Montserrat Espiñeira

Beatriz Herrero

Juan Manuel Vieites

Francisco Javier

Francisco J Santaclara

Validation of End-point and Real-Time PCR methods for the rapid detection of soy allergen in processed products

Keywords: Soy, Soybean, TaqMan, real-time PCR, PCR, ELISA 23 24 25

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

The use of soy protein in the food industry has increased significantly in the last few 30 years, being one of the most important and frequently used vegetable protein additives. 31

Among its functional properties it is worth highlighting the emulsification capability 32 and water and fat absorption, slicing characteristics and organoleptic features such as 33 appearance and firmness [START_REF] Belloque | Analysis of soybean 364 proteins in meat products. A review[END_REF]. In the case of seafood, the water 34 absorption and retention properties of textured soy proteins can be used to bind moisture 35 in fish blocks and reduce fat and jelly deposits liberated during food canning, which 36 results in a firmer final product [START_REF] Endres | Soy Protein Products: Characteristics, Nutritional Aspects, and 379 Utilization[END_REF]. The increase of water retention 37 gives rise to end-products of higher weight, which entails the use of soy protein for the 38 partial substitution of raw material, meat or fish, which is more expensive, making the 39 food products more cost-effective so that there is a considerable decrease in the final 40 cost of the product to the benefit of the manufacturers (MacedoSilva et al 2002). 41 42 On the other hand, food allergies represent an important health problem in industrialized 43 countries [START_REF] Poms | Methods for allergen analysis in food: a 421 review[END_REF], affecting up to 2% of the adult population and up to 8% of 44 children (Woods et al 23002). Anaphylaxis to soy in particular has been reported in 45 several studies [START_REF] Foucard | A study on severe food reactions in Sweden -is soy 382 protein an underestimated cause of food anaphylaxis[END_REF][START_REF] Sicherer | Peanut and soy allergy: a clinical 431 and therapeutic dilemma[END_REF]. Soy is currently recognized as 46 one of the potential allergens that have to be labelled if they are present in foodstuffs, 47 according to the European Directives 2000/13/EC and 2003/89/EC (Directive 48 2000/13/EC; Directive 2003/89EC). 49 50 Due to the previously described arguments, methodology to detect soy protein in food 51 products is necessary in order to ensure food safety, consumer protection and, at the 52 same time, to guarantee fair competition between companies. 53 54 Soy detection is complex in processed products due to the fact that during processing, 55 the food undergoes high temperature treatments or enzymatic processes. This causes 56 alteration in the protein, which is denatured and forms ethanol-water insoluble 57 aggregates and only a small percentage of proteins can be extracted for subsequent 58 detection. For these reasons, the sensitivity obtained by means of immunological 59 methods, such as ELISA technique, is not sufficient to assure safety of food products 60 [START_REF] Meyer | Polymerase chain reaction 409 (PCR) in the quality and safety assurance of food: Detection of soya in 410 processed meat products[END_REF]. DNA-based methods offer many advantages over protein-based 61 3 methodologies, primarily that the target DNA is less degradated than the proteins from 62 food matrices [START_REF] Poms | Methods for allergen analysis in food: a 421 review[END_REF]). In addition, the ELISA technique is more expensive 63 and time consuming than PCR. 64 65 Up to now, several PCR assays have been developed and applied to evaluate the 66 presence of genetically modified soy in food products (Abdullah et al. 2004;Brod et al. 67 2007;[START_REF] Kakihara | Extraction and detection of 398 endogenous soybean DNA from fermented foods[END_REF][START_REF] Moriuchi | Applicability of 413 quantitative PCR to soy processed foods containing roundup ready soy[END_REF] The samples used in the present work were the following: raw soybean; soy protein 82 isolate, concentrate and texturized; and soy flour. Also, 35 commercial processed 83 products of a type potentially containing soy protein were purchased from shops and 84 supermarkets in Spain (Table 1). The sampling included a wide range of foodstuffs with 85 different degrees of processing, some of them showing the presence of soy in the label 86 (meat, seafood and bakery products). Moreover other cereals were also obtained (wheat, 87 corn, oats, rice, rye, barley, triticale, lupin and kamut). 88 89 Genomic DNA was extracted from 30 mg of the samples previously mentioned. For 90 meat, fish and bakery products the amount of sample used was 150 mg. In canned 91 products DNA extraction was performed, from 300 mg of different filtered coating 92 media (water, oil, tomato sauce, pickling brine…) and superficial scraping of the 93 products. 94 Two different DNA extraction methods were evaluated: (1) a method based on silica gel 95 columns using the NucleoSpin Tissue kit (Macherey-Nagel) following the supplier's 96 protocol and (2) a CTAB method previously described [START_REF] Roger | Extraction of DNA from Plant Tissues[END_REF]

3.b. RFLP methodology 140

Restriction maps of the DNA sequences obtained were generated using the software 141

Webcutter 2.0 [START_REF] Heiman | Webcutter 2.0[END_REF]. The enzyme Hinf I was selected for its ability to 142 generate a characteristic restriction profile with band sizes easily distinguishable on 143 agarose gels. About 100 ng of PCR products were digested with 2 units of this enzyme. 144 Digestive reaction mixes were incubated at 37ºC for 2 hours. The results of PCR-RFLP 145 analysis were carried out as described above, except that the electrophoresis was 146 performed on 3% low melting agarose gels (Pronadisa) at 70 V for 110 min. Sizes of 147 fragments were estimated from a 50 bp DNA ladder (GE Healthcare). 148 149

Design of a specific RT-PCR method to detect the presence of soy 150

From sequences of lectin gene obtained with the primers described above, a specific 151 Taqman ® probe was designed for the development of a Real-Time PCR method to 152 detect the presence of soy. 153

The Lectin probe 5' (FAM) CAC ATG CAG GTT ATC TTG GTC 3' (TAMRA) was 154 labeled with the fluorescent reporter dye 6-carboxy-fluorescein FAM at the 5' end and 155 with the quencher 6-carboxy-tetramethyl-rhodamine (TAMRA) at the 3' end. 156

The theoretical specificity of the designed probes was first evaluated by a BLASTN 157 search in the GenBank database for sequences with 100% homology (Altschul et al. 158 1990). 159

In all cases the RT-PCR reactions were carried out in a total volume of 25 µL with the 160 composition previously described, except the amount of primers and probe that were 161 optimized. Optimal amount of primers and probe were evaluated by preparing dilution 162 series. A common range of working stock concentrations of 50, 300 and 900 nM of each 163 primer and 50, 100 and 250 nM of the Taqman ® probe were used to determine the 164 optimal concentrations. Molecular biology grade water (Eppendorf) was used to adjust 165 to the final volume. 166

The reactions were performed in a iQ 96-well PCR plates (BIO-RAD) covered with 167 iCycler iQ TM Optical Tape (BIO-RAD) and run on Bio-Rad iCycler iQ TM Real Time 168 PCR instrument with the following thermal cycling protocol: 95ºC for 30 s followed by 169 45 cycles of 95ºC for 5s and 55ºC-60ºC for 30 s. 170 The specificity of the two assays was evaluated by testing the amplification of DNA 172 from cereals (wheat, corn, oats, rice, rye, barley, triticale, lupin and kamut) and different 173 fish and land animals (tuna, cod, hake, salmon, squid, chicken, pig, cow, rabbit and 174 turkey) . 175 176The sensitivity of the proposed methods was assessed in 3 ways. The first one was 177 established from DNA dilutions. The range of concentration was obtained from raw soy 178 with DNA amounts ranging between 150 ng and 1 pg. The dilutions were prepared by 179 adding DNA from different cereals and animal species to the soy DNA until completing 180 the final amount. 181

The second way used to establish the LOD was by adding soy protein powder to 182 sunflower/olive oil or water in percentages from 1% to 0.01% in tuna cans of 120 mL. 183

These products were autoclaved using a high-pressure steam sterilizer at 121 ºC for 50 184 min. DNA extractions performed from filtered homogenates and superficial scraping of 185 these canned products as described previously were used to establish the detection limit. 186

Finally, corn flour samples containing amounts of soy flour from 10.000 to 10 ppm 187 were also analyzed. The PCR amplification was performed as described above. 188 The aim of the methodological validation was to check whether the manufacturing 205 process which processed food underwent had no influence on the detection of soy 206 protein with the proposed methods. 207 208

Application to commercial products 209

After the validation of the methods developed in the present work, these were applied to 210 35 processed products in order to determine if soy protein was added during the 211 manufacturing process (Table 1). These products were acquired from Spanish 212 supermarkets, and the purpose of the analysis was to evaluate the current status of 213 labelling regarding soy content of foodstuffs on the Spanish market. 214 Two methods for DNA extraction were tested. The best results in terms of DNA yield 219 and purity were obtained with the CTAB method followed by purification on a 220

NucleoSpin© Extract II kit (Macherey-Nagel). Good performances using CTAB-based 221 DNA extraction methods were obtained in previous works for soybean products 222 [START_REF] Gryson | Evaluation and optimisation of five 389 different extraction methods for soy DNA in chocolate and biscuits. Extraction 390 of DNA as a first step in GMO analysis[END_REF][START_REF] Olexová | Comparison of three types of methods 417 for the isolation of DNA from flours, biscuits and instant paps[END_REF]). On the other hand, Mafra et al (2008) 223 obtained best results for DNA extraction from soybean flours and protein isolates using 224 the second method evaluated in the present work, the NucleoSpin Tissue kit Nagel) that showed the best results for DNA extractions, although the CTAB method 226 was generally well suited to all kinds of food matrices tested [START_REF] Mafra | 405 Comparative study of DNA extraction methods for soybean derived food 406 products[END_REF]. 227

The purity of DNA extractions was adequate in all cases (A 260 /A 280 > 1.80). This RFLP is an alternative technique to sequencing and BLAST analysis to confirm the 248 identity of a particular PCR product. PCR followed by digestion of amplification 249 products using informative restriction enzymes was used in several previous works in 250 order to confirm the identity of a PCR product, since it presents several advantages in 251 comparison to sequencing (Ge et al. 2008;[START_REF] Von Buren | Detection of wheat adulteration of spelt 438 flour and products by PCR[END_REF]. For instance, the low 252 cost, and the fact that it is fast and easy. However, some authors advise against using 253 RFLP for forensic identification when there are moderate levels of intraspecific 254 variability, because this could make the RFLP unstable and lead to misidentifications 255 [START_REF] Santaclara | Genetic identification of Squids 427 (Families Ommastrephidae and Loliginidae) by PCR-RFLP and FINS 428 Methodologies[END_REF]). The intraspecific variability can be due to the species or the 256 molecular markers. The housekeeping genes, as Lectin in soy, show very little 257 variability. In this study intraspecific variability was not found in the soy samples 258 analyzed as previously reported (Simon et al 2003). 259

In accordance with the sequence data, the restrictase selected was Hinf I that generated 260 two DNA fragments of 71 and 29 bp, the 71 bp fragment being easily distinguishable on 261 agarose gels (Figure 1). This restriction profile was obtained for all samples analyzed 262 with this technique. Therefore, RFLP represents a suitable alternative technique to 263 sequencing for the assessment of PCR products' identity, since it permits obtaining the 264 characteristic restriction profile. 265

Both RFLP and sequencing techniques are used for confirmation of results. In routine 266 assays for soy detection their application would not be necessary although it is advisable 267 and important throughout the methodological development stage. 268 269

Design of method RT-PCR assay specific to detect the presence of soybean 270

Among the advantages of the real-time PCR technique it is worth highlighting its 271 specificity, sensitivity, reproducibility and rapidity. This technique allows verification 272 of the functioning of the PCR while it is running, saving the time from secondary 273 visualization or identification techniques of the PCR products. The optimization of the 274 PCR conditions allows granting highest level of sensitivity while maintaining the 275 specificity of the technique. 276

The conditions that led to the best results were established by means of primers and 277 probe matrix. The concentrations of 900 nM for both primers and 250 nM for the probe 278 yielded the best results in terms of specificity and sensitivity. 279 The specificity of the primer set LEC IF/LEC IR for soybean was confirmed by PCR 282 amplification using DNA extractions from wheat, corn, oats, rice, rye, barley, triticale, 283 lupin and kamut. No cross-reactivity was detected with any of the samples tested. In this 284 way the optimal annealing conditions were established at 54 ºC and 58 ºC for end-point 285 and Real-Time PCR methods respectively, in order to ensure the higher specificity of 286 the developed methodologies. 287

The detection limit was determined in 3 ways. The first of them defined the minimum 288 amount of DNA necessary to yield a visible band on agarose gel after amplification or a 289 positive fluorescence signal in the end-point and RT-PCR respectively. The detection 290 limit obtained for raw soy DNA extractions was 40 pg and 10 pg for end-point and 291

Real-Time PCR methods respectively (Figure 2 and3). 292

The second way to establish the LOD was through the minimum percentage of soybean 293 powder added to cans of fish of 120 mL. The detection limit thus determined was 294 0.0625% and 0.05% for end-point and Real-Time PCR methods respectively. 295

The last way to establish the LOD from corn flour containing soy flour in variable 296 amounts (10000, 1000, 100 and 10 mg/kg), allowed establishing a detection limit of 100 297 mg/kg and 10 mg/kg for end-point and Real-Time PCR methods respectively. 298

In the case of RT-PCR an important parameter reflecting specificity and sensitivity of 299 this metodology is the threshold cycle (Ct). It is necessary to find the lowest value of Ct 300 that allows maintaining high fluorescence levels together with lowest possible LOD for 301 the technique. In all the cases positive Ct values were between 21 and 38. 302 The results from the specificity and sensitivity evaluation of the developed methodology 303 clearly indicate RT-PCR as the best alternative for soy detection. Besides displaying 304 lower LOD values it is highly specific since this parameter is directly proportional to 305 the specificity of the set primers-probe. The results of this study demonstrated that the 306 developed primers and probe are capable of efficiently amplifying the lectin gene using 307 a real-time fluorescence detector. The assay is rapid, the entire procedure can be 308 completed within 4 h. Moreover, application of the real-time PCR method to food 309 samples has demonstrated the specificity of the method. 310

ELISA technique 312

The kit Soya protein assay kit (Tepnel Biosystems) employs the principle of enzyme 313 immunoassay and is a sensitive and specific assay for soy protein in the presence of 314 vegetable, meat and other proteins. 315

The kit used for the quantitative determination of soy protein provides a detection limit 316 of 5000 mg/kg, far from the detection limit of 10 mg/kg that has been obtained by 317 means of RT-PCR when corn flour and soy flour mixtures are analyzed. 318 However, both conventional and Real-Time PCR developed in this work may be used 319 as screening methodologies at the first step. If the results are negative it may be 320 concluded that soy presence in the sample is below 10 mg/kg. When a positive result is 321 obtained, the ELISA technique may be applied, but only if quantification is necessary. 322

This technique has lower sensitivity, is more expensive, laborious and time consuming 323 than genetic methodologies and may give a negative response if the quantity in the 324 sample does not exceed 5000 mg/kg. Therefore both techniques (ELISA and PCR) may 325 be combined. The PCR alternatives herein developed can be used for screening and 326 ELISA for soy protein quantification in case of positive results, allowing the detection 327 and quantification of soy in a particular sample. 328 329

Methodological validation 330

The aim of the validation step was to evaluate the correct operation of the proposed 331 methodology. The products elaborated in the pilot plant of CECOPESCA were analyzed 332 by the proposed molecular methodologies. The different treatments they were subjected 333 to during the elaboration process allowed us to evaluate the correct DNA amplification 334 in the most extreme cases of degradation (canned products). The soy was detected in all 335 these samples by the methodological approaches developed in the present study. 336 Therefore, it might be applied to canned, frozen, fried, cooked, and battered products. 337 338

Application to commercial products 339

Thirty-five meat, fish and bakery processed products were tested for the presence of soy 340 DNA by using the methods proposed in the present work (Table 1). Extracted DNA 341 showed a good yield and purity optimal in all products analyzed (evaluated by means of 342 optical measurements). Seventeen samples showed a positive result for the presence of 343 soy using the developed methodologies (Table 1). The results obtained for processed Altogether, this work describes the development and validation of two PCR methods to 347 detect the presence of soy protein in a wide rage of foodstuffs. Among the advantages of 348 these techniques it is worth highlighting that these are reliable and sensitive, allowing to 349 detect trace amounts of soybean in processed products. Moreover it is quick and cost-350 saving. Therefore it can be useful in questions regarding the presence of soy protein in 351 processed products, especially to verify the fulfilment of the labelling rules and to 352 protect consumers' rights. 353

  allowing the detection and 68 quantification of different GMO's, among them some soy varieties, with total 69 reliability. 70 71 In the present work two methodologies based on end-point and Real-Time PCR 72techniques using specific primers were developed. These can be alternatively used to 73 detect the presence of soy protein in commercialized processed products by means of 74 the amplification of the lectin gene. The food samples analyzed included a wide range 75 of products that underwent different treatments in order to assess the use of soy protein 76 in the current food industry and check the labelling regarding its presence in foodstuffs.

Information

  primer and molecular biology grade water (Eppendorf) up to adjust to the final volume. 117 PCR was performed in a thermal cycler MyCycler TM (BIO-RAD) under the following 118 conditions: a preheating step at 95 ºC for 10 min, 38 cycles of amplification (95 ºC for 119 30 s, 55-70 ºC for 45 s, 65 ºC for 45 s) and a final extension step of 65 ºC for 5 min. The 120 reactions were run using a range of annealing temperatures between 55 and 70º C. 121The amplified fragments were analyzed by electrophoresis on a 2% agarose gel (Sigma) 122 in 1X TBE buffer (Sigma) with 0.3 µg/mL of ethidium bromide (Sigma) and were 123 visualized as described above. The 50 bp DNA ladder (GE Healthcare) was used as confirm the identity of the amplified PCR products, these were sequenced. 129For that, double-stranded DNA products were purified using the NucleoSpin© Extract II 130 (Macherey-Nagel) according to the manufacturer's instructions. The concentration and 131 purity were measured by means of a NanoDrop™ 1000 spectrophotometer (Thermo 132 Scientific). Subsequently, both DNA extracts were sequenced on an ABI Prism 3130 133 (Applied Biosystems) using BigDye Terminator Cycle Sequencing Ready Reaction v1.1 134 (Applied Biosystems). Next, these sequences were analyzed with Sequencing Analysis 135 Software v5.3.1. (Applied Biosystems) and aligned with Clustal W (Thomson et al 1997) 136 available in the program BioEdit 7.0[START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and 393 analysis program for Windows 95/98/NT[END_REF]. The nucleotide sequences obtained 137 were submitted to the GeneBank database of the National Centre for Biotechnology 138

  and sensitivity of the methods 171

189 6 .

 6 Quantification of the developed methodology using the ELISA technique 190In order to verify the correct operation of the methodology proposed in the present 191 work, the Soya protein assay kit (Tepnel Biosystems) was used for the quantitative 192 determination of soy protein, according to the manufacturer's protocol. 193 194 7. Methodological validation 195In order to evaluate the correct operation of the methodologies proposed in the present 196 work, different food products were elaborated in the pilot plant of CECOPESCA 197 (Spanish National Centre of Fish Processing Technology) adding different types of soy 198 protein (Table2). The most extreme treatment applied to them was sterilization by 199 horizontal retort steel-air at 115 ºC for 50 min and 1.2 bars of overpressure. 200The amount of soy added to the samples employed in the methodological validation 201 corresponded to the minimum amount detected during development of the 202 methodology. Subsequently, the samples were analysed with the methodologies 203 developed in the present work. 204

  228 parameter is important to guarantee high-quality results in PCR amplification, both in 229 end-point and RT-PCR. 230 231 2. Confirmation of identity of PCR products 232 PCR amplification of a 100 bp fragment of the lectin gene using the primer set Lec 233 IF/IR was successfully achieved in all kind of samples (raw soybean; soy protein isolate 234 concentrate and texturized; and soy flour). 235This amplicon was analyzed by means of sequencing and RFLP in order to asses the 236 identity of the PCR products. This approximation allows verifying the specificity of the 237 primers in the conditions of cycling described. 238 2392.a. DNA Sequencing and Blast analysis 240Sequencing followed by Blast analysis is a powerful technique to confirm the identity of 241 PCR products. It is used in laboratories devoted to food control, although it has the 242 drawback of high cost. The results of this approach showed that the obtained sequences 243 were identical to those available in NCBI for soy, achieving a homology score of 100% 244 with the Megablast algorithm in all cases. These sequences were deposited in the

  assessed by means of conventional and Real-Time PCR methods. 281

  of the results obtained by the developed methodology using the 311

  the wide use of soy protein in the food industry for products 345 commercially available in Spain. 346

Figure 1 .Figure 2 .Figure 3 .

 123 FIGURE CAPTION 453 454 Figure 1. RFLP analysis of PCR products obtained with the LEC primer set and DNA 455 template from soybean. Lane PCR: PCR products; Lane 1: PCR product; Lane 2: PCR 456 products after digestion with Hinf I; L-50: molecular size marker (50 bp DNA ladder, 457 GE Healthcare). 458 459 Figure 2. Limit of detection of the End-point PCR assay. Lane L-50: 50 bp DNA 460 Ladder, (GE Healthcare); Lane 1-6: 1 ng, 500 pg, 250 pg, 100 pg, 50 pg and 40 pgof 461 DNA extracted from soybean used as template in the PCR. 462 463 Figure 3. Limit of detection of the RT-PCR assay. A: 100 ng, B: 10 ng, C: 1 ng, D: 100 464 pg, E: 10 pg of total DNA extracted from soybean used as template in the PCR. 465 466

. End-point PCR assay to detect the presence of soy 111

	112	2The primers Lec IF and Lec IR described by Zhang et al (2007) were used to amplify a
	97 113	100 bp fragment of the soy lectin gene.	with
	98 114	slight modifications followed by purification with NucleoSpin© Extract II kit In all cases PCR reactions were carried out in a total reaction volume of 25 µL
	99 115	(Macherey-Nagel). containing 50 ng of DNA template, 0.8 mM of dNTP mix (Bioline), 2.5 µL of 10X
	100 116	buffer, 2 mM of MgCl 2 , 0.75 units of BioTaq TM DNA pol (Bioline), 0.8 µM of each
	101	The extracted DNA was loaded onto a 1% agarose gel containing 5 µg/mL of ethidium
	102 103 104 105	bromide (Sigma), analysed by electrophoresis in 1X TBE buffer (Sigma) at 70 V for 50 min and visualized using the Molecular Imager Gel Doc XR System transilluminator and the software Quantity One® v 4.5.2 (Bio-Rad). The 100-1500 bp DNA ladder (Dominion, MBL) was used as molecular weight marker. F o r
	106 107 108 109	The DNA concentration was determined by measuring the absorbance at 260 nm and P the purity using the absorbance ratio of 260 and 280 nm (A 260 /A 280 ). These measures e were carried out using a NanoDrop™ 1000 spectrophotometer (Thermo Scientific). e r
	110	DNA extracts were appropriately labelled and stored at -20 ºC. R
		e
		v i e
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		O n l
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http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


Rüdiger Schweiss
email: ruediger-bernd.schweiss@sglcarbon.de

Marcus Steeb

Peter M Wilde

Mitigation of Water Management in PEM Fuel Cell Cathodes by Hydrophilic Wicking Microporous Layers

Keywords: PEM Fuel Cell, Water Management, Gas Diffusion Electrode, microporous layer, Performance PEMFC, water management, gas diffusion layer, microporous layers

This study introduces a novel strategy for enhancing the performance of Polymer Electrolyte Fuel Cells at high current densities by means of an advanced gas diffusion layer design. The incorporation of hydrophilic wicking agents into microporous layers of the cathode gas diffusion layer improves water management, thus increasing the maximum power density of Polymer Electrolyte Fuel Cells. Ex-situ measurements with respect to water and vapour transport, and electrochemical polarisation data provide evidence suggesting that the beneficial effect has to be attributed to enhanced liquid water removal through the microporous layer.

Introduction

Polymer electrolyte fuel cells (PEMFCs) constitute promising candidates for future energy systems which will have to pay particular attention to carbon footprint and efficiency. Such requirements are fulfilled by fuel cells which enable a direct conversion of fuel into electrochemical power while only releasing non-toxic effluents from the system. Forthcoming applications are mainly seen in the field of automotive propulsion or backup power. In order to substitute fossil fuel systems, the PEMFC still has to overcome barriers relating to costs and infrastructure and also has to address technical aspects such as durability and performance (which are, in turn, related to costs).

Among the technical issues, water management is one of the critical processes in the operation of Polymer Electrolyte Fuel Cells (PEMFCs) [START_REF]Polymer Electrolyte Fuel Cell Durability[END_REF][START_REF] Li | [END_REF]. Especially in the case of high current density applications such as automotive PEM stacks, mass-transport limitation due to water accumulation is the predominant factor limiting stack performance. In this context, a complex interplay of different competing processes of water generation, water removal, water evaporation, water condensation, and water transport (in the liquid or gas phase) has to be controlled by means of system design, operation mode or fuel cell component properties. From the membrane perspective, a high level of hydration has to be maintained, whereas for the porous media such as the catalyst sites, the gas diffusion layers and the flowfield backings, an efficient removal of liquid water is indispensable. Additionally, the excess water in the porous media not only affects the performance, but also has implications on the degradation of functional components and is further associated with critical events during freeze shutdown/startup of PEMFC stacks [3].

At the component level, different approaches to controlling water transport and removing water from PEMFCs have been described in literature, such as MEA design [3], control of hydrophilicity and microstructure of flow fields [4,5] or the insertion of a water management layer [6]. As the interface between the active areas and the flowfield backing, the gas diffusion layers (GDLs) are not only the key moderators for the different water transport phenomena but also, apart from the flowfields, the main site of water accumulation. Gas diffusion layers which consist of porous carbon fibre webs (papers, nonwovens or cloths) are commonly treated with fluoropolymers and coated with a microporous layer (MPL) in order to perform the complex task of maintaining a certain humidification level of the adjacent catalyst sites and the membrane while avoiding flooding of its porous body which would compromise the reactand gas supply. Additionally, it has been reported that the electroosmotic drag is also influenced by GDL characteristics. At high humidfication levels, the use of a GDL with a hydrophobised base substrate and a microporous layer is common practice [START_REF] Mathias | Handbook of Fuel Cells -Fundamentals Technology and Applications[END_REF][START_REF] Gostick | [END_REF][9]. Water saturation in the GDL was observed to decrease when an MPL is present [START_REF] Gostick | [END_REF]10]. Additionally, the in-plane permeability of a GDL without an MPL is much larger and thus leads to an increased pressure drop which is unfavorable for water removal [11]. Excessive loading of the GDL and the MPL with fluoropolymer, however, was shown to induce flooding of the catalyst layers. A number of studies were recently conducted based on computer modelling approaches or in-situ water imaging with x-ray or neutron radiography in order to arrive at a deeper understanding of the mechanisms underlying water transport in GDLs [12]. A simple description based on capillary-driven flow only was concluded to be insufficient to predict liquid water transport in porous diffusion media, because in this case continuous liquid water flow from the site of high saturation to the areas of lower water saturation would occur [13]. This is why continuous water flow has never been observed in practice.

These findings are supported by water imaging with fluorescent dyes [14], which show that water transport in hydrophobic GDLs is determined by a capillary fingering mechanism with only a few clusters that are released more or less periodically (also referred to as "eruptions" [15,16]). Theoretical considerations taking into account vapour diffusion and thermal gradients across the GDL under real operating conditions suggest, that for an hydrophobic MPL-coated GDL, the majority of the excess water is transported in the vapour phase [17]. This is supported by Synchrotron x-ray radiography studies by Hartnig et al. [18], who showed that water is formed primarily near the channel ribs of the flow field and that, regardless of the current density, hardly any liquid water is present in the microporous layers. Two diffusion barriers were found to be located at the MPL/GDL substrate-interface and at the catalyst-MPL boundary. Neutron imaging similarly yields indications that water is accumulated at the landings and the channel bends [11,19].

The current picture of water transport in porous, hydrophobic gas diffusion media is therefore that

• the liquid water condensation is dependent on the local wettability and the temperature gradient in the GDL • a branched water profile with a certain fraction of dead ends exists in the GDL substrate • there are preferential pathways of liquid water removal that evolve over time • the content of liquid water in the MPL is low • water release from the GDL is described by an eruptive mechanism.

As a result, advanced GDL designs will have to go beyond the simple wet-proofing approach and advance towards an "engineered porosity/hydrophobicity" [20]. In this context, water flow analyses suggest that a bimodal pore size distribution might be an advanced GDL design principle [21]. As a step towards controlled hydrophilic/hydrophobic porosity, this paper introduces a novel strategy of water management mitigation in PEM fuel cells by means of enhancing liquid water transport through the microporous layer of the cathode GDL. This is accomplished by the incorporation of hydrophilic fibres in the microporous layers which act as pathways of preferential liquid water flow. A parametric study was conducted with several reference materials, the emphasis being placed on ex-situ water transport and in-situ measurements.

Experimental

GDL substrates

Rolls of Sigracet® Gas Diffusion Layers GDL (carbon paper grades 24 AA, 25 AA), SGL Technologies GmbH, Meitingen, Germany) were treated with aqueous polytetrafluoroethylene (PTFE) dispersion by means of a dipping process in order to obtain a 5% (w/w) PTFE load of the substrates [22]. The hydrophobised substrates (designated as 24 BA and 25 BA) had open porosities of 89% (25 BA, 35 BA) and 84% (24 BA, 34 BA). The low porosity GDL are therefore recommended for dry operation whereas the highest performance is generally obtained using 25 BC at high humidification levels [23,24].

Microporous Layers

Chopped aluminosilicate fibres (type ALTRA B97 LA C25, Rath GmbH, Mönchengladbach, Germany, fibre diameter 2-4 µm) were treated in a ball mill (Fritsch, Idar-Oberstein, Germany) for 2 mins. The milled fibres obtained had lengths from 20-90 µm( determined by laser diffraction). Thereafter, they were dispersed in deionized water (10 g in 100 g water) The fibre dispersion was added to an aqueous dispersion of acetylene black and PTFE in order to obtain coating paste with a carbon to fibre ratio of 10:1. The paste was applied to a GDL substrate using a continuous blade coating line. This procedure yields a GDL designated as 25 BL. The C-type MPLs (24 BC and 25 BC materials) were prepared accordingly without addition of aluminosilicate fibres. All MPLs (L and C) had a carbon to PTFE ratio of 77:23. Next, the coated GDLs were subjected to a sintering process at 350°C for 10 minutes.

Characterization

The GDLs were characterised with regard to electrical properties, porosity, gas permeability, water vapour transmission and liquid permeability [22,23]. Capillary flow porometry (CFP) was performed with a PMI capillar flow porometer (Porous Materials Inc, USA) and liquid permeability was carried out by means of a PMI liquid permability tester (Porous Materials Inc, USA). Gas permeability was determined using the Gurley method (TAPPI T460, 304 Pa). Water vapour transmission (according to ASTM D6701-01) of GDLs was measured with a Mocon Permatran-W 101K Setup (Mocon Inc, Minneapolis, USA). Single-cell fuel cell tests were performed on a Fuel cell test bench (Hydrogenics Corp Burnaby, CA) using a pneumatically sealed test cell with an active area of 25 cm 2 (Baltic Fuel Cells, Schwerin, Germany). A commercial CCM (Primea 5710®, 0.4 mg cm -2 Pt on cathode, 0.1 mg cm -2 Pt on the anode side, WL Gore Associates, Putzbrunn, Germany) was sandwiched between anodic and cathodic GDLs facing graphite flow fields at a compression load of 1 N mm -2 . Stoichiometry was 1.52 on the anode and 2.50 on the cathode, respectively. Inlet pressure was 50 kPa. Sigracet® 25 BC was used as a standard anode GDL for all of the experiments.

Results and Discussion

Figure 1 shows micrographs of the surface of an MPL with embedded aluminosilicate fibres (GDL 25 BL). Given the total amount of fibres in the MPL and comparing them to microscopic images of several larger MPL areas, it can be concluded that most of the fibres are buried beneath the top surface where they can act as hydrophilic wicks in the microporous layer. Mud cracks were observed for both substrates, but the overall frequency of mud cracks was not found to differ noticeably for both MPLs.

Pore Characteristics

Pore size distribution was determined using capillary flow porometry [24,[START_REF] Jena | 41st Power Sources Conference[END_REF]. Figure 2 shows the flow pore size distribution of different GDLs. Despite the fact that all of the GDLs were coated with the same microporous layer formulation, it was observed that the pore size distribution is still significantly influenced by the GDL base substrate. The GDL based on substrates with the lower porosity (24 BC) consequently shows a mean pore diameter less than GDLs with the 25 base substrate. The addition of hydrophilic fibres to the MPL, in turn, does not have any noticeable effect on the pore size distribution, but only results in a slightly larger bubble point (25 BC = 32 µm, 25 BL = 34µm).

Gas, liquid water and water vapour transport properties

Figure 2 shows a water vapour transmission measurements of GDL reference materials with different substrate thickness and porosity and a standard MPL (C) and the novel GDL with a modified MPL (25 BL). . The water vapour transmission is primarily determined by the diffusion length (GDL thickness) and to a lesser extent by the substrate porosity (as seen by the values obtained for 24/25 and 34/35, respectively). By contrast, no significant effect of the MPL composition on the vapor diffusion was observed.

Since liquid water transport is assumed to be driven by hydraulic pressure [13], liquid permeability measurements [START_REF] Mathias | Handbook of Fuel Cells -Fundamentals Technology and Applications[END_REF] of the GDLs were carried out. As revealed in Figure 4, 25 BL shows a much higher liquid water flux at a given pressure than the 25 BC GDL. This cannot be attributed to differences in the MPL porosity, but is solely the consequence of the hydrophilic wicking effect which is due to the presence of the inorganic fibres. The resistance of the 25 BL against liquid water transport is therefore reduced by factor 5 as compared to the 25 BC. The onset of the flow curve (also referred to as hydrohead pressure [START_REF] Mathias | Handbook of Fuel Cells -Fundamentals Technology and Applications[END_REF] which is the intercept at flux = 0), however, is very similar for both GDLs (0.12 kPa for 25 BL and 0.2 kPa for 25 BC). This means that there are no significant differences in capillary pressure for both GDLs. In other words, the MPL matrix of 25 BL is clearly hydrophobic and the liquid water transport predominantly occurs along a network formed by the hydrophilic fibres.

Fuel Cell Tests

Single-cell fuel cell tests on the different GDL substrates on the cathode side (25 BC was used as a reference on the anode) are depicted in Figure 3. Electrochemical polarisation curves at two different temperatures and humidification levels show distinct differences in the GDL properties. As expected, the low porosity gas diffusion layer (24 BC) shows a mass transport limitation at current densities of more than 0.6 and 1 A cm -2 , respectively. Especially under high humidity conditions (60°C, 100% RH, high current density), the GDL with hydrophilic constituents (25 BL) displays a beneficial effect corresponding to an approximately 8% increase in maximum power as compared to 25 BC. This means that, the 25 BL cathode diffusion layer obviously removes excess water more efficiently than the reference systems. This is due to enhanced water transport in the liquid phase which is caused by the aforementioned hydrophilic wicking mechanism. Interestingly, cathodes based on 25 BL also show a higher power than 25 BC under dry conditions (40% RH) which might be due to the fact that it could retain more liquid water as compared to the 25 BC under low water saturation (dry) conditions.

Conclusions

Gas diffusion media with different properties such as porosity and hydrophobicity were characterised with regard to different water transport mechanisms. A novel concept for tailoring the degree of hydrophilicity of the microporous layer shows a beneficial effect on the water management of cathode GDLs. This is verified by exsitu methods and single-cell fuel cell studies. The increased performance of the novel MPL with hydrophilic wicks demonstrates that a gas diffusion layer with enhanced liquid water permeability is capable of retarding the flooding of the catalyst sites under wet conditions. The beneficial effect of hydrophilic constituents in the microporous layers is twofold. Firstly, an enhanced lateral diffusion of water at the catalyst-MPL interface is expected to counteract excessive flooding of the active areas. Secondly, preferential pathways are generated to guide liquid water through the microporous layers. Evidence of the mechanism is provided by ex-situ water transport measurements and single-cell tests. Further insights into the water transport mechanisms of the different GDLs types are expected from in-situ neutron imaging studies which are currently being performed [START_REF] Mukundan | [END_REF].

In summary, it has been verified that even the addition of hydrophilic compounds to the microporous layer in a relatively arbitrary fashion does indeed improve water management in a fuel cell. In line with a recent study by Park et. al. [START_REF] Park | [END_REF], this study also stresses the importance of a liquid water transport properties of gas diffusions layers for fast drainage and fuel cell performance. Water management can be expected to be further improved by the locally controlled placement of hydrophilic patterns, taking into account the specific flow field geometries.

  Pore size distribution of the GDLs as calculated based on capillary flow measurements (averages of 3 runs) 288x201mm (300 x 300 DPI) Water vapour transmission of GDL reference material and GDL 25 BL. 288x201mm (300 x 300 DPI) Comparison of liquid water permeability measurements for 25 BC and 25 BL 288x201mm (300 x 300 DPI) Electrochemical polarisation curves of single cells with different cathode GDLs under wet conditions (60°C, 100% RH) and dry conditions (80°C, 40% RH). GDL 25 BC was used as standard on the anode.

Acknowledgment

The authors would like to express their gratitude to Werner Buergel for SEM images and to Stefan Woehner (SGL Group, Meitingen) for providing liquid permeability measurements.


Jens F B Rasmussen

Anke Hagen

The Effect of H2S on the Performance of SOFCs using Methane Containing Fuel

Keywords: SOFC, hydrogen, sulfide, Methane, Reforming, poisoning hydrogen sulfide, methane reforming, poisoning, SOFC

In recent years the interest for using biogas derived from biomass as fuel in solid oxide fuel cells (SOFCs) has increased. To maximize the biogas to electrical energy output it is important to study the effects of the main biogas components (CH 4 and CO 2 ), minor ones and traces (e.g. H 2 S) on performance and durability of the SOFC. Single anode-supported SOFCs with Ni-YSZ anodes, YSZ electrolytes and LSM-YSZ cathodes have been tested with a CH 4 -H 2 O-H 2 fuel mixture at OCV and 1 Acm -2 current load (850°C). The cell performance was monitored with electric measurements and impedance spectroscopy. At OCV 2-24 ppm H 2 S were added to the fuel in 24 h intervals. The reforming activity of the Ni-containing anode decreased rapidly when H 2 S was added to the fuel. This ultimately resulted in a lower production of fuel (H 2 and CO) from CH 4 . Applying 1 Acm - 2 current load, a maximum concentration of 7 ppm H 2 S was acceptable for a 24 h period.

Introduction

Technological solid oxide fuel cells (SOFCs) have been demonstrated to operate very well when running on H 2 , synthesis gas or CH 4 -containing fuels for long operation times (e.g., [1]). It has thus become interesting to study the applicability of alternative fuel sources, like biogas derived from biomass. Biogas produced by anaerobic digestion of biomass mainly consists of CH 4 and CO 2 , but also small amounts of impurities, such as for example H 2 S (>30 ppm), halogens, and siloxanes. At the high operational temperature of SOFCs (700-900°C) and the presence of Ni-catalyst electrodes, the main components CH 4 , CO 2 and H 2 O are converted to H 2 and CO (Reaction (1)).

(1)

+ + + + → → → → + + + + + + + +
As both H 2 and CO are fuels to the SOFC the use of biogas in SOFCs seems very attractive. However, the effect of the fuel on performance and durability has to be studied; specifically the main constituents methane vs. hydrogen and impurities such as H 2 S. CH 4 -containing fuels in Ni-YSZ anode based SOFCs have been investigated using oxygen-species O 2 , H 2 O and/or CO 2 , to convert CH 4 with the Ni in the anode acting as catalyst [2][3][4][5][6][7][8]. A critical issue in these studies has been the conversion of CH 4 on the anode's Ni particles without producing coke, as coke lowers the performance of an SOFC. The coke formation can be controlled by different factors, for example the operational temperature and the O:C ratio. A large part of the studies has thus included varying the operational temperature and the O:C ratio to find out how these parameters affect SOFC performance. Typically, increasing the oxygen-species concentration decreases the coke formation, but leads to a decrease in the SOFC efficiency as the oxygen-species dilute the fuel. Furthermore, the Ni particles risk being oxidised at high oxygen-species concentrations in the fuel [9]. It is thus necessary to optimize the fuel component concentrations and the operational temperature to optimize the SOFC performance.

Special attention has been given to the steam reforming of methane, in particular due to the close relations between the typical nickel containing steam reforming catalyst and the nickel containing SOFC anode. In the steam reforming of CH 4 , three main reactions occur, i.e. the steam reforming reaction (Reaction (2)), the water gas shift reaction (Reaction (3)) and the methanation reaction (Reaction (4)):

(2) Having a steam to carbon ratio of 2 at typical SOFC operational temperature (~800°C) is considered to prevent coke formation [10].

2 2 2 H m n CO n O H n H C m n                         + + + + + + + + → → → → + + + + (3)
Impurities like H 2 S in the fuel are critical. From catalysis it is also known that sulphur poisons the reforming activity of Ni-based catalysts and the underlying mechanism is fairly well understood [11]. The natural gas (methane) is usually desulphurized prior to the reforming process in order to prevent the reforming catalyst from being poisoned. Also in the SOFC, H 2 S naturally affects the reforming, as a nickel containing anode is used, and the electrochemical activity as well [10].

Several research groups have experimentally studied the effect of having S-compounds in an H 2 -containing fuel on the performance and the durability of an SOFC [12][13][14], but also the effect of S-compounds in CH 4containing fuels [15,16] has been studied. It was shown that H 2 S had a significant effect on the reforming activity, which in this way also affected the power output of the SOFCs. It has to be noted that the studies were performed in fuel with very low methane concentrations ( [15]: catalytic partial oxidation (CPO) gas with 1.5 vol% CH 4 , [16]: natural gas reformate) and that the direct use of biogas poses a larger challenge due to the high concentrations. It was also concluded that the Ni-sites active for reforming were different from the sites active for the electrochemical reaction [15]. Summarizing, when using biogas as fuel in an SOFC, two effects of H 2 S have to be considered: Poisoning of the reforming and of electrochemical activity and the interplay between these.

In a former study [13], the effect of H 2 S on the electrochemical activity (performance) of a SOFC using an H 2 -H 2 O gas mixture was evaluated. In the current study CH 4 was included in the fuel and thus the aspect of poisoning of reforming activity introduced. Varying concentrations of H 2 S were added at intervals. Electrical measurements and impedance spectroscopy were used to monitor the effect of S-poisoning on the cell voltage and the internal resistance of the SOFCs. Mass spectrometry was used to monitor the steam reforming activity of CH 4 as a function of the H 2 S concentration. This gave the possibility to study the effect of H 2 S on both the electrochemistry and the reforming activity.

Experimental

Cells and Initial Electrochemical Characterization

The used planar SOFCs were produced at the pre-pilot facility located Risø DTU and had a Ni/Yttria-Stabilized-Zirconia (Ni/YSZ) anode support and an active Ni/YSZ anode, a YSZ electrolyte, and a Lanthanum-Strontium-Manganate (LSM)/YSZ cathode [17]. The active area was 4 x 4 cm 2 (total area of 5 x 5 cm 2 ). In the testing setup the cells were placed between Ni containing anode and LSM containing cathode gas distribution layers, and secondly, between current collector foils, i.e. a Ni foil on the anode side and an Au foil on the cathode side. The setup was sealed using glass/ceramic-composite bars. A more detailed description of the setup can be found in [START_REF] Mogensen | High Temperature Solid Oxide Fuel Cells. Fundamentals, Design and Applications[END_REF].

The tests were started by sealing and reducing the cells at 1000 °C followed by an initial electrochemical characterisation (fingerprint) at 850, 800 and 750 o C to verify that the cell performances were as expected. The fingerprint measurements comprised of iV-curves and impedance spectra in 96% H 2 + 4% H 2 O and 80% H 2 + 20% H 2 O fuel composition. In the impedance measurements an oscillating current of approximately 60 mA was applied over the frequency range 0.08 Hz to 82 kHz with a Solartron impedance analyser.

Cell Testing

In Figure 1 The thus obtained two mixtures were combined and led into the cell test house. The total fuel flow at the cell inlet was kept constant at 10 lh -1 . Air was used on the cathode side (140 lh -1 ).

Two different test series were carried out. The aim of the first test series was to measure the effect of H 2 S on the reforming activity in the anode. An SOFC was run at OCV with a quadrupole mass spectrometer (QMS) attached to the fuel inlet and outlet tubes. This way the changes in gas composition as a function of the H 2 S concentration were monitored. In this test series the H 2 S was added in steps for periods of 25-75 h (illustrated in A on the right-hand side of Figure 1).

The second series of tests involved measuring the effect of H 2 S on the performance of the SOFCs. Three SOFCs were run under current load. Illustration B on the right-hand side of Figure 1 shows how H 2 S was added with increasing concentrations for 24 h intervals. The test setup included temperature, current and voltage probes. Furthermore, pO 2 probes were placed in the fuel inlet and outlet tubes to measure the oxygen partial pressure by measuring voltages (pO 2 -in voltage and pO 2 -out voltage, respectively). The pO 2 probes were made of single ended zirconia based tubes, and were supplied with a constant flow of air on the inside. Two Pt wires (one on the inside and one on the outside of the tube) measure the potential difference across the tube wall. By using the Nernst equation the partial pressure of oxygen on the outside (the fuel inlet or outlet) was calculated. A third Pt/Rh wire was welded together with the inside Pt wire, and was used to measured the inlet and outlet fuel temperature. The SOFC performance was evaluated by measuring the cell voltage, the in-plane voltage in anode and cathode (Figure 1) as well as by impedance spectroscopy (see also [13]).

Mass Spectrometry

An OmniStar TM GSD 301 01 quadrupole mass spectrometer (QMS) from Pfeiffer Vacuum was used. It was equipped with an approximately 1 m long stainless steel capillary (ø0.12 mm). The capillary was heated by a heating tape and was connected to the cell house fuel tubes via a ~30 cm long stainless steel tube with ø5 mm. Both transfer lines between the cell testing setup and the QMS were heated (> 100 °C).

3 Results and Discussion

Steam Reforming Activity of the Ni-YSZ Anode

The first test series was carried out to study the effect of H 2 S on the catalytic activity of the Ni/YSZ-anode in an SOFC for the steam reforming. The measurements were carried out at 850°C with a fuel composition of 13% H 2 , 29% CH 4 and 58% H 2 O. The total fuel flow was 10 lh -1 . The measured OCV under these S-free conditions was 984 mV, the pO 2 -in voltage 850 mV and the pO 2 -out voltage 980 mV. These three voltage values can be directly translated to fuel compositions and were used to evaluate the tightness of the testing setup and the reforming activity.

The theoretical cell voltage (Emf) for the SOFC for the present gas mixture was calculated to be ~995 mV. We consider an SOFC to have a gas tight electrolyte and to be sealed sufficiently gas tight from the surroundings if the voltage values do not differ from the Emf by more than approximately 10-15 mV. This demand was fulfilled with the obtained pO 2 -out of 980 mV. We can thus conclude the setup to be sufficiently tight and the influence of leaking gases on the fuel composition and thus mass spectrometry analysis to be negligible.

The OCV and pO 2 -out values of 984 and 980 mV, respectively, correspond to the equilibrium gas mixture, when steam reforming has occurred (Emf: 995 mV). The pO 2 -in at the fuel inlet was smaller, which means that the hydrogen concentration in the fuel before entering the SOFC was lower than under the equilibrium conditions, i.e. the reforming reaction had not taken place before the fuel had contacted the SOFC anode. This finding was expected as it is known that reforming does not proceed spontaneously but needs a catalyst. The cell voltage (OCV) and the pO 2 -out on the other hand were the same and corresponded to the equilibrium gas composition of the reformed fuel. Therefore, reforming happened quite fast after the first contact of the methane containing fuel with the SOFC anode (in absence of H 2 S).

To monitor the effect of H 2 S on the steam reforming activity in the anode, the H 2 S was added in steps of increasing concentration from 2 ppm to 24 ppm. After adding the H 2 S to the fuel, the cell voltage initially dropped fast and then an almost constant cell voltage about 25-75 h after adding the H 2 S was reached. At each of these constant periods iV-curves were measured. The cell voltage development is shown in Figure 2. The measured cell voltages at each step were: 984 mV (0 ppm), 949 mV (2 ppm), 921 mV (4 ppm), 910 mV (7 ppm), 902 mV (9 ppm), 885 mV (20 ppm) and 882 mV (24 ppm) (see also Figure 2). As the H 2 S was turned off, the cell voltage returned to a similar level as before adding the H 2 S.

Figure 2

As the fuel composition was only changed to a small extent (ppm amounts of H 2 S were added), this drop of the cell voltage cannot be related to the H 2 S concentrations in the fuel but indicates a significantly changed fuel composition regarding the other components, i.e. the steam reforming reaction was affected and the fuel composition gradually changed towards the initial fuel inlet composition (850 mV, pO 2 -in) when increasing the H 2 S concentrations. The drop in OCV was thus a direct effect of S chemisorbed on the active sites for the methane reforming. Simultaneously to the voltage measurements under addition of H 2 S, also the gas composition was measured at the fuel outlet using mass spectrometry. In steam reforming, the main reactions ( 2)-( 4) proceed, i.e. an increased amount of CO and CO 2 in the fuel can be directly related to the converted CH 4 .

At the times of constant cell voltage during addition of H 2 S, the fuel gas composition was analysed using the QMS. In Figure 3 the relative ratios of the measured CO, CO 2 and CO+CO 2 intensities (reaction products, see Equation ( 2)-( 3)) to the CH 4 intensity (reactant, see Equation ( 1)) are shown. To avoid having to correct for small pressure differences (and thus differences in absolute intensities) between the single QMS measurements, only ratios of the intensities have been considered. The ratios between product/reactant decreased first rapidly when between 0 ppm and 4 ppm H 2 S were added. At 4 ppm the ratios had dropped by ~90%. This indicated that the steam reforming of CH 4 was strongly affected even at a low H 2 S concentration. With further increasing the H 2 S concentration, this ratio decreased slower.

Figure 3

Based on the measured cell voltage values under H 2 S, the fuel gas composition in the cell was calculated using a program developed in house. In Figure 4 the thus calculated CH 4 conversion is plotted as a function of the H 2 S concentration. Even low concentrations of H 2 S in the fuel (2-4 ppm) have a large effect on the steam reforming activity of the SOFC anode, thereby confirming the observed inhibition of reforming as measured by the QMS (see Figure 3). At 24 ppm H 2 S less than 15% of the CH 4 in the fuel is converted.

Rostrup-Nielsen [START_REF] Rostrup-Nielsen | Catalytic Steam Reforming[END_REF] published a relation between the coverage of S on a Ni catalyst, θ s , , the temperature T in Kelvin and the ratio between the partial pressures of H 2 S and H 2 , Y = pH 2 S/pH 2 (equation ( 5))

(

⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ + + + + ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ - -- - = = = = - -- - - -- - θ θ θ θ
When applying the pH 2 S and pH 2 values used in our CH 4 tests, θ s becomes 0.86 at 850°C and 4 ppm H 2 S (see insert in Figure 4). Having covered the active sites to such a large extent, the observed rapid poisoning of the CH 4 reforming seems quite reasonable.

SOFC Performance

The first test series discussed above was aimed at studying the effect of H 2 S at OCV and reflected thus purely catalytic activity and poisoning. However, current might have a significant effect. Among others Koh et al. [START_REF] Koh | [END_REF] found that less coke was formed on an SOFC under current load compared to an SOFC at OCV. It was speculated that the coke was being reacted off the Ni particles at the three-phase-boundaries (TPB) by O 2--ions. O 2--ions have also been suggested to facilitate desorption of S species from the TPB [14,15,21]. The degree of poisoning of the CH 4 conversion might be moderated when the SOFC is in use, i.e. under current load.

Three SOFCs were tested under current load (1 Acm -2 ) in CH 4 -containing fuel and were exposed to H 2 S for 24 h intervals in order to study the effect of H 2 S on the steam reforming activity and electrochemistry. In all three tests a steam to carbon ratio of ~2:1 was used to prevent coke formation on the Ni particles. The tests were run at a temperature of 850°C. In test 1, a smaller CH 4 concentration was used than in test 2 and 3, in order to study the importance of CH 4 concentration. The fuel compositions, the fuel utilization (FU) and the initial area specific resistance values corrected for FU (ASR cor ) [START_REF] Mogensen | High Temperature Solid Oxide Fuel Cells. Fundamentals, Design and Applications[END_REF] of the cells in test 1-3 (denoted CH 4 tests) are shown in Table 1.

Table 1

The cell voltage developments are shown in Figure 5. After turning on the current load, the SOFCs were run in the CH 4 /H 2 /H 2 O fuel for 300-500 h before adding the first portion of H 2 S. During this period each of the cells degraded (50-60 mV per 1000 h). Addition of H 2 S to the fuel resulted in an initial voltage drop, followed by a nearly constant cell voltage degradation. After turning off the H 2 S the cell voltage regenerated to a level consistent with the overall degradation observed in the absence of H 2 S. The insert in Figure 5 shows these effects after adding the 4 ppm H 2 S to the fuel in test 1 as example. In test 2 and 3, the current load was interrupted for ~100 h due to work on the lab facilities. These interruptions (indicated by horizontal arrows) did not affect the overall cell voltage development.

The cell voltage development had thus much similar characteristics as in the poisoning tests in H 2 fuel [13]. The first voltage drop was ascribed a fast adsorption of S on the Ni surface, blocking the active sites for hydrogen adsorption and oxidation [13,14]. The reason for the following voltage degradation is not completely clear at the moment.

During regeneration from 2 ppm (second time) in test 1, the cell voltage did not return entirely to the overall voltage development, but instead started to decrease progressively. The test was stopped, when a critical cell voltage was reached. When adding 9 ppm H 2 S in test 3 the cell voltage dropped rapidly below 450 mV, which resulted in an automatic turn off of current (cell voltage set to OCV by the system, see vertical arrow in Figure 5). After stopping the H 2 S addition and turning on the current load again, the cell voltage regenerated to a level consistent with the overall development. Adding 7 ppm H 2 S also resulted in a rapid, large voltage drop below 450 mV, which again triggered the system to turn off the current (OCV). Afterwards, it was not possible to apply the same current load (1 Acm -2 ) and the test was stopped.

Therefore the maximum H 2 S amount, which can be allowed in the CH 4 -containing fuels during a 24 h time period under the present conditions, is around/below 7 to 9 ppm. Taking the CH 4 conversion determined at OCV as a guideline (see section above and Figure 4) only 20-30% of the CH 4 was converted at these H 2 S concentrations. As a consequence, the fuel gas in the cell should contain less CO and H 2 and more methane.

During the three tests the pO 2 -in voltage and -out voltage were monitored in order to gather information about the gas compositions entering into and leaving the SOFC. They were both constant except for pO 2 -out in the cases when 4 ppm and 7 ppm H 2 S were added in test 1, and when 4 ppm and 9 ppm H 2 S were added in test 3.

In test 1 the pO 2 -out voltage dropped by 15 mV ± 5 mV when 4 ppm was added and by 32 mV ± 5 mV when 7 ppm was added. In test 3 the pO 2 -out voltage dropped by 18 mV ± 2 mV when 4 ppm H 2 S was added. It was not possible to determine how much the pO 2 -out voltage dropped at 9 ppm as the current was stopped almost immediately. The pO 2 -in voltage remained constant in all three tests as expected, because the fuel gas flows and thus the gas compositions entering the cells were kept constant. The drop of the pO 2 -out voltage at the large H 2 S concentrations is thus a result of the lower concentrations of H 2 and CO in the fuel outlet gas due to a smaller CH 4 conversion, a direct consequence of the decreased reforming activity of the anode.

Figure 5

A lower production of these fuels would also affect the fuel utilization, which might reach values critical for the SOFC anode. The fuel utilization in these cases of strong poisoning was therefore calculated using the gas compositions determined during H 2 S poisoning tests at OCV (see Figure 6).

Indeed, adding 7 ppm and 9 ppm H 2 S in test 3 resulted in a FU >90%, i.e. all of the initially added H 2 and a small amount of H 2 + CO from the remaining steam reforming reaction of CH 4 were converted in the electrochemical reaction. Operating the cells at these large FUs resulted in very low concentrations of H 2 and CO as measured near the outlet part of the cells, which can result in oxidation of the Ni particles (Ni particles become oxidised by O 2--ions from the cathode). This explains the rapid voltage drop below 450 mV in test 3, when 9 ppm H 2 S were added. A larger amount of H 2 in the initial fuel can counteract such over-critical FUs. A gradual poisoning of the CH 4 reforming and thus smaller supply of H 2 and CO has then a less severe effect (see test 1).

Figure 6

During poisoning tests in H 2 fuel, a clear correlation between the initial cell voltage drop and the H 2 S concentration was found [13]. This parameter was also analysed for the CH 4 tests in this study. In Table 2 2). These results suggest that the effects of S-poisoning on SOFCs fuelled with CH 4containing gases are much more severe. From investigations of steam reforming catalysts it was concluded that steam reforming primarily happens at the defect sites (steps), which are also the preferred adsorption sites for S [22]. The electrochemical oxidation of H 2 is less site-sensitive. The S-poisoning effects are thus more significant for the tests with CH 4 -containing fuels, because of the sensitive steam reforming reaction.

After stopping the H 2 S addition, the cell voltage regenerated slowly (Figure 5). The poisoning reflected by the drop pf cell voltage occurred within ~24 h, while the whole recovery occurred over ~200 h in both the CH 4 tests and in the H 2 test [13]. The entire poisoning was thus faster than the entire recovery (see Figure 5). A way to quantify the poisoning and recovery rates is to determine the times for 50% change, i.e. 50% initial voltage drop compared to 50% voltage recovery.

In Figure 7 the rates of the initial poisoning rates (50% voltage drop) and initial recovery rates (50% voltage increase) in the CH 4 tests (top figure) and in the H 2 test [13] (bottom figure) are plotted. In the CH 4 tests the initial poisoning and initial recovery rates were almost the same over the used concentration range, while there was a difference in the H 2 test; the initial poisoning rate was larger than the initial recovery rate (see Figure 7). This difference increased with H 2 S concentration (10 times larger at 100 ppm H 2 S).

Figure 7

Comparing the CH 4 tests and the H 2 test, the initial poisoning rates were almost the same in these, while the initial recovery rate for the test in H 2 was much smaller. This result points to different recovery mechanisms depending on the fuel. Assuming the recovery to be due to desorption of S-species from the Ni particles in the SOFC anode, this process can be in general facilitated by an increase of temperature, a decrease of the pressure or by addition of a displacement agent. The faster recovery in the CH 4 tests could thus be explained by the presence of a higher H 2 O concentration in the fuel in the CH 4 tests, H 2 O acting as an efficient displacement agent.

Impedance spectroscopy was used to obtain more detailed information about the effect of H 2 S on the cells internal resistances. This was done by measuring the impedance of the SOFCs before, during and after each H 2 S cycle. To determine the most relevant times for recording impedance spectra the anode in-plane voltages were followed and impedance spectra were taken at the times of constant levels [13]. In test 1 it was not possible to achieve acceptable resolution on the low frequency part of the spectra (< ~10 Hz), due to small cell voltage oscillations. However, the development of the high frequency arc showed similar development during the poisoning cycles for all the CH 4 tests. The case of 2 ppm H 2 S addition in test 2 is shown in Figure 8 as an example. The Nyquist plots show that the total internal resistance of the cell increased as H 2 S was added to the fuel until the cell was at steady state (the constant level in the in-plane voltage indicates steady state: in the insert). But it returned nearly to the initial level when the H 2 S addition was stopped. The serial resistance increased minimally during the interval (< 0.02 Ωcm 2 ), which was consistent with the overall degradation of the cell voltage. As the cell voltages degraded in the CH 4 tests even before the first H 2 S addition, this increase in series resistance is assigned to the overall voltage degradation. The reason for the overall cell voltage degradation might be related to coke formation on the Ni particles, even though a steam to carbon ratio of 2 was applied.

Mainly the polarization resistance changed under addition of H 2 S. From Figure 8 it can be concluded that the polarization resistance increase was reversible and that mainly the high frequency arc increased. These results thus coincide with those obtained during H 2 S poisoning in H 2 -fuel, although at much smaller H 2 S-concentrations [13]. The poisoning of the electrochemical activity proceeded similarly in methane and hydrogen fuel. The recovery of the cell voltage after stopping the H 2 S exposure was accelerated in the methane/steam containing fuel. Probably, steam facilitates efficient desorption of S from Ni-sites.

Acknowledgement

We acknowledge the project "Effektiv konvertering af vedvarende energi vha. fastoxid-celler" under Det Strategiske Forskningsråds Programkomite for Energi og miljø, sagsnr. 2058-03-0014 for sponsoring this work.

List of Symbols

  Figure 1

Figure 4

 4 Figure 4

Figure 8

 8 Figure 8

  large extent. If not additional hydrogen is added to the fuel, a critical fuel utilization and thus oxidation of the Ni particles leading to a severe degradation of the cell voltage might occur.

Figure 1 :

 1 Figure 1: Left: The test setup. Right: H 2 S was added in two different ways, i.e. in steps of increasing concentrations with the cell at OCV (A) and in intervals with the cell under current load (B).Figure 2: The cell voltage development. H 2 S was added in concentration steps of 2 ppm, 4 ppm, 7 ppm, 9 ppm, 20 ppm and 24 ppm. T = 850°C; OCV, CH 4 /H 2 /H 2 O fuel. Insert: OCV at different H 2 S concentrations (see also values in the text).Figure 3: The CO/CH 4 , CO 2 /CH 4 and (CO+CO 2 )/CH 4 ratios measured at fuel outlet by QMS as function of H 2 S concentration in the fuel. T = 850°C; OCV, CH 4 /H 2 /H 2 O fuel.Figure 4: The calculated CH 4 conversion, based on the measured OCV. T = 850°C; OCV, insert: S-coverage as calculated according to [19].Figure 5: The cell voltage developments. Black: test 1, green: test 2, and blue: test 3. The letters a, b, c and d indicate where the varying concentrations of H 2 S were added to the fuel. The horizontal arrows show the periods under OCV due to maintenance work in the lab. The insert illustrates the initial voltage drop and the degradation rate under 4 ppm H 2 S addition in test 1. T = 850°C; 1 Acm -2 , CH 4 /H 2 /H 2 O fuel (compositions see Table1). Figure6: The calculated FU for the cells in test 1 and 3. T = 850°C; 1 Acm -2 . Figure7: The initial poisoning rates (50% voltage drop) compared to the initial recovery rates (50% voltage increase) as function of H 2 S concentration. Top: CH 4 tests; Bottom: H 2 test[13]. T = 850°C; 1 Acm -2 . Figure8: The internal resistance development (impedance spectra), when 2 ppm H 2 S was added in test 2. T = 850°C; 1 Acm -2 .

Figure

  Figure 1: Left: The test setup. Right: H 2 S was added in two different ways, i.e. in steps of increasing concentrations with the cell at OCV (A) and in intervals with the cell under current load (B).Figure 2: The cell voltage development. H 2 S was added in concentration steps of 2 ppm, 4 ppm, 7 ppm, 9 ppm, 20 ppm and 24 ppm. T = 850°C; OCV, CH 4 /H 2 /H 2 O fuel. Insert: OCV at different H 2 S concentrations (see also values in the text).Figure 3: The CO/CH 4 , CO 2 /CH 4 and (CO+CO 2 )/CH 4 ratios measured at fuel outlet by QMS as function of H 2 S concentration in the fuel. T = 850°C; OCV, CH 4 /H 2 /H 2 O fuel.Figure 4: The calculated CH 4 conversion, based on the measured OCV. T = 850°C; OCV, insert: S-coverage as calculated according to [19].Figure 5: The cell voltage developments. Black: test 1, green: test 2, and blue: test 3. The letters a, b, c and d indicate where the varying concentrations of H 2 S were added to the fuel. The horizontal arrows show the periods under OCV due to maintenance work in the lab. The insert illustrates the initial voltage drop and the degradation rate under 4 ppm H 2 S addition in test 1. T = 850°C; 1 Acm -2 , CH 4 /H 2 /H 2 O fuel (compositions see Table1). Figure6: The calculated FU for the cells in test 1 and 3. T = 850°C; 1 Acm -2 . Figure7: The initial poisoning rates (50% voltage drop) compared to the initial recovery rates (50% voltage increase) as function of H 2 S concentration. Top: CH 4 tests; Bottom: H 2 test[13]. T = 850°C; 1 Acm -2 . Figure8: The internal resistance development (impedance spectra), when 2 ppm H 2 S was added in test 2. T = 850°C; 1 Acm -2 .

  test setup. Right: H2S was added in two different ways, i.e. in steps of increasing concentrations with the cell at OCV (A) and in intervals with the cell under current load (B). 254x190mm (96 x 96 DPI) The cell voltage development. H2S was added in concentration steps of 2 ppm, 4 ppm, 7 ppm, 9 ppm, 20 ppm and 24 ppm. T = 850 o C; OCV, CH4/H2/H2O fuel. Insert: OCV at different H2S concentrations (see also values in the text). 254x190mm (96 x 96 DPI) The CO/CH4, CO2/CH4 and (CO+CO2)/CH4 ratios measured at fuel outlet by QMS as function of H2S concentration in the fuel. T = 850 o C; OCV, CH4/H2/H2O fuel. 254x190mm (96 x 96 DPI) The calculated CH4 conversion, based on the measured OCV. T = 850 o C; OCV, insert: S-coverage as calculated according to [19]. 254x190mm (96 x 96 DPI) The cell voltage developments. Black: test 1, green: test 2, and blue: test 3. The letters a, b, c and d indicate where the varying concentrations of H2S were added to the fuel. The horizontal arrows show the periods under OCV due to maintenance work in the lab. The insert illustrates the initial voltage drop and the degradation rate under 4 ppm H2S addition in test 1. T = 850 o C; 1 Acm -2 , CH4/H2/H2O fuel (compositions see Table 1). 254x190mm (96 x 96 DPI) The calculated FU for the cells in test 1 and 3. T = 850 o C; 1 Acm -2 . 254x190mm (96 x 96 DPI) The initial poisoning rates (50% voltage drop) compared to the initial recovery rates (50% voltage increase) as function of H2S concentration. Top: CH4 tests; Bottom: H2 test [13]. T = 850 o CThe internal resistance development (impedance spectra), when 2 ppm H2S was added in test 2. T = 850 o C; 1 Acm -2 . 254x190mm (96 x 96 DPI)

  the initial voltage drops (∆V ini ) for the cells in the CH 4 tests and for an SOFC with the same test parameters, but running on fuel consisting of only H 2 and H 2 O (referred to as H 2 test[13]), are listed as a function of the H 2 S concentration. At low H 2 S concentration (2 ppm H 2 S) the initial voltage drops were approximately the same for the cells in the CH 4 tests 1 and 2 (present study) as for the H 2 test[13]. At higher H 2 S concentrations the cells in the CH 4 tests started to experience a larger initial voltage drop than in the H 2 test.

After the initial voltage drop the SOFCs degraded (more than the overall cell voltage degradation). In Table

2

the degradation rates (τ deg ) are listed as a function of the H 2 S concentration.

Table 2

 2 Analysing the degradation rates under H 2 S exposure, the CH 4 tests show much larger rates at comparable H 2 S concentrations (Table

	F o r
	P
	e
	e r
	R
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	v i e
	w

4

  ConclusionsIt was studied, how H 2 S affects the reforming and electrochemical activity of a Ni/YSZ anode in a technological SOFC by adding H 2 S to a CH 4 -containing fuel. It was found that H 2 S had a larger effect on the reforming activity than on the electrochemical activity. At 4 ppm H 2 S addition, more than 60% of the reforming activity was eliminated at OCV, which meant that the production of fuel (H 2 and CO) was diminished significantly. When operating an SOFC under current load a maximum H 2 S concentration just below 7-9 ppm was acceptable during a 24 h period. At these H 2 S concentrations the production of CO and H 2 fuel from steam reforming was

Table 1 :

 1 Fuel compositions, FU and ASR cor values for the three CH 4 tests.

	ASR	Area specific resistance
	CPO	Catalytic partial oxidation
	∆V	Voltage drop
	EMF electro motive force
	FU	Fuel utilization
	iV	Current voltage curves
	LSM	Lanthanum strontium manganite
	OCV Open circuit voltage
	pO 2 θ	Partial pressure of oxygen Coverage
	QMS Quadrupole mass spectrometer
	SOFC Solid Oxide Fuel Cell
	τ deg	Cell voltage degradation rate
	TPB	Three-phase boundary
	YSZ	Yttria stabilized zirconia

Table 2 :

 2 The initial voltage drop (∆V ini ) and Degradation rate (τ deg ) of the H 2 test[13] and the three CH 4 tests (Test 1, 2 and 3). T = 850°C; 1 Acm -2 .

	H 2 S conc.	H 2 test		CH 4 tests
	(ppm)	[13]	Test 1	Test 2	Test 3
		∆V ini	τ deg ∆V ini	τ deg ∆V ini	τ deg ∆V ini	τ deg
	2	75	0.1 79	0.7 73	1.1
	4	83	0.2 82	1.3	93	2.8
	7	87	0.2 107	1.2
	9	96	0.3
	18	98	0.3
	42	100	0.5
	100	108	0.7
	∆V

ini : Initial voltage drop (mV) τ deg : Linear degradation rate (mVh -1 )

  2: The cell voltage development. H 2 S was added in concentration steps of 2 ppm, 4 ppm, 7 ppm, 9 ppm, 20 ppm and 24 ppm. T = 850°C; OCV, CH 4 /H 2 /H 2 O fuel. Insert: OCV at different H 2 S concentrations (see also values in the text). Figure 3: The CO/CH 4 , CO 2 /CH 4 and (CO+CO 2 )/CH 4 ratios measured at fuel outlet by QMS as function of H 2 S concentration in the fuel. T = 850°C; OCV, CH 4 /H 2 /H 2 O fuel. Figure4: The calculated CH 4 conversion, based on the measured OCV. T = 850°C; OCV, insert: S-coverage as calculated according to[START_REF] Rostrup-Nielsen | Catalytic Steam Reforming[END_REF]. Figure 5: The cell voltage developments. Black: test 1, green: test 2, and blue: test 3. The letters a, b, c and d indicate where the varying concentrations of H 2 S were added to the fuel. The horizontal arrows show the periods under OCV due to maintenance work in the lab. The insert illustrates the initial voltage drop and the degradation rate under 4 ppm H 2 S addition in test 1. T = 850°C; 1 Acm -2 , CH 4 /H 2 /H 2 O fuel (compositions see Table1). Figure6: The calculated FU for the cells in test 1 and 3. T = 850°C; 1 Acm -2 . Figure7: The initial poisoning rates (50% voltage drop) compared to the initial recovery rates (50% voltage increase) as function of H 2 S concentration. Top: CH 4 tests; Bottom: H 2 test[13]. T = 850°C; 1 Acm -2 . Figure8: The internal resistance development (impedance spectra), when 2 ppm H 2 S was added in test 2. T = 850°C; 1 Acm -2 .

Wiley-VCH Fuel Cells