2.3 Priming experiment and CO2 analysis
A 32-day laboratory incubation experiment was conducted at 20 °C in the dark to determine SOC mineralization and microbial C dynamics with or without glucose/vanillin amendment (n=5). For each microcosm, moist soil (equivalent to 70 g dry mass) sieved through 2-mm was placed in a 500-ml glass flask and pre-incubated at 20 °C in the dark for one week. Subsequently, either a water solution with 13C labeled glucose or vanillin, representing easily degraded and recalcitrant C substrates, respectively, (Sigma-Aldrich, uniformly labeled, 99 atom %, 89.4 µg C g-1 dry soil) or distilled water were sprayed onto the soil to reach a moisture content equivalent to 60% of its water holding capacity (WHC). Soil moisture was maintained at this level by weighing and spraying with distilled water regularly.
CO2 concentrations were determined at 1, 2, 3, 4, 5, 7, 10, 14, 19, 22, 25, 32 days after the treatment application started. To differentiate SOC- and glucose or vanillin-derived CO2, the δ13C value of respired CO2 was measured with an Isotope Analyzer (Picarro TOC-CRDS, US) after dilution with a standard CO2 gas of known concentration and isotopic composition. The δ13C abundance of respired CO2 was corrected for the added CO2 gas using a mass balance approach. At the end of 32-day incubation, soils were extracted to determine the MBC and the amount of13C derived from the added substrate that had been incorporated into microbial biomass. The extraction was performed using the chloroform fumigation extraction method and the isotopic composition was determined using an Isotope Analyzer (Picarro TOC-CRDS, US), as described in our previous studies by Li et al. (2014) and Li et al. (2018a).
The priming effect (PE) induced by substrate (glucose or vanillin) amendment (ug C g-1 soil) was calculated as described by Qiao et al. (2014):
PE= CO2Total - CO2substrate - CO2No substrate
Where CO2Total, CO2substrate, CO2No substrate were the total CO2evolved from soil with glucose or vanillin amendment, CO2 evolved from glucose or vanillin, and CO2 evolved from soil without glucose or vanillin amendment, respectively. The cumulative PE, substrate decomposition, and SOC decomposition was the sum of each corresponding index measured during the incubation period.
Net C balance (NCB) representing C accumulation was calculated as:
NCB = Cin- Csoc-Csubstrate
Where Cin, Csoc, and Csubstrate were substrate-C inputting to the soil, SOC-C loss, and substrate-C loss, respectively.
Microbial CUE was calculated as:
CUE= MB13C / (MB13C +13CO2)
Where MB13C is the amount of substrate C incorporated into the microbial biomass and 13CO2is the substrate-C respired as CO2. The δ13C of total microbial biomass (δ13CMBC) was determined using the following equation as described by Blagodatskaya et al. (2014):
δ13CMBC = (δ13Cf .Cf - δ13Cnf .Cnf) / (Cf - Cnf)
Where δ13Cf and δ13Cnf were the δ13C values of the 24-h chloroform fumigated and non-fumigated samples, respectively, and Cf and Cnf were the amounts of C in the fumigated and non-fumigated K2SO4 samples, respectively.
2.4 Statistical analysis
The influence of N and P addition on the soil properties, cumulative PEs, glucose or vanillin mineralization, SOC mineralization, NCB, and microbial CUE were analyzed with a two-way MANOVA. When there were significant interactions between the addition of N and P, one-way ANOVA’s was used to test the effect of nutrient addition treatment on soil properties, using a Tukey HSD post hoc test (p < 0.05). Linear or quadratic regression models were fitted to describe the relationships between C mineralization, microbial CUE and stoichiometric characteristics of soil and microorganisms. Statistical tests were performed in SPSS 16.0. Graphs were plotted using Sigma Plot 12.5.