molecular characteristics of humic acids isolated from ... · molecular characteristics of humic...

Molecular Characteristics of Humic Acids Isolated fromVermicomposts and Their Relationship to BioactivityDariellys Martinez-Balmori,† Riccardo Spaccini,‡ Natalia Oliveira Aguiar,§ Etelvino Henrique Novotny,#

Fabio Lopes Olivares,§ and Luciano Pasqualoto Canellas*,§

†Departamento de Quımica, Universidad Agraria de La Habana, San Jose de las Lajas, Cuba‡Dipartimento di Agraria, Universita di Napoli Federico II, Via Universita 100, 80055 Portici, Italy§Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Nucleo de Desenvolvimento de Insumos Biologicos para aAgricultura (NUDIBA), Av. Alberto Lamego 2000, Campos dos Goytacazes, 28013-602 Rio de Janeiro, Brazil#Embrapa Solos, Rua Jardim Botanico 1024, 22460-000 Rio de Janeiro, Brazil

*S Supporting Information

ABSTRACT: Vermitechnology is an effective composting method, which transforms biomass into nutrient-rich organicfertilizer. Mature vermicompost is a renewable organic product containing humic substances with high biological activity. Theaim of this study was to assess the chemical characteristics and the bioactivity of humic acids isolated from differentvermicomposts produced with either cattle manure, sugar cane bagasse, sunflower cake from seed oil extraction, or filter cakefrom a sugar cane factory. More than 200 different molecules were found, and it was possible to identify chemical markers onhumic acids according to the nature of the organic source. The large hydrophobic character of humic extracts and thepreservation of altered lignin derivatives confer to humic acids the ability to induce lateral root emergence in maize seedlings.Humic acid-like substances extracted from plant biomass residues represent an additional valuable product of vermicompostingthat can be used as a plant growth promoter.

KEYWORDS: vermicompost, humic acids, NMR, GC-MS, pyrolysis

■ INTRODUCTION

The rising market for humic substances has invoked interest incomposting as a possible economic source for their extraction,thus reducing the reliance on expensive fossil matrices,represented mainly by different kinds of mined lignite (e.g.,leonardite).1 Vermicomposting is the post-thermophilic bio-degradation of organic material through the interactionbetween earthworms and microorganisms.2 The final organicproduct, vermicompost, is a well stabilized, aestheticallypleasing, finely divided peat-like material with excellentstructure, high porosity, good aeration and drainage, and highwater-holding capacity and has the potential to enhance plantgrowth.3 In particular, the mature vermicompost is significantlyenriched in humic acids, which have a well-acknowledgedcapability to induce plant development, especially for rootsystems.4−8 The positive nutritional effects produced byapplying mature vermicompost are well reported, such asenhancement of plant growth and amelioration of the physicalstructure of soil or plant medium. However, these positiveeffects are also related to both the large content and the highavailability of biologically active plant promoter compoundsthat are represented by hormone-like humic substancesproduced during vermicomposting.7,9 The humic-like organicmatter isolated from vermicomposts shows high biologicalactivity, which acts as an effective plant growth promoter.10−18

Furthermore, humic substances isolated from vermicomposteffectively induced the synthesis of plasma membrane (PM)H+-ATPase in a typical auxin-like response, thereby significantlyenhancing lateral root emergence.5 Moreover, an over-

expression of major isoforms of PM H+-ATPase (Mha2) wasrevealed by the application of HS from vermicomposts onmaize plants.16

The effectiveness of humic extracts from vermicompost asplant growth promoters strongly depends on the quality of theraw organic biomass and on the composting stage, which affectthe final molecular composition of humified organic com-pounds. The detailed molecular characterization of humifiedconstituents formed during the composting process appears tobe an essential requirement for evaluating the role of humiccomponents in agricultural and environmental processes.Nondestructive spectroscopic methods such as cross-polar-ization magic-angle spinning (CP-MAS) 13C nuclear magneticresonance (13C NMR) spectroscopy are extensively used toidentify content, distribution, and biochemical modification ofthe molecular components in natural organic substrates andcompost materials.19,20 The physiological responses of plants tohumic acid extracts from 45-day-old vermicomposts fromdifferent organic biomasses have been related to the hydro-phobic molecular characteristic of the humic components, asmeasured by solid-state NMR analysis.20,21 The application of amultivariate statistical approach to the solid-state NMR spectraof VC humic extracts revealed that the main organic functionalgroups associated with plant bioactivity were those related to

Received: September 24, 2014Revised: November 6, 2014Accepted: November 7, 2014Published: November 7, 2014

Article

pubs.acs.org/JAFC

© 2014 American Chemical Society 11412 dx.doi.org/10.1021/jf504629c | J. Agric. Food Chem. 2014, 62, 11412−11419

pubs.acs.org/JAFC

lignin moieties, found at 56, 124, 148, and 153 ppm, as well asto COOH groups at 174 ppm.22

Complementary molecular characterization of complexmatrices may be obtained by the combination of NMR spectrawith structural information provided by thermally assistedhydrolysis and methylation reactions (thermochemolysis)followed by gas chromatography−mass spectrometry (GC-MS).24,25 Pyrolysis in the presence of tetramethylammoniumhydroxide involves the controlled partial cleavage of chemicalbonds with the simultaneous solvolysis and methylation of esterand ether bonds present in natural organic materials, therebyenhancing both thermal stability and chromatographicdetection of different polar functional groups.26 Moreover,the off-line technique allows the analysis of large quantities ofsolid material and thus a more representative sample and moreeffective qualitative and quantitative measurements of structuralcomponents and their relationship with biological activity.27

The aim of this study was to evaluate by 13C NMRspectroscopy and thermochemolysis the molecular character-istics of HAs isolated from five mature vermicomposts ofdifferent composition and to measure their bioactivity by theemergence of lateral roots in maize seedlings.

■ MATERIALS AND METHODSPreparation of Vermicomposts. Five different vermicomposts

were prepared using the following different substrates: (1) cattlemanure; (2) cattle manure and sugar cane bagasse (1:1 w/w as drymass); (3) cattle manure and sunflower cake (1:1 w/w as dry mass);(4) cattle manure, sugar cane bagasse, and sunflower cake (1:1:1 w/w/w as dry mass); and (5) filter cake from a sugar cane factory. Eachorganic residue was placed on a concrete cylinder (100 cm internaldiameter) with a 150 L capacity using two replicates (two cylinders pertreatment). The humidity was maintained at 65−70% by weeklyaddition of water followed by mixing. After approximately 1 month ofcomposting, the earthworm Eisenia fetida was introduced at a ratio of 5kg of worms per cubic meter of organic residue. At the end of thetransformation process, the worms were removed by attracting themto a pile of fresh organic residue (cattle manure) in a corner of thecontainer. The VCs were air-dried, powdered with a ball mill, andsieved through a 500 μm mesh. Total organic carbon (OC) and totalnitrogen (TN) contents were determined by dry combustion using anautomatic CHN analyzer (PerkinElmer 2400 series, Norwalk, CT,USA).Extraction of Humic Acids. The humic acids were extracted and

purified as reported previously.5 Briefly, 10 volumes of 0.5 M NaOHwas mixed with 1 volume of earthworm compost under a N2atmosphere and shaken overnight. After 12 h, the suspension wascentrifuged at 5000g and acidified (pH 1.5) using 6 M HCl. Thissuspension was allowed to settle overnight, the precipitated humicacids were recovered by centrifugation at 5000g, resolubilized in 0.5 MNaOH, and precipitated by acidification (pH 1.5) with 6 M HCl, andthe resulting pellet after centrifugation at 5000g was mixed with 10volumes of a 0.1 M HCl and 0.3 M HF. After centrifugation at 5000gfor 15 min, the precipitate was repeatedly washed with water until anegative test against AgNO3 was obtained. The pH of the finalsuspension was adjusted to pH 7.0 with 0.01 M KOH and thendialyzed against deionized water using a 1 kDa cutoff membrane(Thomas Scientific, Swedesboro, NJ, USA), and lyophilized. Fivehumic acids (HA1, HA2, HA3, HA4, and HA5) were obtained fromthe respective vermicomposts (1−5) described above.Characterization of HA. Offline Thermochemolysis and GC-MS

Analysis. About 100 mg of purified humic acid was placed in a quartzboat and moistened with 0.5 mL of tetramethylammonium hydroxide(25% in methanol) solution. After the mixture had dried under agentle stream of nitrogen for about 10 min, the sample was introducedinto a Pyrex tubular reactor (50 cm × 3.5 cm i.d.) and heated at 400°C for 30 min in a furnace. The products released by

thermochemolysis were continuously transferred by a flow of helium(20 mL/min) into two successive chloroform (50 mL) traps kept in aniced salt bath. The chloroform solutions were combined in a roundflask and concentrated by rotoevaporation under reduced pressure.The residue was redissolved in 1 mL of chloroform and transferred toa glass vial for GC-MS analysis. Two thermochemolysis replicates werecarried out for each humic acid sample. The products ofthermochemolysis were analyzed by GC-MS. Chromatographicseparations were carried out with a GC-MS QP2010 Plus instrument(Shimadzu, Tokyo, Japan). The column used was a 30 m × 0.25 mmid, 0.25 μm, Rtx-5MS WCOT. Chromatographic separation wasachieved with the following temperature program: 60 °C for 1 min(isothermal), raised at 7 °C/min to 100 °C and then at 4 °C/min to320 °C, followed by 10 min at 320 °C (isothermal). The carrier gaswas helium at 1.90 mL/min, the injector temperature was 250 °C, andthe split injection mode had a split flow at 30 mL/min. Mass spectrawere obtained in EI mode (70 eV), and scanning was in the range m/z45−850 with a cycle time of 1 s. Compound identification was basedon comparison of mass spectra with the NIST library database,published spectra, and real standards. For quantitative analysis, due tothe large variety of detected compounds with different chromato-graphic responses, external calibration curves were built by mixingmethyl esters and/or methyl ethers of the following molecularstandards: tridecanoic acid, octadecanol, 16-hydroxyhexadecanoic acid,docosanoic acid, β-sitosterol, and cinnamic acid. Increasing amounts ofstandard mixtures were placed in a quartz boat and moistened with 0.5mL of tetramethylammonium hydroxide (25% in methanol) solution.The same thermochemolysis conditions as for compost samples wereapplied to the standards. The lignin transformation was estimatedusing specific thermochemolysis products in the following indices:26,28

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

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

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

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

Solid-State CP-MAS 13C NMR Spectroscopy. The C functionalitydistributions of the humic acid samples were determined by solid-stateCP-MAS 13C NMR spectroscopy. The spectra were acquired with anAvance 500 MHz (Bruker, Karlsruhe, Germany) spectrometerequipped with a 4 mm wide-bore MAS probe and operating at 13Cand 1H frequencies of 125 and 500 MHz, respectively. The samples(100−200 mg) were packed in 4 mm zirconia rotors with Kel−F caps,which were spun at 13 ± 1 kHz. The spectra were acquired by theramped CP-MAS method, with linear amplitude variation of the 1Hpulse. The experiments were carried out using a cross-polarizationtime of 1.0 ms, an acquisition time of 25 ms, a cycle delay of 2 s, and ahigh-power two-pulse phase modulation (TPPM) proton decouplingof 70 kHz. Bruker Topspin 1.3 software (Bruker Biospin, Karlsruhe,Germany) was used to collect and process the spectra. All of the freeinduction decays (FIDs) were transformed by applying a 4K zerofilling and a line broadening of 75 Hz. The spectra were normalized byarea and integrated in the following 13C chemical shift intervals: 190−160 ppm (carbonyls of ketones, quinones, aldehydes, and carboxyls),160−140 ppm (phenols and O-substituted aromatic C), 140−110ppm (unsubstituted aromatic C and olefinic C), 110−95 ppm(anomeric C), 95−65 ppm (O-alkyl systems), 65−45 ppm (methoxysubstituent; N-alkyl groups), and 45−0 ppm (alkyl C, mainly CH2 andCH3). The relative areas of the alkyl (45−0 ppm) and aromatic (160−110 ppm) components were summed to represent the proportion ofhydrophobic C in the humic samples (degree of hydrophobicity, HB).Similarly, the summation of the relative areas in intervals related topolar groups (190−160 and 110−45 ppm) indicates the degree of Chydrophilicity (HI); the HB/HI ratio was then calculated. The ratiobetween the signal areas in the 45−65 ppm interval (methoxyl-C) overthose in the 140−160 ppm range (O-aromatic-C), denoted the ligninratio, was used to discriminate the contribution of lignincomponents.21

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf504629c | J. Agric. Food Chem. 2014, 62, 11412−1141911413

Bioactivity of HA. Detailed experimental protocols for lateral rootemergence and acidification of the growth medium for the maizeseedling have been reported.21 Briefly, maize seedlings withhomogeneous root length (1 ± 0.2 cm) were treated or not (control)for 48 h with different humic acids applied at a concentration of 2 mMC and adjusted to pH 7.00 with diluted HCl or NaOH. Next, seedlingswere washed abundantly with deionized water and transferred to a 2mM CaCl2 solution at pH 7.00 ± 0.01. After 48 h, the seedlings werecollected. Then, the seedling roots were digitized, and the number oflateral roots was counted. We used five pots as replicates (n = 5) withfive seedlings in each pot.

■ RESULTS AND DISCUSSIONThe total ion chromatograms derived from the thermochemol-ysis products obtained from the different humic acids extractedfrom the vermicomposted plant biomass residues and fromcattle manure are shown in Figures 1 and 2.

Thermochemolysis of humic acids released more than 200different molecules, which were identified as methyl ethers andesters of natural compounds. The list of compounds identifiedin different pyrograms is shown in the Supporting Information,whereas the data in Table 1 summarize the amount anddistribution of the major compound classes. Most of the humic

components originated from higher plant residues that hadbeen biostabilized by microbial activity and were represented bylignin, alkyl biopolymers, nitrogenous compounds, terpenes,and sterol products. The thermochemolysis revealed noticeabledifferences in humic acid composition, presumably reflectingthe variable sources of organic residues for vermicompostproduction. HA2 and HA3 showed the largest amount oflignins and nitrogenous compounds, whereas HA1 and HA4were characterized by a large presence of C-alkyl compounds,and HA5 was characterized by terpenes and sterol derivatives.

Figure 1. Total ion current (TIC) obtained from offline pyrolysis GC-MS of humic acids isolated from vermicompost of (A) cattle manureand (B) sugar cane bagasse.

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



The various lignin components released by thermochemol-ysis from humic acids were associated with the currentsymbolism for basic lignin structures used in thermochemolysis

analysis: P, p-hydroxyphenyl; G, guaiacyl (3-methoxy-4-hydroxyphenyl); and S, syringyl (3,5-dimethoxy-4-hydroxy-phenyl).26 As expected, the most representative lignin

Table 1. Yields (Milligrams per Kilogram) and Composition of Main Thermochemolysis Products Released from Humic Acidsof Different Vermicomposts

compound classa HA1b HA2b HA3b HA4b HA5b

total lignins 950 ± 66.5 4271 ± 213.5 870 ± 69.6 65 ± 3.2 2976 ± 208.3P 223 ± 8.9 1067 ± 74.7 196 ± 5.8 8 ± 0.2 799 ± 33.9G 406 ± 8.1 2179 ± 87.2 532 ± 21.3 41 ± 1.6 1228 ± 98.2S 242 ± 9.4 775 ± 62.0 107 ± 2.1 16 ± 0.3 708 ± 63.7P6/P4 2.3 ± 0.07 0.9 ± 0.02 1.1 ± 0.02 nd 1.4 ± 0.04G6/G4 2.6 ± 0.29 1.6 ± 0.05 2.1 ± 0.04 nd 1.9 ± 0.06S6/S4 4.7 ± 0.4 2.7 ± 0.08 2.7 ± 0.08 4.7 ± 0.1 2.2 ± 0.04ΓG 2.8 ± 0.1 0.6 ± 0.05 0.4 ± 0.02 0.8 ± 0.02 2.3 ± 0.05

alkyl-C (fatty acids as methyl esters, alcohols, alkanes, alkenes) 291 ± 8.7 1274 ± 25.5 239 ± 19.1 228 ± 9.1 942 ± 47.1terpenes and steroids 40 ± 11.8 130 ± 3.9 118 ± 2.4 212 ± 17.0 305 ± 12.7nitrogenous compounds 148 ± 2.6 1618 ± 113.3 409 ± 28.6 49 ± 2.4 818 ± 57.3carbohydrate derivatives 12 ± 0.2 136 ± 5.4 46 ± 2.3 nd 218 ± 15.3aP, p-hydroxyphenyl; G, guaiacyl; S, syringyl; P6/P4, G6/G4, S6/S4, and ΓG are indices to evaluate lignin transformation using specificthermochemolysis products as described under Materials and Methods. The values represent the means from two chromatograms followed bystandard deviation. bHA1, HA2, HA3, HA4, and HA5 are the humic acids isolated from vermicomposts produced with cattle manure, sugar canebagasse, sunflower cake, a mixture of cattle manure, bagasse, and sunflower cake, and sugar cane filter cake residue, respectively.

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



compound found among the pyrolyzed products for all humicacids was a propenoic acid derivative (2-propenoic acid, 3-(4-methoxyphenyl)-methyl ester, P18), which is a basiccomponent of lignified tissues of herbaceous crops and grasses.The lignin compounds decrease in the following manner: HA2> HA5 > HA1 > HA3 > HA4. HA2 obtained from sugar canebagasse and cattle manure showed 65.7 times more lignin thanHA4 obtained from the mixture of bagasse, cattle manure, andsunflower cake (Table 1). A large range of methylated guaiacylderivatives were found in all HAs, although at lowerconcentration with respect to the propenoic acid monomer,whereas limited distribution and lower abundance were shownby syringyl units. Useful specific components found in all humicextracts may be associated with the presence of eithermicrobially processed organic materials or undecomposedplant debris. The extent of lignin decomposition may beestimated by structural indices that are based on the relativeamount of specific thermochemolysis products.26−28 Whereasthe aldehydic (G4 and S4) and acidic (G6 and S6) forms oflignin structures result from progressive degradation of ligninpolymer, involving the ongoing oxidation of propyl chain, thecorresponding homologues with integral hydroxylated sidechains (G14/15, S14/15) are indicative of unaltered lignincomponents, which retain the typical β-O-4 ether intermo-lecular linkages. Therefore, the indices are obtained (Table 1)by dividing the amount found for the acidic structures by thatof, respectively, G4 and S4 aldehydes (Ad/AlG = G6/G4, Ad/AlS = S6/S4) and for the global yield of threo/erythro isomers(ΓG = G6/[G14 + G15]) currently used as indicators of thebio-oxidative transformation of lignin polymers. The larger thevalues of dimensionless indices, the wider the decompositionprocess of lignin substrates. The largest amount of fatty acidsidentified as methyl ester derivatives was observed in HA2isolated from sugar cane bagasse followed by HA5 from filtercake. However, a larger diversity of methyl esters of fatty acidswas found in HA1. The main methyl ester derivatives wereeven- and odd-numbered long chains from plant and bacterialorigin, respectively. HA5 was characterized by the presence ofdocosanol, a long-chain alcohol from carbohydrate enzymaticreactions. Squalene was found in all humic acids, whereas 15-hydroxydehydroabietic acid was found only in HA2 and β-sitosterol only in HA3. Large amounts of cholest-7-ene-3-olacetate were found in HA5 and lanost-8-ene-3,7-dione in HA2.The most common nitrogenous compounds found in all

humic acids, and in larger amounts in HA3, were 2,4-dimethoxyphenylamine, N-methoxycarbonyl, and benzenamine.Proline derivatives were found only in HA1 and HA5, whereaspyrrolidone and pyrimidone derivatives were found only inHA2 and HA3. Few compounds derived directly fromcarbohydrates were found in the samples and only as furanderivatives.

The 13C NMR spectra were characterized by a sharpresonance in the 45−65 ppm range for all humic acids (Figure3). The main signal centered around 55 ppm, associated withthe methoxy substituents in the aromatic ring of guaiacyl andsyringyl components of lignified tissues of plants, which clearlyindicate the incorporation of lignin derivatives in maturevermicomposts. The broad and strong signals found in thealkyl-C region (0−45 ppm) region revealed the largeincorporation of alkyl chains pertaining to different compo-nents. The peaks at 16 and 23 ppm may be derived mainly fromCH3− and CH2− groups of various lipid compounds, such aswaxes, polyesters, and phospholipids. In addition to the peaksbetween 0 and 30 ppm, many distinct signals were shown in thebroad alkyl-C region around 30−45 ppm with signals at 30, 32,39, and 44 ppm, which indicate the simultaneous presence ofdifferent alkyl chains from linear and branched fatty acid andpeptide derivatives. The inclusion of N-containing organiccompounds was stressed by the peaks positioned at 40−44ppm, which were assigned to both Cα and Cβ in amino acids.20

Moreover, a different preservation of nitrogen components inthe humic extracts was also suggested by the comparison of theintense peak at 55 ppm, with a less pronounced intensity of theO-aromatic functional groups in the 140−160 ppm interval,found in the 13C NMR spectra of HA2, HA3, and HA4 (Figure3 and Table 2). Besides the lignin structures, the signalsincluded in the 45−60 ppm chemical shift range may also resultfrom the C−N bonds in amino acid moieties. In this respect thecomparison of signal intensity in the 45−60 ppm interval overthat in the 140−160 ppm range may be helpful for a moreaccurate assignment of methoxyl and phenolic resonances. Thisdimensionless index, hereby denoted the lignin ratio, has beenused to improve discrimination between signals from ligninunits characteristic of other phenolic components or peptidicmoieties.20 Whereas a sharply lower ratio of <1 is usuallyassociated with the inclusion of tannin and polyphenolconstituents to the global O-aromatic-C signals, the oppositeprevalence of the upper fractional part indicates thecontribution of C−N bonds in the 45−60 ppm area. Therefore,the discrepancy between methoxyl-C and phenolic-C signals,summarized by the larger lignin ratio found in HA2, HA3, andHA4 humic extracts (Table 2), suggests a greater incorporationof peptidic moieties and a lesser preservation of ligninderivatives in the stabilized organic fractions of sugar canebagasse, sunflower cake, and mixed mature vermicompost.The different resonances in the O-alkyl-C region (65−110

ppm) are currently assigned to monomeric units in oligo- andpolysaccharide chains of plant tissue. The intense signal around72 ppm corresponds to the overlapping resonances of carbons2, 3, and 5 in the pyranoside structure in cellulose and somehemicelluloses, whereas the signal at 104 ppm is assigned to theanomeric carbon 1 of the glucose unit in cellulose. The

Table 2. Integration Area of 13C CPMAS-NMR Spectra from Different Humic Acids (HAs): (HA1) Cattle Manure; (HA2)Cattle Manure and Sugar Cane Bagasse; (HA3) Cattle Manure and Sunflower Cake Obtained after Oil Extraction; (HA4) CattleManure, Sugar Cane Bagasse, and Sunflower Cake; (HA5) Filter Cake at 90 Days of Vermicomposting Time

chemical shift (ppm)

humic acid 0−45 45−65 65−95 95−110 110−140 140−160 160−190 HB/HI

HA1 25.25 23.07 12.38 6.53 20.15 7.22 5.41 1.79HA2 20.77 21.31 19.61 9.53 17.62 5.86 5.30 0.79HA3 22.68 22.24 18.96 8.87 17.31 5.13 4.81 0.82HA4 19.08 22.81 20.46 8.96 18.53 5.51 4.65 0.76HA5 21.91 17.94 14.64 9.35 20.39 7.48 8.30 0.99



shoulders localized around 62−65 and 84−88 ppm result fromcarbons 6 and 4 of monomeric units, respectively. The low-fieldresonances (higher chemical shift) of each couple indicate thepresence of crystalline forms of cellulose, whereas the high-fieldresonances are assigned to either amorphous cellulose orhemicellulose structures. Besides the signals usually assigned tocellulose, the spectra of different HAs revealed three additionalresonances around 93, 106, and 110 ppm. These signals may berelated, respectively, to the di-O-alkyl-C of monomeric units ofsimple carbohydrates and to the C1 of either hemicellulose orpectic polysaccharides chains contained in cell walls of plants,such as α-1,5 arabinan, β-1,4 galactan, and α-1,4 galacturonan.In the aromatic/olefinic-C region (110−145 ppm), the

different resonances around 120 and 123 ppm are related tounsubstituted and C-substituted aryl carbon pertaining to bothlignin monomers and ring components of polyphenols. Thesignal intensity shown by the specific O-aromatic region (145−160 ppm) in HAs confirms the incorporation of O-substitutedring carbon derived from different lignin structures. Theevident resonances shown at 152 (sharp) and 158 ppmchemical shift ranges are usually assigned to carbons 3, 4, and 5in the aromatic ring of lignin components, with carbon 3 and 5being coupled to the corresponding methoxyl substituents.Finally, the intense signal in the carbonyl region (160−190ppm) at 174 ppm suggests the contribution of carbonyl groupsof peptide bonds of amino acid moieties in all of the humicmaterials. The relative distribution of organic functional groupsand the hydrophobic index indicate that the HAs from cattlemanure and filter cake underwent a more advancedhumification process, with a final large preservation ofrecalcitrant hydrophobic components. Conversely, the ligninratio and the larger amount of polysaccharides found in HA2,HA3, and HA4 (Figure 3) revealed a steady maintenance ofbiolabile compounds in the final mature vermicompost, withthe mixed vermicompost showing a lower hydrophobiccharacter (Table 2).The results of the root growth biological assay represented

by humic acid application are shown in Figure 4. All of thehumic acids from mature vermicomposts showed an improve-ment in the number of lateral roots ranging from 36 to 135%with respect to control plants. However, HA4 showed no

significant differences related to the control by the mean testapplied. The lowest and no significant effect was found for HA4extracted from the mixture of cattle manure, bagasse, andsunflower cake. Among those humic acids that producedsignificant bioactivity effect, we have humic acids re-isolatedfrom bagasse (HA2) and sunflower cake (HA3), whichenhanced the emergence of lateral roots in a very similarrange from 51 to 58%, respectively. HA1 extracted from cattlemanure revealed an increase of 98%, whereas the largerpromotion was associated with HA5 from filter cake vermicom-post.Vermicomposting technology, using earthworms as versatile

natural bioreactors for effective recycling of organic wastes tothe soil, is an environmentally suitable method to convertresidual biomass into nutrient-rich composts for cropproduction.29 Considerable work has been carried out onvermicomposting processes of various organic materials such asanimal manures, agricultural wastes, forestry wastes, city leaflitter and food residues, sewage sludge, and industrial wastessuch as paper pulp and distillery wastes. Vermicompostingrepresents an important environmental service for wasterecycling coupled with the generation of valuable products.29

The results of 13C NMR spectroscopy and offline thermoche-molysis GC-MS indicate that despite the intense biochemicaltransformation process of accelerated humification by earth-worms, the final humic products retained the chemical featuresinherited from the original organic source. Contrary to theindications of 13C NMR spectra, a relatively low yield ofcarbohydrates was detected among the pyrolysis products. Thelack of polysaccharide compounds was already noted inapplications of thermochemolysis on plant woody tissues andsoil organic matter.24,26 These authors pointed out that thelarge amount of thermally unstable hydroxyl functional groupsin polysaccharide chains makes the pyrolysis technique lesssuitable for the effective detection of carbohydrates in complexgeochemical matrices. It is thus conceivable that for compostsamples the setup of thermochemolysis parameters may behighly selective for lignin and alkyl components and mayreduce the simultaneous identification of carbohydrate unitsfrom cellulose. Such selective detection reinforces the use ofdifferent approaches for a more complete compound inventoryassociated with the supramolecular structure of humicsubstances.The different humic acids showed a variable effect on the

emergence of lateral roots (Figure 4) and were characterized byan uneven incorporation of alkyl and aromatic hydrophobiccomponents (Tables 1 and 2). To date, the most hydrophobichumic acids were isolated from cattle manure and filter cakebiomasses, and they provided the best stimulation of lateralroot emergence (Figure 4). In contrast, the root biostimulationeffect was decreased by humic acids from sugar cane bagasse,sunflower cake, and mixed materials, which were characterizedby the progressive lowering of hydrophobic character (Table2). These results are consistent with the previous findings thathumic fractions with a larger hydrophobicity provide a steadyand high bioactivity, and the presence of aromatic and aliphaticcomponents, such as lignin compounds and methyl esteresderivatives, is closely related to the ability of humic acids toinduce lateral root emergence.23,27,30,31 An apparent incon-sistency with these previous findings was presented in thecurrent thermochemolysis results, which showed that thelargest lignin content was for HAs from sugar cane bagasse(Table 1). However, the most abundant aromatic monomers

Figure 4. Number of lateral roots in maize seedlings treated withhumic acids from different vermicomposts: (HA1) cattle manure;(HA2) sugar cane bagasse; (HA3) sunflower cake; (HA4) mixture ofcattle manure, sugar cane bagasse, and sunflower cake; (HA5) sugarcane filter cake. Different letters represent a significant difference bymeans of Duncan’s test (p < 0.01). The coefficient of variation was13.37%.



found in the pyrograms of HA2 were 3-(4-methoxyphenyl)-2-propenoic (P18) and 3-(4,5-dimethoxyphenyl)-2-propenoic(G18) derivatives, which accounted for about 40% of thetotal lignin content (Table 1). These molecules originatedeither from the side-chain oxidation of lignin units or from thearomatic domains of plant biopolymers, the important relativecontribution of which in herbaceous plants may haveexaggerated the total content of lignin in the HAs from sugarcane, compared to those from cattle manure and filter cake.Moreover, the higher values of lignin structural index found inthe pyrograms of HA2 and HA5 (Table 1) suggest theoccurrence of an intense decomposition process of ligninbiopolymers in the humic fraction from cattle manure and filtercake. Therefore, the most advanced humification may havepromoted a preferential accumulation of small and activearomatic fragments bound to the supramolecular humic acidsstructure, which should allow a more prompt physiologicalresponse compared to the partial undecomposed and rigidlignin residues.16

In addition to the content of specific molecules, the ratio ofhydrophobic to hydrophilic moieties is considered an importantcharacteristic for the bioactivity of humic extracts. The role ofhumic hydrophobicity may be explained by the selectivepreservation, in humic recalcitrant compartments, of activebiofragments that may be successively released by conforma-tional changes of humic associations in solution.8,19,21 Thereduced microbial and enzymatic activity provided by thehydrophobic domains is assumed to contribute to theprotection of biolabile hydrophilic compounds, which maybecome available to the plant root system by means of cellexudation in the rhizosphere.28,32 For example, plants treatedwith humic substances showed enhanced exudation of organicacids, thereby leading to a modification of structural arrange-ment and to a release of active molecules in the rhizosphere.22

The present results confirm that mature vermicompost is aviable way to recycle agricultural biomass and to demonstratethat, acting as a source of bioactivity, humic extracts representan additional valuable biological product for a wide range ofagricultural practices.18 Notwithstanding the modification bythe composting processes, the final humic substances retained achemical composition strongly related to the composition ofthe initial biomass. Although the role of hydrophobic humiccomponents needs further evaluation to gain a deeper insightinto the structure−activity relationship, the association ofdetailed molecular characterization and bioactivity assays areunavoidable requirements for a more accurate and valuableutilization of humic material as plant growth promoters and fora comprehensive understanding of the interaction betweenplant and soil organic matter.

■ ASSOCIATED CONTENT

*S Supporting InformationCompounds obtained by GC-MS analysis from different humicacids extracted from vermicomposts. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*(L.P.C.) Phone/fax: +55 22 27397198. E-mail: [email protected].

FundingThe work was supported by Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq), Fundacaode Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ),Instituto Nacional de Ciencia e Tecnologia (INCT) para aFixacao Biologica de Nitrogenio, Internacional Foundation ofScience (IFS), and OCWP.NotesThe authors declare no competing financial interest.

■ REFERENCES(1) Valdrighi, M. M.; Pera, A.; Agnolucci, M.; Frassinetti, S.; Lunardi,D.; Vallini, G. Effects of compost-derived humic acids on vegetablebiomass production and microbial growth within a plant (Cichoriumintybus)−soil system: a comparative study. Agric., Ecosyst. Environ.1996, 58, 133−144.(2) Edwards, C. A.; Arancon, N. Q.; Sherman, R. L. VermicultureTechnology: Earthworms, Organic Wastes, And Environmental Manage-ment; CRC Press: Boca Raton, FL, USA, 2010; p 601.(3) Ali, M.; Griffiths, A. J.; Williams, K. P.; Jones, D. L. Evaluating thegrowth characteristics of lettuce in vermicompost and green wastecompost. Eur. J. Soil Biol. 2007, 43, S316−S319.(4) Muscolo, A.; Bovalo, F.; Giomfriddo, F.; Nardi, S. Earthwormhumi matter produces auxin-like effects on Daucus carota cell growthand nitrate metabolism. Soil Biol. Biochem. 1999, 31, 1303−1311.(5) Canellas, L. P.; Okorokova-Facanha, A.; Olivares, F. L.; Facanha,A. R. Humic acids isolated from earthworm compost enhance rootelongation, lateral root emergence, and plasma membrane H+-ATPaseactivity in maize roots. Plant Physiol. 2002, 130, 1951−1957.(6) Arancon, N. Q.; Lee, S.; Edwards, C. A.; Atiyeh, R. M. Effects ofhumic acids and aqueous extracts derived from cattle, food and paper-waste vermicomposts on growth of greenhouse plants. Pedobiologia2003, 47, 741−744.(7) Arancon, N. Q.; Edwards, C. A.; Atiyeh, R.; Metzger, J. D. Effectsof vermicomposts produced from food waste on the growth and yieldsof greenhouse peppers. Bioresour. Technol. 2004, 93, 139−144.(8) Canellas, L. P.; Piccolo, A.; Dobbss, L. B.; Spaccini, R.; Olivares,F. L.; Zandonadi, D. B.; Facanha, A. R. Chemical composition andbioactivity properties of size-fractions separated from a vermicomposthumic acid. Chemosphere 2010, 78, 457−466.(9) Warman, P. R.; Anglopez, M. J. Vermicompost derived fromdifferent feedstocks as a plant growth medium. Bioresour. Technol.2010, 101, 4479−4483.(10) Nardi, S.; Concheri, G.; Dell’agnola, G. Biological activity ofhumus. In Humic Substances in Terrestrial Ecosystems; Piccolo, A., Ed.;Elsevier: Amsterdam, The Netherlands, 1996; pp 361−40.(11) Masciandaro, G.; Ceccanti, B.; Garcia, C. Soil agroecologicalmanagement: fertirrigation and vermicompost treatments. Bioresour.Technol. 1999, 59, 199−206.(12) Dell’Agnola, G., Nardi, S. On overview of earthworm activity inthe soil. In On Earthworms Selected Symposia and Monographs, Part 2;Bonvicini Pagliai, A. M., Omodeo, P., Eds.; Mucchi Ed.e: Modena,Italy, 1987; pp 103−112.(13) Muscolo, A.; Nardi, S. Auxin or auxin-like activity of humicmatter. In The Role of Humic Substances in the Ecosystems andEnvironmental Protection; Drozd, J., Gonet, S. S., Senesi, N., Weber, J.,Eds.; Polish Society of Humic Substances: Wroclaw, Poland, 1997; pp987−992.(14) Canellas, L. P.; Martinez-Balmori, D.; Medici, L. O.; Aguiar, N.O.; Campostrini, E.; Rosa, R. C. C.; Facanha, A. R.; Olivares, F. L. Acombination of humic substances and Herbaspirillum seropedicaeinoculation enhances the growth of maize (Zea mays L.). Plant Soil2013, 366, 119−132.(15) Atiyeh, R. M.; Lee, S.; Edwards, C. A.; Arancon, N. Q.; Metzger,J. D. The influence of humic acids derived from earthworm-processedorganic wastes on plant growth. Bioresour. Technol. 2002, 84, 7−14.(16) Quaggiotti, S.; Ruperti, B.; Pizzeghello, D.; Francioso, O.;Tugnoli, V.; Nardi, S. Effect of low molecular size humic substances on



http://pubs.acs.org

mailto:[email protected]

mailto:[email protected]

nitrate uptake and expression of genes involved in nitrate transport inmaize (Zea mays L.). J. Exp. Bot. 2004, 55, 803−813.(17) Zandonadi, D. B.; Canellas, L. P.; Facanha, A. R. Indoleaceticand humic acids induce lateral root development through a concertedplasmalemma and tonoplast H+ pumps activation. Planta 2007, 225,1583−1595.(18) Canellas, L. P.; Olivares, F. L. Physiological responses to humicsubstances as plant growth promoter. Chem. Biol. Technol. Agric. 2014,1, 3.(19) Dobbss, L. B.; Canellas, L. P.; Olivares, F. L.; Aguiar, N. O.;Peres, L. E. P.; Azevedo, M.; Spaccini, R.; Piccolo, A.; Facanha, A. R.Bioactivity of chemically transformed humic matter from vermicom-post on plant root growth. J. Agric. Food Chem. 2010, 58, 3681−3688.(20) De Marco, A.; Spaccini, R.; Vittozzi, P.; Esposito, F.; Berg, B.;Virzo De Santo, A. Decomposition of black locust and black pine leaflitter in two coeval forest stands on Mount Vesuvius and dynamics oforganic components analyzed through conventional chemical methodsand NMR spectroscopy. Soil Biol. Biochem. 2012, 51, 1−15.(21) Aguiar, N. O.; Novotny, E. H.; Oliveira, A. L.; Rumjanek, V. M.;Olivares, F. L.; Canellas, L.P. Prediction of humic acids bioactivityusing spectroscopy and multivariate analysis. J. Geochem. Explor. 2013,129, 95−102.(22) Canellas, L. P.; Dobbss, L. B.; Oliveira, A. L.; Chagas, J. G.;Aguiar, N. O.; Rumjanek, V. M.; Novotny, E. H.; Olivares, F. L.;Spaccini, R.; Piccolo, A. Chemical properties of humic matter asrelated to induction of plant lateral roots. Eur. J. Soil Sci. 2012, 63,315−324.(23) Aguiar, N. O.; Olivares, F. L.; Novotny, E. H.; Dobbss, L. B.;Balmori, D. M.; Santos-Junior, L. G.; Chagas, J. G.; Facanha, A. R.;Canellas, L. P. Bioactivity of humic acids isolated from vermicompostsat different maturation stages. Plant Soil 2013, 362, 161−174.(24) Spaccini, R.; Sannino, D.; Piccolo, A.; Fagnano, M. Molecularchanges in organic matter of a compost-amended soil. Eur. J. Soil Sci.2009, 60, 287−296.(25) Zhou, P.; Pan, G. X.; Spaccini, R.; Piccolo, A. Molecular changesin particulate organic matter (POM) in a typical Chinese paddy soilunder different long-term fertilizer treatments. Eur. J. Soil Sci. 2010, 61,231−242.(26) Spaccini, R.; Song, X. Y.; Cozzolino, V.; Piccolo, A. Molecularevaluation of soil organic matter characteristics in three agriculturalsoils by improved off-line thermochemolysis: the effect of hydrofluoricacid demineralisation treatment. Anal. Chim. Acta 2013, 802, 46−55.(27) Martinez-Balmori, D.; Olivares, F. L.; Spaccini, R.; Aguiar, K. P.;Araujo, M. F.; Aguiar, N. O.; Guridi, F.; Canellas, L. P. Molecularcharacteristics of vermicompost and their relationship to preservationof inoculated nitrogen-fixing bacteria. J. Anal. Appl. Pyrolysis 2013, 104,540−550.(28) Song, X. Y.; Spaccini, R.; Pan, G.; Piccolo, A. Stabilization byhydrophobic protection as a molecular mechanism for organic carbonsequestration in maize-amended rice paddy soils. Sci. Total Environ.2013, 458−460, 319−330.(29) Padmavathiamma, P. K.; Li, L. Y.; Kumari, U. R. Anexperimental study of vermi-biowaste composting for agricultural soilimprovement. Bioresour. Technol. 2008, 99, 1672−1681.(30) Canellas, L. P.; Zandonadi, D. B.; Busato, J. G.; Baldotto, M. A.;Simoes, M. L.; Martin-Neto, L.; Facanha, A. R.; Spaccini, R.; Piccolo,A. Bioactivity and chemical characteristics of humic acids from tropicalsoils sequence. Soil Sci. 2008, 173, 624−637.(31) Canellas, L. P.; Spaccini, R.; Piccolo, A.; Dobbss, L. B.;Okorokova-Facanha, A. L.; Santos, G. A.; Olivares, F. L.; Facanha, A.R. Relationships between chemical characteristics and root growthpromotion of humic acids isolated from Brazilian Oxisols. Soil Sci.2009, 174, 611−624.(32) Spaccini, R.; Piccolo, A.; Conte, P.; Haberhauer, G.; Gerzabek,M. H. Increased soil organic carbon sequestration through hydro-phobic protection by humic substances. Soil Biol. Biochem. 2002, 34,1839−1851.



molecular characteristics of humic acids isolated from ... · molecular characteristics of humic...

Documents