Elise Da-Silva

L Barthelmebs
email: barthelm@univ-perp.fr

J Baudart

Development of a PCR-free DNA-based assay for the specific detection of Vibrio species in environmental samples by targeting the 16S rRNA

Keywords: PCR-free, DNA-based assay, Vibrio detection, sandwich hybridization assay, chemiluminescence, environmental analysis

We are thankful to

Introduction

Currently, the water resource is regularly monitored for its bacteriological quality through the detection of fecal indicator bacteria such as Escherichia coli, total coliforms and Enterococci using culture-based methods.

Nevertheless, a large diversity of non fecal pathogenic bacteria is widely distributed in aquatic environments and is potentially harmful for humans and animals. Moreover, due to environmental stresses, microorganisms may enter into a viable but non culturable cell (VBNC) state becoming undetectable using cultural methods.This physiological state has been described for several pathogenic and non pathogenic bacteria, including species belonging to the Vibrio genus [START_REF] Oliver | The viable but nonculturable state in bacteria[END_REF]. This genus, composed of more than 100 species [START_REF] Romalde | New Vibrio species associated to molluscan microbiota: a review[END_REF], is ubiquitous and abundant in aquatic ecosystems, principally in marine and estuarine waters [START_REF] Narracci | Mar Piccolo of Taranto: Vibrio biodiversity in ecotoxicology approach[END_REF]). These bacteria can survive either in the water column, in sediments and also associated to plankton, mainly due to their capability of biofilm formation [START_REF] Shikuma | Marine biofilms on submerged surfaces are a reservoir for Escherichia coli and Vibrio cholerae[END_REF]. Their occurrence in aquatic environments is correlated with several parameters, water temperature and salinity being the most important [START_REF] Lutz | Environmental reservoirs and mechanisms of persistence of Vibrio cholerae[END_REF]. Furthermore, several studies have demonstrated that Vibrio prevalence could be impacted by human activities [START_REF] Okoh | Prevalence and characterisation of non-cholerae Vibrio spp. in final effluents of wastewater treatment facilities in two districts of the Eastern Cape Province of South Africa: implications for public health[END_REF]. Forty four years of monitoring in the Coastal North Sea revealed an increase in prevalence of vibriosis correlated with elevated water temperature as a consequence of global climate change (Vezzuli et al. 2012).

Among Vibrio species, twelve are known to be human pathogen, including V. cholera, V. parahaemolyticus, and V. vulnificus as the most notable and important for public health [START_REF] Tarr | Identification of Vibrio Isolates by a Multiplex PCR Assay and rpoB Sequence Determination[END_REF]. Several other species are considered as pathogens for aquaculture, such as V. anguillarum, V. salmonicida and V. vulnificus that infect fish, V. penaeicida and V. nigripulchritudo infecting shrimps and V. splendidus related strains and V. aestuarianus that are shellfish pathogens [START_REF] Frans | Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention: Pathogen profile of Vibrio anguillarum[END_REF][START_REF] Vezzulli | Aquatic ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus: Ecology of Vibrio splendidus and Vibrio aestuarianus[END_REF][START_REF] Walling | Vibrio nigripulchritudo monitoring and strain dynamics in shrimp pond sediments[END_REF][START_REF] Goarant | Quantification of Vibrio penaeicida, the etiological agent of Syndrome 93 in New Caledonian shrimp, by real-time PCR using SYBR Green I chemistry[END_REF]. During the last decade, food-borne infections caused by the ingestion of uncooked shellfish contaminated with human pathogen Vibrio have significantly increased, especially in the U.S, Canada, Asia and India. While the incidence of these bacteria is currently considered as relatively low in Europe, the potential risk associated with Vibrio infections must be taken seriously, due to their emergence in some European countries [START_REF] Roux | The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis[END_REF]. These last years, with the development of aquaculture, vibriosis has become one of the major causes of biological loss, particularly in France for oyster production [START_REF] Petton | Factors influencing disease-induced mortality of Pacific oysters Crassostrea gigas[END_REF][START_REF] Samain | Summer mortality of Pacific oyster Crassostrea gigas. The Morest Project[END_REF]. Mass mortality outbreaks caused by Vibrio, particularly affect early larval stages and may lead to the decimation of entire populations, becoming one limiting factor for the viability of this economic activity [START_REF] Thompson | Diversity and Dynamics of a North Atlantic Coastal Vibrio Community[END_REF]). However, even if these significant losses in aquaculture can be attributed to the presence of pathogenic agents in seawater, these pathogens are routinely detected in animals but rarely in harvesting waters. Therefore, anticipation of the biological resource contamination is not possible.

Current standardized methods for Vibrio detection and quantification are based on bacteria culture on specific agar media followed by colony counting. These methods are time consuming since 2-3 days are required to obtain the initial results, and at least 7 days are necessary to confirm positive results [START_REF] Cai | Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China[END_REF]. Moreover, the ability of Vibrio to enter into VBNC state may lead to an underestimation of the viable cell number or a failure to isolate pathogens from the sample [START_REF] Griffitt | Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization[END_REF].

A wide range of techniques based on molecular methods have replaced the cumbersome microbiological analysis by targeting specific genetic markers. Within the last years, PCR-based methods have been set up for the detection, quantification and differentiation of several bacteria [START_REF] Oliveira | Detection of Campylobacter jejuni in naturally contaminated chicken skin by melting peak analysis of amplicons in real-time PCR[END_REF][START_REF] Frahm | Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. and Escherichia coli in water samples[END_REF][START_REF] Clifford | Detection of Bacterial 16S rRNA and Identification of Four Clinically Important Bacteria by Real-Time PCR[END_REF], and were applied to the identification of different Vibrio species, such as V. vulnificus, V. cholerae, V. alginolyticus, V. coralliilyticus and V. aestuarianus [START_REF] Tall | Real-time PCR optimization to identify environmental Vibrio spp. strains[END_REF][START_REF] Saulnier | Real-time PCR assay for rapid detection and quantification of Vibrio aestuarianus in oyster and seawater: A useful tool for epidemiologic studies[END_REF][START_REF] Pollock | Detection and Quantification of the Coral Pathogen Vibrio coralliilyticus by Real-Time PCR with TaqMan Fluorescent Probes[END_REF][START_REF] Garrido-Maestu | lolB gene, a valid alternative for qPCR detection of Vibrio cholerae in food and environmental samples[END_REF].

More recently, DNA-based sensors have been proposed as an alternative and effective technique for the detection of DNA or RNA sequences specific to microorganism species (Paniel et al. 2013). These kinds of sensors offer several advantages compared to PCR methods. They could allow the direct detection of genetic markers bypassing the nucleic acid amplification step and thus reducing the drawbacks linked to PCR inhibitors frequently detected in environmental and food samples. Moreover, by incorporating oligonucleotide probes immobilized on a surface, DNA-based sensors have the potential to speed the detection of nucleic acid targets and to increase the specificity and sensitivity of the analysis. To the best of our knowledge, such analytical devices reported for the detection of Vibrio bacteria have been mainly developed for the pathogenic V. cholerae species [START_REF] Low | Electrochemical genosensor for specific detection of the food-borne pathogen, Vibrio cholerae[END_REF][START_REF] Patel | Magnesium oxide grafted carbon nanotubes based impedimetric genosensor for biomedical application[END_REF][START_REF] Liew | Electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere label for detection of Vibrio cholerae[END_REF][START_REF] Yu | Enzymatic electrochemical detection of epidemic-causing Vibrio cholerae with a disposable oligonucleotide-modified screen-printed bisensor coupled to a dry-reagent-based nucleic acid amplification assay[END_REF][START_REF] Patel | A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection[END_REF]. Nevertheless, most of these DNA-based sensors targeted DNA amplified through a PCR reaction, limiting the interest of these assays.

In the present study, a novel PCR-free DNA-based assay for the detection of Vibrio spp. has been investigated.

The developed analytical tool was based on a sandwich hybridization assay in which RNA, as the nucleic acid target, was bound between an immobilized capture probe and a labeled signal probe. The hybridization complex was then revealed by an enzymatic system, leading to a chemiluminescent signal proportional to the entrapped target. Targeting 16S rRNA will not only allow the detection of culturable Vibrio but also the detection of Vibrio cells in VBNC state [START_REF] Coutard | mRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state[END_REF][START_REF] Schauer | Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry: V. cholerae dynamics assessed with solid phase cytometry[END_REF]. Designed probes were first validated with positive control (PC) and detection limit was determined. Then, nucleic acids extracted from a panel of bacteria were used to assess the specificity of the probes, and finally, the assay was tested with environmental samples.

Stability and storage conditions of the assay were also studied.

Material and methods

Bacterial strains and culture conditions

Bacterial strains used in this study were provided by various bacterial culture collections (Microbial Observatory of the LaboratoireArago (MOLA, Banyuls-sur-Mer, France), American type culture collection (ATCC) and Collection de l'Institut Pasteur (CIP, Paris, France) (Supplementary data -Table 1). All environmental strains have been previously identified from the 16S rDNA. Marine strains were grown at 25°C in Marine Broth medium 279110 (MB, Difco™ Becton, Dickinson and Co., USA). E. coli, Citrobacter and Pseudomonas strains were cultivated in Tryptic Soy Broth (TSB) (Biokar, France).

Total RNA extraction from pure cultures

Twenty one Vibrio and 10 non-Vibrio strains were used to evaluate the specificity of the assay. Each strain was grown overnight in appropriate medium at 25°C. In order to optimize the amount of extracted RNA, one milliliter of the overnight culture was added to 500 μL of fresh medium and incubated 4 hours at 25°C before total RNA extraction. Then, 1.5 mL of each culture was centrifuged at 8 000 x g for 5 minutes at 4°C. Total RNA was extracted with Tri-reagent (Sigma, France) as reported by [START_REF] Chomczynski | The single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction: twenty-something years on[END_REF] and then, resuspended in 50 μL DNase-RNase free water. RNA integrity and concentration were checked using the Agilent 2100 Bioanalyzer. Then, RNA extracts were fragmented using a 10 X fragmentation buffer (100 mM ZnCl2, 100 mM Tris HCl pH 7) [START_REF] Gescher | The ALEX CHIP-Development of a DNA chip for identification and monitoring of Alexandrium[END_REF]. Briefly, 1 μL of 10 X buffer was added to 10 μL of RNA and samples were incubated at 70°C for 15 min. Fragmentation reaction was stopped with 1 μL of EDTA 0.5 M pH 8 and samples were stored at -80°C until use.

Environmental samples collection and total RNA extraction

Analytical performances of the assay were tested with environmental samples naturally or artificially contaminated by Vibrio spp. Samples were collected at a French North-western Mediterranean lagoon dedicated to oyster culture (Leucate), and at a North-western Mediterranean marine open bay (SOLA) (42°29N, 03°08E) located ∼ 500 m offshore of Banyuls sur-Mer city, France. A seawater sample (SW) was collected at SOLA and a phytoplankton sample (PK) was collected at Leucate. Fifty liters of SW collected at SOLA were concentrated using an ultrafiltration cartridge (Hemoflow HF80S, Fresenius, Bad Homberg, Germany) with a 20 kDa molecular weight cut off. The cartridge was back-flushed with 1 L of elution buffer prepared with the ultra-filtrated water of the sampling site, 0.01% sodium hexametaphosphate (NaPP, Sigma, France) and 0.5% Tween 80 (Sigma, France). Twenty five milliliters of concentrate was filtered on 0.4 μm polycarbonate filters. Total RNA extraction was directly performed on those filters using the Tri-reagent method. Then, this total RNA extract, named SW RNA, was spiked with total RNA extracted from a pure culture of V. splendidus (CIP 102 893). Each spiked sample was composed of 2 μL of SW RNA in a final volume of 10 μL. SW RNA was spiked with different quantities of V. splendidus RNA to obtain final concentrations of V. splendidus RNA ranging from 0 to 100 ng μL -1 . A control was performed with different concentrations of V. splendidus RNA alone. All the samples were tested in the developed assay.

PK sample was collected at Leucate lagoon using a plankton net (50 μm) allowing the concentration of approximately 25 m 3 of water. The net was immersed for 5 minutes in water at a very low speed of 2 knots. The phytoplankton from the net was collected in polyethylene buckets and a total of 400 mL of concentrate was recovered after sampling. Ten milliliters of the concentrate were subsampled and filtered on 0.4 μm polycarbonate filters. Total RNA extraction was performed on those filters as described above. In parallel, in order to estimate the number of Vibrio cells in the PK concentrate, 100 μL of concentrate were spread at ten-fold dilutions on TCBS agar. Five replicates were performed for each dilution and the number of CFU mL -1 was determined after 24 h of incubation at 25°C. For all environmental samples, RNA integrity and concentration were checked using the Agilent 2100 Bioanalyzer. Before analysis, RNA was fragmented as previously described and stored at -80°C.

Oligonucleotide probes design and use

Capture and signal probes were both designed to hybridize with two adjacent sequences located on the 16S rRNA of all Vibrio species. The probes were checked in silico for the specific recognition of all the members of 1). A 70 bp synthetic oligonucleotide corresponding to the complementary strand of both capture and signal probes was used as positive control (PC). Probes and PC were synthesized by Microsynth (Switzerland) and were diluted in DNase-RNase free water to obtain 100 µM stock solutions. Capture probe solutions were prepared at the appropriate concentration in 0.1 M Tris HCl, 0.3 M NaCl, pH 7.6 and were denatured by 3 min at 90°C, 2 min at 4°C and then 5 min at room temperature, before use.

DNA-based assay development for Vibrio spp. detection

NeutrAvidin was purchased from Thermo Fischer Scientific and all the buffer components from Sigma (France). Hammerstein-grade casein was purchased from MP Biomedicals (France) and anti-digoxigeninhorseradish peroxidase conjugate (Anti-DIG-HRP) was purchased from Roche (France). All solutions were prepared with diethyl pyrocarbonate-treated distilled water to inactivate RNase enzymes in prepared solutions.

Protocol was adapted from Paniel and Baudart (2013). MaxiSorp TM microplates (Nunc, Denmark) were coated with 100 μL of 1 μg mL -1 NeutrAvidin in HEPES 0.1 M, pH 7.2, for 1 h at room temperature. Then, the NeutrAvidin excess was eliminated by 3 washing steps using 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4; pH 7.4).

Three strategies were then assessed. In the first one, pre-hybridization mix containing 25 μL of 4X hybridization buffer (4X HYB buffer: NaCl 0.3 M, Tris 80 mM, SDS 0.04%; pH 8), 10 μL of 1 μM denatured capture probe, 10 μL of 1 μM labeled signal probe, 10 μL of target (i.e PC or RNA or fragmented RNA) and 45 μL of ultra pure water was incubated for 10 min at 65°C. Then, one hundred microliters of this pre-hybrization mix were added to the wells and the microplate was incubated for 30 min at 46°C. Wells were washed 3 times with 1X HYB buffer. Unspecific sites were blocked with 1% Hammerstein casein in binding buffer (PBS 1X, 1 mM MgCl2, 0.01% Tween 20; pH 7.4) for 45 min at room temperature and casein excess was removed by 3 washing steps using 1X HYB buffer.

In the second strategy, pre-hybridization mix containing 25 μL of 4X HYB buffer, 10 μL of 1 μM capture probe, 10 μL of target and 55 μL of ultra pure water was incubated for 10 min at 65°C. One hundred microliters of the pre-hybrization mix were added to each wells and the microplate was incubated for 30 min at room temperature. Wells were washed 3 times with 1X HYB buffer and unspecific sites were blocked as previously described. One hundred microliters of 0.1 μM signal probe were added to the microplate which was then incubated for 30 min at 46°C. Wells were then washed 3 times with 1X POP buffer (NaH2PO4 50 mM, NaCl 0.1 M; pH 6.45).

In the third strategy, one hundred microliters of 0.1 μM denatured capture probe were added to each wells and the microplate was incubated for 30 min at room temperature. Wells were washed 3 times with 1X HYB buffer. Unspecific sites were blocked as previously described. Pre-hybridization mix containing 25 μL of 4X HYB buffer, 10 μL of 1 μM labeled signal probe, 10 μL of target and 55 μL of ultra pure water was incubated for 10 min at 65°C. One hundred microliters of the pre-hybrization mix were added to the wells and the microplate was incubated for 30 min at 46°C. Wells were then washed 3 times with 1X POP buffer.

Then, the revelation of the sandwich hybridization was performed in the same way for the three strategies.

One hundred microliters of anti-DIG-HRP conjugate at 50 mU mL -1 in PBS-T-BSA (PBS 1X, BSA 0.1%, Tween 20 0.05%; pH 7.4) were added to the wells and the microplate was incubated for 20 min at room temperature. Wells were washed using 1X POP buffer.

In this work, two detection methods were used. For colorimetric detection, 100 µL of ready-to-use 3,3′,5,5′-Tetramethylbenzidine substrate (TMB) (Sigma, France) were added to the wells, and after 15 min of incubation at room temperature optical density was measured at 630 nm using the Multiskan Ascent TM . For chemiluminescent detection, 100 μL of luminol-H2O2 (Luminata forte™ ELISA HRP Substrate, Merck Millipore, France) were added to the wells and after 5 min of incubation at room temperature luminescence was measured using the Fluoro-Luminoskan Ascent TM FL (Thermo Scientific, France). For the specificity tests, all the values were standardized as followed: standardized value = measured value / 1 nM of PC value.

Storage and stability study of the DNA-based assay

To minimize experimental variations, all the solutions were prepared at day 0 and used until the end of the experiment. NeutrAvidin, capture probe and casein were successively added to the wells of several strip plates following the third protocol described above. Then, two storage conditions were tested. Strip plates were directly stored at 4°C with casein solution inside each well, or they were washed to eliminate the excess of casein and stored at 4°C without any buffer. For both conditions, a lid was placed on the top of each plate, which was then packed inside an aluminum foil. Then, hybridization and enzymatic revelation were carried out at day 0 as the reference point and at different times during 30 days (d1, d2, d3, d4, d7, d8, d10, d14, d16, d21, and d30) as previously described. Colorimetric detection was used. Results were expressed as: (value at dx * 100) / value at d0 with dx : day of the experiment (d1 to d30).

Results and discussion

Design strategy of the DNA-based assay

In this work, sandwich hybridization format was chosen to optimize the recognition of the target, and thus to improve the specificity of the assay. Three different hybridization strategies were investigated to determine the optimal working conditions. In the first strategy (Fig. 1a), pre-hybridization was carried out by mixing together all the components. Then, this mix was added to the microplate wells for NeutrAvidin binding. In the second strategy (Fig. 1b), the target was firstly pre-hybridized with the capture probe. This solution was then added to the wells for NeutrAvidin binding, and followed by signal probe addition. In the third strategy (Fig. 1c), capture probe was immobilized alone inside the wells. Pre-hybridization between the target and the signal probe was performed, and then this mix was added to the wells.

Comparison of the three strategies was performed using 50 nM of PC. For all experiments, the signals were at least 3 fold higher than the negative control without PC. The best response was achieved when the denaturated capture probe was immobilized alone in each well. This third strategy allowed a signal response 10 and 4 fold higher than the second and the first strategy, respectively (data not shown). These lower results could be explained by the steric hindrance of the biotinylated complex that could decrease the binding to NeutrAvidin, and highlight the importance of the binding step between the biotinylated capture probe and NeutrAvidin to optimize the signal response of our assay. According to those results, the third strategy (Fig. 1c) was selected for subsequent experiments.

Validation of the DNA-based assay

In order to assess the sensitivity of our system, a calibration curve was performed using PC.

Chemiluminescent responses obtained for the different concentrations of PC from 0.1 nM to 25 nM are shown in Fig. 2. As expected, the intensity of the responses was proportional to the PC concentration. Moreover, the results showed that the chemiluminescent responses were linear over the range from 0.1 nM to 2.5 nM of PC (Fig. 2b). The correlation coefficient (R 2 ) value was 0.997, and the relative standard deviation (RSD) average of 8% indicated a good reproducibility of the assay. Due to the low RSD, the detection limit of the assay was determined, based on the threshold value of 3 times the background signal, as 0.1 nM corresponding to 2.17 pg μL -1 of PC.

After optimizing the experimental parameters with PC, assays were performed with 20 ng μL -1 of fragmented and non fragmented RNA extracted from three different species belonging to the Vibrio genus (V. splendidus ATCC 33125, V. pomeroyi CIP 108273 and V. crassostreae CIP 108327). For the three RNA samples, the obtained responses were 7 to 10 fold higher with fragmented RNA than with non fragmented indicating a positive effect of the fragmentation step on the RNA target (data not shown). For subsequent experiments RNA was always fragmented before analysis.

Various concentrations of total RNA from V. splendidus, used as reference strain, were assessed to validate the assay with nucleic acid target and to determine the optimal RNA concentration to be used for the specificity tests. As previously shown with PC, the intensity of the chemiluminescent responses was proportional to the total RNA concentration. Linear responses were observed between 1 and 100 ng μL -1 of total RNA, with a R 2 value of 0.995 (Fig. 3). Based on the threshold value of 3 times the background signal, the detection limit was determined as 5 ng μL -1 of total RNA which was 2 300 times higher than the detection limit observed with PC

(2.17 pg μL -1 ). This observation could be explained by the proportion of target. Indeed, the targeted 16S rRNA only represents a fraction of the total RNA. Moreover, instead of the PC which is a short 70-bp linear sequence, 16S rRNA is longer (1 500 bp) and can present some secondary and tertiary structures as hairpins that can interfere with probes hybridization.

For 20 ng μL -1 , the measured chemiluminescence was 4 fold higher than those measured for the detection limit value. Similar results were obtained for the two other Vibrio strains V. pomeroyi and V. crassostreae (data not shown). Thus, 20 ng μL -1 of total RNA is sufficient to clearly detect Vibrio RNA in the sample.

Specificity evaluation of the DNA-based assay

Regarding our previous results obtained with total RNA, a concentration of 20 ng μL -1 was retained to perform specificity tests. In order to standardize the chemiluminescent responses between experiments, the value obtained for 1 nM of PC was used as the reference.

Twenty one Vibrio strains belonging to seven different clades (Splendidus, Harveyi, Coralliilyticus, Mediterranei, Halioticoli, Rumoiensis and Anguillarum clades) and ten strains belonging to ten others bacterial genera were assessed (Fig. 4).

The results showed that a significant response was observed for the 21 Vibrio strains tested. The lower signal was obtained for V. natriegens, with a luminescence value 55 times higher than the background signal. All the other Vibrio strains tested gave higher signals, with a coefficient factor varying from 1.1 to 6.6 of the signal obtained for V. natriegens. The highest signal was observed for V. gigantis, with a luminescence value 243 times higher than the background signal.

For the 10 non Vibrio strains, observed chemiluminescence values were below the detection limit of the system for 4 strains. For the 6 others, the signal was therefore 2 to 10 times lower than the chemiluminescence value observed for V. natriegens, the Vibrio strain having the lowest signal. Photobacterium lutimaris, the closest relative species to Vibrio [START_REF] Anzai | Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence[END_REF], showed the highest signal. Interestingly, for all the non Vibrio strains showing a weak signal, no perfect match was observed in silico with the two probes. However, both signal and capture probes present mismatches at either the 3' or 5'-end. These observations are consistent with the results obtained by [START_REF] Letowski | Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays[END_REF] demonstrating that probes hybridization could occur even in presence of mismatches located at the probe ends.

Among the 21 Vibrio strains tested, an important variation in the signal intensity can be noticed. We hypothesized that the variation of 16S rRNA operon number between the tested strains could explain these results. Then, the number of 16S rRNA operon was sought from rrn database [START_REF] Wuyts | The European ribosomal RNA database[END_REF]) and collected only for 4 strains of our study. Interestingly, the strains V. splendidus (CIP 107715), V. alginolyticus, V. harveyi and V. natriegens possess 8, 11, 8-11 and 13, 16S rRNA operons respectively but no correlation was observed with the signal responses since luminescence values of 0.713, 0.365, 0.417, and 0.154 were respectively obtained for these strains. Then, another hypothesis could be formulated to explain the obtained results. Since RNA was randomly fragmented with a fragmentation buffer, the length of the generated fragments cannot be controlled and can vary between Vibrio strains due to the differences of sequences. In similar works reported in the literature, specificity tests are routinely performed, but to the best of our knowledge, only on a limited number of species and signal variations observed between targeted species or strains were never explained [START_REF] Low | Electrochemical genosensor for specific detection of the food-borne pathogen, Vibrio cholerae[END_REF][START_REF] Liew | Electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere label for detection of Vibrio cholerae[END_REF].

Nevertheless, we can conclude that a good discrimination between the 21 Vibrio strains and the 10 non Vibrio strains was observed, indicating a high specificity of the developed assay.

Storage and stability study of the DNA-based assay

To investigate the stability of the developed assay, a preservation test of the capture probe coated inside microplate wells was performed during 30 days. Storage conditions were assessed using 2 concentrations of PC, 50 nM and 250 nM. Assay responses over time were compared to the initial response value of day 0 (d0) (Fig. 5). For microplates stored at 4°C with casein in wells, signals were similar the first two days and then decreased from d2 to d7 for both target concentrations. The responses were then stable until d21 before sharply decreasing at d30. Since d3, responses were lower than the initial response. For both PC concentrations, maximal signal was observed at d2 and minimum signal at d30. Between these two days, a signal decrease of 77 % was observed for 250 nM of PC and for 50 nM of PC this decrease reached 93 %. Interestingly, for microplates stored at 4°C without casein, responses between d1 and d30 were a slightly higher than those measured at d0. For both PC concentrations, signals were stable over time. For 50 nM of PC, maximal signal was observed at d2 and minimal signal at d30 corresponding to a signal decrease of 32 % between these two days. For 250 nM of PC, maximal signal was obtained at d3 and minimal signal at d30 corresponding to a signal decrease of 26 %. Furthermore, for both PC concentrations, the measured values were also higher than those obtained at the same time for microplates stored with casein. We could hypothesize that an increase of casein adsorption on well surface could occur over time leading to a decrease in the measured signal from d2 to d30. The second storage condition appeared as the better way to preserve microplates. Indeed, the assay components were stable for at least 30 days and the signal stable over time. These results demonstrated that microplates can be coated beforehand, and stored at 4°C without casein until use allowing a reduction of 3 hours in experiment time. The use of pre-made plates enables the assay to be completed in 2 hours.

Environmental samples analysis

To our knowledge few studies have reported the application of DNA-based biosensors for environmental samples analysis. However, when biosensors are developed for in field application, this step is essential. For complex samples, the performances of such devices could be different from those obtained with bacterial strains growing under laboratory conditions due to matrix interferences. Thus, our developed assay was assessed with two different environmental samples (seawater and phytoplankton) to evaluate the matrix effect, and to validate the suitability of the method for Vibrio detection.

First assay was assessed on SOLA SW sample. A previous analysis by a TaqMan qPCR targeting the 16S rDNA gene of Vibrio spp. was performed on total extracted DNA. No genome unit (GU) was detected in this sample, indicating that the concentration of Vibrio spp. estimated by qPCR was under the quantification limit of the assay (833 GU L -1 of SW) (personal data). In a second step, SW RNA was analyzed using the developed DNA-based assay. To test the matrix effect, a same quantity of SW RNA was spiked with known concentrations of total RNA extracted from V. splendidus ranging from 0 to 100 ng µL -1 , and the results were compared with those obtained with total RNA of V. splendidus alone (Fig. 6a). The analysis performed on SW RNA without addition of V. splendidus RNA showed a positive signal. The positive signal was linked to the presence of RNA from Vibrio in the sample. This genus is known to be a common inhabitant of the coastal marine areas, as demonstrated by a recent study that showed that Vibrio abundances at SOLA site ranged from 7 CFU L -1 to 2.7.

10 4 CFU L -1 over the year (unpublished work). Interestingly, SW RNA sample without spiked RNA was positive for Vibrio spp. analysis using the reported device but negative using qPCR. This negative result could be explained by the dilution procedure applied to extracted DNA before qPCR analysis, required to limit the matrix inhibitors effect, but leading to a decrease of sensitivity. The linearity of the chemiluminescent response was measured for the SW RNA spiked samples and also for the V. splendidus RNA alone, given a R 2 values of 0.997 and 0.989, respectively (data not shown). The signals measured in the spiked samples were 2.2 to 7.2 fold higher than those measured with the RNA from V. splendidus alone confirming the detection of environmental population of Vibrio already present in SW sample using the reported device.

PK samples from Leucate were also assessed since Vibrio bacteria are known to be tightly associated with phytoplankton [START_REF] Huq | Critical Factors Influencing the Occurrence of Vibrio cholerae in the Environment of Bangladesh[END_REF][START_REF] Turner | Plankton composition and environmental factors contribute to Vibrio seasonality[END_REF], and use the phytoplankton extracellular matrix as nutrient sources [START_REF] Thompson | Diversity and Dynamics of a North Atlantic Coastal Vibrio Community[END_REF]). As shown in Fig. 6b, a linear relationship was observed between the measured chemiluminescence and the total RNA concentrations of PK RNA. The R 2 value was 0.980, indicating that our system allowed the specific detection of Vibrio RNA from a total RNA sample extracted from an environmental sample, in the range of 10 to 100 ng μL -1 . However, important standard deviations were observed that could be linked to the sample matrix. These samples were collected by filtering 25 m 3 of seawater to concentrate phytoplankton. Phytoplankton can produce exopolysaccharides [START_REF] Passow | Production of transparent exopolymer particles (TEP) by phyto-and bacterioplankton[END_REF]) and some of these complex molecules could be extracted with RNA. Polysaccharides are well known as PCR inhibitors [START_REF] Schrader | PCR inhibitors -occurrence, properties and removal[END_REF]. They have the ability to co-precipitate with nucleic acids, reducing the ability to resuspend the precipitated RNA [START_REF] Butot | Procedure for Rapid Concentration and Detection of Enteric Viruses from Berries and Vegetables[END_REF]). Furthermore, they can cross link with nucleic acids and change their chemical properties [START_REF] Opel | A Study of PCR Inhibition Mechanisms Using Real Time PCR[END_REF]). In our case, polysaccharides may contribute to heterogeneous RNA solutions and may hide some hybridization sites. This could explain the differences observed between triplicates.

By consequence, volumes of filtered water have to be optimized as well as the filtration method before analysis with the assay.

In order to facilitate evaluation of the assay performance, a detection limit corresponding to a number of cells per sample volume has been estimated for the PK sample. For this sample, Vibrio cell number has been determined by plate counting on TCBS agar as 6.17.10 4 CFU mL -1 of concentrate. Thus, the Vibrio cell number in the 400 mL concentrate was 2.5.10 7 . Since the concentrate has been obtained by filtering 25 m 3 of water, the developed tool allows the detection of 1 000 CFU L -1 .

Conclusion

In this paper, a PCR-free DNA-based assay for the detection of Vibrio spp. in environmental samples was developed. The resulting assay showed an excellent specificity since a high discrimination was observed between the 21 Vibrio strains and the 10 closely related genera tested. The combination of sandwich hybridization format and chemiluminescent detection allowed a direct analysis of the extracted RNA, suppressing the PCR amplification step routinely performed in such analytic techniques. Detection limits of 0.1 nM and 5 ng μL -1 were obtained with synthetic probes and total RNA respectively, indicating a good sensitivity of the reported system.

To optimize experiment time, microplates were coated beforehand and various storage conditions were assessed. Storage at 4°C without casein not only showed that components were stable for at least 30 days, but also decreased the analysis time to 2 hours, allowing a rapid detection of Vibrio spp. using the developed device.

The results with spiked and natural environmental samples indicated that the proposed method appeared as an interesting tool to determine the presence of bacteria in aquatic samples. In future works, optimization of samples preparation and transfer towards an electrochemical format will be performed in order to improve Vibrio detection in aquatic samples. Furthermore, correlation between signal intensities and cell counts from environmental samples will be investigated.

Captions (Table and figures)

Table 1 Synthetic DNA probes and positive control used for the developed DNA-based assay

Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6

 123456 Fig. 1 Schematic illustration of the sandwich hybridization assay. (a) first strategy ; (b) second strategy ; (c) third strategy

  Figure1

  Figure5

  ://blast.ncbi.nlm.nih.gov/Blast.cgi) against GenBank (http://www.ncbi.nlm. nih.gov/genbank/) and Silva (http://www.arb-silva.de/) databases. For the two probes, no perfect match was observed in silico with non Vibrio species. Probes biophysical properties were analyzed using the Oligonucleotide Properties Calculator software (http://www.basic.northwestern.edu/biotools/oligocalc.html). The 35 bp capture probe was biotinylated in 5', and the 35 bp signal probe was labeled with a digoxigenin (DIG) in 3' (Table

	Vibrio	genus	using	the	nucleotide	Basic	Local	Alignment	Search	Tool	(BLAST,

http

Table 1

 1

Table TMB reduced TMB oxidized Substrate Product

 TMB

the Service d'Observation du Laboratoire ARAGO for the sampling campaigns at SOLA. We also thank Christophe Canal and Léa Girard for the phytoplankton sampling at Leucate lagoon and Lisa Morales for her experimental contribution.

Conflict of interest

The authors declare that they have no conflict of interest.

Complience with ethical standards

All authors agreed to be listed and have approved of the manuscript, its content and its submission to Environmental Science and Pollution Research. It has not been submitted or published elsewhere, whether partly or fully. All authors are in agreement with the ethical rules of ESPR.


domain: phys spi

text:
Cornelia Irimiea

Axel Vincent-Randonnier

Jean Pierre Dufitumukiza

Stefano Puggelli

Jean-Baptiste May-Carle
email: jean-baptiste.may-carle@safrangroup.com

Nicholas Treleaven

Thomas Lesaffre

Alexandre Coudray

Etienne Lameloise

Bénédicte Cuenot

Eleonore Riber

Nicolas Fdida

Pascal Cherubini

Xavier Mercier

Experimental and modeling study of soot formation in high-pressure kerosene and SAF combustion

Keywords: SAF, combustion, soot, high pressure, optical metrology, modeling, Large Eddy Simulation

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

Introduction

The aviation industry has to look for renewable energy sources as a short-term solution for reducing gas and particulate emissions due to the stringent standards imposed on emissions and the increasing demand for air traffic [1]. Lately, outstanding progress toward cleaner combustion has been achieved due to the development of lasers and fast acquisition systems and, nonetheless, due to the increased processing power of computers [START_REF] Giusti | Turbulent Combustion Modelling and Experiments: Recent Trends and Developments[END_REF][START_REF] Dreizler | The role of combustion science and technology in low and zero impact energy transformation processes[END_REF]. However, despite these advancements, the characterization and understanding of combustion in industrial-scale combustors and, even more, with sustainable aviation fuels (SAF) is still limited. From an experimental point of view, the coupling of non-intrusive optical techniques, which can provide simultaneous experimental information about the involved physical phenomena (e.g., turbulent flow field, flame, spray, pollutant emissions), is becoming crucial. Implementing extensive optical technique measurements inside aircraft engines is expensive and almost impossible. In this regard, ONERA has dedicated test rig platforms for studying high-pressure and hightemperature combustion conditions similar to those identified in aero-engines. The MICADO test rig, which consists of a single injector combustor with optical access in the reactive zone, belongs to this group. Optical diagnostics are custom developed for characterizing a highly turbulent environment. Complex phenomena such as the evaporation of the fuel, spray dynamics and pollutants formation are monitored with optical techniques such as Mie and Rayleigh scattering, OH* chemiluminescence and laser induced incandescence (LII) for a matrix of selected fuels and blends. In order to complement the information obtained from experimental campaigns, Large Eddy Simulation (LES) is nowadays a valuable solution, especially when analyzing industrial configurations. However, an important volume of work for model validation is required to take confidence in the LES results, especially when pollutants are investigated. The present study is a step forward in realizing a joint experimental/numerical investigation on soot generation from Jet A-1 and SAF combustion in high-pressure and high-temperature conditions. This collaboration was possible in the framework of the ALTERNATE project [START_REF]Alternate Project[END_REF], where ONERA, SAFRAN Tech and CERFACS work jointly to combine experimental data and numerical results to understand soot formation and predict soot levels. Three operating conditions (OCs) representative of Taxi, Approach and Cruise LTO cycles were studied with optical techniques when burning Jet A-1 and Alcohol to Jet (ATJ) fuels in similar fuel-to-air ratios (FAR) at ONERA on the MICADO test rig. Employed optical techniques give access to the spray characteristics (scattering techniques), the flame front position (OH* chemiluminescence) and soot distribution (LII). These data provide information about the flame dynamics, fuel evaporation rates, heat release rate and semi-quantitative information about soot between the tested OCs and fuels. In parallel, LES has been carried out by Safran Tech and CERFACS on one OC for Jet A-1 in order to obtain a first comparison with the available experimental data and gain a better insight into soot generation in the present case. The paper is structured as follows: first, the MICADO test rig and the OCs studied experimentally are described, followed by the numerical setup. After that, the attention is directed toward the description of the experimental data collected with Jet A-1 and ATJ and to the comparison with the LES results for Jet A-1 fuel.

MICADO test rig

The MICADO test rig was used to investigate the high-pressure and temperature conditions that characterize aeronautical combustors [START_REF] Cochet | ONERA test Facilities for Combustion in Aero Gas Turbine Engines, and Associated Optical Diagnostics[END_REF]. This rig comprises the combustor facility, a dedicated optical laboratory placed above the test rig enclosure and a control room. Optical access is obtained thanks to three removable optical port views, i.e., two placed on the sides and one on the bottom of the combustor. Visual access and optical laser diagnostics are possible over a distance of 89 mm from the injector. The combustion chamber is 100 mm × 100 mm tubular square section. The combustor walls are water-cooled, thus allowing continuous running operating conditions for up to two hours after reaching stable conditions. Air and fuel are fed to the combustor through an axial swirl injector able to generate stable aerodynamic conditions. Two injection locations are provided: a main injection that introduces the fuel inside the channel of the swirled air, and a pilot system shown in the sectional side view in Fig. 1. The fuel and the pressure are stabilized with an exit Delavan nozzle and an adjustable throttling plug. The highpressure airflow (240 -492 g•s -1 ) is heated to a temperature of ∼550 K with a heat exchanger coupled to a kerosene burner. The inlet air mass flow entering the MICADO combustor is measured with a Venturi tube placed upstream of the injector inlet. Cooling of the optical windows is realized with 29% of the total airflow deviated with a metallic backplate, and the remaining 71% of airflow passed through the swirler (see Fig. 1). A dedicated LABVIEW acquisition platform ensures the operating control of the installation, as well as the temporal monitoring and record of the mass flow rates, the pressure inside the combustor and temperature. Three OCs were selected for this study. Jet A-1 and ATJ combustion was studied independently for each OC. However, due to MICADO system inertia, the global equivalence ratio (Φ) of Jet A-1 and ATJ for the same operating conditions are slightly different. In particular, Φ of Jet A-1 is slightly higher for conditions OC1 and OC3, while for OC2, Φ is superior for ATJ fuel. The fuel was injected in 100% pilot mode for all OCs. This camera had 94 mm focal length CERCO UV lenses set at f4 aperture. The lenses were equipped with a short bandpass filter with a bandwidth of 10 nm centered at 310 nm. Scattering induced with 527 nm at 10 kHz was recorded simultaneously with OH* by a second Phantom V711 camera during a GW of 0.3 µs. The camera was equipped with 135 mm (F/8) lenses and a narrow-bandpass filter of 10 nm bandwidth centered at 527 nm. The scattered signal was collected perpendicular to the laser sheet. The visible laser sheet at 527 nm was formed from a dual-cavity, frequency-doubled pulsed Nd:YLF laser beam (Dual Darwin 527-80M, 21 mJ/pulse at 10 kHz, 180 ns pulse width). The Gaussian laser beam was shaped into a laser sheet (L=100 mm, H=89 mm and w=1.2 mm) with a set of lenses consisting of a cylindrical negative lens, two spherical lenses and a positive cylindrical lens. LII and flame emission were recorded perpendicular to the IR excitation plane with a third PIMAX4 1024f intensified camera equipped with Sigma 105 mm (F2.8) lenses. A narrow bandpass filter with a bandwidth of 10 nm, centered at 450 nm, collected the transmitted light signal through a second dichroic mirror. LII signal and flame emission were recorded during 50 ns GW in prompt detection at a frequency of 3.33 Hz. A fourth PIMAX2 intensified camera equipped with Sigma 23-300 mm (f/3.5-6.3) lenses collected the flame emission 80 ns before the LII signal during a GW of 50 ns. This camera recorded the images at 3.33 Hz and was synchronized with the PIMAX4. A narrow bandpass filter with a bandwidth of 10 nm, centered at 580 nm, collected the reflected light signal. The corresponding LII laser beam (1064 nm, 9 ns pulse width, 10 Hz, Litron 850G) was shaped into a homogeneous laser sheet (L=100 mm, H=89 mm and w=0.4 mm) with a negative cylindrical lens and two positive cylindrical lenses. The laser fluence at the focus point is 0.25 J/cm 2 . This fluence value is superior to the LII sublimation threshold of ∼0.18 J/cm 2 for mature soot and was chosen to compensate for the attenuation of the laser beam while passing through the dense pressurized sampling volume. All four cameras were flat field corrected, and their focal point was adjusted with a calibrated target before each measurement. After the stabilization of each OC, a total of 10 minutes were dedicated to record images with the coupled optical system. The two high-speed cameras for OH* and scattering were used to recording 15000 images each at 10 kHz simultaneously, followed by the record of the flame emission and LII plus flame emission at 3.33 Hz with 80 ns delay between the intensified cameras. Each PIMAX camera recorded a series of 1500 images. A correction factor was calculated for the flame emission recorded with the PIMAX2 camera to subtract from images recorded with the PIMAX4 camera. This correction factor was estimated while measuring the flame emission with the two cameras without inducing LII signal and flat field measurements.

Numerical methods

The simulations presented hereinafter were performed using the AVBP solver developed by CERFACS [START_REF]CERFACS AVBP website[END_REF]. AVBP solves the fully compressible multi-species Navier-Stokes equations on unstructured grids. The considered computational domain is shown in Fig. 2, where it is pos- sible to distinguish the air inlet which feeds the swirler, the cooling films and the outlet. The boundary conditions were fixed to be consistent with Tab. 1 for the OC1. The Lax-Wendroff advection scheme, second-order in space and time, was employed for all calculations. An unstructured mesh counting 19M elements was initially generated, imposing a uniform sizing inside the swirler channel and the combustion chamber. A first reactive simulation run using this coarse mesh to then apply static mesh adaptation, similarly to [START_REF] Agostinelli | Numerical and experimental flame stabilization analysis in the new spinning combustion technology framework[END_REF]. Next, the zone to be refined was tagged based on the heat release rate field and the local sizing was adapted targeting a flame thickening factor for Dynamic Thickened Flame LES (DTFLES) of 30 in all the flame regions. This led to a mesh counting 57M elements mostly refined in the flame zone, used for all the calculations shown in the following.

The subgrid turbulent contributions were included thanks to the SIGMA model [START_REF] Nicoud | Using singular values to build a subgridscale model for large eddy simulations[END_REF]. The DTFLES model [START_REF] Colin | A thickened flame model for large eddy simulations of turbulent premixed combustion[END_REF] was used to describe the flame propagation, where the efficiency function is computed based on the formulation proposed in [START_REF] Charlette | A power-law flame wrinkling model for les of premixed turbulent combustion part i: non-dynamic formulation and initial tests[END_REF] with β = 0.5. The liquid phase was modeled with a Lagrangian approach. A standard set of modeling choices was selected for the air-fuel interactions, where the drag contribution was included in the equation for the momentum of the liquid phase with the hypothesis of a spherical shape, whereas the evaporation rate was computed based on the model of [START_REF] Abramzon | Droplet vaporization model for spray combustion calculations[END_REF]. A standard correlation for pressure atomizers from [12] was applied to obtain a Sauter Mean Diamter (SMD) of 44 µm, used for a Rosin-Rammler distribution with a dispersion parameter q fixed to 2.5. As far as the chemical description is concerned, two sets of calculations were performed. First, a two-step globally reduced chemistry, derived in [START_REF] Franzelli | A two-step chemical scheme for kerosene-air premixed flames[END_REF], was used to compute the main features of the flame and to draw the first comparison with the experimental data. This calculation is named in the following 2S-BFER.

Global chemistry, albeit able to accurately predict global flame quantities such as the laminar flame speed and the burnt gas state, does not provide an estimation of pollutants, such as soot precursors. Therefore, considering the goals of the present study, the Analytically Reduced Chemistry (ARC) scheme proposed in [START_REF] Gallen | Lagrangian tracking of soot particles in les of gas turbines[END_REF] was employed to predict both the flame structure and the gaseous soot precursors. This simulation with ARC started from the 2S-BFER solution. The ARC scheme counts 29 transported species and 15 species in a Quasi Steady State. After a first computational step to flush out the 2S-BFER initialization, the Lagrangian Soot Tracking (LST) model proposed in [START_REF] Gallen | Lagrangian tracking of soot particles in les of gas turbines[END_REF] was activated to compute soot levels. The LST method, developed by CERFACS, considers soot particles as point sources with their characteristic properties (e.g., temperature, size, velocity). Soot particles are considered spherical at the nucleation stage, then their surface evolves depending on their history (collisions, surface reactions). The reader is referred to [START_REF] Gallen | Prediction of soot particles in Gas Turbine Combustors using Large Eddy Simulation[END_REF] for a complete description of the LST approach. This calculation is named in the following ARC-LST. It should be pointed out that, during the flushout of this computation from the 2S-BFER case it appeared that the ARC flame stabilisation tip was much more sensitive to the mesh resolution and thickening factor. Therefore, keeping the same parameters as in the 2S-BFER case led to a flame detachment from the injector. In order to avoid this, the thickening factor was fixed to one in the zone close to the injector. This adjustment represents a draft solution to focus our attention on soot production and to compare with the available experimental data. However, the problem of stabilizing flames in this configuration with a semi-detailed chemistry is still under investigation to understand and formulate an appropriate method.

For the investigated operating condition, it is considered the flame was stabilized in the first half of the combustion chamber. With this hypothesis, the Flow Through Time (FTT) was estimated to 5 ms. For the ARC-LST calculation, the flush-out was carried out over 3 FTTs and statistics were collected during more than 2 FTTs. As discussed during the description of the results, the flame shows a strong coupling with the liquid phase and consequent important dynamics. The averaging process is ongoing to obtain a more converged set of statistics, especially for soot.

Results

This section evaluates the formation of soot particles for Jet A-1 and ATJ through experimental and modeling tools. First, spray characterization, OH* chemiluminescence and LII signals are presented to identify the main parameters leading to soot formation for the three OC with Jet A-1 and ATJ fuels. Then, the comparison between experiments and LES is proposed, followed by an insight into the soot generation process obtained for OC1 and Jet A-1.

Experimental results

Spray vaporization and dynamics are inferred from instantaneous scattering images that are exhaustively described. Scattered light from multi-phase combustion is attributed to non-vaporized between the spray scattered signal and the detector, similar to shadowgraphy. These dark regions were identified when soot was localized near the sampling focal point. Instantaneous scattering images are predominated by light scattered from the non-vaporized fuel, while light scattered from soot was identified for OC3 with Jet A-1 and ATJ fuels only. This separation is possible when particulates are not interfering with the non-vaporized fuel region, as well as in the case of high local soot concentrations on instantaneous images. Specific features are determined on the instantaneous images, which differentiate mainly the OC and less among the fuels. Similar patterns of the spray angle and coverage area are identified for OC1 and OC2, while OC3 spray shapes are notably different from the first two OCs due to the oscillation of the cone angle and penetration depth from one image to another. Several instantaneous images corresponding to OC1 -Jet A-1 fuel are shown in Fig. 3 (blue-colored area), where the coverage spray zone is compact near the injector and thinner towards the spray branches. Low and high-frequency oscillations of the spray are identified with the 10 kHz recording frequency. For OC1 and OC2, the spray is localized on a 10 mm narrow region next to the nozzle, followed by a ramified branch that expands between 10 to 20 mm from the combustor backplate. Thin droplets of the swirling spray extend from 25 to 45 mm and then impinge on the combustor walls. The spray shape varies considerably for the OC3 case, indicating that this condition is prone to instability. For OC3, the spray penetration depth oscillates between 20 and 45 mm, with a synchronous fan angle oscillating between 140 • and 80 • . Spray oscillations for each OC and fuel were determined after applying fast Fourier Transform (FFT) on summed scattered intensities of the entire frame for a series of 15000 images. The dominant frequency of Jet A-1 fuel in OC1 is 1044 Hz, 1188 Hz corresponds to OC2 and 232 Hz to OC3. Respecting the same OC order, ATJ fuel has the following dominant frequencies: 1043 Hz, 1197 Hz and 230 Hz. The three OCs have a frequency shift between Jet A-1 and AtJ fuel. This frequency shift is negative between Jet A-1 and ATJ for OC1 and OC3 and reversed for OC2, where higher frequencies are detected for AtJ fuel as against Jet A-1. After the FFT analysis, several prominent frequencies were identified for each OC, with four non-harmonic modes for OC1, two for OC2, and six harmonics for OC3. These modes correlate with the changing shape of the spray, its stability and mixing with surrounding hot gas. For instance, the reverse frequency shift between the two fuels in OC2 may be related to the global equivalence ratio's role in the vaporization process rather than the operating conditions. This fact is attributed to the fuel-to-air ratio (FAR), which is smaller for Jet A-1 than ATJ in OC2. FAR values of Jet A-1 in OC1 and OC3 are higher than for ATJ. Identified frequencies can also be attributed to hydrodynamics or thermoacoustic oscillations. The discussion is oriented on averaged images of non-vaporized fuel, presented in Fig. 4. It has to be noted that the flame is slightly asymmetrical, and some regions of the visualization window were damaged or fouled during the experiment. We limit our discussion to the visible regions of the flame through the optical port view. It is also pointed out that on the images with the fields of averaged signals (Fig. 4, 5, and 6), lines and ticks for the simulated case are represented with claret color to highlight the experimental data used for comparison with the numerical method. Spray shape changes with the OC but not significantly with the fuel. On average, for OC1, the spray is localized near the injector (well-defined edges over 5 mm), followed by an opening of 45 • until 45 mm, where the spray is impacting the walls. For the OC3 case, the spray extremities are not touching the walls due to the predominant spray coverage area on the central axis of the chamber and instability. The spray angle near the injector is slightly increasing from OC1 to OC3. At the same time, a slight broadening in the spray fan angle is observed between Jet A-1 and ATJ for the three cases, as referenced in Fig. 4, with green arrows. These changes can rely upon the physical properties of the fuel, and it may be an indicator of different vaporization processes from one fuel to another. Standard error deviation (Std) field images (not shown in the text) corresponding to averaged images, display a higher spread of ATJ scattered signals on the spray region as against the Jet A-1, suggesting again that the vaporization of the two fuels is diverse. Indeed highly scattered signals indicate a dense spray, while weakly scattered signals are typical of a more diluted spray. Overall, averaged intensities from the spray are higher next to the injector region for ATJ fuel in the three OC. Conversely, Jet A-1 spray branches are denser than ATJ cases. Simultaneous detection of OH* chemiluminescence and fuel scattering gives insight into flameflow-spray interactions [START_REF] Kheirkhah | Dynamics and mechanisms of pressure, heat release rate, and fuel spray coupling during intermittent thermoacoustic oscillations in a model aeronautical combustor at elevated pressure[END_REF]. Hence, scattering data is compared with the heat release zones identified via the OH* chemiluminescence to understand fuel vaporization and fuel/air mixing. ings shown in Fig. 5 with a peak of heat release around the combustor centerline and a wide extension in the axial and radial directions. The spray looks also consistent with Fig. 4 with an opening angle of around 45 • and an axial penetration around 15 mm (see the green arrows in Fig. 9). Therefore, from a qualitative point of view, the proposed numerical setup seems capable of retrieving the macroscopic flame behavior. Hence, the attention is now directed toward soot prediction. Fig. 10 reports on the left-side the instantaneous distribution of the soot volume fraction (f v ) combined with the naphthalene mass fraction. The spray trajectory is also displayed with blue points. The whole height of the combustion chamber is here reported (i.e. 50 ≤ y[mm] ≤ 50) with respect to Fig. 6 (i.e. 45 ≤ y[mm] ≤ 45). Additionally, the O2 and OH mass fractions are shown in the center of Fig. 10, together with the soot source terms on the right-side. The source terms do not account here for nucleation and only contain oxidation on top-half and surface growth plus condensation on the bottom-half. Looking at the distribution of soot source terms, a clear correlation with the naphthalene appears: in the near injection zone and at the wall, C 10 H 8 is present, thus enabling the soot nucleation. The soot tends then to be partially oxidized by the oxygen, present in the IRZ, and by the OH near the combustor walls. In that zone, fresh oxygen is also injected at the cooling slots, thus generating a further soot oxidation region. All in all, this leads to an instantaneous soot distribution with two peaks, i.e., the first one in the near injection zone and the second near the walls, similar to what is reported experimentally. Nevertheless, considering the intermittency in the soot generation and the complex flame stabilization process pointed out for this operating condition, a much longer averaging time is required on soot and on the flame structure to reinforce such results and draw a more quantitative conclusion.

Conclusions

The sooting behavior of ATJ was experimentally investigated against Jet A-1 for three operating conditions (OC). It is shown that for similar FAR values, for the three selected OCs, ATJ fuel produces way fewer soot particles than Jet A-1. However, the sooting capacity of ATJ is not linear with the chamber pressure or the FAR. The various parameters that affect the formation of soot particles are the vaporization of fuel, which is different from one fuel to another, although the zones covered by the spray are similar in the two cases, the oxidation and heat release regions and the physical parameters of the fuel. In addition, flame instabilities can affect the production of soot, as observed for OC3, due to unfavorable mixing properties at the shear layer between non-vaporized fuel and surrounding hot gas. For this case, soot values were high for both fuels, but ATJ produced three times less soot and this fact can be related to both the chemical properties of the fuel and hydrodynamic conditions in the combustor. For OC1 and OC2, averaged soot values were near the detection limit (few ppb) for ATJ; conversely, instantaneous images are characterized by localized soot flamelets and pockets with high intensities of soot signals (tens of ppb). A further processing of instantaneous images with advanced statistical tools is envisaged in the future for a better understanding of the processes governing the formation of soot and differences induced by the ATJ and Jet A-1 on small temporal scales. In parallel to experimental findings, a numerical study based on LES was also proposed to characterize the case OC1 with Jet A-1 as a first step. The developed model seems able to capture the macroscopic characteristics of the spray and the flame regions. Similarly to experiments, soot regions are identified near the injector and close to the walls. A longer averaging time is undoubtedly required to draw quantitative conclusions on soot distribution and flame stabilization, which will be pursued in the near future. The present case presents a complex stabilization process with semi-detailed chemistry. As a consequence, an appropriate modeling strategy with respect to the one employed in the present study for this problem is currently under investigation. Ultimately, the model will be extended to multi-component formulation in order to account for SAF composition and directly compare it with the experimental data available with ATJ.

Figure 1 .

 1 Figure 1. Sectional side view and fluid domain of MICADO test rig. One example of the visible flame emission stabilized in OC1 with Jet A-1 is represented in the optical port view area.

Figure 2 .

 2 Figure2. The computational domain used for LES of the MICADO test bench (left-hand side) and mesh obtained after mesh adaptation (right-hand side). The white iso-line represents an instantaneous iso-contour of heat release rate at 2×10 9 W/m 3 .

Figure 3 .

 3 Figure 3. Overlay of instantaneous OH* (red color) and non-vaporized fuel (blue color) images for OC1 -Jet A-1 fuel. The dotted mesh has 10 mm distance between each neighboring dot. Images (I1-4) were randomly selected from the dataset.

Figure 4 .

 4 Figure 4. Average image fields with the scattered signal from non-vaporized fuel. Averaged images were obtained from a series of 2000 instantaneous images.

Figure 8 .Figure 9 .

 89 Figure 8. Instantaneous (top-half) and time-averaged (bottom-half) distributions of (a) axial velocity, (b) gaseous temperature, (c) flame heat release rate and (d) liquid evaporation rate over the chamber mid-plane for the ARC-LST case. The white line on the axial velocity plot represents the iso-contour at u ax = 0m/s, while on the other three figures it shows the zone with a gaseous equivalence ratio 0.8 ≤ φ g ≤ 1.1.

Figure 10 .

 10 Figure 10. Instantaneous soot volume fraction combined with the naphthalene mass fraction (left-hand side), mass fraction of oxygen and OH (center) and soot evolution source terms (right-hand side) from the ARC-LST case. The soot source terms are expressed in [kg /s/m 3 ] as units.

Table 1 .

 1 These tests resulted in six individual OCs with specific operating parameters, as summarized in Tab. 1. The lower heating values (LHV) of Jet A-1 and ATJ are 43.21 MJ/kg and 44.06 MJ/kg. Operating conditions tested during the ALTERNATE project on the MICADO test rig for Jet A-1 and ATJ fuels.

				P ch (MPa)	T ai r (K)		ṁair (g/s)	ṁ f uel (g/s)		Φ
			Avg	Std	Avg	Std	Avg	Std	Avg	Std	Avg	Std
	OC 1	Jet A-1 ATJ	0.45 0.45	3.4×10 -3 2.2×10 -3	540.6 526	0.9 0.4	241.3 244	0.8 1.4	13.2 12.4	0.02 0.08	0.80 0.77	3.2×10 -3 7.4×10 -3
	OC 2	Jet A-1 ATJ	0.79 0.79	6.9×10 -3 1.5×10 -3	555.6 549.6	0.3 0.1	491.7 491.9	1.3 1.0	22.9 24.1	0.10 0.25	0.68 0.72	3.7×10 -3 7.6×10 -3
	OC 3	Jet A-1 ATJ	0.94 0.93	8.5×10 -3 1.8×10 -3	553.3 546.8	0.1 0.7	347.4 347.2	0.9 1.7	18.5 18.3	0.11 0.10	0.78 0.77	5×10 -3 5.7×10 -3

Acknowledgements

This project has received funding from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 875538 in the project ALTERNATE and the ONERA internal research program PR CARBON. This work was granted access to the HPC/AI resources of TGCC under the allocations 2021-A0102B12504 and 2022-A0122B13460.

In particular, OH* chemiluminescence is line-of-sight integrated and is used to infer the bulk dynamics of the flame structure. In addition, spray dynamics can be correlated with the OH* to determine a correlation factor between the vaporization of the fuel, mixing properties and the flame front. On instantaneous images, OH* signals are predominant at the periphery of the spray branches, following their oscillations and propagation. Previous works mentioned that higher OH* intensities are associated with the premixed part of the combustion since there is a higher production yield in these premixed regions of the fuel or next to the thin droplets [START_REF] Kheirkhah | Dynamics and mechanisms of pressure, heat release rate, and fuel spray coupling during intermittent thermoacoustic oscillations in a model aeronautical combustor at elevated pressure[END_REF]. Similar oscillation frequencies of the flame front, as the ones identified from scattering data, were obtained after the FFT analysis of the instantaneous image datasets. The magnitude of the frequency modes is higher for Jet A-1 fuel on the three OCs and the predominance of the modes did not change with respect to the frequency modes identified from fuel scattering. The order of the frequency shift between the non-harmonic and harmonic modes of the two fuels is also respected. Averaged emission OH* for the three cases and two fuels are presented in Fig. 5. Each OC is dominated by a corresponding flame front, which is not changing significantly among selected fuels, except for its magnitude. The heat release rate is expanding almost identically on the vertical axis for OC1 and OC2, and for OC3, the heat release is dominant in the inner recirculation zone of the flame. On the horizontal axis, the shift of OH* intensities from the injector toward the exhaust suggests that the flame is stabilized differently for the three OCs. For OC2 and OC1, the flame is stabilized closer to the injector, with a peak of heat release near 30 mm, indicating a faster fuel evaporation rate and improved mixing. OC3 case is characterized by lower OH* intensities on the averaged image, with a stabilization peak between 15 and 30 mm and a higher turbulence rate since the Std is the highest among the three OCs, and it corresponds to the frequency mode at 244 Hz. Unexpectedly, Jet A-1 fuel combustion is characterized by a higher OH* emission than the one displayed by ATJ in the three OCs. A positive linear correlation exists between the FAR and OH* ratio of the two fuels for OC1 and OC2, but this trend is changing for OC3, the unstable condition. Because the observed difference between the heat release of the two fuels cannot be explained by the fuel LHV only, it is suggested that the local mixing influences the heat release as well, which can affect the evaporation of fuel. It is suggested that differences in the local mixing, which result from the turbulence and the evaporation rate, directly affect the heat release rate and the concentration of soot precursors and soot particles. Soot particle regions were identified with the LII technique, which is highly sensitive (few ppb) for their detection. Average LII signals (flame emission subtracted) are displayed in Fig. 6. Spatially, soot averaged fields are shifted toward the exhaust from the OH* zones for OC1 and OC2. This trend is not kept for OC3, where soot particles are mainly detected in the same region as OH*, due to the highly turbulent behavior of the flame and instabilities. Similar spatial distribution characterizes OC1 and OC2, with reduced average soot volume fractions (few ppb). For these two cases, soot is present near the walls after 30 mm from the combustor back plate. An outer corner recirculation zone (CRZ) is observed near the back plate region, mostly for OC1 and less prominent for OC2. The probability of soot detection on instantaneous images is low (high Std) for the first two cases. Soot filaments and pockets with high intensity (tens of ppb) are identified for OC1. A few in- estimated to be a few ppm on the averaged images. ATJ fuel produces three times fewer soot particles for this OC. The high predominant sooting behavior of OC3 was also captured with the scattering techniques, where soot is mainly identified in the IRZ toward the exhaust when the spray is broader and closer to the back plate. High residence time is identified for soot when the spray has a large fan angle (140 • ) and is localized near the injector; in this case, soot structures persist in the chamber for a few µs until the penetration depth of the spray increases and displaces the soot zones toward the combustor walls. The movement can be associated with low-frequency spray dynamics. High soot formation rates are associated with regions with high equivalence ratios since the fuel is not completely evaporated -data backed up by Mie scattering where the density of the fuel is higher than the other instantaneous images.

Comparison between experimental and LES results

This section analyses the numerical data obtained with Jet A-1 fuel on the OC1. The aim is to assess the capabilities of the selected numerical setup in reproducing the main characteristics of the flame and to provide a better understanding of the soot generation in the considered case. Fig. 8 shows a summary of the instantaneous (top-half) and time-averaged (bottom-half) fields for velocity, temperature, heat release rate and evaporation rate. The chosen view corresponds to the experimental window, where the fresh air duct has been included for clarity. A swirling flow field is generated with the creation of an IRZ, highlighted with the white iso-line at zero axial velocity, which brings burnt gases towards the injector. The flow field is also characterized by the presence of CRZ generated by the shear between the main swirling flow field and the cooling films. The flame is stabilized at the shear layer between the burnt gases in the IRZ and the fresh air coming from the swirler with a resulting V-shape. This zone corresponds to the location of the stoichiometric lines shown on the temperature and heat release rate contour plots. An important part of the flame heat release rate is also located in the near wall zone. The flame stabilization process is explained as follows: once injected, part of the spray almost immediately evaporates and generates a region with high values of equivalence ratio in the near injection zone. This creates the conditions to stabilize the inner flame branch in the shear layer between the burnt gases and the fresh air. Another part of the spray is instead able to pass this region and evaporates further downstream, i.e., near the chamber walls. This generates the second part of the flame in the shear layer between the fresh air and the CRZ. This explanation is supported by the evolution of the liquid phase evaporation source term shown in Fig. 8(d).

Both the instantaneous and time-averaged evolutions of the liquid phase mass transfer are reported. Looking in particular at the time-averaged result, it is evident that the flame extends over the same region where the spray evaporates. This finding suggests an important coupling between the liquid phase and the flame in the present case.

In order to compare the LES results with the experimental data provided in the previous section, Fig. 9 reports the time-averaged line integrated heat release rate and liquid volume fraction over the experimental window. The flame seems located coherently with the experimental find-