Benjamin Duqué

Nabila Haddad

Albert Rossero

Jeanne-Marie Membré

Sandrine Guillou
email: sandrine.guillou@oniris-nantes.fr

Influence of cell history on the subsequent inactivation of Campylobacter jejuni during cold storage under modified atmosphere

Keywords: foodborne pathogen, slaughter process, stresses, food safety, strain variability

Worldwide, Campylobacter infections are the main cause of human bacterial enteritis and broiler meat is considered as the most important source of human campylobacteriosis. Some mitigation strategies have been focused on reduction of Campylobacter at the slaughtering steps. This study aimed to determine the influence of consecutive stresses inspired by slaughtering steps on the subsequent inactivation of Campylobacter jejuni during cold storage under different modified atmospheres. Using a full experimental design, three strains of C. jejuni of poultry origin were submitted to consecutive heat (46°, 50° or 54°C for 4 min) and cold (-4° or 3°C for 2 h) stresses by plunging cultures into baths at appropriate temperatures. Cultures were then stored at 6°C during seven days under modified atmospheres (70% O2 / 30% CO2 or 50% CO2 / 50% N2). Inactivation of C. jejuni induced by cold storage was shown to depend significantly (P<0.0001) upon the heat stress previously applied. It was shown to be the highest under the atmosphere enriched in oxygen, after application of 54°C. Strain inactivation variability was also quantified. These results show that consecutive stresses influence further inactivation of C. jejuni during storage and consequently the contamination level at consumer's plate.

Introduction

Since 2005, Campylobacter has been the main cause of bacterial enteritis worldwide in humans [START_REF] Efsa | The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017[END_REF]. Despite being largely under-estimated, the number of reported confirmed cases of Campylobacteriosis was 246,158 in 2017 with an EU notification rate of 64.8 per 100,000 population [START_REF] Efsa | The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017[END_REF]. Infection with C. jejuni can lead to chronic sequelae, such as Guillain-Barré syndrome (GBS), characterized by the damage of the peripheral nervous system leading to a reversible neuromuscular paralysis [START_REF] Nyati | Role of Campylobacter jejuni infection in the pathogenesis of Guillain-Barre syndrome: an update[END_REF].

The main reservoirs for C. jejuni are avian species and farmed poultry [START_REF] Young | Campylobacter jejuni: molecular biology and pathogenesis[END_REF]). In food, the occurrence of Campylobacter remains high in broiler meat [START_REF] Efsa | The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017[END_REF] and it is considered to be the most important single source of human Campylobacteriosis. In 2017, 37.4% of the 13,445 samples of fresh broiler meat (single or batch, aggregated data from all sampling stages) were found to be Campylobacter positive [START_REF] Efsa | The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017[END_REF].

Several microbiological risk assessment studies have been performed to assess the potential effects of control measures on prevention/reduction of Campylobacter concentration in broiler meat production. Some studies focused more on pre-slaughter stages associated with broiler rearing [START_REF] Hermans | Campylobacter control in poultry by current intervention measures ineffective: urgent need for intensified fundamental research[END_REF][START_REF] Lin | Novel approaches for Campylobacter control in poultry[END_REF] including the use of probiotics [START_REF] Saint-Cyr | Use of the potential probiotic strain Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers[END_REF] or vaccine [START_REF] Meunier | Promising new vaccine candidates against Campylobacter in broilers[END_REF]. Another effective strategy to reduce the number of campylobacteriosis cases may reside in limiting the entry of chicken carcasses highly contaminated by Campylobacter into the market. In this context, the European Commission has just set a microbiological process hygiene criterion for Campylobacter in broiler carcasses (European Commision 2017). A limit of 1000 CFU.g -1 on carcasses after chilling has been defined. The EFSA (European Food Safety Authority) determined that more than 50% human risk reduction could be achieved if broiler carcasses complied with this new microbiological criteria of 1000 CFU.g -1 (European Commision 2017). For food producers, the compliance with this hygienic criterion requires improvements in slaughter hygiene to limit cross-contamination but also investigation of new control measures which may favor Campylobacter inactivation following slaughter. Focusing on the susceptibility of the bacteria to the processing steps may represent one way to tackle Campylobacter risk.

C. jejuni is able to survive to slaughterhouse environments and during poultry processing. In the poultry farm-to-fork chain, several steps may be stressful for bacteria regarding its growth requirements (microaerophily and thermotolerance), such as scalding, chilling and storage. Scalding consists in immersing chicken carcasses into a hot water bath, which may induce heat stress to C. jejuni. At the end of the slaughter process, the chilling step enables quick refrigeration of chicken carcasses, which is necessary to lower the growth rate of pathogenic and spoilage microorganisms [START_REF] James | The primary chilling of poultry carcasses -a review[END_REF], is supposed to generate cold stress to C. jejuni. At last, chicken cuts are generally conditioned under modified atmosphere and stored under chilled temperature until consumption. Depending on the producer, the gas mix of the package may contain high concentrations of oxygen or not. This latter step induces both cold and possibly oxidative stresses. However, if the influence of single stresses on Campylobacter behavior is well-described, the effect of stresses occurring sequentially still needs to be more deeply understood in order to point out conditions of slaughter steps that favor Campylobacter inactivation during poultry products storage.

Indeed, the exposure to a stress may condition the bacterial behavior following the exposure to another stress. Increased resistance has been for example already reported in Bacillus [START_REF] Den Besten | Short-and long-term biomarkers for bacterial robustness: a framework for quantifying correlations between cellular indicators and adaptive behavior[END_REF][START_REF] Desriac | Bacillus cereus cell response upon exposure to acid environment: toward the identification of potential biomarkers[END_REF]. For instance, after being subjected to mild acid or oxidative stress, C. jejuni appeared to be more resistant to a subsequent lethal stress (Murphy et al. 2003a, Murphy et al. 2003b[START_REF] Oh | Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions[END_REF]. Such cross-protection response from one stress to another has also been reported, like genes involved in heat shock response in C. jejuni NCTC 11168 which were upregulated in response to acid stress (Reid et al. 2008b). Additionally, starved C. jejuni cells were able to better withstand heat stress [START_REF] Klančnik | Stress response and pathogenic potential of Campylobacter jejuni cells exposed to starvation[END_REF]).

Besides, it is well established that strains have different behavior when subjected to stress [START_REF] Chan | Survival of Clinical and Poultry-Derived Isolates of Campylobacter jejuni at a Low Temperature (4 C)[END_REF][START_REF] Cools | Survival of Campylobacter jejuni strains of different origin in drinking water[END_REF][START_REF] Newell | Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs[END_REF]) and this is highly relevant for poultry product in the sense that poultry meat is naturally contaminated by different strains of C. jejuni and often more than one species [START_REF] Colles | Genetic Diversity of Campylobacter jejuni isolates from Farm Animals and the Farm Environment[END_REF][START_REF] Johnsen | Genotyping of Campylobacter jejuni from Broiler Carcasses and slaughterhouse environment by amplified fragment length polymorphism[END_REF][START_REF] Rivoal | Genomic diversity of Campylobacter coli and Campylobacter jejuni isolates recovered from free-range broiler farms and comparison with isolates of various origins[END_REF] In the current study, several strains of C. jejuni were submitted to stresses inspired from those encountered during slaughter, and subsequently incubated under modified atmosphere. The effect of cell history i.e. the effect of hot and cold temperature applied following successive heat and cold stresses, on the subsequent inactivation of C. jejuni strains during chilled storage under various modified atmospheres, was then quantitatively analyzed.

Materials and methods

Bacterial strains and culture conditions

Experiments were performed on three C. jejuni strains isolated from poultry products.

C. jejuni C09MJLT205 was isolated from chicken legs distributed at the market level [START_REF] Guyard-Nicodeme | Characterization of Campylobacter spp. transferred from naturally contaminated chicken legs to cooked chicken slices via a cutting board[END_REF]. C. jejuni RM1221 was isolated by [START_REF] Fouts | Major structural differences and novel potential virulence mechanisms from the genomes of multiple Campylobacter species[END_REF]. The strain C. jejuni C97Anses640 was isolated from a poultry product [START_REF] Guyard-Nicodeme | Efficacy of feed additives against Campylobacter in live broilers during the entire rearing period[END_REF]). All strains were stored at -80°C in brain heart infusion (BHI, Biomérieux, Marcy l'Etoile, France) supplemented with 20% (v/v) glycerol. C. jejuni strains were routinely cultured on Karmali (Oxoid, Dardilly, France) agar plates at 42°C for 48-72 h under microaerophilic conditions (5% O2, 10% CO2, and 85% N2). Then, one or two colonies were transferred into 20 mL of Mueller-Hinton broth (MH, Oxoid, Dardilly, France) and incubated for 20 h under the same conditions under agitation (90 rpm). A 1/100 th dilution was transferred into a fresh MH broth and incubated under the same conditions for 18 h, so that the culture reached stationary phase. Finally, a 1/10 th dilution was carried out to obtain what we called the 'starter culture' further used for challenge-test experiments.

To enumerate viable C. jejuni cells, suspensions were serially tenfold diluted and surface plated on Columbia sheep blood agar plates (Biomérieux, Marcy l'Etoile, France) and incubated for 48 h at 42°C under microaerophilic conditions using a SPIRAL plater (EasySpiral Interscience, Saint Nom, France).

Experimental procedure

C. jejuni strains were submitted to stresses inspired from conditions encountered during slaughter chicken process. Slaughter is associated with several steps considered as stressful for C. jejuni. These steps are scalding, chilling, and are followed by cold storage under modified atmosphere. Scalding consists in immersing chicken carcasses into hot water bath. Then, chicken carcasses are submitted to a quick refrigeration during the chilling step.

At last, after cutting carcasses in pieces, chicken cuts are conditioned under modified atmosphere and stored under chilled temperature.

The three previously mentioned stresses were reproduced as closely as possible in the laboratory, and named as heat stress, cold stress and storage stress, respectively. The first two stresses, i.e. heat and cold stresses were assumed to influence the future behavior of C. jejuni during chilled storage under modified atmosphere. As such, they were considered as participating to C. jejuni adaptive history (Fig. 1).

Ten mL of starter culture with an initial concentration of 8 log10 CFU.mL -1 from each strain were transferred into a glass tube and submitted to consecutive heat, cold temperature and storage at 6°C under modified atmospheres according to a full experimental design.

Heat stress consisted in immersing C. jejuni cultures in hot water baths at 46°, 50° or 54°C for 4 min. 54°C was chosen as the maximal observed temperature of scalding baths in French chicken slaughterhouses, 46 °C, the maximum growth temperature [START_REF] Hazeleger | Physiological Activity of Campylobacter jejuni Far below the Minimal Growth Temperature[END_REF] of C. jejuni and 50°C, an intermediate temperature.

After exposure to heat stress, tubes were immediately cooled for 5 min in a water bath at 22°C, corresponding to the conditions of ambient temperature occurring during the five minutes between scalding and chilling steps in slaughterhouse.

Cold stress was applied immediately after cooling by plunging the previous cultures into cold water with ethylene glycol at -4° or 3°C for 2 h. The temperature of 3°C was chosen because it represents the targeted core temperature of poultry carcasses during the chilling step and -4°C is the minimal surface temperature measured during different visits performed in several slaughterhouses. Strains were exposed to cold stress for 2 h similarly as chicken carcasses during the chilling step.

At last, cultures were stored at 6°C for seven days under the modified atmospheres (70% O2 / 30% CO2 or 50% N2 / 50% CO2) commonly used for the packaging of poultry cuts.

These two atmospheres were generated by filling jars with the ad hoc gas mixture (Air Liquide, Carquefou, France). Cultures were placed in bottles with porous silicone caps (Hirschmann Laborgeräte GmbH & Co. KG, Eberstadt, Allemagne) enabling gas exchange.

The temperature of 6°C was chosen according to the average temperature of consumer refrigerators [START_REF] Laguerre | Study of domestic refrigerator temperature and analysis of factors affecting temperature: a French survey[END_REF].

During experiments, the temperature applied during heat and cold stress was recorded using a temperature probe Kistock KTT220 (KIMO, Montpon, France) placed in a control tube. The gas composition (% O2 and % CO2) of the jar atmosphere was measured using the Oxybaby M+ device (WITT-GASETECHNIK GmbH & Co KG, Witten, Germany). It was performed just after filling with gas mixture and regularly over time to check the stability of the gas composition.

After each step, viable counts of C. jejuni were enumerated to determine the viability loss resulting from the application of each step, i.e. ∆log10. A total of 152 (3 strains x 4 repetitions x 12 experimental conditions + 8 controls) experiments were performed.

Statistical analysis

Four independent replicates were carried out and statistical analysis was performed on Δlog10 transformed values. An ANOVA (α=5%) was performed using the software XLSTAT (version 19.03.45028), an Addin of the Microsoft Excel software.

Results

Cumulative effect of subsequent heat, cold and storage stresses on inactivation of C. jejuni (Δlogtotal)

From the starter culture with an initial concentration of 8 log10 CFU.mL -1 , the mean cumulative C. jejuni inactivation, due to the consecutive heat, cold and storage stresses, varied from 0.02 to 4.9 log10 CFU.mL -1 as function of strains and atmosphere conditions.

Detailed results are available in supplementary material Table S1.

During experiments, controls were performed and submitted only to the cold storage under modified atmosphere. The inactivation induced by the only storage step varied from 0.9 to 1.69 log10 CFU.mL -1 with the 70%O2 / 30% CO2 atmosphere and from 0.1 to 0.3 log10 CFU.mL -1 with the 50% N2 / 50% CO2 atmosphere.

For the bacterial samples undergoing all steps, the storage step with the 70%O2 / 30% CO2 atmosphere, had the main impact on C. jejuni inactivation, the bacterial population decrease varied from 1.0 to 3.9 log10 CFU.mL -1 (Fig. 2a). In addition, the highest inactivation during storage was reached after the application of the highest temperature during heat stress. Heat only produced an inactivation following application of 54°C with a mean inactivation between 0.8 and 0.9 log10 CFU.mL -1 , whereas the inactivation was negligible when 50° and 46°C were applied. In our conditions, cold temperature had also a negligible impact on the C. jejuni inactivation, indeed a maximum of 0.3 log10 CFU.mL -1 reduction was achieved. The highest cumulative inactivation was obtained for the C97anses640 strain. This strain appeared to be less resistant than C09MJLT205 and RM1221 strains, especially during storage. For instance, the inactivation of strain C97anses640 at 50°C / -4°C was 3.3 log10 CFU.mL -1 (mean value) compared to 1.2 log10 CFU.mL -1 for both C09MJLT205 and RM1221 strains (Fig. 2b).

Using the 50% N2 / 50% CO2 atmosphere, the cumulative inactivation of C. jejuni was mainly attributable to the storage step and varied from 0.1 to 0.9 log10 CFU.mL -1 (Fig. 2b).

However, especially at 54°C, the heat stress contributed equally with the cold storage to the cumulative inactivation, except for the strain RM1221. Indeed, for the latter, the inactivation in the hot water bath was higher than during the storage step, i.e. mean values of 1.5 and 0.9 log10 CFU.mL -1 , respectively.

3.2 Effect of heat/cold temperature, modified atmosphere, strains and interactions on subsequent inactivation during storage affected by cell history (Δlogstorage)

When subjected to mild stress, bacteria may become more resistant to another stress applied subsequently. In this study, we tested if the application of successive heat and cold temperature enhanced the resistance of C. jejuni during storage. In other words, we studied the effect of cell adaptive story on the subsequent behavior of C. jejuni.

The influence of the different factors and their interaction on inactivation of C. jejuni during cold storage was analysed by ANOVA. The ANOVA highlighted that the temperature of the cold bath did not influence significantly the inactivation during storage. Therefore, a new ANOVA was performed without considering cold stress. The model was significant (p < 0.0001) as indicated in Table 1.The ANOVA showed that there were significant effects of strain, temperature applied during heat stress, and packaging atmosphere. During the cold storage, strains C09MJLT205 and RM1221 exhibited similar inactivation around 1.1 log10 CFU.mL -1 . In contrast, the strain C97anses640 appeared to be less resistant to storage than the two other strains with a mean inactivation of 1.8 log10 CFU.mL -1 (Fig. 3a).

Regarding heat stress, the highest temperature of 54°C induced the highest inactivation associated with storage. Indeed, the mortality reached a mean of 1.9 log10 CFU.mL -1 compared to that obtained at 46° and 50°C (1 log10 CFU.mL -1 ) (Fig. 3b).

Inactivation of C. jejuni during storage was more than five times higher under the modified atmosphere 70% O2 / 30% CO2 than under the atmosphere 50% N2 / 50% CO2 (p < 0.0001), with a mean inactivation of 2.2 log10 CFU.mL -1 and 0.4 log10 CFU.mL -1 , respectively (Fig. 3c).

The ANOVA showed significant interactions between strains and atmosphere, temperature applied during heat stress and atmosphere.

While the mean inactivation resulting from storage under the atmosphere 50% N2 / 50% CO2 was low and similar no matter the strain used, (between 0.3 and 0.5 log10 CFU.mL -1 ), the inactivation was largely higher under 70% O2 / 30% CO2 and different between strains (p< 0.0001). Indeed, strain C97anses640 appeared to be less resistant than the strains C09MJLT205 and RM1221. The mean inactivation of strain C97anses640 was 3.2 log10 CFU.mL -1 compared to that of C09MJLT205 and RM1221 strains, i.e. 1.9 and 1.7 log10 CFU.mL -1 , respectively (Fig. 3d). Besides, this study pointed out that similar inactivation during heat treatment does not necessarily lead to similar inactivation during storage. Indeed, under 70% O2 / 30% CO2 atmosphere, the inactivation during heat stress was similar for each strain but higher for the strain C97anses640 during the storage. Thus, this result highlights the importance of the cell history, and the difficulty to predict future behavior while considering only a previous phenotypic response to a specific stress.

A significant interaction between atmosphere and heat stress was characterized by a similar mean inactivation at 46° and 50°C under the atmosphere 70% O2 / 30% CO2, (1.7 and 2.1 log10 CFU.mL -1 , respectively), compared to that obtained at 54°C, i.e. 3.0 log10 CFU.mL -1 (Fig. 3e). Under the atmosphere 50% N2 / 50% CO2, the inactivation following application of 54°C was also the highest and was 0.8 log10 CFU.mL -1 . In contrast, the inactivation resulting from application of 46°C was slightly higher than that obtained following application of 50°C, i.e. 0.4 and 0.1 log10 CFU.mL -1 respectively.

Discussion

The objective of this study was to assess the effect of the cell history on the subsequent survival of three C. jejuni strains. Stressful steps from slaughterhouses were identified and then reproduced in laboratory.

The first step was to immerse the bacteria in hot water bath at temperature ranging from 46° to 54°C. The inactivation during this step varied between 0 and 1.5 log10CFU.mL -1 according to strain and temperature. From heat inactivation, it was possible to calculate the decimal reduction time or D-value. These D-values corresponds to the time necessary to kill 90% of an initial population or decrease the initial population by one log. The D-value is a common metric and enables to compare more easily results from different studies. In our experimentations, heat stress was applied during 4 min. We measured that one minute was the time necessary to reach the temperature desired. Thus, once reached, the set temperature was maintained during 3 min. For a selected temperature, D-values were calculated by dividing the 3-min constant temperature application by Δlog lost following application of the heat stress.

All collected D-values from different studies in the literature were gathered in Table 2. We can see an important variability between results of decimal reduction times for C. jejuni, mainly at 50°C. It appears that variability was higher at 50°C than at 55°C, which was also mentioned by [START_REF] Mccarthy | pH dependent C. jejuni thermal inactivation models and application to poultry scalding[END_REF]. In our study, C. jejuni was submitted to heat stress in suspension rather than on chicken muscle like in the real scalding step. However, the high variability of collected D-values could not enable to visualize a difference of heat resistance between meat matrices and laboratory media, as it could have been expected. Indeed, it has been shown that the heat resistance was higher for Salmonella when attached to muscle as opposed to free cells [START_REF] Humphrey | Campylobacters as zoonotic pathogens: A food production perspective[END_REF]. C. jejuni cells could also be better recovered from 5-min boiling if they were previously attached to chicken muscle as opposed to carrots [START_REF] De Jong | Extreme Heat Resistance of Food Borne Pathogens Campylobacter jejuni, Escherichia coli, and Salmonella typhimurium on Chicken Breast Fillet during Cooking[END_REF]. D50°C-values varied from 3.5 to 39 min which corresponds to an inactivation from 0.08 to 0.9 log10 in 3 min. D55°C-values varied between 1.5 and 2.8 min and were found in the range of what was observed in previous studies (mean D55°C of 1.5 ± 1.3 min). In light of the variation illustrated in Table 2, heat strain sensitivity, experimental method, and variables such as medium may greatly influence the inactivation of C. jejuni. In our study, for 46°C and 50°C, no inactivation has been observed. Indeed, we considered that inactivation was significant if Δlog was greater than 0.5 log10CFU.mL -1 i.e. the experimental error due to microbial enumeration [START_REF] Pujol | Establishing equivalence for microbial-growthinhibitory effects ("iso-hurdle rules") by analyzing disparate Listeria monocytogenes data with a gamma-type predictive model[END_REF]). These results are in accordance with the very high D-values found by [START_REF] Nguyen | Heat resistance and mechanism of heat inactivation in thermophilic Campylobacters[END_REF].

To our knowledge the effect of cold temperature applied for a short period (i.e. two or several hours) on C. jejuni has not yet been reported. In this study, application of a cold temperature during two hours had no significant effect on the survival of C. jejuni. However, these results have to be interpreted with caution, especially regarding possible extrapolation with the chilling step of the slaughter process. Unlike our experimental conditions, the cold temperature applied during the chilling step is generally associated with desiccation due to ventilation. In laboratory, such conditions could not be mimicked. Nevertheless, the potential influence of desiccation might induce microbial inactivation. For instance, [START_REF] Zoz | Control of Relative Air Humidity as a Potential Means to Improve Hygiene on Surfaces: A Preliminary Approach with Listeria monocytogenes[END_REF] have highlighted the impact of relative humidity on survival of Listeria monocytogenes.

Likewise, a study led by [START_REF] Rivoal | Optimisation des conditions de refroidissement des volailles à l'abattoir pour contrôler la contamination en Campylobacter[END_REF], investigated the relation between temperature, chilling time and air velocity on the inactivation of eight strains of C. jejuni by the use of a miniaturized chilling room prototype. This work highlighted the significant effect of the duration of the chilling step on the inactivation of C. jejuni. A significant interaction between the temperature and the air flow during the process was also pointed out by the authors. However, it is important to reinforce here that carcasses contaminated with more than 3 log CFU.g -1 of Campylobacter could not be significantly decontaminated during the chilling step in slaughtering house.

After the application of hot and cold temperature at the slaughterhouse, C. jejuni was submitted to storage for several days under the same conditions as those found at retailers' and then at consumers' home. During storage, C. jejuni undergoes a combination of cold temperature and modified atmosphere. It was found in the current study that the inactivation varied from -1.0 to -3.9 log10 CFU.mL -1 in the atmosphere 70% O2 / 30% CO2 and from 0 to -0.9 log10 CFU.mL -1 in the atmosphere 50% N2 / 50% CO2. The inactivation of C. jejuni was significantly influenced by the atmosphere, with a higher effect of the atmosphere 70% O2 / 30% CO2. Indeed, Campylobacter are microaerophilic organisms. The presence of O2 in high concentrations is toxic for the pathogen. For many years, studies have investigated the effect of storage during several days under aerobiosis condition or modified atmosphere. On various meat matrices, the reduction of Campylobacter count in aerobiosis at 4°C varied from broth, similar results have also been found [START_REF] Chaisowwong | Physiological Characterization of Campylobacter jejuni under Cold Stresses Conditions: Its Potential for Public Threat[END_REF][START_REF] Chan | Survival of Clinical and Poultry-Derived Isolates of Campylobacter jejuni at a Low Temperature (4 C)[END_REF][START_REF] Garenaux | Survival of Campylobacter jejuni strains from different origins under oxidative stress conditions: effect of temperature[END_REF]. However, the inactivation of C. jejuni was higher in our work compared to what is commonly observed in the literature. Indeed, before the storage step, bacteria were subjected to heat and cold stress which may induce a hurdle effect. Besides, one of the atmospheres used during our experiments had a concentration in O2 much higher than in air. In other studies, it has been found that the inactivation increased with atmospheres enriched in O2 compared to atmospheres containing only CO2 or N2. [START_REF] Boysen | Survival of Campylobacter jejuni in different gas mixtures[END_REF] showed that in broth, the inactivation under atmosphere 70% O2 / 30% CO2 was higher than in atmosphere 70% N2 / 30% CO2 and 100% N2, with a reduction of 0.5 to 2 log10 CFU.mL -1 and 0.3 to 0.8 log10 CFU.mL -1 respectively. Such results have also been found when C. jejuni was submitted to paraquat, a chemical oxidizing agent [START_REF] Garenaux | Survival of Campylobacter jejuni strains from different origins under oxidative stress conditions: effect of temperature[END_REF]). After 7 days of exposition, the population was reduced by 3 log10 CFU.mL -1 .

In this work, thermal steps were applied successively in order to assess the impact of cell history on the subsequent inactivation of C. jejuni during storage. Some studies have already investigated the potential effect of cell history or adaptation of one stress to another stress. Indeed, microorganisms enduring stressful environments can protect or crossprotect themselves to survive subsequent homogeneous or heterogeneous stresses, which is called adaptive tolerance response (ATR). It has been shown that C. jejuni can induce an ATR after aerobic or acid stress [START_REF] Gaynor | The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes[END_REF][START_REF] Jones | Campylobacter jejuni adapts to aerobic metabolism in the environment[END_REF][START_REF] Ma | Stress-induced adaptive tolerance response and virulence gene expression in Campylobacter jejuni[END_REF][START_REF] Martinez-Rodriguez | Physiological changes in Campylobacter jejuni on entry into stationary phase[END_REF], Murphy et al. 2003a, Murphy et al. 2003b, Reid et al. 2008a).

On the other hand, in our study it has been shown that after successive application of heat and cold temperature, if an ATR was induced, it was insufficient to produce an adaptation effect since the inactivation was higher for stressed bacteria compared to unstressed ones.

This study was conducted on three strains isolated from poultry and more particularly on product from retailed market. Tackling strain variability is not easy because the features of the strains which are associated with highest or lowest sensitivity to processing steps are not known. The choice was done in this study to select strains (i) representative of strains commonly found in poultry, and (ii) exhibiting a priori different behaviors regarding selected stresses (data not shown). Firstly, the representativeness of strains in poultry was sought by determining the strain genotype by MultiLocus Sequence Typing (MLST). The strain C97anses640 belonged to the Sequence Type-45 complex (ST-45), the strain C09MJLT205, to the ST-21 complex and the strain RM121, to the ST-354 complex. The ST-21 and ST-45 are the two main clonal complexes encountered in chicken (and poultry meat) worldwide.

Interestingly, they are also commonly found among human isolates [START_REF] Colles | Genetic Diversity of Campylobacter jejuni isolates from Farm Animals and the Farm Environment[END_REF][START_REF] De Haan | Decreasing trend of overlapping multilocus sequence types between human and chicken Campylobacter jejuni isolates over a decade in Finland[END_REF][START_REF] Dingle | Molecular Characterization of Campylobacter jejuni Clones A Basis for Epidemiologic Investigation[END_REF][START_REF] Guyard-Nicodeme | Prevalence and characterization of Campylobacter jejuni from chicken meat sold in French retail outlets[END_REF][START_REF] Habib | Clonal population structure and antimicrobial resistance of Campylobacter jejuni in chicken meat from Belgium[END_REF][START_REF] Levesque | Multilocus sequence typing of Campylobacter jejuni isolates from humans, chickens, raw milk, and environmental water in Quebec, Canada[END_REF][START_REF] Ragimbeau | Multilocus sequence typing, pulsed-field gel electrophoresis, and fla short variable region typing of clonal complexes of Campylobacter jejuni strains of human, bovine, and poultry origins in Luxembourg[END_REF][START_REF] Sheppard | Campylobacter genotypes from food animals, environmental sources and clinical disease in Scotland 2005/6[END_REF]. Secondly, the behavior variability between strains was determined regarding their inactivation during cold storage under modified atmosphere. High response variability between C. jejuni strains has also been reported in various studies [START_REF] Habib | Survival of poultry-derived Campylobacter jejuni of multilocus sequence type clonal complexes 21 and 45 under freeze, chill, oxidative, acid and heat stresses[END_REF][START_REF] Ligowska | Effect of chicken meat environment on gene expression of Campylobacter jejuni and its relevance to survival in food[END_REF][START_REF] Vashin | Influence of temperature on Campylobacter jejuni survival rate in pork meat[END_REF]. These findings highlighted the difficulty and the importance to take into account strain variability in microbial inactivation studies, and, more generally in exposure assessment.

The experimental laboratory conditions applied during the present study differ from what C. jejuni cells really experience during the slaughtering steps because they are not attached on muscle but free in liquid suspension, and because the chilling step does not induce desiccation. Here reside some limitations of laboratory experiments. They offer the possibility of replicating standardized procedures and measures, which is necessary to obtain the proof of concept. Passed this step, a validation step in laboratory on chicken muscle will be necessary and is currently being processed.

In conclusion, to better predict Campylobacter contamination in poultry processing, it is essential to assess its behavior after applying not only one stress but the whole sequence of stresses encountered during the process. Indeed, cell history plays an important role and may induce some physiological responses such as ATR which influences C. jejuni further behavior regarding subsequent stresses. In some cases, the bacteria is able to better resist to another stress whereas sometimes, the application of successive stresses induces a hurdle effect, and in contrast, enhances bacterial inactivation. Finally, this study pointed out that there is no clear correlation between the amount of log inactivation observed in preliminary processing steps and the amount of log inactivation occurring at the latest processing steps such as storage, rendering it very difficult to predict the microbial behavior during the storage at retail and consumer's house from phenotypic information resulting from application of previous slaughtering steps.

A new generation of predictive models, including cell history information, will be necessary to progress along this route, and omics methods might help. These new tools consider not only the bacterial behavioral response, but also the adaptation response depending on the bacterial gene expression and regulation. The investigation of predictive models including gene expression would enable to fine-tune existing exposure assessment models and consider strain-dependent physiological response. interaction between strains and atmosphere (d) and mean interaction between temperature of heat stress and atmosphere all strains combined (e).

Lab medium

A,B,C Different capital letters indicate significant difference according the Least Significant test (P < 0.05) in inactivation.

Figure 1 .

 1 Figures legends:Figure1. Global scheme of experimental procedure. The different factors are represented with their associated levels. After each step, the inactivation (viable bacterial population N lost) was calculated (Δlog).

Figure 2 .

 2 Figure 2. Contribution of factors (temperature applied during hot and cold bath, strain and gas composition of the atmosphere) on the inactivation of C. jejuni induced by each step of the experimental procedure (hot bath in white, cold bath in black, storage at 6°C in grey) under atmosphere 70% O2 / 30% CO2 (a) and atmosphere 50% N2 / 50% CO2 (b).

Figure 3 .

 3 Figure3. Effect of factors and interactions on inactivation during storage at 6°C under modified atmosphere: strain effect (a), mean effect of temperature applied during heat stress all strains combined (b), mean effect of the gas atmosphere composition all strains combined (c),

Figure 1 Figure 2 Figure 3

 123 Figure 1

Acknowledgements

We are grateful to the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) of Ploufragan who realized the MLST analysis.

We also especially thank the Regions Pays de La Loire and Bretagne through the "Pôle Agronomique Ouest" for their financial support in the framework of the Biomics project.


R Besnard

R Winkler

G Arrachart
email: guilhem.arrachart@cea.fr

J Cambedouzou

S Pellet-Rostaing
email: stephane.pellet-rostaing@cea.fr

Ion extraction applications of bilayer-structured hybrid silicas

The self-assembly behaviour of amino-undecyl-triethoxysilane, which can be considered as an amphiphilic organosilane, has been used for the preparation of nanostructured materials. Due to the possibilities offered by this approach, lamellar hybrid materials bearing amine groups can be achieved. In this study, the reactive amino groups at the surface of the hybrid silica were modified for introducing ligands. For this purpose, various glycolamic acid groups have been introduced at the interface between two bilayers on the hybrid material obtained after condensation of silylated amines. The availability of the amino groups and the efficiency of the chemical immobilization of amic acid ligands onto the materials were followed by FTIR, NMR and TGA analysis, and the structural modifications induced by grafting were evaluated by SAXS experiments. The resulting materials exhibit a large number of amic acid groups. The adsorption capacities for rare earth elements were also investigated.

Introduction

There is increasing interest in the development of new solid phase extraction materials based on organosilica. Silica-based organic-inorganic hybrids are attractive materials due to their versatility, which originates from the combination of their inorganic silica network properties and their organic constituents. 1 Organosilica and mainly mesoporous organosilica sorbents have been actively investigated with success for the removal of several inorganic species as heavy metal ions, 2 toxic oxyanions, 3 lanthanide/actinides, 4,5 radionuclides 6 or organic pollutants. 7,8 For these applications, there are three major strategies for the synthesis of organosilica sorbents. 9 The first strategy involves the chemical grafting of specific ligands on the surface ofmesoporous silica. An alternative synthesis procedure consists of a ''one-pot'' approach, in which themesoporous silica is formed and functionalized by the simultaneous co-condensation of tetraethoxysilane (TEOS) with an organosiloxane precursor in the presence of structure-directing agents. Higher organic loading can be achieved using bridging organic groups of the type (R'O)3Si-R-Si(OR')3 as single-source organosilica precursors through the synthesis of periodic mesoporous organosilicas (PMOs), in which the organic bridges are incorporated in the threedimensional network structure of the silica matrix. 10,11 Depending on the selected approaches, drawbacks 12,13 can be problematic for solid phase extraction applications as follows: low functionalization and inhomogeneous distribution of the organic group 12,14 lead to a significant part of functional groups being non-accessible, and the mesoscopic ordering formation can be disturbed. In this context, aiming to overcome these drawbacks and improve the amount of available functional groups, a different approach has been explored to generate organosilicas using organosilane surfactant precursors. [15][16][17] This ''all-in-one'' or ''selftemplating'' approach allows both dense functionalization and accessibility of the functionalities of the silica by taking advantage of the self-assembling and condensable properties of organosilane surfactant precursors. 17 Different structural types of supramolecular materials could be obtained depending on the specific arrangement of the organosilane and on the amount of silica involved during the synthesis without the addition of more external surfactant. 15 For example, various multi-layered organosilicate have been synthesized by a self-assembly process leading to hybrid materials with high adsorption capacity for transition metal cations. 18,19 Recently, we took advantage of this approach using a simple amine (amino-undecyl-triethoxysilane). Organicinorganic hybrid materials with high amine content or aminooxy groups were obtained either with a lamellar or hexagonal periodicity from the self-assembly properties of this precursor under specific conditions. [20][21][22][23][24][25] A primary amino group is one of the most attractive surface functionalities owing to its use as a ligand for various metal ions. 3,26 We demonstrated the accessibility of the functional amine groups in materials prepared from a ''all-in-one'' approach through the performance of the materials engaged in the solid phase extraction of platinum or palladium salts, which are able to form strong complex bonds with amino functions. 27 Solid phase extraction may require a high concentration and availability of functional groups. Taking into account such requirements, the preparation of silica materials bearing a high number of available amines represents a considerable challenge. Indeed, amino functions are useful for further post-synthesis functionalization, allowing various functionalities to be introduced by chemical modifications such as alkylation 3,28 or the formation of amides 5,29,[START_REF] Hermanson | Bioconjugate Techniques[END_REF] and imines. [START_REF] Hermanson | Bioconjugate Techniques[END_REF][START_REF] Utting | [END_REF] In this context, we first studied the availability of amines in amine-bearing silica lamellar materials and their postsynthesis functionalization for the introduction of selective ligands aimed at rare earth extraction. Inspired by the works of Ogata et al., 32,33 we focused on opening ring reactions of anhydride succinic (S), glutaric (G) and diglycolic (D) leading to their respective amic acids (see Fig. 1). The adsorption properties of amino functionalized materials was first compared to that of their analogous aminooxy functionalized materials. The discrepancies in extraction behaviours are discussed. The amine availability in regard to postfunctional groups is calculated from the grafting ratio obtained by FTIR, NMR and TGA analyses. The structural modifications induced by grafting were evaluated by SAXS experiments. Then, the ability of the materials to extract metal ions was also studied by comparing the extraction behaviour in nitric media between d group elements, silver and rare earths elements (REEs).

Results and discussion

After the sol-gel process, thanks to the self-assembly behaviour of amino-undecyl-triethoxysilane, which can be considered as an amphiphilic organosilane, the preparation of lamellar hybrid material bearing easily available amine groups can be achieved. CO2 can be used as assembly agent leading to the formation of material M1 as bis-silylated-alkylammonium carbamate dimers arranged in a lamellar structure. 23,27 The elimination of CO2 by heating allows obtaining materials with free amino groups in which the well-defined lamellar structure is maintained. The possibility of the post-functionalisation of the reactive amino groups at the surface of the hybrid silica was studied through opening ring reactions of anhydride succinic (S), glutaric (G) and diglycolic (D) resulting in the nanostructured M1-S, M1-G, and M1-D.

Post-grafting

FTIR and NMR were used to investigate the grafting to provide evidence of the opening ring on the amino functions. Additionally, TGA and an elemental analysis were used together to quantify the grafting. The FTIR spectra (Fig. 2) of the materials exhibited the characteristic bands at around 2920 and 2850 cm - 1 corresponding to the as(CH) and s(CH) modes, respectively, of the alkyl chains. The intensity of these bands, comparatively to those corresponding to the silica matrix with Si-O-Si framework vibration (as (Si-O)) between 985 and 1160 cm -1 , is representative of the high density of organic parts in the hybrid materials. Fig. 2 shows a specific region included between 1450 and 1850 cm -1 of the FTIR spectra of the materials M1, M1-S, M1-G, and M1-D. The M1 spectrum does not exhibit any intense band in the region included between 1450 and 1850 cm -1 , and grafted materials spectra present three broad bands located at 1550, 1630 and 1700 cm -1 . The bands at 1550 and 1630 cm -1 are usually attributed to the N-H bending mode and the C=O stretching mode of secondary amides, respectively. 34 The band at 1700 cm -1 is known to be associated with the stretching mode of C=O bound of a carboxylic acid. Notably, no band characterizing the carbonyl signal of the anhydride precursors was observed at 1800 cm -1 , indicating that no residual trapped precursors are present in the material.

The solid-state 13 C NMR leads to the same conclusion. Fig. 3 shows the 13 C NMR spectrum of the M1-S material signals corresponding to the carbons of the different carbonyl groups previously mentioned. The carbon of the carboxylic acid is observed at 174 ppm, and the carbon of the amide is observed at 169 ppm. The solid state NMR spectrum of M1 also presents a resonance signal located at 159 ppm, which is attributed to the carbamate carbonyl carbon, confirming the presence of ammonium carbamate salt as already observed in the literature. 21 Fig. 3 (a) 13 C and (b) 29 Si solid-state NMR spectra of the bulk silica hybrid material (M1, M1-D, M1-G, M1-S).

The solid-state 29 Si CPMAS NMR spectra exhibits T 2 (-55 ppm) and T 3 (-65 ppm) silicate centres with the condensation level estimated at around 85%, which indicates that approximately five out of six silanol units in the material are condensed (see Table S1, ESI). 35,36 The materials consist of highly condensed siloxane networks with organic fragments covalently bonded to the silica network. This suggestion was supported by the solidstate 13 C NMR spectra with the absence of signals attributable to residual ethoxy groups.

Spectroscopic measurements allow us to confirm the ring opening of the different precursors on the amine from the M1 material. Although information from these data seems to show that the grafting was complete, a quantitative analysis is required to provide solid evidence.

For this purpose, a thermogravimetric analysis was performed on materials from 30 °C to 1000 °C under air flow. The resulting weight losses for M1 and M1-S are shown in Fig. 4. The thermal profile of M1-G and M1-D are similar to that of M1-S (see Fig. S1,ESI). The results for the M1 material show a CO2 and water release of approximately 10.5 wt% between 70 °C and 120 °C. The CO2 is initially used as the assembly agent leading to the formation of material M1 as bis-silylatedalkylammonium carbamate dimers arranged in a lamellar structure. 23,27 This departure is no longer observed in the case of the M1-S material, reflecting the full substitution of the CO2 during the post-grafting step. This finding confirms that the amic acid groups have been introduced onto the M1 material. In addition, the decrease of the residual weight wf at the end of the analysis from 24.7 wt% to 19.2 wt%, respectively, for the M1 and M1-S materials involves a higher organic content into the initial material. The residual masses after calcination wf can be considered as that of pure silica. Completely condensed silica presents a molar mass of 60 g mol -1 . Considering that each silicon atom of the silica originates from condensed amino precursors or functionalized precursors of xOnSi(CH2)11NH2-yOnSi(CH2)11NH-R zH2O, (where n is the number of oxygen molecules bounded on silicon, and R is the grafted moieties), the average molar mass Mt of the precursor can be deduced with eqn (1):

Mt = M SiO2 𝑤 f (1) Additionally, Mt = xMM1 + yMM1-X+ zMH2O
(2) where MH2O is the molar mass of water, MM1 is the molar mass of M1 at 125 °C, and MM1-X of its grafted analogous M1-X (with X = S, G, D).

Assuming that at 125 °C the silica matrix is also completely condensed and dried, the fraction w1 corresponding to the fraction at 125 °C corresponds to the material of formula xO1.5Si(CH2)11NH2-yO5Si(CH2)11NH-R of average molar mass Mt*.

Mt* = 𝑤 1 M SiO2 𝑤 f = xMM1 + yMtmax* (3)
Additionally, Mt* = xMM1 + yMtmax* (4) where Mtmax* is the molar mass expected for completely grafted and dried materials. The conversion yield y is obtained by resolving eqn (5):

y = 𝑤 1 Mt * -M M1 Mt max * -M M1 (5) x + y = 1
Table 1 shows the picked and calculated data from the thermal analysis of the materials. Completely condensed silica is considered, and the calculated values and grafting yields are minimized. Therefore, as suggested by spectroscopic analysis, the thermal analysis demonstrates a quantitative grafting by ring opening reaction on the functionalized amino@silica. In addition, the elemental analysis on the materials leads to the same conclusion (see Table S2, ESI). Analyses allow for verification that a ring opening occurred in the quantitative yields. The introduction of such functional groups involves all the amine of the initial M1 material available from the reactional media. The availability of the amino groups located at the interfaces of bilayers in such material has been highlighted with the immobilization of platinoid salt complexes in the interlayer space. 27 The chemical modification of the amino groups is therefore expected to involve structural modifications of the resulting grafted materials.

Structural effects

The amino functions are located at the interfaces of the bilayers built during the self-assembly of the aminoundecyltriethoxysilane precursor. The stacking of these bilayers corresponds to a lamellar structure highlighted in the SAXS profile of the material by structure peaks, for which the qn/q1 ratio is an integer n. Fig. 5 shows the SAXS profile of M1 and M1-S, and that of M1-G and M1-D are available in Fig. S2 (see ESI). We have identified the Bragg peaks originating from the lamellar structure. For the M1-S sample, five Bragg peaks denoted as qnS are clearly observed at scattering vectors of 1.6, 3.3, 4.8, 6.4 and 8.1 nm -1 , corresponding to the five successive orders for a lamellar phase of parameter 3.9 nm. In the case of M1, only 3 peaks are resolved, corresponding to the first, second and fourth diffraction order of a lamellar phase of lattice parameter of 3.4 nm. To determine the consistency of the initial lamellar structure, SAXS measurements were performed on each material. All of the results reveal that the lamellar structure is preserved after grafting. However, the lattice parameter characterizing the distance between bilayers is larger than that of M1. The SAXS profile of M1-S is presented in Fig. 5 to illustrate the phenomenon; those of M1-G and M1-D are available in Fig. S2 (ESI). Indeed, all the peaks are shifted towards weaker angles. For instance, we note that q2 peak shifts from 3.7 nm -1 for M1 to 3.3 nm -1 .

Fig. 5 SAXS profiles of M1 and M1-S.

From the q2 peak position, we calculated the swelling of the structure due to the chemical grafting. The interlayer distance d calculated from the q2 peak position, and the expected lengths of the grafted functions Df are summarized in Table 2.

As previously demonstrated by thermal analysis, the CO2 constituting the carbamic acid of M1 has been eliminated during the grafting. Considering that the size of the carbamate groups is negligible and that they are all substituted by grafted functions, the expected swelling gap would be close to 2Df. However, the interlayer distances are close to Df for all of the grafted materials. These results suggest either a tilted lamellar structure or a bilayer structure where functional end chains are interpenetrated. The latest interpretation would likely be governed by hydrogen bounds between amides and acid groups. We showed that the amino functions located at the bilayer interfaces were available for chemical modification. In addition, the functionalization has been completely performed for all the ring precursors without drastically damaging the initial lamellar structure. The ring opening reaction of cyclic anhydrides is thus an efficient way to functionalize amines from hybrid-structured silica. This reaction leads to multifunctional chemical patterns tunable by the nature of the grafted groups. Thus, we obtained amides coupled with terminal carboxylic acids bridged by two or three methylenes for M1-S and M1-G materials and a dimethylether for M1-D.

Table 2 Interlayer distance d (nm) calculated from the q2 peak position and the length Df (nm) of grafted groups for materials M1-S, M1-G and M1-D.

Extraction tests

Extraction properties of M1 and M2 materials. Primary amino and oxyamino groups are one of the most attractive surface functionalities owing to their use as ligands for various metal ions. 3,24,27 In addition, the grafted ligands contain chemical moieties also known to have affinities with specific metal ions. Indeed, if carboxylic acids can complex a large range of metal ions, 4,37 amides 38,39 and amic acid 40,41 seem peculiarly relevant as far as selective rare earth ion extraction is concerned. Thus, we explore the extraction properties of materials in regard to the separation of Y 3+ , Nd 3+ , Eu 3+ and Dy 3+ representing the rare earth elements group. Usually, rare earth elements coming from ores of natural or urban origin are accompanied with iron and other d-block elements. Therefore, iron, copper and cobalt have been added to the simulated leach. Aiming at completing the study, silver has been considered as a monovalent cation.

The main aim of this study was to evaluate the affinities and the apparent selectivity of prepared materials in regard to these eight ions contained in different nitric acid solutions. The concentration in each ion was fixed at 0.1 mmol L -1 . The mass of adsorbent material was 20 mg for M1 and M2 and 25 mg for the grafted material in order to obtain a ratio between ligands (L) and metal ions (M) L/M close to 10. Note that from pH = 3, iron ions precipitate and are eliminated from the solution before ICP-OES analysis. Therefore, the results for iron are not available for solutions containing 0.0001 M of nitric acid. First, we evaluate the extraction capacity of M1 in different acidic condition. To determine the pKa effects on the extraction phenomena, the results are compared to those of M2, which is the analogous lamellarmaterial functionalized by an oxyamine. 23 Fig. 6a exhibits the extraction results of M1 for different nitric acid concentrations. We note that M1 extracts few elements in the two higher acidic concentration values.

Only silver is significantly extracted, reaching 7% from the initial amount of this element for nitric acid concentration of 0.01 M, while at lower acid concentration (pH = 4), copper and silver are quantitatively extracted (93% and 97%, respectively) and heavy rare earths, albeit in lower proportion. In comparison, in the higher acidic conditions, M2 appears to behave as M1 since no significant extraction was observed (Fig. 6a andb). However, while at 0.01 M HNO3, the extraction properties of M1 are low, those of M2 exhibit affinities specifically for both elements coming from block 11 of the periodic table, since only silver and copper are quantitatively extracted at 80% and 60%, respectively (Fig. 6b). For lower acid concentration (0.0001 M), the extraction of silver and copper was complete, but M2 also extracts 60% of cobalt ions and 50% of all of rare earth ions (Y 3+ , Nd 3+ , Eu 3+ and Dy 3+ ). The use of silica-supported aliphatic amines reported by Soliman 42 illustrated that the use of nitrogen donor atoms was an effective means of removing metals such as copper(II) ions.

The extraction properties of M1 and M2 seem to exhibit a similar behaviour to a certain extent, but the behaviours shifted with the pH of the aqueous media. The amino functions of M1 are initially under ammonium carbamate form, while oxyamino functions of M2 are supposed to be free. Moreover, the pKa of M1 amines is estimated to 10.6 against 5 for M2 oxyamines. Two hypotheses can be expressed to explain these behaviour discrepancies between M1 and M2. First, the ammonium carbamate, which hinders the bilayer interface constituting M1 material, is too stable in this thermal condition to allow extraction from solution. Second, in this acidic condition (pH = 2), amino and oxyamino functions are both mostly protonated under ammonium and oxyammonium form respectively for M1 and M2. However, the discrepancy between pKa values involves different equilibrium constants between the formation of ligand-metal species (non-protonated ligands) and non-chelating species (protonated ligands). Equilibrium promoting the formation of ligand-metal species would be observed for M2 at higher pH. Owing to the same shifted extraction behaviour, the second hypothesis is supported. Notably, at a pH value of 2, the calculated silver ion separation factor of M2 with respect to iron, and rare earth ions are included from 200 to 750, involving a higher selectivity.

We have previously demonstrated the accessibility of amine from carbamate interfaces between bilayers in regard to platinoid anionic salt 27 and the possibility of chemical modifications. As previously shown by Alauzun et al. 21 the availability of this function is also extended to cations in the ethanolic solution.

Here, we demonstrate that amino functions from carbamate interfaces are also accessible in nitric aqueous media in a selective way. Owing to the shifted similar extraction behaviour, this selectivity towards copper and silver ions seems to be governed by the pKa of terminal functions and thus by the pH of the solution. Extraction properties of grafted materials. The evaluation of the extraction properties of M1-S, M1-G and M1-D are compared in order to study the influence of the different extractant systems. The effects of bridge size between amides and terminal carboxylic acids will be illustrated by comparing M1-S and M1-G. The effects of bridge nature will be exhibited by comparingM1-G and M1-D. The extraction values for a nitric acid concentration of 0.01 M of these three materials are shown in Fig. 7. M1-S and M1-G have the same affinities in regard to metal ions. Indeed, both extracts have exclusively iron ions in the same proportion, namely, 75% of the initial quantity and, to a lesser extent, silver ions (6-8%). The increase by one carbon atom of the bridge between amide and carboxylic acid is not a dominant parameter to change the extraction properties. This result suggests that the extraction site of iron ion is not located between the abovementioned functions. While in M1-S and M1-G materials the bridges are composed of a methylene group, those of M1-D are ether groups. The M1-D material extracts iron ions in the same proportion, confirming the hypothesis that extraction sites of iron ions are not between the amide and carboxylic acid but rather on the carboxylic acid site, as suggested by the spectroscopic analysis of Ogata et al. 33 M1-D has also significant affinities in regard to all the REEs. Indeed, the extraction of yttrium ions reaches 72%. Among the heavy rare earths, the extraction yields increase with the atomic number (Fig. 7). This result has been already observed by Ogata et al. in hydrochloric acid aqueous media. 32,33 An interesting point in order to selectively extract the rare earth element is to first perform the extraction of the aqueous feed solution using M1-S or M1-G to remove the iron and then perform the extraction of the REEs thanks to the material M1-D. Extraction experiments with a nitric acid concentration of 1 M have been performed and reveal negligible extraction of all the elements in the leach, suggesting that quantitative backextraction is possible. Aiming at recovering the extracted rare earth ions from M1-D, we rinsed the material with a nitric acid aqueous solution at 3 M for 24 h at 25 °C. The reported results in Fig. 8 reveal recovery yields of the extracted elements greater than 86%, which could be considered quantitative. To determine the maximum adsorption capacity of M1-D material in regard to dysprosium ions, we performed an extraction test in nitric acid solution (0.1 mM) with an L/M ratio of 0.5. In light of a structural investigation of the grafted materials, the presumed interpenetrated bilayers structure could have limited the extraction or back-extraction. However, the maximum adsorption capacity of dysprosium ions for M1-D materials was measured at 0.4 mmol g -1 (65 mg g -1 ) involving an L/M ratio of 7.2. Such ratio involves complete availability and accessibility of organic functions to allow trapping ions. Few results involving amic acid loaded onto inorganic supports have been published in regards to adsorption of cations. The introduction of diglycol amic group on silica gel has been proposed by Ogata et al. with adsorption capacity Q E 20 mg g -1 for However, experiments have been realized at pH = 1 under hydrochloric acid. 32 Similarly, adsorption capacity of about 8 mg g -1 with respect to Lu from HNO3 at pH 4 has been reported but using mesoporous sorbents functionalized by phthaloyl diamide. 43 Zheng et al. reported a Q value between 50 and 80 mg g -1 with respect to Gd for an adsorbent based on modified mesoporous silica support with maleic anhydride under comparable conditions (HNO3 pH = 4). 44 Therefore, as compared to the literature, the material M1-D obtained competitive Q values while the material is not based on the commonly modification of mesoporous silica support with chelating ligand.

Experimental

Technical details NMR analyses were performed on a Bruker 400 ultrashield VS spectrometer. Displacements are reported in ppm using the solvent (CDCl3: 7.26 ppm for 1 H; 77.16 ppm for 13 C) as an internal reference. ESI-MS was performed on a Flexar SQ 300 MS instrument. TGA analyses were performed on a Setaram Setsys Evolution 18 instrument under airflow (20 mL min -1 ) with a heating rate of (5 °C min -1 ). The coupled mass spectrometer was a Hiden analytical QGA instrument. Solid-state FT-IR spectra were obtained using an ATR crystal on a Perkin-Elmer 100 spectrometer with a resolution of 4 cm -1 . Solid-state 13 C CP MAS NMR spectra were performed on a Bruker 400 ultrashield VS spectrometer with a 12 kHz MAS spinning rate using Bruker MAS probes (4 mm rotors). The typical contact time was 2-3 ms, and the typical recycle delay was 5-30 s. The degrees of condensation were estimated from the 29 Si CPMAS NMR spectra from the percentages of the different T x units, which were obtained by deconvolution of the spectra using the Dmfit program. 45 SAXS experiments were performed on a setup operating in transmission geometry. A Mo anode associated with a Fox2D multi-shell mirror (XENOCS) delivered a collimated beam of wavelength 0.71 Å. Two sets of scatterless slits46 delimitated the beam to a square section of side length of 0.8 mm. A MAR345 imaging plate detector allowed simultaneous recording of scattering vectors q ranging from 0.25 nm -1 to 25 nm -1 . Samples were placed in glass capillaries with a diameter of 2 mm. Absolute intensities were obtained by measuring a calibration sample of high-density polyethylene (Goodfellow), for which the absolute scattering was already determined. All SAXS profiles are plotted in log-log scale to highlight the dependency of the intensity versus a power law of the scattering vector q. Metal concentrations were measured by inductively coupled plasma/atomic emission spectroscopy (ICP/AES) using a SPECTRO ARCOS spectrometer.

Precursor synthesis

All intermediate reactants and final products P1 and P2 were purified by distillation under a vacuum. 1 H, 13 C NMR, and mass spectrometry were used at each step to verify the structure and purity of the different products; all of the chemical characterizations were in agreement with the literature. [47][48][49] Synthesis of P1. The organic precursor P1 was synthesized starting from 10-undecen-1-ol in four steps (Scheme S1, ESI) in anhydrous conditions through the mesylation of the alcohol, which allow for higher yields than the bromination described in the literature: 47,48 As shown in Scheme S1 (ESI), 1 (34.058 g, 0.2 mol, 1 eq.) and NEt3 (25.5 g, 0.252 mol, 1.26 eq.) were added to 700 Ml CHCl3. Over the course of 90 min, MsCl (27.494 g, 0.24 mol, 1.2 eq.) dissolved in 100 mL of CHCl3 was added dropwise into the stirred solution at room temperature. The temperature was controlled by a water bath. Addition of MsCl produced HCl, which was removed by frequent venting. After 20 h, the solution turned slightly yellow. Half of the reaction mixture was washed with 450 mL water. The aqueous phase was extracted with CHCl3 (2 x 100 mL). The same process was repeated for the second half. All the united aqueous phases were again extracted with CHCl3 (100 mL). The organic phases were reduced to 200 mL and washed with brine (100 mL). CHCl3 was evaporated, and the residue was distilled at 80 °C and 0.5 mbar to remove volatile impurities. The resulting dark oil was again distilled at 160 °C and 0.3 mbar to afford product 2 as translucent brown oil (45.12 g, 0.18 mol, 91%) in the distillate. Under N2-atmosphere, Karstedt catalyst (2.1-2.4 wt% Pt, 0.462 g, 0.02 mmol, 3 x 10 -4 eq.) was added to a stirred mixture of 2 (15 g, 60.05 mmol, 1 eq.) and triethoxysilane (22.99 g, 139.94 mmol, 2.33 eq.) at room temperature. The temperature was kept constant using a water bath. After 20 h, activated charcoal (2 g) was added, and the mixture was stirred at 40 °C for 1 h. The mixture was filtered, and the filtrate was distilled at 170 °C (0.3 mbar) to remove more volatile impurities. Product 3 was obtained as brown oil (21.87 g, 53.06 mmol, 88%). Under N2-atmosphere, NaN3 (7.09, 109.14 mmol, 3 eq.) suspended in CH3CN (100 mL) was heated to 85 °C. 3 (15 g, 36.4 mmol, 1 eq.) in CH3CN (50 mL) was added dropwise to the heated mixture. After 48 h the mixture was cooled to 0 °C filtered, washing the residue with cold CH3CN (2 x 20 mL). After evaporation of CH3CN more volatile compounds were removed by distillation at 110 °C (0.6 mbar) for 2 h. The residue was distilled 200 °C (0.6 mbar) to afford the product 4 as clear, colourless oil (11.94 g, 31.75 mmol, 87%) in the distillate. Reduction of the azido group with palladium (10% Pd/C, Sigma-Aldrich) in anhydrous ethanol under hydrogen atmosphere led to amino-undecyl-triethoxysilane P1. Under an Ar-atmosphere, anhydrous ethanol (500 mL) was added to Palladium supported on carbon (Pd/C, 10 w%) (0.2 g, 0.19 mmol, 0.01 eq.), and 4 balloons filled with H2 were bubbled through. A solution of 4 (7.9 g, 21 mmol, 1 eq.) dissolved in anhydrous ethanol (250 mL) was infused by bubbling through one balloon of H2. The solution containing 4 was then added dropwise under an H2 atmosphere over the course of 1 h. After 2 h of stirring at room temperature, the solution was filtered over Celite, and the solvent of the filtrate was evaporated. The residue was dissolved in pentane, and the resulting solution containing a white precipitate was again filtered over Celite. The pentane was evaporated, and the product P1 was obtained as a slightly yellow liquid (5.5 g, 16.4 mmol, 78%) after fractionated distillation at 200 °C (0.7 mbar). Synthesis of P2. The organic precursor P2 was synthesized in the three steps described in the literature. 49 Bromoundecene (Alpha Aesar) was condensed with N-hydroxy-phthalimide in a Williamson's type reaction. Then, a Gabriel's type reaction using hydrazine led to amino-oxy-undecene. After purification, the latter molecule was reacted with triethoxysilane using a Karstedt catalyst purchased from ABCR to obtain amino-oxyundecyltriethoxysilane.

Materials synthesis Synthesis of M1.

The procedure for preparing the initial material starting from amino-undecyl-triethoxysilane (P1) and containing a carbamate ammonium salt was adapted from the procedure described previously. 27 Amino-undecyl-triethoxysilane (3 mmol) was introduced in a 50 mL flask under pure CO2 gas, resulting in a gel. After 30 min, 20 mL of pentane were poured in the flask and then hydrochloric acid solution was added (81 mL at pH 1.5). The material was aged for 3 days at 30 °C. The resulting material M1 was obtained after filtration followed by a washing step with ethanol and ether before drying under vacuum.

Synthesis of M2.

Samples were prepared as follows: 3 mmol of oxy-undecyl-triethoxysilane (P2) were introduced into 20 Ml of pentane, then 80 mL of hydrochloric acid solution at pH = 1.5 was added. The solution was stirred for ageing for three days before neutralized at pH = 7 with ammonia. The resulting material was washed with ethanol and ether. Then, the powders were dried under a vacuum for 1 hour.

Post-grafting

The opening ring of the different cyclic anhydrides by the amino functionalities of M1 was performed as follows: the M1 material was added to a solution of THF containing 1.1 equivalent of cyclic anhydride (succinic, glutaric or diglycolic) in regard to the amino function contained in M1. The resulting materials are respectively denoted as M1-S, M1-G, and M1-D. The mixture was stirred for 12 hours at 70 °C. After stirring for 12 hours at 70 °C, the mixture was centrifuged at 4500 rpm for 15 min. Then, the resulting materials were rinsed with ethanol and ether. Finally, the powders were dried under a vacuum for 1 hour.

Extraction test

All adsorption experiments were conducted using batch contact methodology in which the powdered adsorbent was contacted with solutions containing the target species in a volume/mass ratio of approximately 400-500 mL g -1 . The adsorbent material (20-25 mg) was added to 10 mL of a solution containing metal ions, and the mixture was stirred at 298 K for 24 h. After centrifugation at 4500 rpm for 15 min, the concentration of metal ions in the filtrate was measured by inductively coupled plasma optical emission spectroscopy. The uptake of metal ions efficiency E% was calculated from the following equation, in which Ci and Cf are the initial and the final concentration of metal ions, respectively:

E% = C i -C f C i × 100 (6)
The cation uptake capacity Q of the samples was calculated using eqn (7), in which Ci is the initial concentration of the metal ion in solution, Cf is the residual metal ion concentration, and V/m is the solution volume to solidmass ratio (V = volume of the treated solution and m = mass of the nanocomposite used):

Q = (C i -C f ). 𝑉 m (7)
The separation factor (SFMa/Mb) or selectivity is given by the following:

SF = Q Ma Q Mb (8)
QMa and QMb are the cation uptake capacity of the competitive ions Ma and Mb, respectively.

Conclusions

We demonstrated the complete accessibility and availability of amines from carbamate interfaces between bilayers in regard to metal ions and the ring opening reaction of anhydrides.

Similar but shifted extraction behaviours of amino and oxyamino@silica materials suggest that their extraction properties are governed by the pKa of the amine moiety.

The ring opening reaction of anhydrides in the M1 material has been quantitatively performed, as indicated by complementary analyses such as TGA and elemental analyses, with three different anhydrides leading to succinamic, glutaramic, and diglycolamic acids. The structure of the resulting materials is swelled by the grafted molecule hindrance but preserved. As for the initial amino@silica material, the interactions at the amic bilayer interfaces do not prevent their accessibility regarding metal ions, allowing quantitative extraction and desextraction in nitric media. Further work will be devoted to the selective extraction of REE with regards to Fe by first performing the extraction of the aqueous feed solution using M1-S or M1-G to remove the iron, and then performing the extraction of the REE thanks to the M1-D material. Indeed, the latter material, prepared by a simple and convenient approach, is a promising adsorbent in regards to its high extraction performance of REEs and their easy desorption.

Fig. 1

 1 Fig. 1 Schematic representation of opening ring reaction of anhydride by lamellar amino functionalized silica, leading to lamellar amic functionalized silica M1-S, M1-G and M1-D.

Fig. 2 FTIR

 2 Fig. 2 FTIR spectra of M1, M1-S, M1-G and M1-D.

Fig. 4

 4 Fig. 4 Thermogravimetric analysis of (a) M1 and (b) M1-S.

Fig. 6

 6 Fig. 6 Acidity dependence of metal ion percent adsorption for adsorbents (a) M1 and (b) M2 in nitric acid solution with a concentration in each ion fixed at 0.1 mM.

Fig. 7

 7 Fig. 7 Metal ion percent adsorption for M1-S, M1-G and M1-D from a 0.01 M nitric acid solution with a concentration in each ion fixed at 0.1 mM.

Fig. 8

 8 Fig. 8 Metal ion percent recovery after rinsing the adsorbent M1-D with nitric acid aqueous solution at 3 M for 24 h at 25 °C.

Table 1

 1 Weight fractions (wf, w1), average dry (Mt*), maximum dehydrated (Mtmax*) molecular weight and conversion rates (y) for M1, M1-S, M1-G and M1-D.

		wf (%)	w1 (%)	Mt* (g mol -1 )	Mtmax* (g mol -1 )	y (%)
	M1	24.7	89.7	218	222	-
	M1-S	19.2	99.8	312	322	90
	M1-G	18.5	99.3	322	336	88
	M1-D	17.6	98.3	335	338	98

Acknowledgements

The authors acknowledge C. Rey and V. Dubois for their technical support.

Conflicts of interest

There are no conflicts to declare.