2 Materials and Methods
2.1 Study site
The study site was located in
the
Zoucheng (ZC) coal-mining area (35°8’12”N–35°32’54”N,
116°46’30”E–117°28’54”E; CGCS2000, Fig. 1) within the Shandong
Province, China. The climate of the study site is within the warm
temperate monsoon climate zone (Csa; Köppen, 1884). The mean annual
rainfall over the period 1979-2018 was 777.1 mm, and the mean annual
temperature was 14.1°C. The soil type is brown fluvo-aquic, with a
22.3% of sand (2-0.02mm), 65.9% of silt (0.02-0.002mm) and 11.8% of
clay (<0.002mm). The soil bulk density is 1.48 g
cm-3 (http://vdb3.soil.csdb.cn/).
The study site is subjected to subsidence (Fig. S1, see supplemental
material) as a result of past coal mining activities. Since 2001,
subsidence has been reverted in the study site through land reclamation,
using coal gangue and topsoil fillings to a depth comprised between 200
and 400 cm, and with a topsoil layer 80 cm deep (Qu et al., 2017). Land
reclamation in the study site was undertaken at different locations over
time. As a result, four distinct reclamation zones can be distinguished
on the basis of the time since reclamation, -i.e. (i) r17 - 17 years
since reclamation; (ii) r14 - 14 years since reclamation; (iii) r11 - 11
years since reclamation; and (iv) r8 - 8 years since reclamation (Fig.
1). After reclamation, land use shifted to farmland, where crops of
wheat and soybean are rotated following a one-year rotation scheme.
2.2 Soil sampling and analysis of key soil
attributes
On 04/05/2019, 15 spatially-distributed, surface soil (i.e. 0-10 cm
below the ground level; b.g.l) samples were retrieved from each
reclamation zone -i.e. r17, r14, r11 and r8 (Section 2.1; Fig. 1),
respectively, following a stratified random sampling approach (Ma et
al., 2020). In addition, 15 surface soil samples were retrieved from
three un-reclaimed plots and used as control (CK; Fig. 1).
A subsample of 20 g of unprocessed, fresh soil was retrieved from the
bulk soil samples. The soil subsamples were frozen and stored at -20oC immediately after sampling for subsequent microbial
analysis (Section 2.3). The remaining soil materials were air-dried,
homogenized and sieved through a 2 mm sieve prior to analyzing eight key
soil attributes. (i) Soil pH and (ii) electric conductivity (EC) were
measured using a pH meter and conductivity meter, respectively (PHC-3C,
DDS-307A, Shanghai leici, China), in a 1:2.5 soil:distilled water
suspension. (iii) Soil organic matter content (SOM) was measured with a
colorimetric method (Lu, 2000), using hydration heat during the
oxidation of potassium dichromate. (iv) Soil total Kjeldahl nitrogen
(TKN) was
measured
with a Kjeldahl analyzer (K9840, Shandong Hanon, China). (v) Soil
extractable phosphorus (OP) was measured with the Olsen method (Lu,
2000). (vi) Soil available potassium (AK) was quantified with the
ammonium acetate–flame photometric method (Lu, 2000). (vii) Soil
enzymatic activity was assessed in the light of the soil dehydrogenase
(DHG), urease (URA), and polyphenol oxidase (PPO) activities. These
enzymatic activities were analyzed using the triphenyltetrazolium
chloride (TTC) method, the sodium-hypochlorite phenol colorimetric
method, and the pyrogallol colorimetric method, respectively (Guan,
1986). (viii) Microbial activity was quantified through measuring the
activity of fluorescein diacetate hydrolase (FDA), using the fluorescein
colorimetric method (Guan, 1986).