The study was conducted in a typical steppe ecosystem on the Mongolian Plateau (43°33′N, 116°40′E). The area is a typical semi-arid continental climate with a mean annual temperature of 1.1°C and average annual precipitation of 345 mm (He et al. 2008). The soil is chestnut type, that is, Calcic kastanozems. The vegetation consists predominantly of grassland plants, such as Leymus chinensis, Stipa grandis, and Cleistogenes squarrosa.
Five grasslands, with similar vegetation and topography across in 2 km × 2 km in extent, comprised a successional series of grassland restoration, where plant communities and soil properties varied predictably after eliminating the large animal disturbance by fences (Table 1). The five plots were categorized as GE0, GE4, GE7, GE11, and GE31. Plot GE0 was subjected to long-term free-grazing and was in a slightly degraded condition in terms of the aboveground community and plant diversity. Plot GE4 was established in 2008 by fencing off a section of previously free-grazing grassland. Plots GE7, GE11, and GE31 were similarly established in 2004, 1999, and 1979, respectively (He et al. 2008). We assumed that changes in vegetation and soil properties among the five plots were mainly a result of grazing intensity and the length of GE, because the five experimental plots were floristically and topographically similar, and all were distributed in the same upper basalt platform.
Table 1. Changes in vegetation and soil properties with grassland succession.
|Grassland type||Aboveground biomass (g m−2)||Soil organic carbon (g kg−1)||Soil total nitrogen (g kg−1)||Microbial biomass C (μg kg−1)||Soil pH||Land-use history|
|Free-grazing grassland (GE0)||60.3 ± 20.6aa||13.41 ± 0.46a||1.43 ± 0.10a||47.52 ± 0.46a||8.17 ± 0.29a||Long-term free-grazing, good condition|
|4-year grazing exclusion (GE4)||162.3 ± 15.0b||15.95 ± 0.55b||1.60 ± 0.03a||42.69 ± 1.73ab||8.07 ± 0.11a||Grassland fenced since 2008, good condition|
|7-year grazing exclusion (GE7)||166.2 ± 13.3b||16.32 ± 2.06bc||1.64 ± 0.16a||38.06 ± 1.59b||7.92 ± 0.16ab||Grassland fenced since 2004, good condition|
|11-year grazing exclusion (GE11)||171.6 ± 9.6b||18.19 ± 0.49c||1.72 ± 0.10a||39.53 ± 1.89b||7.66 ± 0.19b||Grassland fenced since 1999, good condition|
|31-year grazing exclusion (GE31)||148.9 ± 41.3b||17.73 ± 2.18bc||1.48 ± 0.72a||40.02 ± 0.66b||7.19 ± 0.29c||Grassland fenced since 1979, good condition|
Field sampling was conducted in July 2011. In each plot, 4 sampling quadrats (1 m × 1 m) were established at 10-m intervals along a random transect. Aboveground biomass was clipped at ground level. Soil samples in the 0–20 cm soil layer were collected from 10 points in each quadrate. In the laboratory, we manually removed roots and visible organic debris from soil samples, and then measured soil water-holding capacity (WHC, %) and soil gravimetric moisture (%). Approximately 100 g of each soil samples was air-dried for analysis of soil properties (e.g., C, N, and pH). The remaining soil was stored at 4°C.
Laboratory incubation and analysis
The incubation experiment was conducted at six temperatures (0, 5, 10, 15, 20, and 25°C), using four substrates (control (CK), glucose (GLU), mixed grass leaf (GRA), and Medicago falcata leaf (MED). GRA and MED were collected from five plots and mixed evenly. The C concentration in GLU, GRA, and MED was 40.0%, 45.1%, and 44.0%, respectively, and the N concentration was 0%, 2.0%, and 5.2%, respectively. Thus, the N:C ratios of the substrates in increasing order were GLU (0) < GRA (0.043) < MED (0.117). We added 1% of the mass of the incubated soils as GLU, GRA, and MED, which approximated a 2-year input of new SOM from roots and litter in Inner Mongolian grasslands. In total, the incubation experiment comprised 480 samples, with 5 grassland types, 6 temperatures, 4 substrates, and 4 replicates for each treatment.
First, 40 g samples of fresh soils were put into incubation bottles, and the samples were adjusted to 60% WHC. Samples were incubated at 20°C and an instant 80% humidity for 4 days and then incubated at a treatment temperature (0, 5, 10, 15, 20, or 25°C) for 3 days prior to measurement of basal soil respiration. The substrates were subsequently added and mixed evenly. During the 56 days incubation experiment, soil respiration rates were measured 14 times, on days 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, and 56.
An automatic system for measuring soil respiration rates was developed through modification of the continuous gas-flow system reported by Cheng et al. (1993). The system consisted of a Li-COR CO2 analyzer (Li-7000), an electric water bath to control incubation temperature, an air-flow controller, soda-lime equipment to control the initial CO2 concentration, an auto-sampler on a turn-plate, automatic transformation valves to control the sample bottle, and a data collector (Fig. 1). In practice, the system was controlled by the data collector and first automatically lowered the CO2 concentration by using a bypass system of soda lime and then recorded the changes in CO2 concentration as it steadily increased. Soil respiration rates were calculated from the slope of the CO2 concentration as follows:
where R is soil respiration rate (μg CO2 g−1 h−1); C is the slope of the CO2 concentration; V is the volume of the incubation bottle and gas tube; m is the soil weight (g); α is the transformation coefficient of CO2 mass; and β is the transformation coefficient of time.
Figure 1. Schematic representation of the configuration of the automatic measurement system for soil microbial respiration.
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Soil organic C (SOC, %) was measured using a modified Mebius method (Nelson and Sommers 1982). Soil total N (%) was measured with a modified Kjeldahl wet-digestion procedure (Gallaher et al. 1976), using a 2300 Kjeltec Analyzer Unit (FOSS Tecator, Höganäs, Sweden). Soil pH was determined using a pH meter and a slurry of soil mixed with distilled water (1:2.5). Microbial biomass C (MBC) of fresh soil samples was analyzed using the fumigation – extraction method (Vance et al. 1987).
Calculations and statistical analysis
In this study, we selected the data from 1, 7, and 56 days incubations, respectively, to represent the instantaneous-term, short-term, and longer-term effects of the experimental treatments on soil C mineralization. Soil C mineralization at 20°C without substrate addition was used as the base soil respiration (Cmin-20°C).
The temperature sensitivity (Q10) of soil respiration in the 1- and 7-day incubations was calculated using the exponential equations, because 56-day incubation should be too longer to accurately evaluate Q10 due to the respiration rates declining faster at higher temperature with faster depletion of substrate.
where Y is the soil respiration rate (μgC g−1 h−1), T is the temperature (°C); A and B are the constants.
The stimulating effects (SEs) of soil respiration were then calculated to represent the sequestration capacity of grassland soils with fresh SOM input. SEs were calculated from the accumulated C mineralization under different substrates (GLU, GRA, or MED) divided by that of CK; higher values of SEs implied lower sequestration capacity.
One-way analysis of the variance (ANOVA) was used to investigate the differences in vegetation and soil properties among different grassland types. Univariate analysis of three factors (general linear model) was used to determine the effects of grassland types, incubation temperature, and substrates on soil C mineralization, Q10, and SEs. Logarithmic regression was used to identify the changing trend of Q10 and SEs with the duration of GE, MBC, and SOC. Data have been represented as means ± 1 standard deviation (n = 4). Differences were considered to be significant when P < 0.05. All analyses were conducted using SPSS statistical software (v. 13.0, SPSS, Chicago, IL, USA).