## 2 Materials and Methods
### Soil Biological Properties  
Microbial biomass C (C\({}_{\text{MB}}\)) was determined by the fumigation-extraction method [37]: briefly, 5 g of soil was fumigated for 24 h with amylene-stabilized CHCl\({}_{3}\) and extracted in 20 mL of 0.5 M K\({}_{2}\)SO\({}_{4}\). Then, 3.5 mL of chromium reagent [chromium (VI) oxide (0.06% _w/v_)]; sulfuric acid (65% _v/v_)] was added to 2 mL of extract and incubated at 150 \({}^{\circ}\)C for 60 min. The organic C concentration was determined spectrophotometrically at 445 nm. Microbial biomass C was calculated as the difference between the organic C concentration of the fumigated and unfumigated extracts [32]. DNA was extracted from soil (0.25 g) using a DNA PowerSoil(tm) Isolation Kit (MO Bio Laboratories, CA) according to the manufacturer's instructions. Prior to DNA extraction, soil samples were washed twice in 120 mM K\({}_{2}\)HPO\({}_{4}\) (pH 8.0) to wash away extracellular DNA [38]. Extracted DNA concentrations were determined using a TapeStation 4200 (Agilent Technologies). Real-time qPCR was carried out to measure total bacterial (16S rRNA) and fungal (18S rRNA) gene copy abundances [39]. Each 25 \(\upmu\)L reaction mixture contained 1.0 \(\upmu\)L of template DNA, 12.5 \(\upmu\)L of SYBR PremixExTaq (Takara Bio, Inc.), 1.5 \(\upmu\)L of each primer (at a concentration of 30 and 20 \(\upmu\)M for bacteria and fungi, respectively), 0.5 \(\upmu\)L of ROX(tm) dye, and 8.0 \(\upmu\)L of sterile deionized water. Each sample was measured in triplicate. The primers and qPCR conditions are shown in Table S1 [40; 41]. The minimum R\({}^{2}\) value obtained in the calibration curves was 0.986. The minimum efficiency in the PCR was 96.9%. Soil respiration (R) was determined by measuring CO\({}_{2}\) evolution in hermetic flasks incubated at 30 \({}^{\circ}\)C for 72 h according to ISO 16072 [42]. Potentially mineralizable N (N\({}_{\text{PM}}\)) was determined as follows [43]: briefly, deionized water was added to 2.5 g of fresh weight (FW) soil. The suspension was incubated for 7 days in a water bath at 40 \({}^{\circ}\)C. Then, 6.25 mL of 4 M KCl and 1 mL of dichloroisocyanurate(50 mg in 50 mL deionized water) were added to incubated and non-incubated soils. After 30 min at room temperature, absorbance was read at 670 nm. The activities of \(\beta\)-glucosidase (EC 3.2.1.21), \(\beta\)-glucosidaseaminidase (EC 3.2.1.30), xylosidase (EC 3.2.1.37), acid phosphatase (EC 3.1.3.2), L-Ala-aminopeptidase (EC 3.4.11.1) and L-Leu-aminopeptidase (EC 3.4.11.1) were determined according to ISO/TS 22939 [44], using fluorogenic substrates [4-methylumbelliferyl (MUF) and 7-amino-4-methylcoumarin (AMC)] in 96-microwell plates. Briefly, 100 \(\mu\)L of the soil suspension (2.5 g in 150 mL water) was dispensed onto the plates. The four MUF-activities (\(\beta\)-glucosidase, \(\beta\)-glucosidaseaminidase, xylosidase, acid phosphatase) were assayed at pH 6.1 in 150 mM MES [2-(N-morpholino) ethanesulfonic acid] buffer. The two AMC-activities (L-Ala-aminopeptidase, L-Leu-aminopeptidase) were assayed at pH 7.8 in 75 mM Tris-HCl buffer. The plates were continuously stirred and kept at 30 \({}^{\circ}\)C. Fluorescence was measured five times at 20 min intervals. Arylsulphatase (EC 3.1.6.1) activity was determined according to [PERSON] et al. [45]. Urease (EC 3.5.1.5) activity was measured following [PERSON] and [PERSON] [46]. Dehydrogenase (EC 1.1) activity was determined according to ISO 23753-2 [47]. Community-level physiological profiles (CLPPs) of cultivable heterotrophic bacteria were determined with Biolog EcoPlates(tm) following [PERSON] et al. [48]. Data were calculated from Gompertz regressions (three parameters) of the obtained curves as proposed by [PERSON] et al. [49]. To determine the effect of treatments on the structure of soil prokaryotic communities, amplicon libraries were prepared using a dual indexing approach with sequence-specific primers [50] targeting the V4 region of the 16S rRNA gene: 519F (CAGCMGCCGCGGGTAA) adapted from [PERSON] et al.