line), but we can see that the HTO in the soil is mainly from other atmospheric sources (HTO and rainwater, black line).

Over the 2013-2016 period, the mean HTO activity from atmospheric HT reached 0.57 Bq.L -1

(with a peak value of 5 Bq.L -1 ) while the mean value for all sources is 6.21 Bq.L -1 with peak values in excess of 20 Bq.L -1 . Over this period, the contribution of atmospheric HT to the HTO value in Bq.L -1 in soils is calculated as 9.1%. In an environment such as that surrounding the Orano La Hague plant, where near-field atmospheric HT activity is high, the contribution of the oxidation of HT to HTO in soil represents only about 10% of the total available HTO stock in soil.

The aim of this work is to allow for the quantification of the impact of atmospheric HT once converted into HTO and then re-released to the atmosphere, if necessary. The contribution of HT to the radiation dose is lower than that of HTO, therefore it is worth obtaining information on the conversion of HT into HTO in the soil and then subsequently on re-release to the atmosphere as HTO. For the 2013-2016 period, and on the basis of data on HTO in soils produced by the bacterial oxidation of HT (blue line, Figure 4), we used the TOCCATA-χ model to calculate the HTO exchanged at the soil-atmosphere interface (calculation with the limiting condition that the air contains no prior HTO). Figure 5 shows the results obtained, which indicate mean ambient release levels from the soil of 1.5 Bq.m -2 .d -1 with occasional peak values of 15 Bq.m -2 .d -1 over some periods.

This HTO flow released by the soil representing approx. 1.5 Bq.m -2 .d -1 must be compared with the annual quantities of HTO released directly into the atmosphere from plant stacks.

Over the 2012-2019 period, the Orano La Hague fuel reprocessing plant discharged 66.7

TBq.y -1 (± 11%) of tritium on average. Based on weekly measurements at outlets in 2014-2015, mean HTO and HT sources are evaluated at 3.9x10 5 Bq.s -1 and 1.7x10 6 Bq.s -1 respectively over this period. Soil release of HTO generated by the bacterial oxidation of HT was compared with this mean value for the direct ambient release of HTO; for this purpose, we considered a circular surface area with a 2 km radius around the site (i.e. 12.57 km 2 ), which appeared a pertinent value in view of the sea to the north, south and west, at a distance of approximately 2 km. For 12.57 km 2 , 218 Bq.s -1 of HTO was released by the soil, representing only 0.06% of available ambient stock. If we refer to the maximum possible releases from the soil (15 Bq.m -2 .d -1 ) and we extrapolate to produce a constant release over time, the influence of the soil would represent 0.6% of available ambient stock around Orano La Hague. Although this calculation is clearly subject to extensive uncertainty (with respect to the soil surface area to be considered, and the fact that releases in the form of plant transpiration are also ignored), it appears clear that this secondary source term from the soil is insignificant at this specific site, due to the relatively abundant levels of existing HTO in the atmosphere and despite high HT releases, compared to other nuclear sites, which could reach the soil.

Conclusions

This work was started in order to improve our ability to forecast the bacterial oxidation of HT in soils for all soil types. Our second aim was to quantity the amount of HTO produced in soils, and then re-released into the air around a nuclear site generating high levels of atmospheric HT activity (Orano La Hague spent fuel reprocessing plant, North west France, Europe) and to compare this amount with existing HTO activity in the atmosphere and considered in impact calculations.

To begin with, and on the basis of the model prepared by [START_REF] Ota | Measurement and modeling of oxidation rate of hydrogen isotopic gases by soil[END_REF] and laboratory experiments, we proposed an equation which can be used to estimate the frequency factor for bacterial oxidation after calculating soil porosity. These porosity and frequency factor parameters are suitable for the soils found in our working environment and were entered as input for the TOCATTA-χ model (Le Disez et al., 2017[START_REF] Renard | Modélisation à pas de temps horaire des transferts de tritium au sein d'un écosystèlme prairial[END_REF] [START_REF] Kim | HT to HTO conversion and field experiments near Darlington Nuclear Power Generating Station (DNPGS) site[END_REF], even if the approach is not entirely identical.

Our works showed that the soil is not a significant secondary source of HTO for the atmosphere, on a preliminary basis, in the immediate environment of the Orano la Hague reprocessing plant. It is now necessary to focus on a possible marine source. Liquid releases in the marine environment are higher than atmospheric releases. Very low emission levels at the sea -atmosphere interface could be increase HTO activity on the coast.

Symbol Meaning

Units HT concentration in soil air mol.m -3 frequency factor ppm.mol.m -3 reaction activation energy J.mol -1 soil temperature-dependant weighting factor soil water content-dependant weighting factorsoil depth-dependant weighting factor -Michaelis-Menten parameter ppm.mol.m -3 gas constant J.mol -1 .K -1 absolute temperature K initial maximal HT oxydation velocity mol.kg -1 dry soil.s -1 actual HT oxydation velocity mol.kg -1 dry soil.s -1 reference HT oxydation velocity mol.kg -1 dry soil.s -1 temperature and average volumic water content of soil: 12.6°C and 21.5% ; b temperature and average volumic water content of soil: 16.6°C and 15.2%; c temperature and average volumic water content of soil: 11.3°C and 24.5% ; d temperature and average volumic water content of soil: 7.5°C and 28.8% . The data mentionned a , b , c and d come from hourly measurements over the period 2013 to 2019 (Maro et al., 2017).

Figure 1 :

 1 Figure 1 : Schematic representation of the experimental oxidation device. a: HT air canister; b: upstream bubblers (trapping of HTO traces); c: core soil samples (5 cm); d: clip; e: 85 Kr detector (LB123); f: reading acquisition station ( 85 Kr , temperature, water content); g: downstream bubbler.

Figure 2 :Figure 3 :

 23 Figure 2 : Expression of frequency factor A as a function of soil porosity. White circles are data from this study. Grey squares are data from the literature.

Figure 4 :

 4 Figure 4 : Evolution of HTO in soil (Bq.L -1 ) for 2013-2016 period, calculate from TOCATTA-χ model, taken into account only atmospheric measurements of HT in air (blue line) and taken into account all sources of tritium (HTO and HT in air, HTO in rainwater, black line) for site 2 km from Orano La Hague reprocessing plant.

  used by our teams. To begin with, this work demonstrated that bacterial oxidation is the dominant factor, for exposure to a constant value of HT, for a Silty Clay (SiC) soil type, followed by Silty Clay Loam (SiCl), Loam (L) and Silt loam (SiL) with HTO values produced for ambient HT activity of 1 Bq m -3 in excess of 2 Bq HTO.m -2 for soil activity. For the most frequent soil types around the Orano La Hague nuclear plant (Loam and Silt Loam), using the actual HT activities measured over the 2013-2016 period, mean inducted HTO activity exclusively attributable to bacterial oxidation represents 0.6 Bq.L -1 on average in the soil, with peak values of approx. 5 Bq.L -1 . These values represent approx. 10% of the HTO activity measured in Bq.L -1 in these soils. In other words, even in this environment where ambient HT activity is high versus HTO activity (85% of total tritium, i.e. factor 6), the HTO contribution (via the atmosphere and rain) remains dominant over the activity measured in the soil. Secondly, thanks to the TOCATTA-χ model, we were able to calculate bacterial HTO released from the soil into the atmosphere during the 2013-2016 period, in Bq.m -2 .d -1 , by clearly taking a circular area with a 2 km radius around the nuclear site studied. Using this approach, we were able to compare this secondary soil source with the main source of HTO released directly into the atmosphere by the Orano La Hague site. This secondary source is insignificant and represents less than 0.1% of the main source, and less than 1% if we use the highest possible values. These results ultimately reflect the low level of HT converted to HTO, approximately 1%, and are consistent with the results proposed by

Exchange of HTO between soil and atmosphere (Bq.m -2 .d -1 ) for 2013-2016 period, calculate from TOCATTA-χ model, with only atmospheric measurements of HT in air for site 2 km from Orano La Hague reprocessing plant. Positive values indicate flux of HTO from soil to atmosphere, and negative values indicates flux of HTO from atmosphere to soil.

	HTO soil (Bq.L -1 HTO transfert sol-air (Bq.m -2 .d -1 )
	09/06/2013	26/12/2013	14/07/2014	30/01/2015	18/08/2015	05/03/2016
		26/12/2013	14/07/2014	30/01/2015	18/08/2015	05/03/2016

)

Figure :

Table 1 : Description of the parameters used to calculate the oxidation rate of HT in HTO in soils.

 1

Table 2 : Characteristics of the core sampling of the soils used for the oxidation laboratory experiments.

 2

	1
	Experiment Sampling	Latitude/longitude	Location Name	Clay content	Total loam content	Total sand content	Type of soil
		date	(DD)		(kgsands.kgdry soil -1 )	(kgsands.kgdry soil -1 )	(kgsands.kgdry soil -1 )
	1	25/05/2016 49.618°N/-1.351°W	Le Vast (France)	0.183	0.476	0.341	Loam
	2	29/06/2016 49.646°N/-1.644°W	Equeurdreville (France)	0.090	0.399	0.511	Loam
	3	04/04/2016 48.781°N/-0.678°W	Fresnes (France)	0.189	0.567	0.244	Silt loam
	4	15/11/2018 49.618°N/-1.351°W	Le Vast (France)	0.170	0.472	0.358	Loam
	5	19/11/2019 49.618°N/-1.351°W	Le Vast (France)	0.169	0.501	0.330	Loam

Table 3 : Experimental characteristics measured during the laboratory testing of HT bacterial oxidation in the first centimeters from natural soil. Experiment Sample soil surface Duration Massique soil water content Average Air temperature Sample height

 3

	Sample depth
	from surface

Table 4 : Granulometric characteristics, porosity (according to Eq 6) and frequency factor A (according to Eq 5) of the different soils. Soil type Clay content Silt content Sand content Soil porosity Frequency factor A

 4

	-	%	%	%	-	HT ppm.mol.m -3
	Clay	60	20	20	0.416	1.94x10 6
	Silty Clay	45	50	5	0.442	2.19x10 5
	Sandy Clay	40	5	55	0.398	6.75x10 6
	Clay Loam	35	30	35	0.421	1.14x10 6
	Silty Clay Loam	35	55	10	0.446	4.23x10 5
	Sandy Clay Loam	30	10	60	0.401	5.81x10 6
	Loam	20	40	40	0.428	3.92x10 5
	Silt Loam	15	60	25	0.447	5.56x10 5
	Silt	5	90	5	0.475	6.68x10 6
	Sandy Loam	10	25	65	0.412	2.89x10 6
	Loamy Sand	5	10	85	0.396	7.57x10 6
	Sand	4	4	92	0.389	1.00x10 7

Table 5 : Estimation of soil porosity from apparent density and theoretical particle density for soils resulting from HT bacterial oxidation experiments in this study or present in the literature.

 5

	Source	Bulk density	Theorical particule density	Soil porosity
	-	kg.m -3	kg.m -3	-
	Experiment 1	1.52x10 3	2.69x10 3	0.44
	Experiment 2	1.53x10 3	2.67x10 3	0.43
	Experiment 3	1.49x10 3	2.69x10 3	0.44
	Experiment 4	1.52x10 3	2.69x10 3	0.43
	Experiment 5	1.51x10 3	2.69x10 3	0.44
	Conrad et al., 1982	1.44x10 3	2.69x10 3	0.46
	Conrad et al., 1982	1.44x10 3	2.69x10 3	0.46
	Conrad et al., 1982	1.61x10 3	2.66x10 3	0.39
	Conrad et al., 1982	1.61x10 3	2.66x10 3	0.39
	Dong et Layzell 2001	1.62x10 3	2.66x10 3	0.39
	Ota et al., 2007	1.61x10 3	2.66x10 3	0.39
	Piché-Choquette et al., 2016	1.57x10 3	2.67x10 3	0.41

Table 8 : Estimated production of soil HTO (Bq.m -2 ) for different soil types under a La Hague climate type following the 24-hour presence of a HT plume with a concentration of 1 Bq.m -3 .

 8

	Soil Type	Bulk density		Soil HTO
	-	Kg.m -3		Bq.m -2 .d -1
	Season		Spring a Summer b Autumn c Winter d
	Clay (C)	1621	0.73	0.65	0.73	0.68
	Silty clay (SiC)	1530	6.23	6.26	6.16	5.86
	Sandy clay (SC)	1646	0.20	0.16	0.20	0.14
	Clay loam (CL)	1576	1.15	1.00	1.09	0.62
	Silty clay loam (SiCL)	1510	3.19	3.23	3.15	2.99
	Sandy clay loam (SCL)	1626	0.24	0.24	0.23	0.18
	Loam (L)	1539	3.51	3.58	3.46	3.29
	Silt loam (SiL)	1483	2.39	2.46	2.36	2.25
	Silt (Si)	1396	0.19	0.19	0.18	0.18
	Sandy loam (SL)	1572	0.49	0.49	0.48	0.45
	Loamy sand (LS)	1608	0.19	0.19	0.19	0.18
	Sand (S)	1623	0.15	0.15	0.14	0.14
	a

Acknowledgements

The authors thank Orano for their partnership during this research and for providing the stack measurement data.

Since the soil in this study is physically apparent to that described by Conrad et Seiler (1982), this soil is considered to have the same particule size composition; d Average value for this particule size range; e The type of land is issued by Info-sols.ca and the location of the soil sample indicated. The fraction of silica added to the mixture (1/3) is assimilated to pure sand particules.