# Chemical Evolution of Hydrothermal Liquefaction Oils from Microalgae During Hydrotreatment: Insights from GCxGC and FT-ICRMS

Bruno da Costa Magalhães, Ruben Checa, Chantal Lorentz, Théo Voellinger, Jasmine Hertzog, Vincent Carré, Frédéric Aubriet, Pavel Afanasiev, Dorothée Laurenti, Christophe Geantet

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## To cite this version:

Bruno da Costa Magalhães, Ruben Checa, Chantal Lorentz, Théo Voellinger, Jasmine Hertzog, et al.. Chemical Evolution of Hydrothermal Liquefaction Oils from Microalgae During Hydrotreatment: Insights from GCxGC and FT-ICRMS. Energy & Fuels, 2025, Special issue "In Honor of Prof. Umit Ozkan -2024 Recipient of the ACS George A. Olah Award", 39 (27), pp.12936-12948. 10.1021/acs.energyfuels.5c00575. [hal-05134918](https://hal.univ-lorraine.fr/hal-05134918v1)

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Submitted on 29 Jun 2025

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1 Chemical Evolution of Hydrothermal Liquefaction Oils from

2 Microalgae During Hydrotreatment: Insights from GC×GC and FT-

3 ICRMS

4 Published as part of Energy & Fuels special issue "In Honor of Prof. Umit Ozkan -2024 Recipient of the ACS

5 George A. Olah Award".

6 Bruno da Costa Magalhães, Ruben Checa, Chantal Lorentz, Théo Voellinger, Jasmine Hertzog,

7 Vincent Carré, Frédéric Aubriet, Pavel Afanasiev, Dorothée Laurenti, and Christophe Geantet*

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Cite This: https://doi.org/10.1021/acs.energyfuels.5c00575

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Supporting Information

8 ABSTRACT: Hydrothermal liquefaction (HTL) oil derived from
9 Chlorella sorokiniana microalga was upgraded via hydrotreatment in
10 a batch reactor at 375 °C and various reaction times (for 0, 2.5, 5,
11 and 8 h) over a sulfided NiW/Al₂O₃ catalyst. The HTL algal oil and
12 the hydrotreated oils were characterized using global analytical
13 techniques such as elemental analysis (CHNS-O), ¹³C nuclear
14 magnetic resonance (¹³C NMR), gel permeation chromatography
15 (GPC), and molecular characterization techniques such as
16 comprehensive gas chromatography (GC×GC) and Fourier
17 transform ion cyclotron resonance mass spectrometry (FT-ICR
18 MS). Results indicate that longer reaction times enhance oil yield
19 and quality by converting heavy molecules that precipitate at
20 shorter times, reducing heteroatom content, and increasing the H/
21 C ratio of the oil. The elution profile obtained by GPC reveals that heavy compounds were converted into smaller molecules and
22 aromaticity decreases with longer reaction durations. GC×GC analysis showed fatty nitriles in the initial stages, suggesting reaction
23 pathways for converting fatty amides identified in the initial algal HTL oil. Additional experiments using model molecules helped
24 explain the hydroconversion of these nitrogen-containing species. Moreover, GC×GC results confirmed that oxygen- and nitrogen
25 heterocyclic compounds are the most refractory compounds, with their concentration significantly affected by longer reaction times.
26 Lastly, FT-ICR MS data combined with statistical analysis further elucidate the transformation of heavy molecules, showing a
27 decrease in H/C and DBE values over time, indicating the formation of less unsaturated compounds. Therefore, these findings
28 provide valuable insights into the hydroconversion of contaminants present in algal HTL bio-oil, which are crucial for defining
29 process conditions that can be used on an industrial scale to produce biofuel from algal feedstock.

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21 C ratio of the oil. The elution profile obtained by GPC reveals that heavy compounds were converted into smaller molecules and aromaticity decreases with longer reaction durations. GC×GC analysis showed fatty nitriles in the initial stages, suggesting reaction 23 pathways for converting fatty amides identified in the initial algal HTL oil. Additional experiments using model molecules helped 24 explain the hydroconversion of these nitrogen-containing species. Moreover, GC×GC results confirmed that oxygen- and nitrogen 25 heterocyclic compounds are the most refractory compounds, with their concentration significantly affected by longer reaction times. 26 Lastly, FT-ICR MS data combined with statistical analysis further elucidate the transformation of heavy molecules, showing a 27 decrease in H/C and DBE values over time, indicating the formation of less unsaturated compounds. Therefore, these findings 28 provide valuable insights into the hydroconversion of contaminants present in algal HTL bio-oil, which are crucial for defining 29 process conditions that can be used on an industrial scale to produce biofuel from algal feedstock.

1. INTRODUCTION

30 Third-generation biofuels, defined by the use of microalgae
31 feedstocks, were first investigated using the transesterification
32 route, thus mimicking the first generation of FAME biodiesel
33 or new generations of jet fuels.1,2 However, the classical route,
34 involving drying and lipid extraction stages, drastically limits
35 the economic viability of the process. More recent routes
36 involving reactive transesterification with direct conversion of
37 microalgal biomass are under investigation,3 but an alternative
38 is also provided by hydrothermal liquefaction (HTL)
39 technology. Indeed, it utilizes the wet biomass directly,4 and
40 it has the potential to convert the entire microalgal biomass
41 (lipids, carbohydrates, and proteins), thereby expanding the
42 yield of oil and providing an energetic application of
43 microalgae.5,6 The algal slurry is converted mainly into a bio-
44 oil, a solid named char, and aqueous and gas phases through

the HTL process.⁷ The biochemical composition of the algae 45 directly affects the yield and quality of the HTL oil. The 46 resulting HTL bio-oil is a complex mixture of several thousand 47 compounds, containing a high number of heteroatoms and a 48 great molecular diversity or class of compounds.⁸ This 49 complexity originates from multiple reactions, including 50 dehydration, dehydrogenation, deoxygenation, decarboxyla- 51 tion, and deamination of individual components of algae, as 52

Received: February 12, 2025

Revised: June 17, 2025

Accepted: June 18, 2025
well as reactions combining these components, such as
53 cyclization and polymerization.⁹,¹⁰

In this context, hyphenated analytical techniques are often
55 required to access the molecular characterization of algal HTL
56 oil. Wang et al.¹¹ and Barreiro et al.¹² reviewed the strategies
57 for bio-oil characterization, and among these analytical
58 methods, high-resolution mass spectrometry methods provide
59 a unique way to describe the heavy compounds.¹³,¹⁴ In fact,
60 when a detailed analysis of the molecular composition is
61 targeted, comprehensive 2D gas chromatography (GC×GC)
62 and ultrahigh resolution MS, such as Fourier-transform ion
63 cyclotron resonance mass spectrometry (FT-ICR MS) or
64 Orbitrap MS, are considered the most powerful tools.
65

The first technique, GC × GC, provides a detailed and
66 quantified analysis of the volatile organic fraction (typically up
67 to 500 g/mol). Madsen et al.¹⁵ analyzed different HTL bio-oils
68 from macro- and microalgae using GC×GC coupled with a
69 time-of-flight mass spectrometry detector (GC×GC TOF
70 MS). Additionally, other studies using GC×GC have shown
71 that algal HTL oil can contain numerous fatty acids, amides,
72 and cyclic nitrogen compounds such as indoles and have
73 investigated their evolution during hydrotreatment.¹⁶,¹⁷
74

For the heaviest molecules, the FT-ICR MS technique
75 allows obtaining the information at the molecular level. Indeed,
76 with its high mass accuracy and high resolution, it is possible to
77 assign a unique molecular formula to each *m/z* signal and,
78 therefore, to achieve hundreds of thousands of distinct
79 molecular assignments.¹⁸ For algal HTL bio-oils, CHN₂Oₓ or
80 CHN₃Oₓ classes of compounds are often the most abundant
81 ones.¹⁹⁻²⁴ The distribution of CHN₂Oₓ in the HTL oil used in
82 this work has already been described elsewhere,²⁵ as well as the
83 origin of the formation of these compounds.
84

As evidenced by the bio-oil analysis using either global or
85 comprehensive methods, the high amount of heteroatom
86 compounds, viscosity, and acidity prevents its use as a
87 biofuel.²⁶,²⁷ Thus, upgrading is required to make it more
88 suitable for blending with petroleum feeds (coprocessing) or to
89 reach the current fuel legislation. If diesel or jet fuel
90 compositions are desired, the preferred processes are hydro-
91 treating and/or hydrocracking. Dell'orco et al.²⁸ pointed out
92 that the comprehensive analysis of the chemical composition,
93 particularly regarding the speciation of higher molecular weight
94 species, may help to optimize the downstream catalytic
95 process. Besides, FT-ICR MS can also be used to track the
96 behavior of refractory nitrogen compounds upon upgrading
97 steps. Recently, Deng et al.²⁹ characterized a petroleum-based
98 residual oil that underwent a hydrodenitrogenation (HDN)
99 process by electron spray ionization (ESI) FT-ICR MS. Their
100 study revealed that, based on the double bond equivalent, the
101 most refractory nitrogen-containing compounds comprised
102 either multiple naphthenic-aromatic rings or bearing long side
103 chains near the nitrogen atom. In another study, heavy oil
104 fractions subjected to HDN treatment were characterized by
105 FT-ICR MS and ion-mobility mass spectrometry (IM-MS).³⁰
106
107 The CCS values obtained by IM-MS, for some N-containing
108 species before and after the HDN process, ensured the
109 observation of a decrease in isomeric diversity. The authors
110 also made evidence that refractory compounds were
111 characterized by lower CCS values.
112

Although the application of hyphenated techniques for
113 characterizing bio-oils is steadily increasing, studies focused on
114 hydrotreated algal oil remain limited in the literature. Jarvis et
115 al.³¹ characterized the fate of iron porphyrins during

hydroprocessing by using FT-ICR MS. In another study, by
116 applying the same technique in combination with bulk
117 property measurements, simulated distillation, and gas
118 chromatography-mass spectrometry (GC-MS), Jarvis et al.³²
119 compared the chemical compositions of raw and hydrotreated
120 bio-oil samples from pine, microalgae, and sewage sludge.
121 Additionally, Zuber et al.,²³ in a study exploring the upgrading
122 at various temperatures of C. vulgaris HTL oil, evidenced by
123 FT-ICR MS (ESI), demonstrated the removal of O and N
124 hetero compounds in CHN, CHNO, and CHNOP classes of
125 compounds with increasing temperature. With these families,
126 ranging from C₁₀ to C₅₀, the easier removal of O-containing
127 compounds and the resistance of N ones was evidenced.
128

Our previous study on the upgrading of *Chlorella sorokiniana*
129 HTL oil demonstrated that comprehensive GC×GC quantified
130 nearly 40 wt % of the HTL oil, whereas after hydrotreatment
131 for 5 h at 375 °C, this quantification increased to above 80%.¹⁶
132 Under these conditions, deep HDO and HDS conversions
133 could be achieved, but HDN conversion was more challenging.
134 However, the behavior of high molecular weight nitrogen
135 compounds during HDT resulting from condensation
136 reactions is somewhat unclear. Therefore, in the present
137 study, we attempted to investigate the reactivity of the
138 heteroatom-containing molecules, including both the volatile
139 compounds identified by GC×GC and the heavier species
140 characterized by FT-ICR MS combined with chemometrics.
141 These analyses were conducted using conventional approaches,
142 including elemental analysis, carbon nuclear magnetic reso-
143 nance, gel permeation chromatography coupled to a refractive
144 index detector, and a diode array detector, and simulated
145 distillation to provide an overview of the reactivity of algal
146 HTL oil upon HDT. The complementarity of these techniques
147 highlights the diverse effects of hydrotreatment on the removal
148 of heteroatoms and the production of biofuels.
149

## 2. MATERIALS AND METHODS

### 2.1. Microalgae and HTL Algal Bio-Oil Production.
Microalgae strain consisting of *Chlorella sorokiniana* NIES
2173 was grown at the MicroAlgae Processes Platform, CEA
Cadarache. Selection and growth of this strain were reported
153 previously.³³ Then, the continuous HTL experiment was
154 performed at the CEA Grenoble facility, and details about the
HTL unit were published elsewhere,³⁴ while the methodology
155 applied to produce the HTL bio-oil used in this work was
157 reported in detail in our previous study.¹⁶
158

### 2.2. Catalytic Hydrotreatment (HDT) of HTL Algal Bio-Oil.
The catalytic hydrotreatment step was performed using a
160 300 mL Parr stainless steel batch reactor. A commercial NiW/
Al₂O₃ extrudate catalyst (WO₃: 25.7 wt %, NiO: 3.8 wt %,
161 specific surface area: 179 m²/g, and total pore volume: 0.40
162 cm³/g) was sulfided at 400 °C for 3 h under 15% H₂S/H₂ flow
163 (4 NL/h). Then, 0.6 g of the sulfided catalyst, 3.0 g of HTL
bio-oil, and 25 g of *n*-heptane were added to the reactor.
164 Subsequently, the reactor was closed and purged with N₂, and
165 6 MPa was loaded for a leak test. Afterward, N₂ was removed,
166 and the reactor was purged three times with H₂, loaded with 1
167 MPa of H₂, and heated until 375 °C. Once the temperature
168 had been reached, approximately after 40 min, 5 MPa of H₂
169 was added to get a total pressure of 10 MPa, and stirring at
170 approximately 570 rpm was started. Four reactions were
171 performed at different reaction times: 0, 2.5, 5, and 8 h. In
172 other words, these experiments varying the reaction time were
173 carried out at different moments, and the reactor was loaded
174
177 and completely unloaded for each test. Additionally, it is worth 178 noting that for the reaction carried out at 0 h, as soon as the 179 desired temperature and pressure were achieved, the reactor 180 was cooled down. This experiment helped to understand which 181 reactions took place during the heating stage and provided 182 valuable insights regarding the conversion and reaction 183 pathways of less refractory compounds. Lastly, the used 184 catalyst was separated from the hydrotreated oil (HDT-oil) by 185 filtration, and n-heptane was used to wash the reactor in order 186 to recover the residual catalyst and oil. n-Heptane was further 187 removed using a vacuum evaporator, and the HDT-oil was 188 analyzed by different analytical techniques.

189 **2.3. Characterization and Analytic Techniques.**
190 **2.3.1. Elemental Analysis (CHNS-O).** The elemental contents 191 of carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and 192 sulfur (S) were analyzed using a Thermo Scientific Flash 2000 193 series CHONS/O analyzer equipped with a thermal 194 conductivity detector (TCD). The higher heating value 195 (HHV) was calculated based on elemental analysis values 196 according to the equation reported by Channiwala and 197 Parikh.35

198 **2.3.2. Gel Permeation Chromatography (GPC).** Gel 199 permeation analysis was conducted using an Agilent 1200 200 Series HPLC System, which was equipped with two PL gel 201 porous columns of different pore sizes (50 and 500 Å), a 202 differential refractive index detector (RID), and a diode array 203 detector (DAD). The analysis was performed at 35 °C with 204 tetrahydrofuran (THF) as the eluent at a flow rate of 1 mL/ 205 min. Samples were diluted to approximately 1 wt % in THF 206 and filtered through a 0.45 µm filter before analysis. Elution 207 times were converted to molecular weights (Mw) by using a 208 calibration curve derived from a hydrocarbon mixture (Mw 209 ranging from 86 to 1000 g/mol). Consequently, the Mw values 210 are relative to the calibration curve (HC equivalent) rather 211 than absolute. Chromatograms obtained from the RID 212 detector were normalized to the highest peak intensity, while 213 signals from the DAD detector were normalized per mass unit 214 of the sample.

215 **2.3.3. ¹³C Nuclear Magnetic Resonance (¹³C-NMR).** The 216 ¹³C NMR spectra were recorded by using a Bruker AVANCE 217 400 MHz spectrometer and analyzed with TopSpin 3.0 218 software. Measurements were conducted at room temperature, 219 accumulating 4700 scans over approximately 15 h. The 220 samples (60 mg) were diluted with deuterated chloroform 221 (CDCl₃).

222 **2.3.4. Simulated Distillation (Sim-Dis).** Boiling point 223 distributions were determined using Sim-Dis, following the 224 standard ASTM D2887. The analyses were conducted on an 225 Agilent 7890A series gas chromatograph (GC) equipped with 226 an Agilent J&W GC column (dimensions: 10 m × 0.53 mm × 227 2.65 µm, P/N: CP7582) and a flame ionization detector 228 (FID). The liquid products were categorized into five boiling 229 point ranges: gasoline (<175 °C), jet fuel (175–250 °C), 230 diesel (250–350 °C), heavy gas oil (350–500 °C), and 231 residual (>500 °C).

232 **2.3.5. Comprehensive Two-Dimensional Gas Chromatography Coupled to Time-of-Flight Mass spectrometry and to Flame Ionization Detector (GC×GC-Tof MS/FID).** Two 233 GC×GC analytical systems were utilized: one consisting of a 234 cryogenic modulator from Zoex Corporation (USA) coupled 235 with a TOF MS for compound identification (up to C₃₂) and 236 another consisting of a flow modulator coupled with an FID 237 for quantification (up to C₄₀). More details about the GC×GC 238

parameters are provided in Table S1. Modulation parameters 240 were optimized according to Lelevic et al.36 241

Both system tools were used to analyze the HTL and HDT 242 oils obtained at different reaction times. The samples were 243 diluted in THF with a concentration of approximately 1 and 5 244 wt % for TOF MS and FID analysis, respectively. Besides, an 245 internal standard, 3,3'-dimethylbiphenyl (3,3-DMBP), approx- 246 imately 200 ppm, was also added for quantification purposes. 247

2.3.6. Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Samples were first dissolved in 90/ 248 10 methanol/toluene (v/v) to obtain a 1 mg/mL solution. 249 Then, this solution was diluted by 10 in methanol with a 0.1 250 mg/mL sodium acetate solution. This sample preparation was 251 used after optimization on similar samples in previous study.37 252 All bio-oil samples were analyzed in triplicate. 253

The molecular composition of the different bio-oils was 255 obtained by FT-ICR MS with a 7 T FT-ICR mass 256 spectrometer (Solarix 2XR, Bruker Daltonics, Bremen, 257 Germany). The analyses were performed by electrospray 258 ionization (ESI, Bruker Daltonics) in positive detection mode. 259

ESI (+) was used in this study, as it was considered to ionize 260 the majority of the bio-oil components. Indeed, microalgae 261 bio-oils are known to comprise a significant number of basic 262 nitrogen-containing species originating from the thermochem- 263 ical degradation of proteins and pigments.25 Regarding the 264 ionization and detection of carbohydrates, sample preparation 265 with sodium acetate addition was shown to improve the 266 detection of this biochemical class in ESI (+) compared to ESI 267 (−).37 Eventually, sugars and amino acids composing the 268 biomass are subject, during HTL, to Maillard reactions 269 resulting in nitrogen-containing compounds, such as pyrazine 270 and pyridine derivatives that are basic components better 271 detected in positive-ion mode.38 272

Both the ion source and instrument parameters were 273 optimized via software FTMS-Control V2.3.0 (Bruker 274 Daltonics). Prior to acquisition, the mass spectrometer was 275 externally calibrated, and the ICR detection cell was shimmied 276 and gated by using the direct introduction of a 0.1 mg/mL 277 sodium trifluoroacetate solution. The sample was infused into 278 the ESI source at a 2 µL/min flow rate, and the capillary 279 voltage was set to 4.5 kV. The drying gas temperature and flow 280 rate were kept at 200 °C and 0.5 L/min, respectively, and the 281 nebulizer pressure was set to 0.1 MPa. Mass spectra resulted 282 from the accumulation of 500 scans over a 114.7–2000 m/z 283 range and with an 8-megaword time domain. A mass resolution 284 of 1,253000 was achieved at m/z 230. 285

DataAnalysis 5.0 software (Bruker, Daltonics) was used to 286 internally calibrate the FT-ICR mass spectra with a list of 287 known bio-oil compounds with a standard deviation of mass 288 error ≤ 100 ppb. Peak lists with a signal-to-noise ratio ≥ 5 289 from the different mass spectra were exported and aligned into 290 a matrix according to their m/z value in a 0.5 ppm tolerance 291 window. Peak list assignment was performed with Composer 292 software (Sierra Analytics, Modesto, USA). Molecular formulas 293 were assigned within a ± 1 ppm mass error range, considering 294 the C, H, O, and N elements as well as [M + H]⁺ and [M + Na]⁺ ions. Thus, CH, CHOₓ, CHNₓ, and CHNₓOᵧ molecular 295 series were obtained. Compounds were represented according 296 to their oxygen-to-carbon and hydrogen-to-carbon ratios to 297 give van Krevelen's diagram, on which it is possible to 298 distinguish biochemical classes such as lipids and lignin 299 derivatives.39 The DBE (double bond equivalent) versus 300 carbon number graphs represent the aromaticity and 301
<table><caption>Table 1. Elemental analysis, H/C, O/C, N/C, and HHV of *Chlorella Sorokiniana* Microalga, HTL, and HDT Oils</caption><thead><tr><th rowspan="2">Sample</th><th rowspan="2">C</th><th colspan="3">H (wt %)</th><th rowspan="2">N</th><th colspan="3">O (Atomic ratio)</th><th rowspan="2">HHV (MJ/kg)</th></tr><tr><th>H</th><th>O</th><th>N</th><th>H/C</th><th>O/C</th><th>N/C</th></tr></thead><tbody><tr><td>Microalgae</td><td>50.4</td><td>6.8</td><td>33.6</td><td>3.2</td><td>1.62</td><td>0.50</td><td>0.05</td><td>22.1</td></tr><tr><td>HTL bio-oil</td><td>72.7</td><td>9.6</td><td>13.5</td><td>3.3</td><td>1.59</td><td>0.14</td><td>0.04</td><td>35.3</td></tr><tr><td>HDT—0 h</td><td>82.5</td><td>12.8</td><td>3.5</td><td>2.6</td><td>1.92</td><td>0.01</td><td>0.01</td><td>43.5</td></tr><tr><td>HDT—2.5 h</td><td>85.6</td><td>13.8</td><td>1.1</td><td>1.9</td><td>1.93</td><td>0.01</td><td>0.02</td><td>45.9</td></tr><tr><td>HDT—5 h</td><td>85.3</td><td>13.7</td><td>1.3</td><td>1.5</td><td>1.92</td><td>0.01</td><td>0.01</td><td>45.8</td></tr><tr><td>HDT—8 h</td><td>86.1</td><td>14.0</td><td>0.79</td><td>1.2</td><td>1.96</td><td>0.01</td><td>0.01</td><td>46.5</td></tr></tbody></table>

unsaturation degree of a molecule and are calculated as given by eq 1, with $n_C$, $n_H$, and $n_N$ representing the number of carbon, hydrogen, and nitrogen atoms, respectively.

$$ \text{DBE} = 1 + n_C - \left( \frac{n_H}{2} \right) + \left( \frac{n_N}{2} \right) \quad (1) $$

SIMCA-P 9.0 software was used to perform principal component analysis (PCA) on the assigned *m/z* values of the different bio-oil samples analyzed by ESI (+) FT-ICR MS. Origin software was used for the different graphical representations (apart from the PCA score plots).

## 3. RESULTS AND DISCUSSION

### 3.1. Global Analysis of the Samples.
The elemental analysis of the *Chlorella sorokiniana* microalga, HTL, and HDT oils after various reaction times at 375 °C is shown in Table 1. The sulfur content measured in all samples was below the instrument detection limit (0.1 wt %) and has not been reported in the mentioned table. The biochemical composition of *Chlorella sorokiniana* microalga is, respectively, 54.6, 13.5, 319 and 16.1 (wt %) of carbohydrates, lipids, and proteins. At the end of the experiments, a fine black powder that precipitated at the bottom of the reactor was observed. The yield of oils and solids after HDT at different reaction times was around 56–62 wt % and 21–5.5 wt %, respectively (Table S2). The solid yield decreases with increasing reaction time, indicating that the condensed phase tends to be converted under longer operating conditions. Elemental analysis of fresh and used NiWS/Al₂O₃ catalyst and fine powder (Table S3) showed that, basically, no HDN reaction took place at 0 h since the degree of denitrogenation observed was due to the precipitation of nitrogen compounds as solid and not by hydroconversion. In addition, longer reaction time led to greater nitrogen compound conversion and lower nitrogen precipitation.

Moreover, the oxygen and nitrogen contents in the HDT oils significantly decreased when contact time was increased, which confirms the importance of this parameter during the upgrading stage. The deoxygenation obtained after 0 h and 8 h was equal to 74.1% and 94%, whereas the extent of denitrogenation was 21% and 63.6%, respectively. As reported in our previous study,⁴⁰ this illustrates the difficulty of removing nitrogen-containing molecules in algal oils (pyrroles, indoles, carbazoles, for example), whereas deoxygenation of oxygenated compounds (carboxylic acids, phytosterols, and phenols) proceeds more readily under the same reaction conditions.

Although the oxygen amount in the hydrotreated oil at 375 °C during 8 h, reported in Table 1, is below the US DOE's goal (1 wt %), the nitrogen content is still much higher than the desired value (0.05 wt %), which corroborates that more drastic reaction conditions are required or additional hydro- treating stages to achieve the expected denitrogenation level.

Recently, Haider et al.⁴¹ performed a continuous HDT of algal oils under 10 MPa H₂, with a WHSV of 0.5 h⁻¹, at 200–340 °C, over a sulfide catalyst, and reported a maximum denitrogenation of 39%. In addition, oxygen compounds were mentioned to be precursors of coke that can result in catalyst bed plugging or deactivation. Since these molecules can be converted under softer reaction conditions than nitrogen, a first stage at a lower temperature to convert oxygen molecules, followed by a second reactor working at a higher temperature and lower contact time to boost HDN reactions, is suggested.

The ¹³C NMR analysis (see Figure 1) evidenced a reduction in the aromaticity of HDT oils when a higher contact time was

<figure><img src="image_9.png" /><figcaption>**Figure 1.** ¹³C NMR functional group distribution in HTL bio-oil and HDT-oils at different reaction times.</figcaption></figure>

employed, which agrees with the H/C ratio shown above. It was also observed that carbonyl groups were present in the HDT oil treated during 0 h. Previous analyses have shown that, despite the absence of carbonyl groups detected by the ¹³C NMR technique, fatty amides were identified in the GC×GC chromatograms, confirming the importance of employing different analytical techniques to analyze complex feeds such as HTL and HDT algal oils. Therefore, the ¹³C NMR confirms the presence of carbonyl groups in the algal HTL oil and HDT oil at 0 h, but complementary analyses will discuss in detail the composition of these oils.

The GPC-RID elugrams are shown in Figure 2. The main peak in the hydrotreated oils is shifted to the left compared to that in the algal HTL oil, which is associated with a reduction in the average molar molecular weight (Mw). The lower Mw is due to the conversion of carboxylic acids, such as C16 and C18, present in high amounts in this algal HTL oil, as
<figure><img src="image_10.png" /><figcaption>**Figure 2.** GPC-RID analyses of the HTL bio-oil and HDT-oils at different reaction times. The fraction analyzed by GC×GC-TOF MS (below 500 g/mol in eq HC) is shown in the blue area.</figcaption></figure>

evidenced by ¹³C NMR. No significant variation was observed in the elugrams, and Mw was obtained from HDT oils during 2.5 h and 5 h. However, the longer reaction time led to a reduction of molecules with a higher molecular weight and an increase in the light fraction, suggesting that these heavy molecules were either cracked into lighter compounds or converted.

The heavy fraction in algal HTL and HDT oils is difficult to characterize, as these molecules are easily degraded in the GC inlet or are not eluted in conventional gas chromatography. The GC×GC system employed in this work can identify compounds until C₃₂. The fraction analyzed by the comprehensive GC system was estimated and is reported in Figure 2, denoted by the blue area.

The GPC coupled to a DAD detector was also employed to study the aromaticity degree of the bio-oils during the upgrading stage. The polyaromatic compounds provide a UV response between 190 and 450 nm depending on the condensation and functionalization of the rings. The 3D and 2D signals obtained were plotted and presented in Figure S1. Higher reaction time resulted in lower absorbance at the different wavelengths, as evidenced by the 3D plots. This is associated with the reduction of the aromaticity degree, corroborating the elemental (CHN/O) and ¹³C NMR results. Additionally, these results clearly showed that heavier aromatic compounds were cracked or converted into lighter ones or were hydrogenated. This conclusion is also in agreement with the GPC-RID results, which evidenced the reduction of the molecular weight at higher reaction time.

Lastly, the SIMDIS curves and fractional cuts of the algal HTL oil and HDT oils during different reaction times are shown in Figure 3 and Table 2, respectively. Note that for HTL oil, two steps corresponding to the C16 and C18 acids are visible. The calibration curve used for converting retention time in boiling point is based on a mixture of *n*-alkanes. However, the response factor (RF) applied for carboxylic acids using an FID detector is approximately two times lower than the RF for *n*-alkanes used to calibrate the SIMDIS. Therefore, the contribution of these acids in the sample recovered fraction is twice as high. These molecules are rapidly converted into C15, C16, C17, and C18 normal alkanes upon hydrotreating over sulfide catalysts, resulting in an HDT oil that fell mainly in the diesel range. No significant difference was found in the

<figure><img src="image_11.png" /><figcaption>**Figure 3.** Simulated distillation profiles of HTL bio-oil and HDT-oils at different reaction times.</figcaption></figure>

SIMDIS curves of the hydrotreated oils during 2.5 h and 5 h. However, it is shown that after 8 h of reaction, a greater amount eluted in the diesel range, and the mass recovered was higher (85 wt %). These results can be related to the hydroconversion of nitriles and fatty amides, especially C16 and C18, which also result in C15–C18 aliphatic compounds, and to the cracking and/or conversion of heavy molecules. Interestingly, a lower amount of gasoline cut was obtained after the 8 h experiment. Although the variation is low compared to the other samples (<1 wt %), which can be attributed to analysis error, it could also be potentially attributed to more cracking reactions, resulting in the conversion of this light fraction to gas products.

Therefore, the elemental content, combined with ¹³C NMR results, confirms that longer reaction times result in a reduction of the aromatic content, which is attributed to increased hydrogen incorporation. Furthermore, GPC-RID and SIMDIS analyses demonstrated that more than 50% of the diesel cut could be obtained after hydrotreatment, which is suitable for further molecular-level characterization via GC × GC. However, the presence of a heavier fraction requires additional techniques, such as FT-ICR MS. In the next section, a comprehensive analysis of the hydrotreated algal bio-oils will be presented, providing more detailed molecular-level information using GC × GC and FT-ICR MS.

**3.2. Comprehensive Analysis of the Products.** The 2D projected GC×GC chromatogram of the bio-oil obtained from continuous HTL of *Chlorella sorokiniana* microalga has already been published (Figure 2 of ref16). The main compounds were carboxylic acids formed from the lipid hydrolyses and fatty amides, mainly C16 and C18 compounds. These molecules are formed from the reaction between carboxylic acids and nitrogen molecules, such as ammonia, which originated from proteins. In addition, N- and O-heterocyclic compounds such as pyrroles, indoles, and phenols were identified. The mechanism of formation of these compounds is reported in ref25.

**Figure 4** shows the chromatogram of the hydrotreated oils obtained after reaching the reaction temperature (t0) and evidence that many reactions had already occurred during the temperature rise. It is expected that carboxylic acids were converted during this heating stage (t0). Although the catalyst was added to the reactor, stirring was not started since as soon as the temperature was reached, the reactor was cooled. Therefore, in this case, it was mainly a thermal conversion,
<table><caption>Table 2. Fractional Cuts Obtained from the Simulated Distillation of HTL Bio-Oils and HDT-Oils</caption><thead><tr><th>Sample</th><th>Gasoline<br>&lt;175 °C (wt %)</th><th>Jet-fuel<br>175–250 °C (wt %)</th><th>Diesel<br>250–350 °C (wt %)</th><th>Heavy gas oil<br>350–500 °C (wt %)</th><th>Residual<br>&gt; 500 °C (wt %)</th></tr></thead><tbody><tr><td>HTL bio-oil</td><td>0</td><td>1</td><td>12</td><td>21</td><td>66</td></tr><tr><td>HDT-oil—(0h)</td><td>1</td><td>3</td><td>27</td><td>14</td><td>55</td></tr><tr><td>HDT-oil—(2.5h)</td><td>1</td><td>6</td><td>53</td><td>18</td><td>22</td></tr><tr><td>HDT-oil—(5h)</td><td>1</td><td>7</td><td>53</td><td>21</td><td>18</td></tr><tr><td>HDT-oil—(8h)</td><td>0</td><td>7</td><td>60</td><td>18</td><td>15</td></tr></tbody></table>

<figure><img src="image_12.png" /><figcaption>Figure 4. Comprehensive GC×GC-FID/MS chromatogram of HDT oil (0 h).</figcaption></figure>

469 which also resulted in C15–C18 aliphatics but in a lower 470 amount compared to the other experiments, as evidenced by 471 the SIMDIS profiles.

472 Diketopiperazines found in the algal HTL oils were also 473 converted during the HDT at 0 h since these molecules were 474 not found in the 0 h GC×GC chromatogram. This confirms 475 that these compounds with two nitrogen atoms per molecule 476 do not pose a significant issue for the hydrotreatment step. To 477 the best of our knowledge, the reaction pathway of 478 diketopiperazines upon hydrotreatment over sulfide catalysts 479 and the products of this reaction have not yet been reported in 480 the literature yet.

481 Moreover, a family of fatty nitriles with a carbon range from 482 C8 until C22 was identified in the HDT oil (0 h) chromatogram 483 (see Figure 4). It is evidenced that the main compounds in this 484 family are saturated fatty nitriles (DBE = 2). But in the case of 485 the most abundant ones (C16 and C18), nitriles with one 486 unsaturation (DBE 3) are also observed. As shown previously, 487 we identified a high amount of C16 and C18 fatty amides in 488 the GC×GC chromatogram of the algal HTL oils. Few articles 489 have reported the hydroconversion reaction pathway of 490 secondary and tertiary fatty amides over sulfided heterogene- 491 neous catalysts.42,43 However, the hydroconversion of primary 492 fatty amides is still lacking in the literature. Therefore, we 493 performed some experiments using oleanide as a model

molecule upon hydrotreatment with the NiWS/Al₂O₃ catalyst. 494 Our results show that the primary fatty amides are rapidly 495 converted into fatty nitriles by dehydration and carboxylic 496 acids by hydrolysis, as demonstrated in Figure 5.

497 Then, the C18 fatty nitrile was converted into C18 alkane, 498 and the C18 carboxylic acid was converted into C18 or C17 499 alkane by hydrodeoxygenation (HDO) or decarbonylation/ 500 decarboxylation (DCO). The selectivity for C18 *n*-alkane was 501 close to 75% compared with 25% for C17 *n*-alkane, indicating 502 that the dehydration of fatty amides to nitriles is the main 503 pathway. 504

505 Given this, it can be concluded that the C16 and C18 fatty 506 amides in the algal HTL oil were rapidly converted mainly into 507 C16 and C18 fatty nitriles that were more difficult to convert 508 since these molecules also appear in the HDT oil performed 509 during 2.5 h but are almost totally converted after 8 h, as 510 shown in Figure S2. FT-ICR MS analysis evidenced at 0 h the 511 same preponderance of C16 and C18 nitriles for DBE 2. 512 However, as conversion increases, numerous compounds 513 issued from the conversion of nitrogen compounds (for 514 instance, quinoline and carbazole) with the same DBE appear. 515

516 Moreover, the chromatogram of HDT oil during 2.5 h 517 (Figure S2a) evidenced the conversion of esters present in 518 algal HTL oil. Comparing the chromatograms of HDT during 519 2.5 and 8 h (Figure S2a,b) evidences that phenols require a
<figure><img src="image_13.png" /><figcaption>Figure 5. Reaction pathway of oleamide hydroconversion over the NiWS/Al₂O₃ catalyst.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Figure 6. Refractory behavior of the N₁ class (DBE 9) during hydroprocessing at different reaction time.</figcaption></figure>

519 longer reaction time to be converted compared to other 520 oxygen-containing molecules. These results confirm that 521 heterocyclic compounds are more refractory during hydro- 522 treatment.

523 Lastly, higher temperatures led to a significant reduction of 524 nitrogen heterocyclic compounds. The same trend was 525 observed with the reaction time. Indoles were the most 526 nitrogen-containing family identified after 8 h of reaction (see 527 Figure S2b). Based on the results reported in the literature, 528 Figure S3 illustrates the reaction pathway over sulfided 529 catalysts for the main nitrogen heterocyclic compounds 530 identified with our GC×GC analysis. In all cases, these

molecules should be first hydrogenated, which results in the 531 weakening of the carbon–nitrogen bond and a decrease of the 532 equivalent double bond number. Sequentially, the HDN 533 reaction takes place with production of a free nitrogen 534 compound and ammonia. However, as evidenced in Figure 535 S3, the molecules can become acid, basic, or neutral during the 536 hydroconversion. 537

Among N-compounds observed by GC×GC, carbazoles 538 (C12 and DBE 9), even if they are present at a low 539 concentration, are those that present the slowest HDN rates of 540 conversion.44 This is also confirmed by FT-ICR MS, which 541 also allows evidence of long-chain alkylated carbazoles. The 542
<table><caption>Table 3. Composition Description of the Bio-Oil Samples Achieved by ESI (+) FT-ICR MS with a Number of Molecular formulas, Corresponding Percentages, and Weighted Averages for Different Atomic Ratios and Mass</caption><thead><tr><th></th><th></th><th>HTL Bio-oil</th><th>HDT-oil (0 h)</th><th>HDT-oil (2.5 h)</th><th>HDT-oil (5 h)</th><th>HDT-oil (8 h)</th></tr></thead><tbody><tr><td rowspan="5">Heteroatom class</td><td>CH</td><td>0 (0%)</td><td>21 (0.2%)</td><td>55 (0.7%)</td><td>60 (0.9%)</td><td>61 (0.9%)</td></tr><tr><td>CHO<sub>x</sub></td><td>1546 (18.7%)</td><td>957 (11.3%)</td><td>649 (8.8%)</td><td>550 (8.2%)</td><td>570 (8%)</td></tr><tr><td>CHN<sub>x</sub></td><td>744 (9%)</td><td>2052 (24.3%)</td><td>2130 (28.9%)</td><td>2120 (31.5%)</td><td>1981 (27.9%)</td></tr><tr><td>CHN<sub>x</sub>O<sub>y</sub></td><td>5969 (72.3%)</td><td>5410 (64.1%)</td><td>4541 (61.6%)</td><td>4002 (59.4%)</td><td>4476 (63.1%)</td></tr><tr><td><strong>TOTAL</strong></td><td><strong>8259</strong></td><td><strong>8440</strong></td><td><strong>7375</strong></td><td><strong>6732</strong></td><td><strong>7088</strong></td></tr><tr><td rowspan="4">Weighted average</td><td>H/C</td><td>1.43</td><td>1.58</td><td>1.55</td><td>1.53</td><td>1.51</td></tr><tr><td>DBE</td><td>7.21</td><td>6.07</td><td>6.62</td><td>6.88</td><td>6.99</td></tr><tr><td>O/C</td><td>0.12</td><td>0.04</td><td>0.03</td><td>0.03</td><td>0.03</td></tr><tr><td>N/C</td><td>0.05</td><td>0.06</td><td>0.06</td><td>0.06</td><td>0.06</td></tr></tbody></table>

<figure><img src="image_15.png" /><figcaption>Figure 7. Intensity of CHNₓ-CHNₓOᵧ (a) and CH-CHOₓ (b) detected compounds in the different bio-oil samples by ESI (+) FT-ICR MS. Bar plots are colored according to the oxygen atom count.</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Figure 8. Intensity of CHNₓ (a) and CHNₓOᵧ (b) detected compounds in the different bio-oil samples by ESI (+) FT-ICR MS. Bar plots are colored according to the nitrogen atom count.</figcaption></figure>

543 refractory character of DBE 9 compounds, including 544 carbazoles, is due to competition with other nitrogen 545 molecules. Figure 6 illustrates this behavior, evidencing the 546 difficulty of reaching a deep HDN conversion. Note that from 547 our work performed on C. vulgaris HTL oil, a similar analysis 548 was performed, and increasing disappearance with increasing 549 reaction time was observed with this family of compounds, 550 clearly indicating the influence of algae species on the HDN 551 performance.

552 Heteroatom class distribution achieved for the different bio- 553 oil samples in ESI (+) FT-ICR MS is gathered in Table 3. 554 Nitrogen-containing species, CHNₓ and CHNₓOᵧ, represent

between 81 and 91% of the assignments. This aligns with 555 previous studies performed using ESI (+) high-resolution mass 556 spectrometry on bio-oils from HTL of microalgae, where 557 nitrogen-containing compounds were predominantly de- 558 tected.21,45 Interestingly, the total assignment number 559 decreases with the increase in the HDT time. Indeed, the 560 contributions (in %) of the CHOₓ and CHNₓOᵧ series 561 decrease while the proportion of the nonoxygenated 562 compounds, namely, CH and CHNₓ, increases with the 563 reaction time. However, one can notice that this trend is 564 valuable from the raw algal HTL oil to HDT-5h. After that 565 time, the contribution of the oxygen-containing compounds, 566
<figure><img src="image_17.png" /><figcaption>**Figure 9.** (a) PCA score plots of the bio-oil samples analyzed in triplicates by ESI (+) FT-ICR MS. Close to 1500 assignments specific to non- and low-HDT time and to high-HDT-time bio-oils were extracted to be represented according to their heteroatom class distribution (b) and van Krevelen diagram and DBE vs. carbon number graph (c).</figcaption></figure>

CHOₓ and CHNₓOᵧ, and the number of assignments increased a bit while the contribution of the nonoxygenated features decreased. Some molecular descriptors are also informative concerning the global behavior of the bio-oil during the catalytic step. In fact, the H/C value increases, and reciprocally, the DBE value decreases with HDT time, indicating that compounds with less unsaturation are formed or unsaturated ones are converted. This was observed in ¹³C

NMR, where the higher percentage of aliphatics and, reciprocally, the lower proportion of aromatics and olefins increased with HDT time. Regarding the evolution of the O/C value over HDT time, the observed decrease is consistent with the decrease in percentage relative to the oxygen-containing compounds (CHOₓ and CHNₓOᵧ) and with the elemental analysis given in Table 1. Eventually, the N/C values obtained
582 from the FT-ICR MS assignments do not evidence a significant
583 influence of the HDT time.

584 Distributions of the CHNₓ and CHNₓOᵧ series, according to
585 the oxygen atom counts (Figure 7a), show that even after a
586 small contact with the catalyst, the overall intensities of the
587 nitrogen-containing species increase, slightly decrease at 2.5 h,
588 and then increase again at 5 h and 8 h of HDT. Intensities of
589 the CHNₓ compounds (Figure 7a, in gray) are specifically of
590 interest as they significantly increase even after a brief HDT
591 time and are prominent after 8 h. For the CHNₓOᵧ species,
592 their corresponding intensities decrease with time, especially
593 for CHNₓO₂⁻₅ compounds. In addition, the distribution of the
594 HTL bio-oil, according to the molecular formula number, is
595 centered at 3 oxygens. In contrast, for all the HDT bio-oils, the
596 distribution is centered at 1 oxygen.

597 The same graphical representations were created but based
598 on the number of nitrogen atom counts. The evolution of the
599 intensity of the CHNₓ and CHNₓOᵧ compounds according to
600 their nitrogen content follows the same trend as before (Figure
601 8). Indeed, the overall intensity of the CHN₁ compounds
602 dramatically increases in the HDT-0 h to slightly decreases at
603 2.5 h and increases again at 5 h and 8 h (Figure 8a). On the
604 other hand, the distribution for the CHNₓOᵧ class indicates
605 that there is a global decrease in the intensities related to the
606 CHNₓO₂⁻₄ compounds. For CHN₁Oᵧ, there is an increase in
607 the HDT-0 h sample with a decrease at HDT time 2.5 h and 5
608 h, followed by a slight increase in HDT-8 h (Figure 7a).

609 Eventually, Figure 7b clearly shows the effects of HDT on
610 the intensity and distribution of the CH and CHOₓ classes,
611 respectively, according to their oxygen atom count. Indeed, the
612 intensities respective to these molecular series significantly
613 decrease with increasing catalytic time, although there is a
614 slight increase after 8 h. For the oxygen atom count
615 distribution, one of the HTL bio-oil is centered at 4 oxygens
616 and is shifted to lower oxygen values with increasing HDT
617 time. This significant decrease in intensity also explains the
618 relative increase of the nitrogen-containing species with HDT
619 time. In fact, although the number of CHNₓOᵧ features is
620 higher than the CHOₓ one in all samples (Table 3), and Figure
621 7b indicates that the CHOₓ ionization efficiency is much
622 higher than the CHNₓOᵧ one. However, deoxygenation
623 appears to be more effective on the CHOₓ class than on the
624 CHNₓOᵧ class, which explains both the low intensity of the
625 nitrogen-containing compounds in the HTL sample and their
626 increase in the levels of HDT oils.

627 All of these results already indicate the efficiency of the
628 catalytic process, notably concerning deoxygenation. Deni-
629 trogenation can be observed, but it is less pronounced than
630 deoxygenation, as shown in Figure 8.

631 The influence of the HDT time on the molecular
632 composition of the bio-oil was also studied by a statistical
633 approach. The PCA score plot (Figure 9a) allows evidencing
634 the repeatability of the FT-ICR MS analysis with the triplicates
635 clustering together. However, more interestingly, the first
636 component, which accounts for nearly 38% of the sample
637 variability, corresponds to the catalytic time. Between 1400
638 and 1500 features specific to the non- and low-HDT time bio-
639 oils, on the one hand, and to high-HDT time samples, on the
640 other, were extracted and plotted according to their
641 heteroatom class distributions on van Krevelen's diagrams
642 and on DBE vs. carbon number graph (Figure 9b,c). The
643 heteroatom class distribution of the observed intensities for
644 each molecular series shows that CHOₓ species are specific to

non- and low-HDT bio-oils. The corresponding van Krevelen 645
diagram indicates that these species are more oxygenated and 646
saturated than those in the high-HDT-time samples. Overall, 647
among the CHOₓ compounds specific to that sort of sample, 648
there are lipids with O/C < 0.2 and H/C ~ 2. The differences 649
between the samples are even more noteworthy for the 650
nitrogen-containing components. Indeed, the CHNₓ com-
pounds are predominant in the high-HDT-time samples, and 651
the DBE vs. carbon number shows that these specific 652
compounds are more unsaturated with higher DBE values. 653
These results reinforce the previous observations, demonstrat-
ing efficiency in terms of deoxygenation. However, it is 655
noteworthy to highlight that some high-carbon number 657
compounds (C > 30), which are specific to the other samples, 658
are no longer present in the high-HDT-time samples. This 659
suggests that catalytic cracking occurs during the HDT, 660
resulting in a reduction in the mass range, as observed in 661
GPC-RID (Figure 2). 662

4. CONCLUSIONS

In this study, the hydrotreatment of a *Chlorella sorokiniana* bio-
oil obtained from a continuous HTL reaction was performed
663
during different reaction times (0 h, 2.5 h, 5 h, and 8 h) over a
664
sulfide NiW/Al₂O₃ catalyst. Global analysis and molecular
665
techniques, such as GC×GC and FT-ICR MS, were employed
666
to characterize the starting material and hydrotreated samples.
667
668

Our results showed that the increase in reaction time could
669
lead to a higher oil yield, which was associated with the
670
conversion of classes of heavy molecules that precipitated at a
671
lower reaction time, contributing to a higher solid yield. In
672
addition, as expected, the increase in the reaction time resulted
673
in HDT oil with a lower heteroatom content and a higher H/C
674
ratio.
675

GPC coupled with an RID detector showed that heavy 676
compounds were converted or hydrocracked into smaller 677
compounds. At the same time, the results obtained from GPC 678
coupled with DAD confirm the reduction of aromaticity and 679
suggest that heavy aromatic compounds were hydrogenated 680
during a longer reaction duration. 681

By performing GC×GC, it was shown, for the first time, that 682
a family of fatty nitriles in the chromatogram obtained after the 683
heating stage (HDT—0h). Experiments performed with a 684
model molecule, oleamide, under the HDT reaction conditions 685
suggested that fatty amides in the algal HTL oils can be 686
converted into fatty nitriles or carboxylic acids and then into n- 687
alkanes. 688

Moreover, the GC×GC results corroborated that oxygen-
689
and nitrogen-heterocyclic compounds are the most refractory
690
compounds to be converted during hydrotreatment. A longer
691
reaction time significantly affected the concentrations of these
692
compounds. 693

Although carboxylic acids were rapidly converted into n-
694
alkanes under the employed reaction conditions, longer
695
reaction times resulted in a higher diesel recovery mass,
696
which was associated with the conversion of fatty amides that
697
are more difficult to convert and to cracking and/or conversion
698
of the heavy fractions. 699

Lastly, the FT-ICR MS results provided complementary data 700
regarding the heaviest molecules. Chemometric analysis clearly 701
discriminates the sample's evolution. Nitrogen-containing 702
molecules represented the main contribution in the ESI (+) 703
FT-ICR MS analyses. H/C and DBE values decreased with 704
HDT time, corroborating that many compounds with less 705
706 unsaturation are formed, or the unsaturated compounds are
707 converted. Nevertheless, the refractory character of N₁
708 compounds with DBE 9 was evidenced.

709 ■ ASSOCIATED CONTENT

710 ■ Supporting Information

711 The Supporting Information is available free of charge at
712 https://pubs.acs.org/doi/10.1021/acs.energyfuels.5c00575.

713 Detailed parameters for GC×GC analyses, yields of oil,
714 solid, gas, water, and losses obtained during the
715 hydroconversion experiments, elemental analysis of
716 fresh and used NiWS/Al₂O₃ catalyst, GPC-DAD signals
717 for HTL bio-oil and HDT oils, additional GC×GC-
718 FID/TOF MS chromatograms obtained after 2.5 h and
719 8 h of HDT reaction, and reaction pathways of N-
720 heterocyclic compounds, double bound equivalent, and
721 identification by negative or positive electrospray
722 ionization technique (PDF)

723 ■ AUTHOR INFORMATION

724 Corresponding Author

725 Christophe Geantet - Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), UMR 5256, CNRS-Université Claude Bernard Lyon1, Villeurbanne 69626, France; ● orcid.org/0000-0002-3582-9244;
726
727
728 Email: christophe.geantet@ircelyon.univ-lyon1.fr

730 Authors

731 Bruno da Costa Magalhães - Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), UMR 5256, CNRS-Université Claude Bernard Lyon1, Villeurbanne 69626, France

732 Ruben Checa - Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), UMR 5256, CNRS-Université Claude Bernard Lyon1, Villeurbanne 69626, France

733 Chantal Lorentz - Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), UMR 5256, CNRS-Université Claude Bernard Lyon1, Villeurbanne 69626, France

734 Théo Voellinger - Université de Lorraine, LCP-A2MC, Metz F-57000, France

735 Jasmine Hertzog - Université de Lorraine, LCP-A2MC, Metz F-57000, France; Réseau National de Spectrométrie de Masse FT-ICR à très haut champ (FR 3624), Université de Lorraine, Metz F-57000, France; ● orcid.org/0000-0002-7946-1904

736 Vincent Carré - Université de Lorraine, LCP-A2MC, Metz F-57000, France; Réseau National de Spectrométrie de Masse FT-ICR à très haut champ (FR 3624), Université de Lorraine, Metz F-57000, France; ● orcid.org/0000-0002-0719-7366

737 Frédéric Aubriet - Université de Lorraine, LCP-A2MC, Metz F-57000, France; Réseau National de Spectrométrie de Masse FT-ICR à très haut champ (FR 3624), Université de Lorraine, Metz F-57000, France; ● orcid.org/0000-0003-2457-7974

738 Pavel Afanasiev - Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), UMR 5256, CNRS-Université Claude Bernard Lyon1, Villeurbanne 69626, France; ● orcid.org/0000-0002-4494-3495

Dorothée Laurenti - Institut de recherches sur la catalyse et l'environnement de Lyon (IRCELYON), UMR 5256, CNRS-Université Claude Bernard Lyon1, Villeurbanne 69626, France
764
765
766
767

Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.energyfuels.5c00575
768
769

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This project has received funding from ANR for the project 773 RAFBIOALG 18-CE43-009. Bruno da Costa Magalhães thanks the ANR for providing a Ph.D. grant. Financial support from 774 the IR INFRANALYTICS FR2054 for conducting the research 775 is gratefully acknowledged. We thank our partners from CEA-777 CTReg, group GB3G, Adriana Ramirez Romero, and Florian 778 Delrue for providing algae samples and our partners from 779 CEA-Grenoble, Lucie Matricon, and Anne Roubaud for 780 providing HTL algal bio-oil. This article is also dedicated to 781 a dear friend of Pr U. Ozkan, our colleague Dr. J. M. Millet 782 who left us in July 2024. 783

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