P.-Y Jouan

J Keraudy
email: julien.keraudy@liu.se

R D Boyd

T Shimizu

U Helmersson

Phase separation within NiSiN coatings during reactive HiPIMS discharges: a new pathway to grow Ni x Si nanocrystals composites at low temperature

Keywords: Silicon nitride, NiSi nanocrystals, HiPIMS, nanocomposite

Phase separation within NiSiN coatings during reactive

HiPIMS discharges: A new pathway to grow Ni x Si nanocrystals composites at low temperature

Introduction

Nanocomposite thin films made from 3d transition-metal nitride (MeN) nanocrystalline domains (up to 10 nm), randomly dispersed in an amorphous silicon nitride (a-SiN x ) matrix have been extensively investigated for more than two decades due to their unique physical and mechanical properties. [1][2][3][4] They exhibit high indentation hardness, excellent oxidation resistance, as well as high abrasion and erosion resistance which make them ideal candidates for various industrial applications such as protective coatings on cutting tools, biomedical implants and decorative coatings. 5,6 The development of hard nanocomposite coatings relies on the use of a variety of transition metals with high affinity to N, such as Ti, [7][8][9] Cr, [10][11][12] Nb, 4,13,14 and Zr. [15][16][17] Such metals are able to form a hard and thermodynamically stable metal nitride compounds that can be used as the nanocrystalline phase. On the other hand, very few publications are devoted to the growth of SiN x -based nanocomposite thin films with transition metal with low affinity to N, such as Fe, Cu and Ni.

These transition metals can only form soft and thermodynamically metastable nitrogen-poor nitride compounds which make them unsuitable for the applications mentioned above. [18][19][20] However, this low affinity between metal and nitrogen can also offer the opportunity to design new nanocomposite coatings with unique functional properties.

Currently, the only low-affinity metal studied as a nanocomposite together with SiN x , is Ni. [21][22][23][24][25][26] The Ni x Si y N z thin films presented in these publications consists of Si-rich nickel silicide (NiSi x with x ≥ 1) nanocrystals (nc) encapsulated in an amorphous-SiN x matrix. The association of NiSi x -nc and a-SiN x has been demonstrated in having a potential for applications for emerging nonvolatile memory technologies with superior retention characteristics compared to conventional floating-gate memory devices, resulting from coulomb blockade and quantum confinement effects. 21 In the references cited above, the methods of choice for the synthesis of the Ni-containing nanocomposites are reactive magnetron sputter deposition from a silicon-rich NiSi compound targets (70 at.% Si), followed by a rapid high temperature thermal annealing treatment to allow for the NiSi x -nc formation. It was reported by Chen et al. 21,25 that the crystal growth occurred after annealing at 500-600 °C in a nitrogen atmosphere for 100 s while Huang et al. 22 reported growth of NiSi 2 -nc, 4-5 nm in size, after annealing in oxygen ambient at 700 °C for 60 s. In these deposition conditions, the temperature dependence of the NiSi x crystallization is explained by the bulk diffusion and the precipitation of Ni in the amorphous nitride network. 27 Eliminating the annealing process-step, i.e. synthesizing as-deposited NiSi x -nc through a single-step, would be beneficial for the deposition on flexible, temperature sensitive substrates, such as polymers. It would also limit the risk of oxidation and the need for a plasma cleaning step to remove any oxide layer.

In the present study, we report a straightforward strategy for that purpose, by deposition of NiSiN from a Ni-rich composite target (Ni 0.85 Si 0.15 ) at different nitrogen flow rates. At a critical nitrogen flow (N 2 ≥ 6 sccm), without the use of substrate heating or any post-annealing, the resulting film consisted of Ni-rich Ni x Si (x > 1)-nc, 7-8 nm in-sizes, embedded in a-SiN x layer. Under these growth conditions, the formation of Ni-rich nanocrystal is driven via repeated nucleation of subsequently deposited Ni and Si atoms caused by a phase-separation mechanism taking place during the film growth.

Experimental procedures

Experiments were performed in a stainless-steel chamber (0.01 m 2 ) with a base pressure below 10 -5 Pa. A Ni 0.85 Si 0.15 composite disk with a diameter of 50 mm and a thickness of 6 mm was used as a sputtering target and mounted on a strongly unbalanced magnetron. The films were deposited onto conventional single crystal p-doped Si(001) wafers, ultrasonically cleaned in acetone then ethanol and finally dried under nitrogen flow at room temperature. The distance between the target and the substrate holder was 50 mm. Ar (sputtering gas) with a purity of 99.9997 % was introduced into the chamber through a mass flow controller with a flow rate of around 10 sccm. The reactive gas, N 2 with a purity of 99.9995 %, was introduced into the chamber with a flow rate that varied between 0 and 15 sccm. The total pressure was kept constant at 0.6 Pa by the use of a down-stream throttle valve. The average power density was kept constant at 7 W cm -2 , irrespectively of the reactive discharge conditions. Unipolar pulses with a length of 30 µs and a frequency of 1 kHz were supplied by a home-made high-power impulse magnetron sputtering (HiPIMS) pulsing unit fed by a Technic SR 1.5-R-1500 DC power supply. This home-made unit is also equipped with a DC current generator allowing the superposition of a low current (~ 4 mA) to produce of a low-density plasma in-between each high-power pulse ensuring a fast response-time of the discharge current. 28 The discharge current and voltage time characteristics were recorded by a Tektronix DP3014 oscilloscope using a broadband high voltage probe (Tektronix P6015) and a current probe (Tektronix TCP303), respectively, connected close to the power input to the target (see Figure .S1 of the supporting information). The applied voltage delivered to the target was varied depending on the N 2 flow rate keeping the average sputtering power constant at 100 W. High-flux, high-energy ion bombardment of the growing film was utilized with a constant substrate bias at -100 V. The film thickness was kept constant, by adjusting the deposition time, at around 1 µm independent of the N 2 flow.

The crystal structure of the films was identified by x-ray diffraction (XRD) using a Brüker D8 Advance powder diffractometer operating in Bragg-Brentano geometry (Ge monochromator CuK α1 , λ = 1.540597 Å). All XRD scans were recorded between 20 and 69° with a step size of 0.02°. A characteristic length of the Ni nanodomains in the composite films can be extracted from the full width at half maximum of the diffraction peaks using the Scherrer formula after correction to remove the experimental broadening. 29 The chemical composition of the films was evaluated through X-ray photoemission spectroscopy (XPS) spectra recorded on a Kratos Ultra photoelectron spectrometer using a monochromatic Al Kα source (1486.6 eV) operating at 150 W. The pressure in the analysis chamber was kept below 10 -8 Pa. A surface cleaning of Ar + ions impingement at 0.5 kV was applied for 15 min to remove the contaminant oxide layer (~1-5 nm) formed at the top surface by air contamination.

High-resolution spectra of N 1s, Ni 2p and Si 2p core levels were recorded using a pass energy of 20 eV at θ = 0° take-off angle (angle between the surface normal and the detection direction). The spectra were analyzed and fitted using the CasaXPS software. 30 A Shirley type baseline for the background and a pseudo-Voigt function with Gaussian (70%)-Lorentzian (30%) were used to fit each chemical environment composing the exposed deposited films.

Finally, all spectra were calibrated in energy by using adventitious C 1s at 284.7 eV. Scanning electron microscopy (SEM) acquisitions were carried out in a JEOL 7600F microscope with an incident electron-beam of 5 kV. The microstructure of a selected film was investigated by transmission electron microscopy (TEM). Prior to analysis, cross-sectional specimens are prepared by a two-step procedure consisting of mechanical polishing followed by Ar + milling at shallow incidence angle of 4° from the sample surface, with 5 keV ions initially then the ion energy was decreased to 2.5 and 1 keV. All measurements were taken with a FEI Tecnai G2 operated at 200 kV. High angle angular dark field (HAADF) and energy dispersive x-ray spectroscopy (EDS) combined with scanning-TEM (STEM) analysis were taken using a camera length of 140 mm. HAADF images were collected with the annular detector spanning an angular range from 80 to 260 mrad. The thin films' electrical resistivity was measured at room temperature using a four-point probe method in the van der Pauw configuration with a Keithley 237 current source and a Keithley 2700 multimeter. This equipment enabled the measurement of electrical resistivity below 1 x 10 5 Ω cm. The resistivity values reported in this work are average values obtained through 10 measurements.

RESULTS

In order to evaluate the impact of the addition of N 2 on the chemical composition of the coatings, XPS measurements were carried out and the results are displayed in Figure 1.

For all samples, only Ni, Si and N were detected after 15 minutes of Ar + ion etching. These elements were quantified using the Ni 2p, Si 2p and N 1s core level spectra. It was found that increasing the N 2 flow rate in the plasma led to an increasing of the N content into the films, up to ~30 at.% whereas the Ni/Si atomic ratio stayed constant, Ni/Si ~ 6, which is close to the value of the target. [START_REF] Cao | [END_REF] The measured peak positions exhibit a distinct shift to higher 2θ position of both diffraction peaks with increasing N content. At the same time, the full half width maximum (FHWM) of the diffraction peaks increases and their respective intensity decreases with the N content in the film, up to a N content of 20 at.% after which the process stabilizes. Similar behavior, e.g. increasing of FHWM and peak intensity have been previously observed in many other sputtered transition metal carbide-and silicide-based nanocomposite systems and is commonly related to a reduction of the crystallite size. [32][33][34][35] Estimating the crystallite size using the Scherrer formula from the 111 lattice planes confirms a decrease of the grain size from 24 to 7 nm with increasing N content from 0 to 20 at.% in the coatings, see Figure 2.(b).

For higher content, the crystallite size stays constant at around 7 nm.

To investigate the microstructural evolution of Ni 1-x Si x N y layers with increasing N content, three selected films containing 0, 16 and 27 at.% of N were analyzed by crosssectional bright-field TEM (BF TEM) investigations, which are shown in Figure 3. Without N, the Ni 1-x Si x thin film exhibits a dense columnar microstructure, as highlighted by the lowresolution micrograph (top panel, Figure 3.(a)). Such a microstructure is typical for thin films grown using HiPIMS discharges. 36 could not be obtained. It is well-known that the presence, or not, of percolated domains may have a strong impact over the films electric conductivity, which is a crucial importance for microelectronic applications. Supporting evidence, although not conclusive, comes from a high-resolution TEM (HRTEM) micrograph (Figure 4.(c)) illustrating two possible interconnecting points between nano-sized domains. The first with partial attachment with minimal interaction between only 2-3 atomic columns (marked region #1) and second with direct attachment between two crystalline facets (marked region #2).

In order to have a deeper understanding of the chemical atomic bonds within and between the NiSi x -nc and the matrix, high-resolution XPS analyses were carried out. Figure 5.(a) describes, with three representative samples, the influence of an increasing N content on the Si bond structure in the Ni 1-x Si x N y coatings. To see all the collected spectra, please refer to the Figure S2 in the supporting information. Two separate features can be observed in the spectra. As the N content within the film increases, the signals of Si 2p 3/2 and Si 2p 1/2 are decreasing. These two peaks, located at binding energies of 99.3 and 99.7 eV, can be attributed to Si-Ni bonding. [START_REF] Cao | [END_REF]37 Peaks associated to Si-N bonds, located at 100.3, 101.4 and 102 eV, appears for the film with 12 at.% N or higher, and increases with N content.

According to Schmitt et al., 38 the different binding energies of Si-N bonds, observed in the spectrum, originate mainly from the different tetrahedral bond of SiN x , i.e. Si-Si-N, Si-N-N, and to a smaller extent to the presence of nearest-neighbor effects (secondary chemical shifts). . At 27 at.% N, the relative contribution of Si-N represents ~ 40% of the total spectrum. Therefore, it is reasonable to assume that the Si-rich matrix observed in the previous TEM micrographs (see Figure 4.(b)) mainly consist of SiN x , which is commonly observed in other silicide systems, e.g., Ti-Si-N, 35 Cr-Si-N, 12 and Nb-Si-N. 4 Nonetheless, it is important to point out that the contribution of Ni-Si bonds remains the dominant one. This can be attributed to the higher Ni content compared to N in the Ni 1-x Si x N y layer, as emphasized by the XPS analysis shown in Figure 1.

Regarding the Ni 2p photoemission spectra (Figure 5.(b)), all the Ni 2p 3/2 line envelope consisted of three parts: a main emission line located at 852.7 eV eV followed by an asymmetric high binding energy tail at 856.0 eV, and a well-resolved shake-up satellite at 858.2 eV. 37,39 According to the work done by Cao et al., [START_REF] Cao | [END_REF] Recently, Aballe et al. 41 reported on the high diffusivity and chemical reactivity of Ni in the SiN x lattice by occupying lattice interstitials leading to the formation of a localized ternary solid solution (Ni x Si 1-x N y ), even at room temperature. To confirm the presence of this ternary alloy within the Si-rich matrix, a dealloying treatment was carried out on the nanocomposite coating with 27 at.% of N. This approach consists in a preferential etching of the metal alloys domains by a simple treatment in a nitric acid (HNO 3 ), ~5 min. 42,43 In general, the dealloying process is employed to produce nanoporous materials, 44 however in this study it was used to find out the chemical constitution of the Si-rich domains. Crosssection SEM micrographs recorded on Ni 1-x Si x N y with 27 at.% N before and after etching are displayed in Figure 6. After chemical etching, the film thickness was strongly reduced by 21,23 this new feature can be related to the Ni-Si-N ternary bond. Thus, this additional analysis tends to reveal that the nanocomposite structure found in Ni 1-x Si x N y thin films with N content higher than 16 at.% is composed of Ni-rich nanocrystals around an amorphous SiN x with inclusions of a ternary alloy.

The electrical room-temperature resistivity of the as-deposited Ni 1-x Si x N y films versus the N content is shown in the Figure 9. Without N, the value of the electrical resistivity of a 1 µm-thick Ni 0.83 Si 0.14 film is found to be 45 µΩ cm which is higher than a pure 1-µm thick Ni layer (ρ = 13 µΩ cm) but lower than for Ni 3 Si thin films that has been measured to 96 µΩ cm and to 82 µΩ cm for 150 nm and 500 nm-thick films, respectively. 45,46 As the N content increases from 2.5 to 27 at.%, an increase of the resistivity can be noticed, from 45 to 65 µΩ cm. In addition, the most significant increase in resistivity can be observed when the nitrogen content goes above 12 %, which coincide with appearance of the SiN x phase in the Si 2p XPS spectra (see Figure 5.(a)). This increase can be attributed to the finite size effect induced by the decreasing of the crystallite size, as emphasized by XRD analysis (see Figure 2.(b)).

Nonetheless, these electrical measurements reveal that, regardless of the N content, all the NiSiN films exhibit a good electrical conductivity.

DISCUSSION

The results provided from the structural, microstructural and chemical investigations presented above clearly point out that two separated growth mechanisms take place in Ni-rich Ni 1-x Si x N y thin films as a function of the N content incorporated during the film growth.

At low N content (between 0 and 16 at.%), both Ni and Si ad-atoms can diffuse on the surface of the substrate, encounter another adatom and form clusters which then serve as a sink for the subsequent stages of film growth including island nucleation and growth, impingement and coalescence of islands and development of a continuous film. 47,48 Compositional inhomogeneities within the film are unlikely due to the fact that Ni and Si are fully miscible. 49 A calorimetric investigation 50 as well as thermodynamic modeling of the Ni-Si system [51][52][53] report the solid solubility of Si in the fcc phase of Ni at about 10 at.% Si.

Therefore, the slight shift to higher 2θ positions observed for all the samples (see Figure 2.(a)), i.e. smaller interatomic distances than in the pure Ni framework, is likely caused by the incorporation of Si atoms into the lattice, e.g. the Ni-Si bond distances (2.37 Å), 54 are shorter than the Ni-Ni (2.59 Å). 54 Moreover, according to the Ni-Si equilibrium phase diagram, [START_REF] Europe | Ternary Steel Syst. Phase Diagr. Phase Transit. Data[END_REF] the presence of two-phase alloys α-Ni and intermetallic β-Ni 3 Si or possibly a supersaturated α-Ni 1-x Si x solid solution can be presumed at 10-15 at.% Si, which results in a higher electrical resistivity in comparison to the pure Ni coating (see Figure 9). The columnar structure observed in the TEM micrograph (see Figure 3.(a)) is commonly seen for polycrystalline sputtered thin films deposited at low substrate temperature, according to the structure zone model proposed by Thornton. [START_REF] Thornton | [END_REF] In addition, the columns and intercolumnar boundaries observed in our Ni 1-x Si x N y HiPIMS films deposited at low N content are dense, due from the intense low-energy ion irradiation from Ni + , Ar + , Si + and to some extent N 2 + and N + ions during the film growth. Indeed, reactive HiPIMS discharges, even in the absence of an applied substrate bias, are well-known to provide ion-assisted growth, where the deposited flux is composed to a large fraction of metal ions having an average kinetic ion energies of 15-20 eV and a neutral sputtered flux with a kinetic energy of 2 eV. 57,58 The use of a continuous substrate bias, here -100V, can provide additional kinetic energy (+100 eV) to the incident ions species at the substrate surface, resulting in a higher adatom mobility during the film growth and ultimately leads to a reduction of intracolumnar as well as intercolumnar porosity, as shown by Petrov et al. 47 Under such conditions, dense Ni 1-x Si x N y films can be grown even in the absence of substrate heating, which corresponds to zone 3 in the extended structure zone diagram, where energetic ion bombardment is taken into account. 59 Films grown with very low N content, i.e. N < 5 at.% exhibit a strong (111) texture which is usually reported for fcc metallic thin film deposited on a native SiO 2 surface by magnetron sputtering since close-packed (111) planes have the lowest relative surface energy. 60 This result also demonstrates that adding 15 at.% Si in the Ni sub-lattice does not modify the film texture in comparison to pure Ni film. On the other hand, XRD analysis (see strong modification of the thin film formation process, limiting grain coarsening during coalescence and film growth. 64 The resulting texture remains zone 3 with columns extending through the film, but with a lesser degree of preferred orientation and a smaller grain size as shown in Figure 2.(a) and Figure 2.(b), respectively.

At higher N content (from 16 up to 27 at.%), clear phase separation starts to occur leading to a progressive formation of a nanocomposite structure for a N content > 16 at.% (see Figure 3.(c)) with small Ni-rich nanocrystals partially surrounded by a Si-rich amorphous matrix, as seen in the cross-section TEM micrographs (Figure 4). The physical causes for the phase separation are not well understood as many processes can lead to such behavior. 65 Previous studies on the crystallization of NiSi-nc in SiN x reported the appearance of nanocrystals under specific conditions of temperature. Under these deposition conditions, nanocrystal growth took place after a rapid thermal annealing at a temperature of ~ 600 °C during 60 seconds. [21][22][23]26 The average size of the nanocrystal was estimated to be between 4 and 7 nm, with the annealing temperature. In addition, the stoichiometry of all the deposited Ni 1-x Si x N y films were typically Si-rich with Si content up to 70 at.%. Therefore, according to the ternary system nickel-silicon-nitrogen phase diagram proposed by Weitzer and Schuster, 66 the thermodynamically stable structure for these alloying elements would be in coexistence between a metallic phase (either elemental or intermetallic) and SiN x . Even though, as film growth conditions during sputter depositions are far from equilibrium, a metastable solid solution phase Ni x Si 1-x N y are generally reported. Chen et al. 21,23 presented XPS data in the form of Ni 2p core-level spectra, where a single peak was clearly assigned to a ternary Ni x Si 1-

x N y solid solution nitride. Their experimental findings suggest that Ni atoms are uniformly distributed in the bulk of the amorphous network during film growth. As a results, the temperature dependence of the NiSi x crystallization under these conditions can only be explained by bulk diffusion and precipitation of Ni in the amorphous nitride network 27 often termed "metal induced crystallization" (MIC). 67 As the temperature of the thermal annealing increases, accumulated Ni atoms bond to Si atoms and form islands, resulting in the nucleation and subsequent growth of NiSi x nanocrystal with x > 1. 68 The driving force for such a process is the reduction in the free energy due to amorphous-crystalline phase transformation. 69 Similar effects have been observed for the formation of NiSi x nanocrystal in amorphous silicon 68,[70][71][72] and silicon oxide. 73,74 In the present study, the nanocrystal growth occurs in the films, without any additional substrate heating, provided that a critical amount of N 2 was added. Due to the low growth temperature, bulk diffusion can be ruled out and only surface diffusion can occur during the film growth. 75 At the early stage of growth, both Ni and Si ad-atoms can freely diffuse on the substrate surface and form islands which then serves as a sink for the subsequently deposited

Ni and Si atoms. 49,76 Since the flux of the deposited Ni atoms is higher than that of Si, the development of Ni-rich Ni x Si (x > 1) islands will still be dominant. However, one must keep in mind that, in reactive sputtering conditions, N species (N ad-atoms and N 2 ) can also be Our results also demonstrate that Ni atoms can be incorporated in the Si-rich matrix during the growth, forming a ternary solid solution (see Figure 8.(b)). The Ni incorporation in the SiN x phase can be understood in view of the high diffusivity and solubility of Ni in Si-N lattice. 77 Indeed, Ni, like Cu, belongs to the group of 3d transition metals, which are considered as the fastest diffusers in Si at high temperature, 78 but even at room temperature. 79 Finally, the small increase of the room temperature electrical resistivity from about 45 to 65 µΩ with increasing N content suggests that the conduction mechanism of the asdeposited coatings is not strongly affected by the transition to a nanocomposite microstructure. Similar results have been reported by Sandu et al. 80 for nanocomposite ZrN/SiN x thin films deposited using bias voltages of -150 V and a substrate temperature of 250°C and, more recently, by Ramiez et al. 81 for N-deficient TaN/SiN thin films. The authors attributed the weak dependence of the electrical resistivity to the frequent percolation of the nearest-neighbors conducting crystallites due to the small coverage of the SiN x phase. The idea of a percolation conduction path, in our films, is supported by the HRTEM analysis (see the marked region labeled #2 in the Figure 4.(e)), revealing that the surface coverage of SiN x is too small to completely encapsulate the Ni-rich crystallites leading to the percolation of two nearest-neighbor crystallites. This percolation pathway between Ni-rich crystallites offers a highly conducting network for charge carriers to diffuse across the entire film thickness, reducing the electrical resistivity. In addition, due to the small thickness of the SiN x layer, as emphasized by the marked region labeled #1 in the Figure 4.(e), electrical transport could also be occurred through tunneling. 81 Completely isolating the Ni-rich crystallites, and thus increase of the electrical resistivity, should be possible by increasing the Si content within the films.

CONCLUSION

Figure 2 .

 2 Figure 2.(a) displays XRD diffractograms of the Ni 1-x Si x N y coatings with different N contents. The peaks, centered at 44.4° and 51.7° can be attributed to the (111) and (200) lattice planes of fcc-nickel (JCPDS: 71-4655) or of fcc-Ni 3 Si (JCPDS: 32-700), due to the small difference in lattice parameter between Ni (3.524 Å) and Ni 3 Si (3.504 Å).[START_REF] Cao | [END_REF] The measured peak positions exhibit a distinct shift to higher 2θ position of both diffraction peaks

  Selected-area electron diffraction (SAED) pattern (left bottom panel, Figure 3.(a)) reveals an 111-orientated fcc crystal texture, in good agreement with XRD results. Moreover, one can also observed the presence of a weak (211) diffraction ring between (200) and (220), which is characteristic of a Ni 3 Si crystal structure. Highresolution TEM (HR-TEM) (right bottom panel Figure 3.(a)) shows consistently well-defined lattice fringes. Upon incorporation of 16 at.% of N into the films, one can see from the bright field TEM micrograph, see Figure 3.(b), that the columnar nature still is retained. In addition, the SAED pattern is consistent with the one found at 0 at.% N film, with only an additional faint ring in the (111) position. Note that the additional (211) diffraction ring is still apparent. HR-TEM displays both intensity variation and nanosized regions where no lattice fringes are apparent, indicating that here the structure consist of columnar grains which are sprinkled with localized amorphous regions (see, for example the black rectangle, Figure 3.(b)). Finally, Ni 1-x Si x N y coating containing 27 at.% of N displays a completely different microstructure without any trace of columnar grains (Figure 3.(c)). At high resolution, the microstructure is shown to consist of nanocrystallites randomly distributed in an apparently amorphous matrix (Figure 3.(c)), whose size is in the range of 5-7 nm, which is in good agreement with the value extracted from the XRD measurements. The presence of nc domains is further highlighted by the SAED pattern, which consists of almost continuous rings, that can be associated with a randomly orientated a fcc-structure and (211) Ni 3 Si. Further TEM analysis was carried out on the Ni 1-x Si x N y coating containing 27 at.% of N by high angle annular dark field (HAADF) STEM, shown in Figure 4.(a), which indicates that the nanosized domains have an overall higher atomic weight (brighter) compared to the surrounding matrix (darker). The details are revealed by the overlaid colored Ni (green) and Si (red) elemental map, obtained using EDS (Figure 4.(b)), showing the heavier Ni in the domains and the lighter Si in the matrix. Due to the insensitivity of EDS to light elements a reliable N map

Figure 5 .

 5 Figure 5.(a), reveals that the component associated with the Si-N framework increases with N content in detriment to the Si-Ni framework (please refer to the Figure S3 on the supporting information). At 27 at.% N, the relative contribution of Si-N represents ~ 40% of the total

  it is difficult to precisely attribute the Ni 2p 3/2 main peak to the correct chemical environment of Ni atoms due to the overlap between pure Ni and Ni-rich silicide compounds (ex: Ni 3 Si and Ni 31 Si 12 ) peaks. In comparison to the Ni 2p 3/2 peak relative to the pure Ni environment (852.7 eV), the Ni 2p 3/2 core-level shift (ΔE c ) for the Ni-rich silicides compounds is ΔE c = 0.1-0.3 eV, which is more or less the ΔE c associated error for the charge correcting calibration using C 1s (ΔE c = ± 0.2 eV).40 This implies that a correct identification of the chemical environment of Ni atoms by investigating Ni 2p 3/2 core-level spectra is not possible.Based on the information extracted from the XPS core-level spectra analyses, TEM micrographs and EDS analyses, it can be concluded that the Ni 1-x Si x N y nanocomposite thin films deposited at higher N 2 flow (N 2 ≥ 6 sccm) are composed of randomly oriented Nidominated nc embedded in a-SiN x matrix, with for the presence of Si and Ni within the nc domains and amorphous matrix respectively.

  30% and become difficult to image it in the SEM, indicating a low electric conductivity of the film. Similar reduction of the film thickness was already observed by Bouts et al.,44 for a Cu/C nanocomposite, and was explained by a mechanical relaxation of the material occurring as a consequence to the removal of the metal rich nanocrystals from the non-conducting matrix. EDS compositional analysis confirmed the preferential dissolution of Ni atoms during the dealloying process in nitric acid, where after 5 min of etching the Ni:Si ratio drops from 8:1 to 1:1. Bright field TEM analysis, shown in Figure 7.(a), reveals that, after the chemical treatment, the well-defined nanocomposite structure seen previously is no longer evident. At high resolution (Figure 7.(b)), TEM analysis reveals that the Ni-rich nano-domains have been selectively etched and only weak lattice fringes (inset in the Figure 7.(b)) remain which is attributed to a residue of materials from Ni-rich nanocrystals. The low crystalline quality of the film is further confirmed by its associated fast Fourier transform (FFT) pattern (Figure 7.(c)), where very weak spots are detected. Additionally, HAAF-STEM micrograph (Figure 7.(d)) indicates that the size of the brighter nano-sized domains have significantly decrease compared to the ones observed from the film prior to treatment (Figure 4.(a)). All of which indicates the selective removal of the crystalline Ni-rich nanocrystals. After the HNO 3 treatment, the Si 2p core levels, see Figure 8.(a), comprises merely one component arising from Si-N bonds, and Ni-Si bonds are almost absent, confirming the preferential removal of the Ni-based NCs from the film. This result is also supported by the analysis of the Ni 2p core levels spectra, as shown in Figure 8.(b), which after etching exhibits a weak and noisy Ni 2p with the presence of a new peak at 855 eV. According to the literature,

Figure 2 .

 2 Figure 2.(a)) revealed that adding a relatively small amount of N, i.e. N < 10 at.%, has a strong impact on the preferred orientation of the films where a clear transition from (111) to (002) is observed. XRD patterns also revealed that both the (111) and (200) peak positions were shifted to lower diffraction angles as a result of nitrogen incorporation. No nickel nitride phase (Ni 4 N, Ni 3 N or Ni 2 N) were detected whatever the N 2 gas flow added during the present investigation. Similar behavior, e.g. transition from (111) to (002) preferred orientation, has already been observed for NiN x films deposited by conventional magnetron sputtering atdifferent N 2 flow rate, with and without the use of substrate heating.[61][62][63] The exact mechanism for this orientation change is not yet fully understood, but according to Kawamura et al.61 it is considered that the dominant effect controlling Ni nanostructure evolution under these growth conditions is the change in steady-state surface chemistry. The high N 2 + ion fluxes at the substrate surface tends to increase N coverage on Ni(200) surfaces, thereby greatly decreasing the surface energy of the (200) plane such that < and leads to the competitive evolution of (200), rather than (111) orientation. These results suggest that

  adsorbed and diffuse on the growing film surface. Due to the higher affinity of N with Si as compared with Ni, e.g. the enthalpy of formation (ΔH) at room temperature for Si-N and Ni-N are ΔH = -470 and ΔH =-70-85 kJ mol -1 , 23 respectively, will favor the formation of an amorphous SiN x phase (see Figure 5.(a)). As the N concentration is further increased in comparison to the Ni 1-x Si x N y coatings containing 16 at.% N, here the 2D SiN x tissue phase completely covers Ni-rich islands of all orientations at the initial stage of the film growth and coarsening during coalescence is hindered. As observed from the STEM micrograph (Figure 4), the resulting film growth proceeds through repeated renucleation with clear phase separation into Ni-rich and Si-rich domains. Resulting films are composed of small Ni-rich nanocrystals with random orientation (see Figure 2.(a)) dispersed in an amorphous nitride matrix. The nanocrystal growth mechanisms suggested in the present study is consistent with most of the observations reported in the literature for nanocomposite M-Si-N systems (M = Ti, Nb, Zr, Cr) deposited at low temperature. 4 For these systems, the typical nanocomposite microstructural change appears for a Si concentration between 7 and 20 at.%, which is relatively close to our condition, ~12 at.% Si. Thus, the decreasing of the diffraction peak intensity of the Ni-rich phase (Figure 2.(a)) and the decreasing of the crystallite size from 18 to 7 nm derived from the diffraction peak widths (Figure 2.(b)) with increasing N content, results in a broadening of the particle size distribution towards smaller dimensions due to the continuous development of the 2D SiN x tissue phase.

  In the present work, Ni-Si-N thin films were reactively deposited employing HiPIMS from a Ni 0.85 Si 0.15 composite target at different nitrogen gas flow. By adjusting reactive gas both the N concentration, and as a direct consequence, the microstructure of the films can be controlled. A clear transition of a polycrystalline Ni-rich Ni-Si film to a nanocomposite film composed of 7-8 nm Ni-rich Ni x Si nanocrystals embedded in an amorphous SiN x layers was observed when the nitrogen content reached a value above 16 at.%. The formation of Ni-rich nanocrystal is driven via repeated nucleation of subsequently deposited Ni and Si atoms caused by the phase-separation process taking place during the film growth. The proposed scenario is expected be the same for other metals with a low affinity to nitrogen, such as Cu, Co and Fe. This single-step process provides the possibility of in situ fabrication of NiSi nanocrystals without any additional heating, necessary for the development of new nonvolatile nanocrystal memories deposited onto temperature-sensitive substrates.

Figure caption : Figure 1 .

 :1 Figure caption:Figure1. Evolution of the element concentration into Ni 1-x Si x N y layers determined through XPS measurements as a function of the nitrogen flow rate. All the analyses were performed after an in situ cleaning of the surface using an Ar ion gun operating at 0.5 keV.

Figure 2 .

 2 Figure 2. (a) X-ray diffraction patterns and (b) crystallite size dependence estimated from the 111 diffraction peak with the Scherrer equation for NiSiN coatings with different N content on Si(001) at room temperature.

Figure 3 .

 3 Figure 3. Cross-sectional transmission electron microscopy (TEM) analysis of the Ni 1-x Si x N y thin films containing 0 at.% (a), 16 at.% (b) and (c) 27 at.% of N. The top panels are bright-field TEM micrographs from the thin films along with their respective SAED patterns, left bottom panels, fitted to a fcc-structure. Note the presence of a very weak (211) diffraction ring between (200) and (220), which is characteristic of a Ni 3 Si crystal structure. The right bottom panels are high-resolution TEM (HR-TEM) micrographs of a defined region of the microstructure.

Figure 4 .

 4 Figure 4. Advanced microscopy characterization of the Ni 1-x Si x N y thin film containing 27 at.% of N. HAADF-STEM image (a), the corresponding EDS-STEM map (b), with green indicating Ni and red indicating Si, of the same area and HR-TEM image of a single crystallite (c) with two regions indicating possible interconnection to other domains either by three atomic columns (region #1) or direct attachment (region #2).

Figure 5 .

 5 Figure 5. High-resolution XPS core level spectra of the Si 2p (a) and Ni 2p (b) region for Ni 1-x Si x N y coatings containing 0, 16 and 27 at.% N. The spectra were acquired after Ar + sputter cleaning.

Figure 6 .

 6 Figure 6. Cross-sectional SEM micrographs of the Ni 1-x Si x N y film containing 27 at.% of N before etching (a) and after treatment in nitric acid for 5 min.

Figure 7 .

 7 Figure 7. Cross-sectional TEM micrographs of Ni 1-x Si x N y film containing 27 at.% of N recorded after chemical etching in nitric acid for 5 min. (a) Bright field TEM micrographs at low (a) and high magnification (b), its associated FFT pattern (c), and an additional HAADF-STEM micrograph (d). Inset in (b) highlights one of the weak lattice fringes observed in the etched sample.

Figure 8 .

 8 Figure 8. XPS core level spectra of the Si 2p (a) and Ni 2p (b) region for Ni 1-x Si x N y coatings containing 27 at.% N before and after nitric etching. The spectra were acquired after Ar + sputter cleaning.

Figure 9 .

 9 Figure 9. Room-temperature electrical resistivity of the Ni 1-x Si x N y thin films as a function of the N content.

ACKNOWLEDGMENTS

The authors would like to acknowledge H. Machhadani for helpful discussions regarding nitric acid treatment. JK acknowledges financial support from the Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linköping University (Faculty Grant SFO-Mat-LiU No. 2009-00971) and the Swedish Research Council (grant VR 621-2014-4882).

Highlights:

 Deposition of Ni x Si nanocrystals embedded in SiN x matrix by HiPIMS without any intentional heating  Spontaneous formation of Ni x Si nanocrystals and SiN is occurring with N 2 addition during deposition  The phase separation relies on a higher chemical affinity of N with Si


João Vitor

Do Santos

Altair Lais Gomes Fregolente

Odair Pastor Ferreira Benedito Moreira

Stéphane Mounier

Bruno Viguier

Houssam Hajjoul

Márcia Cristina

Santos

Márcia Cristina Bisinoti
email: marcia.bisinoti@unesp.br

Humic-like acids from hydrochars: Study of the metal complexation properties compared with humic acids from anthropogenic soils using PARAFAC and time-resolved fluorescence

Keywords: hydrothermal carbonization, anthropogenic soils, complexation, fluorescence lifetime, soil remediation

come

Introduction

Metal pollution is a worldwide environmental issue in which human activity plays a major role [START_REF] Lesmana | Studies on potential applications of biomass for the separation of heavy metals from water and wastewater[END_REF][START_REF] Li | A review of soil heavy metal pollution from mines in China: Pollution and health risk assessment[END_REF]. Unlike most organic contaminants, metals are not biodegradable and can accumulate in living organisms, thereby causing problems for both human health and the environment [START_REF] Lesmana | Studies on potential applications of biomass for the separation of heavy metals from water and wastewater[END_REF]. Some metals of great concern are cadmium, zinc, copper, and nickel [START_REF] Li | A review of soil heavy metal pollution from mines in China: Pollution and health risk assessment[END_REF][START_REF] Tang | Impact of humic/fulvic acid on the removal of heavy metals from aqueous solutions using nanomaterials: A review[END_REF]. Among them, copper is important for cellular metabolism.

However, this element is highly toxic if swallowed at high concentrations and can cause gastrointestinal problems, hair loss, weight loss, and even death [START_REF] Andreazza | Bacterial stimulation of copper phytoaccumulation by bioaugmentation with rhizosphere bacteria[END_REF][START_REF] Fu | Removal of heavy metal ions from wastewaters: A review[END_REF][START_REF] Tang | Impact of humic/fulvic acid on the removal of heavy metals from aqueous solutions using nanomaterials: A review[END_REF]. Intensified industrial, agricultural, mining, and urbanization activities are the main causes of copper contamination [START_REF] Hladun | Cadmium, Copper, and Lead Accumulation and Bioconcentration in the Vegetative and Reproductive Organs of Raphanus sativus: Implications for Plant Performance and Pollination[END_REF][START_REF] Mackie | Remediation of copper in vineyards -A mini review[END_REF]. Therefore, research is needed to develop techniques for reducing the concentrations of metals in contaminated soils.

It is widely accepted that soil fertility depends on the presence of nutrients and organic matter [START_REF] Stevenson | Humus chemistry: Genesis, composition, and reactions[END_REF]. Humic substances (HS) are the main constituents of soil organic matter and are formed from the remains of plant and animal degradation.

Humic acids (HA) are one of the components of HS. They represent the most hydrophobic part and play important roles in soil fertility, water retention, plant growth promotion, and interaction with organic and inorganic compounds [START_REF] Bento | Humic extracts of hydrochar and Amazonian Dark Earth: Molecular characteristics and effects on maize seed germination[END_REF][START_REF] Canellas | Chemical nature of soil humified fractions and their bioactivity[END_REF][START_REF] Jindo | Influence of biochar addition on the humic substances of composting manures[END_REF]. The bioavailability, distribution, and toxicity of pollutants are affected by their interaction [START_REF] He | Binding characteristics of Cu2+ to natural humic acid fractions sequentially extracted from the lake sediments[END_REF][START_REF] Tang | Impact of humic/fulvic acid on the removal of heavy metals from aqueous solutions using nanomaterials: A review[END_REF]; thus, the use of HA has gained attention for remediation [START_REF] Piccolo | Soil washing with solutions of humic substances from manure compost removes heavy metal contaminants as a function of humic molecular composition[END_REF]. Anthropogenic soils such as TM occur near the Amazon basin and are known for exhibiting high fertility [START_REF] Glaser | Black carbon in density fractions of anthropogenic soils of the Brazilian Amazon region[END_REF][START_REF] Oliveira | Morphological analysis of soil particles at multiple length-scale reveals nutrient stocks of Amazonian Anthrosols[END_REF]. Such soils have gained the attention of researchers who have attempted to reproduce their organic matter by thermochemical methods of biomass conversion, such as pyrolysis and hydrothermal carbonization (HTC). These methods use various biomass types to obtain a material with the characteristics of TM.

The HTC process is a suitable biomass disposal method for the production of carbon-based materials known as hydrochar. In addition, the process water (liquid phase) is generated by this method. Some authors have proposed the use of this process water as a hydrothermal medium in a new carbonization process [START_REF] Kabadayi Catalkopru | Effects of spent liquor recirculation in hydrothermal carbonization[END_REF][START_REF] Stemann | Hydrothermal carbonization: Process water characterization and effects of water recirculation[END_REF][START_REF] Weiner | Influence of Process Water Reuse on Hydrothermal Carbonization of Paper[END_REF] or for liquid fertilizer [START_REF] Fregolente | Toxicity evaluation of process water from hydrothermal carbonization of sugarcane industry by-products[END_REF].

Most HA used in the literature are extracted from soils, water, sediments, or peat [START_REF] Chen | Combination of a Copper-Ion Selective Electrode and Fluorometric Titration for the Determination of Copper(II) Ion Conditional Stability Constants of Humic Substances[END_REF][START_REF] Plaza | Copper(II) complexation of humic acids from the first generation of EUROSOILS by total luminescence spectroscopy[END_REF][START_REF] Zherebtsov | Sorption of copper cations from aqueous solutions by brown coals and humic acids[END_REF]. The formation of these environmental elements under natural conditions requires significant amounts of time and involves several chemical and biological reactions that can last for years (Yang et al., 2019a). However, the formation of HTC needs only hours [START_REF] Melo | Transforming sugarcane bagasse and vinasse wastes into hydrochar in the presence of phosphoric acid: an evaluation of nutrient contents and structural properties[END_REF][START_REF] Silva | Effect of the reaction medium on the immobilization of nutrients in hydrochars obtained using sugarcane industry residues[END_REF]. Humic-like acids (HLA) are defined as extracts of different organic materials that have not undergone the natural process of humification [START_REF] Bento | Humic extracts of hydrochar and Amazonian Dark Earth: Molecular characteristics and effects on maize seed germination[END_REF][START_REF] Jindo | Influence of biochar addition on the humic substances of composting manures[END_REF][START_REF] Jindo | The efficiency of a low dose of biochar in enhancing the aromaticity of humic-like substance extracted from poultry manure compost[END_REF]. Although there is not the natural humification process that occurs in soils, HLA extracted from hydrochars might have characteristics similar to those of natural HA extracted from anthropogenic soils, which are known as a model of organic matter. Knowledge of the chemical structure of HLA is essential for predicting their behavior and benefits in soil.

Because natural HA form complexes with metals [START_REF] Stevenson | Humus chemistry: Genesis, composition, and reactions[END_REF], the study of interaction between metallic ions and HLA is of significant value for predicting whether HLA can assist in the complexation reactions if applied to the soil, and thus the metal bioavailability. A variety of agents has been used for soil washing including biosurfactants as well as synthetic agents such as ethylenediaminetetraacetic acid (EDTA) [START_REF] Mulligan | Heavy metal removal from sediments by biosurfactants[END_REF][START_REF] Mulligan | Recent advances in the environmental applications of biosurfactants[END_REF]. Despite the efficiency of these agents in removing metals from soils, they cause environmental problems and are high in cost, which justifies the need for more studies to develop new washing agents. In previous research, a solution of HLA from manure compost biochar showed the remediation of soils contaminated by multiple metals [START_REF] Kulikowska | Feasibility of using humic substances from compost to remove heavy metals (Cd, Cu, Ni, Pb, Zn) from contaminated soil aged for different periods of time[END_REF]; the same was shown for HLA from composted sewage sludge [START_REF] Piccolo | Soil washing with solutions of humic substances from manure compost removes heavy metal contaminants as a function of humic molecular composition[END_REF]. In addition, the potential of HLA extracted from hydrochars produced with crude waste biomass was presented (Yang et al., 2019a). In that case, HLA from hydrochars produced with by-products of the sugarcane industry were shown to act in complexation reactions between metals and active sites on the HLA surface.

The fluorescence excitation emission matrix (EEM) has gained prominence in the characterization of organic matter and its interaction with metallic species [START_REF] Elkins | Spectroscopic approaches to the study of the interaction of aluminum with humic substances[END_REF]. The analytical potential of fluorescence EEM spectra coupled with chemometric tools such as Parallel Factor Analysis (PARAFAC) has been successfully realized for this type of analysis [START_REF] Mounier | Copper complexing properties of dissolved organic matter: PARAFAC treatment of fluorescence quenching[END_REF][START_REF] Nouhi | Time-resolved laser fluorescence spectroscopy of organic ligands by europium: Fluorescence quenching and lifetime properties[END_REF][START_REF] Stedmon | Characterizing dissolved organic matter fluorescence with parallel factor analysis: a tutorial[END_REF][START_REF] Tadini | Modeling the quenching of fluorescence from organic matter in Amazonian soils[END_REF][START_REF] Yuan | Detection of Copper (II) and Cadmium (II) binding to dissolved organic matter from macrophyte decomposition by fluorescence excitation-emission matrix spectra combined with parallel factor analysis[END_REF]. Moreover, PARAFAC has also been used in the characterization of dissolved organic matter (DOM) of biochars produced with different biomass content [START_REF] El-Naggar | Biochar-induced metal immobilization and soil biogeochemical process: An integrated mechanistic approach[END_REF][START_REF] Rajapaksha | Dissolved organic matter characterization of biochars produced from different feedstock materials[END_REF]Yang et al., 2019b). Further, because different chemical species have different fluorescence lifetimes, it is also possible to study organic matter-metal interaction through timeresolved fluorescence (TRF), particularly in complex samples such as HA and HLA [START_REF] Nouhi | Time-resolved laser fluorescence spectroscopy of organic ligands by europium: Fluorescence quenching and lifetime properties[END_REF].

Thus, the chemical characterization of HLA from hydrochars obtained with sugarcane industry by-products was conducted, and the results were compared with that of HA from TM. In addition, the interaction of HA and HLA with Cu(II) ions by fluorescence quenching was studied to provide quantitative data on the complexation capacity (CC), concentration of binding sites (CL), and conditional stability constant (K). TRF as a complementary technique of fluorescence quenching was used to investigate the interaction by measuring the fluorescence lifetime and its variation with the addition of metal. Thus, the present study can provide a new perspective for analyzing Cu(II)-HLA interactions and the potential of HLA as a complexing agent in the remediation of soils polluted by metals. Traditional Knowledge (SisGen), No. A0018C2.) Soil samples were collected from the 0 to 30 cm deep layer, air-dried, and subsequently sieved through a 2 mm mesh for removal of plant debris and fragments.

Materials and methods

Terra Mulata sampling

Hydrothermal carbonization and recarbonization process

HTC was performed using vinasse and sugarcane bagasse, in which the vinasse was used to produce the hydrothermal medium [START_REF] Melo | Transforming sugarcane bagasse and vinasse wastes into hydrochar in the presence of phosphoric acid: an evaluation of nutrient contents and structural properties[END_REF][START_REF] Silva | Effect of the reaction medium on the immobilization of nutrients in hydrochars obtained using sugarcane industry residues[END_REF].

The HTC processes were conducted in a Teflon® closed reactor coated with stainless steel. Each mixture (Table 1) was heated in a muffle furnace at 230 °C for 13 h. Then, the reactor was cooled in an ice bath, and the suspension was filtered under vacuum filtration to separate the hydrochar from the process water. The hydrochar was washed with distilled water until constant pH was reached and was then dried at 50 °C until constant mass was achieved. The process water generated in the HTC process was used in the recarbonization process to produce the hydrothermal medium for sugarcane bagasse carbonization. The subsequent steps after carbonization, i.e., filtration, hydrochar washing, and drying, were the same.

Table 1 2.3 Extraction of humic acids and humic-like acids

The extractions were performed following the recommendations of the International Humic Substances Society (IHSS) [START_REF] Swift | Methods of soil analysis. Part 3. Chemical methods[END_REF]. HS were extracted from TM at a proportion of 1:10 (soil:extractor). Thus, the extraction was conducted in 100 g of soil to 1 L of solution (NaOH 0.1 mol L -1 ) under nitrogen flow for 4 h. The solution extracted was acidified with HCl (6 mol L -1 ) to reach pH~1. Thus, HA were precipitated, and fulvic acids remained in the supernatant. Subsequently, the HA were separated by centrifugation, dialyzed using a Fisherbrand apparatus with a molecular weight cut-off at 6,000-8,000 Da, and freeze-dried.

The method was modified for the extraction of humic-like substances [START_REF] Jindo | Influence of biochar addition on the humic substances of composting manures[END_REF]. Those from hydrochars were extracted at a proportion of 1:10 (hydrochar:extractor) using NaOH (0.1 mol L -1 ) under nitrogen flow for 4 h. This procedure was repeated, with the same hydrochar portion until the final extract (supernatant phase) showed a lighter color. HLA were obtained by the pH difference, with HCl (6 mol L -1 ) to pH~1, and was separated from the fulvic-like acids by centrifugation. Finally, the extracts of HLA obtained from each extraction step were combined, dialyzed, and freeze-dried.

Characterization of humic acids and humic-like acids

Elemental analysis

Elemental analyses were performed on solids using an elemental analyzer ( 2400Series II CHNS/O, Perkin Elmer, Walther, Massachusetts, USA). The oxygen content was determined according to the difference, i.e., O% = 100 -(C, H, N, S + ashes). The ash content was determined in a muffle furnace at 750 °C for 4 h. The C, H, N, S, and O contents were recalculated on an ash-free basis. The H/C, O/C, and C/N atomic ratios were determined by elemental analysis [START_REF] Giovanela | Chemical and spectroscopic characterization of humic acids extracted from the bottom sediments of a Brazilian subtropical microbasin[END_REF][START_REF] Stevenson | Humus chemistry: Genesis, composition, and reactions[END_REF].

Infrared spectroscopy

Infrared spectra were obtained using an attenuated total reflectance (ATR)coupled spectrophotometer (FTIR; Spectrum Two UATR, Perkin Elmer, Walther, Massachusetts, USA). The solid material was placed directly on the ATR crystal and was analyzed at a spectral range of 4000-400 cm -1 with 20 scans and a resolution of 4 cm -1 .

Ultraviolet-visible spectroscopy

The ultraviolet-visible (UV-Vis) scanning spectra were obtained by employing a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan) at a range of 200-700 nm. HA and HLA solutions (10 mg L -1 ) in total organic carbon (TOC; TOC-VCSN Shimadzu, Japan) were diluted in 0.05 mol L -1 of NaHCO 3 at pH 8. From the spectra, the ratios E 465 /E 665 (E4/E6) and E 270 /E 407 (E2/E4) were calculated. These ratios have been used to describe the degree of aromatic ring condensation and the presence of lignin derivatives, respectively [START_REF] Budziak | Transformações químicas da matéria orgânica durante a compostagem de resíduos da indústria madeireira[END_REF][START_REF] Canellas | Chemical nature of soil humified fractions and their bioactivity[END_REF].

Solid-state 13 C-CPMAS-NMR spectroscopy

Solid-state 13 C-cross-polarized magic angle spinning (CPMAS)-nuclear magnetic resonance (NMR) characterization was performed by employing a Bruker According to the area integration, the aromaticity (ARM), alkyl (A/AO), and hydrophobicity (HB/HI) indices were calculated. [START_REF] Monda | Molecular composition of the Humeome extracted from different green composts and their biostimulation on early growth of maize[END_REF].

Interaction of humic acids and humic-like acids with Cu(II)

Fluorescence quenching

Studies involving interaction with Cu(II) ions were performed using 10 mg L -1 of HA or HLA in a buffer medium with 0.1 mol L -1 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) at pH 7.0. Titration experiments were performed in 13 cuvettes for each HA or HLA sample with various concentrations of Cu(II) (Cu(ClO 4 ) 2 •6H 2 O), ranging from 0 mg L -1 to 11.2 mg L -1 . The equilibrium time of 30 min was determined on the basis of changes observed in the fluorescence signal.

Fluorescence measurements were performed using a spectrofluorometer (F4500, Hitachi, Santa Clara, California, USA) equipped with a 450 W xenon lamp. The spectra were acquired in the scan ranges of 220-700 nm for emission and 220-500 nm for excitation, with both slits fixed at 5 nm. The scan speed was set at 2,400 nm min -1 , and the detector voltage was 700 V. The obtained EEM spectra were treated by using the PARAFAC chemometric model. The number of components responsible for fluorescence was defined by using the core consistency diagnostic (CORCONDIA) [START_REF] Mounier | Copper complexing properties of dissolved organic matter: PARAFAC treatment of fluorescence quenching[END_REF]. The density of binding sites (CL) as well as the conditional stability constant (K) were determined following the metal complexation model proposed by [START_REF] Ryan | Fluorescence Quenching Titration for Determination of Complexing Capacities and Stability Constants of Fulvic Acid[END_REF], as represented by

, ( 1
)
where L is a free complexing site, M is the free or uncomplexed metal, and ML is the complex. Based on this reaction, the conditional stability constant can be calculated as .

(2)

Metal and complexing sites follow a mass balance in solution:

,

where CL and CM represent the total concentration of complexing sites and the metal in the solution system, respectively.

Time-resolved fluorescence

Three samples were selected from the data obtained from the fluorescence quenching experiment, i.e., three total copper concentrations for each HA and HLA interaction. The first contained no concentration of copper (C 0 ), whereas C 0.7 and C 5.6 contained 0.7 mg L -1 and 5.6 mg L -1 , respectively. Prior to each measurement, the prepared solution was bubbled with nitrogen for 10 min to prevent photodegradation.

TRF experiments were performed at 266 nm from the fourth harmonic generation from a nanosecond laser Nd:YAG (Quanta-Ray INDI, Spectra Physic, Germany), and excitation (1024 nm) occurred with a pulse width of 5 ns and a repetition of 20 Hz. Six hundred spectra were accumulated at each time step of 0.2 ns with an optical fiber and were detected by an intensified charge-coupled device (CCD) camera with a 35 ns gate width. Data obtained by TRFS were corrected by energy fluctuation and were treated by using a homemade MATLAB ® program to obtain the spectral deconvolution and the associated lifetime values.

Results and discussion

Characterization of humic acids and humic-like acids

Determining the molecular formula of HA and HLA is still a challenge; however, elemental analysis can provide a general idea about their compositions. The elemental analysis and UV-Vis ratios of soil HA and hydrochar HLA samples are shown in Table 2. In general, HA and HLA showed carbon as the main constituent, at 36.6% to 62.2%, followed by oxygen, hydrogen, nitrogen, and sulfur. In addition, HLA RHH 2 SO 4 had the highest sulfur content of the samples. Regarding the carbon percentage, the HLA samples showed higher values than did HA.

The O/C, H/C, and C/N atomic ratios (Table 2) based on elemental analysis, have been used to describe the content of oxygenated groups in the molecules, the degree of aromatic ring condensation, and the nitrogen content, respectively [START_REF] Giovanela | Chemical and spectroscopic characterization of humic acids extracted from the bottom sediments of a Brazilian subtropical microbasin[END_REF][START_REF] Stevenson | Humus chemistry: Genesis, composition, and reactions[END_REF]. In general, HA showed higher O/C ratios than those of HLA, suggesting a greater abundance of oxygen-containing functional groups [START_REF] Fukushima | Adsorption of pentachlorophenol to a humin-like substance-bentonite complex prepared by polycondensation reactions of humic precursors[END_REF]. The H/C ratio showed that HA, except for the TM II HA, have higher condensation of aromatic rings than do HLA; lower C/N atomic ratios for HA suggest more nitrogen-containing structures (F. Yang et al., 2019).

The UV-Vis spectra of HA have been previously described in the literature and provide little structural information. Generally, however, the overall absorbance decreases with increasing wavelength [START_REF] Uyguner | Evaluation of humic acid photocatalytic degradation by UV-vis and fluorescence spectroscopy[END_REF]. In this study, both HA and HLA showed this behavior; the spectra are shown in the supplementary material (Figure S1). For better interpretation of UV-Vis analysis, the absorbance ratios at certain wavelengths were utilized. The E 465 /E 665 absorbance ratio is related to the degree of condensation of aromatic rings. Values lower than 4 indicate compounds with more condensed aromatic groups [START_REF] Canellas | Chemical nature of soil humified fractions and their bioactivity[END_REF][START_REF] Stevenson | Humus chemistry: Genesis, composition, and reactions[END_REF][START_REF] He | Binding characteristics of Cu2+ to natural humic acid fractions sequentially extracted from the lake sediments[END_REF]. The E 270 /E 407 absorbance ratio refers to the presence of lignin derivatives, of which the content is directly proportional to this value [START_REF] Budziak | Transformações químicas da matéria orgânica durante a compostagem de resíduos da indústria madeireira[END_REF]. This ratio might be indicative for organic matter studies because lignin is involved in the formation of both soils and hydrochars. The obtained parameters are presented in Table 2.

HA showed a higher degree of aromatic condensation than HLA. Furthermore, the lower number of compounds with lignin structures might be linked to greater decomposition of organic matter.

Table 2

The FTIR spectra (Figure 1a and 1b) showed that both HA and HLA have typical features of HS [START_REF] Esteves | Comparative characterization of humic substances from the open ocean, estuarine water and fresh water[END_REF][START_REF] Fernandes | Elemental and spectral properties of peat and soil samples and their respective humic substances[END_REF][START_REF] Giovanela | Chemical and spectroscopic characterization of humic acids extracted from the bottom sediments of a Brazilian subtropical microbasin[END_REF]. In the 3500-3300 cm -1 region, larger bands were observed for HA. However, the bands for HLA were more defined and might be related to N-H stretching of amines or amides and O-H stretching of alcohols and phenols [START_REF] Dick | Chemical Characterization and Infrared Spectroscopy of Soil Organic Matter From Two Southern Brazilian Soils ( 1 )[END_REF]. The same result was observed for the C-H stretching of aliphatic methyl and methylene groups near 2900 cm -1 . In the 1730-1710 cm -1 region, low intensity of C=O stretching of carboxylic acids or ketones was found for HA (Figure 1a), and a slight shoulder was observed for the HLA samples (Figure 1b) [START_REF] Senesi | Humic acids in the first generation of EUROSOILS[END_REF]. Bands in the 1620-1600 cm -1 regions were associated with aromatic C=C stretching [START_REF] Stevenson | Humus chemistry: Genesis, composition, and reactions[END_REF]. C-H bending of CH 3 and deformation of CH 2 and CH 3 structures were observed in the 1420-1380 cm - 1 region for HLA but not for HA. All spectra showed C-O-C stretching at approximately 1250-1030 cm -1 , likely resulting from polysaccharides; stretching in esters, alcohols, and phenols was also observed. In addition, Si-O stretching bands were identified in the 825-750 cm -1 region [START_REF] Giovanela | Chemical and spectroscopic characterization of humic acids extracted from the bottom sediments of a Brazilian subtropical microbasin[END_REF][START_REF] Stevenson | Humus chemistry: Genesis, composition, and reactions[END_REF].

Figure 1

The [START_REF] Mazzei | Quantitative evaluation of noncovalent interactions between glyphosate and dissolved humic substances by NMR spectroscopy[END_REF][START_REF] Monda | Molecular characteristics of water-extractable organic matter from different composted biomasses and their effects on seed germination and early growth of maize[END_REF][START_REF] Spaccini | Molecular characteristics of humic acids extracted from compost at increasing maturity stages[END_REF][START_REF] Tadini | Characterization of typical aquatic humic substances in areas of sugarcane cultivation in Brazil using tetramethylammonium hydroxide thermochemolysis[END_REF].

Chemical shifts near 15 ppm were assigned to short-chain aliphatic structures, whereas those at 30 ppm were assigned to long-chain aliphatic structures attributed to lipid compounds, such as plant waxes and biopolyesters [START_REF] Francioso | Spectroscopic study of humic acids fractionated by means of tangential ultrafiltration[END_REF][START_REF] Monda | Molecular characteristics of water-extractable organic matter from different composted biomasses and their effects on seed germination and early growth of maize[END_REF][START_REF] Spaccini | Bioactivity of humic substances and water extracts from compost made by lignocellulose wastes from biorefinery[END_REF]. These peaks were more intense in the HLA samples (Figure 2). The peak at 54 ppm, related to the presence of methoxyl carbons in both guaiacil and syringyl units of lignin fragments, as well as Cα of oligoand polypeptides [START_REF] Monda | Molecular characteristics of water-extractable organic matter from different composted biomasses and their effects on seed germination and early growth of maize[END_REF], was present for both HA and HLA except in those in which sulfuric acid was used as an additive (Figure 2).

The peaks in the 71-74 ppm range were more evident in HA and are typical of O-akyl-C in mono and polysaccharides, in which the centered peak at 72 ppm is attributed to the overlapping of carbon numbers 2, 3, and 5 in the pyranosidic cellulose and hemicellulose structures, referred to as C-2, C-3, and C-5, respectively [START_REF] Monda | Molecular characteristics of water-extractable organic matter from different composted biomasses and their effects on seed germination and early growth of maize[END_REF][START_REF] Spaccini | Molecular characteristics of humic acids extracted from compost at increasing maturity stages[END_REF]. Unsubstituted aromatic carbons were responsible for the intense signal at 130 ppm [START_REF] Monda | Molecular characteristics of water-extractable organic matter from different composted biomasses and their effects on seed germination and early growth of maize[END_REF] in the HA and HLA structures, whereas their analogs containing -OH, -OCH 3 , and -NH 2 groups were found in the 145-160 ppm region. The signal at 174 ppm corresponds to carboxylic acids, and the low signal at 190-220 ppm refers to carbonyl groups of ketones, amides, and aldehyde [START_REF] Mao | Characterization of humic substances by advanced solid state NMR spectroscopy: Demonstration of a systematic approach[END_REF] in the HA and HLA, as shown in Figure 2.

Figure 2

By employing spectral integration, it is possible to estimate the distribution of carbons in the HA and HLA structures as well as some indices such as A/AO, ARM, and HB/HI, as shown in Table 3. These indices are related to the biochemical stability of different organic compounds. The HA showed aromatic compounds as the main constituents, whereas HLA showed the presence of aromatic and aliphatic carbons (Table 3). The ARM index was similar for both materials. Higher HB/HI index values indicate the incorporation of hydrophobic moieties in HLA [START_REF] Bento | Humic extracts of hydrochar and Amazonian Dark Earth: Molecular characteristics and effects on maize seed germination[END_REF].

Furthermore, the natural HA were more hydrophilic than those HLA obtained from HTC, which is indicated by the decrease in the A/OA index (Table 3). All of these factors, such as presence or absence, greater incorporation of functional groups or not in humic and humic-like acids, influence the binding properties with metal ions.

Table 3 3.2 Interaction of humic acids and humic-like acids with Cu(II)

Fluorescence quenching

The interactions of HA and HLA with Cu(II) ions were evaluated by fluorescence quenching; the concentration of each humic extract was 10 mg L -1 . Other authors have used this concentration value of humic extract in experiments for the same purpose: to avoid inner filters [START_REF] Guo | Characterizing the fluorescent properties and copper complexation of dissolved organic matter in saline-alkali soils using fluorescence excitation-emission matrix and parallel factor analysis[END_REF][START_REF] Yuan | Detection of Copper (II) and Cadmium (II) binding to dissolved organic matter from macrophyte decomposition by fluorescence excitation-emission matrix spectra combined with parallel factor analysis[END_REF]. The quenching results were combined with the PARAFAC multivariate statistical tool, in which the complex signal of the fluorescence spectra was decomposed into simple and independent components.

Thus, a three-component model was found for HA quenching experiments with a good CORCONDIA value (94.75%). The components are shown in Figure 3.

Component 1 (Figure 3a) had a main peak at [λ Ex 300 nm/λ Em 475 nm], which is typical of humic-like substances. Component 2 (Figure 3b) had a secondary peak at [λ Ex 275 nm/λ Em 530 nm], which is associated with low-energy peaks known to be typically linked to terrestrial HA [START_REF] Matthews | Excitationemission-matrix fluorescence spectroscopy applied to humic acid bands in coral reefs[END_REF][START_REF] Stedmon | Tracing dissolved organic matter in aquatic environments using a new approach to fluorescence spectroscopy[END_REF], and a main peak at [λ Ex 475 nm/λ Em 530 nm], which is typical of lignin-derived terrestrial HA. In addition, component 1 showed a blue shift for fluorescence emission intensity, whereas component 2 demonstrated a red shift, characterizing the presence of simple and complex structures, respectively [START_REF] Senesi | Humic acids in the first generation of EUROSOILS[END_REF]. Conversely, component 3 (Figure 3c) showed no variation in its contribution during the quenching experiments, which is likely related to a noise factor. Therefore, this component was not considered in the discussion.

A three-component model was also found for the interaction with HLA, with a CORCONDIA value of 97.46%; these components are also shown in Figure 3.

Component 1 (Figure 3d) showed a main and a secondary peak at [λ Ex 260 nm/λ Em 425 nm] and [λ Ex 340 nm/λ Em 425 nm], respectively. Both components are characteristic of humic substances from terrestrial environments [START_REF] Coble | Characterization of marine and terrestrial DOM in the seawater using exciting-emission matrix[END_REF][START_REF] Matthews | Excitationemission-matrix fluorescence spectroscopy applied to humic acid bands in coral reefs[END_REF][START_REF] Stedmon | Tracing dissolved organic matter in aquatic environments using a new approach to fluorescence spectroscopy[END_REF]. Component 2 (Figure 3e) had a main peak at [λ Ex 275 nm/λ Em 510 nm] and a secondary peak at [λ Ex 400 nm/λ Em 510 nm]. The main peak was classified as typically humic-like, and the secondary peak refers to soil fulvic acid.

Component 3 (Figure 3f) showed no fluorescence variation; therefore, similar to that for HA, it was not considered in the discussion. Figure 4a shows the scores of components 1 and 2 regarding the interaction of TM I HA with Cu(II) ions, and Figure 4b shows those regarding the interaction of HH 2 SO 4 HLA.

All three HA samples showed similar behavior; additional graphs are given in the supplementary material (Figure S2). The four HLA samples also showed similarly behavior, as shown in the supplementary material (Figure S3).

Figure 4

The fluorescence quenching curve for the first component of HA and Cu(II) (Figure 4a) showed a fluorescence intensity decay of approximately 85.6%, and the second component had a value of 92.5%. This significant decay in fluorescence contribution is attributed to the formation of a complex in which the fluorescence quantum yield changed [START_REF] Mounier | Copper complexing properties of dissolved organic matter: PARAFAC treatment of fluorescence quenching[END_REF][START_REF] Tadini | Modeling the quenching of fluorescence from organic matter in Amazonian soils[END_REF]. Moreover, the interaction with Cu(II) was shown to influence the optical properties of organic matter.

For the interaction between HLA and Cu(II) (Figure 4b), the fluorescence intensity decay for the first component remained at approximately 67.3%, whereas that for the second was approximately 58.2%. The decrease in fluorescence intensity of the HA components appeared at the first concentrations of Cu(II). For the HLA components, an increase in copper concentration was necessary for better visualization of the quenching phenomenon.

Optimization of the data using the 1:1 (ligand:metal) complexation model proposed by [START_REF] Ryan | Fluorescence Quenching Titration for Determination of Complexing Capacities and Stability Constants of Fulvic Acid[END_REF] enables evaluation of the equilibrium parameters, particularly CL and K, of which CL is the concentration of binding sites capable of forming a complex and K is the conditional stability constant of the complex formed.

The equilibrium parameters for the HA and HLA experiments, including CL, log K, CC, and the bias values, are given in Table 4. To evaluate the 1:1 complexation model, bias was used as an optimization parameter. Bias is the sum of the absolute value of the difference between the experimental and calculated fluorescence logarithm intensity. In this study, the bias values ranged from 0.27 to 4.18, which are lower than those found in the literature [START_REF] Tadini | Modeling the quenching of fluorescence from organic matter in Amazonian soils[END_REF]. Therefore, the experimental data fit well in the theoretical model (Table 4). The titration curves of the theoretical and experimental values for components 1 and 2 of HA and components 1 and 2 of HLA with Cu(II) can be found in the supplementary material (Figures S4 andS5).

Table 4

For HA components 1 and 2, the log K values remained in the ranges of 5.30-5.41 and 5.58-5.60, respectively; for HLA components 1 and 2, these values were in the ranges of 4.88-4.90 and 5.06-5.09, respectively. The HA components showed higher values for log K and both blue and red shifts in the fluorescence emission wavelengths.

In addition, the fluorophores exhibited different chemical structures and compounds with good binding capacities, which is confirmed by Figure 3a, b. The HLA components showed similar values for log K compared with HA and values either similar to or higher than those found in the literature for other types of HA [START_REF] Fuentes | Main binding sites involved in Fe(III) and Cu(II) complexation in humic-based structures[END_REF][START_REF] Plaza | Copper(II) complexation of humic acids from the first generation of EUROSOILS by total luminescence spectroscopy[END_REF][START_REF] Tadini | Modeling the quenching of fluorescence from organic matter in Amazonian soils[END_REF]. Blue and red shifts were also observed (Figure 3d, e); therefore, the fluorophores have different chemical structures and are weakly bonded compared to HA.

It is noteworthy that TM, which occurs in the Amazon, is different from common soils and has high fertility because of the carbon stock and long-term nutrients.

This fact was demonstrated by good interaction with the metal ion (log K 5.30-5.60).

HLA showed higher log K values than those found in Spodosols, which also occur in the Amazon region (log K 3.79-5.32) [START_REF] Tadini | Modeling the quenching of fluorescence from organic matter in Amazonian soils[END_REF], or peat and leonardite (log K 4.58-5.10) [START_REF] Fuentes | Main binding sites involved in Fe(III) and Cu(II) complexation in humic-based structures[END_REF]. This indicates that the organic matter extracted from hydrochars can also interact satisfactorily with Cu(II) ions to form complexes.

Additionally, by using the CL values, it was also possible to determine the CC for all components 1 and 2 from HA and HLA. This parameter was obtained by dividing the CL values by the carbon content. CC represents the concentration of binding sites per gram of carbon available to interact with Cu(II) ions. Table 4 also shows the CC values for HA and HLA components 1 and 2.

Both HLA components 1 and 2 showed higher concentrations of binding sites per gram of carbon for interaction with Cu(II) ions than those of HA; and the highest value was observed for RHH 2 SO 4 HLA. Regarding log K, the CC values were also higher than those found in the literature for HA from soils, peat, or leonardite [START_REF] Fuentes | Main binding sites involved in Fe(III) and Cu(II) complexation in humic-based structures[END_REF][START_REF] Plaza | Copper(II) complexation of humic acids from the first generation of EUROSOILS by total luminescence spectroscopy[END_REF][START_REF] Tadini | Modeling the quenching of fluorescence from organic matter in Amazonian soils[END_REF]. [START_REF] Tadini | Modeling the quenching of fluorescence from organic matter in Amazonian soils[END_REF] determined the CC of components by fluorescence quenching for HA extracted from Amazonian Spodosols with Cu(II) ions and found CC values ranging from 0.01-0.13 mmol of Cu g - 1 C. The values found in this study for HA extracted from TM were higher, indicating both a larger amount of complexing sites and an affinity of organic matter with the metallic ion. The same result was observed for HLA, indicating that if applied to the soil, it could help in the complexation reactions.

Previous research indicates that the high stability constants of HA are associated with increased oxygen-containing groups such as -COOH and -OH as well as π-π bonds in the C=C of aromatic rings [START_REF] Guo | Study on the spectral and Cu (II) binding characteristics of DOM leached from soils and lake sediments in the Hetao region[END_REF][START_REF] Guo | Characterizing the fluorescent properties and copper complexation of dissolved organic matter in saline-alkali soils using fluorescence excitation-emission matrix and parallel factor analysis[END_REF]. These functional groups were observed for HA and HLA by using both FTIR and 13 C NMR techniques. The results

show that the predominance of conjugate and aromatic structures as well as oxygencontaining groups are responsible for HA complexation, and for HLA, both aromatic and aliphatic groups are responsible. Pearson established the concept of chemical bonding regarding the affinity of different metals to different ligands [START_REF] Pearson | Hard and Soft Acids and Bases[END_REF]. It was reported that chemical species can be classified as "hard" and "soft" or "borderline" acids and bases and that the concepts of "hardness" and "softness" can help to interpret the mechanism of complexation. Soft acids tend to have preferential interaction by soft bases, whereas hard acids tend to bind with hard bases; borderline acids form considerably stable bonds with both hard and soft bases, although it is sometimes difficult to determine the preferential order for bond formation. These concepts partly explain the stability of chemical compounds [START_REF] Pearson | Hard and Soft Acids and Bases[END_REF].

As observed in the 13 C NMR spectra and in the H/C, O/C, and C/N atomic ratios, HA and HLA showed similar ARM, although HA showed a higher percentage of oxygenated groups and nitrogen in its structure. The Cu(II) ion used in this study is a borderline acid and interacts with both hard bases such as oxygen and soft bases such as nitrogen; thus, HA had a stronger interaction with Cu(II) because of their molecular structures. [START_REF] Lu | Characterization of copper complexation with natural dissolved organic matter (DOM) -Link to acidic moieties of DOM and competition by Ca and Mg[END_REF] found that the interaction of DOM with Cu(II) ions is attributed mostly to phenol OH groups, and [START_REF] Hernández | Detection of Copper(II) and zinc(II) binding to humic acids from pig slurry and amended soils by fluorescence spectroscopy[END_REF] showed a positive and significant correlation with carboxylic acid and phenols. Moreover, the presence of large amounts of functional groups and π-π bonds of aromatic rings should confer a greater complexation capacity for HA from TM; however, this did not occur potentially owing to the different molecular arrangements. Further, it is possible that the functional groups and aromatic carbons in HA structures were already occupied by another metal or more condensed aromatic rings. All of these factors can affect the behavior of Cu(II) ions in both soils and hydrochar extracts. Conversely, HLA showed more binding sites available for interaction considering the amount of carbon in the sample, and thus greater complexation capacity.

Time-resolved fluorescence

In addition, fluorescence quenching can be induced by two mechanisms. The first is dynamic quenching, which is caused by collision with a quencher and occurs during the lifetime of the excited state, affecting the fluorescence lifetime. The second is static quenching, which occurs when a ground-state non-fluorescent complex is formed, and the lifetimes of the fluorescent ligand and complex are unaffected [START_REF] Lakowics | Principles of fluorescence spectroscopy[END_REF].

Therefore, TRF is the best method for distinguishing whether static or dynamic quenching occurs during the interaction of organic matter with copper.

Figure 5a and 5b show the fluorescence average lifetime distribution of HA and HLA complexes with copper, respectively. The deconvolution of TRF results in the HA complexes show that the decay was mono-exponential (τ 1 ) decay containing only one lifetime. The average lifetime values observed were shorter, at approximately 5-6 ns, which is associated with the simple structure of HA. This average lifetime can be attributed to component 1 (Figure 3a) observed in PARAFAC. The average lifetime of component 2, with emission at 530 nm (Figure 3b) did not appear in the results because the emission spectra in TRF reached only 510 nm owing to laser leakage at 532 nm.

The best fit for HLA complexes showed a bi-exponential decay (τ 1 and τ 2 ), the average lifetimes of which were approximately 4 ns and 14 ns, respectively. These results fit well with the PARAFAC for HLA, which showed two components, one with a simple structure (component 1) and the other with a complex structure (component 2), reflecting lower and higher lifetimes, respectively. The first average lifetime τ 1 was almost the same as that for HA (Figure 3d). Lifetime τ 2 can be associated with component 2 (Figure 3e), which has a more complex fluorophore structure.

Figure 5

The average lifetime values were determined to be independent of the copper concentration for HA and HLA interaction (Figure 5). This suggests that the mechanism of Cu(II) quenching by fluorescence occurs with a non-fluorescent ground-state complex, i.e., the complexation occurs by static quenching. Nouhi (2018) also reported static quenching and similar lifetime values for interaction between Cu(II) and Eu(III)

with HA extracted from the Saint Lawrence Estuary in Canada [START_REF] Nouhi | Caractérisation Spectrale et Temporelle par Quenching de Fluorescence des Interactions Matière Organique-Eléments Métalliques[END_REF].

In summary, this study demonstrated the potential of fluorescence quenching and TRF for evaluating the interaction between metallic species and organic matter, which provides insight into the interaction mechanism. No previous studies related to fluorescence quenching and lifetime measurements of HA extracted from Amazonian TM and HLA from hydrochars with metallic species are available for comparison with the results achieved here.

Conclusion

HLA were obtained by NaOH aqueous solution extraction from hydrochar produced by the HTC process using sugarcane industry by-products. These HLA showed greater complexation capacity with Cu(II) ions than the HA extracted from TM.

In addition, similar values for stability constants for both HA and HLA were observed.

The interaction with Cu(II) in HLA occurs by both aromatic and aliphatic moieties,

showing that π-π interaction and oxygen groups are not the only factors that play important roles in the CC. Furthermore, the TRF for the interaction of metallic species and organic matter showed that the main interaction mechanism was static quenching.

These results show that HLA extracted from hydrochars can be an efficient approach for interacting with metals and can be a new tool for remediating areas contaminated by metals.

  Soil sampling was performed following the 2000 United States Environmental Protection Agency (EPA) method (US EPA, 2000). For this study, three different areas of TM were selected. The first TM soil was obtained from an open area region containing native vegetation and low vegetation cover (TM I; 3°04′05.45″ S and 58°33′51.11″ W); the second was taken from a closed area region containing native vegetation (TM II; 3°04′05.17″ S and 58°34′11.68″ W); and the third was collected near cultivated areas (TM III; 3°03′59.15″ S and 58°27′04.64″ W). All areas are located near Itacoatiara city in the Amazonas State (Brazil). (SISBio collection was authorized by the Chico Mendes Institute for Biodiversity Conservation, n ° 50042 -2, and registered in the National System for the Management of Genetic Heritage and Associated

  AVANCE 400 MHz spectrometer (Bruker, Billerica, Massachusetts, USA) equipped with a magic angle spin (MAS) probe. A zircon cylindrical rotor with a diameter of 4.0 mm was packed with 80 mg of sample. The spectra were acquired by using a rotor rotation frequency of 10 kHz, recycle time of 1 s, and 5,000 scans. The spectral areas were integrated and divided into seven chemical shifts: C-alkyl (0-45 ppm), Cmethoxyl (45-60 ppm), O-alkyl-C (60-110 ppm), aryl-C (110-145 ppm), phenol-C (145-160 ppm), carboxyl-C (160-190 ppm), and carbonyl-C (190-220 ppm).

  13 C-CPMAS-NMR spectra of HA and HLA are shown in Figure 2. Both samples showed complex chemical compositions, with peaks related to aromatic and aliphatic carbons, substituted or not by heteroatoms, and different intensities. The following peaks were observed in all samples: 0-45 ppm, attributed to the presence of alkyl carbons of methyl and methylene groups; 45-60 ppm for carbons linked to methoxyl groups and nitrogen compounds (C-N); 60-110 ppm, indicating the presence of oxygen-substituted carbons and alkyl groups (O-alkyl); 110-145 ppm, attributed to the presence of aromatic groups; and 145-160 ppm for substituted aromatic carbons, 160-190 ppm for carbonyl groups, and 190-220 ppm for carboxylic groups in the structures of HA and HLA

Figure 3

 3 Figure 3

*

  natural humic acids; ** humic-like acids; # not realized / ARM = aromaticity index [(110 -160) / Σ (0 -45) + (60 -110)]; A/OA = alkyl/O-alkyl index (0 -45) / (60 -110); HB/HI = hydrophobicity index = [Σ (0 -45) + (110-160) / Σ (45 -60) + (60 -110) + (160 -190)].

Fig. 1 .

 1 Fig.1. FTIR spectra for HLA isolated from hydrochar (a) and HA isolated from soil (b).

Fig. 2 .

 2 Figure 1 Click here to download Figure: Figure 1.doc

Fig. 3 .

 3 Figure 2 Click here to download Figure: Figure 2.doc

Fig. 4 .

 4 Figure 3 Click here to download Figure: Figure 3.doc

Fig. 5 .

 5 Figure 4 Click here to download Figure: Figure 4.doc

  Figure 5 Click here to download Figure: Figure 5.doc

Table 1 .

 1 Biomass, additives, temperature, and reaction time used for hydrothermal carbonization and recarbonization.

	Carbonization/			Additive
		Reaction	Biomass		Temp.	Time
	Recarbonization			(4% v/v)
	HH 3 PO 4	1	Vinasse Sugarcane bagasse	Phosphoric Acid	230 °C	13 h
	HH 2 SO 4	2	Vinasse Sugarcane bagasse	Sulfuric Acid	230 °C	13 h
			Process water
	RHH 3 PO 4	3	(from reaction1) Sugarcane	Phosphoric Acid	230 °C	13 h
			bagasse
			Process water
	RHH 2 SO 4	4	(from reaction 2) Sugarcane	Sulfuric Acid	230 °C	13 h
			bagasse

Table 1 Click here to download Table: Table 1.doc

 1

Table 2 .

 2 Carbon (C), hydrogen (H), nitrogen (N), oxygen (O) and sulfur (S) contents; O/C, H/C, and C/N atomic ratios; and absorbance ratios E 465 /E 665 and E 270 /E 407 for HA and HLA samples.

	HA/HLA	C	H	N	O	S	O/C	H/C	C/N	E 465 /E 665 E 270 /E 407
		(%)	(%)	(%)	(%)	(%)
	TM I*	51.24	3.80	3.41	40.61	0.94	0.59	0.89	17.80	3.92	2.81
	TM II*	39.62	4.05	4.04	51.37	0.92	0.97	1.23	11.38	4.33	3.43
	TM III*	50.22	3.69	2.97	42.14	0.98	0.63	0.88	19.95	4.18	2.90
	HH 2 SO 4**	51.95	5.25	1.47	39.88	1.45	0.57	1.21	43.30	5.71	4.06
	HH 3 PO 4**	62.27	5.62	2.60	28.47	1.04	0.34	1.08	28.83	5.33	3.93
	RHH 2 SO 4**	48.90	4.46	1.20	37.99	7.45	0.58	1.09	51.00	3.50	3.33
	RHH 3 PO 4**	53.54	4.82	1.83	35.92	0.89	0.50	1.08	34.31	4.67	3.19

* natural humic acids; ** humic-like acids

Table 2 Click here to download Table: Table 2.doc

 2

Table 3 .

 3 Relative value (% of total area) and indexes obtained from 13 C-CPMAS-NMR spectra of HA and HLA samples.

	13 C NMR region (ppm)

Table 3 Click here to download Table: Table 3.docTable 4 .

 34 Log K, CL, CC, and bias values for components 1 and 2 regarding the interaction of HA and HLA with Cu(II) ions.

	Component 1
	HA/HLA

* natural humic acids; ** humic-like acids

Table 4 Click here to download Table: Table 4.doc

 4

Acknowledgments

This work was supported by the São Paulo Research Foundation (FAPESP) (grants 15/22954-1, 17/26718-6, and 18/15733-7). J.V.S acknowledges a scholarship from FAPESP (grants 17/05408-9 and 18/09914-9). O.P.F acknowledges financial support from FUNCAP (PRONEX PR2-0101-00006.01.00/15). The authors thank Dr.

Maurício Boscolo for offering assistance with the FTIR analysis (process 2017/13230-5) and Dr. Isabella Constatino and Dr. Fabiana Paschoal for their assistance with soil sampling.


Jean-Lou Justine
email: justine@mnhn.fr

Leigh Winsor

First Record of Presence of the Invasive Land Flatworm Platydemus manokwari (Platyhelminthes, Geoplanidae) in Guadeloupe

The land flatworm Platydemus manokwari (Platyhelminthes, Geoplanidae) is recorded for the first time from the island of Guadeloupe (French West Indies) in the Caribbean arc.

Photographs and records were obtained from citizen science and ranged from the end of 2018 to 2020; specimens were deposited in the collections of the Muséum National d'Histoire Naturelle in Paris, France. This is the first record in Guadeloupe and the second for an island in the Caribbean, after Porto Rico.

Introduction

The land flatworm Platydemus manokwari is a highly invasive species, listed among the "100 World's Worst Invader Alien Species" [START_REF] Lowe | Invasive Species Specialist Group (ISSG) a specialist group of the Species Survival Commission (SSC) of the World Conservation Union (IUCN)[END_REF]. Originally described from a locality in Papua New Guinea [START_REF] De Beauchamp | Platydemus manokwari n. sp., planaire terrestre de la Nouvelle-Guinée Hollandaise[END_REF], it has been recorded since in many islands of the Pacific Ocean, Australia, and, more recently, in Porto Rico in the Antilles, Florida in mainland USA [START_REF] Winsor | The invasive land planarian Platydemus manokwari (Platyhelminthes, Geoplanidae): records from six new localities, including the first in the USA[END_REF][START_REF] Winsor | The invasive New Guinea flatworm Platydemus manokwari in France, the first record for Europe: time for action is now[END_REF], and mainland Asia in Hong Kong [START_REF] Hu | First record of the New Guinea flatworm Platydemus manokwari (Platyhelminthes, Geoplanidae) as an alien species in Hong Kong Island, China[END_REF] and Thailand [START_REF] Chaisiri | Distribution of the newly invasive New Guinea flatworm Platydemus manokwari (Platyhelminthes: Geoplanidae) in Thailand and its potential role as a paratenic host carrying Angiostrongylus malaysiensis larvae[END_REF]. We report here its presence in Guadeloupe, an island of the Caribbean arc.

Material and Methods

Records were obtained from Citizen Science; photographs were received from various nonprofessionals, and specimens were solicited and received. Specimens were killed in hot water, stored in ethanol and sent to the laboratory. The species was identified by the examination of photographs of living animals and preserved specimens

Results

Discussion

Platydemus manokwari is an invasive species and a threat to the biodiversity of land animals, including molluscs [START_REF] Gerlach | Predation by invasive Platydemus manokwari flatworms: a laboratory study[END_REF][START_REF] Iwai | Prey-tracking behavior in the invasive terrestrial planarian Platydemus manokwari (Platyhelminthes, Tricladida)[END_REF][START_REF] Ohbayashi | Food habit of Platydemus manokwari De Beauchamp, 1962 (Tricladida: Terricola: Rhynchodemidae), known as a predatory flkatworm of land snails in the Ogasawara Islands, Japan[END_REF] The complete mitogenome of the species has also been sequenced [START_REF] Gastineau | Complete mitogenome of the invasive land flatworm Platydemus manokwari[END_REF].

We expect to provide a molecular account of the specimens from Guadeloupe as soon as the end of the covid-19 lockdown will allow it.

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Platydemus manokwari, photographed in Saint François, Guadeloupe, 7 January 2019; group of flatworms eating a dead lizard. Photograph by François Ferrasson, CC-BY.

Table 1 .

 1 Records of Platydemus manokwari in Guadeloupe. Names of non-professionals who communicated information are given as initials. Observations are from gardens, not wild areas. One observer (MN) also recorded the invasive species Bipalium kewense in the same garden in Baie Mahault where he found Platydemus manokwari. In addition to the information in Table1, Observer FF (François Ferrasson) mentioned that the species was present in his garden in Saint François since the end of 2018 and that he noticed a drastic decrease in the number of Giant African Snails (Achatina fulica) in his garden. He also sent a photograph showing several Platydemus manokwari eating a dead lizard (unidentified Anole) (Figure2).

	Date	Locality	Details	Observer
	07-01-2019	Saint François	Photo only (Figures 1-2)	FF
	09-01-2019	Saint François	Photo only	FF
	08-11-2019	Saint François	Photo and 5 specimens, MNHN	FF
			JL358
	01-01-2020	Baie Mahault	With Bipalium kewense	MN
	06-01-2020	Baie Mahault	Photo only	VD
	05-03-2020	Bouillante	2 specimens	FF

Several records were received (Table

1

). Records based on photographs or specimens were from January 2019 to March 2020, in three localities, Saint François (Figure

1

), Baie Mahault and Bouillante.

  now invaded a large part of Florida and also Texas. In the Antilles Arc, it has been recorded only in Porto Rico[START_REF] Winsor | The invasive land planarian Platydemus manokwari (Platyhelminthes, Geoplanidae): records from six new localities, including the first in the USA[END_REF] and Guadeloupe is thus the second island with a record. It is likely that the species is present in other islands of the Antilles Arc, and there is a probable report from Jamaica[START_REF] Kostik | Interesting finds in Jamaica -Report from the field 2018[END_REF]). However, we received various records of land flatworms in Martinique, another French island close to Guadeloupe: none (yet) was

	2015).	Information	available	from	iNaturalist
	(https://www.inaturalist.org/observations?taxon_id=199342, date: 27-04-2020) suggests
	that it has Platydemus manokwari.
	A study of the COI gene sequence of Platydemus manokwari has shown that two haplotypes
	could be differentiated, but that only one had invaded most of the world (Justine et al. 2015).

; Sugiura 2010; Sugiura & Yamaura 2008). It has been recorded in many islands of the Pacific, and more recently in France, but only in a hothouse (Justine et al. 2014) and was not recorded later in the field (Justine et al. 2015). In North America, it has been recorded in Florida (Justine et al.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 May 2020 doi:10.20944/preprints202005.0023.v1

Acknowledgements

We thank the non-professional observers who sent us photographs and specimens. François Ferrasson kindly signed a permission to have his photographs published under a CC-BY licence. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 May 2020 doi:10.20944/preprints202005.0023.v1