Determination of gross rates of amino acid production and immobilization in decomposing leaf litter by a novel 15N isotope pool dilution technique

Short-term carbon input increases microbial nitrogen demand, but not microbial nitrogen mining, in a set of boreal forest soils

“…Gross protein depolymerization rates were determined following Wanek et al (2010), as modified by Wild et al (2013). Duplicates of fresh soil were amended with a 2 lg ml -1 solution of 15 N-labeled amino acids that contained 12% lysine; 9% leucine and glutamic acid; 8% aspartic acid; 6% arginine and alanine; 5% glycine, proline, glutamine, and asparagine; 4% phenylalanine, serine, valine, and threonine; 3% isoleucine, tyrosine, cysteine, and tryptophane; and 1% histidine and methionine with 96-98 atom% 15 N (Cambridge Isotope Laboratories).…”

“…Samples were derivatized with ethyl-chloroformate (Wanek et al 2010) and analyzed with gas chromatography-mass spectrometry (GC-MS; Thermo Trace GC and DSQ I; OV1701 column, PTV injection, 1.4 ml min -1 helium as carrier, temperature program: 60°C for 1.5 min, first ramp 29°C min -1 to 130°C, second ramp 3°C min -1 to 180°C, third ramp 15°C min -1 to 280°C, 280°C for 5 min). One set of blanks and external amino acid standards (Standard Set 1; one concentration level) was processed together with the samples throughout the entire procedure to account for background concentrations of amino acids and for incomplete recovery of amino acids from the cation exchange cartridges.…”

“…A second set of external amino acid standards (Standard Set 2; eight concentration levels) was amended with the internal standards, derivatized, analyzed together with the samples, and used for calibration of alanine, glycine, leucine, proline, serine, valine, aspartate and asparagine concentrations. Asparagine and aspartate were not distinguished as formaldehyde causes the deamination of asparagine to aspartate (Wanek et al 2010). Concentrations of individual amino acids were corrected using recovery values calculated from Standard Set 1 (alanine: 71%, glycine: 73%, leucine: 27%, proline: 43%, serine: 93%, valine: 27%, asparagine and aspartate: 19%).…”

“…Concentrations of individual amino acids were corrected using recovery values calculated from Standard Set 1 (alanine: 71%, glycine: 73%, leucine: 27%, proline: 43%, serine: 93%, valine: 27%, asparagine and aspartate: 19%). Nitrogen isotopic compositions were calculated by comparing the peak areas of amino acid fragments containing 14 N or 15 N (Wanek et al 2010). Since this GC-MS based method of determining 15 N isotopic values is sensitive to 13 C enrichment such as in the samples amended with 13 C-labeled glucose, we conducted a parallel incubation to this treatment where we amended samples with glucose that was not enriched in 13 C. These non-enriched glucose-amended samples were treated identically to the enriched ones and used to determine gross rates of protein depolymerization and also ammonification without interference by 13 C. Gross ammonification rates were measured following Kaiser et al (2011), by amending duplicates of fresh soil with a 62.5 lM ( 15 NH 4 ) 2 SO 4 solution (25 atom% 15 N, Cambridge Isotope Laboratories; Histosols and Podzol organic layers: 1.0 ml for 2 g soil; Podzol mineral horizon: 0.5 ml for 4 g soil).…”

“…Furthermore, direct evidence linking increases in N-targeting enzyme activities and N mineralization rates to an accelerated breakdown of N-containing SOM polymers is missing. Methods for quantifying gross production rates of oligopeptides or amino sugars by polymer breakdown are lacking, and although a method for gross production rates of amino acids is available (Wanek et al 2010), it has rarely been applied. We are not aware of any study showing an adjustment of gross amino acid production by protein depolymerization to soil C or N availability.…”