2.2 Amendments used and soil incubation
Three soil amendments were used for reclamation purposes, including biochar, nitrogen fertilizer, microbial agent. (ⅰ) Biochar feedstock was crop residue (maize), which was air-dried for 30 days and then was ground to pass a 2 mm sieve. Then, the ground residues were pyrolyzed to form biochar in a muffle furnace with an increase at 5 °C min−1 to 500 °C for 2 h. Biochar was ground to 250 µm for laboratory incubation. Details about biochar amendments are shown in Table 1. (ⅱ) Nitrogen fertilizer was urea (46.67% N). (ⅲ) Microbial agent (obtained from Beijing Danlu Biotechnology Co., Ltd) was mainly prepared with castor as a carrier. The carrier was placed in a polypropylene plastic bag and sterilized at 121 ℃ for 1.5-2 h, and then cooled. Subsequently, the carrier was inoculated with effective microorganism solution, and put it in a 25 ℃ incubator for 4-5 d after mixing. Since we aimed to activate the microbial activity in mine soil by addition of microbial agent, thus, we selected the effective bacteria and fungal with a wide range of adaptability in extreme soil environments. The effective bacteria and fungal composition in the microbial agent (sequencing process according to materials and methods 2.4) contained Ascomycota, Basidiomycota, Chytridiomycota, Mortierellomycota and Proteobacteria, Actinobacteria , Bacteroidetes, Gemmatimonadetes (the relative abundance >1%), and its effective amounts are up to >300 million g-1.
Biochar (C) was used for substrates in all treatments, and was thoroughly mixed with mine soil at an application rate of 30 g carbon kg-1 soil, which was similar to the organic carbon content in the natural grassland soil in the sampling area. Based on the desired C/N ratios of 35:1, 25:1 and 12.5:1, nitrogen fertilizer (N) was added at three levels of 0.86 (N1), 1.2 (N2), and 2.4 (N3) g N kg-1 soil, respectively. Microbial agents (M) were thoroughly mixed with 20 g distilled water into mine soil at a dose of 0.4 (M1) and 0.8 (M2) g kg-1 soil. Thirteen different treatments with three replicates per treatment were applied to the soil samples: C-N0, C-N1, C-N2, C-N3; C-M1-N0, C-M1-N1, C-M1-N2, C-M1-N3; C-M2-N0, C-M2-N1, C-M2-N2, C-M2-N3, and unamended mine soil was used as control (CK) (Fig.1).
Laboratory incubation was carried out with 1000 g of mine soil in a 2 L beaker under aerobic and dark conditions for 90 days in a varying temperature incubator (JYL-253, Jiayu, Shanghai, China), at a constant soil moisture of 50% of water holding capacity and a varying temperature which gradually increased from 5 to 22°C for the first 12 h, and then decreased from 22 to 5°C for the last 12 h (close to the local summer temperature condition). Soils were sampled to monitor pH, soil organic carbon (SOC), total nitrogen (TN), available nitrogen (AN), available phosphorus (AP), available potassium (AK), microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) at 0, 5, 12, 45, 90 days of incubation with. The first sampling point (day 0) was collected just after soil sampling. To observe the effect of treatment on the structure and diversity of microbial communities, bacterial and fungal communities were assayed at the end of the incubation.
2.3 Soil physicochemical and microbial biomass analyses
Amended soil samples were divided into two parts: one part was air dried and shaken on a 2-mm sieves for the measurements of pH, SOC, TN, AN, AP and AK. Detailed soil physicochemical analyzes were described in Chen et al . 2020. The other part was stored at 4 °C in a refrigerator for microbial biomass measurements.
Soil samples stored at 4 °C were measured for MBC and MBN using chloroform fumigation and extraction method (Vance et al., 1987). Briefly, 10 g of oven-dry soil was fumigated with chloroform in the dark for 48 h after which C and N of fumigated and non-fumigated (control) samples was extracted with 0.5 ml K2SO4, and then total dissolved organic C was determined on an organic carbon analyzer (Shimadzu Model TOC), while total extractable N was quantified with a flow-injection instrument. After values in non-fumigated were subtracted from those of fumigated samples, a Kec/Ken factor of 0.45 and 0.54 was applied for MBC both MBN.