ῐ
J. Jpn. Soc. Soil Phys.
ῐ῎῍ῑῒ῏
No. +*., p. +-ῌ,0 ῍,**0῎

Microbial Transformation of Nitrogen in Cattle Slurryas Applied to an Andisol Grassland
Chitoshi MIZOTA*, Yoko YAMAGUCHI** and Kosuke NOBORIO****, ** Faculty of Agriculture, Iwate University, Ueda -ῌ+2ῌ2, Morioka, Iwate *,*ῌ2//**** School of Agriculture, Meiji University, Kawaski, Kanagawa, Japan
Abstract
Organic nitrogen compounds in the animal waste once applied to soil is followed by chemical
modiﬁcation. Microbial transformation of nitrogen in cattle slurry applied to an Andisol grassland with no-cattle grazing over ,* years in northeastern Japan was analyzed with a focus on
temporal changes in chemical composition (ammonium- and nitrate-nitrogen) and the naturalabundance of nitrogen isotopes, d +/N. Most urea and related metabolites, which were majororganic constituents of nitrogen in cattle excreta, were hydrolyzed into ammonium-nitrogen
during storage in an underground closed tank. Nitriﬁcation of the ammonium-nitrogen waslargely delayed under lower soil temperature conditions with average air temperature (.ῌ+.ῌ),
whereas rapid nitriﬁcation occurred under higher air temperature conditions (+1ῌ,1ῌ). A coupledprocess of denitriﬁcation followed by nitriﬁcation was evidenced by a rapid decrease in the nitratecontent with a simultaneous increase in the d +/N values of the residual. Such a process was
facilitated by a combination of the high water retention property of the Andisol with an ample
supply of organic matter.
Key words : cattle slurry, volatilization of NH-, nitriﬁcation, denitriﬁcation, fractionation of nitrogen isotopes

Dairy farming is one of the most important
agricultural industries in the intra-mountainous regions of northeastern Japan, where
Andisol predominates. Cattles have been fed
under housing in a narrow space. In order tosave costly labor, cattle slurry originating fromcattle housing has periodically applied to farmland. Our current concern is the fate of theslurry nitrogen, in particular with respect tothe nitrate contamination of local groundwater.
After the application onto the soil surface,urea, organic metabolites and sometimes ammonium, the major constituents of the slurry,
are subjected to volatilization, mineralization,
nitriﬁcation, denitriﬁcation and ﬁnally leaching, depending on the prevailing soil condi-

tions (Stevenson, +32, ; Aulakh et al., +33,).
Natural variation of d +/N values (and d +2O ofnitrate) in combination with changes in theconcentration of mineral nitrogen would provide direct evidence of a relevant process insoil where diverse microorganisms are involved (Robinson and Conroy, +333). A numberof studies have been conducted on searching
the origin of nitrate in groundwater using
stable isotope technology (Fukada et al., ,**-,and herein references). Nevertheless, detailed
information on the process of microbial transformation that proceeds the formation of nitrate in soil is very limited. Such data are a
prerequisite for elucidating the origin of nitrate in local groundwater.
In humid and cool-to-temperate climatic con-

* Corresponding author : Phone-Fax : *+3ῌ0,+ῌ0+,0, e-mail : mizota@iwate-u.ac.jp῏  +*.

14

ditions, as common in northeastern Japan, thesoil surface is subjected to seasonal ﬂuctuations of temperature and moisture. Such ﬂuctuations would control the fate of slurry nitrogen applied to soil. The objective of the present study is to elucidate the detailed mechanisms involved in the microbial transformation of nitrogen in an Andisol ﬁeld appliedwith cattle slurry, using stable nitrogen isotopic signatures as a tracer.
+.

Materials and Methods+. + Experimental design
A trial ﬁeld (-3.2ῌN, +.+*0ῌE, from -+*ῌ-,*m above sea level), which had a history ofcontinuous application of cattle slurry (exact
rate of nitrogen application could not be estimated due to many variable factors) since+312, was selected from an Andisol located on
the eastern foot slope of Mt. Iwate, Iwate,
northeastern Japan. An outline of the ﬁeld isdocumented by Noborio et al. (,**,). The experimental ﬁeld is a managed pasture. There
has been no cattle-grazing for years. From late
December to late March the soil and vegetation
are covered by deep snow, up to *./ m deep.

Fig. +,**0

The soils are derived from weathering of cumulative pyroclastic deposits ejected from Mt.
Iwate during the Holocene, and are characterized by the predominance of allophone, imogolite, and aluminum-humus complex.
To separate the coarse fecal fragments, amixture of urine and dung deposited on the
housing ﬂoor is mechanically squeezed before
transporting to storage. The resulting cattle
slurry (separated ﬂuid) collected in the underground tank, together with the relatively fresh
urine in the drain channel was periodicallysampled and analyzed for both concentrationand d +/N values of ammonium- and total nitrogen.
Six sampling wells (diameter : 1* mm) were
installed by a hand-auger down to the level ofgroundwater. The geometric layout of thesampling wells is shown in Fig. +. Water ﬂows
constantly from a spring located at the lower
end of the trial ﬁeld. Water samples from thegroundwater and from the spring were periodically collected and analyzed. To obtain areference of natural nitrate derived from precipitation, a sample of spring water virtually
free of human-inﬂuence was collected on Au-

Location map. Numbers indicate the location of sampling wells.

῍ῌ : Microbial Transformation of Nitrogen in Cattle Slurry as Applied to an Andisol Grassland 15

gust -*, ,**. at the crater rim of Mt. Iwate(elevation : +,12* m above the sea-level), andsimilarly analyzed.
Reed canary grass (Phalaris arundinacea) was
the predominant vegetation in the trial ﬁeld.
The matured above-ground parts (leaves together with stem) are cut down three times a
year, in June, August and October, and airdried in situ to make hay. In early spring andafter harvesting each hay, cattle slurry fromthe underground storage tank is applied by amanure sprayer, as is common in elsewhere(Sommer and Hutchings, ,**+). Since excretion, the cattle slurry has been stored in theunderground tanks for at least two months
before application. Samples of the slurry to beanalyzed for chemical characterization werecollected just before application.
Soil temperature and moisture that control
the fate of nitrogen applied to the soil ﬂu-

Fig. ,

ctuated during the year, since the trial ﬁeld
was located at the mid-latitude of the Northern
Hemisphere where slightly cool to temperate
climates alternated. The mean daily ﬂuctuation of air temperature and precipitation during the relevant period of the ﬁeld trial in ,**.
is shown in Fig. ,. Soil temperature conditions
were categorized into two major groups : alower soil temperature period in April and October/November, and a higher temperature
period in June and August (Fig. ,).
+. , Sampling surface soils after applying
cattle slurry
Seven replicates of soil sampling in the ﬁeld
were made after April ,**., covering middle tolower portions of the slope in the trial ﬁeld (Fig.
+). After removing the decaying plant residues,
surface soil samples including the dense root
mat of reed canary grass from * to / cm deep
were collected around the trial wells in the ﬁeld

Daily precipitation and average air temperature in relation to the application of cattle slurry and subsequent sampling of surface soil.

16



(Fig. +). Temporal stagnation of the surface
water was not observed during soil sampling.
The samples were brought back to a laboratory
within + h of sampling, and passed through a ,mm sieve to eliminate grass root by gentle
crushing and sieving.
+. - Sampling grain corn and fresh urine
Grain corn, which is imported from theUnited States of America, constitutes major
feed for housing cattle in Japan. A composite
sample of dry grain corn imported in +323 wascrush and subjected to d +/N analysis. Freshurine samples were collected by syringe fromhousing ﬂoor approximately within two hours
after excrement and placed in a cool container
and then quickly brought to laboratory forchemical analysis.
+. . Analysis of feed, soil, water, and slurry samples
The analytical procedures for determinationand preparation for d +/N determination formineral nitrogen (ammonium and nitrate) weredeveloped in the present study. Fifty g of the
fresh soil sample was shaken with +,/ ml of , M
KCl solution for -* min at +/ and stood overnight at /. Clear supernatant solution was
obtained by centrifugation (.,*** rpm, -* min).
The concentration of ammonium- and nitratenitrogen in the soil extract was determined bysemi-micro Kjeldahl distillation apparatus.
Both ammonium- and nitrate-nitrogen were
ﬁnally converted into ammonium-tetraphenylborate derivates, and the +/N/ +.N ratios were
determined using a continuous-ﬂow mass spectrometer (DELTA plus, Thermoquest Ltd.).
The nitrogen isotopic composition was shownby common d +/N notation, as per mil variation
relative to atmospheric dinitrogen (d +/N*).
Analytical precision during the overall process
is better than*.,. The analytical accuracy
during the isolation of sample nitrogen andsubsequent mass spectrometry was repeatedly
monitored using reagent grade NH.Cl and
KNO-.
Soil moisture was determined gravimetrically by drying in an oven with constant tem-+*.

,**0

perature (+*/) and expressed as a percentageof the maximum water holding capacity (ῌ).
Water samples were passed through a membrane ﬁlter (Toyo Roshi Co., Ltd.) with a pore
diameter of *., mm and concentrated to about/* ml on an electric heater. Concentration andpreparation of borate derivatives for d +/N determinations of the nitrate- and ammoniumnitrogen was carried out in the same way as
described above for soil extracts.
Total nitrogen in samples of cattle slurry andrelatively fresh urine was converted into ammonium-nitrogen with a procedure of Kjeldahl
digestion. Ammonium-nitrogen in the extract,
together with that in cattle slurry and urine
samples was determined by the same procedure as described above.
,.

Results and Discussion

Concentration and d +/N values of nitrogen in cattle slurry
Two fresh urine samples within two h after
excrement contained 1,0** to 2,.3* mg L+ and
00* to +,,** mg L+ of total and ammoniumnitrogen (Table +), respectively. The analyticalresults indicated that the major form of nitrogen in relatively fresh urine was organic, probably in form of urea (Kreitler, +31/).
The concentration of ammonium-nitrogen in
analyzed samples of the cattle slurry ranged
from -,+.* to 1,30* mg L+, whereas that of
total-nitrogen ranged from -,-,* to 1,/** mg L+(Table +). Urea is the major nitrogen constituent in metabolic excreta of higher animals, including cattle. Except for one slurry sample
collected on May ++, ,**., the concentration ofammonium-nitrogen was nearly the same as
total nitrogen, indicating microbial hydrolysis
of urea excreted from cattle occurred during
the storage, as commonly observed in elsewhere (Sommer and Hutchings, ,**+). Cattle
slurry was stored for two months or more
before application and most of the excreted
urea would be hydrolyzed to ammoniumnitrogen, since the slurry used in the present
study was transparent without virtually solid,. +ΐῒ : Microbial Transformation of Nitrogen in Cattle Slurry as Applied to an Andisol Grassland 17

Table +

Concentration and nitrogen isotopic composition of ammonium- and total-nitrogen in samples of cattle slurry and urine

Date of sampling

Concentration
NH.-nitrogen
Total nitrogen

d +/N
NH.-nitrogen
Total nitrogen

October ,2, ,**May ++, ,**.
July +., ,**.
July ,3, ,**.
October +-, ,**.
November ,,, ,**.
January +/, ,**/
February ,, ,**/ῐ
February 2, ,**/
February 2, ,**/ῐ῍῍῍῍ N-mg L῎+ ῍῍῍῍
NDῑ
.52.*
-5+.*
15/**
/5-2*
/500*
.501*
.50**
-5/,*
-5-,*
/530*
/510*
15+0*
051**
+5,**
150**
1530*
0530*
00*
25.3*

῍῍῍῍ ῏ ῍῍῍῍
NDῑ
῍,4.
῍*4,
῍*4+
NDῑ
῍*40
NDῑ
῍+4,
῎*42
NDῑ
ῑ
ND
NDῑ
ῑ
ND
῎+4*
῍+4.
῍+4/
NDῑ
῎+4.
NDῑ
῎,40

ῐ

Relatively fresh urine samples collected from a drain gutter.
Not determined.
pH of the cattle slurry : 3.,ῌ+*.+.
ῑ

constituents. The ammonium-nitrogen content of the slurry samples collected in January,
October, and November exceeded that of totalnitrogen, reﬂecting the analytical errors associated with the heterogeneous nature of theslurry. The low rate of mineralization of urea
into ammonium-nitrogen of a sample collected
on May ++, ,**. was probably resulted from a
relatively lower temperature (from late December to April) during the storage of the cattle
slurry in the underground tank.
The d +/N values of total nitrogen in relatively fresh urine and cattle slurry ﬂuctuatedwithin fairly narrow ranges from ῎,.0 to ῍,..
῏ (Table +). As it is well known, the major partof the current high performance feeds for cattle
housing in the Japanese dairy farming system
is imported grain corn produced in the central
northeast United States. The d +/N values of theanhydrous ammonia and urea, which are major
chemical forms of nitrogen fertilizers in thearea, range from ῎/ to ῍-῏ (Panno et al.,,**+). The d +/N value of a composite grain corn
imported from the U.S.A. in +323 was ῍..3῏.
Incorporation, digestion, and subsequent excretion of urea and related metabolite nitrogenby cows are accompanied by negative nitrogenisotopic fractionation of up to -῏ (Koyama etal., +32/). The observed d +/N values of cattle
slurry were in reasonable ranges in the view of
the isotopic mass balance.
For one slurry sample collected on May ++,,**., there was no discernible di#erence in the
d +/N value between ammonium- and totalnitrogen. It suggests a very limited nitrogenisotopic fractionation associated with the hydrolysis of urea in cattle excreta, as in the casesof NH.῍ production from organic matter decomposition (ammoniﬁcation) (* to /῏) as reviewed by Robinson (,**+).
Reﬂecting the high proportion of ammonium-nitrogen in the cattle slurry, the pH
values were high, ranging from 3.* to 3.0.
Applying the slurry with such high pH values
would be expected to cause marked volatilization. Yang et al. (,**-) observed an emission
loss of up to 0*ῌ of the applied slurry nitrogen
during ﬁve-day periods after application on an
Andisol in southern Kyushu, Japan. Volatilization of NH- into the free atmosphere could be
conﬁrmed by distinct d +/N values of residual
ammonium-nitrogen in soils, as described later.



18

Table ,+*.

,**0

Temporal variation of moisture content of surface soils from the trial ﬁeld
Moisture content relative to the maximum water holding capacity

Date of sampling

,

-

.

April ,-, ,**.
June +/, ,**.
June +2, ,**.
August 0, ,**.
August ++, ,**.
October ,1, ,**.
October -+, ,**.
November /, ,**.

1,
2,
11
23
1,
31
31
23

00
2/
11
31
1/
32
+**
23

031
20
+*20
+*0
+*/
+*,



Sampling site No.
/

0

1

2

῍῍῍῍ ῌ ῍῍῍῍
ND
ND
23
+*,
1.
2+*3
23
23.
+*,
++,
+*0
+*+*+*0

03
20
3,
32
2,
++,
+**
32

ND
ND
ND
ND
ND
+*,
+*0
3+

Not determined.

Soil moisture status during the ﬁeld
trial
Ammonium-nitrogen together with urea
once applied to soil is subjected to volatilization, nitriﬁcation, denitriﬁcation, and leaching,according to the relevant soil conditions.
Table , shows the moisture content of the surface soil during the present ﬁeld trial in ,**..
All the soil samples were characterized byfairly high moisture content in the ﬁeld conditions, as shown by relative values with the
maximum water holding capacity, ranging
from 0- to +*0ῌ (Table ,). Such high water
retention may have been attributed to the presence of allophane, imogolite, and aluminumhumus complex, by which the unique physicalproperty of Andisols is imparted (Maeda et al.,+311). Aulakh et al. (+33,) reviewed the environmental conditions that control the fate ofmineral nitrogen applied to common soils, andemphasized the primary importance of the soil
moisture status, ambient temperature, and concentration of NH. in the soil solution. Depending on the water holding capacity of soils, thecommon optimal status for nitriﬁcation is in arange from 0* to 1/ῌ, under which the nitriﬁcation is retarded, whereas marked denitriﬁcation prevails over the range of 0/ to 1/ῌ. According to the criteria, the soil examined in thepresent study was in an optimal range for,. ,denitriﬁcation, but not for nitriﬁcation.
,. - Temporal changes in the chemical
forms and d +/N values of nitrogen derived from cattle slurry as applied tosoil
Surface application of cattle slurry results ina large kinetic nitrogen isotope fractionationthat is often associated with the microbial
transformation of the chemical species involved in the slurry. The relevant nitrogenisotope fractionation factors are summarized inTable -. Normally a heavy isotope, +/N tends toenrich in a residual phase.
Temporal changes in the chemical forms together with d +/N values of mineral nitrogen
derived from cattle slurry as applied to soils
are shown in Figs. -a, and -b. Fairly highspatial variations could be seen in both concentration and d +/N values of ammonium- andnitrate-nitrogen. It is evident that the chemical
status of the mineral nitrogen in soil as applied
after cattle slurry is strongly a#ected by soil
temperatures. As a whole, the concentration ofammonium-nitrogen in soil at a certain duration after slurry application is higher than thatof nitrate during the trial periods of April(mean air temperature : 3 to +,) and October/
November (mean air temperature : / to +.)when cooler air temperatures prevail. As withmost biological processes, the nitriﬁcation rateΐῒ : Microbial Transformation of Nitrogen in Cattle Slurry as Applied to an Andisol Grassland 19

Table -

Estimates of nitrogen isotope enrichment factors (eN) for major nitrogen cycle processes in soils

Process

Setting

TemperatureeNῐ
Undescribedῑ
῍/ to *

Mineralization of
Microbial transformation
organic nitrogen
under laboratory condition
NH- volatilization

Nitriﬁcation

Denitriﬁcation

Theoretical calculation for,/.+
῍-/equilibrium fractionation
Experimental,/
῍-.
Experimenta : barnyard soils
Undescribed ῍,+40 to ῍,+4*Experimental : decay of agrocybe Undescribed῍.* to ῍-*

Reference

Handley et al. (+333)

Urey (+3.1)
Kirshenbaum et al.(+3.1)
Kreitler (+31/)
Handley et al. (+330)

Pure culture of Nitrosomonas
europaea
Soils : central Illinois
Pure culture of Nitrosomonas
europaea
Pure culture of Nitrosomonas
europaea

Undescribed

῍,04*

Undescribed,2

῍+/
῍-.41῎,4/Feigin et al. (+31.)
Mariotti et al. (+32+),2

῍-- to ῍,/

Yoshida (+322)

Pure culture of Pseudomonas
denitriﬁcans
Soil culture

Undescribed

῍+14-

,*
-*
+*
,*
-*
Diverse῍,34.῎,4.
῍,.40῎*43
῍,/4.
῍,*4/, ῍+34*
῍+/
῍.* to ῍/Submerged soils

Diverseincreases with increasing temperature until an
optimum is reached above which it decreases(Malhi and McGill, +32,).
Soil moisture status is another importantfactor for controlling the transformation ofmineral nitrogen in soils. Optimum moisture
conditions for nitriﬁcation and denitriﬁcation
are known to be around 0*ῌ and more than
2*ῌ, relative to maximum water-holding capacity of soils, respectively (Aulakh et al., +33,).
,. . Chemical and isotopic status of mineral nitrogen in soil under lower temperature conditions (April and October/
November)
The concentration of ammonium-nitrogen insamples collected from sites , and - (2- and +-/mg-N kg῍+ (dry soil)) on six days after slurry
application in April was evidently higher thanthat in samples from sites . and 1 (Fig. -a).

Delwiche and Steyn (+31*)

Delwiche and Steyn (+31*)
Mariotti et al. (+32+)
Mariotti et al. (+32,)

Lehmann et al. (,**-)

Very low concentrations of ammonium-nitrogen in soil from sites . and 1 indicate that theapplication of cattle slurry may not be uniformly applied around the two sample sites.
The d +/N values of ammonium-nitrogen in soil
ranged from ῌ+0.+ to ῌ,+.1ῑ. These values
were clearly higher than those obtained fornitrogen in the cattle slurry (d +/N῏῍*.2 to ῌ,..
ῑ, Table +) and even common organic nitrogenin Japanese arable Andisols (d +/N῏ῌ..1 to ῌ2..
ῑ, Yoneyama, +321). Such an increase in the
d +/N values as observed here is interpreted asindicating a nitrogen isotopic fractionation
associated with the prompt volatilization of
NH-, i.e. selective removal of light +.N relative
to heavy +/N during the application and subsequent deposition of nitrogen onto soil. The
fractionation factors for volatilization of NHobtained under the laboratory conditions are

20

Fig. -+*.

,**0

Temporal changes in the concentration and the d +/ N values of mineral nitrogen soil as
a#ected by application of cattle slurry : a) for April and October/November period underlower air temperature conditions, b) for June and August under higher air temperature
conditions.

reported to be around .* to 0* (Handley et al.,+333). Similarly high d +/N signatures of ammonium-nitrogen in soil, which are resulted fromthe emission of NH-, have been documented forsoil during the microbial decomposition of uricacid deposits derived from seabird’s excreta(Mizutani et al., +32/, +320).
The d +/N signatures of two soil nitrates (sites, and -) collected on six days after slurry application in April showed an identical value of+.1. The apparent nitrogen isotopic fractionations during the nitriﬁcation (nitrogenῑῐ : Microbial Transformation of Nitrogen in Cattle Slurry as Applied to an Andisol Grassland 21

isotopic di#erence between ammonium- andassociated nitrate-nitrogen) observed in thepresent trial ﬁeld was lower than those of previous measurements (-/ to 0*῏, Handley et al.,+333). The sum total of mineral nitrogen decreased to less than +* mg-N kg-dry-soil῍+ inless than two weeks after slurry application(data not shown).
A comparable trend had also been observed
for the status of mineral nitrogen in soil collected in October-November with similarly low soil
temperature regimes (/ to +.῎, Figs. ,, and -a).
The concentration of ammonium-nitrogen insoils was higher, relative to those collected on
the other days. Soil samples collected fromsites ., 0, 1, and 2 one day after slurry application showed very high concentration of ammonium-nitrogen (+,+-* to +,.-* mg kg῍+ (drysoil)), which was one order higher than theother samples. This may be resulted from thelower air temperature (3῎) at the time of
applying cattle slurry and hence limited volatilization of the applied ammonium-nitrogen,or heterogeneous nature of the applied slurry.
It has been well documented that higher air
temperatures promote greater ammonia volatilization (Sommer and Hutchings, ,**+ ; Huijsmans et al., ,**-).
The rate of nitriﬁcation of ammonium-nitrogen applied in October-November underlower air temperature was slow, as seen fromlow content of nitrate-nitrogen, even ten days
after slurry application. The trend was comparable to that of April (Fig. -a). Appreciable
amounts of precipitation (++ mm and .- mm)occurred on ﬁve and ten days after slurry application, respectively. Lower concentrationsof mineral nitrogen as observed for soil samples taken ten days after slurry application
may be due to leaching from the soil system.
Very high concentration of added ammonium-nitrogen in soil as observed for sites ., 0,1, and 2 would suppress subsequent nitriﬁcation (Harada and Kai, +302). The calculated
concentration of the ammonium-nitrogen insoil solution of the soil ranged from /*- mg L῍+for site . soil to /3. mg L῍+ for site 2, which ishigher than those reported as the critical concentration of the inhibitory e#ect for nitriﬁcation, more than ,** mg L῍+ of ammoniumnitrogen (Harada and Kai, +302). A lower rateof nitriﬁcation as observed in October would
be resulted from such an inhibitory e#ect, in
addition to lower ambient temperature.
The d +/N values of ammonium in soil samples after slurry application on OctoberNovember apparently increased from one day
after application (ῌ2.1 to ῌ3.3῏) to ﬁve days
after (ῌ+../ to ῌ,,.2῏) and then ten days after(ῌ,-.0 to ῌ--.,῏), reﬂecting the advance ofnitriﬁcation (Fig. -a). The d +/N values of theassociated nitrate in soil samples ranged from῍+.+ to ῌ-.0῏ on one day, and then decreasedto ῍1.* to ῍/..῏ on ﬁve days after slurry
application. Ten days after slurry application,
the d +/N values of soil nitrate had increased toῌ2.* to ῌ+/..῏. Since there was no discernible
increase in soil nitrate after application of
cattle slurry in October/November, such a nitrogen isotopic trend indicates coupling of nitriﬁcation and subsequent denitriﬁcation insoil. The e#ects of ambient temperature on thenitrogen isotopic fractionation associated withmineralization, nitriﬁcation, and denitriﬁcationof urea and potassium nitrate added to an
Andisol under laboratory conditions will bereported elsewhere.
,. / Chemical and isotopic status of mineral nitrogen in soil under higher temperature conditions (June and August)Relative to April and October-November, theperiod of lower air temperature conditions,chemical processes involving the transformation of mineral nitrogen in soil applied in Junegave unique trends (Fig. -b). Concentration of
nitrate-nitrogen of the soil clearly increased incomparison with the soil obtained in April andOctober-November. There was no precipitation during the ﬁeld trial in June. The marked
trend observed here was that nitriﬁcation insoil from June onwards was accelerated by therise in soil temperatures (average air tempera-

22



ture : +1 to +3). The concentration of ammonium-nitrogen in soil three days after application of cattle slurry ranged from .* to ,1* mg-N
kg+ (dry soil), whereas those of the associatednitrate were from 3* to +1* mg-N kg+ (dry soil).
The concentrations of nitrate in three soil
samples collected from sites -, 0, and 1 werehigher than those of the associated ammonium-nitrogen. The d +/N values of ammoniumnitrogen were high, ranging from +2 to -*.
The observed high d +/N values were resulted
from the marked nitrogen isotopic fractionation associated with the prompt volatilizationof ammonium-nitrogen under higher air temperature at the time of slurry application (airtemperature : ,*), in addition to subsequent
active nitriﬁcation. The d +/N values of nitrateassociated with the ammonium-nitrogen wereclassiﬁed into two major groups, according to
the relevant nitrogen isotopic signatures. The
ﬁrst group is characterized by distinctly lower
isotopic values with negative signature (d +/N
1 to ,) for four sample soils (sites ,, -, .,and /), and the second group by those withhigher, positive values (d +/N0 to 1) for
two other soil samples (sites 0 and 1). Thosedi#erences relate to the relative proportion ofmineral nitrogen in soil samples. The rate ofnitriﬁcation in soil from sites ,, -, . and / wasslower than the other two soils (sites 0 and 1),
probably reﬂecting the micro-scale di#erencein the soil environments.
The concentration of ammonium-nitrogen insoils collected after six days of application on
June decreased relative to that after threedays. The decrease reﬂected the continued
nitriﬁcation. As ammonium-nitrogen content
decreased, the d +/N values increased up to-0, which reﬂected nitrogen isotopic fractionation due to +/N enrichment in the residual
ammonium-nitrogen during the nitriﬁcation.
The d +/N values of the nitrate (d +/N+ to1) increased compared with those collected
after three days of slurry application (d +/N
0.1 to 1), reﬂecting the simultaneous denitriﬁcation of the nitrate formed under high+*.  ,**0

soil moisture conditions (Table ,). The coupledoccurrence of nitriﬁcation and denitriﬁcation
in soil associated with nitrogen fertilization
has been suggested for soils with stagnogleyfeatures in the United Kingdom (Nielsen et al.,+330 ; Abbasi and Adams, +332).
Denitriﬁcationan anaerobic process isstimulated by hypoxia. Denitriﬁcation in theﬁeld is episodic and localized, being restricted
to times when and places where NO- andcarbon are readily available and O, is not (Robinson and Conroy, +333). The soil conditions
under dense rhizome from reed canary grass (acarbon source), and ample supply of nitrate
derived from repeated application of cattle
slurry (nitrate supply), in addition to very highretention of water (oxygen deﬁciency), provide
the conditions for active denitriﬁcation.
Denitriﬁcation of nitrate results in gas formation of dinitrogen together with nitrous
oxide, which escapes from the soil system,
causing marked nitrogen isotopic fractionation.
The nitrogen isotopic fractionation
during denitriﬁcation is reported to be ,2 to-- (Handley and Raven, +33,). The overall
process indicated rapid nitriﬁcation followedby active denitriﬁcation under higher soil temperature regimes as observed in the present
study.
Nearing August, when the average air temperature rose to ,1 (Fig. ,), the comparable
ﬁgure for microbial transformation of slurry
nitrogen in soils was observed (Fig. -b). There
was no precipitation for four days after slurry
application. The concentration of ammoniumnitrogen varied from ++* to -+* mg-N kg+ (drysoil), whereas that of the associated nitrate
ranged from -* to 3* mg-N kg+ (dry soil). The
d +/N values of ammonium-nitrogen were again
high, in a range from ,, to -+. The d +/Nvalues of the associated nitrate showed valuesof + to ++.
Very heavy daily precipitation, amounting
to +*0 mm, occurred just after the ﬁrst soil
sample collection (four days after slurry application) in August. Nitrogen loss may be causedῐ῏ : Microbial Transformation of Nitrogen in Cattle Slurry as Applied to an Andisol Grassland 23

by surface run o# together with percolatingwater. The concentrations of both ammoniumand nitrate-nitrogen in soil ten days afterslurry application were signiﬁcantly low,
showing fairly homogeneous values of ,* to 0*
mg and +** to +1* mg-N kg῍+ (dry soil), respectively, that reﬂected the advanced stage of nitriﬁcation under higher soil temperature conditions. The d +/N values of mineral nitrogenwere in a range from ῌ+* to ῌ,-῎ that tended
to be lower than those four days after theapplication. A somewhat lower d +/N value ofammonium-nitrogen observed for soil sample (ῌ+*.*῎) may be derived from the mineralization of intrinsic organic nitrogen under ahigher temperature condition.
,. 0 Temporal changes in concentration andd +/N values of ammonium- and nitratenitrogen in groundwater
The concentration and d +/N values of miner-

Fig. .

al nitrogen in groundwater samples collectedfrom ﬁve sampling wells and one spring wereperiodically monitored from October to December, ,**. (Fig. .). The ﬁgure contrasts with adistinctly low concentration of nitrate-nitrogen (*.03 mg L῍+) in addition to negative d +/Nvalue (῍2.-῎) for nitrate in one spring at anarea of high elevation (Mt. Iwate) where theonly possible source of nitrate is meteoric in
origin. Major mineral nitrogen in the trial
wells would be derived from local sources, probably from migration of slurry-derived nitrogen.
Nitrate was a major form of mineral nitrogenin the groundwater from the trial wells, whereas two wells (sites . and 0) contained appreciable amounts of ammonium-nitrogen. It is evident that each well has its own chemical together with isotopic characteristics. Well sites, and - and the spring were characterized byTemporal changes in the concentration and the d +/ N values of mineral nitrogen in groundwater samples.



24

an almost constant concentration of nitrateand d +/N values. Two additional samples of thespring water collected on May ++ and June +,,**. showed nearly comparable values (concentration of nitrate-N ; ..0 and ..1 mg L+, d +/N
/.2 and 1.-, respectively). Well sites .
and 0 often contained nitrate-nitrogen characterized by high d +/N values of +. and +0
which were collected on ,2 June. Such highd +/N values were derived from residual nitrate
after denitriﬁcation, as commonly observed foranimal excreta (Wassenaar, +33/). Well 1 wascharacterized by an almost constant concentration of nitrate (.., to ..1 mg L+) and low d +/Nvalues (+.0 to +.1). A comparable d +/Nvalue between residual soil nitrate formed
after certain periods (more than two weeks) of
slurry application and those in the current
groundwater may indicate possible migrationof the local origin of nitrate. For more information on the origin of nitrate in the groundwater, measurement of oxygen isotopic composition (d +2O) of nitrate is required (Silva et al.,,**,). A technical problem would be the isolation of valid nitrate from Andisols where theconcentration of humic substances together
with soluble silica is often high. No successful
protocols are available for the d +2O value measurement of nitrate in such type of soils.
Acknowledgements
We are grateful to Dr. H. Doi (Tohoku University), Mr. Y. Konno (Iwate University), andDr. T. Yamanaka (Kyushu University) for providing technical assistance during the measurement of nitrogen isotope composition of thesample materials. Quantitative determinationof ammonium- and nitrate-nitrogen in groundwater was made by Dr. N. Satta (Iwate University). This research was supported in part by
the Grant-in-Aid for Scientiﬁc Research (B) (No.
+/-2*+0*) and (C) (No. +1/2**/*) of the Japan
Society for the Promotion of Science. The trial
ﬁeld was provided and maintained by Mr. M.
Yokota.

+*.

,**0

References
Abbasi, M.K. and Adams, W.A. (+332) : Loss of nitrogen in compacted grassland soil by simultaneous nitriﬁcation and denitriﬁcation. Plant
Soil, ,** : ,0/ῌ,11.
Aulakh, M.S., Doran, J.W. and Mosier, A.R. (+33,) :Soil denitriﬁcation : Signiﬁcance, measurement, and e#ects on management. Adv. Soil
Sci., +2 : +ῌ/1.
Delwiche, C.C. and Steyn, P.L. (+31*) : Nitrogen isotope fractionation in soils and microbial reactions. Environ. Sci. Tech., ++ : 3,3ῌ3-/.
Feigin, A., Shearer, G., Kohl, D.H. and Commoner, B.
(+31.) : The amount and nitrogen-+/ content of
nitrate in soil proﬁles from two central Illinois
ﬁelds in corn-soybean rotation. Soil Sci. Soc.
Am. Proc., -2 : .0/ῌ.1+.
Fukada, T., Hiscock, K.M., Dennis, P.F. and
Grischek, T. (,**-) : A dual isotope approach toidentify denitriﬁcation in groundwater at ariver-bank inﬁltration site. Water Res., -1 :-*1*ῌ-*12.
Handley, L.L. and Raven, J.A. (+33,) : The use ofnatural abundance of nitrogen isotopes inplant physiology and ecology. Plant Cell Environ., +/ : 30/ῌ32/.
Handley, L.L., Austin, A.T., Robinson, D., Scrimgeour, C.M., Raven, J.A., Heaton, T.H.E., Schmidt,
S. and Stewart, G.R. (+333) : The +/N naturalabundance (d +/N) of ecosystem samples reﬂects
measures of water availability. Aust. J..Plant
Physiol., ,0 : +2/ῌ+33.
Handley, L.L., Brendel, O., Scrimgeour, C.M.,
Schmidt, S., Raven, J.A., Turnbull, M.H. and
Stewart, G.R. (+330) : The +/N natural abundance patterns of ﬁeld-collected fungi fromthree kinds of ecosystems. Rapid Comm. Mass
Spec., +* : 31.ῌ312.
Harada, T. and Kai, H. (+302) : Studies on the environmental conditions controlling nitriﬁcation in soils. I. E#ects of ammonium and total
salts in media on the rate of nitriﬁcation. Soil
Sci. Plant Nutr., +. : ,*ῌ,0.
Huijsmans, J.M.F., Hol, J.M.G. and Vermeulen, G.D.
(,**-) E#ect of application method, manurecharacteristics, weather and ﬁeld conditions
on ammonia volatilization from manure applied to arable land. Atmos. Environ., -1 : -003ῌ
-02*.
Kirshenbaum, I., Smith, J.S., Crowell, T., Gra#, J.
and McKee, R. (+3.1) : Separation of the nitrogen isotopes by the exchange reaction between ammonia and solutions of ammonium.

ῐ῏ : Microbial Transformation of Nitrogen in Cattle Slurry as Applied to an Andisol Grassland 25

J. Chem. Phys, +/ : ..*ῌ..0.
Koyama, T., Sudo, M. and Yoneyama, T. (+32/) :Fractionation of nitrogen isotopes by domestic
animals. Jpn. J. Zootech. Sci., /0 : -0+ῌ-0- (inJapanese).
Kreitler, C.W. (+31/) : Determining the source of
nitrate in ground water by nitrogen isotope
studies. Report of Investigation No. 2-, +ῌ/1.
Bureau of Economic Geology, The Universityof Texas at Austin, Austin, Texas 121+,.
Lehmann, M.F., Reichert, P., Bernasconi, S.M.,
Barbieri, A. and McKenzie, J. (,**-) : Modelling
nitrogen and oxygen isotope fractionation
during denitriﬁcation in a lacustrine redoxtransition zone. Geochim. Cosmochm. Acta,
01 : ,/,3ῌ,/.,.
Maeda, T., Takenaka, H. and Warkentin, B.P. (+311) :Physical properties of allophone soils. Adv.
Agron., ,3 : ,,3ῌ,0..
Malhi, S.S. and McGill, W.B. (+32,) : Nitriﬁcation inthree Alberta soils : e#ect of temperature,moisture and substrate concentration. Soil
Biol. Biochem., +. : -3-ῌ-33.
Mariotti, A., Germon, J.C., Hubert, P., Kaiser, P.,
Lettole, R., Tardieux, A. and Tardieux, P. (+32+) :Experimental determination of nitrogen kinetic isotope fractionation : some principles ; illustration for the denitriﬁcation and nitriﬁcation processes. Plant Soil, 0, : .+-ῌ.-*.
Mariotti, A., Germon, J.C. and Leclerc, A. (+32,) :Nitrogen isotope fractionation associated with
the NO,῍῎N,O step of denitriﬁcation in soils.
Can. J. Soil Sci., 0, : ,,1ῌ,.+.
Mizutani, H., Hasegawa, H. and Wada, E. (+320) :High nitrogen isotope ratio for soils of seabird
rookeries. Biogeochem., , : ,,+ῌ,.1.
Mizutani, H., Kabaya, Y. and Wada, E. (+32/) : Ammonia volatilization and high +/N/ +.N ratio in a
penguin rookery in Antarctica. Geochem. J., +3 :-,-ῌ-,1.
Nielsen, T.H., Nielsen, L.P. and Revsbech, N.P. (+330) :Nitriﬁcation and coupled nitriﬁcation-denitriﬁcation associated with a soil-manure interface. Soil Sci. Soc. Am. J., 0* : +2,3ῌ+2.*.

Noborio, K., Satta, N., Koga, K., Baba, H. andMukaida, Y. (,**,) : Soil as a ﬁlter for nitratetransport in the grass ﬁeld amended withcattle manure. J. Jpn. Soc. Irrig. Drain. Reclam.
Eng., 1* : -+ῌ0-. (in Japanese).
Panno, S.V., Hackley, K.C., Hwang, H.H. and Kelly,
W.R. (,**+) : Determination of the sources ofnitrate contamination in karst springs using
isotopic and chemical indicators. Chem. Geol.,+13 : ++-ῌ+,2.
Robinson, D. (,**+) : d +/N as an integrator of thenitrogen cycle. Trends Ecol. Evol., +0 : +/-ῌ+0,.
Robinson, D. and Conroy, J.P. (+333) : A possible
plant-mediated feedback between elevated
CO,, denitriﬁcation and the enhanced greenhouse e#ect. Soil Biol. Biochem., -+ : .-ῌ/-.
Silva, S.R., Ging, P.B., Lee, R.W., Ebbert, J.C.,
Tesoriero, A. J. and Inkpen E.L. (,**,) : Forensic application of nitrogen and oxygen isotopes in tracing nitrate sources in urban environments. Environ. Forens., - : +,/ῌ+-*.
Sommer, S.G. and Hutchings, N. J. (,**+) : Ammonia emission from ﬁeld applied manure and its
reduction ῌ invited paper. Euro. J. Agron., +/ :+ῌ+/.
Stevenson, F. J. (+32,) : Nitrogen in Agricultural
Soils. Agronomy no. ,,, American Society ofAgronomy. Madison, WI. +ῌ3.*.
Urey, H.C. (+3.1) : The thermodynamic properties
of isotopic substances. J. Chem. Soc., /0,ῌ/2+.
Wassennar, L.I. (+33/) : Evaluation of the origin
and fate of nitrate in the Abbotsford Aquifer
using the isotopes of +/N and +2O in NO-῍. Appl.
Geochem., +* : -3+ῌ.*/.
Yang, Z., Niimi, H., Kanda, K-I. and Suga, Y. (,**-) :Measurement of ammonia volatilization froma ﬁeld, in upland Japan, spread with cattle
slurry. Environ. Pollu., +,+ : .0-ῌ.01.
Yoneyama, T. (+321) : Natural abundance of +-C, +/N,
+2
O, D and -.S in soils and plant : Variations,
implication and utilization. Jap. J. Soil Sci.
Plant Nutr., /2 : ,/,ῌ,02 (in Japanese).
Yoshida, N. (+323) : +/N depleted N,O as a product ofnitriﬁcation. Nature, --/ : /,2ῌ/,3.

./5" ¡ » +*. ¼ T,**0\26

 
*ῌ**ῌ

***

* **
 *,*ῌ2//*  -ῌ+2ῌ2***  ,+0ῌ2/1+  +ῌ+ῌ+,!

"

 !"#$%&'()*+,- ./#012&'3./4"56#789:;:<=>'? @AB5CDE.FGA#012&HIJKLMNO#$%&')*5P4"Q;R<:S+ TUVWXUY3Z[Y)*\ 3)*]^_`abcd Td +/N\ 5efQ;:#gh9ijhH?
IJkK5lm(Y)*no'K*3p5qr"5s3tu- vwAxyVE#z{|#UVW
XUY)*#}~,i2&H?  Axn- UVWXUY)*5Z:
&H? B xn- Z:-2&H? Z:# )- Z[Y)*$ 5m3]f#8HbZ[Y)*|5 d +/ N 5#7892&H? 57m
-CDE.5~, ¡3G¢£4¤¥5("5¦,m§¨S©ª«2&'3#789
2&'3¬­2&H?
#$% : IJKLMNO UVWXU®0 Z: ) )*]^_,¯=°±²³ : ,**0 ± . ² ,2 ³
= ±²³ : ,**0 ± 2 ² , ³*´ :  µ ῌ¶·¸EL : *+3ῌ0,+ῌ0+,0 eo ¹Oº : mizota@iwate-u.ac.jp