2.2 N2O flux measurement
To measure the N2O flux potential in the soil, 30 g dry weight soil was placed into Erlenmeyer flasks (250 mL) and moistened to 50% of water holding capacity. All flasks were sealed with silicone rubber stoppers with three-way valves. Adjusted soil samples were held for 1 week prior to incubation at 25 ℃ and 50% relative air humidity in the dark under aerobic conditions to activate microorganisms. During the incubation period, the samples were stored in an incubator under dark conditions at 25 ℃ and 50% relative air humidity and adjusted to 50% of the water-holding capacity (WHC). The soil moisture content was maintained by adding deionized water every 3 days with a micro pipette to compensate for water loss. The samples were aerated by removing the stoppers for 1 h every day. A total of 84 flasks were divided into four treatments (N0, N150, N225, and N300 treatment), with three independent replicates. Gas samples from each treatment was taken in order to determine N2O flux on 1, 3, 5, 7, 9, 12, and 15 days. Samples were taken using a 50 mL syringe, fitted with a three-way-stopcock and 22G hypodermic needle, and then injected into pre-prepared 50 ml vacuum flasks for determination of the N2O flux. The remaining soil samples were used for the determination of NH4+-N and NO3–N after taking gas samples. The N2O concentration was determined using a gas chromatograph (GC; Agilent 7890, Agilent technologies, Santa Clara, CA, USA). N2O gasstandards were supplied by the National Research Center for Certified Reference Materials, Beijing, China. N2O flux was calculated according to the following formula (3):
F= {(C - C0) × V × M × [273 / (273 + T)]} / (d × m × 22.4 × 1000) (1)
F represents N2O flux (μg·kg-1·d-1). C represents N2O concentration (μL·L-1). C0represents the gas concentration above the culture bottle at the beginning of the culture (μL·L-1). V represents gas volume (mL). M represents the mass of N2O per mole (44). T represents incubator temperature (25℃). The lowercase letter “d” represents incubation time (d/h). The lowercase letter “m” represents dry soil quality (30g).
2.3 Distinguishing between quantifying the contribution of fungi and bacteria to N2O emission
Biological inhibitors were used to distinguish between the contribution of fungi and bacteria to lawn soil N2O emission. Four treatments were established according to the results of previous studies[16,24-26]: (i) cycloheximide (C15H23NO4, a fungicide) treatment at 1.5 mg g-1 was used to inhibit the fungal activity, (ii) streptomycin treatment (C42H84N14O36S3, a bactericide) at 3.0 mg g-1 was used to inhibit the bacterial activity, (iii) cycloheximide at 1.5 mg g-1 and streptomycin at 3.0 mg g-1 were used to inhibit both fungal and bacterial activity, (iv) a no-inhibitor control was used to assess the total microbiological activity contribution to N2O. The contribution of soil fungi to N2O emission was estimated by the equation (1). The contribution to N2O emission of soil bacteria was estimated by the equation (2).
Fungal contribution rate = 100 × (A – B) / (A - D) (1)
Bacterial contribution rate = 100 × (A – C) / (A - D) (2)
A represents N2O fluxes in the no-inhibitor control. B represents N2O fluxes in the cycloheximide treatment. C represents N2O fluxes in the streptomycin treatment. D represents N2O fluxes in the cycloheximide and streptomycin treatment.
Soils were amended with glucose (2 mg C g-1 soil) and KNO3 (100 μg N g-1 soil). All chemicals were dissolved in 2 mL distilled water and added drop to soil to ensure sample homogenization. Deionized water was added to 50% of the soil water holding capacity. The flasks were pre-incubated in a dark climate chamber (25 ℃, 50% relative air humidity, 50%WHC) for 24 h to ensure that biocides had taken effect, and flasks were capped to prevent water evaporation. The flasks were then uncapped for 1 h and resealed for the start of the 48h incubation period in a dark climate chamber (25 ℃, 50% relative air humidity, 50%WHC). During the incubation period, gas samples were collected at 1, 3, 7, 12 and 24 h.