Permafrost

egions can also arise. For the seasonally frozen soil, soil freezing and thawing is known to be
one of the significant events that cause high N2O emission from soil, together with after the
rainfall, compaction and application of N fertilization in arable land. N2O is a climate relevant
trace gas and produced in soils mainly through microbial N transformation processes as a
byproduct and an intermediate product during nitrification and denitrification, respectively.
Denitrification is considered to be highly involved in high N2O emission on seasonally frozen
soil. Field observations suggest that the N2O emission occurs under not frozen but thawing or
diurnal freeze-thaw conditions. Among them, high N2O emission under thawing condition has
been interpreted as a release of accumulated N2O that was entrapped by the frozen surface layer.
However, the origin of the accumulated N2O remained uncertain, whether it was produced in
and diffused from the unfrozen subsurface layer or produced in the frozen surface layer. In our
previous field observations, the highest N2O concentration was observed in the frozen surface
layer, rather than below the frozen surface layer. Therefore, we conducted a laboratory
incubation experiment to determine which part (surface or subsurface) is more responsible for
the production of N2O under the frozen condition of surface soil. Soil samples were collected
from two depths (0-10 cm, 50-70 cm) of three different arable fields (S4, W4S, W8) in the
Shizunai Experimental Livestock farm in Southern Hokkaido, Japan, on May 2005. A sieved (4
mm) and field moist soil sample (7 to 15 g) was placed in eight pieces of test tubes sealed with
rubber stoppers and incubated at +4 . Four of them were subjected to freeze (-3 )-thaw (+4 )
cycle and the others were kept at constant temperature of +4 as unfrozen control. Changes in
N2O concentration in the headspace of the test tubes were monitored throughout the incubation,
which was divided into five periods: initial (+4 ), freezing (+4 to -3 ), frozen (-3 ),
thawing (-3 to +4 ) and thawed (+4 ). Apparent N2O production was not observed in two
subsurface soil samples (W4S and W8) in spite of the incubation temperature, while it was
observed in the other one subsurface (S4) and all three surface soil samples. Two surface soil
samples (S4, W8) produced N2O even in the frozen period, but the N2O production rate
decreased to 56 and 64% of that of each unfrozen control, respectively. In one surface soil
sample (W4S), which had the highest N2O production rate among the samples in the unfrozen
control, the N2O production rate was 13 to 26 times higher in the frozen period than that in the
unfrozen control. In one subsurface soil sample (S4), the N2O production rate decreased to 38%
of that of the unfrozen control in the frozen period. Moreover, negative N2O production
(consumption) was observed in the thawing period, while the unfrozen control showed apparent
N2O production. Collectively, our observation indicated that soil freezing may not terminate
N2O production in surface soils, suggesting that N2O can accumulate in surface frozen soil
layer depending on the condition of the ground surface. In addition, since denitrification is a
sequential reductive reaction from NO3 - to NO2 - , NO, N2O and to N2, the accumulation of N2O
in any given closed space depends on enzymatic activity associated with N2O reduction. Thus,
both apparent N2O consumption and significant increase in apparent N2O production suggested
that denitrification process may be stimulated under frozen condition of soil, in which the ice
formation creates O2 depleted condition into microsite of soil and unfrozen water film on soil
surface that accumulates substrates (NO3 - and dissolved organic C) for denitrifying
communities.
Key words: Trace gas, denitrification, unfrozen water, ice formation, anoxic microsite
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