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Phases of Denitrification Following

Oxygen Depletion in Soil

Article in Soil Biology and Biochemistry · December 1979

DOI: 10.1016/0038-0717(79)90071-3

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 PHASES OF ~~NITRIF~CATION FOLLOWING
OXYGEN DEPLETION IN SOIL
M. SCOTT SMITH* and JAMES M. TIEDJE
Departments of Crop and Soil Sciences and of Microbiology
and Public Health, Michigan State University, E. Lansing, MI 48824, U.S.A.

(Accepted 28 Nocember 1978)

Summary-The short-term response of soil denitrification to reduced aeration was studied using the
acetylene inhibition method for the assay of denitrification. Two distinct phases of denitrification rate
were observed. An initial constant rate, termed phase I, was not decreased by chloramphenicol, was
increased slightly or not at all by organic carbon amendment. and lasted for I-3 h. Phase I was
attributed to the activity of pre-existing denitrifying enzymes in the soil microflora. Following phase
I the denitri~cation rate increased; chloramph~nicol inhibited this increase. In soils without organic-C
amendment a second linear phase, termed phase fl, was attained after 4-8 h of anaerobic incubation.
The linearity of this phase was attributed to the full derepression of denitrifying enzyme synthesis
by the indigenous population and to the lack of significant growth of denitritiers. Phase I rate was
dependent on the initial or in situ aeration state of the soil sample; phase II was not. Therefore,
phase I may be more directly related to field denitrification rates.
Denitrification rate changes following water saturation of soils in aerobic atmospheres were also
examined. Rates were greatly increased by wetting but only after a lag of several hours. Our interpre-
tation is that following wetting of natural soils, anaerobic or partially anaerobic conditions are estab-
lished by respiration and reduced 0, diffusion rate; this first eliminates O2 inhibition then derepresses
the synthesis of denitrifying enzymes, Although denitrifying enzymes are apparently present even in
relatively dry soils, their activity is low until O2 inhibition is eliminated. From this evidence we reason
that most N is lost from soils during brief periods beginning a few hours after irrigation or a rainfall.

INTRODUCTlON MATERIALS AND ME’FNODS

It is generally believed that denitrification occurs in Soils used were Brookston loam, Spinks loamy
agricultural soils predominantly under saturated or sand, Miami sandy loam, and Carlisle muck; they
near-saturated conditions (Focht and Verstraete, have been described by Smith er ul. (1978). The maxi-
1977). Soil denitrification rates have been shown to mum wafer holding capacity, as determined by satu-
increase with added water or reduced aeration rating soil in a filter paper funnel, was 43 ml per 100 g
(Bremner and Shaw, 1958; Ardakani et al., 1977; Pilot for the Brookston and 36 ml per 100 g for the Spinks
and Patrick, 1972). This is consistent with the known and Miami. After collection the soils were stored at
physiology of denitrifiers; 0, both inhibits denitrify- 2-4°C without drying. However, for many experi-
ing enzyme activity and represses synthesis of new ments fresh samples were collected from the same
denitrifying enzymes (Payne, 1973). Nonetheless, there sites immediately before experimentation. All soils
are reports suggesting biological production of were at or below field capacity when collected,
nitrogen gases in well-aerated soils (Starr r# ul., 1974; The C,H, inhibition method was used to measure
Broadbent and Clark, 1965; Bremner and Btackmer, denitrification rates; its validity for measuring short-
1978), though the significance of N loss under these term rates has been established previously (Smith et
conditions has not been determined. The close rela- cd., 1978). The methods used here were similar.
tionship between aeration state and denitrification Briefly: soil to be assayed was made into a slurry
rate indicates that it is important to characterize the and incubated anaerobically in closed flasks. CzH2
short-term soil response to reduced aeration. Until (0.1 atm) was added to inhibit the reduction of
now. methodologjcal limitations have precluded N20 to N,. Nitrate was also added to the soil suspen-
studying the dynamics of the soil response, that is, sions, 1Opg NO;-N.g-’ (soil fresh wt basis) for
how rapidly and to what extent denitrification rates short-term assays and repeated additions of
change following the onset of anaerobic or partially 200 fig ‘g- ’ for longer experiments. Rapid attainment
anaerobic conditions. Nor has the biological com- of anaerobiosis for phase I assays was ensured by
ponent of this response, the short-term reaction of magnetically stirring the suspensions while evacuating
soil denitrifiers to imposed anaerobiosis, been exam- and flushing the flasks; incubation was under a He
ined. In this paper we address these questions which atmosphere. The reported data are means of at least
are important to the basic understanding, and to the three replicates. Rate data were determined from
potential control and prediction of denitrification. linear regressions; also reported is the sample stan-
dard deviation of the regression, s,t 0.05, (Snedecor
and Cochran, 1967).
* Present address. University of Kentucky, Lexington, To determine denitrification rates in aerobic atmos-
KY 40506, U.S.A. pheres. soils were sieved ( < 4 mm) to break up large
261
262 M. SCOTT SMITH and JAMES M. TIEDJE

clods and remove debris. This treatment had little
effect on smaller, stable aggregates. Soil (35 g) was
gently shaken with 0.1 atm CzH2 in a stoppered 50ml
centrifuge tube, then compacted by gently tapping the
tube.
Chloramphenicol(O.27 mg.g-i) was used to inhibit
protein synthesis in soil suspensions. These experi-
ments were done in Brookston soil and with a mix-
ture of 50 g washed silica sand and 5 g Brookston
soil. The sand mixture was incubated anaerobically
with succinate and NO; several days before the ex-
periment to develop denitrifying populations.
Pure cultures of denitrifiers were grown aerobically
to log phase in nutrient broth (Difco, Detroit, MI).
They were harvested on 0.45 pm Millipore filters and
washed with 3 vol of phosphate buffer (pH 7.0,
50 mM). The filters with the cells were placed in flasks
of nitrate broth. The flasks were then made anaerobic
and assayed in the presence of &Hz in the same
manner as the soil suspensions. Some cultures were
also assayed in nitrate broth with 0.5 g .I- 1 Na thio-
glycollate added as a reducing agent. The flasks with
thioglycollate were evacuated and flushed with He
16 h before inoculation. The denitrifiers had been iso-
lated from soils and were fully characterized during
earlier work in this laboratory (T. N. Gamble, unpub-
lished. M.S. Thesis, Michigan State University, 1976).
They were: Pseudomonas juorescens biotype II 72, P.
jhorescens biotype IV 206, P. stutzeri 224, P. aureofa-
cirns 59, and Flacohucterium sp. 175. Numbers refer
to our strain designation.
The concentrations of NzO were measured by g.c.
using h3Ni electron capture and microthermistor
detectors (Smith et N/., 1978). Most probable numbers
of denitrifiers were determined with a microtiter sys-
tem (Rowe et N/., 1977). Disappearance of nitrate and
nitrite from nitrate broth was determined with
diphenylamine.
RESULTS
Two linear phases of denitrification were observed
following the imposition of anaerobiosis on aerobic
soil (Fig. 1). Phase I lasted from approximately
15min, the time for N20 to reach easily measured
concentrations, to between 1 and 3 h. Smith et ul.,
(1978) had shown that this initial period is followed
by an increase in rate until a second linear phase,
phase II, is established. Some phase II data from our
earlier work is included in Fig. 1 (inset) to demon-
strate the biphasic nature of denitrification rates. The
duration of both phases was quite variable among
soils. However, the biphasic pattern was observed
consistently with both fresh and stored (moist) soil
samples when assayed as slurries.
In some experiments attempts were made to ehmin-
ate O2 more rapidly and completely. These included
deaerating the solution and flask before adding
the soil, varying the duration of evacuating and
flushing the flasks, and agitating the suspensions
while evacuating. These procedures did not alter the
bilinear pattern indicating that residual O2 or delayed
establishment of equilibrium was not responsible for
the observed phases. The biological origin of N,O
production was confirmed by finding that autoclaved
soils did not evolve N,O at a significant rate.
The effect of chloramphenicol on denitrification
rate is illustrated in Fig. 2. This compound is an in-
hibitor of protein synthesis but would not be expected
to reduce the activity of enzymes already present.
Phase I rate was not reduced, but very slightly in-
creased by chloramphenicol. This small increase
(which has been consistently observed) could be due
to diversion of carbon supply from protein synthesis
to respiratory processes. Of greater significance is the
effect on the shift from phase I to phase II. Phase
II rates were markedly reduced by chloramphenicol.
Inactivation of chloramphenicol by soil binding and
microbial decomposition would be expected in soil,
so this experiment was also conducted with silica
sand mixed with a small quantity of soil. In this case
the increased rate from phase I to phase II was almost
totally eliminated (Fig. 2). Succinate (l’t;J, a non-fer-
mentable substrate, was added to the sand allowing
growth and thus accounting for the lack of a distinct
linear phase II in the absence of chloramphenicol.

lncubotlon Time (m(n)

Fig. 1. Evidence for two linear periods of denitrification, shown for three soils, described as phase
I and phase II (after 300min). Points are means of three replicates.
 Phases of denitrification in. soil 263

; 210.

vi
‘0 BROOKSTON SOIL
"
c?
cn
0 150-

;
4
F
c
90.

I- PHASE I -I

lncubatlonTime(m!n)

Fig. 2. Effect of chloramphenicol (CHL) on denitrification rates in soil and a sand. Points are the
means of three replicates. Numbers in parentheses are rates (nmoles N,O.g dry soil-‘.rnin- ‘) with
95% confidence limits.

These results indicate that the rate increase following
phase I is a result of the synthesis of denitrifying
enzymes and suggest that phase I, but not phase II,
is a measure of the activity of preexisting denitrifying
enzymes.
This conclusion is supported by the effect of glu-
cose additions on denitrification rates. Though the
effect was quite dependent on the soil sample, the
results in Table 1 are representative of the stimulatory
pattern. Phase I rates were not increased by glucose
in some soils and only slightly increased in others.
Phase I remained linear in glucose-amended soils.
Phase II denitrification was often but not always
stimulated by glucose. The second linear phase was
always of reduced duration and was followed or com-
pletely replaced by a logarithmic increase in rate
when glucose was added. Without an organic carbon
amendment, the denitrification rate remained essen-
tially constant, i.e. phase II persisted, for up to 3 days.
It is concluded that during phase I the enzymatic
capacity to denitrify is a more important limitation
than the supply of electron donor, but that enzyme
synthesis and the potential for growth during con-
tinued incubation increases the demand for electron
donor, making this the more important limiting fac-
tor. The data also suggest that a significant increase
in number of denitrifiers does not occur in anaerobic
soils unless an energy supply is added.
To determine the role of microbial growth in the
denitrification rate changes, denitrifiers were enumer-
ated by an m.p.n. procedure after various periods of
anaerobic incubation. Increasing numbers could be
detected only in soils with added glucose. However,
the methods used are not precise enough to detect
increases of less than an order of magnitude.
Pure cultures of denitrifiers shifted from aerobic to
anaerobic conditions exhibited a distinct lag phase
before beginning to denitrify (Fig. 3). Nitrous oxide
first appeared between 95 and 205 min after the onset
of anaerobiosis; most of the organisms required about
2 h to produce detectable N,O. FIavobucterium sp.
175 grown anaerobically in NO; broth, harvested
and assayed in the same manner as the aerobic cul-
tures began producing NaO immediately. The reduc-
ing agent, thioglycollate. did not decrease lag times
indicating that neither residual 0, nor a high initial
E, were delaying denitrification. The lag times corre-
spond quite well with the time of shift from phase

Table 1. Effect of glucose additions on denitrification rate of Brook-

ston soil

Denitrification rate
Time of anaerobic (nmoles NzO.g dry soil-‘.min-‘)
incubation
(n) No glucose Glucose added

G-l+ 0.21 * 0.03* 0.25 + 0.031

(Phase I)
6-8 0.56 f 0.09 0.56 &- 0.08:
2421 0.67 f 0.06 2.82 k 0.16:
12-75 0.60 & 0.07 1.43 * 0.091

* &,t 0.05.
t 0.15% glucose added.
$ 0.1% glucose added.
 M. SCOTTSMITHand JAMES M. TIEDJE

the sample taken after irrigation was approximately

‘* Time of Anaerobic incubation (min)
Fig. 3. Derepression of denitrifying enzyme synthesis fol-
lowing shift of five aerobically grown denitrifier strains to
anaerobic conditions. Anaerobically grown strain 175 is
a control for prior derepression.
I to phase II in anaerobic soils and further support
the conclusion that the phase I rate is unaffected by
de nova enzyme synthesis.
Since phase I is a measure of the activity of in
situ denitrifying enzymes it should respond to altered
soil aeration in the field. This was demonstrated by
sampling the Miami soil, planted to corn, immedi-
ately before and after 4 h of irrigation. Within 1 h
of sampling the soils were made anaerobic and phase
I and II rates were determined. The phase I rate of
twice that of the sample taken before (Fig. 4). How-
ever, there was no difference in phase II rates. This
comparison serves as a control to show that there
were not significant chemical or physical differences
between the two samples which could account for the
difference in the phase I rates. The increase in phase
1 rate following irrigation can be attributed to the
partial derepression of denitrifying enzyme synthesis
caused by reduced soil aeration. It is interesting that
a significant amount of phase I denitrification capa-
city was detected even in the sample taken before
irrigation. This soil was at 16% water content and
so may be considered reasonably well aerated.
The relationship between phase I rate and soil
aeration was further established by adding various
amounts of water to 5Og Spinks soil in 125 ml flasks.
The flasks were left open for 16 h before phase 1 rates
were determined, I3enitrifying activity was present in
all the treatments but was greatly increased at water
contents above 259:, (Table 2).
Nitrous oxide evolution from soils in an aerobic
atmosphere, with 0.1 atm C2H, added could also be
observed (Fig. 5). Miami soil at 18:; and the same
soil adjusted to 33:/i, water content (approximately
saturation) just before the assay were compared for
patterns of NzO evolution. A very low rate of N,O
evolution, 11 fmoles N,O’ml headspace- “min-‘,
was observed from the drier soil and initially from
the moistened soil. Approximately 6 h after wetting.
the rate began to increase rapidly. Similar results were
observed with several other soil samples. The lag
period ranged from 4 to 7 h. The OJN, ratio in the
headspace, as determined by gc. analysis, was
reduced by less than 5”,, during these experiments.

I
I
Phase I I Phase II
I
I is
I ,’
I
I
I
I
I
I
I
I
I
I
/

100 350 400

incubation Time (min)
* ) I

450
(

Fig. 4. Phase I and phase II denitrification rates of a Miami soil sampled before and after 4 h of
irrigation. Points are the means of three replicates. Numbers in parentheses are rates (nmoles N,O’g
dry soil- ’ ‘min- ‘) with 9.59;,confidence limits.
 Phases of denitrification in soil 265

Table 2. Phase I denitrification rate of Spinks loamy sand preincubated

aerobically at varying water contents

Gravimetric
water content Denitrification rate
(%) (nmoles NzO.g dry soil-’ .min~‘)

6 0.06 i 0.02*
16 0.06 i 0.01
21 0.10 + 0.02
3-l 0.16 f 0.06
58 0.38 * 0.09

* *s,t 0.05.

DISCL’SSION demonstrates that the capacity to utilize electron

donor increases after phase I. Pure cultures of soil
We conclude that the following sequence of events denitrifiers exhibited a lag consistent with the
occurs when a soil or soil microsite becomes anaero- duration of phase I before synthesizing denitrifying
bic: first, the 0, inhibition of native denitrifying enzymes. Payne and Riley (1969) observed a slightly
enzymes is removed, which results in an initial linear shorter lag, 40min, with the marine denitrifier, Pseu-
phase of denitrification (phase I). After a lag of at domonus perfectomarinus. This could be due to a dif-
least 1 h newly synthesized denitrifying enzymes ference in the physiology of this organism or might
become functional and the rate increases. At 4-8 h be related to a difference in habitat.
all denitrifiers are fully derepressed so each cell has Phase I and phase II rates measure completely dif-
attained its maximum capacity to denitrify (phase II). ferent factors in soil denitrification. Phase II rate
At this point denitrification rate can increase only probably corresponds to what is generally called deni-
by an increase in number of cells; significant growth trification potential e.g. Balasubramanian and Kane-
will occur only when the supply of available organic hiro (1976). Our results indicate that this rate is a
carbon is large. These events and their relationship function of the number of soil denitrifiers and the
to the phases observed are summarized in Table 3. limitations imposed on them by the energy and elec-
It should be noted that phases IIa and IIb were tron acceptor supply, pH, and temperature. These fac-
referred to as phases II and III in an earlier brief tors are often altered by the assay conditions. We
summary (Tiedje et ul., 1978). The earlier designation have shown that addition of an energy source can
has been changed to avoid the implication that there have an effect on the rate and the pattern in phase
are always three distinct phases. II. It is also probable that moistening of a completely
The evidence for this explanation of the phases can dry soil has effects similar to adding an energy source.
be summarized as follows: first, various methods of Denitrification potential has been shown to correlate
rapidly removing 0, had no effect, indicating that very well with mineralizable carbon (Bremner and
residual 0, could not account for the lower initial Shaw, 1958). Phase I, in contrast to phase II or deni-
rate. Chloramphenicol reduced phase II denitrifica- trification potential, is sensitive to the aeration state
tion rates but not phase I, suggesting that the increase of the native soil. Phase I is essentially an assay of
was due to enzyme synthesis. The small initial stimu- the cellular denitrifying enzyme activity present in the
lation by glucose relative to subsequent stimulation sample and reflects the immediate biological effect of

Table 3. Generalized description of the phases of soil denitrification after the imposition of
anaerobic conditions

Generalized
time interval
(h)
Characteristics
Phase Start End of rate Explanation

I 0.25 l-3 Constant Activity of pre-existing

enzymes
Transition 1-3 4-8 Increasing Active derepression of
enzyme synthesis
IIa +8 Indefinite Constant Indigenous community
(low available fully derepressed, no
organic carbon) significant growth
IIb 48 Indefinite Logarithmically Growth of denitrifiers
(high available increasing
organic carbon)
266 M. SCOTT SMITH and
changes in soil moisture and aeration~xtremely im-
portant factors for soil denitrification. Therefore,
phase I rate appears to provide more information
about in situ denitrifying activity than does denitrifi-
cation potential and should be a more useful
approach to the study of soil denitrification.
The importance of derepression of denitrifying
enzyme synthesis in nature, as well as in laboratory
incubations, is indicated by the increase in phase I
rate of soil during field irrigation. Also when water
was added to aerobic soils a pattern was observed
similar to that in anaerobic incubation, that is, a low
initial linear rate was followed by an increase in deni-
trification rate. It is tempting to attribute this pattern
to the same causes in aerobic and anaerobic incuba-
tions, Although derepression is undoubtedly involved,
other factors must be considered in the aerobic ex-
periments and in field soils. The time required for
the generation of anaerobic microsites accounts for
the longer initial phase in the aerobic experiments.
The difference between anaerobic phase I and phase
II was usually less than an order of magnitude, while
the rate increase subsequent to wetting of aerobic
soils was significantly greater. This appears to be due
to removal of O2 inhibition of existing denitrifying
enzymes which would be expected to result in much
greater rate increases than would derepression of
enzyme synthesis.
Our results demonstrate the value of working with
simplified soil systems, in this case stirred anaerobic
suspensions. We have been able to isolate and identify
the biological effects of reduced soil aeration by con-
trolling physical variables, particularly O2 and sub-
strate diffusion.
Our data provide some information about when
and under what conditions denitrification could be
expected to occur. We have demonstrated that deni-
trifying enzymes are present even in very dry soils.
Since denitrifying enzymes are not constitutive (Payne.
1973) this implies that either denitrification can occur
z
=: MIAMI SANDYLOAM
: 0.2
2
f
4
0 c
0 -_;._
0 200
lncubatlon
Fig. 5. Lag in denitrification response to adjustment
cates. Eighteen per cent gravimetric water content is 517; o f maximum
approximately field capacity; 33”/b water is 94 “/:, of maximum water holding capacity or approximately
saturation.
JAMES
M. TIEDJE
in anaerobic microsites of well aerated soils or that
denitrifying enzymes may persist in functional form
in the presence of OZ. We have also observed evolu-
tion of N,O from the Miami sandy loam at IV{,
water content in an aerobic atmosphere. If it is
assumed that the rates observed in our assays are
similar to field rates, then a gross approximation of
field N loss can be made. This is done by assuming
that the net ffux of NzO from the soil in the tubes,
i.e., the rate of accumulation in the headspace divided
by the soil surface area, is equal to the flux of N
from the soil in the field. Thus, the rate of N loss
from the drier Miami sample (Fig. 5) would be low,
69mg N.ha-‘.day-‘. Some well-aerated soils we
assayed aerobically evolved N20 at significantly
greater rates, up to 6 g N.haa’.day-‘. These “aero-
bic” rates are too small to be significant in the N
economy of agricultural soils. Yet it is interesting that
N20 evolution was observed from virtually all of the
well-aerated soils examined. We have not yet demon-
strated what fraction of this N,O results from biologi-
cal denitrification; several other mechanisms are poss-
ible. Bremner and Blackmer (1978) have suggested
that nitrification causes tow rates of N20 producti(~n
in aerobic soils, presumably by a process observed
earlier in Nirrosomonas cultures (Yoshida and Alex-
ander, 1970). Non-biological reactions of NO; also
might contribute to N20 production. It is also poss-
ible that the soif aggregates were super-saturated with
N,O. Denitrifying conditions in the field well before
sampling might have caused accumulations of N,O
within aggregates which slowly equilibrate with the
atmosphere. The relative contribution of each of these
mechanisms will be difficult to determine. Preliminary
experiments were not revealing due in part to varia-
bility within and among soil samples, and in part to
the difficulty of experimentally isolating the various
mechanisms of N20 production.
In summary, nitrogen gases can be produced more
or less continuously by soils. Several mechanisms
I8 % water
400 -600 800
Time (min)
of water content. Points are means of five repli-
water holding capacity or
 Phases of denitrification in soil 261

could contribute to this, but only denitrification in BROADBENT F. E. and CLARK F. E. (1965) Denitrification.
wet soils is likely to result in large N losses. Sub- In Soil Nitrogen. (W. V. Bartholomew and F. E. Clark,
sequent to a reduction in soil aeration, denitrification Eds), pp. 344-359. American Society of Agronomy,
Madison.
rate is greatly increased, but only after a lag of several
FOCH? D. D. and VERS~RAETEW. (1977) Biochemical eco-
hours. Anaerobic or partially anaerobic conditions,
logy of nitrification and denitrification. In Adcances in
established by respiration and reduced O2 diffusion Microbial Ecology. (M. Alexander, Ed.), pp. 135-214.
rate, eliminate O2 inhibition then derepress the syn- Plenum Press, New York.
thesis of denitrifying enzymes. Thus we reason that PAYNE W. J. (1973) Reduction of nitrogenous oxides by
most nitrogen loss would occur during brief periods microorganisms. Bacf. Reo. 37, 409-452.
beginning a few hours after irrigation or a rainfall PAYNE W. J. and RILEY P. S. (1969) Suppression by nitrate
and lasting until NO; is depleted or the soil water of enzymatic reduction of nitric oxide. Proc. Sot. exp.
content decreases. Biol. Med. 132, 258-260.
PILOT L. and PATRICK W. H. (1972) Nitrate reduction in
soils: Effect of soil moisture tension. Soil Sci. 114,
Aclinowledgemenrs~We thank Mary Firestone for a por- 312-316.
tion of the chloramphenicol work and her ideas on devel- ROWE R., TODD R. and WAID E. J. (1977) Microtechnique
opment of the phases concept particularly during the in- for most-probable-number analysis. Appl. entlir. Micro.
itiation of this project. This work was supported by a 33, 675-680.
National Science Foundation Grant and USDA Regional SMITH M. S., FIRESTONE M. K. and TIEDIE J. M. (1978)
Research Project NE-39. Published as Journal Article No. acetylene inhibition method for short-term measurement
8604 of the Michigan Agricultural Experiment Station. of soil denitrification and its evaluation using 13N. Soil
Sci. Sot. Am. J. 42, 611-615.
SNEDECOR G. W. and COCHRAN W. G. (1967) Statistical
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