Principal Component Analysis of NMR Spectra of Humic Substances Extracted from Soil Treatments

In order to compare the effect produced by soil treatments on the molecular composition of HS extracts at different experimental times, the areas of C signals in NMR spectra were integrated and their values elaborated with principal component analysis (PCA). The multivariate analysis for the first and third experimentation year at Torino, Piacenza, and Napoli sites of the MESCOSAGR project is shown in Figs. 4.3-4.5, respectively. The relative values (%) associated with the x and y axes in PCA diagrams stand for the total variation of NMR data explained by two statistical principal components (PC1 and PC2), while the dotted lines represent the loading vectors associated with the variables (ppm intervals) obtained from NMR spectra. Tables 4.11-4.13 show the variation of HS structural indexes throughout the three experimental years as calculated from NMR spectra for different soil treatments and experimental sites.

4.5.2.1 Torino Experimental Site

For the first and third experimentation years, PCA score plots of HS from TRA were compared with those from MIN and GMAN treatments (Fig. 4.3a). An overall treatment discrimination was given by PC1 between TRA and MIN and between TRA and GMAN. However, no consistent molecular variation was evident in HS composition among treatments for either replicates or experimental years. The loading vectors associated with signals at 110-60 ppm and 45-0 ppm indicate that HS extracted from MIN after the first year were rich in carbohydrates and poor in alkyl components, respectively. At the final year, an opposite distribution of carbohydrates and alkyl-C was revealed by the corresponding loading vectors in the TRA vs. MIN biplots. Moreover, although the principal components, PC1 and PC2,

First year

160-1

145-110 ppm I

^ 190-160 ppm

110-60 ppm

45-0 ppm 0-145 ppm

Third year

145-110 ppm 110-60 ppm \

0-145 ppm

TRA a f * 190-160 ppm

"50-45 ppm

45-0 ppm

TRA b

• GMAN a

COM1 b

COM1 a» 45-0 pp

110-60 ppm ■ -TRA b-,

TRA a

ppm

Third year

MIN a •

190-

60 ppm

160-145 COM 2 a y

„ 60-45 ppm

• MIN b

TRA b

MIN a b

Ihird year

NoCATb

45-0

-160 ppm

-45 ppm ppm

SI CAT

145-110 ppm ppm 60-45 ppm c ppm

MIN b

45-110 ppm

45 ppm MIN a

Fig. 4.3 Torino experimental site: biplots generated by principal component analysis of NMR spectra of humic substances extracted from soil treatments (a) TRA vs. MIN and TRA vs. GMAN both at first and third year of field experimentation (a and b indicate first and second replicates) (b) MIN vs. COM-1 and COM-2 and CAT vs. NoCAT, at first and third year of field experiments (small letters indicate replicates) (c) COM-2 vs. MIN at first and third year of field experiments (small letters indicate replicates)

First year

Third year

160-146 ppm t

190-160 ppm

145-110 ppm

Third year

TRA b

190-160 ppm

45-0 ppm" " 1 • TRA a 1454K

ppm 160145 ppm

MIN a

a COM2 a 145-100 ppm V V

110-60 ppm1" COM2 b

COM 2 a

"160145 ppm'

f 60-45 ppm

TRA b

First year

Third year

60-45 ppm S

45-0 ppm — ^ - CAT b

145-110 ppm ^ S 160-145 pp

110-60 ppm m

• NoCATb

b 160-145 ppm

190-160 ppm y 145-110 ppm

60-45 ppm

MIN a

160-145 ppm

45 ppm

110-60 ppm

COM 2 J I45-"-45-0 ppm

MIN a •

MIN b

TRA a

MIN a

60-45 ppm

TRA a b

MIN b

110-60 ppm

Fig. 4.4 Piacenza experimental site: biplot generated by principal component analysis of NMR spectra of humic substances extracted from soil treatments (a) TRA vs. MIN and COM-2 at first year of field experiments (small letters indicate replicates) (b) CAT vs. NoCAT and COM-2 vs. MIN at first and third year of field experiments (small letters indicate replicates)

First year

Third year

145-110 ppm S "

190-160 ppm

190-160 _ ppm^ 60-45 ppm \

60-45 ppm * GMAN b

145-110 ppm^ TRA TRA a-

145-110 1

com a 60-45 ppm

COM1 bV * • .--.

TRA a pm

160-145 ppm— „«• 45-0 ppm "'190-16t

110-60 ppm ppm

TRA b

45-0 ppm N

110-60 ppm

190-160 ppm

Third year

60-45

MIN b * » • — —

110-60 ppm

160-145 ppm

' ' TRA a' # MIN a

GMAN a 45-0 ppm

160-145 ppm_ . GMAN b

TRA a

Third year

145-110 ppi COM1a %

60-45 ppm*^ v 160-145 ppm _

"45-0 ppm

COM1b

110-60 ppm • TRA b

PC1 (94.5 %)

45-0 ppm V

TRA a •

60 ppm

• TRA b

Fig. 4.5 (continued)

TRA a

TIA b

MIN a b

First year

Third year

160-145 ppm -160-110 ppm zmw

60-45 ppm CAT b

Third year

45-) ppm • CAT b \ \

NoCATb 1 60-45 ppm

160-145 ppm 1.

NoCATa

-2.0 -1.5 -1.0 1.0 1.5 2.0 -2.0 -1.5 -1.0 1.0 1.5 2.0

• MIN a

145-110 ppm^'' • COM2 bV 190-16

110-60 ppm 0 ppm MIN b

-1.5 -2.0 -2.5

190-160

COM 2 b ^

• MIN b 110-60 ppm

45-0 ppm f

145 ppm

-2.0 -1.5 -1.0 1.0 1.5 2.0 -2.5 -2.0 -1.5 1.5 2.0 2.5

Fig. 4.5 Napoli experimental site: biplot generated by principal component analysis of NMR spectra of humic substances extracted from soil treatments (a) TRA vs. MIN and GMAN at first and third year of field experiments (small letters indicate replicates) (b) TRA vs. COM1 and COM2 at first and third year of field experiments (small letters indicate replicates) (c) CAT vs. noCAT and MIN vs. COM2 at first and third year of field experiments (small letters indicate replicates)

NoCATa

NoCATb

110-60 ppm

145-110 ppm

CAT a represented from 95 to 99% of total variability, no significant differences were found between HS from MIN and TRA in the loading vectors for molecular components.

A similar PCA was derived by comparing NMR spectra of HS extracted from TRA and GMAN (Fig. 4.3a). After 1 year, the main variation was along the PC1 component (84%) and mainly accountable to distribution of phenolic (160-145 ppm) and alkyl (45-0 ppm) compounds, whose amounts were, respectively, larger and lower in HS from GMAN than from TRA. After 3 years, the alkyl-C became more abundant in HS from GMAN, whereas no longer the distribution of phenolic and aromatic compounds represented a significant difference for HS from TRA. With progression of experimental time, NMR spectra of HS from both MIN and GMAN revealed a larger content of hydrophobic molecules than for HS from TRA (Table 4.11). In fact, both the slight HB increase and the more pronounced decrease of Lignin Ratio suggested a progressive incorporation of alkyl and phenolic compounds in HS from MIN and GMAN as compared to those from TRA.

Table 4.11 Torino experimental site, variation with experimental time of structural indexes HB, Lignin Ratio and Aromaticity (%), calculated from 13C-CPMAS-NMR spectra of humic substances extracted from soil treatments for 3 years of experimentation

Treatment

First year

Second year

Third year

HB

Lignin Ratio

HB

Lignin Ratio

HB

Lignin Ratio

Maize

TRA

0.73 (0.02)

4.61 (0.22)

0.76 (0.04)

3.92 (0.35)

0.69 (0.01)

4.19 (0.34)

MIN

0.71 (0.01)

3.95 (0.57)

0.79 (0.01)

3.17 (0.05)

0.83 (0.01)

3.16 (0.19)

GMAN

0.73 (0.01)

3.28 (0.02)

0.75 (0.03)

3.34 (0.44)

0.85 (0.02)

3.40 (0.06)

COM1

0.82 (0.02)

4.70 (0.29)

0.84 (0.03)

2.94 (0.10)

0.91 (0.01)

2.66 (0.39)

COM2

0.82 (0.02)

2.93 (0.03)

1.03 (0.13)

2.66 (0.24)

1.09 (0.01)

2.37 (0.05)

Treatment

First year

Second year

Third year

HB

Aromaticity

HB

Aromaticity

HB

Aromaticity

Wheat

CAT

0.71 (0.01)

12.8 (0.22)

0.76 (0.04)

16.5 (1.01)

0.85 (0.04)

17.5 (0.37)

No-CAT

0.65 (0.01)

13.4 (0.56)

0.76 (0.04)

15.8 (0.49)

0.77 (0.02)

14.6 (0.14)

Number in parentheses indicate standard deviation (n = 2)

Hydrophobic index (HB) = £[(0-45) + (45-60) + (110-160)]/£[(45-60) + (60-110) - (160-190)] Lignin Ratio = (45-60)/(145-160) Aromaticity = (110-160)/(0-190)

Number in parentheses indicate standard deviation (n = 2)

Hydrophobic index (HB) = £[(0-45) + (45-60) + (110-160)]/£[(45-60) + (60-110) - (160-190)] Lignin Ratio = (45-60)/(145-160) Aromaticity = (110-160)/(0-190)

Table 4.12 Piacenza experimental site, variation with experimental time of structural indexes HB, Lignin Ratio and Aromaticity (%), calculated from 13C-CPMAS-NMR data of humic substances extracted from soil treatments for 3 years of experimentation

Treatment

First year

Second year

Third year

HB

Lignin Ratio

HB

Lignin Ratio

HB

Lignin Ratio

Maize

TRA

0.63 (0.01)

6.46 (0.48)

0.74 (0.01)

3.52 (0.27)

0.73 (0.01)

3.61 (0.27)

MIN

0.60 (0.01)

5.86 (0.30)

0.78 (0.01)

3.45 (0.67)

0.66 (0.09)

3.95 (0.25)

COM2

0.71 (0.02)

5.58 (0.15)

0.98 (0.02)

2.36 (0.18)

0.93 (0.02)

2.01 (0.07)

Treatment

First year

Second year

Third year

HB

Aromaticity

HB

Aromaticity

HB

Aromaticity

Wheat

CAT

0.67 (0.01)

16.2 (0.32)

0.74 (0.02)

14.2 (0.07)

0.70 (0.03)

14.3 (0.25)

No-CAT

0.64 (0.01)

10.9 (1.29)

0.76 (0.07)

15.2(1.43)

0.73 (0.01)

14.1 (0.95)

Number in parentheses indicate standard deviation (n = 2)

Hydrophobic index (HB) = £[(0-45) + (45-60) + (110-160)]/S[(45-60) + (60-110) - (160-190)] Lignin Ratio = (45-60)/(145-160) Aromaticity = (110-160)/(0-190)

Number in parentheses indicate standard deviation (n = 2)

Hydrophobic index (HB) = £[(0-45) + (45-60) + (110-160)]/S[(45-60) + (60-110) - (160-190)] Lignin Ratio = (45-60)/(145-160) Aromaticity = (110-160)/(0-190)

The PCA of NMR data for HS extracted from compost-treated plots revealed, with experimental time, an increased incorporation of exogenous hydrophobic organic carbon. After the first year, a significant discrimination (PC1 + PC2 > 96%), was evident between both compost treatments and TRA (Fig. 4.3b). In fact, the loading

Table 4.13 Napoli experimental site, variation with experimental time of structural indexes HB, Lignin Ratio and Ar, calculated from 13C-CPMAS-NMR data of humic substances extracted from soil treatments for 3 years of experimentation

Treatment

First year

Second year

Third year

HB

Lignin Ratio

HB

Lignin Ratio

HB

Lignin Ratio

Maize

TRA

0.80 (0.01)

2.67 (0.12)

0.72 (0.04)

4.07 (0.38)

0.85 (0.01)

3.03 (0.34)

MIN

0.94 (0.13)

2.23 (0.29)

0.85 (0.03)

2.50 (0.02)

0.83 (0.06)

2.85 (0.27)

GMAN

0.92 (0.08)

2.43 (0.32)

0.74 (0.05)

2.67 (0.00)

0.71 (0.05)

3.52 (0.09)

COM1

0.96 (0.06)

2.10(0.11)

0.89 (0.04)

2.59 (0.15)

0.98 (0.01)

2.28 (0.12)

COM2

1.08 (0.02)

2.31 (0.23)

0.95 (0.03)

2.20 (0.20)

1.06 (0.03)

1.64 (0.13)

Treatment

First year

Second year

Third year

HB

Aromaticity

HB

Aromaticity

HB

Aromaticity

Wheat

CAT

1.07 (0.04)

28.1 (0.16)

0.90 (0.07)

24.8 (1.71)

0.90 (0.00)

25.2 (0.66)

No-CAT

0.91 (0.03)

28.1 (0.84)

0.79 (0.02)

22.9 (0.81)

0.90 (0.04)

25.0 (0.71)

Number in parentheses indicate standard deviation (n = 2)

Hydrophobic index (HB) = S[(0-45) + (45-60) + (110-160)]/S[(45-60) + (60-110) - (160-190)] Lignin Ratio = (45-60 ppm)/(145-160 ppm) Aromaticity = (110-160)/(0-190 ppm)

Number in parentheses indicate standard deviation (n = 2)

Hydrophobic index (HB) = S[(0-45) + (45-60) + (110-160)]/S[(45-60) + (60-110) - (160-190)] Lignin Ratio = (45-60 ppm)/(145-160 ppm) Aromaticity = (110-160)/(0-190 ppm)

vectors in the score plot indicated a larger incorporation of alkyl compounds (45-0 ppm) for COM-1 and of both phenolic (160-145 ppm) and aromatic components (145-110 ppm) for COM-2. The NMR spectra of HS from TRA were instead significantly dominated by O-alkyl-C units from carbohydrates and polysaccharides (110-60 ppm). The acquired hydrophobicity of compost-treated soils was also shown by the increased HB index (0.83) for NMR spectra of their HS extracts, as compared to those from TRA, whereas the low values for Lignin Ratio in HS from COM-2 suggested a large incorporation of lignin-like aromatic components (Table 4.11).

The increased hydrophobicity of HS from COM-1 and COM-2 throughout the experimental period shows that compost stabilized material has been progressively incorporated in soil humic fractions (Fig. 4.3b and Table 4.11). After the third year, the PCA of NMR data revealed enhancement of phenolic (160-145 ppm), unsubstituted aromatic (145-110), and alkyl C components (45-0 ppm) in HS from both COM treatments, while the loading vectors associated with NMR signals of hydrophilic C at 160-110 and 60-45 ppm were prevalent in HS from TRA. Furthermore, the large content of C-N bonds from peptidic moieties (60-45 ppm) in HS from TRA was accompanied by the increased Lignin Ratio values as a sign of progressive reduction of hydrophobicity in soil humus from TRA. The contribution of composted matter (mainly alkyl and lignin derivatives) in the molecular composition of stable soil HS was shown by the NMR-derived HB index and Lignin Ratio for COM-1 and COM-2 soil treatments. The enhanced hydrophobicity of HS from COM-2 was also evident in comparison with NMR results obtained for MIN (Fig. 4.3b). The loading vectors associated with alkyl (45-0 ppm) and phenolic and aromatic (160-110 ppm) C regions, for both the first (PC1 90.7%) and third experimental years (PC1 97%) indicated the occurred incorporation of hydrophobic materials in HS from COM-2.

After the first experimental year, only minor differences were found between HS from soil treated with the biomimetic catalyst (CAT) and those from its control (No-CAT) (Fig. 4.3c). Conversely, after 3 years of treatment, HS from CAT revealed a preferential distribution along the PC1 (90.4%) of signals in the 160-145 and 145-110 ppm regions, thereby showing an increased incorporation of phenolic and aromatic C in the soil humic fraction. Such enhanced content of aromatic components in HS from CAT was consistently accompanied by progressive increase of the NMR aromatic index, as compared to that of No-CAT (Table 4.11).

4.5.2.2 Piacenza Experimental Site

No distinct treatment effects on soil humic composition were revealed by the loading vectors along both PC1 and PC2 in the biplots from NMR of HS extracted from either TRA or MIN (Fig. 4.4a). After the first year, the low values for HB and the large ones for Lignin Ratio for HS from both TRA and MIN indicated the predominance of hydrophilic compounds, such as carbohydrates and peptidic moieties. However, at the experiments end, an incorporation of lignified plant material was revealed by the slight decrease of Lignin Ratio for both soil treatments (Table 4.12).

The low hydrophobic character in HS from TRA and MIN was evident when compared with that resulting from NMR data of HS from COM-2 (Fig. 4.4a, b). In fact, for both sampling times, NMR data provided a positive discrimination along PC1 for loading vectors associated with alkyl (45-0 ppm) and overall aromatic (160-145 and 145-110 ppm) carbons in HS from COM-2. Conversely, the HS from COM-2 were negatively correlated with loading vectors associated with carbohydrates signals (110-60 ppm). Table 4.12 supported the occurred incorporation of hydrophobic molecules from alkyl components and lignin derivatives of compost in HS of COM-2. The molecular distribution, in fact, indicates an increase of HB index and a concomitant progressive decrease of Lignin Ratio throughout the experimental period.

After 1 year of soil treatment with biomimetic catalyst, an increase of aromatic C in HS extracts was suggested by the distribution of phenolic (160-145 ppm) and aromatic (145-100 ppm) C signals along biplot PC1 (87%) (Fig. 4.4b) and by the larger Ar index value (16.2%) with respect to that (10.9%) of control (No CAT) (Table 4.12). However, CAT treatment apparently lost effectiveness later in the experimentation, as revealed by the Ar index of HS from CAT, whose value approached that of control, and by the PCA loading vectors which no longer discriminated between CAT and No-CAT (Fig. 4.4b).

4.5.2.3 Napoli Experimental Site

As in the case of Torino and Piacenza, also for Napoli field experiments, few differences were found by PCA among NMR results for HS from TRA, MIN, and GMAN (Fig. 4.5a). In fact, although from 94 to 99% of total variability for the OC distribution was covered by PCA, at every sampling time, no specific distinction was appreciated for the loading vectors associated with different NMR chemical shift regions in HS from TRA, MIN and GMAN.

However, the latter treatments did induce a significant incorporation of hydro-phobic alkyl and lignin compounds in HS, if their values for HB and Lignin Ratio (Table 4.13) were compared to those of the initial soil before the experimentation (Table 4.10). This significant difference may have been due to both the low OC content in the initial undisturbed soil and the large surface area offered by the clay content in this soil. These two conditions may have enhanced the physical— chemical affinity of organic components to soil particles (Staunton and Quiquampoix 1994; van Oss and Giese 1995) and favored the stabilization of the organic molecules that entered the subsoil during cropping seasons (Webster et al. 2001; Piccolo et al. 2004; Winkler et al. 2005).

Hydrophobic molecules from compost were incorporated more in HS from COM treatments than for TRA, as suggested by the PCA of NMR results (Fig. 4.5b). After the first year, this incorporation was already shown by the loading vectors of both aliphatic (45-0 ppm) and phenolic (160-145 ppm) components in HS from both COM-1 and COM-2, though only that associated to alkyl carbon had >95% of statistical significance. Conversely, the loading vector for hydrophilic carbohydrate carbon (110-60 ppm) characterized HS from TRA. After 3 years, the distribution of loading vectors associated to phenolic (160-145 ppm), aromatic (145-110 ppm), and alkyl (45-0 ppm) carbons confirmed the larger hydrophobicity of HS from compost-treated soils, while a persistent content of hydrophilic compounds (110-60 ppm) was found for TRA. The incorporation of alkyl hydro-phobic molecules in compost-amended soil was again evident by comparing the OC distribution in HS from COM-2 and MIN (Fig. 4.5c). In fact, the PC1 discrimination represented 88 and 83% of total variation for initial and final experimental time, respectively, showing a positive correlation of hydrophilic O-alkyl-C in HS from MIN, and of alkyl components in HS from COM-2.

The progressive decrease of HB index in HS from MIN and GMAN and the simultaneous increase of Lignin Ratio (Table 4.13) revealed that reduced tillage or green manuring promoted the progressive loss of stable hydrophobic OM from soil (Piccolo 1996; Spaccini et al. 2006). Conversely, HB index and Lignin Ratio for COM treatments indicated incorporation of hydrophobic organic molecules from compost into soil humic substances (Table 4.13). Moreover, the Lignin Ratio in HS from COM-1 (2.28) and COM-2 (1.64) was lower than that of the very compost used for soil treatments (2.5 in Table. 4.9). This suggests that there must have been an incorporation and sequestration into SOM of additional lignin components derived from crop residues left on soils (Spaccini et al. 2000).

After 1 year of experimentation, the PCA of NMR molecular distribution in HS from CAT and that from No-CAT was not significantly different (Fig. 4.5c). This supports the Aromaticity index that indicated a similar content of total aromatic components for both treatments (Table 4.13). However, a significant increase in HB index was noted for CAT for both the first and second experimentation years (Table 4.13). After 3 years, a positive correlation was found for HS from CAT with the loading vector of unsubstituted and alkyl-substituted aromatic components (145-110 ppm) along PC1 (84% of total variation), though no differences were detected in HS total aromatic content between CAT and No-CAT (Table 4.13).

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