Vincent Montade

Laurent Bremond

Helena Teixeira

Thomas Kasper

Gerhard Daut

Elysée Rasoamanana

Perle Ramavovolona

Charly Favier

Fabien Arnaud

Ute Radespiel

Hermann Behling

Montane rain forest dynamics under changes in climate and human impact during the past millennia in northern Madagascar

Keywords: Palaeoecology, climate changes, pollen, demography, multi-site comparison, megadrought, fire, resilience, bottleneck, late Holocene

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Tropical rain forests are essential to humankind, for goods, services, carbon sequestration, and therefore, for the balance of global biogeochemical cycles [START_REF] Phillips | Evaluating the tropical forest carbon sink[END_REF]. However, a large part of rain forests is facing a rapid increase of anthropogenic pressures mainly through deforestation [START_REF] Unnerstall | Forest Loss -Deforestation of Rainforests[END_REF]. Deforestation is often linked to the expansion of human populations and is frequently the result of the development of agricultural practices carried out at the expense of forests. Madagascar represents a typical example; fast-human population growth coincided with accelerated forest loss and fragmentation over the past decades [START_REF] Vieilledent | Combining global tree cover loss data with historical national forest cover maps to look at six decades of deforestation and forest fragmentation in Madagascar[END_REF]. Most of the remaining rain forests are scattered across eastern Madagascar and in mountainous areas containing small 'pockets' of rain forests. Since Madagascar is a major hotspot of biodiversity [START_REF] Myers | Biodiversity hotspots for conservation priorities[END_REF], these 'pockets' are critical for protecting the unique biodiversity and in particular forest-dependent taxa, but also for securing the water resources of the island. At higher elevations, air masses generate frequent fogs and high orographic precipitation [START_REF] Gradstein | The tropical mountain forest-patterns and processes in a biodiversity hotspot[END_REF]. This moisture is recycled through evapotranspiration processes of rain forests which also prevent soils from runoff and erosion and thus contribute greatly to total water discharges for the surrounding lowlands. Some of the steeper mountain areas in Madagascar still remain relatively unaffected by human impacts, while on their foothills (at lower altitudes) human impacts are generally stronger. This discrepancy may be explained partly by the official protection of certain mountain areas but also by some climatic constraints (i.e., high rainfall and cool temperatures) and steep slopes slowing down the settling of human populations. For assessing the resilience of such forests, it is critical to study their responses to environmental changes with present-day datasets and over long timescales that go beyond historical records, and to analyse a broad range of ecological trajectories. However, obtaining high resolution palaeoecological records in such environments encompassing several millennia is difficult, as it requires sedimentary records from perennial humid areas (e.g., lake, peat bog, marsh) which are not frequent in the tropics. In Madagascar, a first study has been realised in such an isolated mountain rain forest in the northern part of the island in the National Park (NP) of Montagne d'Ambre (Figure 1 and [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF]). This NP represents a highly suitable area for such an approach, as several crater lakes occur at different elevations within the same mountainous area (Figure 1). The first sediment archive, taken from a lake located at 1,250 m above sea level (asl) near the mountaintop, has enabled the reconstruction of the main vegetation changes during the past 25,000 years before present (BP). Vegetation records have revealed a strong influence of the African Humid Period (AHP) in northern Madagascar, which shaped this study site with a replacement of montane forest by evergreen humid forest. The AHP ended at around 5,500 yr BP, marked by a precipitation decline, as evidenced in East Africa [START_REF] Tierney | Abrupt Shifts in Horn of Africa Hydroclimate Since the Last Glacial Maximum[END_REF]. Over the last millennium, a peat bog developed at this study site which currently covers a large part of the lake. Simultaneously, fires are starting to be recorded more frequently and seem to reflect the beginning of human impacts surrounding Mt. d'Ambre NP. This result is consistent with archaeological data showing an increase of human activities recorded in different regions mainly around the last millennium [START_REF] Douglass | A critical review of radiocarbon dates clarifies the human settlement of Madagascar[END_REF]. On the other hand, increase of anthropogenic activities hardly explains the development of the palustrine and peat bog vegetation, which should rather be the result of the filling up of the sedimentary basin and/or the result of an hydrological change. A gold standard in palaeoecology for disentangling drivers of past environmental change on vegetation dynamics is to provide multi-site reconstructions. In this context, our study of a new lake site at a lower elevation in Mt. d'Ambre (Lake Mahasarika,1,073 m asl) aims to better understand and infer the regional responses, and not purely site-related responses, of such a 'pocket' of rain forest to past environmental changes. More specifically, based on multi-site comparison, our detailed analysis of late Holocene (the past 4,000 yr BP) changes aims to disentangle potential different drivers of environmental and forest dynamics. Additionally, the palaeoecological results will be compared with our present knowledge on corresponding demographic dynamics of wildlife from northern Madagascar. These new data from Mt. d'Ambre and comparisons with available data will contribute to answer the following questions: When did humans start to impact ecosystems in northern Madagascar? To what extent have rain forests and wildlife been impacted locally and regionally? Is there evidence for natural climate changes impacting vegetation changes over the last millennia?

Material and methods

(a) Palaeoenvironmental study site Montagne d'Ambre NP is a volcanic massif reaching up 1,475 m asl, that spreads 35 km north-south and 15 km east-west located in northern Madagascar at about 10 km south of Antsiranana. In this region, the climate is characterised by heavy rainfalls in austral summer, which are related to the intertropical convergence zone in northern Madagascar when it moves to its southern boundary [START_REF] Li | A multimillennial climatic context for the megafaunal extinctions in Madagascar and Mascarene Islands[END_REF]. The study site, Lake Mahasarika, is one of the six crater lakes distributed at different elevations on Mt. d'Ambre. Surrounded by a dense humid montane rain forest, Lake Mahasarika is located on the north-eastern flank of the massif at an elevation of 1,073 m asl. Due to its higher elevation, climate conditions in Mt. d'Ambre contrast with surrounding lowlands. Air cooling with increasing altitude generates frequent fogs and important orographic rainfalls (annual precipitation at "La Station Forestière de la Roussette" reaches above 3,000 mm.yr -1 [START_REF] Barat | La montagne d'Ambre (nord de Madagascar)[END_REF]) allowing the development of a dense rain forest which contrasts to the dry forest, savanna and pastures situated in the lowland areas surrounding the mountain. This small isolated patch of forest on the mountain is essential for the local human population in terms of water resources (maintaining soil, etc.) and can be expected to be very sensitive to past precipitation changes. Lake Mahasarika has been selected for its shallow depth (< 10 m) allowing the use of light sediment coring materials on a lake, for its good accessibility, and for its proximity to the ecotone between humid forest and sub-humid/dry forest at about 800 m asl [START_REF] Koechlin | Flexible paleoclimate age-depth models using an autoregressive gamma process[END_REF]. Although the sub-humid/dry forest has almost entirely disappeared near to the park mainly due to human impact over the past decades [START_REF] Vieilledent | Combining global tree cover loss data with historical national forest cover maps to look at six decades of deforestation and forest fragmentation in Madagascar[END_REF], the vegetation dynamics surrounding the lake may at least partially capture the dynamic ecotone changes, and certainly better so than forest located at higher elevations.

(b) Sediment coring and age-depth modelling Fieldwork was carried out in August 2018. Based on bathymetry, the sediment core LMAHA-18 was taken from the central and deepest part of the lake (7 m water depth; 12.53509S, 49.17701E). Core LMAHA-18 was sampled with a Livingstone piston sediment corer operated from a small platform settled on two rubber boats. Using sampling tubes of ca. 5 cm diameter and 120 cm length, a total of 6 sections of about 100 cm sediment length were sampled with overlaps of 10-50 cm length. Preserved in aluminium tubes for transport to the laboratory, sediment was extruded from each aluminium tube and split in two sister cores, LMAHA-18a and LMAHA-18b. Each sister core was stored under cool (4°C) and dark conditions at the laboratory facilities (LMAHA-18a at the Friedrich-Schiller-University Jena and LMAHA-18b at the University of Goettingen, Germany). Cores were photographed and magnetic susceptibility was scanned in 3 mm steps with three replicate measurements using a MS2E surface scanning sensor (Bartington Instruments). Using lithological description, patterns of magnetic susceptibility, and specific marker layers (electronic supplementary material, Figure S1), the different core sections were aligned to a composite master sequence LMAHA-18. The age-depth model is based on 16 AMS radiocarbon dates of 15 bulk sediment samples and 1 plant macro-remain (Table S1), performed at either the Poznań radiocarbon laboratory (Poland) or the LMC14 (Gif-sur-Yvette, France) laboratory. The age of the sediment surface is considered as modern, and was thus set to the year of coring (AD 2018 = -68 calibrated yr BP). The age model was performed as a function of the composite depth with the RStudio software using the R-package "Bacon" (V. 4.0.5) [13] and using SHCal20 calibration curve and bomb 14 C curve [START_REF] Hogg | SHCal20 Southern Hemisphere Calibration, 0-55,000 Years cal BP[END_REF][START_REF] Hua | Atmospheric Radiocarbon for the Period 1950-2010[END_REF].

(c) Palaeoecological analyses

For pollen extraction, 28 subsamples of 0.5 cm 3 from LMAHA-18 were processed following standard chemical techniques with chloridric acid, potassium hydroxide, fluorhydric acid and acetylosis [START_REF] Faegri | Textbook of Pollen Analysis[END_REF]. One tablet of exotic Lycopodium spores (20848 +/-1546, batch number 1031) was added to each sample to estimate pollen concentration. A minimum sum of 300 terrestrial pollen grains was counted for each subsample using a light microscope at 400x magnification, and pollen and fern-spore percentages were calculated on the terrestrial pollen sum. Pollen and spore identification were based on several atlases [START_REF] Gosling | Atlas of the tropical West African pollen flora[END_REF][START_REF] Rasoloarijao | Pollen morphology of melliferous plants for Apis mellifera unicolor in the tropical rainforest of Ranomafana National Park, Madagascar[END_REF][START_REF] Straka | Palynologia Madagassica et Mascarenica[END_REF][START_REF] Schüler | Atlas of pollen and spores and their parent taxa of Mt Kilimanjaro and tropical East Africa[END_REF], the online African Pollen Database (https://africanpollendatabase.ipsl.fr/#/home) and the reference collections from University of Goettingen (http://www.gdvh.uni-goettingen.de/) and the University of Montpellier (https://data.oreme.org/observation/pollen). To investigate compositional changes in the tree community, a Principal Component Analysis (PCA) was performed on square-root transformed relative abundances of arboreal pollen taxa, defined as the percent count of each taxon relative to the total number of arboreal pollen grains in a sample (electronic supplementary material, Figure S3). For extraction of charcoal particles, 1 cm 3 of sediment was taken every cm along the LMAHA-18 core. Each sample was soaked in a 3% NaP 2 O 4 solution plus bleach for several hours to deflocculate the sediment and oxidise organic matter. The samples were sieved through a 160 μm mesh and the carbon particles were counted using a x40 magnification stereomicroscope. Bulk organic δ 13 C was measured against certified standards (L-Prolin, EDTA and USG65) and reported in standard δ notation (‰) against Vienna Pee Dee Belemnite (VPDB) and Air, respectively. Relative errors based on triplicate measurements are 0.05 ‰ for δ 13 C.

(d) Demographic dynamics of wildlife in northern Madagascar

A comprehensive review was conducted on published molecular population demographic studies conducted on Malagasy taxa from northern Madagascar. Studies were collected from the literature using a combination of the following keywords: population genetics, population dynamics, phylogeography, demography, demographic modelling, coalescence, bottleneck, expansion, Quaternary climate change, vegetational shifts and Madagascar. All studies were carefully screened to confirm that the study species were in fact distributed and sampled in northern Madagascar, roughly defined as the area ranging from Nosy Be to the Loky-Manambato region, to achieve the best possible congruence with the palaeoenvironmental records. Studies on species that only occurred outside northern Madagascar were excluded from our review. We reviewed the evidence from studies using different molecular markers (i.e., mtDNA, microsatellite loci and genome-wide SNPs) and various demographic approaches. Studies that fulfilled our criteria were evaluated regarding taxonomic representation (i.e., mammal, bird, amphibian, non-avian reptile, plants) and habitat type (i.e., adapted to dry forest, humid forest or both habitats. Finally, the direction of the detected population demographic change (i.e., constant population size, population bottleneck, expansion or size recovery) was assigned to the palaeoecological periods of interest whenever possible. When multiple studies were available for a single taxon, they were all included in the review for comparative reasons.

Results

Combining pollen, charcoal, sedimentological changes and isotopic data, three distinct periods were distinguished within the Late Holocene which were each characterised by different plant assemblages.

(a) Environmental conditions from 4,000 years to 1,100 yr BP (Period 1) During this period the sediment was dark to light brown, i.e., homogeneous and organic rich. Except for some peaks of magnetic susceptibility, interpreted as event related deposits (electronic supplementary material, Figure S1), the sediment accumulation rate (SAR) was relatively low (0.1 cm/yr) and remained constant showing low erosion. With a relatively low proportion of Poaceae (around 10%, Figure 2b), the rain forest was well-established around the study site, as it is also indicated by the dominance of arboreal pollen (Mallotus, Noronhia, Celtis and Moraceae-Urticaceae; electronic supplementary material, Figure S2 and Table S1). However, the slow decline in PCA Axis-1 values (Figure 2a) reflects that changes in tree composition have occurred during this period. Such changes are marked by, e.g., Noronhia peaks (24%) at ca. 2,550 yr BP. Mallotus increased progressively and reached a maximum (27%) at ca. 1,150 yr BP (electronic supplementary material, Figure S2). Mallotus is a potential indicator of early successional pioneer trees and its increase may provide evidence for increased disturbances. Low values of δ 13 C generally below -24 ‰ also confirm the dominance of C3 forest vegetation in the catchment of the lake (Figure 2d). Furthermore, few charcoal particles are recorded only occasionally, indicating a very low level of fire activity (Figure 2e and2f). (b) Ecological changes and fire increase between 1,100 and 550 yr BP (Period 2) During this period, the sediment had the same texture as in the previous period and SAR remained broadly in the same range with values slightly higher between 0.1 and 0.2 cm/yr (electronic supplementary material, Figure S1). On the other hand, a major vegetation change was recorded and characterised by a two-step increase of Poaceae and Cyperaceae pollen from 1,100 and 900 yr BP up to 30% each (Figure 2b and2c). The abrupt decrease in the values of axis-1 of the PCA indicates a major change in the tree composition from 1,100 yr BP onwards (Figure 2a). This shift was slightly preceded by a change in forest dynamics (electronic supplementary material, Figure S2), with a synchronous decline of Mallotus (<10%) and increase of Trema (>10%). Trema is generally associated with light demanding trees living at forest edges [START_REF] Koechlin | Flexible paleoclimate age-depth models using an autoregressive gamma process[END_REF]. It may have outcompeted Mallotus with increased disturbances right before the increase of herbs. From 900 yr BP, the changes of the tree composition continued (Figure 2a). Arboreal pollen taxa related to low disturbances and humid rain forest habitat decreased (e.g. Celtis, Podocarpus, Pandanus) at the expanse of new tree taxa (Garcinia, Ziziphus, Ilex, Araliaceae) showing that a tipping point was reached and the rain forest composition changed substantially (electronic supplementary material, Figure S2 and Table S2). The presence of Impatiens (electronic supplementary material, Figure S2), typical of small shrubs growing in disturbed rain forest and in the rain forest margins, also provides evidence for an increase of forest disturbance. As pollination of Impatiens is entomophilous (producing less pollen than anemophilous plants), pollen grains of Impatiens have a low dispersal probability. These pollen grains were more likely deposited near the parent plants, indicating that disturbances occurred locally at the study site. The δ 13 C values around -22‰ from 900 yr BP (Figure 2d) indicate an increased proportion of C4 vegetation within the catchment of the lake. This confirms that vegetation changes (increase of Poaceae and Cyperaceae) recorded by pollen data occurred locally at the study site. Charcoal particles also showed a two-step increase in values (Figure 2e and2f). A first increase occurred from ca. 1,070 yr BP onwards and reached rapidly values above 70 particles/cm 3 and 10 particles/cm 2 /yr with a peak around 900 yr BP. The second one occurred from 800 yr BP onwards and reached values above 200 particles/cm 3 and 30 particles/cm 2 /yr with a maximum at around 600 particles/cm 3 and 100 particles/cm 2 /yr at ca. 630 yr BP. The increase in fires might be responsible for increased forest disturbances and development of herbs. If fires would have occurred in the catchment area of the lake, forest fires would have resulted in strong sediment discharges into the lake, since it is characterised by a small catchment area (ca. 2.3 km 2 ) and steep slopes. However, the increase of fires was not combined with synchronous increase of magnetic susceptibility showing no major increase of erosion from 1,100 yr BP onward (Figure 4). Consequently, fires certainly occurred in the region of Mt. d'Ambre but not locally at the lake catchment and do not well explain local vegetation dynamics evidenced by pollen and δ 13 C data (Figure 2d). Another potential explanation of local increase of forest disturbances could be a decrease of the lake level related to a decrease in precipitation. Indeed, an important lake level decrease may have allowed a development of C4-grass vegetation (Poaceae) at the shore of the lake and Cyperaceae in shallow lake margins as it is observed today. This lake level decrease, resulting from reduced precipitation, would also be a possible explanation of tree compositional changes.

(c) Vegetation and fire dynamics since the last 550 yr BP (Period 3) After 550 yr BP, sediments turned to light brown and SAR started to increase rapidly from 250 yr BP, reaching values above 0.2 cm/yr at ca. 100 yr BP, and then values above 1 cm/yr during the past century (electronic supplementary material, Figure S1). Near the core top, the SAR is expected to increase as the sediment compaction is reduced. Among the main vegetation changes, values of Poaceae pollen decreased progressively from 30 to 10% and Cyperaceae fluctuated between 25 and 10% (Figure 2b andc). Except in one sample, values of PCA axis-1 remain in the same range (Figure 2a), showing that the new tree composition was maintained with the dominance of Mallotus, Elaeocarpus, Norhonia, Trema and Araliaceae, between 4 and 10%, respectively (electronic supplementary material, Figure S1 and Table S2). The taxa occurring in the previous period (Ziziphus, Gacinia, Ilex), were still represented with the same range of values between 2 and 4%. The δ 13 C values remained around -22‰, showing a mixture of C3 and C4 plants within the catchment of the lake (Figure 2d). This confirms the relatively high proportion of herbaceous vegetation after 550 yr BP. Although charcoal particles were found in every cm of the core (Figure 2e and2f), the concentration and influx showed a general decrease with values generally below 50 particles/cm 3 and 10 particles/cm 2 /yr. During the last century, charcoal influx increased due to SAR increase with values above 10 particles/cm 2 /yr and with maximum values around 30 particles/cm 2 /yr while charcoal concentration remained in the same range of values.

(d) Demographic dynamics of wildlife in northern Madagascar A total of 17 demographic studies covering 19 studied species distributed across northern Madagascar fulfilled our criteria and were considered here (electronic supplementary material, Table S3). The studies were biased towards mammals (47.4%) and non-avian reptiles (36.8%), with the remaining taxa (amphibian, birds and plants) being represented by a single species each (5.3%). For non-avian reptile species, the timing of the demographic event is either unknown (Phelsuma dorsivittata) or dated back to the late Pleistocene (>12,000 yr BP), precluding conclusions about recent demographic dynamics. Studies on mammals, however, provide important insights about recent demographic dynamics in Malagasy wildlife. Except for three species for which demographic events were not dated (Microcebus tavaratra [START_REF] Sgarlata | Genetic Differentiation and Demographic History of the Northern Rufous Mouse Lemur (Microcebus tavaratra) Across a Fragmented Landscape in Northern Madagascar[END_REF], Eliurus tanala [START_REF] Rakotoarisoa | Late Quaternary climatic vegetational shifts in an ecological transition zone of northern Madagascar: insights from genetic analyses of two endemic rodent species[END_REF] and Microgale brevicaudata [START_REF] Olson | Phylogeography of Microgale brevicaudata (Tenrecidae) and Description of a New Species from Western Madagascar[END_REF]), all other studied terrestrial mammals (i.e., lemurs and rodents) suffered a population decline predating or during the late Holocene (i.e., before or during Period 3; Figure 3 and electronic supplementary material, Table S3). A reduction in population size was also reported for Chaerephon leucogaster [START_REF] Lamb | Genetically and geographically isolated lineages of a tropical bat (Chiroptera: Molossidae) show demographic stability over the late Pleistocene[END_REF], a bat species, during the Late Holocene (Period 3 ;but see [START_REF] Tidière | Species delineation and genetic structure of two Chaerephon species (C. pusillus and C. leucogaster) on Madagascar and the Comoro archipelago[END_REF]). Notably, demographic modelling with the composite-likelihood method implemented in fastsimcoal2 for Microcebus arnholdi [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF] revealed two consecutive population bottlenecks, one predating Period 3 (~5,000 yr BP) and one during Period 2 (~1,000 yr BP). Demographic modelling with the Approximate Bayesian Computation (ABC) for two additional lemur species (i.e., Propithecus perrieri and P. tattersalli [START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF]) also detected a population decline within the last millennium (Period 2, Figure 3). Species demographic response to past environmental vs. human pressures may differ among ecological specialist vs. generalist taxa. The reviewed studies revealed that all mammals adapted to dry habitats exhibited signatures of a population decline (Propithecus perrieri, Eliurus carletoni, Chaerephon leucogaster, Table S3). Except for Furcifer pardalis (that underwent a population expansion in the late Quaternary; [START_REF] Kuhn | Drivers of unique and asynchronous population dynamics in Malagasy herpetofauna[END_REF]) all taxa adapted to humid habitats also underwent a population bottleneck or kept a constant population size (Microcebus arnholdi, Eliurus tanala, Mantella crocea, Phelsuma dorsivittata). In contrast, no clear demographic trend was evident in species adapted to both dry and humid habitats (Microcebus tavaratra, Propithecus tattersalli, Microgale brevicaudata, Myotis goudoti, Dicrurus forficatus, Laliostoma labrosum, Calumma boettgeri, Geckolepis maculata, Mimophis occultus, Zonosaurus madagascariensis), suggesting that ecological specialised taxa are more prone to environmental changes than generalist taxa. Except for [START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF] and [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF], all studies assumed that population structure was negligible, and applied either a single demographic approach (statistical tests or a model-free approach) or a Bayesian Skyline Plot (BSP) coupled with mismatch distribution/ neutrality tests. For Microcebus tavaratra [START_REF] Sgarlata | Genetic Differentiation and Demographic History of the Northern Rufous Mouse Lemur (Microcebus tavaratra) Across a Fragmented Landscape in Northern Madagascar[END_REF] and Eliurus tanala [START_REF] Rakotoarisoa | Late Quaternary climatic vegetational shifts in an ecological transition zone of northern Madagascar: insights from genetic analyses of two endemic rodent species[END_REF], the neutrality tests detected a population expansion, while BSP suggested a constant population size. The neutrality tests were also inconclusive for Eliurus carletoni [START_REF] Rakotoarisoa | Late Quaternary climatic vegetational shifts in an ecological transition zone of northern Madagascar: insights from genetic analyses of two endemic rodent species[END_REF]. In contrast, the demographic methods (i.e., model-free and model-constrained approaches) implemented by [START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF] and [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF] revealed a synchronous population bottleneck for Microcebus arnholdi, Propithecus perrieri and P. tattersalli during the late Holocene (period 2 and 1). S2 for a review of all the studies currently available). The red, blue and yellow bars represent distinct demographic dynamics (population bottleneck, expansion and constant size, respectively). Dashed bars highlight methods that use model-constrained approaches (i.e., fastsimcoal2 and ABC). The remaining studies rely on model-free methods. The late Holocene was divided in the three periods highlighted by the palaeoenvironmental reconstructions of Lake Mahasarika and defined in the text [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF][START_REF] Rakotoarisoa | Late Quaternary climatic vegetational shifts in an ecological transition zone of northern Madagascar: insights from genetic analyses of two endemic rodent species[END_REF][START_REF] Lamb | Genetically and geographically isolated lineages of a tropical bat (Chiroptera: Molossidae) show demographic stability over the late Pleistocene[END_REF][START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF][START_REF] Quéméré | Genetic data suggest a natural prehuman origin of open habitats in northern Madagascar and question the deforestation narrative in this region[END_REF][START_REF] Rakotoarisoa | A phylogeographic study of the endemic rodent Eliurus carletoni (Rodentia: Nesomyinae) in an ecological transition zone of Northern Madagascar[END_REF][START_REF] Ratrimomanarivo | Geographic and Phylogeographic Variation in Chaerephon leucogaster (Chiroptera: Molossidae) of Madagascar and the Western Indian Ocean Islands of Mayotte and Pemba[END_REF].

Discussion (a) The period prior to permanent human settlement in northern Madagascar

The exact schedule for the settlement of humans in Madagascar is still partly debated [e.g., [START_REF] Douglass | A critical review of radiocarbon dates clarifies the human settlement of Madagascar[END_REF][START_REF] Hansford | Early Holocene human presence in Madagascar evidenced by exploitation of avian megafauna[END_REF][START_REF] Anderson | New evidence of megafaunal bone damage indicates late colonization of Madagascar[END_REF]. Recent evidence for early to mid-Holocene (>5000 yr BP) arrival of humans on the island is solely based on indirect indicators (e.g., cut-marks performed on hippopotamus and extinct elephant birds bones [START_REF] Hansford | Early Holocene human presence in Madagascar evidenced by exploitation of avian megafauna[END_REF]), and no archaeological artefacts have so far been found associated with these findings. This suggests that even if humans were already inhabiting Madagascar during these early times, they seem to not have impacted the ecosystems sufficiently enough to leave traces in the existing palaeoecological records. The clear first signals of permanent human settlements on Madagascar are generally available for the time period between 2,000 and 1,000 yr BP [e.g., [START_REF] Douglass | A critical review of radiocarbon dates clarifies the human settlement of Madagascar[END_REF][START_REF] Razanatsoa | Synergy between climate and human land-use maintained open vegetation in southwest Madagascar over the last millennium[END_REF][START_REF] Broothaerts | Vegetation changes and sediment dynamics in the Lake Alaotra region, central Madagascar[END_REF]. However, the earliest proof of human civilization with town establishment in Madagascar has been dated at 1,000 yr BP and it stems from the harbour of Mahilaka in the Northwest. Located at the end of a bay, Mahilaka was inhabited by muslim population and was connected to the Comoro Islands and probably east Africa by maritime trade routes [START_REF] Englong | High-Resolution Multiproxy Record of Environmental Changes and Anthropogenic Activities at Unguja Ukuu, Zanzibar, Tanzania during the Last 5000 Years[END_REF][START_REF] Crowther | Ancient crops provide first archaeological signature of the westward Austronesian expansion[END_REF]. This population seems to be related to the westward Austronesian expansion that had developed maritime trade and agricultural practices based on rice cultivation in the region from 1,250 yr BP onwards [START_REF] Crowther | Ancient crops provide first archaeological signature of the westward Austronesian expansion[END_REF]. Correspondingly, human impact with an increase of anthropogenic burning has been recently suggested on the near-by Malagasy Nosy Be Island from 1,300 yr BP onwards [START_REF] Reinhardt | Rain Forest Fragmentation and Environmental Dynamics on Nosy Be Island (NW Madagascar) at 1300 cal BP Is Attributable to Intensified Human Impact[END_REF]. Our new data from Lake Mahasarika combined with previously published data from Lake Maudit show very low occurrences of fires prior to 1,100 yr BP (Figure 4). This suggests that permanent human settlements did not exist in the areas surrounding the Mt. d'Ambre before this time and that the observed vegetation dynamics would thus have been caused by natural factors such as climate changes. Palaeoclimate records from Mid-to Late Holocene from northwestern Madagascar have revealed a multi-millennial drying trend in this region combined with frequent occurrences of megadroughts [START_REF] Li | A multimillennial climatic context for the megafaunal extinctions in Madagascar and Mascarene Islands[END_REF][START_REF] Wang | The African Humid Period, rapid climate change events, the timing of human colonization, and megafaunal extinctions in Madagascar during the Holocene: Evidence from a 2m Anjohibe Cave stalagmite[END_REF]. Sedimentological changes at higher altitude of Lake Maudit (1,250 m asl) in Mt. d'Ambre in Northern Madagascar are consistent with an overall drying trend that intensified at the end of the AHP from 5,500 yr BP onwards [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF]. Starting from 4,000 yr BP, Mahasarika pollen assemblages reveal that dominant tree taxa were continuously changing (Figure 2) and Mallotus progressively increased until 1,100 yr BP. As a typical taxon of early successional trees, it might indicate that rain forest, adapted to more drought-related disturbances or mesic conditions, may have developed due to the effect of reduced rainfall and occurrences of megadroughts. Forest taxa from Lake Maudit did not show such changes, suggesting that forest was not affected by climate change at higher altitudes [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF]. As humidity increases with elevation in Mt. d'Ambre due to decreasing temperatures, a regional decline in precipitation should have affected the vegetation at lower altitudes, such as at Lake Mahasarika (1,073 m asl), more than rain forests near the mountain top, such as at Lake Maudit (1,250 m asl), where orographic precipitation and cloud cover is maximum. This altitude-dependent effect seems to be confirmed by our study which suggests that ecosystems from lower elevations may be more affected by regional precipitation decline than mountain environments which are under the influence of orographic rainfall that still maintains rain forest. A palaeoecological record from an even lower elevation would allow us to test this hypothesis for this region. The available demographic studies for Northern Madagascar showed that all mammals, birds, amphibians, and non-avian reptiles ranging from Nosy Be to the Loky-Manambato region exhibited a signature of a population size change (expansion or decline) pre-dating the Late Holocene, supporting major environmental changes in the region long before humans impacted these ecosystems. Decadal-resolution time series of BIT index variability from Lake Challa [START_REF] Buckles | Interannual and (multi-)decadal variability in the sedimentary BIT index of Lake Challa, East Africa, over the past 2200 years: assessment of the precipitation proxy[END_REF] and δ 18 O speleothem profile from Anjohibe Cave [START_REF] Li | A multimillennial climatic context for the megafaunal extinctions in Madagascar and Mascarene Islands[END_REF] showing precipitation changes in East Africa and Northwest Madagascar (the dark blue line represents a smooth spline). c and d Comparison of Poaceae percentages, charcoal concentrations and magnetic susceptibility (K) between Lake Maudit [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF], L. Maharika (this study) from Northern Madagascar and L. Amparahibe [START_REF] Reinhardt | Rain Forest Fragmentation and Environmental Dynamics on Nosy Be Island (NW Madagascar) at 1300 cal BP Is Attributable to Intensified Human Impact[END_REF] from Northwest Madagascar. e Black curve represents the number of radiocarbon dates performed on archaeological samples obtained from an archaeological data synthesis [START_REF] Douglass | A critical review of radiocarbon dates clarifies the human settlement of Madagascar[END_REF]. Purple curve represents the estimated changes in the effective human population size [START_REF] Pierron | Genomic landscape of human diversity across Madagascar[END_REF].

(b) Human impact and environmental changes in Northern Madagascar Since 1,100 yr BP, the permanent occurrence of charcoal particles with increase of influx combined with the development of grasses in the Mahasarika record suggests a fire activity increase related to an increase of anthropogenic burning in the surrounding lowland region. On Nosy Be Island, charcoal particles increased strongly together with grasses and were correlated with peaks of erosion, supporting local disturbances in the catchment from anthropogenic burning and this about 200 years earlier than in Mt. d'Ambre (Figure 4). However, the pattern of changes evidenced from the Mahasarika record differ in more than just dating. First, the tree composition changes that suggest increased disturbances with a maximum of Mallotus followed by an increase of Trema, a tree typically growing in disturbed forests and at forest edges, slightly preceded the fire increase. This shows that the fire increase detected by the Mahasarika charcoal record was preceded by increased disturbances and not vice versa. Second, the increase of charcoal particles was not associated with a marked peak of erosion which excludes local fires at the lake catchment. Consequently, the local development of grasses and sedges evidenced by pollen assemblages and δ 13 C results with the establishment of new forest composition cannot be explained by the increase of fire frequency within the lake catchment. By contrast, a lake level decrease, allowing the development of herbs and sedges (reaching maxima between 900 and 550 yr BP) at the lake shore, as it can be seen today, would explain better the observed results from Lake Mahasarika. The complementary results from Lake Maudit suggest the same overall process; the charcoal increase was also not associated with any increase of local erosion during the last millennium (Figure 4). Erosion signal in the central part of the lake (coring site) was even reduced by the development of a peat bog from ca. 1,000 yr BP onwards, also suggesting a lake level decrease at that time combined with the local development of grasses and sedges. Altogether, local changes evidenced in Mt. d'Ambre are initially (ca. 1,100 -1,000 yr BP) better explained by natural precipitation decline and lake level decline , and then by the increase of anthropogenic burning near Mt. d'Ambre. Major precipitation decreases or a megadrought, starting around 1,000 yr BP and culminating at ca. 900 yr BP, is also supported by palaeoclimatic proxies from other sites in Madagascar [START_REF] Li | A multimillennial climatic context for the megafaunal extinctions in Madagascar and Mascarene Islands[END_REF][START_REF] Wang | The African Humid Period, rapid climate change events, the timing of human colonization, and megafaunal extinctions in Madagascar during the Holocene: Evidence from a 2m Anjohibe Cave stalagmite[END_REF][START_REF] Scroxton | Hemispherically in-phase precipitation variability over the last 1700 years in a Madagascar speleothem record[END_REF][START_REF] Voarintsoa | Multiple proxy analyses of a U/Th-dated stalagmite to reconstruct paleoenvironmental changes in northwestern Madagascar between 370CE and 1300CE[END_REF], the Island of Rodrigues [START_REF] Li | A multimillennial climatic context for the megafaunal extinctions in Madagascar and Mascarene Islands[END_REF] and East Africa [START_REF] Buckles | Interannual and (multi-)decadal variability in the sedimentary BIT index of Lake Challa, East Africa, over the past 2200 years: assessment of the precipitation proxy[END_REF]. The Mt. d'Ambre palaeoecological records reveal that the increase of anthropogenic burning and human expansion occurred during the subsequent period of reduced humidity, contemporaneous of the Medieval Climate Anomaly in East Africa and in the Northern Atlantic region (ca. 1,000-700 yr BP [START_REF] Goosse | The Medieval Climate Anomaly in Europe in simulations with data assimilation[END_REF][START_REF] Lüning | Hydroclimate in Africa during the Medieval Climate Anomaly[END_REF]). A combination of natural and anthropogenic factors may subsequently have led to the dramatic ecological changes observed in Northern Madagascar, such as additional animal population declines evidenced by demographic modelling for Microcebus arnholdi (Mt. d'Ambre NP), Propithecus perrieri (Analamerana -Andrafiamena) and P. tattersalli (Loky -Manambato; reviewed here) or megafauna extirpation, as revealed by archaeological data [START_REF] Crowley | A refined chronology of prehistoric Madagascar and the demise of the megafauna[END_REF][START_REF] Railsback | Relationships between climate change, human environmental impact, and megafaunal extinction inferred from a 4000-year multi-proxy record from a stalagmite from northwestern Madagascar[END_REF]. Although a wide range of approaches are currently available and were used to reconstruct species demographic history from molecular data (e.g., neutrality tests, model-free and model-based approaches), the inference of recent population bottlenecks during the last millennia was only successful with model-based approaches, such as fastsimcoal2 and ABC. Moreover, while some studies, implementing mismatch distribution/neutrality tests and Bayesian Skyline Plots, detected different demographic trends, the studies implementing model-free and model-based approaches [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF][START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF] revealed congruent dynamics. Altogether, our demographic review confirms that model-based approaches are a very valuable tool to detect recent demographic changes that are not detected by other demographic approaches, and highlights the importance of using more than one method for demographic inferences. Change in subsistence strategies with populations shifting from hunter-gatherer to herding-farming have also been proposed as a potential explanation of the major ecological changes around 1,000 yr BP [START_REF] Godfrey | A new interpretation of Madagascar's megafaunal decline: The "Subsistence Shift Hypothesis[END_REF][START_REF] Hixon | Late Holocene spread of pastoralism coincides with endemic megafaunal extinction on Madagascar[END_REF][START_REF] Hixon | Drought Coincided with, but Does Not Explain, Late Holocene Megafauna Extinctions in SW Madagascar[END_REF][START_REF] Burns | Rapid human-induced landscape transformation in Madagascar at the end of the first millennium of the Common Era[END_REF]. However, considering the strong hydroclimatic change during this period, and without additional palaeorecords combined with archaeological data to obtain a robust regional synthesis, the causal links between the observed changes, in particular during the critical period of transition, cannot be fully clarified at the island scale. In particular it remains open, if anthropogenic burning increased because precipitation decreased which may also have led human population to use and develop new substance strategies and/or colonise new areas. It should also be considered whether the precipitation decrease may have promoted anthropogenic fire expansion and the occurrence of disasters such as megafires.

(c) Period of the last 550 yr BP Over the past few centuries, data from Lake Maudit have not shown particular changes and vegetation dynamics but instead mainly reflected the development of a large local peat bog [START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF]. In contrast, from 550 yr BP onwards at Lake Mahasarika and, starting from ca. 200 years earlier at Nosy Be, the proportion of grasses decreased in congruence with a decrease of fires. This pattern, particularly the decrease of fires, might be explained in several ways: First, starting approx. 600 yr BP, archaeological data suggested a human population decline in the Northwest at Mahilaka and the surrounding villages of Ampasindava Bay [START_REF] Dewar | The culture history of Madagascar[END_REF][START_REF] Radimilahy | Mahilaka, an eleventh to fourteenth century Islamic port: The first impact of urbanism in Madagascar[END_REF]. Although this remains to be confirmed at the local and regional scale around Mt. d'Ambre with archaeological data, a decline in human occupation in Northern Madagascar could have resulted in a reduction of anthropogenic burning. Second, several precipitation records have revealed an increase in humidity from about 500 yr BP onwards [START_REF] Scroxton | Hemispherically in-phase precipitation variability over the last 1700 years in a Madagascar speleothem record[END_REF]. This may have reduced the risk of catastrophic fire events, such as megafires, and may also explain a general reduction in fires. Third, in case Northern Madagascar was entirely forested, the abrupt increase of fires at a regional scale at the beginning of the last millennium might have produced large amounts of charcoal particles. Subsequently, and as a consequence of more frequent fires, fuel availability for large fires should have decreased, which could also explain the observed decrease in carbon particles emitted. These different hypotheses, or a combination of several of them, seem likely but require additional archaeological and palaeoecological data to be tested. Finally, during the last century, a renewed increase of fires is recorded in the Mahasarika sediment core (Figure 2e andf). Additional study sites are necessary to confirm this trend which is evidenced by charcoal influx. A fire increase during the last century might reflect the effect of the European colonisation in the Mt. d'Ambre region, where Jofreville has been established with development of agriculture from the 1900s [START_REF] Drouhard | Note sur la culture du Quinquina dans la forêt d'Ambre Nord de Madagascar (Diégo-Suarez)[END_REF].

Summary

This multi-proxy and multi-site comparison of lacustrine cores, located in the North of Madagascar in the mountain range of the Mt. d'Ambre NP, revealed a major drought, starting approximately 1,100 years ago, causing a significant drop in the water level at the two lacustrine study sites (Lakes Mahasarika and Maudit). Under low lake levels, palustrine and peat bog vegetation started to develop on the shore of the two lakes. In addition, the rain forest closer to the edge of the montane rain forest zone has been affected by precipitation decrease, as indicated by an increase of pioneer trees. Recorded a few decades later, anthropogenic burning spread throughout the study region, but was limited to the low-altitude areas surrounding the Mt. d'Ambre. Very likely fires were therefore not the driving force behind these early changes observed in the lake catchment areas, as it has also been observed for other study sites, such as on the island of Nosy Be [START_REF] Reinhardt | Rain Forest Fragmentation and Environmental Dynamics on Nosy Be Island (NW Madagascar) at 1300 cal BP Is Attributable to Intensified Human Impact[END_REF]. Although this does not dismiss the importance humans had subsequently in terms of impact on ecosystems, this work demonstrates that the natural drought that intensified regionally about one thousand years ago, significantly impacted the ecosystems independently of anthropogenic activities. The increasing number of inferred population bottlenecks of wildlife at a regional scale during the last millennium, as evidenced by the review of demographic studies, likely resulted from the combination of both human-related impact and environmental changes (i.e., precipitation decline). Remaining important questions, therefore, are not just how precisely humans have influenced their environments, but also whether the natural precipitation decrease caused humans to change their substance strategies and/or to move to new areas. Although the current increase in anthropogenic activities is eroding the resilience of forests, these montane rain forests have shown substantial resilience to climate change during the past millennia. Continuing to protect such areas therefore seems very relevant and essential in the context of global warming. Finally, this work shows that multi-proxy approaches and multi-site comparisons provide essential evidence for distinguishing the different factors driving environmental changes in order to better understand ecosystem functioning over the long term.

Figure 1 .

 1 Figure 1. a Distribution map of natural biomes in Madagascar [9]. b Satellite image of Montagne d'Ambre (source: Google Earth) indicating location of Lake Mahasarika and Lake Maudit.

Figure 2 .

 2 Figure 2. Synthetic palaeoenvironmental reconstructions obtained from the sediment core recovered in Lake Mahasarika (MAHA-18). a Axis-1 of the Principal Component Analysis performed on arboreal pollen taxa reflecting tree compositional changes. b Poaceae pollen grains. c Cyperaceae pollen grains. d δ 13 C measured on bulk organic fraction. e Concentration (black) and f influx (grey) of charcoal particles (>160 μm). The vertical lines demarcate the succession of the three main periods (P) of different environmental conditions.

Figure 3 .

 3 Figure 3. Review results on population demographic dynamics during the past 15,000 years for Malagasy wildlife occurring in northern Madagascar based on molecular datasets. Only studies with dated demographic events are included in the figure (see electronic supplementary material TableS2for a review of all the studies currently available). The red, blue and yellow bars represent distinct demographic dynamics (population bottleneck, expansion and constant size, respectively). Dashed bars highlight methods that use model-constrained approaches (i.e., fastsimcoal2 and ABC). The remaining studies rely on model-free methods. The late Holocene was divided in the three periods highlighted by the palaeoenvironmental reconstructions of Lake Mahasarika and defined in the text[START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF][START_REF] Rakotoarisoa | Late Quaternary climatic vegetational shifts in an ecological transition zone of northern Madagascar: insights from genetic analyses of two endemic rodent species[END_REF][START_REF] Lamb | Genetically and geographically isolated lineages of a tropical bat (Chiroptera: Molossidae) show demographic stability over the late Pleistocene[END_REF][START_REF] Salmona | Climate change and human colonization triggered habitat loss and fragmentation in Madagascar[END_REF][START_REF] Quéméré | Genetic data suggest a natural prehuman origin of open habitats in northern Madagascar and question the deforestation narrative in this region[END_REF][START_REF] Rakotoarisoa | A phylogeographic study of the endemic rodent Eliurus carletoni (Rodentia: Nesomyinae) in an ecological transition zone of Northern Madagascar[END_REF][START_REF] Ratrimomanarivo | Geographic and Phylogeographic Variation in Chaerephon leucogaster (Chiroptera: Molossidae) of Madagascar and the Western Indian Ocean Islands of Mayotte and Pemba[END_REF].

Figure 4 .

 4 Figure 4. Synthesis of environmental changes during the past 3,000 yr BP. a and bDecadal-resolution time series of BIT index variability from Lake Challa[START_REF] Buckles | Interannual and (multi-)decadal variability in the sedimentary BIT index of Lake Challa, East Africa, over the past 2200 years: assessment of the precipitation proxy[END_REF] and δ18 O speleothem profile from Anjohibe Cave[START_REF] Li | A multimillennial climatic context for the megafaunal extinctions in Madagascar and Mascarene Islands[END_REF] showing precipitation changes in East Africa and Northwest Madagascar (the dark blue line represents a smooth spline). c and d Comparison of Poaceae percentages, charcoal concentrations and magnetic susceptibility (K) between Lake Maudit[START_REF] Teixeira | Past environmental changes affected lemur population dynamics prior to human impact in Madagascar[END_REF], L. Maharika (this study) from Northern Madagascar and L. Amparahibe[START_REF] Reinhardt | Rain Forest Fragmentation and Environmental Dynamics on Nosy Be Island (NW Madagascar) at 1300 cal BP Is Attributable to Intensified Human Impact[END_REF] from Northwest Madagascar. e Black curve represents the number of radiocarbon dates performed on archaeological samples obtained from an archaeological data synthesis[START_REF] Douglass | A critical review of radiocarbon dates clarifies the human settlement of Madagascar[END_REF]. Purple curve represents the estimated changes in the effective human population size[START_REF] Pierron | Genomic landscape of human diversity across Madagascar[END_REF].

Acknowledgements

We thank Madagascar National Parks, the director of Mt. d'Ambre National Park, the Direction du Système des Aires Protégées, the Direction Générale du Ministère de l'Environnement et des Forêts de Madagascar, Madagascar's for their permission to conduct the field work in Mt. d'Ambre National Park (N°120/18/MEEF/SG/DGF/DSAP/SCB.Re) and for their support. We thank Romule Rakotondravony for his support during the palaeoecological fieldwork. We thank the guides of Mt. d'Ambre National Park and the local communities for their indispensable help in the field. Five 14 C dates were acquired with the help of Tomasz Goslar (Poznan Radiocarbon Laboratory). Ten 14 C analyses were acquired thanks to the CNRS-INSU ARTEMIS national radiocarbon AMS measurement programme at Laboratoire de Mesure 14 C (LMC14) in the CEA Institute at Saclay (French Atomic Energy Commission). This study was funded by the Deutsche Forschungsgemeinschaft (RA 502/20-1, RA 502/20-3, and BE 2161/30-1) and PALEOMAD project (INSU-LEFE/CNRS).


Bruno Da Costa Magalhaes

Ruben Checa

Chantal Lorentz

Mathieu Prévot

Pavel Afanasiev

Dorothée Laurenti

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

Bruno C Magalhães

Catalytic hydrotreatment of algal HTL bio-oil over phosphide, nitride, and sulfide catalysts

Keywords: biofuel, biomass, heterogeneous catalysis, hydrothermal liquefaction, hydrotreatment

Introduction

Microalgae are potentially biomass sources for producing thirdgeneration biofuels due to their ability to convert solar energy through photosynthesis, capture CO2, and store this carbon in energy-rich compounds such as lipids. [1,2] These microorganisms have high-speed growth rates, resulting in high productivity. [3] Hydrothermal liquefaction (HTL) is a thermochemical process that allows the conversion of wet biomass such as microalgae, avoiding a costly drying step. [4,5] The lipids are hydrolyzed, forming carboxylic acids that can be further converted into dropin fuels for diesel and jet fuel. [6] Besides, the carbohydrates and proteins can also be converted, affecting the quality and properties of the HTL bio-oil as a function of microalgae biochemical composition. [7,8] The algal HTL bio-oil has high oxygen (∼5 -20 wt.%) and nitrogen content (∼2-9 wt.%), as well as sulfur (∼0-1 wt.%) and metals. [9][10][11][12] Therefore, an upgrade is required to convert the HTL bio-oil into drop-in fuels to be compatible with traditional engines. [13] Catalytic hydrotreatment (HDT) is conducted at moderate temperatures (300 -450 °C), under hydrogen pressure (35 -170 bar), with a liquid hourly space-velocity of 0.2-10 h -1 , and in the presence of a heterogeneous catalyst. [14] Supported Mo or W sulfides are traditional hydrotreating catalysts, and some works have demonstrated their efficacy in upgrading algal HTL bio-oil. [15][16][17][18][19] These nanomaterials are usually supported on -alumina and are promoted by nickel or cobalt. [20] Moreover, these catalysts can also be boosted by adding another element, such as phosphorus [21,22] or a chelating agent (e.g., citric acid). [23,24] Biller et al. [25] employed NiMo/Al2O3 and CoMo/Al2O3 sulfide catalysts to upgrade algal HTL bio-oils and reported that both catalysts showed similar activity during hydroprocessing. Later, Guo et al. [26] compared commercial NiMo/Al2O3 and NiW/Al2O3 and concluded that NiMo performed better in removing oxygen and nitrogen. However, the highest sulfur removal was achieved by the NiW catalyst. Moreover, other types of catalysts, such as metal phosphides, carbides, and nitrides, have been considered in the literature as promising for bio-oils upgrading. [27,28] Bowker et al. [29] tested silica-supported Ni phosphide catalysts (Ni2P) for the hydrodenitrogenation (HDN) of carbazole with and without a benzothiophene co-feed. They reported that phosphide catalysts exhibited excellent stability in the HDN reactions and performed better than NiMo/Al2O3 sulfide, even if benzothiophene inhibited the carbazole HDN over the metal phosphide catalysts. Besides, Cole et al. [30] achieved 80% nitrogen conversion using Ni2P/SiO2 to upgrade a blended feed with 3230 N ppm composed of bio-oil from algae HTL and green hydrocarbons from the CO2 hydrogenation. Phosphide catalysts are usually prepared by the temperatureprogrammed reduction (TPR) of phosphate-based precursors in flowing hydrogen at high temperatures. [31] This method can lead to large Ni2P particles (> 10 nm), side reactions between surface P and aluminum in Al-containing support (e.g., γ-Al2O3 and ASA), and can also make difficult the in situ preparation of metal phosphide in the industrial hydrotreating reactors. [32] In this context, d'Aquino et al. [33] reported a preparation method of Ni phosphide over oxide supports from nickel hypophosphite precursors and their reduction at lower temperatures. Their results showed well-dispersed catalysts with small particles sizes (3-4 nm), which were highly active for an HDN and hydrodesulfurization (HDS) of a mixed carbazole (1000 ppm)/benzothiophene (3000 ppm) feed. Ni2P/SiO2 and Ni2P/Al2O3 in situ prepared catalysts showed higher HDN and HDS conversion than commercial NiMo/Al2O3 sulfide. Regarding metal nitride catalysts, Sajkowsk and Oyama [34] compared unsupported Mo nitride against commercial NiMo/Al2O3 sulfide for hydrotreating coal-derived gas oil and residues. They showed that Mo nitride was active under the experimental hydrotreating conditions. Besides, Chu et al. [35] tested a series of NiMoNx/Al2O3 catalysts with different Ni content prepared by the temperature-programmed reaction of NiO.MoO3/Al2O3 with NH3. Those nitride catalysts showed higher pyridine HDN activity than NiMo/Al2O3 sulfide, which was associated with the new phase of Ni3Mo3N and the synergy between metallic Ni and nitride Mo. Mo2N phase and Ni metal were also found in the NiMoN/Al2O3 catalysts. [36] Moreover, Chouzier et al. [37] reported a one-step way to prepare bimetallic nitrides Co(Ni)-Mo from the decomposition of transition metal complexes with hexamethylenetetramine (HMTA) under an inert atmosphere. Then, they studied binary and ternary nitrides prepared via this methodology in the hydrotreatment reaction. [38] Thiophene hydrodesulfurization (HDS) and quinoline HDN tests showed that bimetallic nitrides were highly active HDN catalysts in the absence of sulfur, but no synergy effect was observed for thiophene HDS. Overall, phosphide and nitride catalysts have shown promising results compared to conventional sulfide catalysts, which are highly employed in refineries. However, these investigations were mainly carried out with model molecules, whereas the applicability of phosphides and nitrides to real bio-oil charges remains to be clarified. In order to achieve high yield and selectivity to the desired products, the catalyst plays an essential role during this process, and a careful catalyst screening for upgrading algal HTL bio-oils under hydrotreating (HDT) conditions still needs to be achieved. In this context, supported Ni phosphides, supported and unsupported Mo nitrides catalysts were synthesized and compared with various supported NiMo and NiW sulfide catalysts to upgrade algal bio-oil. The hydrotreated oils were characterized by several analytical techniques to follow the reaction and understand the catalyst impact during the HDT stage.

Results and Discussion

Transmission electron microscopy (TEM) images of metal phosphides, nitrides, and sulfides are reported in the supplementary material, Figure S1. The Ni phosphide catalyst supported on silica showed nanoparticles of Ni2P well dispersed, which was also corroborated by the XRD pattern (See Figure S2). However, the Ni phosphide catalyst supported on alumina showed large particles, which disagrees with the results reported by d'Aquino et al. [33] (3.9 nm ± 0.6). Besides, the XRD pattern for this catalyst evidenced the formation of the Ni2P phase. Ni5P4 impurities was also found. For the unsupported and supported NiMo catalyst, the TEM images reveal isotropic nanoparticles, and the XRD pattern of the NiMo nitride catalyst over alumina confirmed the formation of the Ni2Mo3N phase. The NiW sulfide catalyst, prepared with and without citric acid, showed 3-5 nm WS2 slabs with homogenous distribution of the Ni promoter over the W sulfide (see Figure S1 and Table S1). The HTL algal oil, provided by our partner, was analyzed. The main components in this oil are the C16 and C18 fatty acids (34 wt.%) coming from the hydrolysis of the triglycerides during the HTL process. The yield of oils and solids after HDT over the different catalysts were around 50-70 wt.% and 6-12 wt.%, respectively. The gases were not quantified but missing masses can be attributed to gas mainly in addition to experimental losses and water (Table S2).

The elemental analysis of the HTL and HDT oils with and without catalysts are reported in Table 1. A commercial green diesel composed mainly of C15-C18 aliphatics obtained from the hydroconversion of palm oil was used as a reference for comparison with HDT algal oil. The oxygen content of the HTL oil was highly reduced in the HDT step, even without a catalyst. The high oxygen content in HTL oil is due to the fatty acids, rapidly converted under the HDT experiment conditions by hydrogenolysis reactions or by decarboxylation which can occur without a catalyst. [START_REF] Ramasamy | Hydrothermal Liquefaction: Path to Sustainable Aviation Fuel[END_REF][START_REF] Gosselink | [END_REF] However, only 20% denitrogenation degree was achieved without a catalyst, indicating that these molecules are more difficult to be converted by a thermal process. The Ni phosphide catalysts promoted both HDO and HDN reactions. However, differently to that reported by d'Aquino et al., [33] the ex situ Ni2P/Al2O3 prepared from hypophosphite precursor was more active than Ni2P/SiO2 prepared from phosphate-based precursors. The NiMo nitride catalyst supported over alumina showed an HDN activity (65%) significantly higher than the bulk NiMo nitride catalyst (46%), indicating that the alumina support could promote this reaction by dispersing the active phase. Among the sulfide catalysts tested, NiWS/Al2O3 was the most active, resulting in an HDT oil with lower heteroatom content and greater HHV. No benefit effect was found after a post-treatment of the nickel tungsten catalyst with citric acid (NiWS/Al2O3 + CA) since the results were very similar to those found for the NiWS/Al2O3 without a chelating agent (see Table S3). Besides, the catalyst prepared by the co-impregnation methodology (NiW(CA)S/Al2O3) was tested in a feed with thiophene as the model molecule and showed approximately 30% higher thiophene HDS activity than the NiWS/Al2O3, as reported in Figure S3. However, this catalyst showed poor HDN activity for upgrading algal bio-oil, resulting in an HDT oil with two times more nitrogen compared to the results obtained with NiWS/Al2O3. The NiWS/SiO2-Al2O3 catalyst also showed poor HDN activity compared to the NiWS/Al2O3, even though the metal loadings were very similar. This activity trend can be attributed to the inhibition provoked by the strong adsorption of nitrogen compounds, mainly basic compounds in the algal HTL bio-oil. [41,42] Lastly, the NiMo(P)S/Al2O3 showed close results to the NiWS/Al2O3 catalyst, which performed slightly better. Van Krevelen's diagrams given for O/C and N/C versus H/C (Figure 1) showed a significant improvement in the heteroatom reduction when a catalyst was employed, alumina supported NiWS catalyst being the closest but still far to the green diesel. More severe reaction conditions or the utilization of a second upgrading stage must be used to produce diesel-like oil. [43] To provide a deeper understating of the HDT over different active phases, other analytical techniques were used to characterize the HDT oil obtained over Ni2P/Al2O3, NiMoN/Al2O3, and NiWS/Al2O3. The sulfur content in the algal HTL oil is relatively low compared to other oils. [44] These molecules are derived primarily from amino acids with contain sulfur, such as cysteine and methionine, and sulfate lipids. The structures of the sulfur molecules that can be present in those oils and their reactivity are rarely reported in the literature. [45] In order to go further in this S analysis, a gas chromatography (GC) coupled with a flame photometric detector plus (FPD+), highly selective for sulfur and phosphorous components, was employed to analyze the samples of oil hydrotreated over different catalysts. Thiophene, benzothiophene, dibenzothiophene, and 4,6-dimethyldibenzothiophene (4,6-DMDBT) were used as standards, and their retention time in function of the boiling point was plotted, as shown in Figure 2.

A high linear correlation (R 2 > 0.99) was found between the retention time and the boiling point of the sulfur standards. An extrapolation of this curve was employed to provide insights regarding the sulfur species in HDT oil. As demonstrated by the elemental analysis, the sulfur content in the HDT oil treated over NiMoN/Al2O3 was higher than those obtained with NiWS/Al2O3 and Ni2P/Al2O3, resulting in a more intense FPD+ signal with the first catalyst. Besides, four negative peaks can be observed around 5-and 10-min. These peaks can be attributed to the C15, C16, C17, and C18 n-alkanes, present in high amounts in the HDT oil, as will be discussed further. Several peaks with retention time close to benzothiophene were found (2-7 min). These peaks can be associated with light sulfur compounds and were highly reduced in the function of the sulfur content in the HDT oil. Thin and broad peaks with retention time fitting with dibenzothiophene and higher than the 4,6-DMDBT were also found (7-15 min) and can be associated with heavy sulfur compounds. The peak's shapes indicate that the heavy sulfur compounds are distributed into several molecules, which can justify why these compounds are so difficult to be characterized. Moreover, the Ni2P/Al2O3 resulted in an HDT oil with lower sulfur content compared to the NiMoN/Al2O3, showing a significant reduction of light and heavy sulfur compounds. Lastly, the NiWS/Al2O3 exhibited the highest hydrodesulfurization activity. The residual sulfur present in this HDT oil can be associated mainly with heavy sulfur compounds, suggesting that these molecules are more challenging to be converted than light ones.

-0.005 0.000 0.005 0.010 0.015 0.020 0.025 The 13 C-NMR functional group distribution in the HTL and HDT oils is reported in Figure 3. Both oils showed mainly an aliphatic fraction. However, we observed a considerable amount of unsaturated hydrocarbon components (aromatics, olefins) and carbonyl/carboxyl and ether/alcoholic components in the HTL oil.

During the upgrading stage, the carbonyl compounds (e.g., carboxylic acids, esters, and fatty amides), alcohols, and ethers were converted and not detected by NMR analysis. The amount of olefinic and aromatic carbon decreased for the three HDT oils, which was more significant with the NiW sulfide catalyst than with the Ni phosphide and NiMo nitride catalysts. These results agree with the H/C ratio presented in Van Krevelen's diagrams (Figure 1), which shows that the sulfide catalyst has higher hydrogenation activity, contributing to a lower aromaticity degree. Gel permeation chromatography coupled with a refractive index detector (GPC-RID) was employed to estimate the molecular weight of the HTL and HDT oils (Figure 4). The algal HTL oil showed the main intensity of around 300 g/mol, which can be associated with carboxylic acids (e.g., palmitic and stearic acids) in high amounts in algal oils. [46] Moreover, heavy compounds with molecular weight above 400 g/mol are also present, in agreement with the literature. [47] During the upgrading step, the main peak in the algal HTL oil was shifted to the left, which can be attributed to the conversion of fatty acids to aliphatics by decarboxylation/hydrogenation. Table 2 shows the average molecular weight of the HTL and HDT oils after treatment with and without a catalyst. On the one hand, HDT oil treated over Ni2P/Al2O3 showed a molecular weight close to that of the HDT oil treated without a catalyst. On the other hand, the NiMo nitride catalyst produced an HDT oil with higher molecular weight, suggesting that condensation reactions occurred when this catalyst was employed. Lastly, the NiW sulfide catalyst produced an HDT oil with a lower average molar mass, as can be observed by comparing the HDT oil elugrams in Figure 4 and reported in Table 2, indicating that heavy molecules were partially converted, which is in agreement with literature reports on the FT-ICR-MS analysis of HDT oils processed over sulfide catalysts [48] . Comprehensive two-dimensional gas chromatography coupled with a flame ionization detector and mass spectroscopy (GCGC-FID/MS) was employed to qualify and quantify the semi-volatile organic fraction of the HTL and HTD oils. Figure 5 shows the chromatogram obtained from the HDT oil processed over the Ni phosphide catalyst. Compared to the analysis of the HTL oil that shows mainly fatty acids, amides, and unsaturated (Figure S4), the main fraction was composed of aliphatic hydrocarbons, as corroborated by the 13 C-NMR analysis and reported by Rathsack et al. [49] . A family of mono-aromatics was also identified in all chromatograms. High concentrations of C16 and C18 fatty nitriles and lighter nitriles were found in the HDT oil, processed over Ni2P and without a catalyst. During the HTL reaction, fatty acids from the hydrolysis of the triglycerides can react with ammonia and amines through condensation reactions, forming fatty amides. Some works have reported the presence of these compounds in the algal HTL oil and mentioned that their concentration could be affected by the algae's biochemical composition. [46,47] The 2D and 3D GCxGC chromatograms of the algal HTL oil (Figure S2) showed the presence of fatty amides, mainly composed of C16 and C18 chains. Zhu et al. [50] recently reported that the hydroconversion of tertiary and secondary amides over NiMo/Al2O3 sulfide catalyst occurs via two pathways: deoxygenation followed by denitrogenation of the intermediate amine (main route) and denitrogenation followed by deoxygenation of the alkanol intermediate. Primary fatty amides should react similarly. However, with this kind of amide, it could be expected that the dehydration reaction could lead to fatty nitrile formation. [51] Based on this, it is suggested that the C16 and C18 fatty nitriles reported in Figure 5 were formed from the dehydration of the fatty amides. This reaction also occurred without a catalyst. These nitriles were almost totally converted when NiW sulfide and NiMo nitride catalysts were employed. However, as mentioned before, both chromatograms obtained from the HDT oils treated over Ni2P/Al2O3 and without catalyst showed a high amount of nitriles, mainly C16 and C18. Besides, a similar result was found when the Ni2P/SiO2 catalyst was employed, indicating that the Ni phosphide catalysts were less efficient for converting these compounds. Similar results were reported by Pongsiriyakul et al. [52] , who compared Ni-based catalysts promoted with Cu and Re supported on alumina. They found an HDT oil with high nitrogen content when Ni-Re/γ-Al2O3 was employed, which showed mainly nitrile compounds such as hexadecane nitrile.

The quantification of the cyclic nitrogen compounds (e. g., indoles and carbazoles) and oxygen compounds (e.g., phenols) obtained with the GCGC-FID/MS agreed with the oxygen and nitrogen content reported by the elemental analysis and the H/C ratio of the HDT oils. Therefore, NiWS/Al2O3 catalyst led to a liquid with a lower concentration of these compounds. As reported in the literature, a catalyst with higher hydrogenation activity favors the conversion of these molecules once the C-heteroatomic bond can be easily broken in a fully hydrogenated cyclic compound. [53,54] In summary, GCCG chromatograms of the HDT oils revealed that the Ni phosphide catalyst was less active in converting fatty nitriles and cyclic nitrogen compounds. In contrast, the NiWS catalyst resulted in an HDT oil with lower heteroatomic compounds due to a higher hydrogenation activity. As reported before, the C16 and C18 carboxylic acids were found in high amounts in the HTL oil and were easily converted during the upgrading stage. The first pathway is the decarboxylation/decarbonylation (DCO), leading to the formation of C15 and C17 n-alkanes in the liquid phase, implying the loss of one carbon by eliminating CO or CO2 in the gas phase. The second pathway is hydrodeoxygenation (HDO), which preserves the number of carbon leading to C16 and C18 n-alkanes by removing water with higher hydrogen consumption. [55,56] Based on this, the C16/C15 and C18/17 n-alkanes mass fraction ratios obtained with the GCGC-FID were compared for the catalysts to illustrate the pathways taken for fatty acids conversion (Figure 6). Without a catalyst, the HDO/DCO ratio was lower than 1.It was even lower when the Ni phosphide catalyst was employed, indicating that DCO is favored without a catalyst and with Ni2P. These results agree with Zarchin et al. [57] , who performed the hydroprocessing of soybean oil over a Ni phosphide catalyst supported on silica. They reported a C17 yield two times higher than C18. Besides, a similar trend was observed when Ni2P/SiO2 was employed and specific Ni sites favoring DCO pathway were proposed. [58] It can be an advantage due to the lower H2 consumption. [59] On the contrary, the NiMo nitride catalyst drastically promoted the HDO pathway compared to DCO. Similarly, Monnier et al. [60] reported a molar ratio of n-C17/(nC17 + n-C18) equal to 0.24 when Mo2N/Al2O3 was employed in the hydroconversion of oleic acid, indicating that this catalyst favored the HDO route three times out of four DCO. Besides, the NiW sulfide catalyst showed an intermediate behavior compared to the Ni phosphide and NiMo nitride catalysts, with the two pathways being approximately equal. The simulated distillation profiles of HTL and HTD oils are shown in Figure 7, and the fractional cuts are in Table 3, respectively. The HTL oil curve shows two steps around 350 °C that can be associated with the C16 and C18 carboxylic acids. Note that the interaction of carboxylic acids with the column is different compared to n-alkanes, which results in a false boiling point. The boiling point of palmitic acid is 351 °C, and stearic acid is 361 °C. Using an FID detector, the response factor for carboxylic acids is approximately two times lower than the n-alkane utilized to calibrate the instrument. However, this analysis could also be used to corroborate the results found with GCGC-FID/MS and GPC analysis, which showed the presence of these compounds. As expected, diesel was the main fraction obtained in the HDT oils due to the high concentration of C15-C18 aliphatics formed from the deoxygenation of the carboxylic acids. [61] The NiMoN/Al2O3 catalyst resulted in a lower diesel fraction, probably due to condensation reactions, as mentioned before, since this HDT oil also showed a higher average molar mass than the others. Besides, the diesel fraction amount obtained from the Ni2P/Al2O3 catalyst and without a catalyst should be considered carefully, since the C16 and C18 nitriles, which are still presented in these HDT oils, can elute in the diesel range, evidencing the importance of employing different techniques for the analyses of complex feeds.

Lastly, a heavy fraction composed of heavy gas oil and residual was found in all cases, with a lower amount when NiWS/Al2O3 was employed. As shown by the GPC analysis, the HTL algal biooil and hydrotreated oils are also composed of a heavy fraction that does not elute in the SIMDIS, resulting in a recovered mass close to 80 wt.% for hydrotreated oils. These results suggest that applying these operating conditions, a second stage must be used to convert these heavy compounds and increase the light fraction (jet fuel) that could produce sustainable aviation fuel (SAF).

Conclusion

Several catalysts (Ni phosphide, NiMo nitride, and Ni (Mo, W) sulfides) were tested for the hydroconversion of algal bio-oil obtained from a continuous hydrothermal liquefaction reaction. The initial HTL algal oil was fully characterized, and C16-C18 fatty acids were identified as the main components but many kinds of nitrogen compounds are also present. The hydrotreated oils from experiments with catalysts showed lower oxygen and nitrogen content and higher HHV than that conducted without a catalyst. In summary, NiW sulfide catalysts performed better than NiMo nitride and Ni phosphide, showing a higher HHV, a lower aromaticity degree, and a lower average molar mass, indicating a better conversion of lipids. The Ni2P/Al2O3 was more active than Ni2P/SiO2 for the hydroconversion of algal oil, suggesting that the support can highly affect the catalyst activity in this reaction. Besides, both catalysts promoted decarboxylation or decarbonylation reactions and were less active in converting fatty nitriles formed from the dehydration of fatty amides, indicating poor hydrogenation activity. On the contrary, the NiMoN/Al2O3 promoted the hydrodeoxygenation route and was more efficient than phosphide catalysts in converting fatty amides, as shown by GCGC-FID/MS. The NiWS/Al2O3 was the most active catalyst, showing a higher hydrogenation performance, evidenced by the H/C ratio, which resulted in the hydroconversion of refractory cyclic nitrogen compounds. In contrast, the NiWS/Silica-Al2O3 was the less active sulfide catalyst, probably due to the inhibition/deactivation due to the nitrogen compounds in high amounts of algal oil. Therefore, sulfided catalysts, commonly used in refineries nowadays, showed promising results in the hydroconversion of algal bio-oil. Future research should focus on the stability of these catalysts at different times on stream using continuous reactors, since the presence of metal in the algal HTL oil, as well as the coke formation can affect the catalyst activity. In addition, hydrocracking of the partially hydrotreated fraction, composed mainly of diesel, to increase the production of a lighter fraction such as sustainable aviation fuel (SAF), can also be explored.

Experimental Section Microalgae and HTL bio-oil production

Chlorella vulgaris NIES 227 (NIES-Collection, Japan) was grown in a photobioreactor in a greenhouse at MicroAlgae Processes Platform, CEA Cadarache, France. More details about microalgae cultivation were reported by Chambonniere et al. [62] . The microalgae elemental and biochemical composition are reported in the supplementary material, Table S4. The HTL reaction of microalgae was performed in the continuous pilot-scale setup described elsewhere by CEA. [63] The microalga was dispersed at 10 % of organic matter in water and injected into the reactor through a dual-syringe pump with a flow rate of 1.5 L/h so that the estimated residence time is in the order of 15 min. The pressure was set to 130 bar and the temperature to 300 °C. Then, bio-oil was extracted with dichloromethane.

Catalysts

Phosphide catalysts

Two phosphide catalysts supported on γ-Al2O3 (i) or SiO2 (ii) were prepared. The nominal loading of the catalysts was 25 wt.% for Ni2P/Al2O3 and Ni2P/SiO2. Before the catalyst preparation, both supports were calcinated at 500 °C for 3 h under airflow and dried at 120 °C for 1 h.

(i) The phosphide catalyst supported on sieved γ-Al2O3 (80 -125 µm) was prepared following the methodology reported by d'Aquino et al. [33] . A stoichiometric amount of hypophosphorous acid (H3PO4, Sigma-Aldrich, 50 wt.% in H2O) was added to nickel hydroxide (Ni(OH)2, Sigma-Aldrich) to obtain a P/Ni atomic ratio equal to 2. Distilled water was added, and the solution was kept at 70 °C with stirring to ensure the total solid dissolution.

Then, the green light solution was impregnated onto the support until incipient wetness. Several impregnations were necessary, and the catalyst was dried at 70 °C between them for approximately 1 h. After the last impregnation, the catalyst was dried for 24 h at 70 °C. Following the drying, two reduction steps were performed: the first followed by passivation for catalyst characterization and the second without passivation to measure the catalyst activity during the upgrading step. Approximately 0.7g of the catalyst precursor was transferred to a quartz U-tube and degassed in 50 mL/min N2 for 30 min. Afterward, the precursor was subjected to TPR in H2 flowed at 50 mL/min by heating from room temperature to 500 °C (heating rate of 5 °C/min) after holding for 1 h. The Ni2P/ Al2O3 was cooled to room temperature in continued H2 flow. When a passivation step was performed, the catalyst was purged with N2 for 30 min following passivation with a mixture of 50 mL/min N2 and 10 mL/min air for 2 h.

(ii) The phosphide catalyst supported on SiO2 was prepared following the methodology reported by Bowker et al. [29] . A stoichiometric amount of ammonium dihydrogen phosphate (NH4H2PO4, Alfa Aesar, 98%) and nickel nitrate hexahydrate (Ni(NO3)2•6H2O, Alfa Aesar, 98%) was added to a beaker to obtain a P/Ni atomic ratio equal to 0.9. Sufficient distilled water was added under constant stirring, and the pH of the impregnation solution was adjusted to nitric acid (HNO3, Honeywell Fluka TM , ≥ 65%) until complete solubilization of the precipitate. The resulting impregnation solution was used for the impregnation of the support by the incipient wetness impregnation method. Several impregnations were necessary, and the catalyst was dried between them at 120 °C for approximately 1 h. After the last impregnation, the precursor was dried for 24 h at 120 °C and then calcinated for 3 h at 500 °C (heating rate of 10 °C/min) under air. Following the calcination, two reduction steps were performed: the first followed by passivation for catalyst characterization and the second without passivation to measure the catalyst activity during the upgrading step. Approximately 0.7 g of the catalyst precursor was transferred to a quartz U-tube and degassed in 50 mL/min N2 for 30 min. Afterward, the precursor was subjected to TPR in H2 and flowed at 50 mL/min by heating from room temperature to 650 °C (heating rate of 1 °C/min) after holding for 2 h. The Ni2P/SiO2 was cooled to room temperature in continued H2 flow. When a passivation step was performed, the catalyst was purged with N2 for 30 min following passivation with a mixture of 50 mL/min N2 and 10 mL/min air for 2 h.

Nitride catalysts

Two NiMo nitride catalysts were prepared following the methodology reported by Chouzier et al. [37,38] : a bulk catalyst, named NiMoN, and another supported on γ-Al2O3, named NiMoN/Al2O3. Aqueous solutions of ammonium heptamolybdate ((NH4)6Mo7O24.4H2O, Sigma-Aldrich, 99%), nickel nitrate hexahydrate (Ni(NO3)2•(6H2O), Alfa Aesar, 98%) and hexamethylenetetramine (HTMA) (N4(CH2)6, Sigma-Aldrich, 98%) were mixed under stirring. The atomic ratio Ni/Mo was 0.5; the atomic ratio of carbon from HMTA to the sum of metals C/(Ni+Mo) was 32. The formed precipitate was washed with distilled water and dried under nitrogen flow at 200 °C overnight. Then, the catalyst precursor was placed in a quartz reactor and heated for 2 h at 650 °C (heating rate of 10 °C/min) under N2.

The NiMoN/Al2O3 showed a nominal loading of 16.7 wt% MO3 and 6.7 wt.% NiO.

Sulfide catalysts

A commercial NiW/Al2O3 extrudate catalyst [64] (WO3: 25.7 wt.%, NiO: 3.8 wt.%) was used as a reference, and two catalysts with CA/Ni molar ratio equal to 2 and the same metal content as the reference catalyst were prepared using post-treatment (i) and co-impregnation (ii) as preparation methods.

(i) Sufficient citric acid (C6H8O7•H2O, Sigma-Aldrich, 99%) was added to distilled water with stirring. This solution was used to impregnate the NiW/Al2O3 catalyst, previously dried at 120 °C for 1 h, until incipient wetness. Then, the catalyst aged for 12 h at room temperature and dried at 120 °C for 12 h. This catalyst was named NiW/Al2O3 + CA.

(ii) Ammonium metatungstate hydrate ((NH4)6W12O40.xH2O, Sigma-Aldrich, ≥ 66.5%) was dissolved in distilled water, and this solution was used to impregnate an extrudate γ-Al2O3, previously calcinated at 500 °C for 3 h under airflow and dried at 120 °C for 1 h. Then, the tungsten-alumina base catalyst was aged for 12 h at room temperature and dried at 120 °C for 12 h. Afterward, nickel nitrate hexahydrate (Ni(NO3)2•(6H2O), Alfa Aesar, 98%) was dissolved in distilled water, and citric acid was added. The pH of the solution was adjusted to 5 by adding ammonium hydroxide (NH4OH, Honeywell Fluka TM , 30-33 % NH3 in H2O) to optimize the formation of active NiWS species. [65] Then, the resulting solution was used to impregnate the tungsten-alumina base catalyst until incipient wetness. The resulting catalyst was aged for 12 h at room temperature and dried at 120 °C for 12 h. This catalyst was named NiW(CA)/Al2O3.

Moreover, two other catalysts were tested: NiW/silica-alumina (WO3: 26.5 wt.%, NiO: 3.8 wt.%, SiO2: 19.3 wt.%) and NiMo(P)/Al2O3 (MoO3: 18.6 wt.%, NiO: 3.8 wt.%, P2O5: 4.9 wt.%).

Before characterization and catalytic activity experiments, the catalysts were sulfided ex situ at 400 °C (heating rate of 5 °C/min) for 3 h under 15% H2S/H2 flow (4 NL/h) except the NiW(CA)/Al2O3 which was sulfided at 400 °C (heating rate of 3 °C/min) for 2 h under 15% H2S/H2 flow (4 NL/h).

Catalytic hydrotreatment of HTL algal bio-oil

The upgrading step was performed using a 300 mL Parr stainless steel batch reactor. Firstly, 0.6 g ex situ activated catalyst, 3.0 g of HTL bio-oil, and 25 g of n-heptane were added to the reactor. Subsequently, the reactor was closed, purged with N2, and 6 MPa was loaded for a leak test. Then, the N2 was removed, and the reactor was purged three times with H2, loaded with 1 MPa of H2, and heated to 375 °C. Once the temperature had been reached, 5 MPa of H2 was added to get a total pressure of 10 MPa, and the stirring at approximately 570 rpm was started. The reaction was performed for 5 h, and the system was cooled. The used catalyst was separated from the hydrotreated oil (HDT-oil) by filtration, and n-heptane was used to wash the reactor to recover the residual catalyst and oil. The n-heptane was further removed using a vacuum evaporator, and different analytical techniques were used to analyze the HDT oil.

Characterization of analytic techniques

The C, H, O, N, and S mass fractions were analyzed in a Thermo Scientific Flash 2000 Apparatus. ANTEK 9000NS was used for S analyses, which is more precise (from low ppb until percent levels) and accurate. The HTL bio-oil and HDT-oil were diluted using THF to a concentration of 5 wt.% and 10 wt.%, respectively, then injected into a reactor at 1050 °C. The higher heating value (HHV) was calculated based on elemental analysis values according to the equation reported by Channiwala and Parikh. [66] An Agilent 7890A GC coupled with an FPD+ detector and fitted with a GC column (HP-5: 30 m x 0.32 mm x 0.25 µm) was employed to analyze the sulfur compounds. In addition, thiophene (C4H4S, Sigma-Aldrich, >99%), benzothiophene (C8H6S, Sigma-Aldrich, 98%), dibenzothiophene (C12H8S, Acros Organics, 95%), and 4,6-dimethyldibenzothiophene (C14H12S, Janssen Chimica, 95%) were injected to provide some insights regarding the sulfur compounds in the algal oil.

The gel permeation analyses were performed using an Agilent 1200 series HPLC equipped with two PLgel Columns (50 and 500 Å) and a differential refractive index detector (RID). The analyses were carried out at 35 °C using THF as eluent with a 1 mL/min flow rate. Before analyses, the samples were diluted to a concentration of approximately 1 wt.% in THF and filtered (0.45 μm). The elution time was converted into molecular weight (Mw) using a calibration curve obtained with a hydrocarbon mixture (Mw from 86 to 1000 g/mol). The elugrams obtained from the RID detector were normalized to the highest peak intensity. 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).

The boiling point distributions were determined utilizing simulated distillation (SimDis) according to the standard ASTM D2887. The analyses were performed on an Agilent 7890A GC coupled with an FID and fitted with a GC column (CP-SimDist UltiMetal: 10 m x 0.53 mm x 2.65 µm). 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).

The GCGC analysis was performed using two systems: The first one was a GC Agilent 6890 with a cryogenic modulator from Zoex Corporation (USA) connected to a mass spectrometer (MS) 5975B for the identification of compounds (up to C32) and fitted with two columns (VF-1701-MS: 30 m x 0.25 mm x 0.25 µm and DB-1: 3 m x 0.1 mm x 0.1 µm). The second one was a GC Agilent 7890A with a flow modulator connected to an FID for quantification (up to C40) and fitted with two columns (ZB-1701: 15 m x 0.10 mm x 0.05 µm and DB-1: 5 m x 0.25 mm x 0.25 µm). Modulation parameters were optimized according to Lelevic et al. [67] . The samples were diluted in THF to a concentration of around 10 wt.% and 5 wt.% for MS and FID analysis, respectively. Besides, an internal standard, 3,3'dimethylbiphenyl (C14H14, Sigma-Aldrich, 99%), 200 mg/kg, was also added for quantification purposes.

Figure 1 .

 1 Figure 1. Van Krevelen's diagrams of upgraded HTL bio-oil with and without catalyst: (a) O/C and (b) N/C. A commercial green diesel from palm oil, composed mainly of aliphatics, was used as a reference.

Figure 2 .

 2 Figure 2. FPD+ signal for the hydrotreated oils over different catalysts. thiophene (T), benzothiophene (BT), dibenzothiophene (DBT), and 4,6dimethyldibenzothiophene were used as standard. A linear fitting and their extrapolation correlating the sulfur compounds' retention time and their boiling point were plotted.

Figure 3 .

 3 Figure 3. 13 C-NMR distribution by types of carbons in HTL and HDT oils over different catalysts.

Figure 4 .

 4 Figure 4. GPC-RID analysis of HTL and HDT oils over different catalysts.

  , amides, carboxylic acids) Aromatics, olefins Alcohols, ethers, phenolic-methoxys, carbohydrates sugars Aliphatics HTL bio

Figure 5 .

 5 Figure 5. Comprehensive GCXGC-FID/MS chromatogram of HDT-oil over Ni2P/Al2O3 catalyst.

Figure 6 .

 6 Figure 6. Hydrodeoxygenation and decarboxylation or decarbonylation ratio in function of catalyst.

Figure 7 .

 7 Figure 7. Simulated distillation profiles of HTL and HDT oils.

Table 1 .

 1 Elemental analysis of HTL and HTD-oils.

	Sample	C	H	O	N	S	HHV
			(wt.% or ppm*)		(MJ/kg)
	HTL bio-oil	72.2	9.8	11.7	2.6	2125*	35.5
	Without cat.	82.8	13.0	1.5	2.1	259*	44.1

Table 2 . Average molecular weight for HTL and HDT oils with and without catalysts.

 2

	Sample	Average molecular mass
		Mw (g/mol in HC eq.)
	HTL bio-oil	363
	Without catalyst	287
	Ni2P/Al2O3	286
	NiMoN/Al2O3	333
	NiWS/Al2O3	264

Table 3 .

 3 Fractional cuts obtained from the simulated distillation of HTL and HDT oils (wt.%).

	Sample	Gasoline	Jet fuel	Diesel	Heavy	Residual
					gas oil
	HTL bio-oil	0	1	7	27	65
	Without catalyst	1	5	44	21	29
	NiMoN/Al2O3	1	7	48	18	26

Acknowledgments

This project has received funding from ANR for the project RAFBIOALG 18-CE43-009. Bruno da Costa Magalhães thanks the ANR for providing a Ph.D. grant.

The authors thank CEA Cadarache and CEA Grenoble for providing the microalgae and the HTL bio-oil, respectively.

Entry for the Table of Contents

Supported metal phosphide, nitride, and sulfide catalysts were compared for the hydroconversion of algal oil. Nickel phosphide catalysts promoted the decarboxylation/decarbonylation route for the conversion of carboxylic acids, while NiMo nitride catalysts promoted the hydrodeoxygenation pathway. NiWS/Al2O3 sulfide was the best catalyst resulting in a hydrotreated oil with lower heteroatom content and average molar mass that felt primary in the diesel range.