Molecular Characteristics of Humic Acids Isolated

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Molecularcharacteristics of humic acids isolated from vermicomposts and their relationship to bioactivity Dariellys Balmori-Martinez, Riccardo Spaccini, Natália Oliveira Aguiar, Etelvino henrique Novotny, Fábio Lopes Olivares, and Luciano Pasqualoto Canellas J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504629c • Publication Date (Web): 07 Nov 2014 Downloaded from http://pubs.acs.org on November 11, 2014

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Journal of Agricultural and Food Chemistry

373x183mm (96 x 96 DPI)

ACS Paragon Plus Environment


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Molecular Characteristics of Humic Acids Isolated from Vermicomposts and Their

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Relationship to Bioactivity

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Dariellys Martinez-Balmori†, Riccardo Spaccini‡, Natália Oliveira Aguiar§, Etelvino

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Henrique Novotny#, Fábio Lopes Olivares§ and Luciano Pasqualoto Canellas§*

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†

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Cuba

9

‡

Departamento de Química, Universidad Agraria de La Habana, San José de las Lajas,

Dipartimento di Agraria, Università di Napoli Federico II, Via Università 100, 80055

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Portici, Italy

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§

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Desenvolvimento de Insumos Biológicos para a Agricultura (NUDIBA), Av. Alberto

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Lamego, 2000, Campos dos Goytacazes, 28013-602 Rio de Janeiro, Brazil #Embrapa

14

Solos, Rua Jardim Botânico, 1024, 22460-000 Rio de Janeiro, Brazil

Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Núcleo de

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*

Corresponding author. Tel and Fax: +55 22 27397198; E-mail: [email protected].

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Abstract

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Vermitechnology is an effective composting method, which transforms biomass

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into nutrient-rich organic fertilizer. Mature vermicompost is a renewable organic

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product containing humic substances with high biological activity. The aim of this study

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was to access the chemical characteristics and the bioactivity of humic acids isolated

30

from different vermicomposts produced with either cattle manure, sugarcane bagasse,

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sunflower cake from seed oil extraction, or filter cake from a sugarcane factory. More

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than 200 different molecules were found and it was possible to identify chemical

33

markers on humic acids according the nature of the organic source. The large

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hydrophobic character of humic extracts and the preservation of altered lignin

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derivatives confer to humic acids the ability to induce lateral root emergence in maize

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seedlings. Humic acid-like substances extracted from plant biomass residues represent

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an additional valuable product of vermicomposting that can be used as a plant growth

38

promoter.

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Keywords: NMR; CG-MS; pyrolisis; vermicompost; humic acids.

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2



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Introduction

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The rising market for humic substances has invoked interest in composting as a

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possible economic source for their extraction, thus reducing the reliance on expensive

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fossil matrices, represented mainly by different kinds of mined lignite (e.g. leonardite).1

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Vermicomposting is the post-thermophilic biodegradation of organic material through

47

the interaction between earthworms and microorganisms.2 The final organic product,

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vermicompost, is a well stabilized, aesthetically pleasing, finely divided peat-like

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material with excellent structure, high porosity, good aeration and drainage, high water

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holding capacity, and has the potential to enhance plant growth.3 In particular, the

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mature vermicompost is significantly enriched in humic acids, which have a well-

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acknowledged capability to induce plant development, especially for the root systems.4-8

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The positive nutritional effects produced by applying mature vermicompost are well

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reported, such as enhancement of plant growth and amelioration of the physical

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structure of soil or plant medium. However, these positive effects are also related to

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both the large content and the high availability of biologically active plant promoter

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compounds that are represented by hormone-like humic substances produced during

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vermicomposting.7,9 The humic-like organic matter isolated from vermicomposts shows

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high biological activity which acts as an effective plant growth promoter.

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Furthermore, humic substances isolated from vermicompost effectively induced the

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synthesis of plasma membrane (PM) H+-ATPase in a typical auxin-like response,

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thereby significantly enhancing lateral root emergence.5 Moreover, an over-expression

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of major isoforms of PM H+-ATPase (Mha2) was revealed by the application of HS

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from vermicomposts on maize plants.16

10-18

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The effectiveness of humic extracts from vermicompost as plant growth

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promoters strongly depends on the quality of the raw organic biomass and on the 3


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composting stage, which affect the final molecular composition of humified organic

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compounds. The detailed molecular characterization of humified constituents formed

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during the composting process appears to be an essential requirement for evaluating the

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role of humic components in agricultural and environmental processes. Non-destructive

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spectroscopic methods such as cross-polarization magic-angle spinning (CP-MAS) 13C

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nuclear magnetic resonance (13C-NMR) spectroscopy is extensively used to identify

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content, distribution, and biochemical modification of the molecular components in

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natural organic substrates and compost materials.19,20 The physiological responses of

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plants to humic acids extracts from 45-day-old vermicomposts from different organic

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biomasses has been related to the hydrophobic molecular characteristic of the humic

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components, as measured by solid-state NMR analysis.20,21 The application of a

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multivariate statistical approach to the solid-state NMR spectra of VC humic extracts,

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revealed that the main organic functional groups associated with plant bioactivity were

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those related to lignin moieties, found at 56, 124, 148, and 153 ppm, as well as to

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COOH groups at 174 ppm.22

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Complementary molecular characterization of complex matrices may be

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obtained by the combination of NMR spectra with structural information provided by

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thermally assisted hydrolysis and methylation reactions (thermochemolysis) followed

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by gas chromatography-mass spectrometry (GC-MS).24,25 Pyrolysis in the presence of

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tetramethylammonium hydroxide involves the controlled partial cleavage of chemical

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bonds with the simultaneous solvolysis and methylation of ester and ether bonds present

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in natural organic materials, thereby enhancing both thermal stability and

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chromatographic detection of different polar functional groups.26 Moreover, the off-line

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technique allows the analysis of large quantities of solid material and thus a more

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representative sample, and more effective qualitative and quantitative measurements of 4



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structural components and their relationship with biological activity.27 The aim of this

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study was to evaluate by

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characteristics of HAs isolated from five mature vermicomposts of different

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composition, and to measure their bioactivity by the emergence of lateral roots in maize

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seedlings.

13

C-NMR spectroscopy and thermochemolysis the molecular

97 98

Material and methods

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Preparation of vermicomposts

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Five different vermicomposts were prepared using the following different

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substrates: (1) cattle manure; (2) cattle manure and sugar cane bagasse (1:1 w/w as dry

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mass); (3) cattle manure and sunflower cake (1:1 w/w as dry mass); (4) cattle manure,

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sugarcane bagasse, and sunflower cake (1:1:1 w/w/w as dry mass); and (5) filter cake

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from a sugarcane factory. Each organic residue was placed on a concrete cylinder (100-

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cm internal diameter) with a 150-L capacity using 2 replicates (2 cylinders per

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treatment). The humidity was maintained at 65–70% by weekly addition of water

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followed by mixing. After approximately 1 month of composting, the earthworm

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Eisenia foetida was introduced at a ratio of 5 kg of worms per cubic meter of organic

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residue. At the end of the transformation process, the worms were removed attracting

110

them to a pile of fresh organic residue (cattle manure) in a corner of the container. The

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VCs were air-dried, powdered with a ball mill, and sieved through a 500-µm mesh.

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Total organic carbon (OC) and total nitrogen (TN) contents were determined by dry

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combustion using an automatic CHN analyzer (Perkin-Elmer 2400 Series, Norwalk,

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CT).

115 116

Extraction of humic acids 5


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The humic acids were extracted and purified as reported previously5. Briefly, 10

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volumes of 0.5 M NaOH were mixed with 1 volume of earthworm compost under a N2

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atmosphere and shaken overnight. After 12 h, the suspension was centrifuged at 5,000 g

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and acidified (pH 1.5) using 6 M HCl. This suspension was allowed to settle overnight,

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the precipitated humic acids were recovered by centrifugation at 5,000 g and

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resolubilized in 0.5 M NaOH and precipitated by acidification (pH 1.5) with 6 M HCl

123

and the resulting pellet after centrifugation at 5,000 g was mixed with 10 volumes of a

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0.1 M HCl and 0.3 M HF. After centrifugation at 5,000 g for 15 min, the precipitate was

125

repeatedly washed with water until a negative test against AgNO3 was obtained. The pH

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of the final suspension was adjusted to pH 7.0 with 0.01 M KOH then dialyzed against

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deionized water using a 1 kD cutoff membrane (Thomas Scientific, Swedesboro, NJ),

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and lyophilized. Five humic acids (HA1, HA2, HA3, HA4, and HA5) were obtained

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from the respective vermicomposts (1–5) described above.

130 131

Characterization of HA

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Off-line thermochemolysis and GC-MS analysis

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About 100 mg of purified humic acid was placed in a quartz boat and moistened

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with 0.5 mL of tetramethylammonium hydroxide (25% in methanol) solution. After

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drying the mixture under a gentle stream of nitrogen for about 10 min, the sample was

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introduced into a Pyrex tubular reactor (50 cm × 3.5 cm internal diameter) and heated at

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400 °C for 30 min in a furnace. The products released by thermochemolysis were

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continuously transferred by a flow of helium (20 mL/min) into 2 successive chloroform

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(50 mL) traps kept in an iced salt bath. The chloroform solutions were combined in a

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round flask and concentrated by rotoevaporation under reduced pressure. The residue

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was redissolved in 1 mL of chloroform and transferred to a glass vial for GC-MS 6



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analysis. Two thermochemolysis replicates were carried out for each humic acids

143

sample. The products of thermochemolysis were analyzed by GC-MS. Chromatographic

144

separations were carried out with a GC-MS QP2010 Plus instrument (Shimadzu, Tokyo,

145

Japan). The column used was 30 m × 0.25 mm id, 0.25 µm, Rtx-5MS WCOT.

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Chromatographic separation was achieved with the following temperature program: 60

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°C for 1 min (isothermal), raised at 7 °C/min to 100 °C and then at 4 °C/min to 320 °C,

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followed by 10 min at 320 °C (isothermal). The carrier gas was helium at 1.90 mL/min,

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the injector temperature was 250 °C, and the split injection mode had a split flow at 30

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mL/min. Mass spectra were obtained in EI mode (70 eV), and scanning was in the range

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m/z 45–850 with a cycle time of 1 s. Compound identification was based on comparison

152

of mass spectra with the NIST library database, published spectra, and real standards.

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For quantitative analysis, due to the large variety of detected compounds with different

154

chromatographic responses, external calibration curves were built by mixing methyl

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esters and/or methyl ethers of the following molecular standards: tridecanoic acid,

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octadecanol, 16-hydroxyhexadecanoic acid, docosanoic acid, β-sitosterol, and cinnamic

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acid. Increasing amounts of standard mixtures were placed in a quartz boat and

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moistened with 0.5 mL of tetramethylammonium hydroxide (25% in methanol)

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solution. The same thermochemolysis conditions as for compost samples were applied

160

to

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thermochemolysis producs in the following indexes:26,28

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Ad/AlP (P6/P4):benzoic acid, 4- methoxy-, methyl ester/benzaldehyde,4-methoxy

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Ad/AlS (S6/S4): benzoic acid, 3,4,5- trimethoxy-, methyl ester/benzaldehyde, 3,4,5-

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trimethoxy

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Ad/AlG (G6/G4): benzoic acid,3, 4- dimethoxy-, methyl ester/ benzaldehyde,3,4-

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dimethoxy-

the

standards.

The

lignin

transformation

was

7


estimed

using

specific

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ΓG [G6/(G14+G15)]: benzoic acid, 3,4- dimethoxy-, methyl ester/1,2-dimethoxy-

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4(1,2,3-trimethoxypropyl) benzene

169 170

Solid-state CP-MAS 13C-NMR spectroscopy

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The C functionality distributions of the humic acids samples were determined by

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solid-state CP-MAS 13C-NMR spectroscopy. The spectra were acquired with an Avance

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500 MHz (Bruker, Karlsruhe, Germany) spectrometer equipped with a 4-mm wide-bore

174

MAS probe and operating at

175

respectively. The samples (100–200 mg) were packed in 4-mm zirconia rotors with

176

Kel–F caps, which were spun at 13 ± 1 kHz. The spectra were acquired by the ramped

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CP-MAS method, with linear amplitude variation of the 1H pulse. The experiments

178

were carried out using a cross-polarization time of 1.0 ms, an acquisition time of 25 ms,

179

a cycle delay of 2 s and a high-power two-pulse phase modulation (TPPM) proton

180

decoupling of 70 KHz. The Bruker Topspin 1.3 software (Bruker Biospin, Karlsruhe,

181

Germany) was used to collect and process the spectra. All the free induction decays

182

(FIDs) were transformed by applying a 4 k zero filling and a line broadening of 75 Hz.

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The spectra were normalized by area and integrated in the following 13C chemical shift

184

intervals: 190–160 ppm (carbonyls of ketones, quinones, aldehydes, and carboxyls),

185

160–140 ppm (phenols and O-substituted aromatic C), 140–110 ppm (unsubstituted

186

aromatic C and olefinic C), 110–95 ppm (anomeric C), 95–65 ppm (O-alkyl systems),

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65–45 ppm (methoxy substituent; N-alkyl groups), and 45–0 ppm (alkyl C, mainly CH2

188

and CH3). The relative areas of the alkyl (45–0 ppm) and aromatic (160–110 ppm)

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components were summed to represent the proportion of hydrophobic C in the humic

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samples (degree of hydrophobicity, HB). Similarly, the summation of the relative areas

191

in intervals related to polar groups (190–160 ppm and 110–45 ppm) indicate the degree

13

C and 1H frequencies of 125 MHz and 500 MHz,

8



192

of C hydrophilicity (HI); the HB/HI ratio was then calculated. The ratio between the

193

signal areas in the 45–65 ppm interval (methoxyl-C) over those in the 140–160 ppm

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range (O-aromatic-C), denoted as the lignin ratio, was used to discriminate the

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contribution of lignin components.21

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Bioactivity of HA

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Detailed experimental protocols for lateral root emergence and acidification of

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the growth medium for the maize seedling have been reported.21 Briefly, maize

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seedlings with homogeneous root length (1 ± 0.2 cm) were treated or not (control) for

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48 h with different humic acids applied at a concentration of 2 mM C, and adjusted to

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pH 7.00 with diluted HCl or NaOH. Next, seedlings were washed abundantly with

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deionized water and transferred to a 2 mM CaCl2 solution at pH 7.00 ± 0.01. After 48 h

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the seedlings were collected. Then, the seedling roots were digitalized and the number

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of lateral roots was counted. We used 5 pots as replicates (n = 5) with 5 seedlings in

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each pot.

206 207

Results and Discussion

208

The total ion chromatograms derived from the thermochemolysis products

209

obtained from the different humic acids extracted from the vermicomposted plant

210

biomass residues and from cattle manure are shown in Figures 1 and 2.

211

Thermochemolysis of humic acids released more than 200 different molecules,

212

which were identified as methyl ethers and esters of natural compounds. The list of

213

compounds identified in different pyrograms is showed in supporting information, while

214

the data in Table 1 summarizes the amount and distribution of the major compound

215

classes. Most of the humic components originated from higher plant residues that had

216

been bio-stabilized by microbial activity and were represented by lignin, alkyl 9


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biopolymers,

nitrogenous

compounds,

terpenes,

and

sterol

products.

The

218

thermochemolysis revealed noticeable differences in humic acids composition,

219

presumably reflecting the variable source of organic residues for vermicompost

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production. HA2 and HA3 showed the largest amount of lignins and nitrogenous

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compounds, whereas HA1 and HA4 were characterized by a large presence of C-alkyl

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compounds, and HA5 was characterized by terpenes and sterol derivatives.

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The various lignin components released by thermochemolysis from humic acids

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were associated with the current symbolism for basic lignin structures used in

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thermochemolysis

226

hydroxyphenyl); and S, syringyl (3,5-dimethoxy-4-hydroxyphenyl).26 As expected, the

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most representative lignin compound found among the pyrolyzed products for all humic

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acids was a propenoic acid derivative (2-propenoic acid, 3-(4-methoxyphenyl)-methyl

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ester (P18), which is a basic component of lignified tissues of herbaceous crops and

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grasses. The lignin compounds decrease in the following manner: HA2 > HA5 > HA1 >

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HA3 > HA4. HA2 obtained from sugarcane bagasse and cattle manure showed 65.7

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times more lignin than HA4 obtained from the mixture of bagasse, cattle manure, and

233

sunflower cake (Table 1). A large range of methylated guaiacyl derivatives were found

234

in all HAs, although at lower concentration with respect to the propenoic acid monomer,

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whereas limited distribution and lower abundance were shown by syringyl units. Useful

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specific components found in all humic extracts may be associated with the presence of

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either microbially processed organic materials or to undecomposed plant debris. The

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extent of lignin decomposition may be estimated by structural indexes that are based on

239

the relative amount of specific thermochemolysis products.26-28 While the aldehydic (G4

240

and S4) and acidic (G6 and S6) forms of lignin structures result from progressive

241

degradation of lignin polymer, involving the ongoing oxidation of propyl chain, the

analysis:

P,

p-hydroxyphenyl;

G,

10


guaiacyl

(3-methoxy-4-


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corresponding homologues with integral hydroxylated side chains (G14/15, S14/15) are

243

indicative of unaltered lignin components, which retain the typical β-O-4 ether

244

intermolecular linkages. Therefore the indexes obtained (Table 1) by dividing the

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amount found for the acidic structures over that of, respectively, G4 and S4 aldehydes

246

(Ad/AlG = G6/G4, Ad/AlS = S6/S4) and for the global yield of threo/erythro isomers

247

(ΓG = G6/[G14 + G15]) currently used as indicators of the bio-oxidative transformation

248

of lignin polymers. The larger values of dimensionless indexes, the wider the

249

decomposition process of lignin substrates. The largest amount of fatty acids identified

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as methyl ester derivatives were observed in HA2 isolated from sugarcane bagasse

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followed by HA5 from filter cake. However, a larger diversity of methyl esters of fatty

252

acids was found in HA1. The main methyl ester derivatives were even- and odd-

253

numbered long chains from plant and bacterial origin, respectively. HA5 was

254

characterized by the presence of docosanol, a long chain alcohol from carbohydrate

255

enzymatic

256

hydroxydehydroabietic acid was found only in HA2 and β-sitosterol in HA3. Large

257

amounts of cholest-7-ene-3-ol, acetate was found in HA5 and lanost-8-ene-3,7-dione in

258

HA2.

reactions.

Squalene

was found in

all

humic

acids while

15-

259

The most common nitrogenous compounds found in all humic acids, and in

260

larger amounts in HA3, were 2,4-dimethoxyphenylamine, N-methoxycarbonyl, and

261

benzenamine. Proline derivatives were found only in HA1 and HA5 while pyrrolidone

262

and pyrimidone derivatives were found only in HA2 and HA3. Few compounds derived

263

directly from carbohydrates were found in the samples, and only as furan derivatives.

264

The

13

C-NMR spectra were characterized by a sharp resonance in the 45–65

265

ppm range for all humic acids (Figure 3). The main signal centered around 55 ppm,

266

associated with the methoxy substituents in the aromatic ring of guaiacyl and syringyl 11


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components of lignified tissues of plants, which clearly indicate the incorporation of

268

lignin derivatives in mature vermicomposts. The broad and strong signals found in the

269

alkyl-C region (0–45 ppm) region, revealed the large incorporation of alkyl chains

270

pertaining to different components. The peaks at 16 and 23 ppm may be derived mainly

271

from CH3- and CH2- groups of various lipid compounds, such as waxes, polyesters, and

272

phospholipids. In addition to the peaks between 0 and 30 ppm, many distinct signals

273

were shown in the broad alkyl-C region around 30–45 ppm with signals at 30, 32, 39,

274

and 44 ppm, which indicate the simultaneous presence of different alkyl chains from

275

linear and branched fatty acid and peptide derivatives. The inclusion of N-containing

276

organic compounds was stressed by the peaks positioned at 40–44 ppm, which were

277

assigned to both Cα and Cβ in amino acids.20 Moreover a different preservation of

278

nitrogen components in the humic extracts were also suggested by the comparison of

279

the intense peak at 55 ppm, with a less pronounced intensity of the O-aromatic

280

functional groups in the 140–160 interval, found in the 13C-NMR spectra of HA2, HA3

281

and HA4 (Figure 3 and Table 2). Besides the lignin structures, the signals included in

282

the 45–60 ppm chemical shift range may also result from the C–N bonds in amino acid

283

moieties. In this respect the comparison of signal intensity in the 45–60 ppm interval

284

over that in the 140–160 ppm range, may be helpful for a more accurate assignment of

285

methoxyl and phenolic resonances. This dimensionless index, hereby denoted as lignin

286

ratio, has been used to improve discrimination between signals from lignin units

287

characteristic of other phenolic components or peptidic moieties.20 While a sharp lower

288

ratio of less than 1 is usually associated with the inclusion of tannin and polyphenol

289

constituents to the global O-aromatic-C signals, the opposite prevalence of upper

290

fractional part indicates the contribution of C–N bonds in the 45–60 ppm area.

291

Therefore, the discrepancy between methoxyl-C and phenolic-C signals, summarized by 12



292

the larger lignin ratio found in HA2, HA3, and HA4 humic extracts (Table 2), suggests

293

a greater incorporation of peptidic moieties and a lesser preservation of lignin

294

derivatives in the stabilized organic fractions of sugarcane bagasse, sunflower cake, and

295

mixed mature vermicompost.

296

The different resonances in the O-alkyl-C region (65–110 ppm) are currently

297

assigned to monomeric units in oligo and polysaccharide chains of plant tissue. The

298

intense signal around 72 ppm corresponds to the overlapping resonances of carbons 2,

299

3, and 5 in the pyranoside structure in cellulose and some hemicelluloses, whereas the

300

signal at 104 ppm is assigned to the anomeric carbon 1 of the glucose unit in cellulose.

301

The shoulders localized around 62–65 and 84–88 ppm result from carbons 6 and 4 of

302

monomeric units, respectively. The low-ﬁeld resonances (higher chemical shift) of each

303

couple indicate the presence of crystalline forms of cellulose, while the high-ﬁeld

304

resonances are assigned to either amorphous cellulose or hemicellulose structures.

305

Besides the signals usually assigned to cellulose, the spectra of different HAs revealed 3

306

additional resonances around 93, 106, and 110 ppm. These signals may be related,

307

respectively, to the di-O-alkyl-C of monomeric units of simple carbohydrates and to the

308

C1 of either hemicellulose or pectic polysaccharides chains contained in cell walls of

309

plants, such as α-1,5 arabinan, β-1,4 galactan, and α-1,4 galacturonan.

310

In the aromatic/olefinic-C region (110–145 ppm), the different resonances

311

around 120 and 123 ppm are related to unsubstituted and C-substituted aryl carbon

312

pertaining to both lignin monomers and ring components of polyphenols. The signal

313

intensity shown by the specific O-aromatic region (145–160 ppm) in HAs, confirms the

314

incorporation of O-substituted ring carbon derived from different lignin structures. The

315

evident resonances shown at 152 (sharp) and 158-ppm chemical shift range are usually

316

assigned to carbons 3, 4, and 5 in the aromatic ring of lignin components, with carbon 3 13


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and 5 being coupled to the corresponding methoxyl substituents. Finally, the intense

318

signal in the carbonyl region (160–190 ppm) at 174 ppm suggests the contribution of

319

carbonyl groups of peptide bonds of amino acid moieties in all the humic materials. The

320

relative distribution of organic functional groups and the hydrophobic index indicate

321

that the HAs from cattle manure and filter cake underwent a more advanced

322

humification process, with a final large preservation of recalcitrant hydrophobic

323

components. Conversely the lignin ratio and the larger amount of polysaccharides found

324

in HA2, HA3, and HA4 (Figure 3) revealed a steady maintenance of biolabile

325

compounds in the final mature vermicompost, with the mixed vermicompost showing a

326

lower hydrophobic character (Table 2).

327

The results of the root growth biological assay represented by humic acids

328

application were shown in Figure 4. All of the humic acids from mature vermicomposts

329

showed an improvement in the number of lateral roots ranging from 36 to 135% with

330

respect to control plants. However, HA4 have no significant differences related to the

331

control by the mean test applied. The lowest and no significant effect was found for

332

HA4 extracted from the mixture of cattle manure, bagasse, and sunflower cake. The

333

among those humic acids that produced significant bioactivity effect we have humic

334

acids re isolated from bagasse (HA2) and sunflower cake (HA3), which enhanced the

335

emergence of lateral roots in a very similar range from 51% to 58%, respectively. The

336

HA1 extracted from cattle manure revealed an increase of 98%, while the larger

337

promotion was associated with HA5 from filter cake vermicompost.

338

Vermicomposting technology, using earthworms as versatile natural bioreactors

339

for effective recycling of organic wastes to the soil, is an environmentally suitable

340

method to convert residual biomass into nutrient-rich composts for crop production.29

341

Considerable work has been carried out on vermicomposting processes of various 14



342

organic materials such as animal manures, agricultural wastes, forestry wastes, city leaf

343

litter and food residues, sewage sludge, and industrial wastes such as paper pulp and

344

distillery wastes. Vermicomposting represents an important environmental service for

345

waste recycling coupled with the generation of valuable products.29 The results of 13C-

346

NMR spectroscopy and off-line thermochemolysis GC-MS indicate that despite the

347

intense biochemical transformation process of accelerated humification by earthworms,

348

the final humic products retained the chemical features inherited from the original

349

organic source. Contrary to the indications of 13C-NMR spectra, a relative low yield of

350

carbohydrates was detected among the pyrolysis products. The lack of polysaccharide

351

compounds was already noted in applications of thermochemolysis on plant woody

352

tissues and soil organic matter.24,26 These authors pointed out that the large amount of

353

thermally unstable hydroxyl functional groups in polysaccharide chains make the

354

pyrolysis technique less suitable for the effective detection of carbohydrates in complex

355

geochemical matrices. It is thus conceivable that for compost samples the setup of

356

thermochemolysis parameters may be highly selective for lignin and alkyl components,

357

and may reduce the simultaneous identification of carbohydrate units from cellulose.

358

Such selective detection reinforces the use of different approaches for a more complete

359

compound inventory associated with the supramolecular structure of humic substances.

360

The different humic acids showed a variable effect on the emergence of lateral

361

roots (Figure 4), and were characterized by an uneven incorporation of alkyl and

362

aromatic hydrophobic components (Tables 1 and 2). To date, the most hydrophobic

363

humic acids were isolated from cattle manure and filter cake biomasses, and they

364

provided the best stimulation of lateral root emergence (Figure 4). In contrast, the root

365

biostimulation effect was decreased by humic acids from sugarcane bagasse, sunflower

366

cake, and mixed materials, which were characterized by the progressive lowering of 15


Page 16 of 30

Page 17 of 30


367

hydrophobic character (Table 2). These results are consistent with the previous findings

368

that humic fractions with a larger hydrophobicity provide a steady and high bioactivity,

369

and the presence of aromatic and aliphatic components, such as lignin compounds and

370

methyl esteres derivatives, are closely related to the ability of humic acids to induce

371

lateral root emergence.23,27,30,31 An apparent inconsistency with these previous findings

372

was presented in the current thermochemolysis results, which showed that the largest

373

lignin content was for HAs from sugarcane bagasse (Table 1). However, the most

374

abundant aromatic monomers found in the pyrograms of HA2 were 3-(4-

375

methoxyphenyl)-2-propenoic (P18) and 3-(4,5-dimethoxyphenyl)-2-propenoic (G18)

376

derivatives, which accounted for about the 40% of the total lignin content (Table 1).

377

These molecules originated from either the side chain oxidation of lignin units or from

378

the aromatic domains of plant biopolymers, whose important relative contribution in

379

herbaceous plants may have exaggerated the total content of lignin in the HAs from

380

sugarcane, compared to those from cattle manure and filter cake. Moreover the higher

381

values of lignin structural index found in the pyrograms of HA2 and HA5 (Table 1),

382

suggest the occurrence of an intense decomposition process of lignin biopolymers in the

383

humic fraction from cattle manure and filter cake. Therefore, the most advanced

384

humification may have promoted a preferential accumulation of small and active

385

aromatic fragments bound to the supramolecular humic acids structure, which should

386

allow a more prompt physiological response compared to the partial undecomposed and

387

rigid lignin residues.16

388

In addition to the content of specific molecules, the ratio of hydrophobic to

389

hydrophilic moieties is considered an important characteristic for the bioactivity of

390

humic extracts. The role of humic hydrophobicity may be explained by the selective

391

preservation, in humic recalcitrant compartments, of active biofragments that may be 16



392

successively released by conformational changes of humic associations in solution.8,19,21

393

The reduced microbial and enzymatic activity provided by the hydrophobic domains is

394

assumed to contribute to the protection of biolabile hydrophilic compounds which may

395

become available to the plant root system by means of cell exudation in the

396

rhizosphere.28,32 For example, plants treated with humic substances showed enhanced

397

exudation of organic acids, thereby leading to a modification of structural arrangement

398

and to a release of active molecules in the rhizosphere.22

399

The present results confirm that mature vermicompost is a viable way to recycle

400

agricultural biomass and to demonstrate that acting as a source of bioactivity, humic

401

extracts represent an additional valuable biological product for a wide range of

402

agricultural practices.18 Notwithstanding the modification by the composting processes,

403

the final humic substances retained a chemical composition strongly related to the

404

composition of the initial biomass. Although the role of hydrophobic humic components

405

needs further evaluation to gain a deeper insight into the structure-activity relationship,

406

the association of detailed molecular characterization and bioactivity assays are

407

unavoidable requirements for a more accurate and valuable utilization of humic material

408

as plant growth promoters and for a comprehensive understanding of the interaction

409

between plant and soil organic matter.

410 411

Acknowledgements

412

The work was supported by Conselho Nacional de Desenvolvimento Científico e

413

Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro

414

(FAPERJ), Instituto Nacional de Ciência e Tecnologia (INCT) para a Fixação Biológica

415

de Nitrogênio, Internacional Foundation of Science (IFS) and OCWP.

416 17


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417

Supporting information: list of compounds obtained by CG-MS analysis from

418

different humic acids extracted from vermicomposts.

419

References

420

(1) Valdrighi, M.M.; Pera, A.; Agnolucci, M.; Frassinetti, S.; Lunardi, D.; Vallini, G.

421

Effects of compost-derived humic acids on vegetable biomass production and

422

microbial growth within a plant (Cichorium intybus)-soil system: a comparative

423

study. Agric., Ecosyst. Environ. 1996, 58, 133–144.

424

(2) Edwards, C.A.; Arancon, N.Q.; Sherman, R.L. Vermiculture technology:

425

earthworms, organic wastes, and environmental management. CRC Press, Boca

426

Raton, USA, 2010; p. 601.

427 428

(3) Ali, M.; Griffiths, A.J.; Williams, K.P.; Jones, D.L. Evaluating the growth characteristics of lettuce in vermicompost and green waste compost. Eur. J. Soil Biol. 2007, 43, S316-S319.

429

(4) Muscolo, A.; Bovalo, F.; Giomfriddo, F.; Nardi, S. Earthworm humi matter

430

produces auxin-like effects on Daucus carota cell growth and nitrate metabolism.

431

Soil Biol. Biochem. 1999, 31, 1303–1311.

432

(5) Canellas, L.P.; Okorokova-Façanha, A.; Olivares, F.L.; Façanha, A.R. Humic acids

433

isolated from earthworm compost enhance root elongation, lateral root emergence,

434

and plasma membrane H+-ATPase activity in maize roots. Plant Physiol. 2002, 130,

435

1951–1957.

436

(6) Arancon, N.Q.; Lee, S.; Edwards, C.A.; Atiyeh, R.M. Effects of humic acids and

437

aqueous extracts derived from cattle, food and paper-waste vermicomposts on

438

growth of greenhouse plants. Pedobiologia 2003, 47, 741–744.

439

(7) Arancon, N.Q.; Edwards, C.A.; Atiyeh, R.; Metzger, J.D. Effects of vermicomposts

440

produced from food waste on the growth and yields of greenhouse peppers.

441

Bioresour. Technol. 2004, 93, 139–144. 18



442

(8) Canellas, L.P.; Piccolo, A.; Dobbss, L.B.; Spaccini, R.; Olivares, F.L.; Zandonadi,

443

D.B.; Façanha, A.R. Chemical composition and bioactivity properties of size-

444

fractions separated from a vermicompost humic acid. Chemosphere 2010, 78, 457-

445

466.

446 447

(9) Warman, P.R.; Anglopez, M.J. Vermicompost derived from different feedstocks as a plant growth medium. Bioresour. Technol. 2010, 101, 4479–4483.

448

(10) Nardi, S.; Concheri, G.; Dell’agnola, G. Biological activity of humus. In. Humic

449

Substances in Terrestrial Ecosystems. Piccolo, A. Ed.;. Elsevier: Amsterdam, The

450

Netherlands, 1996; pp 361–40.

451 452

(11) Masciandaro, G.; Ceccanti, B.; Garcia, C. Soil agroecological management: fertirrigation and vermicompost treatments. Bioresour. Technol.1999, 59, 199–206.

453

(12) Dell’Agnola, G., Nardi, S. On overview of earthworm activity in the soil. In: On

454

earthworms selected symposia and monographs. Part 2; Bonvicini Pagliai A.M.,

455

Omodeo P. Eds.;. Mucchi Editore, Modena, Italy, 1987; pp 103–112.

456

(13) Muscolo, A.; Nardi, S. Auxin or auxin-like activity of humic matter. In: The Role

457

of Humic Substances in the Ecosystems and Environmental Protection. Drozd, J.,

458

Gonet, S.S., Senesi, N., Weber, J. Eds.;. Polish Society of Humic Substances,

459

Wroclaw, Poland, 1997; pp. 987–992.

460

(14) Canellas, L.P.; Martinez-Balmori, D.; Médici, L.O.; Aguiar, N.O.; Campostrini, E.;

461

Rosa R.C.C.; Façanha, A.R.; Olivares, F.L. A combination of humic substances and

462

Herbaspirillum seropedicae inoculation enhances the growth of maize (Zea mays

463

L.). Plant Soil. 2013, 366, 119–132.

464

(15) Atiyeh, R.M.; Lee, S.; Edwards, C.A.; Arancon, N.Q.; Metzger, J.D. The influence

465

of humic acids derived from earthworm-processed organic wastes on plant growth.

466

Bioresour. Technol. 2002, 84, 7–14. 19


Page 20 of 30

Page 21 of 30


467

(16) Quaggiotti, S.; Ruperti, B.; Pizzeghello, D.; Francioso, O.; Tugnoli, V.; Nardi, S.;.

468

Effect of low molecular size humic substances on nitrate uptake and expression of

469

genes involved in nitrate transport in maize (Zea mays L.). J. Exp. Bot. 2004, 55,

470

803-813.

471

(17) Zandonadi, D.B., Canellas, L.P., Façanha, A.R., Indoleacetic and humic acids

472

induce lateral root development through a concerted plasmalemma and tonoplast H+

473

pumps activation. Planta 2007, 225, 1583–1595.

474 475

(18) Canellas, L.P.; Olivares, F.L. Physiological responses to humic substances as plant growth promoter. Chem. Biol. Technol. Agric. 2014, 1, 3.

476

(19) Dobbss, L.B.; Canellas, L.P.; Olivares, F.L.; Aguiar, N.O.; Peres, L.E.P.; Azevedo,

477

M.; Spaccini, R.; Piccolo, A.; Facanha, A.R. Bioactivity of chemically transformed

478

humic matter from vermicompost on plant root growth. J. Agric. Food Chem. 2010,

479

58, 3681–3688.

480

(20) De Marco, A.; Spaccini, R.; Vittozzi, P.; Esposito, F.; Berg, B.; Virzo De Santo, A.

481

Decomposition of black locust and black pine leaf litter in two coeval forest stands

482

on Mount Vesuvius and dynamics of organic components analyzed through

483

conventional chemical methods and NMR spectroscopy. Soil Biol. Biochem. 2012,

484

51, 1–15.

485

(21) Aguiar, N.O.; Novotny, E.H.; Oliveira, A.,L.; Rumjanek, V.M.; Olivares, F.L.;

486

Canellas, L.P.. Prediction of humic acids bioactivity using spectroscopy and

487

multivariate analysis. J. Geochem. Explor. 2013, 129, 95–102.

488

(22) Canellas, L.P.; Dobbss, L.B.; Oliveira, A.L.; Chagas, J.G.; Aguiar, N.O.;

489

Rumjanek, V.M.; Novotny E.H.; Olivares F.L.; Spaccini R.; Piccolo A. Chemical

490

properties of humic matter as related to induction of plant lateral roots. Eur. J. Soil

491

Sci. 2012, 63, 315–324. 20



492

(23) Aguiar, N.O.; Olivares, F.L.; Novotny, E.H.; Dobbss, L.B.; Balmori, D.M.; Santos-

493

Júnior, L.G.; Chagas, J.G.; Façanha, A.R.; Canellas, L.P. Bioactivity of humic acids

494

isolated from vermicomposts at different maturation stages. Plant Soil 2013, 362,

495

161–174.

496 497

(24) Spaccini, R.; Sannino, D.; Piccolo, A.; Fagnano, M. Molecular changes in organic matter of a compost-amended soil. Eur. J. Soil Sci. 2009, 60, 287–296.

498

(25) Zhou, P.; Pan, G.X.; Spaccini R.; Piccolo, A. Molecular changes in particulate

499

organic matter (POM) in a typical Chinese paddy soil under different long-term

500

fertilizer treatments. Eur. J. Soil Sci. 2010, 61, 231–242.

501

(26) Spaccini, R.; Song, X.Y.; Cozzolino, V.; Piccolo, A. Molecular evaluation of soil

502

organic matter characteristics in three agricultural soils by improved off-line

503

thermochemolysis: The effect of hydrofluoric acid demineralisation treatment. Anal.

504

Chim. Acta. 2013, 802, 46–55.

505

(27) Martinez-Balmori, D.; Olivares, F.L.; Spaccini, R.; Aguiar K.P.; Araújo, M.F.;

506

Aguiar, N.O.; Guridi, F.; Canellas, L.P. Molecular characteristics of vermicompost

507

and their relationship to preservation of inoculated nitrogen-fixing bacteria. J. Anal.

508

Appl. Pyrolysis 2013, 104, 540–550.

509

(28) Song, X.Y.; Spaccini, R.; Pan, G.; Piccolo, A. Stabilization by hydrophobic

510

protection as a molecular mechanism for organic carbon sequestration in maize-

511

amended rice paddy soils. Sci. Total Environ. 2013, 458–460, 319–330.

512

(29) Padmavathiamma, P.K.; Li, L.Y.; Kumari, U.R. An experimental study of vermi-

513

biowaste composting for agricultural soil improvement. Bioresour. Technol. 2008,

514

99, 1672–1681.

515

(30) Canellas, L. P.; Zandonadi, D. B.; Busato, J. G.; Baldotto, M. A.; Simões, M. L.;

516

Martin-Neto, L.; Façanha, A. R.; Spaccini, R.; Piccolo, A. Bioactivity and chemical 21


Page 22 of 30

Page 23 of 30


517

characteristics of humic acids from tropical soils sequence. Soil Sci. 2008, 173,

518

624–637.

519

(31) Canellas, L.P.; Spaccini, R.; Piccolo, A.; Dobbss, L.B.; Okorokova-Façanha, A.L.;

520

Santos, G.A.; Olivares, F.L.; Façanha, A.R. Relationships between chemical

521

characteristics and root growth promotion of humic acids isolated from Brazilian

522

Oxisols. Soil Sci. 2009, 174, 611–624.

523

(32) Spaccini, R.; Piccolo, A.; Conte, P.; Haberhauer, G.; Gerzabek, M.H. Increased soil

524

organic carbon sequestration through hydrophobic protection by humic substances.

525

Soil Biol. Biochem. 2002, 34, 1839–1851.

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Table 1. Yields and Composition of Main Thermochemolysis Products Released from Humic Acids of Different Vermicomposts Class of Compounds

HA-1

HA-2

HA-3

HA-4

HA-5

-1

.........................................................mg.kg .................................................... Total Lignins

950 ± 66.5

4271 ±213.5

870 ± 69.6

65 ±3.2

2976 ±208.3

P

223 ±8.9

1067 ±74.7

196 ±5.8

8 ±0.2

799 ±33.9

G

406 ±8.1

2179 ±87.2

532 ±21.3

41 ±1.6

1228 ±98.2

S

242 ±9.4

775 ±62.0

107 ±2.1

16 ±0.3

708 ±63.7

P6/P4

2.3 ±0.07

0.9 ±0.02

1.1 ±0.02

Nd

1.4 ±0.04

G6/G4

2.6 ± 0.29

1.6 ±0.05

2.1 ±0.04

Nd

1.9 ±0.06

S6/S4

4.7 ±0.4

2.7 ±0.08

2.7 ±0.08

4.7 ±0.1

2.2 ±0.04

2.8 ±0.1

0.6 ±0.05

0.4 ±0.02

0.8 ±0.02

2.3 ±0.05

291 ±8.7

1274 ±25.5

239 ±19.1

228 ±9.1

942 ±47.1

Terpenes and steroids

40 ±11.8

130 ±3.9

118 ±2.4

212 ±17.0

305 ±12.7

Nitrogenous compounds

148 ±2.6

1618 ±113.3

409 ±28.6

49 ±2.4

818 ±57.3

Carbohydrates derivatives

12± 0.2

136 ±5.4

46 ±2.3

nd

218 ±15.3

ΓG Alkyl-C (fatty acids as methyl esters, alcohols, alkanes, alkenes)

P, p-hydroxyphenyl; G, guaiacyl and S, syringyl; The P6/P4, G6/G4, S6/S4 and ΓG are indexes to evaluate lignins transformation using specific thermochemolysis products as describe in Material and Methods. The values represent the means from two chromatograms followed by standard deviation. HA1, HA2, HA3, HA4 and HA5 are the humic acids isolated from vermicomposts produced with cattle manure, sugarcane bagasse, sunflower cake, the mixture of cattle manure, bagasse and sunflower cake, and sugarcane filter cake residue, respectively.

23


Page 25 of 30


Table 2. Integration area of 13C-CPMAS-NMR spectra from different humic acids (HAs): HA 1) Cattle manure; HA 2) Cattle manure and sugarcane bagasse; HA 3) Cattle manure and sunflower cake obtained after oil extraction, HA 4) Cattle manure, sugarcane bagasse and sunflower cake and HA 5) filter cake at 90 days of vermicomposting time. ----------------------------------------------------------------------Chemical shift (ppm)------------------------------------------------------------Humic acids 0-45 45-65 65-95 95-110 110-140 140-160 160-190 HB/HI HA-1 25.25 23.07 12.38 6.53 20.15 7.22 5.41 1.79 HA-2 20.77 21.31 19.61 9.53 17.62 5.86 5.30 0.79 HA-3 22.68 22.24 18.96 8.87 17.31 5.13 4.81 0.82 HA-4 19.08 22.81 20.46 8.96 18.53 5.51 4.65 0.76 HA-5 21.91 17.94 14.64 9.35 20.39 7.48 8.30 0.99

24



FIGURES CAPTIONS Figure 1. Total ion current (TIC) obtained from off-line pyrolysis GC-MS of humic acids isolated from vermicompost of: A. cattle manure and B. sugarcane bagasse.

Figure 2. Total ion current (TIC) obtained from off-line pyrolysis GC-MS of humic acids isolated from vermicompost of: A. sunflower cake, B. a mixture of cattle manure, sugarcane bagasse, and sunflower cake, and C. sugarcane filter cake.

Figure 3. CP-MAS 13C NMR spectra of humic acids from different vermicomposts: (HA1) cattle manure, (HA2) sugarcane bagasse, (HA3) sunflower cake, (HA4) the mixture of cattle manure, sugarcane bagasse, and sunflower cake, and (HA5) sugarcane filter cake.

Figure 4. The number of lateral roots in maize seedlings treated with humic acids from different vermicomposts: (HA1) cattle manure, (HA2) sugarcane bagasse, (HA3) sunflower cake, (HA4) the mixture of cattle manure, sugarcane bagasse, and sunflower cake, and (HA5) sugarcane filter cake. Different letters represent a significant difference by means Duncan test (p

Molecular Characteristics of Humic Acids Isolated

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