In July 2011, soil samples were collected from control, and N+P plots from within a set of treatments in Moist Acidic Tundra plots established in 1989. A set of Control and N+P plots established on an adjacent hillslope in 2006 with both equivalent (high) and half (low) the fertilization rate of the 1989 plots were also sampled. At the time of sample collection we separated the soil into organic horizon, organic/mineral interface, and the upper 5cm of the mineral soil, and measured the depth of each layer. Field-wet mineral soil samples were broken up along fault lines and pushed through an 8 mm sieve; rocks (> 2 mm in size), live roots, and dead organic matter were removed, and what remained was air-dried. Soils were then transported to the Natural Resource Ecology Laboratory at Colorado State University for further fractionation and laboratory analysis. The sieved air-dried soils were separated into the following four water-stable aggregate size classes using techniques developed by Elliott (1986) and modified by Six et al. (1998): large (>2000 mm) and small (250-2000 mm) macroaggregates, microaggregates (53-250 mm), and a silt + clay fraction (<53 mm). Total organic carbon and nitrogen of all aggregate fractions were quantified by dry combustion using a Leco TruSpec CN analyzer (Leco Corporation, St. Joseph, Michigan). Microaggregates contained within small macroaggregates (250-2000 mm) were isolated using the method of Six et al. (2000). A 10-g subsample was placed on top of a 250-mm sieve along with fifty glass beads (4 mm diam.), shaken on a reciprocal shaker so that the macroaggregates were broken up with the aid of the beads, and washed onto a 53-mm sieve. This separation yields the following three fractions: the material remaining on the 250-mm sieve was considered to be coarse POM; aggregates passing through the 250-mm sieve but retained on the 53-mm sieve are considered occluded microaggregates isolated from macroaggregates; and material passing through the 53-mm sieve is considered clay and silt particles not associated with stable microaggregates. Occluded microaggregates were dried (65˚C), weighed, finely ground, and analyzed for total organic carbon content by dry combustion using a Leco TruSpec CN analyzer (Leco Corporation, St. Joseph, Michigan). Density separations were completed for small macroaggregates (250-2000 mm). Sodium polytungstate (SPT) was used to create a high-density solution to separate light from heavy fractions (HF) using the method of Six et al. (1998). Prior to each LF/HF density separation, aggregate fractions were dried (60ºC) then cooled to room temperature in a desiccator. Sub-samples (5 g) were added to 50-ml centrifuge tubes containing 25 ml of SPT at a density of 1.85 g cm-3, gently mixed in order to avoid aggregate disruption, topped off to 40 ml with SPT, and placed under vacuum (138 kPa) to remove air trapped within aggregates. Tubes were then centrifuged (1250 g) for 60 min to separate light from heavy fractions. Floating material, which comprises the LF, was aspirated onto a 20mm nylon filter and rinsed. Samples were dried, weighed, and analyzed for carbon content using a Carlo Erba (Milan, Italy). The silt and clay fraction was separated from sand by dispersing the HF with sodium hexametaphosphate (0.5% solution by weight), then passing the dispersed fraction through a 53 mm sieve. The carbon content of the <53mm fraction was quantified by dry combustion using a Leco TruSpec CN analyzer (Leco Corporation, St. Joseph, Michigan).

References

Elliott ET (1986) Aggregate structure and carbon, nitrogen, and phosphorus in native and cultivated soils. Soil Science Society of America Journal 50, 627-633.
Six J, Elliott ET, Paustian K, Doran JW (1998) Aggregation and soil organic matter accumulation in cultivated and native grassland soils. Soil Science Society of America Journal 62, 1367-1377.
Six J, Elliott ET, Paustian K (2000) Soil macroaggregate turnover and microaggregate formation: a mechanism for C sequestration under no-tillage agriculture. Soil Biology and Biochemistry 32, 2099-2103.