Structure-function relationship of vermicompost humic ...

Structure-function relationship of vermicompost humic fractions for use in agriculture Andrés Calderín García, Orlando Carlos Huertas Tavares, Dariellys Martínez Balmori, Vitor dos Santos Almeida, Luciano Pasqualoto Canellas, et al. Journal of Soils and Sediments ISSN 1439-0108 J Soils Sediments DOI 10.1007/s11368-016-1521-3

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Author's personal copy J Soils Sediments DOI 10.1007/s11368-016-1521-3

NATURAL ORGANIC MATTER: CHEMISTRY, FUNCTION AND FATE IN THE ENVIRONMENT

Structure-function relationship of vermicompost humic fractions for use in agriculture Andrés Calderín García 1 & Orlando Carlos Huertas Tavares 1 & Dariellys Martínez Balmori 2 & Vitor dos Santos Almeida 3 & Luciano Pasqualoto Canellas 4 & José María García-Mina 5 & Ricardo Luis Louro Berbara 1

Received: 7 March 2016 / Accepted: 2 August 2016 # Springer-Verlag Berlin Heidelberg 2016

1

Departamento de Solo, Laboratório de Biologia do Solo, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rodovia BR 465 km 7, Seropédica, RJ 23890-000, Brazil

2

Departamento de Química, Instituto de Agronomía, Universidad Agraria de La Habana (UNAH), Autopista Nacional km 23 1/2, Provincia de Mayabeque, San José de las Lajas, Cuba

3

Instituto de Química, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rodovia BR 465 km 7, Seropédica, RJ 23890-000, Brazil

4

Núcleo de Desenvolvimento de Insumos Biológicos para a Agricultura (NUDIBA), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Av. Alberto Lamego, 2000, Campos dos Goytacazes, RJ 28013-602, Brazil

tracted following the International Humic Substances Society (IHSS) recommended method, and the solid residue (humified residual (HR)) after extraction of HLAwere characterized using complementary chemical, physic, and spectroscopic technics (elemental composition, UV-Vis and Fourier transform infrared spectroscopy (FTIR) spectroscopies, 13C-CP MAS NMR, and MEV). Biological activity of the three HS was conducted in growth chambers and measured in roots using WinRhizo Arabidopsis software. Principal component analysis (PCA) was used to find a grouping pattern between the structural variables evaluated and the obtained root parameters. Results and discussion Differences were found in elemental composition among HS with larger C/N ratio in HR than in HLA and HLAw. HLA and HLAw FTIR spectra showed carboxyl band at 1714.66 cm−1 better resolved than in HR. Bands at 1642 cm−1 (amide I) and 1510 cm−1 (lignin), were better resolved in HLA. 13C-NMR showed the following order of aromaticity: HLA > HLAw > HR. For HLAw bioactivity, the structures CAlkyl-H,R, CC=O, and CCOO-H,R correlated with the number and growth of smaller root. The aromatic CAr-H,R, CAr-O,N, and aliphatic CAlkyl-O,N, CAlkyl-O, and CAlkyl-di-O structures in HLA, correlated with larger roots growth. HR also stimulated root growth and development in rice plants. Conclusions Aliphatic and oxygenated structures in HLAw showed a relation with induction of initial root emissions, whereas the presence of aromatic compounds in HLA was related with root growth stimulation activity. Higher concentration of HLAw was necessary to produce an equivalent stimulus compared with HLA; it could indicate that, although both fractions showed similar types of structures in their composition, differences in the predominant structures may be determining different effects on the root.

5

Department of Environmental Biology, Agricultural Chemistry and Biology Group—CMI Roullier, Faculty of Sciences, University of Navarra, Pamplona, Spain

Keywords 13C NMR spectroscopy . Bioactivity . Humic substances . Vermicompost

Abstract Purpose The use of humic substances (HS) in agriculture is beneficial and has positive environmental impacts. However, to optimize the use of HS possible links between their structural characteristics and bioactivity must be shown. The goal of this study is to evaluate the bioactivity of different humic fractions extracted from vermicompost (VC) in rice plants and to shed light to possible structure-function relationships. Materials and methods Humic-like fractions were obtained from cattle manure vermicompost processed by African nightcrawlers (Eudrilus eugeniae spp.). Humic-like acid fraction using only water as extractor (HLAw), HLA fraction exResponsible editor: Heike Knicker Electronic supplementary material The online version of this article (doi:10.1007/s11368-016-1521-3) contains supplementary material, which is available to authorized users. * Andrés Calderín García [email protected]

Author's personal copy J Soils Sediments

1 Introduction Vermicompost (VC) use in agriculture has been a sustainable alternative over the last few years (García et al. 2014a). Water extract (liquid humus, compost tea, and leachate) obtained by water extraction from solid vermicompost (Salter and Edwards 2014) are known to have high bioactivity in plants (Arancon et al. 2006; Sahni et al. 2008; Joshi et al. 2009; Doan et al. 2013). Applying such VC extract improves yield, fruit quality (Gutiérrez-Miceli et al. 2007; Zaller 2007; Singh et al. 2008), and plant growth and development (Atiyeh et al. 2000, 2001; Arancon et al. 2004). Vermicompost also contains high quantities of humic substances (HS) in its composition (Atiyeh et al. 2002; BalmoriMartinez et al. 2014), which proven bioactivity when applied to plants (Canellas et al. 2013; Hernandez et al. 2015). Humic acids (HA) isolated from VC are known to stimulate root growth and to exert anti-stress effects on plants (García et al. 2012, 2014b; Aguiar et al. 2013; Olivares et al. 2015). HS bioactivity in plants is currently explained by several modes of action, and the mechanisms by which these substances exert their effects are controversial (Muscolo et al. 2013; Canellas and Olivares 2014). Genetic engineering studies using cDNA-AFLP show that HS affected Arabidopsis through a complex metabolic regulation mechanism involving the expression of genes related to cell metabolism, cell processes, and the stress response (Trevisan et al. 2011). In addition, using gene expression microarrays, Jannin et al. (2012) observed that HA action on Brassica napus occurs through mechanisms involving C, N, and S metabolism as well as stress regulation. HS bioactivity in plants is related to HS structure (Berbara and García 2014). Traversa et al. (2014) reported that the germination in certain plants positively correlated with the phenolic, –OH and oxygen content of HS. Aguiar et al. (2013) observed a positive correlation between methoxy groups, oxygenated aromatic compounds, and carboxyl groups and their bioactivity in plants. Canellas et al. (2012) reported a relationship between the hydrophobicity of HA and their bioactivity and Martínez-Balmori et al. (2013) found this relationship with VC and stimulation of lateral root emissions in plants. Vaccaro et al. (2009) observed that less complex and more hydrophilic humic fragments stimulated root growth and enzymatic activity in maize plants. To optimize agricultural waste management and VC production for agricultural purposes, this knowledge must be integrated. Considering the quantity of information on the effects of HS from VC on plants, comparative studies are necessary to determine how HS structure influences the type and intensity of the humified fraction bioactivity of such substances. Moreover, few studies have reported on the residual solids from liquid extraction of HS from VC. Forming a residual product without an apparent use may limit the large-

scale use of HS extraction. The aim of this study is to evaluate three humic fractions obtained from vermicompost and the relationship between their chemical structure and bioactivity in rice.

2 Materials and methods 2.1 Origin of vermicompost The humic-like acid fractions and residual fraction isolated and characterized were obtained from cattle manure VC processed by African nightcrawlers (Eudrilus eugeniae spp.) in mounds on the ground for 3 months (90 days). The cattle manure used as raw material was from the BEl Guayaval^ ranch located in San José de Las Lajas, Mayabaque Province, Cuba, latitude 22° 59′ 55.95″ N and longitude 82° 10′ 10.27″ W. Vermicomposting was performed according to the procedures described in the technical manuals by Álvarez et al. (1998). In short, plant remains from different types of culture were placed in concrete bed, and the humidity was maintained by supplying water and then the mixture was homogenized. Thereafter, earthworms are added to the mixture and covered with canvas kept for 120 days. 2.2 Extraction of humic fraction (HLA, HLAw, and HR) Humic-like acid (HLA) was isolated and purified from the VC according to the recommendations of the International Humic Substances Society (IHSS 2013) (www.humicsubstances. org). Briefly, a solution of sodium hydroxide (NaOH 0.1 mol L−1) was added to a mass of VC under N2 (g) atmosphere at a 1:10 ratio (m/v) for 24 h. Then, the suspension was centrifuged at 10,000 rpm for 30 min, and the supernatant was collected and filtered through filter paper. The supernatant was adjusted to pH 1.5 by addition of hydrochloric acid (HCl 6 mol L−1) to precipitate the HLA fraction. After 10 h, the fraction of fulvic acids in the liquid fraction was decanted and removed by centrifugation procedures. The precipitated HLA were re-dissolved in NaOH (0.1 mol L−1) and precipitated with HCl (6 mol L−1); this process was repeated three times. After re-precipitation, the HLA were agitated for 24 h in contact with a solution of hydrochloric acid, hydrofluoric acid and water (HCl/HF/H2O) at a ratio of 1:1:98 (v/v) to approximately 100 mL g (HLA) to remove the mineral elements. The suspension was centrifuged again to eliminate the acid solution, and the HLA were washed with distilled water. Then, the HLA were transferred to a dialysis membrane (14 kDa cutoff), and the procedure was performed against distilled water until a negative test for chloride ions (Cl−) using silver nitrate (AgNO3 0.1 mol L−1) was achieved. Next, the HLA were lyophilized and stored in a desiccator.


The humic-like acid fraction using only water as extractor (HLAw) was isolated and purified from the VC following the methodology described by Pinton et al. (1998) with certain modifications. Briefly, a mass of VC was added a volume of deionized water (H2O)d at a VC/(H2O)d ratio of 1:10 m/v, which was maintained on a mechanical stirrer for 48 h under N2 (g) atmosphere. The supernatant was decanted, centrifuged at 10,000 rpm for 30 min and filtered through filter paper. This supernatant was adjusted to pH 1.5 by addition of HCl (6 mol L−1) to precipitate the HLAw. After 10 h, the fraction of fulvic acids in the liquid fraction was decanted and removed by centrifugation. The HLAw was agitated for 24 h in contact with a solution of HCl/HF/H2O at a ratio of 1:1:98 (v/v) to approximately 100 mL g (HLAw) to remove the mineral elements. The suspension was centrifuged again to remove the acid solution, and the HLAw were washed with distilled water. Then, the HLAw was transferred to a dialysis membrane (14 kDa cutoff), and the procedure was performed against distilled water until a negative test for Cl − ions using AgNO3 (0.1 mol L−1) was achieved. Then, the HLAw was lyophilized and stored in a desiccator. The residual solid produced after extraction of the humiclike acid fractions from VC (IHSS-methodology) was washed in acidic medium (HCl 0.1 mol L−1) until the pH approached normality. Next, the solid was washed with an abundant amount of distilled water (H2O)d to completely remove the Cl− ions until a negative test using AgNO3(aq) (0.1 mol L−1) was achieved. The less dense particles (the non-humified fraction) were removed by centrifugation at 10,000 rpm, and the solid was dried at 103 °C (García et al. 2013). 2.3 Elemental analysis and UV-Vis spectroscopy and Fourier transform infrared spectroscopy of HLA, HLAw, and HR The elemental composition (C, H, and N) was determined using a LECO Trupec® Micro CHN analyzer, and the oxygen was measured by difference. Spectroscopy in the UV-Vis region was performed following the methodology of Santos and Camargo (1999). A 2 mg mass of HLA and HLAw was dissolved in 10 mL of a sodium hydrogen carbonate (NaHCO3 0.05 mol L−1) dissolution in a Rayleight UV-2100 UV-Vis spectrometer device with automatic measurement of the absorbance values over the range of 220–800 nm. Specifically, absorbances were measured over the range of 465 to 665 nm to determine the E465/E665 (E4/E6) ratio as an estimation of degree of aromatic rings condensation. 2.4 Characterization by solid-state nuclear magnetic resonance (13C-CP MAS NMR) 13

C-NMR spectroscopy solid-state cross-polarization with magic angle spinning was performed on Bruker AVANCE II

400 MHz NMR spectrometer equipped with a 4 mm Narrow MAS probe and operating at 13C resonance frequencies of 100,163 Mhz. Samples of humified materials were packed in a rotor (sample holder) of zirconium dioxide (ZrO2) with Kel-F caps which were spun at of 8 ± 1 kHz. The spectra were obtained by collecting 2048 data points for the same number of scans to an acquisition time of 34 ms and recycle delay of 5 s. The contact time for the sequence in 1H ramp was 2 ms. The Bruker Topspin 2.1 software was used to collect and process the spectra. The free induction decay (FID) was processed by applying a zero filling equal to 4 k afterward by adjustment exponential function (line broadening) of 70 Hz. Spectra were divided into chemical shifts; the areas were determined after integration of each region and expressed as percentages of total area. The regions were assigned as follows: alkyl C (CAlkyl-H,R) 0–46 ppm; methoxyl and N–alkyl C (CAlkyl-O,N) 46–59 ppm; O–alkyl C (CAlkyl-O) 59–91 ppm; diO–alkyl C (anomeric) (CAlkyl-di-O) 91–110 ppm; aromatic C (CAr-H,R) 110–142 ppm; O,N-aromatic C (CAr-O,N) 142– 156 ppm; carboxyl C (CCOO-H,R) 156–186 ppm; and carbonyl C (CC=O) 186–230 ppm (De la Rosa et al. 2011; Song et al. 2008). The ratio between the hydrophobicity and hydrophilicity indices (HB/HI) was calculated as follows: HB/HI = (0– 46 ppm) + (110–156 ppm) / (46–110 ppm) + (156–186 ppm) (Spaccini and Piccolo 2007; Aguiar et al. 2013). 2.5 Scanning electron microscopy Morphological characterization of the humic-like acid (HLA, HLAw) fractions and residual fraction (humified residual (HR)) was performed by obtaining microscopic images by surface scanning. The samples were covered with a thin layer of gold (5 nm in thickness) with a gold deposition time of 120 s. Then, the samples were analyzed on a Carl Zeiss model EVO MA10 scanning electron microscope under high vacuum using a voltage of 10 kV. 2.6 Biological activity of the different humic fractions in rice plants 2.6.1 Plant material and growth conditions for bioactivity assays of humic fractions Experiments of humic acids bioactivity (HLA and HLAw) in rice plant (Oryza sativa L. cv. Piauí) were conducted in growth chamber with the following growing conditions: light cycle 12/12 h (light/dark), photosynthetic photon flux 250 μm mol m−2 s−1, relative humidity 70 %, and temperature 28 °C/24 °C (day/night). Rice seeds were previously disinfected with sodium hypochlorite (2 %) for 10 min and then washed several times with distilled water. Subsequently, seeds were transferred to pots containing only distilled water and using gauze to prevent seed immersion. Four days after


germination of seeds, the seedlings received a Hoagland’s solution (Hoagland and Arnon 1950) modified to one fourth of the total ionic strength. After 3 days, Hoagland solution was changed to one half of the total ionic strength; this same solution was restored throughout the experiment. Experiments were conducted in order to select the better concentration of humic acids for stimulation of the roots systems. Two days after acclimatization of the plants in the nutrient solution one half the total ionic strength, the rice plants were treated with seven concentrations of humic acids (HLA and HLAw), i.e., 0, 5, 10, 20, 40, 60, 80, 100, and 120 mg (C) L −1 (Fig. S1a, Electronic Supplementary Material). Using the results of the above experiment, the concentrations were selected that showed the highest stimulation of root system; the concentrations for the bioassays were as follows: 40 mg (C-HLA) L−1, 40 mg (C-HLAw) L−1, and 60 mg (C-HLAw) L−1. 2.7 Plant material and growth conditions for bioactivity assays of the residual fraction For the experiments of solid residual (HR) biological activity, we used the same growth conditions that were used in the assays with humic acids. This case a substrate was prepared with washed sand and the HR, which were mixed homogeneously at various ratios (m/m). Furthermore, in all treatments, a nutrient solution of Hoagland with one half of total ionic strength was applied. Experiments were conducted in order to select the better concentration of HR for stimulation of the roots systems. After germination of seeds, seedlings with a size of root of 2 cm were transplanted into pots containing the substrate (sand + RH) at various ratios (m/m), these were 0, 5, 10, 20, 40, 50, 70, and 80 % (sand + HR) (Fig. S1b, Electronic Supplementary Material). Using the results obtained in the previous experiment, which were selected for the following studies, the ratio of 20 % m/m (sand + RH) being the most stimulating plant root system. Experimental design was completely randomized, using five plants per pot with five replications. Statistical analyzes were performed in Statgraphic Centurion XVI. 2.8 Evaluation of roots parameters in rice plants Roots of rice seedlings are uniformly distributed in a layer of water in a transparent acrylic tray (30 cm × 20 cm) for scanning resolution of 600 dpi and a scanning system with Epson Expression 10000XL additional light unit (TPU) was used. Images of the roots are converted to a format in eight-bit grayscale. Five different characteristics roots are analyzed and quantified: length (mm), surface area (mm2), average diameter (mm), root volume (mm3), and number of roots. They will also be defined and measured length (mm) and number of

roots by classification of classes: superfine (0.5 to 1.5 mm), thin (1.5 to 3.5 mm), and thick (>3.5 mm) by use of software WinRhizo Arabidopsis (Regent Instruments, Quebec, Canada Inc.). The limits of the images are defined and then imported into WinRhizo software for analysis of root characters and their quantification. Images are analyzed using a calibration grid as a reference scale and have changed the input settings to pale roots on a black background. Then the data are analyzed via XLRhizo (Regent Instruments, Quebec, Canada Inc.).

3 Results and discussion 3.1 Elemental composition The isolated fractions featured lower hydrogen/carbon (H/C) values than the original VC, indicating selective aromatic enrichment in humified fractions during vermicomposting. The HR had the highest H/C ratio compared with the HLA and HLAw fractions. The HLA fraction had a lower H/C ratio than the HLAw fraction, indicating more marked aromatic characteristics in HLA than in HLAw (Chai et al. 2007; Amir et al. 2010). The oxygen/carbon (O/C) ratio of the HLA and HR fractions remained slightly similar to that of VC; however, an increase in the ratio observed in the HLAw fraction suggests an increased presence of oxygenated structures in this fraction compared with the value for HLA and HR. The carbon/ nitrogen (C/N) ratio of the HR fraction was larger than that of the HLA and HLAw fractions and the starting VC. According to Campitelli and Ceppi (2008), a C/N ratio ranging from 10 to 20 is considered high and suggests the presence of vegetal fragments in the material structure. For both VC and the humic-like acid fractions, these values are similar to those reported in the literature Campitelli and Ceppi (2008) (Table 1). 3.2 UV-Vis and Fourier transform infrared spectroscopy (FTIR) The E4/E6 ratio of the HLA fraction was lower than that of HLAw, confirming the findings for the H/C ratio in the Table 1 Elemental composition and the E4/E6 (UV-Vis) ratio for HLA, HLAw, and HR Elemental Composition

VC HLA HLAw HR

UV-Vis

C H % m/m

N

S

O

H/C

O/C

C/N

E4/E6

48.3 51.0 47.0 50.2

2.8 3.6 4.0 2.0

3.8 0.7 1.7 1.8

38.2 39.1 41.9 39.8

1.51 1.18 1.23 1.48

0.59 0.57 0.66 0.59

20.1 16.5 13.6 29.4

– 4.22 6.02 –

6.1 5.0 4.8 6.2


elemental composition and indicating that the HLA fraction gave an indication that the HLAw fraction can be more aromatic. In the spectra, certain peculiarities in the structure of the humic-like acid fractions can be detected (Fig. 1). For the HLA and HLAw fractions, there was a band at approximately 1714.66 cm−1 that was better resolved than in the HR spectra. The bands at 1604.72 cm−1 and approximately 1510.21 cm−1 indicate the stretching of the C=C aromatic structural skeletons of lignin (Miralles et al. 2015), which are better resolved in HLA. Identities of the bands in the spectra contribute to the understanding of the structural characteristics of humic-like fractions. The bands from 1719 and 1716 cm−1 (HLA and HLAw) correspond to vibrations of C=O group (carboxyls, carbonyls, and ketones) stretching (González-Pérez et al. 2004; Baigorri et al. 2009; Droussi et al. 2009). There was also a predominance of bands from 1649 and 1642 cm−1 corresponding to C=C (aromatic and olefinic) and C=O (amide I, ketones, and quinones) stretching vibrations (González-Pérez et al. 2004; Shirshova et al. 2006; Droussi et al. 2009; Amir et al. 2010). The bands from 1512, 1513, and 1546 cm−1 correspond to C=C (aromatic and lignin structures) stretching, NH deformations, and C–N (amide II) stretching (Shirshova et al. 2006; Droussi et al. 2009; Amir et al. 2010; Li et al. 2011). The HR spectrum showed bands from 1423 cm−1 corresponding to asymmetric CH2 stretching, symmetric COO− (carboxylate) stretching, C–O stretching, O–H (phenols) deformation, and C–H (CH3) stretching (Kumar et al. 2015; Droussi et al. 2009; Li et al. 2011). The bands present from 1330 to 1384 cm−1

Fig. 1 FTIR spectra of the humic fractions isolating from VC. a HLA, b HLAw, and c HR

indicate deformations of C–H bonds from CH2 and CH3 and asymmetric stretching vibrations from COO−. The bands also present from 2220 to 1259 cm−1 indicate C–O (aryl esters and phenols) and C=O (aryl esters) stretching. From 1033 to 1099 cm−1, bands in the spectrum correspond to C–O (carbohydrates and polysaccharides) stretching (Shirshova et al. 2006; Muscolo et al. 2007; Droussi et al. 2009; Amir et al. 2010; Li et al. 2011).

3.3 Nuclear solid-state magnetic resonance spectroscopy (13C-CP MAS NMR) Spectra showed similarities in the spectral Bsignature^ to those obtained in the literature by Nebbioso and Piccolo (2011) for composts, their soluble humified fractions, and humic acid isolated from soil (Fig. 2). The relative distribution of carbon in each region of the spectrum shows a predominance of CAlkyl-H,R, CAlkyl-O,N, CAlkyl-O, and CAr-H,R structures for the HLA fraction. The HLAw fraction had a predominance of CAlkyl-H,R and CCOOH,R structures, whereas the HR fraction had a predominance of CAlkyl-H,R and CAlkyl-O structures. The HLA fraction had a greater presence of aromatic (20.18 %) structures than the HLAw (11.82 %) and HR (8.42 %) fractions, confirming the findings of prior techniques using in this study. The HLAw

Fig. 2

13

C-CP MAS NMR spectra of HLA (a), HLAw (b), and HR (c)


fraction had the greatest presence of carboxylic and carbonylic structures, confirming the O/C ratio findings. The structural properties obtained by 13C-NMR spectroscopy showed the following order of aromaticity: HLA > HLAw > HR. The hydrophobicity index showed the following order: HR > HLAw > HLA (Table 2). In general, the structural characteristics observed for the soluble humic fractions (HLA and HLAw) in this study have also been reported in the literature (Spaccini et al. 2008; Kumar et al. 2015). In terms of structure-function relationship, the results obtained in this study are in line with those shown by Vaccaro et al. (2009), which reported that the compost fractions more hydrophilic and less complex structurally exert positive effects on growth and enzymes activity of nitrogen metabolism in maize plants. 3.4 Scanning electron microscopy The morphology of the humic-like acid fractions and the residual fractions is shown in Fig. 3. SEM revealed morphology for the HLA fraction of structural clustering in layers (Fig. 3a). A compacted surface can be observed ending in irregular elongated shapes forming thinner terminal structures and expanded surfaces with small protrusions, where certain structural collapse events are visible (Fig. 3b). Figure 3c, d shows a different morphology for the HLAw fraction, with more rounded and less laminar agglomerated particles. The surface in the HLAw fraction had features with more collapsed Bholes^ than the HLA fraction. The micrographs shown by Senesi et al. (1996) have demonstrated this type of morphology in humic acid isolated from peat, and Li et al. (2011) have also found these morphological patterns in humic acid isolated from VC. The SEM micrograph of the HR fraction (Fig. 3e, f) showed a surface morphology dominated by a rough agglomeration of particles with sizes less than or equal to 2 μm. It is possible to describe a compacted surface for this fraction but with a predominance of a highly irregular surface morphology. According to the visual information on the images at 10

Table 2 Relative distribution (% of total area) of the chemical shift regions (ppm) in the 13C CP MAS NMR spectra for the HLA, HLAw, and HR fractions

HLA HLAw HR

Chemical shift (ppm) 0–46 46–59 CAlkyl-O,N CAlkyl-H,R 17.43 14.68 37.63 11.82 33.68 11.57

Aromaticity Aliphaticity HB/HI

and 2 μm, the HR fraction appears to be organized as clusters of small particles forming larger particles or clusters. 3.5 Biological activity of humic fractions in rice plants Both the humic acids extracted using the IHSS method (HLA) and using water (HLAw) stimulated root growth in rice plants (Fig. 4). Plants treated with 40 mg (C) L−1 HLA showed statistically significant higher root parameters (main root length, surface area, number of roots, root volume, and emission of smaller roots) than the control treatment. HLAw applied at 40 mg (C) L−1 did not exhibit significant effects on root growth, whereas a 60 mg (C) L−1 application produced similar and, in certain cases, higher root growth than the HLA treatment (40 mg (C) L−1). Root diameter was not affected by applying either HLA or HLAw. HLA and HLAw positively stimulated plant growth and development, but a higher concentration of HLAw (20 mg (C) L−1 higher) was necessary to produce an equivalent effect to that observed for HLA at 40 mg (C) L−1. A principal component analysis (PCA) (99.9 % of variance explained, PC1 + PC2) showed a grouping pattern that related the HLA and HLAw structural characteristics with the root parameters evaluated (Fig. 5). For HLAw, at both 40 and 60 mg (C) L−1, the carbons from saturated aliphatic structures (CAlkyl-H,R) and carbonyl (CC=O) and carboxyl groups (CCOOH,R) were grouped with the number of roots developed by the plants, root volume and diameter, and growth of smaller root (0.5 < T ≤ 1.5, 0.5 < L ≤ 1.5, and 1.5 < T ≤ 3.5) (negative PC1 values 75 %). In contrast, the aromatic (CAr-H,R; CAr-O,N) and substituted aliphatic (CAlq-O,N; CAlkyl-O; CAlkyl-di-O) structures in HLA-40 were grouped with the development of larger roots (1.5 < L ≤ 3.5, L > 3.5, and T > 3.5) (positive PC1 values 75 %). The humified residual (HR) fraction also stimulated root growth and development in rice plants (Fig. 6). Applied at a 1:4 ratio (w/w; sand/HR), HR significantly stimulated root length, surface area, number of roots, and root volume compared with the control treatment.

59–91 CAlkyl-O 15.60 10.75 26.31 HLA 29.04 70.95 0.60

91–110 CAlkyl-di-O 9.17 4.30 7.36

110–142 CAr-H,R 20.18 11.82 8.42 HLAw 14.11 85.88 0.69

142–156 CAr-O,N 8.26 3.22 2.10

156–186 CCOO-H,R 12.84 12.90 8.42 HR 10.52 89.47 0.72

186–230 CC=O 1.83 7.52 2.10


Fig. 3 Micrographs obtained by scanning electron microscopy (SEM) of the humic-like acid fraction. HLA (a and b), HLAw (c and d) and the residual fraction (HR) (e and f)

The HR fraction exhibited a high and significantly posi t i v e e ffe c t o n s m a l l e r ro o t s (0 . 5 < L ≤ 1. 5 , 1.5 < L ≤ 3.5, 0.5 < T ≤ 3.5). These findings with Fig. 4 Root parameters in rice plants by applying of HA fractions (HLA and HLAw). L length, T number of roots classified among the different lengths. Treatments with different letter indicate statistical differences between mean value, according to Tukey’s test (p < 0.05). Bars represent the mean value ± standard error (SE) for five replicates (n = 25)

the decreased root diameter observed from the HR application indicate that HR exerts a more pronounced effect on root emission than on root development.


Fig. 5 Principal component analysis (PCA) performed from the distribution in type of carbon present in the spectra 13C CP MAS NMR of both HA (HLA and HLAw) and its effects on root parameters of rice plants. L length, T number of roots classified among the different lengths

The three studied fractions (HLA, HLAw, and HR) stimulated biomass production from both leaves and roots in rice plants (Fig. 7). However, HR stimulation of biomass production was not as pronounced as stimulation of root development, which indicates more pronounced effects from HR on root emission and growth. Conversely, both HLA (40 mg (C) L−1) and HLAw (60 mg (C) L−1) highly stimulated biomass production in both roots and leaves. We did not observe

Fig. 6 Root parameters in rice plants by applying the residual solid HR. L length, T number of roots classified among the different lengths. Treatments with different letter indicate statistical differences between mean value, according to Tukey’s test (p < 0.05). Bars represent the mean value ± standard error (SE) for five replicates (n = 25)

effects from HLAw at 40 mg (C) L−1 compared with the control and remaining treatments. Root stimulation by HA has been repeatedly reported for HA from different sources. Mora et al. (2010, 2012) when applying leonardite sedimentary HA found increases in root growth as well as root and leaf dry mass in cucumber (Cucumis sativus L. cv. Ashley), and HA isolated from peat and compost increased root emergence and number in Panicum virgatum L. and Arabidopsis (Schmidt et al. 2007; Traversa et al. 2014). However, this study analyzed the bioactivity of HA from VC produced using different extraction methods, how the different extraction methods change the HA structure, and how these changes determine which root parameters are stimulated and the intensity of the stimulation. In addition, the bioactivity of the residual fraction generated by HS extraction from VC has not been previously reported. These results are in line with those reported by Aguiar et al. (2013), which shows that methoxyl, aryl, O-aryl, and carboxyl groups are structures positively correlated with root growth. Moreover, Scaglia et al. (2016) reports that HA from VC with like-auxin activity are related with molecules/substances type carboxylic acids and amino acids. Recent studies published by García et al. (2016) showed that carboxylic and aromatic structures with low structural functionalization in HA from VC and histosoil also correlates positively with growth of

Author's personal copy J Soils Sediments Fig. 7 Root and shoot dry weight of rice plants by applying the residual solid HR, HLA and HLAw. Treatments with different letter indicate statistical differences between mean value, according to Tukey’s test (p < 0.05). Bars represent the mean value ± standard error (ES) for five replicates (n = 25)

roots, whereas in HS, more functionalized structures such as carboxyl, aliphatic chain, and aromatic rings functionalized correlate with the production of roots in rice plants. Taking together these evidences and the results obtained in this study, it is observed that functionalized structures, type carboxyl (–CCOO-H,R), seem to be essential for bioactivity of the HS, at the same way, there seems to be a relationship of the functionalization in different structures with root growth.

4 Conclusions Humic acid fractions isolated from VC using the IHSS methodology (HLA) and water extraction method (HLAw) showed different structural characteristics. HLAw has a lower H/C ratio in its elemental composition and a higher relative amount of aliphatic structures (CAlkyl-H,R) observed by 13C CP-MAS NMR. In contrast, the HLA has a higher relative amount of aromatic structures (CAr-H,R; CAr-O,N) and a lower E4/E6 ratio observed by UV-Vis; also, bands corresponding to C=C aromatic groups observed by Fourier transform infrared spectroscopy (FTIR) were better resolved. This evidence suggests that HLA has greater aromatic characteristic than HLAw fraction. The fractions HLAw and HLA showed larger amount of carboxyl groups (CCOO-H,R) than in HR fraction. FTIR techniques confirmed that the presence of the bands around 1714 cm−1 was better resolved in HLAw than in HLA. The morphology of these fractions shown by SEM was also different, where HR showed a more irregular surface than HLA and HLAw. Interestingly, the structural changes produced from water extraction did not eliminate HLAw bioactivity, but a higher concentration of HLAw was necessary to produce an equivalent stimulus compared with HLA. Our results seem to indicate that although both fractions showed similar types of structures in their composition, differences in the predominant structures determined different effects on the root. Aliphatic and oxygenated compounds were predominant in

HLAw and are likely responsible for stimulating the initial root emission, whereas the more aromatic character of HLA likely stimulates larger root development. The HR solid fraction also showed that aliphatic compounds predominate and stimulate root growth and emission. These observations are useful for agriculture because HR does not have a defined use and few studies have considered HR. The results herein are highly relevant for scientists because they confirm that the intensity and type of HS bioactivity in plants are more related to the complexity than type of structures present. In addition, the results explain the positive effects of water-extracted liquid humus (vermicompost tea and liquid humus) on crops under agricultural production conditions and indicate a possible use for solid HR as a growth substrate, which would complete the use cycle for active vermicompost fractions.

Acknowledgments A.C.G. (sisFaperj 2012028010) thanks FAPERJ for his grant. A.C.G, R.L.L.B, and J.M.G.M thank the CNPq-CAPES for the PDJ scholarship and funding through the project Science without Borders—PVE A060/2013. The authors thank CAPES-MES project no. 46/2013, 215/13.

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