Marina Al Daccache

Mohamed Koubaa
email: m.koubaa@escom.fr

Richard G Maroun

Dominique Salameh

Nicolas Louka

Eugène Vorobiev

Marina Al Daccache

Pulsed electric field-assisted fermentation of Hanseniaspora sp. yeast isolated from Lebanese apples

Keywords: Hanseniaspora sp., fermentation, stimulation, pulsed electric field

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

Introduction

The responses of living organisms under electric field have been attracting great attention for several decades [START_REF] Berg | Possibilities and problems of low frequency weak electromagnetic fields in cell biology[END_REF][START_REF] Hart | The application of electric fields in biology and medicine[END_REF]. These responses include changes in the synthesis of DNA and proteins, cell growth, electroporation of the cell membranes, biochemical reactions, electrofusion of cells, and the inactivation or stimulation of microorganisms [START_REF] Góral | Effect of pulsed electric fields (PEF) on accumulation of magnesium in Lactobacillus rhamnosus B 442 cells[END_REF][START_REF] Liu | Impact of electric pulse treatment on selective extraction of intracellular compounds from Saccharomyces cerevisiae yeasts[END_REF][START_REF] Martínez | Release of mannoproteins during Saccharomyces cerevisiae autolysis induced by pulsed electric field[END_REF][START_REF] Martínez | Pulsed electric fields accelerate release of mannoproteins from Saccharomyces cerevisiae during aging on the lees of Chardonnay wine[END_REF][START_REF] Teissié | Recent biotechnological developments of electropulsation. A prospective review[END_REF]. These effects depend on different factors, including the cell type and size, the application time, and the intensity of the electric field. However, the mechanisms behind these changes are complex and not yet fully understood. Nonetheless, multiple industrial applications have been developed specifically for the inactivation of microorganisms [START_REF] Barba | Mild processing applied to the inactivation of the main foodborne bacterial pathogens: A review[END_REF]El Zakhem, Lanoisellé, Lebovka, Nonus, & Vorobiev, 2006;[START_REF] González-Arenzana | Microbial inactivation and MLF performances of Tempranillo Rioja wines treated with PEF after alcoholic fermentation[END_REF][START_REF] Montanari | Heat-assisted pulsed electric field treatment for the inactivation of Saccharomyces cerevisiae: effects of the presence of citral[END_REF]. This involves the use of high electric field intensity, where the irreversible cell membrane electroporation has been mainly attributed to the inactivation mechanism.

When applying low electric field intensities, considered at sub-lethal levels, the microorganisms are stimulated rather than inactivated [START_REF] El Zakhem | Behavior of yeast cells in aqueous suspension affected by pulsed electric field[END_REF][START_REF] Mattar | Stimulation of Saccharomyces cerevisiae cultures by pulsed electric fields[END_REF][START_REF] Mattar | S. cerevisiae fermentation activity after moderate pulsed electric field pre-treatments[END_REF][START_REF] Mota | Fermentation at non-conventional conditions in food-and bio-sciences by the application of advanced processing technologies[END_REF][START_REF] Wang | A review of sublethal effects of pulsed electric field on cells in food processing[END_REF]. The first study describing the stimulation of microorganisms in presence of electric field was performed by Heinz Doevenspeck in 1962(cited by Sitzmann, (2016)), who reported that the cultures of Escherichia coli being treated with field strengths below 3 kV.cm -1 seemed to increase their growth rates. This impact of electrostimulation has been patented a few years later [START_REF] Doevenspeck | Process and apparatus for changing the charge of particles[END_REF]. Since that, several examples have been described in the literature concerning the stimulation of microorganisms by PEF [START_REF] Ohba | Moderate pulsed electric field treatment enhances exopolysaccharide production by Lactococcus lactis subspecies cremoris[END_REF][START_REF] Yeo | Effect of electroporation on viability and bioconversion of isoflavones in mannitol-soymilk fermented by lactobacilli and bifidobacteria[END_REF][START_REF] Yeo | Effect of electroporation on bioconversion of isoflavones and probiotic properties of parents and subsequent passages of Bifidobacterium longum[END_REF]. Through these works, several observations such as the acceleration of fermentation, better production of the metabolites of interest, and a larger number of cells have been reported compared to the non-stimulated controls. For instance, the stimulation of Saccharomyces cerevisiae yeast by PEF was successfully carried out in batch mode [START_REF] Mattar | Stimulation of Saccharomyces cerevisiae cultures by pulsed electric fields[END_REF][START_REF] Mattar | S. cerevisiae fermentation activity after moderate pulsed electric field pre-treatments[END_REF]. In these works, pulses of 20-2000 V.cm -1 for 10 -5 -1 s [START_REF] Mattar | Stimulation of Saccharomyces cerevisiae cultures by pulsed electric fields[END_REF], and 100 and 6000 V.cm -1 for 100 μs [START_REF] Mattar | S. cerevisiae fermentation activity after moderate pulsed electric field pre-treatments[END_REF] were applied. The stimulation increased in the fermentation rate, mainly in the initial fermentation phase (adaptation phase).

Therefore, the results indicate a great potential for the practical implementation of PEFassisted fermentation technology to improve yeast performances. However, the mechanisms remain yet unclear. There are two possible mechanisms for improved yeast performance by PEF that have been described in the literature, the first one being related to the expression of oxidative stress response genes during PEF treatment [START_REF] Tanino | Analysis of the stress response of yeast Saccharomyces cerevisiae toward pulsed electric field[END_REF], and the second is related to the changes in the activity of certain membrane proteins allowing the assimilation of a larger amount of substrates [START_REF] Vassu | Secondary effects of electroporation with bipolar electric pulses: electrostimulation[END_REF]).

Regarding the great potential behind implementing PEF technology in the fermentation processes, more investigations are required in this field to further study the impact of lowintensity PEF treatments on microorganisms during the fermentation process and not only prior to it. Most of the studies, above-mentioned, were focused on applying a PEF treatment shortly before the inoculation, and not during the fermentation process itself. This work aimed to evaluate the impact of PEF treatment during the fermentation process and by growth phase. Treating the culture medium in batch mode directly in the fermenter is associated with many issues including the increase of temperature inside the fermenter, the difficulty of agitation, the cell aggregation, as well as the scaling-up problems. Furthermore, studying the impact of PEF during the growth phases is crucial for a better understanding of the microbial stimulation mechanisms. In this line, this paper aimed to study the impact of the continuous PEF treatment on the growth of the non-Saccharomyces yeast cells Hanseniaspora sp. The growth and glucose consumption kinetics were recorded during the fermentation processes, depending on the electric field strengths and the growth phases. In fact, the genus Hanseniaspora was identified during spontaneous fermentation of apple juice [START_REF] Pando Bedriñana | Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias[END_REF]Valles, Bedriñana, Tascón, Simón, & Madrera, 2007), and its positive effects in alcoholic fermentation and cider production has been exposed [START_REF] Capozzi | Impact of co-inoculation of Saccharomyces cerevisiae, Hanseniaspora uvarum and Oenococcus oeni autochthonous strains in controlled multi starter grape must fermentations[END_REF]. It has been reported that the use of this strain may contribute to a better aroma profile of the fermented apple juice (de Arruda Moura Pietrowski, dos Santos, Sauer, Wosiacki, & Nogueira, 2012).

Materials and methods

Yeast growth

Hanseniaspora sp. yeast strain used in this work is belonging to the authors' laboratory strains collection [START_REF] Al Daccache | New indigenous yeast strains "Hanseniaspora Meyeri-Libani" for the elaboration of cider[END_REF]. It was previously isolated and identified from spontaneous fermentation of a Lebanese apple juice obtained from the variety "Ace Spur". A Yeast-Extract-Peptone-Dextrose (YPD) medium (1% yeast extract, 2% peptone, 2% dextrose) was used for the culture of Hanseniaspora sp. yeast cells.

Fermentation processes

Hanseniaspora sp. pre-culture

The pre-cultures for the different fermentations (i.e. control and PEF-assisted) were performed by taking one colony of the yeast strain from a YPD agar plate (1 % yeast extract, 2 % peptone, 2 % dextrose, and 2 % agar) into 250 mL YPD liquid medium (1 % yeast extract, 2 % peptone, 2 % dextrose), under sterile conditions (20 min at 121 °C in an autoclave HMC HV-110L (HMC Europe GmbH, Germany)). The incubation was performed during 24 h, at 30 °C temperature, and 250 rpm agitation speed in a shaker (Thermo Scientific, France), which allows the yeast to reach the late exponential phase.

Control fermentations

A volume of 1.5 L of YPD medium (1 % yeast extract, 2 % peptone, 2 % dextrose) was introduced into a 2-L fermenter (LSL Biolafitte S.A.). After sterilization as above-mentioned and cooling to 30 °C, the medium was inoculated by the pre-culture to get an initial concentration of 4.5*10 6 cells.mL -1 , corresponding to an initial OD of 0.1. The control fermentation in batch mode without medium circulation (Figure 1.A) was performed at 250 rpm agitation speed, and temperature maintained at 30 °C. After recording the kinetics of growth and glucose consumption in batch mode without medium circulation, a treatment chamber was designed to allow the circulation of the medium and the yeast treatment by PEF during the fermentation (Figure 1.B). The medium circulation is required since the treatment for a long time during the fermentation may induce the cell aggregation [START_REF] El Zakhem | Behavior of yeast cells in aqueous suspension affected by pulsed electric field[END_REF], and an undesirable increase in the medium temperature (Morales-de la Peña, Elez- [START_REF] Morales-De La Peña | Food preservation by pulsed electric fields: An engineering perspective[END_REF]. For this purpose, two parallel stainless steel electrodes (3 mm width, 6 cm diameter) were used and fixed between two insulators (Figure 1.C and 1.D).

A disk-shaped Teflon ring was placed between the electrodes, forming a channel for the streaming liquid and providing a distance between the electrodes of 1.4 cm. The volume of the treatment chamber was thereby around 40 mL (a ratio of 1:50 between the volume of the fermenter and the volume of the chamber). Two pipe fitting connectors were added to make it operate as a continuous chamber. A peristaltic pump allowed the medium circulation from the reactor to the treatment chamber and back into the reactor. Different circulation flow rates were tested (25, 50, and 75 mL.min -1 ) without PEF application, to get the same growth and glucose consumption kinetics obtained in batch mode without circulation (Figure 1.A). The total length of the tubes used was equal to 90 cm. the treatment chamber and the tubes were sterilized as above-mentioned.

PEF-assisted fermentations

The designed PEF-treatment chamber was used for the electrostimulation of Hanseniaspora sp. yeast. For this purpose, the two electrodes were connected to a PEF generator made by the "Service électronique" of the University of Technology of Compiègne (France) (Figure 2.A). It provided bipolar pulses of nearly rectangular shape, with the respective maximal delivered voltage and current of 400 V and 40 A. Three PEF-assisted fermentations were investigated: 1) PEF-treatment for 6 h during the Lag and Log phases, 2) PEF-treatment for 3 h during the Lag (adaptation) and early exponential phase, and 3) PEFtreatment for 3 h during the Log (exponential) phase. The PEF treatments consisted of applying a succession of trains, each composed of n = 10 pulses during the Lag and Log fermentation phases. Each pulse duration was fixed to ti = 100 μs, with a pulse period time of Δt = 1 ms. The time between the trains was fixed to Δt = 1 s (Figure 2.B). When treating the culture medium during the Lag and Log phases corresponding both to 6 h, the number of trains N applied was 10 716, which corresponded to an effective electric treatment tPEF of 10.716 s (tPEF (s) = N . n . ti (s)). Different electric field strengths E were tested in the range of 72-285 V.cm -1 (Figure 2). For instance, when applying the maximal voltage of 400 V delivered by PEF equipment and taking into account the distance of 1.4 cm between the electrodes, this corresponded to applying 285 V.cm -1 field intensity. All the output data (e.g., current I (A) and voltage U (V)) were recorded using a data logger and software developed by the "Service électronique" of the University of Technology of Compiègne (France). The specific power consumption of PEF treatment W (J.mL -1 ) was calculated according to Equation (1):

𝑊 (J. mL -1 ) = U . I . 𝑡 𝑃𝐸𝐹 𝑉 Equation (1)
where U is the voltage (V), I is the current intensity (A), tPEF is the time of the PEF treatment, tPEF = N • n • ti, N is the number of trains, n is the number of pulses per train, and V is the volume of the culture medium in the bioreactor (mL).

Determination of growth and glucose consumption kinetics

The kinetics of growth and glucose consumption were recorded during the batch fermentation modes with and without culture medium circulation, and during the PEFassisted fermentations.

Determination of the growth kinetics

The growth kinetics were followed by both 1) measuring the optical density (OD) at 600 nm, and 2) determining the cell concentration (cells.mL -1 ). Samples were taken periodically during the fermentation processes, and their OD values were determined using a Thermo Spectronic BioMate 3 UV-visible spectrophotometer (Thermo Scientific, France). For cell counting, serial dilutions were made for each sample, and the cells were counted using a Thoma cell counting chamber (Preciss, France). To detect the viable cells, the samples were mixed volume to volume with a solution of 0.01 % methylene blue. The biomass concentration X (cells.mL -1 ) and biomass yield YX/S (gbiomass.g -1 substrate) were calculated respectively according to equations ( 2) and (3),

𝑋 (cells.mL -1 ) = 𝑎 . 250000 . 𝑑 𝑏 Equation (2)
where a is the number of cells counted, d is the dilution factor, and b is the number of squares counted in the chamber.

𝑌 𝑋/𝑆 (𝑔 𝑏𝑖𝑜𝑚𝑎𝑠𝑠 . 𝑔 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 -1 ) = 𝑋 𝑓 -𝑋 0 𝑆 0 -𝑆 𝑓 Equation (3)
where X0 and Xf are respectively the initial and the final biomass concentrations (g.L -1 ), and S0 and Sf represent respectively the initial and the final glucose concentrations (g.L -1 ).

The maximal specific growth rate µmax (h -1 ) was determined by plotting Ln X (cells.mL -1 ) versus time (h). µmax (h -1 ) values correspond to the slopes of the curves obtained during the exponential phases.

Determination of the glucose consumption kinetics

The content of soluble compounds was followed by measuring the °Brix using a digital refractometer (Anggur DR401, France). The concentrations of glucose were determined using high-performance liquid chromatography instrument LC-20AD (Shimadzu, Japan). The samples taken during the fermentation processes (used for the determination of the growth kinetics) were diluted 10 times in ultrapure water, and 1 µL was injected. The separation was performed in isocratic mode (a mixture of 1:4 (20:80, v:v) water/acetonitrile with a flow rate of 3 mL.min -1 ), and using a Luna 5 μm NH2 (250 x 4.6 mm) column. The separation temperature was set to 40 °C, and the molecules were detected using a low-temperature evaporative light scattering detector ELSD-LT II (Shimadzu, Japan). The compounds' assignation and quantification were performed using pre-established standard curves of glucose (5-30 g.L -1 ).

The glucose consumption rate was calculated according to equation (4).

𝑑𝑆 𝑑𝑡 (𝑔 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 . 𝐿 -1 . ) = 𝑆 0 -𝑆 𝑓 𝑡 𝑓 -𝑡 0 Equation (4)
where S0 and Sf are respectively the initial and the final substrate concentrations (g.L -1 ), whereas t0 and tf are respectively the initial and the final times.

Scanning electron microscopy

The impact of PEF on Hanseniaspora sp. yeasts was followed using an ESEM Quanta FEG 250 Scanning Electron Microscope (Philips, USA). The pictures were taken after 6 h fermentation, for the untreated and the PEF-treated culture at 285 V.cm -1 , respectively. The samples (1 mL) taken from the fermenter were immediately centrifuged at 13000 g for 2 min, and the yeast pellet was washed twice with 1 mL physiologic water and centrifuged under the same conditions to remove the medium traces. Each sample was then resuspended in physiologic water, and ≈20 µL were placed on the analysis plate of the microscope. A vacuum-drying step was then performed at 5 °C and 800 Pa (≈ 6 Torr), and the samples were analyzed by electron beam at 20 kV. All pictures were taken at the physicochemical analysis service of the University of Technology of Compiegne (France).

Statistical analyses

Each fermentation experiment was repeated three times. One-way analysis of variance (ANOVA) was used for the statistical analysis of the data with the help of Statgraphics Plus software (version 5.1, Statpoint Technologies Inc., Warrenton, VA, USA). The significance level at 95% confidence was taken for each analysis. The values presented in the figures correspond to the mean value of the repetition, and the error bars correspond to the standard deviations of each measurement.

Results and discussion

A medium circulation flow rate of 75 mL.min -1 allowed obtaining the same growth kinetic compared to the control fermentation without circulation. It was thereby used for PEF-assisted fermentations. First, PEF was applied between the electrodes of the treatment chamber during the Lag (adaptation) and the Log (exponential) phases, corresponding together to 6 h treatment, and an effective PEF application time of 10.716 s. The results obtained and presented in figure 3 show an increase in the yeast concentrations in the fermenter, proportionally to the PEF intensity, compared to the untreated suspension.

Applying PEF intensities of 72, 143, and 215 V.cm -1 seems to induce a nearly similar stimulation effect on yeasts, with no significant differences between the three growth kinetics. The stimulation effect was more pronounced when an intensity of 285 V.cm -1 was applied (Figure 3.A). In fact, it has been previously reported that the electrostimulation is energy (J.mL -1 ) dependent, and it is not directly related to the intensity of the electric field E applied [START_REF] Mattar | Stimulation of Saccharomyces cerevisiae cultures by pulsed electric fields[END_REF]). Treating the cell suspension at 285 V.cm -1 leads to a significant increase in the final biomass concentration by 75 %, corresponding to an increase in the number of cells from 9.0*10 7 (for the control experiment) to 1.6*10 8 cells.mL -1 . These results

were supported by the measurements of the optical densities (OD values at 600 nm), which demonstrated a significant increase (from 2.43 for the control experiment to 3.23 for the PEFassisted fermentation) at 285 V.cm -1 , and after 8 h of fermentation (Figure 3.B). Besides, the highest specific growth rate µmax (0.47 h -1 ) was observed for the PEF-assisted fermentation at 285 V.cm -1 (Table 1), which indicates a faster division of cells, involving a faster cell growth.

Applying 285 V.cm -1 field intensity to the cell suspension allowed a significant shortening of the Lag phase by around 1 h, compared to the untreated suspension (Lag phase ≈1.5 h). This behavior was previously observed by [START_REF] Castro | The effect of the electric field on lag-phase, ethanol and β-galactosidase production of a recombinant S. cerevisiae growing on lactose[END_REF], who reported similar results when treating a recombinant S. cerevisiae yeast (NCYC869-A3/pVK1.1) at low electric field intensities. Their results showed a greater biomass concentration of almost 3 times compared to the control, and a shorter Lag phase for the treated suspensions. Similarly, S. cerevisiae yeasts were stimulated during the grape fermentation when treated with PEF,

showing an increase in biomass growth and sugar depletion [START_REF] Mattar | Stimulation of grape juice fermentation using moderate pulsed electric field treatment[END_REF]. In another study, the influence of static magnetic fields on S. cerevisiae yeast has been demonstrated,

showing that the rate of the biomass growth in the samples of the magnetized cultures was 3.7 times higher than that of the control cultures [START_REF] Muniz | Influence of static magnetic fields on S. cerevisae biomass growth[END_REF]. A 100% increase in S. cerevisiae growth was also shown under pulses of 0.85 kV.cm -1 [START_REF] Fologea | Increase of Saccharomyces cerevisiae plating efficiency after treatment with bipolar electric pulses[END_REF]. A possible explanation of these observations could be an increase in the yeast cells' division during the PEF treatment, which leads to a higher biomass concentration. In fact, it was reported that the use of PEF could induce some DNA damages, which affects the mitosis checkpoints and the cell cycle phase distribution, compared to the non-treated cells [START_REF] Stacey | Differential effects in cells exposed to ultra-short, high intensity electric fields: cell survival, DNA damage, and cell cycle analysis[END_REF]. [START_REF] Kerns | Electrostimulation of cellulase fermentation by pulsatile electromagnetically induced currents[END_REF] showed that the PEF-induced membrane permeability caused the growth stimulation of Trichoderma reesei at 1.5 mV.cm -1 pulses.

The results of the growth kinetics were supported by the quantification of glucose consumption during the fermentations. Figure 4 presents both the evolution of °Brix and the glucose concentrations (determined by HPLC) for the PEF-assisted fermentations, compared to the control. The results show that the °Brix values concur with that of the glucose concentrations. Both values decreased faster for the PEF-treated samples, compared to the control fermentation, with the fastest glucose depletion obtained for the PEF-treatment applied at 285 V.cm -1 . After 8 h of fermentation, the °Brix decreased from 5.9% (g.100 mL -1 ) for the control to 5.1% (g.100 mL -1 ) for the PEF-assisted fermentation. Thus, for the PEFtreated suspensions, the glucose consumption rates were significantly higher than those of the control fermentation (Table 1). This could be explained by the greater yeast concentration, which induces higher glucose consumption, especially during the Log phase where the cells are exponentially duplicating. The results concur with those of McCabe, [START_REF] Mccabe | Increased efficiency of substrate utilization by exposure of the thermotolerant yeast strain, Kluyveromyces marxianus IMB3 to electric-field stimulation[END_REF], who reported 40% increase in the cellobiose conversion for Kluveromyces marxianus cells under pulses of 0.25 kV for 10 ms. Also, the absorption of nutrients could be enhanced after PEF-treatment by the improvement of the transport through the cell membrane. This could be mainly attributed to the pore formation and/or the activation of membrane proteins [START_REF] Castro | The effect of the electric field on lag-phase, ethanol and β-galactosidase production of a recombinant S. cerevisiae growing on lactose[END_REF]. The biomass yield on glucose was significantly higher for the treated samples after 8 h of fermentation (Table 1), which confirms the abovementioned statements.

Aforementioned, the impact of PEF on the yeast size was studied. Figure 5 shows a significant (p<0.05) size increase of the cells after PEF treatment (5.6±0.4 µm), compared to the untreated cells (4.1±0.2 µm). This observation concurs with the significant increase in the biomass yield (Y (gbiomass.g -1 glucose)) where the mass of biomass per gram increases for one gram of glucose consumed (0.1 gbiomass.g -1 glucose) for the samples treated by PEF at 285 V.cm -1 compared to the untreated ones (0.075 gbiomass.g -1 glucose) (Table 1). The different calculations show an increase by 85% in the amount of biomass in gram compared to 75% increase in the number of cells per mL, which indicates a 10% increase in the cell volume after PEF treatment at 285 V.cm -1 . It could be also noted that the treatment did not induce irreversible deformations of the yeasts. The SEM photos show also that the cells kept their general shape after the treatment. The review of the literature shows that smaller cell size was observed for PEF-treated S. cerevisiae compared to the untreated ones (Araújo, Coelho, Margarit, Vazjunior, & Rocha-Leão, 2004). This observation was not observed for the strain Hanseniaspora sp., studied here.

An important factor influencing the microorganisms' stimulation by PEF is the growth phase. It has been previously demonstrated that PEF has different impacts on microorganisms, depending on the growth phase [START_REF] Mcdonald | Effects of pulsed electric fields on microorganisms in orange juice using electric fields strengths of 30 and 50 kV/cm[END_REF]. In this line, the impact of PEF (285 V.cm -1 ) on the growth and glucose consumption kinetics was studied during each Lag and Log phases of the growth of Hanseniaspora sp. yeast. When the suspensions were treated during the first 3 h (Lag and early exponential phase), an increase in the yeast biomass concentration was observed. In fact, after 8 h fermentation, the biomass concentration was increased by 60 % for the PEFtreated suspensions during this phase (Figure 6). Lower yeast stimulation impact (25%) was observed for the suspensions treated only during the Log phase. Besides, faster sugar depletion was observed during the Lag phase, compared to the Log phase (Figure 6), with a significant difference in the glucose consumption rates (Table 1). Even though, the sugar consumption rates were significantly higher than the control for both PEF-treated suspensions.

The results obtained show that Hanseniaspora sp. yeast cells were sensitive to the PEF treatment during both Lag and Log phases, with more sensitivity during the Lag and early exponential phase. These results concur with those reported by [START_REF] Mattar | Stimulation of grape juice fermentation using moderate pulsed electric field treatment[END_REF] who observed a noticeable enhancement of the fermentation kinetics when stimulating S.

cerevisiae cells by PEF, with a significant acceleration of the sugar consumption during the Lag phase. In addition, it was noted that the electroporation was more effective when the PEF was applied during the first hours of fermentation rather than that after 24 h. It seems that the cells became more resistant to electroporation for longer preliminary fermentation.

Regarding the energy consumption, it was obvious that the treatment at 285 V.cm -1 for 6 h consumed the highest energy, compared to the control and the other PEF-assisted fermentations (Table 1). However, it provided the fastest cell growth and glucose consumption kinetics. Overall, the results obtained in this work demonstrated the great benefit of stimulating Hanseniaspora sp. yeast by PEF as it involves reducing the fermentation time along with increasing the biomass concentration, and the low consumption of energy.

Conclusion

In this work, the effects of PEF electrostimulation on Hanseniaspora sp. yeast cells were studied. For an effective treatment time of 10.716 s, applied during both the Lag and Log phases (6 h), the yeast concentration and the biomass yield increased significantly. The depletion of nutrients was also significantly faster. The yeast cells were more sensitive to PEF treatment during the Lag and early exponential phase rather than during the Log phase.

The yeast concentration and the biomass yield increased significantly during the Lag and early exponential phase. However, the highest stimulation impact was observed using 285

V.cm -1 electric field intensity applied during both phases. Increased growth rates were

B.

Log Log Log

Time (h) Biomass concentration (cells.mL

  accompanied by a faster depletion of glucose. The results obtained in the current study indicate the great potential for the practical implementation of PEF-assisted fermentation technology to improve yeast performances. However, further investigations are required to understand the mechanisms behind microbial stimulation by PEF.

  Figure captions:

Figure 2 .

 2 Figure 2. A. A schematic representation of the PEF-assisted fermentation processes. B. PEF-

Figure 3 .

 3 Figure 3. A. Growth kinetics (viable cells) for the control and the PEF-assisted

Figure 4 .

 4 Figure 4. A. Glucose consumption kinetics for the control and the PEF-assisted

Figure 5 .

 5 Figure 5. Scanning electron microscopy pictures of Hanseniaspora sp. yeast after 6 h

Figure 6 .Figures

 6 Figure 6. A. Growth kinetics of the control and the PEF-assisted fermentations at 285 V.cm -1

  Figure 2.

  Figure 4

Acknowledgments

This work was supported by the Lebanese National Council for Scientific research (CNRS-FS129). The authors wish to thank the Research council of Saint-Joseph University of Beirut, Project FS103. All the authors thank Dr. Nicolas Thiebault as well for his help in performing the HPLC analyses at ESCOM-Chimie (Compiègne, France).

Tables

 Table 1.Energy consumptions, and fermentation process performances (Y (g biomass/ g glucose), dS/dt, µmax) during PEF treatments.

Lag and Log phases treatments

Lag and early exponential phase treated 285 V.cm -1

Log phase treated alone 285 V.cm -1 0 V.cm -1 72 V.cm -1 143 V.cm -1 215 V.cm -1 285 V.cm -1 W (J.mL


Nicolas Gauthier

Cecile Courreges
email: courrègescecile.courreges@univ-pau.fr

Julien Demeaux

Cécile Tessier

Hervé Martinez

Cécile Courrèges

Impact of the cycling temperature on electrode/electrolyte interfaces within Li4Ti5O12 vs LiMn2O4 cells

Keywords: Li-ion Batteries, SEI, LTO, LMO, Temperature, XPS, SAM, ToF-SIMS

INTRODUCTION

Commercial Li-ion batteries (LiBs) based on graphite and LiCoO2 (LCO) have reached their limits regarding cost and safety [START_REF] Etacheri | Challenges in the development of advanced Li-ion batteries: a review[END_REF] . Among the different cell chemistries which could overcome these challenges, the Li4Ti5O12/LiMn2O4 (LTO/LMO) system represents an attractive option especially for lithium-ion batteries used in Hybrid Electrical Vehicles (HEVs) [START_REF] Yi | Recent development and application of Li4Ti5O12 as anode material of lithium ion battery[END_REF] , due to its fast charge capabilities [START_REF] Takami | High-power and long-life lithium-ion batteries using lithium titanium oxide anode for automotive and stationary power applications[END_REF] . Indeed, the three dimensional spinel material LiMn2O4 (LMO) is a promising cobalt-free cathode material which fulfills the criteria of safety, high energy density (due to its high operating voltage at 4.2 V vs. Li/Li + ) and good thermal stability [START_REF] Thackeray | Structural considerations of layered and spinel lithiated oxides for lithium ion batteries[END_REF] . Temperature is one of the major factor on LIBs aging and a strict temperature control is needed to ensure the commercial development of LIBs in electrical vehicles [START_REF] Bandhauer | A critical review of thermal issues in lithium-ion batteries[END_REF] . Concerning lithium titanate, Li4Ti5O12, its capacity of 175mAh/g is lower than graphite (372mAh/g) but its stability in cycling as well as in calendar aging [START_REF] Takami | High-power and long-life lithium-ion batteries using lithium titanium oxide anode for automotive and stationary power applications[END_REF][START_REF] Yuan | Challenges of spinel Li4Ti5O12 for lithium-ion battery industrial applications[END_REF][START_REF] Majima | Development of long life lithium ion battery for power storage[END_REF] and its high potential (1.55 V vs. Li/Li + ) avoiding lithium plating and short-circuits, make it a good candidate, which is already used for electric vehicles [START_REF] Pistoia | Lithium-ion batteries: advances and applications[END_REF] . In addition, the high redox reversibility of LTO/LMO cells results in significantly lower expansions during charge and discharge compared to graphite-based cells. For instance, Zhan et al. [START_REF] Zhan | II) Deposition on Anodes and its Effects on Capacity Fade in Spinel Lithium Manganate-Carbon Systems[END_REF] have reported a better capacity retention for LMO/LTO cells compared to LMO/graphite ones. The damages on the active particles, induced by these volume changes during cycling, are therefore less important in LTO than in graphite [START_REF] Majima | Development of long life lithium ion battery for power storage[END_REF][START_REF] Ohzuku | Zero-strain insertion material of Li[Li1/3Ti5/3]O4 for rechargeable lithium cells[END_REF] . The main drawback of LTO/LMO cells which limits its implementation on the batteries market, is its low energy density. Nevertheless, Majima et al. [START_REF] Majima | Development of long life lithium ion battery for power storage[END_REF] pointed out that LTO based cells, thanks to its smaller expansion factor, would need less packaging than graphite ones. Consequently, if the encapsulation box is included, LMO/LTO cells would have an energy density of 85.5 Wh/kg compared to 94.8 Wh/kg for LMO/graphite ones, which represents only 11% of difference.

Other aging mechanisms could entail capacity fading depending on the intrinsic properties of active materials and their interactions during cycling. For LTO/LMO system, it was proved that manganese (coming from LMO) dissolution in the electrolyte and deposition at the LTO electrode surface, especially at high temperature (over 55°C), significantly affect electrochemical performances of cells [START_REF] Vetter | Ageing mechanisms in lithium-ion batteries[END_REF][START_REF] Wohlfahrt-Mehrens | Aging mechanisms of lithium cathode materials[END_REF][START_REF] Choi | Degradation of spinel lithium manganese oxides by low oxidation durability of LiPF6-based electrolyte at 60[END_REF] . In addition, different studies have reported the formation of a "Solid Electrolyte Interphase" (SEI) and an important gas release at the LTO electrode surface [START_REF] Song | Is Li4Ti5O12 a Solid-Electrolyte-Interphase-Free Electrode Material in Li-ion Batterie? Reactivity between the Li4Ti5O12 Electrode and Electrolyte[END_REF][START_REF] He | Gassing in Li4Ti5O12-based Batteries and its Remedy[END_REF][START_REF] Gieu | Influence of Vinylene Carbonate Additive on the Li4Ti5O12 Electrode/electrolyte Interface for Lithium-ion Batteries[END_REF] coming from the degradation of the electrolyte solvents and the lithium salt, which can lead to a rapid loss of capacity [START_REF] Wu | Influence of manganese ions dissolved from LiMn2O4 cathode on the degradation of Li4Ti5O12-based lithium-ion batteries[END_REF] . These surface layers (5-10 nm) play a key role in batteries efficiency as they have to ensure the passivation of the electrode surface by reducing parasitic reactions, while maintaining a good diffusion of lithium ions. In previous papers, our group revealed that Mn deposition at the LTO electrode surface induce a destabilization of the SEI which is a clear issue for LTO/LMO cells [START_REF] Gieu | New Insights in the Characterization of the Electrode/electrolyte interfaces within LiMn2O4/Li4Ti5O12 cells, by X-ray Photoelectron Spectroscopy, Scanning Auger Microscopy and Time-of-Flight Secondary Ions Mass Spectrometry[END_REF][START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . Although it is well known that the cycling temperature influence the electrochemical performances of batteries [START_REF] Wright | Review on high temperature secondary Li-ion batteries[END_REF][START_REF] Börner | Investigations on the C-Rate and Temperature Dependence of Manganese Dissolution/Deposition in LiMn2O4/Li4Ti5O12 Lithium Ion Batteries[END_REF] , the impact of high temperatures on the chemical composition of solid interfaces and the manganese dissolution in LTO/LMO cells after long cycling has to be highlighted. Gieu et al. have already analyzed LTO interfaces within half cells after the first cycle at high cycling temperatures [START_REF] Gieu | Temperature Effects on Li4Ti5O12 Electrode/electrolyte interfaces at the first cycle: A X-ray Photoelectron Spectroscopy and Scanning Auger Microscopy Study[END_REF] but the SEI characteristics of LTO electrodes in full cells and after long cycling remained unexplored. The objective of this new paper is to complete the work initiated in our laboratory, by characterizing the SEI properties as a function of the temperature in LTO/LMO full cells and after long cycling. To do so, electrochemical performances were correlated to surface analyses after 100 cycles at 25°C, 40°C and 60°C using three main techniques: X-ray Photoelectron Spectroscopy (XPS) which quantitatively determines the chemical environments in interfacial layers, Scanning Auger Microscopy (SAM) which allows the acquisition of highly resolved elemental chemical mappings at the electrode surfaces, and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) which characterizes the in-depth spatial distribution of organic and inorganic molecular species within the SEI of LTO electrodes by carrying out high resolution depth profiling experiments and 3D image reconstructions.

EXPERIMENTAL SECTION

Electrodes formulation. The first step is the preparation of inks which are coated on an aluminum current collector, calendered and then dried (100°C under vacuum for 12h). These inks are obtained by mixing with a T25 digital ULTRA-TURRAX homogenizer (i) 93 wt% of anhydrous active material (LTO or LMO) supplied by SAFT, (ii) 4 wt% of carbon black (CB) as a conductive additive with a specific surface of 65 m 2 .g -1 (99.8% purity, nanometric particles Super C65 TIMCAL from Alfa Aesar), and (iii) 3 wt% of poly(vinylidene fluoride) (PVdF) as a binder from Solvay (Solef 5130). In order to focus on LTO performances, the electrodes capacity is limited by the LTO negative electrode active mass. The loadings of LTO and LMO electrodes are ≈9.0 mg LTO/cm 2 and ≈17.0 mg LMO/cm 2 respectively, corresponding to a theoretical capacity rate of C + /C -≈0.9.

Electrodes with 18 mm of diameter are prepared by punching out the ink foil and porosities of ≈45% and ≈40% for LTO and LMO electrodes are respectively obtained by compression. Finally, all electrodes are dried under vacuum at 110°C for at least 12h in an oven (Büchi, Switzerland) and then transferred into an argon-filled glove box (MBraun, Germany) without exposure to ambient atmosphere.

Batteries cycling.

Electrodes are integrated in coin cells using 0.25 mL of electrolyte and two separators (Celgard, 20µm thickness) in polypropylene. The liquid electrolyte is a mixture of ethylene carbonate (EC, anhydrous, 99% purity) and ethylmethyl carbonate (EMC, purity ≥ 98%) (3:7 in volume), containing LiPF6 lithium salt (purity ≥99.99%, 1 mol.L -1 ). Coin cells are assembled in a glove box, under controlled argon atmosphere with low H2O/O2 levels (< 2 ppm). Cells are then cycled at 25°C, 40°C or 60°C in a thermostatic oven, under galvanostatic mode at C/10 rate from open circuit voltage with a Biologic MPG2 (Biologic SA, Claix, France) apparatus for the first cycle, and under galvanostatic mode at C/2 rate for 98 cycles. Finally, a last cycle is performed at C/10 rate to restore the capacity loss due to high C-rate cycling. Note that the first cycle is carried out at low rate in order to ensure the formation of a passivating layer at the LTO electrode surface [START_REF] Börner | Investigations on the C-Rate and Temperature Dependence of Manganese Dissolution/Deposition in LiMn2O4/Li4Ti5O12 Lithium Ion Batteries[END_REF] . Full cells cycling experiments are monitored based on the LTO electrode potential, between 1.0 V and 2.1 V. The coin cells are limited in capacity by the LTO electrodes (the capacity matching between positive and negative is 0.9).

Electrochemical impedance spectroscopy (EIS).

After cycling, the system is kept at the open-circuit voltage (2.0 V) for 2 hours; then potential-controlled impedance spectra are recorded from 10 kHz to 10 mHz with a perturbation of 10 mV at 25°C (10 points per decade).

Samples cleaning.

After EIS measurements, coin cells are opened in a glove box with low H2O/O2 levels (< 2 ppm). Both LTO and LMO electrodes are carefully extracted and rinsed with a large excess of dimethyl carbonate in order to remove all traces of electrolyte.

X-ray Photoelectron Spectroscopy (XPS). XPS analyses are carried out with a Thermo Scientific K-Alpha Xray photoelectron spectrometer using a focused monochromatized Al-Kα radiation (hν = 1486.6 eV). To avoid any moisture or air exposure, the samples are transferred using a transfer vessel to the XPS machine, which is directly connected to an argon dry glove box working at low H2O/O2 levels (< 4 ppm). Core ionization peaks are recorded with a 20 eV constant pass energy (PE) over an ellipsoidal area of 200×400 µm 2 . Data treatment is performed with CasaXPS software. Peak fitting is processed with a non-linear Shirley-type background and mathematical components are obtained by a weighted least-squares calculation method using 30% Lorentzian and 70% Gaussian line shapes except for the manganese spectra. Indeed, Mn 2p spectra fitting and interpretation are complex due to the multiplet splitting arising from the presence of unpaired electrons in its electronic configuration. This induces asymmetric shapes and close binding energies even for different oxidation states. Therefore, the deconvolution strategy was based on the use of the Mn 2p envelopes of reference compounds in which manganese presents only one oxidation state: Mn 3+ in Mn2O3 and Mn 4+ in Li2MnO3, as previously performed by our group [START_REF] Gieu | New Insights in the Characterization of the Electrode/electrolyte interfaces within LiMn2O4/Li4Ti5O12 cells, by X-ray Photoelectron Spectroscopy, Scanning Auger Microscopy and Time-of-Flight Secondary Ions Mass Spectrometry[END_REF][START_REF] Grissa | Thorough XPS analyses on overlithiated manganese spinel cycled around the 3V plateau[END_REF] . The spectral calibration is done based on the CF2 component (290.7 eV) originated from the PVdF binder in C 1s core peaks. Quantifications are calculated using Scofield's relative sensitivity factors corrected by -0.6 in intensity.

Scanning Electron Microscopy (SEM) and Scanning Auger Microscopy (SAM).

For SEM and SAM analysis at high spatial resolution (~10-100 nm), a JEOL JAMP 9500F Auger spectrometer is used. In order to avoid any sample degradation, samples are transferred from the gloves box to the machine using a transfer vessel. SEM images are recorded using an acceleration voltage of 10 kV and a low target current of 5 nA. Scanning Auger acquisitions are carried out in constant analyzer energy mode to define the useful energy width in order to get a significant intensity difference between the Auger transition and the background shape. An "auto probe tracking" correction (which compares SAM and SEM images recorded after each sequence with the initial reference image) is also used to evaluate and compensate any possible drift during acquisition. The software provided by JEOL is used for processing the chemical mappings. The concentration of each element is materialized individually through a scale of colors (ranging from black for no signal to white for the maximum of signal).

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS).

ToF-SIMS analysis are carried out using a TRIFT V nanoToF II (Physical Electronics, Chanhassen (MN), US) equipped with a 30 kV Au-LMIG. The electrode samples are fixed on the sample holder with a metallic mask and transferred from the gloves box to the spectrometer thanks to a transfer vessel to avoid any air exposure. All mass spectra are obtained using the same experimental conditions, which allows to perform semi-quantitative analysis (comparing the intensity ratios of peaks). For surface analysis, the LMIG gun is tuned to deliver Au3 ++ primary ions with a direct current (DC) of 12 nA over a 50 µm raster size and the number of frames is set to 30 (dose: 2.2x10 12 ions/cm 2 ) over 0 -400 mass range for both polarities. Depth profile experiments (series of 30 "analysis/sputtering" cycles) are carried out using the previous conditions for surface analysis (Au + , 50x50µm 2 raster size) and a 3 kV Ar + gas gun for etching (with an etching time of 20 seconds for each cycle, a DC current of 150 nA and a sputtered area of 500x500 µm 2 ).

Note that [START_REF] Yuan | Challenges of spinel Li4Ti5O12 for lithium-ion battery industrial applications[END_REF] Li + and [START_REF] Majima | Development of long life lithium ion battery for power storage[END_REF] Li + peaks in positive polarity and [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] F -and [START_REF] Gieu | Influence of Vinylene Carbonate Additive on the Li4Ti5O12 Electrode/electrolyte Interface for Lithium-ion Batteries[END_REF] O -peaks in negative polarity appear very intense and have to be blanked during the measurement in order to detect better smaller peaks. Data processing is performed using ToF-DR software provided by PHI company. All negative polarity spectra are calibrated using OH -(m/z 17), C2H -(m/z 25) and LiF2 -(m/z 45) peaks and all positive polarity mass spectra are calibrated using K + (m/z 39), Li2F + (m/z 33) and Ti + (m/z 48) peaks. For depth-profiles graphical representation, the intensity of the secondary ions of interest was normalized related to the total ion counts for both polarities.

RESULTS AND DICUSSION

Electrochemical results

Galvanostatic profiles of LMO/LTO cells at 25°C (in blue), 40°C (in green) and 60°C (in red) for the 1 st cycle, 99 th cycle and 100 th cycle are presented in Figure 1a, Figure 1b and Figure 1c, respectively. After the first charge, LTO capacities at 25°C (173 mAh.g -1 ) and 40°C (172 mAh.g -1 ) are close to the theoretical value (175 mAh.g -1 ) whereas the capacity is a bit higher at 60°C (178mAh.g -1 ). Moreover, after the first discharge, capacity losses of 4 mAh.g -1 , 8 mAh.g -1 and 19 mAh.g -1 are measured at 25°C, 40°C and 60°C respectively, which indicates that Li + ions (supplied by positive electrodes in excess) are irreversibly consumed by parasitic reactions to form surface layers; this phenomenon appears to be more important at 60°C compared to 40°C and 25°C. After the 99 th charge at C/2 (figure 1b), capacities decrease for all temperatures compared to the first charge, especially at 60°C. This observation indicates a decrease of both reversible and irreversible capacities upon cycling. Moreover, capacity values are the same after the 99 th discharge than after the 99 th charge, which evidences a stabilization of the capacity after cycling. A higher C-rate regime induces an accumulation of lithium ions at intercalation sites [START_REF] Thackeray | Structural considerations of layered and spinel lithiated oxides for lithium ion batteries[END_REF] . When the charge is carried out more quickly, some sites will not have the time to be fully occupied by lithium ions, which causes a loss of capacity and a higher polarization of the system. At 40°C and 60°C, the electrons and Li + ions mobility is more limited than at 25°C probably due to the formation of thicker layers at the electrodes surface. The last 100 th cycle is performed at a lower c-rate C/10 (figure 1c) in order to completely discharge the accumulator. Capacities of 8 mAh.g -1 at 40°C and 19 mAh.g -1 at 60°C are restored between charge and discharge, whereas at 25°C there is no difference between the two states of charge of the cell; moreover, the capacity after the 100 th cycle at 25°C reaches 172 mAh.g -1 which is in agreement with a small polarization and a weak fading of the system at this temperature. On the contrary, at 60°C, the polarization remains higher due to an important loss of capacity during cycling, which limits the performance of cells. Figure 1d shows the evolution of charge and discharge capacities as a function of the cycle number for LMO/LTO coin cells at 25°C (in blue), 40°C (in green) and 60°C (in red) and confirms the previous observations. A fastest degradation rate appears at 60°C whereas no obvious capacity fading appears for LMO/LTO cycled at 25°C. Indeed, the capacity retention becomes stable after five cycles for 25°C, whereas it is after 30 cycles for 40°C and no stability in capacity is obtained for 60°C (the coulombic efficiency is under 99.8%). Consequently, the degradation of cells increases with the temperature, in accordance with growth of impedance system. The Nyquist impedance curves of LMO/LTO full cells after 100 cycles at 25°C (in blue), at 40°C (in green) and at 60°C (in red) are shown in Figure S1. An equivalent circuit (inset in figure S1) is used for fitting the electrodes behavior, in which Rs, Rf, and Rct correspond to the ohmic resistance, the film resistance, and the charge transfer resistance, respectively. Constant phase elements CPEf and CPEdl are used to replace the film capacitance (Cf) and the double-layer capacitance (Cdl). The coefficient af relates to the roughness of the electrodes surface [START_REF] Wu | Influence of manganese ions dissolved from LiMn2O4 cathode on the degradation of Li4Ti5O12-based lithium-ion batteries[END_REF] . The fitted parameters are summarized in Table S1. The film resistance increases with the cycling temperature, which may imply a greater accumulation of insoluble compounds. A gradual decrease of af is observed with higher temperature. This might imply that the interfacial film covering electrodes surface at 40°C and 60°C may be less uniform than the ones formed at 25°C.

To further investigate the correlation between electrochemical performances and the cycling temperature, XPS, SAM and ToF-SIMS surface analysis techniques were used to characterize the chemical composition of surface layers formed on both LTO and LMO electrodes. Note that the LTO lithiation (charge of the battery) corresponds to a reduction process of titanium (Ti 4+ → Ti 3+ ) and to an oxidation of manganese (Mn 3+ → Mn 4+ ).

Surface analysis

Reversibility of the redox process: XPS analysis

At the pristine electrodes surface, Ti and Mn 2p signals are split into two components (2p3/2 and 2p1/2) due to spin-orbit coupling. Ti 2p spectra consist of a Ti 2p3/2 component at 458.7 eV and Ti 2p1/2 one at 464.4 eV, in agreement with Ti 4+ ions in an oxygen environment. Concerning Mn 2p spectra, the envelope maxima for each component-doublet, located at 641.4 eV and 642.2 eV, are respectively attributed to Mn 3+ (from Mn2O3 reference) and Mn 4+ (from Li2MnO3 reference) type ions of the LiMn2O4 material.

After cycling, the two components of Ti 3+ signal (Ti 2p3/2 and Ti 2p1/2 at 456.8 eV and at 458.4 eV respectively), related to the lithiation/reduction process of LTO, are still detected and their proportion increase with the temperature (Ti 3+ signal represents 25% of the total detected titanium at 60°C, 20% at 40°C and 7% at 25°C). This is attributed to the irreversible consumption of Li + ions and/or to the slower electrons and ions transportations onto LTO electrodes due to the formation of a thicker interfacial film at higher temperature. Indeed, Ti 2p is less detected at the LTO electrode surface after cycling at 40°C (3.6 at. %) and 60°C (1.2 at. %) compared to 25°C (4.5 at. %). At the end of the hundredth discharge (100 cycles), the Mn 2p spectra are very similar to the one of the pristine electrode for all cycling temperatures, which shows the good reversibility of the electron transfer over cycling at the LMO electrode. The overall quantity of detected manganese at the LMO electrode surface decreases in the same proportion for the three temperatures compared to the pristine one (from 4.6 at. % to ~3.0 at. %), which also suggests a covering of LMO electrodes by a surface layer. All these observations are in agreement with an increase of the capacity loss with high temperature.

that the thickness of the interfacial layers are lower than 10 nm for both systems, as the XPS analysis depth is about 5-10 nm. ROCO2Li, which are the main components of electrolyte solvent decomposition, and at 533.5 eV which is assigned to (fluoro)phosphates ((PO4 3-), PO(RO)3 or LiXPOyFz), resulting from the decomposition of LiPF6 salts and reactions with solvent degradation products [START_REF] Wu | Influence of manganese ions dissolved from LiMn2O4 cathode on the degradation of Li4Ti5O12-based lithium-ion batteries[END_REF] . At the same time, the LTO O 1s component strongly decreases at high temperature; it counts only for 2.7 at. % at 60°C compared to 9.2 at. % at 25°C. This evidences the covering process during cycling of LTO electrodes by a surface layers whose thickness increase with the temperature. In agreement with O 1s spectra, the appearance of new C 1s components after cycling on C 1s spectra (data not shown) at 286.7 eV, 287.8 eV and 289.5 eV, attributed to C-O, C=O and O=C-O/CO3 2-chemical environments respectively, confirms the deposition of organic species at the LTO electrodes surface, which is more important at high temperature.

Characterization of the chemical composition of surface layers: XPS analysis

Concerning F 1s spectrum for the LTO pristine electrode, the two PVdF components, attributed to CF2 and CF3 / CF2-CF2 environments, are observed at 687.9 eV and 689.5 eV respectively. After cycling, the PVdF CF2 component decreases when the temperature increases which proves once again the formation of a thicker surface layer at the LTO electrode surface at high temperature; on the contrary, the intensity of CF3 / CF2-CF2 component slightly increases with the temperature due to probable degradation and rearrangement of PVdF. Moreover, two new F 1s components are detected after cycling at 685.0 eV (light gray) which corresponds to LiF and at 686.5 eV (dark gray) attributed to fluorophosphates (LixPOyFz). The proportion of these last one increases with the temperature (2.0 at. % at 25°C, 4.1 at. % at 40°C and 8.7 at.% at 60°C), whereas no major difference is observed at low and high temperatures for the LiF component. These results confirm a chemical degradation process of LiPF6 salt more important at high temperature.

For LMO electrodes (figure S2), the proportion of surface species also increases with the temperature but in lesser extent. A greater proportion of fluorophosphates is detected at the LMO electrodes surface after cycling, whatever the temperature, which may be due to the cycling at high voltage (4.15V) on the positive electrode side in agreement with the literature [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF][START_REF] Wu | Influence of manganese ions dissolved from LiMn2O4 cathode on the degradation of Li4Ti5O12-based lithium-ion batteries[END_REF][START_REF] Yan | Phosphorus Enrichment as a New Composition in the Solid Electrolyte Interphase of High Voltage Cathodes and Its Effects on Battery Cycling[END_REF] . This result suggests that a dissolution process of fluorophosphates at the LMO electrode surface and their deposition at the LTO electrode surface after migration through the electrolyte, may occur and this phenomenon is more important at high temperature. In the same way, organic species formed at the LMO electrode (by oxidation of the solvents) can also migrate to the LTO electrode which can explain their greater accumulation at high temperature at the negative electrode surface. Indeed, the important decomposition of LiPF6 salt at high temperature could entail a change in the LiPF6 dissociation equilibrium in LiF and PF5 (as a strong Lewis acid) which catalyzes the solvents degradation in the electrolyte, especially EC [START_REF] Campion | Thermal Decomposition of LiPF6-Based Electrolytes for Lithium-Ion Batteries[END_REF][START_REF] Gachot | Deciphering the multi-step degradation mechanisms of carbonate-based electrolyte in Li batteries[END_REF][START_REF] Parimalam | Decomposition Reactions of Anode Solid Electrolyte Interphase (SEI) Components with LiPF6[END_REF] . P 2p core peak (data not shown) also exhibits an increase of phosphates/fluorophosphates proportions at the LTO electrodes surface when the cycling temperature increases from 25°C to 60°C and to a lesser extent at LMO electrodes surface too, which is consistent with the previous observations. Note that fluorophosphates could come from parasitic reactions between POF3 (formed by PF5 decomposition at high temperature) and solvents of the electrolyte, which leads to gaseous releases and severe fading [START_REF] Kraft | Study of decomposition products by gas chromatographymass spectrometry and ion chromatography-electrospray ionization-mass spectrometry in thermally decomposed lithium hexafluorophosphate-based lithium ion battery electrolytes[END_REF] . The quantitative analysis from O 1s, C 1s, F 1s, Li 1s and P 2p core peak is presented in figure 4a and4b for LTO and LMO electrodes respectively. The atomic percentages of LTO electrode (in red), LMO one (in blue) and deposited surface species (in grey) during cycling are reported in figure 4. Note that to determine the atomic percentages of electrodes, the proportions of active materials, carbon black and PVdF components were added; similarly, the amount of surface species corresponds to the sum of organic and inorganic species proportions calculated from XPS spectra. As mentioned before, both LTO and LMO electrodes are covered by surface layers after cycling at 25°C, 40°C and 60°C. The SEI thickness formed at LTO electrodes increases with the temperature, especially at 60°C, whereas the temperature influence is less noticeable for LMO surface layers; these ones are slightly thicker at 60°C, which could be in agreement with the capacity and impedance evolution upon cycling, Manganese is detected by XPS (figure 5) at the LTO electrode surface cycled versus LMO after the 100 th charge and 100 th discharge at 40°C and 60°C but not at 25°C. This manganese, which comes from a dissolution of the LMO active material at the positive electrode, migrates through the electrolyte and is deposited at the negative electrode surface over cycling. Mn 2p spectra at 40°C display a maximum of intensity between 641.3 eV and 642.0 eV with satellites characteristic of manganese in the oxidation state +II [START_REF] Biesinger | Resolving Surface Chemical States in XPS Analysis of first Row Transition Metals, Oxides and Hydroxides: Cr, Mn, Fe, Co and Ni[END_REF] . According to Gieu et al.

and Kim et al. [START_REF] Gieu | New Insights in the Characterization of the Electrode/electrolyte interfaces within LiMn2O4/Li4Ti5O12 cells, by X-ray Photoelectron Spectroscopy, Scanning Auger Microscopy and Time-of-Flight Secondary Ions Mass Spectrometry[END_REF][START_REF] Kim | Re-Deposition of Manganese Species on Spinel LiMn2O4 Electrode after Mn Dissolution[END_REF] this manganese is originated from MnF2 and MnO compounds, which could induce the increase of cells impedance. At 60°C, the amount of detected manganese at the LTO electrode surface is more important and a new peak appears at 638.0 eV which is attributed to metallic manganese Mn 0 . The LiPF6 decomposition more pronounced at high temperature could lead to an acidification of the electrolyte (HF formation) which promotes the manganese dissolution of LMO spinel and may induce its reduction until the metallic state [START_REF] Zhan | II) Deposition on Anodes and its Effects on Capacity Fade in Spinel Lithium Manganate-Carbon Systems[END_REF][START_REF] Gao | Correlation between the growth of the 3.3 V discharge plateau and capacity fading in Li1+xMn2-xO4 materials[END_REF] . These manganese species can favor the re-oxidation/reduction of certain SEI species and the release of lithium ions, leading to a delay in the SEI stabilization [START_REF] Vetter | Ageing mechanisms in lithium-ion batteries[END_REF][START_REF] Kim | Re-Deposition of Manganese Species on Spinel LiMn2O4 Electrode after Mn Dissolution[END_REF][START_REF] Lee | A Comprehensive Study of Manganese Deposition and Side Reactions in Li-Ion Battery Electrodes[END_REF][START_REF] Aoshima | Mechanisms of manganese spinels dissolution and capacity fade at high temperature[END_REF][START_REF] Gilbert | Transition Metal Dissolution, Ion Migration, Electrocatalytic Reduction and Capacity Loss in Lithium-Ion Full Cells[END_REF] . Thus, in the case of LTO/LMO cycled at high temperatures, other mechanisms than the electrochemical reduction of solvents at the LTO electrode surface have to be considered to explain the formation of the SEI layer. On one hand, deposited species come from the dissolution of the positive electrode and migration through the electrolyte (like manganese species and fluorophosphates) and on the other hand, a catalytic phenomenon, increasing the electrolyte degradation due to the presence of manganese species, can occur at the interface. Thereby, the formation of MnO and MnF2 at the LTO electrode surface could come from the reaction between the electrolyte and deposited manganese species.

Inductively-coupled plasma (ICP) measurements were performed on LTO/LMO full cells cycled at 60°C which allow to determine a manganese concentration of around 40 ppm in the electrolyte. This value may be underestimated because some of the dissolved Mn species are deposited on the electrodes surface. Nevertheless, the LMO active material loss caused by dissolution in the electrolyte is not substantial. Besides, as the operating voltage of LMO (at 4.15 V vs. Li + /Li) is much higher than the reduction potential of manganese ions, these ones can only be deposited on LTO anodes and have no significant influence on LMO cathodes. Therefore, the increase of cells resistance is mainly attributed to changes on the LTO anodes side.

Spatial distribution of elemental chemical species at LTO electrodes surface: SAM analysis

SEM images and SAM mappings of LTO electrodes surface after 100 cycles at 25°C a), 40°C b) and 60°C c) are presented in Figure 6. Two different types of particles are distinguishable on the SEM picture: the ones attributed to LTO grains in light grey, and the ones corresponding to the carbon black in dark grey. After cycling, the deposition and growing of the SEI with the temperature cannot be demonstrated by SEM as the obtained images are similar to the pristine electrode one (figure S3). On SAM images, the overlap of titanium and oxygen mappings (figure S3) confirm the localization of LTO particles for all the temperatures. Nevertheless, at 40°C and 60°C, oxygen and carbon are detected besides the LTO and carbon black particles which proves one more time the deposition of organic species on the LTO electrode surface, due to the SEI formation, whose thickness increases at high temperature. However, Titanium is still detected at the end of the hundredth discharge at 60°C, which means that the SEI is thinner than 5 nm. The phosphorus mappings reveal the presence of fluorophosphates mainly localized on LTO particles especially at 40°C and 60°C, which highlights a preferential reactivity of the lithium salt anion with the active material [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] . Concerning fluorine compounds, a more homogeneous repartition is revealed on LTO electrode cycled at 40°C compared to electrodes cycled at 25°C (for which fluorine species are mainly located on black carbon) and at 60°C (for which fluorine species are preferentially deposited on LTO particles). According to XPS analysis at 25°C, SEI fluorine species are mainly originated from LiF, which is preferentially located on the carbon black [START_REF] Gieu | Temperature Effects on Li4Ti5O12 Electrode/electrolyte interfaces at the first cycle: A X-ray Photoelectron Spectroscopy and Scanning Auger Microscopy Study[END_REF] . At higher temperatures, and especially at 60°C, fluorine signal is generated by LiF but also by fluorophosphates which are present in greater quantity; these results are in agreement with the mass spectrometry measurements performed by Gachot et al [START_REF] Gachot | Deciphering the multi-step degradation mechanisms of carbonate-based electrolyte in Li batteries[END_REF] . Finally, despite the detection of manganese by XPS at 40°C and 60°C, SAM manganese mapping is only obtained at 60°C and indicates a heterogeneous distribution over the LTO electrode surface as previously suggested by Börner et al. [START_REF] Börner | Investigations on the C-Rate and Temperature Dependence of Manganese Dissolution/Deposition in LiMn2O4/Li4Ti5O12 Lithium Ion Batteries[END_REF] .

The SEI layer formed at the LMO positive electrode/electrolyte interface was also analyzed by SAM after cycling but the mappings are not presented here for a non-exhaustive presentation. Note that fluorophosphates are preferentially deposited on the LMO active material particles but no difference is noticeable with the temperature increase. Figure 7 shows the mass spectra in negative polarity of cycled LTO negative electrodes at the end of the hundredth cycle at 25°C a), 40°C b) and 60°C c) over 0-120 m/z range. At the end of the hundredth cycle, the main negative secondary ions detected are carbonaceous species like m/z: 12 (C -), m/z: 13 (CH -), m/z: 24 (C2 -), m/z: 25 (C2H -) related to solvents degradation, and fluorine and phosphorus containing species m/z: 45 (LiF2 -), m/z: 63 (PO2 -), m/z: 79 (PO3 -) and m/z: 101 (PO2F2 -), characteristics of the SEI formation from LiPF6 salt decomposition [START_REF] Li | Dynamic behaviour of interphases and its implication on high-energy-density cathode materials in lithium-ion batteries[END_REF] . The most intense peak m/z: 17 (OH -) come from solvent degradation but also from the LTO active material. At 40°C and 60°C, the intensity of peaks attributed to phosphate species is higher than at 25°C, suggesting that the SEI at the LTO electrode cycled at higher temperature is thicker due to more important salt degradation, which is consistent with our previous XPS results. The mass spectra in positive polarity are shown in supporting information (figure S4). Moreover, m/z: 71 (MnO -) secondary ions (as well as m/z: 55 (Mn + ) in positive polarity, data not shown), specific of the LMO active material, are detected at the LTO electrode surface cycled at 60°C [START_REF] Gieu | New Insights in the Characterization of the Electrode/electrolyte interfaces within LiMn2O4/Li4Ti5O12 cells, by X-ray Photoelectron Spectroscopy, Scanning Auger Microscopy and Time-of-Flight Secondary Ions Mass Spectrometry[END_REF][START_REF] Kim | Re-Deposition of Manganese Species on Spinel LiMn2O4 Electrode after Mn Dissolution[END_REF] , which confirms the dissolution process of the LMO electrode and manganese deposition at the LTO electrode surface at high cycling temperature.

In order to have more information about Manganese and SEI species in-depth spatial distribution at the LTO electrode surfaces, depth-profiles experiments (series of "analysis/sputtering" cycles) were performed in positive and negative polarities after the hundredth cycles at 25°C a), 40°C b) and 60°C. Figure 8 presents the normalized TOF-SIMS depth profiles obtained for Ti + (representing the LTO active material, blue line), Li2F + (characteristic of inorganic SEI species, green line), MnO -(coming from LMO dissolution, red line), C3H6O + (specific of organic SEI species, yellow line) and PO2 -(arising from LiPF6 salt decomposition, black line) secondary ions of interest over 400 seconds of etching depending on the cycling temperature.

A distinct rise of the bulk active material species (Ti + ) intensity is observed during etching for all temperatures, but the slope of the curve is more important at lower temperature (0.8 normalized intensity is reached after 150 second for 25°C, after 200 seconds for 40°C and after 275 seconds for 60°C), which clearly evidences the increase of the SEI thickness with higher temperatures. At 25°C, C3H6O + and PO2 -normalized intensities decreases quickly to reach almost zero after the first sputtering phase (30 seconds) while Li2F -signal intensity, initially higher, increases, which proves that organic and phosphate species are mainly located at the extreme surface of the SEI and above Li2F species. The proportion of these last ones gradually decreases during the remaining sputtering time, which evidences that Li2F compounds are present more deeply into the SEI. At 40°C, C3H6O + , PO2 -signals follow the same trend as at 25°C by decreasing mainly during the first sputtering cycle, but the proportion of PO2 -remains slightly higher over a longer etching time than at 25°C, meaning their proportion increases a little bit at 40°C. This time, no initial increase of Li2F -intensity is observed during the first sputtering phase which shows that organic, phosphate and LiF species are mixed together at the extreme surface of the SEI. At 60°C, C3H6O + , PO2 -and Li2F -secondary ions are present in higher proportion at the beginning of the etching compared to 25°C and 40°C, which is consistent with a more important salt decomposition at 60°C, as previously highlighted by SAM and XPS. Like at lower temperatures, C3H6O + and PO2 -signal intensities mainly decrease during the first sputtering phases at 60°C, which means they are located in majority at the SEI extreme surface, but the intensities remain higher over a longer etching time compared to 40°C, meaning they are present in higher proportions and more deeply into the SEI. Concerning Li2F -, its proportion gradually decreases from the beginning of the sputtering (corresponding to SEI top surface) to the end of the etching (related to the inner of the SEI). Finally, concerning manganese related species, the MnO -normalized intensity is much higher at 60°C than at 40°C (for which the signal has been increased x15 for a better visibility), which is again consistent with a dissolution of LMO, migration through the electrolyte and deposition at the LTO electrode surface more important at 60°C. The normalized intensity of MnO -increases after the first sputtering phase and decreases gradually during the remaining sputtering time, which evidences that manganese species are covered by organic and inorganics species at the extreme surface and are present deeply into the SEI.

To better visualize the spatial in-depth distribution of the considered species, 3D images have been reconstructed from depth-profile experiments and are presented under each graph related to one temperature. The signal intensity of Ti + (in blue), Li2F + (in green), C3H6O + (in yellow) and PO2 -(in black) secondary ions is plotted over the analyzed area (50µm x 50µm = x and y dimensions) versus the etching time (the z axis, 400s, which is over dimensioned compared to the other axes) for the three temperatures. When a color is not appearing anymore on the image, it means the signal intensity of the secondary ion is too low in the mass spectrum to be considered.

For 60°C, another 3D image, representing the spatial in-depth distribution of MnO -(in red) and Ti + (in blue) secondary ions, is presented separately for better clarity. Note that the intensity of MnO -at 40°C is too weak to be imaged in 3D.

The 3D map at 25°C clearly highlights that organic and phosphate compounds are located only at the extreme surface of the SEI and that they cover LiF species which insert more deeply into the SEI. The same behavior is observed at 40°C except that LiF species are also present at the extreme surface and phosphate species proportion is slightly higher. At 60°C, the increase of the SEI thickness is clearly visible on the 3D map, where all species are present in higher proportions. The same trend as at lower temperature is obtained, where organic and phosphate species are mainly located in the upper part of the SEI while LiF compounds are continuously present from the top surface to the inner part of the SEI. The manganese species 3D map clearly evidences their presence within the SEI and their covering by the upper part of the SEI (composed of inorganics, organics and phosphate species). All these results are consistent with our previous XPS and SAM results [START_REF] Gieu | New Insights in the Characterization of the Electrode/electrolyte interfaces within LiMn2O4/Li4Ti5O12 cells, by X-ray Photoelectron Spectroscopy, Scanning Auger Microscopy and Time-of-Flight Secondary Ions Mass Spectrometry[END_REF][START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF][START_REF] Gauthier | Probing the in-depth distribution of organic/inorganic molecular species within the SEI of LTO/NMC and LTO/LMO batteries: A complementary ToF-SIMS and XPS study[END_REF] and the literature [START_REF] Gachot | Deciphering the multi-step degradation mechanisms of carbonate-based electrolyte in Li batteries[END_REF][START_REF] Parimalam | Decomposition Reactions of Anode Solid Electrolyte Interphase (SEI) Components with LiPF6[END_REF][START_REF] Li | Dynamic behaviour of interphases and its implication on high-energy-density cathode materials in lithium-ion batteries[END_REF] and confirm that both salt decomposition (inducing an important LiF and phosphate species deposition at the LTO electrode) and manganese dissolution are intensified at high temperature, which explains lower electrochemical performances of LTO/LMO cells at 60°C.

CONCLUSIONS

In conclusion, this work contributes to a better understanding of the impact of the temperature on LTO electrode/electrolyte interfaces in LTO/LMO systems. Thanks to the complementarity of XPS, SAM and ToF-SIMS techniques, it was clearly proved that high temperatures (60°C) increase the SEI thickness and change its chemical composition as well as the in-depth spatial distribution of SEI species. Indeed, the important LiPF6 salt decomposition at 60°C induces the formation and deposition at LTO surface of (fluoro)phosphate species in higher proportions, as well as a more important manganese dissolution at the LMO positive electrode, which migrates through the electrolyte and insert within the SEI; moreover, metallic manganese was detected for the first time by XPS at the LTO electrode surface, which could be linked to the PF5 degradation mechanism at the electrode/electrolyte interface. All these factors destabilize the SEI formed at the LTO electrode surface and can cause the loss of capacity of LTO/LMO cells at high temperature.

Such manganese dissolution detrimental effect will have to be controlled to allow the commercialization of LTO-based LIBs composed of lithium manganese oxides or derivatives. Several options need to be investigated to minimize the fading of LTO/LMO cells at high temperature, like the use of additives in the electrolyte or the replacement of LiPF6 by fluorophosphates free salts; the coating or doping of lithium titanate particles could also be of interest. Moreover, as the NMC electrode presents less active materials dissolution during cycling and better electrochemical performances than LMO when cycled facing LTO [START_REF] Gauthier | Influence of the Positive Electrode on Li4Ti5O12 (LTO) Electrode/Electrolyte Interfaces in Li-Ion Batteries[END_REF] , a blend of NMC and LMO materials could be considered as a good option for the positive electrode.

Figure 1 .

 1 Figure 1. Galvanostatic charge/discharge voltage profiles of LTO/LMO cells at 25°C (in blue), 40°C (in green) and 60°C (in red) a) for the 1 st cycle at C/10, b) for the 99 th cycle at C/2 and c) for the 100 th cycle at C/10 ;

Figure 2

 2 Figure 2 presents XPS Ti 2p core peaks of the pristine LTO negative electrode (first line) and LTO

Figure 2 .

 2 Figure 2. a) Ti 2p XPS spectra of the pristine LTO negative electrode (first line) and LTO electrodes after 100

Figure 3

 3 Figure 3 presents XPS O 1s (a) and F 1s (b) core peaks of the pristine LTO negative electrode (first line)

Figure 3 .

 3 Figure 3. a) O 1s and b) F 1s XPS spectra of the pristine LTO negative electrode (first line) and LTO electrodes

Figure 4 .

 4 Figure 4. Histograms comparing the atomic percentages of surface species (in grey), LTO electrode components

Figure 5 .

 5 Figure 5. Mn 2p XPS spectra of cycled LTO electrodes versus LMO after the 100 th charge and after the 100 th

Figure 6 . 3 . 2 . 5

 6325 Figure 6. SEM images and SAM chemical mappings of fluorine F, phosphorus P and manganese Mn on LTO

Figure 7 .

 7 Figure 7. ToF-SIMS negative ion spectra over a 0-120 m/z range for LTO electrodes cycled at 25°C a), 40°C

Figure 8 .

 8 Figure 8. Normalized ToF-SIMS depth profiles over 400 s of Ti + , Li2F + , C3H6O + , PO2 -and MnO -secondary ions

Figure S2 .

 S2 Figure S2. a) O 1s and b) F 1s XPS spectra of the pristine LMO positive electrode (first line) and the LMO

Table S1 .

 S1 The fitted EIS data of LMO/LTO full cells cycled at 25°C, 40°C and 60°C

	Temperature Re (Ohm) CPE1		Rf / Ohm	CPE2		Rct / Ohm	W
			Qf (10 -6 ) af		Qdl (10 -3 ) adl
	25°C	2.1	82.8	0.8	17.4	2.0	0.9	5.1	2.2
	40°C	2.7	30.0	0.8	50.4	6.6	0.7	4.3	2.1
	60°C	5.0	18.3	0.7	104.3	17.2	0.6	5.4	2.0

ACKNOWLEDGEMENTS

The authors gratefully acknowledge SAFT company, the French CNRS (national center for scientific research) and The French National Research Agency via the project ANR-11-EQPX-0027 MARSS for financial support.