Annual fluxes of C and N were estimated from the measured monthly fluxes. Biomass stocks were estimated annually, while soil stocks were estimated only at the end of the investigation.
Soil C fluxes were characterized by determining the total soil CO2 efflux and heterotrophic respiration, whereas soil N fluxes were characterized by determining the net mineralization and nitrification rates using an in situ incubation method. Total soil CO2 efflux was separated into root- and heterotrophic respiration using a trench method59 (link). A root exclusion barrier (50 cm diameter, 30 cm depth, PVC) was installed in each plot, 4 weeks before the treatments were initiated. Two soil collars (10 cm diameter, 5 cm depth, PVC) were installed inside the rooting barrier to measure heterotrophic respiration and another two were installed outside the rooting barrier to measure heterotrophic + root respiration. Vegetation was removed within the rooting barriers but litterfall was kept. Soil CO2 efflux was measured once a month using an infrared gas analyzer (Model EGM-4, PP-Systems, Hitchin, Hertfordshire, UK) equipped with a flow-through closed chamber (Model SRC-2). Measurements were performed between 10:00 and 13:00 over the study period. Soil temperature was also measured at a depth of 8 cm near the soil CO2 efflux collar using a digital soil temperature probe (K-type, Summit SDT 200, Seoul, Korea).
On each day when the soil CO2 efflux was measured, two soil cores were collected from each plot at a depth of 5 cm using a 100 cm3 core soil sampler. One sample was placed in a plastic bag and the other was returned to the soil and incubated to estimate the net N mineralization rate. We thus collected two soil samples from each plot every month, one fresh and one incubated. Both samples were transported to the laboratory and their fresh weight was measured. A 10 g portion of the fresh soil sample was oven-dried for 48 h at 105 °C to quantify the soil’s gravimetric water content and the rest was kept to determine the concentrations of nitrate and ammonium and the soil pH (measured using a 1:5 soil water suspension with an ion-selective glass electrode; Istec Model pH-220L, Seoul, Korea). The bulk density of each soil sample was calculated from its gravimetric water content and fresh weight.
Ammonium and nitrate were extracted from soil samples (5 g) using 50 ml of a 2 M KCl solution in a mechanical vacuum extractor (Model 24VE, SampleTek, Science Hill, KY, USA). The resulting solutions were then immediately placed in a cooler at 4 °C for storage. The ammonium and nitrate concentrations of the solutions were determined using an auto analyzer (AQ2 Discrete Analyzer, Southampton, UK). The mineral N concentration was measured throughout the period when fertilizer was applied, from April 2011 to April 2014. The net rates of ammonification and nitrification were estimated based on the differences in the ammonium and nitrate contents of the soil, respectively, between before and after the incubation. The sum of these two rates was taken as the net mineralization rate. If the net mineralization rate was negative, it was regarded as a net immobilization rate.
Additional soil samples were collected from four randomly selected points in each plot at depths of 0–15 cm both 4 weeks before starting the treatments and at the end of the investigation (2014). Samples were pooled within a plot and their concentrations of C, N, and available P were determined to capture the soil’s initial condition and the treatment response. C and N were analyzed with an elemental analyzer (vario Macro cube, Elementar Analysensysteme GmbH, Germany). The available P concentration was determined by extraction using NH4F and HCl solutions60 and analyzed using a UV spectrophotometer (Jenway 6505, Staffordshire, UK). C and N stocks were estimated by measuring the concentrations of both elements in a 100 cm3 core and dividing by the bulk density of the soil in the 0–5 cm layer.
Soil C fluxes were characterized by determining the total soil CO2 efflux and heterotrophic respiration, whereas soil N fluxes were characterized by determining the net mineralization and nitrification rates using an in situ incubation method. Total soil CO2 efflux was separated into root- and heterotrophic respiration using a trench method59 (link). A root exclusion barrier (50 cm diameter, 30 cm depth, PVC) was installed in each plot, 4 weeks before the treatments were initiated. Two soil collars (10 cm diameter, 5 cm depth, PVC) were installed inside the rooting barrier to measure heterotrophic respiration and another two were installed outside the rooting barrier to measure heterotrophic + root respiration. Vegetation was removed within the rooting barriers but litterfall was kept. Soil CO2 efflux was measured once a month using an infrared gas analyzer (Model EGM-4, PP-Systems, Hitchin, Hertfordshire, UK) equipped with a flow-through closed chamber (Model SRC-2). Measurements were performed between 10:00 and 13:00 over the study period. Soil temperature was also measured at a depth of 8 cm near the soil CO2 efflux collar using a digital soil temperature probe (K-type, Summit SDT 200, Seoul, Korea).
On each day when the soil CO2 efflux was measured, two soil cores were collected from each plot at a depth of 5 cm using a 100 cm3 core soil sampler. One sample was placed in a plastic bag and the other was returned to the soil and incubated to estimate the net N mineralization rate. We thus collected two soil samples from each plot every month, one fresh and one incubated. Both samples were transported to the laboratory and their fresh weight was measured. A 10 g portion of the fresh soil sample was oven-dried for 48 h at 105 °C to quantify the soil’s gravimetric water content and the rest was kept to determine the concentrations of nitrate and ammonium and the soil pH (measured using a 1:5 soil water suspension with an ion-selective glass electrode; Istec Model pH-220L, Seoul, Korea). The bulk density of each soil sample was calculated from its gravimetric water content and fresh weight.
Ammonium and nitrate were extracted from soil samples (5 g) using 50 ml of a 2 M KCl solution in a mechanical vacuum extractor (Model 24VE, SampleTek, Science Hill, KY, USA). The resulting solutions were then immediately placed in a cooler at 4 °C for storage. The ammonium and nitrate concentrations of the solutions were determined using an auto analyzer (AQ2 Discrete Analyzer, Southampton, UK). The mineral N concentration was measured throughout the period when fertilizer was applied, from April 2011 to April 2014. The net rates of ammonification and nitrification were estimated based on the differences in the ammonium and nitrate contents of the soil, respectively, between before and after the incubation. The sum of these two rates was taken as the net mineralization rate. If the net mineralization rate was negative, it was regarded as a net immobilization rate.
Additional soil samples were collected from four randomly selected points in each plot at depths of 0–15 cm both 4 weeks before starting the treatments and at the end of the investigation (2014). Samples were pooled within a plot and their concentrations of C, N, and available P were determined to capture the soil’s initial condition and the treatment response. C and N were analyzed with an elemental analyzer (vario Macro cube, Elementar Analysensysteme GmbH, Germany). The available P concentration was determined by extraction using NH4F and HCl solutions60 and analyzed using a UV spectrophotometer (Jenway 6505, Staffordshire, UK). C and N stocks were estimated by measuring the concentrations of both elements in a 100 cm3 core and dividing by the bulk density of the soil in the 0–5 cm layer.
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