Humic extracts of hydrochar and Amazonian Dark Earth: Molecular characteristics and effects on maize seed germination

Journal Pre-proofs Humic extracts of hydrochar and Amazonian Dark Earth: Molecular characteristics and effects on maize seed germination Lucas Raimundo Bento, Camila Almeida Melo, Odair Pastor Ferreira, Altair Benedito Moreira, Stéphane Mounier, Alessandro Piccolo, Riccardo Spaccini, Márcia Cristina Bisinoti PII: DOI: Reference:

S0048-9697(19)34992-7 https://doi.org/10.1016/j.scitotenv.2019.135000 STOTEN 135000

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Science of the Total Environment

Received Date: Revised Date: Accepted Date:

19 August 2019 14 October 2019 14 October 2019

Please cite this article as: L. Raimundo Bento, C. Almeida Melo, O. Pastor Ferreira, A. Benedito Moreira, S. Mounier, A. Piccolo, R. Spaccini, M. Cristina Bisinoti, Humic extracts of hydrochar and Amazonian Dark Earth: Molecular characteristics and effects on maize seed germination, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.135000

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Humic extracts of hydrochar and Amazonian Dark Earth: Molecular characteristics and effects on maize seed germination Lucas Raimundo Bentoa, Camila Almeida Meloa, Odair Pastor Ferreirab, Altair Benedito Moreiraa, Stéphane Mounierc, Alessandro Piccolod, Riccardo Spaccinid, Márcia Cristina Bisinotia* a

São Paulo State University (UNESP), Institute of Biosciences, Humanities and Exact

Sciences, São José do Rio Preto, Brazil b

Laboratório de Materiais Funcionais Avançados (LaMFA), Departamento de Física,

Universidade Federal do Ceará, P.O. Box 6030, 60455-900 Fortaleza, Ceará, Brazil c

Laboratoire MIO, CNRS-IRD-Université de Toulon-AMU – CS 60584, 83041

TOULON, CEDEX 9, France d

Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per

l’Ambiente, l’Agroalimentare ed i Nuovi Materiali (CERMANU), Università di Napoli Federico II, Via Università, 100, 80055 Portici, Italy

*

Corresponding author

Name: Márcia Cristina Bisinoti E-mail: [email protected] Address: Laboratório de Estudos em Ciências Ambientais, Departamento de Química e Ciências Ambientais, Universidade Estadual Paulista, Cristóvão Colombo, 2265, São José do Rio Preto, São Paulo State, Brazil, 15054-000.

1

Abstract Inspired by the presence of anthropogenic organic matter in highly fertile Amazonian Dark Earth (ADE), which is attributed to the transformation of organic matter over thousands of years, we explored hydrothermal carbonization as an alternative for humiclike substances (HLS) production. Hydrothermal carbonization of sugarcane industry byproducts (bagasse and vinasse) in the presence and absence of H3PO4 afforded HLS, which were isolated and compared with humic substances (HS) isolated from ADE in terms of molecular composition and maize seed germination activity. HLS isolated from sugarcane bagasse hydrochar produced in the presence or absence of H3PO4 comprised both hydrophobic and hydrophilic moieties, differing from other HLS mainly in terms of phenolic content, while HLS isolated from vinasse hydrochar featured hydrophobic structures mainly comprising aliphatic moieties. Compared to that of HLS, the structure of soil-derived HS reflected an increased contribution of fresh organic matter input and, hence, featured a higher content of O–alkyl moieties. HLS derived from lignocellulosic biomass were rich in phenolics and promoted maize seed germination more effectively than HLS comprising alkyl moieties. Thus, HLS isolated from bagasse hydrochar had the highest bioactivity, as the presence of amphiphilic moieties therein seemed to facilitate the release of bioactive molecules from supramolecular structures and stimulate seed germination. Based on the above results, the hydrothermal carbonization of lignocellulosic biomass was concluded to be a viable method of producing amphiphilic HLS for use as plant growth promoters. Keywords: hydrothermal carbonization, biochar, sugarcane bagasse, vinasse, germination bioassay

2

1

Introduction

The effective utilization of organic residues and byproducts of agro-industrial activities in the production chain has recently attracted much attention. International organizations strongly support the reutilization of organic biomass and the recycling of bio-waste into organic fertilizers to transform waste management problems into economic opportunities and thus improve environmental resilience and circulate the agricultural sector economy (FAO, 2017). Therefore, the processing of local biomasses into innovative products, e.g., through pyrolysis and hydrothermal carbonization (HTC), may provide an incentive to widen the environmental and economic valorization of organic materials for the sustainable management of agro-ecosystems. Pyrolysis refers to thermochemical treatment of dried biomass at high temperatures in an oxygendeficient environment to afford char (pyrochar) rich in carbon (Kambo and Dutta, 2015). In contrast to pyrolysis, HTC is applied to wet biomass, as the involved reactions occur in an aqueous medium, and is performed at a lower temperature to afford hydrochar (Kambo and Dutta, 2015; Reza et al., 2015). Biochar, denoting hydrochar and/or pyrochar intentionally used for agricultural purposes, has been used to improve soil water holding capacity (Bento et al., 2019; Mangrich et al., 2015; Narzari et al., 2017; Zheng et al., 2013), cation exchange capacity (Mukherjee et al., 2011), nutrient release properties (Bento et al., 2019; Mukherjee and Zimmerman, 2013), metal/organic contaminant adsorption capacity (Pratiwi et al., 2016; Zhu et al., 2019), and carbon storage ability. Hydrochars can be produced from a wide range of raw materials and therefore feature diverse applications such as liquid/gas adsorption, heterogeneous catalysis, energy storage in batteries, and supercapacitor fabrication (Reza et al., 2014a; Titirici et al., 2012; Wang et al., 2018). Both products of HTC, namely process water and hydrochar, 3

have been evaluated in terms of their effects on germination and plant growth (Bargmann et al., 2013; Fregolente et al., 2018; Melo et al., 2019, 2018), which depend on hydrothermal treatment conditions, biomass, and temperature. Previously, sugarcane bagasse–vinasse mixtures were subjected to HTC at different temperatures (150–230 °C) in the presence of various additives with the goal of increasing the carbonization degree and the nutrient incorporation ability of the resulting hydrochars (Fregolente et al., 2019; Melo et al., 2017; Silva et al., 2017). These hydrochars have been shown to well retain water and release nutrients, additionally being capable of stocking a part of carbon released in an oxisol during a short soil-column experiment (Bento et al., 2019). The use of biochar for soil amendment was inspired by the presence of anthropogenic organic matter in Amazonian Dark Earth (ADE), which is ascribed to the waste management activities of Amazonian pre-Colombian populations. The practice of burning wastes such as plant- and animalderived residues generated char- and nutrient-containing soils with an anthropogenic horizon rich in organic matter (Clement et al., 2015; Fraser et al., 2011; Glaser and Birk, 2012; Kern et al., 2019; Novotny et al., 2009). This organic matter mainly comprises aromatics, which increase soil resilience and cation exchange capacity. Such soils differ from the surrounding soil (SR), which is poor in organic matter, weathered, and acidic (Kern et al., 2019; Novotny et al., 2015). Thus, supplementation of soil with biochar can be used to mimic soils containing anthropogenic organic matter. The isolable part of organic matter naturally present in the environment is denoted as humic substances (HS), which comprise molecules (<1000 Da) held together by hydrophobic interactions (Van der Waals, π-π, CH-π) and hydrogen bonds to afford a large supramolecular structure (Drosos et al., 2018; Nebbioso and Piccolo, 2011; Piccolo et al., 2018). The term humic-like substances (HLS) refers to the extracts of 4

different organic materials that have not undergone the natural process of in-soil humification. Both HS and HLS can facilitate plant growth by exerting hormone-like effects and enhance the biological fertility of soil. In addition, it interacts with organic and inorganic pollutants (Canellas and Olivares, 2014; Huang et al., 2018; Monda et al., 2017; Piccolo et al., 2019; Savy et al., 2016; Spaccini et al., 2019; Sun et al., 2017). As chars produced by both HTC and pyrolysis have an extractable fraction (Huang et al., 2018; Novotny et al., 2009), HTC may be a new technological means of producing HS similar to those found in soil for use as plant growth promoters, since under natural condition requires a quite long time (Yang et al., 2019). The HTC with autogenous pressures, low oxygen environment achieves higher carbon yield different from a natural process that involves biotic and abiotic reactions in an open environment (Yang et al., 2019). In turn, the extraction residue can be used in well-established applications, e.g., as a soil remediation agent, solid fuel (Bian et al., 2019; Lee et al., 2019) or an adsorbent (Qian et al., 2018). A profound knowledge of the molecular composition of humic extracts is required to shed light on their structure-activity relationships and enable practical applications. Hence, this study aimed to (i) evaluate the HTC as new way to produce bioactive HLS through the molecular features and their relationship with maize seed germination and (ii) compare these properties with those of HS isolated from ADE and SR soil samples as reference material. 2

Material and Methods

2.1

Hydrochar preparation

Hydrochar was prepared following Melo et al. (2017) and Silva et al. (2017) using sugarcane bagasse (HB), sugarcane bagasse with phosphoric acid (HBH3PO4), vinasse 5

(HV), vinasse with phosphoric acid (HVH3PO4), sugarcane bagasse and vinasse mixture (HBV) and sugarcane bagasse and vinasse mixture with phosphoric acid (HBVH3PO4). The biomass mixture was used to incorporated nutrients from vinasse in the hydrochar. The phosphoric acid used was concentrated H3PO4 (65%, w/v) and 4% was added in relation to the total volume of reaction (400 mL). The reaction was conducted in a stainless steel coated Teflon® closed reactor (600 mL maximum capacity) and placed in a muƫe furnace at a steady temperature of 230 ± 10 °C for 13 h, with self-generated pressures. The reactor was then withdrawn and cooled in an ice bath to stop the reaction. The hydrochar was separated by filtration and washed with deionized water until pH was constant (indicating that all the acidic from the process water were eliminated). Then, the hydrochar was dried at 50 °C in an oven until a constant mass was achieved. A brief summary of raw material composition is provided in the Supplementary Material and in the Table S1. 2.2

Alkaline-extractable organic matter

Although HLS do not have a history of having undergone humification, they exhibit some characteristics of natural organic matter. Herein, HLS were extracted from six hydrochars produced with different matrices: sugarcane bagasse (HLS-HB), sugarcane bagasse + phosphoric acid (HLS-HBH3PO4), vinasse (HLS-HV), vinasse + phosphoric acid (HLS-HVH3PO4), sugarcane bagasse + vinasse (HLS-HBV), and sugarcane bagasse + vinasse + phosphoric acid (HLS-HBVH3PO4). HLS isolation was conducted in 100 g of each hydrochar to 1 L of solution (0.1 M NaOH), under nitrogen using four 4-h extraction procedures for each hydrochar, until the final extract has a lighter color in relation to the first extracts as described by Jindo et al. (2016). The soluble fraction was separated by centrifugation 10,000 g for 15 min at 10 °C. The extracts of each hydrochar were combined, dialyzed (Fisherbrand, molecular weight cut-off = 6,000– 6

8,000 Da) in deionized water (18.0 MΩ cm) until constant conductivity, lyophilized, and analyzed. In this study the humic extracts were not fractionated into humic-like and fulvic-like, both fraction were studied together. HS were isolated from ADE and SR for comparison using an identical procedure. A description of soil origin is given in the Supplementary Material. 2.3

Characterization of HS and HLS

2.3.1 Elemental composition Carbon, nitrogen, and hydrogen contents were determined using an elemental analyzer (Fisons, EA 1108, USA). Oxygen content was calculated from ash content and C, H, N contents as 100% − (C, H, N + ash). In turn, ash content was determined by thermogravimetric analysis (PerkinElmer TGA-4000) of 4-mg HLS samples in an atmosphere of oxygen. Samples were stabilized for 1 min at 30 °C and heated to 700 °C at 10 °C min−1. The residue obtained after thermal degradation was treated as ash. The contents of C, H, N, and O were recalculated on an ash-free basis. 2.3.2 Fluorescence and UV-vis analysis To avoid inner filter effects, we optimized HLS and HS concentrations used for fluorescence measurements (solutions with total organic carbon (TOC) concentrations of 5, 10, 15, 20, 25, 50, and 100 mg L−1 were studied). The optimal TOC concentration equaled 10 mg L−1, as determined by a TOC analyzer (Shimadzu, TOC-VCSN, Japan). Solutions were prepared in 0.05 M NaHCO3 with pH 8.0. Fluorescence measurements were performed using a luminescence spectrometer (Thermo Scientific, Lumina), and excitation–emission matrix (EEM) spectra were acquired for the emission wavelength range of 300–600 nm and an excitation wavelength range of 250–500 nm. The scan step and rate equaled 5 nm and 1,200 nm min−1, respectively. EEM graphics were processed by canonical polyadic/parallel factor (CP/PARAFAC) analysis (Bro, 1997; Luciani et 7

al., 2009; Mounier et al., 2011, 1999). UV-vis analysis was performed in a 1-cm-pathlength quartz cell using a UV–vis spectrophotometer (Shimadzu, UV-2600, Japan) for HLS and HS solutions (TOC = 10.0 mg L−1) in 0.05 M NaHCO3 with pH 8.0 to obtain absorbances at 465 and 665 nm and determine their ratio (E4/E6). 2.3.3 Solid-state 13C magic angle spin nuclear magnetic resonance spectroscopy (13C CPMAS NMR) HLS and HS were analyzed by 13C CPMAS NMR using a Bruker AV 300-MHz spectrometer (Bruker, Billerica, MA, USA) equipped with a magic angle spin probe with a bore of 4.0 mm. Spectra were obtained using 4,000 scans, a rotation frequency of 10 kHz, a recycle time of 1 s, a contact time of 1 ms, and an acquisition time of 20 ms. Zircon cylindrical rotors (diameter = 4.0 mm) were packed with ~80 mg of the sample and sealed with a Kel-F® cap. For the interpretation of 13C-CPMAS-NMR spectra, the overall chemical shift range was divided into the following main regions: alkyl-C (0–45 ppm); methoxyl-C and Nalkyl-C (45–60 ppm); O-alkyl-C (60–110 ppm); unsubstituted and alkyl-substituted aromatic-C (110–145 ppm); O-substituted aromatic-C (145-160 ppm); carboxyl-C (160–190 ppm) and carbonyl-C (190-220 ppm). The area of each functional group was divided by the sum of all spectral zones, to obtain a relative amount (MestreNova 6.2.0 software, Mestre-lab Research, 2010). In order to summarize the structural differences of humic extracts, dimensionless structural indexes were calculated from the relative amount of C distribution in the NMR spectra. Hydrophobicity, aromaticity, and alkyl indices were calculated as shown in Equations 1–3, respectively (Monda et al., 2018). The indexes were obtained by the integrated relative areas of NMR spectra: 1. Hydrophobicity index (HI) was obtained by the integrated relative areas of hydrophobic carbons (0–45 + 110–160 ppm) over hydrophilic carbons (60–110 + 60–110 + 160–190 8

ppm); 2. Aromaticity index (AI) was obtained by the integrated areas of aryl moieties (110–160) over alkyl moieties (0–45 + 60–110 ppm); 3. The alkyl index (Al/O-Al) was obtained by the integrated areas of alkyl-C (0–45 ppm) to those of O-Alkyl-C (60–110 ppm). ȭሺͲെͶͷ’’ ሻ൅ሺͳͳͲെͳ͸Ͳ’’ ሻ

Hydrophobicity index (HI) = >ȭሺͶͷെ͸Ͳ’’ ሻ൅ሺ͸ͲെͳͳͲ’’ ሻ൅ሺͳ͸ͲെͳͻͲ’’ ሻሿ, Aromaticity index (AI) =

ሺͳͳͲെͳ͸Ͳ’’ ሻ

,

>ȭሺͲെͶͷ’’ ሻ൅ሺ͸ͲെͳͳͲ’’ ሻሿ

Alkyl index (Al/O–Al) ൌ

ሺͲ–‘ Ͷͷ’’ ሻ

.

ሺ͸Ͳ–‘ ͳͳͲ’’ ሻ

(1) (2) (3)

2.3.4 Offline thermal methylation-assisted hydrolysis coupled with gas chromatography and mass spectrometry (TMAH-thermochemolysis GC-MS) TMAH-thermochemolysis GC-MS was performed on 100-mg humic extract samples. The samples were placed in a quartz tube, moistened with 300–600 µL tetramethylammonium hydroxide solution (25 wt% in methanol), naturally dried, introduced into a Pyrex tubular reactor (tube furnace model F21100), and heated at 400 °C. The products released during thermochemolysis were continuously transferred by a flow of helium into two successive chloroform traps. The thus obtained chloroform solutions were combined in a flask and concentrated in vacuo. The sample was recovered, placed in a vial, and blown dry with nitrogen. The vial was charged with 500 µL of chloroform, and the obtained solution was subjected to GC-MS analysis (PerkinElmer, Auto System XL) using an RTX-5MS WCOT capillary column (Restek, 30 m × 0.25 mm; film thickness = 0.25 μm) coupled to a PE Turbomass-Gold quadrupole mass spectrometer. The inlet temperature equaled 250 °C, and the initial temperature of 60 °C was held for 1 min, raised to 100 °C (at 7 °C min−1) and then to 300 °C (4 °C min−1), and held for 10 min. Helium was used as a carrier gas at 1.00 mL min−1 with a split ratio of 1:30. Mass spectra were obtained in EI mode (70 eV), and 9

scanning was performed in the range of m/z 45–650 for a cycle time of 0.2 s. The pyrolytic process was performed in duplicate for each sample, and chromatograms were submitted for interpretation according to the NIST database. 2.3.5 Germination test Germination tests were performed in a BOD-type germination chamber (MA 403, Marconi) for five days (120 h) at 25 °C in the dark. The experiments were conducted in Petri dishes (15 × 2 cm) in agar medium (Sigma Aldrich), as described by Fregolente et al. (2018). Maize seeds (Zea mays, Poaceae; 85% germination) purchased from Seminis were sterilized by soaking in 2.0% (w/v) NaClO for 30 min followed by rinsing and 12h soaking in water. The agar medium was prepared using an agar:deionized water ratio of 1:100 (w/v). Stock solutions of HLS and HS (100 mg each) in deionized water were subjected to TOC determination (Shimadzu, TOC-VCSN, Japan) and diluted to TOC levels of 1, 10, and 50 mg L−1 (pH was adjusted to 6.0 with 0.1 M KOH or 0.1 M HCl). For each concentration, 10 maize seeds were placed in each of the five Petri dishes. The same experiments were performed with HS isolated from soils. Control experiments were performed exactly as described above, in the absence of humic extracts. After experiment completion, the seedlings were photographed, and radicle, lateral seminal root, and coleoptile lengths were measured for each seed using ImageJ (v1.51i) software. 2.4

Data analysis

The statistical difference between the concentrations of humic extracts in relation to the control was verified by analysis of variance (ANOVA). Data were first evaluated in terms of parametricity/non-parametricity and were then shown to have a normal distribution. Subsequently, one-way ANOVA and Tukey's test were used for validation,

10

and values of p < 0.05 were considered to indicate statistical difference. The statistical differences between the elemental analyses were also evaluated with ANOVA. Humic extract data were explored using principal component analysis (PCA). The data matrix (X) was self-scaled, and the scores and loadings were calculated using Octave software. Significant variables were chosen by taking absolute values of ≥ 0.22. 3

Results

3.1

Elemental and UV-vis analyses

Table 1 lists the elemental compositions, atomic ratios, and E4/E6 ratios of HLS isolated from hydrochar and HS isolated from soils. All HLS presented similar C and H contents and were rich in carbon (63.0 to 67.0%). The above carbon content was more similar to that of ADE-derived HS than to that of SR-derived HS. The presence/absence of H3PO4 did not affect the HLS H/C and O/C ratios, i.e., the contents of aromatic and carbonlinked oxygen moieties, respectively. HLS isolated from bagasse hydrochar had lower nitrogen contents than the others HLS because of the composition of the former raw material (p < 0.05) (Melo et al., 2017; Silva et al., 2017). The addition of H3PO4 decreased HLS nitrogen content, and hence, increase the C/N ratio (p < 0.05). The H/O ratio is a measure of humic material polarity and oxidation degree (De Paolis and Kukkonen, 1997). Thus, soil-derived HS were more hydrophilic than HLS, showing lower H/O ratios and higher O/C ratios. The E4/E6 ratio is a measure of aromaticity, with low values suggesting preferential absorbance at high wavelengths and, hence, arrangements mostly comprising aromatic units (Chen et al., 1977). For HLS, the above ratio decreased when H3PO4 was added, which suggested the presence of different aromatic domains and an increased degree of polycondensation. Table 1 11

3.2

Fluorescence analysis

Figure S1 of Supplementary Material shows the components identified in the EEM for HLS isolated from H3PO4-treated and non-treated hydrochars and for HS isolated from ADE and SR. The obtained results allowed us to perform CP/PARAFAC analysis, in which two fluorescent components were obtained for samples with a core consistency diagnostic (CORCONDIA) of 99.83 % (Bro and Kiers, 2003). Two components were chosen, as the introduction of a third one would decrease CORCONDIA to 50.0%. Component 1 had λexcitation/λemission = 310/450 nm and was identified as a humic-like fluorophore. Component 2 had λexcitation/λemission = 310 and 470/540 nm, and was concluded to be structurally complex because of the shift to higher wavelengths (Matthews et al., 1996; Mounier et al., 1999). Figure S2 of Supplementary Material shows the fluorescence intensity variation of components identified by CP/PARAFAC. HLS had a lower contribution to fluorescence intensity than that of soil-derived HS, while HS-ADE presented lower intensity than that of HS-SR (Fig. S2a in the Supplementary Material). HLS isolated from H3PO4treated and non-treated hydrochar also presented differences in fluorescence intensity (Fig. S2b in the Supplementary Material). The differences between fluorescent contributions were correlated with the structural differences of HLS and HS. HLS extracted from H3PO4-treated hydrochar featured a more rigid arrangement, i.e., comprised linearly condensed aromatic rings contributing to a decrease in fluorescence intensity, while the presence of the hydroxyl, methoxy, and N-containing groups in lower-complexity structural components increased fluorescence intensity (Senesi and Miano, 1991; Traversa et al., 2014). The higher fluorescence intensity of HLS extracted from non-acid-treated hydrochar was due to the presence of a mixture of substituents 12

and hydrophobic moieties (aromatic and alkyl). Moreover, the raw material molecules were less degraded in HLS isolated from non-acid-treated hydrochar and featured smaller (e.g., phenolic) structures, which may have contributed to the increase of fluorescence intensity. HS-ADE showed a low contribution to fluorescence intensity, which was indicative of a more linearly condensed arrangement, while HS-SR seemed to comprise small molecules. 3.3

13

C CPMAS NMR of HLS and HS

The 13C CPMAS NMR spectra of HLS and HS are shown in Fig. 1. Resonances between 0 and 45 ppm were assigned to methylene and methyl groups in the alkyl chains of either natural lipids such as plant waxes and polyesters (Monda et al., 2017; Spaccini et al., 2019) or of hydrocarbons produced during the charring of recycled biomass (Chen et al., 2017). The signal centered at 56 ppm (Fig. 1) was assigned to the methoxy carbons (O–CH3) on the aromatic rings of lignin guaiacyl and syringyl units as well as to C–N bonds in peptides and amino acids (Spaccini et al., 2008). Signals in the central region (61–110 ppm) were ascribed to O–alkyl–C moieties pertaining to monomeric components of oligo- and polysaccharide structures. The intense band at 73 ppm observed for soil-derived HS corresponded to the overlapping resonances of carbons 2, 3, and 5 in the pyranoside structure (Spaccini et al., 2019). The shoulder at ~64 ppm represented the carbon nucleus in position 6, whereas the signal at 106 ppm was related to anomeric carbon 1 in the glycosidic bonds of sugar units. The resonance of aromatic carbons at 111–145 ppm included the contributions of non-substituted and C-substituted phenyl carbons of both plant-derived and pyrolytically condensed aromatic moieties, while the downfield signals within the 145–160 ppm region belonged to aromatic–aryl–C units bound to either hydroxy or methoxy groups (e.g., phenolic and lignin compounds). The most deshielded resonances at 161–190 ppm and 13

191–220 ppm were ascribed to carboxylic and other carbonyl groups (such as aldehydes and ketones), respectively (Dignac et al., 2003). Figure 1 The different C distributions found in HLS and HS samples highlighted the specific origin of humic extracts (Table 2). The large content of O–alkyl–C (61–110 ppm) and the abundance of alkyl–C derivatives (0–45 ppm) in the NMR spectra of ADE and SR samples indicated the input of polysaccharides and lipids from plant litters (Tadini et al., 2015a; Tadini et al., 2015b). On the other hand, the significant amount of aromatic carbon and carboxylic groups suggested an advanced humification of organic components typical of ADE. Moreover, the lack of distinct signals in the 82–88 ppm range due to the carbon nucleus in position 4 of pyranoside structures linked through 1o4 glycosidic bonds (Fig. 1) suggested ether bond cleavage and progressive transformation of polysaccharides within soil humus (Spaccini et al., 2019). Conversely, hydrochar-derived humic extracts showed a predominance of aryl (111–160 ppm) and alkyl compounds (0–45 ppm) with relative abundances of 32–43% and 33–47%, respectively, with corresponding lower relative areas in the 60–110 ppm region (Table 2). The NMR spectra of humic-like fractions isolated from hydrochars were indicative of an increased content of phenolic derivatives, which was ascribed to the use of a medium favoring the extraction of polar molecules. The molecular characteristics of humic materials may be described using the dimensionless structural parameters currently used to assess the biochemical stability of different organic materials, such as the indexes HI, AI and Al/O-Al (Monda et al., 2018, 2017; Spaccini et al., 2019). In particular, the HI index is commonly related to the biochemical stability of different organic materials. In fact, the relatively large values indicate an increased process of organic matter stabilization, as associated to selective 14

accumulation of hydrophobic compounds. Moreover, the calculation of AI and Alkyl ratio is helpful to highlight the role of aliphatic or aromatic hydrophobic moieties in the structural properties of humic assembly. Structural modification induced by thermal treatment resulted in a sharp increase of overall HI in HLS, as compared to the case of soil-derived HS (Table 2). In this respect, the AI and the Al/O–Al ratio indicated the preferential extraction of aromatic moieties for bagasse-derived HLS, while vinassederived HLS contained hydrocarbon moieties. Furthermore, the shift from O–alkyl compounds to apolar aliphatic molecules seemed to be stimulated by the addition of H3PO4 in the case of HLS isolated from biomass mixture–derived hydrochar. Table 2

3.4

Offline thermochemolysis GC-MS of HLS and HS

The thermochemolysis of HLS and HS released ~160 aliphatic, cyclic, and aromatic compounds (Table S3 in the Supplementary Material), which were grouped into main classes and characterized in terms of relative yields (Fig. 2). The thermochemolysis resulted in a carbohydrate yield significantly lower than that observed for NMR analysis, which was attributed to the lower efficiency of offline pyrolysis for detecting the carbohydrate units of polysaccharides in complex matrices. The thermal behavior and pyrolytic rearrangement of polyhydroxy compounds combined with the basic reaction conditions of TMAH are believed to negatively affect the release of polysaccharides. The most abundant thermochemolysis products were represented by benzene derivatives, aliphatic esters, and phenolics (Fig. 2). Benzene and phenolic units were mainly derived from lignin monomers (hydroxyphenyl, guaiacil, and syringyl) (Cao et al., 2014), while sterols, terpenes, and aliphatic compounds were associated with external plant structures such as waxes and biopolyesters (Monda et al., 2018). The 15

largest relative content of phenols was determined for HLS-HB and HLS-HBH3PO4 (Fig. 2) followed by other HLS samples. These data confirm the results of NMR analysis, in which case an intense phenol peak at 146.9 ppm was observed for humic substances isolated from bagasse hydrochar (Fig. 1). The intensity of this peak progressively decreased for substances extracted from the products of vinasse and mixed biomass decomposition (Table 2). The presence of polycyclic aromatic hydrocarbons (PAHs) was indicative of aromatization, as HCT induced the decomposition of polysaccharides present in raw biomass to afford furfural and promoted their condensation, polymerization, and aromatization (Reza et al., 2014b). There are no structural indications that the identified phenanthrene, anthracene, and naphthalene were either present as "free" compounds or as fragments of polycondensed aromatic rings (black carbon). In fact, the relative area of PAH signals was low compared to that of low-molecular-weight compounds. Therefore, bioassays such as the germination experiment are useful for evaluating the effects of molecular composition, although the presence of PAH was already pointed out in studies on the humic extracts of soil (Chiapini et al., 2018; Schellekens et al., 2017). Contrary to the results of NMR analysis, the pyrograms of HLS isolated from acidtreated biomass revealed a decreased abundance of global aromatic moieties such as benzene and phenol derivatives along with minor components. This finding was ascribed to the structural arrangement of HLS isolated from chars obtained in the presence and absence of acid, and, consequently, to the different responses to analytical techniques. HTC in the presence of H3PO4 further enhanced the conversion of polysaccharide structures and provided HLS with higher structural order and increased polycondensation degree (Zhou et al., 2017), as revealed by the corresponding low 16

fluorescence intensity (Fig. S2b). Hence, it was concluded that the aromatic core is less susceptible to subsequent thermochemolysis, i.e., exhibits greater thermal stability (Rosa et al., 2012). On the other hand, HLS produced under acid-free conditions featured a slightly larger content of O–alkyl structures (Table 2). The thermochemolysis of HLS released less complex aromatic compounds, while hydroxyl-bearing groups possibly underwent pyrolytic rearrangement and aromatization to afford phenols and benzene derivatives (Nebbioso and Piccolo, 2012). Thus, the higher abundance of O– alkyl moieties accounted for the increased presence of benzene derivatives in HLS produced under acid-free conditions and in HS-SR (Table 2). Figure 2 3.5

Bioassay in maize

Figure 3 shows the lengths of coleoptiles, radicles, and lateral seminal roots of maize seedlings treated with HLS and HS solutions at TOC levels of 1, 10, and 50 mg L−1. No differences were found between the germination efficiencies of HLS- or HS-treated seeds and that of the control sample (data not shown), which indicated that the added organic materials did not exert any toxic influence. HLS solutions of different concentrations exerted an overall positive effect on the analyzed maize tissues (Fig. 3), the length of which increased in the order of 1 mg L−1 > 50 mg L−1 > 10 mg L−1. The application of HLS-HB (1 and 50 mg L−1) and HLSHBH3PO4 (1 mg L−1) resulted in the most significant elongation of coleoptiles, radicles, and lateral seminal roots in relation to control and soil-derived HS samples. The application of HLS-HV promoted the elongation of radicles at concentrations of 1 and 50 mg L−1, while the application of other HLS did not afford any statistical differences in relation to the control. The large variability of data reduced the statistical significance of the observed developments. 17

HLS-HB and HLS-HBH3PO4 were identified as the most bioactive HLS with the strongest promotional effect on maize tissues elongation (compared to other HLS and HS). Although the direct application of hydrochar to soil negatively affect plants (Bargmann et al., 2014, 2013), our results suggest that hydrochar and soils afforded extracts with distinct properties, with each specific molecular composition interacting with maize seedlings in a different way, as has been shown by Monda et al. (2018), Olaetxea et al. (2018), and Spaccini et al. (2019). Figure 3 3.6

Multivariate analysis (PCA)

Figure 4 shows the results of PCA application to thermochemolysis GC-MS and elemental analysis data as well as to the integrated relative areas of NMR signals, revealing separations among HLS chemical compositions. The first component (PC1) corresponded to 51.89% of the total variance and the HLS-HB and HLS-HBH3PO4 scores were neatly separated by phenolic compounds (negative scores) from those of HLS-HV, HLS-HVH3PO4, HLS-BV, and HLS-HVH3PO4 by alkyl content (positive scores). The second component (PC2) corresponded to 16.76% of the total variance and positively separated the HLS-HB, HLS-HV, and HLS-HVH3PO4 scores based on H content, N content, H/C atomic ratio, CC content, HS content, and SE content. HLSHBH3PO4, HLS-HBV, and HLS-HBVH3PO4 scores were negatively separated by ES content. Figure 4 PCA analysis was also performed for HLS and HS, as shown in the score plots of Fig. 5 and the loads in Fig. S3 in the Supplementary Material. PC1 corresponded to 42.57% of the total variance and separated HS from soil by O–alkyl compounds (positive scores) from HLS by O–aryl and carbon contents (negative scores), showing that HLS 18

comprised numerous oxygenated aromatics, while HS featured an increased content of aliphatic species linked to oxygenated functionalities (polysaccharide structures). PC2 corresponded to 23.97% of the total variance and separated HLS-HB, HLS-HBH3PO4, HS-ADE, and HS-SR scores from other HLS by phenolic content (positive scores), suggesting that sugarcane bagasse HLS resembled HS in terms of phenolic content, while HLS scores derived from vinasse and the bagasse-vinasse mixture were separated by heterocyclic oxygen (negative scores). Figure 5

4

Discussion

HLS composition depended largely on hydrochar type, e.g., HLS isolated from sugarcane bagasse hydrochar contained amphiphilic structures in contrast to those isolated from sugarcane vinasse hydrochar. This difference was ascribed to the greater content of alkyl moieties in HLS from vinasse hydrochar, which afforded a more hydrophobic structure. The differences are also highlighted by phenols released by thermochemolysis from humic extracts that clearly differentiated sugarcane bagasse HLS from the others HLS (Fig. 4). These results agreed with the presence of an intense peak of phenol at 149.0 ppm in the NMR spectra of HLS-HB (Fig. 1), while vinasse HLS mainly contained long-chain esters. The addition of phosphoric acid increases HLS hydrophobicity and aromaticity by promoting dehydration reactions and the generation of more hydrophobic hydrochars (Wang et al., 2018; Zhou et al., 2017). Consequently, HLS extracted from hydrochar obtained in the presence of H3PO4 featured more ordered aromatic cores, indicated by the low contribution of components C1 and C2 to fluorescence intensity (Fig. S2) and lower E4/E6 ratios. The decreased contribution of oxygen functionalities and the 19

increased abundance of linearly condensed structures helped differentiate HLS from HS. Although, HLS-HB and HLS-HBH3PO4 featured phenolic contents similar to those of soil-derived HS, as shown by PC2 in Fig. 5, the O–alkyl group contents of HLS-HB and HLS-HBH3PO4 differed from that of HS. HS were extracted from the soil top layer and thus contained abundant polysaccharides (O–alkyl) originating from fresh organic matter. The maize seed bioassay revealed that HLS isolated from hydrochar treated and nontreated with H3PO4 promoted a higher elongation of coleoptiles, radicles, and lateral seminal roots in relation to HS. The elongation appeared to be correlated with the composition of HLS extracts, which were predominantly hydrophobic and featured a higher content of aromatic and phenolic moieties than that of HS. Furthermore, HLSHB and HLS-HBH3PO4 exhibited the strongest positive effect on maize seed germination. This effect was mostly attributed to the relative content of phenols, as determined by thermochemolysis (Fig. 2) and NMR spectroscopy (Fig. 1), which was a distinguishing feature of these HLS (Fig. 4). The biological activity of humic materials is mostly associated with hormone-like molecules and their auxin-like and gibberellin-like effects (Canellas et al., 2010; Canellas and Olivares, 2014; Zandonadi et al., 2007). Auxin promotes the elongation of roots and coleoptiles, and increases the activity of plasma membrane H+-ATPase, while gibberellins enhance hypocotyl elongation, affect flower and fruit development, and interrupt seed dormancy (Zhao and Zhong, 2013). Phenolic and aromatic compounds can exert auxin-like effects (Savy et al., 2017; Tanase et al., 2019). Experiments on organic extracts derived from vermicomposts and biorefinery by-products (MartinezBalmori et al., 2014; Savy et al., 2017) revealed the promotional effects of humic acids or water-soluble extracts enriched in lignin molecules on maize shoot and root 20

elongation. This plant growth–promoting interaction was mainly ascribed to the hormone-like effects of phenolics and lignin derivatives. The effects of different humic materials and their concentrations on maize seedlings were ascribed to molecular and conformational properties. The bioactivity of humic materials depends on the supramolecular conformation and dynamic interactions of hydrophobic and hydrophilic domains pertaining to exogenous organic inputs (Canellas and Olivares, 2014). The hydrophobic cores of humic superstructures may act as effective sinks able to incorporate, preserve, and transport active molecules, mainly represented by low-molecular-weight compounds including both apolar molecules and bioavailable polar components, such as lignin fragments, phenol derivatives, peptides, and aromatic acids (Canellas et al., 2010; Piccolo et al., 2018). In this respect, it is known that exudates can influence plant growth through interactions and thermodynamic rearrangements of humic molecules (Canellas et al., 2019; Spaccini et al., 2000). In rhizoplane environments, a large concentration of root exudates increases the chance of physicochemical interactions with weakly bound supramolecular structures (Olaetxea et al., 2018; Canellas et al., 2019). The more dynamic and flexible amphiphilic humic conformation may undergo a favorable thermodynamic rearrangement more easily than a hydrophobic one, releasing molecules capable of initiating bioactivity in close proximity to root membranes (Canellas et al., 2019). HLS extracted from bagasse hydrochar positively affected elongation compared to HS-ADE, which represents model organic matter in terms of fertility and soil carbon storage (Novotny et al., 2009). This finding was ascribed to the content of Fe and Al bonded in soil-derived humic extracts (Table 2S in the Supplementary Material). The higher ionic strength of HS solutions may have limited the uptake capacity of seedling roots. Moreover, the Al and Fe presence increased the molecular rigidity of humic aggregates, 21

thereby suppressing the possible release of bioactive components (Nebbioso and Piccolo, 2009). Conversely, the structural modification of HLS derived from sugarcane bagasse hydrochar may have ensured a more flexible arrangement favoring the prompt displacement of bioactive phenolic derivatives and the promotion of plant growth (Canellas and Olivares, 2014; Monda et al., 2017). On the other hand, the high aliphatic group content of HLS from vinasse hydrochar, such as long-chain esters, may they have induced a close interaction of apolar alkyl chains within the supramolecular structure in aqueous solution, thus decreasing amphiphilicity and disassembling properties (Chilom et al., 2013). Humic material conformation in aqueous solution seemed to influence the response observed for increasing concentrations. It is conceivable that at a low concentration of 1 mg L−1, the humic extracts easily underwent the above-sketched structural rearrangement with a suitable unfolding of bio-stimulants depending on the specific molecular composition. The application of higher concentrations (50 mg L−1) possibly promoted a closer aggregation of hydrophobic structures with a small chance of structural modification. In this respect, additional or alternative indirect mechanisms of plant tissue development have been related to the activation of an array of physiological responses, such as reduced root hydraulic conductivity, to stress signals (Olaetxea et al., 2018). The low stimulating effect observed for the intermediate concentration of 10 mg L−1 suggested the occurrence of a limiting interference of the two mechanisms. The addition of H3PO4 and the subsequent increase of HLS hydrophobicity may have affected the release of bioactive molecules. The use of hydrochar extracts may be a new way of stimulating plant growth, depending on hydrochar type, as extracts of lignocellulosic biomass–derived hydrochar featured aromatics (aryl and O–aryl moieties) in a supramolecular structure able to release bioactive molecules, while vinasse HLS were unable to release bioactive molecules. 22

5

Conclusion

HLS extracted from hydrochar showed two types of molecular arrangements, namely amphiphilic (O–aryl–C, aryl–C, and alkyl–C moieties) and hydrophobic (comprising aryl-C and alkyl–C (major) and O–alkyl–C (minor) moieties). Molecular composition affected the structural flexibility of supramolecular structures, i.e., the amphiphilic arrangement had a supramolecular structure capable of interacting with other organic compounds and was therefore categorized as a flexible structure able to release bioactive molecules. HLS isolated from bagasse hydrochar comprised amphiphilic moieties, while HLS isolated from vinasse hydrochar were more hydrophobic because of the higher content of alkyl moieties. The most bioactive HLS was extracted from bagasse hydrochar, and this enhanced bioactivity was attributed to the presence of abundant aromatic and phenolic units promoting the growth of coleoptiles, radicles, and lateral seminal roots. In contrast, the hydrophobic domains did not improve maize seed germination (the hydrophobic structures generated a stronger apolar interaction unable to release bioactive molecules). All HLS differed from both HS in terms of hydrophobicity and polysaccharide content, with the latter parameter being higher in the case of HS. The results of this study are expected to inspire further investigations on HTC as a new tool to produce amphiphilic humic-like extracts suitable for use as plant growth promoters.

Acknowledgements This work was supported by the São Paulo Research Foundation (FAPESP) (grants 15/22954-1 and 18/15733-7) and the National Council for Scientific and Technological Development (CNPq). L.R.B. acknowledges a scholarship from FAPESP (grants 17/05575-2 and 17/17991-0). O.P.F acknowledges financial support from FUNCAP 23

(PRONEX PR2-0101-00006.01.00/15). We would also like to thank Dr. Roland Redon (Laboratoire MIO, Université de Toulon, France) for providing statistical analysis software (CP/PARAFAC) and Erick Paiva Cancella for his help with the bioassay.

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potential and dissolved organic matter molecular characteristics. Sci. Total Environ. 659, 655–663. doi:10.1016/j.scitotenv.2018.12.399

31

Fig. 1. 13C CPMAS NMR spectra of HLS and HS.

32

Fig. 2. Relative abundance (%) of main classes of compounds identified in HLS produced by HTC in the presence and absence of H3PO4 and HS from soil. BD = benzene derivatives, HN = heterocyclic nitrogen-containing compounds, ES = aliphatic esters, CC = cyclic compounds, HS = heterocyclic sulfur-containing compounds, PH = phenolic compounds, HO = heterocyclic oxygen-containing compounds, PAH = polycyclic aromatic hydrocarbons, AL = alkanes/alkenes/alkynes, DA = dicarboxylic acids, AC = alcohols, SE = steroids, and ST = sterols.

33

10

Coleoptile length (cm)

8

1 mg L-1 -1 10 mg L -1 50 mg L

* * * *

* 6

4

2

0

g p (cm) ( ) Radicle length

10

1 mg L-1 10 mg L-1

8

6

-1 50 mg L

* * *

4

2

Lateral seminal roots length (cm)

0 10

-1 1 mg L 10 mg L-1

8

-1 50 mg L

*

6

*

*

4

2

0

Fig. 3. Effects of applying humic extracts (TOC = 1, 10, and 50 mg L−1) on radicle, coleoptile, and lateral seminal root length (cm). Columns (mean ± standard deviation) followed by an asterisk (*) and a triangle (Δ) indicate statistical differences of p < 0.05 between HLS and the control and those between HLS and ADE-HS, respectively. “Δ” in ADE columns indicates differences relative to HS-SR. 34

Fig. 4. PCA score plots obtained based on the results of HLS elemental analysis, classes of compounds identified by thermochemolysis GC-MS, and relative areas of NMR signals.

35

Fig. 5. PCA score plots based on the results of HLS and HS elemental analysis, classes of compounds identified by thermochemolysis GC-MS, and relative areas of NMR signals.

36

Table 1. Elemental compositions, atomic ratios, and E4/E6 ratios of (i) HLS isolated from hydrochars in the presence and absence of H3PO4 and (ii) HS isolated from ADE and SR.

C (%)

H (%)

N (%)

O (%)

H/C

C/N

O/C

H/O

E4/E6

HLS-HB

63.13 ± 0.42

5.97 ± 0.02

0.58 ± 0.02

30.32 ± 0.45

1.13

126.57

0.36

3.16

4.58

HLS-HBH3PO4

63.82 ± 0.03

5.85 ± 0.41

0.25 ± 0.01

30.08 ± 0.37

1.09

295.74

0.35

3.11

4.30

HLS-HV

63.10 ± 0.10

6.90 ± 0.21

4.44 ± 0.00

25.56 ± 0.11

1.30

16.65

0.30

4.30

4.68

HLS-HVH3PO4

67.48 ± 0.17

7.22 ± 0.02

2.85 ± 0.03

22.45 ± 0.22

1.27

27.76

0.25

5.14

4.05

HLS-HBV

66.52 ± 0.10

7.26 ± 0.04

4.03 ± 0.01

22.19 ± 0.13

1.30

19.34

0.25

5.22

5.81

HLS-HBVH3PO4

64.93 ± 0.10

6.76 ± 0.12

2.54 ± 0.01

25.77 ± 0.03

1.24

29.97

0.30

4.16

5.38

HS-ADE

52.07± 0.07

5.10 ± 0.01

3.63 ± 0.04

39.20 ± 0.10

1.17

16.82

0.57

2.07

4.35

HS-SR

44.50 ± 0.04

5.66 ± 0.00

2.89 ± 0.05

46.96 ± 0.01

1.51

18.06

0.79

1.91

4.29

37

3.09

3.70

1.93

6.25

2.35

4.66

4.53

HLS-HBH3PO4

HLS-HV

HLS-HVH3PO4

HLS-HBV

HLS-HBVH3PO4

HS-ADE

HS-SR

4.37

4.66

7.51

6.42

5.83

7.82

7.10

5.35

4.37

4.36

9.86

9.34

10.79

10.29

12.65

12.26

C NMR region (ppm) Carboxylic acid O-Aryl (δ190—160) (δ 160—140)

13

16.40

21.18

23.00

22.96

26.55

22.22

29.94

27.67

C-Aryl (δ 140—110)

35.15

28.97

5.63

7.00

4.61

6.58

6.48

8.18

O-Alkyl (δ 110—60)

11.56

9.81

7.04

7.98

9.04

6.58

8.02

10.06

O-CH3 (δ 60—45)

19.21

20.56

46.95

43.58

42.36

42.80

32.72

33.02

Alkyl (δ 45—0)

0.55

0.72

2.76

2.61

3.12

2.29

2.02

0.38

0.52

0.63

0.64

0.80

0.66

1.09

0.97

0.55

0.71

8.33

6.22

9.19

6.50

5.05

4.04

C NMR structural indexesb HI AI Alkyl/OAlkyl

1.86

13

Hydrophobicity index (HI) = Σ [(0-45 ppm)+(110-160 ppm)]/ Σ[(45-60 ppm)+(60-110 ppm)+(160-190 ppm) +(190-220 ppm)]

Alkyl index (Al/O-Al) = (0-45 ppm) /(60-110 ppm) 38

Aromaticity index (AI) = (110-160 ppm) / Σ[(0-45 ppm)+(60-110 ppm)]

b

The total area of each spectrum was considered as 100% and was divided into seven regions (220—190, 190—160, 160—140, 140—110, 110—60, 60—45, 45—0 ppm), the values on the table are the percentage of each resonant carbon nucleus

a

3.45

Carbonyl (δ220—190)

HLS-HB

indexes.

Table 2. Integrated relative areas (%)a along the chemical shift of main peaks (ppm) in the 13C CPMAS NMR spectra of HLS, HS and structural

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Please check the following as appropriate:

o

All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version.

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Author’s name

Affiliation

Lucas Raimundo Bento – São Paulo State University Camila de Almeida Melo– São Paulo State University Altair Benedito Moreira– São Paulo State University Odair Pastor Ferreira- Universidade Federal do Ceará Alessandro Piccolo- Università di Napoli Federico II Riccardo Spaccini - Università di Napoli Federico II Stephane Mounier- Université de Toulon Márcia Cristina Bisinoti– São Paulo State University

39

GRAPHICAL ABSTRACT

Highlights • Humic extracts of hydrochar and Amazonian Dark Earth were isolated and characterized • Extract molecular structure was correlated with maize seed germination activity • HLS isolated from bagasse hydrochar had the highest bioactivity • Hydrothermal carbonization was concluded a new tool to produce HLS

40