Incubation experiment preparation
Soil samples were taken from a surface soil (0-10 cm) under a productive
grassland restored from cropland for about 30 years and a subsoil at a
depth of > 2 m in a cropland cultivated by soya/maize in
rotation for about 30 years at the Hailun Experimental Station, Chinese
Academy of Sciences, Hailun, Heilongjiang Province (47°26′N, 126°38′E).
The soil was developped from sedimentary matierals of loess-like
materials and classified as a Pachic Haploboroll according to USDA Soil
Taxonomy or Phaeozems according to the World Reference Base for Soil
Resources. The subsoil consisted of 420 g kg-1 clay
(< 2 μm) and 356 g kg-1 silt (2-20 μm), and
its clay mineralogy was dominated by vermiulite, illite and a mixed
layer mineral of vermiculite and illite43.
The surface soil was used to extract microbial inoculum at a soil: water
ratio of 1: 15 (mass: volume)45. The soil suspension
was shaken with glass beads for 2 hours to better slake the soil and
centrifuged (1000 g ) for 12 minutes to separate soil solution
from the soil. The supernatant was then transferred into a new vial and
centrifuged at 3470 g for 30 minutes. These procedures were
repeated twice, and the mixed supernatants were used as the inoculum.
The subsoil was used to extract the silt+clay (< 53 μm)
fraction by wet sieving. The silt+clay fraction of the subsoil was
heated at 500 oC for 2 hours to remove soil organic
carbon and microbes to derive a carbon-free natural soil material that
was dominant withvermiulite, illite and a mixed layer mineral of
vermiculite and illite. This soil material and other three commercially
available pure clay minerals (vermiculite, illite or kaolinite)
(Supplementary Table 1) were used to prepare four model soils, which
were composed of single soil material or pure clay minerals with silt-
(5-6.3 µm) and sand- (200-100 µm) sized quartz at a clay: silt: sand
ratio of 5: 4: 1, reproducing the particle size distribution of the
original subsoil. We mixed these four model soils with either maize
(Zea mays L.) or soya (Glycine max L.) straw, and with one
inoculum, then incubated the model soils at constant moisture and
temperature for 120 days. Briefly, aliquots of 90 g of each model soil
and 3 g of a litter were added into each of 24 Erlenmeyer flasks (240
mL) ensuring a similar volume or soil bulk density by slight packing in
each flask, providing three replicates for each gas and soil sampling.
Both maize litter and soya litter had been harvested three months after
sowing and chopped into < 0.25 mm particles. The C/N ratio was
29 for maize litter and 14.9 for soya litter. Then 20.6 mL distilled
water and 5.4 mL inoculum from the surface soil were added and
thoroughly mixed by spoon for 5 minutes in each flask. The flasks were
sealed with rubber stoppers and incubated in the dark at
25oC for 120 days.