LIFE CarbOnFarm Progress report Annex 7.1 Deliverables

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1 Report for C. 2 Action: first year The data are related to the field soil samples from project sites of Piemonte (Tetto Frati and Grugliasco) and Campania, (Castel Volturno and Prima Luce) after the application of SOM managements. Project site: Tetto Frati Field tests: TRAD mineral fertilization; PORF biomimetic catalyst; 0N without Nitrogen; SS A and SS B: solid separate high and low levels; CMP A and CMP B mature compost high and low levels Project site: Grugliasco Field tests: TRAD mineral fertilization; 0N: without Nitrogen; SS A and SS B: solid separate high and low levels; CMP A and CMP B mature compost high and low levels Project site: Castel Volturno Field tests: TRAD mineral fertilization; PORF; biomimetic catalyst; CMP A and CMP B on farm compost high and low levels Project site Prima Luce Field tests: A control no fertilization; B traditional organo-mineral ; C on-farm compost low dose; D on-farm compost high dose completed analyses soil aggregate stability SOC and total Nitrogen of bulk soils and soil aggregates for Tetto Frati, and Castel Volturno analyses in progress: SOC and total Nitrogen of bulk soils and soil aggregates: Grugliasco; Prima Luce 13C-OC of bulk soils and soil aggregates phospholipid fatty acids (PLFA) SOM characterization by THM GC-MS Analytical methodologies - Soil aggregate stability A modified procedure of the classical method described by Kemper and Rosenau (1986) was used to separate the water-stable aggregates. Forty grams of the <2.00 mm, air-dried soil samples were put in the topmost of a nest of three sieves of 1.00, 0.50 and 0.25mm mesh size and pre-soaked in distilled water for 30 min. Thereafter the nest of sieves and its contents 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 the soil particles on the topmost sieve were always below the water surface during each oscillation. After wet-sieving, the resistant soil materials 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. The percentage ratio of the aggregates in each sieve represents the water-stable aggregates of size classes: , , and <0.25 mm. Mean-weight diameter in water (MWDw) of water-stable aggregates was calculated as follow 1

2 n MWDw= Σ Xi Wi i=1 where Xi is the mean diameter of the ith sieve size and Wi the proportion of the total aggregates in the ith fraction. - PLFA and NLFA analyses Selected fatty acids in the soil phospho-lipid (PLFA) and neutral lipid (NLFA) fractions, as biomarkers for different soil microbial groups, are analyzed using the modified Bligh-Dyer technique. Total soil lipids are extracted from 2 g of soil by a chloroform/methanol (MeOH)/citrate buffer (1:2:0.8 v/v). Separation of lipid classes is conducted in silica gel columns: neutral, glycoand phospho-lipids fractions were obtained by sequentially eluting silica gel columns with chloroform, acetone, and methanol, respectively. While the glycolipid fraction is discarded, neutral and phospho-lipid fractions are dried under a N 2 flow at 37 C, and stored at 20 C. Fatty acid methyl esters were formed by a mild alkaline methanolysis. Thirty microliters of methyl nonadecanoate fatty acid (19:0; Sigma Aldrich) is then added as an internal standard and the methylated samples are dried under a N 2 flow. Samples dissolved in 200 µl of hexane are analysed using a Perkin-Elmer Autosystem XL (GC) equipped with a PE Turbomass-Gold quadrupole mass spectrometer. Chromatographic separation was achieved through 60m Supelco Capillary column SLB-5ms under helium as carrier gas (1 ml min 1 ). Samples (2.5 µl) are injected in splitless mode in a injector held at 250 C. The initial oven temperature, 100 C, was held for 5 min, raised to 210 C at a rate of 2 C min-1, then raised from 210 C to 250 C at a rate of 5 C min 1, and held for 12 min. Mass spectra were obtained in EI mode (70 ev), scanning in the range of m/z , with a cycle time of 1 s. The abundance of individual PLFA was derived from the relative area under each chromatographic peak, as compared to that of internal standard (19:0) and related to the calibration curve of the 19:0 standard fatty acid dissolved in hexane. Each PLFA content was expressed as nmol of PLFA per gram of dry soil. Fatty acids are identified according to the ω-designation described as follows: total number of carbons; the number following the colon indicates the amount of double bonds; the symbol ω indicates the position of the first double bond from the methyl end of the molecule. Cisand trans-configurations are indicated by c and t, respectively; Iso and anteiso forms of methylbranched fatty acids are indicated by i- and a-, respectively. 10Me indicates a methyl group placed on the tenth C atom from the carboxyl end of the molecule; cy refers to cyclopropane fatty acids. Individual fatty acids will be used as signatures for various groups of microorganisms. The 18:2ω6c and 18:1ω9c PLFA are representative biomarkers for fungal biomass. The a15:0, i15:0, i16:0, i17:0, a17:0 PLFA represent Gram-positive bacteria, while the 16:1ω7c, 18:1ω7c, 18:1ω5c, cy17:0, cy19:0 PLFA are related to Gram-negative bacteria, and the 10Me16:0, 10Me17:0, 10Me18:0 PLFA indicated activity of Actynomicetes. Both 16:1ω5 NLFA and PLFA are index of arbuscular mycorrhizal fungi (AMF) content. The 16:1ω5 NLFA is specific but indicates storage material rather than biomass, therefore the 16:1ω5 NLFA-to-PLFA ratio will be used as a measure of AMF prevalence in communities, with ratios >1 reflecting prevalence of AMF. Total PLFA concentration will be calculated from all identified PLFAs (listed above). The ratios of fungal to bacterial (F:B) PLFA, Gram-positive to Gram-negative bacteria (GP:GN) PLFA and AMF to saprotrophic fungi is calculated by dividing the respective sums of marker fatty acids and represent the relative abundance metrics of these groups. 2

3 Results After the field test application, no significative differences among treatments, were found in respect to aggregate distribution and stability index for each project site (Figs 1a, 2a, 3a and 4a). The final slight increase in the MWDw values observed in the soil addition with high dose of solid separate (SS-A) at Tetto Frati soils (Fig. 1a), may be related to a temporary stimulation of microbial activity promoted by the addition of fresh decomposable organic materials and to the possible consequent inclusion of fines particle sizes (< 0.25 mm), usually associated with microorganisms and microbial by-products, in the upper intermediate aggregate class ( mm), thereby promoting an increase of stability index. No significative correlations were found among soil treatments and SOM content in the analysed samples (Figs 1b,c, 1b,c and 3b,c), although for the project sites of Tetto Frati and Castel Volturno, the field plots with OM addition showed a slight increasing trend of OC and N content in either bulk samples and cumulative size-aggregates (Fig. 1b,c and 3b,c). Moreover for the project site of Tetto Frati, a lower N recovery was found in soil aggregates of TRAD and 0N (Fig. 1c) treatments, thereby suggesting a lower physical retention capacity of biolabile organic components, in respect to soil treatments with exogenous OM addition. Soil distribution (%) MWDw PORF Figure 1a. Distribution (%) of water-stable aggregate sizes (mm) and stability index (MWD) under different treatments at Tetto Frati 3

4 Soil organic carbon g kg -1 PORF Figure 1b SOC content (g kg-1) in bulk soil and soil aggregates for different treatment at Tetto Frati Figure 1c Total N content (g kg-1) in bulk soil and soil aggregates for different treatment at Tetto Frati 4

5 MWDw Figure 2a. Distribution (%) of water-stable aggregate sizes (mm) and stability index (MWD) under different treatments at Grugliasco Soil organic carbon g kg -1 Soil distribution (%) Figure 2b SOC content (g kg-1) in bulk soil and soil aggregates for different treatments at Grugliasco 5

6 Figure 2c SOC content (g kg-1) in bulk soil and soil aggregates for different treatments at Grugliasco Soil distribution (%) MWDw PORF Figure 3a. Distribution (%) of water-stable aggregate sizes (mm) and stability index (MWD) under different treatments at Castel Volturno 6

7 Soil organic carbon g kg -1 PORF Figure 3b SOC content (g kg-1) in bulk soil and soil aggregates for different treatments at Castel Volturno Figure 3c Total N content (g kg-1) in bulk soil and soil aggregates for different treatments at Castel Volturno 7

8 For the project sites of Prima Luce the field replicates were left split to each other, in order to correlate the specific sampling point with the corresponding soil properties, as indicated in the report for the field activities included in the present report -Annex 7.5 Report Action B.3 Soil distribution (%) MWDw Figure 4a. Distribution (%) of water-stable aggregate sizes (mm) and stability index (MWD) under different treatments at Prima Luce 8