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.