Telegang Chekem
email: vincent.goetz@promes.cnrs.fr

Thermal Activation of persulfates for wastewater depollution on pilot scale solar equipment

Keywords: Wastewater, -pollutant, persulfates, AOP, pilot, solar energy

Thermal activation of new oxidant chemicals like Persulfate (PS) has recently gained attention in driving advanced oxidation processes (AOP) for a number of applications. Persulfate (PS) is an oxidant chemical likely to undergo thermal activation to drive advanced oxidation processes (AOP) with respect to a number of applications. On the other hand, addressing energy demand with carbon free and sustainable energy solutions represents an asset for new technologies in the modern society. This paper then investigates on the removal of pharmaceuticals residues in wastewater (WW), conducted with PS advanced oxidation reactions on a solar thermal pilot equipment. IIndoor preliminary investigations on small scale laboratory experimentations shows how increasing temperature has a direct positive impact on pollutants degradations rate (kapp value ranging from 0.4•10 -3 s -1 at ambient to 10.4•10 -3 s -1 at 75°C)., However, after an experimental screening methodology, carried out with a screening methodology, lead to 65 °C and 200 µM appear as the appropriate target temperature and PS oxidant dosage to cope with both activation of PS oxidant and degradation of ten target micropollutants typically found in WW stream. Outdoor experimentations on the solar pilot equipment were operated conducted according to a batch running mode at a treatment capacity scale of a cubic meter. 95 % pollutant removal rate is achieved within 2 hours once the temperature has raised up to the target value (65 °C) in the bulk treated WW effluent. Series of treatment cycles under the natural day/night period demonstrate the reproducibility of the performance. As an early step result of the process optimization approach, heat recovery demonstration on the pilot equipment improves significantly the energetic balance over the now innovative thermal solar water treatment.

Highlight

 Efficiency ofTthermal induced activation of persulfate is effective in removing biorefractory micropollutants in WW effluents.

 Less than 2 hours needed to removeUp to 95% abatement of pollutants cocktail with 200 µM of persulfate and, under 65 °C target temperature, on a solar pilot equipment. and 200 µM of persulfate.

 Degradation of ten target pollutants is achieved upon a solar pilot equipment running thermal activation of persulfate.

 SSmart heat management system system to tackle solar energy intermittency.to improve the efficiency of the solar process with respect to energy balance.

Highlight graph

Introduction

Advanced oxidation processes (AOPs) are based upon the formation of highly reactive free radicals species from different routes. Owing to the high redox potentials, the way free radicals react and degrade soluble organics is highly relevant regarding environmental remediation [START_REF] Brienza | Use of solar advanced oxidation processes for wastewater treatment: Follow-up on degradation products, acute toxicity, genotoxicity and estrogenicity[END_REF][START_REF] Marjanovic | Effect of μM Fe addition, mild heat and solar UV on sulfate radical-mediated inactivation of bacteria, viruses, and micropollutant degradation in water[END_REF][START_REF] Shawaqfeh | Photocatalytic treatment of water soluble pesticide by advanced oxidation technologies using UV light and solar energy[END_REF][START_REF] Vilar | Treatment of textile wastewaters by solar-driven advanced oxidation processes[END_REF]. Typically, free radicals catalyzed chain reactions readily make possible degradation and/or mineralization of a wide range of hardly biodegradable organic molecules. Combined with the direct use of solar energy for radicals production, they offer a unique opportunity to design an oxidative treatment process, at almost no additional energy cost, in agreement with the concepts of green chemistry and sustainable development [START_REF] Malato | Decontamination and disinfection of water by solar photocatalysis: The pilot plants of the Plataforma Solar de Almeria[END_REF][START_REF] Strazzabosco | Solar PV adoption in wastewater treatment plants: A review of practice in California[END_REF]. For many years since the discovery of free radicals potential for water decontamination, hydroxyl radicals (OH˚) have been considered as the most attractive [START_REF] Brienza | Relevance of a photo-Fenton like technology based on peroxymonosulphate for 17β-estradiol removal from wastewater[END_REF]Duran et al., 2010;[START_REF] Liu | Insight into electro-Fenton and photo-Fenton for the degradation of antibiotics: Mechanism study and research gaps[END_REF]. In the late 1990s, OH˚ is successfully used to run in situ chemical oxidation reaction, based upon utilization of oxidant reactants such as ozone, potassium permanganate and hydrogen peroxide [START_REF] Cai | Degradation and mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) by thermally activated persulfate oxidation[END_REF]. Since then, sulfate radicals (SO4 -°) , generated from a primary oxidant such as persulfate (PS) ions (S2O8 2- ) have also been progressively studied regarding major advantages such as the high redox potential (E 0 = 2.6 V/SHE) and the longer lifetime of corresponding sulfate free radicals, i.e. 1000 fold more than OH˚ radicals [START_REF] Cai | Degradation and mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) by thermally activated persulfate oxidation[END_REF][START_REF] Matzek | Activated persulfate for organic chemical degradation: A review[END_REF]. For any of these options, running the process with solar energy upon the well-known photo-Fenton (Fe 2+ -H2O2, Fe 2+ --S2O8 2-), has been so far the most popular and sustainable way to achieve radicals production [START_REF] Cuervo Lumbaque | Solar photo-Fenton-like process at neutral pH: Fe(III)-EDDS complex formation and optimization of experimental conditions for degradation of pharmaceuticals[END_REF][START_REF] Malato | Decontamination and disinfection of water by solar photocatalysis: The pilot plants of the Plataforma Solar de Almeria[END_REF][START_REF] Soriano-Molina | Mechanistic modeling of solar photo-Fenton process with Fe3+-EDDS at neutral pH[END_REF].

However, the acidic pH conditions required to avoid iron ions precipitation for achieving Fenton reaction, now appear as a serious bottleneck for process dissemination [START_REF] Clarizia | Homogeneous photo-Fenton processes at near neutral pH: A review[END_REF][START_REF] Johnson | Persulfate Persistence under Thermal Activation Conditions[END_REF].

A number of publications have then addressed on the possibility to run AOPS under less acidic or mild pH conditions. In the event of using Fe 2+ chelates under neutral pH conditions, the efficiency to achieve free radical chain reactions is still yet to be attractive [START_REF] Clarizia | Homogeneous photo-Fenton processes at near neutral pH: A review[END_REF][START_REF] Ike | Critical review of the science and sustainability of persulphate advanced oxidation processes[END_REF][START_REF] Matzek | Activated persulfate for organic chemical degradation: A review[END_REF][START_REF] Wu | Toward a Better Understanding of Fe(III)-EDDS Photochemistry: Theoretical Stability Calculation and Experimental Investigation of 4-tert-Butylphenol Degradation[END_REF]. Iron free direct generation of OH° from hydrogen peroxide has also been addressed very recently and seems promising, as far as solar driven AOPs are concerned [START_REF] Aguas | Reclamation of Real Urban Wastewater Using Solar Advanced Oxidation Processes: An Assessment of Microbial Pathogens and 74 Organic Microcontaminants Uptake in Lettuce and Radish[END_REF][START_REF] Ferro | Cross-Contamination of Residual Emerging Contaminants and Antibiotic Resistant Bacteria in Lettuce Crops and Soil Irrigated with Wastewater Treated by Sunlight/H2O2[END_REF]. Regarding sulfate radicals (SO4 -°), iron free heat activation route of persulfate (schematically represented with Eq. 1) is also reported [START_REF] Cai | Degradation and mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) by thermally activated persulfate oxidation[END_REF][START_REF] Ghauch | Naproxen abatement by thermally activated persulfate in aqueous systems[END_REF][START_REF] Olmez-Hanci | Bisphenol A treatment by the hot persulfate process: Oxidation products and acute toxicity[END_REF] but few research studies addressed solar heat activation of PDS and the real advantages asset it could provide as a comparison with more conventional existing AOPs.

      4 2 8 2 2SO Heat O S 1
The few recent papers dealing with the issue showed how successful is the activation of persulfate (single used reactant) towards a wide range of biorefractory organic pollutants in water [START_REF] Cai | Degradation and mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) by thermally activated persulfate oxidation[END_REF][START_REF] Ghauch | Ibuprofen removal by heated persulfate in aqueous solution: A kinetics study[END_REF][START_REF] Johnson | Persulfate Persistence under Thermal Activation Conditions[END_REF][START_REF] Yang | Degradation of azole fungicide fluconazole in aqueous solution by thermally activated persulfate[END_REF]. The results obtained on lab scale experiments are quite promising but the demonstration of the technique on real pilot equipment needs further investigation, especially in the case of solar irradiation used as the only source of energy. Indeed, regarding heat energy needed to achieve relevant target temperature, energy efficiency/profitability of what could be the future real processes is definitely relevant. Unlike conventional "in direct sunlight" photocatalysis scheme, thermal activation based process appears more "robust" with respect to heat energy collected (from sunlight) being stored over time for optimal usage, including during less fair weather episodes.

This paper investigates on the capacity of a pilot equipment to run solar driven heat activation of PS for water depollution. The system efficiency is investigated regarding emerging contaminants found downstream wastewater (WW) plants. To the best of the author knowledge, this is the first time a pilot scale solar demonstration is used to run PS thermal activation for the removal of micropollutant in real WW stream. In the south west of Europe, some areas face overexploitation of water resources together with lack of precipitations. In the perspective of achieving the new circular economic paradigm of water reuse, emerging and biorefractory microcontaminants need to be discarded from WW effluents. However, most of these microcontaminants are biorefractories molecules like pharmaceuticals and pesticides constituents, which are hardly removed by existing biological treatments [START_REF] Devi | In-situ chemical oxidation: Principle and applications of peroxide and persulfate treatments in wastewater systems[END_REF][START_REF] Faraldos | Environmental applications of titania-graphene photocatalysts[END_REF][START_REF] Oh | Generation of sulfate radical through heterogeneous catalysis for organic contaminants removal: Current development, challenges and prospects[END_REF]. At the meantime, the high toxicity level and bioaccumulation fate of a number of microcontaminants represent a real threat to public health and environmental protection as well [START_REF] Brienza | Enantioselective reductive transformation of climbazole: A concept towards quantitative biodegradation assessment in anaerobic biological treatment processes[END_REF][START_REF] Miralles-Cuevas | EDDS as complexing agent for enhancing solar advanced oxidation processes in natural water: Effect of iron species and different oxidants[END_REF].

The main objective of this study is to achieve PS thermal activation and then removal of emerging pollutants from WW, on a pilot scale newly designed solar equipment. Prior to real pilot scale demonstrations, degradation of ten target molecules was lab scale investigated on small wastewater samples. This was necessary to collect information on the influence of relevant experimental parameters on the depollution process. The ultimate aim was then to set favorable conditions for running the pilot equipment under solar irradiated and optimal basis.

Preliminary experiments

This section deals with indoor experiments, carried out in the laboratory prior to real solar pilot scale experimentations. Working first on low WW volumes (500 mL) was necessary to understand how impactful experimental parameters on degradation process are.

Wastewater and reagents

WW was used in this study to keep the pilot set up under "real lifelike" experimental conditions. WW was collected downstream of a WW treatment plant in the south of France.

Suspended materials were removed after an additional sand filtration and WW was stored for less than 2 days at 4 °C before usage. On Table 1 presents are the average values of some physicochemical parameters of WW samples. For depollution experiments, WW samples were spiked with a cocktail of ten target molecules (Table 2) at µg.L -1 concentrations range. The molecules belong to different families of pharmaceuticals reportedly found in WW effluents in the south west of Europe [START_REF] Brienza | Use of solar advanced oxidation processes for wastewater treatment: Follow-up on degradation products, acute toxicity, genotoxicity and estrogenicity[END_REF]. They were obtained from Sigma Aldrich at more than 98% purities, together with potassium iodide (99.5%) and sodium bicarbonate (99,,7%). Potassium persulfate (99%) was purchased from Acros Organics. All the chemicals were analytical reagent grade.

For liquid chromatography, the reagents used in this study were of ultra-pure HPLC grade.

Table 2. List of emerging contaminants used as target molecules

Analytical Apparatus and measurement procedures

An ultra-high performance liquid chromatography -electrospray -orbitrap mass spectrometer (UHPLC-MS/MS) was used to determine the concentrations of contaminants in WW samples, under both positive and negative modes of the ion spray source. The UPLC compartment (Agilent 1290 infinity) is equipped with a C-18 analytical column under 0.2 mL.min -1 flow rate of binary solvent mixture (water and acetonitrile). This was coupled to a MS/MS tandem 6460 triple quad mass spectrometer. The accuracy and sensitivity of the analysis system and then the corresponding protocol comply with the detection of pollutants at very low concentrations (down to ng/L concentrations range).

The residual persulfate concentration was determined according to a new spectrometric method recently detailed in the literature [START_REF] Liang | A rapid spectrophotometric determination of persulfate anion in ISCO[END_REF][START_REF] Zhao | Rapid and simple spectrophotometric determination of persulfate in water by microwave assisted decolorization of Methylene Blue[END_REF]. The method is straightforward and less time consuming as a comparison with the conventional well known iodometric dosage. A UV-vis spectrophotometer (VL-320 model) equipped with quartz cuvettes

(1 cm beam path) was used to read the absorbance of solutions after reaction of PS with potassium iodide (KI). Calibration was carried out on a series of prepared standard solutions (20 mL) with PS concentration ranging from 0 to 10 mg/L while keeping constant KI and NaHCO3 concentrations (respectively 1 and 0.1 g in 20 mL solutions, respectively). The water/KI/PS/ NaHCO3 mixture was hand shaken and then allowed to react for about 30 min.

The intensity of the resulting yellowish color depending on the amount of PS which reacted with the KI excess concentration. It's worth mentioning that NaHCO3 is also added to prevent air oxidation of KI reactant [START_REF] Liang | A rapid spectrophotometric determination of persulfate anion in ISCO[END_REF]. Absorbance reading was carried out at 400 nm after sample solutions have followed the same protocol of calibration standards except PS addition step. Dilution factors in the measurement solutions were considered for the determination of concentrations.

TOC measurements were performed with a TOC meter equipment (Shimadzu TOC-VCSH/CSN) after total and inorganic carbons have been determined.

Depollution experiments

For indoor lab scale experiments, contaminated WW samples contained in 500 mL flasks were prepared after proper dilution of corresponding reagents. For the 10 target molecules of this work, the concentration in the treated solutions was set at between 2.2 -25 µg.L -1 , i.e. close to naturally occurring concentrations [START_REF] Brienza | Use of solar advanced oxidation processes for wastewater treatment: Follow-up on degradation products, acute toxicity, genotoxicity and estrogenicity[END_REF] significant as the temperature of the treated solution increases. This is in agreement with PS concentration profiles at different temperatures (Fig. 2). They suggest that thermal activation of persulfate for generation of free radical species is temperature dependent as reported in previous studies in the literature [START_REF] Cai | Degradation and mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) by thermally activated persulfate oxidation[END_REF][START_REF] Johnson | Persulfate Persistence under Thermal Activation Conditions[END_REF]. Fig. 3 is the first order rate representation of kinetic profiles at different temperatures. One can see that the more the temperature applied, the higher the pseudo-first-order rate constant of pollutants disappearance kinetics. The exponential trend on the increase of kapp rate constant (from 0.4•10 -3 s -1 at 20 °C to 10 -2 s -1 at 75 °C) is consistent with the fate of sulfate free radicals appearance under thermal activation conditions, reported in the literature [START_REF] Deng | Thermally activated persulfate (TAP) oxidation of antiepileptic drug carbamazepine in water[END_REF][START_REF] Johnson | Persulfate Persistence under Thermal Activation Conditions[END_REF]. Regarding PS thermal decomposition under different experimental conditions, Johnson et al. [START_REF] Johnson | Persulfate Persistence under Thermal Activation Conditions[END_REF] showed highlighted a pseudo first order profile that of PSpersulfate thermal activation follows a pseudo first order profile. They also obtained an exponential increase of rate constant regarding sulfate ion production from PS within 30 -70 °C temperature range. Considering the short lifetime of sulfate radicals (SO4 -°), it is worth mentioning that degradation of pollutants is correlated to the capacity of the system to generate SO4 -° free radicals on a continuous basis. Activation energy has been calculated after setting a correlation (R 2 = 0.86) with respect to 1/T and then ln(kapp) according to Arrhenius equation,

T R E A kapp Ln a    / ln ) ( 2
where A being the Arrhenius constant, Ea the activation energy (kJ.mol -1 ), T the absolute temperature (K) and R the universal gas constant (8.314 J.mol -1 .K -1 ). The linear regression obtained from experimental points is represented on Fig. 3 inserted panel. The increase of pollutants removal rate with PS concentration is in agreement with papers in the literature [START_REF] Cai | Degradation and mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) by thermally activated persulfate oxidation[END_REF][START_REF] Marjanovic | Effect of μM Fe addition, mild heat and solar UV on sulfate radical-mediated inactivation of bacteria, viruses, and micropollutant degradation in water[END_REF][START_REF] Yang | Degradation of azole fungicide fluconazole in aqueous solution by thermally activated persulfate[END_REF]. Indeed, increasing oxidant concentration is favorable to organic removal as higher concentrations of sulfates radicals are available for oxidation. However, the utilization of PS reactants should take into account environmental norms and standards regarding release of sulfates in natural streams, some health disorders being reportedly associated to high level of sulfates in water. Few hundreds mg of sulfate ions are most often admitted in wastewater [START_REF] Heizer | Intestinal Effects of Sulfate in Drinking Water on Normal Human Subjects[END_REF]. Later on in this paper, only 200 µM of PS oxidant will be considered. A maximum of 40 mg/L of sulfate ions is likely to be released out, should initial PS oxidant being completely converted.

-Comparison of the fate of coexisting chemical species Fig. 6 is a comparison between kinetic breakthroughs of coexisting chemical species in the treated solution i.e. target pollutants, PS oxidant and organic charge represented by TOC.

After 6 hours of reaction, less than 10 % of the gross initial TOC load (about 12.2 mg.L -1 ) is removed. At the meantime, the target pollutants are completely discarded when 60 % of oxidant initially present has been consumed. This is a relevant example of the selective activity of sulfates free radicals towards target pollutants (in µg.L -1 concentrations range). Sulfate selectivity is already reported in a number of studies in the literature. [START_REF] Cai | Degradation and mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) by thermally activated persulfate oxidation[END_REF], achieved a complete degradation of 2.4-dichlorophenoxyacetic acid from contaminated WW with 65 % remaining TOC at the end of reaction. Working on winery wastewater, Amor et al. [START_REF] Amor | Winery wastewater treatment by sulphate radical based-advanced oxidation processes (SR-AOP): Thermally vs UV-assisted persulphate activation[END_REF] obtained similar results but succeeded in removing total TOC after combining thermal and UVC/iron activation of persulfates. In their research work with Titania photocatalyst, [START_REF] Shawaqfeh | Photocatalytic treatment of water soluble pesticide by advanced oxidation technologies using UV light and solar energy[END_REF] needed two times longer period to achieve TOC load as compare to time needed for removing some typical micropollutants. In this study, after 5 hours of reaction, 60 % of initial PS concentration is being consumed. Assuming PS being completely consumed over degradation reactions, this accounts for about 20 mg.L -1 residual concentration, very low value regarding standards on WW effluents release [START_REF] Heizer | Intestinal Effects of Sulfate in Drinking Water on Normal Human Subjects[END_REF].

Pilot scale experiment

Pilot experimental set up

A picture of the pilot is presented in figure 7, together with a descriptive diagram. It is mainly composed of two tanks (upper and lower) of 1 m 3 capacity, three flat solar panels (3 x 2 m 2 light harnessing surface) set in series and a heat exchanger (plate type model). Irradiation conditions were monitored with a radiometer (Kimo CR-110) and temperatures of WW in the upper tank, at the outlet of the panels and the inlets/outlets of the heat exchanger were measured with thermocouples (K type) connected to a data acquisition system. For simulation of naturally occurring conditions, the inlet concentration of the ten target pollutants stood between 12 -22 µg.L -1 .

Treatment consisted in two successive phases. First, heating of the WW effluent to a target temperature and then oxidation reactions conducted with PS reactant, introduced from a reagent vessel. For running the process, a volumetric pump (Pumprec) allows WW recirculation over a closed fluid loop connecting the upper tank with the thermal receiver (solar heating panels). For optimization purposes regarding solar energy utilization, recirculation takes place only when WW temperature straight at the thermal receiver outlet is above a representative value in comparison to the temperature in the upper tank (ΔT threshold setting). The same regulation approach is used in traditional solar water heating system for preventing preheated effluent to undergo reverse cooling off under poor irradiation condition (cloudy periods & nights). Raising WW to 65°C was the target to achieve before injection of PS oxidant (solenoid dosage with Verderdo VE1-C pump). Periodically, few amounts of WW effluent were collected from the system for analysis. First, the treated WW effluent in the treatment tank (upper tank) is transferred to a lower tank.

From the lower tank, treated WW effluent is cooled down before leaving the system and the energy released is used for heating new contaminated WW volume filling in the upper tank at the meantime. This "heat energy management system" is a reliable way to tackle the issue of energy saving and low investment on nominal surface area of thermal panel.

Wastewater depollution on solar pilot

Figure 8 shows the variation of WW temperature over six days in the row, on the month of June in Perpignan. The corresponding solar irradiation (W.m 2 ) is also presented. For better illustration and interpretation of results, night periods data have been removed as well as earlier time in the morning and later in the afternoon, i.e when irradiation is quite poor (looping pump off). Day 1 starts first by filling the treatment tank with 800 L WW prior to data collection (Fig. 8).

Starting from ambient temperature of WW (25 °C), a day of irradiation was not sufficient to achieve 65 °C threshold of WW temperature. At the end of the first day, the treated effluent caps at around 60°C and because of heat losses over the equipment during night window, a slight decrease (2-3 °C) is observed before solar heating resumed the second day (Fig. 8).

Thereafter, 3 to 4 hours were needed to reach the expected 65°C. Theoretically, about 37 kWhth of thermal energy are expected for elevation of 800 L of WW from 25 to 65 °C. In practice, the required solar energy backing this temperature elevation is the amount of radiation received right on top of active surface of the solar panels, according to Eq. 3,

     S t G Q i i e 3
where S (m 2 ) is the solar receiver surface, Gi (W.m -2 ) the global irradiation registered within ti (s) period of time.

The calculated value of Qe corresponding to the first treated batch was about 52 kWh.

Assuming that heat losses and thermal inertia of the tank are negligible, the thermal efficiency of the whole system was estimated to around 0.7. This value is in agreement with the use of flat collector thermal panels at working temperature within the range from ambient to few tens of Celsius degrees.

As PS oxidant is injected after 65 °C has been reached, up to 95 % pollutants removal rate was achieved right away after 1.5 h of reaction (Fig. 9). However, one can observe that more than 60 % global removal rate (regardless individual pollutant) was rapidly achieved within the first 15 min of treatment, indicating relative high rate of degradation oxidation reactions. As expected from preliminary studies, the degradation of pollutants is very effective should the required condition of temperature is met.

If persulfate was injected below 65 °C, preliminary experiment (results not presented) showed an important activity regarding oxidant consumption as WW bulk temperature increased slowly under solar streaming heating. By the meantime, very little activity and low rate degradation of pollutant was obtained. This result points out the necessity of injecting the oxidant at the right temperature to guarantee a minimum rate of sulfate radicals production. The issue will be discussed with more details on a future paper.

After completion of treatment on Day 2, solar energy accumulation kept going on until the end of the day, together with elevation of the effluent temperature (Fig. 8). For day 3 and 4 (Fig. 8), the mean value of the amount of solar energy collected to drive pollutant degradation (i.e. periods between time of injection on day n-1 and the injection time on day n) is about 38 kWh, corresponding to 47 Wh/L. This value is 25% smaller than solar energy needed on a process without heat energy recovery (52 kWh required for the first treated batch). This is a promising finding of what stands as the energy optimization approach.

However, more efficiency is likely to be achieved regarding energy balance, together with some improvements over the heat management system. Some of these improvements deal with a more suitable sizing of heat exchanger and minimization of energy losses due to inertia of materials the effluent is in contact with, and, more basically, an improvement of the system compactness.

. On the other hand, it's worth mentioning that the heat management system copes with the discontinuous feature of sun light energy as applicable on Day 5 (Fig. 8). Despite a poor solar irradiation conditions of this day, energy collected by the system was sufficient to raise WW effluent up to the target temperature, and to treat a fourth batch of 800 L WW (Fig. 11.b). More than 80% of pollutant global charge is removed after PS oxidant injection.

Conclusion

Persulfate thermal activation can be effective in removing emerging contaminants in water via advanced oxidation reactions. In this paper, persulfate is thermally activated on a solar pilot to drive degradation of ten target pollutants typically found in wastewater streams. Preliminary studies were first carried out prior to outdoor experimentations on real solar pilot equipment of 1000 L capacity. Indoor preliminary experiments highlighted how increasing temperature and oxidant concentration has a positive effectimpact on pollutants degradations rate. A slight variation of degradation rate can be observed from one individual pollutant to another. However, out of the ten, only Cafeine and Tramadol is still found in very few residual amount in the treated solution after 2 hours of treatment (at 70°C with 200 µM PS concentration).

Regarding pilot scale equipment, experiments were carried out on batches of several hundreds of liters to make a proof of concept demonstration under real solar irradiation. The degradation of pollutants is effective onver a bulk treated volume (800 L) heated under solar irradiation above the 65 °C target temperature. Similarly to the small scale laboratory tests, around two hours were needed to achieve high removal rate of the 10 micropollutants cocktail within a real waste water effluent. The heat recovery system on the pilot definitely plays a key role to improve the energy balance and a smart use of solar energy collected on the top of basic solar panels. Since the heat energy collected can be stored over time, it allows an optimal running of the system, in particular during days with less fair weather episodes. The next critical step will be the optimization of energy balance to make the process competitive for the treatment of micropollutants in WW, with low investment on the extent of solar panels. Anyway, the general principle of a solar thermal activation of persulfate is experimentally demonstrated on a pilot scale set up.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Kinetic profiles of pollutants removal (average concentration) at different temperatures (Initial PS concentration 200 µM, Co average value= 5.2 µg.L -1 )

Fig. 3 .

 3 Fig. 3. First order representation of kinetic profiles of pollutants elimination at different temperatures (Initial PS concentration 200 µM, Co = 2.2 -25 µg.L -1 )

Fig. 4 .

 4 Fig. 4. Kinetic profiles of elimination of individual target molecules (a) with and (b) without oxidant. PS oxidant concentration 200 µM -Temperature 75 °C.

Fig. 5 .

 5 Fig. 5. Kinetic profiles of pollutant removal at different concentrations of PS oxidant -Temperature 65 °C.

Fig. 6 .

 6 Fig. 6. Kinetic profiles of coexisting chemical species in treated solution. PS oxidant 200 µM, Temperature 75 °C.

Fig. 7

 7 Fig. 7 Water treatment solar pilot picture and descriptive diagram

Fig 8 .

 8 Fig 8. Effluent temperature and solar irradiation in 6 days in the row, heat and oxidant management between one batch treatment to another (800 L of WW effluent)

Fig. 9 .

 9 Fig. 9. Pollutants kinetic profile on Day 2 (Fig. 8)

Fig. 10 .

 10 Fig. 10. Temperature profile over the heat exchange system with volumetric flow rates of the fluids equal to 200 L.h -1 .

Fig. 11 .

 11 Fig. 11. Pollutants kinetic profiles on (a) Day 3 and (b) Day 5 (Fig. 8)

Table 1 .

 1 Physicochemical properties of wastewater samples

	MES (g.L -1	)	pH ( -)	Cond. (µS.m -1 )	TOC (mg.L	-1 )	DCO (mg.L	-1 )	NTK (mg.L	-1	)	NGL (mg.L	-1	)	2-SO 4 (mg.L	-1 )
	8.5		7.4	669	12.2		28.1		7			9.2			195

Acknowledgements

. The results published in this research paper are part of the findings of the INTERREG FEDER project called 4Kets4Reuse. The authors are thankful to the European project funding.


Stéphane Erb

S Cianférani

Julien Marcoux

Hands on Native Mass Spectrometry Analysis of Multi-protein Complexes

Keywords: Structural Mass Spectrometry, Non Covalent Mass Spectrometry, Oligomeric states, Stoichiometry, Subcomplexes

. Besides, heterogeneous samples, both in terms of oligomeric states and ligand-bound species can be fully characterized. Here we thoroughly describe the analysis of several oligomeric protein complexes ranging from a 16 kDa dimer to a 801 kDa tetradecameric complex on different instrumental setups.

Introduction

Mass spectrometry (MS) plays a pivotal role for the characterization of multi-protein complexes. Routinely coupled with reversed-phase liquid chromatography (LC-MS), it allows purity and homogeneity assessment of biological complexes. Classical LC-MS workflow require organic solvents (usually H 2 O/acetonitrile) and acidic conditions (e.g. acetic or formic acid) to achieve best chromatographic resolutions along with optimal MS detection. However those experimental conditions induce multi-protein noncovalent complexes' dissociation. In the early 90s, MS approaches performed in ammonium acetate buffer maintaining quaternary structures of multi-protein complexes have been first described [2-4], opening the way for a new field of application called noncovalent or "native" MS [5,6]. By transferring intact noncovalent assemblies into the gas phase of the mass spectrometer, native MS enables multi-protein complex stoichiometry assessment thanks to accurate mass measurements. Mostly performed on TOF and Q-TOF [START_REF] Sobott | A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies[END_REF] instruments in its early years, it has recently benefited from technological improvements to reach high resolution native MS [START_REF] Rose | High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies[END_REF]. Native MS is currently implemented in most structural biology departments to complement conventional structural biophysical techniques like crystallography, electron microscopy, NMR or SAXS. The methodology is versatile as any type of multi-protein complex can be analyzed, ranging from homo-oligomeric proteins [5] to hetero-oligomers [9,10] or even protein/nucleic assemblies [START_REF] Martinez-Zapien | Intermolecular recognition of the non-coding RNA 7SK and HEXIM protein in perspective[END_REF][START_REF] Saliou | Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity[END_REF] and membrane complexes [START_REF] Barrera | Micelles protect membrane complexes from solution to vacuum[END_REF][START_REF] Chorev | Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry[END_REF]. Latest methodological breakthroughs in the field include the direct analysis of complexes from cell lysates [START_REF] Gan | Native Mass Spectrometry of Recombinant Proteins from Crude Cell Lysates[END_REF] or from native membranes [START_REF] Chorev | Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry[END_REF] and the release of a new instrument enabling the complete analysis of very large complexes with unprecedented resolution [START_REF] Van De Waterbeemd | Dissecting ribosomal particles throughout the kingdoms of life using advanced hybrid mass spectrometry methods[END_REF].

In this chapter we illustrate the possibilities of native MS on different types of multi-protein complexes and different MS platforms to assess stoichiometries. Data interpretation is usually performed by comparing the masses of the individual subunits of the complexes obtained by classical MS analysis in denaturing conditions and the mass of the intact noncovalent multi-protein assembly measured in native conditions. Prepare all solutions using deionized water and analytical grade reagents. Prepare and store all reagents at 4°C. Operate the instrument in positive mode with a m/z range from 1,000 to 10,000. Apply 120 V and 5 V for the sampling cone and extraction cone, respectively. The source temperature is set at 90°C but the capillary voltage and the gas flows are not used in the TriVersa Nanomate TM interface setup. 3. Adjust the backing pressure to 6 mbar (Note 7). 4. Start a 2 min acquisition with 4 sec scans. 5. Average the signal over the 2 min in the Chromatogram panel and in the Spectrum panel, select "Tools" then "Make Calibration" and select "CsI_esi.ref" profil. Adjust calibration points for the m/z range targeted and click on File\Save Spectrum then click OK and finally accept the calibration after closing the window. 6. Do not forget to load this calibration file (e.g. named CsI_date) before starting the first acquisition of the analyzed sample. To do so, click on "Acquire", then select "calibration" and load the calibration file. Finally perform the acquisition.

Maxis II

1. Using the HESI source and the 500µL Hamilton™ syringe, deliver cesium iodide calibration mix (see 2.2) with a flow rate set to 3 µL/min. 2. In the MS Tune window, manually set the following parameters:

Operate the instrument in positive mode with a m/z range from 1,000 to 8,000. Apply 20 eV and 5 eV for isCID and CE parameters, respectively. Furthermore, native experiments involve adapting transfert and prepulse storage times (Note 8): set to 320 µs and 15 µs, respectively. Concerning the source parameters, set the capillary voltage at 4 kV, the nebulizer at 0.4 bar, the dry gas at 4.0 L/min and the temperature at 220°C. 3. To perform the calibration, go to the "Calibration" tab and select the CsI reference file, then check that the calibration mode is on "Enhanced Quadratic Mode". Finally, click to "Calibrate" and accept the calibration.

3.4.5 Exactive Plus EMR 1. Using the HESI source and the 500 µL Hamilton™ syringe, deliver cesium iodide calibration mix (see 2.2) with a flow rate set to 10 µL/min. 2. In the MS Tune page manually set the following parameters: Operate the instrument in positive mode with a m/z range from 1,000 to 20,000. Apply 25 eV and 100 eV for the CID and CE parameters, respectively. The source temperature is set at 250°C, the capillary voltage is set to 4 kV and the gas flows is set to 10 u.a. The trapping gas pressure is set to 7 u.a. Set the ion optics (injection, inter and bent flatapoles) at 4 V, the nominal resolution at 17,500 and activate the EMR mode. 3. To perform the calibration, wait for a stable TIC (<12 %) then select "Calibrate" and check "EMR MS Mass Calibration (pos)". Save these parameters as an .ipr file. 4. Press "acquire" to start a 2 min TOF-MS acquisition in the 1,00-10,000 m/z range. 5. The corresponding averaged and smoothed (20 times) raw data is represented in Fig. 2A. 6. Manual or software-driven (see 3.5.5) deconvolution of the data identifies different species corresponding to the three monomers of the CAK complex: namely Cdk7 (MW: 40.1 kDa), Cyclin-H (MW: 37.7 kDa) and MNAT1 (MW: 35.9 kDa) (Fig. 2B andC). A species corresponding to the heterodimer Cdk7-Cyclin-H at 77.9 kDa is visible, confirming their direct interaction. Finally, the physiologically active heterotrimeric CAK is identified at 113.9 kDa. Save these parameters as an MS tune file (.ipr file). 7. In MassLynx, create a sample list with the corresponding MS file, Inlet file, MS tune file. Specify the sample position (Bottle) and the volume to be injected (10 µL). 8. Start the acquisition. 9. The chromatogram shows two main peaks at 6.14 min and 7.32 min (Fig. 5A), corresponding to the tetramer (Fig. 5C) and monomer (Fig. 5B) of the ADH, respectively.

3.10

Protein/DNA complex analysis on a Q-TOF mass spectrometer -Synapt G2 HDMS 1. Desalt 50 µL of ERRα DBD-BE26PSIR3 complex [START_REF] Mohideen-Abdul | Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor[END_REF] at 28 µM on a Zeba™ spin column equilibrated with 200 mM ammonium acetate at pH 7.1 as explained in section 3.1. 2. Use the TriVersa Nanomate TM (see 3.3) coupled to the mass spectrometer and infuse the protein complex at 5 µM. 3. In the Tune page, start with the parameters used for the CsI calibration and adapt the cone voltage at 140 V, the extraction cone at 5 V and the backing pressure at 6 mbar (Note 7). 4. Start a 2 min acquisition in the 1,000 to 6,000 m/z range. 5. The average full MS spectrum obtained is presented in Fig. 7.A. 6. The corresponding deconvoluted spectrum (see section 3.5.5) is represented in

3.11

High molecular weight homo-oligomeric protein on a high resolution Orbitrap mass spectrometer -Exactive Plus EMR 1. Use the freshly prepared and desalted GroEL protein (see 2.4). Fig. 8B is a zoom where ions signal are detected between 11,000-13,000 m/z. After data processing (see 3.5.5), a unique species corresponding to the 14-mer complex is identified (Fig. 8C) at 801.6 kDa. Otherwise, condensation will occur, water will fill the wells and the plate will have to be discarded the next day. 5. The gas pressure and voltage to apply are the main two parameters to adjust.

The stability of the spray can be visualized in the Spray Optimization window, by pressing "Graph". If the intensity of the electrospray is dropping, incrementally increasing the gas pressure (no more than 0.5 psi) or the Voltage to apply (no more than 1.80 kV) might help. If the spray is not coming back, use another nozzle by pressing "Next Nozzle". Reducing the gas pressure and Voltage to apply while spraying usually results spray loss. The optimum Voltage to apply is protein-dependent and has to be carefully optimized, as higher values usually increase the ion transmission (more intense signal) and buffer removal (better resolved signal) but might result in complex dissociation. Ticking "Return unused sample to tray" enables to recover most of the sample after data acquisition. 6. Reference files for higher m/z ranges can be custom made. In the case of CsI, increments of +259.81 Da can be directly added to the initial CsI calibration file. 7. The backing pressure is a key parameter on Waters instruments (LCT, Synapt), that needs to be optimized for each protein complex. Generally, this value is set around 6 mbar and can be adjusted if necessary. This is possible by adding a homemade system with two external valves that reduce the suction of the primary pump. 8. In order to optimally accumulate and transfer heavy ions, transfer time and prepulse storage time parameters should be increased compared to small ions analysis. These two parameters are linked but the transfer time has to be higher than the prepusle storage time. 9. Waters TOF or Q-TOF mass spectrometers can be upgraded (MS vision, the Netherlands) by modifying electronics and gas pressure in order to improve the transmission of high molecular mass species in non-denaturing conditions. On our upgraded LCT, the combination of an additional gas arrival (argon) in the first hexapole housing through a needle valve and the modification of different electronic parts (e.g. higher voltages applied to sample and extraction cones) result in an improvement of high molecular mass transmission. Typically, the sensitivity for GroEL 14 analysis is increased six-fold by argon admission if the pressure is set around 4.10 -2 mbar. A micro-Pirani gauge is installed to control the pressure applied 10. n-Dodecyl β-D-maltoside can be used at two-times the critical micellar concentration (0.02%) in order to stabilize very labile soluble complexes. It can also be used, at the same concentration to spray membrane proteins [23,24]. 11. The m/z range on Waters Q-TOF depend on the type of Quadrupole that is installed: up to 16,000 m/z for a 4,000 m/z quadrupole, up to 32,000 m/z for an 8,000 m/z quadrupole and up to 128,000 m/z for a 32,000 m/z quadrupole. 12. The sample cone is a key parameter on Waters instruments (LCT, Synapt…), that needs to be optimized for each protein complex. Similarly, to the voltage applied to the nozzle of the TriVersa Nanomate TM , higher values usually increase the intensity of the signal and provide better resolved peaks by removing residual buffer. However, high sample cone values might result in complex dissociation.

Here again, an optimum value has to be found that provides good signal without undermining the multiprotein complex integrity. A good strategy is to start with a maximum Sampling Cone voltage (150 V) and progressively reduce it by 10V steps. 13. The trap collision energy is another key parameter on Waters Q-TOF. In the case of membrane proteins or very large multiprotein complexes (> 500 kDa), increasing the trap collision energy might be helpful or even mandatory in order to thermodynamically cool down the ions (large complexes) or remove the remaining detergent molecules. Simultaneously and incrementally increasing the Trap gas flow (from 2 mL/min up to 8 mL/min) might improve the transmission of these particular large or membrane protein complexes.

Fig. 1 .

 1 Fig.1. Native MS analysis of the E. coli RNA polymerase complex. A Full MS spectrum of E. coli RNA polymerase complex showing charged states 35+ to 41+ in the 9,000 to 12,000 m/z range corresponding to the hetero-oligomer composed of five subunits (orange triangle) and 16+ to 33+ under m/z 2,500 corresponding to the α-subunit (green circle). B Non denaturing mass spectrum of E. coli RNA polymerase after deconvolution with UniDec highlights the presence of the hetero-oligomeric protein (390,315 Da) and the monomeric α-subunit (36,509 Da).

Fig. 3 . 3 . 6 . 2

 3362 Fig.3. Native MS analysis of the p8 subunit from the human TFIIH complex A Raw MS spectrum of p8 showing charged states 4+ to 9+ corresponding to the monomer (purple) and 6+ to 11+ corresponding to the dimer (red). The inset shows a close-up view of the 2069.01 peak, revealing an overlap of the monomeric 4+ and dimeric 8+ charged states. B Native MS spectrum of after deconvolution with UniDec, showing the presence of monomeric (8272 Da) and dimeric (16544 Da) species. 3.6.2 The Yeast Alcohol Dehydrogenase (ADH) 1. Weight 1.6 mg of alcohol dehydrogenase from Saccharomyces Cerevisiae (Sigma-Aldrich). Resuspend in 280 µL of 200 mM ammonium acetate to reach a tetrameric concentration of 40 µM. 2. Connect the TriVersa Nanomate TM to the Q-Exactive TM Biopharma Orbitrap as explained in 3.4 3. In Excalibur Tune window manually set the following parameters: Operate the instrument in positive mode with an m/z range from 2,500 to 8,000. Intact protein mode: off; HRM mode: on; resolution: 15,000; Micro Scan Count: 10; Max. Ion Time: 200 ms; AGC target: 3e6, S-Lens RF Level: 200, Capillary Temperature: 320°C, Capillary Voltage: 4.4 kV. Save these parameters as an .mstune file. 4. Start a 2 min acquisition in the 2,500-8,000 m/z range. 5. The corresponding averaged raw data is represented in Fig.4A 6. Manual or software-driven (see 3.5.5) deconvolution of the data identifies different species corresponding to the monomeric (MW=36,879 Da) and tetrameric (MW=147,516 Da) ADH protein (Fig.4B). The effective resolution ranges from 2,000 (m/z 5,674) to 4,600 (m/z 2,844).

Fig. 4 .

 4 Fig.4. Native MS analysis of the of the ADH from Saccharomyces cerevisiae A Raw MS spectrum of the ADH showing charged states 10+ to 14+ corresponding to the monomer (purple) and 23+ to 28+ corresponding to the tetramer (red). B Native MS spectrum of p8 after deconvolution with UniDec, showing the presence of monomeric (36,879 Da) and tetrameric (147,516 Da) species.

3. 8

 8 SEC Native MS coupling (ADH) 1. Connect the ACQUITY UPLC H-Class Bio to the SynaptG2Si. 2. Connect the ACQUITY UPLC Protein BEH SEC Column (200 Å, 1.7 µm, 4.6 mm x 150 mm) and equilibrate with 200 mM ammonium acetate at 300 µL/min.

3 .

 3 Weight 1.6 mg of alcohol dehydrogenase from Saccharomyces Cerevisiae (Sigma-Aldrich). Resuspend in 280 µL of 200 mM ammonium acetate to reach a tetrameric concentration of 40 µM. 4. In MassLynx, setup an MS file with the following parameters: Start Time: 0 min; End Time: 10 min; Polarity: Positive; Analyser Mode: Sensitivity; Dynamic Range: Normal; Sensitivity: Normal; Low Mass: 1000 Da; High Mass: 10,000 Da; Scan Time: 1 sec (Continuum); No Trap or Transfer CE. 5. In MassLynx, setup an Inlet file with the following parameters: Flow rate 200 µL/min with 100% solvent A (200 mM ammonium acetate) for 10 min. 6. In MassLynx MS Tune windows manually set the following parameters: Nanoflow+ panel: Capillary: 3 kV; Sampling Cone: 150 V. Source Offset: 80V. Source Temperature: 100°C. Cone Gas: 50 L/h; Nano Flow Gas: 0 Bar; Purge Gas: 100 L/h. Instrument: Trap and Transfer Collision Energy are turned off (4V and 2V, respectively by default).

Fig. 5 .

 5 Fig.5. SEC-Native MS analysis of the ADH from Saccharomyces cerevisiae A Total Ion Chromatogram (TIC) corresponding to the SEC separation of the monomer and tetramer of the ADH. B Native MS spectrum of the monomer (36.9 kDa) before (left) and after (right) deconvolution with UniDec. C Native MS spectrum of the tetramer (147.5 kDa) before (left) and after (right) deconvolution with UniDec.3.9 Homo-oligomeric Concanavalin A analysis on a high-resolution Q-TOF mass spectrometer -Maxis II

Fig. 6 .

 6 Fig.6. Native MS analysis of Concanavalin A from Canavalia ensiformis protein. A Full MS spectrum of Concanavalin A showing charged states 24+ to 20+ in the m/z range from 4,000 to 5,500 corresponding to the homo-oligomeric protein (blue circle). B Non denaturing mass spectrum of Concanavaline A after deconvolution with UniDec highlights the tetrameric state of the protein complex (102,707 Da).

Fig. 7 .

 7 C and confirms the 1:1 stoichiometry between protein and DNA species (27,892.5 Da for full length DNA). Other species corresponding to truncated DNA associated to the protein with a 1:1 stoichiometry are also detected (27,740.6 Da and 27,586.3 Da).

Fig. 7 .

 7 Fig.7. Native MS analysis of the ERRα DBD-BE26PSIR3 complex. A Full MS spectrum of protein/DNA complex. Two charge states distributions in the 2,000 to 4,000 m/z range are present, corresponding firstly to the 6+ and 7+ charge states of DNA alone in the 2,000 to 3,000 m/z range and secondly to the 9+ and 8+ charge states of the protein/DNA complex in the 3,000 to 3,800m/z range. B Zoom in the 3,000 to 3,600 m/z range showing the protein/DNA complex 9+ and 8+ charge states revealing the presence of two truncated DNA species. C The associated deconvolution of native mass spectrum with UniDec software highlights a 1:1 stoichiometry for the protein/DNA complex (27,892.5 Da) and confirms the presence of two truncated DNA species which are also able to interact with the protein (27,740.6 Da and 27,586.3 Da).

2 .

 2 Connect the TriVersa Nanomate TM (see3.3) to the mass spectrometer. Infuse the sample concentrated at 5 µM of oligomer. 3. In the Tune page, set the following parameters: Positive mode with a 1,000 to 20,000 m/z range. Activate the EMR mode and set the trapping gas pressure at 7 u.a., the ion optics at 4 V (inter, injection and bent flatapoles), the S-lens RF voltage at 200 V, the temperature at 250°C, the fragmentation parameters at 25 eV and 150 eV for CID and CE values, respectively. Select a nominal resolution of 17,500. 4. Start a 2 min acquisition with a stable TIC. 5. The resulting averaged mass spectrum is presented in Fig.8A(full mass range).

Fig. 8 .

 8 Fig.8. Native MS analysis of Chaperonin 60 (GroEL) complex. A Full MS spectrum of GroEL complex showing a unique charge states distribution around m/z 12,000. B Zoom in the 10,000 to 14,000 m/z range where charge states 64+ to 71+ of the protein complex are detected. C Associated deconvolution obtained after data processing using UniDec software in the 785 kDa to 815 kDa mass range, showing one species corresponding to the 14-mer homo-complex at 801.6 kDa.

  Browse to select the previous acquisition file. Click on History and select the last AccMass2 line. 10. Close the 4 windows by clicking OK and close the Mass Calibration Profile Editor. 11. Click Next, Next and Start to launch the calibration. 3.4.2 Q-Exactive TM Biopharma Orbitrap 1. Using the syringe pusher to deliver the ammonium hexafluorophosphate calibration mix at 5 µl/min. 2. In the Calibration Panel, select "HMR Mode calibration (pos)". 3.4.3 LCT (upgraded for high mass detection by MS Vision) 1. Using the TriVersa Nanomate TM (see 3.3), deliver 5 µl of cesium iodide calibration mix (see 2.2). 2. In MassLynx MS Tune window, manually set the following parameters:

	4. Equilibrate the columns by adding 500 µL (Micro Bio-Spin TM ) or 300 µL (Zeba TM )
	of 200 mM ammonium acetate solution, centrifuge for 1 min at 1,000 g (Micro Bio-
	Spin TM ) or at 1,500 g (Zeba TM ) and discard the eluate.
	5. Repeat step 3 thrice.
	2.1 Desalting 6. Load between 20-75 µL (Micro Bio-Spin TM ) or 30-130 µL (Zeba TM ) of sample.
	1. Ammonium acetate: 200 mM solution in water (Note 1). Weigh 1.54 g ammonium Loading smaller volume may affect recovery (Note 3).
	acetate (>98%) and transfer to a 100 mL cylinder. Add water to a volume of 95 7. Place the column in a 1 mL eppendorf tube and elute the desalted proteins by mL. Mix and adjust pH to 7.4 with ammonium hydroxide (see Note 2). Make up to 100 mL with water. centrifuging 4 min at 1,000 g (Micro Bio-Spin TM ) or 2 min at 1,500 g (Zeba TM )
	3.2 Ultrafiltration desalting 2.2 Cesium iodide calibration mix 1. Operate at +4°C. Resuspend cesium iodide powder in 50/50 isopropanol/water (v/v) to a final 2. After selecting the appropriate MWCO of the ultrafiltration device, equilibrate its concentration of 2 mg/mL. membrane with 500 µL of 200 mM ammonium acetate. Typical devices include
	2.3 Ammonium hexafluorophosphate calibration mix Amicon TM Ultra 0.5 mL or Vivaspin TM 500 centrifugal concentrators (Merck).
	Resuspend ammonium Hexafluorophosphate powder in 50/50 isopropanol/water Centrifuge at 10,000 g for 30 sec, discard the eluate and repeat thrice.
	(v/v) to a final concentration of 1 mg/mL. 3. Load your sample (up to 500 µL)
	4. Concentrate down to 50 µL at 10,000 g. Make up to 500 µL with 200 mM
	2.4 GroEL reconstitution ammonium acetate and repeat five times this dilution/concentration cycle.
	GroEl must be freshly prepared.
	1. Prepare 9.445 mL of 1M Tris-acetate by weighting 1.21 g of Tris-base and atop 3.3 Direct infusion of the sample with the TriVersa Nanomate TM (Advion) up to 10 mL with acetic acid. 1. In the interface setting panel, set the controller power On and the temperature to
	4°C (Note 4).
	2. Load 10 µL of desalted protein sample to the 96 or 384-well plate.
	3. In the Spray Optimization panel, set the Sample Volume to 5 µL, the gas pressure
	3. Prepare a 200 mM ammonium acetate solution pH 6.9 by weighting 770.8 mg in to 0.35 psi, the Voltage to apply to 1.60 kV and press "Deliver Sample" (Note 5).
	50 mL. 4. Reconstitute GroEL (Chaperonin 60 from Escherichia coli, Sigma-Aldrich 3.4 Calibration
	3.4.1 Synapt G2 and G2Si reference n°C7688) powder (1 mg) by adding 80 µL of refolding buffer and 20 µL of methanol directly into the vial. 1. Using the TriVersa Nanomate TM (see 3.3), deliver 5 µl of cesium iodide
	5. Shake for 2 h a room temperature. calibration mix (see 2.2).
	6. Transfer the entire volume into an Eppendorf, add 100 µL acetone and agitate 2. In MassLynx MS Tune windows manually set the following parameters:
	gently. Operate the instrument in positive and sensitivity modes with a m/z range from 7. After 2 min, centrifuge for 1 min at 11,000 rpm: a white precipitate should be 1,000 to 8,000. present. Delete the supernatant. Then, add 200 µL of refolding buffer and agitate Nanoflow+ panel: Sampling Cone: 150 V. Source Offset: 30V. Source gently until complete dissolution. Temperature: 80°C. The Capillary Voltage and the gas flows are not used in the 8. Shake for 1 hour at room temperature. Centrifuge for 1 min at 1,500g and take TriVersa Nanomate TM interface setup. the supernatant to perform the first desalting step as explained in 3.1 (on Zeba™ Instrument: Trap and Transfer Collision Energy are turned off (4V and 2V, spin column). respectively by default). 9. The second desalting step is performed as explained in 3.2 with 10 cycles (each one takes between 8-15 min) at 4,500 rmp with a 50 kD MWCO membrane. Save these parameters as an .ipr file.
	Finally, check the protein concentration by UV absorbance at 280 nm. Typically, 3. Press "acquire" to start a 2 min TOF-MS acquisition in the 1000-8,000 m/z
	approximatively 10 µM of 14-mer should be obtained (ε = 10,430 M -1 .cm -1 for range.
	GroEL monomer at 280 nm). 4. Average the signal over the 2 min in the Chromatogram panel and in the
	Spectrum panel, select Process\Automatic Peak Detection with the "Set Peak
	Detection Parameters Automatically" option activated.
	3 Methods 5. Click on File\Save Spectrum and click OK. 3.1 Benchtop Size Exclusion Chromatography desalting 6. In the Acquity UPLC Console, click on Intellistart, select "Create Calibration" 1. Operate at +4°C. 2. You can use 6K MWCO Micro Bio-Spin TM 6 (Biorad) or 0.5 mL 7K MWCO Zeba TM and click Start.
	(ThermoFisher Scientific) spin columns.
	3. Remove the storage solution by 2 min centrifugation at 1,000 g for Micro Bio-
	Spin TM columns and 1 min at 1,500 g for Zeba TM columns.

2. Prepare 10 mL of reconstitution buffer, composed as follows: 200 µL Tris-acetate at 1M previously prepared, 50 µL EDTA at 100 mM, 10.7 mg magnesium acetate tetrahydrate, 5.5 mg ATP, 37.3 mg potassium chloride to reach final concentrations of 20 mM, 0.5 mM, 5 mM, 1 mM and 50 mM, respectively. 7. In the calibration profile editor, select the right Mass Calibration Profile (here CsI in the 1,000-8,000 m/z range), right click and reset it. 8. Right Click and edit the Mass Calibration Profile. Below the Positive Polarity chart, click on edit. Select the right Reference Compound (here "CsI_1000-8000 (positive) with reference masses ranging from 1,172.1450 Da to 7,927.2031 Da (Note 6). 9.

1A and

  Infuse the sample at 5 µM with the TriVersa Nanomate TM (see 3.3). 3. Start with the same parameters as the ones used for CsI calibration but set the cone voltage to 150 V and the extraction cone to 50 V. Increase the backing pressure up to 7 mbar (Note 7). Adjust the pressure inside the first hexapole ion guide to 0.11 mbar using argon (available for the upgraded LCT) in order to optimise the transmission of high molecular species for native MS application (Note 9). 4. Start a 2 min acquisition in the 1,000 to 20,000 m/z range. 5. The corresponding smoothed full mass spectrum is presented in Fig.

	3.5 High mass detection: Analysis of E. coli RNA polymerase on a modified ESI-TOF
	(LCT)
	1. Desalt approximatively 100 µL of E. coli RNA polymerase [17] at 30 µM in 500
	mM ammonium acetate at pH 6.9 as explained in 3.2 using a Vivaspin™ 100 kDa
	cut-off membrane at 15,000 g and +4°C.
	2.

the corresponding deconvoluted spectrum in Fig.

1B

. In order to perform this deconvolution with UniDec [18,19] (http://unidec.chem.ox.ac.uk/), first export the spectrum list (Edit\Copy spectrum list), then paste it in a text editor and save it as a .txt file. Import the .txt file in the UniDec software. Process the data with the following parameters: m/z range 1000 to 12,000 Th; Subtract Curved: 0; Gaussian Smoothing: 10; Bin Every: 10. Deconvolute the processed spectrum with the following parameters: Charge Range: 20 to 50; Mass Range: 20,000 to 500,000 Da; Sample Mass Every 1 Da; Peak FWHM 15 Th; Peak Shape Function: Gaussian.Detect and label the deconvoluted species with the following parameters: Peak Detection Range: 50; Peak Detection Threshold: 0.1; Peak Normalization: Max. As shown in Fig1, the hetero-oligomer of E. coli RNA polymerase (390,315 Da) composed of five subunits and the α-subunits (36,509 Da) species are detected.

  Volatility of the buffer is key for native MS. Beware to avoid any trace of nonvolatile salts such as sodium chloride. Ammonium acetate resuspended at 200 mM in water is usually at pH 6.9. If you want to adjust pH below 6.9, do not use HCl but formic acid or acetic acid instead. Alternatively, you can dilute a commercial 7.5 M ammonium acetate solution (Sigma). Conversely, to increase the pH, use liquid NH3 and not NaOH. 3. For smaller volumes, 7K MWCO Zeba TM Micro Spin columns (2-12 µL, ThermoFisher Scientific) can be used. 4. Do not forget to turn off the cooling of the plate after use of the nanomate.

	4 Notes
	1. The optimum ammonium acetate concentration is protein-dependent and can be
	optimized by testing concentrations ranging from 10 mM up to 1 M. If needed,
	volatile additives can be added, such as dithiothréitol or tris(2-carboxyethyl)
	phosphine for disulfide bond reduction, imidazole, some detergents (see Note X)
	or metallic ions in a volatile buffer (calcium acetate, zinc acetate…).
	2.

Acknowledgement

The authors would like to thank Arnaud Poterszman (CAK complex), Albert Weixlbaumer (E. coli RNA polymerase) and Isabelle Billas Massobrio (ERR complexes) from the IGBMC (Strasbourg, France) and Virginie Gervais & Alain Milon from IPBS (Toulouse, France) for kindly providing samples. This work was supported by the French Ministry of Research (Investissements d'Avenir Program, Proteomics French Infrastructure, ANR-10-INBS-08) and the FEDER (Fonds Européens de Développement Régional), Toulouse Métropole and the Région Midi-Pyrénées.


Lucian Florin

Constantin
email: fl.constantin@univ-lille1.fr

Terahertz Frequency Conversion with a Photomixer

The nonlinear electrical response of a low temperature grown GaAs photomixer driven with the optical beat of two continuous-wave lasers is exploited in optical frequency downconversion for THz-wave modulation and heterodyne detection.

Introduction

The terahertz-waves have a recognized potential for several applications including medical research, atmospheric and astrophysical observation, high-speed communications, chemical sensing and security screening [START_REF] Dhillon | The 2017 terahertz science and technology roadmap[END_REF]. The optical frequency down-conversion with a photoconductive antenna (photomixing) allowed continuous-wave (cw) THz generation [START_REF] Brown | Photomixing up to 3.8 THz in low-temperature-grown GaAs[END_REF]. The approach exploited the optical beat of two cw lasers emitting at frequencies f 1 and f 2 to drive an ultrafast photodetector with quadratic response to the optical fields. Upon application of a dc bias voltage, the photocurrent, oscillating at the difference-frequency f THz =|f 1 -f 2 | in the terahertz domain, was coupled to an antenna to generate THz-waves. Compact, room-temperature operated, cost-effective and low-power consuming THz photonic systems with optical frequency control based on photomixers provided narrow spectral linewidth, wide frequency tunability, high dynamic range and high signal-to-noise ratio. This contribution exploits nonlinear photoconduction in low temperature grown GaAs (LTG-GaAs) for THz-wave modulation and detection by photomixing. Precisely, the optical frequency down-conversion is performed with an alternative electric field applied to the photomixer electrodes either by modulating the bias voltage or by an incoming THz radiation received by the antenna. The nonlinear electrical response of the LTG-GaAs allows frequency mixing in the photoconductor of the optical beat with the alternative electric field, enabling direct current modulation of the photomixing signal [START_REF] Constantin | Direct-current modulation of a photomixing signal[END_REF] or, alternatively, down-conversion to the microwave domain of an incoming THz-wave with a suitably tuned optical beat [START_REF] Constantin | Phase-coherent heterodyne detection in the terahertz regime with a photomixer[END_REF].

Results

The optical frequency down-conversion is performed with two spatially superposed coherent optical waves, detuned by an angular frequency ω and carrying the same optical power P, that enter at normal incidence in a LTG-GaAs photoconductor. The photomixing model developed in this contribution is based on uniform unidimensional carrier transport directed along an electric field oriented in parallel with the surface of the photoconductor. The continuity equations allow to derive the expression of the current density inside the photoconductor :

( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ωτ + ω + ω ωτ + τ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ωτ + ω + ω ωτ + τ ϖ η = 2 2 1 1 1 1 2 E t cos t sin E E v E E t cos t sin E E v E Ad P e t j p p p p n n n n h (1)
where the laser beams, carrying nearly the same energy , make an optical beat which is focused on a photoconductive area A, absorbed within a depth d, with a quantum efficiency η. The dependences of the carrier lifetimes τ n,p (E) with the electric field [START_REF] Zamdmer | Increase in response time of low-temperature-grown GaAs photoconductive switches at high voltage bias[END_REF] are linearized here, by taking into account only the carrier heating :

ϖ h ( ) ( ) [ ] T k eE E B p , n p , n 2 λ 3 1 τ τ 0 0 + ≈ (2)
in function of the Boltzmann constant k B , the electron charge e, the room temperature T=300 K, the low-field carrier lifetimes τ 0n,0p and the distance λ between Coulomb barriers in LTG-GaAs. The carrier drift velocities have a nonlinear dependence on the electrical field [START_REF] Brown | A photoconductive model for superior GaAs THz photomixer[END_REF][START_REF] Brown | Milliwatt output levels and superquadratic bias dependence in a low-temperature-grown GaAs photomixer[END_REF], that reads :

( ) ( ) [ ] E v v E v p , n sp , sn sp , sn p , n 0 0 µ 1+ = (3)
where v sn,sp are the saturation velocities and µ 0n,0p are the low-field mobilities for electrons and holes. The electric field in the photoconductor is expressed in the parallel-plate approximation as an alternative electric field (amplitude E AC =V AC /r, angular frequency ω AC , electrode spacing r) superposed to a bias electric field (E DC =V DC /r ) :

( ) ( ) ( t cos r V r V t E AC AC DC ω × + = ) (4)
The electrical nonlinearity arising from v n,p (E) and τ n,p (E) dependences yields a current density with spectral components at angular frequencies ω±nω AC (n integer). The amplitudes and phases of these spectral components may be analytically calculated with eq. (1-4) for a given E DC using a power series expansion in terms of E AC . The alternative electric field induced by an incoming THz-wave on the antenna electrodes, The experiment (Fig. 1.A) is based on a LTG-GaAs photomixer with a log-spiral antenna driven by the optical beat of two extended cavity laser diodes emitting at 820 nm. The extremities of the antenna are connected to a microwave line addressed with a bias-T, that allows to couple the modulation signal superposed to the bias voltage and to measure the photomixing signal on a microwave spectrum analyser. First, the modulation of the photomixing current is observed by coupling a radiofrequency signal to the photomixer operated with an optical beat in the microwave domain. The spectrum of the photomixing signal (Fig. 1.B) displays the carrier and the sidebands at ω±ω AC with nearly the same amplitude and other less intense sidebands. The amplitudes of the spectral components are calculated with eq. (1-4) using the modulation parameters, the LTG-GaAs photoconductor parameters from refs. [START_REF] Zamdmer | Increase in response time of low-temperature-grown GaAs photoconductive switches at high voltage bias[END_REF][START_REF] Brown | A photoconductive model for superior GaAs THz photomixer[END_REF][START_REF] Brown | Milliwatt output levels and superquadratic bias dependence in a low-temperature-grown GaAs photomixer[END_REF], and the set of photomixer parameters. The results are multiplied with a common factor in order to match the amplitudes of the most intense spectral components and plotted with bars. The calculated and measured amplitudes agree for ω±2ω AC sidebands. The model may be improved by taking into account spatial dependence of the current density, optical power and spatial charge separation effects in the photoconductor. Alternatively, the photomixer is exploited for heterodyne detection of a THz-wave generated with a frequency multiplier driven by a microwave synthesizer (Fig. 1.C). A signal at 102.54 GHz, down-converted to ~0.749 GHz, was recorded with a signal-to-noise ratio of 7.3 in 300 kHz bandwidth. The responsivity is at the 10 -3 A/W level. The linewidth of the signal is related to the spectral purity of the diode lasers locked with the Pound-Drever-Hall technique to a Fabry-Perot cavity. The heterodyne detection signal is sensitive to the THz-wave amplitude and phase and, via antenna's geometry, to the spatial mode and polarization state. The photomixing approach enables integration of the optical beat generation and electrically-driven optoelectronic frequency conversion in a photonic circuit for THz-wave generation and detection.

  -electric field Ε and the effective vector length l . THz frequency downconversion may be performed by heterodyning a THz-wave with the optical beat exploited as local oscillator (LO).

Fig. 1 .

 1 Fig. 1.A. The experimental setup. DL1, DL2 diode lasers, SA spectrum analyzer, LF radiofrequency synthesizer, MW microwave synthesizer. B. Spectrum of a modulated photomixing signal. Optical power 2 × 20 mW, optical beat at ω = 2π × 1178 MHz. Modulation voltage V AC =0.5 V at ω AC = 2π × 2 MHz, bias voltage V DC =0.517 V. The calculated amplitudes of the spectral components are plotted with blue bars. Photomixer parameters used in the calculations: r=2 µm, d=2 µm, A=12.6 µm 2 , e ϖ h =1.5 V, η=0.68. RBW=300 kHz, sweep time 4 ms, 2 video averages. C. Spectrum of the heterodyne detection signal of a THz-wave at ω AC = 2π × 102.54 GHz. Bias voltage V DC =1 mV. Optical beat power 2×14.5 mW. RBW=300 kHz, sweep time 10 ms, 10 video averages. Lorentzian adjustment (red line) with a full-width at half-maximum linewidth of 74 kHz.


M.S Mattia ; Pasqua
email: mattia.pasqua@utc.fr

Ph.D Ulysse Pereira
email: ulysse.pereira@utc.fr

Ph.D Antonietta Messina
email: antonietta.messina@inserm.fr

M.S Claire De Lartigue
email: claire.de-lartigue@utc.fr

Pascale Vigneron
email: pascale.vigneron@utc.fr

M.D Anne Dubart-Kupperschmitt

Cécile Legallais

Mary Ann

Ph.D Cecile Legallais
email: cecile.legallais@utc.fr

Cecile Legallais

HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver

Keywords: Bioartificial liver, Acute liver failure, HepaRG spheroids, Cell encapsulation, Tissue engineering

Impact statement

It is recognized that culturing cells in spheroids is advantageous since they better recapitulate the threedimensional physiological microenvironment. This approach can be exploited in bioartificial liver applications, where getting a functional hepatic biomass is the major pitfall. Our study describes an original method of culturing hepatic cells in alginate beads that allows the autonomous formation of spheroids after three days of culture. In turn, cells adequately differentiate and display a large set of hepatic features. They are also able to treat a model of pathological plasma. Finally, this setup can be easily scaled-up for the treatment of acute liver failure.

Introduction

Fulminant liver failure is a life-threatening critical illness with an extremely high mortality rate, even if intensive care is provided (1). To date, liver transplantation is the only available treatment for patients with end-stage liver disease such as acute liver failure (ALF) [START_REF] Starokozhko | Challenges on the road to a multicellular bioartificial liver[END_REF]. The growing gap between the number of patients on waiting list and the number of donor organs available has highlighted the need for alternative therapies as a bridge to transplantation or liver regeneration. Extracorporeal liver support systems could represent a valid solution for this problematic and nowadays two types of devices are considered: artificial (ALs) and bioartificial liver support systems (BALs). ALs are designed to remove water-soluble toxins from patient's plasma in order to improve clinical state [START_REF] Pless | Artificial and bioartificial liver support[END_REF] while BALs are meant to fulfil the maximum number of synthetic and detoxification liver functions (4). The key component of a BAL is the bioreactor, the cell-housing component. Its expected role is to allow a patient in ALF to survive until an available liver donor or to stimulate autonomous liver regeneration.

Access to a bioactive mass (or biomass) is the main pitfall in the context of human BALs. Although primary human hepatocytes are still considered the gold standard, their limited availability, the phenotypic instability [START_REF] Van Wenum | Bioartificial livers in vitro and in vivo: tailoring biocomponents to the expanding variety of applications[END_REF], as well as logistical issues, hamper their use in BALs. In the past, most of the BALs in clinical or preclinical studies relied on primary porcine hepatocytes, due to their easy availability and high functional activities (6) , [START_REF] Mundt | A method to assess biochemical activity of liver cells during clinical application of extracorporeal hybrid liver support[END_REF] , [START_REF] Van De Kerkhove | Phase I clinical trial with the AMC-bioartificial liver[END_REF]. However, transfer of zoonotic diseases [START_REF] Yang | Fluidized-bed bioartificial liver assist devices (BLADs) based on microencapsulated primary porcine hepatocytes have risk of porcine endogenous retroviruses transmission[END_REF], proteinprotein incompatibility between species and possible immune responses generated during treatment remain challenges for the use of xenogeneic hepatocytes [START_REF] Velde | Immunological Consequences of the use of Xenogeneic Hepatocytes in a Bioartificial Liver for Acute Liver Failure[END_REF]. Recently, the focus has been put on the use of induced pluripotent stem cells and embryonic stem cells as innovative cell source for BAL, especially for their great proliferative capacity and their potential to show metabolic functions close to those of human hepatocytes. Despite great efforts in this direction, today these cell types are not able yet to exhibit full functions of mature hepatocytes in terms of metabolic performance and efforts have still to be made to allow their clinical use [START_REF] Chen | Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells. Gastroenterology [Internet[END_REF].

Currently, human cell lines keep thus a great potential for BAL application. The main advantages of hepatocyte cell lines are their almost unlimited proliferative capacity and the relatively cheap culture process. They demonstrate some metabolic functionalities comparable to human hepatocytes and also to be safe and non-tumorigenic [START_REF] Chamuleau R A Fm | Bioartificial liver: its pros and cons[END_REF]. HepG2 and its sub-clone present limited metabolic functions (5)

F o r P e e r R e v i e w O N L Y / N o t f o r D i s t r i b u t i o n 4
for BAL applications, although encouraging results were recently presented by the group of Selden on a pig model of ALF. HepaRG is a hepatic progenitor cell line able to differentiate into hepatocyte-like cells after 14 days of specific culture [START_REF] Gripon | Nonlinear partial differential equations and applications: Infection of a human hepatoma cell line by hepatitis B virus[END_REF]. Investigators already demonstrated that HepaRG cell line was suitable for BAL application for their proliferation capacity and their enhanced hepatic metabolism in three-dimensional (3D) configuration [START_REF] Hoekstra | The HepaRG cell line is suitable for bioartificial liver application[END_REF].

In the context of BAL, hepatic cell microencapsulation in alginate porous beads has been recognized as an interesting alternative to classical cell immobilization in hollow fibre membranes. This material is quite inert, not toxic regarding cells and ensures an adequate biocompatibility. In addition, its relatively low cost and gelation capacity by divalent cations such as Ca 2+ makes the process suitable for hepatocyte encapsulation [START_REF] Legallais | Bioengineering Organs for Blood Detoxification[END_REF]. In vitro liver models combined with tissue engineering approaches provide a 3D microenvironment which can be expected to mimic in vivo conditions. In the process of encapsulation, cells are entrapped within spherical alginate microbeads that protect them from mechanical stress while ensuring exchanges of nutrients or waste molecules within the surrounding medium. The immunoisolation provided by alginate encapsulation is undoubtedly the major advantage of this technology [START_REF] Tran | Alginate hydrogel protects encapsulated hepatic HuH-7 cells against hepatitis C virus and other viral infections[END_REF].

For BAL application, the challenge is to induce/maintain the hepatic functions over time starting from an initial biomass, taking into account parameters such as costs, manipulation ease for the operator, and scaling-up feasibility. Recently, some authors successfully enhanced biosynthetic and xenobiotic performance of HepaRG functions, cultivating cells as spheroids (SPHs) before encapsulation, without the use of dimethyl sulfoxide (DMSO) [START_REF] Rebelo | HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism[END_REF]. Nevertheless, the technologies available nowadays for the production of large amounts of spheroids (such as rotating or shaking reactors) may lead to an unacceptable loss of cells that reduces considerably the initial biomass. Indeed, the majority of these studies are focused on toxicology approaches in which it is important to evaluate the metabolic activity "per cell". In the context of BAL, the challenge is not to determine the best activity "per cell" but rather "per bioreactor", taking into account logistic hurdles or costs due to additional manipulation.

Therefore, in this study we propose to analyse cell differentiation and metabolic performance of an initial biomass of encapsulated HepaRG in alginate 1.5%, for further use in fluidized-bed BAL.

Materials and Methods

F

o r P e e r R e v i e w O N L Y / N o t f o r D i s t r i b u t i o n 5 2.1. 2D cell culture
HepaRG from Biopredic (Rennes, France) cells were expanded in two-dimensional (2D) monolayers following the indications reported by the supplier. Cells were passaged every 2 weeks until passage 18, with proliferation culture medium William's E (WE, with sodium bicarbonate, without L-glutamine and phenol red, Sigma-Aldrich, with added Biopredic 710 proliferation media) replenishment thrice a week.

The cultures were maintained in a humidified environment at 37°C, 5 % CO 2 . After that, cells were detached by trypsin-ethylenediaminetetraacetic acid (EDTA) 0.25% (ThermoFisher scientific) from the culture flasks and used for the cell encapsulation process (explained in paragraph 2.2).

Alginate microencapsulation

Cells were encapsulated in alginate (Manucol LKX from FMC BioPolymer) 1.5% (w/v), sterilized by successive filtrations (0.8, 0.45, and 0.22 µm pore size membrane filters). The encapsulation was performed with a home-made system based on a coaxial air flow extrusion method [START_REF] Gautier | Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated c3a cells in bioartificial liver applications[END_REF]. Briefly, the alginate solution containing cells was extruded through a 24 G nozzle and the droplets fell into a gelation bath (NaCl 154 mM, HEPES 10 mM and CaCl 2 115 mM, pH 7.4). Droplets produced were allowed to settle for 15 min in the gelation bath to ensure gel formation. After that, the microbeads were washed three times in WE medium and then resuspended in proliferation culture media. Finally, the encapsulated cells were transferred in culture dishes and maintained for 14 days in continuous orbital shaking (60 rpm) in a humidified environment at 37 °C, 5 % CO 2 . Proliferation culture medium was replaced every 2 days. Empty microbeads (without cells) were also produced as control for the following metabolic tests.

Cell aggregates diameter measurement

The diameter of the cellular aggregates was determined by measuring their diameter using ImageJ software version 1.52h. For each experiment, the diameter of 10 aggregates in different beads was measured orthogonally by optical microscopy. Mean and standard deviation were calculated.

Cell performance experimental setup

F o r P e e r R e v i e w O N L Y / N o t f o r D i s t r i b u t i o n 6
The experimental setup designed to assess cell metabolic performance is shown in figure 1. At day 0, cells were encapsulated in alginate 1.5%. Cell differentiation was studied via immunofluorescence staining (paragraph 2.6) at day 0, 7, 10 and 14 post-encapsulation and metabolic test (albumin synthesis) between days 0-1, 6-7, 9-10, 13-14 post-encapsulation. At day 7 and 14 post-encapsulation, the cell performance was tested through metabolic and xenobiotic tests. The latter were conducted in intervals of up to 2 hours of incubation and, at each time-point (time 0 and time 2 hours), aliquots of supernatant were collected for further analysis. At the end of each experiment, microbeads were recovered and assessed for cell viability. The quantity of marker analysed was calculated and normalized by the number of hours of incubation and the number of cells seeded. Moreover, in order to avoid artefacts, all the metabolic activities tested were normalized by the quantity at time zero and the control (empty microbeads).

In order to study the differentiation of cells, at day 0, 7, 10, and 14 post-encapsulation the subcellular localization of proteins typically expressed by hepatoblasts and mature hepatocytes was assessed by immunofluorescence (figure 4). At day 0 post-encapsulation, hepatoblasts are present since we observe their typical markers such as AFP, HNF 6 and CK-19. During the differentiation process, the level of hepatoblasts markers decreases while the mature hepatocytes markers show up (ALB, HNF 4α). The CYP 3A4 is constantly expressed and functional at day 7 and 14. Those qualitative observations are recapitulated in table 2. In parallel, synthesis of albumin remains overall stable over 14 days of culture with a tendency to increase at day 10 (data not shown).

Discussion

In this study, we analysed the metabolic activities of a biomass of micro-encapsulated HepaRG for subsequent treatment of acute liver failure. Such engineered liver tissues could be used in fluidized-bed BAL, a technology developed in our laboratory and in others [START_REF] Legallais | Design of a fluidized bed bioartificial liver[END_REF]. With a similar bioreactor hosting HepG2 grown in alginate beads, Selden's group recently published the improvements of clinical parameters in a surgical porcine model of liver failure [START_REF] Selden | A clinical-scale BioArtificial Liver, developed for GMP, improved clinical parameters of liver function in porcine liver failure[END_REF]. Zhou et al., 2016 also obtained prolonged survival time for pigs with D-galactosamine induced ALF using a fluidized bed BAL hosting porcine primary hepatocytes encapsulated in alginate-chitosan beads.

F o r P e e r R e v i e w O N L Y / N o t f o r D i s t r i b u t i o n 7
As stated in the introduction, further clinical applications request the production of large amounts of encapsulated cells to get a functional and cost effective biomass at human scale. The required hepatic cells' functions are mainly protein synthesis (albumin and clotting factors), ammonia and lactate elimination (both implicated in hepatic encephalopathy and cerebral oedema), xenobiotic detoxification (enzyme machinery involved in cleaning toxins accumulating during ALF) [START_REF] Van Wenum | Bioartificial livers in vitro and in vivo: tailoring biocomponents to the expanding variety of applications[END_REF]. In the present study, we demonstrated that HepaRG microencapsulated in alginate beads were able to produce albumin, detoxify ammonia and lactate, and to express the enzymes involved in the xenobiotic machinery, with highest activity at day 14.

Our results show that cell viability was affected neither by the encapsulation process nor by the longtime culture cells, nor by the exposition to a toxic culture media, containing typical ALF toxins.

Interestingly, within the alginate microbeads, cells were able to rearrange forming cell spheroids, a phenomenon already observed with HepG2/C3A that are highly proliferative [START_REF] Coward | Proliferation rates of HepG2 cells encapsulated in alginate are increased in a microgravity environment compared with static cultures[END_REF] [START_REF] Capone | Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation[END_REF]. In addition, mature hepatocytes markers were present, demonstrating that, when encapsulated, these progenitor cells were able to fully differentiate over time, in the absence of differentiation agents such as growth factors or DMSO, that are classically employed in vitro. The 3D reorganization and the alginate environment are thus promoting cell differentiation in contrast with 2D culture.

Regarding cells functions, we performed a large set of assessment. It is of note that encapsulated HepaRG are responsive to the ICG tests. The uptake of this dye is carried out by the transporters OATP1B3 and NTCP that are expressed at the basolateral plasma membrane of hepatocytes [START_REF] Graaf W De | Transporters involved in the hepatic uptake of and indocyanine green Tc-mebrofenin[END_REF] and excretion is regulated by the transporters MDR3 and MRP2 [START_REF] Cusin | Hepatocyte Concentrations of Indocyanine Green Reflect Transfer Rates Across Membrane Transporters[END_REF], expressed in the hepatocytes apical (canalicular) membrane [START_REF] Oude Elferink | Function and pathophysiological importance of[END_REF]. Moreover, F-actin accumulating at cellular junctional sites of the cell aggregates resembled interconnected bile canalicular structures. This observation has also been noticed by [START_REF] Rebelo | HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism[END_REF] Both facts indicate a highly polarized cellular organization favouring cell maturation [START_REF] Blau | The role of cellular interactions in the induction of hepatocyte polarity and functional maturation in stem cell-derived hepatic cells[END_REF]. Cell aggregates' dimension increased over the days of culture but remained in a range (maximum 120 µm) where mass transfer was not affected, and where oxygen gradient can take place [START_REF] Leedale | In silico-guided optimisation of oxygen gradients in hepatic spheroids[END_REF]. In addition, it has been observed that when HepaRG in 2D configuration are cultured in orbital shaking, hepatic differentiation and metabolic functions are both positively affected due to higher mitochondrial biogenesis [START_REF] Adam | A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines[END_REF]. Also in our experimental setup, the encapsulated cells are kept in orbital

F o r P e e r R e v i e w O N L Y / N o t f o r D i s t r i b u t i o n 8
shaking. These last two phenomena described could induce a gain of function in the whole cell metabolic performances that can explain why some of our activities are stronger in comparison with other authors.

The group of Chamuleau seeded HepaRG in the Amsterdam Medical Center Bioartificial Liver (AMC-BAL), a membrane based bioreactor in which cells are in direct contact with plasma. They reported ammonia (around 0.04 nmol/h/10 6 ) and lactate (around 9.3 nmol/h/10 6 ) detoxification with lower rates in comparison with our results (respectively 119±20 nmol/h/10 6 and 167±24 nmol/h/10 6 ). These differences can be explained by the fact that in our system cells formed spheroids, on the one hand, and are protected from the shear stress by the alginate structure, on the other hand. In addition, these data were obtained with total plasma and the authors suspected that large molecules in plasma could have a deleterious effect on cells' functions [START_REF] Van Wenum | Effects of Healthy-and Acute Liver Failure Plasma on Differentiated Human Heparg Progenitor Cells in Monolayers and Bioartificial Livers[END_REF]. Compared to HepG2/C3A, we can observe that albumin production was in the order of magnitude, and that all phases of enzymatic biodegradation were present, although they are lower than those of primary human hepatocytes just after their collection. Considering Selden's promising results in preclinical studies with HepG2, the same could be expected with HepaRG. As a cell line, issued from hepatocarcinoma, the same protection should be envisaged for safety use in patients, such as the presence of filters downstream the bioreactor [START_REF] Selden | A clinical-scale BioArtificial Liver, developed for GMP, improved clinical parameters of liver function in porcine liver failure[END_REF].

Going back to the encapsulation process, Rebelo et al., 2014 also entrapped HepaRG in alginate beads, with reported high performances regarding biotransformation, interesting toxicological issues. However, they encapsulated pre-formed spheroids and not individual cells, as proposed in the present work. When encapsulating a biomass for long time cultures, it is important to consider different logistical parameters influencing the whole process of manipulation for a future scaling-up for clinical BAL application. From our own experience (Supplementary data), we observed a loss of cells when preparing aggregates/spheroids before encapsulation, especially using processes that can be upscaled such as rotating or shaking reactors. This loss is limited with hanging drop or microfluidic techniques [START_REF] Mehta | Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy[END_REF], but these latter cannot treat billions of cells as requested for a human BAL size. In addition, aggregates pre-formation request at least 3 days of specific culture, before encapsulation, and additional handling that make the GMP production more complex and not cost-effective. As our preliminary results did not show any evidence of better performances of encapsulated pre-formed HepaRG spheroids, we

F o r P e e r R e v i e w O N L Y / N o t f o r D i s t r i b u t i o n
9 conclude that their self-reorganization with alginate beads with appropriate density should be preferred in a clinical application strategy.

In conclusion, in this study we demonstrated that HepaRG cells were able to self-organize as spheroids when entrapped in 1.5% alginate beads. After 14 days of culture, these encapsulated cells presented a wide range of functions (protein synthesis, enzymatic activities and biotransformation of toxins) that are in the range or above those presented in other studies about bioartificial liver. Waiting for fully mature hepatic cells issued from pluripotent stem cells, this HepaRG based bioconstruct demonstrated its potential for further use for extracorporeal treatment of acute liver failure. The next step will thus be to characterize the biomass activity in fluidized-bed BAL, a setup known to favour the mass exchange between the plasma of a patient in ALF and the immobilized cells.

Acknowledgement

We thank Baptiste Hirsinger, Marwa Hussein and Augustin Lerebours for their technical assistance. The HepaRG cell line, media and supplements used for this investigation has been supported by Biopredic International (funded by ANR 16-RHUS-0005 in the framework of iLite program), for which the authors express their appreciation. This cell line has been patented by Inserm, under the number WO 03/004627.

Disclosure Statement

No competing financial interests exist.

Funding

This work was supported by PIA-RHU iLite (ANR 16-RHUS-0005)

1.

Tuñón MJ, Alvarez M, Culebras JM, González-Gallego J. An overview of animal models for investigating the pathogenesis and therapeutic strategies in acute hepatic failure. World J.

Gastroenterol. In a preliminary study, pre-formed spheroids were generated in order to test their metabolic performance at day 7. In particular, after cell amplification in 2D monolayers, cells at passage 18 were detached, mixed with the alginate solution at density of 5x10 6 cells per mL and encapsulated. In order to obtain cell spheroids, 5x10 6 cells were inoculated in glass culture dishes (50 mm x 15mm), coated with an antiadhesive coating (Sigmacote ® , SL2 Sigma-Aldrich). Cells were subjected to continuous orbital agitation at 60 rpm with oscillation amplitude of 16 mm (SSL1 orbital shaker, Stuart) in a humidified environment at 37°C and 5% CO2. Proliferation culture medium was replaced one time during the spheroid's formation. After 3 days of aggregation, spheroids were encapsulated. Aggregate size was determined by measuring Feret's diameter using ImageJ software version 1.52h.

Here we report a comparison between the diameter of pre-formed spheroids and the spheroids formed inside the beads, discussed in this article, at day 7 (figure 1 and 2 supplementary data).

Fig. 1

 1 Fig. 1 experimental setup (see text for the details of the protocol)

Figure 1 supplementary Figure 2 supplementary

 2 Figure 1 supplementary

  spheroids (A) and cells in beads rearranged in spheroids (B) (bright field microscopy). Scale bar: 100 µm 109x36mm (300 x 300 DPI) Page 28 of 28 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, of pre-formed spheroids and cells in beads rearranged in spheroids at day 7 post-encapsulation 99x59mm (300 x 300 DPI) Page 29 of 28 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801 Tissue Engineering

  Tissue Engineering Manuscript Central: http://mc.manuscriptcentral.com/ten large set of hepatic features necessary for the treatment of a patient in acute liver failure. These activities include albumin synthesis, ammonia and lactate detoxification and the efficacy of the enzymes involved in the xenobiotic metabolism (such as CYP1A1/2).

	F o r P e e r R e v i e w O N L Y / N o t f o r D F o r F o r F o r P P P e e r e e r e e r R e v i R e v R e v e w i e w i e w O N L O N O N Y / L Y / L Y / N o t N o N o f t t o r f o f o D r r
	i s t r i b u t i o n i s t r D i s D i s i t t b u t r i b r i b i o u t u t n i o i o
	n n
	2

Table 1 :

 1 list of antibodies

	15(25), 3086, 2009

Table 2 :

 2 evolution of cell differentiation over time

	Cell state	Marker	Day 0	Day 7	Day 10	Day 14
		HNF 6
	Hepatoblasts	CK-19
		AFP
		HNF 4α
	Mature
		ALB
	Hepatocytes
		CYP 3A4
	Legend:	increasing expression,	decreasing expression,	constant expression

  cells self-rearranged in aggregates: diameters over time (A). F-actin (phalloidin, in green) and nuclei (DAPI, in blue) staining at day 14 (B) (confocal microscopy). Scale bar: 20 µm 159x52mm (300 x 300 DPI)

	Page 21 of 28	Tissue Engineering Tissue Engineering Tissue Engineering Tissue Engineering Tissue Engineering Tissue Engineering Tissue Engineering	Page 20 of 28
	8 cell viability after exposition to BAL function test medium at day 7 and 14 (confocal microscopy). In green (Calcein AM): viable cells, in red (ethidium homodimer-1): dead cells, in blue (Hoechst 33342 dye): cell nuclei. Scale bar: 100 µm o F r P e e r R e v i e w O N L Y / N o t f o r D experimental setup (see text for the details of the protocol) 139x62mm (300 x 300 DPI) F o r P e e r R e v i e w O N L Y / N o t f o r D 100 µm 99x69mm (300 x 300 DPI) F o r P e e r R e v i e w O N L Y / N o t f o r D F o r P e e r R e v i e w O N L Y / N o t f o r D F o r P e e r R e v i e w O N L Y / N o t f o r D F o r P e e r R e v i e w O N L Y / N o t f o r D F o r P e e r R e v i e w O N L Y / N o t f o r D F o r e r e v i w N L / t r r D o f o t f o N o / N Y L Y N O O w e i e v e R R r e e e P P r o F 1. Supplementary
		i s t r i b u t i o n i s t r i b u t i o n i s t r i b u t i o n i s t r i b u t i o n i s t r i b u t i o n i s t r i b u t i o n i s t r i b u t i o n i s t r D i s i t b u t r i b i o u t n i o
		Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801	n
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beads morphology (bright field microscopy) and cell viability (confocal microscopy). In green (Calcein AM): viable cells, in red (ethidium homodimer-1): dead cells, in blue (Hoechst 33342 dye): cell nuclei. Scale bar: immunofluorescence staining over time (confocal microscopy). Hepatoblast markers (Hb): HNF 6 in red, CK-19 in green, AFP in green, nuclei in blue. Mature hepatocyte markers (MH): HNF 4α in red, ALB in green, CYP 3A4 in green, nuclei in blue. Scale bar: 20 µm 139x95mm (300 x 300 DPI) Page 22 of 28 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801 metabolic activities of encapsulated cells at day 7 and day 14. Albumin secretion rate (A, significance analysed by Wilcoxon matched-pairs signed-ranks test), ICG releasing rate by the transporters MDR3 and MRP2 (B), CYP1A1/2 activity (C), CYP3A4 activity (D) 160x154mm (300 x 300 DPI) Page 23 of 28 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801 CYP1A1/2 activity after induction by β-naphthoflavone 100 µM and rifampicin 10 µM at day 7 (A) and 14 (B), significance analysed by Mann-Whitney Test 160x80mm (300 x 300 DPI) Page 24 of 28 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801 metabolic activities of encapsulated cells in BAL function test medium at day 7 and 14. Albumin synthesis rate (A), ammonia (B) and lactate (C) detoxification rate 159x58mm (300 x 300 DPI) Page 25 of 28 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801 cell viability after exposition to BAL function test medium at day 7 and 14 (confocal microscopy). In green (Calcein AM): viable cells, in red (ethidium homodimer-1): dead cells, in blue (Hoechst 33342 dye): cell nuclei. Scale bar: 100 µm 99x99mm (300 x 300 DPI) Page 26 of 28 Mary Ann Liebert, Inc.,140 Huguenot Street, New Rochelle, NY 10801