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Changes in soil humic pools after soil application of two-phase olive millwaste compost

N. Serramiá a, M.A. Sánchez-Monedero a,⁎, A. Roig a, M. Contin b, M. De Nobili b

a Department of Soil and Water Conservation and Organic Waste Management, Centro de Edafología y Biología Aplicada del Segura, CSIC, PO Box 164, 30100 Murcia, Spainb Department of Agriculture and Environmental Sciences, University of Udine, Via delle Scienze 208, 33100 Udine, Italy

a b s t r a c ta r t i c l e i n f o

Article history:Received 21 February 2011Received in revised form 3 July 2012Accepted 23 August 2012Available online xxxx

Keywords:C sequestrationHumic acidsOlive mill wasteCompost application

The use of appropriate amendments derived from twophase olivemillwastes (TPOMW) can represent a suitableoption to maintain and restore C levels in agricultural soils under Mediterranean climates. We evaluated soil or-ganic matter stabilisation pathways among different humic pools in a Calcaric Cambisol amended with 2%(40 Mg ha−1) of TPOMWcompostingmixtures of different composition and at different degrees of stabilisation:starting mixture, after 14 weeks of composting (thermophilic stage) and after 30 weeks (mature compost).Non-humified soil organic C and two different fractions of humic acids (HA), namely free HA (biochemicallystabilised) and bound HA (biochemically and chemically stabilised) were obtained by sequential extractionwith NaOH and alkaline Na4P2O7 after 90 and 150-days incubation. HA were characterised by thermal analysis,size exclusion chromatography (HPLC-SEC), FTIR and 13C CPMAS-TOSS NMR. Amendments promoted incorpora-tion of altered lignin structures, carbohydratemoieties and N-containing compounds into free HA and to a lesserextent into the bound HA, and increased the proportion of high MW fractions. There was an average increase of40% for non-humic C in the free C fraction even after 90 days of incubation under optimum conditions formineralisation. Augmentation of bound C resulted in an average increase of about 0.7 Mg ha−1 of humic C inamended soils. This increase is important as it contributes to one of the more inert soil C pools and could repre-sent a useful indicator for soil C stabilisation.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The application of organic amendments, such as crop residues,manures and composts, is a well established management practiceto maintain and increase the levels of soil organic matter (SOM) in ag-ricultural ecosystems. This strategy is particularly important in Med-iterranean areas, where a large proportion of agricultural lands havebeen degraded by loss of organic matter and other processes that re-duce soil productivity (Batjes, 1996; Foley et al., 2005). Furthermore,typical climatic conditions in these areas, characterised by relativelyscarce yearly rainfall during their colder season and periods from 4to 6 months without any significant precipitation during the warmerpart of the year, affect the net primary production of natural vegeta-tion and rain fed crops, so that C inputs are definitively scarce andlimit effective organic C stabilisation.

Organic amendments are incorporated into SOM and stabilised bydifferent mechanisms (Baldock and Skjemstad, 2000; Krull et al.,2003; Oades and Waters, 1991): physical protection by aggregates,chemical stabilisation, which is the result of cation bridge formationor physico-chemical binding on mineral surfaces, and biochemical

stabilisation through the formation of recalcitrant compounds (DeNobili et al., 2008; Six et al., 2002).

Biochemical stabilisation results from the biotic or abiotic produc-tion (humification) of complex organic structures which progressive-ly acquire resistance to microbial decomposition and whichcontribute, through condensation reactions and complex formation,to the stabilisation of easily decomposable organic matter compo-nents (Krull et al., 2003; Piccolo et al., 2004). Biochemical stabilisationalso refers to the inherent recalcitrance related to aromatic structuresin complex macromolecules such as lignin or modified lignin, whichare selectively preserved during composting. Therefore, the use ofamendments of appropriate composition (such as lignocellulosicwastes) can promote organic C stabilisation in soil.

The physico-chemical characteristics of olive mill wastes makethese materials a suitable and valuable source of SOM in the Mediter-ranean Basin (Altieri and Esposito, 2008; López-Piñeiro et al., 2007;Nastri et al., 2006; Sánchez-Monedero et al., 2008). Two-phase olivemill waste (TPOMW) is nowadays produced in enormous quantitiesby the olive oil processing industry which processes about threemillion tons of olive oil per year, 76% of which are produced in South-ern Europe (FAOSTAT, 2009). This by-product is characterised by alarge content of organic matter (>850 g kg−1), mainly of lignocellu-losic nature, a reasonable amount of nutrients and the presence of po-tentially phytotoxic substances such as polyphenols, lipids and salts

Geoderma 192 (2013) 21–30

⁎ Corresponding author. Tel.: +34 968396364; fax: +34 968396213.E-mail address: [email protected] (M.A. Sánchez-Monedero).

0016-7061/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.geoderma.2012.08.032

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(Alburquerque et al., 2004; Roig et al., 2006). The direct use of olivemill wastes can have a negative impact on soil due to the antimicro-bial properties of polyphenols (Capasso et al., 1992) and the inhibi-tion of key enzymes involved in soil nutrient cycles (Piotrowska etal., 2006, 2011) and in lignin degradation (Sayadi et al., 2000). How-ever, TPOMW transformation through composting minimises thesenegative impacts and ensures an adequate degree of stability and ma-turity before land application.

An intense organic matter humification has been described duringcomposting of olive mill wastes with other agro-industrial wastes(Droussi et al., 2009) and after soil application (Brunetti et al., 2005,2007). The lignocellulosic composition and the presence of polyphenols,precursors in the synthesis of humic substances, play an important rolein the humification processes (Ait Baddi et al., 2009; Francioso et al.,2007; Serramiá et al., 2010), giving rise to humic-likematerial featuringa prevalent aliphatic character and a marked presence of proteinaceousmaterials, partially modified lignin moieties and polysaccharides-likestructures (Senesi et al., 2007). However, and despite the extensivecharacterisation of the humic-like material in TPOMW composts andin amended soils, there is only scarce information about pathways of Cstabilisation after application of these composts to soil.

The purpose of this workwas to evaluate soil stabilisation pathwaysof C from lignocellulosic amendments based on TPOMW compostingmixtures, prepared by mixing TPOMW with different agro-industrialwastes and at different degrees of stabilisation, by studying quantitativeand qualitative changes of soil humic fractions.

2. Materials and methods

2.1. Soil and organic amendments

Soil was sampled from the arable layer (0–20 cm depth) of anolive orchard located in Jaén (South Spain). The soil is classified asCalcaric Cambisol (WRB-FAO), with sandy loam texture (70.5% sand,15.8% silt and 13.7% clay), low total organic carbon and total nitrogen:respectively 8.7 and 2.0 g kg−1, alkaline pH (8.61), low electrical con-ductivity (0.186 dS m−1) and a large CaCO3 content (395 g kg−1).

The organic amendments used in the incubation experiment wereproduced by composting three different mixtures (TS, TOS and THU)prepared with TPOMW and other agro-industrial wastes used asbulking agents and N-sources. TS (33% TPOMW+67% sheep manure,expressed as dry weight proportions) represented a common mixtureemployed by typical composting plants in Southern Spain. TOS (20%TPOMW+20% olive tree prunings+60% sheep manure) was prepared

by replacing some of the TPOMW by another largely available lignocel-lulosic material such as olive tree prunings. THU (33% TPOMW+67%horse manure+14 kg t−1 urea) was prepared by using a different Nsource and enriched with mineral N.

The performance of the composting processes was described bySerramiá et al. (2010). Sampling was performed at three differentstages of composting: initial (I), thermophilic (T; after 14 weeks ofcomposting) and mature (M; after 30 weeks). The main characteristicsof the nine organic amendments are reported in Table 1. Compostingsamples were air dried and ground to pass a 0.5 mm sieve.

2.2. Incubation experiment and humic and humic-like substancesextraction

Composting mixtures were added (2% w/w; equivalent to40 Mg ha−1) to 180 g (oven dry basis) of conditionedmoist soil sam-ples at 40% of water holding capacity. Amended soils and controlswithout amendment were placed in 800 mL plastic boxes togetherwith a 10 mL vial containing water to maintain a constant moisturecontent. Samples were incubated aerobically at 25 °C in the dark for150 days. Each treatment was replicated three times. After 90 and150 days of incubation, 60 g of soil (oven-dry basis) were air driedand stored for subsequent extraction of the humic substances.

Free and bound C fractions were obtained from air-dried soil sam-ples (45 g) by sequential extraction (1:10 w/v) as described by DeNobili et al. (2008). Free humic and non-humic C fractions wereextracted in 0.5 M NaOH and bound humic and non-humic C fractionswere extracted from the residue with 0.1 M Na4P2O7 plus 0.1 MNaOH, as shown in extraction scheme reported in Fig. 1. A first extrac-tion with sodium hydroxide allows solubilisation of non-humichydrolysable components (free non-humic C) and of biochemicallystabilised C, not bound to the mineral constituents of soil (free humicC fraction). A second extraction with alkaline sodium pyrophosphatesolubilises part of the chemically stabilised C that is bound to mineralconstituents, by dissolving metal-organic complexes via chelation ofpolyvalent cations such as Ca2+, Fe2+ and Al3+ (bound C fraction).Both free and bound humic fractions include native soil humic sub-stances coextracted with compost derived humic-like substances. Bothextractions were carried out under N2 flux for one hour. The extractswere centrifuged and supernatants were filtered through 0.4 μm cellu-lose filters. In both free and bound C fractions, the non-humic C(hydrolysed polysaccharides, proteins, peptides, etc.) and the humic C(humic plus fulvic acids) were separated from the extracts by solidphase extraction (SPE) on cross-linked polyvinylpyrrolidone (Aldrich)

Table 1Selected characteristics of the composting mixtures at different stabilisation degree (I=initial mixture; T=thermophilic stage, M=mature compost).

Sample OMa NTOTb C/N CWS

c Phenolsd CHA/CEXTe Lipids Lignin Holocellulose

g kg−1 g kg−1 g kg−1

TS mixture: TPOMW+sheep manureTSI 522 18.0 15.6 23 2.9 69.3 1.8 283 282TST 443 18.1 13.2 25 4.6 78.9 1.7 258 196TSM 440 17.8 12.1 30 3.6 81.1 1.5 262 182

THU mixture: TPOMW+horse manure+ureaTHUI 557 17.2 18.3 28 2.0 48.0 2.1 249 343THUT 406 14.4 16.9 7 0.5 85.0 1.6 219 197THUM 390 14.8 13.8 6 0.4 85.8 1.7 220 160

TOS mixture: TPOMW+olive tree prunings+sheep manureTOSI 729 12.8 30.2 56 5.3 54.8 7.3 369 461TOST 649 16.4 21.3 22 3.0 75.0 2.0 367 404TOSM 630 18.7 16.4 29 2.7 79.0 1.9 345 315

a Organic matter.b Total nitrogen.c Water‐soluble carbon.d Water‐soluble phenols (expressed as % caffeic acid).e Percentage of humic acid‐like (×100): humic acid‐like carbon to alkali extractable carbon ratio. The coefficient of variation of the chemical analysis was lower than 5%.

22 N. Serramiá et al. / Geoderma 192 (2013) 21–30


(De Nobili et al., 2008) and the organic C was quantified in bothnon-humic and humic fractions by wet oxidation with K2Cr2O7.

Free and bound humic acids (HA) were precipitated at pH 1.5 fromthe corresponding extracts and washed with acidified distilled water,then redissolved in 0.5 M NaOH and treated with acid-washedAmberlite IR-120H+ until neutral pH before freeze drying. Treatmentof the alkaline extract with a cation exchange resin largely preventsthe co-precipitation of the co-extracted carbohydrates with the HA(Cheshire, 1979).

2.3. Analytical methods

Organic matter content of the organic amendments was deter-mined by loss on ignition at 430 °C for 24 h. Total nitrogen (NTOT)and total organic C (CTOT) of soil and organic amendments weremea-sured by automatic elemental microanalysis after carbonates remov-al. Water-soluble organic carbon (CWS) was determined in theaqueous extract (1:20, w/v) by automatic microanalysis after bicar-bonate removal (Sánchez-Monedero et al., 1996). Extractable carbon(CEXT) of the organic amendments was measured in a 0.1 M NaOHextraction (1:20, w/v) and humic-like acid carbon (CHA) was deter-mined after precipitation at pH 1.5 according to Sánchez-Monederoet al. (1996). Lignin was determined by the American National Stan-dards methods (ANSI, 1977) and hollocellulose by Browning's meth-od (Browning, 1967).

Elemental composition (C, H, and N) of freeze-dried HAwas deter-mined in duplicate by automated elemental microanalysis andexpressed on a dry ash free basis. Oxygen content was calculated bydifference and can therefore include trace fractions of S and/or P.

Thermal analysis was performed with a SDT-2960 simultaneousDSC-TGA thermal analyzer (TA instruments) on solid freeze-driedHA. Experimental conditions for TG were: static air atmosphere, tem-perature equilibration at 30 °C followed by heating from 30 to 105 °Cat 5 °C min−1 to calculate moisture content of HA. After 10 min, thetemperature was increased from 105 to 680 °C (5 °C min−1). Theash content of HA was calculated from the inorganic residueremaining at the end of the ramp. Main weight losses occurred at110–350 °C and 350–550 °C. Mass loss ratio (R1), used as an indexof thermal lability of humic structures, was calculated from the ratio

between mass loss at 350–550 °C and mass loss at 110–350 °C(Plante et al., 2009).

Molecular weight (MW) distributions of HA were obtained by sizeexclusion chromatography (HPLC-SEC) on a Biorad BioSil 250 columncalibrated with fractions of humic substances of reduced molecularweight polydispersity and polystyrenesulfonate standards with thefollowing MW (kDa): 77, 17, 8, 6.8 and 5. The eluent was 0.75 mMtris-hydroxymethyl-aminomethane (Tris) adjusted to pH 7.3 withphosphoric acid. Samples of HAwere dissolved in a minimum amountof 0.1 M NaOH, diluted with 0.75 mM Tris down to a concentration of0.1 mg mL−1 and the pH adjusted to 7.3. The UV-Vis detector was setat 320 nm for free HA and at 400 nm for bound HA.

Infrared spectra were recorded with a Perkin-Elmer 16F PC FT-IRspectrophotometer on KBr pellets made by mixing 1 mg of dried HAwith 300 mg of pre-dried and pulverised spectroscopic grade KBr.

Solid state 13C CPMAS-TOSS (Total Suppression of Side-bands) NMRspectra were obtained with a Varian-300 operating at a resonance fre-quency of 75.42 MHz for 13C. The experiments were performed usingcross-polarisation magic angle spinning (CPMAS) applied with aspectral width of 50 kHz with 90° pulse of 6.7 ms, contact time of1.5 ms, spinning rate of 4 kHz, a pulse delay of 4 s and acquisitiontime of 35 ms. Spectra obtained after acquiring 20,000 scans weredivided into the following chemical shift regions (in ppm): 0–45 (alkylC); 45–65 (N-alkyl and methoxyl C); 65–95 (O-alkyl C); 95–108(acetal C); 108–160 (aromatic C); 160–210 (carboxyl, amide, esterand carbonyl C). Areas were estimated in the deconvoluted spectra bystandard Brüker Topspin 20 software to avoid the interference ofcarbonates.

Results were subjected to analysis of variance (ANOVA). Multiplemean separations were performed using Duncan's multiple rangetest at P 0.05 using the SPSS 17.0 program for Windows.

3. Results and discussion

3.1. Description of the organic amendments

The organic amendments were prepared from compostingmixturesat different degrees of stabilisation, all of them characterised by a richlignocellulosic composition (up to 830 g kg−1 of lignin+holocellulose

Soil

Supernatant

1st extraction:0.5M NaOH

2nd extraction:0.1M NaOH

+0.1M Na4P2O7

pH < 2

SOLUBLEFRACTION

(FA+Non HS)

INSOLUBLE FRACTION

(HA)

PVP

Non retained

Retained

FREENON-HUMIC C

FA

FREEHUMIC C

(HA+FA)

Eluted0.5M NaOH

pH < 2

SOLUBLEFRACTION

(FA+Non HS)

INSOLUBLE FRACTION

(HA)

PVP

Non retained

Retained

BOUNDNON-HUMIC C

FA

BOUNDHUMIC C

(HA+FA)

Eluted0.5M NaOH

Discard

Solid residue

Supernatant

Solid residue

Fig. 1. Scheme of sequential extraction and separation of non-humified and humified (HA+FA) extractable free and bound organic C fractions by means of solid phase extraction(SPE) on polyvynilpyrrolidone (PVP). Non-humic substances (non-phenolic) are not retained; after washing with 0.01 M H2SO4, phenolic substances (FA) were eluted with 0.5 MNaOH and added to HA.

23N. Serramiá et al. / Geoderma 192 (2013) 21–30


in the case of mixture TOSI) (Table 1). The use of an exhaustivelyextracted TPOMWoriginated organic amendmentswith low concentra-tions of lipids (between 1.5 and 7.0 g kg−1), compared to typical lipidconcentrations in TPOMW, around 12 g kg−1 (Alburquerque et al.,2004). Changes in the typical maturation indexes such as C/N ratio,water-soluble C concentration and the evolution of the humificationindex (about 80% of the extractable C was humic-like C at the end ofthe process) reflected the increased biochemical stability gainedthrough the process (Table 1). Consequently, the starting mixtureswere generally characterised by larger amounts of soluble C, lipidsand holocellulose than the more stabilised materials, especially in thecase of THUI and TOSI. The lower amount of OM in mature composts,compared to the starting mixtures, reflected the marked biodegrada-tion of the holocellulose fraction during composting, whereas the levelsof lignin remained almost unchanged during the process (Table 1).

3.2. Changes in total and extractable soil organic C fractions

Table 2 shows the CTOT and NTOT contents of the control and theamended soils after 90 and 150 days of incubation under optimummoisture and temperature conditions. Addition of the organic amend-ments significantly increased the concentrations of CTOT and NTOT

depending on the composition of the composting mixtures. Even after150 days under intense mineralisation conditions, TOS-amended soilsmaintained a larger CTOT concentration (15.8–17.0 mg g−1) than TSand THU amended soils (13.0–14.6 mg g−1) due to the large organicmatter and lignin concentrations in the TOS mixture, prepared witholive tree prunings (Table 1).

The different extractable soil organic C fractions after 90 and150 days of incubation are shown in Fig. 2. The addition of the differ-ent organic amendments caused an increase in free and bound C frac-tions. After 90 days, total extractable free C (first extraction withsodium hydroxide) varied from 0.82 mg g−1 in control soil up to1.69 mg g−1, while it increased from 2.3 up to 3.4 mg g−1 in thebound C fraction (second extraction with alkaline sodium pyrophos-phate). The increase in these soil organic C fractions was observedeven after 150 days of incubation under optimummineralisation con-ditions. This means that at the given rate (40 Mg ha−1) the organicamendments were able to induce changes in the composition of thesoil humic pool, which are likely to persist in the soil.

In the case of the free C fraction, the average increase was about 40%for non-humic C content. Non-humic C was affected by the degree of

stabilisation of the composting mixtures, as the amount labile Cprovided by initial (I) composting mixtures (Table 1) was generallylarger than that supplied by the other treatments. This was especiallyimportant in the case of the soil amended with TOSI where the amountof non-humic C after 90 days was still twice than that of the control(Fig. 2). The soil amended with TOSI and TOST mixtures, containinglarge amounts of water-soluble C, extractable C, cellulose, hemicellu-loses, proteins and lipids (Table 1), maintained significantly largeramounts of non-humic C than the rest of the amended soils, evenafter 150 days of incubation. However, addition of the most stabilisedcompostingmixtures did not cause any significant effect in the amountsof free non-humic C compared to control soil, due to the intensemineralisation of labile C and biochemical stabilisation that had oc-curred during composting (Table 1).

Opposite to the free non-humic C fraction, there were only smallchanges in the bound non-humic C contents. The average increasein the bound non-humic C was only about 10%, whereas the boundhumic C fraction significantly increased in all treatments by 22–30%compared to control soil. In the case of the total bound organic C,both humic and non-humic fractions were not significantly affectedby the initial composition of the composting mixtures. Only in thecase of the more stabilised TOS mixtures, there was a significantlyincreased the amount of bound humic C in the amended soils evenafter prolonged soil incubation (150 days).

Bound HA was the largest fraction extracted from the soil employedin this experiment (free C/bound C ratiob1). De Nobili et al. (1999), ex-amining soils from the long term experiment at Rothamsted, found thatthe bound humic C fraction, related to chemically stabilised humicmaterials, predominated in arable soils that had reached equilibrium Clevels at low C inputs. As expected, bound C remained stable throughoutthe experiment in all treatments resulting in an average increase of0.7 Mg ha−1 of humic C in amended soils, despite the optimum condi-tions for mineralisation during soil incubation. The increase of thebound HA fraction (stabilised by polycations and bonding via cationicbridges to soilminerals) has important implications on soil C stabilisationsince this fraction contributes to one of the more inert soil C pools andcould represent a useful indicator for C sequestration processes in thesoil.

3.3. Changes in the chemical structure of humic acids

Tables 3 and 4 respectively show the elemental composition andatomic ratios of free and bound HA isolated from the control andamended soils after 90 and 150 days of incubation. The addition ofthe organic amendments caused an increase of the C and N concen-trations of free HA after 150 days of incubation, whereas the HAbound fraction, generally characterised by higher C and lower Nthan free HA, only showed minor alterations of the chemical compo-sition. The addition of TS and THU caused the largest increase in Nconcentrations in free HA, up to 70 mg g−1 after 150 days of incuba-tion, compared to soil control (54.6 mg g−1). The initial compositionand/or stabilisation degree of the composting mixtures did not showany significant effect of the elemental composition of the bound HAextracted from the amended soils.

Free and bound HA isolated from the control and amended soils at90 and 150 days of incubation originated thermograms (data notshown) characterised in all cases by an endotherm at low tempera-ture (60 °C), generally representative of dehydration reactions, andtwo main exothermic reactions between 200–550 °C, due to a two-step thermal oxidative decomposition of organic components ofincreasing thermal stability. The ratio between mass losses associatedwith the second and the first exothermic reactions (R1) is indicativeof the relative stability of organic matter (Plante et al., 2009). Ther-mograms of bound HA were dominated by the second exotherm(410–436 °C) related to breakdown of more thermostable aromaticcompounds (Plante et al., 2009), leading to higher R1 ratio than free

Table 2Carbon andnitrogen contents in control and amended soils at 90 and150 days of incubation.

Treatment 90 days 150 days

CTOT NTOT C/N CTOT NTOT C/N

mg g−1 mg g−1

Control soilS 11.8e 1.4g 8.2 11.7e 1.3h 9.0

Soil+TS (TPOMW+sheep manure)S+TSI 13.5d 1.6de 8.3 13.2d 1.8cd 7.5S+TST 14.1d 1.7cde 8.5 14.0d 1.7cd 8.1S+TSM 14.1d 1.8bc 8.0 13.9d 1.7cde 8.5

Soil+THU (TPOMW+horse manure+urea)S+THUI 14.6cd 1.7cde 8.4 14.3d 1.6de 8.7S+THUT 13.7d 1.6ef 8.5 14.6cd 1.7cde 7.9S+THUM 13.7d 1.7cde 8.0 13.0d 1.5fg 8.5

Soil+TOS (TPOMW+olive prunings+sheep manure)S+TOSI 16.9ab 1.8cd 9.4 15.8bc 1.7cd 9.1S+TOST 17.4a 2.0a 8.9 15.9bc 2.0a 8.0S+TOSM 17.1ab 1.9ab 8.9 17.0ab 2.0a 8.5

CTOT: Total organic carbon. NTOT: Total nitrogen. I=initial mixture; T=thermophilicstage; M=mature compost. For each variable, values followed by the same letter arenot significantly different according to the Duncan test (Pb0.05).



HA, which thermograms were dominated by the first exothermic re-action (thermal degradation more labile compounds such as carbohy-drates). The R1 values of free and bound HA were generally lower in

amended soils than in control, especially in the case of bound HAthat decreased from 1.53 in control to values close to 1 in amendedsoils after 150 days of incubation. The low R1 reflects the persistence

S I T M I T M I T M I T M I T M I T M

free

C (

mg

g-1)

0

1

2

3

4

90 days

S

S

boun

d C

(m

g g-1

)

0

1

2

3

4

S+TS

I T M

S+THU

I T M

S+TOS

I T M S

S+TS

I T M

S+THU

I T M

S+TOS

I T M

Humic CNon-humic C

150 days

F F FCDECDE CD CDE CDE

EFA CD AB

DEF DEFBC

EF EF BC BC BC

A A

E E

ABABCABC

BC CDABC ABC ABC ABC ABC

BC ABC ABCD

AB AB

fgabc cde cdef cd efg efg

abcd bcd

def defcd

g

ab abdef

abcd

abcdabcd abcd

a

abcd abcdabcd

d abcdabcd

abcdabcd

fgcd def

cd

cdd

cd

abab

Fig. 2. Free and bound non-humic and humic organic C in control and amended soils (S=control; S+TS=TPOMW+sheep manure compost amended soil; S+THU=TPOMW+horse manure+urea compost amended soil; S+TOS=TPOMW+olive tree prunings+sheep manure compost amended soil; I=initial; T=thermophilic; M=maturecompost) at 90 and 150 days of incubation. For each fraction (free and bound C), bars with the same letter are not significantly different according to the Duncan's test (Pb0.05).

Table 3Elemental composition and ratios from thermal and FT-IR analysis of free HA from con-trol and amended soils (S=control; S+TS=TPOMW+sheep manure compostamended soil; S+THU=TPOMW+horse manure+urea compost amended soil;S+TOS=TPOMW+olive tree prunings+sheep manure compost amended soil; I=initial; T=thermophilic; M=mature compost) at 90 and 150 days of incubation.

Treatment Mass (mg g−1, ash freebasis)

Atomic ratios R1c RIR

d

C N H Oa N/Cb H/C

90 daysS 511 57 66 366 9.6 1.5 0.47 1.66S+TSI 523 63 70 343 10.4 1.6 0.70 1.67S+TST 527 64 66 343 10.5 1.5 0.49 1.86S+TSM 520 63 67 349 10.5 1.5 0.64 1.75S+THUI 524 68 70 339 11.1 1.6 0.38 1.57S+THUT 520 68 67 345 11.2 1.6 0.46 1.45S+THUM 528 66 70 336 10.7 1.6 0.47 1.69S+TOSI 539 56 70 334 9.0 1.6 0.31 1.50S+TOST 548 57 71 325 8.9 1.5 0.38 1.55S+TOSM 544 64 72 320 10.0 1.6 0.40 1.57Spe 15 3 8 18 0.2 0.2

150 daysS 508 55 63 374 9.2 1.5 0.69 1.73S+TSI 558 69 75 298 10.6 1.6 0.40 1.77S+TST 577 72 73 278 10.6 1.5 0.37 1.56S+TSM 558 70 73 299 10.7 1.6 0.40 2.02S+THUI 535 66 57 341 10.7 1.3 0.48 1.42S+THUT 541 69 67 323 11.0 1.5 0.45 1.75S+THUM 539 72 63 327 11.4 1.4 0.45 1.60S+TOSI 545 59 73 322 9.3 1.6 0.44 1.54S+TOST 544 61 75 319 9.7 1.7 0.44 1.65S+TOSM 552 62 69 317 9.7 1.5 0.43 1.61Sp 10 2 5 11 0.1 0.2

a Calculated by difference.b (×100).c Ratio between the mass losses associated with the second and the first exothermic

reactions of thermal analysis.d Ratio between aromatic (1620 cm−1) to aliphatic (2925 cm−1) FT‐IR peaks.e Pooled standard deviation (Sp) of the chemical analysis (McNaught andWilkinson,

1997).

Table 4Elemental composition and ratios from thermal and FT-IR analysis of bound HA fromcontrol and amended soils (S=control; S+TS=TPOMW+sheep manure compostamended soil; S+THU=TPOMW+horse manure+urea compost amended soil;S+TOS=TPOMW+olive tree prunings+sheep manure compost amended soil; I=initial; T=thermophilic; M=mature compost) at 90 and 150 days of incubation.

Treatment Mass (mg g−1, ash freebasis)

Atomic ratios R1c RIR

d

C N H Oa N/Cb H/C

90 daysS 585 51 72 294 7.5 1.4 1.25 2.53S+TSI 616 49 74 261 6.8 1.4 1.20 2.10S+TST 573 58 75 294 8.7 1.6 1.18 2.08S+TSM 575 55 76 293 8.3 1.6 1.10 2.04S+THUI 579 49 77 295 7.2 1.6 0.98 2.22S+THUT 529 47 67 357 7.6 1.5 0.93 2.00S+THUM 575 53 74 298 8.0 1.6 0.94 2.25S+TOSI 578 51 76 294 7.6 1.6 0.90 1.88S+TOST 578 52 79 291 7.8 1.6 0.98 2.01S+TOSM 586 53 77 284 7.7 1.6 0.99 2.23Spe 4 1 4 6 0.2 0.2

150 daysS 562 41 68 329 6.3 1.4 1.53 3.10S+TSI 586 47 73 294 6.9 1.5 1.00 2.91S+TST 572 50 74 304 7.5 1.5 0.92 3.73S+TSM 600 50 71 280 7.1 1.4 0.91 3.50S+THUI 550 43 70 338 6.7 1.5 0.82 2.93S+THUT 572 49 80 300 7.4 1.7 0.92 3.59S+THUM 578 49 78 295 7.3 1.6 1.05 3.53S+TOSI 585 46 78 291 6.7 1.6 0.90 2.36S+TOST 576 46 78 299 6.9 1.6 1.00 2.81S+TOSM 587 47 81 285 6.9 1.6 1.05 3.13Sp 3 1 3 5 0.2 0.2

a Calculated by difference.b (×100).c Ratio between the mass losses associated with the second and the first exothermic

reactions of thermal analysis.d Ratio between aromatic (1620 cm−1) to aliphatic (2925 cm−1) FT‐IR peaks.e Pooled standard deviation (Sp) of the chemical analysis (McNaught andWilkinson,

1997).



of the components of lower thermal stability derived form thehumic-like material added with the composting mixtures. These ra-tios were not significantly affected by the degree of stabilisationand/or the initial composition of the composting mixtures added tothe soil.

The structural changes of HA were also studied by the evolution ofdefined FT-IR peaks during the incubation (Chefetz et al., 1996). Relativeintensities (RIR) of absorption at 1600–1620 cm−1 (aromatic C=C andH-bonded C=O of conjugated ketones) and at 2900–2940 cm−1 (ali-phatic C–H stretching) were used to compare aromaticity of free andboundHA fromcontrol and amended soils. BoundHAwere characterisedby a larger proportion of aromatic structures (larger RIR) than free HA.The addition of the organic amendments generally caused a temporal de-crease of this ratio in freeHAafter 90 days of incubation compared to soilcontrol, except for the TS treatment, but no significant differences wereobserved after 150 days of composting. In the case of bound HA, the RIRdisplayed a tendency to increase with the degree of stabilisation of theadded composting mixtures.

The apparent MW distributions of free and bound HA extractedfrom the control and amended soil after 90 and 150 days of incuba-tion are presented in Fig. 3. Free and bound HA showed a predomi-nance of relatively small molecules (0.3–5 kDa) in all treatments.Bound HA also contained a small amount of large apparent MW

components, in the range between 5 and 20 kDa. An increased pro-portion of components of larger apparent MW was found in allamended soils in the free HA fraction and only in bound HA of soilsamended with the TS mixtures. MW distributions were highly repro-ducible and not affected by incubation time and/or stability degree ofthe amendment except again for the TS treatments.

The 13C CPMAS NMR spectra of free and bound HA extracted fromthe control and amended soils after 90 and 150 days are given inFig. 4. The relative area distribution of the different chemical shift-regions together with ratios of alkyl C to O-alkyl C, alkyl C toN-alkyl+methoxyl C and O-alkyl C to aromatic C are reported inTables 5 and 6, for free and bound HA fractions, respectively. Spectraof free HA were typical of mineral soils and had a more unfavourablesignal to noise ratio as compared to bound HA. Free HA also displayeda peak at 176 ppm caused by inorganic carbonate that was excludedfrom calculations.

The alkyl C chemical-shift region (0–45 ppm) accounted for 30–35% of the total signal intensity and appeared as a dominating signalat 30 ppm attributable to polymethylene C (–CH2–) in long chain al-iphatic structures (e.g. fatty acids, waxes). The alkyl C region of thefree HA was also characterised by a signal at 40 ppm that arisesfrom quaternary groups, suggesting the presence of highly branchedaliphatics (Quideau et al., 2001). This alkyl C fraction can be

TS

App

aren

t MW

dis

trib

utio

n (%

)

0

20

40

60

80

100TS

THU

0

20

40

60

80

100THU

TOS

log MW

0

20

40

60

80

100TOS

0 1 2 3 4 5 0 1 2 3 4 5

S + M

90 days 150 days

Free HA : S S + I S + T S + M

Bound HA : S S + I S + T

Fig. 3. Apparentmolecular weight (MW) distributions of free and bound HA fractions from control and amended soils (S=control; S+I=soil amended with initial composting mixture;S+T=soil amended with thermophilic composting mixture; S+M=soil amended with mature compost; TS=TPOMW+sheep manure compost; THU=TPOMW+horsemanure+urea compost; TOS=TPOMW+olive tree prunings+sheep manure compost) at 90 and 150 days of incubation.



selectively preserved in soil as a component of biomacromolecules orcan be formed by oxidative polymerisation of low-molecular weightlipids, representing a precursor of recalcitrant and stabilised aliphaticsoil organic C (Lorenz et al., 2007).

The sharp signal centered at 56 ppm is characteristic of methoxylgroups from hemicelluloses and from guaiacyl and syringyl units oflignin (Hatcher, 1987; Kögel-Knabner, 2002). Lignin and modified lig-nins, but also N-alkyl C in peptides (Kögel-Knabner, 2002), derivedfrom the composting mixtures, caused an increase in the relative in-tensity of this signal in all amended soils. The proportion of N-alkyl+methoxyl C in free HA (between 15–18%) was larger thanthat in bound HA (between 11–13%). The presence of N compoundsinto the free HA pool was also observed in the elemental analysis of

free HA extracted from amended soils (Tables 3 and 4), but therewas a general decrease of the relative area of this signal from 90 to150 days of incubation, suggesting the temporal protection of thesefractions in the biochemical stabilised free HA pool.

Aliphatic alcohols and ether moieties such as those present instructural polysaccharides (cellulose and hemicelluloses) resonatein the chemical-shift region between 50–110 ppm but several struc-tural elements of lignin also produce signals in this same range(Kögel-Knabner, 2002). This region was dominated by the signal at73 ppm, attributed to ring carbons of carbohydrates, probably fromhemicelluloses or lignin-hemicellulose complexes added with the or-ganic amendments. This signal was much more intense in free HAisolated from amended soils. Among them, it is worth to underline

Free HA / 90 days

200 150 100 50 0

200 150 100 50 0

200 150 100 50 0

200 150 100 50 0

S

S

Free HA / 150 days

Bound HA / 90 days Bound HA / 150 days

Chemical shift (ppm) Chemical shift (ppm)

Chemical shift (ppm) Chemical shift (ppm)

S

S + T S I

S

S + T S T

S + T S M

S + T H U I

S + T H U T

S + T H U M

S + T O S I

S + T O S T

S + T O S M

S + T H U I

S + T H U T

S + T H U M

S + T O S I

S + T O S T

S + T O S M

S + T S M

S + T S T

S + T S I

Fig. 4. 13C CPMAS-TOSSNMRspectra of free and boundHA fromcontrol and amended soils (S=control; S+TS=TPOMW+sheepmanure compost amended soil; S+THU=TPOMW+horsemanure+urea compost amended soil; S+TOS=TPOMW+olive tree prunings+sheepmanure compost amended soil; I=initial; T=thermophilic;M=mature compost) at 90 and150 daysof incubation.



the larger relative area of this signal in the TOSI and TOST treated soils(23–24% of the total signal intensity compared to the 13–16% of theother treatments; Table 5) which reflected a large presence ofO-alkyl C into the free HA, probably as a mixture of carbohydrate

moieties and the pre-existing soil HA. The incorporation or protectionof carbohydrates into the soil humic pool may be also reflected by theresonance at 63 ppm, only visible in the spectra of TOSI and TOSTamended soils, which arises from the C-6 (in hexoses) or C-5 (in

Table 5Relative area distributions (%) of solid state 13C CPMAS NMR spectra of free HA from control and amended soils (S=control; S+TS=TPOMW+sheep manure compost amended soil;S+THU=TPOMW+horsemanure+urea compost amended soil; S+TOS=TPOMW+olive tree prunings+sheepmanure compost amended soil; I=initial; T=thermophilic;M=maturecompost) at 90 and 150 days of incubation.

Treatment Alkyla Methoxyb N-alkyl O-alkyl Aromc Carboxyl, amide,ester, carbonyld

Alkyl/O-alkyl Alkyl/N-alkyl O-alkyl/Arom

0–45 45–65 65–110 110–160 160–210

90 daysS 31.2 10.5 13.0 16.1 18.2 2.40 2.97 0.81S+TSI 28.6 14.9 14.2 14.6 20.0 2.02 1.92 1.05S+TST 26.6 16.6 12.8 14.3 20.5 2.07 1.61 0.90S+TSM 31.7 17.9 15.5 16.1 18.7 2.04 1.77 0.96S+THUI 33.5 16.3 16.3 11.0 17.4 2.06 2.06 1.47S+THUT 32.0 17.8 16.6 15.2 18.4 1.93 1.80 1.09S+THUM 31.6 15.7 14.5 14.9 18.0 2.18 2.00 0.97S+TOSI 30.2 16.6 24.2 10.8 16.1 1.25 1.82 2.23S+TOST 30.4 16.5 23.6 12.4 15.7 1.29 1.84 1.91S+TOSM 34.0 15.7 12.5 11.6 19.7 2.73 2.16 1.07


a Alkyl C.b Methoxyl C.c Aromatic C.d Carboxyl, carbonyl, amide and ester C.

Table 6Relative area distributions (%) of solid state 13C CPMAS NMR spectra of bound HA from control and amended soils (S=control; S+TS=TPOMW+sheep manure compost amended soil;S+THU=TPOMW+horsemanure+urea compost amended soil; S+TOS=TPOMW+olive tree prunings+sheepmanure compost amended soil; I=initial; T=thermophilic;M=maturecompost) at 90 and 150 days of incubation.

Treatment Alkyla Methoxyb N-alkyl O-alkyl Aromc Carboxyl, amide,ester, carbonyld

Alkyl/O-alkyl Alkyl/N-alkyl O-alkyl/Arom

0–45 45–65 65–110 110–160 160–210



a Alkyl C.b Methoxyl C.c Aromatic C.d Carboxyl, carbonyl, amide and ester C.



pentoses) carbon atoms of hemicelluloses (Kögel-Knabner, 2002). Asimilar behaviour was observed in the defined signal at 103 ppmpresent in free HA from amended soils (not found in control soil).This signal was very sharp in TOS amended soils, characterised by arich lignocellulosic composition, probably due to syringyl units fromlignin (C2 and C6 ring carbon atoms)(Hatcher, 1987), or to anomericC1 carbon atom of polysaccharides (Kögel-Knabner, 2002) or evenfrom tannins, confirmed the presence of such carbohydrate andpoliphenol moieties along side the native soil HA.

The high proportion of O-alkyl C in the soils amended with theTOSI and TOST mixtures (Table 5) caused a decrease in the alkyl C toO-alkyl C ratio in the free HA fraction. A decrease in this ratio indi-cates larger potential degradability in the amended soils comparedto control soil (Baldock et al., 1997; Webster et al., 2001). On theother hand, these changes persisted even after 5 months of incuba-tion under conditions that favour intense mineralisation.

Signals in the aromatic C region (110–160 ppm) at respectively130 and 150 ppm correspond to C-substituted aromatic C and pheno-lic C from lignin structures. HA from the bound C fraction and controlsoil had, in general, a larger proportion of aromatic C (15.4–18.5%)than free HA (9.2–15.5%) and presented larger aromatic character(Tables 5 and 6). The phenolic group signal (145–160 ppm) detectedin both the free and bound fractions of amended soils was even moreclearly distinguishable after 150 days. The O-alkyl C to aromatic Cratio has been suggested as an indicator of the stabilisation degreein humic substances (De Nobili et al., 2008). This index is muchlower in bound HA (Table 6) and shows the preferential binding of ar-omatic structures to soil minerals. This ratio decreased during incuba-tion in the free HA fraction (Table 5) suggesting increased structuralcomplexity.

The region between 160 and 210 ppm, attributed to the resonanceof carboxyl- (also esterified), carbonyl- and amide C, showed a dom-inating signal at 175 ppm. Bound HA were enriched with thesegroups compared to free HA (Tables 5 and 6) indicating a larger con-tent of peptidic moieties and/or the presence of carboxyl groups in al-iphatic acids or resistant O–C groups from alkyl polyesters. Thisspecific region was not significantly affected by the initial composi-tion or degree of stabilisation of the organic amendments.

4. Conclusions

The qualitative and quantitative changes observed in the differenthumic fractions of amended soils demonstrate the partial and pro-gressive incorporation of components from the applied TPOMWcomposting mixtures. The increase in both free and bound extractableC fractions reflected the incorporation of the humic-like materialadded with the organic amendments into soil humic pool, beingprotected against degradation during the incubation period. Freeand bound HA display distinctive different chemical and spectroscop-ic features: free HA where characterised by 13C CPMAS NMR spectrawith a larger proportion of N-alkyl and O-alkyl C and much lower RIand RIR values than bound HA extracted from the correspondingtreatments. The lignocellulosic rich composition of TPOMW and thepresence of active water- and alkali-soluble C fractions in the organicamendments promoted the incorporation/protection of altered ligninstructures and carbohydrates moieties into the pre-existing soil HA.The addition of TOS amendments, characterised by the largest ligninand holocellulose concentrations, markedly increased the proportionof labile C (O-alkyl C) biochemically stabilised in the free humic frac-tion. The selective incorporation of alkyl-C fraction into the humicpool may reflect the stabilisation of recalcitrant biomolecules, whichis coherent with the observed increase of the proportion of highMW fractions. Further investigations should determine the potentialsaturation level of the HA binding capacity of these soils. This willprovide useful information to design field experiments to evaluate ef-fective sequestration potentials of low C input Mediterranean soils.

Acknowledgements

This work was supported by the Spanish Ministry of Science andInnovation, research projects Ref. CTM2005-05324 and CTM2009-14073-C02-02. The staff of IFAPA Centro “Venta del Llano” is grateful-ly acknowledged for its assistance in compost preparation, especiallyDr. A. Fernández-Hernández and Dr. C. García-Ortíz Civantos. Wethank Mr. Aldo Bertoni and Mr. Andrea Cudini for their skilful techni-cal assistance.

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