Niki Baccile

Lisa Van Renterghem

Patrick Le Griel

Guylaine Ducouret

Martha Brennich

Viviana Cristiglio

Sophie L K W Roelants

Wim Soetaert

Lisa Van Renterghem

Bio-based glyco-bolaamphiphile forms a temperature-responsive hydrogel with tunable elastic properties

Introduction:

Flory classified hydrogels as lamellar and particulate structures, but also physical and covalent polymer networks, [START_REF] Flory | Faraday Discuss[END_REF] are studied in depth for tissue engineering, [2][3][4][5][6] drug release, 7 biomedical injectable fillers, 8 and other more advanced applications, like art conservation, 9 confined reaction vessels 10 and ionic conductors. 11 Floryˈs classification has been challenged in the past two decades by new types of gels, namely molecular gels. These are obtained by the selfassembly of Low Molecular Weight Gelators (LMWG) into a Self-Assembled Fibrillar Network (SAFiN). 12 These soft materials are highly promising: gelation is reversible due to their stimuliresponsiveness (towards pH, T, ionic force, light, mechanic …), which is an advantage compared to most polymer-based hydrogels, for targeted applications in the biomedical field, [12][13][14][15][16] in wastewater treatment 17 and even antibacterial use. 18 Most LMWG are a combination of the π-π stacking and hydrogen bonding of functional groups in the same molecule, e.g., fluorenylmethoxycarbonyl (Fmoc), L-cystine or amino acid derivatives. Through molecular engineering one can obtain rapid (in the order of seconds up to few minutes) gelation with elastic moduli in the 10 kPa -10 2 kPa range, for concentrations in the mM range or even below. [19][20][21][22] However, engineered molecular systems often require complex organic synthesis involving multi-step protection/deprotection processes and, in any case, the smartest molecular design does not guarantee success in terms of hydrogelation, which is often driven by serendipity 12,19 and more down-to-earth physico-chemical parameters like temperature, heating/cooling rate, pH, etc… [23][24][25][26][27][28][29][30][31] Sustainability in the development of hydrogels is becoming a popular approach, 17 especially for potential applications in cosmetics, food science and biomedicine. 16 Glycosylated LMWG have shown some nice examples of hydrogel-forming systems, [32][33][34][35] exhibiting elastic moduli above 10 kPa 21 . Such compounds are derived from sugars, which are widely available and renewable biobased molecules, and can also be obtained from side stream process and lignocellulosic biomass. 21,[32][33][34][36][37][38][39] However, this class of compounds is still obtained through sugar organic chemistry, which is notoriously time and energy-consuming due to the lengthy protection/deprotection procedures. Instead of developing a new complex chemical synthesis, we have focused our efforts on bio-based-hydrogels produced by a microbial fermentation approach.

Fermented glycosylated lipids, a class of molecules often referred to as biosurfactants, are historically developed for biodegradable detergent formulations, 40,41 even if recent developments in this field show a much higher potential. [42][43][44][45][46] Despite the large benefit of this approach (biocompatibility, biodegradability) one must face limited structural heterogeneity. To overcome this problem a strong effort in terms of genetic engineering [START_REF] Lodens | Synthetic Biology -Series Methods in Molecular Biology[END_REF] has shown that molecular variability, [START_REF] Van Renterghem | [END_REF] higher uniformity and purity, production scale-up and process development [49][50][START_REF] Roelants | Microbial Biosurfactants and their Environmental and Industrial Applications[END_REF] can be obtained by new strains. [START_REF] Van Bogaert | [END_REF][53][54] We have recently shown that the fibrillation properties in water of acidic sophorolipids 55,56,57 and hydrolyzed cellobioselipids, 58 but their gelation properties are not demonstrated so far. Here, we illustrate the hydrogel properties of a new generation of symmetrical bolaform sophoroside 44 biosurfactant. The common carboxylic acid present in sophorolipids is replaced with an additional sophorose unit [START_REF] Van Bogaert | [END_REF] and palmitic alcohol is employed, instead of standard fatty acids, as substrate to generate the chemically-stable ω-1 hydroxylated C16:0 symmetrical bolaform sophoroside (sBola C16:0 SS, Figure 1a). [START_REF] Van Renterghem | [END_REF] This compound was produced at gram-scale with the genetically-engineered S. bombicola strain ∆at ∆sble ∆fao1, derived from the S. bombicola strain ∆at ∆sble (producing non-symmetrical bola acidic sophorolipids) with an additional knock out (∆fao1).

We assume that this new compound has similar fibrillation properties as found in acidic sophoro-and cellobioselipids, but with a stronger tendency to form an extended H-bonding network, which could enhance hydrogel formation, a property which is not reported for the acidic compounds, yet. In addition, the loss of the carboxylic acid confers a better stability towards pH. 44 In this work, we combine Small Angle X-ray Scattering (SAXS), cryo-Transmission Electron Microscopy (cryo-TEM), time and temperature-resolved rheology and solution [START_REF] Flory | Faraday Discuss[END_REF] H Nuclear Magnetic Resonance (NMR) to show that sBola C16:0 SS forms a SAFiN hydrogel at concentrations as low as 1 wt%, with an elastic modulus which can be tuned between 100 Pa and 20 kPa under controlled conditions: these properties are comparable with hydrogels obtained using synthetic gelators. [19][20][21][22] This work consists of a first thorough study of the aqueous self-assembly and hydrogel-forming properties of a genetically-engineered, bio-based, glycolipid; considering the potential biocompatibility toward cells (red blood count test) for this class of compounds, [START_REF] Van Renterghem | [END_REF] we open new ways for microbially-derived soft materials in the field of biomedical and health-care applications.

Materials and Methods:

Products. Symmetrical bola sophoroside (SS) with a C16:0 backbone (sBola C16:0 SS) were produced as described by Van Renterghem et al. using S. bombicola strain ∆at ∆sble ∆fao1. [START_REF] Van Renterghem | [END_REF] The production occurred in shake flask cultures and purification is performed as described by the same authors. Chemical structure by 1D and 2D NMR and purification of sBola C16:0 SS has also been described in ref [START_REF] Van Renterghem | [END_REF] . HPLC-ELSD and LC-MS data, confirming the purity of the product, are reported and commented in Figure S 1.

Self-assembly. sBola C16:0 SS is dissolved in milliQ grade water at the desired concentration at 25°C and strongly vortexed for about 30 s to 1 min until the powder has entirely dissolved and a translucent colloidal suspension is obtained. The apparent viscosity of the solution is quite low after vortexing, but it slowly increases with time. In a second set of experiments, the as-prepared sBola C16:0 SS solution is heated above 30°C (please, refer to text for more details) for five minutes until the solution becomes clear. The temperature is then lowered to 25°C or below. This procedure is detailed in the section titled Temperature-dependent mechanical properties of the hydrogel.

Small Angle X-ray Scattering (SAXS): SAXS experiments are performed at 25°C immediately after sample preparation on the BioSAXS BM29 beamline at the ESRF synchrotron facility (Grenoble, France) using 12.5 KeV energy and a sample-to-detector distance of 2.867 m, imposed by the beamline standard configuration. The energy is calibrated by measuring the LI and LIII edges of platinum and the sample-to-detector distance is determined using silver behenate (d ref = 58.38 Å). 59,60 For this experiment, we employ the automatic sample changer for liquids using the 96-well plates and about 100 µL of each sample. 61 The liquid sample is automatically loaded into a 1.8 mm quartz glass capillary and ten acquisitions of 1 s each are taken as the sample passes the beam.

Individual frames are manually controlled for systematic changes and averaged for better statistics if none are found. Eventual changes can be either due to intrinsic sample heterogeneity or radiation damage. The signal of the Pilatus 1M 2-D detector, used to record the data, is integrated azimuthally with PyFAI to obtain the I(q) vs. q spectrum (𝑞𝑞 = 4𝜋𝜋 sin 𝜃𝜃 𝜆𝜆 � , where 2θ is the scattering angle) after masking systematically wrong pixels and the beam stop shadow. 62 Absolute intensity units were determined by measuring the scattering signal of water (0.0163 cm -1 ).

Small Angle Neutron Scattering (SANS):

SANS was performed at the Institut Laue-Langevin (ILL) on the D16 beamline using a neutron wavelength, λ= 4.5 Å, a sample-to-detector distance, d= 955 mm, sample and detector rotation angles: ω= 5.5° and 2θ= 11°, and an acquisition time= 60 min.

Neutron absorber: Boron Carbide (B4C), thickness of quartz Hellma cells: 1 mm. [START_REF] Baccile | Institut Laue-Langevin (ILL)[END_REF] The sample is thermalized at 25°C and 40°C. D2O is used as solvent for the self-assembly experiments. Direct beam, empty cell and H2O are also recorded. The background corresponded with 99.9 % D2O.

Direct beam, empty cell and H2O were also recorded for both spectrometers Fourier Transform InfraRed spectroscopy (FTIR): FTIR experiments have been done in the ATR mode using a Perkin Elmer Spectrum 400 instrument.

Rheology: viscoelastic measurements were carried out on a stress controlled rheometer Haake RS 600 equipped with a sandblasted, stainless steel cone and plate geometry (diameter 35 mm, angle 2°, truncation 104 mm). First, the sample was submitted to a short stress sweep at 1 Hz to determine the viscoelastic regime. In order to preserve the sample, the stress sweep was stopped before the end of the viscoelastic plateau. Next, the frequency sweep was performed covering a frequency (f) range from 1.5 10 -2 to 15 Hz. The elastic (Gˈ) and loss (Gˈˈ) moduli were measured in the linear viscoelastic regime. In order to prevent the sample from drying, the geometry is protected by a homemade device, although drying could occur for experimental time longer than about 20 h -24 h. Measurements were repeated on several samples to check the reproducibility. the time-resolved acquisition starts when T= 25°C is reached, where a delay of about 3 min occurs during temperature variation. A slightly longer delay (5 min) occurs between T= 40°C and lower temperatures. In any case, such a delay is always taken into account in all time-resolved plots and the zero-time, t0, is systematically taken at T= 40°C. This is not important for the kinetic experiments at T= 25°C, but it becomes crucial at lower temperatures, where the sol-gel transition occurs during the dead time. Finally, an internal coaxial insert capillary containing a TMSPd4 solution in D2O as internal reference is used to calibrate both the chemical shift and intensity variation throughout the experiment. Absolute values of the peak area as a function of time is obtained using the "integration" and "relaxation" moduli of the Topspin™ 3.5 pl7 version of the software, while the Full Width at Half Maximun (FWHM) profiles have been automatically

Cryo-Transmission

obtained by using of DMFit software, available free of charge at the developer's website. 65,66 We have observed that phasing problems due to lowering the temperature and phase transition may affect the peak of H2O. Since this is the most intense peak, poor phasing can affect the baseline in the vicinity of the sugar CH region of sBola C16:0 SS between 3 ppm and 4.5 ppm. This unavoidable fact strongly affects the actual value of the peak area. For this reason, we mainly present the time-resolved evolution of the aliphatic peak area, contained between 0.5 ppm and 2.5 ppm. Peak area normalization is performed with respect to the spectrum recorded at t0 in the sol phase, which is at T= 40°C, when one can detect the entire sBola C16:0 SS population. Finally, the volume of the solution is always kept at 500 μL for all the experiments.

High-Performance

Results and Discussion

Hydrogel-forming properties of sBola C16:0 SS.

Upon mixing sBola C16:0 SS with milliQ grade water at room temperature without pH adjustment, a turbid solution is obtained. Several concentration values between 1 and 10 wt% were Pa, respectively, for solutions at 1 wt% and 10 wt%. The classical gel-like behavior for solutions between 1 wt% and 5 wt% (t= 15h) is shown in Figure 2. Both elastic modulus Gˈ and loss modulus Gˈˈ are constant, and Gˈ is at least a decade higher than Gˈˈ on the entire frequency range. The viscoelastic properties of the sample does not depend on the frequency, unlike conventional polymers suspensions 67 or other low molecular gelators, which display viscous behavior at low frequency. 14 Typical longer rest times may improve the overall Gˈ value, although this aspect cannot be trustily measured due to water evaporation for a sensibly longer waiting time. The magnitude of Gˈ found for sBola C16:0 SS hydrogel is comparable with standard LMWG found in the literature, where Gˈ≥ 1 kPa. 19,20,21 FmocY (fluorenylmethoxycarbonyl) derivatives are characterized by Gˈ values between 1 kPa and 4 kPa below 2 wt% at 37°C and in a pH range between 6 and 9, MAX1 has Gˈ in the order of 1 kPa at 2 wt%, pH 9 and 25°C, 19 while carbohydrate-based compounds 20,21,32,33,36 have Gˈ values above 10 kPa in the 1 wt%-2 wt% range.

b) c d) e

The formation of a stable gel over time is expected due to the self-assembly of sBola in water. Figure 1d and Figure 1e show the nano-and mesoscale structure of sBola C16:0 SS in water, respectively by Small Angle X-ray Scattering (SAXS) and cryo-TEM. Concentration-dependent SAXS experiments performed at t= 0 h show a nice scattering signal for all samples between 0.3 and 5 wt%. Whatever the concentration values, the signal looks the same and it is characterized by low-q scattering profile, which is strongly similar to the SAXS pattern of self-assembled ribbons, commonly found for the stearic acid (C18:0) derivative of acidic sophorolipid. 55 In particular, the power law exponent (slope) measured for our data lies between 1.6 and 1.7; these values are close to 2, generally expected for (helical) ribbons both theoretically and experimentally. 55,68,69 The small discrepancy between the theoretical and experimental value of the exponent in this system can be attributed to fiber aggregation, as shown before. 56 Cryo-TEM experiments performed on a 1 wt% sample at t= 0 h (Figure 1e) confirm that the sample is composed of fibers, which are most likely twisted (cross-section below 20 nm), as previously described in our own 55 and other works. 70 Since we cannot guarantee that all fibers in the systems are twisted, the general term fibers and twisted ribbons will be both used to refer to the fibrillary phase of the sample.

SAXS data also display a broad Bragg diffraction peak at q= 2.53 nm -1 , which corresponds to a correlation distance of d= 2.48 nm, and it highlights a periodic molecular arrangement of sBola C16:0 SS within each ribbon, as also described for other sophorolipid systems, 55 and found for other sugar bolaamphiphile-derived fibers. 71 Since the full-extended size of sBola C16:0 SS is expected to be well above 2.5 nm (according to the Tanford formula, [START_REF] Tanford | The hydrophobic effect: Formation of micelles and biological membranes[END_REF] the fully extended length of C16 is about 2.1 nm while the size of one sophorose unit is about 1 nm), the inter-lipid distance is then coherent with a tilted arrangement of sBola C16:0 SS, as described for other bolaform glycolipids. 71 To this regard, the literature is not clear whether the diffraction peak in our SAXS data can be unambiguously attributed to a crystalline or liquid crystalline order within the ribbons; [START_REF] Barclay | [END_REF] since it is not our intent to solve this matter here, we will simply refer to a Bragg diffraction peak. The attribution of the second diffraction peak at 4.46 nm -1 (d= 1.41 nm) is less straightforward: its d-spacing value, which is 1.8 times the first peak, could suggest the presence of a hexagonal packing within the ribbon (expected ratio is √3). Nonetheless, most studies performed on lipid systems self-assembled in chiral fibers have shown the presence of orthorhombic, triclinic or lamellar packing, instead. [74][75][76][77] Other studies run on self-assembled peptides rather attribute the d-spacing range between 1.3 nm and 1 nm to an interfibers periodic stacking. [78][79][80] Our data do not allow us to have a clear-cut interpretation and further work, out of the present scope, will be needed to clarify this point.

From the data in Figure 1, we can safely argue that the gelling properties of sBola C16:0 SS derive from the spontaneous formation of a dense network of self-assembled fibers in water.

Considering the frequency-independent profile of the viscoelastic moduli in Figure 2, characterized by an infinite relaxation time, which is classical in fiber-based gels, 81 we can consider that the fiber are de facto "unbreakable", and in this case the elastic properties are driven by entanglements, in analogy to polymer gels. 14 To confirm this assumption, we can follow the evolution of the elastic properties of the gel as a function of concentration, Gˈ(C), in log-log scale (Figure 1c), in terms of the power law 𝐺𝐺 ∝ 𝐴𝐴𝐶𝐶 𝑛𝑛 , well-known for colloidal and polymer gels: [82][83][START_REF] Flory | Principles of polymer chemistry[END_REF] A is a constant and n an empirical exponent, which characterizes the type of aggregation. Just after mixing, t= 0 h, we can identify a Gˈ(C) with an exponent close to 4 (n= 3.9 ± 1.1, Figure 1c), similar to those found in weakly aggregating polymer colloids, where it can vary between 3.7 and 4.5. 83 After 15 h, Gˈ(C) scales with a power law of 2.6 ± 0.4, a value which is close to what one finds in entangled polymer gels in a good solvent in a semidilute regime, and for which Gˈ(C) on the plateau is expected to have n= 2.25, as described by de Gennes. 82 n values around 2 are found in both synthetic and biopolymer hydrogels, 85-87 but also in fibrillary hydrogels, for instance composed of bacterial cellulose. 88 The analogue Gˈ(C) behavior described by entanglements and observed both in polymer and fibrillary systems may seem surprising, but it is worth noting that self-assembled filaments can be described as polymers with a significant bending rigidity. [START_REF] Kierfeld | Semiflexible Polymers and Filaments: From Variational Problems to Fluctuations[END_REF] To this regard, the theory developed by de Gennes on the dynamics of entangled polymer gels 82 (the frequency range used to measure the mechanical properties must be higher than the disentanglement rate and lower than the characteristic frequency associated with the motion of the fiber between entanglement points) should be valid also in filamentous systems. This is observed experimentally in our and other systems. 88 In summary, sBola C16:0 SS spontaneously self-assembles into weakly interacting colloidal fibrils at room temperature; the fibrils grow over time into fibers and their elastic properties are driven by entanglements, as observed for polymers.

Time-dependent mechanical properties of the hydrogel.

The time-dependent evolution of the elastic properties of the hydrogel have been investigated in more detail by rheology, SANS and cryo-TEM. range probed with respect to SAXS (Figure 1d), all data are characterized by the same Bragg diffraction peak and scattering profile at low-q, thus indicating that the morphology of, and molecular packing within, the self-assembled fibers do not vary with time. The presence of fibers is also observed at very low concentration (0.3 wt%) at t= 0 h and after 12 h, when the solution is fluidic at any time and no hydrogel is formed, as shown by the corresponding SANS data (Figure S 3), the shape of which is analogous to the system at higher concentration. Cryo-TEM data SAFiN with different viscoelastic properties. When nucleation is homogeneous and fiber growth is "infinite", gel network is strong and characterized by transient entanglements, in analogy with polymers materials. 82 Whenever growth prevail over nucleation, highly branched, spherulitic, networks are rather characterized by permanent junctions and the corresponding gel has overall poorer mechanical properties. 24,[START_REF] Liu | [END_REF][91][92] Rheology and cryo-TEM data show that the hydrogel studied in this work has the structural features of a homogeneous gel network rather than a spherulitic one.

Temperature-dependent properties of the hydrogel.

Figure 3a shows the evolution of Gˈ and Gˈˈ with temperature from 5°C to 35°C at fixed stress and frequency, where each temperature is reached after dispersion of the sample in water at 25°C, at which fibers readily form, as discussed above (Figure 1 and Figure 2). The present study concerns a temperature evolution from T= 25°C to Tf, where 5°C < Tf < 35°C. Three regions can 6 Pa shows that the gel formed is very fragile (Figure 3b).

These data can be explained by SANS in Figure 3c, which show a typical micellar scattering profile above T= 40°C at any concentration, if compared to the discussed fiber-like scattering profile at T= 25°C. Similar SANS profiles of micellar aggregates have been described before for acidic sophorolipids. 93,94 A standard Guinier analysis [START_REF] Glatter | Small Angle X-ray Scattering[END_REF][START_REF] Guinier | [END_REF] of the micellar scattering profile performed below q< 1 provides an average radius of gyration, Rg= 0.9 ± 0.1 nm which, in the hypothesis of spherical objects, corresponds to a sphere of radius, R= 1.2 ± 0.1 nm (Figure S 8a). This is confirmed by the numerical modelling of the SANS curve (details concerning the modelling are provided on Page S9, in the Supporting Information) using a homogeneous sphere form factor, providing R= 1.3 ± 0.1 nm (Figure S 8b). This size could be compatible with a micelle constituted of interpenetrated sBola C16:0 SS, analogous to what we have observed before on a similar acidic sophorolipid, [START_REF] Van Renterghem | [END_REF] although one must assume a bent configuration of sBola C16:0 SS within the micelle, because its fully-stretched size (> ~4 nm) is longer than the micellar radius.

Finally, a closer look at Figure 3c shows that the 10 wt% sample shows a noticeable Bragg diffraction peak, characteristic of the self-assembled fibers, and indicating that the micelle-fiber equilibrium is both temperature and concentration dependent. To study the kinetic and thermodynamic parameters of the hydrogel formation, we study hereafter the sol-gel transition in the 40°C-5°C temperature interval. 24,[START_REF] Liu | [END_REF]97 become measurable; the gel time occurs when Gˈ overtakes G" (Figure S 9), that is 42 < tgel/min < 60, reported on Figure 4, while a plateau is reached already after 4 h, with Gˈ~ 4x10 3 Pa. These data indicate that, if the exact value of tgel may fluctuate within a range of 15 min to 20 min, the plateau value reached for the complex moduli are in all cases above 10 3 Pa. This value range indicates the formation of a stiff hydrogel with a comparable storage modulus obtained by direct dispersion of the sample in water at 25°C (rest 15 h) (Figure 1c).

The heating-cooling cycle is robust, as shown by its reversibility presented in Figure 5a and Figure 5b: 20 temperature cycles have been carried out between 25°C and 35°C, during a period of 24 h. In order to combine a gel recovery providing satisfying mechanical properties with the highest number of cycles within 24 h, so to exclude a water evaporation, a recovery time of 1 h was chosen. Under these conditions one finds Gˈ= 995 ± 577 Pa, a value which shows that, despite its large variability due to the uncompleted recovery of the gel (which needs at least 4 h, Figure 4), the sol-gel transition for sBola C16:0 SS is highly reproducible and robust.

Quantification of the fiber fraction.

Solution NMR probes mobile molecular species, of which the homonuclear interactions (dipolar coupling in particular) are averaged by Brownian motion. When the mobility is reduced due to a phase transition (e.g., liquid-to-solid), the transverse relaxation time becomes too short (inversely proportional to the peak width) to be detected by solution NMR and the slower species become de-facto "invisible" (the peaks become too broad). 39,35,98,77,99 Under this assumption, one can assume that the normalized peak area is associated to the micellar fraction, XM; as a consequence, we use NMR to monitor the fraction of fibers, XF= (1-XM), in solution at all times, and in particular its evolution with time and temperature. 100 With the intention of quantifying the sol-gel transition, and in particular the micelle-tofiber phase change, we have run a time and temperature-resolved combined 1 H NMR experiment, presented in Figure 6. When a sBola C16:0 SS water solution is heated above 25°C, a liquid micellar phase forms; its typical 1 H NMR signal is shown in Figure 6a When the temperature is cooled instantly from T= 40°C to T= 25°C inside the NMR coil, the first spectrum is recorded as t= 0 h at T= 25°C, then each spectrum is recorded every 30 min over 34 hours (please note that in this case evaporation is limited and contained because the NMR tube is closed). When looking at the first spectrum at t= 0 h, one can already observe a slight broadening of the linewidth if compared to the spectrum at T= 40°C; at longer times, t= 10 h and 20 h, all signals have lost their initial resolution and they have massively broadened, indicating the ongoing micelle-to-fiber phase transition. It is crucial to note that the signal is always attributed to sBola C16:0 SS in a micellar environment; the mobility is reduced more and more by the presence of the fibrillary phase, which could only be characterized directly by solid-state NMR. 55,101 Under these circumstances, we follow the decay of the integrated signals of sBola C16:0 SS in their micellar phase over time (Figure 6b) and quantify the extent of the micelle-to-fiber phase transition. From Figure 6b we conclude that all signals have the same decay profile, which indicates that the entire sBola C16:0 SS molecule is involved in the phase transition; the signal of TMSPd4, which has no time-dependent evolution, is also shown as a control. If one associates the normalized peak area to XM, the fraction of molecules in a micellar environment, one can then easily say that XF= (1-XM) corresponds to the fraction of molecules in the fiber phase. By associating XF measured by NMR to the viscoelastic properties measured by rheology under analogous conditions, one can determine: 1) the minimal fraction of sBola C16:0 SS in the fiber phase that are needed to form a gel and 2) the total fraction of molecules in the fiber phase when the gel can be considered to be at equilibrium. These data are shown in Figure 6c, where the G*(t) profile (black empty circles in Figure 4) having a tgel= 1 h has been selected to represent the viscoelastic behavior of the sample.

Figure 6c shows that only ~20% of sBola C16:0 SS is in the fiber form at tgel and not more than 40% when G* enters the plateau. In fact, even after 35 h, the molecular population in the fiber phase does not exceed 60%, thus showing that the micelle-to-fiber phase transition is not quantitative, although this is enough to obtain a stiff hydrogel (Gˈ~ 4x10 3 Pa). Since the G* plateau is reached after about 4 h, one can suppose that the 10% increase in the NMR signal between 5 h and 35 h can be explained by fiber growth, although with a limited impact on the rheological properties.

Mechanism of fiber formation at T= 25°C.

The sol-gel transition between 40°C and 5°C has been used to quantify further the micelleto-fiber transition. Determination of the sol-gel transition temperature, Tg, and the minimum gel The Avrami equation is commonly used to determine the nucleation and growth mechanism of bulk crystals 102,103 and it has been successfully applied to the study of SAFiN nucleation and growth in LMWG systems both in water and organic solvents. 24,[START_REF] Liu | [END_REF]97 The general form of the Avrami equation is 𝑋𝑋 𝑐𝑐𝑐𝑐 = 1 -𝑒𝑒 -𝑘𝑘𝑡𝑡 𝑛𝑛 , 𝑋𝑋 𝑐𝑐𝑐𝑐 is the volume fraction of the crystalline phase at a given time of the reaction, k is the kinetic constant, t is the time and n is the type of nucleation (heterogeneous or instantaneous) and dimensionality of crystal growth, and where n is commonly contained between 1 and 4, indicating a 1-D or fiber-like, 2-D or platelet-like and 3-D growth.

Finally, the Avrami plot is generally applied in the nucleation and growth phase, so to avoid complex crystallization effects. 104 Plotting ln{-ln[(1-XF)]} against ln(t) gives access to n (slope) and ln(k) (intercept). 𝐺𝐺 * (𝑚𝑚𝑚𝑚𝑚𝑚)-𝐺𝐺 0 * , for rheology experiments (Figure 4). 24,29 In the latter, 𝐺𝐺 * (𝑡𝑡) and 𝐺𝐺 * (𝑚𝑚𝑚𝑚𝑚𝑚) are taken as average values, 29 respectively at t= 0 and on the plateau, and time can be safely replaced by (t-tgel), because the Avrami plot is used to describe mainly the nucleation and growth phase, that is when gelling occurs. 24,[START_REF] Liu | [END_REF] The value of n that we found by NMR is systematically below 1, where one can even observe a two-slope behavior (Figure S 11b), with n= 0.8 and n= 0.3. The Avrami plot obtained from rheology experiments confirm values of n below 1 (n= 0.6). Although unexpected, values of n below unity and multiple slope behavior of the Avrami plot are far from being uncommon: typical interpretation has been attributed to a diffusion-controlled crystallization growth and heterogenous nucleation, where shorter times (ln(t)< 8) are characterized by nucleation and longer times (ln(t)> 8) by growth. 29,97,104,105

Control of hydrogel elastic properties at T= 5°C

The mechanical properties of SAFIN are known to depend on the temperature variation, which controls fiber growth through modulation of the supersaturation. 92 This is observed in the system studied in this work. When the temperature is lowered from 25°C to 5°C, the elastic modulus of the hydrogel is smaller (Figure S 2), and several hypotheses can be formulated. The elastic properties of self-assembled fibrillary systems displaying a Maxwellian behavior was shown to improve at lower temperatures; 14 obviously, this is not the case here, and one can reasonably account for the existence of a gel-to-sol transition temperature below room temperature, as discussed for other systems. 106,107 However, this hypothesis has to be excluded because the system has the classical visual appearance of a gel at temperatures as low as 5°C. The FWHM with time from Tsol (40°C ) to Tgel (5°C and 15°C, Figure S 11a) shifts from about 10 Hz at 40°C to 35 Hz at 5°C, which is also typical sign of gel formation. Interestingly, the FWHM at 25°C at equilibrium (50 Hz) is larger than at 5°C, a fact which is in agreement with the better mechanical properties of the hydrogel prepared at 25°C; nonetheless, the kinetics of gel formation is faster for strong undercooling. At low undercooling, ΔT (Tgel -Tsol)= -15°C, 1.5 h are necessary to achieve a FWHM of 25 Hz, while this value is reached after only few minutes for strong undercooling (ΔT = -35°C).

The second hypothesis for a weaker hydrogel at 5°C can be formulated in terms of structural arguments as a function of the undercooling protocol, which is known to be a crucial parameter in the formation of SAFiN. Under strong undercooling (ΔT = -35°C) conditions, homogeneous nucleation and limited growth dominate the crystallization process, thus resulting in the significant formation of short-sized sBola C16:0 SS fibers. This is confirmed by the value of XF close to unity at T= 5°C (Figure S 12a): almost the entire micellar population transforms into fibers in few minutes. The corresponding Gˈ, Gˈˈ(f) for a sample undergoing strong undercooling is shown in Figure 7 (squares): Gˈ does not exceed few hundred Pascal. This is higher than what was found in Figure 3a (transition from pre-formed fibers at 25°C to 5°C), but the order of magnitude is the same. Interestingly, upon gradual heating to 25°C (Figure S 12a), the fiber-tomicelles phase transition partially takes place and the final proportion of fibers lies in the vicinity of 0.6, as previously found. The corresponding FWHM for this set of experiments is evaluated to be between 27 Hz and 30 Hz, for any temperature explored (Figure S 12c). Given the impact of the undercooling protocol, we have run another experiment in which the system is cooled down from 40°C to 5°C, but with low undercooling (15°C < ΔT < 10°C).

From the NMR data in Figure S 12b and Figure S 12d one finds that XF at 25°C and 5°C are respectively of 60% and 90%, that is comparable to XF obtained with strong undercooling (Figure S 12a). However, the values of the FWHM is higher, and it reaches 30 Hz at 25°C and 37 Hz at 5°C. Under these conditions, the corresponding Gˈ(f) (Figure 7, circles) increases by a factor 100 and it reaches 20 kPa, confirming the NMR data. The activation energy during the cooling cycle at small ΔT is evaluated at Ea= 87.2 ± 8.0 kJmol -1 (Figure S 13), which is in agreement with the activation energy values for the micelle-to-fiber phase transition classically measured in SAFiN, 108,109 between 80 kJmol -1 and 100 kJmol -1 . These experiments show that both temperature and kinetics of the micelle-to-fiber phase transition are able to control the elastic modulus of sBola C16:0 SS hydrogel and values above 10 kPa at 3 wt% can be obtained. Under strong undercooling (ΔT> -30°C), massive fibrillation occurs but growth in time is either limited, thus justifying the poor mechanical properties 20 or, as commonly found for SAFiN, a higher degree of branching is developed. The branching distance is generally inversely proportional to the extent of undercooling: the higher ΔT, the smaller the branching distance, so the network is less homogeneous and contains more fractal spherulites, and branched gels signature can be obtained. A common feature in this case is the smaller value of the Avrami coefficient n, which is also found to be smaller in this system at 5°C (n< 0.1, estimated from the time evolution of XF at T= 5°C and measured by 1 H NMR, data not shown) than at 25°C (n= 0.3, Figure S 11b). 24,92 Branched gels are known to have lower elastic moduli with respect to homogeneous entangled fibrillar gels, 25,91 a fact which can explain the worst elastic properties of sBola C16:0 SS hydrogels at 5°C under strong undercooling. On the contrary, low temperature combined with gentle cooling and small gradual undercooling provide a large extent of fibrillation with minimized branching, leading to higher homogeneity, thus favouring the formation of stronger gels.

Conclusion

In this work we study the hydrogel-forming properties of a new biobased bolaform biosurfactant produced from a modified strain of S. bombicola. When the compound is dispersed in water below 28°C, it self-assembles into twisted fibers having a diameter below 20 nm and length in the order of magnitude of 100 nm. The fiber length increases with time, which induces entanglement and consequential increase of the viscosity. At equilibrium, the elastic modulus as a function of concentration scales as a power law with an exponent of 2.4, which is very close to the predicted value of 2.25 found in entangled polymers gels by de Gennes. 82 Under these conditions, these materials exhibit elastic modulus in the order of the kPa. micelles

X F ~ 60% @ 25°C X F ~ 90% @ 5°C
X F : fiber fraction

S. bombicola ∆at ∆sble ∆fao1

Bolaform sophoroside 1000x

The system is found to have a gel-transition temperature of 28°C and a minimal gel concentration of 0.25 wt%. Above the Tgel, micelles are formed. Below Tgel, the fiber phase is stable, although both phases coexist in the order of 60:40 fibers:micelles at 25°C, while at 5°C 90% of the compound assembles into fibers. The elastic modulus of the hydrogel can be controlled by the undercooling protocol between the gel and sol phase. At strong undercooling (-35°C), the hydrogel is much weaker than for small, gradual, undercooling of about 10°C/15°C, where Gˈ can reach 20 kPa at 5°C. The thermodynamic and kinetics analysis show that the fibers undergo a diffusion-limited growth during the sol-gel transition with an activation energy below 100 kJ.mol - The fit in Figure S 8b is intended to provide another way to estimate the radius of the micelles and it is not intended to precisely describe the structure of the micelle, most likely expected to be core-shell, as found in similar system. 3 For this reason, we have used a simple homogeneous sphere form factor model with the following parameters: Scattering Length Density (SLD) of the solvent (D2O): 6.34 * 10 -4 𝑛𝑛𝑛𝑛 -2 (fixed). Scale: 0.03 (fixed). In the model proposed by SasView, this parameter is equivalent to the volume fraction, which corresponds to 3 wt%. SLD of the sphere: 2.97 * 10 -4 𝑛𝑛𝑛𝑛 -2 (fitted). The SLD of the micelle is expected to be contained between ~4 * 10 -4 𝑛𝑛𝑛𝑛 -2 , corresponding to the SLD level of glucose in D2O (including a full OH/OD exchange), and ~10 -5 𝑛𝑛𝑛𝑛 -2 , corresponding to the level of an aliphatic chain in water. The SLD values are estimated using the SLD calculator tool in the 3.1.2 version of Sasview software.

Radius of the sphere: 1.3 𝑛𝑛𝑛𝑛 (fitted). Background: 0.017 cm -1 (adjusted through fitting).

  . The background sample (D2O) signal was subtracted from the experimental data. Data treatment is done with the home-made software package provided at the beamline. Absolute values of the scattering intensity are obtained from the direct determination of the number of neutrons in the incident beam and the detector cell solid angle. The 2-D raw data were corrected for the ambient background and empty cell scattering and normalized to yield an absolute scale (cross section per unit volume) by the neutron flux on the samples. The data were then circularly averaged to yield the 1-D intensity distribution, I(q).

  tested, and the obtained solution is generally fluid at any concentration while mixing. However, upon a rest time of few minutes, the viscosity increases, and whatever the concentration, gels are translucid, as shown by the classical inverted tube test shown on Figure1b. Concentration-and time-dependent rheology (Figure1c) experiments show that the storage modulus, Gˈ, of the solution few minutes after mixing (t= 0 h) is systematically below 1 Pa for the 1 wt% system, which characterizes a fluidic solution, while Gˈ increases at about 10 3 Pa for a 10 wt% solution at t= 0 h. Upon rest overnight (t= 15 h), Gˈ strongly increases and it varies between 10 2 Pa and 10 4

Figure 1 -Figure 2 .

 12 Figure 1 -a) Chemical structure of sBola C16:0 SS. b) Inverted tube test method visually showing the hydrogel formation as a function of concentration (in wt%) at preparation time, t= 0 h. c) Concentration-dependency of the plateau storage modulus, Gˈ at two different rest time values and T= 25°C. A selection of Gˈ(f) are given in Figure 2. d) SAXS data collected as a function of concentration (wt%) at room temperature and at t= 0 h. e) Cryo-TEM micrograph obtained on solution at C= 1 wt%, room temperature and t= 0 h.

Figure 2 -

 2 Figure 2 -Measurement of the storage and loss moduli for different sBola C16:0 SS concentration (t= 15 h ; T= 25°C) values as a function of the frequency. Experiments were carried out at at selected τ values so to be in the linear domain (0.2-1 Pa).

  Figure S 2a shows the timedependent evolution of sBola C16:0 SS at 3 wt% at rest (f= 0 Hz) and at f= 1 Hz over a period of 20 h, without evaporation, showing no specific difference. The hydrogel displays a Gˈ value one order of magnitude higher than Gˈˈ, the loss modulus from t= 0 h and until the end of the experiment, indicating an elastic behavior. The storage modulus Gˈ starts at about 200 Pa at t= 0 h and increases by a factor ten over 20 h. Beyond 20 h and due to evaporation, the experiment could not be pursued any longer; however, although a plateau is not reached yet after 20 h, the slope of Gˈ(t) in the range 15 < t (h) < 20 is 0.03, a value about 16 times smaller than in the first hour of the experiment, thus indicating that the kinetics has considerably reduced and the gel can be de-facto considered to be close to equilibrium. The time-dependency of the mechanical properties raises questions about the nano and mesoscale structure. SANS experiments are presented in Figure S 2b. Despite the truncated q-

  these hypotheses:Figure S 4 shows that the sample at 3 wt% and t= 0 h is significantly composed of self-assembled fibers displaying the same morphological features described for the sample at 1 wt% (Figure1eand Figure S 5). After t= 15 h (C= 1 wt%, Figure S 6), when the hydrogel has the highest storage modulus (Figure 1c), cryo-TEM confirms that the mesoscale morphology does not vary while the length increases, confirming that both fiber growth and entanglement are responsible for the gel formation. The high and low-magnification cryo-TEM images in Figure S 6 show that the sBola C16:0 SS SAFiN is constituted by a dense network of entangled bundles of fibers, most of which seem to be composed of twisted ribbons having a pitch of about 250 nm and a cross-section below 20 nm. LMWG fibers can nucleate and grow in at least three different ways, each one providing a

be identified: 1 )

 1 at T< 20°C the hydrogel is weak, characterized by Gˈ~ 10 2 Pa (Gˈ and Gˈˈ at T= 5°C as a function of time are given in Figure S 7 and Figure S 2 in the 20°C < T < 35°C region, the hydrogel is the stiffest, with Gˈ~ 10 3 Pa; 3) at T> 35°C, Gˈ abruptly drops down and cannot be properly measured: the sample becomes a solution. The strength of the gel is studied as a function of stress applied between 10°C and 30°C. The short length of the viscoelastic linear plateau in the range of 0.8 Pa to 1.

Figure 3 -

 3 Figure 3 -Experiments performed on sBola C16:0 SS hydrogel at t= 15 h and C= 3 wt%. a) Evolution of the storage and loss moduli with temperature at f= 1 Hz, τ= 0.5 Pa. b) Evolution of the storage modulus as a function of the applied stress at fixed frequency (f= 1 Hz) for temperature range between 10°C and 30°C. c) SANS data recorded as a function of concentration and temperature (T= 25°C and T= 40°C).

Figure 4

 4 shows the time-dependency of the complex modulus G*, obtained from the storage and loss moduli, Gˈ and Gˈˈ according to the standard relationships tan 𝛿𝛿 = 𝐺𝐺 ′′ 𝐺𝐺 ′ , 𝐺𝐺 ′ = 𝐺𝐺 * cos 𝛿𝛿 and 𝐺𝐺 ′′ = 𝐺𝐺 * sin 𝛿𝛿; the values for Gˈ and Gˈˈ are given in Figure S 9. Experiments have been run at T= 25°C after cooling a sBola C16:0 SS solution (3 wt%), initially set at T= 35°C for 10 minutes. At the outset, G* is very low, as the material is very fluid. In the interval between 30 min and 50 min, Gˈ and Gˈˈ b) c)

Figure 4 -Figure 5 -

 45 Figure 4 -Time-dependent evolution of the complex modulus, G* (f= 1 Hz, τ= 1 Pa), recorded on a sBola C16:0 SS hydrogel (C= 3 wt%) heated at T= 35°C for 10 min and brought to T= 25°C. Time scale starts at T= 25°C. The red and black empty circles represent two different samples under the same experimental conditions. The corresponding Gˈ and Gˈˈ are shown in Figure S 9. Values for tgel are reported for each curve and they are determined at the crossing between Gˈ and Gˈˈ (Figure S 9). Sampling was done as follows: 60 measurements/h up to 1h, then 1 measurement/h until the end of the experiment.

  at T= 40°C. The aliphatic signal is between 1.0 and 2.0 ppm, the sophorose signal is detected between 3.0 and 4.1 ppm while the anomeric proton of sophorose is at 4.55 ppm. The sharp signal between 4.5 and 5.0 ppm is attributed to water and a signal at 0.0 ppm (not shown in the figure) belongs to TMSPd4, used as a reference throughout the experiment and contained in an independent coaxial insert, to avoid sample. In the micellar phase at T= 40°C, the spectrum is characterized by wellresolved peaks, which reflect the typical fingerprint of a fast-tumbling molecular motion,

Figure 6 -

 6 Figure 6 -a) 1 H solution NMR spectra of the sBola C16:0 SS (C= 3 wt%) sol-gel transition in D2O. The sample is equilibritated at T= 40°C in the NMR tube outside the probe and then inserted in the probe set at T= 40°C. After recording the first spectrum, the probe temperature is lowered in-situ at T= 25°C. The time t= 0 h starts at T= 25°C. b) Time evolution of the 1 H NMR normalized peak area values sampled every 30 min and measured for the chemical shift values given on the decays profiles. The black hyphens refer to TMPSd4 dissolved in D2O inside an insert coaxial tube, used as an external control both for chemical shift and peak area.

  concentration, Cgel, at T= 25°C are determined in Figure S 10a and Figure 8b by mean of both 1 H solution NMR. Evolution of the FWHM and normalized peak area signals recorded on the alkyl resonance at 1.3 ppm of sBola C16:0 SS indicate a break in their temperature-dependent evolution between 28°C and 29°C (Figure S 10a). A Tg= 28.3 ± 0.2 °C is estimated, although the nucleation and growth probably start at higher temperature, in the vicinity of 35°C, where the peak area starts to decrease while the FWHM starts to increase. These data are in very good agreement with the estimation of Tgel by rheological measurements. Figure S 10c and Figure S 10d presents the timedependent evolution of Gˈ and Gˈˈ across the gel-to-sol and sol-gel transition, respectively. One can measure an average value of Tgel= 28.1 ± 1.0 °C, where the larger error depends on the different methodology used to estimate Tgel (please, refer to Figure S 10c,d). It is worth noting that in the gel-to-sol transition, the estimated Tgel corresponds to the inflection point where Gˈ decreases, whereas Gˈˈ>Gˈ at T= 32.5°C. The combined set of NMR and rheology data show that in the solgel transition, nucleation probably starts above 30°C, while growth starts in the vicinity of 28°C. On the contrary, in the gel-to-sol transition, the fibers start to break in the vicinity of 28°C, while above 30°C the system loses its gel behavior. Finally, one can estimate Cgel as a function of concentration using NMR: the narrow values of the FWHM indicate a sol phase. The evolution of the FWHM with concentration at T= 25°C (Figure S 10b) identifies a Cgel= 0.25 ± 0.04 wt%.

Figure S 11b

  Figure S 11b and Figure S 11c show the Avrami plots obtained from NMR and rheology data at T= 25°C respectively, and where Xcr≡ XF= (1-XM)= (1-A1.3), 100 where A1.3 is the normalized peak area at δ= 1.3 ppm for NMR experiments (Figure 6b), while 𝑋𝑋 𝑐𝑐𝑐𝑐 ≡ 𝑋𝑋 𝐹𝐹(𝑐𝑐ℎ𝑒𝑒𝑒𝑒) 𝐺𝐺 * (𝑡𝑡)-𝐺𝐺 0 *

Figure 7 -

 7 Figure 7 -Frequency-dependence of the elastic Gˈ and loss Gˈˈ moduli of sBola C16:0 SS hydrogel at T= 5°C. This temperature is achieved by a large (ΔT < -35°C, squares) and low (ΔT= -15°C, -10°C, -10°C, circles) undercooling. Starting temperature is T= 40°C. All data are recorded in the linear domain regime.

  * § §: ΔT= -35°C (τ= 0.6 Pa) *: ΔT= -15°C, -10°C, -10°C (τ= 15 Pa) 40°C  5°C

Figure 8 -

 8 Figure 8 -Schematic summary of the sol-gel transition in water of the microbial sBola C16:0 SS glycobolaamphiphile. Temperature controls the volume fraction of the fibers (90% at 5°C), while undercooling controls the elastic modulus (Gˈ> 10 kPa for small gradual undercooling).

Figure S 1 Figure S 2 Figure S 3 Figure S 4 Figure 13 S2Figure S 1 -S3Figure S 2 x10Figure S 3 -S5Figure S 4 -S6Figure S 5 -S7Figure S 6 -

 123413123456 Figure S 1 Figure S 2 Figure S 3 Figure S 4

Figure S 4 ,

 4 Figure S 4, Figure S 5 and Figure S 6 show that morphology changes are not responsible for the increase in the mechanical properties of the hydrogel. The low-magnification cryo-TEM images for the 3 wt% (Figure S 4) and 1 wt% (Figure S 5) samples at t= 0 h obviously show a higher density of the fibers in the 3 wt% sample, which can explain the higher storage modulus at t= 0 h for this sample. However, the time-dependent increase of Gˈ can be explained by an increase in length of the fibers themselves over time, thus confirming from a visual point of view the analysis of the time-dependent evolution of Gˈ(C). At t= 0 h, one can fit the length distribution of the 1 wt% sample with a log-normal function having mean and standard deviation respectively of μ= 436.6 ± 30.8 nm and σ= 0.6 ± 0.1 (Figure S 5). Despite the limited number of sampled population (N= 160), due to the strong aggregation of the fibers on the cryo-TEM grid, a factor which made it impossible to analyze the length distribution at 3 wt%, one should retain the presence of fibers with an overall finite size when the sample is prepared. On the contrary, the length of the fibers strongly increases and it becomes "infinite" (with respect to the cross-section) after 15 hours (lowmagnification image in Figure S 6. Cryo-TEM confirms the rheology assumptions: fiber growth and entanglement are responsible for the gel formation and its elastic properties.

Figure S 7 -

 7 Figure S 7 -Measurement of the time-dependent (f= 1 Hz, τ= 1 Pa) storage and loss moduli for sBola C16:0 SS at C= 3 wt% and T= 5°C

Figure S 9 -Figure S 10 Figure S 11 Figure S 12 Figure S 13

 910111213 Figure S 9 -Time-dependent evolution of the storage and loss moduli (f= 1 Hz, τ= 1 Pa) recorded on a sBola C16:0 SS hydrogel (C= 3 wt%) heated at T= 35°C for 10 min and brought to T= 25°C. Time scale starts at T= 25°C. a) and b) are two different samples, recorded on the same system so to show the fluctuations in terms of tgel.

Table 1 -Summary of the major structural and mechanical parameters for the sBola C16:0 SS hydrogel obtained after dispersion in water at T= 25°C. C is concentration, LF is the fiber length (median of log-normal distribution), 𝑮𝑮 𝒎𝒎𝒎𝒎𝒎𝒎 ′ is the value of elastic modulus 𝑮𝑮 ′ at equilibrium.

 1 The entire set of data collected so far, which are summarized in Table1and Table2and represented in Figure8, indicate that the magnitude of gelation is temperature-dependent but not process-dependent, because XF is practically the same at any temperature, independently of ΔT and heating/cooling cycles as shown in FigureS12. Nonetheless, both NMR and rheology show that the strength of the hydrogel increases when the sol-gel transition is approached with short and

	C/wt%	time/h	LF/nm	𝑮𝑮 𝒎𝒎𝒎𝒎𝒎𝒎 ′	/kPa	Technique
	3 10	15	-	2 44	Rheology
	1	0	436.6 ± 30.8	-	Cryo-TEM
	gradual undercooling: low undercooling favours an heterogeneous nucleation and a diffusion-
	controlled mechanism, which promotes an infinite 1D fiber growth, as classically found in
	fibrillary gels. 81

Table 2 -Summary of the major structural, mechanic and thermodynamic parameters for the sBola C16:0 SS hydrogel (C= 3 wt%) obtained after a sol-gel transition (starting temperature is 40°C). T is temperature, C is concentration, t is time. Tgel is the hydrogel-forming temperature at Gˈ,Gˈˈ crossover, ΔT is the undercooling defined as (Tgel-Tsol), Cgel is the hydrogel-forming concentration, tgel is hydrogel-forming time, n is the Avrami exponent, XF is the fraction of the compound in the fiber phase, Ea is the activation energy, 𝑮𝑮 𝒎𝒎𝒎𝒎𝒎𝒎 ′ is the value of elastic modulus 𝑮𝑮 ′ at equilibrium.

 2

	Sol-gel transition	T/°C	ΔT/°C	Technique
	Tgel/°C	28.3 ± 0.2	-	-	NMR
	Tgel/°C	28.1 ± 1.0	-	-	Rheology
	Cgel/wt%	0.25 ± 0.04	25	-	NMR
	tgel/h	0.85 ± 0.15	25	-15	Rheology
	n	0.3 ± 4.10 -4 0.6 ± 0.1	25	-15	NMR Rheology
	XF	60 %	25	-15	NMR

  [START_REF] Flory | Faraday Discuss[END_REF] , as expected for low molecular weight gelators.This works shows that a new structural variation of the well-known sophorolipid biosurfactant spontaneously forms a hydrogel in water in the 5°C -28°C temperature range and with tuned elastic modulus between 100 Pa and 20 kPa. These properties have never been reported neither for sophorolipids nor for any other biobased glyco-amphiphile. This research should open the perspectives of employing this new class of biobased amphiphiles in a broad field of applications going from cosmetics to tissue engineering.

Acknowledgements

This work received financial support from the Institut Laue-Langevin (ILL), Grenoble, France, under the experiment number 9-12-481, and by the European Synchrotron Radiation Facility (ESRF), Grenoble, France, under the experiment number MX1821. This work benefited from the use of the SasView application, originally developed under NSF award DMR-0520547. SasView contains code developed with funding from the European Unionˈs Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement No 654000. Dr. François Ribot (LCMCP) is kindly acknowledged for his assistance on the NMR experiments. Finally, also financial support was received from European Horizon 2020 Bio-Based Industries (BBI) Consortium Project Carbosurf (grant number 669003).


Member, IEEE M Gaillardin
email: marc.gaillardin@cea.fr

M Martinez

Senior Member, IEEE S Girard

Member, IEEE, P. Paillet V Goiffon

Senior Member, IEEE J L Leray

Fellow, IEEE P Magnan

Member, IEEE Y Ouerdane

A Boukenter

C Marcandella

O Duhamel

Member, IEEE M Raine

N Richard

F Andrieu

S Barraud

Member, IEEE O Faynot

P Paillet

High Total Ionizing Dose and Temperature Effects on Micro-and Nano-Electronic Devices

Keywords: Annealing, bulk silicon, fully depleted (FD), high temperature, MGy irradiation, partially depleted (PD), silicon on insulator (SOI), total ionizing dose (TID)

This paper investigates the vulnerability of several micro-and nano-electronic technologies to a mixed harsh environment involving high total ionizing dose at MGy levels and high temperature. Such operating conditions emerge today for several applications like new security systems in existing or future nuclear power plants, fusion experiments, or deep space missions. In this work, the competing effects of ionizing radiations and temperature are characterized in elementary devices made of MOS transistors from several technologies. First, devices are irradiated using a radiation laboratory X-ray source up to MGy dose levels at room temperature. Devices are either grounded or biased during irradiation to simulate two major circuit cases: a circuit which waits for a wake up signal, representing most of the lifetime of an integrated circuit operating in a harsh environment, and a nominal circuit function. Devices are then annealed at several temperatures to discuss the post-irradiation behavior and to determine whether an elevated temperature is an issue or not for circuit function in mixed harsh environments.

I. INTRODUCTION

E LECTRONIC systems designed for harsh environments are used in various applications, such as high energy physics instruments, fusion reactors or space missions. Since recent events which occur in Fukushima Daichii, they have been pointed out as critical issues for safety systems in nuclear facilities, when used to monitor parameters such as ionizing radiation level, temperature or pressure levels either in the nuclear core or in the spent-fuel pools of nuclear power plants [START_REF] Fleetwood | How to protect nuclear reactor electronics[END_REF]. Radiation tolerant electronic components dedicated to other applications like space exist [START_REF] Johnston | Space radiation effects and reliability considerations for micro-and optoelectronic devices[END_REF], [START_REF] Lacoe | Designing radiation hardened CMOS microelectronic components at commercial foundries: Space and terrestrial environments and device and circuit techniques to mitigate radiation effects[END_REF]. They are designed to meet the specifications required for the targeted environment, which commonly implies lower Total Ionizing Dose (TID) levels than those encountered by electronic components in the case of an accident in nuclear power plant. For instance, most space missions consider that devices and Integrated Circuits (ICs) have to withstand less than 1 kGy while electronic systems in nuclear power plants have to be hardened up to several MGy ionizing doses. Specific studies should thus be performed to identify technologies which may present enough hardening potential to meet such requirements. Only few papers have been dedicated to high TID effects in electronic devices [START_REF] Leray | Activation energies of oxide charge recovery in SOS or SOI structures after an ionizing pulse[END_REF], [START_REF] Leray | Limiting factors for SOI VLSI high-level hardness : Modeling and improving[END_REF], [START_REF] Leray | CMOS/SOI hardening at 100 Mrad(SiO2)[END_REF], [START_REF] Faccio | Radiation-induced edge effects in deep submicron CMOS transistors[END_REF]. Most of them discussed the hardening level of either bulk or SOI technologies in the frame of the Large Hadron Collider using dedicated test structures. More recently, a review of the MGy dose effects induced in a wide range of modern and innovative technologies including bulk, Partially Depleted (PD) Silicon On Insulator (SOI), Fully Depleted (FD) SOI and FinFET structures has been presented [START_REF] Gaillardin | Investigations on the vulnerability of advanced CMOS technologies to MGy dose environments[END_REF].

This paper being more focused on nuclear facilities, it considers both high TID and high temperatures occurring in nominal and accidental conditions. Few papers have already addressed such mixed temperature and irradiation issues but considering ionizing doses of about few Mrads [START_REF] Fleetwood | High temperature silicon-on-insulator electronics for space nuclear power systems: Requirements and feasibility[END_REF], [START_REF] Shaneyfelt | Effects of irradiation temperature on MOS radiation response[END_REF], [START_REF] Fleetwood | Effects of isochronal annealing and irradiation temperature on radiation-induced trapped charge[END_REF], two orders of magnitude lower than recent requirements specified for nuclear power plants particularly in accidental conditions. To the authors' knowledge, the combination of MGy irradiation and elevated temperature is considered as the most constraining scenario for accidental conditions. Test standards [START_REF]Assessment and management of ageing of major nuclear power plant components important to safety : In containment instrumentation and control cables[END_REF], [START_REF] Nf M | Procédure de qualification des matériels électriques installés dans l'enceinte de confinement des réacteurs à eau sous pression et soumis aux conditions accidentelles[END_REF] to qualify equipments designed for such an environment thus recommend performing a two-step experimental procedure, the first one being devoted to irradiations up to MGy doses, followed by a thermodynamic test. The main issue discussed in this paper is to determine how the temperature affects the TID response of elementary test structures fabricated in various technologies. The silicon film thickness, the front-end and the back-end of line processes are similar in all cases, but such ICs can be fabricated on SOI substrates with various Buried OXide (BOX) thicknesses nm, nm or nm. This may affect the FDSOI device response to MGy dose levels since it was already demonstrated to significantly modify the TID response of such integrated devices at 10kGy [START_REF] Gaillardin | Impact of SOI substrate on the radiation response of ultra-thin transistors down to the 20 nm node[END_REF]. Table I summarizes the nominal voltages used for each technology.

II. EXPERIMENTAL DETAILS

All devices are mounted in standard Dual-In Line packages for both irradiation and temperature experiments.

B. Irradiation and Annealing Tests Description

First, devices are irradiated using a 10 keV X-ray laboratory source at a constant dose rate of 50 Gy/s. The TID is deposited in several steps up to a maximum of 1 MGy. Electrical measurements are performed at each TID step. Static drain-current vs gate-voltage electrical characteristics are measured with a HP 4145 parametric analyzer through a Keithley 707 switching matrix. All parameter extractions presented in this paper come from these static measurements including the threshold voltage , the subthreshold slope , the transconductance , the drive current ( at ) and the leakage current ( at V and ). Devices are either grounded or biased during irradiation. In the following, these two bias configurations are referred as the NULL-case and OFF-case respectively. In the NULL-case, all terminals are grounded. The device can thus be considered in a stand-by mode signal. In contrast, it is in its nominal bias configuration when the device is biased during irradiation such as in the OFF-state. In this bias case, only the drain electrode is biased to the nominal voltage of the technology when other terminals are grounded. These two configurations aim at representing most of the device functions to simulate the main profile of use of electronic devices in such harsh operating conditions, especially in accidental conditions, for which an IC is most of the time in a stand-by mode. Then, isochronal annealing experiments are performed using a dedicated setup to investigate the effect of temperature. Three annealing steps are done at 150 , 200 and 250 , each of them during 1 hour. Electrical measurements are always performed at room temperature. A simple schematic of the annealing steps is plotted in Fig. 1. The experimental setup is optimized to reduce to only few minutes the rise time and the fall time between each temperature steps.

The first annealing step (1 hour, 150 ) is done to be consistent with requirements described in standards for test of in-containment instrumentation in accidental conditions [START_REF]Assessment and management of ageing of major nuclear power plant components important to safety : In containment instrumentation and control cables[END_REF], [START_REF] Nf M | Procédure de qualification des matériels électriques installés dans l'enceinte de confinement des réacteurs à eau sous pression et soumis aux conditions accidentelles[END_REF]. The two others are performed to get additional insights on temperature effects induced in highly irradiated silicon-based devices and to discuss the phenomena at stake in a wider range of operations.

III. MGY DOSE SENSITIVITY OF SI-BASED TECHNOLOGIES

Two major TID induced effects in MOS devices are reported in the literature [START_REF] Schwank | Total dose effects in MOS devices[END_REF], [START_REF] Oldham | Total ionizing dose effects in MOS oxides and devices[END_REF], [START_REF] Ma | Ionizing radiation effects in MOS devices and circuits[END_REF]: on the one hand the trapping of radiation-induced generated charges in the oxide bulk, and on the other hand the generation of interface traps at silicon-dielectric interfaces. Physical mechanisms leading to oxide-charge trapping and interface traps generation are widely studied since it is needed to mitigate their effects on electronic device characteristics. Their respective contributions are usually estimated using the charge separation technique described in [START_REF] Mcwhorter | Simple technique for separating the effects of interface traps and trapped-oxide charge in metal-oxidesemiconductor transistors[END_REF] which allows to attribute a voltage, either or , and then a density or for oxide-trapped charge and interface traps respectively.

In most cases, radiation-induced trapped charges are positive in MOS devices. It leads to the modification of electrical characteristics of electronic devices and ICs depending on their technology, architecture, fabrication process and geometry. This may shift the electrical characteristics I-V of MOS transistors when the total amount of trapped charge is enough to change the electrostatic potential in the active silicon layer. All main electrical parameters ( and ) can thus be modified due to ionizing radiation. Furthermore, oxide-trapped charges can also trigger uncontrolled parasitic conduction paths such as parasitic lateral transistors due to trapping in field oxides [START_REF] Shaneyfelt | Challenges in hardening technologies using shallow trench isolations[END_REF], enhanced narrow channel effects [START_REF] Faccio | Radiation-induced edge effects in deep submicron CMOS transistors[END_REF], [START_REF] Gaillardin | Enhanced radiation induced narrow channel effects in a commercial m bulk technology[END_REF] or parasitic back transistors in SOI technologies [START_REF] Schwank | Radiation effects in SOI technologies[END_REF]. This could also shift the main electrical characteristics of FDSOI devices due to both charge trapping into the BOX and coupling effects inherent to this specific SOI technology [START_REF] Lim | Threshold voltage of thin film silicon-oninsulator (SOI) MOSFET's[END_REF], [START_REF] Gaillardin | Impact of SOI substrate on the radiation response of ultra-thin transistors down to the 20 nm node[END_REF], [START_REF] Jenkins | Radiation response of fully-depleted MOS transistors fabricated in SIMOX[END_REF], [START_REF] Schwank | New insights into fully-depleted SOI transistor response after total-dose irradiation[END_REF], [START_REF] Paillet | Total ionizing dose effects on deca-nanometer fully depleted SOI devices[END_REF].

In contrast, interface traps are either negative or positive in NMOS or PMOS transistors respectively. In both cases, they degrade the subthreshold slope of I-V characteristics.

A. Bulk Technology

The TID behavior of I/O transistors fabricated using a standard m bulk technology is investigated as in [START_REF] Gaillardin | Enhanced radiation induced narrow channel effects in a commercial m bulk technology[END_REF]. Fig. 2 shows the characteristics obtained on a standard Open Layout Transistor (OLT) with a wide design (transistor width m and gate length m) from pre-rad (black squares) up to 1 MGy (red circles). It highlights the most relevant parameters which will be used in the following of this study.

The open layout transistor (Fig. 2(a)) exhibits a significant increase of the leakage current ; this is due to the buildup of oxide-trapped charge in field oxides which triggers the parasitic lateral conduction [START_REF] Shaneyfelt | Challenges in hardening technologies using shallow trench isolations[END_REF]. By contrast and as expected, no significant shift of the threshold voltage is observed, meaning that Radiation-Induced Narrow Channel Effect (RINCE) [START_REF] Faccio | Radiation-induced edge effects in deep submicron CMOS transistors[END_REF], [START_REF] Gaillardin | Enhanced radiation induced narrow channel effects in a commercial m bulk technology[END_REF] does not occur in the wide OLT (Fig. 2(a)). Even so, radiation-induced effects occur at MGy dose levels despite the fact that devices are grounded during irradiation. The device response is less impacted than when devices are biased during irradiation [START_REF] Gaillardin | Investigations on the vulnerability of advanced CMOS technologies to MGy dose environments[END_REF] but the effect remains significant. Using an enclosed geometry [START_REF] Faccio | Radiation-induced edge effects in deep submicron CMOS transistors[END_REF] in the transistors' design (ELT, Fig. 2(b)) strongly mitigates these effects since oxide-charge trapping only occurs in the thin gate oxide. In that case, characteristics are mostly changed due to the buildup of interface traps, which only degrades the subthreshold slope and then the drive current . Fig. 3 summarizes the leakage current increase with TID measured on bulk devices. As expected, the ELT (red circles) presents high TID tolerance with very limited modifications of its electrical characteristics; devices being grounded (filled red circles) or biased (open red circles) during irradiation. This geometry features no interface between active silicon and thick field oxides which intrinsically removes the parasitic lateral conduction leading to the observed increase on the OLT (black squares). Using ELT also mitigates the RINCE which can occur in narrow transistors.

In the same time, Fig. 2 shows that the drive current decreases by about 30% at 1 MGy in both the OLT and the ELT. This significant lowering of the drive-current with TID must be considered in the circuit design to get margins enough to prevent circuit failure due to the lowest drivability obtained after irradiation. Actually, the circuit won't be able to operate in nominal conditions: a slower speed than the nominal one should be used to ensure reliable circuit functions.

From the circuit design's standpoint, the main drawback remains the significant area penalty induced by this hardening by design technique for both PMOS and NMOS transistors.

B. Partially Depleted SOI Technology

PDSOI technologies have already demonstrated their capabilities to withstand MGy dose irradiations. In the late eighties, pioneering works performed by Leray et al. [START_REF] Leray | Activation energies of oxide charge recovery in SOS or SOI structures after an ionizing pulse[END_REF], [START_REF] Leray | Limiting factors for SOI VLSI high-level hardness : Modeling and improving[END_REF], [START_REF] Leray | CMOS/SOI hardening at 100 Mrad(SiO2)[END_REF] demonstrated the hardening potential of SOI technologies to MGy dose levels for Large Hadron Collider applications. More recently, PDSOI devices designed with external body contacts exhibited a very promising tolerance to MGy dose [START_REF] Gaillardin | Investigations on the vulnerability of advanced CMOS technologies to MGy dose environments[END_REF] either grounded or biased during irradiation. In such devices, the buildup of oxide-trapped charge in the BOX induces a parasitic back conduction which increases the leakage current for devices biased during irradiation. By contrast, it was demonstrated that when devices are grounded during irradiation, the amount of trapped charge in the BOX is not enough to modify the electrical characteristics even after 3 MGy. This is confirmed by the parameter extractions plotted in Fig. 4 for body contacted devices only. Actually, all other electrical parameters are almost unaffected by TID.

The current slightly increases from 0.3 MGy for devices grounded (filled squares) or biased (open squares). The leakage current shifts reach less than nA at 1 MGy which stays within very low leakage current range. In this particular case, is larger when the device is grounded during irradiation than when it is OFF-biased. Actually, the OFF-case does not seem to be the worst case for such a device. Indeed, larger modifications have been presented in [START_REF] Gaillardin | Investigations on the vulnerability of advanced CMOS technologies to MGy dose environments[END_REF] for similar devices biased in the ON-state during irradiation (gate biased to the nominal voltage, other terminals grounded). However, one should note that the presented in Fig. 4 are still small despite the high TID value. Such a technology appears highly tolerant to TID all the more because the drive current stays within the same value (less than 2% change, not shown here, see Fig. 7 in Section III-D for more details) even after a 1 MGy irradiation.

By contrast, PDSOI transistors with a standard design without external body contacts, named as floating body transistors, behave in a different manner which will be close to the one of bulk transistors studied in the previous section. Floating body transistors feature interfaces between the active silicon and field isolations contrary to body contacted devices. Such devices then suffer from the inherent parasitic lateral conduction due to buildup of oxide-trapped charge in field oxides leading to potential additional increase.

C. Fully Depleted SOI Technology

Finally, the FDSOI technology offers better scalability for nanometer scale era than both bulk and PDSOI technologies. This technology provides high performances, low power consumption and improved process variability [START_REF] Fenouillet-Beranger | Impact of a 10-nm ultra-thin box (UTBOX) and ground plane on FDSOI devices for 32 nm and below[END_REF], [START_REF] Brunet | New insights on Vt stability of HK/MG stacks with scaling in 30 nm FDSOI technology[END_REF] which are all key issues for highly integrated technologies intended for commercial market. However, its TID sensitivity is strongly governed by oxide-charge trapping in the BOX which modifies the main electrical characteristics due to electrostatic coupling effects inherent to the FDSOI structure [START_REF] Lim | Threshold voltage of thin film silicon-oninsulator (SOI) MOSFET's[END_REF]. Some papers [START_REF] Gaillardin | Impact of SOI substrate on the radiation response of ultra-thin transistors down to the 20 nm node[END_REF], [23], [START_REF] Schwank | New insights into fully-depleted SOI transistor response after total-dose irradiation[END_REF], [START_REF] Paillet | Total ionizing dose effects on deca-nanometer fully depleted SOI devices[END_REF] show that the TID sensitivity of FDSOI technologies is mainly driven by the body doping level, designed with or without body contacts, the gate length and above all the buried oxide thickness. All studies previously published were dedicated to TID below 10 kGy, so the following investigations will focus on the TID behavior of FDSOI transistors fabricated on various SOI substrates, i.e. various BOX thicknesses, at MGy dose levels. All devices were fabricated using a similar process leading to the same active silicon thickness, gate stack and doping levels. Their TID responses can thus be directly compared.

Fig. 5 summarizes the characteristics of FDSOI devices on three different SOI substrates: nm (black squares), nm (red circles) and nm (blue triangles). Results for transistors with both a short gate (filled symbols) and a long gate (open symbols) are displayed.

Short gate transistors all exhibit significant modifications of their electrical performances with TID. The drive currents increase by several decades when almost decreases with TID, especially on the device with nm (filled black squares). Decreasing the BOX thickness down to the thinnest value nm enhances the TID tolerance by reducing the shifts reported both on and compared to the other SOI substrate options. This is even more obvious on transistors with a long gate length ( m, open blue triangles). Only the drive current exhibits variations but with a leakage current which stays within the same order of magnitude. Furthermore, results obtained on devices processed with Ultra-Thin BOX (UTB) substrates ( nm and 11 nm) are characteristic of their overall TID behavior since it was previously demonstrated that this behavior does not change with bias configuration during irradiation [START_REF] Gaillardin | Impact of SOI substrate on the radiation response of ultra-thin transistors down to the 20 nm node[END_REF]. These promising results indicate that FDSOI devices may withstand high TID levels with appropriate substrate and design options, both if grounded or biased during irradiation. Obviously, standard thick BOX ( nm) devices do not present such characteristic, the NULL case being the one presenting the weakest TID induced modifications. So, the TID response of nanometer-scaled transistors with nm either grounded (black squares) or biased (red circles) during irradiation processed on two different SOI substrates is presented in Fig. 6 with TID, but their functionality do not suffer from any drive-current degradation. Fig. 6 basically shows the opposite behavior with an improvement of with TID which can be attributed to both the radiation-induced shift of the electrical characteristics and the stronger coupling effects with BOX thinning. This specific property may be beneficial to enhance the FDSOI devices' TID tolerance at MGy levels since the transistor drivability is not degraded contrary to what is observed on thick BOX devices.

If the requirements needed by the final application can manage increase of the leakage current, thus of the power consumption, such technology may present interesting properties to operate in harsh environment with a reduced cost related to the design hardening.

D. Discussion on the Hardness Potential of Modern Technologies Submitted to High TID

All investigated technologies show TID behaviors directly linked to their architecture, fabrication process, design and geometry. However, using the appropriate design options allows enhancing their TID tolerance. Fig. 7 summarizes the plots obtained on the most TID tolerant device for each As a main conclusion, results presented in Fig. 7 show that all devices are still functional after a 1 MGy irradiation. Their electrical characteristics change with TID but in all cases they could be managed using the appropriate margins in the circuit design. The entire characteristics must be considered, and not only the pre-and post-irradiation results, which do not necessarily give the largest TID-induced modifications. In the case of an accident in a nuclear facility, the choice of a technology will probably be driven by the circuit function and reliability rather than by its power consumption. Indeed, one may consider that in such environment, the power consumption of a stand alone circuit will not be the first critical parameter compared to its functionality and reliability. The requirements on the drive current stability with TID would be a greater issue than the one of the leakage current.

IV. IMPACT OF ELEVATED TEMPERATURE ON MGY DOSE SENSITIVITY

The experiments should represent the real environment as much as possible. Elevated temperatures have thus to be considered in addition to ionizing radiations for security systems in nuclear facilities. Standards for tests in such conditions recommend to anneal irradiated devices for one hour at 150 to qualify equipments submitted to mixed harsh environments [START_REF]Assessment and management of ageing of major nuclear power plant components important to safety : In containment instrumentation and control cables[END_REF], [START_REF] Nf M | Procédure de qualification des matériels électriques installés dans l'enceinte de confinement des réacteurs à eau sous pression et soumis aux conditions accidentelles[END_REF]. Such tests have been performed on all irradiated devices presented previously but they are also completed with additional experiments performed up to 250 in order to discuss the annealing behavior of electronic devices in a wider range of operations.

Elevated temperatures in MOS devices usually induce recovery of radiation-induced effects [START_REF] Schwank | Total dose effects in MOS devices[END_REF]. Radiation-induced oxide-trap charges could be detrapped when point defects responsible for interface traps may anneal. So, elevated temperature may be an efficient way to recover electrical characteristics of pristine integrated circuits. However, using elevated temperatures is also a way to perform dynamic "life tests" which speeds up the ageing of electronic components. Irradiated devices may apparently recover electrical characteristics close to their initial ones but they could also suffer from a degraded reliability and thus a strongly lower remaining lifetime than at room temperature.

Fig. 8 presents the typical recovery observed on irradiated MOS transistors after annealing experiments at elevated temperatures: the subthreshold slope retrieves its initial value, meaning that most interface traps are annealed after 250 (red circles) in this transistor. The characteristics of the ELT annealed at 250 superimposed the characteristic measured on this transistor before irradiation (not shown for the sake of clarity) meaning that the major part of radiation-induced degradation are annealed. Such plot cannot be presented for the PDSOI transistor since no reliable parameter extractions can be done due to the weak impact of ionizing radiations induced on electrical characteristics of these transistors. Actually, no significant oxide charge and interface traps buildup occurs in the tested PDSOI devices.

FDSOI devices exhibit I-V characteristics which retrieve their initial shape after annealing in Fig. 9. It highlights that the I-V curve of the device that experienced a 1 MGy irradiation followed by 250 annealing (blue triangles) is closely shaped like the pristine one (black squares). It means that a strong annealing of both the oxide-trap charge and the interface traps occurs in the gate oxide and in the buried oxide and at each silicon-oxide interfaces respectively. These phenomena are enough to recover electrical characteristics close to the pristine one after the 150 annealing step (green hexagons). These results are very promising since the elevated temperature mostly counterbalances the ionizing dose effects reported in FDSOI devices even in the nominal qualification test conditions.

Elevated temperatures strongly reduce the influence on the interface traps buildup in MOS devices even at MGy dose levels. This is summarized in Fig. 10 where the characteristics obtained on the three tested technologies are plotted. All characteristics retrieve values close to their initial ones for all tested devices. However, one should consider the entire "story" of the devices characteristics' instead of only looking at the pre-irradiation, post-irradiation and post-annealing results. If so, most of the information on the TID and annealing behaviors would have been lost. This may lead to take circuit design margins which do not include the largest modifications induced by TID and temperature on the electrical characteristics.

Furthermore, elevated temperatures may degrade the intrinsic reliability of electronic devices and then reduce their lifetime by several years. This should be quantified by additional dedicated tests to foresee the failure probability of security systems and their replacement periodicity.

Finally, the test procedure presented in this paper only involves "irradiation then temperature" tests. It never takes into account the effect of ionizing radiation with temperature at the same time which represents the more realistic test case compared to real accidental conditions. However, it was demonstrated recently by Girard et al. [START_REF] Girard | Combined high dose and temperature effects on multimode silica-based optical fibers[END_REF] that it may strongly affect the radiation response of optical fibers which show higher TID induced degradation of their optical propagation properties contrary to what is usually assumed. It should then be checked using dedicated experiments in electronic devices as well to ensure that "irradiation THEN temperature" tests are still relevant than their "irradiation AND temperature" counterparts for modern and innovative technologies. Finally, these first results obtained using 10 keV x-rays on modern technologies should be by to gamma radiation experiments which are more representative to the real environment.

V. CONCLUSION

This paper reviews the TID behavior of several micro-and nano-electronic devices at MGy dose levels combined with elevated temperatures. The experimental results show that design rules specific to each technology are required to ensure device functionality at MGy dose levels under elevated temperature. This paper also highlights that one has to take the entire electrical characteristics shifts' induced by ionizing radiations and elevated temperatures to take margins enough to prevent any circuit function failure instead of taking only the pre-and post-TID/temperature test results. At first glance, elevated temperatures lower MGy ionizing dose-induced effects but their impacts on the intrinsic reliability and lifetime of electronic devices have to be quantified to properly qualify electronic systems designed for mixed harsh environments. Furthermore, dedicated studies should be performed to ensure that usual test procedures to qualify electronic devices operating in a mixed environment involving ionizing radiation and high temperature are still relevant for innovative and technologies.

  Three different technologies are studied in this paper to estimate their relative strengths and weaknesses in high TID and temperature environments: mconventional bulk I/O technology featuring Metal-Oxide-Semiconductor (MOS) transistors designed either with a standard Open Layout (OLT) or an Enclosed Layout (ELT). The nominal voltage of the technology is 3.3 V. -130 nm PDSOI technology featuring MOS transistors processed on a SOI substrate with two designs: standard Floating Body (FB) and Body Contacted (BC) devices. The silicon film thickness is enough to prevent coupling effects [14] occurring in the following FDSOI technology. The nominal voltage of the technology is 1.2 V. -20 nm FDSOI technology featuring open layout transistors. One characteristic of this technology is that devices and ICs can be processed on different base SOI substrates.

Fig. 1 .

 1 Fig. 1. Schematic of annealing steps performed during experiments. Electrical measurements are performed at room temperature between each temperature step.

Fig. 2 .

 2 Fig. 2. Drain current vs gate voltage characteristics of two bulk NMOS transistors: an Open Layout Transistor (OLT, top, a) and an Enclosed Layout Transistor (ELT, bottom, b) before irradiation (black squares) and after 1 MGy (red circles). Transistors are grounded during irradiation.

Fig. 3 .

 3 Fig. 3. Leakage current shifts vs TID in Open Layout Transistors (OLT, black squares) and Enclosed Layout Transistors either grounded (ELT, filled red circles) or biased (ELT, open red circles) during irradiation.

Fig. 4 .

 4 Fig. 4. Leakage current shifts induced after 1 MGy on a Body Contacted transistor either grounded (filled squares) or biased (open squares) during irradiation.

Fig. 5 .

 5 Fig. 5. characteristics as a function of TID on FDSOI transistors processed on SOI substrates with a BOX thickness nm (black squares), nm (red circles) and nm (blue triangles). Transistors with both a short gate length ( nm, filled symbols) and a long gate length ( m, open symbols) are plotted for each SOI substrate option. Devices are grounded during irradiation.

Fig. 6 .

 6 Fig. 6. characteristics as a function of TID on FDSOI transistors processed on SOI substrates with a BOX thickness nm (filled symbols) and nm (open symbols), grounded (squares) or biased (circles) during irradiation. Transistors have an extremely short gate length nm.

  within an plot. Devices are fabricated on substrates with a BOX thickness of either nm (filled symbols) or nm (open symbols). Fig. 6 clearly highlights the bias dependence of the FDSOI devices' TID response at MGy dose levels. Thick BOX devices (filled symbols) exhibit a clear bias dependence of their TID responses when the one of thin BOX devices (open symbols) is not anymore governed by the bias configuration during irradiation as already observed at lower TID in [15]. characteristics of thick BOX devices ( nm) are clearly separated when measurements performed on transistors processed on the SOI substrate with nm (open symbols) are nearly superimposed. Thin BOX devices present a significant increase of the leakage current

Fig. 7 .

 7 Fig. 7. characteristics as a function of TID on FDSOI transistors for bulk (black squares), PDSOI (red circles) and FDSOI (blue triangles) technologies. Devices are grounded during irradiation.

  studied technology: bulk (black squares), PDSOI (red circles) and FDSOI (blue triangles).

Fig. 8 .

 8 Fig. 8. characteristics of the bulk ELT transistor after a 1 MGy irradiation (black squares) and after the 250 annealing step (red circles). The transistor is grounded both during irradiation and annealing.

Fig. 9 .

 9 Fig. 9.characteristics of a long gate FDSOI transistor before irradiation (black squares), after 1 MGy (red circles), after MGy (green hexagons) and after MGy (blue triangles) The transistor is grounded both during irradiation and annealing.

Fig. 10 .

 10 Fig. 10. characteristics as a function of TID on FDSOI transistors for bulk (black squares), PDSOI (red circles) and FDSOI (blue triangles) technologies. Annealing results are presented for each technology with open symbols for 150 ,200 and 250 . Devices are grounded during irradiation and annealing experiments.