drivers of soil respiration of root and microbial ... - Unitus DSpace

Soil enzymatic activity
Criteria for choosing enzyme assays were based on their sensitivity to soil management,
importance in nutrient cycling and SOM decomposition.
Enzyme activity was measured according to the method of Marx et al. (2001) and
Vepsäläinen et al. (2001), partially modified, and based on the use of fluorogenic
methylumbelliferyl (MUF)- and (AMC)- substrates. The soil was analysed for β-cellobiohydrolase
(exo-1,4-β-glucanase, EC 3.2.1.91), N-acetyl-β-glucosaminidase (EC 3.2.1.30), β-glucosidase (EC
3.2.1.21), α-glucosidase (EC 3.2.1.20), acid phosphatase (EC 3.1.3.2), β- xylosidase (EC 3.2.2.27),
arylsulphatase (EC 3.1.6.1) and leucine-aminopeptidase (EC 3.4.11.1). The respective substrates
were 4-MUF-β-D-cellobioside, 4-MUF- N-acetyl-β-glucosaminide, 4- MUF - β-D-glucoside, 4-
MUF - α-D-glucoside, 4- MUF -phosphate, 4- MUF -7-β-D-xyloside, 4- MUF -sulphate and L-
leucine-7- amino-4-methylcoumarin. 2.0g of fresh soil were homogenised in 100ml of 0.5M acetate
buffer, pH 5.5, using an Ultra Turrax IKA for 3 minutes at 9600 rev/min. Aliquots of 100 µl of
diluted soil were pipetted in a 96 multiwell plate with three replicates. Each substrate was added in
each well in aliquots of 100 µl for a final concentration of 500 µM; then the microplates were
incubated at 30 °C for 3 hours, with fluorescence readings occurring every 30 minutes. For the
calibration curve 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1 nmoles of MUF or AMC, for leucine-
aminopeptidase, were added to the same aliquots of soil suspension to take into account the
quenching effect on fluorescence intensity (Freeman et al., 1995). Fluorescence readings (excitation
360 nm; emission 450 nm) were performed using a Fluoroskan Ascent (Thermo electron)
fluorometer.
The synthetic enzymatic index (SEI) was calculated following Dumontet et al. (1998) and
using specific enzyme activities releasing the same reaction product (MUF).
Soil N mineralization (Short Nitrification Assay)
Potential nitrification was measured after inhibition of N-NO2 - oxidation with sodium
chlorate (10mM) according to the short nitrification assay (SNA) reported by Hopkins et al. (1988).
Briefly, 2g of soil (60% WHC) were taken from each soil sample and were extracted with
(NH4)2SO4 solution during 24h shaking. Extracts were centrifuged at 3800 rev per 20 min, then 1ml
from each sample was transferred to 50 ml flasks together with diazotizing and coupling reagents.
Colorimetric readings were performed on the spectrophotometer (UV mini 1240, UV-vis
spectrophotometer, Shimadzu). To construct a calibration curve 0, 0.25, 0.5,1, 2, 3, and 4 ml were
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