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.