Amino acids derivatization and analysis: Sediment slurries were centrifuged to separate the sediments from the medium and the sediments were freeze-dried and homogenized. 50 mg of samples spiked with the internal reference standard norleucine were decalcified and acid hydrolyzed (6M, 110℃) for 15 h. The hydrolysate was centrifuged to remove the sediments and subsequently defatted with hexane/ DCM mixture (V/V, 2:1) three times. The purification and derivatization of amino acids were performed by following the protocols described by Takano et al.53 and Chikaraishi et al.54, respectively. In brief, the hydrolysates were evaporated under N2 gas to dryness and purified using Dowex 50WX8 200 400 mesh cation exchange resin to eliminate the matrix effects. The purified amino acids were then isopropylated with a mixture of isopropanol and acetyl chloride (4/1, v/v) at 100 ℃ for 2 h. The solution was then evaporated to dryness and followed 3 times with DCM addition and evaporation to remove any remaining reagent. The AA isopropyl esters were then acylated using a mixture of pivaloyl chloride and DCM (1/1, v/v) at 100 ℃ for 2 h to obtain pivaloyl-isopropyl ester (Pv/AA/iPr). The Pv/AA/iPr solution was then evaporated to dryness and followed 3 times DCM addition and evaporation to remove any remaining reagent. Liquid-liquid extraction was performed by using MiliQ water and a hexane/DCM mixture (2/1, v/v). The Pv/AA/iPr were stored frozen (-20℃) and dissolved in ethyl acetate before analysis.
DIC measurement: an aliquot of the slurry was filtered with a 0.2-µm filter to remove cells and other particles. After filtration, 1 mL of samples were acidified with 100 μL 45% phosphoric acid overnight in an Exetainer® vial pre-purged with CO2-free air before analysis. All samples were measured with a Delta Ray Isotope Ratio Infrared Spectrometer (IRIS) with URI Connect and autosampler (Thermo Fisher Scientific, Germany) with an analytical error of ±1‰. All isotopic values are reported in the delta notation as δ13C relative to the Vienna PeeDee Belemnite (VPDB) standard. Deviations of triplicate isotopic measurement of sample DIC were between ± 1‰ and ± 1,000‰ (for DIC with label uptake of >10,000‰). DIC concentration was calculated from the released amount of CO2 by calibration with sodium hydrogen carbonate solution.
Incubation time was controlled based on the development of δ13C values of CO2 in the headspace. δ13C of CO2 was measured by Thermo Finnigan Trace GC coupled to a Thermo Finnigan Delta plus XP isotope ratio mass spectrometer (IRMS). Deviations of triplicate isotopic measurement of δ13C of CO2 were between ± 1‰ and ± 1,000‰ (for CO2 with label uptake of >10,000‰). Slurries were harvested at five time points for detailed analyses.
Fluxes of carbon (C) from soil to the atmosphere and of nitrogen (N) to the plants are mainly driven by turnover of soil organic matter (SOM). The release of organic substances by living roots into the soil (rhizodeposition) and the stimulation of microbial activity can provoke changes in the rate of SOM-decomposition termed 'rhizosphere priming effects' (RPE). The aim of this project is to provide comprehensive understanding of RPE by assessing factors, mechanisms, intensities and dynamics of RPE in field and laboratory experiments and by investigating the relevance of RPE for agroecosystems. Two main factors significantly affecting RPE will be intensively studied: the amount of the primer and the mineral N status of the soil. By combining various isotopic approaches (13C natural abundance, 15N labeling and 15N dilution) we will simultaneously trace C and N derived from SOM-decomposition in the field, thus, improving the understanding of priming effects at field scale. The combination of 13C continuous labeling with a high-resolution d13C analysis of soil CO2 will provide short term dynamics of RPE and will allow to determine their dependence on plant growth stages and associated therewith, on root properties. Digital images of the distribution of exudates analyzed by 14C imaging and enzyme activities along single root segments (by zymography) will allow to study spatial dynamics of RPE. Furthermore, a new approach for estimating microbial groups involved in RPE will be tested. Based on the mechanisms of priming effects the functioning of C and N turnover in rhizosphere will be clarified.