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Article
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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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§*
6 7
†
8
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
11
§
12
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
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Solos, Rua Jardim Botânico, 1024, 22460-000 Rio de Janeiro, Brazil
Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Núcleo de
15 16
*
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
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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
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promoter.
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Keywords: NMR; CG-MS; pyrolisis; vermicompost; humic acids.
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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
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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
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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).
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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
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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
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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
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sample. The products of thermochemolysis were analyzed by GC-MS. Chromatographic
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separations were carried out with a GC-MS QP2010 Plus instrument (Shimadzu, Tokyo,
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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
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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
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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
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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
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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
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MAS probe and operating at
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respectively. The samples (100–200 mg) were packed in 4-mm zirconia rotors with
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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
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were carried out using a cross-polarization time of 1.0 ms, an acquisition time of 25 ms,
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a cycle delay of 2 s and a high-power two-pulse phase modulation (TPPM) proton
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decoupling of 70 KHz. The Bruker Topspin 1.3 software (Bruker Biospin, Karlsruhe,
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Germany) was used to collect and process the spectra. All the free induction decays
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(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
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intervals: 190–160 ppm (carbonyls of ketones, quinones, aldehydes, and carboxyls),
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160–140 ppm (phenols and O-substituted aromatic C), 140–110 ppm (unsubstituted
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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
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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
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in intervals related to polar groups (190–160 ppm and 110–45 ppm) indicate the degree
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C and 1H frequencies of 125 MHz and 500 MHz,
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of C hydrophilicity (HI); the HB/HI ratio was then calculated. The ratio between the
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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
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The total ion chromatograms derived from the thermochemolysis products
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obtained from the different humic acids extracted from the vermicomposted plant
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biomass residues and from cattle manure are shown in Figures 1 and 2.
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Thermochemolysis of humic acids released more than 200 different molecules,
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which were identified as methyl ethers and esters of natural compounds. The list of
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compounds identified in different pyrograms is showed in supporting information, while
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the data in Table 1 summarizes the amount and distribution of the major compound
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classes. Most of the humic components originated from higher plant residues that had
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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
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thermochemolysis revealed noticeable differences in humic acids composition,
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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
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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
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sunflower cake (Table 1). A large range of methylated guaiacyl derivatives were found
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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
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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
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degradation of lignin polymer, involving the ongoing oxidation of propyl chain, the
analysis:
P,
p-hydroxyphenyl;
G,
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(3-methoxy-4-
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corresponding homologues with integral hydroxylated side chains (G14/15, S14/15) are
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indicative of unaltered lignin components, which retain the typical β-O-4 ether
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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
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(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
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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
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acids was found in HA1. The main methyl ester derivatives were even- and odd-
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numbered long chains from plant and bacterial origin, respectively. HA5 was
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characterized by the presence of docosanol, a long chain alcohol from carbohydrate
255
enzymatic
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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-
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The most common nitrogenous compounds found in all humic acids, and in
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larger amounts in HA3, were 2,4-dimethoxyphenylamine, N-methoxycarbonyl, and
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benzenamine. Proline derivatives were found only in HA1 and HA5 while pyrrolidone
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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
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lignin derivatives in mature vermicomposts. The broad and strong signals found in the
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alkyl-C region (0–45 ppm) region, revealed the large incorporation of alkyl chains
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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
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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
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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
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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
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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
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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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Supporting information: list of compounds obtained by CG-MS analysis from
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different humic acids extracted from vermicomposts.
419
References
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Júnior, L.G.; Chagas, J.G.; Façanha, A.R.; Canellas, L.P. Bioactivity of humic acids
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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.
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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
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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