Bruno Da Costa Magalhães

Ruben Checa

Chantal Lorentz

Pavel Afanasiev

Dorothée Laurenti

Christophe Geantet
email: christophe.geantet@ircelyon.univ-lyon1.fr

Catalytic hydroconversion of HTL micro-algal bio-oil into biofuel over NiWS/Al2O3

Introduction

The global demand for energy in the world and the environmental concerns to supplying this energy are constantly increasing. Consequently, the production of transportation fuel from renewable resources is receiving more attention from the scientific community. Among the species that constitute the biomass, microalgae display extremely varied biodiversity and are rich sources of new molecules and lipidic materials. Microalgae represent an almost untapped resource (It is estimated that there are about 200,000-800,000 species in many different genera, of which only about 50,000 species are described.) This feedstock seems to be a potential raw material for third-generation fuel production due to its high growth rate and its abundant production that exceeds the best oilseed crops, e.g., biodiesel yield of 58,700 L/ha for microalgae containing only 30% oil by wt.% compared with 1190 L/ha for rapeseed or Canola.

Besides, this microorganism has the potential for CO2 fixation through photosynthesis, storing this carbon in energy rich-compounds such as lipids, carbohydrates, and proteins. Its high lipid content can be converted into drop-in fuels for diesel and jet fuel. Life cycle assessment has shown a reduction in greenhouse gas emission by 76% for algal bio-jet fuel production, compared to conventional jet fuel [START_REF] Singh | Commercialization potential of microalgae for biofuels production[END_REF][START_REF] Santosa | Impact of storage and blending of algae and forest product residue on fuel blendstock production[END_REF][START_REF] Ramírez-Romero | Chlorellaceae Feedstock Selection under Balanced Nutrient Limitation[END_REF][START_REF] Fortier | Life cycle assessment of bio-jet fuel from hydrothermal liquefaction of microalgae[END_REF].

Hydrothermal liquefaction of microalgae is one of the most promising alternatives for producing biofuels as this process is performed in water, it does not require any algae drying and leads to an interesting yield of liquid. Nevertheless, the biocrude obtained still contains nitrogen and oxygen compounds. The total acid number (TAN) tends to be approximately 60 mg KOH/g oil which comes from the fatty acids [START_REF] Ramasamy | Hydrothermal Liquefaction: Path to Sustainable Aviation Fuel[END_REF]. Besides, the density is around 0.96 g/mL and the viscosity can vary from 40 to 400 cSt @ 40 °C. Thus, requires an upgrading stage to obtain a compatible transport fuel. Upgraded algal HTL oils showed a TAN close to zero, density and viscosity roughly 0.77 g/mL and 4 cSt @ 40 °C, respectively [START_REF] Elliott | Process development for hydrothermal liquefaction of algae feedstocks in a continuous-flow reactor[END_REF][START_REF] Albrecht | Impact of heterotrophically stressed algae for biofuel production via hydrothermal liquefaction and catalytic hydrotreating in continuous-flow reactors[END_REF][START_REF] Jarvis | Assessment of Hydrotreatment for Hydrothermal Liquefaction Biocrudes from Sewage Sludge, Microalgae, and Pine Feedstocks[END_REF]. In 2016, Lopez Barreiro et al. [START_REF] Barreiro | Hydrothermal liquefaction (HTL) of microalgae for biofuel production: State of the art review and future prospects[END_REF] reviewed the literature on upgrading biocrude oils obtained by HTL of microalgae and concluded the need to improve processes, catalysts, and underlined the importance of algae species. Xu et al. [START_REF] Xu | Catalytic hydrothermal liquefaction of algae and upgrading of biocrude: A critical review[END_REF] and Djanda et al. [START_REF] Djandja | Progress in Hydrothermal Liquefaction of Algal Biomass and Hydrothermal Upgrading of the Subsequent Crude bio-oil: A Mini Review[END_REF] complemented this review by adding technico-economic studies which also suggest that algae composition strongly impacts the economics of the process. Catalytic upgrading is usually performed by using either hydrotreatment catalysts or acid catalysts active for catalytic cracking (or both functions in hydrocracking). However, sulfur and nitrogen compounds present in the biocrude require robust thio-and azo-resistant, and low-cost catalysts. Metal sulfide hydrotreating catalysts are often considered for industrial application in bio-oil hydroconversion. The most recent studies in this domain use either NiMo or NiW supported on alumina catalysts [START_REF] Rathsack | Analysis of hydrogenation products of biocrude obtained from hydrothermally liquefied algal biomass by comprehensive gas chromatography mass spectrometry[END_REF][START_REF] Guo | Hydrothermal liquefaction of Chlorella vulgaris and Nannochloropsis gaditana in a continuous stirred tank reactor and hydrotreating of biocrude by nickel catalysts[END_REF][START_REF] Fang | Hydrotreatment of model compounds with catalysts of NiW/Al2O3 and NiWP/Al2O3 to simulate low temperature coal tar oil[END_REF][START_REF] Haghighat | Hydrotreating of Hydrofaction TM biocrude in the presence of presulfided commercial catalysts[END_REF][START_REF] Haider | Catalytic hydrotreatment of microalgae biocrude from continuous hydrothermal liquefaction: Heteroatom removal and their distribution in distillation cuts[END_REF][START_REF] Haider | The Art of Smooth Continuous Hydroprocessing of Biocrudes Obtained from Hydrothermal Liquefaction: Hydrodemetallization and Propensity for Coke Formation[END_REF]. The scalability of HDT unit for HTL bio-oil upgrading was recently demonstrated at Pacific Northwest National Laboratory (PNNL), as well as two-stage upgrading [START_REF] Thorson | Scaleable Hydrotreating of HTL Biocrude to Produce Fuel Blendstocks[END_REF][START_REF] Haider | Two-stage catalytic hydrotreatment of highly nitrogenous biocrude from continuous hydrothermal liquefaction: A rational design of the stabilization stage[END_REF], and the feasibility of co-processing in FCC unit was also assessed [START_REF] Santillan-Jimenez | Co-processing of hydrothermal liquefaction algal bio-oil and petroleum feedstock to fuel-like hydrocarbons via fluid catalytic cracking[END_REF]. However, there is still a significant challenge in the growth and algae strain selection with an obvious target of high lipid contents since they will provide ideally long-chain alkanes after upgrading. However, the complexity of the microalgae bio-oils obtained by HTL makes difficult the understanding of their reactivity during upgrading.

Catalysts have various roles in the conversion of bio-oils [START_REF] Guilhaume | The role of catalysis in the valorization of woody biomass fast pyrolysis liquids: Overview and contribution of IRCELYON[END_REF]. Regarding the upgrading stage, the hydrogenolysis properties for removing oxygen from oxygenated compounds (mostly phenols) are similarly required for algae HTL oils. As compared with pyrolytic oils, algae biocrude contains different species. The presence of nitrogen-containing compounds in the algal bio-oils constitutes another challenge since an almost complete removal of nitrogen is required for a fuel application. This denitrogenation issue was underlined by Leng et al. [START_REF] Leng | Nitrogen in bio-oil produced from hydrothermal liquefaction of biomass: A review[END_REF]. Other specific components of algae feedstocks might be problematic. For instance, using FT-ICR-MS, Jarvis et al. [START_REF] Jarvis | Impact of iron porphyrin complexes when hydroprocessing algal HTL biocrude[END_REF] evidenced that iron porphyrins can remain after HTL, and like porphyrins in petroleum, it may cause catalyst deactivation. A model molecule study demonstrated that chlorophylle, which can represent up to 5 wt% of algae content, can release, during the HTL process, phytols that are further converted into several unsaturated compounds [START_REF] Changi | Reaction kinetics and pathways for phytol in hightemperature water[END_REF]. After upgrading, these molecules can also produce long-chain alkanes (C20). A detailed semi-quantitative analysis by comprehensive GC of the hydrogenation products of an algal biocrude was provided by Rathsack et al. [START_REF] Rathsack | Analysis of hydrogenation products of biocrude obtained from hydrothermally liquefied algal biomass by comprehensive gas chromatography mass spectrometry[END_REF]. These authors underlined the possibility of converting phenols but difficulties to further reduce nitrogen compounds such as indoles or anilines. In the present work, the reactivity of HTL micro-algal bio-oil over a NiW/Al2O3 sulfided catalyst will be investigated at different temperatures and characterized with several analytic tools such as 13 C-NMR, GPC-RID/DAD, Sim-Dis, and GCxGC-MS/FID. Thus, we will describe the behavior of compounds and families of compounds identified in the bio-oil and quantify them. Removal of heteroatomic compounds (O, N, and S), which can significantly affect the quality of the biofuel, is also considered. Careful attention is given to lipids and the evolution of long-chain compounds on different stages, from the initial microalga until final biofuel. Lastly, the transfer of the metal present in the microalga to the used hydrotreating catalysts will be also discussed.

Materials and methods

Microalgae production

The microalga strain consists of Chlorella sorokiniana NIES 2173 and was grown at MicroAlgae Processes Platform, CEA Cadarache, France. a Based on dry algae b Calculated using a nitrogen-to-protein conversion factor of 5.0 as reported by Templeton and Laurens [START_REF] Templeton | Nitrogen-to-protein conversion factors revisited for applications of microalgal biomass conversion to food, feed and fuel[END_REF].

The amino acids profile of a batch of microalgae grown under similar conditions is given in supplementary material Table S1.

Extraction and transesterification of lipids.

Lipids extraction from dry microalga was performed with a mixture of chloroform and methanol (volume ratio 2:1) in an automatic solvent extractor VELP Scientifica SRE 158, following the Bligh and Dyer method. As Ryckebosch et al. [START_REF] Ryckebosch | Optimization of an analytical procedure for extraction of lipids from microalgae[END_REF] mentioned, two extractions in series could be performed to ensure that all lipids are extracted, irrespective of the microalgae species. Therefore, after two extractions, 11 wt.% (dry-ash-free basis) of extract were recovered from nearly 8 g of dry microalga. The transesterification reaction was performed with a mixture of methanol, sulfuric acid, and THF at 60°C for 2h using a stirring of 500 rpm according to the procedure described by Nguyen et al. [START_REF] Nguyen | High biodiesel yield from wet microalgae paste via in-situ transesterification: Effect of reaction parameters towards the selectivity of fatty acid esters[END_REF]. This analysis aims to qualify and quantify carbon chains.

HTL bio-oil production

Hydrothermal liquefaction reaction was performed in a 300 mL Parr stainless steel reactor. The reactor was loaded with the microalga and water (20 wt.% -dry-ash-free basis) and purged with N2 to remove the air inside. Subsequently, 0.1 MPa of N2 pressure was applied, and the reactor was heated to 300 °C with a heating rate of approximately 10 °C/min. When the target temperature was reached, the stirring (370 rpm) was started, and the reaction was performed for 30 min. The reactor total pressure at 300°C was approximately 10 MPa. After the reaction, the biocrude and the aqueous phase were separated by filtration, and the reactor was washed with ethyl acetate to recover the residual biocrude. Then, all the biocrude was mixed with ethyl acetate and heated until 40 °C for 1h to favor the solubilization and extraction of the bio-oil.

The bio-oil and char were separated by vacuum filtration, and the solvent was removed using a rotary evaporator. This protocol was repeated ten times to produce enough bio-oil for upgrading experiments. The total bio-oil mass was approximately 50 g, and the average yield over the HTL reactions was 24% (dry-ash-free basis).

Catalytic hydrotreating (HDT) of HTL bio-oil

The hydrotreating step was performed using the same reactor than for the HTL experiments.

The commercial NiW/Al2O3 catalyst (WO3: 25.7 wt.%, NiO: 3.8 wt.%, specific surface area: 179 m 2 /g, and total pore volume: 0.40 cm 3 /g) was previously sulfided at 400°C for 3h under a 15% H2S/H2 flow (4 NL/h) [START_REF] Gachet | Optimization of the composition of Ni-W/A12O3 hydrotreating catalysts using model molecules and real feedstock conversion studies[END_REF] . Firstly, the reactor was loaded with 3.0 g of bio-oil, 0.6 g of pre-sulfided catalyst, and 25 g of n-heptane, which was used to enable an efficient agitation during the experiment. Then, the reactor was purged three times with N2 and was pressurized until 6 MPa for a leak test. Subsequently, the N2 was removed, and the reactor was purged three times with H2. At room temperature, 1 MPa of H2 was added, and the reactor was heated until the desired temperature. Three different upgrading temperatures were used to study the reactivity of the compounds in the HTL micro-algal bio-oil: 350, 375, and 400 °C. Once the desired temperature had been reached, 5 MPa of H2 was added to get a total pressure of 10 MPa at the reaction temperature. All experiments were performed for 5h. After that, the reactor was cooled down, and the catalyst and hydrotreated bio-oil were separated by filtration. The reactor was washed with n-heptane to remove the residual oil and catalyst. The n-heptane was later removed using a vacuum rotary evaporator, and the oil was characterized with different analytical techniques described after.

Characterization and analytical techniques 2.5.1. Thermogravimetric analysis (TGA)

Thermogravimetric analysis of dry microalga (2-7 mg) was carried out under air or N2 (60 mL/min) from 30 °C until 800 °C (10 °C/min) on a Mettler Toledo TGA/DSC 1 as described in the literature by Cheng et al. [START_REF] Cheng | Hydrothermal liquefaction of high-and low-lipid algae: Mass and energy balances[END_REF].

Metals, phosphorus, and sulfur content

The metals, phosphorous, and sulfur were firstly identified by X-ray fluorescence (XRF) analysis performed on a Panalytical Epsilon 4 instrument and, subsequently, the metals and phosphorus were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES) performed on an Activa (Jobin Yvon) apparatus.

Elemental analysis (CHONS)

The C, H, O, N, and S mass fractions were analyzed in a Thermo Scientific Flash 2000

Apparatus. The formed gas was analyzed via a TCD detector. For the analysis of low S and N concentrations in liquid products, ANTEK 9000NS apparatus, which is more precise (from low ppb until percent levels) and accurate, was also used. The bio-oil and HDT-oil were diluted using THF to a concentration of ≈ 5 wt.% and ≈ 10 wt.%, respectively, then injected at 1050 °C. The sample was burned, and S and N were converted into SO2 and NO2, respectively, and analyzed by two detectors in series. Two calibration curves, one with thiophene and another with pyridine, for S and N analysis, were made to convert the signal response into concentration (ppm).

The degree of deoxygenation (de-O), denitrogenation (de-N), and desulfurization (de-S) were calculated by the following equation (1):

de I % 1 x 100 (1)
where "I = O, N, and S", "de-I" is the degree of deoxygenation, denitrogenation, or desulfurization, "IHDT-oil", and "Ibio-oil" is the heteroatoms content of hydrotreated oil (HDT-oil) and bio-oil, respectively.

The higher heating value (HHV), equation ( 2), based on the elemental mass fraction, was estimated through the correlation proposed by Channiwala and Parikh [START_REF] Channiwala | A unified correlation for estimating HHV of solid, liquid and gaseous fuels[END_REF]:

HHV 0.3491C 1.1783H 0.1005S 0.1034O 0.0151N (2)

Gel permeation chromatography (GPC)

The GPC analysis was performed using an Agilent 1200 series HPLC equipped with two PLgel Columns (50 and 500 Å), a refractive index detector (RID), and a diode array detector (DAD).

The analysis was carried out at 35 °C using tetrahydrofuran (THF) as eluent with a 1 mL/min flow rate. The samples were diluted to a concentration of ≈1wt.% in THF and filtered (0.45 μm) before analysis. Two molecular weight (Mw) standards are available to convert the elution time into Mw: hydrocarbon mixture (Mw from 86 to 1000 g/mol) or polystyrene mixture (Mw from 162 to 55100 g/mol). The hydrocarbon mixture was used as a calibration curve to present more remarkable similarities with the analyzed samples and the chromatograms were normalized to the highest peak intensity. When the DAD detector was used, the signal was normalized per mass unit of sample to compare different samples.

Simulated distillation (Sim-Dis)

The boiling point distributions were determined utilizing Sim-Dis, according to the standard ASTM D2887 [START_REF]Standard Test Method for Boiling Range Distribution of Petroleum Fractions by Gas Chromatography[END_REF]. The analysis were performed on an Agilent 7890A series gas chromatograph fitted with an Agilent J&W GC column (dimensions 10 m × 0.53 mm × 2.65 µm P/N: CP7582) and two detectors: flame ionization detector (FID) and flame photometric detector (FPD Plus), specific for sulfur compounds. The instrument was calibrated with a reference gas oil sample (initial boiling point of 114 °C and final boiling point of 475 °C) and a calibration mix (mixture of C6-C44) following the standard method. The liquid products were defined by five boiling point ranges: gasoline (< 175 °C), jet-fuel (175-250 °C), diesel (250-350 °C), heavy gas oil (350-500 °C), and residual (> 500 °C).

2.5.6. 13 C nuclear magnetic resonance ( 13 C-NMR)

13

C-NMR spectra were recorded with a Bruker AVANCE 400 MHZ spectrometer and analyzed using the software TopSpin 3.0. The measurements were performed at room temperature with an accumulation of 4700 scans for approximately 15 h. The samples (60 mg) were diluted with deuterated chloroform (CDCl3).

Comprehensive two-dimensional gas chromatography (GCxGC)

Two GCxGC analytical systems have been used: a cryogenic modulator from Zoex Corporation (USA), described in the supplementary material (Table S2), connected to a mass spectrometry (MS) for the identification of compounds (up to C32), as reported elsewhere [START_REF] Toussaint | Comprehensive 2D chromatography with mass spectrometry: A powerful tool for following the hydrotreatment of a Straight Run Gas Oil[END_REF], and a flow modulator connected to an FID for quantification (up to C40). Modulation parameters were optimized according to Lelevic et al. [START_REF] Lelevic | Quantitative performance of forward fill/flush differential flow modulation for comprehensive two-dimensional gas chromatography[END_REF]. The same column set of reversed polarity, consisting of a polar column in the first dimension and apolar in the second dimension, were utilized for both systems. Such configuration allows a better separation of the families of polar compounds [START_REF] Omais | Investigating comprehensive two-dimensional gas chromatography conditions to optimize the separation of oxygenated compounds in a direct coal liquefaction middle distillate[END_REF][START_REF] Pu | Lignin catalytic hydroconversion in a semi-continuous reactor: An experimental study[END_REF][START_REF] Staš | Two-Dimensional Gas Chromatography Characterization of Pyrolysis Bio-oils: A Review[END_REF].

Both analytical tools were used to analyze the extracts from microalga, transesterified lipids, HTL bio-oil and HDT-oils obtained at various temperatures. The samples were diluted in THF with a concentration of ≈ 10wt.% and ≈ 5wt.% for MS and FID analysis, respectively. Besides, an internal standard, 3,3'-dimethylbiphenyl (3,3-DMBP), ≈ 200 ppm, was also added for quantification purposes. For better discrimination of double bonds position in the transesterified lipids, a specific column ZB-FAME (30 m x 0.25 mm x 0.20 µm) was used.

Quantification of compounds and families

A quantification of the compounds on the different steps, from extracts of initial microalga until HDT-oil upgraded at different temperatures, was performed to evaluate the evolution of lipids chains and conversion of refractory nitrogen and oxygen compounds present in the bio-oil.

Standard compounds such as hexadecane, oleamide, stearic acid, methyl stearate, and cholesterol were analyzed, and a calibration curve was done. However, due to the complexity of bio-oil, some standards could not be easily purchased or synthesized in the lab [START_REF] Guo | A review of bio-oil production from hydrothermal liquefaction of algae[END_REF].

Moreover, a calibration curve preparation could be time-consuming and costly. For the compounds that did not have a calibration curve, the relative response factor (RRF) for FID analysis could be estimated from their chemical structure. A methodology to predict the RRF was proposed by Sternberg et al. [START_REF] Sternberg | Mechanism of response of flame ionization detectors[END_REF], introducing the effective carbon number (ECN) concept.

They showed that the effect of heteroatoms on the ECN of a molecule depended on the functional group's nature and empirically quantified each functional group's contribution to the ECN. These values were also reported by Scanlon et al. [START_REF] Scanlon | Calculation of flame ionization detector relative response factors using the effective carbon number concept[END_REF] and used in this work to calculate the ECN value. The ECN value was calculated by equation ( 3), and the relationship between RRF and ECN is given by equation ( 4), as reported in [START_REF] De Saint Laumer | Quantification in gas chromatography: Prediction of flame ionization detector response factors from combustion enthalpies and molecular structures[END_REF]:

ECN ∑ N x Cnt (3)
where "N" is the number of atoms or functional groups in the compound i, and "Cnt" is the ECN contribution coefficient reported by Scanlon et al [START_REF] Scanlon | Calculation of flame ionization detector relative response factors using the effective carbon number concept[END_REF].

RRF

where "ECNi" is the effective carbon number of a compound or family i, "MWIS" is the molecular weight of the internal standard, "MWi" is the molecular weight of compound or family i, and "ECNIS" is the effective carbon number of internal standard.

Note that the theoretical contribution of nitrogen for ECN in cyclic compounds can be difficult to estimate. For this reason, the quantification of cyclic nitrogen compounds, such as pyrrole, indole, and carbazoles, was made considering the experimental ECN value reported by Jorgensen et al. [START_REF] Jorgensen | Prediction of Gas Chromatography Flame Ionization Detector Response Factors from Molecular Structures[END_REF].

Results and discussion

Algal composition

The thermogravimetric curves and their derivatives under N2 and air are given in supplementary material (Figure S1).

Lipids that can be extracted from dry microalga correspond to many compounds such as neutral lipids (triacylglycerol and free fatty acid), polar lipids (glycolipids and phospholipids), unsaponifiable matters, chlorophylls (and related metabolites), and others [START_REF] Yao | Microalgae lipid characterization[END_REF]. Therefore, liquid extraction with chloroform-methanol mixture led to a large distribution of compounds containing heavy and lighter compounds, as illustrated by the GPC analysis reported in Figure 1. Extracted lipids exhibit three main contributions, which can be attributed, respectively, from the highest to the lowest Mw, to triglycerides, diglycerides, and free fatty acids or other compounds. These assumptions are comforted by using tristearin and methyl stearate as references. The extracted lipids were converted into fatty acid methyl esters (Fame) during the transesterification reaction in the presence of methanol and sulfuric acid. The GPC curve obtained for the Fame was shifted to the lower masses and close to the methyl stearate reference, which can be associated with the presence of C18 methyl esters and also C16 methyl esters, as will be discussed further. GPC with hydrocarbon calibration is also helpful to indicate the fraction of compounds that can be observed by gas chromatography (typically below 450 g/mol), thus corresponding to the first contribution of GPC analysis. Figure S2(a) illustrates the nature of extracted lipids identified by GC×GC-FID/MS. The compounds with high Mw (diglycerides and triglycerides, per example), observed with GPC, cannot be analyzed with this GC system as they are too heavy.

We observed the C16 and C18 fatty acids as the main components in the extraction liquid, as well as the presence of methyl esters of these fatty acids. During the chloroform and methanol extraction, some free fatty acids might be converted into C16 and C18 methyl esters by transesterification reaction, as already reported by Biller et al. [START_REF] Biller | Catalytic hydrothermal processing of microalgae: Decomposition and upgrading of lipids[END_REF]. These authors mentioned that esterification reactions could occur under these moderate conditions, due to the presence of alkali salts that can act as a catalyst in the microalgae and are expected to esterify free fatty acids only. Phytosterols and phytol derivatives were also found in the extract. Figure S2(b) presents the Fame chromatogram after the transesterification reaction. As reported in Table 3, in the micro-algae, the lipids are composed basically of C16 and C18 chains. C16 chains are mainly saturated, while C18 chains are unsaturated, as reported by Qiu et al. [START_REF] Qiu | Effects of pH on cell growth, lipid production and CO2 addition of microalgae Chlorella sorokiniana[END_REF] for the same algae.

HTL micro-algal bio-oil composition

Hydrothermal liquefaction of microalgae involves many reactions. Hietala and Savage [START_REF] Hietala | A molecular, elemental, and multiphase kinetic model for the hydrothermal liquefaction of microalgae[END_REF] recently proposed a kinetic model based on 16 reaction pathways illustrating the complexity of families of compounds reacting together, leading to a viscous bio-oil. C, H, O, N, S atoms are redistributed between gas, liquid (aqueous and organic phase) and solid phases during the process.

The lightest compounds of the bio-oil obtained by HTL were characterized by GC×GC-MS/FID (Figure 2). Several families of compounds can be identified, similar to those listed by Torri et al. [START_REF] Torri | Hydrothermal treatment (HTT) of microalgae: Detailed molecular characterization of HTT oil in view of HTT mechanism elucidation[END_REF] and Madsen et al. [START_REF] Madsen | Characterizing Semivolatile Organic Compounds of Biocrude from Hydrothermal Liquefaction of Biomass[END_REF], such as C16 and C18 carboxylic acids, amides, and esters, phytosterols, cyclic nitrogen and oxygen compounds. The HTL yield was 24%, approximately two times higher than lipids content in microalga, indicating that carbohydrates and proteins were also converted into an organic phase during HTL reaction. This hypothesis is confirmed by GCxGC chromatogram, where compounds such as phenols, pyrroles, indoles, carbazoles, and its alkyl-substituted homologues formed from the decomposition of carbohydrates and proteins, for example by Maillard reaction can be found [START_REF] Changi | Hydrothermal Reactions of Biomolecules Relevant for Microalgae Liquefaction[END_REF]. Diketopiperazines (DKPs) that are coming from the degradation of proteins and their recombination were also detected in the bio-oil [START_REF] Toor | Hydrothermal liquefaction of biomass: A review of subcritical water technologies[END_REF]. These compounds would affect biofuel quality and must be removed during the hydrotreatment step. The C16 and C18 carboxylic acids formed from the hydrolysis of di-and tri-glycerides represent 41% of HTL bio-oil. Fatty amides were also formed obviously from the reaction between fatty acids and ammonia coming from proteins degradation. The products coming from the hydrothermal transformation of phytol, such as phytane and dihydrophytol, were also found. As reported by Changi et al. [START_REF] Changi | Reaction kinetics and pathways for phytol in hightemperature water[END_REF], chlorophyll is a complex molecule, usually formed by a hydrophilic chlorine ring and the hydrophobic ester-linked to the phytol side chain, which can be readily hydrolyzed to release a phytol and products during HTL conditions [START_REF] Rontani | Phytol degradation products as biogeochemical tracers in aquatic environments[END_REF].

A family of compounds with polarity close to C16 and C18 carboxylic acids and amides, but with a higher boiling point was also observed in the GCxGC-FID chromatogram. However, their identification was not possible with MS detector due to the fragmentation of the molecule and low probability using the NIST MS data base.

Hydroconversion of HTL bio-oil

In order to improve the quality of the organic phase, the HTL bio-oil was upgraded under hydrogen pressure at 350, 375, and 400 °C for 5h over sulfide NiW/Al2O3 catalyst. A section considering the transfer of the metal present in the microalga to the hydrotreating catalysts used is reported in the supplementary material.

The upgraded oil yield, deoxygenation, denitrogenation and desulfurization degree are reported in Figure 3 for the three temperatures. The yield in upgraded oil is around 70% at 350°C and decreases slightly to 65% at 375 and 58% at 400°C, which can be associated to cracking reactions, that form gases, as reported by Haider et al. [START_REF] Haider | Catalytic hydrotreatment of microalgae biocrude from continuous hydrothermal liquefaction: Heteroatom removal and their distribution in distillation cuts[END_REF], who worked with sulfide NiMo and under close operating conditions. High conversion of oxygenated compounds was already achieved (90%) at 350 °C, while barely 40% of nitrogenated compounds were converted.

However, after a hydroconversion performed at 400 °C, more than 90% of de-oxygenation, denitrogenation, and desulfurization was achieved, indicating that a higher temperature is required to convert nitrogen compounds. The elemental analysis of microalga, HTL, and HDT-oils at different temperatures is presented in Table 4. HDT-oil yield de-O de-N de-S upgrading step, which is indicative that hydrotreatment can be used to ameliorate the quality of HTL bio-oil from microalgae. Guo et al. [START_REF] Guo | Hydrothermal liquefaction of Chlorella vulgaris and Nannochloropsis gaditana in a continuous stirred tank reactor and hydrotreating of biocrude by nickel catalysts[END_REF] reported a C. vulgaris HTL bio-oil hydroconversion using close operating conditions and the same type of catalyst (sulfide NiW and NiMo). Although the HHV of microalga and HTL bio-oil were similar to the one reported in this work, the HHV of HDT-oil was lower, approximately 43 MJ/kg after upgrading at 400 °C. This difference in HHV can be associated with the high nitrogen content in the C. vulgaris bio-oil (6.9 wt.%), that is two times higher than the value reported in this paper for the bio-oil from C. sorokiniana, and that can inhibit the catalytic activity. The difference could also come from the effect of the supercritical state of n-heptane solvent, in used operating conditions.

According to Xu et al. [START_REF] Xu | Upgrading of Athabasca vacuum tower bottoms (VTB) in supercritical hydrocarbon solvents with activated carbon-supported metallic catalysts[END_REF], the use of supercritical fluid (SCF) can improve the diffusion rates in catalyst pore mouth and hence minimize the mass-transfer limitations and coke formation.

Besides, hydrocarbons in the supercritical state have the "hydrogen-shuttling" ability and enhanced solvent effects. Recently, Fang et al. [START_REF] Fang | Upgrading of palmitic acid over MOF catalysts in supercritical fluid of n-hexane[END_REF] showed that the catalyst's performance was superior in SCF of n-hexane than in the conventional media for the hydrogenation of hexadecanoic acid, which is, with octadecanoic acid, the main compounds in HTL bio-oil.

The broad distribution of compounds in the HTL bio-oil is illustrated by the distribution of molar masses given by the GPC analysis of the bio-oil (Figure 4). The prominent peak is close to 300 g/mol, attributed to the contribution of C16 and C18 carboxylic acids, which represent a considerable fraction of HTL bio-oil and will be discussed further with GCxGC-MS/FID analysis. The Mw was reduced when the upgrading temperature was increased, as reported in Table 5. Consequently, the distribution is shifted to the left. As written before, this Mw reduction can be associated with carboxylic acid and amides converting into alkanes and cracking reactions. Moreover, it was also possible to observe the conversion of heavy molecules. However, even after an upgrading step at 400 °C, molecules with Mw higher than C32, GCxGC-MS instrument limit, can be still found in the HDT-oils. This is in agreement with the studies of Jarvis et al. [START_REF] Jarvis | Assessment of Hydrotreatment for Hydrothermal Liquefaction Biocrudes from Sewage Sludge, Microalgae, and Pine Feedstocks[END_REF] and Zuber et al. [START_REF] Zuber | Analysis of Hydrogenation Products of Biocrude Obtained from Hydrothermally Liquefied Algal Biomass Using Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry[END_REF], which mentioned the presence of heavy molecules by FT-ICR-MS analysis after an upgrading step of algal bio-oil under severe reaction conditions. Molecular weight (g/mol in eq. HC)

HTL bio-oil HDT -350 °C HDT -375 °C HDT -400 °C DAD projection, the temperature increase during the upgrading step led to a shift of intensity for a higher retention time (Figure S3). The 13 C-NMR gives information about the functional group distribution in the HTL bio-oil and HDT-oils (Figure S4). Carboxylic groups are present in the HTL bio-oil and can be associated with the carboxylic acids formed by hydrolyses of di and triglycerides during the HTL reaction [START_REF] Biller | Catalytic hydrothermal processing of microalgae: Decomposition and upgrading of lipids[END_REF]. Then, these compounds were converted during the upgrading step, probably related to the significant decrease in oxygen content in the HDT-oils compared to the HTL bio-oil, as reported by elemental analysis. The carboxylic acids can be easily converted during the upgrading step under the reaction conditions employed, as reported by Toba et al. [START_REF] Toba | Hydrodeoxygenation of waste vegetable oil over sulfide catalysts[END_REF], which achieved approximately 100% of HDO of waste vegetable oil using the same type of sulfide catalyst at 300 °C. Moreover, the degree of aromaticity reduced when the temperature was increased as confirmed by GPC-DAD analysis and can be correlated to the H/C. The conversion of aromatics compounds during the hydrotreatment step is essential since these compounds have a negative effect on the diesel's cetane number.

Figure 6 shows the simulated distillation profiles of oils hydrotreated at different temperatures, and the fractional cuts are presented in Table 6. The hydrotreated oil from microalga falls primarily in the diesel range corresponding to approximately 60% of the biofuel, in agreement with Elliot et al. [START_REF] Elliott | Process development for hydrothermal liquefaction of algae feedstocks in a continuous-flow reactor[END_REF], who mentioned that about 80-85% of hydrotreated HTL bio-oil would be blendable into the diesel pool. Furthermore, within the diesel range, four distinct steps are present in the Sim-Dis curves, which are associated, respectively, with the n-alkanes C15, C16, C17, and C18, as will be discussed further. The increase in the upgrading temperature enhanced cracking reactions, promoting the formation of compounds with lower boiling points, such as jet-fuel, and favored the conversion of heavier compounds like heavy gas oil and residual. This tendency is corroborated by the GPC results, which showed an Mw reduction when the temperature was increased, in accordance with the works of Castelo et al. [START_REF] Castello | Catalytic upgrading of hydrothermal liquefaction biocrudes: Different challenges for different feedstocks[END_REF] and Biller et al.

[57], who reported similar results.

Gasoline

The GCxGC coupled to an MS and FID detector was used to identify and quantify different families presented in the hydrotreated oil (Figure 7). A family of aliphatic hydrocarbons formed by n-, iso-, and cycloalkanes from C8 until C37 was observed in agreement with Rathsack et al. [START_REF] Rathsack | Analysis of hydrogenation products of biocrude obtained from hydrothermally liquefied algal biomass by comprehensive gas chromatography mass spectrometry[END_REF], who reported hydrocarbons chains from C7 until C30. The longer hydrocarbons are presumably formed by a sequence of reactions beginning with a ketonization reaction between two fatty acids involving CO2 removal and leading to larger ketones. In the next step, ketones are hydrogenated toward hydrocarbons. C16 and C18 carboxylic acids were easily converted using the employed conditions, as observed by 13 C-NMR analysis, and corroborated by Castello et al. [START_REF] Castello | Catalytic upgrading of hydrothermal liquefaction biocrudes: Different challenges for different feedstocks[END_REF], who mentioned that this type of compound is relatively easy to upgrade into the corresponding paraffin, even at mild conditions. Mono, di, poly-aromatics, phytosterols and phenols, and cyclic nitrogen compounds, such as pyrroles, indoles, and carbazoles, were identified in the HDT bio-oil, and their concentration changed in function of the temperature employed during the experiment. C16 and C18 alcohols and nitriles were also found in the hydrotreated bio-oil obtained at 350 °C. As reported by Senol et al. [START_REF] Şenol | Reactions of methyl heptanoate hydrodeoxygenation on sulphided catalysts[END_REF] with a model molecule, the carboxylic acid can be hydrogenated to aldehyde, subsequently to alcohol, and finally to alkane. The fatty amides can be rapidly hydrogenated to nitriles and then converted to imine, amine, and alkane, as reported by Pongsiriyakul et al. [START_REF] Pongsiriyakul | Effective Cu / Re promoted Nisupported γ-Al2O3 catalyst for upgrading algae bio-crude oil produced by hydrothermal liquefaction[END_REF], over NiRe catalyst. It is worth emphasizing that no imine or amine were found during our experiments, suggesting that maybe those intermediates are rapidly converted or not detected with our GC system. Therefore, a higher temperature (≥ 375 °C) was required for the total conversion of C16 and C18 alcohols and nitriles. Although nitriles were not found in the experiments at 375 °C and 400 °C, fatty amides are still present after upgrading even if higher temperatures are employed. The 3D GCxGC-FID/MS chromatograms of bio-oil and HDT-oils that illustrate better the concentration of the various families are shown in the supplementary material (Figure S5). During the hydrotreatment step, the C16 and C18 carboxylic acids can be converted mainly via two pathways: decarboxylation/decarbonylation (DCO), which implies the loss of carbon by eliminating CO or CO2 in the gas phase and forming C15 and C17 n-alkanes in the liquid phase, or hydrogenation (HDO), which preserves the number of carbon leading to C16 and C18 nalkanes by removing water, but consumes more hydrogen [START_REF] De Brimont | Deoxygenation mechanisms on Ni-promoted MoS 2 bulk catalysts: A combined experimental and theoretical study[END_REF][START_REF] Snåre | Heterogeneous catalytic deoxygenation of stearic acid for production of biodiesel[END_REF]. Effectively, the main nalkanes in the hydrotreated oil were C15, C16, C17, and C18 (Figure 8), in accordance with previous works [START_REF] Rathsack | Analysis of hydrogenation products of biocrude obtained from hydrothermally liquefied algal biomass by comprehensive gas chromatography mass spectrometry[END_REF][START_REF] Castello | Catalytic upgrading of hydrothermal liquefaction biocrudes: Different challenges for different feedstocks[END_REF]. These results are also consistent with the Sim-Dis analyses that showed that approximately 60% of biofuel from microalga is in the diesel range (Figure 6).

Temperature increases during the hydrotreatment step promoted cracking reactions, leading to the formation of lighter n-alkanes (bellow C14) and the reduction of C16 and C18 n-alkanes (also reported in Figure 8). This trend can be explained by the fact that increasing temperature favors the decarbonation pathway, as reported by Arora et al. [START_REF] Arora | Kinetic study of hydrodeoxygenation of stearic acid as model compound for renewable oils[END_REF]. This is also confirmed by the C16/C15 and C18/C17 n-alkanes ratio that decreases slightly from 350°C to 375°C and more strongly after 375°C (Figure 9). Therefore, it is possible to vary the distribution of nalkanes by changing the operating conditions. On metal sulfide catalysts, phenol can react by two routes: direct deoxygenation route (DDO) forming benzene, or by a consecutive route including hydrogenation (HYD) with sequent elimination of oxygen, to produce cyclohexene which can be further hydrogenated to cyclohexane or converted to methylcyclopentane, as shown by Bui et al. [START_REF] Bui | Hydrodeoxygenation of guaiacol with CoMo catalysts. Part I: Promoting effect of cobalt on HDO selectivity and activity[END_REF]. These compounds were fully converted at 400 °C, which agrees with Rathsack et al. [START_REF] Rathsack | Analysis of hydrogenation products of biocrude obtained from hydrothermally liquefied algal biomass by comprehensive gas chromatography mass spectrometry[END_REF]. These authors reported 2% of phenols after an upgrading step performed at 340 °C, and this value decreased to 0.1 when a temperature of 400 °C was employed. Concerning the hydrodeoxygenation of phytosterols, our results showed a similar tendency proposed by Anthonykutty et al. [START_REF] Anthonykutty | Catalytic upgrading of crude tall oil into a paraffin-rich liquid[END_REF]. Sterane is the main product of this reaction and, like phenolics, more severe operating conditions are required for the deoxygenation of all phytosterols. Although more than 90% denitrogenation degree was achieved at 400 °C, some nitrogen compounds remain in the upgraded bio-oil. To better discuss the refractory compounds, the concentration of different nitrogen families was evaluated and reported in Figure 12. Diketopiperazines present in the algal bio-oil were not detected anymore in the upgraded bio-oil, indicating that these compounds were fully converted. As mentioned before, five-membered N-ring such as pyrroles, indoles, carbazoles, and their alkyl-substituted homologues were identified in the algal bio-oil. Usually, the HDN reaction of these types of compounds can proceed by hydrogenation of the aromatic ring to which the amino group is attached, followed by C-N bond scission [START_REF] Abu | HDN and HDS of model compounds and light gas oil derived from Athabasca bitumen using supported metal phosphide catalysts[END_REF][START_REF] Nguyen | Indole hydrodenitrogenation over alumina and silica-Alumina-supported sulfide catalystscomparison with quinoline[END_REF]. Possible HDN products of N-containing heterocycles are shown in Figure 11.

According to Stern [START_REF] Stern | Reaction networks in catalytic hydrodenitrogenation[END_REF], the conversion rates decrease as the five-membered ring of pyrroles is increasingly substituted; carbazoles being less reactive than indole and pyrrole, respectively, which is in agreement with the conversion range obtained for these families. Pyrroles and indoles concentration were reduced when a higher temperature was employed and they were almost entirely converted at 400 °C. However, carbazoles content was approximately the same when an upgrading step was performed at 350 °C and 375 °C and started to be converted just when more severe conditions (400°C) were employed. Surprisingly, fatty amides were found in the hydrotreated bio-oil at 400 °C. These results contrast to the one reported by Rathsack et al. [START_REF] Rathsack | Analysis of hydrogenation products of biocrude obtained from hydrothermally liquefied algal biomass by comprehensive gas chromatography mass spectrometry[END_REF], who mentioned an almost total conversion of fatty amides at 380 °C. However, they used a much higher hydrogen pressure (249 bar) and found a low concentration of carbazoles in their upgraded oil.

In contrast to oxygenated and nitrogenated compounds, the concentration of mono-aromatics and iso-and cyclo-alkanes increased with temperature (Figure 11). As mentioned, cracking products were also observed. According to the literature, on acidic catalysts such as zeolites, zirconia, and tungsten oxide supported on zirconia, n-alkanes first isomerize to mono-branched isomers, then into di-branched isomers, and subsequently are hydrocracked into smaller chains [START_REF] Akhmedov | Recent advances and future aspects in the selective isomerization of high n-alkanes[END_REF]. The increase of iso-alkanes concentration can be attributed to the operating conditions (rising temperature) enabling cracking reactions. Moreover, cyclo-alkanes and aromatic compounds, such as decylcyclohexane and 2-phenylundecane, were also identified in the upgraded bio-oil chromatogram. According to Snare et al. [START_REF] Snåre | Heterogeneous catalytic deoxygenation of stearic acid for production of biodiesel[END_REF], alkane chains can be cyclized and dehydrogenated to aromatics. Therefore, our results suggest that these reactions were promoted by temperature since the concentration of cyclo-alkanes and mono-aromatics increased (the latter compounds being also obtained by hydrogenation of polyaromatics).

Lastly, HDO and HDN products and the conversion of heavy cyclic molecules into smaller compounds could also contribute to the increase of mono-aromatics concentration. The sulfur level in the HTL bio-oil (0.3 wt.% in this study) is much higher than the values required by current environmental legislation for fuels [START_REF] Srivastava | An evaluation of desulfurization technologies for sulfur removal from liquid fuels[END_REF]. These compounds are primarily derived from sulfur-containing amino acids, such as cysteine and methionine. They can be removed during the upgrading step by hydrodesulfurization reaction (HDS), forming sulfurfree compounds and hydrogen disulfide. The literature related to sulfur in HTL micro-algal biooil remains scarce and requires deep exploration. Duan and Savage [START_REF] Duan | Upgrading of crude algal bio-oil in supercritical water[END_REF] mentioned that the only sulfur-containing compound detected in the algal bio-oil, 1-methyl-2-piperidinethione, was removed after the upgrading process. No S compounds were identified during our analysis of bio-oil and hydrotreated oil with GCxGC-MS. Therefore, the samples were analyzed with a GC coupled with an FPD Plus detector, which is highly sensitive for sulfur compounds. Reference sulfur-bearing molecules, generally found in petroleum, like thiophene (T), benzothiophene (BT), dibenzothiophene (DBT), and 4,6-dimethyldibenzothiophene (4,6-DMDBT), were injected to verify their retention time and enable a better identification. Figure 13(a) shows the sulfur speciation in HTL algal bio-oil and Figure 13(b) in the hydrotreated bio-oils. To estimate the volatility of sulfur compounds, the boiling point curve of n-paraffins, from C7 until C44 obtained with the FID detector, was also included in Figure 13. We observed that the sulfur compounds in initial and hydrotreated bio-oils are numerous and probably of various nature, as they are eluted between 2 and 15 min. Once the concentration of initial and hydrotreated oil samples is similar, we see that the intensity of the sulfur signal reduces after the upgrading step, indicating that part of the sulfur compounds was removed, as verified by ANTEK analysis.

Figure 1 .

 1 Figure 1. GPC-RID analysis (in HC calibration) of extracted lipids, Fame, and reference compounds (tristearin and methyl stearate).

Figure 2 .

 2 Figure 2. Comprehensive GCxGC-FID/MS chromatogram of HTL bio-oil.

Figure 3 .

 3 Figure 3. HDT-oil yield, degree of deoxygenation (de-O), denitrogenation (de-N), and desulfurization (de-S) in the function of temperature.

  As expected, the H/C increased in the function of temperature due to the promotion of hydrogenation reaction. On the other hand, O/C and N/C decreased due to the conversion of oxygenated and nitrogenated compounds. The HHV was significantly improved after yield, de-O, de-N, and de-S (%) Temperature (°C)

Figure 4 .Table 5 .

 45 Figure 4. GPC-RID analysis of the HTL bio-oil and HDT-oil at various temperatures.

Figure 5 .

 5 Figure 5. 3D projection of GPC-DAD signals for (a) HTL bio-oil and (b) HDT-oil at 400 °C.

Figure 6 .

 6 Figure 6. Simulated distillation profiles of oils hydrotreated at different temperatures.

Figure 7 .

 7 Figure 7. Comprehensive GCxGC-FID/MS chromatogram of HDT-oil at 350 °C.

Figure 8 .

 8 Figure 8. n-Alkanes distribution of bio-oil hydrotreated at different temperatures.

Figure 9 .Figure 10 .

 910 Figure 9. HDO/DCO ratio in function of upgrading temperature.

Figure 11 .

 11 Figure 11. Illustration of the of HDO or HDN reactivity of the various families of compounds.

Figure 12 .

 12 Figure 12. Nitrogen compounds concentration after hydrotreatment step at different temperatures.

Furthermore, comparing the

  obtained signal with the retention time of sulfur model molecules, it can be concluded that sulfur compounds heavier than 4,6-DMDBT, with boiling point (bp) close to 500 °C as benzo[b]naphtho[1,2-d]thiophene (bp: 434 °C) and dinaphtho[1,2-b:2',1'd]thiophene (bp: 523 °C), are present in the bio-oil. These are the most refractory molecules, as their signal is more intense than the signal of light sulfur compounds, and it remains intense even after upgrading step (Figure 13(b)). These observations shed light on the fact that these molecules are difficult to detect by GCxGC-MS due to their diversity, low concentration, and high molecular weight.

Figure 13 .

 13 Figure 13. N-paraffin's boiling point curve obtained from FID detector and sulfur compounds signal obtained from FPD Plus detector: (a) HTL micro-algal bio-oil and (b) HDT-oil.

Table 1 .

 1 Chlorella sorokiniana feedstock analysis.

	Component	Content (%)
	Moisture	41.2
	Ash a	2.4
	Carbohydrates a	31.4
	Lipids a	13.3
	Proteins a,b	16.5

Table 2

 2

. It is worth emphasizing that the Mw values calculated with the hydrocarbon calibration present a deviation of 7% for methyl stearate (Mw = 298.5 g/mol) and 26% for tristearin (Mw = 841.5 g/mol). Although this error is not negligible, the average Mw values are reported to compare different samples.

Table 2 .

 2 Molecular weight for extracted lipids, fame, and reference compounds (tristearin and methyl stearate) using RID detector (in HC equivalent).

	Sample

Weight average mass Mw (g/mol)

		100			Tristearin
					Extracted
	Signal RID normalized (%)	40 60 80			lipids Methyl stearate Fame
		20
		0
		200	400	600	800	1000
			Molecular weight (g/mol in eq. HC)
		Tristearin			659
	Extracted lipids			504
	Methyl stearate			279
		Fame			250

Table 3 .

 3 Fatty acid profiles of microalga Chlorella sorokiniana (%).

		C16:0 C16:1 C16:2 C16:3 C18:0 C18:1 C18:2 C18:3
	Fatty acids (%)	26.9	7.2	7.1	2.8	1.8	12.4	31.2	5.0

Table 4 .

 4 Elemental analysis of microalga, HTL, and HDT-oils at different temperatures.

	Sample	C	H	O (wt.% or ppm*)	N	S	H/C	O/C (Atomic ratio)	N/C	HHV (MJ/kg)
	Microalga	50.4	6.8	33.6	3.2	0.2	1.61	0.50	0.05	22.5
	HTL bio-oil	74.5	9.4	11.6	3.3	0.3	1.52	0.12	0.04	35.9
	HDT -350 °C	83.5	13.7	1.3	1.9	613*	1.97	0.01	0.02	45.2
	HDT -375 °C	83.8	14.0	0.7	1.2	337*	2.00	0.01	0.01	45.6
	HDT -400 °C	84.4	14.4	0.5	0.2	61*	2.05	0.00	0.00	46.4

Table 6 .

 6 Fractional cuts obtained from the simulated distillation of HDT-oil at different temperatures.

		100
		< 175	°C	Jet-fuel 175-250 °C	Diesel 250-350	°C	Heavy gas oil 350-500 °C
	Sample recovered (wt.%)	40 60 80
		20					HDT -350 °C
							HDT -375 °C
		0					HDT -400	°C
		100 150 200 250 300 350 400 450 500
				Boiling Point (°C)
		Gasoline	Jet-fuel		Diesel	Heavy gas oil	Residual
	Sample	< 175 °C	175-250 °C	250-350 °C	350-500 °C	> 500 °C
		(wt.%)	(wt.%)		(wt.%)	(wt.%)	(wt.%)
	HDT -350 °C	1		7			59	21	12
	HDT -375 °C	2		9			61	20	8
	HDT -400 °C	4		17			66	12	1

Mis en forme : Anglais (États-Unis)Mis en forme : Anglais (États-Unis) Mis en forme : Anglais (États-Unis)

Acknowledgments

This project has received funding from ANR for the project RAFBIOALG 18-CE43-009.

We thank our partners from CEA-CTReg, group GB3G, Adriana Ramirez Romero, and Florian Delrue for providing algae samples.

Appendix A. Supplementary material

Supplementary data associated with this article can be found in the online version at

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.


Keywords: Deep neck infection, Intensive care unit, Airway management, Weaning of mechanical ventilation

Airway management in patients with severe deep neck infections: from intubation to ventilator weaning in ICU Background: Deep neck infections (DNI) cause swelling and obstruction of the airway, which can make securing the airway challenging. We aimed to describe the various airway management techniques used in this population, identify risk factors for failed intubation, and describe strategies for mechanical ventilation weaning.

Method: This retrospective study analyzed all adult patients who underwent surgical treatment for DNI and required invasive mechanical ventilation for more than 24 hours in the Intensive Care Unit (ICU) of two centers, over seven years.

Results: Among the 91 analyzed patients, 61% were male, with a median age of 52 years and a median SAPS score of 29 on admission. Intubation was performed using direct laryngoscopy (DL) (36%), awake fiberscopy (AF) (28.9%), or video-laryngoscopy (VL) (30%). The choice of intubation technique varied between preoperative evaluation of the mouth opening, centers and changed over time. The rate of first-pass intubation failure was 22% with more desaturation, and no predictive factors for failure could be identified. In ICU, factors associated with extubation failure were pharyngeal or tonsillar origin (p=0.003), mediastinitis (p=0.02), number of surgical revisions (p<0.001) and septic shock (p=0.013). Patients who experience extubation failure have an increased risk of complications, longer mechanical ventilation, and length of stay in the ICU.

Conclusion:

With awake fiberscopy, videolaryngoscopy could be another appropriate technique to control the airway in patients with severe deep neck infection. Clinical and radiological findings are not reliable predictors of intubation failure on the first attempt.

As such, decision-making criteria for airway management should focus on factors such as mouth opening, the expertise of the anesthesiologist, and equipment preference.

REMERCIEMENTS

• Jury : A Madame le Professeur Claire Roger, je vous remercie de me faire l'honneur de présider ce jury de thèse. Votre parcours professionnel, votre implication dans l'encadrement de notre formation, votre bienveillance mais aussi votre gestion humaine des équipes font de vous un modèle pour notre génération d'internes. Soyez assurée de ma reconnaissance et de mon admiration.

A Monsieur le Professeur Pierre-François PERRIGAULT, je vous remercie de votre présence dans ce jury. Dans une réanimation de plus en plus technique, vous n'en oubliez pas l'homme derrière ces machines. Cette combinaison technique et gestion de l'être humain a marqué ma formation. Veuillez trouver ici l'expression de mon profond respect.

A Monsieur le Docteur Valentin Favier, merci de faire partie de ce jury de thèse.

Merci aussi d'avoir pu apporter votre expertise et vos conseils lors de l'élaboration de ce travail et cela avec une grande réactivité. Soyez assuré de ma considération et de ma reconnaissance.

A Madame le Docteur Flora Djanikian, merci de m'avoir encadré pour ce travail de thèse. Merci pour ton accessibilité, ta réactivité sur mes nombreuses interrogations, tes relectures multiples, ta rigueur dans le travail et surtout ta bienveillance tout au long de ce projet. C'est justement pour ces deux dernières raisons que je t'ai demandé de diriger mon travail de thèse il y a maintenant 2 ans. Je t'exprime ma profonde reconnaissance et ma profonde gratitude.

• Aux intervenants ayant contribués à ce travail :

A Monsieur le Docteur Kevin Chalard, merci pour les conseils et le temps consacré à la relecture de ce travail.

A Océane Garnier, une aide précieuse tout au long du projet de thèse, merci de m'avoir partagé ton expérience de la recherche clinique, merci pour les heures passées à trouver les nombreuses incohérences dans mon tableur.

A Maha, pour ton aide précieuse avec la relecture des imageries, et surtout ta gentillesse. En effet tu as toujours su garder ton calme malgré mes nombreuses relances. En espérant avoir la chance de travailler ensemble dans nos carrières futures. [START_REF] Celakovsky | Deep neck infections: risk factors for mediastinal extension[END_REF][START_REF] Chow | Life-threatening infections of the head and neck[END_REF].

Airway management in patients with

In addition to antibiotic therapy, surgical drainage of the collections under general anesthesia is required. However, the oedema can modify anatomical relationships, reduce cervical mobility and limit mouth opening, increasing ventilation and intubation difficulties [START_REF] Karkos | Challenging airways in deep neck space infections[END_REF]. Therefore, securing the airway remains the main goal for the anesthesiologist but predicting difficult intubation and choosing the best device is challenging because clinical or radiological predictors of failed intubation are not well defined in this specific population.

The management of upper airways varies between centers and over time.

Conventional endotracheal intubation using direct laryngoscopy (DL) may lead to acute airway collapse. For a long time, tracheotomy using local anesthesia was the gold standard but the invasiveness and potential long-term complications including swallowing disorders and tracheal stenosis led to the use of alternatives techniques [START_REF] Sethi | Deep neck abscesseschanging trends[END_REF][START_REF] Hansel | Videolaryngoscopy versus direct laryngoscopy for adults undergoing tracheal intubation[END_REF]. Awake fiberscopy (AF) and, more recently, videolaryngoscopy (VL) showed improved success rates on the first intubation attempt, particularly in known or predicted difficult airways but have not been evaluated in DNI population.

In the most severe patients representing 7% to 50% of cervico-facial cellulitis cases, airway oedema also compromises extubation in the recovery room [START_REF] Arulkumaran | Videolaryngoscopy versus direct laryngoscopy for emergency orotracheal intubation outside the operating room: a systematic review and meta-analysis[END_REF][START_REF] Cho | Airway management in patients with deep neck infections[END_REF]. In these patients, physicians face a difficult choice between early tracheotomy or delayed extubation attempt. By selecting the first option, the length of stay in the intensive care unit (ICU) could be reduced and the ventilator-associated pneumonia (VAP) prevented [START_REF] Tapiovaara | Comparison of intubation and tracheotomy in patients with deep neck infection[END_REF][START_REF] Chen | Factors Affecting the Necessity of Tracheostomy in Patients with Deep Neck Infection[END_REF][START_REF] Potter | Tracheotomy Versus Endotracheal Intubation for Airway Management in Deep Neck Space Infections[END_REF]. By choosing the second one, long-term complications due to tracheotomy do not occur, but the risk of extubation failure and prolonged mechanical ventilation are significant [START_REF] Degouy | Risk factors for difficult ventilatory weaning in intensive care patients with cervical cellulitis[END_REF].

The primary objective of this study is to describe the airway management techniques used in patients with DNI requiring mechanical ventilation for more than 24 hours. The secondary objectives are to identify the risk factors for intubation failure on the first attempt, to describe mechanical ventilation weaning strategies, and to retrieve risk factors for extubation failure.

MATERIALS AND METHODS

Study population

This retrospective study was conducted at two university hospital centers in Montpellier and Nîmes, France, between January 2015 and December 2021. All adult patients admitted in the ICU for community DNI and required mechanical ventilation for more than 24 hours were included. Patients who were minors, adults under legal protection, non-French speaking and those who expressed opposition to the study were excluded.

Ethics and consent

This retrospective research has been approved by the local Institutional Review Board of the University Hospital Center of Montpellier (IRB-MTP_2022_06_202201141).

An information note was sent to every patient before data collection and they were allowed to refuse their study participation, according to French law [START_REF] Toulouse | French legal approach to patient consent in clinical research[END_REF].

Data Collection

We collected data from patients' medical files. Demographics, medical history, the onset time of symptoms, the use of antibiotics or anti-inflammatory drugs (nonsteroidal anti-inflammatory drugs or corticosteroids) and relevant gravity score as American Society of Anesthesiologists status (ASA) and Simplified Acute Physiology Score (SAPS II) were described. Subjective symptoms, finding on clinical examination and biological results (CRP levels, WBC count) at admission in the emergency department were recorded.

A cervical and upper thoracic computer tomography (CT) was performed in all patients before surgery. The DNI etiology, the infection spreading along the cervical spaces, the relationship between DNI and the airway (with obstruction being classified as mild to moderate if less than 50% and severe if more than 50%) and the presence of mediastinitis were described [START_REF] Cassagneau | Exploration radiologique des infections cervicofaciales[END_REF] .

Airway management data specified the first intubation technique used as well as the last successful technique. The intubation failure at the first attempt was defined as failure to achieve tracheal intubation on a single laryngoscope or nasofiberscope insertion. For the analysis, patients were separated into 3 groups according to the technique used to secure the airway:

-direct laryngoscopy (DL) included direct Macintosh laryngoscopy with or without bougie use.

-videolaryngoscopy (VL) included all types of VL available, with or without bougie use.

-awake fiberscopy (AF) for nasotracheal intubation.

Complications related to intubation procedure were defined by oxygen desaturation less than 90%, severe oxygen desaturation less than 80% or cardiac arrest occurrence.

In the ICU, the weaning strategy was described with the time to first extubation attempt, the use of pre-extubation corticotherapy and the result of cuff leak test.

Extubation failure was defined as the need for reintubation or death within 48 hours following extubation. Finally, information related to surgical revision, occurrence of ICU complications, duration of mechanical ventilation, lengths of stay and mortality were collected. Ventilator-associated pneumonia was defined thanks to the Clinical Pulmonary Infection Score (CPIS) criteria.

Patient management

After clinical examination by an otorhinologist, all patients were intubated for surgical drainage of the cervico-facial collections. Intubation was performed by an anesthesiologist in either the operating room or ICU. The choice of the technique was at the discretion of the anesthesiologist according to the available device at the given time.

In the ICU, the strategy of ventilation weaning between an early tracheotomy within seven days or an extubation attempt was determined by the intensive care team in consultation with the surgeon in each centers.

Statistical Analysis

After data collection, we used descriptive statistical analyses to summarize patients' characteristics. Quantitative variables were expressed as medians [25th-75th percentiles] and compared using Kruskal-Wallis test. Qualitative variables were expressed as numbers (%) and the absolute numbers were compared using the chisquared test or the Fisher's exact test as appropriate. The statistical analysis was performed by the medical statistics department of the Montpellier University Hospital thanks to RStudio version 4.2.1.

RESULTS

Patients' characteristics

The flow chart of the study is shown in Figure 1. During the inclusion period, 94 patients were treated surgically and admitted in ICU for deep neck infection requiring more than 24 hours of mechanical ventilation. The patients' characteristics and clinical details of the 91 patients analysed are summarized in Table 1. Risk factor analyses for intubation and extubation success were each performed on 90 patients. Subjects' median age was 52 years old, male represented 61% of cases. Patients had few comorbidities (48% of them had an ASA score equal to 1) and the median SAPS II severity score was 29.

Pharyngo-tonsillar and odontological origins were the two main etiologies, with respectively 40% and 37% of DNI. A third of subjects were concerned by more than one collection and a quarter of them had mediastinal dissemination. The most frequent symptoms observed upon admission to the emergency department were related to obstruction of the oro-tracheal pathway. Indeed, 66% of patients presented limited mouth opening, 68% had dysphagia, 22% dysphonia and 31% of them were affected by dyspnea. Only 8% of DNI were complicated with septic shock.

First intubation technique used

Intubation was performed in 92% of cases in the operating room (OR) and 7% in the emergency room of the ICU. Three techniques for first-line approach were used in broadly similar proportions, with 36% using DL, 30% using VL and 28.9% using AF. The choice of the intubation technique was influenced by several factors presented in Table 2. Firstly, the choice changed between centers, with indirect techniques being more frequently used at center 1, while direct techniques are preferred at center 2 (27% versus 66% of DL used respectively in first intention). Moreover, we observe a variation in the techniques used over time, with an increasing use of the VL in the last years (Figure 2).

In addition, the choice of intubation technique depends on the preoperative evaluation of the oro-tracheal route. Indirect techniques were more commonly used in patients with dysphagia (VL and AF in 70% and 86% respectively p=0.05). Limited mouth opening was also a significant factor in the choice of intubation technique. AF was used preferentially for patients with mouth opening less than 20mm (61.54%) and DL for patients with mouth opening greater than 35mm (57.6%). The etiology and the findings in the preoperative CTscan did not seem to influence the choice of intubation technique.

. The univariate analysis of patients' characteristics and procedure description according to the success or failure at the first-pass intubation is presented in

Direct

Ventilator weaning strategy

The weaning of mechanical ventilation in ICU involved early tracheotomy performed within seven days for 14 patients (16%) and attempting extubation for 76 patients (84%), with a secondary tracheotomy in case of failure of extubation (3/6 patients, see Figure 1). We found no demographic characteristics or clinico-radiological factors associated with the choice of ventilator weaning strategy. However, this choice differed between centers with respectively 5% and 44% of early tracheotomy in center 1 and 2 (p<0.001).

Among the 76 patients who underwent an extubation attempt, the failure rate concerned 6 patients (8%). All these patients were reintubated with success at the first attempt, without major complication. Factors significantly associated with extubation failure described in particularly notice that neither classical clinical characteristics, nor the etiology, nor the radiological airway obstruction permit to predict the difficulty of intubation in our cohort.

These observations are in line with the literature. Indeed, Cho et al. suggests that even if an infection involves multiple spaces and is large, it may still not complicate airway management (9) .

Our bicentric cohort description proves the major role of the center in the choice of weaning strategy [START_REF] Chen | Factors Affecting the Necessity of Tracheostomy in Patients with Deep Neck Infection[END_REF][START_REF] Degouy | Risk factors for difficult ventilatory weaning in intensive care patients with cervical cellulitis[END_REF]. Fifteen percent of the patients were early tracheotomised but these proportions were highly different between the two centers. Early tracheotomy could probably decrease ICU stay duration, or mechanical ventilation duration like in studies of Potter's or Tapiovaara [START_REF] Tapiovaara | Comparison of intubation and tracheotomy in patients with deep neck infection[END_REF][START_REF] Potter | Tracheotomy Versus Endotracheal Intubation for Airway Management in Deep Neck Space Infections[END_REF]. But this technique also conveys substantial risk of infection spread to the mediastinum, and tracheal stenosis as a late complication. The comparison between these two strategies could be interesting but is not permitted in our retrospective study.

Eight percent of patients experienced extubation failure, a rate comparable to that generally observed in critically ill patients [START_REF] Epstein | Effect of failed extubation on the outcome of mechanical ventilation[END_REF][START_REF] Seymour | The outcome of extubation failure in a community hospital intensive care unit: a cohort study[END_REF]. All patients were easy to reintubated,

CONCLUSION

With awake fiberscopy, videolaryngoscopy could be another appropriate technique to control the airway in patients with severe deep neck infection. The rate of failed intubation on the first attempt in these patients is high. Neither clinical nor radiological findings can predict intubation failure at the first-pass. Regarding weaning from mechanical ventilation in ICU, failed extubation is rare but associated with prolonged hospital stay. Further studies are needed to evaluate which airway management method would be optimal. We suggest the mouth opening, the anaesthesiologist expertise and equipment preference as main decision-making criteria.

  American Society of Anesthesiologists status -BMI = Body Mass index -CI = Confidence Intervals -CPIS = Clinical Pulmonary Infection Score -CRP = C reactive protein -CT = Computed Tomography -DNI = Deep Neck infection -DL = Direct laryngoscopy -ICU = Intensive care unit -SAPS II = Simplified Acute Physiology Score -VAP = Ventilator-associated pneumonia -VL = Video Laryngoscopy -WBC = White blood cell INTRODUCTION Deep neck infection (DNI) is a serious bacterial disease that can originate from odontogenic or pharyngo-tonsillitis sources (1,2). The infection can spread along the potential spaces and fascial planes of the neck, causing upper airway oedema, mediastinitis by caudal propagation or septic shock. Due to the rapid worsening of airway obstruction, early diagnosis of DNI is essential

Figure 2 :

 2 Figure 2: Intubation techniques used over years.

Figure 3 :

 3 Figure 3: Sankey diagram depicting the relationship between the first and last intubation techniques used.

  without any serious complications. No patients required emergency rescue tracheotomy after extubation failure. Extubation failure did not lead to immediate complications but were associated with a significantly bad outcome with longer ICU and hospital stay. The initial difficulty of intubation does not seem to be directly linked to extubation failure in our population. The significant predictive factors of extubation failure shown by our study are in connection with etiology and severity of the DNI. Indeed, 83% of the patients requiring reintubation had DNI from pharyngeal or tonsillar origin, versus 30 % in the group extubation success. The association in univariate analysis between mediastinitis, the numbers of surgical revisions, VAP, septic shock and the extubation failure suggest causality between severity of the cervicofacial damage and extubation failure. This should be explored in multivariate analysis on a larger cohort.Our study has several limitations that should be considered when interpreting the results. First, it is a retrospective study, which carries the risk of selection bias and confounding factors. During intubation, some interesting adverse events are missing in this retrospective study, like pneumothorax, dental trauma, gastric inhalation epistaxis or hypotension. The second limitation concerns the broad range of VL devices available over seven years. This variability of the intubation equipment may have led to variation in performance, not measured in our work. The third limitation concerns the experience of the operator that was not recorded in our work.

Table 1 :

 1

	N=91

Baseline characteristics and preoperative clinical details. Values are expressed as median [25th-75th percentiles]. 1 number of patients under 91; BMI Body mass index; ASA American Society of Anesthesiologists status; CRP C reactive protein; SAPS II Simplified acute physiology score II; CTscan Computed tomography scan

Table 2

 2

	: Clinical data of patients with deep neck infection according to the airway management techniques used at the first
	attempt. Values are expressed as median [25th-75th percentiles]. 1 number of patients under 86; 2 Chi-Square Test of
	Independence; 3 Kruskal-Wallis test;

4

Fisher's exact test. DL Direct laryngoscopy; VL Videolaryngoscopy; AF Awake fiberscopy; BMI Body mass index; ASA American Society of Anesthesiologists status; SAPS II Simplified acute physiology score II; CTscan Computed tomography scan.

Table 3 .

 3 The significant predictive factors for failure were the non-use of anti-inflammatory drugs before hospitalization (p=0.043) and young age (p=0.043). Severity of the symptoms or radio

	clinical findings demonstrating an impact on airway, such as reduced mouth opening,
	dysphagia, dyspnea, multiple collections and airway obstruction according the CTscan
	were not significantly different between the two groups.
	Concerning the devices used, there was a low rate of intubation failure with VL
	(3/27, 11%) but the statistical analysis did not reveal an inhomogeneous distribution of
	intubation failures according to the technique used (p=0.202). Failure during the first
	intubation attempt was significantly associated with an increased risk of desaturation with
	21% of desaturation below 90 % and 26% below 80% compared to 13% and 7%
	respectively in the group of intubation success (p=0.019). No cardiac arrest during
	intubation attempt was found in our study.

Table 3 :

 3 Outcome in the ICU ICU evolution and clinical outcome of the 91 patients analysed are summarized in

Predictive Factors for first-attempt intubation failure. Values are expressed as median [25th-75th percentiles]. 1 number of patients under 90; 2 Chi-Square Test of Independence; 3 Kruskal-Wallis test; 4 Fisher's exact test. BMI Body mass index; ASA American Society of Anesthesiologists status; SAPS II Simplified acute physiology score II; CTscan Computed tomography scan.

Table 4 .

 4 Forty-three patients (47%) had one or more surgical revision. Subjects' median length of stay was 10 days in the ICU and 16 days in hospital. One patient is dead after withdrawal of life-sustaining treatment in ICU, before any extubation attempt.

	N=91

Table 4 :

 4 Complications in the intensive care unit and outcome. Values are expressed as median [25th-75th percentiles].

Table 5

 5

	were pharyngeal or tonsillar DNI's origin (p=0.03),
	mediastinitis (p=0.02), number of surgical revisions (p<0.001), VAP (p<0.001), and septic
	shock during ICU stay (p = 0.013). Failure at the first intubation attempt was not
	statistically associated with extubation failure (p=0.331). The outcome was significantly
	worse in case of extubation failure with 50 % of secondary tracheotomy, an increase of

Table 5 :

 5 Intensive care unit evolution and outcome according to extubation failure. Values are expressed as median [25th-75th percentiles].

1 number of patients under 76; 2 Fisher's exact test; 3 Kruskal-Wallis test; BMI Body mass index; SAPS II Simplified acute physiology score II; VAP Ventilator associated pneumonia; ICU Intensive care unit.

DISCUSSION

Severe deep neck infections with no possibility of immediate extubation and need for mechanical ventilation for more than 24 hours represent between 7% and 50% of cervico-facial cellulitis treated in emergency settings [START_REF] Tapiovaara | Comparison of intubation and tracheotomy in patients with deep neck infection[END_REF][START_REF] Riekert | Severe odontogenic deep neck space infections: risk factors for difficult airways and ICU admissions[END_REF]. This serious condition can be life-threatening, especially because of the challenging urgent airway control. However, there is currently no universal agreement on the ideal intubation method for this specific population. The aim of our study was to describe the airway management techniques used in patients with severe DNI and to identify the risk factors for intubation and extubation failure. The composition of our bicentric cohort is in line with the cohorts previously described in the literature, which ensures the external validity of the analyses presented below [START_REF] Degouy | Risk factors for difficult ventilatory weaning in intensive care patients with cervical cellulitis[END_REF]. Indeed, the patients had few comorbidities, their illness severity was mainly related to the impact of the cervico-facial oedema on the upper airways rather than the systemic consequences of the sepsis. Despite an extended stay in ICU, mortality rate was very low.

In this 7-years bicentric cohort, we highlighted a rather balanced use of the 3 main airway control techniques (direct laryngoscopy, videolaryngoscopy and awake fiberscopy), with a more frequent use of VL over years, as much for the first-attempt intubation as for the backup technique. This evolution can be explained by greater accessibility of the VL for routine or emergency intubation with a benefit on intubation success marked even for inexperienced operators [START_REF] Lewis | Videolaryngoscopy versus direct laryngoscopy for adult patients requiring tracheal intubation: a Cochrane Systematic Review[END_REF]. We identified the limited mouth opening as the only clinical factor guiding the choice of the first-intubation attempt method. Indeed, the laryngoscopies are preferred as soon as the mouth opening permit it but AF is still widely use in limited mouth opening situations although this technique is challenging for young anaesthesiologists in emergency conditions. Moreover, the intubation practices heterogeneity observed suggests that the choice of the intubation technique is mainly related to the anaesthesiologist & otorhinologist duo expertise and device availability in each center.

In our study, the intubation first-attempt failure rate was 22%. These intubation failures were associated with deep desaturations under 80% but we did not find any serious complications. The incidence of difficult intubation in the emergency context is 10% on average, highly variable across studies, ranging from 1 to 23%. This intubation failure rate is probably affected by both patients related and operators related factors. In univariate analysis, prior anti-inflammatory drugs use appears to facilitate intubation, perhaps by reducing cervical swelling through the limitation of inflammatory oedema. We SERMENT ➢ En présence des Maîtres de cette école, de mes chers condisciples et devant l'effigie d'Hippocrate, je promets et je jure, au nom de l'Etre suprême, d'être fidèle aux lois de l'honneur et de la probité dans l'exercice de la médecine.

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