Philippe Poizot

Lydia Laffont-Dantras

Jacques Simonet
email: jacques.simonet@univ-rennes1.fr

Palladized silver as new cathode material: Evidence of a one-electron scission for primary alkyl iodides

Keywords:

Palladium silver alloy Silver electrodes Cleavage of carbon-iodine bonds Homo-coupling a b s t r a c t Silver electrodes modified by ''palladization" thanks to an immersion in acidic solutions of Pd 2+ salts, exhibit exceptional catalytic capabilities especially in the cleavage of primary alkyl halides used as standard systems in order to evaluate the catalytic activity. Such chemically modified surfaces characterized by the formation of an Ag/Pd-based alloy showed large potential shifts compared to the use of glassy carbon or even smooth silver cathodes. Specifically, these palladium-doped silver surfaces were successfully used for cleaving carbon-iodine bonds. Thus, preliminary results have revealed that palladized silver electrodes behave quite differently from smooth silver and showed exceptional catalytic capabilities. Cathodic reductions were achieved in dimethylformamide, acetonitrile, and propylene carbonate. While smooth silver cathodes generally entailed two-electron cleavage reactions (evidence of two clearly separated steps in propylene carbonate), palladized silver interfaces always yield catalytic cleavages of carbon-iodine bonds according to single one-electron processes with the transient formation of a free alkyl radical leading to homo-coupling processes.

Introduction

The discovery of ideal and multi-purpose electrodes in the field of organic electrochemistry remains nowadays an important goal to specifically achieve new reactions in which the heterogeneous character of the electrode surface could play a crucial role [START_REF] Shäfer | Encyclopedia of Electrochemistry[END_REF]. Basically, the use of mercury is now banned for environmental reasons [START_REF] Brown | Physical Chemistry of Organic Solvent Systems[END_REF] whereas both cost and weak hydrogen over-voltage make the use of platinum within the cathodic domain difficult. Alternately, different carbon surfaces such as graphite or glassy carbon provide universal solid working electrodes but their neutrality towards electrochemical ions insertion and their tendency to graft electrogenerated species were often mentioned (e.g., like highly reactive free radicals within a well defined potential range) [START_REF] Coulon | [END_REF]. Similarly, electrodes covered with functionalized conductive polymers could be used in interfacial synthesis by mimicking the principle of organic chemistry on solid support [START_REF] Steckhan | Organic Electrochemistry[END_REF] especially when organo-metallic centers and/or metallic micro aggregates are incorporated into the matrix. Consequently, some new insights in electrochemical synthesis may be linked to the development of solid electrodes with very specific properties. Following this approach, pure silver [START_REF] Rondinini | [END_REF] as well as other solid metal electrodes modified by adatoms [6] could offer interesting prospects when tailored for specific reactions.

Especially within the cathodic range, two-electron cleavage reactions are of importance in organic synthesis because some of them can be applied to deprotection processes [START_REF] Kossai | Sulfones and aryl tosylates can be efficiently cleaved by means of electrochemical reduction[END_REF]. However, the success in achieving such deprotections by reductive scission reactions depends on the electrochemical potential necessary to cleave these carbon-heteroatom bonds. Most of the reported reactions of this type occurs at potentials lower than À2 V vs. SCE, and consequently does not allow the use of aqueous or even wet organic solvents at most polarized solid metals.

The group from Rennes underlined during the past three years the interest in using smooth palladium and palladized surfaces [8] to improve the ease of carbon-halide bonds scission [9], in particular those of alkyl iodides. More recently, the use of palladium associated with copper was successfully developed [10][11][12]. In the present preliminary paper, we similarly develop an easy way to produce a thin layer of an Ag/Pd alloy particularly useful to strongly speed up bonds scission often described as having high activation energy to achieve the first electron transfer [13][14][15][16][17][START_REF] Savéant | Advances in Physical Organics[END_REF]. Thus these new modified electrodes have been simply formed by chemical palladization of both smooth silver and silvered solid conducting substrates, the latter being produced simply by electrodeposition of Ag 0 . Here are gathered the first results on the cleavage of a large series of primary alkyl iodides RI. Reactions on palladized silver substrates are compared to those currently observed at glassy carbon electrodes. The advantages of these Ag-Pd surfaces in terms of catalytic character, stability, simplicity of doi:10.1016/j.jelechem.2008.06.004 use and formation are preliminarily underlined. The use of such interfaces is presented in terms of potential shifts, chemical stability of the surface, and catalytic efficiency. Our results were also compared to the use of smooth silver recently developed [START_REF] Mussini | [END_REF][20][21][22][23][24] using, however, organic tertiary and benzylic halides already known to be more reactive than the ones studied in the present work.

Experimental

Chemical doping of silver by palladium and characterization

The modification of silver by palladium at the nanometric scale was simply obtained by contact of smooth silver metal with a fresh aqueous solution of palladium sulphate in sulphuric acid. One gram of Pd(SO 4 ) Á 2H 2 O (Alfa Aesar) was dissolved in 100 mL of 0.1 N H 2 SO 4 solution. After a few seconds, a shiny layer progressively covered the silver surface due to the displacement of Ag 0 by palladium cations. Palladium nitrate could be used as well. The layer thickness appeared to strongly depend on the time of contact with the palladium solution.

Characterization of the as-produced layers was carried out by a combined SEM, HRTEM and EDS study. Morphology of the layer formed onto the electrode was first investigated by SEM using a FEI QUANTA F200P coupled with an Oxford Link ISIS instrument for EDS analysis. The TEM and HRTEM imaging were performed using a FEI TECNAI F20 S-TWIN whereas the elemental composition was also determined by EDS analysis at nanometer resolution. Since the displacement reaction occurs whatever the silver substrate used, TEM characterizations were directly performed using a homemade silver sample-holder (silver grids for microscope commercially unavailable). The diffraction patterns were performed using the selected area diffraction (SAED) mode or by Fourier transform of the HRTEM imaging.

In order to be used as an electrode (micro-or working-electrode), the metallic substrate was rinsed with water, then alcohol and at last acetone. Finally each electrode was dried with a hot air flow during about 30 s. Under such conditions, electrodes were easily re-used and gave coherent data providing they were rinsed according to the above procedure. Along this study, the resulting electrolysis products such as free radicals are suspected to be more or less strongly adsorbed onto the surface, and disturb the following experiments.

It also appeared of interest to make electrodes from silvered surfaces. These electrodes surfaces were obtained by galvanostatic deposits of silver from a solution of AgNO 3 (10 g L À1 in a 0.1 N HNO 3 solution). These deposits, elaborated onto glassy carbon, smooth silver or platinum, were achieved thanks to fixed current electrolyses (0.5 mA mm À2 ). Silver deposition corresponded to electricity amounts between 10 À3 and 4 Â 10 À3 C mm À2 .

Electrochemical procedure; salts and solvents

In all of the experiments, the supporting salt concentration was fixed at 0.1 M. In this study, the results mainly concern the solutions of tetra n-butylammonium tetrafluoroborate (TBABF 4 ) and tetra n-butylammonium hexafluorophosphate (TBAPF 6 ) into different dipolar solvents. The purity of these salts (at least 98%) was considered suitable to achieve experiments, and was used without any further purification. These salts were purchased from Aldrich. The used solvents were dimethylformamide (DMF) and acetonitrile (quality for analyses) purchased from SDS and propylene carbonate (PC) purchased from Merck (quality for syntheses). It is worth mentioning that procedures given hereafter do not require especially dry solutions. However, if one wishes to reach potentials as low as À2 V vs. Saturated Calomel Electrode (SCE), the solution needs to be dried more efficiently to limit the hydrogen evolution (reduction of the remnant water), and therefore increase the electrical yield of the overall organic cleavage. All electrochemical experiments were performed under inert atmosphere thanks to a bubbling of dry argon.

Electrochemical instrumentation and procedures

All potentials are given vs. aqueous Saturated Calomel Electrode (SCE). The electrochemical instrumentation has been described in previous publications [9,10].

Working electrodes

All the electrodes used in voltammetry had an apparent surface area of 0.78 mm 2 . Silver, glassy carbon, pure palladium and platinum disks when used as substrates were always carefully polished with silicon carbide paper (Struer) or with Norton polishing paper (type 02 and 03). Before use, the electrodes were twice rinsed with water then alcohol and finally acetone. Lastly, they were dried in a hot air stream. Palladized platinum and palladized glassy carbon electrodes -used for comparison -were obtained by means of a galvanostatic deposit from a solution of palladium chloride (e.g., PdCl 2 at the concentration 10 g L À1 in aqueous 0.1 N HCl). In general, such galvanostatic deposits correspond to amounts of electricity from 10 À3 to 2 Â 10 À2 C cm À2 with currents of the order of a few hundreds micro-amps. The procedure of palladization gave a quasi-instantaneous dull deposit onto the smooth silver surface apparently due to the displacement of silver by palladium (E°(Pd 2+ /Pd) = 0.92 V/NHE and E°(Ag + /Ag) = 0.799 V/NHE) concomitantly with an unexpected reaction leading to a specific Ag-Pd interface. The determination of the resulting alloy is described hereafter (see Section 3).

Coulometry and electrolyses

Coulometric measurements and electrolyses of alkyl iodides were carried out using three-electrode system cells with a total catholyte volume of about 5-10 mL. The anodic compartment was separated by a fritted glass of weak porosity. The experiments were completed on small substrate amounts (typically 0.1 mM). In order to avoid the disturbance resulting from the possible presence of silver oxide, which could depend on the history of solid substrate, a pre-electrolysis of the solution was systematically achieved prior to adding the RX compound to the cell. Efficient argon bubbling was completed in all cases in the course of voltammetries and coulometries, and enabled quite a good reproducibility of data especially in voltammetry.

Chemicals

All the alkyl halides studied in the present work were obtained from Aldrich, with a minimum purity of 95%. They were used as such.

Results and discussion

Textural and structural characterization of the Ag-Pd interface

With quite short times of contact (e.g., less than 1 min) the asproduced layer appeared to be stable, and the sonication procedure had no visible effect on the adhesion of micro crystals covering the silver substrate. A selection of SEM images (Fig. 1) shows the spectacular surface evolution of the substrate as the ''palladization" reaction prolongs. In the initial stages of the film growth, very small grains of spheroid-like shape cover the surface of the substrate. As expected, the particle size progressively increases with much longer contact times yielding a quasi covering of the substrate after 5 min (Fig. 1c). Although Pd and Ag exhibit similar energy diagrams, EDS analyses of the layer were performed since the Pd-L a1 radiation, located at 2.8387 eV in energy, is not overlapped.

For comparison, Fig. 2 shows two spectra, before and after surface modification, leading to the conclusion that those spheres contain palladium after the chemical treatment. However, it was difficult to evaluate the Ag/Pd ratio of spheres since the thickness of the layer is not high enough to suppress the silver substrate contribution.

So a TEM study was performed to determine the texture, the structure and the composition of this layer. The bright field image (Fig. 3a) shows a dendritic-like growth of the palladium-based layer obtained with the solution of palladium sulphate (size < 20 nm). The HRTEM image (Fig. 3b) of one part of the bright field image represents some agglomerated nanoparticles, which composed the dendrite. These nanoparticles have homogeneous morphology and dimension (around 5-10 nm) and are well crystallized. The SAED pattern (Fig. 3c) of the corresponding Fig. 3a is composed of diffraction circles due to the fact that all nanoparticles are oriented in different directions. Due to the software 'Process Diffraction', a line profile of the electron diffraction pattern enables to plot a graph similar to the X-ray diffraction (Fig. 3d), and to determine that the layer is composed of an Ag/Pd alloy [START_REF] Pearson | A Handbook of Lattice Spacings and Structures of Metals and Alloys[END_REF] (lattice parameter: 4.0499(8) Å) and also of PdO and Ag. Indeed, as recently mentioned [START_REF] Wang | [END_REF], Ag/Pd alloys can slightly decompose under air into PdO and Ag by oxidation. The EDS analysis (not shown here) obtained on this sample indicates systematically an alloy composition of 80% Ag and 20% Pd. As reported by Rao [27], the lattice parameter of Ag/Pd alloys changes with temperature, which is in good agreement with the alloy composition that we have found (i.e., Ag 0.8 Pd 0.2 ) since the beam heating is around 200 °C.

Cathodic reduction of primary alkyl iodides at silver and palladized silver interfaces

Electrochemical data found for Ag-Pd surfaces with a series of primary alkyl iodides (see Table 1, propylene carbonate being chosen as solvent due to the lack of disturbing inhibition phenomena) were quite similar to those previously reported for other kinds of Pd-based surfaces such as palladized platinum or palladized glassy carbon [8]. As underlined in Table 1, it seems clear that the potential shift regarding the reduction step at an Ag-Pd surface is significant by comparison to both glassy carbon (DE > 0.9 V) and smooth silver (DE > 0.1 V). This effect does not depend much on the length of the alkyl chain. For comparison, voltammetries at smooth silver in different solvents were found to be often complex probably because a non-conducting layer is produced by contact between silver and organic iodide (Fig. 4) in particular in both acetonitrile and DMF. This kind of inhibition to the heterogeneous electron transfer leads to a relative shift toward more negative potentials (forward scan) while the backward one gives an interesting curve-crossing, probably due to the chemical formation of the non conductive layer has no time to occur. The inhibition cause could be clarified by dipping smooth silver into the primary alkyl iodides RI (or its concentrated solution in a polar solvent like DMF): one may evidence a reaction between the metal and the iodide. Prolonged contacts (e.g., more than 30 min) bring very large discrepancies at the silver surface. For example, a silver surface maintained in contact of 1-iodobutane or 1-iodooctane exhibited a huge structural change as shown by SEM analysis (Fig. 5). Large black zones are widespread onto the surface, which can be assigned to adsorbed product of silver with alkyl iodide as well asproduced free radicals since a huge peak of carbon is present on the EDS spectra. So, such results confirm the reactivity of silver toward RI compounds. This reaction was recently discussed [28].

The importance of silver implication depends on the contact time between Ag and RI. We can expect from these preliminary experiments that primary RIs will be slowly chemically reduced by silver. The weak but effective reducing capabilities of silver metal towards carbon-iodine bonds already permit to propose a reduction scheme (see Scheme 1) where the first and second elec-tron transfers can be differentiated under quite well defined experimental conditions. Especially, experiments achieved in propylene carbonate enable to support the existence of a one-electron step with the formation of an alkyl free radical at the level of peak 1 (see Fig. 6, curve B and coulometry in Table 1). This first peak is followed by a second peak, which accounts for the reduction of the free radical. The potential range found for peak 2 located at around À1.5 V (Fig. 6) quite agrees with the standard potentials given by Lund [29] for a large panel of similar free alkyl radicals. In general, the second step is rather small, and it is expected that the free radicals formed during the first reduction process are strongly adsorbed at the metal surface. However, fixed potential electrolyses at smooth silver at applied potentials lower than E p2 enabled to demonstrate that the global reduction may reach 2 F mol À1 . It is Table 1 Voltammetric and coulometric data relative to some primary alkyl iodides in 0. remarkable that the reduction of RI compounds can occur in two separate steps. Interestingly, examples of dimers obtained with tertiary alkyl iodides by coupling of the free radical R at silver cathodes under comparable (but not identical) conditions can be found in a recent literature [23]. It is quite possible that adsorption of free radicals at the silver surface may inhibit their own reduction (level of peak 2). At this point, let us recall that the addition of free radicals onto metallic interfaces has frequently been observed [START_REF] Coulon | [END_REF]30]. The effective separation of two reduction steps with RIs could be valuable for the modification of silver surfaces. Lastly, the adsorption of free radicals onto silver could cause the pre-step (to step 1) found here with all the primary alkyl iodides in propylene carbonate. If we refer to the classification by Laviron [31][32][33] on polarographic and voltammetric currents, this would be caused by the fast adsorption of the reduction product (i.e., free alkyl radical). Electrolyses at the level of step 1 of the modified interface by dipping of Ag in RI lead to the merging of crystals (Fig. 5d) clearly identified as dimers R-R issued from R . Note that the example reported in Fig. 5 corresponds to 1-iodooctane; its homo-dimer hexadecane is weakly soluble in PC.

E p /2 (V) I p (lA) E p /2 (V) I p (lA) E p /2 (V) b I p (lA) E (V) Q (F mol À1 ) E (V) Q (F mol À1 ) 1 1-Iodopropane ( 10
Voltammetric curves may also depend on the nature (and preparation) of the silver surface and the level of moisture present in the DMF solution. Thus, with small RI amounts dissolved in super-dry electrolyte solutions (obtained by addition in situ of neutral activated alumina), voltammetric currents show no complication brought by adsorption, and curves are well reproducible (see Fig. 7, case of 1-iodobutane). It is likely that the low level of moisture at the silver interface favors the solubility of the silver salt essentially lipophilic, and therefore strongly diminishes the occurrence of the inhibition of the first electron transfer always evidenced at large RI concentrations. This technique would, in principle, permit to monitor the alkyl iodides reduction (potential at the level of the first peak, Fig. 7B) in order to obtain a pure oneelectron step. However, the use of activated alumina remains a heavy technique, and forces to a constant activation of alumina.

On the contrary, palladized silver surfaces do not exhibit any apparent chemical reaction towards RIs. As a matter of fact, only one step under diffusion controlled and proportional to the organic iodide concentration was obtained with Ag-Pd electrodes for concentrations smaller than 10 mM. Because of the absence of inhibition at the silver surface, the resulting peak was found to be shifted at less cathodic potentials towards the one already described at pure silver surface. This absence of inhibition at Ag-Pd surface with RI compounds can be related to the absence of pure silver zones at the interface. Fig. 6 with hexadecane iodide, curve C and Fig. 7, case of 1-iodobutane, curve C underline that the step current at Ag-Pd electrodes corresponds roughly to half of the current obtained at glassy carbon, well known to enable two-electron processes (classical hydrogenolysis of the C-I bond). This point would mean that the use of palladized silver enables specific one-electron reductions. This proposal is well checked by means of coulometry at Ag-Pd cathodes (Table 1, reductions in DMF at about À1.5 V vs. SCE, entries 2-5), which perfectly establish that RIs reduction under these conditions obeys a pure one-electron process whatever the polar organic solvent. The applied potential does not matter much since the reduction step obtained at Ag-Pd surface is the lone step and well separated from the cathodic limit at À2 V, which is specific of such palladium surface under the defined experimental conditions. This means that free radical reduction at palladium and palladized interfaces (possibly owing to a strong chemisorption of those radicals leading to an efficient coverage of the interface) does not occur fast within a potential range until $0.7 V beyond the peak potential. These results are in full agreement with recent data [8] observed with palladized electrodes with RI compounds, whatever the mode of Pd deposition.

Additionally, the presence of a free alkyl radical is efficiently checked thanks to the spin trapping method in ESR; the latter is due to the formation of a paramagnetic nitroxide radical when the reduction was completed in the presence of nitrone (N-tert-butyl-a-phenylnitrone) or nitroso compounds (tert-butyl-nitroso). This method has been successfully developed with other palladized surfaces [11,12], and is in full agreement with a one-electron overall process (real trapping, in this case, of the free radical R by the nitrone).

Thus, the use of Ag-Pd surfaces always brought a real improvement of the catalytic effect both in potential shift and electrolysis specificity.

Reduction products at macro smooth silver and Ag-Pd cathodes

For comparison, macro-reductions were achieved both at large silver and silver-palladium surfaces (surface areas of about 2 cm 2 ) with several alkyl iodides. As shown in Table 1, results in DMF containing TBA + salts confirm the one-electron character of the global cathodic process with Ag-Pd electrodes. Also at smooth silver surfaces, one-electron process can be also easily observed on the provided condition that macro-electrolyses are achieved in PC at moderate potentials (between À1.2 V and À1.4 V vs. SCE). Preliminary data (see Table 2) achieved with RIs are in agreement with the transient formation of a free radical R capable to dimerize or/ and disproportionate. Thus, preliminary GC-MS analyses on electrolyses mixtures showed the major formation of homo-dimers R-R and the monomers R(-H) and R(H) roughly in equal amount. The specificity of silver-palladium cathodes obeys probably interfacial catalytic process put forward in Scheme 2, and based on a possible synergy between Ag and Pd. Ratios dimer/monomers vary upon experimental conditions but it should be stress that dimer yields are excellent or quasi quantitative. It seems, however, too RAg + , I -/A -+ e - R • + I -/A -+ A g 0 (firststep , E 1 )

R • + e - R -Proton source R H (second step, E 2 ) M + A - (electrolyte) RI + Ag 0 slow R Ag + , I - RAg + , A - (1)
(2) early to draw definitive conclusions about a mastering of the dimer production.

Conclusions

The aim of the present work was to propose a new cathodic material in order to achieve, at moderate potentials, one-electron processes with primary alkyl iodides taken here as target substrates. Thus, for the first time, a silver-palladium alloy was used as modifier of smooth silver surfaces. Its chemical structure has been totally defined and its building is particularly easy and fast. The preliminary results relative to organic iodides listed in the present paper are extremely attractive. As a matter of fact, this new cathodic material combines two important properties: (i) the catalysis specific of silver towards primary alkyl iodides with substantial potential shifts towards data previously shown at glassy carbon [START_REF] Peters | Organic Electrochemistry[END_REF] and (ii) the role of silver-palladium modifier for specifically orienting cleavages of carbon-iodine bonds to one-electron reduction processes. These preliminary results have pointed out that the use of such Ag-Pd electrodes could be at first considered as a source of free alkyl radicals as shown by means of coulometries, spin trapping technique, and fixed potential electrolyses. Moreover, voltammetries achieved in several dipolar solvents, showed that Ag-Pd surfaces certainly privileged obtaining of well defined one-electron reduction steps. These reduction steps were found to be generally diffusion controlled and electrolyses could be achieved stepwise. The stability of the surface appeared to be good and macro-electrolyses (current densities up to 50 mA cm À2 ) allowed several runs by using the same electrode. Let us stress also that the necessary promotion of Ag-Pd electrodes needed beforehand a wider scope of the intrinsic behaviour of silver towards organic halides. Thus, the present work has additionally clarified the reason for the catalysis already observed with the alkyl iodides.

However, until now, the intrinsic reason for obtaining an electro-catalysis at such electrodes is not totally understood and the unexpected synergy [Ag + Pd] evidenced within the present work needs additional experiments. A general scope of other kinds of organic substrates (possessing chemical bond capable to cleave under electron transfer like primary alkyl bromides) is being undertaken with success.

Fig. 1 .

 1 Fig. 1. SEM images of (a) the pristine silver surface, (b) after immersion in a PdSO 4 solution during 30 s, and (c) during 5 min.

Fig. 2 .

 2 Fig. 2. EDS spectrum of a smooth silver sample (a) and a modified silver surface by palladium after a contact time of 5 min (b).

Fig. 3 .

 3 Fig. 3. (a) Bright Field image of the Ag/Pd alloy. (b) HRTEM picture showing the nanoparticles of the alloy. (c) SAED pattern of the TEM picture showing the polycrystalline nature of the layer. (d) Line profile of the SAED pattern characteristic of the Ag 0.8 Pd 0.2 alloy, PdO and Ag.

Fig. 4 .

 4 Fig. 4. Recurrent scans measured at a smooth silver electrode using 1-iodohexane (concentration, 2.7 mM) in 0.1 M TBAPF 6 /DMF. Apparent surface areas, S = 0.78 mm 2 ; Scan rate, 50 mV s À1 . (A) Inert glassy carbon taken as a standard. (B) Five first scans at a smooth silver electrode (sweeps between À0.7 V and À2.5 V). (C) New scan immediately after the 5th sweep of (B)).

Fig. 5 .

 5 Fig. 5. SEM image of (a) the pristine silver surface, (b) after 30 min contact with 1-iodooctane, (c) after 30 min contact with 1-iodopropane, and (d) same treatment as in (b) followed by an electrochemical reduction at À1.4 V vs. SCE.

Fig. 6 .

 6 Scheme 1.

Fig. 7 .

 7 Fig. 7. Voltammetries obtained at different working electrodes using 1-iodobutane (concentration, 9 mM) in 0.1 M TBAPF 6 /DMF. Super-dry conditions have been obtained by adding freshly activated neutral alumina to the electrolyte. Apparent surface areas, S = 0.78 mm 2 ; scan rate, 50 mV s À1 . (A) Glassy carbon. (B) Smooth silver. (C) Ag-Pd electrode.

  Scan rate, 50 mV s À1 . Potentials are referred to aqueous SCE. Apparent surface area of all electrodes, 0.78 mm 2 . The treatment of the Ag electrode was achieved by a dipping time of 1 min in Pd sulphate (see Section 2). At À1.5 V, electrolyses achieved both in DMF and PC yield in all cases to two-electron processes, 2 ± 0.2 F mol À1 .

		mM)	À1.92	14.0	À1.24	12.1	À1.17	11.2	À1.6	1.1	-	-
	2	1-Iodobutane (8.8 mM)	À1.93	10.6	À1.22	9.1	À1.10	8.3	À1.6	1.1	À1.2	1.1
	3	1-Iodohexane (7 mM)	À1.91	8.2	À1.20	7.2	À1.14	6.6	À1.5	1.2	À1.1	1.0
	4	1-Iodooctane (7 mM)	À1.91	6.1	À1.24	5.8	À1.10	6.0	À1.6	1.3	À1.2	1.1
	5	1-Iodohexadecane (5 mM)	À1.83	7.4	À1.23	4.1	À1.12	5.5	À1.5	1.1	À1.3	1.0

a Potential of the first peak. b Appearance of a lone peak. c Coulometries achieved in DMF. d Coulometries achieved in PC.

Table 2

 2 The experimental conditions are those reported in Table1

	RI	Experimental conditions	R(H) (%)	R(-H) (%)	R-R (%)
	nC 6 H 13 -I	Electrode: smooth silver;	3.6	3.2	93.1
	nC 8 H 17 -I	electrolyte: PC + TBAPF 6 ;	2.7	5.1	92.2
	nC 10 H 21 -I	applied potential: À1.30 V	3.2	3.3	93.4
	nC 16 H 33 -I				7.7	6.7	85.6
	Ph(CH 2 ) 3 -I				-	-	>99
	nC 8 H 17 -I	Electrode: Ag-Pd;	-	0.1	99.9
	nC 10 H 21 -I	electrolyte: DMF + TBABF 4 ;	1.1	1.6	97.3
	Ph(CH 2 ) 3 -I	applied potential: À1.25 V	-	-	>99
	R I	Ag-Pd interface	[RI] ads		[R	Pd ≡ Ag	I] ads
	-I -e -	[R • ] ads + A g-Pd		sol R •
				R-R (coupling)
	2 R • sol
				R (H) + R (-H) (disp)
				Scheme 2.

Acknowledgments

The authors want to deeply thank Dr. Joseph Le Lannic and Dr. Sandra Casale for their help in achieving SEM experiments relative to surface analysis and Prof. V. Jouikov (Université de Rennes) for his interest in this work. They are also grateful to Michèle Nelson for helpful assistance.


Jun Li

Kuo Zeng

Dian Zhong

Xin Chen

Ange Nzihou

Haiping Yang

Hanping Chen

Algae pyrolysis in alkaline molten salt: Products transformation

Algae pyrolysis in alkaline molten salt is a promising technology for hydrogen production, but the pyrolysis products transformation has not clearly been described up-to-date. Moreover, the complex bio-oil especially heavy bio-oil fraction directly influences the products transformation pathways, while limiting the hydrogen production yield and application. Both the light and heavy bio-oil fractions are investigated in detail and the effect of alkaline molten salt on products transformation is obtained. Molten salt promotes the cyclization and aromatization of linear alkenes, thereby increasing aromatic compound content and hydrogen production. The decarbonation of Na 2 CO 3 and the deoxygenation of NaOH lead to the methane release, in turn reducing hydrogen production. The heavy compounds tend to polymerize at higher temperatures into compounds with higher molecular weights and unsaturation, while generating hydrogen. As the temperature rises, char reacts with molten salt to form hydrogen and disappears at 650 • C, while aromatics in light compounds condense into heavy compounds, which then grow to form soot-like at 600 • C, accompanying hydrogen release. Based on the relationship between products transformation, hydrogen release and alkaline molten salt effect, strategies to improve the hydrogen yield and purity of AMSP process are proposed.

Introduction

Promoting clean and sustainable energy is of great significance for ensuring energy security and promoting environmental protection [START_REF] Xu | The influence of key reactions during hydrothermal carbonization of sewage sludge on aqueous phase properties: a review[END_REF][START_REF] Xu | Hydrothermal carbonization of sewage sludge: effect of aqueous phase recycling[END_REF]. In recent years, biomass has been recognized as a prospective renewable energy source to address these challenges, among which algae is attracting increasing attention due to its fast growth rate, high photosynthetic efficiency and global distribution [START_REF] Choi | Production of brown algae pyrolysis oils for liquid biofuels depending on the chemical pretreatment methods[END_REF][START_REF] Kouhgardi | Current status and future prospects of biofuel production from brown algae in north america: progress and challenges[END_REF]. The production of biofuels such as bioethanol and biohydrogen is the foremost option when considering algae refining strategies [START_REF] Wang | A review on optimistic biorefinery products: biofuel and bioproducts from algae biomass[END_REF][START_REF] Zeng | Solar pyrolysis of heavy metal contaminated biomass for gas fuel production[END_REF]. For algae refining processes, thermochemical processes are favored over biochemical processes because of their advantages of high utilization rate, low cost and fast kinetics [START_REF] Yang | New insights into the carbon neutrality of microalgae from culture to utilization: a critical review on the algaebased solid biofuels[END_REF]. As hydrogen is a clean-renewable energy carrier with high energy density, many researchers have produced algae-based hydrogen using pyrolysis, gasification and liquefaction, but these methods are subject to high energy consumption, high operating pressure, salt precipitation and low hydrogen yield etc. [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF][START_REF] Cherad | Hydrogen production from the catalytic supercritical water gasification of process water generated from hydrothermal liquefaction of microalgae[END_REF][START_REF] Duman | Hydrogen production from algal biomass via steam gasification[END_REF][START_REF] Lin | Microwave plasma studies of spirulina algae pyrolysis with relevance to hydrogen production[END_REF].

Molten salt has been widely used as an excellent catalytic and heating medium for the thermochemical conversion of carboncontaining materials to accelerate the reaction rate and regulate the product composition [START_REF] Xie | Study on CO 2 gasification of biochar in molten salts: reactivity and structure evolution[END_REF][START_REF] Li | Biomass gasification in molten salt for syngas production[END_REF]. It can also retain a certain amount of ash in algae to prevent slagging or salt precipitation at high temperature [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF]. Recently, our group investigated the potential of algae pyrolysis in alkaline molten NaOH-Na 2 CO 3 (referred to as AMSP, as shown in reaction [START_REF] Xu | The influence of key reactions during hydrothermal carbonization of sewage sludge on aqueous phase properties: a review[END_REF] for hydrogen production and obtained a stable high hydrogen yield of 71.48 mmol/g-algae in continuous operation [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF]. Algae not only act as a hydrogen source, but also as a source of carbon and water source (generated by algae pyrolysis [START_REF] Chen | Algae pyrolytic polygeneration: influence of component difference and temperature on products characteristics[END_REF]) to extract hydrogen from NaOH. More importantly, the CO X (CO and CO 2 ) product reacts with molten NaOH to form Na 2 CO 3 , significantly reducing the carbon footprint, and the used salt can be subsequently regenerated by industrial waste calcium carbide slag or used as valuable chemical in a wide range of applications [START_REF] Ishida | One-step production of co-and co2free hydrogen from biomass[END_REF]. Moreover, molten salt can be used to store solar heat and heat released from biomass decomposition for algae pyrolysis [START_REF] Yin | Harvesting capacitive carbon by carbonization of waste biomass in molten salts[END_REF], saving the cost of energy consumption needed for heating.

C 1 H 1.69 O 0.56 N 0.14 S 0.006 + aNaOH→bNa 2 CO 3 + cH 2 + salt + char + liquid + gas [START_REF] Xu | The influence of key reactions during hydrothermal carbonization of sewage sludge on aqueous phase properties: a review[END_REF] Studies have shown that the effects of component interaction of biooil (light and heavy fraction) can affect the transformation and composition of pyrolysis products [START_REF] Xiong | Effects of the component interaction on the formation of aromatic structures during the pyrolysis of bio-oil at various temperatures and heating rates[END_REF][START_REF] Xiong | Evolution of coke structures during the pyrolysis of bio-oil at various temperatures and heating rates[END_REF]. And the transformation of hydrogen-containing compounds further affects hydrogen production. optimized design resulted in some changes in product distribution and gas yield compared to previous work [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF].

Prior to the AMSP experiment, N 2 (250 mL/min) was introduced through the feed tube for 30 min to exhaust the air in the enclosure. Another N 2 (200 mL/min) was introduced through the bubbling tube to stir the molten salt as soon as the temperature reaches 400 • C. After the target temperature (500 • C, 550 • C, 600 • C, 650 • C, 700 • C and 750 • C) was reached, ten algae pellets were fed into 110 g of molten NaOH-Na 2 CO 3 to undergo pyrolysis, and the reaction lasted 25 min. Each experiment was performed 3 times. The liquid product containing biooil and water was condensed in the ice-water bath, the gases were collected by a sample bag (Dalian Delin Gas Packing Co., Ltd), and the remaining char in the crucible was soaked in deionized water, filtered and collected. The methods for calculating the yield of each product was described in a previous work [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF]. In addition, Inconel series (Inconel 625) and Hastelloy-N (GH3535) are suggested to be used as the materials of reactor and pipelines to provide resistance to the corrosion of alkaline molten salt in industrial applications [START_REF] Pooja | High temperature corrosion behaviour of stainless steels and inconel 625 in hydroxide salt[END_REF][START_REF] Yin | Corrosion behavior and mechanism of gh3535 alloy in naoh, hf and h2o2 solution[END_REF].

It should be noted that at temperatures above 550 • C, there was solid products formed in the ice-water bath, referred to as soot-like in this paper, and was also collected for subsequent analysis. In addition, for comparison, the pyrolysis experiments (SP) were performed on this apparatus under the same reaction temperatures. SPT and AMSPT are used to represent the experimental conditions, where T represents the reaction temperature.

Characterization

The gas products were analyzed by an off-line gas chromatography (Panna A91, China) equipped with a 5A molecular sieve column, a Porapak Q column and an Al 2 O 3 /KCl capillary column. CO, H 2 , CH 4 and CO 2 were detected by a thermal conductivity detector, while olefins and alkanes (C 2+ ) were detected by a flame ionization detector. The columns were first maintained at 80 • C for 4 min, then heated to 150 • C at 10 • C/ min, and finally held at 150 • C for 6 min.

The ultimate analysis of solid products and algae was measured using an CHN/O elemental analyzer (Vario Micro Cube, Germany). For char and soot-like, a field emission scanning electron microscopy (FE-SEM, ZEISS Gemini 300, Germany) was used to investigate the morphology, an X-ray diffraction spectroscopy (XRD, SmartLab-SE, Japan) with Cu Kα radiation at λ = 1.5406 Å was used to analyze the microcrystalline structure, and a Raman spectroscopy (inVia-Reflex, China) equipped with Nd-YAG laser (wavelength 532 nm) was employed to characterize the carbon skeleton with the spectrum integrated three times to improve the signal-to-noise ratio. For both XRD and Raman tests, each sample was tested three times in different zones to reflect the overall properties of solid products.

The liquid products were first dehydrated using anhydrous magnesium sulphate and tested by a gas chromatograph-mass spectrometer (GCMS, Agilent 7890 A series GC coupled with a 5975 MS detector) with a capillary column (HP-5MS, 30 m × 0.25 mm i.d. × 0.25 um d.f.). The detail settings of the GCMS were as follows: the injection temperature was 280 • C, the split ratio was 20:1, and the carrier gas in the column was set as 1 mL/min. The temperature in oven was first maintained at 40 • C (2 min), and then heated to 200 • C at 5 • C/ min (held for 5 min), and finally heated to 300 • C at 10 • C/ min (held for 5 min). All the compounds were identified using a National Institute of Standards and Technology Library (NIST 14.L).

The heavy components in the bio-oil were analyzed using a FT-ICR-MS (7.0 T Solarix, Germany) equipped with ESI in positive-ion mode. The bio-oil samples were first dehydrated with anhydrous magnesium sulfate and then diluted with methanol to 0.35 mg/ml. Before analysis, the bio-oil solution was spiked with 0.5 vol% of formic acid to enhance the ionization of organic compounds. The emitter voltage, capillary voltage and end plate offset were set as 4.0 kV, 4.5 kV and -0.5 kV, respectively. The flight time was set as 0.5 ms and a total of 128 scans However, the pyrolysis products transformation (especially heavy biooil) during the AMSP process has not clearly been described up-todate. Studies have explored the effect of molten alkaline salt (mostly carbonates) during the pyrolysis of carbon contained feedstock [START_REF] Xu | The characteristics and evolution of nitrogen in bio-oil from microalgae pyrolysis in molten salt[END_REF][START_REF] Zeng | Molten salt pyrolysis of biomass: the mechanism of volatile reforming and pyrolysis[END_REF][START_REF] Ratchahat | Combined molten salt-Ni/Al 2 O 3 as synergistic medium for high-quality syngas production[END_REF], but little research has been done on alkaline molten salt (hydroxides). Although both molten carbonates and molten hydroxides are basic and have similar alkali metal ions, they provide different effects during the pyrolysis. For example, hydroxides can serve as a hydrogen donor for hydrogen production and be used to destroy the original structure of biomass [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF][START_REF] Kumar | Lignocellulose biomass pyrolysis for bio-oil production: a review of biomass pre-treatment methods for production of drop-in fuels[END_REF].

Typically, the heavy components constitute over 50 wt% of the total species in bio-oil [START_REF] Shashkov | Development of comprehensive analysis of pyrolysis products for lignocellulose raw materials and sludge sediments by chromatographic methods[END_REF][START_REF] Ye | Feasibility study on recycled vegetable oil waste and recycled polyethylene for the modification of aged asphalt[END_REF]. However, the inherent properties of heavy components (molecular weight > 200 Da) together with high boiling points and complex structures make their investigation challenging [START_REF] Chaiwong | Study of bio-oil and bio-char production from algae by slow pyrolysis[END_REF][START_REF] Xiong | Effects of aaems on formation of heavy components in bio-oil during pyrolysis at various temperatures and heating rates[END_REF]. Recently, the advanced Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) equipped with electrospray ionization (ESI) has been successfully applied to identify the heavy biooil compounds derived from the pyrolysis of various biomass [START_REF] Xu | The characteristics and evolution of nitrogen in bio-oil from microalgae pyrolysis in molten salt[END_REF][START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF][START_REF] Zhong | Characteristics and evolution of heavy components in bio-oil from the pyrolysis of cellulose, hemicellulose and lignin[END_REF][START_REF] Qiu | Effects of cellulose-lignin interaction on the evolution of biomass pyrolysis bio-oil heavy components[END_REF]. Consequently, the bio-oil transformation, especially heavy bio-oil fraction, during the AMSP process could be investigated in insight.

In this work, the experiments were conducted in the temperature range of 500-750 • C (thermal stabilization interval of alkaline molten salt) to study the products transformation with emphasis on hydrogen production [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF]. A combination of GC-MS and FT-ICR-MS was used to identify the light and heavy bio-oil compounds in depth. Based on the detailed bio-oil composition, the transformation pathways of pyrolysis products as well as strategies to further improve hydrogen yield and purity were proposed.

Material and methods

Materials

Nannochloropsis sp. with particle size less than 0.6 mm was first dried at 105 • C for 24 h and then pressed in a cylindrical mold to produce pellets (0.23 ± 0.005 g) with a diameter of 6 mm and a height of 6.5 mm. The purpose of pelleting is to reduce transportation costs caused by the low volume and energy density of raw algae. The ultimate and proximate analysis results of feedstock were presented in Table S1. Details on chemical composition of algae, as well as the test methods of ultimate and proximate analysis can be found in detail in our previous studies [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF][START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF]. Sodium carbonate and sodium hydroxide powder were bought from the Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The mixture of sodium hydroxide and sodium carbonate (mass ratio 8:2, with a melting point of approx. 289 • C) was not pre-melted for homogeneity, as it is strongly absorbent.

Experimental methods

The AMSP experiments of algae were carried out on the apparatus as shown in Fig. S1, which mainly comprises a graphite crucible (carbon content > 99.9, 250 mm height, 40 mm i.d. and 44 mm o.d.), an enclosure (Hastelloy C276, 70 mm i.d.), a feeding unit, an electric furnace with temperature control system and a volatile collection system. The graphite crucible acted as a reactor to contain the molten salt, and the feeding unit stored algae pellets, which were then fed through a feed tube (Hastelloy C276, 10 mm i.d.) into the molten salt for pyrolysis. N 2 was used to stir the molten salt through a bubble tube (Hastelloy C276, 1.7 mm i.d.) to obtain homogeneous molten salt similar to that achieved by pre-melting. The openings for the feed and bubble tubes were placed 15 mm and 8 mm above the bottom of molten salt, respectively, while the perforated plate was positioned 8-10 mm below the upper surface of the molten salt. It should be mentioned that the pore size of the perforated plate was changed from 1.2 mm (previous work [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF]) to 0.5 mm for sufficient reaction of some small char particles. This was applied. Finally, the obtained data was analyzed by a custom MATLAB script. More detail information about the parameter settings of instrument and the programming principles of the MATLAB script can be found in our previous work [START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF]. All the results were calculated based on the ash-free dry basis of algae.

Results and discussion

Pyrolysis products distribution

The products distribution for SP and AMSP of algae are presented in Fig. 1. In the case of SP, as the temperature increases, the gas yield increases steadily to 57.84 wt%, the char yield decreases continuously to 22.90 wt%, and the liquid yield reaches the peak of 50.06 wt% at 550 • C. The variation of liquid yield is attributed to more proteins and lipids decomposing into liquids at temperatures below 550 • C [START_REF] Wang | Pyrolysis characteristics and pathways of protein, lipid and carbohydrate isolated from microalgae nannochloropsis sp[END_REF], while the secondary cracking of volatile at temperature above 550 • C leads to a reduction in liquid yield [START_REF] Chen | Algae pyrolytic polygeneration: influence of component difference and temperature on products characteristics[END_REF]. In the case of AMSP, the gas and liquid yield decrease to 38.77 wt% and 2.99 wt% at 750 • C, respectively, due to the reaction between CO X and molten salt reaction (2-3) and the secondary cracking of volatiles promoted by molten salt [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF][START_REF] Zeng | Molten salt pyrolysis of biomass: the mechanism of volatile reforming and pyrolysis[END_REF][START_REF] Ishida | Production of cox-free hydrogen from biomass and naoh mixture: effect of catalysts[END_REF]. The total yield of char and soot-like is consistently below 5 wt%. With the increase of temperature, char disappears at 650 • C due to the enhanced reaction with molten NaOH-Na 2 CO 3 reaction (3-4), while soot-like appears at 600 • C probably due to the recombination and polymerization of volatile [START_REF] Cherad | Hydrogen production from the catalytic supercritical water gasification of process water generated from hydrothermal liquefaction of microalgae[END_REF][START_REF] Nagase | Kinetics and mechanisms of the reverse boudouard reaction over metal carbonates in connection with the reactions of solid carbon with the metal carbonates[END_REF][START_REF] Trubetskaya | Effects of several types of biomass fuels on the yield, nanostructure and reactivity of soot from fast pyrolysis at high temperatures[END_REF]. In addition, the remaining algae substance is transferred to molten salt and its composition and transformation path were described in our previous work [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF].

2NaOH (l) + CO (g) →Na 2 CO 3(l) + H 2(g)
(2)

4NaOH (l) + C (s) + CO 2(g) →2Na 2 CO 3(l) + 2H 2(g) (3)
C (s) + Na 2 CO 3(l) →Na 2 O (s) + 2CO (g) (4)

Properties of gas and solid products

The composition and content of gas products at different reaction temperatures are presented in Fig. 2 and Fig. S2 respectively. Gas yield per unit weight of algae was used to compare the gas composition (especially H 2 , CO and CO 2 ) of different processes. In the presence of molten NaOH-Na 2 CO 3 , H 2 yield increases sharply, CH 4 and C 2+ yield increase slightly, while CO and CO 2 production decrease sharply, which is consistent with the previous work [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF]. The reduction in the pore size of the perforated plate reduces the incomplete reaction caused by the floating of small char particles during the AMSP process. As a result, CO X is almost completely consumed by molten salt (reaction (3) and ( 5)) [START_REF] Ferguson | Novel approach to hydrogen production with suppressed cox generation from a model biomass feedstock[END_REF] and its yield is less than 0.25 mmol/g-algae at the temperature studied (the CO X yield in Fig. 2 has been artificially increased to facilitate reading). As the temperature rises, the H 2 yield increases from 26.75 mmol/g-algae to 72.14 mmol/g-algae (750 • C), with H 2 accounting for 83.23 vol% of the total gas, CH 4 for 11.36 vol% and C 2+ for 5.17 vol%. Since CH 4 and C 2+ occupy a high proportion in gas products, it is desirable to convert CH 4 and C 2+ simultaneously by suitable means in the subsequent bio-oil reforming process to further improve hydrogen yield and purity. The elemental composition and content of char and soot-like produced from AMSP are listed in Table 1. Char has higher carbon content than soot-like, because char reacts with molten salt for a longer time, and Na + promotes the cleavage of C-C bond [START_REF] Blasi | Influences of the chemical state of alkaline compounds and the nature of alkali metal on wood pyrolysis[END_REF]. The hydrogen content of soot-like is significantly lower than that of char, implying that sootlike is formed by dehydrogenation of volatile, which is similar to the mechanism of soot formation reported in the literature [START_REF] Wang | Nano-scale soot particle formation during the high-temperature pyrolysis of waste plastics in an entrained flow reactor[END_REF]. Both higher temperatures and molten NaOH-Na 2 CO 3 promote the decrease of nitrogen content of char, and the decrease of nitrogen content in soot-like may be related to the decrease of N-containing compounds in volatiles [START_REF] Tay | Preparation and characterization of activated carbon from waste biomass[END_REF][START_REF] Li | Solar pyrolysis of algae in molten salt for capacitive carbon preparation[END_REF]. In addition, the molar H/C of soot-like is in the range of 0.1-0.2, slightly higher than the molar H/C (0.05-0.01) of mature soot from xylan, cellulose and lignin pyrolysis reported by Deng et al. [START_REF] Deng | Differences in soot produced from rapid pyrolysis of xylan, cellulose and lignin under pulverized-fuel conditions[END_REF]. The morphology of typical char and soot-like observed by SEM is shown in Fig. 3. It is clear that char and soot-like have quite different morphologies, char appears as irregularly stacked lamellae with broken borders due to the reaction with molten salt, while soot-like is presented as particles and spheres.

2NaOH (l) + CO (g) →Na 2 CO 3(l) + H 2(g) (5)
Fig. S3 shows the Raman spectrum of typical char and soot-like with the D peak (1350 cm -1 ) and the G peak (1580 cm -1 ) used to represent certain properties. The I D /I G , calculated from the intensity ratio of the D and G peaks, reflects the degree of graphitic disorder, and the results for char and soot-like are given in Table 2. As the temperature rises, the I D / I G values for both char and soot-like decrease, suggesting an increase in graphitization, which matches well with the trend of molar H/C in Table 1. In addition, char has a relatively higher degree of graphitization due to its higher I D /I G values than those of soot-like. The crystalline structure of typical char and soot-like is further revealed by the XRD as displayed in Fig. S4. Only one broadened diffraction peak (21-25 • ) appears on the spectrum, indicating that both char and soot-like are amorphous carbon without a three-dimensional long-range ordered structure. Compared to char, soot-like has a smaller peak angle and larger crystal spacing (according to the Bragg equation) [START_REF] Lu | Char structural ordering during pyrolysis and combustion and its influence on char reactivity[END_REF], and therefore a relatively lower degree of graphitization, which is consistent with the Raman results. More information about the differences of the gas and liquid products derived from SP and AMSP as well as the related reasons can be found in our previous work [START_REF] Li | Algae pyrolysis in molten NaOH-Na2CO3 for hydrogen production[END_REF].

Properties of light compounds in bio-oil

The light organic compounds in bio-oil derived from SP and AMSP are divided into eight groups, as shown in Table 3 and Table 4 respectively, and the trend of the five most abundant compounds as a function of temperature is presented in Fig. S5. For SP, the light compounds consist mainly of aliphatic hydrocarbons (3.20-29.20

Table 3

The main groups of light organic compounds for SP. The aliphatic hydrocarbons are divided into linear alkanes (e.g., pentadecane) and linear alkenes (e.g., 1-octadecene). With the increase of temperature, the linear alkanes and linear alkanes decrease continuously in content, possibly because higher temperatures promote their cracking to non-condensable gases (e.g., C 2 H 2 ) or their aromatization to form aromatics [START_REF] Baldwin | Current research on thermochemical conversion of biomass at the national renewable energy laboratory[END_REF][START_REF] Ledesma | Formation and fate of pah during the pyrolysis and fuel-rich combustion of coal primary tar[END_REF]. Molten NaOH-Na 2 CO 3 contributes little to alkanes conversion, but promotes alkenes conversion dramatically at temperatures higher than 550 • C, due to the fact that alkenes are more reactive than alkanes. Since only the content of aromatic hydrocarbons and oxygenated compounds increases above 550 • C, it is presumed that molten salt intensifies the aromatization or oxidation of linear alkenes.

The content of N-heterocyclic aromatic compounds (e.g., indole) for AMSP is significantly reduced (dropping to zero above 600 • C) compared to that for SP. Studies have shown that the N-heterocyclic aromatic compounds in bio-oil are mainly derived from the cracking of amino acids, as well as the secondary cracking of pyridine-N and pyrrole-N in char [START_REF] Chen | Thermochemical conversion of low-lipid microalgae for the production of liquid fuels: challenges and opportunities[END_REF][START_REF] Debono | Nitrogen products and reaction pathway of nitrogen compounds during the pyrolysis of various organic wastes[END_REF]. Reactions between molten NaOH-Na 2 CO 3 and amino acids may be the main reason for the decrease in the content of Nheterocyclic aromatic compounds [START_REF] Chen | A review on the nox precursors release during biomass pyrolysis[END_REF]. In addition, the above reactions are also responsible for the conversion of nitriles and amines/amides at temperatures above 600 • C, as nitriles and amines/amides are mainly derived from the conversion of protein fragments (amino acids and peptides) and the dehydration of amides [START_REF] Debono | Nitrogen products and reaction pathway of nitrogen compounds during the pyrolysis of various organic wastes[END_REF][START_REF] Chen | Transformation of nitrogen and evolution of n-containing species during algae pyrolysis[END_REF].

The higher content of aromatic hydrocarbons for AMSP implies that molten NaOH-Na 2 CO 3 promotes the hydrogen formation from the aromatization of light compounds. According to the number of benzene rings, the aromatics hydrocarbons are further categorized as one/two/ three/four/five-rings aromatics to show the relationship between the composition of aromatics hydrocarbons and the hydrogen yield at different temperatures (Fig. 4). In the presence or absence of molten salt, the main compounds are essentially the same, such as naphthalene, 2methylnaphthalene, anthracene and pyrene. At 550 • C, the one-ring aromatics (e.g., toluene) can be formed from the long-chain fatty acids by C-C bond breaking, cyclization and aromatization [START_REF] Chen | Algae pyrolytic polygeneration: influence of component difference and temperature on products characteristics[END_REF], and the tworing aromatics (mainly naphthalene) may be evolved from benzene through the fusion of alicyclic hydrocarbons [START_REF] Bensabath | Polycyclic aromatic hydrocarbon (PAH) formation during acetylene pyrolysis in tubular reactor under low pressure carburizing conditions[END_REF]. As temperature rises, the aromatization of aromatics with fewer rings intensifies to form aromatics with more rings and release large amounts of hydrogen [START_REF] Zeng | Molten salt pyrolysis of biomass: the mechanism of volatile reforming and pyrolysis[END_REF]. Compared with SP, the content of aromatics with fewer rings (one and/ or two rings) from AMSP decreases, while that of aromatics with more rings (three, four and five rings) increases at temperatures above 600 • C. The aromatics with more rings in the volatile tend to condense (forming condensed aromatics in heavy compounds, refer to 3.4) and grow to form soot-like eventually at higher temperatures. On the other hand, Na + in molten salt can promote the conversion of small aromatic ring systems to large aromatic ring systems, while the high heating rate provided by molten salt has the opposite effect [START_REF] Xiong | Effects of aaems on formation of heavy components in bio-oil during pyrolysis at various temperatures and heating rates[END_REF][START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF].

For the phenolic compounds, molten salt significantly increases the relative content of phenol especially at higher temperatures, and the content of phenols reaches 31.44 area % at 750 • C, including 30.45 area % for 2,2′-methylenebis(4-methyl-6-tert-butylphenol) and 0.99 area %

Table 4

The main groups of light organic compounds for AMSP.

for 3,5-bis(tert-butylphenol). The existing studies have shown that the decarbonation by carbonate promotes the formation of methoxyphenols, and OH -can further promote the deoxygenation of methoxyphenols to form alkylphenols or phenols [START_REF] Özbay | Pyrolysis of water buffalo manure: influence of temperature and alkali hydroxide additives on the quality of bio-oil[END_REF][START_REF] Peng | Pyrolysis of lignin for phenols with alkaline additive[END_REF]. Moreover, it has found that methoxyphenols can be completely converted to alkylphenols in molten salt (monomer of 2,2′-methylenebis(4-methyl-6-tertbutylphenol)) at temperatures above 450 • C in our previous study [START_REF] Zeng | Molten salt pyrolysis of biomass: the mechanism of volatile reforming and pyrolysis[END_REF]. Therefore, large quantities of 2,2′-methylenebi(4-methyl-6-tert-butylphenol) with high purity was produced by the polymerization of alkylphenols (3,5-bis(tert-butylphenol)) with the effect of molten NaOH-Na 2 CO 3 , while releasing a certain amount of CH 4 [START_REF] Xie | Solar pyrolysis of cotton stalk in molten salt for bio-fuel production[END_REF][START_REF] Stefanidis | In-situ upgrading of biomass pyrolysis vapors: catalyst screening on a fixed bed reactor[END_REF].

Properties of heavy compounds in bio-oil

Fig. 5 presents the molecular weight distribution of heavy components in bio-oil at different temperatures. Overall, the heavy components show a bimodal distribution (as shown as the red lines representing AMSP550) in molecular weight, and the components with molecular weight greater than 500 Da are more abundant, especially at higher temperatures. As the temperature increases, the molecular weights of the left and right peaks of SP increase slowly from 250 to 300 Da and 550-600 Da to 300-350 Da and 600-650 Da, respectively. And for AMSP, the molecular weight of the left peak increases sharply from 250 to 300 Da to approximately 500-550 Da, and that of the right peak remains roughly unchanged (500-550 Da). Molten salt enhances the polymerization (which is also promoted by higher temperatures) of heavy components with smaller molecular weight, while promote the decomposition of heavy components with larger molecular weight, which is attributed by the integrated effect of Na + , high heating rate and soot-like formation [START_REF] Xiong | Effects of aaems on formation of heavy components in bio-oil during pyrolysis at various temperatures and heating rates[END_REF][START_REF] Trubetskaya | Effects of several types of biomass fuels on the yield, nanostructure and reactivity of soot from fast pyrolysis at high temperatures[END_REF][START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF].

The distributions of carbon atoms and double bond equivalents (DBE) of heavy components are presented in Fig. 6 to evaluate the core structure of detected species. DBE is an essential index used to evaluate the unsaturation of compounds, and its calculation method was reported elsewhere [START_REF] Koch | Molecular formulae of marine and terrigenous dissolved organic matter detected by electrospray ionization fourier transform ion cyclotron resonance mass spectrometry[END_REF]. It can be noticed that most of the heavy compounds (from the 10 % line and 90 % line) have carbon atoms number (CN) and DBE mainly distributed between 13 and 47 and 2-50, respectively, for both SP and AMSP. There is an overall upward trend (as illustrated by the 50 % line) in both CN and DBE as the temperature rises, suggesting a stronger condensation associated with unsaturated carbon bonds occurs at higher temperatures. The addition of molten salt reduces the growth rate of CN and DBE at temperature higher than 650 • C, suggesting that molten NaOH-Na 2 CO 3 can inhibit the condensation of heavy components at higher temperatures (this result can also be illustrated by the Van Krevelen diagrams below).

CHO compounds and nitrogen-containing compounds (both present at high levels in bio-oil) could affect the catalyst activity in the bio-oil reforming process [START_REF] Xiong | Effects of vapor-/solid-phase interactions among cellulose, hemicellulose and lignin on the formation of heavy components in bio-oil during pyrolysis[END_REF][START_REF] Mohamed | Advanced ni tar reforming catalysts resistant to syngas impurities: current knowledge, research gaps and future prospects[END_REF], so the content of CHO compounds as well as the average nitrogen number (ANN) in heavy components are calculated (the method can be found in [START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF]) with the results listed in Table S2. shown in Fig. 7. With reference to the existing literatures [START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF][START_REF] Ohno | Ultrahigh resolution mass spectrometry and indicator species analysis to identify marker components of soiland plant biomass-derived organic matter fractions[END_REF], the heavy components are mainly divided into lipids, unsaturated hydrocarbons, peptides, lignin, condensed aromatics and carbohydrates.

For SP, the major compounds are composed of lipids, unsaturated hydrocarbons and lignin at 550 For AMSP, the addition of molten salt seems to facilitate the pyrolysis process, as AMSP650 and SP750 share a very similar Van Krevelen diagram, especially the region where the green lines are located and the disappearance of stretching lines associated with the diffusion point (O/ C = 0, H/C = 1). This is because molten salt accelerates the heat transfer from the surface to interior of the algae pellet, greatly reducing the temperature gradient inside the pellet. It is clear that there are more heavy compounds belonging to the region of lipids, which was reported to mainly consist of fatty acids, resin acids and other wood extractives [START_REF] Hertzog | Toward controlled ionization conditions for esi-ft-icr-ms analysis of bio-oils from lignocellulosic material[END_REF]. The main conversion pathways for heavy fatty acids are chainbreaking and cyclization [START_REF] Chen | Algae pyrolytic polygeneration: influence of component difference and temperature on products characteristics[END_REF], but the acid content detected by GC-MS for both SP and AMSP is low, suggesting that molten NaOH-Na 2 CO 3 inhibits the cyclization rather than chain-breaking of large fatty acids.

Transformation pathways of pyrolysis products

The transformation pathways of pyrolysis products related to hydrogen-containing gases release in molten NaOH-Na 2 CO 3 are proposed based on the above discussions (shown in Fig. 8). The light compounds in bio-oil consist mainly of aliphatic hydrocarbons, phenols and aromatic hydrocarbons, and the content of phenols and aromatic hydrocarbons reaches up to 95.5 % at 750 • C. Linear alkenes of aliphatic hydrocarbons tend to form aromatics with H 2 release through cyclization and aromatization above 550 • C. Alkylphenols (3,5-bis(tert-butylphenol) are produced by the decarbonation of Na 2 CO 3 and the deoxygenation of NaOH, and are subsequently converted into 2,2′methylenebi(4-methyl-6-tert-butylphenol) by polymerization with CH 4 release. Besides, molten salt promotes the cracking of protein fragments (e.g. amino acids) into NH 3 , substantially reducing the nitrogencontaining compounds derived from these protein fragments.

The heavy compounds consist mainly of lipids, peptides, lignin and unsaturated hydrocarbons. As temperature rises, the heavy compounds undergo polymerization to form compounds with higher molecular weights and higher unsaturation, leading to an increase in CN and DBE. Char reacts with molten salt to form H 2 and disappears at 650 • C, while aromatics in light compounds condense into heavy compounds, which then grow to form soot-like and release H 2 at 600 • C. Moreover, molten salt promotes the cleavage of C-C bonds in char, leading to a lower carbon content in char than that in soot-like, while higher temperatures enhance the dehydrogenation of soot-like, resulting in a decrease in molar H/C and I D /I G values.

Future development of AMSP

In order to further improve the hydrogen yield and purity production by the AMSP of algae, impurity gases and bio-oil vapors entrained in the hydrogen-rich gas need to be removed or converted as much as possible. In terms of gas composition, despite the high hydrogen yield and purity (72.14 mmol/g-algae and 83.23 vol%) obtained, 11.36 vol% CH 4 and 5.17 vol% C 2+ are still contained in the gas product. Therefore, it is desirable to convert CH 4 and C 2+ simultaneously during the AMSP process to further improve hydrogen yield and purity, such as steam reforming. In terms of bio-oil composition, the higher aromatic and phenolic content (95.5 %) allows Ni-based catalysts (e.g., Ni-ZrO 2 , Ni-MgO, etc.) suitable for bio-oil steam reforming, because these Nibased catalysts have significant capacity for C-C,C-H and O-H bond cleavage [START_REF] Santamaria | Influence of the support on ni catalysts performance in the in-line steam reforming of biomass fast pyrolysis derived volatiles[END_REF][START_REF] Santamaria | Stability of different ni supported catalysts in the in-line steam reforming of biomass fast pyrolysis volatiles[END_REF]. In addition, the content of active CHO compounds and nitrogenous compounds in the heavy bio-oil obtained from ASMP changes little compared to that obtained from SP, suggesting that conventional catalysts considering carbon deposition or poisoning are applicable for the bio-oil cleaning process of AMSP.

Conclusion

Algae pyrolysis in molten NaOH-Na 2 CO 3 produces 72.14 mmol/galgae of H 2 at 750 • C with a purity of 83.23 vol%. As the temperature rises from 500 • C to 750 • C, char reacts with molten salt and disappears at 650 • C, while aromatics in light compounds condense into heavy compounds and then grow to form soot-like at 600 • C, both processes releasing large amounts of H 2 . Molten salt promotes the cyclization/ aromatization of linear alkenes and the cracking of protein fragments

Fig. 1 .

 1 Fig. 1. Products distribution for: (a) SP and (b) AMSP at different temperatures.

Fig. 2 .

 2 Fig. 2. Gas composition under different temperatures.

Fig. 3 .

 3 Fig. 3. FE-SEM images of (a) char obtained at 500 • C and (b) soot-like obtained at 750 • C.

Fig. 4 .

 4 Fig. 4. The relationship between the composition of aromatics hydrocarbons and hydrogen yield (entity: SP, shadow: AMSP).

Fig. 5 .

 5 Fig. 5. The molecular weight distribution of heavy components.

Fig. 6 .

 6 Fig. 6. The distributions of CN and DBE in heavy components (50% line represents the median line, and the rest parameters are similar in principle).

  Obviously, SP and AMSP share a similar trend in CHO content and ANN. With the increase of temperature, the ANN increases, while the CHO content remains basically unchanged. Overall, molten NaOH-Na 2 CO 3 contributes slightly to the removal of nitrogen, as well as the formation of CHO compounds, suggesting that the available catalysts for bio-oil (derived from SP) reforming can be referenced for AMSP process when only the heavy compounds are considered.The heavy components are further plotted in the Van Krevelen diagram based on their elemental composition (O/C and H/C value), with the size of circles representing the abundance of each compound, as

Fig. 7 .

 7 Fig. 7. Van Krevelen diagrams of heavy components in bio-oil.

  • C and 650 • C, and more condensed aromatics are formed at 750 • C due to the aromatization (e.g., hydrocarbons aromatization, refer to the line of O/C = 0), which is promoted by high temperature. Besides, a large number of new compounds are produced at 750 • C, which is linearly distributed on the Van Krevelen diagram, as shown as the green lines. It is speculated that these compounds are formed by a combination of the deoxygenation of carbohydrates (due to the presence of a diffusion points with O/C = 1, H/C ≈ 1.6) and the oxidation of nitrogen-containing polycyclic compounds (e.g., C 65 H 29 N 5 and C 50 H 20 N 5 ) at higher temperatures.

Fig. 8 .

 8 Fig. 8. Transformation pathways of pyrolysis products in molten NaOH-Na 2 CO 3.

Table 2

 2 The I D /I G values based on the Raman spectra of char and soot-like.

	Sample	Temperature( • C)	I D /I G
			Value	Error (-)	Error (+)
	Char	500	0.884	0.018	0.013
		550	0.851	0.024	0.015
		600	0.834	0.022	0.017
	Soot-like	600	0.916	0.017	0.025
		650	0.905	0.018	0.020
		700	0.895	0.014	0.015
		750	0.884	0.019	0.009

Table 1

 1 The element composition and content of solid products produced from AMSP.

	Classification	Content (area %)
		500 • C	550 • C	600 • C	650 • C	700 • C	750 • C
	Aliphatic	29.20	22.83	20.78	12.57	7.34	3.20
	hydrocarbons
	Linear alkanes	10.68	7.52	5.61	2.37	1.07	0.60
	Linear alkenes	18.53	15.31	15.16	10.20	6.27	2.40
	Aromatic	6.08	9.70	19.50	45.20	56.64	65.27
	hydrocarbons
	Alicyclic	11.95	13.60	11.06	2.42	-	-
	hydrocarbons
	N-heterocyclic	25.43	27.86	21.96	23.18	24.12	23.68
	aromatic
	compounds
	Amines/amides	5.17	4.21	4.45	1.42	-	-
	Nitriles	8.99	9.31	8.29	5.69	5.50	3.55
	Phenols	1.39	5.50	10.61	7.32	4.69	3.11
	Oxygenated	11.80	6.99	3.36	2.21	1.71	1.19
	compounds*

* consisting of alcohols, carboxylic acids, esters and aldehydes; -, not detected. aliphatic hydrocarbons (1.11-36.60 area %), phenols (2.07-31.44 area %) and aromatic hydrocarbons (19.99-64.06 area %) are the major components of light compounds, and the most abundant compounds are 2,2′-methylenebi(4-methyl-6-tert-butylphenol), 2-methylnaphthalen, naphthalene, cholesta-3,5-diene and dimethyl phthalate.

  consisting of alcohols, carboxylic acids, esters and aldehydes; -, not detected.

	Classification	Content (area %)
		500 • C	550 • C	600 • C	650 • C	700 • C	750 • C
	Aliphatic	36.60	27.56	10.69	2.57	2.13	1.11
	hydrocarbons
	Linear alkanes	12.39	8.13	6.51	2.57	2.13	1.11
	Linear alkenes	24.21	19.43	4.18	-	-	-
	Aromatic	19.99	26.15	55.44	59.36	63.40	64.06
	hydrocarbons
	Alicyclic	12.47	9.26	2.78	-	-	-
	hydrocarbons
	N-heterocyclic	8.71	7.93	2.80	-	-	-
	aromatic
	compounds
	Amines/amides	5.49	4.32	-	-	-	-
	Nitriles	9.91	9.11	1.64	-	-	-
	Phenols	2.07	8.32	18.30	20.65	29.33	31.44
	Oxygenated	4.76	7.35	8.35	17.43	5.17	3.40
	compounds*

*

Acknowledgement

Authors acknowledge funding from the National Natural Science Foundation of China (52261135626, 52111530296, 52076098), and the support of National Natural Science Funds for Distinguished Young Scholar (52125601). In addition, authors would like to thank the Analytical and Testing Center in Huazhong University of Science & Technology (http://atc.hust.edu.cn) and the shiyanjia lab (https:// www.shiyanjia.com) for the test. There are no conflicts to declare.

Data availability

Data will be made available on request.

into NH 3 . Alkylphenols are produced via the decarbonation by Na 2 CO 3 and deoxygenation by NaOH, and then further polymerized to release CH 4 . The heavy bio-oil compounds, with CN and DBE mainly in regions of 18-44 and 2-41. As the temperature increases, these components tend to polymerize into compounds with higher molecular weights and unsaturation, leading to an increase in CN and DBE. Based on the products transformation, the ex-situ steam reforming processes using Nibased catalysts are proposed to further improve the hydrogen yield and purity.

CRediT authorship contribution statement

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.fuel.2023.129868.


Keywords: Cell cycle, circadian clock, model coupling, mutations

The existence of links between the cell division cycle and the circadian clock has been recently discovered. In this research report, we perform a small in silico analysis of how mutations on the clock genes composing the mammalian circadian clock affect the phenotype of the cell cycle. For this purpose, we use a coupled model of the mammalian circadian clock and the mammalian cell cycle where the latter one is entrained by the former one via a common protein kinase WEE1. Our in silico experiments exploit the modeling environment BIOCHAM.

Model-based Predictions of the Influence of Circadian Clock Genes Knock-Outs on the Cell Cycle

Résumé : L'existence de liens entre le cycle de division cellulaire et l'horloge circadienne a été mise au jour récemment. Dans ce rapport de recherche, nous menons une petite analyse in silico sur comment les mutations des gènes de l'horloge circadienne composant l'horloge circadienne des mammifères affectent le phénotype du cycle cellulaire. A cette fin, nous utilisons un modèle couplé de l'horloge circadienne et du cycle cellulaire des mammifères dans lequel ce dernier est entrainé par le premier via la protéine kinase WEE1. Nos expériences in silico exploitent l'environnement de modélisation BIOCHAM.

Mots-clés : Cycle cellulaire, horloge circadienne, couplage de models, mutations

Introduction

In this research report we aim at studying how mutations on the clock genes composing the mammalian circadian clock affect the phenotype of the cell cycle. For this purpose, we take into consideration the coupled model of the mammalian circadian cycle and the mammalian cell division cycle proposed by Calzone and Soliman in [START_REF] Calzone | Coupling the cell cycle and the circadian cycle[END_REF], following Matsuo et al. [START_REF] Matsuo | Control mechanism of the circadian clock for timing of cell division in vivo[END_REF].

As for the circadian cycle, we refer to the model described by Leloup and Goldbeter in [START_REF] Leloup | Toward a detailed computational model for the mammalian circadian clock[END_REF], that expresses the regulatory effects exerted on gene expression by the PER (period), CRY (cryptochrome), BMAL1, CLOCK, and REV-ERB α proteins1 and post-translational regulation on these proteins by reversible phosphorylation. To account for entrainment of the circadian clock by light-dark cycles, the effect of the light is incorporated in the maximum rate of PER mRNA expression. The model gives rise to sustained circadian oscillations with a period close to 24 hours. BMAL1 mRNA (mBmal1 ) oscillates in antiphase with PER and CRY mRNAs (mPER and mCRY, respectively). The proteins undergo similar oscillations and follow their mRNA by a few hours.

As for the cell division cycle, we consider a model based on that of Qu, MacLellan, and Weiss in [START_REF] Qu | Dynamics of the cell cycle: checkpoints, sizers, and timers[END_REF], where a generic signaling module is used to represent either the G1/S or the G2/M transitions. The focus we consider here is on the G2/M transition, leading to a two-phase cell-cycle model with G1-S-G2 and M phases (see [START_REF] Calzone | Coupling the cell cycle and the circadian cycle[END_REF] for more details).

The kinetic differential equations present in the two papers allowed Calzone and Soliman to encode both models in the rule-based language of Biocham2 . The link between the two models comes from the experiments of [START_REF] Matsuo | Control mechanism of the circadian clock for timing of cell division in vivo[END_REF] and is reflected through a direct influence of CLOCK-BMAL1 (Bmal1 ) on the synthesis of WEE1 mRNA (mWee1 ), a kinase that delays or prevents entry into mitosis by phosphorylation of MPF.

We would like to underline that the models we use are not fitted to real data yet; however, they allow us to get an initial insight on the effects of circadian mutations on the cell cycle as a basis for discussion with biologists.

In Figure 1 there is a 100 hours-simulation of the coupled circadian/cell cycle model (entrainment by light-dark cycles is taken into account), where it is possible to observe that all the displayed compounds undergo oscillations whose period is approximately 23 hours (if the cell division cycle model is considered alone, the oscillation period is about 23 hours too).

The first three columns of Table 1 show the oscillation amplitude and period and the mean value of some selected compounds observed towards the end of a 650-hours simulation.

In Silico Circadian Clock Genes Knock-outs

Hereafter we describe how the cell cycle reacts to circadian gene/protein mutations in our coupled model. First we consider single mutations, then we study the combination of different mutations. As for single compound mutations, we explore what happens when a given compound (for genes we will concentrate on the mRNA) is missing, that is, its concentration equals zero. To this aim, it is possible either to set the compound synthesis at zero, or to make the compound be absorbed by a "super-inhibitor" (e.g., the knock-out of a given compound C can be modeled by inserting in the model the rule Inhibitor + C → Inhibitor-C, where the initial concentration of Inhibitor is very high). As a matter of fact, both the alternatives have the same impact on the behavior of the coupled model. The mutations we take into consideration concern mPER, mCRY, mB-mal1, and Bmal1_nucl. The simulations we provide in the following are up to 650 hours. mPER=0. As graphically depicted in the plot of Figure 2, in this case the circadian rhythmicity is lost. The concentration of many compounds, including mCRY, mBmal1, Wee1, mWee1, and Bmal1_nucl, becomes constant after some oscillations of decreasing amplitude with a period of 40-50 hours lasting approximately 500 hours. CRY_cyto distinguishes itself because it reaches a constant value of 78.7. The other compounds, including MPF, C25P, APC, and CRY_ nucl-PER_ nucl, approach 0 after a few hours from the beginning of the simulation. A notable remark is that the mean value of the mitosis inhibitor mWee1 (and of Wee1 ) is higher with respect to normal conditions.

More schematically, the amplitude, period, and mean value assumed by the mentioned compounds towards the end of a 650-hours simulation are reported in the third, fourth and fifth columns of Table 1. Since in this case there are no oscillations, the amplitude is omitted and the period is conventionally set to ∞. mCRY=0. In this case the behavior of the model is approximately the same of the previous one (see Figure 3), that is, we can distinguish a first class of compounds whose concentration becomes constant after 500 hours, e.g., mB-mal1, Wee1, mWee1, and Bmal1_nucl, and a second class of compounds whose concentration approaches 0 a few hours after the beginning of the simulation, e.g., MPF, C25P, APC, and CRY_ nucl-PER_ nucl. The difference is that setting mCRY value to 0 causes a big increase of mPER, whose value goes over 30, and a huge increase of PER_cyto, whose value goes over 1835 3 . mBmal1=0. As the plot of Figure 4 shows, when mBmal1 is not present it is possible to distinguish the following behaviors: all the cell cycle compounds except mWee1 continue to oscillate with a period of approximately 20 hours (the amplitude of Wee1 and Wee1∼ {p} is very low, that is, 0.005 and 0.006, respectively), mWee1 concentration stabilizes at 0.005, and all the circadian compounds approach 0 after a few hours from the beginning of the simulation (actually, CRY_cyto, CRY_cyto-PER_cyto, and CRY_nucl-PER_nucl approach 0 after an initial peak of amplitude greater than 5). The oscillation amplitude and period and the mean value of the compounds towards the end of a 650-hours simulation are reported in the last three columns of Table 1.

Bmal1_nucl=0. In this case the assumed behavior is the same that we get when mBmal1=0, that is, all the cell cycle compounds except mWee1 oscillate intensely while mWee1 and all the circadian compounds approach 0 after a few hours from the beginning of the simulation, with the difference that now mBmal1 grows rapidly, getting over 20.

The simultaneous mutation of more than one gene is considered in the following.

mPER=mCRY=0. When both mPER and mCRY equal 0, we get the same behavior that we have when either mPER or mCRY equals 0. mCRY=mBmal1=0. As in the previous case, the model acts as the one where only mBmal1 is set to 0. mPER=mCRY=mBmal1=0. Again, the behavior of this model is assimilable to the one of the model where only mBmal1 is set to 0.

Comparison with Literature about Circadian Clock Genes Mutations

In the following we itemize some facts we found in literature concerning the dependence of the cell division cycle on circadian rhythmicity/mutations and, when possible, we discuss the consistency with our results.

• Decreasing the level of CRY by halving the rate of CRY synthesis does not abolish the oscillations, but decreases the period (in continuous darkness) from 23.9 to 22.1 hours [START_REF] Leloup | Toward a detailed computational model for the mammalian circadian clock[END_REF]. In our coupled model we obtain the same conclusion by halving the rate of CRY synthesis in continuous darkness.

• In absence of PER mRNA or PER protein it is possible to observe sustained oscillations in the circadian cycle [START_REF] Leloup | Toward a detailed computational model for the mammalian circadian clock[END_REF]; according to our results it is only possible to observe oscillations up to 500 hours.

• The expression of several mammalian cell-cycle genes, including c-myc, Cyclin-D1, and mWee1, is regulated in a circadian manner [START_REF] Hunt | Riding tandem: Circadian clocks and the cell cycle[END_REF].

• Overexpression of PER1 leads to apoptosis whereas inhibition of PER1 inhibits apoptosis. It appears that PER1 antagonizes the cell cycle in an oscillatory fashion similar to the manner in which it antagonizes the function of Clock-Bmal1 [START_REF] Hunt | Riding tandem: Circadian clocks and the cell cycle[END_REF]. According to our experiments, a PER inhibition produces an increase of mWee1, an thus a mitosis inhibition.

• PER1 and TIM seem implicated on the DNA-damage response because both can be found complexed with the ATM and ATR kinases and the checkpoint kinases Chk2 and Chk1, respectively [START_REF] Hunt | Riding tandem: Circadian clocks and the cell cycle[END_REF].

• The double mutation of PER1 and PER2 may result in an alteration in the timing of cell division [START_REF] Hunt | Riding tandem: Circadian clocks and the cell cycle[END_REF].

• The oscillatory expression of c-myc is abolished in mPER2 mutant mice, which could then result in an alteration of the p53 function [START_REF] Hunt | Riding tandem: Circadian clocks and the cell cycle[END_REF].

• In CRY deficient cells, the circadian rhythmicity is lost [START_REF] Van Der Horst | Mammalian cry1 and cry2 are essential for maintenance of circadian rhythms[END_REF], Wee1, overexpressed, and MPF, less active, loses rhythmicity [START_REF] Matsuo | Control mechanism of the circadian clock for timing of cell division in vivo[END_REF]. According to our results, in case of a CRY knock-out the circadian compound concentrations become constant after some oscillations with a period of 40-50 hours lasting approximately 500 hours. The effect on Wee1 and MPF are roughly consistent with data.

Conclusion

In particular, these are some of the conclusions in clear accordance with our in silico experiments:

• when either mPER or mCRY are missing, the cell cycle rhythm is slowed down;

• the absence of mBmal1 causes an acceleration of the cell cycle;

• when either mPER or mCRY are missing, mitosis inhibition is stronger than in normal conditions;

• when mBmal1 is not present, mitosis inhibition is very slight.

The confrontation of these results with more precise experimental data is the next step for us to fit our models and refine them where necessary.

Figure 1 :

 1 Figure 1: Simulation of the coupled model without mutations.

Figure 2 :

 2 Figure 2: Simulation of the coupled model with mPER=0.

Figure 3 :

 3 Figure 3: Simulation of the coupled model with mCRY=0.

Figure 4 :

 4 Figure 4: Simulation of the coupled model with mBmal1=0.

Table 1 :

 1 Oscillation amplitude and period and mean value towards the end of 650-hours simulations in the cases of no mutations, mPER=0, and mBmal1 =0.

			wild type			mPER=0			mBmal1=0
	Compound	Amplitude	Period	Mean Value	Amplitude	Period	Mean Value	Amplitude	Period	Mean Value
	MPF	1.015	24	0.5	-	∞	0.006	0.856	20.4	0.43
	C25P	0.74	24	0.37	-	∞	0.009	0.627	20.4	0.32
	Wee1	1.33	24	0.66	-	∞	4.02	0.005	20.4	0.003
	APC	0.99	24	0.5	-	∞	0.0004	0.986	20.4	0.5
	mWee1	0.99	24	0.5	-	∞	2.68	-	∞	0.005
	mPER	5.83	24	2.9	-	∞	0	-	∞	0
	mCRY	2.72	24	1.36	-	∞	6.22	-	∞	0
	mBmal1	9.99	24	9.16	-	∞	5.88	-	∞	0
	CRY_ nucl-PER_ nucl	6	24	3.04	-	∞	0	-	∞	0
	Bmal1_nucl	1.9	24	1.04	-	∞	1.07	-	∞	0
							INRIA

For the sake of simplicity, we consider a version of the model where the role of REV-ERB α in the indirect negative feedback exerted by BMAL1 on the expression of the Bmal1 gene is not explicitly taken into account.

For a review of Biocham syntax, which is very similar to SBML one, the reader can refer to[START_REF] Calzone | BIOCHAM: An environment for modeling biological systems and formalizing experimental knowledge[END_REF].RR n°7064

RR n°7064

A similar behaviour is obtained when either PER_cyto=0 (in this case, as well as in the case when mPER=0, CRY_cyto value goes over 78) or CRY_cyto=0 (in this case, as well as in the case when mCRY =0, PER_cyto value goes over 1835).INRIA

Acknowledgements

We acknowledge support from the EU FP6 Strep project TEMPO and fruitful discussions with Franck Delaunay, Jean Clairambault and Francis Lévi.