In the Freeze 1 project, samples were collected three times each year
(May, August and Octobler), to correspond with key plant phenological
stages: pre-green, peak green, and senescence. For the Freeze 2
project soils were first sampled in June 2002 (prior to freezing
treatment). Frozen soil samples were obtained in February 2003.
Subsequent sampling occurred during snowmelt and summer of 2003 and
2004.
Samples were obtained using a split-PVC corer. In this method, a 5 cm
diameter split PVC corer is used to take all samples. A split PVC
corer consists of a piece of 2 inch (5 cm) PVC pipe, about 15-20 cm
long, split lengthwise on both sides. The corer is actually in two
pieces. We put the corer together along the cuts, and duct-tape one
side -- the "hinge" side. Holding the corer firmly together,
we hammer it 10-15cm into the ground. The corer is removed and then
opened with the intact soil core inside. The exception to this was the
winter 2003 samples which were obtained using a diamond tip sampling
auger. The soil is split into two layers, organic and mineral
horizons. The organic samples were comprised of the Oi, Oe and Oa
horizons. Mineral samples generally consist of the top 10 cm of
mineral soil beginning with the A horizon. Each horizon is measured
and placed into a sample bag. We typically collect 2-8 cores per plot,
compositing all cores by horizon.
LABORATORY PROCEDURES
Samples were stored at 4o C between sampling and analysis (from less
than 1 week to up to three weeks). Soils were manually homogenized:
all large rocks, roots, and other non-decomposed organic material were
removed, and samples were thoroughly mixed. No more than three minutes
were spent homogenizing any sample. 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.
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), dextrose or 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 Robertson
et al. (1999).
Soil pH was measured by weighing 10 g of soil at field moisture into a
small beaker. 20 ml of deionized water was added and swirled to make a
slurry. After 30 minutes the pH of the slurry was measured using a pH
meter calibrated using pH 4 and pH 7 buffers.
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.
REFERENCES
Jenkinson, D. S., and D. S. Powlson. 1976. "The effects of
biocidal treatments on metabolism in soil – V: A method for measuring
soil biomass. Soil Biology & Biochemistry 8:209-213.
Robertson, G.P., C.S. Bledsoe, D.C. Coleman and P. Sollins, editors.
1999. Standard Soil Methods for Long Term Ecological Research. Oxford
University Press, New York.
Smith, M.S., and J. M. Tiedje. 1979. Phases of denitrification
following oxygen depletion in soil. Soil Biology & Biochemistry
11:262-267.
