Molecular evaluation of soil organic matter characteristics in three agricultural soils by improved off-line thermochemolysis: The effect of hydrofluoric acid demineralisation treatment

Analytica Chimica Acta 802 (2013) 46–55

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Molecular evaluation of soil organic matter characteristics in three agricultural soils by improved off-line thermochemolysis: The effect of hydrofluoric acid demineralisation treatment Riccardo Spaccini a,b,∗ , XiangYun Song b,1 , Vincenza Cozzolino b , Alessandro Piccolo a,b a

Dipartimento di Agraria, Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy Centro Interdipartimentale di Ricerca sulla Risonanza Magnetica Nucleare per l’Ambiente, l’Agro-Alimentare ed i Nuovi Materiali (CERMANU), Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy b

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• The composition of soil organic matter in three agricultural fields was determined by off-line pyrolysis. • Clay minerals of heavy textured soils heavily affect the results of thermochemolysis. • Soil treatment with hydrofluoric acid enhanced the performance of thermochemolysis. • The thermochemolysis of demineralised soils revealed significant differences among soil treatments.

a r t i c l e

i n f o

Article history: Received 1 July 2013 Received in revised form 15 September 2013 Accepted 17 September 2013 Available online 27 September 2013 Keywords: Molecular characterization Soil organic matter Off-line thermochemolysis Clay minerals Hydrofluoric acid

a b s t r a c t The molecular composition of soil organic matter (SOM) in three agricultural fields under different managements, was evaluated by off-line thermochemolysis followed by gas chromatography mass spectrometry analysis (THM-GC-MS). While this technique enabled the characterization of SOM components in coarse textured soil, its efficiency in heavy textured soils was seriously affected by the interference of clay minerals, which catalyzed the formation of secondary artifacts in pyrolysates. Soil demineralization with hydrofluoric acid (HF) solutions effectively improved the reliable characterization of organic compounds in clayey soils by thermochemolysis, while did not alter significantly the results of coarse textured soil. A wide range of lignin monomers and lipids molecules, of plant and microbial origin, were identified in the pyrograms of HF treated soils, thereby revealing interesting molecular differences between SOM management practices. Our results indicated that clay removal provided by HF pretreatment enhanced the capacity of thermochemolysis to be a valuable and accurate technique to study the SOM dynamics also in heavy-textured and OC-depleted cultivated soils. © 2013 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: Dipartimento di Agraria, Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy. Tel.: +39 0812539176; fax: +39 0812539186. E-mail address: [email protected] (R. Spaccini). 1 Present address: Institute of Resources, Ecosystem and Environment of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China. 0003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2013.09.031

The molecular characterization of soil organic matter (SOM) is a basic requirement for the investigation on composition and dynamics of soil organic carbon (SOC) and to study the role of SOM management in the sustainability of agro ecosystems. Accumulation and decomposition of SOC closely depend, in fact, not only on the quantity of incorporated SOM, but also on its chemical composition that, in turn, controls SOM functions [1–3]. Various approaches

R. Spaccini et al. / Analytica Chimica Acta 802 (2013) 46–55

47

Table 1 Soil classification, OC content (g kg−1 ), textural (%) and clay composition of different experimental fields. Field sites

Soil type

OC

Soil texture

Sand

Silt

Clay

Clay typea

Torino Piacenza Napoli

Typic Ustifluvent Udifluventic Haplustept Vertic Haploxeralf

7.7 12.3 8.9

Silt loam Silty clay loam Sandy clay loam

36.9 17.9 47.0

56.2 47.1 20.1

6.9 35.0 32.9

C÷V, K, I, S(-) S, S/I I, K

(-) Minor component. a C, chlorite; K, kaoline; I, illite; S, smectite; V, vermiculite; S/I, interstratified minerals.

and methodologies have been applied to analyze the various SOM components and fractions, which are characterized by specific origin and different molecular characteristics [4–6]. Although each technique may provide useful information to clarify composition and functionalities of SOM pools, most analytical methodologies show peculiar advantages and drawbacks according to samples and isolation procedures. Thus, no widely shared protocols have been established for univocal or unambiguous determination of SOM pools [7,8]. Pyrolysis followed by gas chromatograph–mass spectrometry (Pyr-GC-MS) is a powerful tool for the analysis of OM, since it can be directly applied on complex matrices (soils, humic substances, plant tissues, composts), thus limiting the inconveniences related to extraction, fractionation and purification steps. Although the utilization of high temperatures may promote the possible occurrence of artifacts [9,10], the application of Pyr-GC-MS provided valuable information to correlate SOM composition with soil morphology, land use, cropping sequences and soil management [11–13]. However, notwithstanding the effectiveness of Pyr-GC-MS on SOM studies, also this technique may undergo serious limitations depending on textural and mineral composition of soil samples. The analyses of sedimentary OM have shown that the presence of clay minerals heavily affect the results of pyrolysis [14,15]. Furthermore, previous studies, based on model organic compounds, revealed that clay surfaces can act as catalyst against the thermally unstable organic functional groups (alcohols, phenols, aliphatic and aromatic acids, aminoacids). The interference of clays promotes the formation of secondary organic derivatives (like alkenes/alkanes, alkyl-nitriles, poly-aromatic compounds, alkylbenzenes) that hinder the identification of the original organic materials [16–18]. Soil treatment with hydrofluoric acid (HF) solutions is an effective procedure to remove clay minerals from soil samples and is currently applied on SOM analyses based on solid state NMR spectroscopy [19,20]. Despite the feasibility of HF treatment for soil demineralisation, only few works have investigated its effect on the pyrolysis of soil samples. The studies on SOM composition after acid hydrolysis have mainly focused on the application of classical on-line Pyr-GC-MS methods [21,22] and were limited to the analysis of coarse textured sandy soils and C-enriched samples of forest soil or specific soil fractions [23,24] In respect to on-line flash-pyrolysis method, the applications of off-line pyrolysis in the presence of tetra-methyl ammonium hydroxide (TMAH) is regarded as a more suitable technique for the analysis of complex samples, such as those represented by organic materials, sediments and agricultural soils [3,25,26]. The thermochemolysis (THM) conducted in off-line mode allows the utilization of a large amount of sample and provides an effective quantitative determination of pyrolytic products [27,28]. Moreover the addition of an alkylation reagent, like TMAH, involves the solvolysis and methylation of ester and ether bonds present in the organic materials, thereby enhancing the thermal stability of polar functional groups. The requirement of lower temperatures combined with the occurrence of solvolysis and methylation reactions, are believed to limit the interactions of clay surfaces and avoid the formation of secondary pyrolytic rearrangements [18].

In the present work the off-line thermally assisted hydrolysis and methylation followed by gas chromatography and mass spectrometry (THM-GC-MS), was applied before and after soil demineralisation with HF treatment, to analyze the molecular composition of SOM in three OC-depleted agricultural soils. The aim was to assess the effectiveness of HF treatment for the application of thermochemolysis on soil samples and to evaluate the effect of acid hydrolysis on yield and distribution of SOM components. 2. Materials and methods 2.1. Soil samples and management practices Soils from the experimental farms of the Italian Universities of Torino, Piacenza and Napoli, were subjected to the following field managements during a three year experiment, using maize (Zea mais L.) as a monoculture annual crop: 1. Traditional (Tra) tillage: plowing at 35 cm depth, surface harrowing and addition of mineral fertilizers; 2. Minimum (Min) tillage: no plowing, surface harrowing and addition of mineral fertilizers. Each soil treatment was performed on a 4 m × 4 m plot with four replicates, in a randomized block experiment. Details of the experimental design are reported elsewhere [29]. Classification (USDA Soil Taxonomy), textural (pipette method), organic carbon content (Wakley-black method) and clay composition (X-ray diffractometry) of soil samples are shown in Table 1. After three year of cultivation, soil samples from the ploughed horizon (30 cm) of each plot were randomly sampled, air-dried and sieved at 2 mm. Soil samples were then sonicated at low energy (170 J g−1 ) to break down macro-aggregates (>0.25 mm). Briefly, 35 g of dry weight soil sample were placed in a 150 mL glass beaker with 100 mL of deionised water and sonicated at 50 J s−1 for 120 s. After sonication the moist soil samples were then centrifuged at 7000 × g for 30 min and the supernatant discarded. This procedure helps to remove the undecomposed light coarse organic debris, which was discarded with the supernatant and maintains only the OM closely associated with soil fractions [30]. The soil samples were finally oven-dried at 40 ◦ C, reduced to powder by an electrical agate-ball mill (Retsch PM 200, Retsch GmbH & Co., Haan, Germany) and sieved at 500 mm for the subsequent analyses. 2.2. HF treatment For the demineralisation treatment 10 g of each soil replicates were placed in 200 mL centrifuge tube and added with 100 mL of hydrofluoric acid (HF-39.5%) water solution (5%, v/v), corresponding to about 1.12 M of HF, and shaken overnight. The solution was then centrifuged at 7000 × g for 30 min and the supernatant discarded. The HF treatment was repeated twice, and the final residue was extensively washed with deionised water till pH 5–6. 2.3. Off-line pyrolysis TMAH-GC-MS The suitable operative conditions applied for off-line pyrolysis (soil amount, TMAH/soil ratio, temperature program, run time, He flow) were determined in previous experiments [3,25,30]. About

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R. Spaccini et al. / Analytica Chimica Acta 802 (2013) 46–55

Table 2 Identified productsa released by THM of untreated samples from the field site of Torino. RTa

Assignmentb

Characteristic ions m/z

5.5 5.8 6.0 8.0 8.7 9.0 9.9 11.3 11.6 11.9 12.4 14.3 14.8 15.0 15.8 16.4 17.1 17.5 18.0 18.3 18.7 19.3 19.8 20.6 20.8 21.5 21.8 22.1 22.9 23.6 24.0 24.2 24.5 25.1 25.4 25.8 25.9 26.0 26.2 26.5 26.8 28.1 28.2 28.2 28.4 28.6 28.7 28.8 29.0 29.3 30.1 30.5 31.0 31.3 31.5 31.7 32.0 33.3 33.5 34.0 34.2 34.6 35.4 36.0 36.2 36.7 37.4 37.8 38.0 38.2 38.6 39.0 39.8 40.1 40.4 40.8

Benzene, 1-CH3 O Lg P1 1H-Pyrrole, 2-ethyl-4-methylBenzene, 1-methoxy-4-methyl Lg P2 Methoxy pyridine Benzene, 1-ethenyl-4-CH3 O Lg P3 1,2-Di-CH3 O benzene Lg G1 CH3 O benzaldheyde Lg P4 Benzoic acid, 4-CH3 O, ME Lig P6 Indole, 2-methylCarbohydrate derivative Carbohydrate derivative 1,2,3-Tri-CH3 O benzene Lg S1 Carbohydrate derivative Carbohydrate derivative 1H-Isoindole-1,3-dione, 2-methyl4-CH3 O-1-methylindole Carbohydrate derivative 3,4-Di-CH3 O benzaldehyde Lg G4 Carbohydrate derivative Carbohydrate derivative C12 FAME Nonanedioic acid, DIME 3,4-Di-CH3 O acetophenone Lg G5 Benzoic acid, 3,4-di-CH3 O ME Lg G6 3,4,5-Tri-CH3 O-benzaldehyde, Lg S4 cis-2-(3,4-Di-CH3 O phenyl)-1-CH3 O ethylene Lg G7 trans-2-(3,4-Di-CH3 O phenyl)-1-CH3 O ethylene Lg G8 cis-1-(3,4-Di-CH3 O phenyl)-3-CH3 O 1-propene Lg G9 trans-3-(4-CH3 O phenyl)-3-propenoic acid ME Lg P18 N compound 3,4,5-Tri-CH3 O benzoic acid ME Lg S6 C14 FAME cis-1-(3,4-Di-CH3 O phenyl)-1-CH3 O-1-propene Lg G11 trans-1-(3,4-Di-CH3 O phenyl)-3-CH3 O-1-propene Lg G13 cis-1-(3,4,5-Tri-CH3 O phenyl)-2-CH3 O ethylene Lg S7 Thr/Eryth.1-(3,4-di-CH3 O phenyl)-1,2,3-tri CH3 O propane Lg G14 trans-1-(3,4,5-Tri-CH3 O phenyl)-2-CH3 O ethylene Lg S8 C15 iso FAME Mic-PLFA Thr/Eryth.1-(3,4-di-CH3 O phenyl)-1,2,3-tri CH3 O propane Lg G15 cis-1-(3,4,5-Tri-CH3 O phenyl)-1-CH3 O prop-1-ene Lg S10 C15 anteiso FAME Mic-PLFA trans-1-(3,4,5-Tri-CH3 O phenyl)-3-CH3 O-1-Propene Lg S13 trans-3-(3,4-Di-CH3 O phenyl)-3-propenoic acid ME Lg G18 cis-1-(3,4-Di-CH3 O phenyl)-1,3-di-CH3 O-1-Propene Lg G16 C16 iso FAME Mic-PLFA Thr./Eryth.1-(3,4,5-tri-CH3 O phenyl)-1,2,3-tri CH3 O propane Lg S14 C16: 1 FAME Mic-PLFA Th./Eryth.1-(3,4,5 tri CH3 O phenyl)-1,2,3-tri CH3 O propane Lg S15 C16:1 FAME C16 FAME C17 iso FAME Mic-PLFA C17 anteiso FAME Mic-PLFA trans-3-(3,4,5-Tri-CH3 O phenyl-3-Propenoic acid ME Lg S18 C17 cy FAME Mic-PLFA cis-1-(3,4,5-Tri-CH3 O phenyl)-1,3-di CH3 O prop-1-ene Lg S16 C17 n-FAME C18 iso FAME Mic-PLFA C18:1 FAME Mic C18:1 FAME C18 FAME Carbohydrate derivative C16, 16 CH3O, FAME Carbohydrate derivative cy C19 FAME Mic-PLFA C19 FAME C16 dioic acid DIME C16, 8(9,10)-16 diCH3O, FAME C23 alkane Carbohydrate derivative C18:1, 18 CH3O, FAME C20 FAME Carbohydrate derivative C24 alkane C18:1 dioic acid DIME Carbohydrate derivative C18 dioic acid DIME

65, 78 M+ 108 94 M+ 109 77, 91, 107 M+ 122 79, 108 M+ 109 65, 91, 119 M+ 134 77, 95, 123 M+ 138 77, 92, 135 M+ 136 77, 92, 135 M+ 166 77, 130 M+ 131 101, 129, 161 M+ nd 101, 129, 161 M+ nd 110, 125, 153 M+ 168 88, 101, 130 M+ nd 88, 101, 130 M+ nd 76, 104 M+ 161 118, 146 M+ 161 101, 129, 161 M+ nd 151, 165, M+ 166 101, 129, 161 M+ nd 88, 101, 130 M+ nd 74, 87, 187, M+ 214 55, 152, 185 M+ 216 137, 165, M+ 180 165, 181, M+ 196 125, 181, M+ 196 151, 179 M+ 194 151, 179 M+ 194 91, 177 M+ 208 133, 161 M+ 192 98 M+ nd 195, 211 M+ 226 74, 87, 211 M+ 242 165, 193 M+ 208 91, 177 M+ 208 181, 209 M+ 224 166, 181 M+ 270 181, 209 M+ 224 74, 87, 213 M+ 256 166, 181, M+ 270 195, 223 M+ 238 74, 87, 199, 256 91, 207 M+ 238 191, 207 M+ 222 176, 207 M+ 238 74, 87, 227 M+ 270 181, 211 M+ 300 55, 74, 236 M+ 268 181, 211 M+ 300 55, 74, 236 M+ 268 74, 87, 239 M+ 270 74, 87, 241 M+ 284 74, 87, 227 M+ 284 221, 237 M+ 252 55, 69, 250 M+ 282 206, 237 M+ 268 74, 87, 253 M+ 284 74, 87, 253 M+ 298 55, 69, 264 M+ 296 55, 69, 264 M+ 296 4, 87, 267 M+ 298 101, 111, 187, 219 M+ nd 55, 74, 268, 285 M+ 300 101, 111, 187, 219 M+ nd 55, 69, 278 M+ 310 74, 87, 281 M+ 312 74, 98, 241, 283 M+ 314 71, 95, 87, 201, 215 M+ 330 57, 71, 85, M+ 324 101, 111, 187, 219 M+ nd 55, 67, 81, 262, 294 M+ 326 74, 87, 295 M+ 326 101, 111, 187, 219, 279 M+ nd 57, 71, 85, M+ 338 55, 69, 97, 308 M+ 340 101, 111, 187, 219, 279 M+ nd 74, 98, 241, 311 M+ 342

R. Spaccini et al. / Analytica Chimica Acta 802 (2013) 46–55

49

Table 2 (Continued) RTa

Assignmentb

Characteristic ions m/z

41.2 41.6 42.3 42.8 43.4 43.9 44.1 44.4 44.8 45.4 46.0 46.3 46.5 47.0 47.3 47.7 47.9 48.5 48.6 48.8 49.1 49.4 49.7 50.0 50.6 50.9 51.2 51.5 51.8 52.1 52.2 52.6 54.0 54.4 55.8 56.7 57.1

Abiet 7 en 18 oic acid ME C25 alkane C22 FAME C20, 20CH3 O, FAME C26 alkane C24-CH3 O C18, 9-10 epoxy 18CH3 O, FAME C20 dioic acid DIME C22, 2-CH3 O, FAME Mic C27 alkane C24 FAME C23, 2-CH3 O, FAME Mic C22, 22-CH3 O, FAME C28 alkane Squalene C26-OMe C24, 2-CH3 O, FAME Mic C22 dioic acid DIME Abieta 8 11 13-triene 3,13 diCH3 O Phytosterol (tetracyclcic) C29 alkane C26 FAME C25, 2-CH3 O, FAME Mic C24, 24-CH3 O, FAME Phytosterol (tetracyclcic) C28-CH3 O Phytosterol (tetracyclcic) C24 dioic acid DIME Phytosterol (tetracyclcic) C31 alkane C28 FAME Triterpenol (pentacyclcic) C30-CH3 O C33 alkane C30 FAME Triterpenol (pentacyclcic) Triterpenol (pentacyclcic)

121, 259, 303 M+ 318 57, 71, 85, M+ 352 74, 87, 323 M+ 354 55, 74, 292, 324, M+ 356 57, 71, 85, M+ 366 57, 69, 83, 346, M+ 368 74, 87, 185, 282, 312 M+ 342 74, 98, 241, 339 M+ 370 57, 71, 97, 325 M+ 384 57, 71, 85, M+ 380 74, 87, 351 M+ 382 57, 71, 97, 339 M+ 398 55, 74, 320, 352 M+ 384 57, 71, 85, M+ 394 69, 81, 136, 341 M+ 410 57, 69, 83, 364, M+ 396 57, 71, 97, 353 M+ 412 74, 98, 241, 367 M+ 398 297, 315 M+ 330 107, 255, 329, 368 M+ 400 57, 71, 85, M+ 412 74, 87, 379 M+ 410 57, 71, 97, 367 M+ 426 55, 74, 348, 380 M+ 412 213, 255, 289, 382 M+ 414 57, 69, 83, 392, M+ 424 255, 271, 351, 394 M+ 426 74, 98, 241, 395 M+ 426 213, 255, 329, 396 M+ 428 57, 71, 85, M+ 440 74, 87, 407 M+ 438 204, 218, 301, 316 M+ 440 57, 69, 83, 420, M+ 442 57, 71, 85, M+ 468 74, 87, 435 M+ 466 189, 207, 218, M+ 440 165, 218, 275, 410 M+ 442

a b

Retention time (min). cy, cyclopropane; CH3 O, methoxy; DIME, dimethyl ester; FAME, fatty acid methyl ester; Lg, lignin; ME, methyl ester; Mic, microbial; nd, not determined.

2 g of each untreated and demineralised soil sample were placed in a quartz boat and moistened with 3 mL of TMAH solution (25% in methanol). After drying the mixture under a stream of nitrogen for about 10 min, the sample was introduced into a Pyrex tubular reactor (50 cm × 3.5 cm i.d.) and heated (ramp 20 ◦ C min−1 ) up to 400 ◦ C (10 min isothermal) for total run time of about 30 min (Barnstead Thermolyne 21100 furnace). The released products of thermochemolysis were continuously transferred by a helium flow (30 mL min−1 ) into two successive chloroform (50 mL) traps kept in ice/salt baths. The chloroform solutions were combined in a round flask and concentrated by roto-evaporation. The residue was dissolved in 0.2 mL of chloroform and transferred in a glass vial for GC–MS analysis. The GC–MS analyses were conducted with a Perkin-Elmer Autosystem XL equipped with an RTX-5MS WCOT capillary column (Restek, 30 m × 0.25 mm i.d.; film thickness = 0.25 ␮m) and coupled, through a heated transfer line (250 ◦ C), with a PE Turbomass-Gold quadrupole mass spectrometer. Chromatographic separation was achieved with the following temperature program: 60 ◦ C (1 min isothermal), raised at 7 ◦ C min−1 to 100 ◦ C and then at 4 ◦ C min−1 to 320 ◦ C (5 min isothermal), for a total run time of about 61 min. Helium was used as carrier gas at 1.90 mL min−1 , the injector temperature was at 250 ◦ C, and the split injection mode had a 30 mL min−1 of split flow. Mass spectra were obtained in EI mode (70 eV), scanning in the range of m/z 45–650, with a cycle time of 0.2 s. Compound identification was based on comparison of mass spectra with the NIST library database, published spectra, and real standards. For quantitative analysis [25,26,30], external calibration curves were built by mixing methyl-esters and/or methyl-ethers of the following standards: heptadecane, tridecanoic acid, cinnamic

acid, octadecanol, 16-hydroxy hexadecanoic acid, docosandioic acid, and beta-sitosterol. Increasing amounts of standards mixture were placed in the quartz boat and moistened with 0.5 mL of TMAH (25% in methanol) solution. The same thermochemolysis conditions were applied to the standards.

3. Results and discussion 3.1. Untreated bulk soils The total ion chromatograms (TIC) derived from the thermochemolysis of untreated soils are shown in Figs. 1 and 2, while the compounds identified in the pyrograms are listed in Table 2, for the field site of Torino, and in Tables S1 and S2 of supporting information for the experimental fields of Piacenza and Napoli in the order. The thermochemolysis applied to the coarse textured soils of Torino released more than hundred recognizable different molecules, which were identified as methyl ethers and esters of natural compounds (Fig. 1 and Table 2). The majority of these compounds originated from higher plants and microbial by-products and was represented by lignin components, fatty acids, aliphatic biopolymers, hydrocarbons and alcohols. The large yield of THM GC-MS products enabled a feasible quantitative determination of the organic compounds detected in Torino soils. Amount and distribution of the most representative organic molecules found in soil samples of Torino (Table 3) were comparable with previous results obtained from the thermochemosys of organic materials, plant tissues and soil samples [25,26,31]. This finding validates

R. Spaccini et al. / Analytica Chimica Acta 802 (2013) 46–55

Lg P18

Ch

%

0

8.4

Ch

13.4

Lg G6

100

LgG14/15

50

TRA Ch

18.4

23.4

28.4

33.4

0

8.4

13.4

Lg G6

Ch

%

18.4

43.4

48.4

Lg G10

MIN

28.4

23.4

58.4 Time

53.4

Ch

Lg P18

100

38.4

33.4

38.4

43.4

48.4

53.4

58.4 Time

Fig. 1. Total ion chromatograms of thermochemolysis products released from the untreated soil samples of experimental site of Torino. (䊉) alcohol; Ch, carbohydrate; () alkyl-dioic acid DIME; () FAME; () hydroxy-FAME; Lg, lignin.

0

5.9

10.9

15.9

Di-Methyl Naphthalene

100

20.9

25.9

C6 Benzene

%

Piacenza-MIN

Anthracene

tri-Methyl Naphthalene tri-Methyl Naphthalene

100

Alkyl Pyrene

30.9

35.9

40.9

45.9

50.9

Methyl Phenathrene Methyl Phenathrene

55.9

60.9

Time

Napoli-MIN

%

0

5.9

10.9

15.9

20.9

25.9

30.9

35.9

40.9

45.9

50.9

55.9

60.9

Time

Fig. 2. Representative total ion chromatograms of thermochemolysis products released from the untreated soil samples of experimental sites of Piacenza and Napoli. () alkene/alkane.

the effectiveness of direct application of the THM-GC-MS for the SOM investigation in soils or soil horizons with low amount of clay content. The lignin monomers released by the field plots from Torino are inherited from the structural components which build up the lignified tissues of herbaceous plants [3,26]. The specific compounds have been determined by the main fragmentation pattern (Table 2) and were associated to the current symbols used to distinguish the different structural units [31,32]: P, phydroxyphenyl; G, guaiacyl (3-methoxy, 4-hydroxyphenyl); S, syringyl (3,5-dimethoxy, 4-hydroxyphenyl). The lignin molecules found in the soil samples of Torino (Table 2) indicated the

presence of, both, fresh decaying plant residues and that of microbial processed organic materials. The latter derivatives included the oxidized products of both di- and tri-methoxy phenylpropane molecules, with the aldehydic (G4, S4), ketonic (G5, S5) and benzoic-acid (G6, S6) forms as main components. Conversely the concomitant release from the thermochemolysis of soil samples, of 1-(3,4-dimethoxyphenyl)-1(3)-methoxy-propene (G10/11, G13) and 1-(3,4,5-trimethoxyphenyl)-1(3)-methoxy-propene (S10/11, S13), as either cis or trans isomers (Table 2), may be related to the incorporation on SOM of slightly decomposed plant debris [26,31]. Moreover the identification of the enantiomers of 1-(3,4-dimethoxyphenyl)-1,2,3-trimethoxypropane (G14 and

R. Spaccini et al. / Analytica Chimica Acta 802 (2013) 46–55 Table 3 Compositiona and yields (␮g g−1 )b of main thermochemolysis products released from the field managements of untreated soil of Torino. Compounds

TRA

Lignin Linear fatty acids C12–C30 (C18:1) Long chain (>C20) % Hydroxyl fatty acids C16–C26 (C16, C18) Alkane dioic acids C16–C24 (C18:1) Alkanes C21–C31 (C27) Alcohols C24–C30 (C26) Phytosterols PFLAc C15–C19 (C15, 17 iso/anteiso) ␤-Hydroxy acids C22–C26

920 850 25.2 606 188 163 445 125 225 253

MIN 1015 920 33.0 780 236 145 410 220 408 350

a Total range varying from Ci to Cj; compounds in parentheses are the most dominant homologues; numbers after colon refer to double bond. b n = 3, overall coefficient of variations lower than 10%. c Phospholipid fatty acids.

G15) and 1-(3,4,5-trimethoxyphenyl)-1,2,3-trimethoxypropane (S14 and S15), confirmed the persistence of not decomposed lignified plant tissues [33]. Among the last eluted lignin monomers, the 3-(4,5-dimethoxyphenyl)-2-propenoic (G18) and the 3-(3,4,5trimethoxyphenyl)-2-propenoic (S18) acid forms, may have originated from either the side chain oxidation of guaiacyl and siringyl units or from the partial decomposition of aromatic domains of suberin biopolymers in plant tissues. The various alkyl molecules found in the pyrograms of field managements from Torino soil, were mainly composed by aliphatic and alicyclic lipid compounds of plant and microbial origin (Table 2). The most abundant compounds were the methyl ester of linear fatty acids, dominated by the hexadecanoic and octadecanoic saturated and unsaturated homologues. Not withstanding the multiple possible origins of the C16 and C18 acids, the amount of heavier molecules (>C20, Table 3) found in both soil managements (33 and 25% for TRA and MIN samples, respectively) and the predominance of even carbon atoms, indicated the plant waxes as prevalent source of the straight chain aliphatic acids. These compounds may derive from the breakdown of long chain ester as well as from the terminal oxidation of other components such as linear hydrocarbons and aliphatic alcohols. The prevailing role of plant input in soil lipid composition was also suggested by the detection of the C24, C26 and C28 aliphatic alcohols (Table 2), which are common components of wax layer of non-lignified tissues. This finding was confirmed by the observed distribution of long-chain hydrocarbons (Table 2), marked by the peculiar prevalence of heavier odd-numbered alkanes. The offline pyrolysis of Torino soils, produced also a notable yield of the methylated form of ␻-hydroxy alkanoic acids and alkan-dioic acids (Tables 2 and 3). These molecules are the main constituents of the external protective barriers of fresh and lignified plant tissues, namely cutin and suberin. No clear predominance of particular monomer was revealed by both of these compound classes, which instead showed an almost uniform distribution of even carbon-numbered long chain components (Table 2). Conversely, the di- and tri-hydroxy substituent of the C16 and C18 homologues were the unique components of mid-chain-hydroxy alkanoic acids found in Torino soil samples (Fig. 1; Tables 2 and 3). The 9,16/10,16-dihydroxyhexadecanoic isomers, and the 9,10 epoxide 18 hydroxy-octadecanoic (Table 2) acid were the most abundant representative monomers of these important structural units of plant cuticles, frequently used also as plant biomarkers [35]. The relatively least abundant lipid compounds were the high molecular weight tetra- and pentacyclic triterpenes (Tables 2 and 3). The sterol and triterpenol molecules have been tentatively identified as methyl ethers and esters of both methyl/ethyl cholesten-3-ol structures, and of ursane, lupeane and oleanane derivatives that

51

are characteristic lipid components of aerial and root plant tissues. The contribution of microbial input to soil lipids was shown by the inclusion of various structural components of microbial cells [36], such as phospho-lipid fatty acids (PLFA) and 2-hydroxy aliphatic acids (Table 2). The most representative PLFA monomers were, in order of elution, the 12- and 13-methyl tetradecanoic (iso/anteiso pentadecanoic), the 14- and 15-methyl hexadecanoic (iso/anteiso heptadecanoic) acids and the cyclopropane-(2-hexyl)octanoic acid (C17 cy FAME), which are common microbial constituents of natural organic matter in soil and sediments [37]. A relative lower amount of carbohydrates derivatives were found among the pyrolysis products of the field management from Torino soil. This finding has been related to the lower efficiency of off-line pyrolysis techniques to detect carbohydrate units of polysaccharides in complex matrices [38,39]. The thermal behavior and pyrolitic rearrangement of poly-hydroxy compounds combined with the basic reaction condition of TMAH reagent solution, are believed to negatively interfere in the release of polysaccharides. However, despite the expected low response of carbohydrates, various methylated forms of mono- and oligosaccharides components were still found among thermochemolysis products of Torino soil from both field treatments (Fig. 1 and Table 2). These compounds may be mainly associated to xylans and cellulose moieties of coarse ligno-cellulosic debris of plant residues. The unusual release of such compounds may have resulted from the larger amount of sample subjected to off-line pyrolysis, that might have then partially overcome the cited limits of thermochemolysis in the detection of carbohydrates. Contrary to the data obtained for the coarse textured soil of Torino, completely different results were revealed by the thermochemolysis applied to the clayey soils from Piacenza and Napoli (Fig. 2). The pyrolysed products (Tables S1 and S2 of supporting information) were almost exclusively composed by short chain branched hydrocarbons, aromatic, polyaromatic and alkyl aromatic compounds and from a succession of alkene/alkane doublets. The large part of these molecules could not be directly inherited from the main classes of natural organic structures (lipids, carbohydrates, lignin, polyphenols, peptides) usually found in the SOM pools. Conversely the majority of the compounds accounted for the occurrence of secondary structural rearrangements of natural organic components, promoted by the pyrolytic process. Previous studies focused on analytical pyrolysis of SOM, have pointed out that the natural organic compounds, subjected to pyrolytic conditions, may undergo fast and uncontrolled condensation and aromatization reactions [10]. The yield of aromatic and alkyl aromatic moieties observed in the pyrolysates of organic matter in soils and sediments has been attributed to the rough structural rearrangement of the thermally unstable functional groups of SOM units [9]. Moreover the investigations on the pyrolytic behavior of aliphatic and alycyclic organic compounds, revealed that the presence of different clay minerals and associated cations, strongly promote the formation of aromatic, polyaromatic and alkylaromatic derivatives [16,40]. The combination of high temperature and active clay surfaces catalyze an initial cleavage of carbon–carbon bonds followed by the subsequent cyclisation and rearrangement of pyrolytic products and the final formation of secondary aromatic structures. Unlike the aromatic artifacts, the inclusion of alkene/alkane clusters among the pyrolysis products of SOM, have been often associated to the presence of highly resistant non-hydrolyzable aliphatic components, which are believed to represent the alkyl core of recalcitrant OM pools in soils and sediments [13,41]. However it has also been shown that the pyrolysis of aliphatic compounds, bearing unprotected polar functional groups (e.g. fatty acids, alcohol, wax esters), may promote the formation of alkyl

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radical through the breakage of terminal C C bonds and the loss of polar heads [17,18]. The subsequent intramolecular transfer of the unstable H-radical throughout the carbon chain (scrambling), followed by the cleavage of the associated allylic bonds, lead to the progressive fractionation of the long-chain molecules thereby generating the homologous distribution of alkene/alkane doublets in pyrograms [16]. Also this process appear to be catalyzed and strongly supported by the interaction of clay surfaces and associated cations which may, thereby, hinder the detection of polar alkyl compounds. In order to avoid the occurrence of structural rearrangements, it has been postulated that a previous sample addition with an alkylating reagent might effectively prevent the loss of polar functional groups and hence limits the interaction with clay minerals [42–44]. Such pre-treatment should allow, both, the preservation of functional groups during pyrolysis and the detection of unmodified compounds by GC–MS analysis. Faure and coworkers [40] showed that the addition of TMAH before pyrolysis of model clay–humus complexes strongly reduced the cyclization and aromatization processes and inhibited the effect of clay minerals in the generation of analytical artifacts. On the contrary the almost exclusive yield of secondary by-products obtained, in the present experiment, by the thermochemolysis of the two clayey soils from Piacenza and Napoli, indicated that the sample treatments with TMAH failed to counteract the effect of clay minerals and did not allow an effective analysis of the SOM components. This contrasting result may be explained with the low amounts of OM held in the soil samples of Piacenza and Napoli (Table 1) as compared to the large OM (10%, w/w) content in the clay–humic complexes used by Faure and coworkers. The most favorable humus/clay ratio could had overcome the saturation capacity of clay surfaces, thus limiting their catalytic effects. Moreover the synthetic complexes prepared by the simple mixing of various clay types with humic acids, are expected to have a different response to thermochemolysis in respect to real SOM samples. The organo-mineral interactions in soil depends on various physical-chemical parameters such as aging, dry-wetting cycles, pH and ionic strength of soil solution, hydrophobic effect and electrostatic forces, that strengthen the association between organic and inorganic soil fractions. It is then conceivable that the close interaction of clay particle-size and SOM, in real bulk soil samples, may reduce the effectiveness of solvolysis effect of TMAH on the polar functional groups of organic molecules, and hence bias the results of thermochemolysis analyses. 3.2. HF-treated soils The soil hydrolysis with hydrofluoric acid is currently applied in the studies on SOM based on solid state NMR analysis of bulk soils and soil particle size fractions. This treatment is aimed to, both, improve the intrinsic low sensitivity of CPMAS-NMR technique and to remove the interference of paramagnetic elements. However the effective improvement of NMR spectra after soil treatments with concentrated HF solutions is often associated with a significative loss of TOC content [24,45]. In the present experiment, in order to prevent an excessive decrease of the TOC content of original samples (Table 1), the soil hydrolyses have been accomplished with two subsequent mild HF treatments. Irrespective of soil type and field management, a similar removal of soil mass was found in the various soil samples after the HF hydrolyses, with final yields that ranged from 59 to 66% of initial weight (Table 4). Conversely an uneven response to HF treatment was shown for the TOC content of different soil samples. An almost steady level of bulk OC was found, at the end of HF treatment, for both the field managements of the coarse textured soils of Torino, which retained about the 97% of initial TOC amount

(Table 4). On the contrary the two clayey soils of Piacenza and Napoli, after an initial slight decrease of TOC content, underwent a marked loss of SOM with the second HF treatment, regardless of the field managements (Table 4). The large variability observed in SOC maintenance following a HF hydrolysis is attributed to differences in operational protocols, soil type and horizon and initial OC content [24,45]. In this study the larger SOC removal from the heavy textured soils of Piacenza and Napoli, should be accounted to the concomitant solubilization of SOM closely associated with the fine colloidal clay fractions dissolved by the HF treatment The effectiveness of soil demineralisation for the molecular characterization of SOM was revealed by the results of the THM performed on the soil samples after the HF treatments (Fig. 3). The methyl ester and ether derivatives of lignin, lipids and carbohydrates moieties identified in demineralised samples of each soil treatments of all experimental sites, closely resemble the thermochemolysis products found in the untreated bulk samples of Torino soil (Table 2). The list of organic components released by HF treated soils is shown in Table S3 of supporting information. Minor differences were shown by the off-line pyrolysis of the HF treated soils of Torino as compared to the original untreated samples (Tables 2 and S3). As indicated by the observed maintenance of TOC (Table 4), no remarkable effects were produced by soil demineralisation on the qualitative and quantitative composition of thermochemolysis products (Table 5). Contrasting results have been reported on the possible selective removal of specific soil organic component produced by the HF treatments, which effects varied with soil type, soil horizons and acid strength of applied solutions [24,46]. For Torino soil both yield and distribution of molecular components found in pyrograms of demineralised samples, closely resembled those obtained from the original field managements (Tables 3 and 5). The main differences were related to the decrease, after HF treatment, in the released amount of hexadecenoic and octadecenoic acids and the disappearance of the unsaturated C18 dioic- and of 9-10 epoxy 18 hydroxy-alkanoic acids. While the possible hydrolysis of double bonds, followed by an oxidation of resulting fragments into short chain dioic acids, may be claimed for the modification of unsaturated compounds, the identification of 9,10, 18 trihydroxy C18 acid in the pyrograms of HF treated soils (Table S3) ascertain the occurrence of hydrolytic conversion of the corresponding epoxide C18 precursor [34]. A suitable improvement of SOM characterization was revealed by the survey of the pyrograms obtained from the clayey soils of Piacenza and Napoli after the HF treatment. The Total ion chromatograms obtained from the thermochemolysis of demineralised samples (Fig. 3) showed an almost complete disappearance of alkylaromatic and polyaromatic by-products and of the alkane/alkene clusters, which were predominant in the corresponding untreated soils. The pyrolytic artifacts were replaced by the methylated products of various organic molecules (Table S3), whose overall distribution was similar to that found from the coarse-textured soil of Torino (Table 5). The main aromatic compounds were represented by the typical lignin monomers pertaining to the different basic structure originated from the lignified tissues of higher plants, dominated by, both, Guayacil and Siryngil derivatives (Table S3). Also, the most abundant alkyl molecules corresponded to the common aliphatic and alicyclic lipid molecules deriving from plant waxes and plant biopolyesters, composed by linear fatty acids, mono- and poly-hydroxyl acids, long chain alkyl-dioc acids, aliphatic alcohols and tetra and penta-cyclic trirpenes (Table S3). The lower but detectable yield of PFLA components and of ␤-hydroxy fatty acids indicated the presence of microbial compounds, while a notable but uneven distribution was revealed by the methyl-ether forms of mono and oligosaccahrides moieties. This finding combined with the SOC preservation observed

R. Spaccini et al. / Analytica Chimica Acta 802 (2013) 46–55

53

Table 4 Mass yielda and total organic carbon (TOC) contenta from different soil and managements after the HF treatments. Torino

Piacenza

Napoli

TRA

MIN

TRA

MIN

TRA

MIN

Soil mass (g)

Initial I HF II HF

10 8.1 (0.2) 6.3 (0.6)

10 7.9 (0.1) 6.6 (0.3)

10 7.8 (0.2) 6.2 (0.1)

10 7.8 (0.2) 5.9 (0.2)

10 8.4 (0.2) 6.4 (0.2)

10 8.1 (0.2) 6.1 (0.2)

TOC (g kg−1 )

Initial I HF II HF

7.4 (0.4) 9.0 (0.2) 11.3 (0.5)

7.5 (0.5) 9.2 (0.2) 11.0 (0.5)

12.7 (0.3) 14.7 (1.2) 17.0 (0.6)

13.6 (0.4) 16.9 (0.6) 21.1 (0.1)

8.2 (0.4) 9.4 (0.6) 11.0 (0.7)

9.4 (0.4) 10.9 (0.5) 13.7 (0.5)

TOC lossb (%)

I HF II HF

1.5 (0.1) 3.4 (0.1)

3.1 (0.1) 3.2 (0.2)

2.3 (0.3) 16.8 (2.5)

3.0 (0.2) 8.6 (1.9)

3.2 (0.1) 14.0 (1.0)

5.8 (1.7) 11.1 (1.6)

Numbers in parenthesis indicate the standard deviation (n = 4). TOC loss is calculated with respect to initial TOC content.

Ch

%

0

8.4

13.4

C24, 24 CH3O, FAME

Ch

Lg S14/15

Ch

C18:1

C15 iso/a-iso FAME

Lg G13

100

Lg S4

a b

18.4

23.4

28.4

33.4

38.4

43.4

48.4

Torino-MIN

53.4

58.4

Time

C17 iso/a-iso FAME 100

Lig P18

Lg G6

Lig S14/15

Ch

Lg G4

%

Piacenza-MIN

Ch

Sterol

0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0 Time

Ch

%

Lg G14/15

Ch

Lg S4 Lg G7 Lg G10

100

Napoli-MIN Ch Sterol

0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

Time

Fig. 3. Representative total ion chromatograms of thermochemolysis products released from the HF treated soil samples. (䊉) alcohol; Ch, carbohydrate; () alkyl-dioic acid DIME; () FAME; () hydroxy-FAME; Lg, lignin.

in the soil samples of Torino suggests that the soil hydrolysis with mild HF treatment did not alter significantly the accuracy and the reproducibility of thermochemolysis results. Moreover, the results of off-line pyrolysis applied to the demineralised samples revealed slight but significative differences, in the quality of incorporated SOM, between the two soil management systems. In particular, specific lignin components, currently associated

to the presence of either microbial processed organic materials or to undecomposed plant debris, may be used to evaluate the extent of lignin decomposition [31]. In fact, while the aldehydic (G4, S4) and acidic forms (G6, S6) of lignin structures result from progressive oxidation processes, the corresponding homologues with the integral hydroxylated side chain (G14/15, S14/15) are indicative of unaltered lignin components, which retain the

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Table 5 Compositiona and yields (␮g g−1 ) of main common thermochemolysis products released from the field managements of HF treated soils. Compounds

Ligninc Ad/AlG G Ad/AlS S Linear FAME C14–C30 (C18:1) Long chain (>C20) % Hydroxyl FAME C16–C26 (C16, C18) Alkane dioic acids C16–C24 Alkane C21–C31 (C27, C29) Alcohols C22–C28 Phytosterols PFLAd C15–C19 (C17 iso/a.iso) ␤-Hydroxy acids C22–C25 a b c d

Torino

Piacenza

sdb

Napoli

TRA

MIN

TRA

MIN

TRA

MIN

490 2.4 2.0 4.1 1.9 1200 26.5 820 170 191 550 160 320 288

520 0.9 1.2 2.1 1.3 1330 32.1 1172 290 167 620 290 510 380

834 2.7 2.2 3.2 2.9 1551 28.5 1038 236 422 680 214 557 433

1193 1.4 1.4 1.8 1.5 1757 27.2 1683 338 310 720 620 1166 837

414 2.2 2.3 3.1 3.5 1195 25.4 800 182 479 450 235 250 340

660 1.4 1.8 1.7 1.7 1308 30.4 1340 287 510 395 480 520 360

35.5 0.4 0.2 0.6 0.5 45.5 92.5 27.0 50.5 70.5 37.0 80.5 64.0

Total range varying from Ci to Cj; compounds in parentheses are the most dominant homologues; numbers after colon refer to double bond. Standard deviation is relative to the results of Torino soil. Structural indices: Ad/Al = G6/G4, S6/S4;  G = G6/(G14 + G15);  S = G6/(G14 + G15). Phospholipid fatty acids.

propyl ether intermolecular linkages (Fig. S1). Therefore the ratio of peak areas of acidic structures over that of, both, the corresponding aldehydes (Ad/AlG = G6/G4, Ad/AlS = S6/S4) and over the sum of peak areas for the threo/erythro isomers ( G = G6/[G14 + G15];  S = S6/[S14 + S15]), are considered to be good indicators of the biooxidative transformation of lignin polymers in soil [3,30]. Although the detection of these specific monomers is extremely dependent on both sample handling and analytical conditions [47], the preventive soil demineralization with HF hydrolysis allowed an accurate evaluation of these SOM components. In fact, notwithstanding the similar total yield of lignin monomers found in the HF treated samples of both field managements of each experimental site, the evaluation of decaying structural indexes for continuous minimum tillage systems indicated, either, a preferential incorporation or selective preservation of fresh and slightly decomposed lignin derivatives in the field plots cultivated with this most conservative SOM practice (Table 5). Besides the lignin components, an higher yield of decomposable molecules, represented respectively by phytosterols, hydroxy-acids and PLFA components, were found in soil treatments under minimum tillage as compared to traditional ploughed soils (Table 5). The lower preservation capacity commonly shown by these plant and microbial components, has been related to a more sensitive potential reactivity to decomposition pathways [48,49]. Despite the overall biochemical decomposability and the liability to experimental conditions of these SOM components, the present results point out the effectiveness of HF treatment as complementary valuable tool to improve the application of thermochemolysis on soil samples.

The removal of clay minerals accomplished by soil pretreatment with hydrofluoric acid solutions effectively limited the occurrence of pyrolytic rearrangements, thereby improving the identification of molecular organic components in all soil samples. A wide range and detectable amounts of lignin monomers and lipid compounds, of plant and microbial origin, were released by the thermochemolysis of field managements of heavy textured soils after the HF treatment. On the other hand, minor differences were found in yield and composition of pyrolyzed products before and after the HF treatment in coarse-textured soil, thereby indicating the suitability of mild HF treatments for the evaluation of SOM dynamics. The application of HF treatment and thermochemolysis enabled to point out the molecular differences determined in SOM by two different management practices. Moreover the pyrolysates of the HF treated samples revealed the steady inclusion in SOM of the hardly analysable carbohydrates derivatives and of minor biolabile components, such as phytosterols and microbial biomarkers. Although these results need a further validation in order to be extended to a large array of soil type and SOM managements, the observed analytical improvements strengthen the usefulness of the combination of HF hydrolysis and off-line thermochemolysis as valuable technique for the detailed analysis of SOM in C-depleted agricultural soils.

4. Conclusion

Appendix A. Supplementary data

The results of SOM characterization further confirmed that the off-line thermochemolysis is a rapid and effective method to obtain a reliable direct qualitative and quantitative evaluation of molecular OM components in C-depleted agricultural soils. However, the suitability of thermochemolysis for the analysis of SOM in soil and soil fractions require a previous evaluation, according to textural and mineralogical composition of soil samples. While this technique enables the direct molecular characterization of SOM in coarse textured soils, its efficiency in heavy textured soils may be seriously affected by the presence of clay minerals. In the present experiments the catalytic effect of clays during pyrolysis led to the release of several analytical artifacts that bias the accuracy of molecular signature of SOM components.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.09.031.

Acknowledgements This work was supported by the National FISR-MIUR project: “MESCOSAGR-Sustainable Methods for Organic Carbon Sequestration in Agricultural soils”. The second author is grateful to IRREA and CERMANU for the support during his scientific work in Italy

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