SAMPLING DESIGN
Four 10x10 cm samples of organic soil were collected from each plot
1-6 (n= 24), with one sample from each quadrant. Based on the quadrant
from which a sample was removed, it was assigned a "Sample
ID" (X1-X4, where X = Plot and the second digit refers to the
quadrant that was sampled). These "Sample IDs” align with frost
tube, snow depth, and soil respiration data.
A 10x10 cm cardboard template was used to determine proper size, and a
soil knife was used to carefully cut around the template. A trowel was
then used to gently lift the organic layer out of the soil. The entire
organic layer was collected, regardless of depth (depth was measured
on each side of the removed section of organic soil and these four
values were averaged). Sample collection took place between 430 and
530 PM. All samples were sealed in a plastic bag and stored on ice
overnight.
Samples were processed the following day in the lab at Boston
University from 9am - noon. Roots were picked from each sample during
initial stages of soil seiving (8 mm mesh) and homogenization. Roots
were removed for further analysis and seived soil was subsampled for
soil moisture and enzyme activity. These samples were shipped to the
Groffman Lab at the Cary Institute for Inorganic N and microbial N and
C analysis.
LABORATORY PROCEDURES
All samples were held at field moisture before analysis. Soil water
content was determined gravimetrically.
Microbial biomass C and N content was measured using the chloroform
fumigation-incubation method (Jenkinson and Powlson 1976). Soils were
fumigated to kill and lyse microbial cells in the sample. The
fumigated sample was inoculated with fresh soil and sealed in a jar,
and microorganisms from the fresh soil grew vigorously using the
killed cells as substrate. The flushes of carbon dioxide
(CO2) and 2 M KCl extractable inorganic N
(NH4
+ and
NO3
-) released by
the actively growing cells during a 10-day incubation at field
moisture content were assumed to be directly proportional to the
amount of C and N in the microbial biomass of the original sample. A
proportionality constant (0.41) was used to calculate biomass C from
the CO2 flush in the fumigated samples. Biomass
N is the total inorganic N flush in the fumigated samples.
Inorganic N and CO2 production were also
measured in "control" samples. Control samples were prepared
in the same fashion as those listed above, but were not fumigated.
These incubations provided estimates of microbial respiration and
potential net N mineralization and nitrification. Microbial
respiration was quantified from the amount of
CO2 evolved over the 10-day incubation.
Potential net N mineralization and nitrification were quantified from
the accumulation of
NH4
+ plus
NO3
- and
NO3
- alone during
the 10-day incubation. We measured 2 M KCl extractable inorganic N in
the fresh soil samples to determine the initial soil
NO3
- and
NH4
+ concentrations.
Carbon dioxide was measured by thermal conductivity gas
chromatography. Inorganic N was measured colorometerically using an
autoanalyzer (Lachat Quikchem 8000).
Denitrification enzyme activity was measured using the short-term
anaerobic assay described by Smith and Tiedje (1979). Sieved soils
were amended with
NO3
- (100 mg N
kg-1), glucose (40 mg
kg-1), chloramphenicol (10 mg
kg-1) and acetylene (10 kPa) and were
incubated under anaerobic conditions for 90 minutes. Gas samples were
taken at 30 and 90 minutes, stored in evacuated glass tubes and
analyzed for N2O by electron capture gas
chromatography. For more information on any of the methods described
above, refer to Standard Soil Methods for Long-Term Ecological
Research (1999).
CALCULATIONS
All results are expressed on a per gram of dry soil basis. Values can
be converted to a “per area” basis using data on the mass of different
soil horizons found elsewhere on the data page of this website.
