Soil Extractable C and N Pools

For measurements of extractable C and N pools fresh soils (aliquots of 4 g) were extracted for 30 min with 30 ml 0.5 M K₂SO₄, subsequently filtered through ash-free cellulose filters and extracts kept frozen at −20°C for later analyses. Total dissolved N and dissolved organic carbon (measured as non-purgeable organic carbon) were measured by high temperature catalytic oxidation on a Shimadzu TOC-VCPH with TNM-1 unit and ASI-V autosampler (Shimadzu Austria, Korneuburg, Austria). Ammonium and nitrate were determined with colorimetric methods, NH₄⁺ with a modified indophenol reaction and NO₃⁻ with the VCl₃/Griess assay, as described in detail by Hood et al. [88] . Amino acid concentrations were quantified by a modified fluorometric OPAME procedure based on Jones et al. [89] , optimized for free amino acid measurement in protein hydrolysates. The modified OPAME reagent was made up of 10 mg o-phthaldialdehyde dissolved in 1 ml of HPLC grade methanol to which 20 µl of 3-mercaptopropionic acid was added. This reagent was mixed with 40 ml potassium borate buffer (0.2 M; pH 9.5) and left to stand overnight. Sample aliquots (50 µl) were mixed with 200 µl reagent in the microtiter plate and measured after 10 min at an excitation wavelength of 340 nm and emission wavelength of 450 nm with a fluorescence microplate reader (Tecan Infinite M200, Tecan Austria GmbH, Grödig, Austria). As NH₄⁺ in the soil extracts also gives a fluorescence signal, NH₄⁺ concentration had to be determined [88] and its fluorescence contribution was determined via a NH₄⁺ concentration series and subtracted from the total fluorescence of the samples. Total free amino acid-N concentration was then calculated using glycine as a standard and assuming that each mol of amino acid contains 1.37 mol N. Proteins were quantified in K₂SO₄ extracts by acid hydrolysis and subsequent amino acid analysis (modification of [89] –[90] ; see amino acid determination above). Protein hydrolysis to free amino acids was performed in 6 M HCl and heating was performed for 15 hours at 100°C in a drying oven with Bovine serum albumin (BSA containing 15.8% N) as internal standard. Samples were then dried in an N₂ stream and redissolved in deionized water. Determination of the NH₄⁺ concentration to correct the amino acid fluorescence was done as in the case of amino acid determination, with the exception that the sodium nitroprusside solution (the color reagent) contained 1.5 M NaOH instead of the original 0.3 M NaOH to neutralize residual acid in the hydrolysates.