Hydrolyzable N pool size and 15N atom % of natural and enriched soils collected from Imnavait watershed in summer of 2005.
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Soil sampling and extraction
Four randomly-located soil cores (diameter range: 5-6cm) were collected from each 15N addition plot after 2 yr (16 and 20 Jul 2005). At each collection, cores were taken to the maximum possible depth, defined as either the full core length (40 cm), the bottom of the seasonally thawed “active layer”, or to a rock (at Crest only). Upon collection, the length of the core was measured and the core was separated in the field into the following layers by cutting with a knife on a clean plastic sheet:
(1) Green Layer, the surface live moss-detritus-plant layer including lichens, graminoid foliage < 2 cm above the ground, and groundcover species (e.g., Vaccinium vitis-idaea, Empetrum nigrum, forbs).
(2) 2nd layer or 1st Organic Layer, the upper ~10 cm of organic soil beneath Green layer
Within 12 hrs of collection, soil samples were weighed, homogenized, and live roots and rocks hand-picked. Subsamples were dried at 50 ºC to determine moisture content. Subsamples of the 1st Organic Layer was chemically fractionated by acid hydrolysis. To obtain natural 15N abundance for these fractions, three additional soil cores were collected from outside of the plots at six topolocations (CR, MS-WT, MS_NWT, FS_WT, FS_NWT, and R), processed as described above, then composited by the topolocations (total number of subsamples =6). All soils were kept frozen until analysis.
Pool size and d15N signature of hydrolyzable-N pools
The pool size and % 15N atom excess (determined as a difference in atom% between natural abundance soil and 15N added soil) were determined by chemically fractionating soil N into three hydrolysable labile-N pools; hydrolysable NH4+ (HNH4+), amino acids (HAA), and amino sugars (HAS).
Soils were thawed and five replicates (2–10 g) of ground samples were hydrolyzed in hot 6 N HCl for 12 h under reflux according
to Mulvaney and Khan (2001). The hydrolysates were neutralized with NaOH (Mulvaney and Khan 2001). Hydrolysable NH4+ was determined by a hypochlorite-alkaline phenol method. To determine d15N in this pool, aliquots (~10 mmol-N) of the hydrolysates were diffused in a 450-mL Mason jar with MgO at 50 °C for 6 hr onto an acid trap according to Mulvaney and Khan (2001), but replacing their “wet” acid trap (a beaker containing 5 mL of 4% H3BO3) with a “dry” acid trap (an acidified GF/D filter disc encapsulated in Teflon tape) so that N on the trap could be directly analyzed for d15N by isotope ratio mass spectrometry.
Concentration and d15N of hydrolysable amino sugar and amino acid were determined by a sequential diffusion of the neutralized hydrolysates. For concentration, the hydrolysate was diffused for 8 hrs into a wet acid trap, then re-diffused with a new wet acid trap for 6 hrs after converting amino acids to NH4+ by a ninhydrin reaction under an acidic condition (Mulvaney and Khan 2001). Both first (HAS) and second (HAA) acid traps were analyzed for NH4+concentration as for hydrolysable ammonium.
To determine d15N of the HAS and HAA pools, aliquots of the hydrolysate (~10 mmol-N) were sequentially diffused with dry acid traps in place of wet traps. Because total soil N is a sum of hydrolysable-labile N (HNH4+, HAS, and HAA) and non-hydrolysable-labile N (non-HLN), pool sizes and d15N signatures of non-HLN fraction was calculated by mass balance of known N pools (total soil N, HNH4+, HAS, and HAA).
** Mulvaney, R. L., and S. A. Khan. 2001. Diffusion methods to determine different forms of nitrogen in soil hydrolysates. Soil Science Society of America Journal 65: 1284-1292.
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