MD Jérôme Dumortier
email: jerome.dumortier@chu-lyon.fr

MD Tomas Dedic

MD Domitille Erard-Poinsot

MD Christine Rivet

MD Olivier Guillaud

MD Christine Chambon-Augoyard

MD Alexie Bosch

MD Alain Lachaux

MD Eduardo Couchonnal

MD Olivier Thaunat

MD Olivier Boillot

MD Valérie Dubois

Pregnancy and donor-specific HLA-antibody-mediated rejection after liver transplantation: "Liaisons dangereuses"?

Keywords: liver transplantation, rejection, anti-HLA antibodies, pregnancy Humoral rejection and pregnancy

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

INTRODUCTION

Antibodies-mediated rejection (AMR) is related to donor-specific anti-HLA antibodies (DSA) and has been extensively studied in all kind of solid organ transplantations including recently liver transplantation (LT) [START_REF] Terasaki | A personal perspective: 100-year history of the humoral theory of transplantation[END_REF]. Outside the field of LT, many studies have shown that both preformed and de novo DSA were associated with increased risk of acute rejection and graft failure [START_REF] Terasaki | Four-year follow-up of a prospective trial of HLA and MICA antibodies on kidney graft survival[END_REF][START_REF] Morales-Buenrostro | A single human leukocyte antigen-antibody test after heart or lung transplantation is predictive of survival[END_REF][START_REF] Cantarovich | Posttransplant donorspecific anti-HLA antibodies negatively impact pancreas transplantation outcome[END_REF][START_REF] Abu-Elmagd | Preformed and de novo donor specific antibodies in visceral transplantation: long-term outcome with special reference to the liver[END_REF]. Nevertheless, it has been suggested since the early 90's that positive crossmatch could increase risk of early graft loss after LT [START_REF] Takaya | Liver transplantation in positive cytotoxic crossmatch cases using FK506, high-dose steroids, and prostaglandin E1[END_REF][START_REF] Ogura | High one-month liver graft failure rates in flow cytometry crossmatch-positive recipients[END_REF][START_REF] O'leary | Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation[END_REF][START_REF] Musat | Pretransplant donor-specific anti-HLA antibodies as predictors of early allograft rejection in ABO-compatible liver transplantation[END_REF]. Similarly, in the more recent years, several studies have shown that post-LT DSA may play a role in acute and chronic rejection [START_REF] Kasahara | Postoperative flow cytometry crossmatch in living donor liver transplantation: clinical significance of humoral immunity in acute rejection[END_REF][START_REF] Musat | The significance of donor-specific HLA antibodies in rejection and ductopenia development in ABO compatible liver transplantation[END_REF][START_REF] Kozlowski | Sinusoidal C4d deposits in liver allografts indicate an antibody-mediated response: diagnostic considerations in the evaluation of liver allografts[END_REF][START_REF] Kaneku | De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients[END_REF].

Risk factors for the development of anti-HLA antibodies include history of blood transfusion, organ transplantation (modulated by post-transplantation immunosuppression), and pregnancy. Huyn et al. evaluated how different sensitization events affect the panel-reactive antibody status in 674 (354 males and 320 females) solid organ transplant candidates [START_REF] Hyun | Effects of different sensitization events on HLA alloimmunization in solid organ transplantation patients[END_REF]. Reactive antibody (class I and/or II)positive rates were significantly higher in patients with transfusion (33.0%), pregnancy (71.4%), or transplantation events (76.9%) than in patients without any sensitization events (5.6%).

Transplantation had the strongest immunization effect, especially for class II HLA antigens. Female compared with male patients (60.3% vs. 34.2%) and retransplantation, compared with first transplantation candidates of kidney transplantation (80.2% vs. 41.1%) showed significantly higher rates. The aim of the present study was to assess the impact of pregnancy on the occurrence of de novo DSA and the impact of these DSA on graft outcome in a large population of young female LT recipients.

PATIENTS AND METHODS

Study design

The present single center retrospective study included all female patients who underwent a first LT between January 1990 and December 2010 at the Edouard Herriot University Hospital (Lyon, France) who were of childbearing age during post-LT follow-up (i.e. 18 to 40 years old) and who had both at least one screening for anti-HLA antibodies at day of transplantation (Day 0) and another screening after LT. Pre-and post-LT pregnancies and transfusions were recorded for all patients.

All transplantations were ABOc LT. The initial immunosuppressive regimen was based on calcineurin inhibitors: ciclosporine or tacrolimus. In addition, patients received 500 mg of methylprednisolone after reperfusion. Induction with polyclonal or monoclonal antibodies was performed in some recipients and in cases of renal failure at the time of the LT for delayed CNI introduction. On postoperative day 1, we began tapering methylprednisolone which was maintained at 20 mg/day thereafter, then tapered to a maintenance dose of 0 to 5 mg/day after 12 months post-transplantation.

Azathioprine, mycophenolate mofetil, or everolimus (EVR) were either administrated as a part of initial triple immunosuppressive regimen, or introduced during follow-up as maintenance immunosuppressive agents.

Screening was performed retrospectively on historical serum samples for the anti-HLA antibodies before 2010, and prospectively after 2010. DSA were considered as de novo when not present at D0. Timing between LT and DSA screening was calculated as the interval between transplantation, and either the last negative DSA screening or the first positive DSA screening. Concerning DSA screening from historical serum, it was usually performed at 1-, 5-and 10-years protocolized evaluation after transplantation (serum were obtaines from our virology department).

Liver function tests (serum aspartate aminotransferase -AST, alanine aminotransferase -ALT, gamma-glutamyl transpeptidase -GGT, alkaline phosphatase -ALP, total bilirubin -TBil, direct bilirubin -DBil, and indirect bilirubin -IBil) and measurement of immunosuppressive drug blood levels were performed by the hospital's laboratory during hospitalizations, and externally before routine patient visits.

Protocol liver biopsies were planned at 1, 5 and every 5 years thereafter from transplantation. All biopsies performed for liver function test abnormalities and protocol biopsies were reviewed. For diagnosis and grading of allograft rejection, Banff classification published in 1997 and updated in 2000 [START_REF] O'leary | Antibody-mediated rejection as a contributor to previously unexplained early liver allograft loss[END_REF] was used. All liver biopsies with features of rejection were reviewed and the histological diagnosis of AMR, including C4d staining, was made from the Banff update of 2016 [START_REF] Couchonnal | Deleterious impact of C3dbinding donor-specific anti-HLA antibodies after pediatric liver transplantation[END_REF].

To identify risk factors for de novo DSA and their impact, the DSA-positive and DSA-negative populations were compared with regards to clinical data.

Immunological analyses

All analyses were performed in a blinded fashion by a trained immunobiologist (V.D. at the Etablissement Français du Sang, Lyon, France). HLA typing of donors and recipients was performed by Luminex PCR-SSO reverse (One Lambda, Canoga Park, CA, US). Serum samples were analyzed using Luminex Single Antigen Flow Beads assays (LSA class I and class II, Lifecodes, Immucor, Norcross, GA, US). The mean fluorescence intensity (MFI) was measured on a LABscan IS 200, and all the specificities were evaluated with the company's defined threshold such as MFI ≥1500 and AD-BCR ≥5 to define positivity (AD-BCR is a ratio of adjusted MFI to the quantity of coated antigen per bead). The positivity of these two criteria together allows us to consider the corresponding specificities as a preformed DSA. In some few cases, only the AD-BCR is higher than 5, with MFI between 500 and 1500. In these cases, the DSA is validated only for post transplantation follow-up (de novo DSA) when the corresponding graft mismatch Antigen is not included in a CREg positive reaction.

Statistical analysis

Categorical variables were expressed as percentages and compared with the Chi-squared or Fischer's exact tests. Continuous variables were expressed as mean ± SD and compared using the Student's t test or the Mann-Whitney's test in case of non-Gaussian distributions. Patient survival was calculated from the date of LT to that of death or the final clinical visit. Graft survival was calculated from the date of LT to that of retransplantation, death or last visit if there was no retransplantation. Survival curves were constructed using the Kaplan-Meier method and compared with the log-rank test. The predictive factors for developing de novo DSA were determined by univariate and multivariate regression analyses. A p value less than 0.05 was considered statistically significant. Statistical analyses were performed using SPSS software, version 13.0 (IBM, Armonk, NY, US).

RESULTS

Study population

During the 1990-2016 periode, a total of 101 female LT recipients of childbearing age were followed in our center. Among these, 73 were screened for anti-HLA antibodies on the day of transplantation, and also had at least one other screening after LT and were included in the study population. The mean age at LT was 20.9 years (0.6-39.9); 32 patients were transplanted during childhood. After LT, 33 patients experience one or more pregnancies (including miscarriages or interrupted pregnancies).

Immunosuppressive regimen at the time of pregnancies consisted in tacrolimus (n=27, 81.8%), cyclosporine (n=6, 18.2%), MMF (n=6, 18.2%), azathioprin (n=3, 9.1%), corticosteroids (n=7, 21.2%). For patients under MMF, pregnancies were unexpected.

From the study population, 3 patients (4.1%) had preformed DSA (all had history of pregnancy before LT), and 31 patients (42.5%) developed de novo DSA, after a median delay of 15.5 years (1-25) of follow-up after LT. Most de novo DSA were anti-class II alone (90.3%), and included anti-DQ for 80.6%. Median maximal MFI was 7,500 (600-17,500) for de novo DSA.

Risk factors for DSA

Post-LT presence of de novo DSA was significantly more frequent in patients with history of pregnancy and in pediatric LT recipients (Table 1). Recipient age, at the time of LT was also significantly lower in patients who developed de novo DSA. Living donor LT (vs. cadaveric), history of massive red blood cells transfusion, history of platelets transfusion, initial immunosuppressive therapy, maintenance immunosuppressive therapy or trough level of tacrolimus were not associated with the presence of de novo DSA. Multivariate analysis disclosed that history of pregnancy (OR=6.37; 95%CI, 2.17-18.52) and age at LT (OR=1.05; 95%CI, 1.01-1.09) were significantly associated with de novo DSA.

AMRs

Among the 31 patients who had de novo DSA, the diagnosis of antibody-mediated rejection was made in 8 patients (23.6%), after a median delay of 74 months after LT; 6/8 (75.0%) had history of post-LT pregnancy. These patients had class II DSA, with MFI ranging from 1,500-28,600, and liver graft dysfunction. In 4 patients, treatment consisted of steroids, rituximab, plasmapheresis, and immunoglobulins. During follow-up, 3 of these 8 patients lost their grafts and died.

Survival

According to Kaplan-Meier estimates, patient and graft survivals were not significantly different according to the presence or not of de novo DSA (Figure 1A andB).

In the group of patients without DSA, causes of death/graft loss were chronic rejection (n=3), ischemic cholangiopathy (n=1), or recurrent initial disease (n=1). In the group of patients with DSA, causes of death/graft loss were chronic rejection (n=4), recurrent initial disease (n=2), or lung carcinoma (n=1).

DISCUSSION

We report herein that post-LT pregnancy is associated with an increased risk for the development of de novo DSA, and thereafter DSA-mediated rejection, with poor outcome in some cases. The impact of DSA on patient and graft survival after LT has been more extensively studied in the recent years, and it has been suggested that presence of DSA could be associated with higher risk of rejection and decreased liver allograft survival after adult or pediatric LT in most [START_REF] O'leary | Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation[END_REF][START_REF] Musat | Pretransplant donor-specific anti-HLA antibodies as predictors of early allograft rejection in ABO-compatible liver transplantation[END_REF][START_REF] Kaneku | De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients[END_REF][START_REF] Castillo-Rama | Preformed antibodies detected by cytotoxic assay or multibead array decrease liver allograft survival: role of human leukocyte antigen compatibility[END_REF][START_REF] Goh | Human leukocyte antigen crossmatch testing is important for liver retransplantation[END_REF][START_REF] Kozlowski | Liver allograft antibody-mediated rejection with demonstration of sinusoidal C4d staining and circulating donor-specific antibodies[END_REF][START_REF] O'leary | High mean fluorescence intensity donor-specific anti-HLA antibodies associated with chronic rejection Postliver transplant[END_REF][START_REF] O'leary | Antibody-mediated rejection as a contributor to previously unexplained early liver allograft loss[END_REF][START_REF] O'leary | Impact of IgG3 subclass and C1q-fixing donor-specific HLA alloantibodies on rejection and survival in liver transplantation[END_REF][START_REF] Bello | De novo Donor-specific anti-HLA antibodies mediated rejection in liver-transplant patients[END_REF][START_REF] Bello | Prevalence, incidence and risk factors for donor-specific anti-HLA antibodies in maintenance liver transplant patients[END_REF][START_REF] Couchonnal | Deleterious impact of C3dbinding donor-specific anti-HLA antibodies after pediatric liver transplantation[END_REF], but not all studies [START_REF] Ruiz | Implications of a positive crossmatch in liver transplantation: a 20-year review[END_REF][START_REF] Taner | Prevalence, course and impact of HLA donor-specific antibodies in liver transplantation in the first year[END_REF][START_REF] Kim | Preformed donor-specific antibodies do not affect the 1-year allograft survival in living donor liver transplantation[END_REF][START_REF] Vandevoorde | Prevalence, Risk Factors, and Impact of Donor-Specific Alloantibodies After Adult Liver Transplantation[END_REF]. In our present study, there was a numerical difference in patient death and/or graft loss according to the presence or not of DSA, and the lack of statistical significance was probably related to the small number of patients. Results and interpretation may depend on DSA type (preformed or de novo), DSA class (I or II) and cut-off value with Luminex assays, together with the period of follow-up after LT (1-, 5-, 10-, 15-, 20-years). In the large study of Kaneku et al., 8.1% of LT recipients developed de novo DSA with a MFI >5000 1-year after transplantation, and almost all de novo DSA were against HLA class II antigens (the majority against DQ antigens). This strongly recalls our present results. In addition, multivariable analysis disclosed that the use of cyclosporine (as opposed to tacrolimus) and low calcineurin inhibitor levels increased the risk of de novo DSA formation, while a calculated MELD score >15 at the time of LT and recipient age >60 years old reduced the risk.

Our results strongly support that pregnancy could play a major role in DSA-mediated rejection after LT. This is probably rare since young women able to become pregnant after LT are in very limited number. One of our unfavourable cases seems to be caricatural since the woman was transplanted with a liver graft from a living donor, her husband. Such cases of accelerated rejection following offspringto-mother and husband-to-wife transplants have been previously reported [START_REF] Rosenberg | Accelerated rejection following offspring-to-mother and husband-to-wife transplants[END_REF]. Rebibou et al. reported that about 50% of women develop anti-HLA antibodies after 3 pregnancies [START_REF] Rebibou | Flow cytometric evaluation of pregnancy-induced anti-HLA immunization and blood transfusion-induced reactivation[END_REF].Interestingly, Ayna et al. studied DSA in the sera of patients planned for living kidney transplantation from their spouse or children [START_REF] Ayna | Flow cytometric evaluation of pregnancy-induced anti-donor immunization[END_REF]. The study population included 37 wives (group I) and 48 husbands (group II). Among group I were 48.6% of positive cross-match vs. 10.4% for group II (p=0.001). In addition, anti-HLA antibodies were detected in 94.4% of the patients with positive cross-match in group I vs. 60% among group II. Honger et al. investigated the frequency and determinants of pregnancy-induced childspecific sensitization shortly after full-term delivery of 301 non transplanted mothers and their children [START_REF] Honger | Frequency and determinants of pregnancy-induced child-specific sensitization[END_REF]. The overall frequency of pregnancy-induced sensitization ranged between 45% (MFI > 1000 cut-off) and 76% (ratio cut-off). The rate of child-specific sensitization at the HLA-A/B/C/DRB1 loci was between 28% (MFI > 1000 cut-off) and 38% (ratio cut-off). The number of live birth was associated with a higher frequency of sensitization. There was a clear hierarchy of sensitization among the investigated loci 31%; A-locus: 26%; DRB1-locus: 20%; C-locus: 15%; p < 0.0001). Some mismatched paternal HLA-antigens led to a significantly higher rate of sensitization than the average (e.g. HLA-A2, HLA-B49, HLA-B51, HLA-C*15). We cannot investigate this effect because we did not obtain the father's typing in most cases. When we got it, we showed that common HLA Ag between father and graft is an additional factor to develop DSA and antibody mediated rejection after pregnancy [START_REF] Ducreux | Monitoring efficiency of humoral rejection episode therapy in liver transplantation: any role for complement binding Luminex Single Antigen assays?[END_REF]. Furthermore, the mother's own HLA-phenotype (especially HLA-A/B homozygosity) was also associated with a higher rate and broadness of sensitization. The number of mismatched HLA-A/B/C eplets between both parents, strongly correlated with the rate of child-specific class I sensitization. Interestingly, it has recently been suggested, in kidney transplantation, that pregnancy-induced DSA could be more reactive than antibodies induced by prior transplantation [START_REF] Higgins | Pregnancy-induced HLA antibodies respond more vigorously after renal transplantation than antibodies induced by prior transplantation[END_REF]. In addition to the role of pregnancy, we confirm herein that younger age is also a predictive factor for DSA development. For instance, we reported a rate of 33.0% 10-years after LT in pediatric recipients, vs. 19.5% in adults [START_REF] Couchonnal | Deleterious impact of C3dbinding donor-specific anti-HLA antibodies after pediatric liver transplantation[END_REF][START_REF] Vandevoorde | Prevalence, Risk Factors, and Impact of Donor-Specific Alloantibodies After Adult Liver Transplantation[END_REF]. We were not able to investigate the role of non-adherence to immunosuppressive treatment, but it has been strongly demonstrated that younger recipients (especially adolescents) of organ transplant have higher risk to be non-adherent [START_REF] Dobbels | Adherence to the immunosuppressive regimen in pediatric kidney transplant recipients: a systematic review[END_REF][START_REF] Meng | Adherence to medical regimen after pediatric liver transplantation: a systematic review and meta-analysis[END_REF][START_REF] Yazigi | Adherence and the pediatric transplant patient[END_REF]. In summary, the high incidence of de novo DSA in our population could be due to a long follow-up, the young age of our patients (including pediatric recipients), non-adherence (associated with younger age), and also the high rate of pregnancies.

Since the diagnosis of DSA-mediated rejection can be highly suspected from clinical, biological, immunological and histological features, the question of adapted treatment is the major goal, and is matter of debate. Reports of different protocols and regimens have been published, each with different outcomes and sometimes contradictory findings. In the past recent years, multiple reports using IVIG, rituximab and plasmapheresis, as single agents or in combination, have emerged [START_REF] Singh | Antibody-mediated rejection: treatment alternatives and outcomes[END_REF]. Nevertheless, very few data are available in LT recipients, reported as individual cases [START_REF] Kamar | Early plasmapheresis and rituximab for acute humoral rejection after ABO-compatible liver transplantation[END_REF][START_REF] Barth | Antibody-mediated rejection of renal allograft in combined liver-kidney transplant[END_REF][START_REF] Lee | Bortezomib is effective to treat acute humoral rejection after liver transplantation[END_REF]. We used such therapy in 4 severe cases of AMR we observed, and the outcome was favourable in 3. In one case, improvment was partial probably because of severe liver damage and pre-existing cirrhosis, leading to the indication of retransplantation, but the patient died before retransplantation. Probably, the treatment should be started as soon as possible in order to prevent irreversible liver graft lesions.

Finally, our study has some limitations. First, except in some rare cases of husband living donors, HLA grouping of children's fathers was not available, to confirm that father and graft had some common antigens associated with DSA. The determination of the HLA of the father before pregnancy could better inform the woman about the possible impact of pregnancy on the risk of developing DSA.

Second, regarding the retrospective nature of our study, we cannot exclude that some pregnancies (only miscarriages or interrupted pregnancies) have not been recognized. Third, we cannot be sure that DSA appeared after pregnancy since we have not systematic DSA screening both before and after pregnancy. Nevertheless, the difference we observed between women with history of pregnancy vs. not (71.0 vs. 26.2%) strongly supports the major impact of pregnancies. Recalling the design of our study, Hebral et al. analysed 61 pregnancies that occurred in 46 kidney transplanted women [START_REF] Hebral | Pregnancy after kidney transplantation: outcome and anti-human leucocyte antigen alloimmunization risk[END_REF]. De novo DSA were detected after only 5.9% of pregnancies, but 27.0% of the women had DSA before pregnancy.

In conclusion, DSA-medited rejection is probably a rare but well recognized entity in LT recipients.

Close monitoring of at risk patients should be recommended, including young women after pregnancy.

Legends for figures

Figure 1 :

 1 Figure 1: Patient (A) and graft (B) survival, according to the presence of DSA (no significant

Table 1 :

 1 (A)The probability of patient survival was 97.6%, 97.6%, 93.4%, 93.4% and 93.4% at 5, 10, 15, 20 and 25 years, respectively, for patients without DSA, vs. 100.0%, 92.9%, 88.9%, 84.2% and 77.2% at 5, 10, 15, 20 and 25 years, respectively, for patients with DSA (p=0.20). (B) The probability of graft survival was 97.6%, 97.6%, 93.4%, 88.7% and 77.6% at 5, 10, 15, 20 and 25 years, respectively, for patients without DSA, vs. 100.0%, 89.6%, 81.5%, 76.9% and 69.9% at 5, 10, 15, 20 and 25 years, respectively, for patients with DSA (p=0.45). Risk factors for de novo DSA

	de novo DSA	no de novo DSA	p-value
	(n=31)	(n=42)

* at the time of DSA screening

funding source for this study. Lifecodes (Immucor) donated reagents but was not involved in analysis of data, writing of the report, and in the decision to submit the paper for publication.


S Rodosik

J.-P Poirot-Crouvezier

Y Bultel
email: yann.bultel@grenoble-inp.fr

Impact of humidification by cathode exhaust gases recirculation on a PEMFC system for automotive applications

Keywords:

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

Introduction

With the growing interest in the hydrogen economy, Proton Exchange Membrane Fuel Cells are promising components for systems dedicated to transport applications. Indeed, their low operating temperature and high energy density make them suitable for the automotive industry. However, these systems are still facing several challenges on their way to mass market production. Reaching a durability of 5000 h recommended by the U.S. Department of Energy and decreasing system cost are major hurdles to their development.

Gases humidification in PEMFC systems is necessary to ensure best performance as well as long-term stability. It allows establishing a proper water management that is to say, operate with stable conditions avoiding cell flooding and drying in the stack, and favor a longer lifetime. Despite its large volume and thermal inertia, the most used technology at the cathode side is a passive humidification technique, called in this study "classic humidification". This technology allows transport to the cathode inlet of water collected at the cathode exhaust, thanks to selective mass transfer through hollow fiber membranes. It can be classified among "external humidification solutions", where humidification of the gaseous reactant flow is realized before the fuel cell inlet. Even though fuel cell durability is strongly dependent on water management [START_REF] Borup | Scientific Aspects of Polymer Electrolyte Fuel Cell Durability and Degradation[END_REF][START_REF] Yousfi-Steiner | A review on PEM voltage degradation associated with water management: Impacts, influent factors and characterization[END_REF][START_REF] Pei | Main factors affecting the lifetime of Proton Exchange Membrane fuel cells in vehicle applications: A review[END_REF], relative humidity control with such component is hardly feasible without affecting the operating conditions and stack voltage. Many other external humidification solutions have been developed, including liquid water transport through membranes, bubblers, enthalpy wheels, or direct water injection, in liquid or vapor form. The latter offers a possibility to control the water amount injected at the cathode inlet whereas passive techniques are completely dependent on the fuel cell operating conditions.

In some cases, no external humidification solution is used in fuel cell systems, which is designated as "internal humidification solutions". These latter consist in self-humidification of the fuel cell, where the reactant flows are humidified directly inside the fuel cell by the water produced by the electrochemical reaction. They can be improved by modifications of the cell components material composition in either the MEA or the gas diffusion layer or the cell design, more specifically channel design [START_REF] Williams | Operation of Nafion®-based PEM fuel cells with no external humidification: influence of operating conditions and gas diffusion layers[END_REF] [START_REF] Park | Effect of the hydrophilic and hydrophobic characteristics of the gas diffusion medium on polymer electrolyte fuel cell performance under non-humidification condition[END_REF]. Self-humidification can also be achieved by special operating control strategies such as low temperature operation or high pressure [START_REF] Eckl | Experimental analysis of water management in a self-humidifying polymer electrolyte fuel cell stack[END_REF] [7] focusing on water transport through the fuel cell membrane.

Several external and internal humidification solutions have been studied in literature. Büchi and Srinivasan [START_REF] Büchi | Operating Proton Exchange Membrane Fuel Cells Without External Humidification of the Reactant Gases Fundamental Aspects[END_REF] tested a single cell of 50cm² active area during 1800h with an internal humidification solution, i.e. fed with dry hydrogen and air. They observed 20 to 40% performance losses compared to an external humidification solution enabling the injection of gases saturated with water vapor. Besides, they noticed that the maximum temperature to establish a stable behavior of the fuel cell with the self-humidification mode was 70°C.

Otherwise, the cell tended to dry out. They also concluded that diffusion of water through the membrane was sufficient to ensure water management in a wide range of operating conditions. Recently, Martin et al. [START_REF] Martin | Long-term operation of a proton exchange membrane fuel cell without external humidification[END_REF] tested several Pt loading electrodes prepared by the electrospraying deposition technique. These cells were dedicated to self-humidification operation. They achieved long-term operation but at a maximum temperature of 60°C and obtained low performance: 600mV at 0.2A/cm². Hwang and Kim [START_REF] Hwang | An experimental study on the cathode humidification and evaporative cooling of polymer electrolyte membrane fuel cells using direct water injection method at high current densities[END_REF] studied the coupled effect of humidification and cooling using direct liquid water injection inside the fuel cell. The experiments were held at high current densities with a 5-cells PEMFC stack with 250 cm² MEA. They concluded that the method was more efficient for cell humidification at the lowest temperature (i.e. 60°C) and obtained better performance than with an external humidification system enabling an inlet relative humidity (RH) of 60%. Migliardini et al [START_REF] Migliardini | Experimental comparison between external and internal humidification in proton exchange membrane fuel cells for road vehicles[END_REF] studied three different external humidification solutions at the cathode including bubblers, heat and mass exchange by selective polymeric membranes and active humidification by water injection.

They compared it with an internal humidification solution. In their study, self-humidified fuel cells were considered as the best solution for automotive applications, with high control possibilities but at a maximum cell temperature of 55°C. Sanchez et al [START_REF] Sanchez | Analysis of the Influence of Temperature and Gas Humidity on the Performance Stability of Polymer Electrolyte Membrane Fuel Cells[END_REF] studied the performance and stability of the cell voltage with temperature and relative humidity variations. They compared voltage responses at 60°C and 80°C on a single cell of 142cm².

At equivalent relative humidity levels (50/50 at both sides), they recorded a voltage improvement of 4.8% at 80°C. However, decreasing cathode RH to 20% was sensitive and caused a drying behavior at higher temperature due to a flowrate higher at the cathode than at the anode. Indeed, the voltage response could drop of 21%. They noticed that drying could be compensated by a sursaturated flow at the anode side and recorded a voltage drop of only 8%. They finally recommended to operate at very high relative humidity at the anode when the humidification at the cathode is inferior to 50%. Iranzo et al. [START_REF] Iranzo | Investigation of the liquid water distributions in a 50 cm2 PEM fuel cell: Effects of reactants relative humidity, current density, and cathode stoichiometry[END_REF] performed a parametric study on a 50-cm² cell including RH, current density and air stoichiometric ratio variations and concluded that cathode RH had a stronger influence on the cell performance than anode RH. Neyerlin [START_REF] Neyerlin | Effect of Relative Humidity on Oxygen Reduction Kinetics in a PEMFC[END_REF] studied the Oxygen Reduction Reaction kinetics under low relative humidity and concluded that a minimum RH level around 50-60% was necessary for the ORR to occur independently from RH values.

All of these studies show that water management with either internal or external humidification solutions is complex. An accurate humidification can be achieved with selfhumidification in variable conditions but only at low temperature, whereas external humidifiers allow to achieve better cell performance at high temperature. Nevertheless, the latter are not suitable to ensure best water management following power variations, such as automotive conditions, because of their slow dynamic. Besides, low temperature operation lead to a higher cooling subsystem sizing. As automotive system volume tend to be reduced in order to ease their integration, operation temperature should be increased. Removing cathode external humidifiers from the system is highly desirable in the objective of decreasing the system mass and volume. However, in order to prevent membrane dehydration, we suggest a system composed of a recirculation loop at the cathode side. This architecture is called the Cathode Exhaust Gases Recirculation (CEGR) or cathodic recirculation. CEGR appears as a suitable solution to humidify air at the cathode side using water vapor produced by the fuel cell. Besides, cathodic recirculation has the advantage to modulate the relative humidity at stack inlet by varying the amount of water recirculated from the stack outlet. It can be considered as an active water management solution since the humid gas flow mixed with fresh air before the stack inlet is controlled with a compressor speed. In contrast, during operation with passive humidifiers, relative humidity at stack inlet is mainly imposed by operating pressure, temperature and stoichiometric ratio ("Sto").

Various recirculation architectures have been patented in recent years, as in [START_REF] Knoop | Apparatus for recirculation of a cathode gas in a fuel cell arrangement, method for shutting down such a fuel cell arrangement[END_REF][START_REF] Hild | Cathode humidification of a PEM fuel cell through exhaust gas recirculation into a positive displacement compressor[END_REF][START_REF] Goebel | Fuel cell shutdown and startup using a cathode recycle loop[END_REF][START_REF] Garrettson | Integrated recirculating fuel cell system[END_REF][START_REF] Greszler | Shutdown strategy to avoid carbon corrosion due to slow hydrogen/air intrusion rates[END_REF][START_REF] Pelch | Système de pile à combustible à recirculation intégrée[END_REF][START_REF] Goebel | Fuel cell shutdown and startup using a cathode recycle loop[END_REF].

However in literature only few studies deals with CEGR [START_REF] Yang | Study on self-humidified PEMFC with reactant circulation[END_REF][START_REF] Kim | Studies on the cathode humidification by exhaust gas recirculation for PEM fuel cell[END_REF][START_REF] Junming | Water management in a self-humidifying PEM fuel cell system by exhaust gas recirculation[END_REF][START_REF] Cheng | Air supply system model with exhaust gas recirculation for improving the life of fuel cell[END_REF][START_REF] Jiang | Experimental study on dual recirculation of polymer electrolyte membrane fuel cell[END_REF][START_REF] Xu | Self-humidification of a proton electrolyte membrane fuel cell system with cathodic exhaust gas recirculation[END_REF][START_REF] Jenssen | Improved PEM fuel cell system operation with cascaded stack and ejector-based recirculation[END_REF][START_REF] Zhao | Study on voltage clamping and selfhumidification effects of pem fuel cell system with dual recirculation based on orthogonal test method[END_REF][START_REF] Alizadeh | The experimental analysis of a dead-end H2/O2 PEM fuel cell stack with cascade type design[END_REF]. To our knowledge, Yang et al. [START_REF] Yang | Study on self-humidified PEMFC with reactant circulation[END_REF] were the first to study a new stack architecture allowing humidification at the both sides of the membrane with reactant recirculation. They worked with three cascaded cells.

Therefore, the cells are connected in series, each of them humidifying the next one. A recirculation loop at both anode and cathode allows to humidify the first cell from the last one. Their study was mainly focusing on start-up at 30°C, 1 bar. They concluded that the cathode recirculation had more effect than the anode one. Moreover, they noticed that cathode flooding could be avoided thanks to the recirculation. More recent work from Kim and Kim [START_REF] Kim | Studies on the cathode humidification by exhaust gas recirculation for PEM fuel cell[END_REF] introduced the recirculation ratio defined as the ratio of the molar recirculated gas flow on the total gaseous flow at the cathode outlet. They suggested two different architectures of CEGR to remove external humidifiers. The first one included only one compressor for the mix of fresh and recirculated air. The second architecture had two compressors, one for fresh air compression and the second one for the recirculated flow.

They only modeled the first architecture and used simulation results to obtain the cathode gas inlet composition and carry out performance experiments on a 5-cells stack. They observed a voltage drop around 7% at 1.0 A/cm² with a low recirculation ratio. They also compared the effect of oxygen dilution and relative humidity at stack inlet on the cell voltage, and observed that the first one had a higher impact. Unfortunately, no experiment was performed with high recirculation. On the same architectures, Junming et al. [START_REF] Junming | Water management in a self-humidifying PEM fuel cell system by exhaust gas recirculation[END_REF] modeled only one system with a single compressor similarly to Kim and Kim [START_REF] Kim | Studies on the cathode humidification by exhaust gas recirculation for PEM fuel cell[END_REF] and proposed a control strategy for water management with cathode EGR. Cheng [START_REF] Cheng | Air supply system model with exhaust gas recirculation for improving the life of fuel cell[END_REF] was interested in oxygen dilution by recirculation for voltage clamping, while Jiang et al [START_REF] Jiang | Experimental study on dual recirculation of polymer electrolyte membrane fuel cell[END_REF] and Xu et al. [START_REF] Xu | Self-humidification of a proton electrolyte membrane fuel cell system with cathodic exhaust gas recirculation[END_REF] studied recirculation at both sides of the membrane. All of them concluded that recirculation could achieve the same relative humidity levels as external humidifiers, but that voltage could be reduced due to the oxygen dilution at the cathode inlet. This can be an advantage in startup or idle states, because it could avoid degradations occurring at voltage levels higher than 0.8V. However, all of these studies have been conducted at very low current and high stoichiometric ratios.

To our knowledge, very few experimental works have been performed with multi-scale analysis. On a real-scale system equipped with CEGR architecture, we focused on the system efficiency as well as the stack performance, and the different cells behaviors through the stack thanks to in-line electrochemical impedance spectroscopy. Some experimental studies have been performed on real-scale systems by Kim et al. [START_REF] Kim | Parametric study on interaction of blower and back pressure control valve for a 80-kW class PEM fuel cell vehicle[END_REF] on a 80-kW system, or Wahdame et al. [START_REF] Wahdame | Study of a 5 kW PEMFC Using Experimental Design and Statistical Analysis Techniques[END_REF] on a 5-kW system but both were only focused on stack and system performance. In our study, a 5 kW-system built for operation representative of automotive conditions has been used: pressure below 1.5 bar and temperature above 65°C. In this work, the system was equipped with a 70-cells stack. We studied the impact of CEGR under various current densities, temperatures, pressures, stoichiometric ratios and recirculation ratios, and a comparison with passive humidification was done. A design of experiments has been performed and analyzed to look for optimum operating conditions and evaluate the interest of cathode exhaust gases recirculation.

Material and methods 2

System and stack architecture description

The fuel cell stack used in this work is a 70-cells stack. Commercial MEAs with active area 220cm² have been assembled with serpentine design bipolar plates. More details about cell design have been developed in previous work [START_REF] Nandjou | A pseudo-3D model to investigate heat and water transport in large area PEM fuel cells -Part 1: Model development and validation[END_REF]. Table 1 presents the system specifications. Anode with some modifications to avoid water and nitrogen accumulation in the cells.

Operating conditions usually obtained with anode recirculation are achieved without the need of a pump or an ejector. The complete sequence is described in Figure 2. In Figure 2 (a)

hydrogen is fed at the left half stack. A solenoid valve opens the left circuit, feeding the left half-stack from its H 2 inlet. All the cells of this half-stack are fed in parallel from their H 2 inlet.

Since the outlet manifolds of both half-stacks are connected in the central plate, the right half-stack is fed from its H 2 outlet, and its cells are fed in parallel from their own H 2 outlet.

Then in Figure 2 A radiator and fan control on a deionized water-cooled loop ensures thermal management.

During operation, stack outlet temperature is maintained stable within ±1°C tolerance. The cooling loop is also used to preheat hydrogen before the anode inlet in a tubular heatexchanger.

For the purpose of comparison of different humidification solutions at the cathode side, two passive humidifiers FC-150 series, mounted in parallel, from Permapure® (not presented on Figure 1) are also installed on the test bench. They are used when the cathode recirculator is turned off, and by-passed when the recirculation mode is used. Thus, experiments carried out with the passive humidification mode are performed on the same system as recirculation experiments. Therefore, the impact of system components is equivalent and the results with both architectures can be compared.

Full factorial design of experiment

In order to deeply study the effect of cathodic recirculation on the PEMFC behavior, a complete Design of Experiments (DoE) has been built including 5 parameters. The current density, pressure (similar at anode and cathode), stack temperature, air stoichiometry as well as the recirculation ratio were identified as variables. For each one, three levels (low, medium and high) are investigated. All parameters and DoE levels are gathered in Table 2.

The limitations of the test bench in terms of fresh air flow and pressure, mentioned in Table 1, restricted their variation range.

In order to decrease system volume, automotive systems tend to increase temperature to decrease the radiator sizing. That is the reason why a temperature of 70°C was chosen as the maximum level in the DoE. The minimum temperature studied here is 60°C, considering that a lower temperature is not realistic for automotive application. So, the three temperature levels are 60, 65 and 70°C. The pressure range was chosen as large as possible, considering the capabilities of the compressor. Consequently, the three pressure levels were fixed at 1.2, 1.35 and 1.5 bar.

System stoichiometry in recirculation mode was intentionally set lower than with classic humidification since recirculation increases the flowrate at stack inlet. Therefore, to reduce the power consumption of compressor 1, system stoichiometry is set from 1.4 to 1.8 instead of 2 in classic humidification process. A definition of the recirculation ratio "R", similar as Kim and Kim's study [START_REF] Kim | Studies on the cathode humidification by exhaust gas recirculation for PEM fuel cell[END_REF], was used in the DoE and will be presented in the modelling section:

3.1. Recirculation ratio values could not be similar for the different current densities because of the limitations of the recirculation pump, which are both its maximum flowrate (20 Nm 3 /h) and its maximum pressure ratio (1.12). For each current density value, the maximum recirculation ratio reachable with the recirculation pump was determined: 0.60 for 0.3 A/cm² and 0.33 for 0.5 A/cm². It was also evaluated equal to 0.16 for a higher current density of 0.7 A/cm². However, it corresponds to a very limited range of recirculation ratio. As a consequence, this current density value was not studied further. Then three levels of recirculation ratio were determined for the remaining current density levels. The low, medium and high recirculation levels are respectively 0.19, 0.34 and 0.48 at 0.3A/cm² and 0.13, 0.22 and 0.32 at 0.5A/cm². The recirculation flow is limited by the dimensioning of recirculation components, and more precisely of the recirculation pump. When the recirculation flow increases, the pressure drops inside the air pipes and in the stack also increase. In the present study case, the pressure ratio of the recirculation pump is high enough to obtain recirculation ratios of 0.48 at 0.3 A/cm² and 0.32 at 0.5A/cm².

Modelling of cathode exhaust gases recirculation 3

CEGR model description

In order to be able to predict the relative humidity at stack inlet for any operating conditions and demonstrate the benefits of the use of cathodic recirculation in a system, a simplified model based on mass balances was built. The model uses the following assumptions:

-Calculations on the gas phase is performed assuming ideal gas equations -Phase separators remove 100% of liquid water (or separator yield is 1) -Temperature is assumed constant in all components, except in compressors, and is equal to stack temperature -Gas and water transports through the fuel cell membrane are neglected -The system exhaust flow rate is saturated of water vapor To be able to control the recirculated flow, a new variable has to be defined as the recirculation ratio "R". This variable is defined as the ratio of the recirculated molar flow on the gaseous stack outlet molar flow , (only considering gas flow) to obtain a value ranging from 0 to 1:

= , (1)
Assuming no gas transport through the membrane, nitrogen is conserved during operation at the cathode side. Introducing the system stoichiometric ratio , using the Faraday's law, and the usual molar fraction for the air composition, the nitrogen flow at the system outlet , is:

, = = • • • ."# $%• . & (2)
Where is the nitrogen molar flow at the system inlet coming from the ambient air, ' (( the number of cells in the stack, ) the current and * the Faraday constant.

Similarly, the oxygen flow in the exhaust gases + , can be defined using Faraday's law for oxygen consumption:

+ , = + -+ = • $% - -1/ (3)
As water condensation occurs at the stack outlet, the gaseous mix at the system outlet is saturated. And so, the water molar fraction at the system outlet 0 1 + , can be expressed thanks to the saturation pressure at stack temperature 2 -3 / and the total pressure 2 , at the phase separator outlet:

0 1 + , = 4 56 -7 6 / 4 , (4)
Then, the gaseous flow 8 , can be written thanks to a combination of equation ( 1) to (4): (5) At the cathode inlet, the molar flow rate of each specie A (oxygen and nitrogen) is expressed as:

B ,B = B ,B + • B , = + @ &=@ • B , (6)
Where B ,B is the molar flow rate of specie A at the system inlet and B , the molar flow in the exhaust gases.

Using Faraday's law for oxygen consumption and using the stoichiometric ratio at the system inlet , the equation ( 6) becomes for oxygen:

+ ,B = • $% D D1 + @ &=@ E - @ &=@ E (7)
The stoichiometric ratio at the system inlet is a value classically used to control the air flow at the stack inlet. However, in our case the air flow at the system inlet is different from the stack inlet because of the recirculation. Consequently, a new definition of the stoichiometric ratio at stack inlet is defined as follows:

= D1 + @ &=@ E - @ &=@ (8)
This definition shows that when recirculation is set at maximum namely R=1, → +∞ and when R=0 i.e. without recirculation, = .

The amount of water vapor reinjected at stack inlet may be expressed based on the equation ( 6) and a balance at the phase separator. In constant operation, all the water produced is rejected at the system outlet, either with the other gases in vapor form or in liquid form at the phase separator. Thus, the molar fraction of water 0 1 + ,B at the cathode inlet can be written independently from the water production rate but from the recirculation ratio, saturation pressure at stack temperature (in 0 1 + , ) and operating parameters:

0 1 + ,B = > ?:9 5 • 6 6 5 ;@•> ?:9 , • , 6 6 ,H =
The relative humidity at the stack inlet is then simply derived from the water fraction at stack inlet:

T ,B -3 / = > ?:9 ,H •4 ,H 4 56 -7 6 / * 100 [START_REF] Hwang | An experimental study on the cathode humidification and evaporative cooling of polymer electrolyte membrane fuel cells using direct water injection method at high current densities[END_REF] Finally, considering the balance of all species and with similar development to the equation ( 9), the oxygen molar fraction at stack inlet can be given by:

0 + ,

Model results and validation

The expressions developed in the model show that water and oxygen content are dependent on the recirculation ratio and operating conditions but independent from the consumption/production rate.

The Figure 3 (a) shows the decrease of the oxygen molar fraction at the stack inlet with the increase of both the recirculation ratio and the stoichiometric ratio at the system inlet. It is worth mentioning that the consumption of oxygen during operation induces a decrease of the oxygen molar fraction at stack outlet, while nitrogen, which is an inert specie in the process, dilutes the other species. Therefore, at stack inlet, a higher system stoichiometric ratio increases the oxygen molar fraction, whereas a higher recirculation ratio decreases drastically the oxygen molar fraction. For example, the latter can drop down to 12.2%mol at Sto sy =1.4 and 0.8 recirculation ratio. Therefore, oxygen starvation effect might occur with high recirculation ratio impacting the stack performance. The Figure 3 (b) presents the simulated relative humidity levels reached with increasing recirculation and system stoichiometric ratio. It is observed that a relative humidity higher than 50% can be achieved with recirculation ratio higher than 0.4. According to the model results, a wide relative humidity range could be achieved varying the recirculation ratio (R).

For instance, at fixed Sto sy = 1.8, relative humidity could be set between 10 and 80% with R varying from 0.05 to 0.8. The Figure 4 shows the simulated RH at two different current densities (i.e. 0.3A/cm² and 0.5A/cm²), varying the recirculation ratio (R). In both cases, a very good agreement is observed between the model and experimental data obtained on the range of recirculation ratio feasible with the test bench. We can notice a gap at high recirculation ratio probably due to the pressure drops in the recirculation line, for which the accuracy of the model at high recirculation ratio can be discussed. Additionally, according to the model, no condensation is supposed to occur when mixing the recirculated flow with the fresh compressed air. Indeed, the fresh air at the system inlet being heated through the compressor 1, its temperature is close to the stack temperature. So, mixing both flows at similar temperature levels is achieved without liquid water formation. Relative humidity at stack inlet is thus only dependent on the amount of water recirculated in the gaseous phase. For instance, when using our test bench with passive humidification in standard conditions (1.2 bar, 60°C, 0.3A/cm², Sto=2), the relative humidity is around 85%. The lowest relative humidity achievable is about 35% and may be reached with a by-pass of one of the two humidifiers and an increase of the stack temperature from 60°C up to 80°C. The latter temperature increase may reduce the cell durability.

Experimental result analysis and discussion 4

Impact of the operation conditions on the stack performance

This section investigates the influence of the operating conditions on the stack performance with CEGR mode to compare it with passive humidification. Temperature, pressure as well as system stoichiometric ratio may have an impact on the stack performance.

The Figure 5 presents the influence of temperature, pressure and system stoichiometric ratio, on the cell voltage averaged over the stack and the measurement period for the three recirculation levels selected in the DoE definition. Regardless of the recirculation ratio, the stack performance decrease when increasing the stack temperature. Moreover, Figure 5 (a,b) highlights a severe voltage drop at 70°C at a minimum recirculation level. Indeed, the recirculation at a minimum level induces a lower relative humidity at stack inlet. This behavior is more pronounced at higher temperature, where the drying process is reinforced. The Figure 5 (a) shows an increase of the stack performance at a current density of 0.3 A/cm² when increasing R. It is different in Figure 5 (b), with a current density of 0.5 A/cm², where the cell voltage seems independent from the R level at 60 and 65°C. These low variations could be explained by the recirculator sizing since recirculation ratio range at 0.5A/cm² is quite small. However, performance at 70°C drastically drops for both current density cases and is even unstable at very low recirculation ratio (a stable voltage point cannot be obtained at 0.5A/cm²). At this higher temperature, the minimum relative humidity drops to 20% at 0.3 A/cm² for low R and is even lower at 0.5A/cm². Increasing the recirculation ratio to the maximum, the relative humidity reached at 0.3A/cm² is around 50% but only 30% at 0.5A/cm². So, water amount is not sufficient to obtain a correct hydration level of the membrane whatever the R value at 0.5 A/cm² and high temperature (i.e. 70°C). A higher recirculation ratio would be necessary to bring a higher water content with the aim of stabilizing the voltage at higher temperature. This is consistent with existing data in literature at low RH values [START_REF] Neyerlin | Effect of Relative Humidity on Oxygen Reduction Kinetics in a PEMFC[END_REF], [START_REF] Gößling | Analysis of liquid water formation in polymer electrolyte membrane (PEM) fuel cell flow fields with a dry cathode supply[END_REF].

The Figure 5 (c) and (d) show the positive impact of a pressure increase on stack voltage, respectively at 0.3 A/cm² and 0.5 A/cm². At 0.3A/cm², the low pressure case meets the optimum conditions at medium recirculation ratio. However, at higher pressure, the increase of oxygen partial pressure seems to slightly move the optimum to higher recirculation ratios than the one measured. At 0.5A/cm², the voltage increases with the recirculation ratio at low pressure, when the partial pressure of water is low. On the contrary, with higher pressure, the recirculation level seems to have no effect due to the increase of water partial pressure, reducing the drying effect.

Finally, Figure 5 (e) and (f) present the influence of the system stoichiometric ratio on stack voltage, respectively at 0.3 A/cm² and 0.5 A/cm². Maximum performance is observed at Sto sy =1.8 at 0.3A/cm² and Sto sy =1.6 at 0.5A/cm². In additions, optimum performance is obtained at medium recirculation ratio at Sto sy =1.8 for both current densities and Sto sy =1.6 at 0.5A/cm².The existence of an optimum behavior will be analyzed in part 4.2. Indeed, higher Sto sy might increase stack voltage by increasing the oxygen content at the stack inlet, but also induces a voltage drop due to cell drying (i.e. too much dry air is fed in the total air flowrate at the cathode inlet). The drying effect was effectively observed at high current density (0.5A/cm²) when best performance is obtained at Sto sy =1.6. Indeed, in this case, relative humidity was 4% higher than the case at Sto sy =1.8. The behavior at Sto sy 1.6 and low current density seems to show that the optimum is not reached and would be at a higher recirculation ratio than 0.48. A rise of R can increase the relative humidity at stack inlet, but will decrease oxygen partial pressure too. Combining both phenomena, we can observe optimum performance with a set of stoichiometric ratio/recirculation ratio.

At low current density, rising the stoichiometric ratio increases the voltage for any recirculation ratio. The optimum voltage of 710 mV is observed at medium recirculation ratio with Sto sy =1.8. It is different at higher current density where, a Sto sy of 1.6 with medium recirculation ratio gives the best performance: 586 mV.

bar and 65°C

In order to show the limited impact of temperature and pressure on RH with passive humidifiers, several experiments have been performed. The influence of the pressure was first investigated, the other operating parameters remaining constant: 0.5A/cm², 65°C, Sto sy =2. The results showed a relative humidity decrease of 8% with a cell voltage increase from 611 mV to 628 mV when the pressure rises from 1.35 up to 1.5 bar. Secondly, a temperature step of 5°C was imposed, from 65 to 70°C with constant parameters: 0.3A/cm², 1.5 bar, Sto=2. Similarly, a low relative humidity decrease of 4% has been recorded with a 3mV voltage loss, namely from 703 mV to 700 mV. These experiments highlight the limitations of water management with passive humidifiers since it is mainly designed for a narrow operating range. It could be enlarged with cathodic recirculation, thanks to RH modulation.

Impact of recirculation on cell performance around optimum conditions

This second section investigates more deeply the influence of the recirculation ratio on the cell performance for both current densities namely 0.3 and 0.5 A/cm². The Figure 6 illustrates the behavior of mean cell voltage (stack voltage divided by cell number) as well as the lowest cell voltage in the stack and the highest one when increasing the recirculation ratio.

All three values are averaged over the measurement period. Three different domains can be distinguished as a function of the recirculation ratio. At very low recirculation levels referred to as zone I in Figure 6 (a) and (b), performance is at its maximum level. However, the gap between the mean and lowest cell voltage value is also maximum, while the difference between mean and highest value is low. This indicates that decreasing recirculation ratio increases cell voltage heterogeneity in the stack. The high difference between lowest and mean voltage, contrasting with the low difference between highest and mean voltage, confirms the hypothesis of cells drying or flooding. However, flooding occurs usually with high relative humidity levels. This latter behavior is probably due to cells drying, because the relative humidity at stack inlet is below 43% at 0.3A/cm² and 51% at 0.5A/cm² when R is lower than 0.3. Some cells located near the air inlet, and potentially subject to a higher air flow than the average, are suspected to be dried.

At moderate or high recirculation rates, the cell voltages are more uniform while the mean cell voltage follows a decreasing tendency. In zone II with recirculation levels between 0.3 and 0.4, as shown in Figure 6 (a), higher stack homogeneity is achieved at 0.3 A/cm². The homogeneity is confirmed by the reduced cell voltage difference between the highest and the lowest value, and the central position of the mean value inside this voltage range. The voltage level is likely comparable to the previous case at low recirculation ratio because of oxygen dilution at the stack inlet. The corresponding relative humidity values are consistent with the cells behavior. Namely, RH was measured at 43% at R=0.3 and RH was recorded at 57% at R=0.4. The same behavior can be observed in Figure 6 (b) with the same recirculation ratios. However, the profile is shortened due to the recirculation compressor limitations. The beginning of zone II can be identified from R=0.3 while the gap between the maximum and minimum cell voltages is reduced and mean cell voltage start to decrease. At higher recirculation levels in zone III as referred in Figure 6 (a), the mean cell voltage drop is more visible (-0.5% to -2%) due to the reduction of oxygen partial pressure (see Figure 3). Nevertheless, relative humidity values are still increased with R from 57% to 87% at R=0.4 to 0.57 respectively. It appears that stack homogeneity is similar to the previous recirculation zone. It can be concluded that CEGR ensures an efficient cells humidification in zones II and III.

At low current density, namely 0.3A/cm², optimum conditions highlighted through the DoE are reached at 60°C, 1.5 bar and Sto sy = 1.8. The experiments performed at various recirculation ratios show that maximum performance is observed at R=0.2, which appears to be a good compromise. With lower recirculation ratio, the decrease of the relative humidity lowers the performance. With higher recirculation ratio, a performance decrease is observed due to oxygen depletion. However, a better homogeneity among the cells is obtained with a higher recirculation ratio around 0.3. Indeed, higher stack stoichiometric ratio and relative humidity resulting from a higher R favor a higher cells homogeneity. That is why we choose to operate with a slight performance loss at a recirculation ratio close to 0.3 in these conditions. Further investigations can be observed in Figure 7 for a specific test at low recirculation ratio (zone I) where both dried and flooded cells can be detected. The operating conditions of the test can be considered as dry conditions, with a low recirculation ratio of 0.17, a high system stoichiometric ratio of 1.8, medium temperature and pressure of 65°C and 1.35 bar respectively, and a low current density of 0.3 A/cm². The relative humidity at stack inlet is only 22% in this conditions. The mean cell voltage, averaged over the complete experiment, is 626 mV. Figure 7 (a) shows the cell voltage profile along the stack with mean cell voltages recorded during the experiment. Three particular cell behaviors were compared, corresponding to three different locations inside the stack: close to the air inlet, where cell drying is often observed in dry conditions (cell n°66), between the two half-stacks on the central distribution plate (cell n°36) and in the middle of the other half-stack (cell n°21).

Accordingly, the cell voltage versus time profile of these three cells are presented in the Figure 7 (b). Cell number 21 is considered healthy since its mean voltage (633 mV) is higher than the mean cell voltage of the experiment (626 mV). Its location is not impacted by the stack architecture, as it is neither close to the air or hydrogen inlets, nor close to a terminal plate or central plate, where side effects might occur (refer to Figure 2 for flow feed locations). Even if the cell number 21 seems to be healthy, cells number 36 and 66 might be considered as flooded and dried because of their low cell voltage and location in the stack.

Cell number 36 is considered flooded because its voltage profile during measurement was periodically rising after each anode purge. As one of the main function of the purge is liquid water removal, it can be anticipated that anode flooding occurs in this cell. The cell number 66, for which the cell voltage is 14 mV lower than the healthy cell, is assumed dried, considering its location near the air manifold inlet, with relatively dry operating conditions. It has to be noticed that thermal side effects have an influence on the cell voltage behavior.

Voltages recorded for cells number 68 to 70 are higher than cell 66. Their temperature maybe colder than cell 66 that can explain a lower drying effect.

To confirm these hypotheses, Electrochemical Impedance Spectra have been performed.

The frequency ranged between 5000 and 0.1 Hz with 10 points per decade by using a single sine signal in galvanostatic mode (66 A with 10% amplitude). Figure 7 (c) shows the EIS spectra obtained for cells 21, 36 and 66 with their respective mean voltages obtained in the measurement conditions. On the EIS spectra, low frequency loop may be distorded due to the system instabilities. Ping-Pong alternating feed frequency as well as anode purges generate pressure and relative humidity instabilities which can have an impact on the cell behavior. Therefore, only the high frequencies are used for this analysis. So, high frequency resistance measured at 854 Hz characterizing the membrane resistance [START_REF] Zhang | Electrochemical Impedance Spectroscopy in PEM fuel cells[END_REF][START_REF] Springer | Characterization of Polymer Electrolyte Fuel Cells Using AC Impedance Spectroscopy[END_REF] are compared in the Figure 7 (d). For the healthy cell 21, the high frequency resistance measured at 854 Hz is 0.973 mΩ. For the flooded cell 36, the high frequency resistance measured at 0.645 mΩ confirms this behavior since the value is lower than the healthy cell.

As the membrane conductivity is improved with a good hydration level, the resistance is reduced with a higher water content. Therefore, a low resistance value coupled to a low and unstable voltage profile points out a flooding behavior.

For the cell number 66, the high frequency resistance is measured at 1.1 mΩ which is 0.138 mΩ higher than the healthy cell and the typical 45° branch, which looks like a warburg impedance in the high frequency region for dry spectrum, hints a drying behavior.

These analyses highlight the heterogeneous cell voltage behaviors occuring at low recirculation ratio (in zone I in Figure 6). As relative humidity at stack inlet is reduced, some cells may suffer from drying. Additionally, as the flowrate at cathode inlet is reduced, some particular cells can suffer from flooding.

Impact of recirculation on stack performance at 70°C

The Figure 8 presents two polarization curves measured with both architectures: passive humidification and recirculation. The common operating conditions for both architectures were set at the same values: temperature at 70°C, pressure at 1.2 bar. These conditions were determined considering automotive conditions. In order to maximize system efficiency, pressure has to be lowered to avoid the energy loss due to the compressor efficiency.

Temperature should also be higher to minimize the radiator size.

In order to obtain a stack stoichiometric ratio close to a value of 2 for both cases, a system stoichiometric ratio of 1.8 was imposed for the recirculation mode, with high recirculation ratio. The highest recirculation ratio was also chosen to reach a relative humidity level as high as possible for the complete polarization curve since RH level achieved with classic humidification was close to 85% in this case. The first polarization curve was recorded with classic humidification mode using the passive humidifiers. The second one was recorded with CEGR as humidification solution. An impact of the recirculation process on the fuel cell performance is observed, leading to a small stack voltage loss. During polarization curves, all parameters have to be kept stable, and the speed of the compressor 2 was set to maintain a maximum recirculation ratio for each current density value ranging from R=0.7 to 0.33.

At 70 A (i=0.32A/cm²), we observed a voltage difference of 50 mV. This gap is partly explained by the oxygen dilution and by the reduced relative humidity achieved at 70°C. As highlighted in the Figure 3

Impact of recirculation on the system performance at 70°C

Performance decrease with recirculation architecture has also an impact on the system efficiency. System efficiency a is calculated as follows: a = 4 65 b =4 5cR d1e ?: •% ?:

Where P stack is the stack power P aux , the power consumption of ancillaries and LHV H2 the Lower Heating Value of hydrogen and 1 hydrogen molar flow.

Power consumption of the water pump, solenoid valves or sensors are assumed negligible compared to compressors consumption and so only the latter are considered for calculations (P aux =P comp ). For sake of simplicity, ideal compressor efficiencies are considered to be free from real component specifications as set in equation ( 13):

2 f = &.$ &.$=& B ,B • • 3 gD 4 4 5 E L.NML L.N -1h (13)
Where B ,B is the air flow rate at the system inlet in moles, the perfect gas constant, 3 and 2 the ambient temperature and pressure respectively, 2 the outlet pressure of the compressor.

and variable recirculation ratios

The Figure 9 presents the system efficiencies obtained with both architectures (passive humidification and recirculation) for two different current densities. In the case of CEGR, the recirculation ratio was ranging from 0.19 to 0.48 at the 0.3A/cm² and from 0.13 to 0.32 at 0.5A/cm². As passive humidification experiments have been performed previously for a polarization curve at 70°C and 1.2 bar and Sto=2, cell voltage measurements in CEGR mode have been extracted from the DoE at the same temperature and pressure conditions with variable recirculation ratios and stoichiometric ratios to compare their system efficiencies.

A minimum gap of 3.7% on the system efficiency is observed at a current density of 0.3 A/cm² with R=0.48 and Sto sy =1.8 whereas a larger one of 10.1% is observed at a current density of 0.5 A/cm² with R=0.32 and Sto sy =1.6. The best performance is obtained with a high recirculation ratio instead of medium because of the operating temperature. The sensitivity analysis in part 4.2 was performed at 60 and 65°C where relative humidity could be slightly lowered without affecting cell performance and stability. In the present case temperature is 70°C, which requires a higher water content at the stack inlet and thus a higher R.

The efficiency difference between passive humidification and recirculation system can be explained with three elements, related to oxygen dilution, relative humidity at stack inlet, and compressor power. First, oxygen dilution at stack inlet induces a voltage drop due to the decrease of oxygen activity in the catalyst layer of the cell. Second, lower inlet gas humidification at the cathode inlet when decreasing the recirculation ratio has a detrimental effect on the stack performance. Finally, theoretical power consumed to compensate stack and air loop pressure drops can represent 4% to 20% of total power consumption for air compression slightly decreasing the system efficiency. The stack power and system efficiency are therefore lower than the one obtained in passive humidification mode.

To visualize the impact of compression power during operation, the consumption of both compressors is modeled based on the isentropic case written in equation ( 13) for compressor 1 (fresh air compression). Figure 10 presents the total power consumed by air compression as a function of both the recirculation ratio and the system stoichiometric ratio.

It is observed in Figure 10 (a) that ideal recirculation compression is limited compared to fresh air compression and particularly when R is lower than 0.35. Indeed, the compression ratio of the recirculation pump being low compared to the first compressor, the ancillaries power consumption is not very far from the reference case without recirculation. Besides, as

Sto st is increased with recirculation, system stoichiometry could be reduced to limit the first compressor consumption and achieve a total power consumption close to the system without recirculation. As an example, a case without recirculation at a Sto sy of 2 has a power consumption equivalent to a recirculation case at Sto sy =1.75 and R=0.5. Therefore, a simulation with reduced stack pressure drops has been implemented to observe the benefits on total power consumption. Figure 10 (b) presents the power consumed with stack pressure drops reduced of a third. It is observed that power consumed is lower than the previous case, the decrease being more pronounced at higher recirculation ratio. For instance, at Sto sy =1.8 and R=0.6, the total power consumption is 123W in the first case and 110W in the second case allowing a 10% gain. This highlights the fact that pressure drops in the air loop have to be minimized. The recirculation induces a higher air flowrate at the cathode side and therefore higher pressure drops in the fuel cell. An optimization of the cell design reducing the total stack pressure drops could drastically reduce the power consumption of compressor 2 with a positive impact on the system efficiency.

Impact of recirculation on the system dynamic

Another advantage of CEGR for integrating in automotive systems compared to classic humidification would be the dynamics. Indeed, relative humidity can be controlled thanks to the recirculation ratio as already presented in part 3.2 and handle large RH ranges.

Additionally, CEGR has a faster dynamic than passive humidification. Actually, the water vapor production follows the fuel cell dynamics and is almost immediately recirculated. On the contrary, the dynamic behavior of the passive humidifier is limited by the water transport through the hollow-fiber membranes at its operating temperature and pressure. In Figure 11 (b) is presented a temperature increase from 70°C to 80°C and at the same time a pressure variation from 1.2 to 1.3 bar with passive humidification. It is observed in Figure 11 (b) that the temperature reached the set point by a ramp and the relative humidity followed the same trend. Indeed, temperature variations cannot be achieved with steps. On the contrary, as observed in Figure 11 (a), relative humidity can be adjusted instantaneously with recirculation ratio steps.

Besides, with passive humidification, the pressure and temperature changes have a limited effect on relative humidity with a decrease from only 47% to 39%. In contrast, recirculation mode can handle a relative humidity variation from 85 to 26%. The large relative humidity range obtained with recirculation could be a useful tool for water management during fuel cell operation. Indeed, drying could be reduced by recirculation ratio increase to reach relative humidity close to 85%. Additionally, flooding could be avoided by reducing the recirculation ratio.

Conclusion

Despite their large volume and thermal inertia, passive humidifiers are classically used in PEMFC systems to humidify the fresh air before the cathode inlet. Besides, their slow dynamic make them not suitable to ensure the best performance following power variations enforced by automotive conditions. In this work, we studied experimentally and theoretically the possibility to reduce the system volume replacing passive humidifiers by a specific architecture at the cathode side, while achieving best performance and stability. This architecture is called the Cathode Exhaust Gases Recirculation (CEGR) and uses the water vapor produced by the fuel cell to humidify the cathode inlet flow. Contrary to passive humidifiers, CEGR offers a possibility to control the water amount injected at the cathode inlet whereas passive techniques are completely dependent on the fuel cell operating conditions. The impact of CEGR was investigated on a 5-kW-system under various current densities (0.3 and 0.5A/cm²), temperatures (60 to 70°C), pressures (1.2 to 1.5 bar), stoichiometric ratios (1.4 to 1.8) and recirculation ratios. It was also compared with performance and stability with passive humidification through a complete Design of Experiment. At first, a simplified model based on mass balances was built in order to predict relative humidity levels at the cathode inlet. The model was validated, and predicts a relative humidity higher than 50% for a recirculation ratio higher than 0.3 and achieve a relative humidity range as large as 60% at 0.3A/cm².

Behavior of the stack under various recirculation ratio conditions was investigated for a fixed temperature, pressure, and system stoichiometric ratio. Three zones were identified as a function of the recirculation ratio value. The first one at low recirculation ratio allows the best performance but also the worst stability since the water amount is not sufficient to humidify the membrane which started to dry out. In the second zone at medium recirculation ratio, performance is almost equal to the previous one but stability is highly increased. A suitable hydration level was achieved. The third one at high recirculation ratio gives the best stability but performance decrease is higher than in the previous zone (-2%) due to the higher oxygen dilution, even if relative humidity was in a high range. Although performance is lower than with passive humidifiers and current densities higher than 0.5A/cm² were not tested due to system components limits, we can recommend using a medium or high recirculation ratio (i.e. zone 2 or 3) for long terms performance and stability for transport applications.

Air humidification thanks to CEGR could also be of great advantage in long-term operation since voltage is reduced by the oxygen dilution. Indeed, carbon corrosion as well as catalyst degradation mechanisms are likely to occur at high cell potential. Decreasing cell voltages when operating at low current may reduce the performance loss and longer the fuel cell lifetime. Durability studies should be performed in order to complete this investigation on the benefits of the architecture.

  (b), Hydrogen feeds of the right half stack. 1.1 second after the first sequence in (a), the left solenoid valve closes and the right one opens. The same process beginning by the right half-stack arises. Right cells are fed from their inlet and left cells from their outlet. Finally, in Figure 2 (c) is presented the purge sequence. As liquid water and nitrogen accumulate, every 50 seconds, all 3 solenoid valves open to remove efficiently the non-desired species. With purge period set at 50 seconds and purge duration 0.5 second, anode stoichiometric ratio is 1.02 and equal to DEA anode stoichiometric ratio in the same conditions. Figure 2 (d) presents the air feed configuration. The air is fed by the terminal plate close to cell number 70. Then air flows through all cells in parallel and exhaust gases are removed at the same side as the air feed.

Figure 1 :Figure 2 :

 12 Figure 1: Schematic of the fuel cell system

Figure 3 :

 3 Figure 3: Simulation with constant parameters 0.3A/cm², 1.35 bar, 65°C and air feed at ambient temperature (20°C, 40% RH, Patm) (a) Oxygen molar fraction variations at stack inlet and (b) Relative humidity variations at stack inlet with recirculation ratio and system stoichiometric ratio.

Figure 4 :

 4 Figure 4: Model validation of the relative humidity levels reached with recirculation ratio variations at operating conditions: 1.5 bar, 60°C, Sto sy =1.8, (a) 0.3 A/cm², (b) 0.5 A/cm²The relative humidity range available with recirculation ratio control is very interesting compared to the passive humidification possibilities. In the examples presented in the Figure4, variation range can reach 60% at 0.3A/cm² and 38% at 0.5A/cm² respectively. The RH range is reduced when the current density increases due to the limitations of the recirculation device in terms of flow and pressure ratio. With passive humidifiers, variations of the relative humidity can only be hardly established by varying all the operating conditions of the stack.

Figure 5 .

 5 Figure 5. Mean cell voltages obtained with variable recirculation levels: (a) impact of stack temperature at 0.3A/cm², 1.35bar and Sto sy =1.8, (b) impact of stack temperature at 0.5A/cm², 1.35bar and Sto sy =1.8, (c) impact of stack pressure at 0.3A/cm², 70°C and Sto sy =1.8, (d) impact of stack pressure at 0.5A/cm², 65°C and Sto sy =1.8, (e) impact of Sto sy at 0.3A/cm², 1.2 bar and 60°C, (f) impact of Sto sy at 0.5A/cm², 1.2 bar and 65°C

Figure 6 :

 6 Figure 6: Voltage response of minimum (red dashed line), maximum (green dotted line) and mean (black squares) cells in the stack as a function of the recirculation ratio at 60°C ; 1.5 bar ; system stoichiometric ratio = 1.8, (a) 0.3 A/cm², (b) 0.5 A/cm²

Figure 7 :

 7 Figure 7: Stack and cell behaviors in dry conditions at 0.3A/cm², 65°C, 1.35 bar, system stoichiometric ratio = 1.8 and recirculation ratio = 0.17 with (a) cell voltage profile along the stack during experiment, (b) Cell voltage temporal measurement for cells 21, 36 and 66 (c) Electrochemical Impedance Spectra of 3 particular cells in the stack : cells 21 healthy, 36 flooded and 66 dried (d) Parameters evaluated from impedance spectra at high frequency : resistance measured at 854 Hz and 45° branch for the dry cell

  , oxygen partial pressure is reduced due to dilution with recirculation: Y Z[ \,] ^_`= 0.15.

Figure 8 :

 8 Figure 8: Polarization curves for classic humidification (black triangles) and maximum recirculation (red circles), operating conditions 1.2 bar, 70°C, Sto syst = 2 for classic humidification and Sto syst = 1.8 for recirculation mode and recirculation ratio variation (dotted line)

Figure 9 :

 9 Figure 9: System efficiency with and without recirculation taking into account only compressors as ancillaries for system efficiency calculations. Cell voltage measured at 70°C, 1.2 bar, for classic humidification (red circles) at fixed stoichiometry=2, and for recirculation mode (black triangles) Sto sy = 1.4 to 1.8 and variable recirculation ratios

Figure 10 :

 10 Figure 10 : Theoretical power consumed in Watt by both fresh air compressor and recirculation pump with recirculation ratio and system stoichiometric ratio at the cathode side for a simulated current density at 0.3A/cm², 1.35 bar, 65°C (a) normal case based on pressure drops measurements during operation (b) reducing 33% of stack pressure drops While an increase of the recirculation ratio improves cell voltage stability, it also increases the parasitic power consumed by the recirculation component. One of the main factors influencing this consumption is the value of the pressure drops along the recirculation loop.

Figure 11 :

 11 Figure 11: Humidification dynamics comparison between (a) recirculation steps from R=0.6 to 0.15 at 0.3A/cm², 1.5bar, 60°C and (b) Passive humidification with a pressure step from 1.2 to 1.3 bar, temperature step from 70 to 80°C and i=0.4A/cm². The Figure 11 compares the dynamic behavior of the relative humidity with either recirculation or classic humidification. In Figure 11 (a), Recirculation ratio was decreased from 0.6 to 0.15 in several steps with a current density of 0.3A/cm², a temperature at 60°C and pressure at 1.5 bar set as constant parameters.

Table 1 : System characteristics

 1

	Parameter	Value
	MEA	Commercial
	Cell number	2×35
	Max power	5 kW
	Operating temperature	60 to 70°C
	Operating pressure	1.2 to 1.5 bar
	Maximum fresh air flow	20 Nm3/h

Table 2 : Design of Experiment parameters and levels used for recirculation characterization

 2

	DoE values	Lower level	Medium level	Higher level
	Current density	0.3 A/cm²	0.5 A/cm²	-
	Temperature	60°C	65 °C	70°C
	Pressure	1.2 bar	1.35 bar	1.5 bar
	Stoichiometry	1.4	1.6	1.8
	Recirculation ratio	low	Medium	high

The DoE gathered 162 operating points, which were performed randomly for each temperature value since thermal steady state was long to establish. At start-up, the test bench steady state was considered to have been reached after operating 40 minutes at fixed operating conditions. Between each operating point, steady state was considered to have been reached after 15 minutes. In every case, the measurement was recorded during 800 seconds after stabilization.

Acknowledgments

The authors are thankful to Mathieu Giustini, who largely contributed to the experimental work.