Adnan Sayegh

Guillaume Longatte

Olivier Buriez

Francis-André Wollman

Manon Guille-Collignon

Eric Labbé

Jérôme Delacotte

Frédéric Lemaître

Diverting Photosynthetic Electrons from Suspensions of Chlamydomonas reinhardtii Algae -New Insights using an Electrochemical Well Device

Keywords: photosynthesis, quinones, electrochemistry, Chlamydomonas reinhardtii algae, photocurrent

In the last years, many strategies have been developed to benefit from oxygenic photosynthesis in the present context of renewable energies. To achieve this, bioelectricity may be produced by using photosynthetic components involved in anodic or cathodic compartments. In this respect, harvesting photosynthetic electrons from living biological systems appears to be an encouraging approach. However it raises the question of the most suitable electrochemical device. In this work, we describe and analyze the performances of an electrochemical device based on a millimeter sized well involving a gold surface as a working electrode. Photocurrents were generated by suspensions of Chlamydomonas reinhardtii algae using quinones as mediators under different experimental conditions. Chronoamperometry and cyclic voltammetry measurements gave insight into the use of this device to investigate important issues (harvesting and poisoning by quinones, photoinactivation…). Furthermore, by introducing a kinetic model originally developed for homogeneous catalytic systems, the kinetics of the electron diverting from this system (Chlamydomonas reinhardtii algae + 2,6-DCBQ + miniaturized setup) can be estimated. All these results demonstrate that this experimental configuration is suitable for future works devoted to the choice of the best parameters in terms of long lasting performances.

Introduction

Producing electricity from natural photosynthesis is still a burgeoning field. [START_REF] Blankenship | Comparing Photosynthetic and Photovoltaic Efficiencies and Recognizing the Potential for Improvement[END_REF] Photosynthesis is the process used by plants, cyanobacteria and algae to convert sunlight into chemical energy eventually stockpiled as carbohydrates. Briefly, light absorption by chlorophyll antenna induces excitation of the Photosystem II (PSII that transfers its energy to the PSII primary donor, P680 (chlorophyll dimers)). The subsequent charge separation results in water oxidation and in some electron transfer steps along the chain (plastoquinone (PQ) / plastoquinol (PQH2) pool → cytochrome b6f (b6f) → plastocyanin (PC) → Photosystem I (PSI) → Ferredoxin (Fd) → Ferredoxin-NADP + reductase (FNR)) that lead to the NADP + reduction (Figure 1A). The consecutive ATP production by ATP synthase finally leads to CO2 reduction by the mean of the Calvin cycle.

Taking advantage of natural photosynthesis generally occurs by using an outer polarized electrode for collecting a fraction of the electron flow along the photosynthetic chain. This harvesting strongly depends on the biological target, as summarized in several reviews. [START_REF] Sekar | Recent advances in photosynthetic energy conversion[END_REF][START_REF] Voloshin | Photoelectrochemical cells based on photosynthetic systems: a review[END_REF][START_REF] Mccormick | Biophotovoltaics: oxygenic photosynthetic organisms in the world of bioelectrochemical systems[END_REF] In fact, using isolated photosystems (PSII and PSI) as photochemical converters is one of the best strategies to favor electron transfer between the photosynthetic target and the collecting electrode. [START_REF] Badura | Wiring photosynthetic enzymes to electrodes[END_REF][START_REF] Kato | Protein film photoelectrochemistry of the water oxidation enzyme photosystem II[END_REF][START_REF] Tel-Vered | Photo-bioelectrochemical Cells for Energy Conversion, Sensing, and Optoelectronic Applications[END_REF] Nevertheless, it raises the question of the stability/denaturation of these systems outside their biological environment. This is why isolated thylakoid membranes or chloroplasts are also considered. [START_REF] Calkins | High photo-electrochemical activity of thylakoid-carbon nanotube composites for photosynthetic energy conversion[END_REF][START_REF] Hasan | Photoelectrochemical Communication between Thylakoid Membranes and Gold Electrodes through Different Quinone Derivatives[END_REF][START_REF] Hasan | Photobioelectrocatalysis of Intact Chloroplasts for Solar Energy Conversion[END_REF][START_REF] Pinhassi | Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures[END_REF][START_REF] Rasmussen | High performance thylakoid bio-solar cell using laccase enzymatic biocathodes[END_REF] Yet, the lack of cell proliferation of these two strategies is an important issue and pushes toward the investigation of intact photosynthetic organisms. [START_REF] Hasan | Evaluation of Photocurrent Generation from Different Photosynthetic Organisms[END_REF][START_REF] Sekar | Photocurrent generation by immobilized cyanobacteria via direct electron transport in photo-bioelectrochemical cells[END_REF][START_REF] Grattieri | Bioelectrochemical Systems as a Multipurpose Biosensing Tool: Present Perspective and Future Outlook[END_REF][START_REF] Laureanti | Photosynthetic Microbial Fuel Cells[END_REF][START_REF] Strik | Microbial solar cells: applying photosynthetic and electrochemically active organisms[END_REF] However, the more complex the target is, the more difficult is the electron transport ways toward the electrode. [START_REF] Zhang | Photoelectrochemistry of Photosystem II &ITin Vitro&IT vs&IT in Vivo&IT[END_REF] Indeed, direct harvesting from a photosynthetic organism is rather scarce. [START_REF] Kim | Patterned Nanowire Electrode Array for Direct Extraction of Photosynthetic Electrons from Multiple Living Algal Cells[END_REF][START_REF] Ryu | Direct Extraction of Photosynthetic Electrons from Single Algal Cells by Nanoprobing System[END_REF][START_REF] Kim | Scalable long-term extraction of photosynthetic electrons by simple sandwiching of nanoelectrode array with densely-packed algal cell film[END_REF] This is why electron shuttles including soluble mediators (quinones, Fe(CN)6 3-…), redox polymers and nano-objects are used as complementary tools to enhance photocurrent production. [START_REF] Sekar | Recent advances in photosynthetic energy conversion[END_REF] Of course, the distance between the photosynthetic target and the electrode is another important issue that determines the experimental configuration (immobilization vs. suspension).

In this context, we have recently investigated an experimental configuration where a Chlamydomonas reinhardtii algae suspension was mixed with exogenous quinones. [START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF][START_REF] Longatte | Mechanism and analyses for extracting photosynthetic electrons using exogenous quinones -what makes a good extraction pathway?[END_REF] Living algae suspension was used because it potentially ensures its own culture and proliferation during the electron extraction process. To collect the photosynthetic electrons a polarized carbon gauze electrode (geometric surface ~1 cm 2 ) was used to oxidise hydroquinone (QH2) on its surface. [START_REF] Fu | Redesigning the Q(A) binding site of Photosystem II allows reduction of exogenous quinones[END_REF][START_REF] Longatte | Electrochemical Harvesting of Photosynthetic Electrons from Unicellular Algae Population at the Preparative Scale by Using 2,6-dichlorobenzoquinone[END_REF] However, though performances in terms of current magnitude (up to 60 µA cm -2 ) were quite encouraging, quinones exhibited an antagonistic behaviour. [START_REF] Longatte | Electrocatalytic Mechanism Involving Michaelis-Menten Kinetics at the Preparative Scale: Theory and Applicability to Photocurrents from a Photosynthetic Algae Suspension With Quinones[END_REF] Indeed, quinones acted as good electron carriers but also as poisoning agents. [START_REF] Longatte | Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time[END_REF] Chronoamperometry measurements helped in the identification of different poisoning pathways, but several issues are difficult to address with this set-up. On the one hand, the relatively large size of the carbon gauze electrode led to high equilibration times that required long timeframes (~1h) to be neglected before the proper experiment. On the other hand, it corresponded to quite large time constants that required long (and possibly photoinactivating) irradiation times to reach a steady state photocurrent. Furthermore, even though it is slightly less crucial, the preparative scale involved the use of forced convection conditions that are energy consuming. As a consequence, these main drawbacks prevent the use of this former set-up to achieve both systematic and comprehensive analyses of quinones.

Because the role of quinones derivatives as photosynthetic electron cargos is still a matter of interest, [START_REF] Hasan | Photoelectrochemical Communication between Thylakoid Membranes and Gold Electrodes through Different Quinone Derivatives[END_REF][START_REF] Hasan | Evaluation of Photocurrent Generation from Different Photosynthetic Organisms[END_REF][START_REF] Longatte | Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time[END_REF][START_REF] Grattieri | Understanding Biophotocurrent Generation in Photosynthetic Purple Bacteria[END_REF] we therefore report on a device aimed at decreasing the size of the photoelectrochemical cell. The use of a miniaturized well-type electrochemical device (Figure 1B) results in diminishing the experimental average time and allows exhaustive parameter analyses. Furthermore, the use of a mechanistic model originally devoted to classic homogeneous systems allows fast qualification of the performances of a given algae/mediator couple in a semi-quantitative way. Here the main properties of the generated photocurrents can thus be described and analyzed as a function of several experimental parameters in order to have in the future a better understanding of phenomena occurring in both algae and extracellular medium during electron harvesting.

Experimental

2.1.Cell culture and preparation

Chlamydomonas reinhardtii algae (wild-type or mutants) were grown in Tris Acetate Phosphate aqueous medium (TAP) containing Tris base (20 mmol L -1 ), NH4Cl (7 mmol L -1 ), MgSO4 (0.83 mmol L -1 ), CaCl2 (0.45 mmol L -1 ), K2HPO4 (1.65 mmol L -1 ), KH2PO4 (1.05 mmol L -1 ), CH3CO2H (0.3 mmol L -1 ) at 25°C under rather dim light conditions (50 µE m -2 s - 1 ) to a cell suspension at a concentration of 2.10 6 cells mL -1 , then centrifugated at 4000 rounds min -1 before being re-suspended into Tris-minimal medium (TAP without acetate) for electrochemical experiments (see below) to reach a final steady concentration of 2.10 7 cells mL -1 that corresponds to stable conditions for algae. In specific cases, Tris-minimal medium supplemented with Ficoll 400 (10% w/w) was used and prepared according to the reference [START_REF] Nawrocki | State transitions redistribute rather than dissipate energy between the two photosystems in Chlamydomonas[END_REF].

Chemical materials and solutions preparation

All chemicals including quinones have been purchased from sigma Aldrich and have been used without any further purification. In this work, 2,6-dichlorobenzoquinone (2,6-DCBQ) and 2,6-dimethylbenzoquinone (2,6-DMBQ) were selected owing to their different ability to harvest photosynthetic electrons from algae. [START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF][START_REF] Fu | Redesigning the Q(A) binding site of Photosystem II allows reduction of exogenous quinones[END_REF] Practically, quinones were dissolved in absolute ethanol in order to make mother solutions (10 mmol L -1 ). Appropriate small volumes of these solutions were thus directly added to the algae suspension for subsequent electrochemical experiments (see below).

Electrochemical experiments 2.3.1. Fabrication of the ITO-Au device

An ITO/Au modified glass slide was used as the working electrode. This electrode was obtained by sputtering a thin indium tin oxide (ITO) film (thickness 10 nm) followed by another film of gold (50 nm) onto a glass slide (Deckglaser Menzel-Glaser microscope cover slides, Fisher Scientific; 24 mm x 48 mm x 170 µm) using a sputtering system EMITECH K675X. ITO layer allowed good adhesion between glass and gold film. A PDMS (polydimethyloxane RTV-615; Momentive Performance Materials France) piece with a 9 mm diameter hole inside was attached on the ITO/Au modified glass slide by the mean of a treatment with oxygen plasma at 400 mtorr (100 W) for 3 min (Harrick Plasma, NY, USA). The final device thus corresponds to a PDMS well (volume ∼0.5 ml) possessing the ITO/Au working electrode (9 mm in diameter) in the bottom. Appropriate electrical connections were made by using a copper wire and a silver paint (Radiospares) covered by an epoxy glue.

Cyclic voltammetry experiments

A platinum wire (30 x 1 mm) and a Ag/AgCl wire (35 x 0.5 mm) with chloride anions in solution (~8 mmol L -1 ; see above) were used as counter and reference electrodes, respectively. Experiments were performed with aqueous solutions (500 µL) containing quinones and/or algae within the gold PDMS well using a Parstat 2273 potentiostat (Princeton Applied Research). In the case of quinone/algae mixture, this corresponds to an excess of quinones since the mean ratio of photosystems and quinones is around 1:1000 (for instance with a concentration of algae equal to 2.10 7 cells mL -1 and a PSII concentration of 4.10 6 PSII cells -1 [START_REF] Kirst | Truncated Photosystem Chlorophyll Antenna Size in the Green Microalga Chlamydomonas reinhardtii upon Deletion of the TLA3-CpSRP43 Gene[END_REF] and a quinone concentration equal to 100 µmol L -1 ). Actinic white light was provided by a Scott KL1500 LCD halogen lamp. The lamp was switched on 30 min before shutter opening to allow temperature stabilization. Once algae were incubated with quinones, the suspension was left in the well at open circuit voltage (OCV) for 5 seconds before launching the measurement. All the measurements with/without algae were carried out with constant scan rate ranging from 1 mV.s -1 to 100 mV s -1 (5 kHz filter).

Photocurrent recording

The same set-up was used for chronoamperometry measurements. Quinones were added to the algae suspension and kept 5 seconds at OCV for equilibration. Then the working electrode was polarized at 0.38 V vs Ag/AgCl. After stabilization of the baseline, the light source shutter was opened, a faradic photocurrent being instantly generated. Light is turned off two minutes before the end of the experiment to allow the current to reach the baseline.

All plots and statistical analyses (average values ± s.e.m.) were performed using SIGMA Plot 9.0 software (Systat Software Inc., Richmond, CA, USA).

Results and discussion

Validation of the miniaturized device

Chronoamperometry measurements were carried out using the well device containing algae suspension with 2,6-dichlorobenzoquinone (see experimental section). The electrode potential was appropriate to oxidize the hydroquinone (QH2 = Q + 2e -+ 2H + ) resulting from the electron harvesting by the quinone form (Figure 2A). The background current observed after electrode polarization canceled after 90 s. This current magnitude decay (too long for a pure capacitive effect) may be ascribed to the faradaic re-oxidation of the fraction of hydroquinones generated by the algae metabolism in the presence of quinones under dark conditions. [START_REF] Fu | Redesigning the Q(A) binding site of Photosystem II allows reduction of exogenous quinones[END_REF][START_REF] Longatte | Electrochemical Harvesting of Photosynthetic Electrons from Unicellular Algae Population at the Preparative Scale by Using 2,6-dichlorobenzoquinone[END_REF][START_REF] Longatte | Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time[END_REF] Of note, the baseline current remained stable beyond this equilibration time (Figure 2B). As a consequence, the miniaturization of the electrode enhanced noticeably the time response compared to the previously described setup with a carbon gauze electrode (a few minutes vs one hour respectively) according to a smaller electrode surface area in the new device. The illumination of algae in the medium supplemented with quinones triggered an instant photocurrent generation (Figure 2B) that directly results from the photosynthetic electron diverting from algae (see control experiments without algae or quinones in Figure S1). As expected, this photocurrent increased until reaching a steady state value. For given experimental conditions (quinone concentration, light illumination, algae concentration), reproducibility is quite good and is likely related to cell variability. For instance, chronoamperometry measurements (Figure 2B) from a suspension (2.10 7 cells mL -1 ) illuminated at P = 60 mW cm -2 in presence of 2,6-DCBQ (100 µmol L -1 ) gives a maximum current value (Imax) equal to (22.3 ± 3.9) µA. Photocurrents appeared sensitive to many experimental parameters (see below) and varied in the range of 10-30 µA, i.e. 16-50 µA cm -2 .

These are common values for comparable biophotoelectrochemical systems. Subsequently, the lack of forced convection raised the question of the sedimentation of the algae suspension.

Algae sedimentaion did not affect photocurrent dynamics

A first way to evidence a possible sedimentation of the algae suspension during electron harvesting is to investigate the effect of the medium viscosity. Therefore, we performed chronoamperometry measurements with algae suspensions in minimum medium supplemented with Ficoll. [START_REF] Nawrocki | State transitions redistribute rather than dissipate energy between the two photosystems in Chlamydomonas[END_REF] Indeed, Ficoll 400 is a high-mass (~ 400 kDa) and hydrophilic polysaccharide that is easily soluble in aqueous solution. It is known to hinder the cell motility without exceeding physiological osmolality and it was therefore used to prevent the suspension of sedimentation. [START_REF] Qin | Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids[END_REF] As displayed in Figure 3A, the recorded steady-state photocurrents with 2,6-DCBQ were lower in Ficoll supplemented medium ((22.3 ± 3.9) µA vs (13.8 ± 2.1) µA respectively. 2,6-DCBQ is an efficient electron acceptor so the same experiment was performed with 2,6-DMBQ whose ability to harvest electrons from the photosynthetic chain is rather moderate. [START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF][START_REF] Fu | Redesigning the Q(A) binding site of Photosystem II allows reduction of exogenous quinones[END_REF] The same trend was thus observed (Figure S2). The ratio of the photocurrents with and without Ficoll is about (1.6 ± 0.1) in both cases. This is globally consistent with the viscosity of a 10% w/v Ficoll solution (intrinsic viscosity being 17 cm 3 g -1 ). Furthermore, normalized chronoamperograms (Figure 3B and Figure S2) demonstrate that the whole shape of the i = f(t) curve did not strongly depend on the medium as also evidenced by the half-time values (2,6-DCBQ : (135 ± 25) s with Ficoll vs (115 ± 15) s without Ficoll; 2,6-DMBQ : (395 ± 40) s with Ficoll vs (350 ± 30) s without Ficoll). This suggests that sedimentation of the algae suspension did not significantly occur during electron diverting and that differences between the currents might be related to other parameters like quinones diffusion. Furthermore, this trend was confirmed by additional experiments where effects of natural and forced convection were investigated in presence of the reduced form of a dichloroquinone. Of note, this kind of experiments could not be achieved with dichloroquinone due to its ability to poison the biological system, as reported elsewhere. [START_REF] Longatte | Electrochemical Harvesting of Photosynthetic Electrons from Unicellular Algae Population at the Preparative Scale by Using 2,6-dichlorobenzoquinone[END_REF][START_REF] Longatte | Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time[END_REF] Thus two pre-treatments before applying the detection potential and light illumination were compared, i. Therefore, these results show that the transient and steady-state phases are not significantly affected by sedimentation but are mainly due to the start of the diverting by the quinones (partitioning phenomena, electron harvesting along the chain and biological adjustment) before reaching a pseudo-equilibrium state between the extraction and oxidation reaction rates.

Cyclic voltammetry experiments

The device shown in Figure 1B was also used to perform cyclic voltammetry (CV) experiments on an algae suspension with 2,6-DCBQ (Figure 4A). Under dark conditions, the quasi-reversible CV of the QH2/Q redox couple is observed as expected for a case where the electron harvesting by quinones did not take place. After illumination over 1 min, taking into account the faradic signals, the reduction peak (Q → QH2) decreases in favor of the oxidation peak (QH2 → Q). This shows the quite fast ability of quinones to accept electrons from the algae even when they are in such large excess and confirms the formation of the hydroquinone form during this reaction.

A way to investigate the process of current production is to consider cyclic voltammogramms of algae with hydroquinone species. CVs of algae suspension with dichlorohydroquinone are displayed in Figure 4B. At very low scan rates and under illumination (see also Figure S3), the electrogenerated quinones can interact with algae. The corresponding cyclic voltammogramm is only observed under light and is due to an electrocatalytic cycle involving two steps under illumination (QH2 = Q + 2e -+ 2H + and reduction of Q by the algae).

It is challenging to model in depth this system involving heterogeneous components (aqueous solution, algae, membranes, electrode…). First of all, the introduction of the electrochemical data collected from our quinone-algae system within a catalytic model developed for soluble species was not aimed at providing an accurate set of kinetic constants related to the photo-generation of QH2. Our ambition here is to check whether the model provides turnover characteristics in keeping with the observation, thus allowing to use it for rough qualification of the performance of a given algae-quinone system. Therefore, several orders of magnitude can be extracted by means of some assumptions. In average, Chlamydomonas reinhardtii is a sphere of 3 µm radius. [32] Considering diffusion coefficients of quinones in aqueous solution (2.10 -9 m 2 s -1 ) and lipids (~ 10 -16 m 2 s -1 ), [START_REF] Ilani | DIFFUSION-LIMITED AND REACTION RATE-LIMITED REDOX PROCESSES MEDIATED BY QUINONES THROUGH BILAYER LIPID-MEMBRANES[END_REF] the time for a quinone to cross a lipidic membrane (thicknesses ~ 4 nm) and aqueous compartments of an alga is around 180 ms. This is lower than the characteristic time (~ 5 s) deduced from the usual convection-diffusion thickness (100 µm). It therefore suggests that diffusion within algae can be neglected as a first approximation so the algae may be assimilated to uniform macrosystems catalyzing the reduction of exogenous quinones under illumination. As a consequence and because the quinone concentration is relatively low, electron harvesting may be roughly modeled as a pseudo-first order law (v = k'[Q]) by considering the excited site (PSII complex) concentration as a mean constant value. Combining these assumptions to the relatively low effect of sedimentation, the system may be globally considered as homogenenous and described as an electrocatalytical cycle where Q is reduced by algae to QH2 and QH2 is oxidized at the electrode surface to reform Q. Therefore, at low scan rates, the resulting S-shaped catalytic wave should be independent of scan rate according the following equation adapted to our experimental case: [34-36]

2 2 0 2 'D 2 2 1 exp ( ( / )) QH QH C k i FS F E E QH Q RT =   + - -°    (1)
Where C°QH2 is the added hydroquinone concentration, DQH2 is the diffusion coefficient of QH2 and S is the electrode surface area.

For potential values higher than E°, the current remains constant according to :

2 2 0 2 'D steady state QH QH i FSC k = (2)
In absence of substrate to reduce the electrogenerated Q, i.e. for a QH2/algae mixture under dark conditions, the current follows the Randles-Sevcik's equation and depends on the scan rate "v" :

2 2 0 0 2 0.446 2 QH p QH D Fv i FSC RT = × (3)
Finally, one obtains : 0 ' 2.24 ' 0.254 2

steady state p i RT k k i Fv v = = (4)
This ratio directly depends on v -1/2 . However, it requires that the current plateau observed at 1 mV s -1 is the maximum value. Complementary amperometric measurements with the dichlorohydroquinone lead to a steady-state value "isteady state" of 15.3 µA. This value will be coupled with the oxidation peaks "i°p" from the CVs recorded as a function of the scan rate.

The appropriate range of scan rates corresponding to an electrocatalytic behavior can be figured out by depicting the ratio of currents under illumination and dark conditions as a function of -log(v) (see Figure S4). Displaying isteady state/i°p as a function of v -1/2 results in a straight line shape (Figure S5). The slope is equal to (0.163 ± 0.02) V 1/2 s -1/2 and leads to k' = (0.41 ± 0.05) s -1 . In this view, k' is an apparent first order constant that takes into account the mean rate of electron harvesting among the reaction-diffusion layer. It is formally equal to the real rate constant multiplied by the substrate concentration (i.e. excited PSIIs) so it is actually related to the turnover frequency (TOF) of the catalyst. k' therefore accounts for the total number of excited PSIIs which will release their charge through the catalyst by unit of time.

Of note, k' represents a global rate constant that encompasses all the electron extraction steps (including the quinone partitionning with membranes and cell compartements [START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF]). The corresponding half-time is therefore t1/2 = ln2/k' ~ 1.7 s. In other words, 90 % of quinones are reduced after 6 s in accordance with the cyclic voltammograms mentioned above confirming an efficient catalysis. Furthermore, one can determine the diffusion-reaction layer according to µ = (D/k') 1/2 ~ 70 µm. As expected, the current production is due to reactions occurring within a small equivalent volume of ~ 5 µL (i.e. 1 % of the volume of solution).

Of note, reducing this complicated biophotoelectrochemical system to a homogeneous electrocatalytical mechanism is an oversimplification. However, considering the timescales under study, this approximation finds its reliability in the fact that it gives access to interesting orders of magnitude to understand the phenomena involved in the electron diverting.

Complementary views can be provided by investigating the effects of several experimental parameters.

Effects of experimental parameters

Quinone concentration

Photocurrents were recorded as a function of the concentrations of two exogenous quinones (2,6-DMBQ and 2,6-DCBQ) that behave differently in terms of their ability to harvest photosynthetic electrons from Chlamydomonas reinhardtii algae (2,6-DCBQ is significantly more efficient than 2,6-DMBQ). [START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF][START_REF] Fu | Redesigning the Q(A) binding site of Photosystem II allows reduction of exogenous quinones[END_REF] The curves are displayed in Figure 5.

In both cases, the steady state current reaches a saturation point at high concentration. This is consistent with a Michaelis-Menten like behavior (Iss = ImaxCQ/(KM+CQ)), as evidenced by the quite good correlations between the experimental and theoretical curves. This correlation is consistent with the ability of PSII to catalyze the reduction of exogenous quinones.

Furthermore, this has been already demonstrated for other cases (isolated PSII, cyanobacteria…) [START_REF] Kasuno | Characterization of the photoinduced electron transfer reaction from the photosynthetic system in Rhodobacter sphaeroides to an exogenous electron acceptor[END_REF][START_REF] Torimura | Electrochemical investigation of cyanobacteria Synechococcus sp PCC7942-catalyzed photoreduction of exogenous quinones and photoelectrochemical oxidation of water[END_REF][START_REF] Brinkert | Photocurrents from photosystem II in a metal oxide hybrid system: Electron transfer pathways[END_REF] and confirms that the photocurrent production is globally controlled by the electron diverting by quinones. [START_REF] Longatte | Electrochemical Harvesting of Photosynthetic Electrons from Unicellular Algae Population at the Preparative Scale by Using 2,6-dichlorobenzoquinone[END_REF] In fact, at high concentrations, the extraction process is determined by the electron transfer itself and leads to a saturation of the current.

Conversely, the harvesting is limited by the quinone arrival and leads to the current increase at low concentrations. Furthermore, the results obtained are consistent with the intrinsic

Algae concentration

Effects of algae concentration were also investigated. As displayed in Figure 6, the steady state photocurrents (recorded at t = 900 s) strongly depend on algae concentration. As expected, significant photocurrents were detected for high algae concentration in agreement with a larger number of available extraction sites within the suspension. However, a threshold is observed for algae concentrations of 8.10 7 cells mL -1 . In this case, oxygen may become scarce and consumption by algae becomes more difficult. It may therefore induce anaerobiosis that may limit the ability of the suspension to perform photosynthesis. It is worth mentioning that low quinones-algae ratios should disadvantage the electron diverting due to the ability of quinones to be sequestered in cell compartments before accessing the photosynthetic chain. [START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF] Finally, considering time experiments over 1000 s demonstrated that the photocurrent progressively decreases at long timeframes (Figure 6A) according to a gradual poisoning of the suspension.

[27]

Stability -day/night cycles

The progressive decrease occurring during the photocurrent production depends on the quinone concentration (Figure 7A). The photocurrent is especially stable for low added quinone concentrations (none or slow decrease after the maximum current for 2,6-DCBQ concentrations ranging from 50 to 100 µmol L -1 ) in accordance with the poisoning effect of quinones that is concentration-dependent. [START_REF] Longatte | Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time[END_REF] Conversely, higher 2,6-DCBQ concentrations lead to an immediate decrease after the maximum current value (slopes of -(4.5 ± 0.6) 10 -3 µA s -1 and -(8.3 ± 1.2) 10 -3 µA s -1 for 150 and 200 µmol L -1 respectively).

However, the stability of the electron harvesting process is also related to the illumination and induced photoinactivation phenomena. This can be featured by comparing experiments with a continuous illumination and day/night cycles. In the case of 2,6-DCBQ (Figure 7B), the decrease at long time frames is more significant for a continuous illumination, thus demonstrating that photoinactivation is also involved in the whole performance decrease of the electron harvesting. In the case of 2,6-DMBQ (Figure S6), the photocurrent reaches a steady state values under the same experimental conditions, as expected for this relatively low poisoning quinone. [START_REF] Longatte | Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time[END_REF] Nevertheless, currents recorded by means of day/night cycles are still higher than those measured under a continuous illumination. This shows again that photoinactivation occurs during the extraction. However, discriminating quinone poisoning and photoinactivation is not obvious. Indeed, electron harvesting mainly takes place within the diffusion-reaction layer, i.e. close to the electrode surface. Near the electrode surface and under illumination, the electrochemical oxidation of QH2 leads to high local concentrations of quinones that both favor poisoning and extraction from algae. Under dark conditions, the electrogenerated quinones can diffuse out of the reaction layer limiting their possible toxic effect. The effect of quinones poisoning could only be investigated by recording chronoamperograms after a pre-incubation period (Figure S7). In the case of 2,6-DCBQ, the control experiment (no incubation) corresponds to the expected increase of current.

Furthermore, the recorded currents at different incubation times gave better results at shorter times in accordance with the accumulation of quinones within the algae and the production of hydroquinones ready to be oxidized when applying light. Conversely, currents recorded at larger timeframes rapidly diminished because of the poisoning that occurred during the incubation. In all cases, this derivation appears to be harmful to the biological system whether by poisoning or photoinactivation. This result raises the question of the possible alleviation of the photosynthetic chain in the presence of exogenous quinones.

May quinones alleviate the photosynthetic chain?

Analyses involving this device confirmed that photoinactivation and quinone poisoning can occur during the extraction itself. However, this does not necessarily mean that electron diverting is not able to alleviate the photosynthetic chain under light illumination and prevent photoinactivation, this fact being a major concern for the stability of any bioelectrochemical photosynthetic system. This is why the previously described electrochemical experiments were also achieved with preliminary highly illuminated algae suspension. In this case, ∆f1 mutants were considered because they lack b6f complex. [START_REF] Rimbault | Identification of the initiation codon for the atpB gene in Chlamydomonas chloroplasts excludes translation of a precursor form of the beta subunit of the ATP synthase[END_REF] The photosynthetic chain is therefore interrupted downstream of PSII complex making the algae more prone to undergo photoinactivation. As displayed in Figure 8, photocurrents with 2,6-DCBQ (one of the best PSII acceptors due its high redox potential [START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF]) are higher when no previous illumination was applied. It therefore suggests a protective effect at least at short times against photodamages and shows that the proper choice of quinone structure (as well as effective diverting achieved by relevant concentrations) is necessary to achieve long-lasting experiments while also avoiding poisoning effects.

Conclusion

In this work, we reported on the use of a device involving a millimeter-sized gold electrode for investigating electron harvesting from a photosynthetic algae suspension. As long as no stirring wais applied, significant photocurrents were recorded. In this configuration, extraction was demonstrated to mainly occur near the electrode surface in a tiny reaction-diffusion layer, thus enabling to estimate a global extraction rate. Moreover, the relatively low surface area of the device helped to strongly decrease the equilibration time (once the potential was set at a value allowing the hydroquinone to be oxidized) compared to the previously reported set-up involving a centimeter sized carbon gauze. As a consequence, this device enabled us to perform larger number of experiments for investigating various effects of experimental parameters (quinone and algae concentrations, stability…). Because this analytical strategy confirmed some previous trends already observed at the preparative scale (poisoning, photoinactivation), it paves the way for setting future criteria toward the choice of quinone structure in order to attain maximum photocurrent stability over the longterm and performances for bioelectrochemical photosynthetic systems.

  Figure 3C and show that natural and forced convections before electron diverting had no significant effect (Imax = (19.8 ± 3.5) µA and t1/2 = (20 ± 5) s without any previous stirring s vs Imax = (19.7 ± 3.0) s and t1/2 = (15 ± 5) s after a pre-stirring; please note that half-times are faster in this case because the electrochemical oxidation of QH2 near the electrode surface before illumination will lead to high local concentrations of quinones that increase extraction from algae.).

  capability of both quinones to interact with the photosynthetic chain (maximum steady-state value Imax = 44.5 and 14.8 µA; Michaelis constant KM = 71.3 and 61.0 µmol.L -1 for 2,6-DCBQ and 2,6-DMBQ respectively).[START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF]

Figure 1 .

 1 Figure 1

Figure 2 .

 2 Figure 2. A) Scheme of the electrocatalytical cycle inducing the production of a photocurrent from an algae suspension under light illumination in presence of exogenous quinones. The photosynthetic chain is embedded in the chloroplast and consists of thylakoid membranes and different complexes (see text). B) Representative chronoamperometric trace (EW = 0.38 V vsAg/AgCl) from a suspension of Chlamydomonas reinhardtii algae (2.10 7 cells mL -1 ) with 2,6-DCBQ (100 µmol L -1 ) under illumination (P = 60 mW cm -2 ; white rectangle).

Figure 3 .

 3 Figure 3. Chronoamperograms from a suspension of Chlamydomonas reinhardtii algae (2.10 7cells mL -1 ) under illumination (P = 60 mW cm -2 ). A) Current = f(t) with 2,6-DCBQ (100 µmol L -1 ) : with (solid line) and without (dashed line) Ficoll ; B) Normalized current = f(t) with 2,6-DCBQ (100 µmol L -1 ) with (solid line) and without (dashed line) Ficoll. C) Current = f(t) with 2,5-DCHQ (100 µmol L -1 ) with (dashed line) or without (solid line) any pre-stirring. Please note that 2,6-DCHQ is not commercially supplied. Because 2,6-DCBQ and 2,5-DCBQ have a similar behavior,[START_REF] Longatte | Evaluation of photosynthetic electrons derivation by exogenous redox mediators[END_REF][START_REF] Longatte | Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time[END_REF] the commercially available 2,5-DCHQ is used in this peculiar experiment (see text).

Figure 4 .

 4 Figure 4. Cyclic voltammograms of a suspension of Chlamydomonas reinhardtii (2.10 7 cells mL -1 ). A) With 2,6-DCBQ (100 µmol L -1 ) : under dark conditions (solid line) or under illumination (dashed line; 60 mW cm -2 ) over 1 min. v = 100 mV s -1 B) With 2,5-DCHQ (100 µmol L -1 ) : under dark conditions (dashed line) or under illumination (solid line; 60 mW cm -2 ). v = 1 mV s -1 .

Figure 5 .

 5 Figure 5. Steady state photocurrents recorded with a suspension of Chlamydomonas reinhardtii algae (2.10 7 cells mL -1 ) under illumination (P = 60 mW cm -2 ) as a function of added quinone concentration for two exogenous quinones : A) 2,6-DCBQ ; B) 2,6-DMBQ. The experimental curves were modeled by means of a Michael-Menten like behavior (Iss = ACQ/(B+CQ); R 2 = 0.97).

Figure 6 .

 6 Figure 6. A) Chronoamperograms from several suspensions of Chlamydomonas reinhardtii algae under illumination (P = 60 mW cm -2 ) with 2,6-DCBQ (100 µmol L -1 ). The rectangle

Figure 7 .

 7 Figure 7. A) Chronoamperograms from a suspension of Chlamydomonas reinhardtii algae (2.10 7 cells mL -1 ) under illumination (P = 20 mW cm -2 ) with different 2,6-DCBQ concentrations (50-200 µmol L -1 ). B) Chronoamperograms from a suspension of Chlamydomonas reinhardtii algae under the same conditions with 2,6-DCBQ (100 µmol L -1 ) : solid line : day/night cycles ; dashed line : continuous illumination.

Figure 8 .

 8 Figure 8. A) Chronoamperograms from a suspension of Chlamydomonas reinhardtii algae (∆f1) under illumination (P = 60 mW cm -2 ) with 2,6-DCBQ (100 µmol L -1 ) after different times of pre-illumination. B) Steady state photocurrents recorded as a function of the pre-illumination time.

Figure S5 .

 S5 Figure S5. Ratio of the steady state current under illumination (15.3 µA; 60 mW cm -2 ) and the current peak under dark conditions (i°p) as a function of v -1/2 for a suspension of Chlamydomonas reinhardtii (2.10 7 cells mL -1 ) with 2,5-DCHQ (100 µmol L -1 ).

Figure S6 .

 S6 Figure S6. Chronoamperograms from a suspension of Chlamydomonas reinhardtii algae (2.10 7 cells mL -1 ) under illumination (P = 60 mW cm -2 ) with 2,6-DMBQ (100 µmol L -1 ). Solid line : day/night cycles ; dashed line : continuous illumination.

Figure S7 .

 S7 Photocurrent (µA)

Acknowledgements

This work has been supported in part by CNRS (UMR 8640, UMR7141), Ecole Normale Supérieure, French Ministry of Research, Faculté des Sciences et Ingénierie -Sorbonne Université and the "Initiative d'Excellence" program from the French State (Grant "DYNAMO", ANR-11-LABX-0011-01). G.L. and M.G.-C. thank Institut Universitaire de France Fellowship Program.

Supporting Information

Diverting Photosynthetic Electrons from Suspensions of Chlamydomonas reinhardtii

Algae -New Insights using an Electrochemical Well Device

Adnan Sayegh, Guillaume Longatte, Olivier Buriez, Francis-André Wollman, Manon Guille-Collignon, Eric Labbé, Jérôme Delacotte, Frédéric Lemaître


Martin Turbet

Ha Tran

Olivier Pirali

François Forget

Christian Boulet

Jean-Michel Hartmann
email: jean-michel.hartmann@lmd.polytechnique.fr

Far infrared measurements of absorptions by CH 4 +CO 2 and H 2 +CO 2 mixtures and implications for greenhouse warming on early Mars

Keywords: Mars, spectroscopy, measurement, methane, hydrogen, collision induced absorptions, climate

We present an experimental study of the absorption, between 40 and 640 cm -1 , by CO 2 , CH 4 and H 2 gases as well as by H 2 +CO 2 and CH 4 +CO 2 mixtures at room temperature. A Fourier transform spectrometer associated to a multi-pass cell, whose optics were adjusted to obtain a 152m path length, were used to record transmission spectra at total pressures up to about 0.98 bar. These measurements provide information concerning the collision-induced absorption (CIA) bands as well as about the wing of the CO 2 15 µm band. Our results for the CIAs of pure gases are, within uncertainties, in agreement with previous determinations, validating our experimental and data analysis procedures. We then consider the CIAs by H 2 +CO 2 and CH 4 +CO 2 and the low frequency wing of the pure CO 2 15 µm band, for which there are, to our knowledge, no previous measurements. We confirm experimentally the theoretical prediction of Wordsworth et al. 2017 that the H 2 +CO 2 and CH 4 +CO 2 CIAs are significantly stronger in the 50-550 cm -1 region than those of H 2 +N 2 and CH 4 +N 2 , respectively. However, we find that the shape and the strength of these recorded CIAs differ from the aforementioned predictions. For the pure CO 2 line-wings, we show that both the χfactor deduced from measurements near 4 µm and a line-mixing model very well describe the observed strongly sub-Lorentzian behavior in the 500-600 cm -1 region. These experimental results open renewed perspectives for studies of the past climate of Mars and extrasolar analogues.

1.Introduction

It is well known that, in the atmospheres of many planets, the far wings (FWs) of molecular absorption lines (e.g. those of CO 2 in Venus, of CH 4 in Titan and Jupiter) and collision-induced absorptions (CIAs, e.g. that by CO 2 pairs in Venus; of N 2 pairs in Titan; of H 2 pairs in Jupiter) significantly contribute to the radiative budgets. This has motivated numerous experimental and theoretical laboratory studies of the CIA of various molecular systems, from the visible to the far infrared (see reviews in [START_REF] Frommhold | Collision Induced Absorption in Gases[END_REF][START_REF] Hartmann | Collisional effects on molecular spectra: Laboratory experiments and models, consequences for applications[END_REF][START_REF] Richard | New section of the HITRAN database: Collision-induced absorption (CIA)[END_REF], Hartmann et al. 2018band Karman et al. 2018). The same remark stands for the FWs, but the latter were essentially studied for H 2 O and CO 2 only (see Hartmann et al. 2008, Hartmann et al. 2018b andreferences therein).

Understanding how the early Martian climate could have been warm enough for liquid water to flow on the surface remains one of the major enigmas of planetary science [START_REF] Wordsworth | The climate of early Mars[END_REF][START_REF] Haberle | The Early Mars Climate System[END_REF] and no consensus scenario has yet been reached. Thick, CO 2 -dominated atmospheres do not provide the necessary greenhouse effect to warm the surface of early Mars above the melting point of water [START_REF] Forget | 3D modelling of the early martian climate under a denser CO 2 atmosphere: temperatures and CO 2 ice clouds[END_REF], Wordsworth et al. 2013). In this case, longwave radiation losses essentially occur in the 200-600 cm -1 region [START_REF] Wordsworth | Infrared collision-induced and far-line absorption in dense CO 2 atmospheres[END_REF] where absorption and emission are dominated by the CO 2 rototranslational CIA and the FWs of the intense 15 µm-band lines. The radiative budget in this spectral region is significantly affected by the assumption made on the shape of these FWs [START_REF] Turbet | Comment on "Radiative transfer in CO 2 -rich atmospheres: 1. Collisional line mixing implies a colder early Mars[END_REF], and no measurements are, to our knowledge, available to constrain it.

In reducing atmosphere conditions, absorptions can arise from the CIAs of H 2 -CO 2 and CH 4 -CO 2 pairs. Such absorptions can potentially absorb in the 200-600 cm -1 region corresponding to a carbon dioxide transparency window and thus produce a strong greenhouse warming in a CO 2 -dominated atmosphere. Up to now, the modelings of these two contributions were based, due to lack of relevant data, on the CIAs of H 2 -N 2 and CH 4 -N 2 pairs, respectively (e.g. [START_REF] Ramses | Warming early Mars with CO 2 and H 2[END_REF]. [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] recently calculated that the collision-induced absorptions by H 2 +CO 2 and CH 4 +CO 2 mixtures should be significantly stronger than those for H 2 +N 2 and CH 4 +N 2 , respectively. These calculations have important implications for our understanding of the climate of early Mars, at the time when valley networks were carved by liquid water. Indeed, with these new CIAs, only few % of CH 4 and/or H 2 in a CO 2 -dominated atmosphere could suffice to warm early Mars enough for surface liquid water to become stable [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF][START_REF] Ramirez | A warmer and wetter solution for early Mars and the challenges with transient warming[END_REF]). These calculated CIAs, if confirmed by laboratory experiments, have the power to reconcile the Martian geology and mineralogy with our knowledge on atmospheric sciences (see Wordsworth et al. 2017 and references therein).

The remainder of this paper is organized as follows. Section 2 describes the experiments and the way the recorded spectra of CO 2 , CH 4 , H 2 , H 2 +CO 2 and CH 4 +CO 2 were analyzed. The results obtained for the CIAs and for the low frequency wings of the pure CO 2 lines are presented in Sections 3 and 4, respectively. Discussions of the results and the consequences for studies of the past climate of Mars are the subject of Section 5. Final conclusions and possible directions for future studies are given in Section 6.

Experiments and their analysis

Spectra recordings

The spectra used in this study have been recorded at the AILES line of the Soleil synchrotron facility in a way similar to that used in [START_REF] Pirali | High-resolution infrared absorption spectroscopy of thermally excited naphthalene. Measurements and calculations of anharmonic parameters and vibrational interactions[END_REF][START_REF] Hartmann | Molecular dynamics simulations for CO 2 spectra. II. The far infrared collision-induced absorption band[END_REF]. A Bruker IFS 125HR Fourier transform spectrometer (FTS) and a 2.5 m long multipass cell with 60 µm thick polypropylene windows, were used. The globar source inside the FTS was used, together with a 6 µm thick mylar beam splitter, a band-pass filter, and a Si bolometer detector cooled down to 4.2 K by liquid helium. Measurements were made at room temperature (≈ 296 K, since the cell temperature cannot be varied) for a path length of 151.75 m, with an unapodized spectral resolution of 1 cm -1 . In total, 1000 scans were co-added to generate each of the spectra. Due to the optics and detector used, the signal is significant and reliable between 40 and 680 cm -1 . Within this range, each recording provides about 2650 spectral values with a step of 0.24 cm -1 . The pressures were measured and continuously monitored with both a 1000 millibar (Pfeifer) and a 1000 Torr (Edwards) gauges. Their readings agreed in all cases to better than 1 %. CO 2 , H 2 and CH 4 gases provided by Air Liquide were used for the recording of pure samples as well as mixtures. The latter were made by first introducing H 2 (or CH 4 ) and then adding CO 2 up to a total pressure of about 0.95 bar. After waiting for about 1 hour to let the mixture homogenize a first spectrum was recorded, then the pressure of each initial gas sample was progressively reduced with a vacuum pump to yield spectra of the same mixture (or pure gas) at several lower pressures. In addition, reference (100% transmission) spectra were obtained for similar pressures by introducing pure argon into the cell. The list of recorded spectra and their conditions are given in Appendix A.

Note that we had to limit the total pressure in the cell to slightly below 1 bar in order to avoid possible damage of the cell and/or of its windows since they are not designed to go above atmospheric pressure. The product of the path length and gas densities is thus limited to about 1.3 10 4 cm amagat 2 (1 amagat corresponds to 2.69 10 19 molec/cm 3 ) for studies of pure gases and 0.33 10 4 cm amagat 2 for mixtures, which induces large uncertainties on the determination of weak CIAs, as shown in Secs. 3 and 4. We also draw the reader's attention to the fact that the SOLEIL-AILES facility is extremely sought and busy and that we only had access to it for a few days, limiting the number of spectra we could record in this first experimental study of the CIAs by H 2 -CO 2 and CH 4 -CO 2 pairs. We hope that this will stimulate further thorough experimental investigations, hopefully under more absorbing conditions.

Spectra treatments

The raw spectra recordings were treated using the five steps procedure detailed below.

Step 1: For all samples, the recorded spectra show absorption by water vapor lines due to small amounts of H 2 O present in the gases used. The first step of the treatment of the collected data was thus to remove their contributions. For this, H 2 O transmissions were computed and used, as explained in Appendix B, in order to remove the absorption features due to water lines from the raw spectra and obtain "dried out" spectra.

Step 2: For a given sample, the transmission spectrum was then obtained by dividing the associated recording cleared from the H 2 O contribution by a pure argon spectrum, also cleared of the H 2 O lines. Among the various recorded pure Ar spectra (see Appendix A), we chose a linear combination of two Ar spectra for which the broad-band features resulting from the absorption and interference patterns generated by the propylene windows are the closest to those observed in the sample gas spectrum. Indeed, since these features are different from one spectrum to another (likely because of changes of the temperature and/or window curvature induced by pressure), ratioing adapted recordings is essential to minimize the residual broadband oscillations in the resulting transmission spectrum. However, baseline variations due to changes of the globar source emission, bolometer responsivity and mechanical deformations of the multipass cell itself may also occur from one recording to another. This introduces an error on the 100% transmission level which directly affects the weak and broad-band absorption continua searched for. The way we dealt with this difficulty is described in step 5 below.

Step 3: For the part of this study devoted to the collision-induced absorption continua discussed in Sec. 3 (but not to the far line wings discussed in Sec. 4), the third step was to remove the contributions of the local lines of CO 2 and CH 4 . These contributions were computed as explained in Appendix B, yielding the associated transmission by local lines by which the spectrum obtained from steps 1 and 2 is divided so that only the CIA remains.

Step 4: The transmissions obtained from the preceding treatments show a few sharp features (outliers) essentially found close to the local lines (of H 2 O, CH 4 and CO 2 ) that have been removed in steps 1 and 3. Analysis shows that this is due to small differences between the measurements and the calculation of these narrow features. In order to "clean" the spectra from these outliers, we performed the following procedure. First, a moving average of each spectrum over 5 cm -1 was computed. Then, the mean distance between this averaged spectrum and the original one was computed at each wavelength. All data points of the original spectrum that are further away from the averaged spectrum than half of this mean distance were rejected. Note that this 5 cm -1 -averaged spectrum is used only for the rejection of outliers and that the next steps of the analysis use the values of the original spectra that have not been rejected by this filtering.

Step 5: Starting from each transmission spectrum obtained from steps 1-4, we compute the negative value of its natural logarithm and divide the result by the optical path length (15175 cm). This provides the measured absorption coefficient 2 ( , , ) meas X CO P P α σ at wave number σ that, since local line contributions have been removed and only CIA remains, should be equal to:

2 2 2 2 2 2 calc 2 2 ( , , ) ( ) ( ) ( ) X CO CO CO CO X CO CO X X X X P P P C P P C P C - - - α σ = σ + σ + σ , (1)
where C CO2-X (σ) is the pressure-normalized binary collision-induced absorption coefficient for CO 2 -X pairs. In order to determine its spectral values, two procedures have been used: -In the first procedure, we assume that the 100% transmission baseline is correct. For pure gases, we determine ( )

X X i
C -σ from a simultaneous least square fit of all spectra, by minimizing the quantity

2 meas 2 ( , ) ( ) X i i X X X X i P P P C - σ   α σ - σ ∑ ∑   . For CO 2 -X mixtures, we minimize { } 2 2 2 2 2 2 2 meas 2 2 2 ( , , ) ( ) ( ) ( ) X CO i i X CO CO CO CO i X X X i P P X CO CO X i P P P C P C P P C - - σ -   α σ - σ - σ ∑ ∑ ∑     - σ  
by floating the values of the 2 ( )

CO X i C -σ , those of ( ) X X i C -σ and 2 2 ( ) CO CO i C - σ being fixed.
For the latter, we used the data of [START_REF] Ho | Far-infrared collision-induced absorption in CO 2 . I. Temperature dependence[END_REF] for pure CO 2 , of [START_REF] Codastefano | Far infrared absorption spectra in gaseous methane from 138 to 296 K[END_REF] for pure CH 4 and of [START_REF] Abel | Collision-induced absorption by H 2 pairs: from hundreds to thousands of Kelvin[END_REF] for pure H 2 , because they are more precise than the ones determined in the present study. These least square fits also provide the 1-σ statistical (rms) uncertainties on the fitted parameters [ ( )

X X i C -σ and 2 ( ) CO X i C -σ ].
Note that 2σ was adopted for the error bars displayed in the figures below.

-In the second procedure, we introduce a baseline correction as a linear function of wave number. This function is assumed to be the same for a series of consecutive spectra at different pressures (i.e. those for a given mixture or a pure gas). In this case, for pure gases, we minimize the quantity

2 meas 2 ( , ) ( ) X i i X i X X X i P P A B P C - σ   α σ --σ - σ ∑ ∑  
by floating the values of A, B and C X-X (σ i ). For CO 2 -X mixtures, since we recorded spectra at several pressures for each sample composition (i.e. each value of 2 / X CO P P , see Table 1), we minimize the quantity

{ } 2 2 2 2 2 2 2 2 2 meas 2 2 2 ( , , ) ( ) ( ) ( / ) ( / ) ( ) X CO i i X CO CO CO CO i X X X i P P X CO X CO i X CO CO X i P P P C P C A P P B P P P P C - - σ -   α σ - σ - σ ∑ ∑ ∑     - - σ - σ  
by floating the values of 2 ( / )

X CO A P P , 2 ( / )

X CO B P P , and 2 ( )

CO X i C -σ .
We thus assume here that A and B have a unique value for each mixture relative composition. Note that there are about 1400 points in each fitted spectrum and that, for each mixture, nine spectra (3 pressures for each one of the three mixtures, see Table 1) were recorded. We thus have a total number of ~12600 absorbance values. They provide enough information to deduce, from their adjustments, the ~1400 pressure-normalized absorptions 2 ( )

CO X i C -σ
CO X i C
-σ remain noisy and uncertain, a moving spectral average will be made in order to deduce the final values listed in Appendix C and plotted in Fig. 7, as explained at the end of Sec. 3.2.

The Collision-Induced Absorptions (CIAs)

3.1 The test cases of the CIAs of pure CO 2 , CH 4 and H 2 Although measuring the CIAs of pure gases is not the objective of this study since several measurements and calculations have been made before (see below), we measured them to test and evaluate the accuracy of our experimental and data analysis procedures. Indeed, since the previous experimental studies have been made under much more favorable conditions (stronger absorptions thanks to higher pressures), they are likely more accurate than our determinations.

For the pure CO 2 CIA, measurements were reported in [START_REF] Harries | The temperature variation of the far infrared absorption in compressed CO 2[END_REF][START_REF] Ho | Far-infrared collision-induced absorption in CO 2 . I. Temperature dependence[END_REF][START_REF] Hartmann | Molecular dynamics simulations for CO 2 spectra. II. The far infrared collision-induced absorption band[END_REF], for instance, and theoretical calculations can be found in [START_REF] Gruszka | Roto-translational collision-induced absorption of CO 2 for the atmosphere of Venus at frequencies from 0 to 250 cm -1 , at temperatures from 200 to 800[END_REF][START_REF] Gruszka | Computer simulation of the far infrared collision induced absorption spectra of gaseous CO 2[END_REF][START_REF] Hartmann | Molecular dynamics simulations for CO 2 spectra. II. The far infrared collision-induced absorption band[END_REF]. A comparison of our results, obtained from fits of the five spectra at pressures from about 0.20 to 0.95 bar with and without introduction of a baseline, with the measured values of [START_REF] Ho | Far-infrared collision-induced absorption in CO 2 . I. Temperature dependence[END_REF] (uncertainty stated as better than 10 %) and the calculated results of [START_REF] Gruszka | Roto-translational collision-induced absorption of CO 2 for the atmosphere of Venus at frequencies from 0 to 250 cm -1 , at temperatures from 200 to 800[END_REF] is plotted in Fig. 1 (#) . As can be seen, adjusting a baseline in the fit significantly reduces the error bars (and rms of the fit), corrects for the negative values in the high frequency wing, and improves the agreement between our results and the previous determinations. In this case, the area obtained from the integration of our results between 60 and 250 cm -1 is 2.74 ± 0.32 10 -3 cm -2 /amagat 2 (the uncertainty given here being directly obtained from the error bars in Fig. 1) while 3.11 ± 0.31 10 -3 cm -2 /amagat 2 is obtained from the measurements of [START_REF] Ho | Far-infrared collision-induced absorption in CO 2 . I. Temperature dependence[END_REF], and 3.73 10 -3 cm -2 /amagat 2 from the calculated values of Gruszka & Borysow 1997. Our determination thus agrees, within uncertainties, with the previous measurement of [START_REF] Ho | Far-infrared collision-induced absorption in CO 2 . I. Temperature dependence[END_REF]. Note that, when compared to the other gas samples discussed below, the pure CO 2 case is the most favorable since, for the most optically thick sample investigated (at ~0.95 bar), the transmission is 55% at the peak near 50 cm -1 . (#) The Amagat density unit, generally used in studies of CIA, corresponds to the number density of molecules at P 0 =1 atm ad T 0 =273.15 K, i.e. Loschmidt's number n 0 =2.686 10 19 molec/cm 3 . For an ideal gas at pressure P(atm) and temperature T(K), the density in amagat is (P/ P 0 )( T 0 /T). [START_REF] Gruszka | Roto-translational collision-induced absorption of CO 2 for the atmosphere of Venus at frequencies from 0 to 250 cm -1 , at temperatures from 200 to 800[END_REF] which are available in the HITRAN CIA database section [START_REF] Richard | New section of the HITRAN database: Collision-induced absorption (CIA)[END_REF]. The results in the left and right hand-side panels have been obtained without and with the baseline adjustment, respectively.

The CIA of pure CH 4 was investigated experimentally in [START_REF] Codastefano | Far infrared absorption spectra in gaseous methane from 138 to 296 K[END_REF][START_REF] Codastefano | Temperature dependence of the far infrared absorption spectrum of gaseous methane[END_REF][START_REF] Birnbaum | Far infrared collision-induced spectrum in gaseous methane. I. Band shape and temperature dependence[END_REF][START_REF] Dagg | Collision-induced absorption in gaseous methane at low temperatures[END_REF][START_REF] El-Kader | New insights into collision-induced rototranslational absorption and scattering spectra of gaseous methane at different temperatures[END_REF], Borysow & Frommhold 1987aand Borysow & Frommhold 1987b provide examples of theoretical calculations. A comparison between some of these previous data and the results obtained from our three spectra (pressures between ~0.60 and 0.98 bar) is plotted in Fig. 2. As for pure CO 2 , introducing a baseline in the fit reduces the uncertainties (and rms), improves the CH 4 -CH 4 CIA high frequency wing, and leads to a better agreement with previous determinations. In this case, the area obtained from the integration of our results between 70 and 600 cm -1 is 2.2 ± 0.2 10 -3 cm -2 /amagat 2 . It is slightly lower than the experimental value of 2.97 ± 0.31 10 -3 cm -2 /amagat 2 from [START_REF] Codastefano | Far infrared absorption spectra in gaseous methane from 138 to 296 K[END_REF]. Note that, for the most optically thick sample investigated (at ~0.98 bar), the peak absorption in our measurements is significantly smaller than for pure CO 2 with a transmission of about 90% near 250 cm -1 . Fig. 2: Pure CH 4 CIAs at room temperature. The symbols with error bars are the present measurements, the green line is a moving average of the latter over 10 cm -1 , the red line represents the measurements of Codastefano et al. 1985 (digitized from Fig. 1 of this reference), and the blue curve denotes the calculated values from Borysow & Frommhold 1987a which are available in the HITRAN CIA database section [START_REF] Richard | New section of the HITRAN database: Collision-induced absorption (CIA)[END_REF]. The results in the left and right hand-side panels have been obtained without and with adjustment of the baseline, respectively.

The CIA of pure H 2 was investigated experimentally in [START_REF] Birnbaum | Far-infrared absorption in H 2 and H 2 -He mixtures[END_REF][START_REF] Bachet | The translational rotational absorption spectrum of hydrogen[END_REF]. Theoretical calculations were made in [START_REF] Birnbaum | Collision-induced absorption of H 2 -H 2 and H 2 -He in the rotational and fundamental bands for planetary applications[END_REF][START_REF] Gustafsson | Collision-induced absorption in the rototranslational band of dense hydrogen gas[END_REF][START_REF] Abel | Collision-induced absorption by H 2 pairs: from hundreds to thousands of Kelvin[END_REF], Karman et al. 2015b, and references therein. A comparison between some of these previous data and the results of our measurements is plotted in Fig. 3. Note that in this case, the treatment applied to the spectra was slightly different from that used for all other samples. In fact, a quick look at the transmissions obtained after step 4 revealed that they differed from unity at small wave numbers (where the absorption should be negligible [START_REF] Birnbaum | Far-infrared absorption in H 2 and H 2 -He mixtures[END_REF] for our recording conditions) with significant inconsistencies between the three spectra. This explains the very large uncertainties and negative values of the CIA obtained when no base line is introduced in the fit (see the left panel of Fig. 3). Therefore, we pre-treated each transmission spectrum by dividing it by the averaged value at about 100 cm -1 where the absorbance under our conditions should be nearly zero. Then, the procedure described in step 5 of Sec. 2 was applied with the fit of a single base line for all spectra. This leads to the results plotted in the right hand side panel of Fig. 3 which show several obvious improvements with respect to those in the left hand side panel. However, significant differences with previous determinations remain. These differences can be likely explained by the fact that, for the most optically thick sample investigated (i.e. about 0.95 bar of H 2 ), the peak absorption is very small with a transmission of about 94% near 600 cm -1 (according to the values of [START_REF] Birnbaum | Far-infrared absorption in H 2 and H 2 -He mixtures[END_REF].

Fig. 3: Pure H 2 CIA at room temperature. The symbols are the present measurements, the green line is a moving average of the latter over 10 cm -1 , the red line represents the measurements of Birnbaum 1978 (digitized from Fig. 2 [START_REF] Abel | Collision-induced absorption by H 2 pairs: from hundreds to thousands of Kelvin[END_REF] which are available in the HITRAN CIA database section [START_REF] Richard | New section of the HITRAN database: Collision-induced absorption (CIA)[END_REF]).

of this reference), and the blue curve denotes the calculated values from

The CIAs of CH 4 +CO 2 and H 2 +CO 2 mixtures

Now that the comparisons between our results for pure gases and previous determinations have validated our experimental and data analysis procedure (at least when the peak absorption is large enough, thus excluding the pure H 2 case) we consider the mixtures for which no other measurements are available. Note that, while squared pressures up to ~1 bar 2 could be used for pure gases, the case of mixtures is much less favorable since 2 X CO P P is now limited to ~0.25 bar 2 . Finally, since the results of Sec. 2 show that adjusting a linear baseline correction leads to significant improvements, only the results obtained with this approach are presented below. However, for the two considered mixtures, results obtained without adjustment of baselines are fully consistent when error bars are considered.

The CIA of CH 4 +CO 2 has never been measured before, but theoretical predictions have been made [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF]. A comparison between these predictions and the results of our measurements is plotted in Fig. 4. We measure a significant CO 2 -CH 4 CIA in the 50-500 cm -1 spectral range that has, as predicted [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF], two spectral regions of higher absorption around 50 and 270 cm -1 . However, our experiments lead, on average, to significantly smaller values with an area between 50 and 500 cm -1 of 9.9±2.7 10 -3 cm - 2 /amagat 2 , about 1.7 times smaller than that obtained from the prediction of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF]. Despite the rather large uncertainties, the experimental results show that the theoretical prediction of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] overestimates this CIA. Indeed, our CH 4 +CO 2 CIA leads to peak absorbances for the optically thickest conditions investigated (~0.47 bar of CH 4 + ~0.47 bar of CO 2 ) of 0.16 and 0.10, corresponding to a transmission of about 85 % and 90 % near 50 and 270 cm -1 respectively, which gives relatively "comfortable" conditions. Fig. 4: CH 4 +CO 2 CIAs at room temperature. The symbols with error bars are the present measurements, the green line is a moving average of the latter over 10 cm -1 , the blue line represents the calculated values of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] As for the CIA of CH 4 +CO 2 , that of H 2 +CO 2 has never been measured before, but theoretical predictions have also been made [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF]. These calculated data and the results of our experiments are plotted in Fig. 5. We measure a significant CIA throughout the entire spectral range (corresponding to an absorbance of ~0.07 for the optically thickest conditions) which increases with wavenumber at high frequencies, as predicted in [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF]. However, large differences can be observed in Fig. 5 between our measurements and the predictions, both for the shape and magnitude, for which we have no explanation at this step. Finally note that our determinations are limited to wavenumbers lower than 550 cm -1 because of the strong contribution of CO 2 absorption lines beyond this frequency (see Fig. 6). In addition, it is possible that a part of the absorption that we measure comes from the H 2 -broadened wings of the 15 µm CO 2 band lines, whose contributions are, as the CIA, proportional to In order to generate "the best" datasets of measured CH 4 +CO 2 and H 2 +CO 2 CIAs we started from the values with error bars in Figs. 4 and5. For the mean values, we computed a moving average over 25 cm -1 and tabulated its values with a step of 5 cm -1 . Determining the uncertainties is, as it is well known, a very difficult task, even in cases where numerous measurements have been made for reliable statistics. In the present study, we computed the moving average, over 25 cm -1 , of the error bars plotted in Figs. 4 and5. The obtained results were then multiplied by 1.5 in order to take into account other sources of uncertainties (mostly due to base line stability within the same recording series). Since the error bars in Figs. 4 and5 already correspond to the 2-σ value (95% confidence interval), we believe that the total uncertainty estimated this way is (very) conservative considering the measured data at our disposal. The final set of data for the CH 4 +CO 2 and H 2 +CO 2 CIAs are given in Table 2 of Appendix C. We recall the reader that these should be used with great care as their uncertainties are rather large.

The low frequency wing of the CO 2 ν 2 band

The recorded pure CO 2 spectra enable to study the absorption in the low frequency wing of the intense ν 2 band (centered at 667 cm -1 ), from about 500 to 600 cm -1 . While there are, to our knowledge, no previous measurements available, comparisons with calculations are possible. Indeed, the spectra can be predicted using the line-mixing model of [START_REF] Tran | Measurements and modeling of high pressure pure CO 2 spectra from 750 to 8500 cm -1 . I-Central and wing regions of the allowed vibrational bands[END_REF] or Voigt profiles corrected in the wings by using a χ factor. Three sets of such factors have been proposed, deduced from measurements in the high frequency wings of the ν 2 (σ>750 cm -1 ) [START_REF] Tran | Measurements and modeling of high pressure pure CO 2 spectra from 750 to 8500 cm -1 . I-Central and wing regions of the allowed vibrational bands[END_REF]) and ν 3 (σ>2400 cm -1 ) [START_REF] Perrin | Temperature-dependent measurements and modeling of absorption by CO 2 -N 2 mixtures in the far line-wings of the 4.3 µm CO 2 band[END_REF][START_REF] Tran | Measurements and modeling of high pressure pure CO 2 spectra from 750 to 8500 cm -1 . I-Central and wing regions of the allowed vibrational bands[END_REF] bands. Note that the line-mixing model of [START_REF] Tran | Measurements and modeling of high pressure pure CO 2 spectra from 750 to 8500 cm -1 . I-Central and wing regions of the allowed vibrational bands[END_REF] and the χ factor of [START_REF] Perrin | Temperature-dependent measurements and modeling of absorption by CO 2 -N 2 mixtures in the far line-wings of the 4.3 µm CO 2 band[END_REF] were used in Turbet & Tran 2017 for radiative transfer calculations in the far infrared for rich CO 2 atmospheres but that they had not been validated in this spectral region yet. Comparisons between measured transmissions and those computed with the four models mentioned above and by using purely Voigt profiles (no line-mixing and no χ-factor correction, i.e. χ(∆σ) = 1) are plotted in Fig. 6, calling for the following remarks. The first is that calculations with pure Voigt line shapes (blue) lead to very large errors showing the strongly sub-Lorentzian behavior of the line wings. The second is that the three χ-factors used (red curves) lead to indiscernible results on this plot, all in very good agreement with the measured spectrum, except in a narrow interval around the Q branch centered at 545 cm -1 . This local effect of line-mixing is very nicely reproduced by the line-mixing model of [START_REF] Tran | Measurements and modeling of high pressure pure CO 2 spectra from 750 to 8500 cm -1 . I-Central and wing regions of the allowed vibrational bands[END_REF] which is also in excellent agreement with the χ-factor predictions (as also shown in [START_REF] Turbet | Comment on "Radiative transfer in CO 2 -rich atmospheres: 1. Collisional line mixing implies a colder early Mars[END_REF]) and the measured spectra throughout the entire spectral range.

Discussion

On the CIAs of CH 4 +CO 2 and H 2 +CO 2

A comparison between our final experimental determinations of the CH 4 +CO 2 and H 2 +CO 2 CIAs and the corresponding predictions of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] as well as with the CIAs when CO 2 is replaced by N 2 is plotted in Fig. 7. [START_REF] Borysow | Theoretical collision-induced rototranslational absorption spectra for modeling Titan's atmosphere H 2 -N 2 pairs[END_REF]) and N 2 -CH 4 [START_REF] Borysow | Far infrared CIA spectra of N 2 -CH 4 pairs for modeling of Titan's atmosphere[END_REF], respectively, taken from the HITRAN CIA database section [START_REF] Richard | New section of the HITRAN database: Collision-induced absorption (CIA)[END_REF].

First, both our measurements and the predictions indicate that the effect of interactions of CH 4 and H 2 with CO 2 induce much stronger absorptions than with N 2 . This could actually be expected by simply considering the long range induction mechanisms which are likely dominant here and are governed by the isotropic polarizability and the electric quadrupole moment (of N 2 or CO 2 ). As shown by the data given in [START_REF] Li | Long-range, collision-induced dipoles of T d -D ∞h molecule pairs: Theory and numerical results for CH 4 or CF 4 interacting with H 2 , N 2 , CO 2 , or CS 2[END_REF], the values of all these parameters are, for CO 2 , from 1.5 to nearly 3 times greater than for N 2 . Reminding that the square of the dipole is involved, and even though the intermolecular potential and its anisotropy also play a role, it is quite obvious that the X-CO 2 should be significantly larger than the X-N 2 CIA.

For the differences between our measurements and the predictions of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF], which are significant and concern both the magnitude and the shape of the CIAs, we have no explanation at this step. Trying to understand their origin(s) is beyond the scope of the present paper. Obviously, independent checks of our results by new measurements are needed, if possible under more absorbing conditions in order to reduce the uncertainties resulting from a poor knowledge of the 100% transmission level. Ideally, we recommend performing future investigations of CO 2 -CH 4 and CO 2 -H 2 CIAs for products of the path length and gas densities up to at least 2 10 4 cm amagat 2 . Furthermore, measurements at several temperatures (between at least 200 and 300 K) would be of considerable interest since they would provide the planetary science community with CIAs that can be used directly in numerical climate models. However, such experiments are difficult and there are not many (if any) existing setups suitable to carry them. Note that the predictions of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] were obtained using a very simple approach based on a combination of pure gas CIA data and not on direct calculations of the absorption spectrum of the mixture (detailed in their supporting information, Section 1.1.). Therefore, their quality needs to be investigated and this could be done by using various alternative theoretical approaches. These include fully classical computations based on molecular dynamics simulations (e.g. [START_REF] Gruszka | Computer simulation of the far infrared collision induced absorption spectra of gaseous CO 2[END_REF][START_REF] Hartmann | Molecular dynamics simulations for CO 2 spectra. II. The far infrared collision-induced absorption band[END_REF], Hartmann et al. 2018a), semi-classical approaches based on line shapes (e.g. [START_REF] Birnbaum | Theory of line shape in pressure-induced absorption[END_REF][START_REF] Hunt | Lineshape analysis of collision induced spectra of gases[END_REF]) and the so-called "isotropic approximation" (e.g. [START_REF] Leforestier | Temperature dependences of mechanisms responsible for the water-vapor continuum absorption. II. Dimers and collision-induced absorption[END_REF], Hartmann et al. 2018a), and fully quantum scattering computations assuming isotropic intermolecular forces (e.g. [START_REF] Borysow | Theoretical collision-induced rototranslational absorption spectra for modeling Titan's atmosphere H 2 -N 2 pairs[END_REF][START_REF] Borysow | Far infrared CIA spectra of N 2 -CH 4 pairs for modeling of Titan's atmosphere[END_REF] or taking the intermolecular potential anisotropy into account (e.g. Karman et al. 2015a, Karman et al. 2015b). Note that, in the absence of relevant measurements, theory may give information on the temperature dependences of the CO 2 -CH 4 and CO 2 -H 2 CIAs which are, as mentioned above, crucially needed.

Implications for the climate of early Mars

Although our measurements have large uncertainties, we can discuss their implications for the climate of early Mars. In the 200-600 cm -1 spectral window of early Mars, our measurements confirm that the CO 2 -CH 4 and CO 2 -H 2 CIA are much stronger than those of N 2 -CH 4 and N 2 -H 2 pairs, respectively. However, they also show that the calculations of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] overestimate on average the CO 2 -CH 4 and CO 2 -H 2 CIAs by a factor of about 1.7 and 1.6, respectively.

Our measurements indicate that [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] and Ramirez 2017 overestimated the effect of these CIAs in their numerical climate simulations and that they therefore underestimated the amount of H 2 and CH 4 required to warm the surface of early Mars above the melting point of water, by a factor up to 2-3. Similarly, calculations of the boundaries of the Habitable Zone based on hydrogen [START_REF] Ramirez | A volcanic hydrogen habitable zone[END_REF] or methane [START_REF] Ramirez | A Methane Extension to the Classical Habitable Zone[END_REF] should be revised accordingly. Future experiments and calculations of these CIAs combined with numerical climate model simulations will eventually and hopefully better constrain these numbers.

Conclusion

We performed the first measurements of the far infrared CIAs of CO 2 -CH 4 and CO 2 -H 2 mixtures as well as of the absorption by the (low frequency) wings of the pure CO 2 15 µm band lines. We confirm the theoretical prediction of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] that the H 2 +CO 2 and CH 4 +CO 2 CIAs are significantly stronger in the 40-600 cm -1 region than those of H 2 +N 2 and CH 4 +N 2 , respectively. However, our results for H 2 +CO 2 and CH 4 +CO 2 significantly differ from the predictions, both in terms of the magnitude and shape of the CIAs. In the expected 200-600 cm -1 spectral window of early Mars, our measurements show that the calculations of [START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF] overestimate in average the CO 2 -CH 4 and CO 2 -H 2 CIAs by a factor of about 1.7 and 1.6, respectively. The amount of H 2 and CH 4 required to warm the surface of early Mars above the melting point of water should be revised accordingly. For the pure CO 2 15 µm-band line-wings, we show that both the χ-factor deduced from measurements near 4 µm and a line-mixing model very well describe the observed strongly sub-Lorentzian behavior in the 500-600 cm -1 region. The results of this first experimental investigation obviously need confirmation by independent measurements and we hope that the results presented in this paper will stimulate new laboratory investigations. Similarly, it would be of great interest to compare the results of our measurements and of the only prediction available with those obtained from alternative models and calculations.

Fig. 1 :

 1 Fig.1: Pure CO 2 CIAs at room temperature. The symbols with error bars are the present measurements, the green line is a moving average of the latter over 10 cm -1 , the red line represents the measurements ofHo et al. 1971 (digitized from Fig. 1 of this reference), and the blue curve denotes the calculated values from[START_REF] Gruszka | Roto-translational collision-induced absorption of CO 2 for the atmosphere of Venus at frequencies from 0 to 250 cm -1 , at temperatures from 200 to 800[END_REF] which are available in the HITRAN CIA database section[START_REF] Richard | New section of the HITRAN database: Collision-induced absorption (CIA)[END_REF]. The results in the left and right hand-side panels have been obtained without and with the baseline adjustment, respectively.

Fig. 5 :

 5 Fig.5: H 2 +CO 2 CIAs at room temperature. The symbols with error bars are the present measurements, the green line is a moving average of the latter over 10 cm -1 , the blue line represents the calculated values of[START_REF] Wordsworth | Transient reducing greenhouse warming on early Mars[END_REF]

Fig. 6 :

 6 Fig. 6: Pure CO 2 transmission spectra for three pressures. The black circle denotes the measured values (not all plotted). The red lines are the (indistinguishable) results obtained with the three χ-factors (see text). The blue line is obtained with purely Voigt line shapes (χ = 1). The green line denotes the results of calculations obtained with the pure CO 2 line-mixing model of[START_REF] Tran | Measurements and modeling of high pressure pure CO 2 spectra from 750 to 8500 cm -1 . I-Central and wing regions of the allowed vibrational bands[END_REF]

Fig. 7 :

 7 Fig. 7: In black, H 2 +CO 2 CIA (top panel) and CH 4 +CO 2 CIA (bottom panel) measured at room temperature (symbols with error bars). The blue lines are the calculated valued of CO 2 -H 2 and CO 2 -CH 4 , respectively, taken from Wordsworth et al. 2017. The red lines are the CIA of N 2 -H 2[START_REF] Borysow | Theoretical collision-induced rototranslational absorption spectra for modeling Titan's atmosphere H 2 -N 2 pairs[END_REF]) and N 2 -CH 4[START_REF] Borysow | Far infrared CIA spectra of N 2 -CH 4 pairs for modeling of Titan's atmosphere[END_REF], respectively, taken from the HITRAN CIA database section[START_REF] Richard | New section of the HITRAN database: Collision-induced absorption (CIA)[END_REF]).

Acknowledgements

The authors thank R. Wordsworth for providing the calculated CH 4 +CO 2 and H 2 +CO 2 CIA spectra plotted in Fig. 2 of the Supporting Information file of Ref. (Wordsworth et al. 2017). They are grateful to R. Gamache for providing the data for the broadening of H 2 O lines by CO 2 . They also thank the SOLEIL-AILES beamline staff for their technical support during this project and C. Janssen and L. Manceron for lending pressure transducers.

Appendix A -Conditions of the recorded spectra

We performed various measurements of transmission spectra of Ar, CO 2 , CH 4 , H 2 as well as of H 2 +CO 2 and CH 4 +CO 2 mixtures at room temperature (23 °C). In total, we recorded 39 spectra:

-7 spectra of pure Argon between 100 and 950 mbar.

-5 spectra of pure CO 2 between 200 and 950 mbar.

-3 spectra of pure CH 4 between 600 and 980 mbar.

-3 spectra of pure H 2 between 600 and 950 mbar.

-9 spectra of CO 2 +CH 4 between 500 and 950 mbar, for three different mixtures (30,50 and 70% of CH 4 ). -10 spectra of CO 2 +H 2 between 400 and 950 mbar, for three different mixtures (30,50 and 70% of H 2 ). The experimental conditions of the recorded spectra are detailed in In order to remove the contribution of H 2 O lines from the raw recorded spectra, the absorption coefficient of this species under the temperature, pressure and mixture compositions of the measurements was computed using Voigt line shapes with a cut-off 3 cm - 1 away from the line center, using the kspectrum code [START_REF] Eymet | kspectrum: an open-source code for high-resolution molecular absorption spectra production[END_REF]. We used the line positions and intensities from the 2012 edition of the HITRAN database [START_REF] Rothman | The HITRAN2012 molecular spectroscopic database[END_REF]. For the pressure-broadening coefficients, we used data from [START_REF] Brown | CO 2 -broadened water in the pure rotation and ν 2 fundamental regions[END_REF] for H 2 O-CO 2 , a factor of 1.14 applied to the air-broadening coefficients from HITRAN for H 2 O-CH 4 [START_REF] Nwaboh | Line strength and collisional broadening coefficients of H 2 O at 2.7 µm for natural gas quality assurance applications[END_REF]. For H 2 -broadening coefficients of H 2 O lines, a factor of 1 was used [START_REF] Steyert | Hydrogen and helium pressure broadening of water transitions in the 380-600cm -1 region[END_REF]), as well as for Ar-broadening. Note that the accuracy of the broadening coefficients used is not crucial since the spectral resolution of the measured spectra (1 cm -1 ) is much larger than the line half-width (about 0.1 cm -1 at 1 atm). The transmission was then computed and convolved by the Fourier transform spectrometer instrument line shape (a cardinal sine function with a resolution of 1 cm -1 ) and the relative H 2 O amount was floated until the best agreement between measured and calculated absorptions around H 2 O lines was obtained. The measured spectrum was then corrected by dividing it by the computed transmission of water vapor. Note that the retrieved H 2 O relative amounts are small (between 1.2 10 -6 and 8.0 10 -6 mol/mol), making the contributions of the self and foreign water vapor continua [START_REF] Mlawer | Development and recent evaluation of the MT_CKD model of continuum absorption[END_REF][START_REF] Ma | A far wing line shape theory and its application to the foreignbroadened water continuum absorption[END_REF]) fully negligible here.

The same approach was used in order to remove the absorption by local CH 4 and CO 2 lines from the transmissions deduced from the spectra after their "cleaning" from the water vapor lines. CO 2 and CH 4 local lines were computed with the kspectrum code [START_REF] Eymet | kspectrum: an open-source code for high-resolution molecular absorption spectra production[END_REF]) by using the HITRAN 2012 database [START_REF] Rothman | The HITRAN2012 molecular spectroscopic database[END_REF] and Voigt line shapes, with a cut-off 3 cm -1 away from the line center. We used the self and air-broadening coefficients from HITRAN 2012 for all mixtures, except for the pressure broadening of CH 4 by CO 2 , where we applied a factor of 1.3 on the air-broadened Lorentzian half width at half maximum (deduced from [START_REF] Fissiaux | CO 2 -broadening coefficients in the ν 4 fundamental band of methane at room temperature and application to CO 2 -rich planetary atmospheres[END_REF][START_REF] Rothman | The HITRAN2012 molecular spectroscopic database[END_REF]. We used the known partial pressures of CH 4 and CO 2 to remove the local contributions of CH 4 and CO 2 lines from the recorded spectra because this yields a very good result. This good match validates the procedure used to make the gas mixtures and determine their compositions.

We did not remove H 2 quadrupole transitions from our recorded transmission spectra because they make a totally negligible contribution to our measured spectra because of their very small intensities and of the 1 cm -1 spectral resolution used.

Appendix C -Measured CH 4 +CO 2 and H 2 +CO 2 CIAs

H 2 -CO 2 CIA CH 4 -CO 2 CIA Wavenumber (cm -1 ) Abs. coef. (cm -1 /amagat -2 ) Uncertainty (cm -1 /amagat -2 ) Wavenumber (cm -1 ) Abs. coef. (cm -1 /amagat -2 ) Uncertainty (cm -1 /amagat -2 )