Mouatassim Charai
email: charai.m@yahoo.com

Haitham Sghiouri

Ahmed Mezrhab

Mustapha Karkri

El Kamal

Hammouti

Comparative study of a clay before and after fired brick-making process

Keywords: Brick-making, clay, clay minerals, mineralogical, insulation, red-bricks, sintering, thermophysical

By using X-ray diffraction, SEM/EDS spectroscopy, FT-IR spectroscopy and thermogravimetric analysis, this paper explores the structural, mineralogical and thermochemical transformations of a clay brick before and after baking process under real brick-making plant conditions. The investigated soil is a clayey soil used in brick manufacture, with a clay fraction of 59% dominated by silica. The thermal stability and the crystallinity state of the clay and its brick produced at firing temperatures of 880 °C were examined. The mineralogical transformation of kaolinite, quartz, illite and calcite were discussed and confirmed. The sintering phenomenon of clay was also observed. The thermal transport properties, including the thermal conductivity, the thermal diffusivity and the specific heat capacity were determined using a Transient Plane Source (TPS) method. A comparison of the values for the unfired clay and its associated fired brick can also be seen. It was confirmed that firing process has a negative effect on the thermal insulation of clay bricks due to the sintering process. Therefore, further studies are encouraged to be conducted to identify optimal firing temperatures for best pore interconnections in order to develop thermally efficient clay bricks with high porosity and good mechanical strength.

Introduction

The firing of clay dates back centuries as a standard manufacturing process for improving the durability and the mechanical properties of pottery and clayey building materials. However, the investigation of the thermochemical and the mineralogical changes of clay during the firing treatment has emerged at the end of the 20th century [START_REF] Bergaya | Handbook of clay science[END_REF] and has become essential for optimizing industrial processing, especially in fired clay brick-making industry. Indeed, the mastering of clay sciences has allowed researchers to study the effect of clay minerals and the heat treatment on the material behaviours in order to develop efficient products with controlled properties. Aras et al. [START_REF] Aras | [END_REF] studied the impact of common clay minerals on the mineralogy of fired products and interested in the modifications that take place during the heating process. García-Ten et al. [3] investigated the correlation between the mineralogical composition and the microstructure of materials on their thermo-mechanical parameters. It was found that the thermal conductivity and the mechanical strength strongly depend on two main factors, namely, the degree of sintering and the mineralogical composition of raw materials, which can be controlled respectively through firing and the preparation mixture phase. Recently, a review was conducted by V. A. Drebushchak et al. [4] to examine the thermal transformations of clay minerals under firing treatment.

To advance the understanding, this paper will examine the thermophysical, the chemical and the mineralogical modifications of a local clay used in hollow brick-making manufacture during its baking process.

Nomenclature a

Thermal diffusivity (mm 2 /s) cp Specific heat capacity (J/KgK) λ

Thermal conductivity (W/mK) ρ Density (Kg/m 3 )

Materials

Raw clay

The studied raw material is a local clayey soil used for the manufacture of fired hollow bricks, which was supplied by ARGILUX factory. The particle size distribution of soil was carried out using humid sieving and hydrometer sedimentation according to NF EN ISO 17892-4 standard, and showed 93.5% of particles finer than 65 μm. The Atterberg limits of soil was determined using Casagrande apparatus in accordance with the French standard NF P94-051. The chemical composition of soil was also investigated by Energy-Dispersive Spectroscopy (EDS) analysis coupled with Scanning Electron Microscopy (SEM) to identify the main elements present that may be of help in the explanation of results.

A summary of the physical and the chemical properties of the collected soil are given in Table 1. In addition, the X-Ray Diffraction (XRD) of the studied soil was performed using the SHIMADZU XRD-6000 diffractometer operating at 40 kW / 30 mA with a wavelength λ = 0.154 nm of Cu Kα radiation with range 10-100° (2θ) at a rate of 0.02°/min to establish the mineralogical identity of used soil. The powder XRD pattern, shown in Fig. 1, confirmed the EDS's results and showed that the raw material was essentially composed of quartz, kaolinite, illite, goethite and a small amount of calcite.

K aolinite Calcite Illite Illite + Quartz Illite Calcite K aolinite Q uartz Quartz Q uartz Q uartz Illite + Q uartz Illite Q uartz Illite Total Clay Calcite + Quartz Calcite Q uartz Q uartz Q uartz Plagioclase Quartz Illite K aolinite Goetite G oetite + Q uartz Total Clay

Fired clay

After the clay has passed through the production chain (i.e. preparation, shaping, drying and firing), the final hollow bricks (Fig. 2) were collected for the comparative analyses. It is worth noting that the hollow bricks were dried in a dryer room at 60°C for three days to prevent shrinkage cracks, and fired in a furnace for 24 hours at selected temperature of 880°C. Then, the studied samples were transported for laboratory tests.

Methods

In the following, we will discuss the testing methods used in this paper to study the clay changes in the industrial manufacture of fired clay bricks. This comparative study can be divided into three main parts:

Mineralogical comparison

As the raw clay, the powder of the fired clay brick was prepared for investigating its crystalline state. The analysis was performed using the same X-Ray diffractometer and under the same diffraction angle to evaluate the crystalline changes of our clay. In addition, a thermogravimetric analysis TGA was performed using on both samples to evaluate the thermal stability of clay before and after the baking process.

Microstructural comparison

The morphological modifications of our clay samples were carried out using the SEM technique. As a complementary analysis, a Fourier Transform Infrared Spectroscopy (FT-IR) was performed on the raw clay and its associated fired brick to determine the molecular functional groups present on the sample's surface. The studied powder samples are prepared and premixed with 1% of the KBr compound. The infrared spectra are obtained by means of the SHIMADSU FTIR-8400S spectrophotometer with transmission mode in the wave number range between 400 cm -1 and 4000 cm -1 .

Thermophysical comparison

Since the studied clay was used for building material manufacturing, it is worthwhile to compare the thermal performance between our fired and unfired bricks. To this end, the thermophysical properties of both fired and unfired clay bricks were measured using the Hot Disk TPS 2200 apparatus that measures the thermal conductivity property of samples according to ISO22007-2.2. The measurement principle is based on a transient plan source technique, which was developed by Gustafsson [5]. As illustrated in Fig. 3, a double-spiral nickel sensor is sandwiched between two identical samples. The TPS sensor served to heat the samples with a constant electrical power and then to monitor their time-dependent temperature response. Finally, the thermal conductivity, the thermal diffusivity and the specific heat capacity are simultaneously calculated based on the recorded thermal response of samples studied.

Results and discussion

Fig. 4 illustrates the powder XRD pattern of the raw clay and its associated fired clay brick. The X-ray diffraction analysis showed that the clay was subjected to four main changes during the backing phase, resulting in the modification of the crystalline structure of the clay. The slight modification of the quartz peak can be explained by the transformation of the α-quartz to β-quartz. The disappearance of both kaolinite and calcite peaks were observed. A total disappearance of calcite was confirmed by the EDS analysis of the fired clay brick. These modifications represent respectively the chemical transformations given in Table 2. The TGA thermograms obtained of the raw clay and its fired brick are shown in Fig. 5. The thermal stability was investigated by firing both samples from 50 °C to 900 °C in air atmosphere with a heating rate of 20 °C/min. The results show that the unfired and fired clay has respectively a total mass loss of 10% and 2%, highlighting the high thermal stability of fired clay bricks compared to the unfired ones. While the small change in mass of fired clay bricks can be attributed to the dehydration of adsorbed water, the unfired clay was subjected to five main decomposition listed in Table 3. These results showed a good agreement with the XRD analysis. Fig. 6 shows the infrared spectrometry of clay before and after its firing. The modifications (i.e. I and II) of the clay under the firing brick-making treatment was clearly observed. The FT-IR confirmed the presence and the decomposition of Kaolinite and Calcite in the clay and its fired brick, respectively. The results of the FT-IR analysis are summarized in Table 4. The finding results were well corroborated by the previous analyses.

I I

Transmittance (%) Wavenumber (cm-1) AFTER I Fig. 6. FT-IR spectra of clay before (black) and after (red) firing process. The SEM images of clay before and after the firing are presented in Fig. 7. The SEM micrographs shows clearly the sintering phenomenon of clay minerals due to the thermal treatment of clay. This phenomenon presents the main objective of baking clay-based materials to enhance the mechanical strength of clayey products by eliminating microspores shown in Fig. 7a. In addition, the EDS results confirmed the total disappearance of calcite due to the baking processing.

In the following, we will discuss the effect of these changes on the thermal performance of clay bricks by investigating the thermal transport properties of clay before and after the industrial thermal treatment. Table 5 shows the measured thermophysical properties of unfired and fired clay. From Table 5 it can be seen that the values of the thermal conductivity and the thermal diffusivity of clay were increased by 10.8% and 14.8% after baking, respectively. In addition, the apparent density was increased by 7% due to the compaction of clay after the thermal sintering process. Therefore, and by dividing the volumetric heat capacity (i.e. ρcp) by the density, the thermal capacity of clay was reduced from 701.5 to 668.5 J/mK. In a thermal point of view, the results demonstrate the effectiveness of unfired clay bricks compared to their associated fired bricks for developing high-performant building envelopes with low environmental impact.

Conclusion

In this work, a raw clay and its associated fired brick were studied. The results obtained from the test measurements show that the industrial backing process changes the microstructure and the chemical properties of the clay. Although the mechanical improvement, the thermophysical characterization of the studied clay and its fired clay shows that the thermal treatment of clay negatively affects the thermal performance of clay bricks and reduces their thermal insulation property. According to the carried out analyses, we can draw the following conclusions:

• During firing process, the physical, mineralogical, chemical characteristics of clay change.

• Firing process enhances the mechanical strength while reduces the thermophysical properties of clay.

• SEM analysis reveals the sintering phenomenon of clay.

• From the thermal point of view, the unfired clay bricks are effective compared to the fired clay.

From this paper, further studied are encouraged to identify optimal firing temperature in order to ensure best pore structure, or to develop unfired clay bricks with enhanced mechanical properties using environmentally friendly strategies.

Fig. 1 .

 1 Fig. 1. XRD spectra of soil.

Fig. 2 .

 2 Fig. 2. Studied samples: (a) fired and unfired clay bricks (b) view of bricks during backing.

Fig. 3 .

 3 Fig. 3. Hot Disk TPS Tests.

Fig. 4 .

 4 Fig. 4. XRD spectra of clay before (black) and after (red) firing process.

Fig. 5 .

 5 Fig. 5. TG-DTG pattern of clay before (right) and after (left) firing process.

Fig. 7 .

 7 Fig. 7. SEM images of clay (a) before and (b) after thermal treatment.

Table 1 .

 1 Soil characteristics.

	Property	%
	Atterberg limits
	Liquid limit	40
	Plastic limit Plasticity index	21 19
	Particle size distribution
	Sand	6.5
	Silt	34.5
	clay	59
	Chemical composition
	SiO2	51.17
	Al2O3	19.57
	Fe2O3	10.88
	CaO	1.55

Table 2 .

 2 Main transformations of clay during industrial baking

	Transformation	Reaction	Temp (°C)	Reference
	Dehydroxylation of Kaolinite to metakaolinite	Al4Si4O10(OH)8 → 2Al2Si2O7 + 4H2O	450 °C -700 °C	[6]
	Transition of α-Quartz into β-quartz	α-quartz → β-quartz	570 °C	[7]
	Decomposition of Calcite	CaCO3 →CaO + CO2	800 °C	[8]

Table 3 .

 3 Thermogravimetric analysis of clay.

	Temperature (°C)	Mass loss (%)	Interpretation
	86	2.01	Dehydration
	285	1.22	Goethite decomposition
	525	3.12	Quartz transformation (α-Quartz → β-Quartz)
	680	2.89	Decomposition of Kaolinite and Illite
	830	0.41	Decomposition of Calcite

Table 4 .

 4 FTIIR interpretation of clay.

	Band cm -1 description	Interpretation
	I
	3697,66	Valence vibrations of the O-H bond	Vibrations of structural hydroxyl groups' characteristic of kaolinite
	3620,51	Valence vibrations of the O-H bond	Stretching vibrations of the OH group of the adsorbed water
	3421,83	Valence vibrations of the O-H bond	Stretching vibrations of the OH group of the adsorbed water
	II
	1637,62	Flexion of water molecules H-OH	Deformation vibrations of the OH group of the adsorbed water
	1431,23	C-H3 deformation	Characteristic band of carbonates

Table 5 .

 5 Thermophysical properties of Clay before and after firing.

	Clay	λ [W/mK]	a [mm 2 /s]	ρcp [MJ/m 3 K]	ρ [Kg/m 3 ]
	Unfired	0.807	0.581	1.389	1980
	Fired	0.972	0.682	1.424	2130

Acknowledgements

The authors would like to thank the "National Center for Scientific and Technical Research" for funding this work through the PPR project "Promotion of solar energy and energy efficiency in the oriental region of Morocco". The authors would also like to thank the Laboratory of Mineral and Analytical Solid Chemistry of Mohammed 1st University is acknowledged for performing XRD, TGA and FTIR analyses. MAScIR Laboratory is also acknowledged for performing the SEM/EDS analysis. Professor Elkhadir Gharibi is acknowledged for discussing the results of the mineralogical analysis.


Xavier Altafaj

Weijun Cheng

Eric Estève

Julie Urbani

Didier Grunwald

Jean-Marc Sabatier

Roberto Coronado

Michel De Waard

Michel Ronjat

Altafaj Xavier

Est Ve È Eric

Urbani Julie

Grunwald Didier

Sabatier Jean-Marc

Coronado Roberto

De Waard Michel

Ronjat Michel

Pathologies

Maurocalcine and domain A of the II

Keywords: Adenosine Triphosphate, chemistry, Animals, Binding Sites, Binding, Competitive, COS Cells, Calcium Channels, L-Type, chemistry, Caveolin 1, Caveolins, chemistry, Chromatography, Cloning, Molecular, Cryoelectron Microscopy, Microscopy, Fluorescence, Muscle, Skeletal, metabolism, Peptides, chemistry, Plasmids, metabolism, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, chemistry, Ryanodine, chemistry, Ryanodine Receptor Calcium Release Channel, chemistry, Scorpion Venoms, chemistry, pharmacology, Transfection

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

INTRODUCTION

In skeletal muscle cells, the activation of the plasma membrane dihydropyridine receptor induces, through the ryanodine receptor (RyR1), a massive release of the calcium stored in the terminal cisternae of the sarcoplasmic reticulum (SR). The set of events, that starts with the depolarization of the plasma membrane, sensed by the DHPR, and ends with the opening of the RyR1, is called excitation-contraction (EC) coupling. Several results have led to the conclusion that the skeletal muscle EC coupling is based on a mechanical process that is made possible by the physical interactions of the two Ca channels ( ). The transmission of information from 2+ 1 the DHPR to the RyR1 has been termed orthograde signal. In addition, the same physical interactions, which occur between RyR1 and DHPR, have also been proposed to be responsible for a retrograde signal in which the DHPR calcium channel activity is controlled by RyR1 ( , ). The identification of protein domains involved in these interactions between both DHPR and RyR1 and of proteins regulating 2 3

these interactions remains today an important field of research.

The DHPR is composed of four different subunits , , and ( ). The subunit has been shown not only to form the pore region

α 1 β α 2 δ γ 4 α 1
and to carry the voltage sensitivity of the channel but also to be directly involved in the functional coupling of the DHPR with RyR1 ( ). 5

Indeed, the cytoplasmic loop that links the domains II and III of the subunit has been proposed to be responsible for the mechanical α 1 coupling between the DHPR and the RyR1 ( ). Within this cytoplasmic loop, two regions (domains A and C, respectively) have been 6

shown to regulate ryanodine binding and channel gating of RyR1 ( ). Other regions of the subunit have been found to interact in vitro 7 α 1 with RyR1 but the functional role of these interactions is less essential or remains to be established ( ). 8 Ryanodine receptors are formed by the homotetramer assembly of a 560 kDa polypeptide. RyR1 activity is regulated by a in vitro number of chemical compounds such as ATP, Ca , Mg , and proteins such as calmodulin and FK506 binding protein (FKBP12) ( ).

2+ 2+ 9
Nevertheless, due to i) the extremely large size of RyR1, ii) the small number of high affinity effectors and iii) the absence of atomic resolution structure, the mapping of the functional sites of RyR1 is still far from completion. Expression in dyspedic cells (lack RyR1) of chimeric RyR1 channels, that carry sequences of different RyR isoforms, allowed the identification of domains engaged in the EC coupling process ( ). At the same time, binding sites for different effectors and partner proteins have been identified. As part of the 10-12 search for molecules able to strongly modify RyR1 properties, toxins isolated from scorpion venoms have been described as being able to induce important modifications in RyR1 channel behavior. Nowadays, these toxins represent the most effective effectors of RyR1. Among them, imperatoxin A (IpTx A) and maurocalcine (MCa) have been extensively characterized both in terms of their effects on RyR1 and their three-dimensional structure (

). Interestingly, these two peptide toxins present some amino acid sequence homology with the 13-18 domain A of DHPR subunit and structural studies strongly suggest that the beta-sheet structure of these toxins could mimic that of the α 1 domain A of the DHPR subunit ( , ). In a previous work, we demonstrated that nanomolar concentrations of MCa induces a dramatic α 1 16 17 conformational change of RyR1, witnessed by the increase in H -ryanodine binding (7-fold) and the induction of long-lasting channel

[ 3 ]
openings ( , ). The observed homologies in amino acid sequences and in 3-D solution structure have prompted different authors to 13 14 make the hypothesis that IpTx A and MCa could share a common binding site with the domain A of the DHPR subunit on the RyR1.

α 1
We have therefore undertaken the identification of the amino acid region of RyR1 that carries the binding site for MCa and/or the domain A of the skeletal DHPR subunit.

α 1
In this work, a set of polypetides, covering the entire sequence of RyR1, was expressed and tested individually for their ability in vitro to interact with either MCa or peptide A (a synthetic peptide corresponding to the domain A of the skeletal DHPR subunit). We sk α 1 identified a single domain of RyR1 interacting with both MCa and peptide A . We also showed that MCa and peptide A compete for the sk sk interaction on this domain. In order to asses the functional relevance of this domain for the effect of MCa on RyR1, we expressed a RyR1 channel that lacks the MCa binding domain in COS-7 cells and measured the effect of this deletion on the activation of ryanodine binding by MCa. Taken together, our results clearly establish that MCa and domain A of the DHPR subunit share a common binding site on

α 1
RyR1.

MATERIAL AND METHODS

Purification of RyR1

The skeletal heavy SR vesicles were prepared from rabbit leg and back muscles by differential centrifugation, as previously described ( ). Vesicles were then solubilized in the presence of CHAPS, and RyR1 was purified by centrifugation of the solubilized proteins on a 19

sucrose density gradient ( ) and stored in liquid nitrogen. 20

Peptide syntheses

Peptides used in this work were synthesized as previously described ( , ), with the addition of an exogenous biotin on the according to the manufacturer s instructions. For pull-down assays, 5 l of the translation product were incubated with polystyrene ' μ magnetic-beads coated with the peptide of interest as described above. After overnight incubation at 4 C, beads were washed with 1 °volume of buffer A. The associated proteins were eluted in denaturing buffer and analyzed by autoradiography after separation on SDS-PAGE.

Expression of His-tagged RyR1 fragments in bacteria

RyR1-F7 fragment was sub-cloned into three shorter segments (F7.1, F7.2 and F7.3) by a PCR-based approach. Briefly, the cDNA was amplified using primers containing a linker encompassing a I site (in the 5 end of the forward primer) and a I (in the 3 end of Nco ′ Xba ′ the reverse primer). The PCR product was cloned using the I and I sites in the pMR78 plasmid that possesses a His-tag sequence Nco Xba upstream of the polylinker ( ). E. Coli. BL21 pLys(DE3) bacteria were transformed with RyR1-F7( ) containing plasmid. Protein 23 1-3 synthesis was induced by the addition of 1 mM IPTG (Sigma) to the culture media. 3 h after induction, the bacteria were collected by centrifugation (3,000 for 10 min at 4 C) and suspended in MCAC buffer (20 mM Tris/HCl, 500 mM NaCl, 10 v/v glycerol, pH 8.0) g °% supplemented with protease inhibitors (Complete , Boehringer Mannheim). Cell lysis was induced by adding Triton X-100 (0.1 v/v),

™ %

DNAse I (50 g) and MgCl (10 mM). After 10 min incubation at room temperature, insoluble material was removed by centrifugation ( 15μ 2 min at 20,000 and 4 C). Solubilized proteins were then passed through a column of Ni-NTA agarose beads (Qiagen) equilibrated with g °MCAC buffer. After wash with four volumes of MCAC buffer containing 60 mM imidazole, his-tagged RyR fragments were eluted in 0.5 ml fractions with MCAC buffer containing 250 mM imidazole and detected by Western blot analysis with antibodies directed against the histidine tag (Sigma) used at a dilution factor of 1/10,000.

Transfection of COS-7 cells with RyR1 constructs and cytoimmunofluorescence

Wild

Microsomes preparation

COS-7 cells were harvested in ice cold buffer containing 320 mM sucrose, 5 mM Hepes pH 7.4 and protease inhibitors, using a cell-scraper. Cells were then homogenized using a Teflon potter and centrifuged for 5 min at 7,000 at 4 C. Microsomes were then g °collected by centrifugation at 100,000 for 1 h at 4 C and resuspended in the same buffer. After quantification of protein concentration by g °Bradford assay, samples were stored in liquid nitrogen. -ryanodine (10 nM) and caffeine (0 or 6 mM) or MCa (0 or 30 nM). Free and bound H -ryanodine were separated by filtration of the

[ 3 ]
microsomes on Whatmann GF/B filters and H -ryanodine measured by liquid scintillation. Non-specific H -ryanodine binding was

[ 3 ] [ 3 ]
measured in the same conditions in the presence of 10 M unlabeled ryanodine.

μ

RESULTS

presents the primary structure of MCa, the domain A and the domain C of the II-III loop of the skeletal muscle DHPR. In order to identify the amino acid sequences of RyR1 involved in the interaction with MCa and peptide-A , we generated a contig of b sk nine clones encompassing the full length RyR1 cDNA in plasmids ( ). Each of these clones was expressed separately in an Figure 1C in cell-free translation system in the presence of radiolabeled S -methionine. RyR1 fragments (F1 9) were incubated in the presence vitro ). Corresponding polypeptides were then expressed in E. coli, affinity-purified trough their histidine tags, and Figure 2A tested for their interaction with MCa and peptide-A . Interacting fragments were detected by immunolabeling with antibodies directed b sk against the his-tag, after separation on SDS-PAGE.

shows that only the fragment F7.2, encompassing amino acid residues Figure 2A 3351-3501, retains the ability to interact with both MCa and peptide-A . No interaction was observed with fragment F7.1 or F7. We next investigated the importance of the F7 region in the sensitivity of RyR1 to MCa. For this purpose, COS-7 cells were co-transfected with two plasmids encoding the DsRed protein and the entire RyR1 (RyR1wt) or the RyR1 deleted of residues 3241-3661

(RyR1 F7) (
). Immunolabeling of DsRed-positive cells with antibodies directed against the C-terminal domain of RyR1

demonstrates that RyR1wt and RyR1 F7 exhibit a similar cell distribution. The distribution essentially consists in a cytoplasmic labeling Δ most likely representing the endoplasmic reticulum ( ). Fluorescence analysis showed that about 10 of cells co-express DsRed Figure 3A % and RyRwt or DsRed and RyR F7. A weak labeling with anti-RyR1 antibodies was also observed in cells that do not express DsRed Δ protein, suggesting that some cells could express exogenous RyR1 without DsRed or that they possess a low level of endogenous RyR1 expression (however barely detected by Western blot analysis,

). Figure 3B In order to establish the functional relevance of the F7 region in RyR1 regulation by MCa or peptide-A , we characterized the binding sk of H -ryanodine on RyR1wt or RyR1 F7 expressed in COS-7 cells and tested the effects of both caffeine and MCa on this binding. For this purpose, we prepared microsomes from cells transfected with either RyR1wt or RyR1 F7. Immunoblot analysis of the different Δ microsome preparations using antibodies directed against RyR1 ( upper panel) supports the localization of expressed RyR1wt Figure 3B and RyR1 F7 in reticulum membrane of transfected cells. These results also show that RyR1wt and RyR1 F7 are expressed at a similar Δ Δ level and remain stable in transfected cells. Finally, endogenous RyR1, if present, is barely measurable. Using these microsome preparations, we then measured H -ryanodine binding on expressed RyR1wt and RyR1 F7 in the presence or absence of caffeine or

[ 3 ] Δ
MCa. No significant specific H -ryanodine binding was observed on microsomes obtained from non-transfected cells ( ). In

[ 3 ] Figure 3B
addition, no effect of caffeine or MCa was observed on these control microsomes (data not shown). This data confirmed immunological and Western blot data showing a low level if any of endogenous RyR1 in COS-7 cells. In contrast, H -ryanodine binding was observed

[ 3 ]
on microsomes populations obtained from COS-7 cells transfected with RyR1 constructs. Specific binding was 116 57 fmol/mg and 113 ± 31 fmol/mg for microsomes obtained from RyR1wt and RyR1 F7 transfected cells, respectively. Taken together with the Western blot

± Δ
analysis, these results indicate a similar H -ryanodine binding ability of expressed RyR1wt and RyR1 F7. Activation of H -ryanodine

[ 3 ] Δ [ 3 ]
binding by caffeine and MCa is presented in . In the presence of 6 mM caffeine, we observed a 197 5 and 218 32 Figure 3B ± % ± % increase of H -ryanodine binding on RyR1wt and RyR1 F7 microsomes, respectively. In contrast, 30 nM MCa induced a 202 28

[ 3 ] Δ ± %
increase of H -ryanodine binding on RyR1wt microsomes but no significant change of H -ryanodine binding on RyR1 F7 microsomes

[ 3 ] [ 3 ] Δ ( 107 3
). These results clearly demonstrate that deletion of amino acids 3241 to 3661 of RyR1, included in fragment F7, induces a ± % complete loss of RyR1 sensitivity to MCa while it does not modify its sensitivity to caffeine.

DISCUSSION

Recently, we characterized the effect of MCa on RyR1 ( , ). MCa is a 33 amino acid toxin initially isolated from the venom of the 13 14 scorpion , and subsequently produced by chemical peptide synthesis ( ). Based on sequence similarities, MCa Scorpio maurus palmatus and IpTxA (another scorpion toxin interacting with RyR1) were proposed to share with the domain A of the skeletal DHPR subunit a α 1 common binding site(s) on RyR1. In this work, we identified two sequences of RyR1, fragment F3 encompassing amino acids 1021-1631 and fragment F7 comprising the residues 3201-3661, that are able to interact with both MCa and domain A of the II-III loop of the in vitro DHPR subunit. Among these two sequences, fragment F7 displays the strongest interaction with MCa and peptide-A . We then α 1 sk generated a RyR1 construct that lacks the amino acid region corresponding to fragment F7. This RyR1 construct with its internal deletion displays an expression profile and a caffeine sensitivity that is perfectly similar to the wild-type RyR1. In contrast, deletion of the F7 region completely abolished the stimulatory effect of MCa on ryanodine binding, highlighting the critical role of this domain for MCa regulation. A molecular dissection of fragment F7 allowed us to identify a discrete domain corresponding to amino acids 3351-3501 (fragment F7.2) responsible for the interaction of MCa and peptide-A with RyR1. These results clearly demonstrate that, in agreement sk with earlier expectations, MCa and domain A interact with the same RyR1 region within the 3351-3501 stretch of amino acid residues.

Binding of MCa and peptide-A to the same RyR1 site was confirmed by the complete inhibition of the interaction of peptide-A with the sk sk fragment F7.2 in the presence of an excess of MCa.

Using an affinity chromatography approach, Leong and MacLennan identified a domain of RyR1 that interacts with the entire II-III loop of the DHPR subunit ( ). This sequence (amino acid residues 1076 to 1112) is included within our fragment F3 that we found to

α 1 25
interact more weakly with both peptide-A and MCa. In contrast, these authors did not observe any significant interaction of the entire II sk -III loop with a polypeptide comprising amino acid residues 3158-3724 (which includes fragment F7). Our previous results, together with those presented here, showing that domain C of subunit does not interact with any sequence of RyR1, suggest that domain A is the sk Several studies using either RyR1/RyR2 or RyR1/RyR3 chimeras led to the identification of different domains of RyR1 involved in functional and structural interaction of DHPR with RyR1 ( , ). Interestingly, fragment F7 carrying the MCa and peptide-A binding 11 12 sk domain is included in one of these domains formally defined as domain R9 or Ch-9. Similarly, the deletion of residues 1272-1455 of RyR1

(included in the F3 fragment) has been shown to induce a loss of depolarization-induced Ca release ( ). Therefore, regardless of the 12 approach, the two regions of RyR1 spanning fragments F3 and F7 have been shown to play important roles in the functional communication between RyR1 and DHPR. In agreement with previous studies suggesting that noncontiguous regions of RyR1 may act together to control the functional coupling between RyR1 and DHPR, the F3 and F7 fragments may form a unique functional domain. This possibility is supported by three-dimensional reconstruction of RyR1 indicating that regions including the F3 and F7 fragments could be close from each other in the RyR1 tetramer ( ). 26

In conclusion, we identified the binding domains of MCa on RyR1. We also introduce the first biochemical evidence that MCa and domain A of the DHPR subunit interact with identical sequences on RyR1. In view of the important effects of MCa on RyR1 gating

α 1
properties ( , ), the RyR1 sequences that we identified in this report ought to play an important function in RyR1 calcium channel 13 14 behavior. The fact that domain A of the II III loop of DHPR binds onto the same RyR1 sequences re-opens the question of the role of the domain A in the cont rol of RyR1 calcium channel activity in the context of the DHPR/RyR1 complex. Our results suggest that the two sequences of RyR1 (F3 and F7), that are involved in the binding of MCa, could fold together to create a unique MCa binding site.

Cryoelectron microscopy studies of the RyR1-MCa complex should permit to test this hypothesis and to refine the correspondence between primary structure and 3-D reconstruction images of RyR1.

Ackowledgements:

This , Functional effect of the F7 deletion on C RyR1 binding properties. H -ryanodine binding experiments on purified cell microsomes showed that, while both constructs responded to caffeine administration (6 mM), the deletion of the F7 fragment abolished MCa effect (30 nM).

  -type RyR1 (RyR1wt) construct was cloned into a modified pCI-neo (Promega). Briefly, Nde (nt 387) and Nhe (nt 1085) sites I I on the pCI-neo vector were destroyed. Within the RyR1 cDNA, EcoR (nt 9726) and Nhe (nt 10014) sites were introduced and used for V I cloning into the modified pCI-neo vector, for further expression studies in mammalian cells. RyR1?F7 construct was generated from the pCI-neo/RyR1 clone (containing unique EcoR , Nhe and Nde restriction sites), by cutting out the sequence between nucleotides 9730 V I I and 10983. COS-7 cells were transfected with the RyR1wt or RyR1 F7 containing plasmid using the linear polyethylenimine jetPEI reagent Δ ™ according to the manufacturer s instructions (Qbiogene). Expression of RyR1wt and RyR1 F7 was controlled by immunofluorescence ' Δ using antibodies directed against the C-terminal domain of RyR1 ( ). 48 h after transfection, cells were fixed in 4 paraformaldehyde for 19 % 10 min, washed 3 times with PBS and permeabilized with 0.1 Triton X-100. Cells were then incubated for 1 h at room temperature in the % presence of 1 BSA and 1 goat serum before adding anti-RyR1 antibodies (dilution 1:200 in PBS-0.1 Tween 20, PBS-T). After 2 % % Cter % h, the cells were washed four times with PBS-T and incubated for 30 min at room temperature with Alexa488-conjugated anti-rabbit IgG (dilution 1:800 in PBS-T). Cells were then washed four times with PBS-T and mounted. Samples were analyzed by confocal laser scanning microscopy, using a Leica TCS-SP2 operating system. Alexa , and DsRed fluorescence were excited and collected sequentially 488 (400 Hz line by line) by using the 488 nm line of an argon laser for Alexa , and the 543 nm line of an helium-neon laser for DsRed 488 excitation. Fluorescence emission was collected from 598 to 541 nm for Alexa , and from 554 to 625 nm for DsRed.488

[ 3 H 2 °[ 3 ]

 323 -ryanodine binding assay] Microsomes preparation (100 g of proteins) obtained from control non-transfected COS-7 cells, and RyR1 or RyR1?F7-expressing μ cells were incubated at room temperature for 1 h in presence of 1 M recombinant FKBP12 in a buffer containing 150 mM NaCl, 2 mM μ EGTA and 2 mM CaCl (pCa 5), 20 mM Hepes, pH 7.4. Microsomes were then incubated for 150 min at 37 C in the presence of H

α 1

 1 Biotinylated peptides, named MCa , peptide-A (pA ) and peptide-C (pA ) corresponding to these different sequences were b Sk sk Sk sk synthesized and immobilized on polystyrene beads coated with streptavidine. shows the SDS PAGE analysis of the bound RyR1 Figure 1B fraction following incubation of beads covered with each of the peptide with purified RyR1 as described in Materials. A polypeptide band corresponding to RyR1 is only observed in the bound fraction obtained with beads covered with MCa , peptide-A . No signal was b Sk detected when beads were covered with peptide-C or biotin. These results confirm the specificity of the interaction between the skeletal Sk sequence of domain A and RyR1 and represent the first biochemical evidence of a direct interaction of MCa with RyR1.

%

  either with MCa , peptide-A , peptide-C or biotin. The fragments interacting with the different peptides were then b sk sk detected by autoradiography after separation on SDS-PAGE. shows that among the nine translated fragments, F7 fragment Figure 1C encompassing RyR1 amino acid residues 3201-3661 interacts strongly with MCa and with peptide-A . Fragment F3 encompassing the b sk RyR1 amino acid residues 1021-1631 consistently shows a weak interaction with both MCa and peptide-A . In agreement with what is b sk observed with purified RyR1, no significant interaction with peptide-C was observed with any of the nine RyR1 fragments. Similarly, no sk interaction was observed of any fragment with beads covered with biotin (not shown). Statistical analysis of the amount of complex formed with each of the fragment shows that about 50 of the F7 fragment interacts with MCa or peptide-A , while only about 15 of % b sk % the F3 fragment was retained by MCa or peptide-A . For all other fragments, the amount of complex formed with MCa or peptide-A b sk b sk was not significantly different from what observed with peptide-C and represented less than 5 of the input. sk To further define the RyR1 sequence that interacts with MCa and peptide-A , the cDNA corresponding to F7 was subcloned into b Sk three smaller fragments, named F7.1, F7.2 and F7.3, encompassing RyR1 amino acid residues 3201-3350, 3351-3501 and 3502-3661, respectively (

  3. The b sk specificity of the interaction of the F7.2 fragment with MCa and peptide-A was confirmed by the lack of signal with beads covered with b sk peptide-C or biotin (data not shown). We therefore investigated the ability of MCa to inhibit the interaction of fragment F7.2 with sk b peptide-A . Incubation of fragment F7.2 for 2 h in the presence of non-biotinylated MCa induces a complete loss of interaction of this sk fragment with immobilized peptide-A ( ) strongly suggesting that MCa and peptide-A interact on the same site on the F7.2 sk Figure 2B sk fragment.

α 1 sk

 1 only sequence of II III loop interacting with RyR1 ( ). The difference between the two sets of results may reflect an effect of the distal -21 part of the II III loop on its conformation thereby possibly modifying the accessibility of domain A to RyR1.

: 1 CACNA1S:Figure 1

 11 Figure 1 Identification of RyR1 fragments interacting with MCa and domain A , Sequence alignment of maurocalcine (MCa), domain A (pA ) and domain C (pC ) of the II-III loop of the subunit of the DHPR. Grey A sk sk

  representing the percentage of binding of translated RyR1 fragments to MCa , estimated by densitometry and expressed as in vitro b the means S.E.M. from at least three independent experiments.

±

Figure 2 1 ,

 21 Figure 2 Maurocalcine and domain A of the II-III loop of the subunit bind onto the same site of RyR1 α 1 , Fine dissection of RyR1 fragments that interact with MCa and pA . , schematic representation of the RyR1-F7 sub-fragments A sk Upper panel (F7.1, F7.2, F7.3) cloned into pMR78-his tag vector for bacteria expression and affinity chromatography purification. , Western Lower panel blot with anti-his antibodies, showing the specific pull-down of the F7.2 fragment of RyR1 with both MCa and pA . b sk , Western blot B resulting from a competition experiment, where the RyR1-F7.2 fragment was preincubated with an excess of MCa (1 M) and then incubated μ with 16 nM of pAsk previously immobilized on streptavidine-magnetic beads. While the fragment F7.2 was able to bind to pAsk (left lane), b the presence of MCa abolished this interaction (right lane), showing a competition of these two peptides for the same binding site on RyR1.

Figure 3

 3 Figure 3 Deletion of the F7 region abolishes the effect of maurocalcine on H -ryanodine binding onto RyR1 expressed in COS-7 cells

[ 3 ],

 3 RyR expression pattern in COS-7 cells cotransfected either with DsRed and RyR1wt (upper panels) or with DsRed and RyR1-F7 (lower A Δ panels) in COS-7 cells. Immunofluorescent signal in left panels (green signal) show the distribution pattern of RyR1 and red signal in central panels correspond to DsRed protein. Right panels correspond to merged images. , Expression of RyR1 constructs in COS-7 cells. Western B blot with anti-RyR1 (C-terminal) antibodies, showing a lack of endogenous RyR1 expression (lane 1) in control cells and equal protein amounts of RyR1 wt (lane 2) and RyR1-F7 (lane 3) in 50 g of microsomes, 48h after transfection.Δ μ

and expression of RyR1 fragments

						14 21
	C-terminal amino acid of i) MCa synthetic peptide, ii) peptide A , corresponding to residues Thr -Lys of the DHPR 1s subunit, and sk 671 690 α
	iii) peptide C , corresponding to the residues Glu -Pro of the DHPR 1s subunit. sk 724 760 α
	Pull-down experiments
		Polystyrene magnetic beads (500 g) coated with streptavidine (Dynal) were incubated for 30 min at room temperature in the presence μ
	of biotinylated peptide (100 g/ml in buffer A; 150 mM NaCl, 2 mM EGTA, 2 mM CaCl , pCa5 and 20 mM HEPES, pH 7.4). Beads were μ 2
	then washed three times with buffer A and incubated for 2 h at room temperature with purified RyR1 (100 nM in buffer A). After washing
	twice with buffer A, proteins bound to the immobilized peptide were eluted by boiling the beads for 5 min in 2.5 SDS and analyzed by %
	Western blot using antibodies directed against RyR1 after separation on 8 SDS-PAGE.
		RyR1 fragments F1-F8 were cloned in pSG5 vector (Stratagene) by a PCR-based method using specific primers with a linker for
	further enzymatic digestion and insertion into the plasmid backbone ( ). Fragment F1 (from aminoacid 1 to 526) was cloned in 22	Eco	RI
	and	Not	I restriction sites; F2 (aminoacids 527-1021) in	HI-Bam Not	I sites; F3 (aminoacids 1022-1631), F4 (aminoacids 1632-2169), F5
	(aminoacids 2170-2697), F6 (aminoacids 2698-3200) and F7 (aminoacids 3201-3661) were cloned between	Eco	RI and	Bam	HI restriction
	sites; F8 (aminoacids 3662-4244) was cloned between	Bam	HI and	Not	I. Fragment F9 (aminoacids 4007-5038) was produced by cutting
	the original pCIneo-RyR1 plasmid (kindly provided by Dr. Allen) with	Xho	I, followed by self-ligation.
		35	S-labeled RyR1 fragments were expressed using a coupled	in vitro	transcription and translation system (TNT kit, Promega), ™

%

Cloning


Rafik Mallek

Christelle Miqueu

Matthieu Jacob

Christophe Dicharry
email: christophe.dicharry@univ-pau.fr

-loaded porous activated carbon particles immersed in brine

Keywords: CP hydrates, CP-adsorption/desorption, activated carbon, energy compensation, hydrate-based processes, desalination

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

Experimental evaluation of the partial thermal energy compensation of hydrate crystallization from cyclopentane

Introduction

Hydrate-based desalination (HBD) is a potential water-desalination technology that was first proposed by A. Parker in 1942 [START_REF] Parker | Potable water from sea-water[END_REF] and has received marked attention since the 1960s in order to strengthen the water-energy nexus [START_REF] Babu | A Review of Clathrate Hydrate Based Desalination To Strengthen Energy-Water Nexus[END_REF]. It is based on the formation of gas hydrates which, once formed, are separated and dissociated to obtain desalinated water due to the fact that salt ions do not participate in the hydrate structure [START_REF] Zheng | Progress and trends in hydrate based desalination (HBD) technology: A review[END_REF]. Gas hydrates are ice-like compounds formed by the association of water cages and small chemical compounds known as guests.

They generally form at relatively high pressures and low temperatures [START_REF] Sloan | Clathrate Hydrates of Natural Gases[END_REF]. Some of them, such as cyclopentane (CP) hydrates, can however form at atmospheric pressure. Hydrate crystallization has been widely studied in the literature in different contexts such as natural sediments [START_REF] Tohidi | Improving the accuracy of gas hydrate dissociation point measurements[END_REF][START_REF] Atig | Contactless probing of polycrystalline methane hydrate at pore scale suggests weaker tensile properties than thought[END_REF][START_REF] Qanbari | Storage of CO2 as hydrate beneath the ocean floor[END_REF], flow assurance [START_REF] Baek | Inhibition effects of activated carbon particles on gas hydrate formation at oil-water interfaces[END_REF][START_REF] Liu | Research on flow assurance of deepwater submarine natural gas pipelines: Hydrate prediction and prevention[END_REF][START_REF] Olajire | Flow assurance issues in deep-water gas well testing and mitigation strategies with respect to gas hydrates deposition in flowlines-A review[END_REF] and hydrate-based processes [START_REF] Babu | A Review of Clathrate Hydrate Based Desalination To Strengthen Energy-Water Nexus[END_REF][START_REF] Zheng | Progress and trends in hydrate based desalination (HBD) technology: A review[END_REF][START_REF] Cha | Increasing Gas Hydrate Formation Temperature for Desalination of High Salinity Produced Water with Secondary Guests[END_REF][START_REF] Fakharian | Desalination of high salinity produced water using natural gas hydrate[END_REF][START_REF] Ho-Van | Cyclopentane hydrates -A candidate for desalination?[END_REF][14][START_REF] Torré | CO2 capture by hydrate formation in quiescent conditions: In search of efficient kinetic additives[END_REF]. A significant number of water-guest systems and experimental set-up designs have been studied, mainly to describe hydrate formation mechanisms [START_REF] Nguyen | Critical Review on Gas Hydrate Formation at Solid Surfaces and in Confined Spaces-Why and How Does Interfacial Regime Matter?[END_REF][START_REF] Linga | A Review of Reactor Designs and Materials Employed for Increasing the Rate of Gas Hydrate Formation[END_REF] or hydrate crystal morphology [START_REF] Babu | Morphology of Methane Hydrate Formation in Porous Media[END_REF][START_REF] Delroisse | Effect of a Hydrophilic Cationic Surfactant on Cyclopentane Hydrate Crystal Growth at the Water/Cyclopentane Interface[END_REF][START_REF] Linga | Gas Hydrate Formation in a Variable Volume Bed of Silica Sand Particles[END_REF], to promote [START_REF] Li | Promotion effect of graphite on cyclopentane hydrate based desalination[END_REF][START_REF] Mallek | Effect of porous activated carbon particles soaked in cyclopentane on CP-hydrate formation in synthetic produced water[END_REF] or inhibit [START_REF] Baek | Inhibition effects of activated carbon particles on gas hydrate formation at oil-water interfaces[END_REF][START_REF] Zylyftari | Salt effects on thermodynamic and rheological properties of hydrate forming emulsions[END_REF] hydrate formation, and to reduce the energy required for hydrate crystallization [START_REF] Babu | Hydratebased desalination (HyDesal) process employing a novel prototype design[END_REF][START_REF] He | A novel conceptual design of hydrate based desalination (HyDesal) process by utilizing LNG cold energy[END_REF][START_REF] Chong | Economic evaluation of energy efficient hydrate based desalination utilizing cold energy from liquefied natural gas (LNG)[END_REF][START_REF] Montazeri | Energy saving in carbon dioxide hydrate formation process using Boehmite nanoparticles[END_REF][START_REF] Lee | Conceptual design and economic analysis of a novel cogeneration desalination process using LNG based on clathrate hydrate[END_REF]. The optimization of energy consumption for hydrate crystallization, which is an exothermic reaction, has essentially been studied in the context of an economically viable hydrate-based desalination process. In the studies published, the authors generally tried to make the most of the endothermic energy produced by a reaction which is exogenous to the system. For instance, [START_REF] He | A novel conceptual design of hydrate based desalination (HyDesal) process by utilizing LNG cold energy[END_REF] and [START_REF] Babu | Hydratebased desalination (HyDesal) process employing a novel prototype design[END_REF] attempted to make use of LNG regasification to cool the water to be desalinated [START_REF] Babu | Hydratebased desalination (HyDesal) process employing a novel prototype design[END_REF][START_REF] He | A novel conceptual design of hydrate based desalination (HyDesal) process by utilizing LNG cold energy[END_REF]. He et al. (2018) achieved a decrease in specific energy consumption of 27.42% and 24.61% for 3.5wt% NaCl and 7 wt% NaCl brines respectively [START_REF] He | A novel conceptual design of hydrate based desalination (HyDesal) process by utilizing LNG cold energy[END_REF].

The hydrate formation stage of a recently patented HBD process [START_REF]Method for crystallising clathrate hydrates, and method for purifying an aqueous liquid using the clathrate hydrates thus crystallised[END_REF], which has the particularity to use porous activated carbon particles (PACP) loaded with CP, was studied in a previous paper [START_REF] Mallek | Effect of porous activated carbon particles soaked in cyclopentane on CP-hydrate formation in synthetic produced water[END_REF]. The main results showed that the PACP act as both kinetic promoters and a source of CP for hydrate formation. However, a relatively small amount of hydrates was obtained (< 50% of the theoretical maximum amount of hydrates that could be formed) [START_REF] Mallek | Effect of porous activated carbon particles soaked in cyclopentane on CP-hydrate formation in synthetic produced water[END_REF].

This was proven to be related to the fact that the CP found in the small pores of the PACP remains trapped [START_REF] Mallek | Experimental characterization of cyclopentane adsorption/desorption in microporous activated carbons[END_REF][START_REF] Mallek | Investigation on Cyclopentane-hydrate formation from CP-loaded porous activated carbon particles[END_REF]. Based on this observation, PACP with predominantly large pores were recommended for enhancing the process. In the present work, we focus on the energy aspect of the hydrate formation stage in this HBD process, in which the energy supplied by hydrate crystallization -exothermic reaction (-115.4 kJ/mol of CP [START_REF] Delroisse | Determination of thermophysical properties of cyclopentane hydrate using a stirred calorimetric cell[END_REF]) -could possibly be partially compensated by a reaction endogenous to the system, namely the desorption of CP from the PACP, which is endothermic (+28 to +57 kJ/mole of CP [START_REF] Mallek | Experimental characterization of cyclopentane adsorption/desorption in microporous activated carbons[END_REF]).

The main objective of the present work is to experimentally prove this partial thermal energy compensation and determine its value. To do so, CP hydrates were crystallized from CPsaturated PACP immersed in brines of 3.5 wt% NaCl and 8 wt% NaCl, at a subcooling (difference between the hydrate equilibrium temperature and the experiment temperature) of 6°C. These salt concentrations mimic those of seawater [START_REF] Kang | Seawater desalination by gas hydrate process and removal characteristics of dissolved ions (Na+, K+, Mg2+, Ca2+, B3+, Cl-, SO42-)[END_REF] and produced water [START_REF] Cha | Increasing Gas Hydrate Formation Temperature for Desalination of High Salinity Produced Water with Secondary Guests[END_REF] respectively. The experiments were carried out using both differential scanning calorimetry (DSC) and refractive index measurements.

Experimental section

Materials and Apparatus

CP (purity of 97%) was supplied by Sigma Aldrich. Deionized water (resistivity of 18.2 MΩ.cm) was produced by a Purelab laboratory water-purification system. Sodium chloride (NaCl) (purity of 99.5%) was purchased from Acros Organics. A grade of PACP called BGX, supplied by Chemviron SA (Belgium), was used for the hydrate formation experiments. BGX is granular activated carbon with an average particle size of about 1 mm and a pore size distribution that has the specificity of being developed in both the meso-and macroporous regions in addition to the more standard microporous range (see SM-1 and SM-2). Their CPadsorption capacity is 0.96 ± 0.02 g of CP/g of BGX at 1°C (see SM-3). A closed vessel and a set of resealable glass flasks with a 5-ml capacity were used for the CP-saturated PACP preparation. Plastic syringes (3-mL capacity) and cellulose acetate syringe filters (0.8-µm mesh size), used to sample and filter the unconverted water at the end of the hydrate formation experiments, were purchased from Sodipro.

A vacuum preparation apparatus called VacPrep™ (from Micromeritics) was used to purify the PACP samples under primary vacuum at 200°C. The samples were weighed using an ALC-110.4 balance (from Accurlab ®) with an uncertainty of 10 -4 g. A universal convection incubator (from Memmert®) was used to impose the chosen temperature on the closed vessel, in which the PACP are placed to be saturated with CP (cf. paragraph 2.2.). Hydrate formation was performed in the measurement cell (7-cm 3 capacity) of a Tian-Calvet BT2.15 calorimeter (from Setaram Instrumentation). The uncertainty with regard to the measured energy is 4% (see SM-4).

The refractive index nD (at a wavelength of 589.3 nm) of the water samples was measured using an Abbemat 550 refractometer (from Anton Paar) with an accuracy of ± 0.00001.

Preparation of the CP-saturated PACP

The saturation of PACP with CP was performed as follows: 0.54 g of BGX were purified at 200°C under primary vacuum for 12 hrs. Using a glass funnel, 0.500 ± 0.005 g of this hot sample was immediately transferred to a resealable glass flask, which was then introduced into the closed vessel partially filled with liquid CP (Fig. 1). This in turn was placed in the incubator at 1.0 ± 0.5°C. The PACP were left in contact with saturating CP vapor for 48 hrs to saturate their pores with CP. For this experiment, 0.500 ± 0.005 g of CP-saturated BGX and 4.15 g (or 4.35 g) of 3.5 wt% NaCl (or 8 wt% NaCl) solution were introduced into the measurement cell of the BT2.15 calorimeter along with a few CP-hydrate crystals prepared beforehand (see Fig. 2). These hydrate crystals were added to initiate hydrate crystallization by using the so called "memory effect". Memory effect is a phenomenon resulting from the non-complete dissociation of hydrate structure which reduces the time required to initiate hydrate formation [START_REF] Sloan | Clathrate Hydrates of Natural Gases[END_REF]. The PACP, NaCl solution and cell were all precooled to the equilibrium temperature Teq (5.5°C and 2.8°C for the 3.5 wt% NaCl and 8 wt% NaCl solutions respectively [START_REF] Mallek | Effect of porous activated carbon particles soaked in cyclopentane on CP-hydrate formation in synthetic produced water[END_REF]). The cell was then introduced into the calorimeter, which was set at a temperature 0.2°C above Teq in order to prevent (or at least limit) the added hydrate crystals from melting. The system was maintained at this temperature for one hour (stage (a) in Fig. 3) so that the thermal signal (heat flow) caused by the introduction of the cell into the calorimeter should reach the baseline and the added hydrate crystals should melt slowly. Then, the system temperature was decreased to the target temperature (Teq -6°C), at a cooling rate of 1°C/min, and maintained for 24 hrs (stage (b)). After 24 hrs of hydrate formation, the cell was removed from the calorimeter, a sample of the residual (unconverted) NaCl solution was collected and filtered, and its refractive index was measured (stage (c)).

𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 , deduced from the direct measurements, was obtained by integrating the thermal peak generated by subtracting the "blank" thermogram (obtained in a similar fashion but without CP) from the "experiment" one (see Fig. 4). The "blank" thermogram (brown curve in Fig. 4) only takes into account the heat flow deviations due to the temperature programming and mass difference between the "measurement" and "reference" cells. Thus, the resulting thermogram "Experiment minus Blank" (blue curve in Fig. 4) only shows the heat flows due to both the CP desorption from the PACP and the hydrate crystallization, and its integration gives 𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 .

𝐸 𝑐𝑟𝑦𝑠 , deduced from the indirect measurements, was obtained by first determining the salinity [NaCl]24h of the unconverted solution -concentrated with salt as hydrates formed -from the measured refractive index and a previously generated "nD-[NaCl]" calibration curve (see SM-5). Based on this, the water-to-hydrate conversion (WHC) and the water-to-hydrate amount (WHA) were determined using equations 1 and 2 respectively. The stoichiometric ratio of CP to water in the CP hydrates (1 mole of CP:17 moles of water) served to determine the amount of CP-to-hydrates obtained (CPHA) (equation 3). The hydrate formation energy was deduced from the CP-hydrate dissociation enthalpy (115,400 ± 7,600 J/mole of CP (Delroisse et al., 2018)) using equation 4.

𝑊𝐻𝐶(%) = 100 - 100 * [𝑁𝑎𝐶𝑙] 𝑖 * (100-[𝑁𝑎𝐶𝑙] 24ℎ ) (100-[𝑁𝑎𝐶𝑙] 𝑖 ) * [𝑁𝑎𝐶𝑙] 24ℎ (1)
𝑊𝐻𝐴 (𝑔) = 𝑚 𝑖𝑤 * 𝑊𝐻𝐶 (%) (2)
where

The uncertainties as regards 𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 were determined as the sum of experiment repetition and calibration-control deviations, whereas the uncertainties as regards 𝐸 𝑐𝑟𝑦𝑠 were calculated from the experiment-repetition deviation, the calibration curve deviation, the refractometer accuracy, and the uncertainty regarding the dissociation enthalpy.

Results and discussion

Fig. 4 shows the resulting thermal peak (green curve) obtained after subtracting the "blank" thermogram (brown curve) from the "experiment" one (blue curve) for both NaCl solutions.

The superposition of the thermal peaks obtained in experiments 1 and 2 shows a good reproducibility for each salinity (Fig. 5). The average values of 𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 obtained by integrating the thermal peaks are also given in Fig. 5. As can be seen in Table 2, 𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 is lower than 𝐸 𝑐𝑟𝑦𝑠 . The difference in energy (𝐸 𝑐𝑟𝑦𝑠 -𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 ) is in fact the balanced energy. Since the PACP are initially saturated with CP, meaning that the CP is found only within the pores, the balanced energy can thus be fully attributed to CP desorption. While assuming that all the CP desorbed from the PACP is converted to hydrates (i.e. the desorbed-CP amount is equal to CPHA), the balanced energy value, calculated as the ratio of (𝐸 𝑐𝑟𝑦𝑠 -𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 ) to CPHA (in mol), is between 9 (=28- [START_REF] Delroisse | Effect of a Hydrophilic Cationic Surfactant on Cyclopentane Hydrate Crystal Growth at the Water/Cyclopentane Interface[END_REF] kJ/mol of CP and 47 (=28+19) kJ/mol of CP. The balanced energy can be compared to the differential enthalpy of CP desorption (the opposite of the differential enthalpy of CP adsorption on PACP), which ranges from 28 to 57 kJ/mol of CP. The differential enthalpy of CP desorption was measured in a previous paper for two different activated carbons (see SM-6) [START_REF] Mallek | Experimental characterization of cyclopentane adsorption/desorption in microporous activated carbons[END_REF] using the calorimetric-manometric technique [START_REF] Mouahid | Mise au point point d'un dispositif couple manométrique calorimétrique pour l'étude de l'adsorption de fluides supercritiques dans des milieux microporeux et[END_REF]. The similarity of the curves indicates a general range of values for CP adsorbed on an activated carbon. For the sake of comparison, the balanced energy range and the differential enthalpy of CP desorption from PACP are plotted in the same figure (Fig. 6). As can be seen in Figure 6, the former (green area) does not cover the entire range of the latter (dots), which confirms that part of the CP did not desorb from the PACP (and therefore did not participate in the formation of hydrates). The high enthalpy values (48 to 57 kJ/mol of CP)

of the uncovered dots correspond to the filling of pores at the lowest pressures (0 < Peq/Psat <~ 0.02) [START_REF] Mallek | Experimental characterization of cyclopentane adsorption/desorption in microporous activated carbons[END_REF] i.e. the smallest pores, mainly micropores, where the prevailing interactions are fluid-solid. The non-desorbed CP was therefore trapped in these pores. The enthalpy values of the covered dots (28 to 48 kJ/mol of CP) correspond to medium-to-high-pressure filling of the meso-and macropores (~ 0.02 < Peq/Psat < 1) [START_REF] Mallek | Experimental characterization of cyclopentane adsorption/desorption in microporous activated carbons[END_REF]. Hence, CP that desorbed from the PACP to form hydrates was contained in these pore ranges where the prevailing interactions are solidfluid-fluid and fluid-fluid interactions. One can see that the balanced energy range obtained was almost the same for both salinities (3.5 wt% NaCl and 8 wt% NaCl), suggesting no significant effect of salt on this phenomenon. This result makes sense since the balanced energy was found to be related to the CP-desorption energy. The balanced energy (+9 to +47 kJ/mol of CP) implied a thermal energy compensation of between 8% and 40% of the energy supplied by hydrate crystallization. The (positive) balanced energy (+9 to +47 kJ/mol of CP) implied a thermal energy compensation of the (negative) energy supplied by the hydrate crystallization (-115.4 kJ/mol of CP). Its value, calculated as the ratio of the balanced energy to the dissociation enthalpy of CP hydrates, is between 8% (=100×9:115.4) and 40% (=100×47:115.4).

The investigation into the thermal energy compensation together with the assumption that (some) water molecules can enter the pores of PACP [START_REF] Nguyen | Critical Review on Gas Hydrate Formation at Solid Surfaces and in Confined Spaces-Why and How Does Interfacial Regime Matter?[END_REF][START_REF] Mallek | Investigation on Cyclopentane-hydrate formation from CP-loaded porous activated carbon particles[END_REF] lead us to propose a mechanism for CP-hydrate formation from CP-saturated PACP (Fig. 7). In the smallest pores, the fluidsolid interactions prevent CP from desorbing and thus from participating in hydrate formation.

In larger pores, the prevailing interactions (solid-fluid-fluid and fluid-fluid interactions) allow the desorption of CP (or at least part of it). In this case, hydrate formation can occur outside and/or inside the pore.

It should be noted that the surface chemistry of the PACP is reported to impact (promote or inhibit) hydrate crystallization [START_REF] Baek | Inhibition effects of activated carbon particles on gas hydrate formation at oil-water interfaces[END_REF][START_REF] Nguyen | Critical Review on Gas Hydrate Formation at Solid Surfaces and in Confined Spaces-Why and How Does Interfacial Regime Matter?[END_REF]35]. However, in the case of our investigated system, the low-size pores of PACP, where the specific surface is significant and thus also the surface chemistry effects could be, do not participate to hydrate formation as previously explained.

Therefore, the role of surface chemistry is probably not critical compared to that of pore size.

117Fig. 1 .

 1 Fig. 1. Preparation of CP-saturated PACP

  [𝑁𝑎𝐶𝑙] 𝑖 ,[𝑁𝑎𝐶𝑙] 24ℎ 𝑎𝑛𝑑 𝑚 𝑖𝑤 are the initial water salinity (3.5 wt% NaCl or 8 wt% NaCl), the final water salinity (after 24 hrs.) and the initial pure-water mass respectively.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Preparing the system for the hydrate formation experiment in the calorimetric cell of

Fig. 4 .Fig. 5 .

 45 Fig. 4. Thermal energy peak (green curve) generated by subtracting the "blank" thermogram

Fig. 6 .

 6 Fig. 6. Comparison of the balanced energy obtained in the case of hydrate formation from CP-

Fig. 7 .Fig. S1 . 2 Fig. S2 .

 7S12S2 Fig. 7. Proposed prevailing mechanisms of hydrate formation from CP-saturated PACP

  The thermal energy values 𝐸 𝑐𝑟𝑦𝑠 deduced from the indirect measurements for the same systems are given in Table1. The refractive indexes (nD) measured after 24 hrs of hydrate formation in experiments 1 and 2 are in good agreement which indicates a good reproducibility (difference of less than 0.002%).

	Table 1. Indirect measurement of hydrate formation energy
			3.5 wt% NaCl	8 wt% NaCl
	nD	Exp. 1	1.340402	1.348213
	at t = 24 h	Exp. 2	1.340376	1.348220
		Average	1.340390 ± 0.00001	1.34822 ± 0.00001
	[NaCl]24h (wt%)	4.23 ± 0.01	8.71 ± 0.01
	WHC (%)		18.0 ± 0.4	8.70 ± 0.25
	WHA (g)		0.72 ± 0.02	0.35 ± 0.01
	CPHA (g)		0.160 ± 0.004	0.080 ± 0.002
	𝐸 𝑐𝑟𝑦𝑠 (J)		271 ± 24	131 ± 12

Table 2 .

 2 Determination of the balanced energy per mole of CP

		3.5 wt% NaCl	8 wt% NaCl
	𝐸 𝑑𝑒𝑠+𝑐𝑟𝑦𝑠 (J)	204 ± 16	100 ± 7
	𝐸 𝑐𝑟𝑦𝑠 (J)	271 ± 24	131 ± 12
	CPHA (g)	0.16	0.08
		(2.3 10 -3 ) *	(1.1 10 -3 ) *
	Balanced energy (J)	67 ± 40	31 ± 19
	Balanced energy (kJ/mol CP) 29 ± 17	28 ± 19

* value of CPHA in mol

Acknowledgments

This research was financially supported by Total. The authors would like to thank Manuel Ildefonso (UPPA), Pascal Le Mélinaire (BGH), and Patrick Baldoni-Andrey, Hervé Nabet, and Stéphane Nowe (Total) for their valuable scientific inputs and discussions. C. Miqueu acknowledges the E2S UPPA hub Newpores supported by the "Investissements d'Avenir" French programme managed by ANR (ANR-16-IDEX-0002).

SM-3

Determination of the CP-adsorption capacity of BGX

To perform CP-adsorption capacity measurements, the PACP were purified at 200°C for 12 hrs under primary vacuum. Using a glass funnel, the hot purified sample was directly transferred into a small closable flask (5-mL capacity), which was then weighed and introduced into the CP adsorption chamber (closed-vessel partially filled with a flask of liquid CP). The PACP were only in contact with CP vapor (cf. Figure S1). The CP adsorption chamber was placed in the incubator at 1.0 ± 0.5°C. After 48 hrs of adsorption, the flask was weighed again and the CP-adsorption capacity of the PACP was determined. The experiment was repeated 3 times to check the results.

SM-4

SM-6

Gbac and ChemA are the notation used for G-BAC-G70R and Chemviron-A, which are two different PACP manufactured by Keruha Gmbh and Calgon, respectively. The differential enthalpies of CP-adsorption on these PACP were measured in a previous work [START_REF] Mallek | Experimental characterization of cyclopentane adsorption/desorption in microporous activated carbons[END_REF], and they