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).