Eva Roubeau Dumont

Arnaud Elger

Céline Azéma

Hiram Castillo

Suzy Surble

Camille Larue
email: camillelarue@ensat.fr

Cutting-edge spectroscopy techniques highlight toxicity mechanisms of copper oxide nanoparticles in the aquatic plant Myriophyllum spicatum

Keywords: copper, distribution, macrophyte, nanoparticle, toxicity

Copper oxide nanoparticles (CuO-NPs) have been increasingly released in aquatic ecosystems over the past decades as they are used in many applications. Cu toxicity to different organisms has already been highlighted in the literature, however toxicity mechanisms of the nanoparticulate form remain unclear.

Here, we investigated the effect, transfer and localization of CuO-NPs compared to Cu salt on the aquatic plant Myriophyllum spicatum, an ecotoxicological model species with a pivotal role in freshwater ecosystems, to establish a clear mode of action. Plants were exposed to 0.5 mg/L Cu salt, 5 and 70 mg/L CuO-NPs during 96 hours and 10 days. Several morphological and physiological endpoints were measured. Cu salt was found more toxic than CuO-NPs to plants based on all the measured endpoints despite a similar internal Cu concentration demonstrated via Cu mapping by micro particle-induced X-ray emission (µPIXE) coupled to Rutherford backscattering spectroscopy (RBS).

Biomacromolecule composition investigated by FTIR converged between 70 mg/L CuO-NPs and Cu salt treatments after 10 days. This demonstrates that the difference of toxicity comes from a sudden massive Cu 2+ addition from Cu salt similar to an acute exposure, versus a progressive leaching of Cu 2+ from CuO-NPs representing a chronic exposure. Understanding NP toxicity mechanisms can help in the future conception of safer by design NPs and thus diminishing their impact on both the environment and humans.

Introduction

The last decades have seen an exponential increase in the use of engineered nanoparticles (NPs), i.e.

particles with at least one dimension below 100 nm [START_REF] Boverhof | Comparative assessment of nanomaterial definitions and safety evaluation considerations[END_REF]. Their small size confers them properties which are highly valuable for both domestic and industrial purposes, such as in electronics, cosmetics, drug engineering or agriculture [START_REF] Bundschuh | Nanoparticles in the environment: where do we come from, where do we go to?[END_REF]. They can be found in many products of our daily life, such as paints, sunscreens, toothpaste and clothing, making them omnipresent in our environment. Products containing NPs increased by 91-fold between 2005 and 2020 [START_REF] Environment | Search Database -The Nanodatabase[END_REF]. As an example, the global annual production of copper oxide NPs (CuO-NPs) was approximately 570 tons/year in 2014 and is predicted to be 1600 tons/year by 2025 [START_REF] Keller | Global life cycle releases of engineered nanomaterials[END_REF]. CuO-NPs are mainly used as biocides [START_REF] Ivask | Mechanisms of toxic action of Ag, ZnO and CuO nanoparticles to selected ecotoxicological test organisms and mammalian cells in vitro : A comparative review[END_REF], [START_REF] Tamayo | Release of silver and copper nanoparticles from polyethylene nanocomposites and their penetration into Listeria monocytogenes[END_REF], and they are included in several applications in agriculture, such as fungicides and herbicides, but also as growth regulators and fertilizers [START_REF] Xiong | Copper Oxide Nanoparticle Foliar Uptake, Phytotoxicity, and Consequences for Sustainable Urban Agriculture[END_REF]. Such extensive use has led to a direct contamination of many ecosystems whose extent started to be acknowledged by the end of the 2000's in both aquatic and terrestrial environments [START_REF] Lee | Toxicity and bioavailability of copper nanoparticles to the terrestrial plants mung bean (phaseolus radiatus) and wheat (triticum aestivum): plant agar test for water-insoluble nanoparticles[END_REF]- [START_REF] Dimkpa | Fate of CuO and ZnO nano-and microparticles in the plant environment[END_REF]. The aquatic environment is known to be especially at risk as it acts as a sink for pollutants [START_REF] Bashir | Concerns and Threats of Contamination on Aquatic Ecosystems[END_REF]. CuO-NPs are released into aquatic ecosystems through indirect pathways, such as runoffs and leaching from industrial and agricultural sites, and also through direct pathways, with the use of antifouling paints [START_REF] Adeleye | Release and detection of nanosized copper from a commercial antifouling paint[END_REF], [START_REF] Muller-Karanassos | Environmental concentrations of antifouling paint particles are toxic to sediment-dwelling invertebrates[END_REF], raising a global concern for ecological impacts of such contamination [START_REF] Wu | Assessment of Cu and CuO nanoparticle ecological responses using laboratory small-scale microcosms[END_REF].

Negative impacts have been reported on several organisms, although the toxicity range varies depending on the species and environmental factors. Indeed, water quality, organic matter and pH will influence the colloidal stability of metal-based NPs, and thus influence their potential toxicity [START_REF] Peng | Transformation of CuO nanoparticles in the aquatic environment: Influence of pH, electrolytes and natural organic matter[END_REF]. It is therefore challenging to assess their effects on aquatic ecosystems. Several studies have been performed on aquatic species to assess the mode of action and toxicity of CuO-NPs and if it stems from ionic leaching or particle-specific toxicity. No consensus has been reached so far. Especially, the mechanisms behind toxicity in aquatic plants remain unclear. Few studies found that aquatic plant species were more sensitive to the nanoparticulate form than the ionic one [START_REF] Song | Effects of CuO nanoparticles on Lemna minor[END_REF]- [START_REF] Zhang | Uptake and accumulation of CuO nanoparticles and CdS/ZnS quantum dot nanoparticles by Schoenoplectus tabernaemontani in hydroponic mesocosms[END_REF], while another study on duckweed attributed toxicity to ionic leaching from CuO-NPs [START_REF] Perreault | Effect of soluble copper released from copper oxide nanoparticles solubilisation on growth and photosynthetic processes of Lemna gibba L[END_REF]. Finally, one study performed on a submerged rooted aquatic plant species, Elodea nuttaliii, showed similar effects between Cu salt and CuO-NP exposure on growth and down-regulation of a Cu transporter gene, COPT1, after 24h of exposure [START_REF] Regier | Effects of copper-oxide nanoparticles, dissolved copper and ultraviolet radiation on copper bioaccumulation, photosynthesis and oxidative stress in the aquatic macrophyte Elodea nuttallii[END_REF], suggesting that toxicity was due to ionic leaching. In order to understand better the toxicity mechanisms, we need to access Cu spatial distribution which is not investigated in most studies. For this purpose, biophysical techniques represent a great asset even though they are not often used in environmental sciences. Indeed, spectroscopic techniques have proven to be effective tools to assess metal uptake and distribution in organisms at high spatial resolution [START_REF] Larue | Innovative combination of spectroscopic techniques to reveal nanoparticle fate in a crop plant[END_REF]. The combination of micro particle-induced X-ray emission (µPIXE) with Rutherford backscattering spectroscopy (RBS) provides unique in situ information on elemental mapping in plant tissues and is highly relevant to assess uptake of metal based NPs [START_REF] Castillo-Michel | Practical review on the use of synchrotron based micro-and nano-X-ray fluorescence mapping and Xray absorption spectroscopy to investigate the interactions between plants and engineered nanomaterials[END_REF].

In this study, we aimed at making use of cutting-edge spectroscopic techniques to bring new and original data to answer the question of the mechanisms of action implied in CuO-NP toxicity in aquatic plants in comparison with Cu salt. Myriophyllum spicatum (L.), a submerged OECD model species (OECD TG 238, 239), was used to link dissolution, adsorption, absorption, and Cu localization to CuO-NP mode of action. Cu distribution was analysed using µPIXE coupled to RBS to avoid mixing the Cu signal coming from Cu adsorbed at the surface of leaves with Cu really absorbed inside the leaf tissues, allowing to map Cu distribution in leaf cross-sections. Additionally, Cu toxicity was assessed through "traditional" biomarkers (such as growth, dry matter content and photosystem efficiency) and by evaluating its impact on plant biomacromolecule composition via Fourier-transformed infrared spectroscopy (FTIR) analysis.

Material and methods

Plant growth and Cu exposure

M. spicatum L. (Haloragaceae) was chosen as a model species of aquatic freshwater species (see supporting information for more details about plant growth). In total, two concentrations of CuO-NPs were used: 5 and 70 mg/L; along with one control concentration (0 mg/L) and one CuSO4 concentration containing 0.5 mg/L Cu 2+ , with n = 10 per concentration (see supporting information for more details about exposure protocol) for either 96h or 10 days of exposure. CuO-NP concentrations were chosen based on literature and preliminary experiments allowing proper Cu visualization inside leaf tissues by µPIXE/RBS. Although no information on environmental CuO-NP concentration can be found, studies have highlighted Cu 2+ concentrations up to 100 mg/kg in European topsoils [START_REF] Ballabio | Copper distribution in European topsoils: An assessment based on LUCAS soil survey[END_REF] and Cu concentration of 40 mg/kg in freshwater sediments are considered environmentally-relevant [START_REF] Ahmed | Environmental concentrations of copper, alone or in mixture with arsenic, can impact river sediment microbial community structure and functions[END_REF], [START_REF] Rader | The Fate of Copper Added to Surface Water: Field, Laboratory, and Modeling Studies[END_REF].

Nanoparticle characterization

The nominal diameter of NPs was determined using Transmission Electron Microscopy (TEM, Jem-1400, Jeol, USA), and imageJ software for image analysis (n = 45). Nanoparticle hydrodynamic diameter in suspension was assessed by dynamic light scattering (DLS; Zetasizer Nano ZS90, Malvern Panalytical, UK) and the software Zetasizer (n = 3). The zeta potential in plant culture medium was measured with 12 runs for both suspensions (Zetasizer Nano ZS90, Malvern Panalytical, UK).

Nanoparticle dissolution at the end of exposure in plant exposure medium was assessed by ICP-OES (see supplementary materials for more details).

Copper leaching and concentration in plant samples

Copper concentrations in the media were measured by sampling water at the beginning and at the end of exposure from experimental units of both Cu exposure times (96 h and 10 days) in order to assess effective concentrations. Copper concentration in plants was measured at the end of Cu exposure after acid digestion of dry plant material. For more details about sample preparation see supporting information.

Copper and other elemental (Ca, K, P, Fe, S, Mg, Mo, Mn, Zn) concentrations in both plants and media were measured using ICP-OES (Iris Intrepid II XLD, Thermo Electron, MA, USA) with a detection limit of 0.0012 mg/kg for water samples and 0.00169 mg/kg for plant samples. Different controls were analysed to ensure the quality of the measurements.

Exposure endpoints

Growth-related endpoints

Fresh mass was measured at the beginning and at the end of exposure after having gently dried the plants with blotting paper, to calculate the relative growth rate based on biomass production (RGR).

Samples were oven-dried at 70 °C during 72 h before weighting again to measure their dry matter content (DMC).

RGR was calculated for each experimental unit as follows:

𝑅𝐺𝑅 𝑖-𝑗 = (ln(𝑁𝑗) -ln(𝑁𝑖)) / 𝑡
where RGRi-j is the relative growth rate from time i to j, Ni and Nj is the endpoint (fresh mass) in the test or control vessel at time i and j, respectively, and t is the time period in days from i to j. DMC in % was calculated as:

%𝐷𝑀𝐶 = ( 100 × 𝐷𝑀 𝐹𝑀 )
where FM is the fresh mass of a plant sample, DM is its corresponding dry mass.

Physiological endpoints

Oxidative stress was evaluated through lipid peroxidation measurements, using the production of malondialdehyde (MDA) in the samples to assess membrane integrity according to Parveen et al.

(2017) [START_REF] Parveen | Biochemical adaptations of four submerged macrophytes under combined exposure to hypoxia and hydrogen sulphide[END_REF] (more details in supporting information).

Quantum efficiency of photosystem II was estimated at the end of exposure for each experimental unit using the Fv/Fm ratio, which is the ratio of the variable (Fv) to the maximum chlorophyll fluorescence (Fm), i.e. the maximal ability of the plant to harvest light [START_REF] Murchie | Chlorophyll fluorescence analysis: A guide to good practice and understanding some new applications[END_REF]. Measurements were conducted using a Diving-PAM fluorometer (Heinz Walz GmbH, Germany) in a dark chamber, 30 min after dark acclimatization of the plant to ensure that all reaction centers were opened for new photons. The basic settings of the Diving-PAM, namely intensity of measuring light (50: MEAS-INT) and amplification factor (49: GAIN) were set to 8 and 2, respectively.

Biomacromolecule composition

Biomacromolecule composition was determined by Fourier transformed infrared spectroscopy (FTIR).

Plant samples were dried for 48 h at 105°C, then ground in thin powder (> 20 mg). Samples were analysed using a FTIR microscope in attenuated total reflection (ATR) mode (Thermo Nicolet NEXUS 470 ESR, ThermoFisher™, Massachusetts, USA) over the frequency range of 4000 -400 cm -1 with a spectral resolution of 4 cm -1 . One spectrum was an average of 64 scans per sample. Each powdered plant was placed on the sample plate and three independent technical replicates for each sample (10 biological replicates per treatment) were acquired. OMNIC software was used to export experimental spectra after ATR correction (OMNIC™ FTIR Software, ThermoFisher™, Massachusetts, USA).

FTIR data treatment was performed using Orange software [START_REF] Demšar | Orange: Data mining toolbox in Python[END_REF]. Briefly, data were pre-processed which implies selection of the region of interest (including most of the variance among samples), vector normalization and smoothing by Savitzky-Golay filter. Using the second derivative, a principal component analysis (PCA) was carried out for the different exposure times (n=30 per time of exposure × condition with technical replicates). The components permitting to explain at least 70% of the variance were used to perform a subsequent linear discriminant analysis (LDA). This approach permitted to plot the samples and detect differences among groups of samples. When a difference was detected, a logistic regression was applied to the pre-processed data to identify wavenumbers contributing to the difference detected among groups by the PCLDA.

Spatial distribution and semi-quantification of Cu

Absorption and adsorption of Cu in leaves were mapped and measured using a nuclear microprobe. A combination of micro-particle induced X-ray emission (μPIXE) and Rutherford backscattered spectroscopy (RBS) was used for elemental mapping and semi-quantification. Leaves were thoroughly washed three times with deionized water to take off Cu lightly bound to the surface and immersed in a droplet of resin (Tissue Teck Sakura ® ) to be immediately cryo-fixed by plunging the sample in isopentane cooled with liquid nitrogen. Samples were then cut in thin cross-sections (40 μm) using a cryo-microtome (Leica, Germany) and finally freeze-dried (48 h, -52°C, 0.01 mbar). Freeze-dried sections were analyzed at the nuclear microprobe available at the Atomic Energy Commission (CEA)

Center of Saclay (France) with a proton source of 3 MeV, a beam focused to 2.5 μm and a current intensity of 500 pA. Data processing was performed using Rismin software [START_REF] Daudin | Development of 'position-charge-time' tagged spectrometry for ion beam microanalysis[END_REF] to define regions of interest and extract spectra, and SIMNRA [START_REF] Mayer | SIMNRA, a simulation program for the analysis of NRA, RBS and ERDA[END_REF] and GUPIX [START_REF] Campbell | Guelph PIXE software package III: alternative proton database[END_REF] codes to fit RBS and PIXE data, respectively. The Cu/(K+Ca) ratio was used as a Cu enrichment indicator as K and Ca are the most abundant endogenous elements.

Statistical analyses

Results were analyzed using the R studio software (R Core Team (2016) V 3.3.1) and analyses were performed within each exposure time (96 h and 10 days). Homoscedasticity was tested using Bartlett test. Data normality was assessed with Shapiro-Wilk test on ANOVA residuals, with logtransformation when normality assumption was not met. Two-way ANOVAs were performed on results showing normal distribution, with or without log transformation, to assess the interactive effects of Cu concentrations and time of exposure. Tukey HSD post-hoc tests were used to identify significant differences among Cu concentrations and exposure times. Kruskal-Wallis tests were used in dataset when no normality was found despite log-transformation. The differences in plant inorganic composition resulting from exposure were assessed using a linear discriminant analysis (LDA, n = 10, ade4 package [START_REF] Dray | Package 'ade4[END_REF]). The significance of the discriminant analyses was assessed using Monte-Carlo tests with 1000 repetitions.

Results

CuO-NP characterization, Cu 2+ concentration and leaching

Nominal diameter of CuO-NPs was on average 64.9 ± 8.5 nm according to TEM images (n = 45, Fig. 1A). Sedimentation was visually observed with NP deposition on the leaves of M. spicatum after the first 2 hours of exposure, forming a thin black layer (Fig. S1). Hydrodynamic diameter measured at different times showed agglomeration of NPs from the beginning of exposure with no strong evolution over time, except after 10 days at the highest concentration (Fig. 1B). Hydrodynamic diameter based on Z-average was 331 nm after 96 h of exposure for both concentrations, and 59 nm and 321 nm after 10 days of exposure at 5 mg/L and 70 mg/L CuO-NPs, respectively. Finally, the zeta potential of CuO-NPs in Smart & Barko was -21.7 ± 3.5 mV.

Total Cu in the medium for all concentrations at the beginning of exposure were 0, 0. concentration in the medium of 0.5 mg/L. After 10 days of exposure, the Cu 2+ concentration in the medium decreased by 86% and 79% for 5 mg/L and 70 mg/L CuO-NP suspensions, respectively, likely due to NP sedimentation on sediment (Fig. S1) and to plant adsorption/absorption of Cu 2+ . 2E). Cu accumulation decreased in parenchyma and vascular cylinder and was similar for both treatments. Our results showed that Cu from both treatments was internalized by the plants in parenchyma and vascular tissues.

Copper concentration and distribution in plants

Copper toxicity to plants

Copper toxicity based on "traditional" endpoints

Relative growth rate was only significantly impacted by Cu salt exposure, inhibiting growth by 57% after 96 h and by 80% after 10 days of exposure (2-way ANOVA, F3,72 = 12.64, P < 0.0001, Fig. 3A).

CuO-NP exposure inhibited growth by 30% at 70 mg/L after 10 days of exposure, however the variation among replicates was too high to highlight a significant difference.

Dry matter content significantly increased after 10 days of exposure at 0.5 mg/L Cu salt, with 14% of DMC in exposed plants against 8% for others (2-way ANOVA, F3,71 = 6.423 P = 0.0149, Fig. 3B).

Lipid peroxidation levels were significantly increased at both exposure times for 0.5 mg/L Cu salt, and after 10 days of exposure to 70 mg/L CuO-NPs (2-way ANOVA, F3,63 = 21.559, P < 0.001, Fig. 3C).

An interactive effect was found between treatments and exposure time (2-way ANOVA, F3,63 = 5.333, P = 0.002), with a significant effect of CuO-NPs on lipid peroxidation only after 10d of exposure compared to Cu salt.

Finally, no significant effect of CuO-NPs was observed on Fv/Fm at any concentration despite the deposition of a thin black layer on plant leaves, whereas Cu salt significantly decreased Fv/Fm by 12% after 96 h of exposure (2-way ANOVA, F3,72 =12.956, P < 0.001, Fig. S1 &S2).

Copper toxicity based on biomacromolecule composition

Biomacromolecule composition significantly changed among treatments at both exposure times (Fig. 4). The significant differences and the peak interpretations are listed in Table 1. After 96 h of exposure, plants at 0.5 mg/L Cu salt segregated from other treatments based on the first and third dimensions of the PCA toward the up-right corner, and plants exposed to 70 mg/L CuO-NPs segregated along the first dimension on the right side (Fig. 4A,B). Plants exposed to Cu segregated from the control based on the first dimension, with an overlap between 5 and 70 mg/L CuO-NPs treatments. These differences in composition were observed mostly in proteins, phenolic compounds, carbohydrates and cellulosic compounds (Table 1). After 10 days of exposure, plants exposed to 5 mg/L CuO-NPs remained the closest to control plants in terms of composition and remained on the left side of the first dimension, whereas composition from plants exposed at 0.5 mg/L Cu salt and 70 mg/L CuO-NPs converged on the right side of the same dimension (Fig. 4C,D). The plants exposed to these two treatments exhibited higher absorbances for the peaks representing polysaccharides, carbohydrates and proteins compared to control plants (Table 1).

Effect of Cu exposure on plant ionome

Plant exposure to ionic Cu and CuO-NPs significantly influenced their inorganic composition after 96 These results were similar to those found by µPIXE/RBS in plant leaves after 10 days (Fig. S5).

Calcium concentrations were significantly higher for plants exposed to ionic Cu, and found rather accumulated in parenchyma and epidermis tissues (Fig. S5A). Sulfur was found significantly more abundant in plants exposed to CuO-NPs with no difference in distribution among tissues due to variation among cross-sections (Fig. S5B) and Zn was found at higher concentrations in plants exposed to CuO-NPs at 70 mg/L, with no difference in distribution among plant tissues (Fig. S5C).

DISCUSSION

Our results, based on an original combination of spectroscopic techniques (FTIR and µPIXE/RBS) and a thorough characterization of NP dynamics in suspension, suggest that Cu ion leaching is the main driver of CuO-NP toxicity even though a specific nano form effect cannot be excluded but would remain minor. These conclusions confirmed some data available in the literature on different organisms. Indeed, few studies suggest that the most prominent mode of action of CuO-NPs on organisms is the leaching of ionic Cu 2+ from NPs, inducing reactive oxygen species (ROS) production and subsequent stress in organisms [START_REF] Wu | Assessment of Cu and CuO nanoparticle ecological responses using laboratory small-scale microcosms[END_REF], [START_REF] Malhotra | Review of copper and copper nanoparticle toxicity in fish[END_REF]. This is shared by most heavy-metal based NPs which are prone to dissolution, such as Ag-NPs or ZnO-NPs [START_REF] Ivask | Mechanisms of toxic action of Ag, ZnO and CuO nanoparticles to selected ecotoxicological test organisms and mammalian cells in vitro : A comparative review[END_REF], [START_REF] Aruoja | Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata[END_REF]- [START_REF] Kahlon | Impact of heavy metals and nanoparticles on aquatic biota[END_REF], although some NPs, such as TiO2 and CeO2, behave differently [START_REF] Hou | Toxicity and mechanisms of action of titanium dioxide nanoparticles in living organisms[END_REF], [START_REF] Bour | Toxicity of CeO2 nanoparticles at different trophic levels -Effects on diatoms, chironomids and amphibians[END_REF]. This mode of action was shown from single-cell organisms [START_REF] Gunawan | Cytotoxic Origin of Copper ( II ) Oxide Nanoparticles : Comparative Studies and Metal Salts[END_REF]- [START_REF] Wang | Where does the toxicity of metal oxide nanoparticles come from: The nanoparticles, the ions, or a combination of both?[END_REF], to more complex organisms such as zooplankton, fish and aquatic plant species [START_REF] Yue | Interaction of CuO nanoparticles with duckweed (Lemna minor. L): Uptake, distribution and ROS production sites[END_REF]- [START_REF] Ozmen | Ecotoxicity of Nanomaterials in Aquatic Environment[END_REF].

A big difference in toxicity can be found from one study to another in the literature even within a same species, which may be related to environmental conditions [START_REF] Wu | Assessment of Cu and CuO nanoparticle ecological responses using laboratory small-scale microcosms[END_REF], [START_REF] Zhao | Uptake, Distribution, and Transformation of CuO NPs in a Floating Plant Eichhornia crassipes and Related Stomatal Responses[END_REF].

Indeed, the behavior of NPs and the subsequent ionic leaching is directly linked to water physicochemical parameters [START_REF] Ozmen | Ecotoxicity of Nanomaterials in Aquatic Environment[END_REF]. In our exposure conditions, CuO-NPs were prone to quickly form agglomerates as a result of low repulsive forces, high surface energy and high ionic strength of the medium leading to a high sedimentation rate, as demonstrated by the NP deposition on leaves. The same kind of results was obtained in the literature for other heavy-metal based NPs [START_REF] Khan | Coagulation and dissolution of CuO nanoparticles in the presence of dissolved organic matter under different pH values[END_REF]- [START_REF] Song | Phytotoxicity and accumulation of zinc oxide nanoparticles on the aquatic plants Hydrilla verticillata and Phragmites Australis: leaf-type-dependent responses[END_REF]. The presence of CaCl2 in the Smart & Barko medium likely increased the formation of agglomerates and sedimentation speed, as Ca 2+ is known to form bridges in solution [START_REF] Sousa | Aggregation kinetics and surface charge of CuO nanoparticles: The influence of pH, ionic strength and humic acids[END_REF]. The black deposition on the plant leaves visually observed suggests that CuO-NPs were tightly adsorbed at the plant surface as it was not eliminated with several washing steps. [START_REF] Shi | Phytotoxicity and accumulation of copper oxide nanoparticles to the Cu-tolerant plant Elsholtzia splendens[END_REF] noticed a similar black deposition on the roots of Elsholtzia splendens, a terrestrial plant, after hydroponic exposure which resulted in very high concentrations at the surface of plant roots [START_REF] Shi | Phytotoxicity and accumulation of copper oxide nanoparticles to the Cu-tolerant plant Elsholtzia splendens[END_REF].

The Cu 2+ concentration leached from CuO-NPs after 96 h was similar between the two CuO-NP treatments and equivalent to the Cu salt concentration in solution (i.e. 0.5 mg/L). After 10 days of exposure, Cu 2+ leached from CuO-NPs was still similar between the two concentrations, but strongly decreased compared to the concentration found at 96 h. This could be explained by the continuous uptake of Cu 2+ by plants, leading to Cu decrease in the water column. Furthermore, underwater photosynthesis changes the pH over time through the release of HCO3 -, which can influence colloidal stability and increase the formation of agglomerates, decreasing ionic leaching [START_REF] Pedersen | Underwater photosynthesis of submerged plants -recent advances and methods[END_REF], [START_REF] Qiu | The aggregation and sedimentation of two different sized copper oxide nanoparticles in soil solutions: Dependence on pH and dissolved organic matter[END_REF]. This, combined with the formation of hetero-agglomerates with organic matter produced by M. spicatum, can decrease further the colloidal stability and ionic leaching, as it is proportional to the surface area to volume ratio [START_REF] Shi | Phytotoxicity and accumulation of copper oxide nanoparticles to the Cu-tolerant plant Elsholtzia splendens[END_REF], [START_REF] Sousa | Aggregation kinetics and surface charge of CuO nanoparticles: The influence of pH, ionic strength and humic acids[END_REF], [START_REF] Qiu | The aggregation and sedimentation of two different sized copper oxide nanoparticles in soil solutions: Dependence on pH and dissolved organic matter[END_REF]- [START_REF] Gross | Release and ecological impact of algicidal hydrolysable polyphenols in Myriophyllum spicatum[END_REF].

In our study, CuO-NPs were less toxic to M. spicatum than Cu salt, especially after 96 h where no effect of NPs was observed, whereas Cu 2+ concentration in the medium was similar between Cu salt and NPs. An increase in lipid peroxidation was observed after 10 days of exposure to 70 mg/L CuO-NPs and was the only significant sign of toxicity when copper was provided under a NP form. On the other hand, Cu salt strongly decreased growth and increased lipid peroxidation at both exposure times, and increased DMC after 10 days as a result of stress [START_REF] Michalak | Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress[END_REF], [START_REF] Elger | Leaf dry matter content as an integrative expression of plant palatability: the case of freshwater macrophytes[END_REF]. This result could be surprising as Cu bulk concentration was higher (by a factor of 9) in plants exposed to 70 mg/L CuO-NPs compared to plants exposed to Cu salts.

However, Cu bulk concentration does not give any information as to Cu internalization and localization, especially as CuO-NP accumulation was visible on plant surface. The data provided by the µPIXE/RBS analysis confirmed that Cu mostly accumulated on the plant surface (epidermis) with a factor of 3 between Cu salts and CuO-NPs 70 mg/L, even if this difference was not significant due to high variation among replicates. Furthermore, when focusing on the internalized Cu, a similar concentration was found in plants exposed to Cu salt and to 70 mg/L CuO-NPs. This is also in line with the fact that equivalent ionic Cu concentrations were measured in the medium suggesting an internalization which is mainly occurring under ionic form. Previous work has shown that ionic internalization is detected through an homogenous distribution with µPIXE, whereas a dot-like distribution is linked to nanoparticulate form [START_REF] Larue | Innovative combination of spectroscopic techniques to reveal nanoparticle fate in a crop plant[END_REF]. In our study, internalization of NPs themselves cannot be excluded as some highly concentrated sub-micrometric to micrometric spots were detected by µPIXE inside leaf parenchyma, compared to the more homogeneous Cu distribution in the vascular tissues. This phenomenon has also been demonstrated in other studies on both terrestrial and aquatic species [START_REF] Zhao | Uptake, Distribution, and Transformation of CuO NPs in a Floating Plant Eichhornia crassipes and Related Stomatal Responses[END_REF], [START_REF] Zhang | Uptake and accumulation of CuO nanoparticles and CdS/ZnS quantum dot nanoparticles by Schoenoplectus tabernaemontani in hydroponic mesocosms[END_REF], [START_REF] Yuan | Internalization and Phytotoxic Effects of CuO Nanoparticles in Arabidopsis thaliana as Revealed by Fatty Acid Profiles[END_REF], [START_REF] Dai | Interaction of CuO nanoparticles with plant cells: Internalization, oxidative stress, electron transport chain disruption, and toxicogenomic responses[END_REF], but further investigations using X-ray absorption spectroscopy would be needed for speciation confirmation. Indeed, some studies demonstrated that aquatic species, such as Chlorella pyrenoidosa and Eichhornia crassipes, were able to transform CuO-NPs into other Cu species, such as Cu2S or Cu2O-NPs, highlighting the need to go deeper into the speciation of CuO-NP once it enters biological barriers as speciation can influence both its translocation and toxicity [START_REF] Zhao | Uptake, Distribution, and Transformation of CuO NPs in a Floating Plant Eichhornia crassipes and Related Stomatal Responses[END_REF], [START_REF] Zhao | Interactions of CuO nanoparticles with the algae Chlorella pyrenoidosa: adhesion, uptake, and toxicity[END_REF].

These findings explain why such a high Cu concentration in plants exposed to CuO-NPs was found by ICP, but do not explain why Cu salt was more toxic to the plants despite a similar internal concentration found by µPIXE/RBS. This difference in toxicity between Cu salt and CuO-NP treatments can be explained by the sudden addition of soluble Cu salt compared to the progressive leaching of ionic Cu from CuO-NPs [START_REF] Perreault | Effect of soluble copper released from copper oxide nanoparticles solubilisation on growth and photosynthetic processes of Lemna gibba L[END_REF]. The results for biomacromolecule composition support these findings as a gradual convergence between plants exposed to 70 mg/L CuO-NPs and Cu salt was observed during exposure. A strong effect of Cu salt was observed on plant composition after 96 h compared to CuO-NP treatments (5 and 70 mg/L) whereas the overall ionic Cu concentration in the medium was similar. This lower impact of NPs despite similar ionic concentration as Cu salt suggests that the leaching was progressive, and the subsequent stress was of lower amplitude and mitigated over the duration of our exposure. A change in phenolic compounds, proteins and cellulosic compounds was observed in plants exposed to Cu salts, likely as the result of a stress, and possibly corresponding to an antioxidant response [START_REF] Yue | Interaction of CuO nanoparticles with duckweed (Lemna minor. L): Uptake, distribution and ROS production sites[END_REF], [START_REF] Hasanuzzaman | Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator[END_REF], [START_REF] Sivaci | Changes in antioxidant activity, total phenolic and abscisic acid constituents in the aquatic plants Myriophyllum spicatum L. and Myriophyllum triphyllum Orchard exposed to cadmium[END_REF], as well as a mechanism to maintain membrane integrity [START_REF] Dai | Interaction of CuO nanoparticles with plant cells: Internalization, oxidative stress, electron transport chain disruption, and toxicogenomic responses[END_REF], [START_REF] Wang | Photodegradation Elevated the Toxicity of Polystyrene Microplastics to Grouper (Epinephelus moara) through Disrupting Hepatic Lipid Homeostasis[END_REF]. Additionally, ICP-OES and µPIXE/RBS analyses showed changes in concentrations of S or Zn mostly impacted by ionic Cu and by CuO-NPs to a smaller extent. It could be linked to shifts in the antioxidant balance, as these elements act as co-factors for several detoxification enzymes and Cu regulation pathways [START_REF] Han | C2H2 Zinc Finger Proteins: Master Regulators of Abiotic Stress Responses in Plants[END_REF]- [START_REF] Hossain | Molecular Mechanism of Heavy Metal Toxicity and Tolerance in Plants: Central Role of Glutathione in Detoxification of Reactive Oxygen Species and Methylglyoxal and in Heavy Metal Chelation[END_REF]. An interesting response was the increased Ca concentration and its distribution primarily in epidermis tissues. Studies have shown that Ca was an important part of the signaling pathway and stress response in plants, for instance with the calmodulin pathways for signaling or the formation of egg box structures for heavy metal regulation in cell walls [START_REF] Jalmi | Traversing the links between heavy metal stress and plant signaling[END_REF], [START_REF] Kosakivska | Molecular mechanisms of plant adaptive responses to heavy metals stress[END_REF].

More specific assays would be necessary to assess the extent of the stress response triggered by exposure, and its specific pathways, by targeting mechanisms such as enzymatic activities, transcription, and production of antioxidant compounds.

Several studies on different aquatic micro and macro-organisms have found similar results in which Cu internal concentration resulting from CuO-NP exposure was not correlated to the observed toxicity, and the ionic counterpart was much more toxic for a similar or lower internal concentration, supporting our finding [START_REF] Song | Effects of CuO nanoparticles on Lemna minor[END_REF], [START_REF] Regier | Effects of copper-oxide nanoparticles, dissolved copper and ultraviolet radiation on copper bioaccumulation, photosynthesis and oxidative stress in the aquatic macrophyte Elodea nuttallii[END_REF], [START_REF] Gunawan | Cytotoxic Origin of Copper ( II ) Oxide Nanoparticles : Comparative Studies and Metal Salts[END_REF], [START_REF] Perreault | Effect of soluble copper released from copper oxide nanoparticles solubilisation on growth and photosynthetic processes of Lemna gibba L[END_REF], [START_REF] Arratia | Comparative toxic effects of copper-based nanoparticles and their microparticles in Daphnia magna by using natural freshwater media[END_REF]. For instance [START_REF] Wu | Assessment of Cu and CuO nanoparticle ecological responses using laboratory small-scale microcosms[END_REF] demonstrated no correlation between toxicity and Cu concentration in three aquatic organisms exposed to CuO-NPs, compared to

Cu salt [START_REF] Wu | Assessment of Cu and CuO nanoparticle ecological responses using laboratory small-scale microcosms[END_REF]. [START_REF] Adam | The chronic toxicity of CuO nanoparticles and copper salt to Daphnia magna[END_REF] found a higher toxicity in Daphnia magna exposed to Cu salt whereas Cu concentration was higher in organisms exposed to CuO-NPs. The toxicity of CuO-NPs was attributed to the Cu ions formed during NP dissolution [START_REF] Adam | The chronic toxicity of CuO nanoparticles and copper salt to Daphnia magna[END_REF]. Similarly, the marine bacteria Vibrio anguillarum showed a lower sensitivity to CuO-NPs than to Cu salt, and toxic effects were attributed to progressive Cu ions leaching from NPs [START_REF] Rotini | Salinity-based toxicity of CuO nanoparticles, CuO-bulk and Cu ion to vibrio anguillarum[END_REF].

Overall, exposure to Cu salt can be considered as an acute exposure to ionic Cu, triggering a rapid and strong change in plant physiology, whereas exposure to CuO-NPs corresponds to a chronic exposure to ionic Cu, inducing progressive physiological adjustments of lower amplitude. The antioxidant balance can mitigate a chronic exposure over time by inducing progressive physiological changes [START_REF] Hasanuzzaman | Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator[END_REF], [START_REF] Wang | Photodegradation Elevated the Toxicity of Polystyrene Microplastics to Grouper (Epinephelus moara) through Disrupting Hepatic Lipid Homeostasis[END_REF],

whereas an acute exposure could lead to a tipping point where the stress can no longer be copped with [START_REF] Regier | Effects of copper-oxide nanoparticles, dissolved copper and ultraviolet radiation on copper bioaccumulation, photosynthesis and oxidative stress in the aquatic macrophyte Elodea nuttallii[END_REF], [START_REF] Keller | Comparative environmental fate and toxicity of copper nanomaterials[END_REF].

CONCLUSION

This study assessed the toxicity of CuO-NPs compared to Cu ions from CuSO4 salt on M. spicatum, a model aquatic plant species. Based on our observations, the toxicity of CuO-NPs appeared driven by a progressive ionic leaching from NPs and was found less toxic than Cu salt, as the plant was able to adapt and mitigate stress over time through physiological changes. Cutting-edge techniques showed that most of the Cu leached from NPs was adsorbed at the plant leaf surface rather than absorbed, which would have not been possible with other bulk analyses such as ICP-OES. µPIXE/RBS provided a new perception of the links between toxicity and accumulation regarding heavy metal-based NPs.

Thanks to high-throughput FTIR technique, we were able to visualize global biomacromolecule composition shifts resulting from exposure. In future experiments, we will investigate more specific response patterns such as phenolic compound production and cell walls components. These findings do not exclude a nanospecific toxicity mechanism, as NP internalization was suggested by µPIXE, but further studies on speciation within organisms remain to be done.

Table 1. Spectral assignment and significant differences in biomacromolecule composition of M. spicatum exposed to Cu salt at 0.5 mg/L and CuO-NPs at 5 and 70 mg/L during 96 hours and 10 days. Peak numbers refer to Figure 4, the significant differences are either between (-) concentrations, or one treatment differs from all others (alone). Significant differences were set at p-value < 0.05 and were calculated with ANOVAs on Orange software for each wavenumber. Numbers in A, C refers to the peaks listed in Table 1.

Peaks

29

  Significant differences in bulk Cu concentrations in plants were observed among treatments (2-way ANOVA, F3,72 = 3784.903, P < 0.0001) but not between times of exposure. On average at both exposure times, Cu concentrations in plants were 4.7 ± 0.5, 8.1 ± 1.1 and 41.7 ± 4.7 mg/g dry weight (DW) of plants exposed to 0.5 mg/L Cu salt, 5 and 70 mg/L CuO-NPs, respectively (Fig.2A).To go further into Cu internalization and localization in plant leaf, spatial distribution analysis was performed on plants exposed to 0, 0.5 mg/L Cu salt and 70 mg/L CuO-NPs for 10 days. Homeostasis level of Cu was found in control plants for basal metabolism (Fig.2B, C), whereas significantly higher accumulation of Cu was found at 0.5 mg/L Cu salt (Fig.2D) and 70 mg/L CuO-NPs (Fig.2E, 2-way ANOVA, F2,38 = 340.64, P < 0.0001). The highest Cu accumulation was detected on leaf epidermis for both treatments, with more than 3 times Cu level in plant sections exposed to 70 mg/L CuO-NPs compared to plants exposed to 0.5 mg/L Cu salt (Fig.

h and 10

 10 days, as revealed by the linear discriminant analyses (LDA) based on ICP-OES measurements realized on the whole plants (Monte-Carlo test, P = 0.001, 22.47 % and 22.62% of inertia explained, respectively, Fig S3). A significant increase of Ca concentrations was observed at both times of exposures for plants exposed to ionic Cu (2-way ANOVA, F3,72 = 40.41, P < 0.001), whereas plants exposed to 70 mg/L CuO-NPs showed a significantly higher Ca concentration only after 96 h (Fig. S4A). Magnesium concentration significantly increased in plants exposed to ionic Cu after 96 h, and increased both in ionic Cu and 70 mg/L CuO-NPs treatments after 10 days (Kruskal-Wallis, df = 3, P < 0.001, Fig. S4B). Sulfur concentration in plants significantly decreased after 10 d of exposure to ionic Cu (2-way ANOVA, F2,50 = 5.61, P < 0.001, Fig. S4C) whereas Zn concentration was significantly higher for plants exposed to 70 mg/L CuO-NPs at both times of exposure (Kruskalwallis, df = 3, P < 0.005, Fig. S4D).

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. (A) Cu concentrations in Myriophyllum spicatum plants measured with ICP-OES n = 10 and

Figure 3 .

 3 Figure 3. (A) Relative growth rates with Cu 2+ leached from CuO-NPs in mg/L displayed in italic, (B)

Figure 4 .

 4 Figure 4. Biomacromolecule composition of M. spicatum analyzed by FTIR exposed to Cu salt at 0.5

Figure 1

 1 Figure 1 680

Figure 4

 4 Figure 4

ACKNOWLEDGMENTS

This research was funded by the French Ministry of research and higher education through a Doctoral Fellowship awarded to Dr Eva Roubeau Dumont. We thank Emmanuel Flahaut and Vincent Baylac from the CIRIMAT, Toulouse, for their contribution on NP characterization regarding the trainings on Turbiscan and DLS measurements, Lucas Vigier for his implication in methodological developments of MDA protocols and Clarisse Liné for the TEM pictures. Authors have no competing interest to declare.


Laura Carolina Cardenas Velandias

Anne Elisabeth Fontaine

Denis Loquet

Ruben Checa

Chantal Lorentz

Bruno Bujoli

Nolven Guilhaume

Christophe Geantet

Emmanuel Chailleux

Clémence Queffélec

Dorothée Laurenti

Catalytic Hydrothermal Conversion of Algal Residue to bio-Bitumen

Keywords: Microalgae, Hydrothermal Liquefaction, Oxide Catalysts, Bio-binders, Bitumen

After extraction of high-added value components of Spirulina sp., the remaining residues can be valorized by hydrothermal liquefaction to produce an oily water insoluble viscous phase (biocrude) which is very similar to petroleum bitumen. This hydrophobic fraction is composed of various N-,O-containing cyclic compounds, fatty acids, amides, nitriles, alkenes and alkanes.

In order to optimize the rheological properties of the biocrude, we studied the addition of different catalysts with various loadings: ZrO2 or CeO2, or Ce(NO3)3.6H2O as well as the effect of residence time during HTL. The biocrude has been systematically characterized by GCxGC, GPC, elemental analyses and dynamic shear rheology and compared to a petroleum bitumen.

The different catalysts tested had little influence on the composition of the HTL hydrophobic fraction in the chosen operating conditions. In contrast, increasing the reaction time (from 1 to 7 h) was found to promote condensation reactions, thereby increasing the average molecular weight of the biocrude, leading to a viscoelastic material very similar to a petroleum bitumen.

Introduction

Similarly to transportation fuels, alternatives bio-resources have to be investigated for the production of bitumen substitutes in order to be more sustainable and address the uncertainty of the fossil feedstocks. Recently, it has been shown that the hydrophobic fraction obtained by hydrothermal liquefaction (HTL) of Scenedesmus sp. [START_REF] Audo | Subcritical Hydrothermal Liquefaction of Microalgae Residues as a Green Route to Alternative Road Binders[END_REF] or Spirulina sp. residues residues led to a viscous hydrophobic phase that presented rheological properties similar to those of a conventional bitumen. In contrast, spirulina residues led to an oily phase similar to an elastomer-rich bitumen, in terms of viscoelastic properties. Very recently, lignocellulose [START_REF] Ding | Development of bio oil and bio asphalt by hydrothermal liquefaction using lignocellulose[END_REF] or food waste [START_REF] Mahssin | Hydrothermal liquefaction bioproduct of food waste conversion as an alternative composite of asphalt binder[END_REF] were transformed by HTL and the obtained bio-oil were mixed at various percentage with conventional asphalt binders and their rheological properties were closed with those of petroleum-based asphalt.

In the literature, many catalysts have been investigated during HTL conversion and they were shown to affect the composition of the hydrophobic phase that lead in some cases to new interesting properties. The different catalysts previously used in HTL were either homogeneous (water soluble) or heterogeneous (water insoluble) catalysts. [START_REF] Nagappan | Catalytic hydrothermal liquefaction of biomass into bio-oils and other value-added products -A review[END_REF] Homogeneous catalysts were mainly water alkali salts or organic and inorganic acids [START_REF] Zhang | Effect of acidic, neutral and alkaline conditions on product distribution and biocrude oil chemistry from hydrothermal liquefaction of microalgae[END_REF]. One of the most commonly used is sodium carbonate (Na2CO3) that usually increases the overall biocrude yield by promoting the decomposition of carbohydrates [START_REF] Ross | Hydrothermal processing of microalgae using alkali and organic acids[END_REF][START_REF] Biller | Catalytic hydrothermal processing of microalgae: Decomposition and upgrading of lipids[END_REF][START_REF] Biller | Potential yields and properties of oil from the hydrothermal liquefaction of microalgae with different biochemical content[END_REF], although it may also induce saponification reactions in the presence of lipids [START_REF] Biller | Potential yields and properties of oil from the hydrothermal liquefaction of microalgae with different biochemical content[END_REF][START_REF] Zou | Production and characterization of bio-oil from hydrothermal liquefaction of microalgae Dunaliella tertiolecta cake[END_REF]. As regards heterogeneous catalysts, supported metals, metal oxides and zeolite catalysts were widely used in the HTL treatment of microalgae for biofuels production. For this application, the main goal is to improve the yield of the oily phase by decreasing the size of its molecular components and to reduce its oxygen and nitrogen contents [START_REF] Biller | Catalytic hydrothermal processing of microalgae: Decomposition and upgrading of lipids[END_REF][START_REF] Jena | Comparison of the effects of Na2CO3, Ca-3(PO4)(2), and NiO catalysts on the thermochemical liquefaction of microalga Spirulina platensis[END_REF][START_REF] Duan | Hydrothermal Liquefaction of a Microalga with Heterogeneous Catalysts[END_REF][START_REF] Yang | Bio-oil from hydro-liquefaction of Dunaliella salina over Ni/REHY catalyst[END_REF][START_REF] Chen | Catalytic Hydrothermal Liquefaction of D. tertiolecta for the Production of Bio-Oil over Different Acid/Base Catalysts[END_REF][START_REF] Xu | Hydrothermal liquefaction of Chlorella pyrenoidosa for bio-oil production over Ce/HZSM-5[END_REF][START_REF] Rahman | Effect of Li-LSX-zeolite on the in-situ catalytic deoxygenation and denitrogenation of Isochrysis sp. microalgae pyrolysis vapours[END_REF][START_REF] Xu | Catalytic hydrothermal liquefaction of algae and upgrading of biocrude: A critical review[END_REF][START_REF] Li | Microalgae hydrothermal liquefaction and derived biocrude upgrading with modified SBA-15 catalysts[END_REF]. According to Galadima and Muranza, the main role of heterogeneous catalysts in this case is to promote rapid bond cleavages, allowing the production of light molecular weight compounds [START_REF] Galadima | Hydrothermal liquefaction of algae and bio-oil upgrading into liquid fuels: Role of heterogeneous catalysts[END_REF].

The novelty of the present study is to assess the effect of different catalysts on the rheological properties of the biocrude obtained after HTL from algal residues. In contrast, Since the objective of the present study was to produce bio-bitumen, high molecular weight constituents have to be preserved to provide a suitable viscosity and rheology of the bio-oils.

For this purpose, we wished to increase the carbon chain size and also to remove some heteroatoms and small molecules. For instance, the coupling of two carboxylic acids via a ketonization reaction might produce longer chains ketones or hydrocarbons [START_REF] Chen | Catalytic Hydrothermal Liquefaction of D. tertiolecta for the Production of Bio-Oil over Different Acid/Base Catalysts[END_REF][START_REF] Maag | Catalytic Hydrothermal Liquefaction of Food Waste Using CeZrOx[END_REF].

Therefore, promoting ketonization reactions through the use of a catalyst seemed appropriate since these reactions have been reported as a possible pathway to obtain high molecular weight compounds, through the reactive condensation between two carboxylic acids or esters that are converted into a linear ketone with the loss of carbon dioxide and water [START_REF] Gaertner | Catalytic coupling of carboxylic acids by ketonization as a processing step in biomass conversion[END_REF]. Ceria-zirconia mixed oxides were successfully used for bio-oil upgrading [START_REF] Gärtner | Catalytic Upgrading of Bio-Oils by Ketonization[END_REF]. For example, Lee et al. [START_REF] Lee | Ketonization of hexanoic acid to diesel-blendable 6undecanone on the stable zirconia aerogel catalyst[END_REF] have reported the ketonization reaction of hexanoic acid to obtain 6-undecanone and investigated the performances of different catalysts: silica-alumina, Al2O3, TiO2, ZrO2, CeO2, MgO and MnOx. The basic oxides MgO and MnOx were found the most active but resulted in a high leaching of metal cations in the reaction medium and a deactivation of the catalyst.

Various zirconia catalysts were then tested including sulfated, aerogel and xerogel zirconias and classical low surface area zirconia [START_REF] Lee | Ketonization of hexanoic acid to diesel-blendable 6undecanone on the stable zirconia aerogel catalyst[END_REF]. The amphoteric character of ZrO2 led to higher conversions without leaching, especially in the case of high surface area.

In this work, HTL experiments were performed on new Spirulina residues, called Spirugrass, and the influence of several type of catalysts was investigated, including oxides like such as ZrO2 or CeO2 known to promote ketonization reaction, or salts such as Ce(NO3)3.6H2O which is soluble in water and can be transformed in situ into cerium oxide under HTL operating conditions. The chemical characterization of the resulting hydrophobic fraction was carried out using various advanced techniques, such as bi-dimensional gas chromatography (GCxGC) with MS and FI detectors, elemental analysis (CHNS-O), gel permeation chromatography (SEC/GPC). Finally, the rheological properties of the different hydrophobic fractions were measured and compared to a conventional petroleum bitumen.

Materials and methods

Materials

The Spirulina sp. residue, so called "Spirugrass" was provided by the AlgoSource company (Alpha-Biotech, Asserac, France). AlgoSource markets Spirulina sp. extracts as food supplement. In a typical process, the crude Spirulina biomass is harvested, freezed, brought back to room temperature and centrifuged. The aqueous supernatant which contains about 50 wt.% of the initial dry biomass is mainly composed of proteins, carbohydrates and mineral salts, but no lipid. After a purification process which is kept confidential, the resulting drinkable solution is commercialized as food complement with a guaranteed phycocyanin content (0.08 wt.%). After the centrifugation step, a solid residue is obtained which is usually eliminated as a waste. The latter was used for this study and was freeze-dried prior to use.

The Spirulina sp. composition can vary depending on the growth conditions; a representative example is: proteins (65 wt%), carbohydrates (20 wt%) and lipids (15 wt%) [START_REF] Borghol | Biosourced analogs of elastomer-containing bitumen through hydrothermal liquefaction of Spirulina sp microalgae residues[END_REF]. This Spirugrass is a solid fraction waste which contains lipids. This solid was crushed and grinded to be used as feedstock in hydrothermal liquefaction experiments.

When performing the catalytic hydrothermal liquefaction of Spirugrass, one of the following catalysts was added: zirconium oxide (ZrO2, Norpro) which was crushed and sieved to obtain a particle size around 125-200 μm, cerium oxide (CeO2, Solvay) already in powder form and cerium nitrate hexahydrate from Sigma Aldrich. Specifications of zirconium oxide and cerium oxide catalysts, such as specific surface area, are presented in Table 1. HPLC grade solvents (i.e. dichloromethane, tetrahydrofuran, chloroform, methanol…) used for products separation or analysis were obtained from Carlo Erba and used without further purification.

Solvent extraction of microalgae residues

In order to quantify the lipid fraction present in the spirugrass residue, solvent extraction was carried out using a Velp Scientifica SER 158 automatic solvent extractor. For each extraction, approximately 10 g of crushed Spirugrass were deposited in a cellulosic thimble, then 100 mL of a chloroform-methanol mixture (2:1 in volume using the same mixture and proportion as Audo et al. [START_REF] Audo | Subcritical Hydrothermal Liquefaction of Microalgae Residues as a Green Route to Alternative Road Binders[END_REF] ), was placed in the beaker where the thimble was immersed. This apparatus works following 5 successive steps: immersion (50 min), removing (20 min), washing (30 min), recovery (30 min) and cooling (10 min).

Thermogravimetric analysis (TGA)

The thermal decomposition of the residue was studied using a TGA/DTA SETARAM with a Setsys Evolution 12 apparatus. Approximatively 7 mg of sample was deposited in a platinum crucible and heated up until 750 °C with a heating rate of 5 °C/min under an air flow of 50 mL/min. The weight loss at 100 °C corresponds to the water content, whereas the remaining weight at the end of the measurement corresponds to the ash content.

Gel permeation chromatography (GPC/SEC)

The size exclusion analysis was carried out with an Agilent apparatus (1200 series) equipped with two PL gel porous columns of different porous size (500-50 Å), using tetrahydrofuran (THF) as the mobile phase and a differential refractive index (DRI) detector. Each sample was diluted at 5 wt % in THF and filtered using a syringe filter with a pore size of 0.45 μm.

The system was calibrated using hydrocarbons (HC) standards with a range of molecular weight from 86 to 1000 g/mol. The linear alkane calibration is more appropriate than usually used polystyrene standards taking into account the presence of fatty chains in the algal oil. However, because of the solubility of alkanes, this calibration ends at 1000 g/mol which is not enough to evaluate the whole molecular weight distribution. Therefore, the distribution is not absolute, it is relative to the calibration curve. For an easier comparison, the chromatograms were normalized according to the most intense peak.

Elementary analysis (CHNS-O)

The C, H, N, O and S content was measured using a Thermo Scientific Flash 2000 series CHNS/O Analyzer, equipped with a thermal conductivity detector (TCD). The analysis was performed twice for each sample. The C, H, N and S percentages were measured after a flash combustion of the sample, generating a gas mixture composed of N2, CO2, H2O and SO2, which flows through a GC column to be analyzed by the TCD detector. The oxygen content was measured separately, by the catalytic pyrolysis of the sample under He, where CO is produced and quantified.

Bi-dimensional gas chromatography (GCxGC)

The analyses of the oily part of the hydrophobic fractions were carried out with an Agilent 6890 apparatus, equiped with a ZOEX thermal modulation system ( cryogenic Nitrogen ). The modulation period was set to 11.63 sec.. The first column is a semi-polar capillary column ZB-35 (phenomenex) of 30 m length (35 % phenyl and 65 % dimethylpolysiloxane) and the second one is a nonpolar capillary column DB-1 (Agilent) of 2 m length (100 % dimethylpolysiloxane).

Two detectors were used to analyze the samples: mass spectrometry (MS) detector, carried out on a single quadrupole mass spectrometer Agilent 5975B, for qualitative analysis and flame ionization detector (FID) for quantitative analysis, where decane was used as internal standard (3-10 μL). Samples were diluted in THF or DCM, in a weight percentage among 5-10 %, and all the samples were filtered (0.45 mm) before the measurement. In the case of the hydrophobic phase and the extracts, the method was carried out with an injection volume of 0.5 μL at 310 °C and a split ratio of 50:1, while for the aqueous phase the injection volume was 0.1 μL at 310 °C and a split ratio of 10:1 (considering the expansion volume of water in the gas phase). The scan mass spectra were measured in the range of 33-280 m/z. The temperature program used for the analysis started at 50 °C for 5 min., followed by a 2 °C/min ramp to 350 °C for the first oven. The heating program of the second oven containing the second column was identical to the first oven. This equipment is controlled by Mass Hunter software (Agilent) and the 2D data were processed by GC-Image Software.

Ashes composition

The ash content residue obtained (orange) after TGA was characterized by scanning electron microscopy (SEM-EDX), in order to determine its qualitative composition and then ICP-AES was performed to quantify the identified elements.

Elemental analysis SEM-EDX

Scanning electron microscopy with energy dispersive x-ray spectroscopy (SEM-EDX) was performed on a JEOL JEM2010 apparatus, to identify the mineral content in the Spirugrass ashes.

Elemental analysis ICP-AES

The inductively coupled plasma with atomic emission spectroscopy (ICP-AES) technique was carried out on a Activa apparatus from Horiba Jobin Yvon, to quantify the various elements identified thanks to SEM-EDX of the Spirugrass ashes.

Hydrothermal Liquefaction (HTL) experiments

The hydrothermal liquefaction of Spirugrass was performed either (i) in a 300 mL or a 1000 mL stainless steel batch Parr reactor. Both reactors were equipped with a stirring and heating system composed of an electric oven covered with a glass wool jacket to reduce the heat losses. The pressure inside the reactor was measured through a manometer and the temperature was taken recorded inside the reacting solution, using a thermocouple system connected to the Parr 4842 controller.

Almost all experiments were performed under inert atmosphere (N2) and following the same protocol: (i) in the 300 mL reactor (resp. 1000 mL), 20 g of crushed Spirugrass (resp. 96 g) and 80 mL of distilled water (resp. 385 mL) were introduced corresponding to a ratio of 4 mL of water per gram of biomass. The reactor was degassed with a flow of N2 in order to remove the air and the oxygen possibly dissolved in water. Before heating the reactor, a leak test was carried using N2 to pressurize the system until around 50 bar for several minutes. Subsequently, the reactor was heated up to 260 °C (subcritical conditions, autogenous pressure of approx. 53 bar) with an approximate rate of 8 °C/min and a stirring speed of 500 rpm. Several reaction times were tested (between 15 mn and 7 h); at the end of the reaction, the heating was stopped and the system was cooled down to ambient temperature.

Catalytic Hydrothermal Liquefaction (HTL) experiments

Following the above protocol, various catalysts were added in the initial feed. The amount of catalyst is given based on the initial biomass, and the different assays are presented in Table 2.

Experiments without catalysts and with cerium nitrate catalyst were repeated five times. The accuracy in yield determination was less than 3%.

Products recovery

At the end of the HTL experiments, two distinct phases were obtained, the hydrophobic phase on the bottom of the reactor and the supernatant aqueous phase. The aqueous phase was directly transferred from the reactor to a storage flask and the sticky hydrophobic phase remaining in the reactor was recovered using dichloromethane. After this step, no filtration was done except for one experiment, which means that the solid fraction was left inside the hydrophobic phase. Subsequently, the solvent was evaporated under reduced pressure and the hydrophobic fraction was weighted to calculate the bio-oil yield, based on the initial biomass, applying equation 1. When a catalyst was employed, as it remained mixed with the solid fraction, its initial mass was substracted to assess the reaction yield. to 0°C) and at various frequencies (from 0.01 Hz to 10 Hz). Measurements were performed in the small strain domain in order to remain in the linear viscoelasticity theory framework.

Results and discussion

Spirugrass characterization and lipid extraction

Spirugrass was characterized by elemental analysis and TGA to determine the CHONS, moisture and ashes content (Table 3 and Figure S1 in the Suppl. Inf.). After TGA, the recovered ashes of Spirugrass were analysed by SEM to identify the major inorganic elements that were then quantified using ICP-AES (Figure S2). The elements detected by SEM-EDX in ashes were mainly Ca, Fe, P and Si and the ICP-AES measurement confirmed this result. The high content in Ca and P, respectively 21 and 10.4 wt%, is attributed to the nutrients added during the growing phase of microalgae that are trapped in the wet initial biomass and subsequently concentrated in the dry residue.

Lipids extraction of Spirugrass in CHCl3/MeOH mixture (2/1 v/v) was carried out using a solvent extractor following Bligh and Dyer adapted procedure [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] and gave 16 wt% yield after removal of the solvent under reduced pressure. This extract was analyzed by elemental analysis (CHONS content, Table 3), GPC and GCxGC-MS. The concentration of nitrogen (5.0 ± 0.28 wt %) in the extract might originate from phycocyanin (a blue pigment present in large amount in Spirulina sp.) and/or from chlorophyll as the colour of the extract is blue-green [START_REF] Antelo | Thermal degradation kinetics of the phycocyanin from Spirulina platensis[END_REF].

Figure S3 displays the GCxGC analysis of the volatile compounds present in the lipid-rich extract. As a general comment, few compounds were detected by GCxGC-MS because most of the extract is composed of non-volatile matter. As already reported by Paoletti for Spirulina sp. [START_REF] Paoletti | Unsaponifiable matter of green and blue-green algal lipids as a factor of biochemical differentiation of their biomasses: I. Total unsaponifiable and hydrocarbon fraction[END_REF] the presence of alkanes was detected (i.e. pentadecane, hexadecane) among which heptadecane was one of the most abundant in this study. C16 and C18 carboxylic acids were the main other detected compounds [START_REF] Babadzhanov | Chemical composition of Spirulina platensis cultivated in Uzbekistan[END_REF][START_REF] Muhling | Variation in fatty acid composition of Arthrospira (Spirulina) strains[END_REF], with small amounts of unsaturated fatty alcohols and acetate. The 3D-view also given in Figure S3 allowed to evaluate the relative intensity of each corresponding peaks in relation with their concentration in the extract, showing that hexadecanoic acid is the main observable component.

The GPC molecular mass distribution of the Spirugrass extract (Figure S4) displayed with stearic acid (C18:0) and glycerylstearate (C57) as references, showed two main zones with the alkane calibration (HC equivalent). The first one, centred at 305 g/mol HC eq., included the stearic acid peak and can thus correspond to C16-C18 fatty acids and to higher molecular masses compounds, up to a mass of 420 HC eq. The second one, centred at 535 g/mol HC eq., corresponded to triglycerides, with a mass distribution lower than stearine but higher Mw compounds were also present. The GCXGC analysis described above only allowed to detect the compounds with molecular masses in the range 100-420 g/mol.

HTL conversion of Spirugrass

In our former work [START_REF] Borghol | Biosourced analogs of elastomer-containing bitumen through hydrothermal liquefaction of Spirulina sp microalgae residues[END_REF], we have demonstrated that the HTL treatment of Spirulina sp. residues led to a hydrophobic fraction showing viscoelastic properties similar to an elastomer-rich bitumen. Interestingly, the Algosource Technologies Company provided us with Spirugrass, which is characterized by a lipid content lower than previously. We have thus studied in this paper the HTL conversion of this new residue under different operating conditions and in the presence of catalysts, to tune the rheological properties of the different bio-binders. For all HTL experiments presented here, the temperature was fixed at 260 °C and we have studied the influence of (i) the addition of a lipid-rich Spirugrass extract, (ii) the presence of a catalyst, (iii) the biomass loading for a same catalyst and (iv) the reaction time (from 15 min to 7 h).

The yields obtained for HTL reactions and repeatability were assessed as explained in the Materials and Methods section. The obtained yields (Figure 1) were in the same range as those reported (42-48 wt %) for HTL of Spirulina sp. [START_REF] Gai | An investigation of reaction pathways of hydrothermal liquefaction using Chlorella pyrenoidosa and Spirulina platensis[END_REF][START_REF] Toor | Hydrothermal liquefaction of Spirulina and Nannochloropsis salina under subcritical and supercritical water conditions[END_REF].

As shown in Figure 1, the presence of a catalyst had little influence on the yield of the hydrophobic fraction and no significant difference was observed among the different catalysts (ZrO2, CeO2, cerium nitrate). The highest yields (ca. 48 wt %) were obtained using 1 wt % of Ce(NO3)2.6H2O.

To confirm the interest of dispersed catalysis, oxide precursors like Ba(NO3)2 and Fe(NO3)3•9H2O were tested (they are transformed in situ into the metal oxides or hydrohydes during HTL) but both hydrophobic fractions obtained exhibited a solid texture and did not turn into viscous liquids even if they were heated up to 100 °C. Consequently, these samples could not be characterized in terms of rheology, and are useless as a road binder.

Chemical characterization of the biocrude

Each HTL hydrophobic fraction oil was analyzed by CHONS elemental analysis and the resulting O/C, H/C and N/C atomic ratio were plotted in a general van Krevelen diagram (Figure 2) and compared with the initial Spirugrass and the Spirugrass solvent extract.

A decrease in the N/C atomic ratio from 1.16 to ca. 0.8 was invariably observed for all HTL hydrophobic fractions (see size of the balls in Figure 2), whatever the experimental conditions in terms of reaction time, and presence or absence of any catalyst, suggesting the degradation of some nitrogen compounds under HTL conditions (Table S1). It can be also noticed that the O/C atomic ratio of the different hydrophobic fractions after HTL were lower than in the initial Spirugrass (a reported advantage of the HTL process), and even lower when no catalyst was used. This last result is not totally in accordance with previous studies where heterogeneous catalysts like supported CoMo, Ni or Pt catalysts were found to lower O/C ratio and improve HHV [START_REF] Biller | Catalytic hydrothermal processing of microalgae: Decomposition and upgrading of lipids[END_REF][START_REF] Duan | Hydrothermal Liquefaction of a Microalga with Heterogeneous Catalysts[END_REF] but it could be originated from the lower temperature used here for the HTL. While extraction of Spirugrass with CHCl3/MeOH also reduced the nitrogen and oxygen content, the hydrogen content was however higher compared to HTL. However, the entrapment of the catalyst in the hydrophobic fraction might result in potential errors in the measurement of the C,H,O,N, and S content by elemental analysis, making not obvious the comparison of the results when a catalyst is used or not. The O/C value is important for estimating the degree of deoxygenation taking place during the HTL of the microalgae residue, although the obtained values are still far from those reported for a conventional petroleum bitumen (O/C: 0,01) [START_REF] Lesueur | The colloidal structure of bitumen: Consequences on the rheology and on the mechanisms of bitumen modification[END_REF].

In conclusion, it is difficult to rationalize the elemental analysis results as the presence of catalyst increased inaccuracies in the sampling. The only clear tendency is the strong decrease of O and N in the HTL oil compared to the Spirugrass residue.

Then, the oily part of the hydrophobic fractions was analysed by GCxGC-MS. This technique is very powerful to separate and identify components in complex mixtures but limited to volatile compounds, therefore only the light part of the samples could be analysed. The detected components represented 1 to 4 wt% of the oil thus they were not quantified by using a FI detector for that reason. In nearly all cases, except when the HTL experiments were run for 7h, the GCxGC analysis displayed approximately the same compounds and families: alkanes (C17), alkenes (C18-C20), N-cyclic compounds such as, pyrroles, pyrazines, piperazines, diketopiperazines, pyrrolidines, and fatty molecules like hexadecanoic acid (palmitic acid), ketones (e.g., C18H36O), amides, esters and morpholine (Figure 3 and Figures S5 andS6). This qualitative analysis on a restricted part of the oil, does not show significant change when using any of the catalysts after 1 h reaction. Most of these compounds can originate from the different reactions occurring between the bio-molecules under HTL conditions. For instance, most of alkanes and alkenes likely resulted from the decarboxylation of fatty acids. However, here as we are using a residue, and since heptadecane was shown to be present in the Spirugrass extract obtained by CHCl3/MeOH extraction, part of alkanes are probably already present in the residue and were just released and not much affected by HTL. Pyrazines usually comes from the Maillard reaction [START_REF] Qiu | Biocrude Oil Production through the Maillard Reaction between Leucine and Glucose during Hydrothermal Liquefaction[END_REF][START_REF] Fan | Hydrothermal liquefaction of protein-containing biomass: study of model compounds for Maillard reactions[END_REF][START_REF] Zhang | Enhancing the performance of Co-hydrothermal liquefaction for mixed algae strains by the Maillard reaction[END_REF][START_REF] Changi | Hydrothermal Reactions of Biomolecules Relevant for Microalgae Liquefaction[END_REF] between sugars and amino acids or amines, and pyrroles derive from the thermal degradation of chlorophyll and proteins. Diketopiperazines typically comes from the depolymerization and cyclization of peptides, while some ketones might be formed from the vicinal hydroxy groups in carbohydrates after dehydration and isomerization or from the ketonization reaction between two carboxylic acids [START_REF] Chen | Catalytic Hydrothermal Liquefaction of D. tertiolecta for the Production of Bio-Oil over Different Acid/Base Catalysts[END_REF][START_REF] Maag | Catalytic Hydrothermal Liquefaction of Food Waste Using CeZrOx[END_REF][START_REF] Gaertner | Catalytic coupling of carboxylic acids by ketonization as a processing step in biomass conversion[END_REF][START_REF] Gärtner | Catalytic Upgrading of Bio-Oils by Ketonization[END_REF][START_REF] Torri | Hydrothermal Treatment (HTT) of Microalgae: Detailed Molecular Characterization of HTT Oil in View of HTT Mechanism Elucidation[END_REF][START_REF] Zhang | Hydrothermal Liquefaction to Convert Biomass into Crude Oil[END_REF]. The increase of the reaction time with both CeO2 and ZrO2 catalysts had a deeper impact on the HTL oil composition (Figures S7 andS8) and that could be attributed to the slow kinetics when using catalyst in this middle. Effectively, after 7 h of reaction, the number of detected volatiles compounds decreased strongly. Even if it was not quantified as mentioned above, we can observe that notably the N-cyclic compounds including pyrazinediones had been mostly removed, while alkanes/alkenes and fatty amides/morpholines remained. This suggests that more condensation reaction with the lipidic fraction took place or on the contrary that longer reaction time favoured degradation of the small molecules present after 1 h. Some samples of HTL aqueous fractions were also qualitatively analysed by GCxGC-MS,

showing that the reaction time has also an influence on their composition, when using a ZrO2

or CeO2 catalyst (Figure S9). After 1 h, the main components of the aqueous fraction were piperazinediones with alkyl and benzyl substituents similarly observed in the hydrophobic fraction, in addition to pyrazines and pyrrolidinediones. In contrast, after 7 h, the amount of carboxylic acids, pyrazines and pyrrolidinedione families was respectively higher while lower for piperazinedione.

The molecular weight distribution of the bio-oil is a very important feature regarding its application as asphalt binder for aggregates. GPC was used to investigate the influence of the different catalysts as well as the effect of the HTL reaction time (Figure 4 and Figure S10).

In the presence of catalyst and after 1 h HTL reaction, the distribution of the molar masses does not show significant differences with the HTL realized without catalyst. The HTL of Spirugrass enriched with lipid fraction presents the same distribution than in Spirugrass HTL experiments (Figure S10).

Interestingly, an increase of the amount of high molecular weight products was observed using a CeO2 catalyst for 7 h of reaction (Figure 4), and to a lower extent in the case of Ce(NO3)3 and ZrO2 (not shown for clarity). The same trend as ZrO2 and Ce(NO3)3 was also found when increasing reaction time in the absence of catalyst, suggesting that this phenomenon is rather resulting from a longer reaction time which favours condensation reactions not necessarily promoted by catalysts. This was in full agreement with the average molecular weight (Mw) which slightly increased when increasing the reaction time (Table S2). In addition, most of the HTL samples displayed a similar distribution with a major contribution located at 304 g/mol HC. eq. (stearic acid area) like for the Spirugrass extract. However, the Spirugrass extract displayed a major contribution at 535 g/mol (triglyceride area, Figure S4) while the HTL experiment with and without catalyst showed a large contribution in the 400-800 g/mol HC.

eq. range with a significant peak around 505 g/mol. When a Spirugrass extract was added to the initial biomass, with no catalyst, the resulting HTL oil showed an increase of the Mw value due to the higher concentration of initial lipids which might participate in condensation reactions.

In conclusion, the role of the chosen catalysts are still not clear at this stage, and contrarily to some previous papers, we did not observe much difference by using Ce-and Zr-based oxide catalysts except at longer reaction time, that is consistent with low kinetics rates of the catalysts. Due to the difficulties to characterize well the hydrophobic fractions in regards with the limits of the techniques used, it would be risky to make conclusions about the role and the choice of the catalysts. The literature evidences that other types of acidic, basic or metallic catalysts [START_REF] Biller | Catalytic hydrothermal processing of microalgae: Decomposition and upgrading of lipids[END_REF][START_REF] Chen | Catalytic Hydrothermal Liquefaction of D. tertiolecta for the Production of Bio-Oil over Different Acid/Base Catalysts[END_REF][START_REF] Xu | Hydrothermal liquefaction of Chlorella pyrenoidosa for bio-oil production over Ce/HZSM-5[END_REF] can change the chemical bio-oil composition but the evidence was only made on the volatile part of the bio-oil that usually represent only a few percentage of the whole biocrude. Thus, to quantify the chemical composition changes, further analysis such as FTICR-MS [START_REF] Zhang | Effect of acidic, neutral and alkaline conditions on product distribution and biocrude oil chemistry from hydrothermal liquefaction of microalgae[END_REF] are necessary but all the chemical characterization depends greatly of the nature of the feed and operating conditions used. Finally, we have seen by the analysis of the ashes content that inorganics as Ca, Fe, Mg and P are present in the residue (Figure S1 andS2). These species may have a catalytic effect or may poison the catalytic sites depending of the exact nature of the original components. This could explain the few differences observed between the hydrophobic fractions obtained with and without oxide catalysts.

Rheological properties

The rheological properties of the hydrophobic HTL fractions were measured as described in the Materials and Methods section. Under some conditions (10% of CeO2 at 1 h, 2% of Ce(NO3)3.6H2O at 1 h, 1% of Ce(NO3)3.6H2O at 15 min) a non-fusible material was obtained, making impossible the measurements of their rheological properties. Then, the shear modulus |G*[ of these non-fusible fractions will be considered as constant with a value equal to 1 GPa (organic matter modulus in the glassy state). Moreover, considering that HTL experiments with heterogeneous catalysts (ZrO2 and CeO2) were performed using a 300 mL reactor whereas HTL experiments with an homogenous catalyst (Ce(NO3)3.6H2O) were done in the 1000 mL reactor, the thermal reaction conditions could not be strictly considered as equal.

For this reason, the two series of rheological results were interpreted independently.

Concerning HTL experiments with heterogeneous catalysts, hydrophobic fractions obtained with 10% of ZrO2 during 1 h and 7 h and 10% CeO2 during 7 h exhibit viscoelastic signatures, over a large temperature and frequency domain, similar to conventional bitumen. Indeed, as observed for conventional bitumen, the rheological data plotted in the Black space (Figure 5)

showed a continuous curve from 90 °C, where the material is liquid, to 0 °C, where the material is an elastic solid. The continuity of the curve indicated that the material molecular structures were thermally stable. However, even though the overall viscoelastic signature was similar to conventional bitumen, the stiffness of the HTL fractions measured at 1 Hz was largely higher than for bitumen (Figure 6) whatever the catalyst or the reaction time.

Concerning HTL experiments performed for 1 h in the presence of a homogeneous catalyst, the rheological data plotted in the Black space (Figure S11) showed again a viscoelastic liquid behaviour similar to conventional bitumen with a thermal molecular structure stability.

However, the amount of Ce(NO3)3.6H2O added to the reaction medium appeared to influence this viscoelastic signature leading to more elasticity for the hydrophobic fractions (showed by a phase angle decrease a given modulus value). This had a direct effect on the stiffness, as shown in Figure S12, increasing with the Ce(NO3)3.6H2O content until a non-fusible material was obtained (i.e. above 1% of catalyst).

The influence of the reaction time was also investigated in the presence or without any catalyst. Interestingly, isochrones related to HTL performed in the presence of ZrO2 (Figure 6)

showed a stiffness reduction when increasing the reaction time. This was confirmed in the case of HTL experiments without catalyst and in the presence of 1% of Ce(NO3)3.6H2O, for which a two decades modulus decrease (at 50 °C) was observed between 1 h and 7 h reaction times (Figure 7), leading to a rheological behaviour very close to conventional bitumen when experiments were carried out for 7 h.

Finally, whatever the conditions applied (type of catalyst, amount of catalyst, reaction time), the complex modulus of each hydrophobic fraction was found to be higher than for petroleum bitumen, leading to stiffer materials with the only exception of the binder obtained after 7 h of HTL in the absence of catalyst, for which the stiffness was lower (Figure 8) and the rheological signature very close to bitumen. Reaction time, with and without catalyst, is the most predominant parameter on the hydrophobic fraction by reducing the stiffness.

Considering the fact that the GPC shows an increase in the average molecular weight (Mw) for the 7 h experiment and, at the same time a decrease in the stiffness, one hypothesis would be that the solid residues (not analyzed in GPC) are hydrolyzed during HTL and finally were solubilized into the oily phase. Thus, even with an increase in the average molecular weight of the oily phase, the solid / oil ratio decreases and leads to a reduction in stiffness.

Conclusion

The treatment of Spirulina sp. residues was undertaken by HTL with various oxide catalysts and the reaction time was varied (1 h to 7 h). Addition of zirconia or ceria during HTL had a mild impact on the composition of the hydrophobic fraction but allowed an enhancement of the average molecular weight when increasing the reaction time. The overall rheological signatures of the hydrophobic fractions obtained in the presence of the different catalysts or without any catalyst were not far from a classical bitumen. Indeed, Black diagrams showed that both bio-and conventional bitumen behave like Newtonian liquid at high temperatures and like an elastic solid at low temperatures. For both materials, the transition between liquid and solid state is smooth and continuous (isotherms in the Black diagrams overlapped). Finally, the economic viability of this technology is under investigation and addresses different aspects : scaling-up of the process, studies on bio-bitumen aging, life-cycle analysis, and global cost analysis of the process and compared to petroleum bitumen.

  Yield = (Whydroph -Mcat)/ (Wi) * 100 (Eq. 1) With Whydroph = Weight (g) of the hydrophobic phase Mcat = Mass of catalyst introduced in the reactor (g) Wi = Mass of Spirugrass introduced in the reactor (g) 2.12. Rheological analyses The rheological properties of the HTL hydrophobic fractions were measured with a Kinexus pro + rheometer in shear mode, according to the European Standard EN 14770. The samples were loaded, in the melted state at 100 °C, on a plate-plate geometry (with a diameter of 8 mm) with 1 mm gap. The phase angles d (°) and norm of complex modulus (|G*|) were measured using dynamic shear rheometry (DSR), at different temperature values (from 80°C

However, absolute values of the stiffness measured at 1

 1 Hz for the different biobinders (at 20 °C, 1 Hz, |G*| > 4E7 Pa) remained largely above the value obtained for petroleum bitumen (at 20 °C, 1 Hz, |G*| ≈ 5E6 Pa). The reaction time had important effects on composition and was found to promote condensation reactions. Besides, increasing reaction time led to diminution of the stiffness of the biobinder (at 20 °C, 1 Hz, |G*| ≈ 5E6 Pa) which is very similar to a petroleum bitumen. Reaction time seems to have a huge importance on the solid to oil ratio in the biocrude which appeared to be the key parameter for the rheological properties.

Figures CaptionsFigure 1 .Figure 2 .

 12 Figures Captions

Figure 3 .Figure 4 .

 34 Figure 3. GCxGC-MS analysis of the volatile part of HTL bio-oils after 1 h residence time with ZrO2 Figure 4. GPC chromatogram of the various HTL oils after 1 h, with and without catalyst.

Figure 5 .

 5 Figure5. Phase angle versus norm of the complex modulus for the hydrophobic fractions obtained from HTL in the presence of heterogeneous catalysts (ZrO2, CeO2) and comparison with a conventional bitumen.

Figure 6 .Figure 7 .

 67 Figure 6. Norm of the complex modulus as a function of temperature, measured at 1 Hz for the hydrophobic fractions obtained from HTL in the presence of heterogeneous catalysts (ZrO2, CeO2), and comparison with a conventional bitumen

Figure 8 .

 8 Figure 8. Norm of the complex modulus as a function of temperature, measured at 1 Hz for the hydrophobic fractions obtained from HTL in the presence of a homogeneous catalyst (Ce(NO3)3.6H2O), by comparison with a conventional bitumen. Influence of the reaction time for a given amount of catalyst

Figure 1 .Figure 2 .

 12 Figure 1. Yields of hydrophobic fractions after HTL reactions with Spirugrass residue

Figure 3 .

 3 Figure 3. GCxGC-MS analysis of the volatile part of HTL bio-oils after 1 h residence time with ZrO2 catalyst

Figure 4 .

 4 Figure 4. GPC chromatograms after 1 and 7 h reaction with and without Ce catalysts

Figure 5 .

 5 Figure 5. Phase angle versus norm of the complex modulus for the hydrophobic fractions obtained from HTL in the presence of heterogeneous catalysts (ZrO2, CeO2) and comparison with a conventional bitumen.

Figure 6 .Figure 7 .

 67 Figure 6. Norm of the complex modulus as a function of temperature, measured at 1 Hz for the hydrophobic fractions obtained from HTL in the presence of heterogeneous catalysts (ZrO2, CeO2), and comparison with a conventional bitumen

Figure 8 .

 8 Figure 8. Norm of the complex modulus as a function of temperature, measured at 1 Hz for the hydrophobic fractions obtained from HTL in the presence of a homogeneous catalyst (Ce(NO3)3.6H2O), by comparison with a conventional bitumen. Influence of the reaction time for a given amount of catalyst

Table 3 .

 3 Chemical data related to the Spirugrass used in this study and its extraction products using a (MeOH/CHCl3) mixture.

			Spirugrass	Spirugrass extract (MeOH/CHCl3)
	Moisture (wt%)	5.6	-
	Ashes wt%	6.5	-
	C		45.5	61.9
	H		6.4	9.0
	N	(wt%)	8.5	5.0
	S		0.4	0.3
	O		25.1	16.2
	O/C		0.41	0.20
	H/C	At.	1.68	1.74
	N/C		0.16	0.07

Acknowledgement

This work received the financial support of ANR (Algoroute project, grant ANR-16-CE08-0017).

Algosource company is acknowledged for providing the Spirugrass residue.

Supplementary data

Supplementary material related to this article can be found, in the online version, at xxx.