Natural Abundance Method

The use of 13C NAM in soil studies provides a highly accurate method to trace carbon transfer among pools, and has been successfully used in studies on SOC dynamics (Martel and Paul 1974; Nissenbaum and Schallinger 1974; Leavitt et al. 1994; Paul et al. 2001; Stevenson et al. 2005). NAM is based on the discrimination of 13C and 12C isotopes during CO2 assimilation by plants due to different photosynthesis pathways, which lead to plants with distinct d13C values. Plants with a C3 photosynthetic pathway have d13C values ranging from about —32 to —22% (mean —27%), whereas those with a C4 pathway range from —17 to —9% (mean —13%) (Boutton et al. 1998). The isotopic composition of soil organic C reflects the plant materials from which it is derived. The NAM is based on the cultivation of C3 plants in a soil developed under C4 vegetation, or vice versa, and the estimation of rhizodeposition according to the d13C value in soil C pools or CO2 evolved from soil. This method can be considered as a variation of continuous labelling, because plants and soils are permanently labelled. However, the labelling of plants and soils is the result of natural processes, rather than the artificial procedures used in the case of pulse or CLMs described above. NAM can easily be used under field conditions (Rochette and Flanagan 1998) because special equipment for plant labelling and separation from atmosphere is not necessary. The latter feature and future development of mass-spectrometry will promote the use of this method in forthcoming investigations.

The natural (d13C) isotopic difference (about 14%) between C3 and C4 plants enables new carbon derived from one pathway (e.g., C3) to be traced in SOM that is derived from plants having the other pathway (Balesdent and Mariotti 1996; Gleixner et al. 2002; Lobe et al. 2005).

Balesdent and Mariotti (1996) proposed a method to calculate the replacement of old soil carbon by the new vegetation carbon. They propose to derive the contribution of plant B to the total C content using the expression:

where F is the fraction of new carbon in soil, and A and B represent the different photosynthetic pathway types (e.g., C3 and C4). C = CA + CB is the total soil carbon content; CA and CB are equal to the organic carbon contents from the old (A) and new (B) vegetation, and dAB is the isotopic composition of the soil C under mixed vegetation:

dAB (Ca + Cb) = ^ab(C) = 8aCa + ¿bCb, dB and dA are the d13C values of vegetation A and B.

Because dA and dB cannot be measured directly in the mixed cropping system, dB is estimated by the d13C value of the new vegetation (dVEG B), replacing also the dA values with d13C of a control site that still has the original vegetation (dVEG A) and soil d13CA value by that of the control soil (dREF A), respectively (Balesdent and Mariotti 1996). Finally the new portions of vegetation B are estimated from:

The limitations of the 13C NAM are caused by soil-plant pairs. Situations, where C3 plants grow in a C4 soil, or vice versa, are unnatural. Hence, this method is restricted to places where soils developed under C3 vegetation allow the growth of C4 plants and vice versa. Moreover, high-resolution and high-sensitivity mass-spectrometry is necessary for 13C analyses because a maximal range of only 14% is available for all variations of the 13C/12C ratio. At the same time, the variability of d13C values in soil or plant is about ±1-2% (Cheng et al. 1993). For the latter two reasons, only a rough estimation of rhizodeposition in soil and in carbon pools with large exchange rates with root-derived C is possible (e.g., microbial biomass, dissolved organic C, active pools of SOM, etc.). Finally, a limitation of all methods based on C tracers is that organic C-pools may interact with inorganic C-pools in soil (carbonates and bicarbonates).

5.6 Nitrogen Tracers in the MESCOSAGR Project

A study on the stable isotope 15N was set up to (1) evaluate the contribution of compost to N nutrition of maize (Ndfc) as well as the fertilizer use efficiency (%FUE) of compost-derived nitrogen for 3, 2 and 1 years amendments; (2) assess the ability of an 15N enrichment technique to trace the N flows from compost to maize and separate the effects of different amendment years even after repeated application; (3) quantify the incorporation of N from compost into SOM.

15N-labelled compost (Table 5.1) was prepared at the University of Basilicata and applied to the unlabelled soil at the experimental field of the University of Torino each year (TO site already described in Chap. 3) where maize (Zea mays L.) was grown in a 3-year experiment (2006, 2007, 2008). The experimental design consisted of four treatments (Table 5.2), laid out with four replicates in a complete

Table 5.1 Chemical properties of composts used each year

Year

Total N (kg ha—

1) C/N

d15N (%)

Lignin N (%)

Lignin ± cellulose N (%)

C/P

I - 2006

130

12.4

242.0

6.8

11.8

39.8

II - 2007

166

37.7

193.1

13.7

41.8

49.6

III - 2008

128

24.3

136.6

11.3

23.4

59.6

Table 5.2 Description of plot treatments (A-D) and nitrogen (kg ha ) added in each plot treatment in different forms (labelled compost or unlabelled urea) Year Experimental plot

I - 2006 15N compost (COM1)

II - 2007 15N compost (COM2)

Urea (130)

block design. The treatments were applied to individual plots of 12 m2, selected within a main plot of 48 m2.

Maize epigeal biomass production (grains, stalk plus leaves and cob plus husks) was determined each year, while root biomass production was determined only 1 year and the shoot/root ratio in the other 2 years. Each year aboveground and root biomass samples were analysed for total N content (Carlo Erba NC2500 elemental analyser) and atom% 15N excess (Finnigan Delta-Plus isotope ratio mass spectrometer).

5.6.1 Contribution to Maize Nitrogen Nutrition of Compost

The fraction N taken up by maize and deriving from compost (%NpfC) was estimated following a modified model [(5.6), consisting of an application of (5.1)] derived from Shearer and Kohl (1993) and taking into account the isotopic discrimination during plant absorption of soil and fertilizer N:

%Npfc atom% 15N excess labelled maize — atom% 15N excess non — fertilized maize

atom% 15N excess maize grown on labelled fertilizer — atom% 15N excess non - fertilized maize

Isotopic discrimination during compost mineralization and plant N uptake was evaluated for one vegetative cycle of maize fertilized only with compost in a pot experiment. Since the compost applied differed over the 3 years, the calculated shift of isotope percentage was used in the other 2 years to estimate the atom% 15N excess of maize grown only on labelled compost. The ANI was considered null (Powlson and Barraclough 1993) and the contribution of seed nitrogen was considered insignificant.

As compost was repeatedly added, the residual compost mineralization in subsequent years of application was estimated by comparing the %Npfc of treatments and assuming that the addition of new compost did not influence the mineralization rate of residual compost.

5.6.2 Soil Sampling, Aggregate Fractionation and Isotope Determination

Soil samples were collected from topsoil (0-30 cm) and the method described by Kemper and Rosenau (1986) and Spaccini et al. (2004) was used to separate water-stable aggregates. Twenty grams of <4.75 mm air-dried soil sample was put in the topmost sieve of a nest of three sieves of 1.00, 0.50 and 0.25 mm mesh size and pre-soaked in distilled water for 30 min. Thereafter the nest of sieves and their content were oscillated vertically in water 20 times using a 4-cm amplitude at the rate of one oscillation per second. Care was taken to ensure that soil particles on the topmost sieve were always below the water surface during each oscillation. After wet-sieving, the soil materials left on each sieve and the unstable (<0.25 mm) aggregates were quantitatively transferred into beakers, dried in the oven at 50°C for 48 h, weighed and stored for the analyses of total and organic C, and total N. The percentage ratio of the aggregates in each sieve represented the water-stable aggregates in the following size classes: 4.75-1.00, 1.00-0.50, 0.50-0.25 and <0.25 mm. The mean-weight diameter (MWD) of water-stable aggregates was calculated as mm n

where Xi is the mean diameter of the ith sieve size, and Wi the weight of total aggregates in the ith fraction.

After aggregate fractionation, soil aggregates were finely ground in an agathe mortar to a fine powder (<200 mesh), and duplicate subsamples (~25 mg) of soil were analysed for d15N using a Finnigan Delta-Plus isotope ratio mass spectrometer linked to a Carlo Erba NC2500 elemental analyser located at the University of Basilicata.

The proportion (f) of soil N derived from the 15N-labelled compost was calculated using the 15N atom% values of the 15N-enriched samples against the 15N natural abundance samples (control-D) by the isotope dilution method:

atom% 15N excess sample — atom% 15N excess natural abundance atom% 15N excess labelled material — atom%15N excess natural abundance'

where 15N sample = 15N atom% for the sample of interest; 15N-labelled material = 15N atom% of compost; 15N natural abundance = 15N atom% of soil from the same plot collected before the addition of 15N-labelled compost.

5.6.3 Results and Discussion 5.6.3.1 Enrichment by 15N in Maize Plant

The effect of the 15N-labelled compost amendments for 15N enrichment in plants was different among treatments after 3 years of experiments (Fig. 5.1). Results indicate that the d15N enrichment in maize plants was larger for all compost treatments than for controls. Moreover, the d15N of maize increased by continuous compost application each year. The d15N values in grains of treatment A was significantly greater than those of treatments B, C and D and there were significant differences among all treatments.

The first year mineralization of compost was quantified to be about 20% of applied N, with decreasing values in the second and third subsequent years. A great variability was found in the compost mineralization rates in the first year depending on compost maturity and composition (data not shown). These results are in line with the findings of Sikora and Enkiri (2001), who observed a 25% total availability of added compost. Similarly, Hargreaves et al. (2008) indicated a 10-22% availability of compost N.

5.6.3.2 d15N in Different Soil Aggregate Fractions

Table 5.3 shows the values of soil d15N (%) in different treatments and soil aggregates after a continuous 3-year experiment.

Abundance of stable N isotopes varied among soil aggregate fractions and treatments after three experimentation years. The 15N values were more variable

Grains Stalks and Leaves Cob and Husks

Plant Parts

Fig. 5.1 Changes in d15N (%) values of aboveground maize parts as affected by labelled organic amendment in different treatment plots (a-d). The bars with different letters within each plant part are statistically significant at P < 0.05

Grains Stalks and Leaves Cob and Husks

Plant Parts

Fig. 5.1 Changes in d15N (%) values of aboveground maize parts as affected by labelled organic amendment in different treatment plots (a-d). The bars with different letters within each plant part are statistically significant at P < 0.05

Table 5.3 Mean d15N (%) values in bulk topsoil (0-30 cm) and aggregate sizes (mm) after the third experimentation year

Soil sample Experimental plots

Table 5.3 Mean d15N (%) values in bulk topsoil (0-30 cm) and aggregate sizes (mm) after the third experimentation year

Soil sample Experimental plots

d15N

±a

BULK

12.35

1.78

9.13

1.25

5.14

1.10

4.31

1.38

4.75-1

21.38

12.90

14.31

3.47

6.17

1.67

2.95

0.38

1-0.5

11.04

2.60

8.93

4.78

4.78

0.29

2.87

1.76

0.5-0.25

9.94

1.62

5.67

0.45

5.92

1.20

3.34

0.72

<0.25

8.39

0.63

5.33

2.24

4.44

1.95

2.43

1.69

Table 5.4 Mean d15N (%) values in treated plot A for bulk topsoil (0-30 cm) and aggregate sizes (mm) over experimentation years (0, I, II and III years)

Soil sample

A (0) d15N

±a

A (I) d15N

±a

A (II) d15N

d15N

±a

BULK

4.31

1.38

10.66

2.20

7.65

1.35

12.35

1.78

4.75-1

2.95

0.38

9.54

4.85

7.74

1.89

21.38

12.90

1-0.5

2.87

1.76

6.61

2.12

6.94

3.07

11.04

2.60

0.5-0.25

3.34

0.72

7.20

2.42

8.67

0.54

9.94

1.62

<0.25

2.43

1.69

4.57

0.70

8.00

1.46

8.39

0.63

Table 5.5 Soil d15N (%) enrichment in bulk topsoil (0-30 cm) and aggregate sizes (mm) as affected by 15N-labelled compost additions in different treatment plots Soil sample Experimental plots

(A) 2006

(B) 2007

(C) 2008

(0) Control

d15N

±a

d15N

±a

d15N

±a

d15N ±s

BULK

10.66

2.20

5.30

1.05

5.14

1.10

4.31 1.38

4.75-1

9.54

4.85

3.23

1.32

6.17

1.67

2.95 0.38

1-0.5

6.61

2.12

5.21

1.33

4.78

0.29

2.87 1.76

0.5-0.25

7.20

2.42

5.15

1.32

5.92

1.20

3.34 0.72

<0.25

4.57

0.70

4.38

0.58

4.44

1.95

2.43 1.69

in A and B treatments than in C and D treatments. In most cases the d15N values increased with increasing soil aggregate size (Table 5.4).

Data indicate that 15N enrichment in bulk soil and all soil aggregates increased with time, as compared to plot soil at time 0. The soil 15N enrichment following yearly 15N-labelled compost amendment in different plots (A-C) over time is reported in Table 5.5. Different enrichments were obtained, possibly due to different chemical compositions of added composts (Table 5.1).

The macro-aggregate fraction was shown to be very sensitive and responsive to management (Elliott 1986; Six et al. 2000). This phenomenon was observed also in our study, where most changes for the 15N-labelled material were associated to macro-aggregates. Smaller and slower changes in soil 15N organic matter were observed with micro-aggregates and silt-and-clay fractions.

Table 5.6 Percent of soil-N derived from 15N-labelled compost materials in bulk topsoil (0-30 cm) and aggregate-sizes (mm) in different treatment plots over the experimentation time

Soil samples

Experimental plots

(A) COM1, 2006

(B) COM2, 2007

(C) COM3, 2008

2006

BULK

2.68

4.75-1

2.76

1-0.5

1.57

0.5-0.25

1.62

<0.25

0.89

2007

BULK

0.99

0.52

4.75-1

1.89

0.15

1-0.5

0.73

1.22

0.5-0.25

1.49

0.94

<0.25

1.52

1.01

2008

BULK

1.38

2.12

0.61

4.75-1

3.11

4.35

2.36

1-0.5

0.91

2.21

1.41

0.5-0.25

1.81

0.14

1.90

<0.25

1.29

0.47

1.48

Nitrogen added with compost is bound to be progressively incorporated in the SOM. The use of an 15N tracer enabled to monitor the SOM evolution under compost addition despite the short duration of the experiment (only 3 years). The percent of soil nitrogen derived from 15N-labelled compost in bulk soil and aggregate-sizes is reported in Table 5.6. Estimation of total compost-derived N sequestered in soil resulted to be 34.2, 38.2 and 42.5 percent of total N added with compost, for 1-year amendment (148 kg ha—1), 2-year amendments (314 kg N ha—1), and 3-year amendments (442 kg ha—1), respectively (data not shown).

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