Ham's

This is to certify that the
thesis entitled

TRANSFORMATIONS OF 15N LABELED AMMONIUM SULFATE AND
UREA FERTILIZERS IN SOILS (WITH SPECIAL REFERENCE
TO EARLY IMMOBILIZATION AND NITRIFICATION PROCESSES)

presented by

Gilbert Uwahamaka Okereke

has been accepted towards fulfillment
of the requirements for

Ph.D. Crop and Soil Sciences

degree in

 

 

 

V, A .
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Major professor

Date August 6, 1980

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TRANSFORMATIONS OF 15N LABELED AMMONIUM SULFATE AND
UREA FERTILIZERS IN SOILS (WITH SPECIAL REFERENCE TO EARLY
IMMOBILIZATION AND NITRIFICATION PROCESSES)

By

Gilbert Uwahamaka Okereke

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Science

1980

ABSTRACT

TRANSFORMATIONS OF 15N LABELED AMMONIUM SULFATE AND UREA
FERTILIZERS IN SOILS (WITH SPECIAL REFERENCE TO EARLY
IMMOBILIZATION AND NITRIFICATION PROCESSES)

By

Gilbert Uwahamaka Okereke

The primary objective of the investigation was to provide evidence
that nitrogen (N) immobilization occurs within a matter of hours after
application of N fertilizers. The secondary objective was to study some
of the factors that influence early immobilization and nitrification of
applied labeled fertilizers in soils. The investigation was therefore
conducted as a laboratory incubation study under controlled conditions to
obtain a comparison between the rate of immobilization and nitrification
of ammonium sulfate (NH4)2804 and urea fertilizers applied to organic
soils and arable and forest mineral soils. Fertilizers labeled with 15M
were used to distinguish organic-N and NOB-N produced by immobilization
and nitrification, respectively, of fertilizer N from the soil organic-N
and N03-N already present. Nitrogen immobilization was measured by the

15

appearance of N in the organic fraction while nitrification was

measured by the appearance of 15N in the soil N03- fraction.
Comparison of immobilization and nitrification processes in the

soils show that marked differences may exist in the ability of the

Gilbert U. Okereke
various soils to immobilize and nitrify labeled fertilizers when
evaluated under comparable conditions. For the lower pH organic soil,
10.4% of the labeled (NH4)2504 fertilizer was recovered as organic-N
while for the lower pH arable mineral soil and forest mineral soil, 1.4%
and 5.7%, respectively,were recovered as organic -N after 12 hours of incu-
bation. Application of labeled urea resulted in immobilization of 8.6% and
1.8% of the applied urea fertilizer in the lower pH organic soil and the
lower pH arable mineral soil, respectively, after 12 hours incubation.
After the same period of incubation, 12.4%, 2.3% and 1.4% of the applied
labeled (NH4)2504 was nitrified in the lower pH organic soil, lower pH
arable mineral soil and forest mineral soil, respectively. Addition of
urea caused l5.3% and 2.1% of the labeled urea fertilizer to be nitrified
in the lower pH organic and lower pH arable mineral soils, respectively,
These data indicate that early immobilization and nitrification oflSN
labeled (NH4)2504 and urea fertilizers does exist.

In an effort to further study the influence of pH and
readily-available carbon (C) on microbial activity and subsequent H
transformation, several additional experiments were conducted. Changes
in pH due to the addition of a fertilizer source exercised a considerable
effect on the rate and amount of N immobilized and nitrified. In general,
a higher pH favored immobilization and nitrification. Immobilization of
urea fertilizer in the presence of readily available glucose-C occurred
immediately and was a rapid process. In the presence of glucose, the net
immobilization was 97.0% in the lower pH (pH 5.7) arable mineral soil.
For the higher pH (pH 6.5) arable mineral soil, addition of glucose—C

caused 88% immobilization compared to 6. % in unamended soil while in the

Gilbert 0. Okereke
higher pH (pH 6.8) organic soil, 90% was immobilized by the glucose
amended soil compared to 26.0% in unamended soil. After addition of
glucose-C and incubation for 96 hours, the net nitrification was 10.3% of
the applied urea-N in the higher pH (pH 6.8) organic soil while in the
lower and higher pH arable mineral soils, it approached zero. These data
show that the state at which N is found in the soils studied is
determined to a high degree by the presence or absence of a

readily-available C source, i.e. energy.

TO MY WIFE AND CHILDREN

This thesis is dedicated to my beloved wife, Victoria
and children for their courage, prayers and

encouragement all these years.

ii

ACKNOWLEDGEMENTS

The writer is indebted and very grateful to his major Professor, Dr.
V. w. Meints for patient assistance in completion of this study and in
preparing the manuscript. He is particularly grateful to Dr. 8. G. Ellis
and Dr. V. w. Meints who made arrangements for financial assistance for
this study.

Special thanks are due to Dr. B. D. Knezek who was instrumental in
arranging for initial financial assistantship for a graduate study
(Ph.D.) at Michigan State University. He wishes to express his
appreciation to Dr. J. Hart and Dr. B. G. Ellis for their enlightening
contribution to the preparation of this manuscript. He is also thankful
to Dr. J. M. Tiedje for the opportunity to use his Isotope Ratio Mass
Spectrometer and his contribution to this study.

He is grateful to the International Fertilizer Development Center in
Muscle Shoals, Alabama for financial support which enabled the writer to
prepare for, initiate and complete this study.

Appreciation goes greatly to his wife Victoria for continuous

support and care for his children during his research.

iii

TABLE OF CONTENTS

LIST OF TABLES .

LIST OF FIGURES

INTRODUCTION

LITERATURE REVIEW .
Immobilization
Nitrification
Ureolysis .

MATERIALS AND METHODS
Soils
Soil sampling and characterization
Incubation and extraction
Fractionation of soil N

Extraction of exchangeable NH

NO3-N from incubated soils

Extraction of urea .

4-N, NOZ-N and

Distillation procedures for analysis of various soil
N fractions . . . . . . .

Determinations of organic-N and nonexchangeable NH4-N
Total Kjeldahl hydrogen flouride-N

Total Kjeldahl N (TKN)

Analysis of 15N in samples

15

Preparation of distillates for N analysis .

iv

Page

vii

ix

12
14
14
16
16
18

I9
20

20

22

22
23
23

15N analysis in V. G. Micromass

622 isotope ratio mass spectrometer .
Calculations of results . . . . .

Atom percent 15

N in samples
Correction for isotOpe dilution calculation

Calculation of fertilizer N in different
soil N fractions

Statistical analysis .

RESULTS AND DISCUSSION

Preliminary experiment to determine NH4+ fixation
capacities of soils . . . . . .

Overall recovery of tracer N

Transformations of (NH4)2504 and urea fertilizers
in soils . . . . . . . . .

Ammonium sulfate transformations in soils
Urea transformations in soils

SO

Comparison of immobilized 13N labeled (NH4)2 4

and urea fertilizers in soils

Influence of changes in soil pH on immobilization of
labeled (NH4)2504 and urea fertilizers

Influence of initial pH of a soil on immobilization of
labeled urea fertilizer in soils - . .

Effect of addition of glucose-C on immobilization of
labeled urea fertilizer in soils - -

Agronomic implication of immobilization in the presence
of easily available carbonaceous material -

Effect of fertilizerlscurce and initial soil pH on
nitrification of .1 labeled (NH4)ZSO4 .and urea
fertilizers in soils

Page

24
26
26
27

28
29

33

33
33

37
37
43

45

49

52

53

59

63

Effect of addition of glucose-C on the rate of
nitrification of urea fertilizer
SUMMARY AND CONCLUSIONS
RECOMMENDATIONS FOR FUTURE RESEARCH
BIBLIOGRAPHY
APPENDICES
A. Mean and standard deviations of 15Nlabeled
fertilizers immobilized during laboratory
incubation period
8. Mean and standard deviations of 15M labeled
fertilizers nitrified during laboratory
incubation period
C. Linear regression coefficients relating
percent (H SO fertilizer
immobilized 4dari‘rig different time intervals
0. Linear regression coefficients relating percent

urea fertilizer immobilized during different
time intervals .

vi

Page

70

73

75

76

84

84

85

86

87

Table

LIST OF TABLES

Chemical and physical prOperties of soils used.

Clay fixation of labeled (NH
in soils.

4)2804 fertilizer

Total recovery of N added as (NH4)ZSO4 and urea
fertilizer applied to soils. .

Changes in pH due to addition of labeled urea and
(NH4)ZSO4 fertilizers.

Immobilization of 15N labeled (NH4)2304

and urea fertilizers after 12 hours
incubation.

Comparison of rate of immobilization of fertilizer

N in the presence and absence of glucose-C.

Nitrification of 15N labeled (NH4)ZSO4

and urea fertilizers after 12 hours
of incubation.

Comparison of rate of nitrification of
fertilizer-N in the presence and

absence of glucose-C.

vii

Page

15

31

32

42

44

57

62

68

Table Page

9. Net nitrification and immobilization of fertilizer
N in the presence and abscence of glucose-C - - - - .59

viii

LIST OF FIGURES

Figure

1.

7.

Transformations of 15N labeled (NH4)ZSO4

and urea fertilizers in soils.

15

Fractionation of soil N for N analysis.

Sample preparation and le analysis in IsotOpe
Ratio Mass Spectrometer.

Changes in NH4-, N03- and organic-N after
addition of (NH ) SO to soil M818.
4 2 4
Changes in NH -, N03- and organic-N after

addition of labeled (NH SO to soil Nl

4)2 4

Changes in NH -, N0 - and organic-N after
addition of labeled (NH4)2504 to soil F2 ,

Changes in urea-, NH4-, N03-, and organic-N after
addition of labeled urea fertilizer to soil N1.

Changes in NH4-, NO3- and organic-N after
addition of labeled urea fertilizer to soil NZ.

ix

Page

17

25

34

35

36

38

39

Figure Page

9. Changes in urea-, NH -, N03- and organic-N after
addition of labeled urea fertilizer to soil MBl8. . - - 40

l0. Changes in NH4-, N03- and organic-N after addition
of labeled urea fertilizer to soil MClS. . . . . . . 41

ll. Comparison of immobilized labeled (NH4)2504 fertilizer
in various soils . . . - 46
l2. Effect of fertilizer source on immobilization of

fertilizer N in soils Nl and MBl8. . . . . . . . . 47

TB. Influence of initial soil pH on immobilization of
labeled urea fertilizer in various soils . - - - - - 5l

l4. Effect of addition of glucose-C on immobilization
of labeled urea fertilizer in soil W1 . . . . . . . 54

l5. Effect of addition of glucose-C on immobilization
of labeled urea fertilizer in soil W2 . . . . . . . 55

l6. Effect of addition of glucose-C on immobilization
of labeled urea fertilizer in soil MClS. . . . . . . 56

T7. Effect of fertilizer source on nitrification of labeled
(NH4)ZSO4 and urea fertilizers in various soils . . . .60

18. Effect of initial soil pH on nitrification of labeled
urea fertilizer in various soils . . . . . . . . .5]

19. Effect of addition of glucose-C on nitrification of
labeled urea fertilizer in soil Nl. . . . . . . . .55

Figure Page

20. Effect of addition of glucose-C on nitrification of
labeled urea fertilizer in soil NZ. . . . . . . . 66

21. Effect of addition of glucose-C on nitrification of
labeled urea fertilizer in soil MCIS. . . . . . . 67

xi

INTRODUCTION

It has been well established that nitrogen (N) is an essential plant
nutrient; thus, the series of transformations of applied fertilizer N is
very important in plant nutrition.

The application of fertilizer N in agriculture and forestry has
increased recently without quantitatively assessing the efficiency,
economy and environmental effects of these increasing rates of N
application. A clearer understanding of fertilizer N transformations in
soils, especially immobilization and nitrification, are therefore
needed.

Most studies in this area have been conducted with N fertilizer
added to mineral agricultural soils. To my knowledge, little such work
has been done using organic soils or forest mineral soils. Organic soil
constitutes an important potential soil reserve in Michigan and other
parts of the world. There are 4-1/2 million acres of organic soils in
Michigan of which less than 5% is farmed. (Davis and Lucas, 1959).
Forest soils also represent a major soil reserve since 50.7% of Michigan
is forested (Somers, 1977). Forest soils have an accumulation of organic
N which results from N cycling and forest floor development. The N
transformation activities are closely associated with forest soil
productivity as it is influenced by forest succession, stand development,

forest fertilization, harvesting, weed control and site preparation

 

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activities.

Although immobilization does not represent loss of N from the soil,
it does compete with plant uptake. 0n the other hand, it reduces
volatilization, denitrification and Teaching of the applied N
fertilizer.

Nitrification, the microbial oxidation of ammonium (NH4+) to nitrite
(N027) and nitrate (N03-),is of great environmental significance for many
reasons. Except for fertilizers and certain chemical reactions that form
N03' from oxides of N in the atmosphere, biological nitrification is the
sole natural source of N03- in the biosphere. The importance of the
process is that it produces an oxidized form of N which may participate
in denitrification reactions, resulting in the loss of readily available
N from the soil environment.

Under conditions favoring a high degree of microbial activity, the
addition of inorganic N to soils causes an adjustment in the equilibrium
of the system resulting in an interchange between inorganic and organic
forms of N (Kirkham and Bartholomew, 1955). The extent of the adjustment
depends on soil microorganisms, C/N ratio, temperature, moisture, pH,
etc. (Hideaki, et al., 1969).

A diagram showing some of the recognized processes involved in
fertilizer N transformations under aerobic conditions is presented in
Figure l. The diagram shows that immobilization and nitrification make
use of the same substrate with different end products.

The primary objective of the investigation was to provide evidence
that N immobilization occurs within a matter of hours after application
of N fertilizers. The study was modified to provide information on the

relationship between immobilization and other competing processes which

utilize inorganic soil N. A knowledge of the magnitudes of these
competing processes is essential in determining the availability of N
applied as fertilizer to plants. The secondary objective was to study
some of the factors that influence immobilization and nitrification under
laboratory incubation conditions.

The investigation was conducted as a laboratory incubation study
under controled conditions to obtain comparison between the rate of
immobilization and nitrification of labeled ammonium sulfate [(NH4)2504]
and urea fertilizers.

Nitrogen immobilization was measured by the appearance of 15N in the
organic fraction. Nitrification was measured by the appearance of 15N in

the soil N03- fraction.

LITERATURE REVIEW

Immobilization

 

"Immobilization" generally denotes the process of conversion of
inorganic N to the organic form during decomposition (Hutchinson and
Richards, 1927).

Bierema (1909, cited by Jansson, 1958) was the first to establish
that a variety of micororganisnc present in soils were able to immobilize
NH4- and NO3-N. Initially, this immobilization of N was regarded as
being detrimental to cr0ps as they consumed N that could be available to
plants. Further research gradually accumulated evidence that showed that
the detrimental effect was temporary and changed into a favorable
effect.

Tracer and non-tracer experiments reviewed by Allison (1966)
indicate average recoveries of fertilizer N under field conditions in a
single harvest to range between 50 and 70%. A review by Kundler (1970)
of research utilizing 15H- labeled fertilizers reports first year
recovery in the crop of 30 to 70% with 10 to 40% retained in soil, 5 to
10% removed by leaching and 10 to 30% unaccounted for and presumed lost.
Similar ranges of values were reported in a review by Hauck (1971) in
which N balance data are given for different cropping systems and
experimental methods. Studies with 15N-labeled fertilizers demonstrate
that much of the fertilizer N not recovered by the initial.cr0p becomes
immobilized and slowly available to succeeding crops (Legg, et al., l97l)

5

Several investigators in recent years have indicated that rapid
interchange between added fertilizer N and organic N in soil occurs.
According to Stewart, et al., (1963) most of the N initially immobilized
appears in the amino acid fraction. Low plant recovery of tagged N
fertilizer in several greenhouse and field experiments is evidence that a
substantial part of a N fertilizer application becomes at least
temporarily unaviailable to crops (MacVicar, et al., 1950; Bartholmew, et
al., 1950; and Bartholomew, 1957). Tyler and Broadbent (1958) used

15N which had been immobilized

successive crops of ryegrass to recover
into organic form and found that uptake of fertilizer N was substantially
greater from newly synthesized organic forms than would be expected if
this N had been uniformly diluted with the total N present. Jansson
(1958) and Tyler and Broadbent (1958) have suggested that a small portion
of the total soil organic matter may be acting as an "active" fraction.
This "active" fraction is only a minor fraction of the soil N and a large
portion of immobilized fertilizer N will enter this fraction.

Broadbent (1966) and Broadbent and Nakashima (1965) showed that if
the level of soil inorganic N is measured during a period when conditons
favor net immobilization, there is at first a rapid decrease followed by
a somewhat more gradual increase in soil N. The length of the
intervening period of N depression may vary from a day or two (Broadbent
and Tyler, 1962) to several months (Bartholomew, 1965) depending on the
nature of the organic matter undergoing decomposition in soil.

Work with 15N-tagged fertilizer, however, indicates that highly

stabilized forms of organic N are produced in relatively short periods

(Legg and Allison, 1967). Yoneyama and Yoshida (1977) found that under

lowland conditions, the amount of N immobilized was small during the
first week, but became large after two or three weeks. Nhile under
upland conditions, the immobilized N reached its maximum during the first
week, but the amount was not as large as under lowland conditions.
Broadbent and Tyler (1962) found that immobilization attained a maximum
in 6 to 10 days after addition of tracer N to two different soils.

A number of environmental conditions have been shown to influence
the rate of N immobilization and subsequent mineralization. The
influence of moisture on N tie-up was studied by Jansson and Clark
(1952). Their data showed that more accumulation of N in organic form
occurred during a 10 day incubation period under a moisture content of
1/6 of the saturation capacity than at full saturation.

In soil, the optimum moisture content for decompositon of C was
reported by Stotklassa and Ernst (1907) to be about 50 percent of the
saturation capacity. Bollen (1947), on the other hand, reported the
optimum to be at 75 percent of the saturation capacity. De and Digar
(1954) reported that at the higher moisture contents, especially in
waterlogged soil, N is generally lost in a gaseous form.

Norman (1931) studied the effect of H+ ion concentration on the rate
of immobilization of N and found that the process was more rapid under
slightly alkaline conditions and ultimately more N was retained. He
attributed the observed effects to differences in character of the active
microflora induced by changes in pH. Nommick (1968) found that liming an
originally acid raw humus substantially increased immobilization of
mineral N.

Richards and Shrikhande (1935) were the first to demonstrate that
there is preferential utilization of NH4+ over N03- by heterotroohic

microorganisms in the decomposition of straw.

Bremner and Shaw (1955), Broadbent and Norman (1947) showed that
application of carbonaceous materials to the soil with or without N tied
up the N and that its release occurred after a time.

Waksman (1924) studied the immobilization of N from decomposing
plant residue and concluded that organic materials with N content of 2.0
to 2.5 percent or more tend to decompose with the immediate production of
NH3, whereas materials with less N will show a lag period before N
liberation; or in the case of low N residues will fail to liberate any

NH because all the N will be immobilized by the microorganisms which

3
carry out the decomposition.

Net immobilization of N during the process of plant residue
decomposition has been shown to reach a maximum very quickly after
addition of a C source to soil, (Allison and Klein, 1962, Windsor and
Pollard, 1956). They concluded that the N requirement in the
decomposition process depended on composition of the materials, on
environmental conditions which affect the nature of the microflora and
rate of decay, and on the time of incubation. In this regard, Hiltbold,
et al., (1950) noted that two to four times more N was immobilized in
cr0pped than in fallowed soils. In a N balance field study, it was
observed that considerable applied N was immobilized by microorganisms
during the growing season (Kissel, et al., 1976).

A soil perfusion technique was used by Lees (1948) to study
immobilization of NO3-N in the presence of organic compounds. With
glucose and sucrose, immobilization of N reached its maximum in two to
four days. The relationship between C/N ratio and N transformations in

the soil is influenced by the ease of decomposition of the various

constituents present (Rubins and Bear, l942).

Allison (1927) noted that an immediate harmful effect resulted from
adding materials of a wide C/N ratio to soil. However, the ultimate
effect of this action was beneficial provided sufficient time was allowed
for the N03' supply to return to normal. He attributed these results to
a temporary increase in biological activity followed by a slowing up of
this activity until a point was reached where the proteins assimilated in
microbial cells were made available to plants through their death and the
subsequent ammonification and nitrification of the microbial remains.

Nitrification

 

The classic forerunner of all studies in this field was by
Schloesing and Muntz (1877, cited by Jansson, 1958). After demonstrating
the microbial nature of NH4+ oxidation, these workers went on to show
that almost as much nitrification occurred in soils at an oxygen (02)
concentration of 11 percent as at 21 percent. Work done by Gainey and
Metzler (1917) explored the relationship between 02 concentration and
nitrification. Their results substantiated those of Schloesing and Muntz
(1877) and showed that soil air does not vary greatly in composition with
depth in the profile. They concluded that conditions are rarely met
where there is not sufficient 02 potentially available to insure maximum
nitrification.

Studies by Amer and Bartholomew (1951) confirmed the observation
that decreasing the 02 concentration from 20 to 11 percent has a
negligible effect on the nitrification rate. They also stated that a
minimum level of 02 concentration exists somewhere between 0.2 and 0.4
percent where nitrification does not occur. The partial pressure of 02

in the soil air is rarely more than 1 or 2 percent lower than that in the

IO

atmOSphere, except in water-logged soil or in heavily compacted subsoils.
Nitrification is evidently an aerobic process since it does not occur in
the absence of 02. The process is, however, less sensitive to conditions
of a limited 02 supply than would be expected (Woldendorp, 1975).
Nitrifying bacteria can survive for long periods in anoxic environment
even though they cannot grow (Painter, 1970).

Nitrosomonas has a broad pH optimum which may vary with strain, but

 

usually lies between pH 6.0 and 9.0 (Engel and Alexander, 1958).

Nitrobacter strains have optima between pH 6.3 and 9.4 (Winogradsky and

 

Winogradsky, 1933). Chen, et al., (1972) found no appreciable
nitrification in acid, soft water lake sediment.

)co.

Upon hydrolysis, urea is converted to ammonium carbonate (NH 3

4
This leads to a high concentration of NH4+ and a rise in pH near the
fertilizer application. These conditons adversely affect the activity of
Nitrobacter Spp. and an accumulation of N02' may result (Aleem and
Alexander, 1960; Alexander, 1965; and Pang, et al., 1973). Such
accumulations of N02' have been reported to result in losses of N from
the soil system through chemodenitrification (Broadbent and Clark, 1965;
Reuss and Smith, 1965). In acid soils, the increased alkalinity can
stimulate nitrification (Stojanovic and Alexander, 1958). Turk, (1939),
showed that the nitrifying capacity of two acid soils (pH 4.3 and 3.4)
was increased about fourfold by liming.

Temperature has a marked effect upon nitrification. Generally

higher temperatures accelerate nitrification. Nitrobacter seem to be

 

 

more sensitive to cool temperatures than Nitrosomonas since N02"
accumulates more at 5°C than at 25°C (Gasser, 1964). Urea hydrolysis is

also temperature sensitive. Hydrolysis increases as the temperature

11

increases (Broadbent, et al., 1958 and Fisher and Parks, 1958) and at
10°C hydrolysis may be the rate limiting step in the nitrification of
urea (Fisher and Parks, 1958).

Soil water content has a significant effect upon nitrification.
Nitrification is reported to be an almost linear function of water
content between 0.2 and 15 bars of soil moisture tension (Reichman, et

al., 1960). Nitrification of 150 ppm NH -N in a slightly basic loam soil

4
was completed in about 56, 28, 20 and 12 days at 15, 10, 7 and 1 bars
water tension, respectively (Justice and Smith, 1962).

Nitrifiers are mostly autotrOphs and it is probable that they are
not sensitive to growth inhibition by small organic molecules, which is
not surprising because they generally exist in rich organic environment.
Characteristically, they adhere to particles, probably many of them
organic, in soil (Gray, et al., 1968). It is necessry for nitrifying
bacteria to be in a region where NH4+ is available and close to a zone of

organic decomposition. It has been shown that Nitrobacter is capable of

heterotrophic growth (Smith and Hoare, 1968). Nitrosomonas is also not a

 

true autotroph, in that its growth is stimulated by pyruvate and amino
acids, (Clark and Schmidt, 1967).

In most organic soils, a valuable product of organic matter
decomposition by bacteria is N03' (Davis and Lucas, 1959). The rate of
formation depends upon soil moisture, temperature, aeration and total N
content.

Roberge and Knowles (1966) found that over a 42 day period, there
was no appreciable NOB-N production in Black spruce humus. Total absence
of nitrification is unusual in agricultural soils (Broadbent, et al.,
1957) but has been reported by other workers, especially in forest soils

(Harmsen and Van Schreven, l955).

12

The PH optimum for N03 oxidation was shown to be over the range

6.8 to 8.2 in phosphate buffer (Silver, 1961). Similar results were also
obtained by Lees and Simpson (1957).

Lack of nitrification in a forest soil may be due to the absence of
nitrifying microorganisms rather than the presence of substances
inhibitory to nitrification (Nakos, 1975). The population of nitrifying
organisms is an important factor affecting the amount of nitrification
occuring in soils (Anderson and Purvis, 1955; Frederick, 1957). Theobald
and Smith, (1974) showed that soil incubation tests indicated that the

microflora of two forest soils had a weak capacity to produce N0 Their

3.
results agree with those of Maftoun and Pritchett, (1970) and Smith, et
al., (1971) who detected negligible amounts of nitrification in other
forest soils of the low coastal plain.
Ureolysis

Urea is unique among commonly used N fertilizer materials in that it
is an organic compound which generally requires an enzymatic hydrolysis
to make its N available to plants (More, 1967). Urea as a fertilizer has

been commonly considered to resemble N0 ' in its susceptibility to

3
leaching and to be like NH3 in transformations following hydrolysis
(Broadbent, et al. 1958). Gibson (1938) noted that although urea is not
absorbed by soils, its rapid conversion to NH3 will generally prevent
losses by leaching.

The N transformations occuring following addition of urea-N and of
(NH4)2C03-N (3,500 ppm) to the raw humus in laboratory experiments have
been reported by Roberge and Knowles (1966). They found that ureolysis

was complete after 2 days; that in spite of the high C/N ratio,

13

immobilization was small and that in most treatments, nitrification was
negligible. Hydrolysis would occur within 3 to 4 days or less under
favorable temperature conditions (Broadbent, et al., 1958).

Many micro-organisms possess the enzyme urease, the catalyst
responsible for hydrolyzing urea.

Bacteria, fungi and actinomwcetes synthesize urease and therefore

can use urease as an N source for growth (Alexander, 1977).

)

CO(NH2 2

+ H20 ___) HZNCOjJ HH4
+
2NH3 C02
Following hydrolysis of urea biochemical transformations begin

immediately. These are nitrification and immobilization of the NH4-N

produced.

MATERIALS AND METHODS

Soils

 

Soil samples were collected to represent agricultural mineral soils,
(W1 and NZ); a forest mineral soil, (F1); and organic soils, (M818 and
MC15). The agricultural mineral soils, W1 and NZ, were collected from
plots 40 and 24, respectively, of an experimental area formerly utilized
by Dr. A. R. Wolcott on the Michigan State Old Soils Farm. Soil WZ
received applications of dolomitic agricultural lime in the spring of
1965 and the spring of 1966. Annual applications of sodium nitrate
fertilizer at the rate of 136 kg N acre.1 yr.1 were made between 1959
and 1972. Soil W1 did not receive any lime and fertilizer during the
same period of time. Details about the history, fertilizer treatments
and crops grown in these plots are contained in an M. S. thesis by
Burutolu (1977). Soil F2 was collected from Baker Woodlot, a research
area under the supervision of the Department of Forestry, Michigan State
University. This area is predominantly a hardwood forest that has been
clearcut in the last 100 years. Tree species presently growing at the
sample site include beech, maple, red oak, black cherry and basswood.

Soils M818 and MC15 are muck soils collected from the Michigan State
University Muck Farm supervised by Department of Cr0p and Soil Sciences,
Michigan State University. Soil M818 was planted to potatoes which
received 68 kg N acre-1 as urea fertilizer in 1978. Soil MC15 was
planted to sod which received no fertilizer in 1978. In 1979, the sod

was plowed. 14

15

 

 

 

Table 1. Chemical and physical properties of the soils used.
Soils
Properties WI WZ F2 MClS MB18
Texture Sandy Sandy Sandy Organic Organic
Loam Loam Loam
pH 5.1 6.5 5.7 6.8 5.7
Classification Typic Typic Typic Typic Typic
Hapludalf Hapludalf Hapludalf Mediprists Medisa-
prists
Z Organic-N 0.05 0.07 0.13 2.15 2.12
Z Organic-C 1.4 1.2 3.2 45.0 47,4
C:N 29:1 17:1 24:1 20:1 22:1-
Ammonium-N(Ppm) 4.9 0.04 5.1 0.04 1.0
Nitrate-N(Ppm) 3.2 3.8 2.8 393.4 373.6
Z moisture at 1/3
atms. 15 14.8 17.0 158.0 175.0

 

16

Soil samplinggand characterization

 

Bulk samples of the t0p soil (0—15 cm) were collected from each site
in the fall of 1979, sieved (2 mm screen), thoroughly mixed in a small
cement mixer and stored in their moist condition in a cold room at 2°C
until used.

Some relevant physical and chemical prOperties of the soils are
summarized in Table 1. The method used to determine these are as
follows: The pH values refer to a water suspension (soilzwater ratio,
1:1 for mineral soils and 1:2.5 for organic soils). The field moisture
capacity of the soils was determined by use of pressure membrane plates
that were allowed to equilibrate at'a tension of 1/3 atmosphere for 48
hours after which the moisture content was determined gravimetrically.
The method used was a modification of a method used by Richards and
Weaver (1943). Organic C was determined using Leco carbon analyzer
(Model 750-100, Laboratory Equipment Corporation, St. Joseph, Michigan).
Ammonium-N and N03-N were determined by alkaline distillation of KCl
extract of the soil with MgO and Devarda's alloy,respectively, (Bremner
and Keeney, 1966).

Incubation and extraction

 

An incubation and extraction method was used that allowed
determination of various forms of soil N at regular intervals over an
extended period of time. Unless otherwise stated, the general procedure
was to add 2 mg N as labeled (NH4)ZSO4 (90.2 atom percent 15N) or urea

(96 atom percent 15

N) in solution to soil equivalent to 20 g of oven dry
soil weighed in duplicate or triplicate into 300 ml Erlenmeyer flasks.

Additions corresponded to 100 ppm N on the soil basis (2 mg N/20 g oven

17

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18 .

dry soil). Soils were adjusted to their l/3 atmosphere moisture
percentage, thoroughly mixed with a spatula and evenly distributed over
the bottom of the flask by tapping the flask gently. The flasks were
closed with cotton to allow gaseous exchange with the atmosphere and at
the same time control excessive evaporation of water from the incubated
soil. A constant temperature cabinet was used for incubation.
Preliminary study showed that by providing basins of water in the
incubation chamber during the incubation period, the moisture content on
a dry weight basis did not decline more than 1 percent per flask. The
flasks were incubated at 27°C for periods of either 0, 3, 6, 12, 24, 48,
96 and 120 hours or 0, 12, 24, 48, and 96 hours. Analyses were made
after each time interval.

In order to provide comparisons of urea transformations among
samples differing in pH but otherwise having similar properties, soil
samples with varied pH collected within the same general area were used
in one of the incubation experiments. The method of incubation was
similar to the one described above except that only labeled urea
fertilizer was applied to the soil.

Ammonium sulfate and urea transformations in soils in the absence
and presence of glucose were studied in much the same manner as described
previously, except that 4 mg glucose-C per gram soil was added to each
soil prior to addition of fertilizer solution.

Fractionation of soil N
A diagram showing the analytical procedure adopted for fractionation

of soil N into its components and for its subsequent 15

N analysis is
presented in Figure 2. The fractionation procedure adopted was based on

the fact that in soil systems, the problem of evaluating N fertilizer

19

transformations is complicated and better understood by analyzing all the
possible soil N fractions. It is known that NH4+ sources of N are
utilized preferentially by the heterotrOphic population, (Jannson et al.,
1955) and that an NH4+ source cannot be analyzed independently under
normal conditions since it is rapidly converted to N03'. Disappearance

of NH4-N cannot be used as a reliable estimate of immobilization in soils

+

capable of nitrification and fixation of significant quantities of NH4

into nonexchangeable forms. Hence, the initial experiments involved
determination of the various possible N fractions.
(a) Extraction of exchangeable NH,+-N, NOET-N and N0,'-N from incubated

soils

 

At the end of each incubation period, the soil samples were brought
out from the incubation chamber for extraction of the mineral N with 2N
KCl. The extraction and distillation procedures used were similar to
those described by Bremner and Keeney, (1966). Sahrawat, (1979) recently
confirmed that 2N KCl is the best extractant when recovery of a known
amount of NH4-N applied to soils was used as a criterion for evaluating
extracting efficiency.

The soil extracts were prepared by adding 200 ml of 2N KCl into the
entire contents of the 300-ml Erlenmeyer flask without subsampling, thus
avoiding possible sampling errors due to uneven distribution of inorganic
N. Then 0.8 ml of chloroform was added to the flasks before being
steppered and shaken on a mechanical shaker for one hour. Addition of
chloroform served to inhibit microbial transformations of inorganic N
forms. After shaking, the suspension was transfered to Buchner funnels
containing 11-cm Whatman No. 42 filter paper which had been moistened and

sealed firmly by suction on Buchner funnels mounted on 500-ml Buchner

20

flasks. The soil was washed three times with a total of 90 ml of 2N KCl
solution. Finally, the soil extract was transfered to 300-ml volumetric
flasks and the volume made up to mark. The extracts for use in the
determination of mineral N were stored in polyethylene plastic bottles.
Determinations were always performed immediately after extraction. The
remaining KCl extracts were stored at -20°C for possible recheck of
results.

The washed soils were immediately frozen in a mixture of dry ice and
acetone or liquid N. The frozen soils were stored in a freezer at -20°c
until dried in a freeze dryer (Repp Industries Inc., Gardiner, New York)
for 48 hours. After freeze drying, the soils were packaged and stored in
a freezer at ~20°C for subsequent determination of organic N and
nonexchangeable NH4-N.

Extraction of urea

 

In experiments involving treatment of the soil with urea, 20 ml of
2N potassium chloride - phenylmercuric acetate (2N KCl-PMA) solution was
added to each incubation flask containing the 20 9 soil. Data by Douglas
and Bremner (1970) showed that in the 2N KCl-PMA solution adopted for
extraction of urea from soils does not interfere with extraction and
steam distillation of exchangeable NH4+, N02“ or N03". Their data also
show that phenylmercuric acetate completely inhibits urease. A
preliminary study was conducted to prove the effectiveness of the
phenylmercuric acetate inhibitor before adopting it in the major
experiments. The rest of the procedure was the same as that used above
for extraction of mineral N in the soils treated with (NH)2504.

Distillation Procedure for Analysis of Various Soil N Fractions

 

The distillation apparatus used was designed to take a

micro-Kjeldahl distillation flask (100 or 250 ml capacity). The

21

apparatus was a modification of the one shown by Bremner and Edwards
(1965). The distillation procedure was that of Keeney and Bremner (1966)
and (1967). The steam generation rate during distillation was adjusted
to 7 to 8 ml distillate per minute and the water flow through the
condenser was regulated to ensure that the temperature of the distillate
did not exceed 22°c. Plastic tubes were connected to each steam outlet
such that each distillation tube was approximately 4 mm from the bottom
of the distillation flask. Preliminary experiments were carried out to
determine the percentage recovery of known amounts of NH4-N before use in
the distillation of experimental samples.

Before distillation of any batch of samples, 30 ml of ethanol was
first distilled through the apparatus for 10 min. to remove any traces of
NH4+
analyses 20 ml of each soil extract were pipetted using wide tip pipettes

present in the distillation apparatus. For most of the mineral N

into 100-ml distillation flasks made alkaline with 0.2 g ignited M90.
The NH3 was distilled into 10 ml of 2% boric acid solution mixed with
bromocresol green-methyl red indicator contained in 125 ml Erlenmeyer

flasks. The N03'- plus N02 -N was determined by addition of 0.2 g of
Devarda's alloy to the same flask after the removal of NHZ-N and
distilling the NH3 produced into 10 ml of 2% boric acid indicator
solution. Urea-N was determined colorimetrically according to the method
of Douglas and Bremner (1970).

The distillation for all forms of mineral N was stopped after
collecting about 35 ml of the distillate. Cross contamination of samples

which can lead to serious error in 15

N analyses was prevented by
detaching the sample flask and immediately distilling 15 ml of ethanol

into the receiver flask up to the 65 ml mark. To further reduce any

22

potential errors, samples expected to have low 15

N content were always
distilled before those with expected high 15N content. Titrations were
carried out (to determine the NH4-N in the distillate) with 0.005N or
0.025N H2504 from a 5 ml microburette graduated at 0.01 ml intervals.
For organic N, the higher concentration of H SD was used for titration.

2 4
Determination of organic-N and nonexchangeable NH41N

 

Total N in the soil samples from which NH4-N, urea-N, N03-N and

NOZ-N had previously been extracted was determined by a modification of

the Kjeldahl procedure (Stewart and Porter, 1963, Meints and Peterson,

1972). The N extracted by this method is referred to in this thesis as

+
4

(fixed) was determined by the procedure of Silva and Bremner (1966).

total Kjeldahl hydrogen flouride-N (TKHF-N). Nonexchangeable NH

Organic N in the soil was obtained by subtracting nonexchangeable NH4-N
from the TKHFN.
Total Kjeldahl-hydrogen floride—N

This method involved pretreatment of soil samples with an HF-HCl
acid mixture before the normal Kjeldahl digestion. One gram of the
mineral soils or 0.2 g of the organic soils and 20 ml of 5N HF-IN HCl
solution were shaken in a centrifuge tube for 24 hours on a mechanical
shaker. The mixture (both soil and solution) was then transfered into a
100-ml Kjeldahl digestion flask. The catalyst mixture (100 g of K2304,
10 g of CuSO4 .SHZO, and 1 g of Se) and concentrated HZSO4 (4 ml) were
added to the flask. A few drops of octyl alcohol were added to prevent
bumping and frothing during digestion. The samples were digested for
three hours (one hour after clearing). After completion of digestion 10
ml of water was added to the flask to bring any insoluble material into

suspension. The contents were transfered into a 300-ml Kjeldahl flask

23

marked to indicate a volume of 60 ml. The digestion flasks were
subsequently washed three times with about 20 ml of water to complete the
transfer. Water was added to the flask to bring it up to the 60-ml mark.
The digested soil was distilled with 13N NaOH (20 ml of cold 50% NaOH)
into 10 ml boric acid indicator solution contained in a 125-ml Erlenmeyer
flask. The amount of N in the distillate was determined by titration
with H2504 as previously described.

Total Kjeldahl N (TKN)

 

Because the soils used had relatively low NH4-N fixing capacities,
TKHF-N and nonexchangeable NH4-N determinations were discontinued after
the first group of experiments. For the rest of the experiments
organic-N to include NO3-N and NOZ-N were determined according to the
method by Bremner (1965). The procedure was a micro-Kjeldahl method that
included N03' and N02' using one tenth of the amounts of soil and
reagents recommended by Bremner in his regular macro-Kjeldahl methods.
The size of soil sample was 0.2 gm for organic soil and l or 2 g for
mineral soils. Four milliliters of concentrated H2504 and 1.5 9 catalyst
mixture were used with a digestion time of 6 hours. Titration of the
distillate was the same as previously described.

15

ANALYSIS OF N SAMPLES

 

15N analysis

Preparation of distillates for
The distillate from each N determination was prepared for mass
spectrometer analysis according to the procedure described by Porter and
O'Deen (1977). To avoid loss of NH4+ upon evaporation, the distilled and

titrated samples were slightly acidified with 2 ml of 0.1N H2504 before

24

drying, (Edwards, 1978). To provide sufficient N for 15N analysis on a
V. G. Micromass 622 isotope-ratio mass spectrometer, 1 ml of unenriched
4)ZSO4 solution containing 3 mg N ml.1 was added to distillates with
low N content (Tiedje, et al., 1980). One source of reagent grade

(NH

(NH4)ZSO4 contained in one bottle was used for all dilutions. For

4)2504 solution

(ranging from 3 mg N ml'1 to 15 mg N ml'l) was added to dilute the

highly-enriched distillates, 1 ml of unenriched (NH

distillates to less than 3 atom percent 15N. The exact amount of

unenriched (NH SO added to the distillates therefore depended on the

4)2 4
concentration of N and the expected 15N enrichment in each distillate as

determined by preliminary experiments.

After addition of the unenriched (NH $0 the distillates

4)2 4’

contained in the 125-ml Erlenmeyer flasks were vigorously mixed before
and during drying. The contents were dried down to about 5 ml on a hot
plate (80°C) and then transfered to 10 ml disposable vials. The samples
in the vials were evaporated to dryness and analyzed immediately or
covered with parafilm and stored in a refrigerator for later analysis.

15N analysis in the V. G. Micromass 622 Isotope Ratio Mass Spectrometer

 

Isotopic assay of the N gas was performed in a Micromass 622
isotope-ratio mass spectrometer. The Micromass 622 is a 900 sector
magnetic collection instrument of 6 cm radius with twin Faraday bucket
collectors coupled to a ratio recording output stage for the
determination of precise isotope ratios. The Micromass 622 is designed
for operation in two distinct modes, namely single beam and ratio. The
single beam mode of operation is used for background spectra, and
checking for leaks and contamination. In the ratio mode, the ratio of

the minor signal to the major signal is displayed directly. The signals

25

 

 

[ N SAMPLE [N STANDARDS 1
I

ACIDIFY AND CONCENTRATE

 

 

 

 

 

 

 

 

 

 

[TRANSFER INTO VIALS [e

 

 

[DRY ON H01 PLATE [

I

[INSERT VIAL INTO N- [

 

CONVERSION APPARATUS f

I EVACUATE VIAL [

[ADD LIOBR [

l I

A ADNIT GAS THROUGH COLD [

 

 

TNN’INNIMN§SSHZHRDNJER

I

[ READ RATIO IN [

 

MASS SPECTROMETER

 

 

[ CALCULATE 1 OF 15N IN SAMPLE [

 

15

Figure 3. Sample preparation and N analysis in isotope ratio

mass spectrometer.

26

of the twin head amplifiers are fed to the analogue ratio unit where the
major and minor signals are attenuated and the resulting signals
displayed on the major and minor meters, respectively. Figure 3 shows
the analytical steps involved in sample preparation and its isotope ratio
analysis.

Checks on the precision of the instrument were performed by several
determinations of both labeled and unlabeled standard (NH4)ZSO4 salts
over a period of three months. Several reagent (NH4)ZSO4 reference
samples were run each time a batch of unknown samples were analyzed. To

minimize errors in 15

N ratio due to possible leakage of air into the mass
spectrometer, the 02 peak (m/e 32) was checked after each sample run.
The effect of partial pressure of N on isotope ratios was also
determined. The effect of partial pressure is important, because it is
related directly to sample size at a given volume. Throughout the
analysis, the N pressure of standards and samples were adjusted to a
constant and similar pressure, using the variable reservoir on the mass
spectrometer.

To minimize memory effects during isotope ratio analysis, samples
with high expected 15N content were analyzed after those with lower 15N

content.
CALCULATION OF RESULTS

Atom_percent 15N in samples

 

From the measured ratios of the samples, an apparent percentage of
15N was calculated after correction for the major and minor gain settings

used. The equation used in calculation of apparent atom % 15N at low

27

abundance where it was difficult to measure the mass peak 30 with

accuracy was as follows:

Atom % 15N = 2&31'

. 1
Where R = Intens1ty of 28 _ 4N14N

Intensity of’29 " 14N15N

R is corrected for gain setting used

07‘

o 15 _ R _ 29N
Atom % N --§e;—§ Where R - 28N'

Correction for isotope dilution calculation

Correction for isotope dilution was made in cases where natural
abundance NH4-N was added to the distillates before drying as previously
explained. From the ratio measured, the atom % 15N in the equilibrated
NH4+ fraction was calculated by means of the equation shown above. The
15

N of the labeled NH4-N before dilution was calculated

using the equations below. These equations are based on isotope dilution

actual atom %

principles as explained by Gest, et al., (1946).

AX + BY = (A + B) Z
X = (A + B) Z - BY

where:

14 15

A = milliequivalents of N in sample ( N + N) before dilution.

28

X = actual atom % 15N in sample A before dilution
8 = milliequivalents of natural abundance N added.
Y = Atom % 15N in the added natural abundance N.

Z = isotope ratio of A and B after equilibration.

All calculations of N were made on equivalent mass basis.

Calculation of fertilizer N in different soil N fractions

 

The quantity of labeled fertilizer N found in each soil N fraction
was caluclated using the equation stated below (Stojanovic and Broadbent,
1956, and Edwards, 1978). The 15N content of the standard with which all

samples were compared in computing milligrams fertilizer N was found to

be 0.367 atom % 15R.
Y = Z(c-a)
TIT-3)
where:
a = fraction 15N coming from natural compound
b = fraction 15N in added N [(NH4)ZSO4 or urea].
c = fraction 15N in recovered N (NH4-N, N03-N or organic -N).
Y = quantity N from added source (mg. fertilizer N)
Z = quantity N recovered in given fraction

Based on the above calculations, the percentage total recovery of
applied tagged fertilizer at each sampling period were computed as
follows:

Total Fertilizer N Recovered

°/ =
” Total Recovery Total Fertilizer N Applied

 

29

The percentage of fertilizer-N found in each soil N fraction was

determined as follows:

% Recovery of Fertilizer-N = mg fertilizer-N present X 100
in Nitrogen Fraction mg fertil1zer—N applied

Statistical analysis

A statistical analysis was conducted on data obtained from two or
three replications as indicated in Appendixes A and B. Fishers least
significant difference (FLSD) was employed to statistically evaluate
differences between treatment means. The statistical analysis procedures
were those outlined in Steel and Torrie (1960). Regression analysis and
all other statistical analysis were performed using the Michigan State
University's computer facilities. The objectives of these analysis were
to show the relationship between the amount of fertilizer N recovered in
the various fractions and the length of incubation period.

The regression models used were as follows:

Y = a+bT

Y = a+bT+cT2

where:

T = incubation period (hours)

Y = Percent (%) of applied N fertilizer recovered as NO -N

3
or organic-N

a, b and c are regression coefficients in the equation
The quadratic term (T2) was added only in cases where additional sums

of squares was significant. Values for R2 for both linear and quadratic

3O

equations including other statistical tests of these equations are shown
in figures to be presented later. Pairwise comparison of the different
curves were also made. Simple correlation coefficients (r) between the
different N forms and incubation time, the dependent variable, were

calculated.

31

Table 2. Clay fixation Oflabeled (N34)2504 fertilizer in soils.

 

Fertilizer-N Recovered as Clay-fixed NBA-N

 

 

Soil Zero 48
Houra Hoursa

WL 0.005 0.01

52 0.01 0.04

M818 0.02 0.07

 

a, Hours of incubation

Tab

le 3.

32

Total recovery of N Added as (NH4)ZSO

and urea fertilizer

 

 

 

applied to soils. 4
Total Fertilizer Recovered in Soilsa
MBle sz 1111b 141C MBle
Hours Z
- 0 95.911.7* 94.110.23* 94.8:0.l* 99.612. 100 :1.
3 N.D.e N.D. N.D. 96.7:1. 98.8:1.
6 94.910.32* 99.0:O.1 94.9:O.1* 100.4:1. 99.411.
12 97.2:2.8 98.7:O.4 96.7il.4 95.8:1. 100.4:1.
24 94.111.7* 94.9:O.5* 95.2:O.2* 91.7:0. 97.714.
48 96.2:1.9 92.011.5* 95.510.22 94.2:1. 98.3iO.9
96 93.811.8* 96.5i1.1 97.0:1.24 98.111. 101.6:2.
144 94.110.13* 93.411.2* 95.7t1.2* N.D. N.D.

 

 

 

a, Mean and standard deviations (of 3 replications) of percentage
total fertilizer recovered at each sampling period

b, Treated with

c, Treated with

15N labeled (NH fertilizer (100 ppm NH4-N)

4)2SO4
15N labeled urea (100 ppm urea-N) and glucose

(4000 ppm glucose-C)

d, Treated with

N labeled urea (100 ppm urea-N)

e, N. D., Not determined
* Statistically different from 100 percent recovery PC:0.05)

RESULTS AND DISCUSSION

Preliminary experiment to determine NH4+ fixation capacities of soils

Preliminary experiments (Table 2) indicated that in all the soils
the amount of labeled NH4+ fertilizer fixed within 48 hours was not
significant (less than 1%). Similar results about NH4+ fixation were
observed by Broadbent (1965) whose data show that ion exchange between
fixed and exchangeable NH4+ and between exchangeable NH4+ and amino N was
negligible. Sowden et al., (1977) reported that in a clay fixation
experiment most of the NH4+ fixation was complete in 2 hours with a much
slower rate of fixation in the next 3 days.

In view of the results of this preliminary experiment, clay-fixed
NH4+ was not determined as a component of the N fertilizer

transformations reported in this research.

Overall recovery of tracer N

 

In tracer experiments dealing with interchange between inorganic and
organic forms of N, it is essential to provide a complete accounting of
tracer N in the system. This serves as a check on the reliability of the
analytical procedures and indicates whether losses are taking place.

As shown in Table 3, the 15

N labeled fertilizer (NH4)ZSO4 and urea
were quantitatively recovered as mineral N at zero time and subsequently
at other sampling times during the period of incubation. The recovery
ranges from 92 to 102%. These recoveries indicate that clay fixation of

the tagged NH4+ fertilizer was negligible during the incubation period.

33

34

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36

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Statistical analysis shown in Table 3 indicate the recovery values that
were significantly different from 100% recovery. Low recoveries in some

cases may be due to experimental errors involved in 15

N analysis. There
is no evidence with the present data to attribute these low recoveries to
volatilation or denitrification. The recovery data compares favorably
with similar recoveries by Broadbent (1966) and Craswell and Martin,

(1975)..

 

Transformations of (N54)2504 and urea fertilizers in soils

The aim of this section is to show an overall view of the trans-
formations of fertilizer N added as labeled (NH4)ZSO4 and urea in
soils without any soil amendments. This is to present the soil as a
continuously functioning biological system carrying out the series of
transformations upon which the higher plants are highly dependent in
obtaining the N necessary for their development. Specific details of
the individual transformations are discussed later in the thesis.

Ammonium sulfate transformations in soils

 

Figures 4, 5 and 6 show the NH -, N0 - and organic-N from labeled

3
(NH4)2504 fertilizer as a function of time in the soils used. As shown
in Figures 4, 5 and 6, the rate of disappearance of fertilizers NH4-N
varied depending on the type of soil and the associated microbial pro-
cesses which have taken place in the soil. In the case of soil MBl8,
the NH4-N disappeared at a rapid rate (1.5% per hour) with no signi-
ficant lag period, (Figure 4). At the same time, NO3-N began to in-

crease rapidly almost at the same rate (l.2% per hour) but the increase

in NO3-N did not equal the decrease in NH4-N. This net decrease in

38

.F3 PPOm o» LmNPqurme ewe: umpmnep no cowu_cum cwumm z-u_:mmao use -moz .1 :2 .1omtz cw mmmcocu .5 meamwa

Ammsozv nofimmm zofip<m=uz_

 

 

 

mm mm m: :N
2-8222820 x.
2-moz +
2-222 nu
2-5% 0 a
.c
m[

 

 

 

om

om

OOH

8321111833 N 03IlddV 30 (Z) 1N3383d

39

.N3 pwom o» Lm~w_wu2me our: umanm_ mo cowuwuum rupee z-uwcmmeo new 1moz .- 22 :_ mmmcocu .w mrsmwa

Ammaozv comm“; zomp<m=uz~

(‘4
I’\

mm

{D

1r

2-0222220
2-mo2

2-222

1P

+

mm

H 4'

0

(O

3

 

cm

on

om

ow

sea

8321111833 N 03IlddV 30 (Z) 1N3083d

4O

.mpmz Fwom o» cmNPFVHLme owe: vmfimnoF no cowuwuum cmuwm z-uwcmmeo use 1moz .1222 .1mmL: cw mmmcmcu .m acumen

Amazozv nommma zo~p¢m=uzfi

 

 

mm NR m2 am e
0 4 D Q n > [i _ o
. Hum
2-8222220 o2
z-moz
2-222
2-2222
Ago
. Rum

8321711833 N 03IlddV 30 (Z) 1N3383d

 

 

chem

41

.m_uz ~_0m on Lm~w_wurme ewe: umanm_ mo cowu_uum emote z-uwcmmco cam 1moz .- 22 :2 mwmcmgu

Amazozv momma; onH<m=qu

mm mm m: cm

’ ’

b F P
‘ J1 1 d

‘i‘

 

’J

2-0122220 +
2-moz D

21512 O a.

a

 

 

OOH

.o_ oe=a_a

832Illl833 N 03IdeV 30 (Z) 1N3383d

42

Table 4- Changes inPHdue to addition to labeled urea
and (NH4)2504 fertilizer.

 

Soil Reaction

 

 

 

 

Incubation Period Soil Wlb Soil wc Soil 111318C
pH
0 5.1 5.1 5.7
(Before fertilizer
addition)
0 4.8 5.1 5.7
(After fertilizer
addition)
3 4.8 5.4 5.7
6 4.7 5.5 5.7
12 4.7 5.6 5.7
24 4.7 6.1 5.7
48 4.7 6.0 5.6
96 4.7 6.0 5.9

 

a, Mean of duplicate determinations

b, Labeled (NH

) SO added at the rate of 100 ppm fertilizer N
(w/w basisl.

2 4

c, Labeled urea fertilizer added at the rate of 100 ppm fertilizer-N.

43

mineral N was due to immobilization of the NH4+-N because the rest
of the fertilizer was found in the organic fraction. After 96 hours of
incubation, the percentage of fertilizer N in each fraction appears to
remain relatively constant with NOB-N and organic-N comprising 76% and
l8%, respectively, of the N applied.

Ammonium sulfate transformations in soil Wl and F2, (Figures 5 and
6) were small even though they were statistically different from zero
hour readings. In general, there was little disappearance of fertilizer
NH4'N or appearance of the fertilizer NO3-N and fertilizer organic-N.
This indicates early nitrification and immobilization, even though they
are minimal when compared to soil MBl8. The interesting thing is that
significant changes in the N03-N and organic-N were observed in soil
MBl8 as early as 6 hours while in the case of soils Wl and Fl, the
significant increases were observed at l2 to 24 hours. The reasons for
these differences are explained in later paragraphs.

Urea transformations in soils

 

In general, the urea applied was very readily hydrolyzed and much of
the added urea N was transformed to NH4-N in 2 days (Figures 7, 8, 9 and
10). The pH of soil Wl increased during this urea hydrolysis. The pH
of MBl8, the organic soil, did not significantly change because of its
high buffering capacity (Table 4).

In soil Wl (initial pH of 5.1), net nitrification and immobilization
of urea at 96 hours of incubation were 10% and 6% of the applied fer-
tilizer, respectively. Disappearance of NH4-N produced by hydrolysis
of urea was greater than in treatments where (NH4)ZSO4 was applied.

In soil WZ (Figure 8), after 24 hours the majority of applied urea

was in the form of NH4-N. After 24 hours, there was a greater disappearance

44

... . 1
Table 5. Immobilization of 5N—labeled (NH4)2801 and urea
fertilizers after 12 hours of incubation.

 

 

 

Soil Fertilizer and Fertilizer N
Carbon Treatment Immobilizeda
Z
' +
W1 (NH4)2304 1.4 _ 0.03
W1 Urea 1.8 i 0.06
W1 Urea + Glucose-Cb 17.8 i 1.3
,. /
F2 (NH4)2804 5 7 _ 0.04
4.
M818 (N114)ZSO4 10.4 _ 0.25
M318 Urea 8.6 t 0.8
WZ Urea 2.3 t 0.03
WZ Urea + Glucose-Cb 21.7 i 0.01
MC15 Urea 9.7 t 0.4
MClS Urea + Glucose-Cb 24.8 i 0.01

 

a, Mean and standard deviations of percent labeled fertilizer
recovered as organic-15N

b, Applied at the rate of 4000 ppm glucose-C.

45

of NH4-N because of rapid transformations of NH4-N to oxidized forms of
N and the immobilization of some of the NH4-N into the organic-N

In soil MC15 and M818, the NH4+ form produced after hydrolysis
decreased very rapidly and totally disappeared by the end of the incubation
period and NO3-N became the major form of N from urea (Figures 9 and 10).
These two soils appear to have identical trends in their transformations of
applied urea.

Results of urea hydrolysis show that the reaction is rapid and varies
in rate from one soil to another. Urea added to soils is apparently
hydrolyzed in a matter of a few days (Overrein, 1967). The ammonical-N
resulting from this enzymatic process becomes at least partly equilibrated
with the mineral-N pool of the soil. After the rapid hydrolysis of urea, a
sizeable pool of tracer NH4-+N developed in the soil and its subsequent
behavior depended on the biological properties of the soil. Similar
results were obtained by Broadbent, et al., (1958) who stated that urea as
a fertilizer has been considered to resemble NH3 in transformations
following hydrolysis.

Comparison of immobilized 15N labeled (NH4)HSO and urea fertilizers in

(’4"
soils

 

The percent (NH4)ZSO4 and urea fertilizer immobilized in
various soils after 12 hours of incubation with and without glucose
amendments is shown in Table 5. Mean and standard deviations of

labeled fertilizer immobilized during laboratory incubation of
different soils throughout the incubation period are shown in Appendix A.
These data were obtained by adding an enriched (NH4)ZSO4 or urea fertilizer
and following the distribution of the isotope in the organic form at

definite intervals as discussed previously.

46

    
   
 
       

 

28 ~ 112
o MBl8 0.84
8 F2 0.91
24 ~ 0 WI 0.95
Z
e'.
E 20 ~
0
0:
O
2 o
C! -
a 16 _ Y-9.7 + 0.07T
”:1
+—
a:
LLJ
L1.
2 12 "
o A
53
g 11:5.03 + 0.041
<1
2 8 ’
O
:3
q Y=O.8 + 0.041

 

PERCENT (

    

g

0 6 12 24 48 72 95 144
INCUBATION PERIOD (HOURS)

Figure ll. Comparison of immobilized labeled (NH4)2504 fertilizer in various soils.

1

PERCENT (Z) OF APPLIED N FERTILIZER AS ORGANIC-N

28

24

20

16

12

I

 

M818 + UREA

MBl8 + (NH4)ZSO4
Wl + UREA
W] + (NH4)2504

3X I) D» (3

47

0.88
0.91

0.91
0.95

 
    

Y=5.6+O.I6T

Y=9.0 + 0.09T

 

x::='
0' 6 12 24 48 72 96
INCUBATION PERIOD (HOURS)
Figure 12. Effect of fertilizer source on immobilization of labeled fertilizer

N in soils Wl and MBl8.

48

In Figures 11 and 12, incubation time has been plotted against the
percent fertilizer N immobilized. The results show a linear relationship.
Inclusion of the zero hour reading for soils M818 and F2 treated with
labeled (NH4)ZSO4 (Figures 11 and 12) did not permit the prediction of N
for the first 6 hours after the addition of the labeled fertilizer.
Regression equations with the zero hour reading for such soils tend to
underpredict the values at O to 6 hours. Because the provision for
separate reactions appeared to be anomolous and a departure from the trend
of the remainder of the data, it was necessary to delete the zero point in
the regression analysis. Based on the data of this experiment, this
anomoly may be attributed to the various experimental errors associated
with the different steps in sample preparation and analysis.

Appendices C and 0 show that better prediction was achieved when the
zero hour was deleted. The most practical use of this information would be
for times between 6 to 96 hours. A significant increase in immobilization
(P50.05) occurred in all the soils during the incubation period.

The high correlation coefficients show a high association between the
immobilized N and time of incubation. For soil M818, 10.4% of the applied
labeled (NH4)ZSO4 fertilizer was recovered as organic-N while for soils
W1 and F2, 1.4% and 5.7%, respectively, were recovered as organic-N
12 hours of incubation.

The rate of immobilization of (NH4)ZSO4 fertilizer indicated by the
linear regression coefficients are as follows, M818, 0.07% N hour'l; F2,
0.04% N hour“1 and WI 0.04% N hour-1. Soil M818 was found to be
significantly different from soils W1 and F2 using a pairwise comparison

procedure.

49

Application of urea resulted in immobilization of 8.6% and 1.8% of the
applied urea fertilizer in soils M818 and W1, respectively, after 12 hours
of incubation (Table 5). The rates of immobilization of urea fertilizer

1 and v1 0.06% N hour'1 (Figure 12).

were as follows, M818 0.2% N hour-
Tests for statistical significance made at the 5 percent probability level
showed that all the regression curves in Figures 11 and 12 were
statistically significant.

The range in the amount of (NH4)ZSO4 and urea fertilizers immobilized
during the incubation period suggests very different levels of microbial
activity in the various soils. The higher rate of immobilization of
(NH4)2304 and urea fertilizers seen in soils M818 and M015 (organic soils)
could be attributed to a higher pOpulation of heterotrophic organisms and
the presence of a readily-available C source for microbial activity.

In an experiment to be reported later, all the soils effectively
immobilized the urea fertilizer almost at the same rate when treated with
glucose-C. This shows that the soils had the potential to immobilize N,
but differences seen may have been due to differences in the immediate
availability of a C source.

In an effort to further study the influence of pH and
readily-available C on microbial activity and subsequent immobilization,
several additional experiments were conducted.

Influence of changes in soil pH on immobilization of labeled(NH4)250%:agg

urea fertilizers

 

The pH effect is an important consideration as protons are liberated
3 O
poorly buffered environments, it may cause inhibition of nitrification.

in the oxidation of NH4+ to NO When acid conditions are produced in

The changes in pH during the incubation period as affected by the type

50

of fertilizer, were shown previously in Table 4 for soils W1 and M818. In
soil W1, application of urea fertilizer resulted in an initial marked
increase in pH, while (NH4)ZSO4 application resulted in a slight decrease

) SO treated soil, and

4 2 4
after 12 hours on the urea treated soil, the pH remained relatively

in pH. After the initial decrease in pH on the (NH

constant throughout the rest of the incubation period. There were
relatively minor changes in the pH of the organic soil (MBl8) with the
addition of urea which is probably due to the higher buffering capacity of
this soil. In soil W1, after 24 hours of incubation, the pH had increased
from initial value of 5.1 to 6.1 due to hydrolysis of the urea.

The net effect of these increases in soil pH are evidenced in the
increased rate of immobilization (Figure 12). There was a significant
increase (P5 0.05) in N immobilization for soil W1 when urea was applied.
After 96 hours of incubation, application of urea and (NH4)ZSO4 resulted in
immobilization of 6.1% and 4.2%, of the applied N, respectively.

Net immobilization of fertilizer N was greater in the soil M818 that
received urea fertilizer. Initially, in the early hours of incubation, the
amount of fertilizer N immobilized in the (NH4)ZSO4 treated soil seemed to
be greater though this was not statistically significant (P 50.05). This
may have been due to the time factor involved in the hydrolysis of urea to
NH4-N. As soon as this process was underway, the immobilization became
higher in urea amended soils and continued to be higher until the end of
incubation.

From the results presented, it seems that changes in H+ ion
concentration exercise a considerable effect on the rate and amount of N
immobilization. The wide range of organisms present in the soil and the

cooperative nature of their activities make possible a large number of

PERCENT (Z) OF APPLIED N FERTILIZER AS ORGANIC-N

32

28

24

20

16

12

 

51

 

 

 

 

urea fertilizer in various soils.

R pH
1. MClS 0.99 6.8
C) MBl8 0.88 5.7
(3 W2 0.99 6.5
_ x WI 0.91 5.l
T
v=2.1+0.7T-0.004T2
CI
” Y=5.6+0.2T
O
F C)
_ o
v=0.4+0.2T-O.00112
. ,I
I
. Y=0.9+0.06T
- x
I
.411"
pr
0 12 24 48 72 96
INCUBATION PERIOD (HOURS)
Figure l3. Influence of initial soil pH on immobilization of labeled

52

balance reactions, each capable of bringing about the same ultimate result,
though the route and rate of achieving them may differ. The effects and
differences observed are undoubtedly due to a modification in nature of the
active microflora. Similar results were obtained by Norman (1931) who
found that under slightly alkaline conditions, immobilization was more
rapid than in either neutral or slightly acid conditions. He attributed the
observed effects to differences in the character of the active microflora

influenced by changes in pH.

Influence of initial soilng on immobilization of labeled urea fertilizer
in soils

 

Soils with similar physical and chemical properties collected within
the same general area were used for this comparison. It was assumed that
the only variable factor in these soils was the difference in their pH
caused by prior agricultural practices, even though this assumption may not
have been completely correct.

The second assumption was that because the soils were formed from the
same parent material, they had comparatively similar microbial
transformations under identical conditions. The soils were selected to
include a low and a high pH. For the mineral soils, W1 had a pH of 5.1 and
NZ a pH of 6.5. For the organic soils, the pH of soil MBl8 was 5.7 while
that of MC15 was 6.8. All the soils received the same quantity of
fertilizer N (100 ppm urea-N) and were incubated under the same
conditions.

Results as shown in Figure 13 indicate significant differences (PS
0.05) in the rate of immobilization of fertilizer N in identical soils with
differing pH. In general, immobilization of N was greater in the organic

soil series (MC15 and M818) than in the mineral soil series (W2 and W1).

53
These differences were all statistically significant (P.g 0.05). In the
absence of direct microbiological data of the soils used, it was not
possible to assess the extent to which the relationship between the pH of
the soils and immobilization of N was due to differences among the
microorganisms present. However, there is abundant evidence that the pH
of the soil does influence the numbers of and types of organisms present
in soils and it is generally considered that acid conditions favor fungi
rather than bacteria. In the case of the acid soil, the immobilization
may have been due mainly to the work of fungi, the growth of which will
be favored by such conditions. In the more alkaline soil, MC15, the
condition is more suitable for bacterial development and less favorable
for fungi. It is not surprising that the immobilization rate was
consistently higher than under the conditions of higher pH since in
general, bacteria have a higher N content than fungi. Waksman (1922)
showed that addition of lime stimulated the develOpment of bacteria and
actinomyces, but not fungi. By analogy, it is possible that the
relationship between pH and immobilization of N in the present
investigation was in part due to differences among the soil organisms
present.

Effect of addition of glucose-C on immobilization of labeled urea

 

fertilizer in soils

 

In view of the low rate of immobilization in some of the soils
reported previously, an explanation to this was sought: At least the
previous experiments mentioned in this thesis Show that low pH was partly
responsible for the low rate of immobilization. In a subsequent
experiment, glucose-C applied at the rate of 4 mg glucose-C gm-1 soil
(4000 ppm glucose-C) was added to soils W1, NZ and MC15 after the
addition of 100 ppm of urea-N. This resulted to a C/N ratio of 40/1

PERCENT (Z) OF APPLIED N FERTILIZER AS ORGANIC-N

100

70

20

10

 

54

 

 

 

urea fertilizer in soil WI.

1.
"O
L.
v=-2.6+3.OT-0.0212
0
R2
0 Wl+GLUCOSE-C 0.95
' c» Wl 0.90
O
Y=O.9+0.06T '
+
0 12 24 48 72 96
INCUBATION PERIOD (HOURS)
Figure l4. Effect of addition of glucose—C on innnbilization of labeled

PERCENT (Z) OF APPLIED N FERTILIZER AS ORGANIC-N

100

90

80

70

60

50

40

30

20

10

 

55

 

 

urea fertilizer in soil NZ.

r
b ()
v=-1.4+3.01-0.0212
L C)
_ R2
o Wl+GLUCOSE-C 0.95
‘3 WZ 0.99
h-
C)
" v=0.4+0.2T-0.00112
———4D
I g l L
0 12 24 48 72 96
INCUBATION PERIOD (HOURS)
Figure l5. Effect of addition of glucose-C on immobilization of labeled

PERCENT (Z) OF APPLIED N FERTILIZER AS ORGANIC-N

100

90

80

70

60

40

30

20

10

 

56

 

 

O
I’ O
2
Y=2.3+2.6T-0.02T
C)
- R2
O MCl5+GLUCOSE-C 0.96
O MC15 0.99
_ v=2.1+0.7T-0.00412
C) 1.
1 l 1 1 J
0 12 24 48 72 96
INCUBATION PERIOD (HOURS)
Figure 16. Effect of addition of glucose-C on immobilization of labeled

urea fertilizer in soil MClS.

57

Table 6. Comparison of rate of immobilization of fertilizer-N
in the presence and absence of Glucose-C

 

Soil Fertilizerb Rate of ImmobilizationC _
Source Percent of Applied N hour

 

No Carbon With Carbon

 

 

 

W1 Urea 0.6 2.3
WZ Urea 0.2 2.3
MC15 Urea 0.7 2.0

 

a, Applied at the rate of 4,000 ppm glucose
b, Added as 100 ppm urea-N

c, Represents the rate between 12 and 24 hours incubation period

58

15 15

(glucose-C/urea-N). Determinations of NH - M, NO -]5N and organic- N

4 3
were made at intervals on duplicate flasks of soil in the manner previously
described.

The results, expressed in percentage recovery of fertilizer in Organic-N
fractions in the soil, have been plotted against the period of incubation
up to 96 hours as shown in Figures 14, 15 and 16. These figures show
comparisons with and without addition of glucose-C. As shown in these
figures, the addition of a readily-available C source caused very rapid
rates of immobilization which, in all cases, were found to be significantly
different (P 50.001) when compared to their unamended counterparts. Table
6 shows that when the three different soils are compared with respect to
the rate of immobilization (between 12 and 24 hours) after glucose-C
addition, they all show a similar rate of immobilization: soil W1, 2.3% N
hour‘l; soil WZ, 2.3% N hour-1; and soil MC15, 2.0% N hour-1. In the
presence of glucose, the net immobilization was 97.0% in soil W1 after the
96 hour incubation period, compared to 6.0% in the absence of glucose
amendment. For soil WZ, addition of glucose-C (after a 96 hour incubation
period) caused a net immobilization of 88.0% compared to 6.0% in unamended
soil, while in soil MCIS 90.0% was immobilized by the glucose amended soil
as compared to 26.0% in unamended soil.

In the three soils studied, immobilization of urea fertilizer in the
presence of readily-available glucose-C occurred immediately, and was a
rapid process, reaching its maximum in these soils within 48 hours.
Although the results indicate that unamended soils have lower rates of
immobilization, the immobilization potential under optimum conditions
(presence of readily-available C) was high and similar for all soils (Table

6).

 

59

Immediate and rapid immobilization of N in soils NI, NZ and MC15
amended with glucose-C was due to the ready availability of this C source
to microorganisms for performance of a complex series of reactions. When
the glucose-C was added, heterotrophic organisms of the soil likely used
the readily-available source of energy and increased rapidly in numbers.
Part of the added C was evolved as CO2 while part was used by the organisms
in the synthesis of their own cell substance. This synthesis of microbial
tissue was responsible for the immobilization of fertilizer N.

In a similar experiment but using a soil perfusion technique, Lees
(1948) showed that with addition of glucose and sucrose immobilization of N
reached its maximum in two to four days. Rubins and Bear (1942) also
indicated that the relationship between C/N ratio and N transformations in
the soil is influenced by the ease of decomposition of the various

constituents present.

Agronomic implication of immobilization in the presence of

 

available carbonaceous material

 

Under agricultural situations where plant materials which contain a
wide C to N ratio are added to soils, the size of the decay population and
the quantity of N immobilized in microbial cell materials may increase
temporarily to levels which seriously deplete the soil of the mineral N
(principally N03-) for plant growth. Nitrogen immobilization has a
significant bearing on the interpretation of any chemical or biological
tests used to make recommendations on crop management practices and N
fertilizer recommendations. Because of immobilization, the results of
chemical or biological tests for N will vary, not only with the time of

year when soil samples are taken, but also with the point in the rotation

 

6O

  
  
  
  
    

 

 

 

 

 

100 '
R2
A MBl8+UREA 0.95
90 . 0 MBl8+(NH4)2504 0.99
o Wl+UREA 0.97
o Wl+(NH4)2504 0.98
80 ' x F2+(NH4)2804 0.41 A
0
5:
E 70 “
<(
C!
p—
5 A
L!)
< 60 _.
Efi
I: Y=6.0+0.9T Y=3.2+0.8T
.J
E l-
3 50
Z
D
:3
_..|
e 40 t
q
LI.
0
g
H 30 ”
Z
Lu
U
0:
Lu
0.
20 r
0
10 Y=0.7+0.lT
D , YsLetQJEu;_. 4-—<|
v=1.2+o.0091 u
k, .. --° 1" ' X n
0 " A4 4 1_ 4 I
0 6 12 24 48 72 96

INCUBATION PERIOD (HOURS)

Figure l7. Effect of fertilizer source on nitrification of labeled (NH4)ZSO4
and urea fertilizers in various soils.

PERCENT (%) OF APPLIED N FERTILIZER AS NITRATE-N

100

90

80

7O

60

SO

40

30

20

10

61

   
 
   

 

 

F
R2 INITIAL pH
0 MBl8 0.95 5.7
x MClS 0.96 6.8
C) NZ 0.99 6.5
. o m 0.97 5.1
I
)(
Y=l.8+0.8T
.
Y=0.8+0.2T ' Y=0.7+0.IT
.
C
. ‘TT‘——. .
0 6 12 24 48 72 96

INCUBATION PERIOD (HOURS)

Figure 18. Effect of initial soil pH on nitrification of labeled urea
fertilizer in various soils.

   
  
  
 

62

Table 7. . Nitrification of 15N labeled (NH4)ZSO4 and urea
fertilizers after 12 hours incubation.

 

 

 

Soil Fertilizer and Carbon Fertilizer N Nitrifieda
Treatment-
‘7.
+
W1 (NH4)ZSO4 2.3 - 0.15
W1 Urea 2.1 i 0.28
F2 (NH4)ZSO4 1.4 i 0.12
.4 i .

M318 (NH4)ZSO4 12 1 10
MBl8 Urea 15.3 i 1.0
WZ Urea 3.1 i 0.68
WZ Urea + Glucose—Cb 2.8 i 0.21
MC15 Urea 5.7 i 0.0
MC15 Urea + Glucose-Cb 9.0 i 0.47

 

a, Mean and standard deviations of percentage labeled fertilizer
recovered as NO3-13N after 12 hours of incubation

b, Applied at the rate of 4000 ppm glucose-C

63

when they were taken. It appears, therefore, that any direct correlation
between chemical and biological soil tests and cr0p response to N will be
difficult to achieve unless ways are found to evaluate the immobilizing
potential of carbonacious residues in the soil.

15

Effect of fertilizer source and initial soil pH on nitrification on N

~

labeled (NH,),SOA and urea fertilizers in soils

Figure 17 shows the effect of different fertilizer sources (urea and
(NH4)ZSO4) on nitrification of 15N-labeled fertilizer N in different soils,
while Figure 18 shows the effect of initial soil pH on the nitrification of
15N-labeled urea fertilizer in soils MBl8, MC15, WZ and W1.

Nitrification of (NH4)2504 and urea fertilizers was significantly
greater in soils MB18 and M615 than in soils W1, F2 and WZ (Ps 0.05). At

an application rate of 100 ppm NH -N as (NH4) 30 , soil MBl8 had converted

4 2 4
74% of the NH4+I to N0344 by the end of the 96 hours of incubation. At
the end of the same time period (96 hours), 2.6% and 5.5% of the N applied
was recovered as N03+l in soils F2 and W1, respectively. At the same
application rate of 100 ppm N as urea 79.6%, 75.3%, 16.2% and 10% of the
applied urea+l was converted to N03+l at the end of the 96 hours of
incubation in soils MBl8, M615, W2, and W1, respectively.

The percentages of applied (NH4)ZSO4 and urea fertilizers nitrified
within 12 hours of incubation are shown in Table 7. It is apparent that

the different soils did not nitrify the (NH ) SO and urea fertilizers at

424

the same rate.

Ammonium sulfate and urea, in general, exhibited similar nitrification
behavior in all soils; however, specific differences in the rates relate
directly to the influence of acidity. Urea as a fertilizer has been
considered to be like NH3 in transformations following hydrolysis

(Broadbent, et al., 1958).

64

In the case of soil W1 treated with (NH
1

4)2504, the nitrification rate
while the nitrification rate for the same soil treated
I

was 0.04% N hour-

with urea was 0.1% N hour- (Figure 17). Data on pH changes (already

4)2504 decreased

the pH of this soil, while urea treatment increased the pH of the same soil

reported) due to addition of fertilizer show that the (NH

upon hydrolysis. There was also a higher rate of nitrification in the urea
treatment of soil MBl8 (0.9% N hour'l) compared to the same soil treated
with (NH4)2504 (0.8% N hour-1). This observation indicates that soil
acidity may have limited, or even prevented the nitrifying bacteria from
oxidizing the NH4+ to N03- in the acidic soils. Since soil W1 was

initially acid, the rise in pH accompanying the addition of urea and its
hydrolysis apparently provided a somewhat more favorable environment for
nitrifying microorganisms. This contributed to the earlier and higher rate
of nitrification of labeled NH4+-N from the urea application compared to the
(NH4)2504 application.

Roberge and Knowles (1966) found that over a 42 day period, there was
no appreciable N03-N production in black spruce humus. Total absence of
nitrification is unusual in agricultural soils (Broadbent et al., 1957) but
has been reported by other workers especially in forest soils (Harmsen and
Van Schreven, 1955).

In the experiment reported in this study, there was no evidence to
prove that the slow rate of nitrification in these soils (W1 and F2) was
due to lack of nitrifying bacteria or due to the existence of inhibitory
substances. The only evidence to prove that it was a pH effect was the
noticeable but not statistically significant increase in nitrifying
capacity of soil W1 when urea was applied compared to an application of

SO

(NH4)2 4.

PERCENT (%) OF APPLIED N FERTILIZER AS NITRATE—N

lO

 

65

 

 

T
0»
R2
(3 Wl 0.97
. O Wl+GLUCOSE—C 0.l4
L
b Y=0.7+0.lT .
Ci
_ ll
0
° Y=0.9-0.007T
? o
g 1 I I I I
0 12 24 48 72 SE
INJUBATIW PERICD (RIBS)
Figure l9. Effect of addition of glucose-C on nitrification of labeled urea

fertilizer in soil Wl.

PERCENT (%) OF APPLIED N FERTILIZER AS NITRATE-N

66

 

 

 

2C) '
R2
4. wz 0.99
16 _ OWZ+GLUCOSE-C 0.14
12 -
Y=0.8+0.2T
8 -
u u-
2 P-
Y=l.3-0.0lT
li/ . 9 <3 . *2“
0 0 12 24 48 72 96

INCUBATION PERIOD (HOURS)

Figure 20. Effect of addition of gluclose-C on nitrification of labeled
urea fertilizer in soil WZ.

PERCENT (%) OF APPLIED N FERTILIZER AS NITRATE-N

100

90

8O

7O

60

50

4O

30

20

10

P (D

 

 

67

MC15 0.98
MC15+GLUCOSE-C 0.55

  
   

2 (I

  

Y=-2.4+l.3-0.005T

 

 

fertilizer in soil MCIS.

Y=3.2+0.2T~0.00lT2 A

r)

8 . .

0 12 24 48 72 96
INCUBATION PERIOD (HOURS)

Figure 2l. Effect of addition of glucose-C on nitrification of labeled urea

68

Table 8. Comparison of rate of nitrificatign of fertilizers-N in the
presence and absence of Glucose-C

 

Soil Fertilizerb Rate of Nitrification
Source

 

No Carbon With Carbon

 

Z

 

 

w1 Urea 0.1c -o.7x10‘2C
wz Urea 0.2c -o.01C
MC15 Urea 1.12d --o.17d

 

a, Applied at the rate of 4000 ppm glucose-C
b, Added as 100 ppm urea—N
c, Represents the rate between 0 and 96 hours incubation period

d, Represents the rate between 0 and 24 hours incubation period

69

Table 9. Net nitrification and immobilization of urea fertilizer in
the presence and absence of glucose-C3.

 

Fertilizer N

 

 

SOIL Immobilizedb Nitrifiedc Immobilizedb Nitrifiedc
no Carbon with Carbon

m 6.11- 0.13 10 i" 1.1 97 :2.0 0.191008

wz 6.5-i- o.34 15.2 1- 2.0 87.8-1- 0.5 0.49:0.00

MC15 26.2-1- 0.20 75.3 1- 3.2 89.8-14.0 10.301026

 

a, Applied at the rate of 4000 ppm glucose—C

b, Mean and standard deviations of percent labeled
fertilizer recovered as organic-N at 96 hours

c, Mean and standard deviations of percent labeled
fertilizer recovered as NO -N at 96 hours

3

70

For the comparison of nitrification of urea in an initially acid soil
with a counterpart that was initially less acid, soils WI and W2 were used
as well as soils M818 and M015. The nitrification rate for soil WZ (pH
6.5) was greater than that for soil W1 (pH 5,1) (Figure 18). On the other
hand, the rate of nitrification in soil M818 (pH 5.7) was greater than that
in soil M015 (pH 6.8). The result for soils W1 and WZ demonstrate the pH
effect discussed earlier. The only explanation for the opposite effect
shown by soils M818 and M015 is that in these two soils, the differences in
rate of nitrification may not necessarily have been controlled by pH alone,
but by other soil factors as well. One of these factors may have been an
initially greater microbial population in soil M818 than in soil M015.
Effect of addition of glucose-C on the rate of nitrification of urea

fertilizer

 

The rate of nitrification of urea in the presence of glucose-0 was
determined on soils W1, W2, and M015. These results are presented in
Figures 19, 20, and 21 which show that in absence of C, the urea-N
nitrified very readily. When glucose-C was added, the nitrification rate
was slowed considerably in soil M015 and completely reduced in soils W1 and
.WZ (Table 8). After 96 hours, net nitrification was 10.3% of applied
urea-N in soil M015 while in soils W1 and WZ, it approached zero (Table
9).

It is impossible to know for sure from these data, but it seems that
some nitrification of the labeled urea fertilizer occurred initially in

soils W1 and NZ treated with glucose-0 but that the N0 -N produced was

3
later immobilized during the incubation. After 12 hours of incubation, 2%

of the fertilizer N applied was recovered as NO -N in soil Wl while for

3

71

the subsequent incubation period, the amount decreased until it was about
0.1% after 96 hours of incubation. A similar observation was noticed in
soil WZ where 3% fertilizer N was recovered as N03-N after l2 hours
incubation and only 0.5% after 96 hours. In soil M015, no further NO3'
seems to have been produced after 12 hours. The net NOB-N produced
during 12 hours was 9% and this remained almost the same throughout the
incubation period.

These results show that when glucose-C was added to the soils after
addition of labeled urea fertilizer, the nitrifying bacteria were unable to
compete effectively with heterotrOphic microorganisms immobilizing the
added N. In soil M015 in the absence of glucose-C, the nitrifying bacteria
were able to compete effectively for NH4-N since a substantial part of
the tracer N added as urea-N was recovered in the NOB-N fraction. The
increase reached a maximum shortly after the NH4+ had disappeared, which
required only 96 hours.

The interesting thing in this experiment was that when sufficient
energy was available in the form of a readily-available 0 source, the
energy demanding steps of the N cycle predominate, i.e. immobilization of N
(Table 9). In the absence of energy, nitrification is the predominant
process leading to the most oxidized state of N, viz. N03-N (Woldendorp,
1975). From the foregoing, it is clear that the state in which N is found
in the soil is determined to a high degree by the presence or absence of a
readily available 0 source, i.e. energy. Nitrifiers are autotrophic
bacteria and are very inefficient, relative to heterotrophs, in converting
the energy available from their respective oxidations into cellular
materials (Fenchel and Blackburn, 1979). This suggests that the quantity

of N nitrified in a given situation will depend to a considerable extent

72

upon the activity of the heterotrOphic flora in relationship to that of the
nitrifiers.

A point of difference with Jansson's (1958) findings is in relation to
his conclusion that in competition between heterotrophic flora and the
nitrifiers for NH4-N, the nitrifiers are left with the N not needed by the
heterotrophs. The experimental data on soil M818 and M015 previously
discussed showed that nitrifiers competed effectively for NH4-N in the
absence of added 0, but in the presence of an available 0 source, the
heterotrophic organisms outcompeted the nitrifiers. Broadbent and Tyler,
(1962) in a similar experiment with Sacramento clay that received

(NH 304 plus straw, found that nitrifying bacteria were able to compete

4)2
effectively with the immobilizing flora for N whereas in the Moreno soil,

this was not the case.

SUMMARY AND CONCLUSION
These data show significant immobilization and nitrification of 15N
labeled (NH4)ZSO4 and urea fertilizers applied to soils after 12 hours of
incubation. Urea applied to soils was very rapidly hydrolyzed and much of

the added urea N was transformed to NH -N in two days. Following

4
hydrolysis, urea transformations resembled that of NH3. Marked differences
existed in the ability of the various soils to immobilize and nitrify

4)2504 and urea fertilizers in soils in the absence of a
glucose-C amendment when evaluated under comparable conditions.

labeled (NH

Changes in the pH of the soil which resulted from fertilizer
application exercised a considerable effect on the rate and amount of N
immobilized and nitrified. Alkaline reaction caused greater immobilization
and nitrification while an acidic reaction slowed them down. Soils with
initial high pH had greater immobilization and nitrification than those
with initial low pH. In one case, nitrification in soil M818 (pH 5.7) was
greater than that in soil M015 (pH 6.8). This shows that nitrification may
not necessarily have been controlled by pH alone, but by other soil factors
as well.

In conclusion, the quantity of ammonical fertilizer nitrified in a
given situation will depend in part on the rate at which it is immobilized
into organic-N. In these experiments with organic soil (M818 and M015),
the nitrifying bacteria were able to compete effectively for NH4+ with the

heterotrophic flora, but the Opposite was the case in the presence of an

73

74

available 0 source. In addition, the quantity and rate of fertilizer N
transformation are dependent not only on pH and available 0 source, but

also on other soil factors.

RECOMMENDATIONS FOR FUTURE RESEARCH

Laboratory studies do not adequately reflect the importance of some
major soil factors under natural field conditions. For more reliable
estimates of N transformations, field experiments should be developed to
obtain practically useful prediction of the pattern of N transformations
after fertilizer application. The time taken for immobilized fertilizer
N to be re-mineralized is something about which very little is known.
This requires incubation experiments at extended periods of time.

Long soil columns can be used to simulate field conditions in N
transformations experiments that involve amendment of soils with natural
organic materials after application of fertilizer N.

It may also be interesting to study the effect of other nutrients on
N transformations. For example, addition of readily available organic
matter stimulates microbial activity. This is accompanied by the
immobilization of nitrogen, which requires phosphorous (P) and other
elements essential to growth. Such a data can explain the relationship
between the different N transformations and the levels of P and other

elements in the soils.

75

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83

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APPENDICES

84

coHumcflspmumm oz .Q.z .u

 

 

 

 

 

 

.ZIUHcmwuo mm vmum>oomu umNfikuumm vmamnma mwmucmuuma wo Amcowumuwaamu mV macaumw>mv vumvamum mam ammz .n
.ZIUHcmwuo mm mmpm>oumu umnaafiuumm vwamnma mmmucmuuom we Amcowumuwaamu NV mcoflumfi>mm uumvcmum mam cmwz .m
o.o m.H ~.H m.o o.~ w.w o.m H.H m.~ 5.0 Amo.ov.o.m.s
Km.o ae.m m.ohe.AH en.oh~.HH .o.z .a.z .o.z .o.z .a.z .o.z .o.z 44H
mo.o n~.q mN.Hen.AH N.Hh©.¢ e.shw.om 05.0“ ~.Am m.oaw.ew mH.ohN.cN m.Hh H.e m.Nho.oN sm.oh 4.8 cm
H.oH m.~ sm.~nm.mm H.Hhm.A o.hhs.mn w.ahme.mm wH.oae.m “.mho.m~ ms.oa H.s Na.0nm.mH 3.0“ H.e we
wo.on ¢.H s~.ohe.HH mH.oae.m so.oha.so oe.0n~.- A.Nhs.oA em.Nho.mH H.0h e.~ o.Nno.Nz mm.oh m.s an
no.0“ s.H mm.oas.:H «c.0aa.m Hc.chm.ew m.Hzm.nH Ho.0h “.mw «H.0he.a 80.0“ m.~ A“.ohe.w no.0“ m.~ NH
H.ohma.fi Hm.0an.m mm.oho.s .a.z .o.z .a.z .o.z .o.z N.ohw.m ma.z o
~c.oAHN.o nH.oho.H N~.ohm.o o.oho.H mo.ohN.o so.ohem.w. No.0Hm.H so.ohem.o mw.ohm.m No.oHAm.o Amwaomv
mHz swam: emu ammo: pm: aw; . mafia: pas swam: aw:
cOmNA¢IzV + Hwom inmOUSHo + mm»: + HMOm mmm: + Hwom COMMWMMMCH

 

ZIUHcmwuo mm umNHHfluumm mmamnmq

 

.mafiom ucmumwwfin mo
vofiumm cowumnsucH >u0umuonm4 wcHuao mmnwawnoeeH umuwafluumm vwawnmq mo mcofiumw>mo mummcmum mam cam:

.< xwmcmaa<

5
8

cowumcwspmuwa oz .o.z .u

 

 

 

 

 

 

Zimoz mm noum>ouou uwNHHfiuumm mmamnma wmmucwuuwa mo Amcofiumuwaamu mv mCOfiumfi>mm upmmCMum mam cmmz .n
Zimoz mm nmum>ouwu uoswawuumu voamnma mwmucmuuma we Amcofiumuwaamu NV macaumfi>mn mummcmum mam amwz .m
N.o N.H 5.0 so.H cm.o om.o m.m H.H o.~ o.HAmo.ov.Q.m.A
so.on.m mm.owa.on on.0wfl.~ .o.z .Q.z .o.z .o.z .o.z .o.z .Q.z «8H
N.on.m mc.omq.qm oo.owo.m o~.oH m.oa mo.oHoH.o 0.0qu.o q.mwm.mm mo.HH m.m 0.8 we.mm o.~fl N.©H om
«0.0Ho.m m~.owo.cq oQ.OMN.H om.oHom.n oqo.on~.o HH.0mmm.o N.Huq.am oo.oH w.q mm.oflmc.mo H.ofl o.m we
No.omn.~ H.HH©.ON me.0wo.a mq.oflom.o mmo.oHow.o coo. “mm.o oo.on.mN mq.ou n.m mq.oHc.wN N.oH m.q cm
mH.OMm.~ H.HM¢.NH NH.qu.H ma.0m m.m NH.“ mn.a H~.onm.N 0.0“ m.m wN.oH H.~ mq.omm.mH 5.0“ H.m NH
-.ohm.a sm.oaa.o HH.oa~.H .o.z .o.z .o.z .o.z .a.z em.oa o.m 0.8.2 o
mo.oHM.H oo.oHo.N Ho.ohm.o HH.0mmo.o Nwo.oflmmo.o Hmo.oflmo.o N.on~.o ca.owmm.o NH.OMNH.O m.0mo¢.o o
N Amusozv
pa; swam: nmm mmauz nH3 mNB mmaoz 3H3 swam; QM:
«OmNAqmzv + Hwom Qimmousau + amp: + Afiom mmu: + Hwom newumm
newumnsocH

 

newumm coflumnzocH zuOUmuonmq wcHuzo vmwuwuuwz uwmflawuumm mmamnmq mo mcofiumw>ma mummcmum mam cmwz

2: oz mm umuwafiuumm mmamnmq

.aaaom stateless 00

.m xwmcmam<

Appendix C.

Linear regression coefficients relating percent (NH

86

4)2804

fertilizer immObilized during different time intervals.‘

 

Soils

Time Interval (Hours)

 

 

0-144 6-144 12-144

M818a R2 0.69 0.84 0.82
7.55 9.74 10.50

0.09 0.07 0.06

F2 R2 0.78 0.91 0.90
a 3.89 5.03 5.09

b 0.06 0.04 0.04

2

w1 R 0.95 0.96 0.95
0.82 1.05 1.11

0.04 0.03 0.03

 

a, Intercept

b, Linear regression coefficient.

Appendix D. Linear regression coefficients relating percent (NH )80
and urea fertilizer immobilized during different

time intervals.

87

2 .

 

Time Interval (Hours)

 

 

Soils
0-96 6-96 12-96
MBl8 + Urea 0.88 0.89 0.88
5.58 6.66 7.83
0.16 0.14 0.13
M318 + (NH4)2804 0.72 0.91 0.90
6.64 8.95 9.53
0.13 0.06 0.09
W1 + Urea 0.91 0.91 0.89
9.90 1.25 1.40
0.06 0.05 0.05
W1 + (NH4)ZSO4 0.95 0.99 0.99
0.82 1.01 1.06
0.04 0.03 0.03

 

a, Intercept

b, Linear regression coefficient