Sampling and measurement during the incubation experiment
During the 120-day incubation, soil respiration was recorded through
measurement of CO2 evolution in the flask headspaces.
Gas samples were collected at 3, 6, 9, 12, 18, 22, 27 and 30 days in the
first month, and weekly in the following months. Three blank Erlenmeyer
flasks for each sampling time and mineral treatment were analyzed for
background CO2 concentrations. After each gas sampling,
all remaining Erlenmeyer flasks were opened for 20 minutes to replace
their CO2-enriched headspace with fresh air. Then soil
moisture content in each flask was adjusted by weighing and adding a
specified water volume to re-establish the original moisture content.
The CO2 concentration in each gas sample was measured by
titration using 0.1 N NaOH and corrected by subtracting the background
CO2 concentration. The corrected CO2concentration was used to calculate daily emission.
At the end of the incubation, soil samples were taken for measuring C
and N contents, X-ray diffractograms, PLFAs and amino sugars and to
extract mineral-associated SOM for measuring C and N contents and
chemical structures using solid-state 13C cross
polarization/total sideband suppression (CP/TOSS) NMR spectroscopy. The
mineral-associated SOM was extracted by hydrofluoric acid treatment and
centrifugation. Briefly, 5-g air-dried soil samples were mixed with 40
mL 2% hydrofluoric acid in 100 mL plastic tubes, shaken for 2 hours at
25oC at 180 rpm and centrifuged at 1000 g for
10 minutes. Before and after centrifugation, visible floating materials
was removed, and the supernatant was discarded. The treatments were
repeated four more times. The final remaining soil residues were washed
four times with deionized water to remove hydrofluoric acid, oven dried
at 80oC until constant weight, and measured for C and
N contents and chemical structures by using 13C NMR.
Based on the C contents before and after the treatment of 2%
hydrofluoric acid, C loss was estimated, being from 4.7% to 16.8% in
all model soils except for the vermiculitic soil added with soya litter
(37.6%) (Supplementary Table 2).