Mélanie A Cron

Solène Maillard

Fabien Delisle

Nolwenn Samson

Frédérique Truffault

Maria Foti

Elie Fadel

Julien Guihaire

Sonia Berrih-Aknin

Rozen Le Panse
email: rozen.lepanse@upmc.fr

Analysis of microRNA expression in the thymus of Myasthenia Gravis patients opens new research avenues

Keywords: AChR: acetylcholine receptor DGCR8: DiGeorge syndrome Critical Region gene 8 miRnome, miR-7, miR-125, CCL21, FMR1, WDR1

In early-onset Myasthenia Gravis (MG) with anti-acetylcholine receptor antibodies, thymic abnormalities associated with ectopic germinal centers are frequent. miRNAs by acting as post-transcriptional regulators are involved in autoimmunity. To investigate the implication of miRNAs in thymic changes associated with early-onset MG, we performed a miRnome study and data were analyzed with different approaches. miRNAs of interest were further investigated by RT-PCR and transfection experiments for functional tests.

First, analyzing specific dysregulated miRNAs, we focused our attention on miR-7-5p and miR-125a-5p, and confirmed by RT-PCR their respective down-and up-regulation in MG thymuses. miR-7 was the most down-regulated thymic miRNA in MG and we observed an inverse correlation between its expression and CCL21 mRNA expression. We next showed that miR-7 down-regulation was due to thymic epithelial cells and by transfecting these cells with miR-7, we demonstrated that it controlled CCL21 release. As CCL21 is essential for germinal center development, we suggested that miR-7 could be involved in thymic changes associated with MG. miR-125a was up-regulated in MG thymuses and is of great interest as it is known to regulate FoxP3 expression, and to modulate the different inflammatory signaling pathways.

Thanks to this thymic miRnome study, we also showed the specific dysregulation of miRNA clusters. In particular, we observed that miRNAs localized at the extremity of the X chromosome were down-regulated. This effect seemed linked to their close localization to the fragile X mental retardation 1 gene (FMR1) and the DNA methylation status.

Altogether, this miRnome analysis demonstrated that specific thymic miRNAs can be associated with MG and provides novel insights into the pathogenesis of MG. HIGHLIGHTS  Dysregulated thymic miRNAs are associated with early-onset MG  miR-7-5p, the most down-regulated miRNA, decreases CCL21 expression  Down-expression of miRNA clusters on the X chromosome  Increased expression of miR-125, a key regulator of inflammation signaling pathways

INTRODUCTION

Acquired Myasthenia Gravis (MG) is a rare neuromuscular disease caused by autoantibodies against components of the neuromuscular junction. Over 85% of patients develop autoantibodies against the acetylcholine receptor (AChR). MG patients suffer from invalidating fluctuating skeletal muscle weaknesses. MG is a prototype autoimmune disease and if the target organ is the muscle, the effector organ is the thymus [START_REF] Berrih-Aknin | Myasthenia gravis: A comprehensive review of immune dysregulation and etiological mechanisms[END_REF] [START_REF] Marx | The different roles of the thymus in the pathogenesis of the various myasthenia gravis subtypes[END_REF]. AChR-MG patients with an early-onset form of the disease (mostly females under 45-50 years old) show thymic abnormalities as the thymus displays the characteristics of tertiary lymphoid organs: chronic inflammation, neoangiogenic processes with high endothelial venule and lymphatic vessel development, chemokine overexpression, such as CXCL13 and CCL21, favoring peripheral-cell recruitment and leading to ectopic germinal center development [START_REF] Méraouna | The chemokine CXCL13 is a key molecule in autoimmune Myasthenia Gravis[END_REF] [START_REF] Berrih-Aknin | CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia[END_REF]. These changes seem mainly orchestrated by the abnormal expression of interferon (IFN)-β that is overexpressed in the MG thymus [START_REF] Cufi | Central role of interferonbeta in thymic events leading to myasthenia gravis[END_REF] and this could be related to a pathogen infection [START_REF] Cavalcante | Etiology of myasthenia gravis: innate immunity signature in pathological thymus[END_REF].

Actually, the MG thymus contains all the components for the anti-AChR response: AChR expression by thymic epithelial cells (TECs) and myoid cells, presence of B cells producing anti-AChR antibodies and of anti-AChR autoreactive T cells [START_REF] Berrih-Aknin | Myasthenia gravis: A comprehensive review of immune dysregulation and etiological mechanisms[END_REF].

MicroRNAs (miRNAs), by acting as post-transcriptional regulators, have a role in physiological and pathophysiological processes, including autoimmune diseases [START_REF] Chen | The role of microRNAs in the pathogenesis of autoimmune diseases[END_REF]. They are transcribed as primary miRNAs, cleaved into pre-miRNAs by DROSHA and its partner DGCR8 (DiGeorge syndrome Critical Region gene 8) in the nucleus and transported to the cytoplasm by the duo DICER/TRBP1. Mature miRNAs coupled with the RNA-induced silencing complex (RISC, composed among other proteins of DICER, TRBP1, AGO2…) are guided to mRNA targets, leading to their degradation or to the inhibition of their translation, according to the perfect or imperfect miRNA-mRNA matching, respectively [START_REF] Winter | Many roads to maturity: microRNA biogenesis pathways and their regulation[END_REF]. As a given miRNA can have several targets and a mRNA can be targeted by several miRNAs at the same time, regulation by miRNAs is very subtle and complex to apprehend [START_REF] Ebert | Roles for microRNAs in conferring robustness to biological processes[END_REF]. The level of cytoplasmic miRNAs available to regulate mRNA expression is dependent on different regulation processes, as detailed by Gulyaeva and Kushlinskiy [START_REF] Gulyaeva | Regulatory mechanisms of microRNA expression[END_REF].

A few studies have investigated the global role of miRNAs in normal thymuses. In conditional knockout (KO) mice for Dicer or Dgcr8 in TECs, a profound disruption of the thymic architecture and an alteration in TEC cellularity are observed. An important decrease in the number of thymocytes is also observed [START_REF] Papadopoulou | The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-alpha receptor[END_REF] [START_REF] Khan | Canonical microRNAs in thymic epithelial cells promote central tolerance[END_REF]. The lack of mature miRNAs in TECs can affect thymopoïesis [START_REF] Zuklys | MicroRNAs control the maintenance of thymic epithelia and their competence for T lineage commitment and thymocyte selection[END_REF], the expression of the autoimmune regulator (AIRE) and of tissue-specific antigens [START_REF] Ucar | An evolutionarily conserved mutual interdependence between Aire and microRNAs in promiscuous gene expression[END_REF]. Surprisingly, in conditional Dicer-TEC KO mice, epithelial voids appear due to epithelial apoptosis and an unusual presence of CD19 + B cells is observed [START_REF] Papadopoulou | The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-alpha receptor[END_REF]. Specific miRNAs can also play key roles in thymus homeostasis and thymopoïesis and their regulation must be tightly controlled. Environmental factors, such as infections, are able to modify the expression of miRNAs and eventually alter the thymic function [START_REF] Belkaya | Dynamic modulation of thymic microRNAs in response to stress[END_REF] [START_REF] Linhares-Lacerda | Differential Expression of microRNAs in Thymic Epithelial Cells from Trypanosoma cruzi Acutely Infected Mice: Putative Role in Thymic Atrophy[END_REF]. For example, in mice miR-29a is an important regulator of the type I IFN (IFN-I) signaling pathway by targeting a subunit of its receptor (IFNAR1) in TECs and by reducing cell-sensitivity to IFN-I and consequently to pathogen infections [START_REF] Papadopoulou | The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-alpha receptor[END_REF].

We then hypothesized that dysregulated miRNAs could play a role in thymic changes leading to MG. By investigating for the first time the global expression of miRNAs and clusters of miRNAs in the thymus of early-onset MG patients, we bring new knowledge in posttranscriptional mechanisms associated with thymic changes in MG and opens new research avenues on myasthenia gravis.

MATERIALS AND METHODS

Patient selection and thymic samples

Thymic biopsies were collected from early-onset MG patients undergoing thymectomy and healthy donors undergoing cardiovascular surgery at the Marie Lannelongue Surgical Center (Le Plessis-Robinson, France). Briefly, MG and control thymuses from female donors were used for the miRnome study and RT-PCR (controls (n=6, 15-33 years old); untreated MG (n=12) and corticoid-treated MG (n=8) patients were detailed in table 1). MG and control thymuses from female and male donors were used for thymocytes extraction (controls (n=5, from 9 days to 27 years old); MG (n=12-Table 1)) and for TEC cultures (controls (n=10, 10-35 years old); MG (n=13-Table 1)). MG patients were only treated with cholinesterase inhibitors except for a group of corticosteroid-treated patients for Fig. 4B (Table 1). Patients had no other known diseases including thymoma. All the studies on thymuses were approved by a local ethics committee (CPP, authorization number ID RCB 2010-A00250-39) and informed consent forms have been collected.

RNA preparation

Total RNA from thymic biopsies was extracted using the mirVana miRNA Isolation Kit, according to the manufacturer's instructions (Life Technologies, Villebon-sur-Yvette, France). Thymic fragments were lysed directly in the Lysis/Binding Solution provided in the kit using the FastPrep FP120 instrument (Qbiogen, Illkirch, France). RNA quality was assessed (Bioanalyzer 2100, Agilent Technologies, Les Ulis, France) and samples with a RNA Integrity Number (RIN) over 7 were used in our experiments.

miRNA profiling

Global miRNA expression analyses were performed using the Affymetrix GeneChip miRNA 3.0 Array (Santa Clara, USA). Chips contained 3391 human miRNAs (1658 pre-miRNAs and 1733 mature miRNAs) and 2216 human snoRNAs, CDBox RNAs, H/ACA Box RNAs and scaRNAs.

Briefly, 1 µg of total RNA was labeled using the flashTag Biotin HSR RNA Labeling Kit (Affymetrix), followed by the hybridization overnight according to the manufacturer's instructions. Afterwards, miRNA chips were then scanned with the Affymetrix GeneChip Scanner 3000.

miRNA data analyses

The quality control of the scanned data was first estimated by confirming the order of the signal intensities of the Poly-A and hybridization controls using the Affymetrix GeneChip Expression Console. Then raw expression values were generated as ".CEL files" and ".CHP files" and were analyzed into the Transcriptome Analysis Console Software (TAC). The average for the intensity of fluorescence for each small RNA is calculated with the Tukey's Biweight method that calculates a robust average unaffected by outliers. The Tukey's Biweight average values are given in tables 2A-B in a log2 scale. TAC computes and summarizes a traditional unpaired One-Way Analysis of Variance (ANOVA) for each pair of condition groups. The fold changes (FCs) are given in linear values for MG versus control data. We selected small human RNAs that were dysregulated with a FC over 1.5 or -1.5 and p-value ≤ 0.05. Other miRNAs of interest could have also been extracted if we had lowered the fold change threshold and these miRNAs can be found in the supplemental table S1.

To search for miRNA clusters susceptible to be dysregulated in MG patients versus controls, we compared for each chromosome the number of dysregulated miRNAs with a χ2 test in table 2A to the number of mature human miRNAs spotted on the Affymetrix GeneChip miRNA 3.0 Array.

Combined miRNA and mRNA data analyses

To sort out dysregulated miRNAs that could modulate gene expression in the thymus of MG patients, we have used the web resource MAGIA2 (gencomp.bio.unipd.it/magia2/) [START_REF] Bisognin | MAGIA(2): from miRNA and genes expression data integrative analysis to microRNA-transcription factor mixed regulatory circuits (2012 update)[END_REF]. This web tool allows the analyses of miRNA and mRNA expression data for the detection of inversely correlated miRNA and mRNA pairs. The integration of target predictions with miRNA and gene expression profiles improves the detection of functional miRNA-mRNA relationships.

We have used transcriptomic data obtained on thymus samples from MG patients and controls. This study has been published in 2006 [START_REF] Panse | Microarrays reveal distinct gene signatures in the thymus of seropositive and seronegative myasthenia gravis patients and the role of CC chemokine ligand 21 in thymic hyperplasia[END_REF] and data are available on the ArrayExpress database -accession no. E-MEXP-518 (www.ebi.ac.uk/arrayexpress).

For the mRNA matrix, we uploaded dysregulated gene transcripts with a FC of 1.8 and a pvalue under 0.05 (97 up-and 154 down-regulated mRNAs) [START_REF] Panse | Microarrays reveal distinct gene signatures in the thymus of seropositive and seronegative myasthenia gravis patients and the role of CC chemokine ligand 21 in thymic hyperplasia[END_REF]. For the miRNA matrix, as fold changes were less discriminative, all mature miRNA (1733) expression data were uploaded.

For non-matched miRNA and mRNA expression data, a meta-analysis based on the inverse χ2 distribution identified oppositely variable miRNA-target gene pairs. Two target prediction databases that are regularly updated, were used with a medium stringency: 1) Targetscan predicts miRNA targets by searching for conserved and non-conserved 8-to 6-mer sites in the 3'UTR of protein-coding transcripts [START_REF] Agarwal | Predicting effective microRNA target sites in mammalian mRNAs[END_REF], 2) DIANA-microT identifies miRNA targets in the 3'UTR but also in the coding sequence of protein-coding transcripts [START_REF] Paraskevopoulou | DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows[END_REF].

Retro-Transcription and Polymerase Chain Reaction (RT-PCR)

RT-PCR on mRNA were performed as previously described [START_REF] Cufi | Central role of interferonbeta in thymic events leading to myasthenia gravis[END_REF]. miRNAs were retro-transcribed using TaqMan® MicroRNA RT Kit (Life Technologies) according to the manufacturer's instructions. PCR reactions were carried out on a Light Cycler 480 (Roche, Meylan, France) using the TaqMan® Universal Master Mix II, no UNG (Life Technologies). The PCR cycle conditions were as followed: a cycle of denaturation of 10 minutes at 95°C, 50 cycles of amplification of 15 seconds at 95°C and 1 minute at 60°. miRNA expression was normalized to 28S rRNA or GAPDH mRNA expression. Primer sequences and references are listed in tables S2A-B.

Primary TEC cultures for transfection or methylation experiments

Primary thymic epithelial cells (TECs) were cultured from thymic explants and correspond mainly to medullary TECs as previously described [START_REF] Cufi | Implication of double-stranded RNA signaling in the etiology of autoimmune myasthenia gravis[END_REF]. TECs were transfected with Lipofectamine RNAiMax (Life Technologies). Briefly, 40pmol of miR-7-5p or scramble RNA (Eurogentec, Seraing, Belgium) were diluted in 50µl of X-vivo15 medium (Lonza, Switzerland).

In parallel, 4µl of Lipofectamine RNAiMax were diluted in 50µl of X-vivo15 medium. Then, both solutions were mixed and incubated for 20 minutes at room temperature. The resulting mixes were added to cultured cells (4 x10 5 cells per well) at a ratio of 1:4 of X-vivo15 medium in 12well plates for 48h.

For methylation experiments, TECs were seeded in RPMI containing 10% of fetal calf serum (5 x10 5 cells per well) and treated after 24 hours with 5µM of 5-Azacytidine for 48 hours. TECs were then lysed for total RNA extraction.

CCL21 ELISA

CCL21 antibody (AF366, R&D Systems, Lille, France) was diluted at 1 µg/ml in coating buffer (50 mM NaHCO3 and 50 mM Na2CO3, pH 9.4) and incubated overnight at 4°C. TEC culture medium (1.5mg/mL of proteins) were incubated for 3 hours, and subsequently, 0.25 µg/ml of biotinylated anti-CCL21 (BAF366, R&D Systems), and streptavidin-horseradish peroxidase (Life Technologies) were added. Tetramethylbenzidine (BioLegend, United Kingdom) was used for color development and plates were read at 450 nm on a SPARK 10M microplate reader (TECAN Life Sciences, Grödig, Austria).

Statistical analyses

In dot graphs, 2-by-2 comparisons were done using the non-parametric Mann-Whitney or Wilcoxon tests as indicated in the figure legends. For correlation analyses, significance was assessed using a Pearson's correlation test.

RESULTS

Analysis of differentially expressed miRNAs in the thymus of MG patients

Thymic miRNA expression profiles were compared between MG patients and age-matched female controls. Table S1 lists all miRNAs analyzed on the Affymetrix miRNA arrays. Analyzing all human miRNAs that were dysregulated in MG thymuses (FC ≥1.5 and p≤0.05), we found 74 miRNAs (Tables 2A-B and Fig. 1A): 61 mature miRNAs (24-up and 37 down-regulated) and 13 pre-miRNAs (10 up-and 3 down-regulated) (Table 2B). 5 of the pre-miRNAs (3 up-and 2 downregulated) were also found dysregulated as mature miRNAs: miR-548w, miR-4538, miR-4539 and miR-30e, miR-363 (Tables 2A-B).

To confirm the expression profile of the most down-regulated miRNAs (miR-429-3p, miR-7-5p and miR-1-5p) or the most up-regulated miRNAs (miR-138-1-3p, miR-486-5p and miR-4455-5p) (Fig. 1B-G), we analyzed by RT-PCR their level of expression. Although the decrease or the increase of these miRNAs were consistently confirmed, the differences between MG and controls were only significant in RT-PCR for miR-486-5p and miR-7-5p (Fig. 1H-M).

Analysis of miR-7 expression in the MG thymus

From the miRnome analysis, miR-7-5p was one of the most down-regulated miRNAs in the thymus of MG patients and we confirmed by RT-PCR its down-regulation in MG thymuses compared to controls (Fig. 1I). We then demonstrated that miR-7-5p was even more downregulated in the thymus of patients with a high degree of thymic hyperplasia (Fig. 2A). In corticoid-treated patients, miR-7-5p was still down-regulated compared to non-MG donors but a slight increase was observed when comparing corticoid-treated patients with MG patients with a high degree of hyperplasia (Fig. 2A). To further investigate the miRnome data, we also measured the level of expression of other miR-7 members: miR-7-1-3p and miR-7-2-3p. We confirmed the specific down-regulation of miR-7-5p (the leading strand) in MG thymuses, as we did not observe any change for miR-7-1-3p and miR-7-2-3p (the passenger strands) expression in the thymus of MG and control donors (Fig. 2B-C). We next analyzed if the down-regulation of miR-7-5p was observed in the two main thymic cell types: TECs obtained from thymic explants after 7 days in culture and thymocytes freshly obtained from thymic biopsies. miR-7-5p was expressed in both cell types but very weakly in thymocytes. We observed a down-expression of miR-7-5p in TECs from MG patients compared to TECs from controls (Fig. 2D). In contrast, in freshly isolated thymic cell suspension, we observed a higher expression level of miR-7-5p in cells from MG patients compared to controls (Fig. 2E). This increase was not correlated with the presence of B cells in thymic-cell suspensions from MG patients as no correlations were observed compared to CD19 mRNA expression (data not shown). All these data confirmed that miR-7-5p was down-regulated in the thymus of MG in relation to a decrease in TECs.

Effect of miR-7 on CCL21 expression in thymic epithelial cells

From predictive databases, we determined that miR-7-5p was of particular interest as it targets mRNAs of several genes involved in MG (Fig. 2F-G). As we observed a direct inverse correlation between miR-7-5p expression and CCL21 mRNA in the thymus (Fig. 2H), we investigated whether miR-7-5p could inhibit CCL21 expression in TECs. We then carried out transfection experiments in TECs and analyzed the level of CCL21 released in the culture medium after 48h. First, we demonstrated the efficiency of our transfections in primary TEC cultures by evaluating the intracellular level of miR-7-5p after transfection (Fig. 2I). Next, we demonstrated that CCL21 protein levels were decreased from 9 to 20% when TECs were transfected with miR-7-5p compared to a scramble RNA (Fig. 2J). These results sustain that miR-7 has an effect on CCL21 protein expression and a decrease in miR-7-5p expression could be responsible for the overexpression of CCL21 in MG thymuses [START_REF] Berrih-Aknin | CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia[END_REF].

Search for inverse correlation for miRNA and mRNA expression data

To sort out dysregulated miRNAs that could modulate gene expression in the thymus of MG patients, we have used the web resource MAGIA2 as fully detailed in the method section. We identified mature miRNAs and genes possibly regulated by transcription factors, whose expression levels were inversely correlated in the thymus of MG patients. The target prediction databases, Targetscan and DIANA-microT extracted 6 miRNAs (1 up-and 5 downregulated) and 16 miRNAs (4 up-and 12 down-regulated), respectively. Among them, miR-486-5p was again put forward as an up-regulated miRNA in MG patients. miR-125a-5p, miR-34c-5p, miR-1-5p and miR-429-3p were identified by both predictive databases (Fig. 3A-B and Table 3). Among these dysregulated miRNAs, we analyzed by PCR the expression of miR-125a-5p and confirmed its up-regulation in the thymus of MG patients (Fig. 3C). Among miR-125a-5p targets listed in table 3, we were particularly interested in WDR1 (WD repeat-containing protein 1), a molecule recently implicated in auto-inflammatory processes [START_REF] Kim | Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1beta[END_REF]. We then showed that WDR1 mRNA expression was significantly decreased in the thymus of MG patients and that its expression was indeed inversely correlated with miR-125a-5p expression (Fig. 3D-E).

Analysis of chromosomal cluster dysregulation

In the list of dysregulated mature miRNAs (Table 2A), we observed a high representation of miRNAs from the chromosomes 14 and X. Using a χ2 test, we demonstrated for up-regulated mature miRNAs a significant overrepresentation of chromosomes 4, 8, 14 and X. For chromosomes 4 and 8, miRNAs were found up-regulated but they were spread along each chromosome (Fig. 4A and Table 2A). In contrast, for miRNAs up-regulated on chromosome 14, miR-4538-5p, miR-4539-3p, miR-4710-5p were all located in the 14q32.33 region on the negative DNA strand. Moreover, pre-miRNAs for miR-4539 and miR-4538 were also found upregulated (Table 2B). All these miRNAs belong to a miRNA cluster well referenced in miRbase.

We found for down-regulated mature miRNAs a significant overrepresentation of miRNAs located on the negative DNA strand at the extremity of the long arm of the X chromosome (Fig. 4A and Table 2A): miR-20b-3p*, miR-363-3p and miR-509-3p, miR-890-5p and miR-892b-3p. miR-20b-3p* and miR-363-3p belong to a referenced cluster in the Xq26.2 region, while miR-890-5p, miR-892b-3p and miR-509-3p belong to two other miRNA clusters in the Xq27.3 region. These miRNAs were all found down-expressed (Table 2A). We selected 2 of these miRNAs for validation by RT-PCR: miR-20b-3p* and miR-892b-3p, and confirmed their decreased expression in MG thymuses compared to controls (Fig. 4B-E).

This chromosomal region corresponds to a fragile site where the fragile X mental retardation 1 (FMR1) gene locates. Analyzing the level of expression of FMR1 in the thymus, we observed also a significant decreased expression in MG patients (Fig. 4F). FRM1 expression level is linked to the degree of methylation of its 5'UTR sequence that could also impact the level of expression of miRNAs that are encoded nearby [START_REF] Wheeler | Associated features in females with an FMR1 premutation[END_REF]. We then checked on TEC cultures if FMR1, miR-892b-3p and miR-20b-3p* were conjointly regulated by DNA methylation. TEC cultures were treated for 48h by 5-Azacytidine, a molecule causing hypomethylation of DNA. Changes in miR-20b-3p* expression could not be analyzed as this miRNA was hardly expressed in TECs. However, we observed a concomitant increased expression of FMR1 and of miR-892b-3p after demethylation (Fig. 4G-H). Altogether, these results suggest that the degree of methylation of this chromosomal region could explain the low level of FMR1 as well as miRNAs, and could play a role in MG.

DISCUSSION

MG patients with anti-AChR antibodies very often display thymic abnormalities with neoangiogenic processes and follicular hyperplasia characterized by the development of ectopic germinal centers. Here, we investigated dysregulated miRNAs that could help to understand the impact of post-transcriptional mechanisms on events leading to thymic changes associated with MG.

Specific dysregulated miRNAs in MG

We used two different approaches to search for dysregulated miRNAs in the MG thymus: a classical approach based on the fold change of miRNA expression and another approach taking into account the inverse correlation between the miRNA and mRNA expression levels. We validated by PCR the overexpression of miR-486-5p and miR-125a-5p, and the downexpression of miR-20b-3p*, miR-892b-3p and miR-7-5p. Among these miRNAs, we focus our interest on miR-7-5p as detailed below (paragraph 4.2), but miR-125a-5p and miR-20b-3p* are also of interest in the context of MG and autoimmunity. A decreased expression of miR-20b-3p* has already been observed in the serum of early-onset MG patients [START_REF] Chunjie | Disease-specific signature of serum miR-20b and its targets IL-8 and IL-25, in myasthenia gravis patients[END_REF]. Xin et al. also showed a decreased expression of miR-20b in the serum of thymoma-associated MG, and in thymoma compared to non-thymomatous adjacent tissues. They demonstrated that the decrease of miR-20b could favor T-cell proliferation [START_REF] Xin | miR-20b Inhibits T Cell Proliferation and Activation via NFAT Signaling Pathway in Thymoma-Associated Myasthenia Gravis[END_REF] but a lower expression level of miR-20b could also favor Th17-cell differentiation as observed in multiple sclerosis [START_REF] Zhu | miR-20b suppresses Th17 differentiation and the pathogenesis of experimental autoimmune encephalomyelitis by targeting RORgammat and STAT3[END_REF]. Lately, miR-20b has been characterized as an inhibitor of the inflammation by acting on NLRP3 (NLR Family Pyrin Domain Containing 3), a key protein of the inflammasome [START_REF] Lou | MiR-20b inhibits mycobacterium tuberculosis induced inflammation in the lung of mice through targeting NLRP3[END_REF]. Therapies aiming at restoring miR-20b expression in MG but also in other autoimmune conditions could have a beneficial impact on disease evolution as described with Natalizumab in multiple sclerosis [START_REF] Ingwersen | Natalizumab restores aberrant miRNA expression profile in multiple sclerosis and reveals a critical role for miR-20b[END_REF].

Here, we observed that miR-125a-5p was overexpressed in the thymus of early-onset MG patients and this miRNA has also been described as up-regulated in thymoma-associated MG.

It is suggested that it could be involved in the regulation of FoxP3 expression which is decreased in thymoma-associated MG [START_REF] Li | Altered expression of miR-125a-5p in thymomaassociated myasthenia gravis and its down-regulation of foxp3 expression in Jurkat cells[END_REF] [START_REF] Nishi | Thymoma in Patient with Myasthenia Gravis Has Significantly Fewer Forkhead Box P3 Positive Lymphocytes than that without One[END_REF]. FoxP3 is an indispensable transcription factor for the generation and the regulatory function of regulatory T cells. The suppressive activity of regulatory T cells is severely impaired in early-onset MG patients and associated with a decreased expression of FoxP3 in CD4 + T cells [START_REF] Balandina | Functional defect of regulatory CD4(+)CD25+ T cells in the thymus of patients with autoimmune myasthenia gravis[END_REF]. Altogether, this suggests that miR-125a could play a role in the decreased functionality of regulatory T cells in MG patients. Here, we demonstrated an inversed correlation in the expression of miR-125a-5p and of WDR1, one of its mRNA target extracted with MAGIA2. WDR1 has been implicated in auto-inflammatory processes associated with IL-18 expression [START_REF] Kim | Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1beta[END_REF]. This observation is of particular interest as IL-18 is overexpressed in the serum of MG patients [START_REF] Jander | Increased serum levels of the interferon-gamma-inducing cytokine interleukin-18 in myasthenia gravis[END_REF] and the administration of an anti-IL-18 antibody to myasthenic rats leads to the alleviation of clinical symptoms [START_REF] Souroujon | Suppression of myasthenia gravis by antigen-specific mucosal tolerance and modulation of cytokines and costimulatory factors[END_REF]. miR-125a could also target other mRNAs such as TNFAIP3 (Tumor Necrosis Factor Alpha-Induced Protein 3), also known as A20. TNFAIP3 is essential for the development and function of immune cells and has been implicated in the pathogenesis of various autoimmune diseases [START_REF] Zhang | Roles of A20 in autoimmune diseases[END_REF]. TNFAIP3 is a key molecule in the negative regulation of NF-κB (Nuclear Factor κB) and also TLR (Toll-Like Receptor) signaling pathways [START_REF] Kim | MicroRNAs miR-125a and miR-125b constitutively activate the NF-kappaB pathway by targeting the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20)[END_REF] [START_REF] Hamerman | Negative regulation of TLR signaling in myeloid cells--implications for autoimmune diseases[END_REF]. Consequently, increased expression of miR-20b and miR-125a could modulate inflammatory pathways and could play a key role in MG and in other autoimmune diseases [START_REF] Zhang | Roles of A20 in autoimmune diseases[END_REF] [START_REF] Avidan | VAV1 and BAFF, via NFkB pathway, are genetic risk factors for myasthenia gravis[END_REF].

Consequently, these observations suggest that miRNAs such as or miR-125a-5p can modulate inflammatory pathways and could play a key role in MG and in other autoimmune diseases.

miR-7 downregulation is involved in thymic changes

We validated that miR-7-5p was the most down-regulated miRNA in MG thymuses and this was due to its down-regulation in TECs. We showed that the level of miR-7 expression was even more decreased in highly hyperplastic thymuses. Interestingly, in corticoid-treated patients, miR-7 expression was higher than in hyperplastic thymuses. Altogether, this suggests that miR-7 could vary along with treatments and the hyperplastic state of the thymus. miR-7 was initially described in cancers but more recently its potential implication in autoimmune conditions has been highlighted. miR-7 expression is downregulated in the serum of patients with dermatomyositis and neuromyelitis optica [START_REF] Oshikawa | Decreased miR-7 expression in the skin and sera of patients with dermatomyositis[END_REF] [START_REF] Vaknin-Dembinsky | Circulating microRNAs as biomarkers for rituximab therapy, in neuromyelitis optica (NMO)[END_REF]. However, we did not observe such decrease in the serum of MG patients (data not shown). miR-7 expression seems to be decreased in inflammatory tissues. As observed in MG thymuses, miR-7 is also downregulated in the skin of dermatomyositis patients [START_REF] Oshikawa | Decreased miR-7 expression in the skin and sera of patients with dermatomyositis[END_REF] and in actively inflamed Crohn's disease colonic tissues [START_REF] Nguyen | MicroRNA-7 modulates CD98 expression during intestinal epithelial cell differentiation[END_REF].

Using prediction databases, we found that miR-7-5p can interact with the 3'-UTR region of CCL21. More, we observed that miR-7-5p and CCL21 mRNA expression were inversely correlated in the thymus of MG patients. CCL21 plays an important role in thymopoïesis. It is mainly secreted by TECs promoting the recruitment of pro-thymocytes and later on the migration of CD4 + thymocytes towards the medulla region [START_REF] Kurobe | CCR7-dependent cortex-tomedulla migration of positively selected thymocytes is essential for establishing central tolerance[END_REF]. In the MG thymus, CCL21 is abnormally overexpressed and this is mainly due to ectopic lymphatic vessels [START_REF] Berrih-Aknin | CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia[END_REF], but also to a lesser extent by TECs. Indeed, IFN-β that is overexpressed in MG thymuses can induce CCL21 expression in lymphatic endothelial cells and TECs [START_REF] Cufi | Central role of interferonbeta in thymic events leading to myasthenia gravis[END_REF]. As miR-7-5p down-regulation was observed in MG TECs, we investigated its effects by transfecting miR-7-5p in primary TEC cultures. We showed that miR-7-5p could decrease CCL21 release by TECs. This suggests that decreased miR-7-5p expression in MG thymuses could affect CCL21 expression and disturb intrathymic-cell migration. We did not investigate the impact of miR-7-5p transfection in thymic lymphatic endothelial cells that have been shown to highly expressed CCL21 in MG thymuses. The level of post-transcriptional regulation of mRNAs by miRNAs is probably celldependent and the effects of miR-7-5p could be higher in lymphatic endothelial cells.

Altogether our results suggest that an abnormal expression of miR-7 could participate in the abnormal expression of CCL21 associated with thymic changes observed in MG patients.

Dysregulated miRNA clusters suggest the implication of X chromosome in MG

Analyzing the chromosomal localization of dysregulated miRNAs, we observed a significant overrepresentation of two chromosomal regions. miRNAs overexpressed in 14q32.33 correspond to a cluster of miRNAs, including miR-4537, miR-4538, miR-4539 and miR-4507. This chromosomal region was also found significantly overrepresented in a thymic transcriptome study of MG patients [START_REF] Panse | Microarrays reveal distinct gene signatures in the thymus of seropositive and seronegative myasthenia gravis patients and the role of CC chemokine ligand 21 in thymic hyperplasia[END_REF]. It corresponds to the immunoglobulin heavy chain locus and its overrepresentation likely reflects the increased number of B cells in MG thymus.

The miRNAs encoded by this cluster are not considered as intragenic miRNAs but they could be transcribed together with immunoglobulin heavy chain genes as they are encoded on the same DNA strand and on overlapping regions.

Analyzing specific dysregulated miRNA from table 2A, we also found a significant overrepresentation of down-regulated miRNAs encoded by the Xq27.3 and Xq26.2 regions.

The importance of miRNAs in the Xq27.3 region was also pointed out by our analysis of inverse correlation for miRNA and mRNA expression (Table 3). The dysregulated miRNAs in these chromosomal regions has not yet been linked to autoimmunity and inflammation, except miR-20b, as detailed in paragraph 4.1. As many autoimmune diseases, MG is predominantly observed in females, and autoimmunity is much higher in patients with X-linked disorders [START_REF] Larizza | Autoimmune stigmata in Turner syndrome: when lacks an X chromosome[END_REF]. The Xq27.3 chromosomal region encodes for the FMR1 gene implicated in the Fragile X syndrome, a form of X-linked mental retardation. This syndrome results from the expansion of CGG repeat sequences in the 5′ untranslated region of the FMR1 gene. Normal alleles are considered to have between 5 and 44 CGG repeats, alleles with 45 to 54 repeats are intermediate (grey zone), alleles with 55 to 200 repeats constitute premutated FMR1 and over 200 repeats alleles present a full mutation. Individuals with FMR1 full mutation or premutations display immune-mediated disorders and in particular a higher risk to develop autoimmune diseases [START_REF] Winarni | Immune-mediated disorders among women carriers of fragile X premutation alleles[END_REF] [START_REF] Pretto | Differential increases of specific FMR1 mRNA isoforms in premutation carriers[END_REF].

The regulation of FMR1 expression is very complex and dependent on the number of CGG repeat sequences in 5'UTR and the subsequent DNA methylation level, especially for individuals with the full mutation genotype. However, the presence of a methylation boundary region upstream FMR1 promoter and of four CTCF binding sites are also important for FMR1 expression. CTCF is an important transcriptional regulator of genes harboring trinucleotide repeats and it is mostly active in chromatin organization [START_REF] Naumann | A distinct DNA-methylation boundary in the 5'-upstream sequence of the FMR1 promoter binds nuclear proteins and is lost in fragile X syndrome[END_REF] [START_REF] Lanni | Role of CTCF protein in regulating FMR1 locus transcription[END_REF]. Analyzing FMR1 mRNA expression, we observed a significant decreased expression in the thymus of MG patients. Of note, the expression of FMR2/AFF2, a gene localized near FMR1 was also strongly decreased in the thymus of MG patients (data not shown). We also demonstrated that demethylation with 5-Azacytidine of control TEC cultures increased FMR1 expression concomitantly with the increased expression of miR-892b-3p. miRNAs that were found down-regulated in our study could have been impacted by the higher methylation status of DNA in this region even if there are on opposite strands [START_REF] He | Demethylation of miR-10b plays a suppressive role in ccRCC cells[END_REF]. This suggest that genes and miRNAs encoded in this chromosomal region could be conjointly regulated and it raises the issue of a link between autoimmunity and this chromosomal region.

CONCLUSION

Studies have been carried out to investigate the implication of miRNAs in physiological and pathophysiological mechanisms. Here for the first time, we investigated with different approaches dysregulated miRNAs in the thymus of MG patients. We demonstrated that specific miRNAs were implicated in thymic changes associated with MG, in particular, miR-7-5p but others such as miR-125a-5p and miR-20b-3p* are also of interest. We also demonstrated a decreased expression of genes and miRNA clusters localized at the extremity of the X chromosome with a potential link with autoimmunity. Altogether this detailed analysis of the thymic miRnome is of great interest bringing new knowledge in the etiological and pathophysiological mechanisms involved in early-onset MG.

Figure 1 :

 1 Figure 1: Dysregulated mature and precursor miRNAs in MG thymuses compared to

Figure 2 :

 2 Figure 2: Down-regulation of miR-7-5p in the thymus of MG patients

Figure 3 :

 3 Figure 3: Inverse correlation for miRNA and mRNA expression data

Figure 4 .

 4 Figure 4. Expression of miRNAs and FMR1 mRNA located ion the same region of the X

Acknowledgments

We would like to thank Perrine Cufi for her technical assistance, Dr Vincent de Montpreville and Sylvie Planté from the anatomy and histology department from the Marie Lannelongue Surgical Center and Lilia Lamrani for the management of thymus collect. Dr Florent Dumont and Dr Micaela Polay-Espinoza for their advice regarding miRnome analyses. We also would like to thank Dr Nadine Dragin and Marine Guilbaud for helpful discussion and Damien Amelin for his technical advice. This work was supported by grants from the European Community (FIGHT-MG/HEALTH-2009-242-210) and from the "Association Française contre les Myopathies" (AFM).

Authorship

MAC, SM, FD, NS performed and analyzed the experiments, FT collected samples and provided patient information, MF performed the miRnome experiment, EF and JG provided thymic biopsies, SB-A and SM read and revised the manuscript, MAC and RLP designed the study, analyzed the experiments and wrote the manuscript.

Conflict of interest

The authors have no conflict of interest to declare.

TECs treated with 5-Azacytidine (5µM) for 48h. mRNA and miRNA expression levels were given in arbitrary units and were normalized to GAPDH. P-values were assessed by the Mann-Whitney test and only p-values < 0.05 are indicated (*p<0.05, **p<0.01).

Table 1

(1) Degree of thymic hyperplasia: low hyperplasia (with 2 or fewer GCs per section) or high hyperplasia (with 3 or more GCs per section). ( 2


Marianne Boix
email: marianne.boix@ensiacet.fr

Ludovic Montastruc

Manuel Ramos

Olivier Gentilhomme

Serge Domenech

Benefits analysis of optimal design of eco-industrial parks through life cycle indicators

Keywords: life cycle assessment, industrial symbiosis, process simulation, optimization, multicriteria decision making

Industrial symbiosis offers to companies the possibility to make economic benefits and to minimize environmental impacts by sharing flows and increasing inter-enterprise exchanges. However, even if some studies have demonstrated the benefits of the development of eco-industrial parks (EIP), there is no consensus to evaluate their benefits in a global point of view and there is a lack of integrated indicators for the assessment of EIPs. The aim of this study is to propose a holistic approach to evaluate the global impacts of an EIP. To reach this goal, the potential eco-industrial park of Mongstad in Norway has been chosen. Several steps are considered: a simulation through Aspen Properties®, then the superstructure optimization problem solved within GAMS® environment by minimizing the total cost of the EIP is done. Finally, an evaluation of the optimal solution through a life cycle approach is carried out. The results show that companies included in the EIP have environmental impacts reduced from 45% to 80% compared to the impacts of stand-alone companies.

Introduction

Following several famous environmental summits, and more particularly, at a national scale, the French Environmental Conference that occurred in 2013, France defined some guidelines for its ecological transition. The main research axis that has been defined is the development of a circular economy in order to design new ways of producing goods and services that allow decreasing resources consumption (water, energy and raw materials). In this context, the development of industrial and territorial ecology has become one of the big international issues. One of the answer to overcome this issue consists in implementing eco-industrial parks (EIP) [START_REF] Boix | Optimization methods applied to the design of eco-industrial parks: a literature review[END_REF]. Indeed, industrial symbiosis offers to companies the possibility to make economic benefits as it is the case in the eco-industrial park of Kalundborg, in Denmark, for example. Other successful examples even more numerous are built all over the world. Most of them were built in industrialized countries of North America, Europe, or Australia but more recently it is in developing countries that many parks are born (such as China, Brazil and Korea for example). As it can be inferred, a basic condition for an EIP to be economically viable is to demonstrate that benefits of each industry involved in it by working collectively is higher than working as a stand-alone facility. However, even if some studies have demonstrated Antonio Espuña, Moisès Graells and Luis Puigjaner (Editors), Proceedings of the 27 th European Symposium on Computer Aided Process Engineering ESCAPE 27 October 1 st -5 th , 2017, Barcelona, Spain the benefits of some punctual environmental indicators such as water consumption [START_REF] Alnouri | Synthesis of industrial park water reuse networks considering treatment systems and merged connectivity options[END_REF], global warming potential or energy consumption [START_REF] Valenzuela-Venegas | Sustainability indicators for the assessment of eco-industrial parks: classification and criteria for selection[END_REF], there is no consensus to evaluate the benefits of eco-industrial parks in a global point of view. Furthermore, there is a lack of integrated indicators for the assessment of EIPs. EIP optimization models are far from being single-objective classical optimization problems. [START_REF] Zhang | Model-Centered Approach to Early Planning and Design of an Eco-Industrial Park around an Oil Refinery[END_REF] provided a general procedure allowing the early planning and design of EIPs. Furthermore [START_REF] Sokka | Industrial symbiosis contributing to more sustainable energy use an example from the forest industry in Kymenlaakso, Finland[END_REF] proposed to compare stand-alone environmental impacts to an industrial symbiosis system by a life cycle assessment. In the present work, the proposed systemic methodology consists in a combination of modern engineering tools, such as process simulation, mathematical modelling and optimization and life cycle approach to evaluate the solution. The aim of this study is to propose a systemic approach for the design of eco-industrial parks that allows taking into account the multi-participant aspect at the beginning of the project; but also that allows evaluating environmental impacts of the proposed solution that minimizes the cost.

Methodology

The generic methodology deployed in this work is illustrated in Figure 1 for several modelling scales.

Figure 1. Generic methodology developed

Indeed, whatever the scale of the system it is possible to overcome the data limitations by means of thermodynamics modelling or process simulation to obtain data that allows building the superstructure. For example, specific details from the molecule level are traduced by thermodynamics modelling. After processing the data, a mathematical modelling is necessary to optimize the whole system regarding an economic criterion. The optimization step is done according to the previous work of [START_REF] Ramos | Water integration in ecoindustrial parks using a multi-leader-follower approach[END_REF] by using a multi-leader-follower game (MLFG). The game theory approach is proven reliable compared to traditional multi-objective optimization (MOO) methods because it provides a numerical Nash equilibrium solution [START_REF] Ramos | Water integration in ecoindustrial parks using a multi-leader-follower approach[END_REF]. In this model, the enterprises of the park are considered as Leaders and each one minimizes its own cost while an EIP authority, which is the Follower, is responsible for minimizing resources consumption. In this work, the solution obtained is then environmentally evaluated through a life cycle approach.

Application of the methodology to the case study

This holistic method is applied on a case study located at Mongstad, in Norway already studied by [START_REF] Zhang | Model-Centered Approach to Early Planning and Design of an Eco-Industrial Park around an Oil Refinery[END_REF]. The system is composed of several companies established in a same geographical area that do not collaborate for the moment (Figure 2). Indeed, [START_REF] Zhang | Model-Centered Approach to Early Planning and Design of an Eco-Industrial Park around an Oil Refinery[END_REF] -The final step is to evaluate the optimal solution to compare environmental impacts of the integrated EIP to stand-alone companies. This environmental evaluation has been carried out through a life cycle approach where the system boundaries are limited to the whole EIP (Figure 4), thus, it consists in a gate-to-gate analysis. The inventory was conducted according to the norm ISO Standard 14044:2006. The system includes the production of raw materials, wastes and effluents and fuels and electricity used by the different companies. Calculations of impacts were conducted using Simapro 8.0 and IMPACT 2002+ method as well as the EcoInvent database. The functional unit of the study is one-year production of the whole symbiosis.

Results and discussion

Process simulation and optimization of the potential EIP

Each process of the EIP has been defined and simulated with ASPEN Properties®. Given the energy requirements obtained by the simulations, different utilities were considered for the potential EIP: water (at different temperatures) and steam (at low and high pressures).

Figure 3. Inputs and outputs for the system studied.

After these simulations, the superstructure of the utility network has been raised in order to implement the game theory approach. The optimal solution is calculated and the different flow exchanges are summarized in Figure 3. In this solution, some internal exchanges are proposed but it remains a lot of inputs and outputs for the whole system because this solution aims at minimizing the own cost of each company of the EIP. Inter-enterprises flows are represented but they will not affect the environmental impacts. The power plant needs the great majority of the utilities consumed, natural gas, and petroleum that will greatly cause damages on the environment. As expected, utility consumption decrease in the optimal solution and the refinery is the lead CO2 producing plant. However, in order to fulfil a significant analysis, it is important to quantify the environmental benefits proposed by this solution.

Environmental impacts of the optimal solution

Environmental impacts of the solution have been calculated and compared to the case where companies work in stand-alone configuration. Mid-point impact categories are reduced from 4.8% for Respiratory organics to 94.5% for Ionizing radiations categories (Figure 4). These benefits are mainly due to the fact that 116T/h of CO2 are recuperated from coal gasification and sent to the CO2 capture plant within the EIP instead of being released as emissions into the environment. This is the same case for the petroleum of the powerplant partly recycled as an inlet of the refinery. Finally, Figure 5 illustrates the impact points regarding end-point damage categories. The resources consumption remains the principal damage caused because petroleum, natural gas and water are used as inlets of the standalone companies. However, in the EIP, although this damage category is drastically reduced (from 480 to 40 points), it could be interesting to design an alternative that proposes the utilization of renewable energies in order to reduce these damages. The climate change damages are also reduced with the EIP option thanks to the recycling of CO2 at the outlet of the coal gasification plant.

Figure 5. Damage categories generated by the EIP compare to stand-alone companies.

Conclusion

In this work, a holistic method has been developed by coupling several tools: process simulation, optimization through game theory and an environmental evaluation of the optimal solution. Usually applied to a product or process, a life cycle approach is here used as a post-optimization evaluation tool applied to the whole EIP system. A comparison of the environmental impacts generated by the companies individually and when included in the park helps to demonstrate concretely the environmental benefits of an EIP. These results show that for each mid-point and end-point categories, the environmental impacts of industrial activities when they are included in the EIP are reduced from 45% to 80% compared to the impacts of stand-alone companies. Furthermore, this integrated approach also permitted to design an EIP solution where significant gains are reached because the total cost is reduced of 25%. In future works it will be interesting to integrate LCA indicators during the optimization step. However, this kind of approach with game theory needs to be formulated as a multi-leader multifollower game (MLMF) where all the followers aims at minimizing each indicator.

  have already identified this case study as a potential EIP and explored what kind of flows the different companies could share. The main activities were selected to be part of the EIP, the selected ones are those with internal processes, as well as energy exchanges and mass flows relevant for the proper development of the park, such as the refinery or the power plant. Modules as water treatment or aquaculture have been excluded. The selected activities for this study are Coal Gasification, CO2 Capture, Methanol and Dimethyl Ether (DME) Synthesis, Refinery Plant, Power Plant and Air Separation.

Figure 2 .

 2 Figure 2. Potential EIP participating plantsIn this work, the aim is to propose an optimal configuration for the design of this EIP where every participant is satisfied by its own situation and a good environmental evaluation of this solution. Several steps have been carried out to reach this goal: -The first step consists in the transformation of a real problem with limited data into a mathematical optimization model. Inputs and outputs of each company need to be exactly quantified so that proposal of exchanges can be made. At this step, a process

Figure 4 .

 4 Figure 4. Benefits generated from the EIP configuration compared to stand-alone companies regarding mid-point impact categories (in percentage).


Maillé Laurence

L Maillé

Y Le Petitcorps

A Guette

G Vignoles

J Roger

Synthesis of carbon coating and carbon matrix for C/C composites based on a hydrocarbon in its supercritical state

Keywords: supercritical fluid, Chemical Vapor Deposition (CVD), Chemical Vapor Infiltration (CVI), C/C composites

This study addresses a new process for the fabrication of a C/C composite for aeronautic and aerospace industries. The infiltration of a carbon fibrous preform by means of a hydrocarbon in the supercritical fluid state is carried out at high temperature in order to get a carbon matrix. The selected hydrocarbon is methane. The microstructure of the pyrocarbon coating is characterized by optical microscopy, Transmission Electron Microscopy and Raman Micro-Spectroscopy. The experimental parameters (temperature, hydrocarbon pressure, residence time) are tuned in order to improve the carbon matrix yield. The feasibility of a dense composite from a precursor in its supercritical state, reducing the infiltration duration as compared to Chemical Vapor Infiltration (CVI), is demonstrated.

Introduction

Ceramic Matrix Composites are developed for aeronautic and spatial applications [START_REF] Naslain | SiC-Matrix Composites: Non-brittle Ceramics for Thermo-Structural Application[END_REF] for their refractoriness and their low density. Starting from a woven carbon fiber preform, C/C composites are usually obtained by a long Chemical Vapor Infiltration (CVI) step, using as precursors gaseous hydrocarbons such as propane [START_REF] Naslain | SiC-Matrix Composites: Non-brittle Ceramics for Thermo-Structural Application[END_REF] . However, CVI is a rather timeconsuming process, which is a huge drawback. As a result, the manufacturing cost of these composites is very high. In order to reduce the cost, other processes can also be used to prepare the matrix, such as liquid phase routes with mineral charges [ 2 -8 ] .

In this paper, we explore a new and original densification process in order to prepare a Carbon/Carbon (C/C) composite: the Chemical Supercritical Fluid Infiltration. A supercritical fluid is a substance taken at a temperature and pressure above its thermodynamic critical point. Its properties are simultaneously those of gases and liquids [START_REF] Le Trequesser | Supercritical fluid deposition of compositionally uniform yttria stabilized zirconia films[END_REF] . extraction / fractionation [START_REF] Eckert | Supercritical fluids as solvents for chemical and materials processing[END_REF][START_REF] Cooper | Polymer synthesis and processing using supercritical carbon dioxide[END_REF][START_REF] Richter | Accelerated solvent extraction: A technique for sample preparation[END_REF] ; ii) purification / recycling [START_REF] Erkey | Supercritical carbon dioxide extraction of metals from aqueous solutions: a review[END_REF][START_REF] Dillow | Bacterial inactivation by using near-and supercritical carbon dioxide[END_REF][START_REF] Sunarso | Decontamination of hazardous substances from solid matrices and liquids using supercritical fluids extraction: A review[END_REF] ; and iii) synthesis of materials [START_REF] Blackburn | Deposition of Conformal Copper and Nickel Films from Supercritical Carbon Dioxide[END_REF][START_REF] Matson | Rapid expansion of supercritical fluid solutions: solute formation of powders, thin films, and fibers[END_REF] . Supercritical fluids have the unique ability to diffuse through porous solids like a gas, and to dissolve species like a solvent. This combination of the solvent ability and the high diffusivity should enable a rapid infiltration and a good densification of the porous preform.

Application to pyrocarbon infiltration results in a rapid densification of fiber preforms [START_REF] Guette | Method for the rapid densification of a porous substrate comprising the formation of a solid deposit within the porosity of the substrate, patent US[END_REF][START_REF] Maillé | Comblement des macropores d'un substrat fibreux par du pyrocarbone[END_REF][START_REF] Maillé | Comblement des macropores d'un substrat fibreux par nucléation en phase homogène[END_REF][START_REF] Maillé | Comblement des macropores d'un substrat fibreux avec un mélange de fluides supercritiques[END_REF] .

Preliminary results revealing the potential of this technique are reported and discussed in the next sections of this article, considering either deposition on a dense substrate or infiltration in a porous substrate.

Preliminary study

Before performing experiments, the potential gains of working in supercritical conditions instead of classical CVI conditions are investigated with a thermochemical study. Also, a first guess of the influence of the operating parameters is expected from these computations.

The stability of all the chemical components in the experimental conditions (temperature and pressure), starting from pure methane, was assessed by a calculation of the thermochemical equilibrium of carbon and several hydrocarbons using the Thermocalc 3.0 software [START_REF] Andersson | [END_REF][START_REF] Carlès | Thermophysical approach of supercritical fluids[END_REF][START_REF] Coquelet | New trends in inorganic materials science using supercritical fluids: from nanopowders to more complex nanomaterials[END_REF] . The relative equilibrium amounts of the different phases (Supercritical Fluid SF and solid carbon) are analyzed, in order to quantify thermodynamically the carbon deposition yield (i.e. the number of carbon moles compared to the initial number of methane moles), the SF yield (i.e. the number of SF moles compared to twice the number of methane moles). Fig. 1 represents the evolution of the equilibrium yield of SF and solid carbon with pressure (P∈ [10; 100] bar), together with the mole fractions of H 2 and CH 4 in the final SF, for temperatures ranging from 800 to 1200°C. The influence of the pressure on the fluid composition at 1200°C is shown Figure 2, in which the thermodynamic stability of CH 4 is seen to increase with pressure. The mole fractions of CH 4 and H 2 sum up to close to unity, since the equilibrium partial pressures of all other hydrocarbons are negligible (below 2.10 -4 in all cases). The system behaves as if there were only a single equilibrated reaction:

CH 4 (sf)  C (s) + H 2 (sf) (1)
The thermodynamic calculations results of Fig. 1 show a decreasing stability of the solid phase and a decreasing yield of SF with increasing pressure. This is in accordance with Le Châtelier's principle [START_REF] Liu | Application of the Le Châtelier principle on gas reactions[END_REF] . On the other hand, since reaction (1) is endothermic, the yield increases with temperature. Therefore, high temperatures and low pressures would be the most adequate conditions on a thermodynamic point of view. So it seems better to work at a pressure close to the critical pressure (46 bar). However, the actual choice of appropriate temperature will result from a trade-off between the thermodynamic predictions on the one hand and on kinetics considerations on the other hand. Experimentally, solid carbon formation appears only above 850°C at an initial pressure of 50 bar.

Materials and experimental procedures

Two types of materials were used: (i) graphite pipes (6 cm length, 0.6 cm diameter) were used as a substrate for the kinetic studies of the deposition, and (ii) 3D porous carbon fibrous preforms (2 cm length, 1.5 cm diameter) for infiltration study. The initial open porosity of the preforms was around 77%.

A sealed cold wall reactor made of Inconel alloy was used at high pressure, in batch mode. It is schematically represented in Figure 3. The graphite pipe was heated by Joule effect. The temperature was measured by a thermocouple whose wiring was protected by an alumina tube and whose extremity was inserted in a graphite tube. Pressure (bar ) For kinetic studies, thin films were deposited using methane (CH 4 ) as the carbon precursor (T c = -82.6°C and P c = 46 bar) on the graphite pipes during 10 minutes with an initial pressure of 50 bar at 950°C.

H 2 CH 4 C 2 H 6 C 2 H 4 C 2 H 2 C 3 H 8
For infiltration studies, 3D porous carbon fibrous preforms were used, with the same carbon precursor. The durations and temperatures selected for the single-step (batch) infiltration varied between 10 and 60 minutes and 950°C and 1050°C respectively (Table 1, sample A-B and C), with an initial pressure of 50 bar.

Another experiment with five successive runs of infiltration (semi-continuous feeding), described in Table 1 (sample D), was carried out to improve the matrix densification, with an initial pressure of 50 bar. Thin films and composites were observed with a Reichert-Jung MeF3 apparatus on polished surfaces by reflection optical microscopy. Observations under natural light were made to estimate the thickness of thin film and to detect a possible gradient of densification from the internal to the external part of composites. The degree of anisotropy of pyrocarbons was determined under crossed polarizers by the classical extinction angle method.

Table 1. Infiltration conditions of the 3 D carbon preforms.

(*) For each step, the vessel is evacuated and then refilled with the CH 4 precursor at a constant pressure of 50 bar.

Transmission electron microscopy (TEM) analyses were carried out with a Philips CM30ST TEM (LaB 6 operated at 300kV) with a point resolution of 0.2 nm and an information limit of 0.12 nm. Selected area electron diffraction patterns and (002) dark field mode images were acquired on the sample. A JEOL Ion Slicer (EM-09100IS) device was used to prepare the sample.

Raman microspectrometry analyses were performed on the same polished cross-sections as with optical microscopy, with a Labram HR spectrometer (HORIBA Jobin Yvon) using a 632 nm emission line and an analyzer for polarized light. The spectra were interpreted as recommended by Vallerot et al. [START_REF] Vallerot | Quantitative structural and textural assessment of laminar pyrocarbons through Raman spectroscopy, electron diffraction and few other techniques[END_REF] and compared to known families of pyrocarbons [START_REF] Bourrat | Low temperature Pyrocarbon: a review[END_REF] .

Results and discussion

Kinetic study of pyrocarbon coating on a graphite pipe

With a sufficient residence time, it is possible to obtain an important thickness of pyrocarbon: 33 µm for 10 min. It confirms the relevance of using a supercritical fluid. By comparison, several hours are necessary to reach the same thickness with the CVI-process at a similar temperature (~ multiplied by 1000) [START_REF] Kaplas | Synthesis of pyrolytic carbon films on dielectric substrates[END_REF] . The thickness of the pyrocarbon coating varies significantly along the pipe due to the presence of a temperature gradient (Fig. 4). As in classical CVI, the temperature is a very influential parameter. The morphology of the pyrocarbon coating (Fig. 5) is similar to the one created with the classical CVI-process: it is characterized by a cone-like growth structure [START_REF] Christin | Design -fabrication and application of thermostructural composites (TSC) like C/C -C/SiC and SiC/SiC composites[END_REF][START_REF] Bourrat | Regenerative laminar pyrocarbon[END_REF] .

Figure 5. Cross section of a pyrocarbon layer deposited on the carbon pipe (pyrocarbon elaborated for 10 minutes, at 50 bar and 950°C).

Influence of temperature on the infiltration of a preform

Two composites were prepared at an initial pressure of 50 bar for 10 minutes at 950°C (Figure 6) and 1050°C (Figure 7). The mass gains obtained in these experiments are similar: 42% at 950°C and 43% at 1050°C, indicating a possibly weak role of temperature at these values; however, the morphology of the infiltrated matrices differs. On the one hand, at both temperatures, close to the graphite pipe (Figure 7), the densification of the composite is of good quality and consists in a thick, adherent and homogeneous pyrocarbon coating around each fiber. This phenomenon can be explained by the fact that the temperature is higher in the vicinity of the graphite tube, yielding an inside-out infiltration gradient.

On the other hand, the presence of another type of carbon, resulting from homogeneous nucleation, is observed at high temperature only. It is located in the initially most porous areas of the preform. The infiltration of this distinct form of pyrocarbon compensates a larger

Graphite pipe Carbon fibers

Pyrocarbon coating

Graphite pipe Carbon fiber Coating on carbon fiber

Homogeneous nucleation developed in the pores gradient of deposit thickness around the fibers, therefore yielding similar overall amounts of degree of pore filling despite the morphological differences.

Influence of infiltration time on the densification of a preform

With temperature and initial pressure fixed at 950°C and 50 bars respectively, infiltration cycles with several durations have been run.

The first experiments show a good densification (up to 0.5 mm depth of the preform) for a short infiltration time (10 minutes). The infiltration depth is 1.5 mm after 60 min in the same conditions. However, at that moment, the densification is not yet complete, as can be seen in Fig. 8: various pores are still present between some fibers even close to the surface of the preform. Anyway, after 10 or 60 minutes, the pyrocarbon deposit is homogeneous in thickness and adherent to the fibers. In order to further improve the densification, additional experiments were performed, using the semi continuous feeding reactor (table 1, sample D). Five successive infiltration runs have been carried out, with a total duration equivalent in time to 42 min of a batch processing. The temperature was increased from 950°C to 1050°C between steps 3 and 4.

The use of the semi continuously fed reactor allowed the matrix densification to be greatly improved. After the five runs, the mass gain of the sample increased to 90%, whereas it had varied from 42% to 43% between 10 and 60 minutes of batch processing (table 1, samples A and B). A good densification is observed up to a depth of 1.5 mm in the preform; a thin pyrocarbon coating is still visible around each fiber up to 2.5 mm deep in the preform (figure 9).

Figure 9.

Cross section micrographs of sample D prepared in five steps by semi-continuous reactor feeding described in table 1.

Figure 9 shows several micrographs of the infiltrated sample. As for sample C (Fig. 7), two areas can be found in it: i) a fibrous area with a pyrocarbon coating around each fiber and ii) a fiber-free area where pore filling results from homogeneous nucleation.

Characterizations of the deposited pyrocarbons

The pyrocarbon resulting from homogeneous nucleation has been observed by TEM, associated to SAED, as reported in Fig. 10. At micrometer resolution it is possible to distinguish nuclei surrounded by laminar pyrocarbon layers, with various types of textures.

The topmost layer has a more granular texture. Electron diffraction confirms that the degree of organization is rather important, with neatly apparent (10) and ( 11) peaks; however, graphitic order is not present. In the laminar pyrocarbon layers, the SAED pattern reveals a high degree of anisotropy, with a (002) arc opening angle close to 22°. Measurements by Raman microspectrometry have been carried out on the grains produced by homogeneous nucleation, and on the pyrocarbon coatings deposited around the fibers. Figure 11a displays the spectra of both regions. Comparison with respect to known families of pyrocarbons is made by measuring the D band half-with, characteristic of the density of inplane defects, and the D peak anisotropy ratio, characteristic of the degree of texture [27;31-32] .

It is found that the pyrocarbon deposited around the fibers belongs to the Regenerative laminar (ReL) type [START_REF] Bourrat | Regenerative laminar pyrocarbon[END_REF] , whereas the one formed by homogeneous nucleation, with a lesser anisotropy and density of defects, lies halfway between the ReL and the Smooth Laminar (SL) types. The latter fact is not surprising, taking into account that this PyC is a mixture of nuclei and laminar, highly anisotropic carbons. The rather high degree of anisotropy obtained by Raman spectroscopy is confirmed by polarized light optical microscopy, with extinction angles around 21° for the fiber coating. Figure 12 ReL is clearly distinct from Rough Laminar (RL) though having the same degree of anisotropy and being frequently confounded with it under the global denomination of "Highly-Textured" PyC [START_REF] Farbos | Nanoscale structure and texture of highly anisotropic pyrocarbons revisited with transmission electron microscopy, image processing, neutron diffraction and atomistic modeling[END_REF] . ReL PyC is usually obtained by CVD and not by CVI [START_REF] Vignoles | CVD and CVI of pyrocarbon from various precursors[END_REF] . Indeed, its formation is linked to an important degree of hydrocarbon maturation, i.e. for high residence times and low surface/volume ratios [START_REF] Vignoles | The control of interphases in carbon and ceramic matrix composites[END_REF] . It is striking to encounter ReL here, infiltrated inside a fibrous preform: this shows that the kinetics of gas-phase maturation is much faster in the chosen supercritical conditions than in classical CVI conditions. Moreover, in CVD or in gas-phase pyrolysis of hydrocarbons, for very long residence times, homogeneous nucleation may appear. It seems to be the case in the current process at high temperatures, confirming once again the faster kinetics of the gas-phase reactions. The expected consequence would have been soot formation, but instead of this, it turns out that a limited amount of nuclei appear directly inside the largest pores and grow by laminar PyC deposition, which prevents further nucleation and helps in rapidly plugging these large pores.

Conclusion

The feasibility of infiltrating a fiber preform with pyrocarbon in minutes using supercritical methane has been demonstrated. A device has been set up for this purpose allowing to work under a pressure of 50-100 bar at 950-1050°C. At high temperature, the infiltration depth of the preform could reach 1.5 mm; fiber coatings were observed at up to 2.5mm in depth. A nearly full infiltration was achieved in a semi-continuous operating cycle by increasing the temperature when re-feeding the reactor with gaseous precursor. The pyrocarbons obtained by this process are of two types: one, directly deposited on the fibers, is of the Regenerative Laminar type (highly anisotropic but defect-rich); the other one, arising from homogeneous nucleation and growth, is less anisotropic due to the synthesis mechanism itself.

Considering the short infiltration times, the good infiltration quality, and the high anisotropy of the infiltrated pyrocarbon, this process has a great potential for the manufacture of carbon/carbon artifacts. For this, more process studies are necessary, including scale-up and process modeling. This is the object of undergoing work.

Figure 1 .Figure 2 .

 12 Figure 1. Computed equilibrium yields of solid carbon (C) and supercritical fluid (SF) and methane (CH 4 ) and dihydrogen (H 2 ) mole fractions at selected temperatures versus pressure.

Figure 3 .

 3 Figure 3. Schematic representation of the cold wall reactor (A) and photograph of the interior of the reactor (B).

Figure 4 .

 4 Figure 4. View of graphite pipe inside the reactor (A) and influence of the temperature gradient of the graphite pipe on the thickness of the pyrocarbon deposit along the graphite pipe after 10 minutes of deposition (B).

Figure 6 .

 6 Figure 6. Micrographs of a composite produced at 50bar and 950°C during 10 min (sample B).

Figure 7 .

 7 Figure 7. Micrographs of a composite produced at 50bar and 1050°C during 10 min (sample C).

Figure 8 .

 8 Figure 8. Cross sections of the infiltrated C/C composite fabricated at 50 bar and 950°C at various times of the methane: left 10 minutes (sample B), right: 60 minutes (sample A).

Figure 10 .

 10 Figure 10. TEM observation of sample D fiber-free area in dark field mode and selected area electron diffraction patterns.

  is a PLOM micrograph showing the characteristic Maltese cross edges of the ReL PyC type obtained in the deposit around the fibers.

Figure 11 .

 11 Figure 11. A) Raman spectrum of sample D; the analyzed area was 1µm × 1µm × 100nm. B) Location of PyC coating around fiber (circle) and PyC from homogeneous nucleation (triangle) on the Raman based 2D classification proposed by Bourrat et al.[27;30;33]

Figure 12 .

 12 Figure 12. Optical microscopy micrograph under crossed polarizers of C/C composite obtained at 1050°C.


Olivier Monfort
email: olivier.monfort@ensc-rennes.fr

Ewa Dworniczek

Leonid Satrapinskyy

Alicja Seniuk

Daniela Nyblova

Gustav Plesch

Efficient Nb-modified BiVO 4 film for photo-induced bacterial inactivation and photocatalytic removal of organic pollutant

Using Nb-modified BiVO 4 film, the photo-induced properties in Methicillin-resistant Staphylococcus aureus (MRSA) inactivation under simulated solar light reached 43% after 1 hour irradiation while 72% of Rhodamine B was degraded after 3 hours. To evaluate the efficiency of Nb-BiVO 4 , its photoperformance was compared with that of BiVO 4 /TiO 2 composites and pure BiVO 4 .

Introduction

Bismuth vanadate (BiVO 4 ) is a promising solar-light driven photocatalyst which exhibits energy band gap at 2.4 eV. 1,2 For this reason BiVO 4 materials, and more generally Bi-based oxide materials, are intensively studied for photodegradation of organic pollutant and photoproduction of O 2 by water splitting. [1][2][3][4][5][6] However BiVO 4 photocatalyst exhibits high recombination rate of electron-hole pairs that limits its performance. 1,2 Therefore it is necessary to modify BiVO 4 photocatalyst to increase its efficiency in photochemical processes by designing doped or composite systems. 1,2 Modification using silver or carbon-based materials can be cited. [3][4][5][6] On the other hand, BiVO 4 -based photocatalysts are mainly studied in the form of powder suspensions [7][8][9][10] rather than supported systems [11][12][13][14][15] for photo-oxidative degradation of organic pollutant. Supported systems such as films are more practical for obvious reason: the readily post-separation of the photocatalyst with the polluted media. 16 Different photocatalytic mechanisms were reported according to the way of BiVO 4 modification. Indeed variously doped and numerous BiVO 4 composite systems could exhibit either photogenerated holes or radicals as main oxidative species. [3][4][5][6][7][8][9][10][11]15 In addition, among the works devoted to photo-induced antimicrobial properties of bismuth vanadate-based materials, most of them are focused on E. coli inactivation. [17][18][19][20][21][22][23][24][25][26] In this work, bismuth vanadate films are prepared by metal organic decomposition and modified using niobium(V) and TiO 2 . The photocatalytic efficiency in Rhodamine B degradation was compared using different BiVO 4 -based films and the study of inactivation of MRSA strain is, for the first time, demonstrated under solar-like irradiation. MRSA (Methicillin-resistant Staphylococcus aureus) are chosen in this study because such bacteria are clinically important and more resistant than E. coli bacterial strains. Indeed, the Staphylococcus aureus is the most common bacterial pathogen worldwide. 27 Its methicillin-resistant variants (MRSA) generate serious concern over the loss of antibiotics susceptibility. 27 The observed photo-induced properties as well as mechanisms are also discussed here, especially for Nb-modified BiVO 4 . Such photocatalytic films could find promising environmental applications but also could be developed for clinical or food technology.

Experimental

Pure bismuth vanadate films were prepared using a precursor solution based on vanadium(IV) acetyalacetonate and bismuth(III) nitrate pentahydrate. 13,14 For the preparation of Nb-BiVO 4 , the precursor solution was modified by 10 at% of Nb(V) chloride while BiVO 4 /TiO 2 composites were prepared using the BiVO 4 precursor solution and a TiO 2 sol-gel. 13,14 Films were deposited using doctor blade method by casting the precursor solution between Scotch® tape using glass Pasteur pipette on Si/SiO 2 substrates. Crystalline films were obtained after subsequent metal organic decomposition at 500°C for 5 minutes. For the composites, two different layered configurations were designed: top coated-BiVO 4 over TiO 2 (top-BiVO 4 /TiO 2 ) and top coated-TiO 2 over BiVO 4 (top-TiO 2 /BiVO 4 ). The films were approximatively 1 µm thick.

The composition of crystalline phases in the films was analyzed by X-ray diffraction using PANalytical XPert Pro MRD diffractometer. The surface morphology was characterized by scanning electron microscopy using Tescan Lyra III. The energy band gap was evaluated by diffuse reflectance spectroscopy using Shimadzu model 2600 spectrophotometer.

Photocatalytic experiments were performed for 180 minutes under solar-like irradiation using a Bclass solar simulator with spectral characteristics similar to natural sunlight (HQI TS -OSRAM 400 W; λ max = 525 nm) where the decolorization of 10 -5 M Rhodamine B (RhB) solution was followed by UV-vis spectrophotometer (Jasco V-530). The mechanism of photooxidative degradation was determined indirectly using charge scavengers of the different species possibly involved: ammonium oxalate for photogenerated holes (h + ), iso-propanol for hydroxyl radicals (OH • ) and p-benzoquinone for superoxide radicals (O 2

•-). The experiments were performed in triplicates.

To evaluate the antimicrobial effectiveness at the surface of the samples, modified JIS Z 2801: 2010 test method, was used. Briefly, 100 µL of MRSA K324 which represents (at t = 0 min) 3•10 5 CFU/mL (Colony Forming Unit) was instilled onto each BiVO 4 -based film (1 cm 2 ) kept in a humid atmosphere to avoid extensive drying of the suspension and irradiated for 30 min and 60 min (Xenon lamp -15 mW/cm 2 ). Control experiments were carried out in the dark, under the same conditions. At appropriate time, the test bacteria were washed out with sterile saline solution, to release the inoculum from the surface. Quantification of the viable cells was performed by a standard plating method and incubation at 37 o C for 24-48h. Bactericidal activity of the samples was evaluated using logarithmic reduction of number of microbial cells. Each antimicrobial test was performed in duplicate.

Results and discussion

The crystalline phase composition of freshly prepared materials is analyzed by XRD (Electronic Supplementary Information). All the photocatalysts exhibit crystalline monoclinic scheelite BiVO 4 structure (PDF 00-014-0688). In addition, no other phase is detected in Nb-BiVO 4 indicating that niobium is probably present in the form of oxide either amorphous or at nanosize level. This observation is confirmed by EDX (Energy Dispersive X-ray Spectroscopy) measurement which detected niobium in Nb-BiVO 4 sample. Both BiVO 4 /TiO 2 composites exhibit additional anatase phase (PDF 00-021-1272). The energy band gap of pure and Nb-modified BiVO 4 is evaluated at 2.4 eV approximatively for both materials by using the Tauc's method, i.e. they absorb light in the visible region (at λ ≤ 500 nm).

However, the composites exhibited a blue shift in the energy band gap (2.5 eV) due to screening effect of TiO 2 that is absorbing only in the UV region.

Strong differences appear in the morphology of film surface. In Fig. 1, it is clear that Nb-BiVO 4 (Fig. 1c) exhibits a porous surface morphology presenting a hierarchical structure while pristine and composite samples (Fig. 1a,b) have a more dense and homogeneous surface. Concerning the both composites, top-BiVO 4 /TiO 2 sample exhibits similar morphology than pure BiVO 4 (Fig. 1a) while top-TiO 2 /BiVO 4 has similar surface than pure TiO 2 (Fig. 1b). The photocatalytic degradation of RhB was followed under simulated solar irradiation and the corresponding degradation rates are presented in Fig. 2. It is worth to notice that the photocatalysts remain intact after their use since XRD patterns did not change (Electronic Supplementary Information).

Comparing the tested photocatalysts, Nb-BiVO 4 exhibits the best photodegradation efficiency with a removal of 72% of RhB after 180 min irradiation. Such performance is due to the beneficial morphology of the film surface that exhibits high porosity and thus higher active surface area than the other materials (Fig. 1). Both composites exhibit also excellent photoxidative properties by degrading from 56 to 61% of pollutants which is better than the single components (Fig. 2a). It means that composites are constituted of efficient heterojunction between titania and bismuth vanadate that improves electronhole pair separation and transport within the material: photogenerated e -are accumulated in TiO 2 while h + are concentrated in BiVO 4 . 13 It is also noteworthy that top-TiO 2 /BiVO 4 is more efficient than top-BiVO 4 /TiO 2 (Fig. 2a). The photocatalytic mechanism was evaluated using different charge carriers (Fig. 2b). It is obvious that photocatalytic degradation proceeds mainly through photogenerated holes using films exhibiting BiVO 4 -based material at their surface (i.e. pristine BiVO 4 , Nb-BiVO 4 and top-BiVO 4 /TiO 2 composite) while films with titania at the surface oxidize essentially through superoxide and hydroxyl radicals. Indeed, the photocatalytic activity of top-TiO 2 /BiVO 4 and pure TiO 2 is strongly inhibited in presence of O 2

•-and OH • scavengers indicating that they are the main oxidative species (Fig. 2b).

Similarly, the other photocatalysts show a strong decrease in photooxidative degradation rate of RhB in presence of ammonium oxalate which are thus the main reactive species in this case (Fig. 2b).

The antimicrobial properties of BiVO 4 -based films are summarized in Fig. 3 after simulated solar irradiation and in the dark. It is clear that in absence of light, the series of BiVO 4 -based photocatalysts are not able to inactivate the MRSA strain (Fig. 3a). There is also no photolysis of bacterial cells under irradiation alone. Under our conditions, 30 min irradiation time is too short to put in evidence any antimicrobial effect (Fig. 3b). On the other hand, after 60 min irradiation (Fig. 3b), Nb-BiVO 4 and top-BiVO 4 /TiO 2 composite exhibit significant photo-induced antimicrobial properties (43 and 39% inactivation, respectively) while the remaining samples show poor bactericidal activity. It is remarkable that, in case of top-TiO 2 /BiVO 4 composite, only weak photo-disinficient properties are observed although this composite has good photooxidative power with the production of superoxide and hydroxyl radicals. So far, we cannot explain this observation. But it could be assumed that inactivation of the bacterial strains depends not only on the degradation mechanism but also on other factors related to the bacterial cell such as its wall structure. Thus it is probable that the affinity of MRSA is higher toward BiVO 4 surface than titania surface. In our case, the two antimicrobial active samples (Nb-BiVO 4 and top-BiVO 4 /TiO 2 ) exhibit a photocatalytic power through holes and their bactericidal activity improves by the fact that (1) Nb-BiVO 4 film exhibits porous structure with high surface area (Fig. 1c) and ( 2) top-BiVO 4 /TiO 2 composite has improved electron-hole pair separation giving rise to increased lifetime of holes. 14 In addition it has been reported that photogenerated holes from BiVO 4 can act as efficient oxidative agents toward bacterial cells if close contact between the photocatalyst and the bacteria is achieved. 18,23,24 Therefore from the above discussion, it could be assumed that the high antimicrobial activity of Nb-BiVO 4 is the result of fullfilled conditions for an efficient attack of the cell membrane by photogenerated holes: high surface area combined with good affinity of MRSA at bismuth vanadate photocatalyst surface.

Conclusion

Conflicts of interest

There are no conflicts to declare.

Figure 1 .

 1 Figure 1. Surface morphology of (a) pristine BiVO 4 and (inset) top-coated BiVO 4 over TiO 2 composite, (b) pure TiO 2 and (inset) top-coated TiO 2 over BiVO 4 composite and (c) Nb-BiVO 4 .

Figure 2 .

 2 Figure 2. (a) Degradation rates of RhB after 180 min irradiation under solar-like light and (b) the corresponding effects of charge scavengers on the removal efficiencies of RhB.

Figure 3 .

 3 Figure 3. Survival of MRSA strain on the different BiVO 4 -based materials (a) in the dark, (b) after 30 and 60min solar-like irradiation.

Different supported BiVO 4 -

 4 based materials in the form of films were tested in both the photocatalytic degradation of RhB and the inactivation of MRSA strain under simulated solar irradiation. It was found that the best material was Nb-BiVO 4 due to its beneficial morphology which results in high degradation of pollutant and substantial bacterial inactivation. The mechanism of photo-induced properties using Nb-BiVO 4 involved directly the photogenerated holes. It is the first time that both photocatalytic and antimicrobial activities of different supported BiVO 4 systems are simultaneously reported under the same conditions, especially for the inactivation of clinically important MRSA which achieves here 43% in presence of Nb-modified BiVO 4 .

Acknowledgements

Authors wish to acknowledge the financial supports granted by the Scientific Grant Agency of the Slovak Republic and the Statutory Research of the Wroclaw Medical University through the projects VEGA 1/0276/15 and ST.A130.16.032, respectively. Authors gratefully thank Tomas Roch for XRD measurements. This work was also supported by the Research and Development Operational Program funded by the ERDF (project ITMS:26240220027).


A Guibert

P Loisel

D Heitz

N Auvinet

T Bioteau

L Blondel

G Nunes

P Peu

J Buffet

Measurements of methane leaks from biogas plant based on infrared camera

Keywords: Methane emissions, infrared imaging, optical flow, biogas plant, leak

Minimization of methane leakages in biogas plants is a challenge to ensure the environmental benefit of anaerobic digestion systems. Current estimation methods highlight uncertainties mainly due to spatial and temporal variations of leakages. Therefore, a real-time detection and quantification technology would be the achievement of a great bet to limit such uncertainties. The presented novel method approaches this optimum. The technique is based on a turbulent gas flow estimator model adapted to infrared image sequences. After a lab calibration phase, on-site experiments were conducted to validate the new method and compare its results with traditional measurements techniques.

INTRODUCTION

Targeted benefits of anaerobic digestion through the development of biogas plants are the reduction of methane emissions inherent to conventional manure management, the recycling of organic waste, the contribution to renewable energy, and the substitution of mineral nitrogen by the digestate. To ensure the environmental benefit of anaerobic digestion, biogas leaks should be as low as possible. According to [START_REF] Borjesson | Environmental systems analysis of biogas systems -Part II: The environmental impact of replacing various reference systems[END_REF] leaks of biogas plant are normally less than 2%, but can vary between 0.2% and 13%. To reduce biogas emissions it is important to gather precise knowledge about the biogas leaks occurring in the production chain. [START_REF] Holmgren | Measurements of methane emissions from biogas production[END_REF] showed large variations in the estimation of leaks when comparing more or less classical methods. In this study we propose an alternate approach by extending Optical Gas Imaging (OGI), classically used for detection, to gas leak quantifications. [START_REF] Sandsten | Volume flow calculations on gas leaks imaged with infrared gas-correlation[END_REF] and [START_REF] Shao-Hua | Gas Diffusion Optic Flow Calculation and Its Applications in Gas Cloud Infrared Imaging[END_REF] developed infrared gas diffusion flow estimators, demonstrating the interest of these new nonintrusive techniques. In the present study, we propose a novel turbulent fluid dedicated optical flow approach to estimate the leaks. For this purpose controlled laboratory experiments and on-site measurements have been carried out.

MATERIALS AND METHODS

Infrared cameras for gas leaks detection

Especially for safety reasons, the use of OGI is frequent in the detection of leaks for the oil and gas industry. This technique is also being developed in the field of biogas plants. OGI is based on infrared absorption of a specific gas of interest. In this study, we used Flir GF320 camera which is largely used for its usability and which provides real time methane leak visualization. Flir GF320 camera's resolution is 320 x 240 pixels at 30 frames per second. Detection threshold is low, about 2.5 Nl/h (B. Thomas et al., 2012). A detection campaign pinpointed more than 60 leaks in 12 different biogas installations. This gave us an overview of the types of leakages encountered in agricultural sector.

New gas leaks quantification method by infrared cameras

The proposed Methane Flow Estimator (MFE) is based on methane clouds velocity and its concentration-length. The velocity vectors field -as shown in figure 1 and corresponding to the gas motion between two consecutive infrared images-is estimated with a turbulent fluid dedicated optical flow approach (P. [START_REF] Héas | Power laws and inverse motion modeling: application to turbulence measurements from satellite images[END_REF][START_REF] Héas | Bayesian estimation of turbulent motion[END_REF]. Concentration-length is estimated from pixels' luminance. Therefore, the MFE is a combination of velocity profiles and concentration-length profiles along the gas cloud.

Lab-scale experiment

The estimator was calibrated and validated in laboratory experiments. Controlled leaks were generated by mixing methane and carbon dioxide with a 60/40 proportion. Two Bronkhorst mass flow meter provided methane and carbon dioxide flow with high accuracy. Leaks were generated in a wind tunnel where the air temperature and wind speed were controlled. After a first campaign of validation in wind tunnel, a series of tests were conducted outdoor with controlled leaks. In the last step of lab-scale experiments, a prototype was developed to improve acquisition conditions.

On-site measurements

On-site campaigns were conducted at a small-scale agricultural biogas plant (150 kWe) located in Brittany (France). Such experiments were carried out to validate the novel method, i.e. OGI extended to quantification thanks to the MFE described above. The validation was done through two complementary approaches, a standard reference method which is bagging and a tracer gas method. Ten leaks were detected around the digester. In this paper, we focused on one leakage located on a digester trapdoor. This leak was chosen to make possible synchronised measurements between OGI+MFE estimation and the bagging technique involving a gas analyser and a hot wire anemometer.

RESULTS AND DISCUSSION

Lab-scale validation

Figure 2A shows estimator accuracy for 12 flows rate generated in the wind tunnel. In these optimal experimental conditions, mean error was of 5% up to 120 l/h. For highest leakages the gas flow was underestimated. This behavior was due to the large gas cloud displacement between two frames (higher than 20 pixels). It's possible to reduce this effect by moving the camera back. Instantaneous gas flow estimation is shown in figure 2B for a fixed flow at 60 l/h. As we can see, the technique exhibits rather low variations of the instantaneous estimated flow. 45 tests were performed to characterize the method in different conditions like background temperature, gas humidity, wind speed, and camera distance. The accuracy of the results is mainly affected by the large displacements and by the difference between background temperature and gas temperature (dT). The sensitivity of the estimator to such operational parameters deducted from the lab is important to be known before an on-site experiment. Hence, we developed a prototype to improve measurements conditions when necessary.

On-site validation

Figure 3 shows for one leak the gas flow estimation given by the infrared camera and the bagging technique. The mean flow estimation was 38 l/h for the infrared technique and 51 l/h for the bagging technique. By extrapolating the measurements to one year, this leak represents 332 NM 3 of methane, corresponding to less than 0.1% of the annual production of the installation considered.

Figure 1 .

 1 Figure 1. Infrared gas image with superposed velocity vector field.

Figure 2 .

 2 Figure 2. Methane flow estimation (blue) and reference (red) at the laboratory: (A), Mean flow estimation for several leakage rates; (B), Instantaneous flow estimation for one leakage rate.

Figure 3 .

 3 Figure 3. Infrared measurement (blue) and bagging technique (red) for one leak at biogas plant.

ACKNOWLEDGEMENT

We are very grateful to the French Environment & Energy Management Agency (ADEME) for its support and funding for this project named TrackyLeaks.

CONCLUSIONS

In this study, a novel infrared methane flow estimation technique was presented for biogas leaks measurements. The method was developed and validated through controlled laboratory experiments and on-site measurements. Results showed that our method provided very accurate estimations when the gas cloud displacements in the images are not too large and with sufficient thermal contrast between gas and background. Further developments will tackle large displacement and thermal contrast issues, and improve on-site measurement context. On-site measurements confirmed the ability of the technique to estimate biogas leak with encouraging accuracy. Process of all detected leaks of an installation is currently in progress to provide the overall emission rate for one typical biogas plant.