EPA 660/2-73-002
August 1973 Environmental Protection Technology Series
NITRATE AND NITRITE
VOLATILIZATION BY
MICROORGANISMS IN
LABORATORY EXPERIMENTS
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
degradation from point and non-point sources of
pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet environmental quality
standards.
EPA Review Notice
This report has been reviewed by the Environmental Protection
Agency and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies
of the Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or
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EPA-660/2-73-002
August 1973
NITRATE AND NITRITE VOLATILIZATION BY MICROORGANISMS
IN LABORATORY EXPERIMENTS
Jean-Marc Bo Hag
Pennsylvania State University
University Park, Pennsylvania 16802
Project No. R-800997
(formerly 16080 EIT,
formerly WP-01536)
Program Element 1B2045
Project Officer
Richard E. Thomas
National Water Quality Control Research Program
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1
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ABSTRACT
Microbial nitrate arid nitrite volatilization was considered as a means
to eliminate nitrogen from soil and water in order to inhibit the
accumulation of nitrogenous substances as pollutants or health hazard-
ous compounds. Therefore it was attempted to compare nitrate-reducing
microorganisms in their reactions to different environmental conditions
in laboratory experiments.
Changing oxygen concentration, pH, temperature, nitrate or nitrite con-
centration affected differently the denitrification process of various
isolated microorganisms. Unfavorable growth conditions led to the
accumulation of nitrite if nitrate served as substrate.
It was found that certain soil fungi are also capable of volatilizing
nitrogen as nitrous oxide.
Biological and chemical factors were evaluated during nitrite transfor-
mation in autoclaved and non-autoclaved soil by determination of the
evolvement of nitrogenous gases. During chemical nitrite volatiliza-
tion, which occurred essentially at a low pH, the major gases evolved
were nitric oxide and nitrogen dioxide, but if biological activity was
predominant in a neutral and alkaline environment, nitrous oxide and
molecular nitrogen were formed.
The validity of laboratory observations in relation to field studies in
the domain of denitrification is discussed and evaluated.
This report was submitted in fulfillment of Project Number R-800997
(16080 HIT - formerly; WP-01536 - formerly), Contract 68-X-0100 under
the sponsorship of the Office of Research and Monitoring, Environmental
Protection Agency. The experiments were performed in the Laboratory of
Soil Microbiology, Department of Agronomy, The Pennsylvania State
University, University Park, Pennsylvania 16802.
11
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CONTENTS
Abstract ii
List of Figures v
List of Tables vi
Acknowledgements viii
Sections
I Conclusions 1
II Recommendations 3
III Introduction 5
IV Methods Used for the Determination of the Denitrifying
Activity 9
V Isolation of Denitrifying Bacteria and Some of Their
Physiological Characteristics 13
VI Comparative Denitrification of Selected Microorganisms
in Culture Media and in Autoclaved Soil 19
VII Environmental Factors in the Denitrifying Process
Denitrification at different temperatures 25
Effect of pH on denitrification 25
Influence of different nitrate and nitrite
concentrations 27
Interactions of microorganisms during
denitrification 31
VIII Nitrous Oxide Release by Soil Fungi
Experimental procedure 33
Experiments with growing cultures 35
Resting cell experiments 39
111
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IX Denitrification in Soil Samples
Experimental procedure 43
Biological versus chemical nitrite decomposition 45
Influence of oxygen on nitrite volatilization 49
Effect of inoculation of sterilized soil on
nitrite disappearance 52
X Inhibition of Methane Formation in Soil by Various
Nitrogen-containing Compounds
Influence of nitrogen-containing compounds on
methane formation 56
Influence of organic substances on methane
formation 59
XI References 61
XII Publications 65
IV
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LIST OF FIGURES
No.
1 Tracing of a Chromatogram Revealing the Appearance of
Gases Liberated after 3 Days Incubation of a Growth
Culture of _F_. oxysporum in 'Semi-anaerobic1 Conditions 11
2 Nitrate Reducing Characteristics of the Isolated Micro-
organisms During Growth under Anaerobic Conditions 16
3 Denitrifying Activity of Different Bacteria under
Anaerobic Conditions in Culture Media and in Soil 21
4 Effects of pH on Growth and Denitrification (Samples
Taken from 2-day-old Anaerobic Cultures) 28
5 Influence of Different Nitrate and Nitrite Concentrations
on the Growth and Denitrifying Characteristics of the
Isolated Bacteria (Samples Taken from 3-day-old Anaerobic
Cultures) 30
6 Denitrifying Characteristics During Combined Growth of
the Investigated Bacteria (The Letters Indicate the Com-
bination of the Tested Isolates.) 32
7 Formation of Nitrogenous Gases from Nitrite During
Anaerobic Incubation of Leached Sterile and Non-sterile
Soils with Different pH Values 47
8 Denitrification and Methane Formation in Soil (At the
Start of the Experiment the Soil Contained 325 yg of
NO '-N/g Soil and 2.5 ug Glucose/g Soil was Added.) 57
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LIST OF TABLES
No.
1 Morphological and Biochemical Characteristics of the
Investigated Nitrate Reducing Bacteria 15
2 Percentage of Total Nitrogen (Kjeldahl Determination)
in Culture Medium after 3 Days' Growth under Aerobic
and Anaerobic Conditions 18
3 C02 Formation under Anaerobic Conditions in Giltay's
Medium and in Soil 23
4 Effect of Temperature on Denitrification by Anaerobically
Grown Isolates 26
5 Effect of Growth of Fusarium Isolates under Aerobic and
Initially Aerobic Conditions for 6 Days at 30°C on the
Disappearance of Nitrate and Nitrite from the Growth
Medium 37
6 Nitrite Disappearance and Nitrous Oxide Formation During
Growth of Fusarium oxysporum under Initially Aerobic and
Anaerobic Conditions 38
7 Nitrite Reduction and Nitrous Oxide Production by Resting
Cells of F^. oxysporum 40
8 Production of Nitrogenous Gases from Autoclaved and Non-
autoclaved Soil at pH 7 under Anaerobic Conditions 46
9 Nitrate and Nitrite Transformation and Formation of
Nitrogenous Gases in Sterile and Non-sterile Soil, at
Different pH Values, in Leached and Unleached Samples
Incubated under Anaerobic Conditions 50
10 Nitrite Disappearance under Aerobic and Anaerobic Condi-
tions from Sterile and Non-sterile Soils with Different
pH Values 51
11 Effect of Inoculation of Sterilized Soil at Different pH
Values with Denitrifying Bacteria on Nitrite Disappearance
and Nitrogenous Gas Evolution after 4 Days under Anaerobic
Conditions 53
12 Influence of Different Nitrogen-containing Compounds on
Methane Production in Soil 58
VI
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No.
13
Effect of Glucose, Starch, and Alfalfa on CH. and CO
Evolution from Soil
60
VII
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ACKNOWLEDGEMENTS
The support and assistance of the Project Officer, Mr. Richard E.
Thomas, of the Robert S. Kerr Water Research Center, Environmental
Protection Agency, is acknowledged with sincere thanks.
The author also wishes to acknowledge the assistance and skillful
work in performing the reported experiments by the following persons:
Mary Lou Orcutt, Brigitte Bollag, Stanislaw Drzymala, Gabrielle Tung,
Stanislaw Czlonkowski, and Cheryl Nash, as well as the typing of
Donna Zimmerman. The author is also thankful to Dr. L. T. Kardos for
valuable advice and discussions.
viii
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SECTION I
CONCLUSIONS
1. Although microbial denitrification appears to be a potential solu-
tion to the problem of nitrogen elimination from soil and water (lakes,
rivers, etc.), the available knowledge is still fragmentary and inade-
quate in order to manage efficiently the denitrification process in the
various ecosystems. The reported data in this investigation present a
contribution to the problem of optimizing denitrification, but con-
siderable more research is needed in order to be able to use the basic
research findings under the numerous different natural conditions.
2. There are many indications that unfavorable growth conditions may
have a greater effect on the nitrite-reducing enzyme system than on the
enzyme which transforms nitrate. Consequently, a temporary or remain-
ing nitrite accumulation can occur when nitrate is reduced in a non-
optimal environment.
3. The optimum pH for nitrate reduction and growth of bacteria was
found to be in the neutral area. However, the metabolic activity on
nitrite has a different pH range for the investigated microbes. Con-
sequently, the interactions which occur in soil by changing pH are
complex.
4. Soil fungi can also participate in the volatilization of nitrogen.
It could be shown that fungi from the genus Fusarium can reduce nitrite
with the simultaneous release of nitrous oxide. Although only approxi-
mately 10% of the supplied nitrite substrate was detected in gaseous
form, fungi may be of importance in influencing the nitrogen content of
the soil since they can account for the largest portion of the total
microbial protoplasm in certain soils.
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5. It is possible to conclude from our experiments with soil samples
that nitrite is converted to gaseous products under aerobic conditions
by chemical reactions, whereas at a neutral and alkaline pH the conver-
sion is caused mainly by biological activity. During chemical nitrite
volatilization the major gases are nitric oxide and nitrogen dioxide,
but if biological activity is predominant, nitrous oxide and molecular
nitrogen are formed.
6. Various nitrogen-containing compounds interfere with methane-
producing microorganisms. Nitrate caused the strongest inhibition of
methane formation followed with decreasing efficiency by nitrite,
nitric oxide and nitrous oxide. This observation could provide a means
to control methane formation if this process causes an ecological
problem.
7. Most laboratory incubation studies leave doubt as to whether the
data obtained apply to field conditions. Artificial conditions of the
laboratory are, however, requisite if the factors determining the paths
of a biological reaction are to be critically evaluated. The study
comparing denitrifying microorganisms in liquid media and in soil
demonstrates that the physiological reactions of the bacteria are
similar in the two media, and consequently basic studies performed in
the laboratory may provide valuable indicative results for further
evaluation under field conditions.
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SECTION II
RECOMMENDATIONS
Additional research on the nitrogen transformation resulting from
raicrobial activities in soil, streams and lakes should be undertaken.
Considerable more basic knowledge on denitrifying microorganisms is
needed if one is interested to monitor their activity under the various
environmental conditions, in order to use the denitrification process
for nitrogen removal.
Research should be performed under field conditions as well as in
well-controlled laboratory studies. The investigation in the labora-
tory is still .an important prerogative since the conditions in a
specific ecosystem like soil are so complex that it is extremely diffi-
cult to receive conclusive results concerning a specific activity of
microorganisms.
Different approaches in research should be used in order to find
effective means for optimizing or stimulating conditions for extensive
denitri fication:
Nutritional factors which may be selective for the growth and activity
of denitrifiers should receive special consideration. Certain organic
compounds which serve better as energy sources and more significantly
satisfy the nutritional demands of the denitrifying microflora should
be added at the appropriate time to stimulate the nitrogen removal
process.
It may also be assumed that certain growth factors or other specific
substances are required and can be identified by their selective action
on the denitrifying group among the microbes.
-------
Compounds which interfere in the nitrogen cycle, i.e., which inhibit
nitrogen fixation or nitrification, are also of special interest. It is
often assumed that nitrifiers and denitrifiers keep the nitrogen level
in soil at a certain equilibrium. An interference in an ecosystem may
result in the dominance of one group and consequently alter the physio-
logical balance.
The use of denitrification for removal of nitrogen can also be applied
on a small scale. Therefore it would be valuable to develop devices in
which the denitrification process can be used in homes and farms for
removal of nitrate and nitrite from drinking water.
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SECTION III
INTRODUCTION
The accumulation of nitrogen in soil and water causes one of the major
problems in pollution of the environment. In soil systems with high
concentrations of nitrate-nitrogen, the nitrate-nitrogen can leach
into the groundwater where it may present a health hazard. Drinking
water with nitrate-nitrogen levels above 8 to 9 ppm causes methemo-
globinemia or cyanosis in infants (Report of the 'Environmental Pollu-
tion Panel of the White House1, 1965). For water given to livestock,
a nitrate concentration above 5 ppm nitrate-nitrogen is regarded as
unsafe, and high concentrations may result in methemoglobinemia, loss
of milk production, vitamin A deficiency, thyroid disturbances, and
reproductive difficulties and abortions.
The use of a land disposal system for sewage effluents appears to be
a promising method for renovation of waste water, but the removal of
nitrogen presents one of the limiting factors for continuous and
increasing applications of waste.
Microbial denitrification appears to be a potential solution to the
problem of nitrogen elimination. This process, which has been used by
modifying a conventional biological sewage treatment system, may have
a considerable importance in soil as well as in an aqueous ecosystem.
In order to exploit the denitrification reaction for these specific
aims it is necessary to understand more clearly the physiological
mechanisms which lead to this reaction. The literature on denitrifica-
tion is voluminous. However, there exist many conflicting reports, and
we are still ignorant about essential steps during denitrification,
especially its dependence on ecological factors.
It must be remembered that two alternatives for the reduction of
nitrites and nitrates by bacteria exist: 1. Assimilative Reduction;
-------
formation of ammonia from NO ~ or NO ~, which is then transferred into
the anabolic cell-metabolism. This reductive process merely accumu-
lates the nitrogen in the soil and leaves it available for re-oxidation.
2. Respiratory Reduction, i.e., denitrification in which nitrates or
nitrites replace oxygen as final electron acceptors in respiration.
The end product of this respiration may be molecular nitrogen or the
oxides of nitrogen. The term denitrification has sometimes been used
in a much broader sense, but will be used in this report according to
the above definition which was approved by the Soil Science Society of
America (Proceedings 26, 307).
Microbial reduction of nitrates and nitrites is brought about by a
number of species of facultatively anaerobic bacteria. Most of them
use oxygen preferentially as a hydrogen acceptor but may also use
nitrates and nitrites as substitutes. The end products of this res-
piration are—as outlined above—nitrogenous gases which are lost from
the soil by diffusion into the atmosphere.
It is this mechanism which provokes concern in agricultural practice
since the loss of nitrogen in gaseous form was regarded as a loss of
fertilizer. Most of the applied research in agriculture has therefore
been directed toward eliminating denitrification.
It was the major purpose of this project to find methods to stimulate
the microbial denitrification process. This would provide a biological
method for nitrate removal in gaseous form when an aqueous or a soil
ecosystem is overloaded with nitrogen-rich material from waste dis-
posal. In recent years it has also been recognized that nitrogen-loss
in soil involves a combination of biological as well as chemical
reactions. Although it was our intention to study especially the
biological mechanism, certain chemical processes were also evaluated.
Field investigations of the denitrification process have met with many
difficulties. The conditions in a soil ecosystem are so complex that
-------
it is extremely difficult to receive conclusive results concerning a
specific microbial process. Especially in the nitrogen cycle it is
difficult to know or to distinguish under field conditions if, for
example, the denitrification process is partially hidden by nitrifying
organisms or if a non-biological factor contributes to additional
release of nitrogenous gases. Therefore, it appeared to us that in
order to study denitrification and the major influencing factors, it is
necessary to have well-controlled conditions and also pure microbial
cultures for obtaining conclusive results. The drawback is obvious
since the conditions designed in the laboratory have to be quite dif-
ferent from those existing in the microenvironment within the soil.
Nevertheless, it appeared that results obtained from pure microbial
cultures in liquid solutions or in a soil environment are comparable,
and consequently may provide indicative results for further evaluation
under field conditions. The artificial conditions of laboratory
experiments are requisite if the many ecological factors determining
the paths of a biological reaction are to be critically studied.
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SECTION IV
METHODS USED FOR THE DETERMINATION OF THE DENITRIFYING ACTIVITY
Nitrate determination
Nitrate was initially measured by the brucine method (American Public
Health Association, 1965), but later this determination was abandoned,
since the use of the nitrate-electrode proved to be a much less time-
consuming procedure and simultaneously the sensitivity and accuracy was
quite comparable. A high nitrite concentration interfered especially
with the brucine determination. When this happened, the amount of
nitrate was determined and interpolated after nitrite determination
according to established standard curves. It was very rare that
nitrite accumulated in the investigated samples to such an extent that
interference constituted a problem.
Nitrite determination
Nitrite was always measured in the aqueous phase by the naphthylamine-
sulfanilic acid procedure (American Public Health Association, 1965).
Measurements of nitrogenous and other gases
Gas samples were withdrawn from the flasks with a gas-tight syringe
(Hamilton Corp., Whittier, Cal.) and injected into a gas chromatograph
(Varian Aerograph 1820) using two parallel columns (3 mm o.d.) simul-
taneously at 50°C: Porapak Q (600 cm; 50-80 mesh) and Molecular sieve
5A (450 cm; 45-60 mesh). The detectors were dual thermal conductivity
cells at a temperature of 200°C. The inlet temperature was 70°C.
Helium was used as a carrier gas at a flow rate of 40 ml per minute
under 2.95 kg/cm2 pressure. The filament current was 200 mA for the
Porapak Q column and 150 mA for the Molecular sieve 5A column. The
quantity of gases was determined by the use of an integrator (Digital
Integrator, Model 477, Varian Aerograph) and calibrated with known
amounts of the various gases. During incubation of the different
-------
samples, gases were usually formed which caused a positive pressure in
a flask. The increase in gas pressure was measured with a manometric
device, and the amount of the formed and detected gases was calculated
accordingly. The column of Porapak Q clearly indicated peaks of carbon
dioxide (CC>2), nitrous oxide (N20), and nitric oxide (NO), whereas the
other column of Molecular Sieve 5A separated oxygen and nitrogen (see
example in Figure 1).
In certain experiments nitrogen dioxide (N02) and nitric oxide (NO)
were determined by absorption of these gases in 1 N^ NaOH-solution con-
taining 125 mMol of KMnO per liter. Depending on the expected for-
mation of NO- or NO, one or two vials containing 4 ml of the KMnO.
solution were placed in the incubation flask. For further analysis
this solution was decolorized according to the procedure of Anderson
(1965) and subsequently neutralized and the amounts of nitrite and
nitrate were determined.
10
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RETENTION TIME (MIN)
Figure 1: Tracing of a chromatogram revealing the appearance of gases
liberated after 3 days incubation of a growth culture of
F. oxysporum in 'serai-anaerobic' conditions.
11
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SECTION V
ISOLATION OF DENITRIFYING BACTERIA AND SOME OF THEIR PHYSIOLOGICAL
CHARACTERISTICS
Though there exists a considerable literature on denitrification,
many reports on the physiological and biochemical characteristics of
denitrifying microbes appear contradictory. Many studies have been
performed with a single bacterial strain or atypical denitrifier
(e.g., E_. coli) and consequently general conclusions have been mis-
leading. Therefore, it seemed desirable to isolate various denitri-
fiers from soil and to compare their physiological activity.
A large number of nitrate-reducing bacteria strains were enriched from
soil (Hublersburg silt loam soil) by using anaerobic conditions in a
culture solution (Giltay's medium) containing 1.0 g KNO,, 1.0 g
1-asparagine, 8.5 g Na-citrate, 1.0 g KH2P04> 1.0 g MgSO^y^O, 0.2 g
CaCl'6H 0, and 0.05 g FeCl -6H 0 per liter of distilled water which
was adjusted to a final pH of 7.2 with NaOH. After several transfers
in the liquid medium, the microbes were isolated using the dilution
technique and plating on agar. After incubation for several days at
30°C, single colonies were selected and purified by further plating.
The resulting pure cultures were transferred at 2-month intervals to
fresh slants of nitrate agar. After growth at 28°C, these slants were
maintained at 3-5°C and were used for preparation of the liquid
cultures.
The experiments under anaerobic conditions were carried out in 250 ml
suction flasks fitted with a rubber stopper containing a glass tube
that extended into the culture medium. On top of the glass tube and
at the suction outlet rubber tubing was attached and sealed airtight
with a pinchcock clamp. All flasks contained 90 ml of Giltay's medium
and a test tube containing 4 ml of 8N NaOH for absorbing carbon
dioxide. In order to provide anaerobic conditions, argon or helium was
13
-------
forced through the glass tube for 5 to 10 minutes and allowed to escape
through the suction outlet. Then the flask was immediately sealed.
The flushing of the incubation flasks was performed at the beginning of
an experiment and daily thereafter when samples were withdrawn. All
cultures were incubated at 30°C.
Cultures grown in air were obtained by inoculating a 250 ml Erlenmeyer
flask containing 60 ml of medium on a gyrotory shaker (200 to 250
oscillations/minute) at 30°C.
Each test was replicated several times; average values are reported.
Culture samples were withdrawn daily and observed for growth and gas
production. Gas appearance was regarded as an indication of denitrifi-
cation, but the only certain proof of denitrification in these studies
was the actual measurable loss of nitrogen as indicated by total
nitrogen determination (Kjeldahl method, Bremner and Shaw, 1958).
After the isolation of approximately 60 nitrate reducing microorganisms
from soil, the denitrifying characteristics were established in pre-
liminary experiments. Only four isolates were selected for further
investigation since they varied considerably in their nitrate reducing
features. Since the microbes were not identified, they will be
labelled in this report as Isolate A, D, G, and H. Some morphological
and physiological characteristics of these bacteria are summarized in
Table 1.
When the selected isolates were cultivated under anaerobic conditions,
all nitrate disappeared from the culture medium within 48 to 72 hours'
incubation at 30°C. Nitrite was usually not detected in the medium of
Isolates A and D. However, nitrite invariably accumulated in the
growth medium of Isolates G and H. Nitrite disappeared only with the
bacterial strain G after further incubation (Figure 2).
14
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TABLE 1: MORPHOLOGICAL AND BIOCHEMICAL CHARACTERISTICS OF THE INVESTIGATED NITRATE REDUCING BACTERIA.
Cell Gram Acid formation from Gelatin Litmus Requirement Nitrate
Bacterium Morphology Motility reaction Glucose sucrose Lactose li(luefaction milk Pigment for oxygen reduction
Isolate A rod non-motile negative
Isolate D rod non-motile negative
Isolate G rod motile negative
Isolate H
rod
motile negative
alkaline
alkaline
facultative
facultative
alkaline orange facultative +
(on potato) temporary
nitrite
formation
alkaline dark
orange
(on nitrate
agar)
facultative
nitrite
formation
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70
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ISOLATE G
ISOLATE D
--x
0.8
0.4
ISOLATE H
0.8
O
CJl
o
0.4
NITRATE
DAYS
o NITRITE
x GROWTH
Figure 2: Nitrate reducing characteristics of the isolated micro-
organisms during growth under anaerobic conditions.
16
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In order to determine if the disappearance of nitrate and nitrite and
the observed development of gases by the isolates could be used as
indicators for the denitrifying activity, the total nitrogen was deter-
mined in each growth culture under aerobic and anaerobic conditions.
It is evident from Table 2 that a considerable loss of nitrogen
occurred with Isolates A and D when comparing the cultures which were
exposed to oxygen with those cultured anaerobically. Isolate G showed
indications that some nitrogen may be lost even under aerobic
conditions.
During anaerobic growth it was observed, as in previous reports, that
there was an "alkaline drift" from the original pH of 7.2. The final
pH after 3 days' incubation was measured as 8.4, 7.9, 7.6, and 8.1,
respectively, for the Isolates A, D, G, and H. Isolate H showed a
slight shift to the acid side during the early stages of growth.
17
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TABLE 2. PERCENTAGE OF TOTAL NITROGEN (KJELDAHL DETERMINATION) IN
CULTURE MEDIUM AFTER 3 DAYS' GROWTH UNDER AEROBIC AND
ANAEROBIC CONDITIONS.
CULTURE CONDITIONS
Aerobiosis Anaerobiosis
Isolate A
Isolate D
Isolate G
Isolate H
Control medium
%N
4.76
4.91
4.10
5.11
5.01
%N
2.04
1.89
3.04
2.93
4.91
(uninoculated)
18
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SECTION VI
COMPARATIVE DENITRIFICATION OF SELECTED MICROORGANISMS
IN CULTURE MEDIA AND IN AUTOCLAVED SOIL
It was also attempted to compare the denitrifying characteristics of
the isolated bacteria and of some laboratory cultures in the liquid
artificial growth medium and in a sterile soil system in order to
clarify if the mere change from a prepared liquid to the solid and com-
plex structure of a soil causes an essential change of denitrification
activity by the tested microbes.
Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis
which were also used for this study were obtained from the culture col-
lection of the Department of Microbiology at The Pennsylvania State
University. The liquid culture solution was 'Giltay's medium1 (des-
cribed in the previous section) and the soil used in these experiments
/
was a Hagerstown silt loam soil with a neutral pH; the indigenous
nitrate-nitrogen concentration was 75 yg/g of soil; organic-carbon,
1.80%; and the sand-, silt- and clay-contents were 8.5%, 63.4%, and
28.1%, respectively. Twenty grams of soil were placed into each incu-
bation flask and 10 ml of distilled water containing 5 mg nitrate-
nitrogen were added. Sterilization of the soil was accomplished by
autoclaving the samples three times for 30 minutes over a 2- to 3-day
period with at least 12 hours' interval. To inoculate the soil, 1.0 ml
of the selected stock culture grown in Giltay's medium was added to
each sample.
Incubation of anaerobic samples was observed in 125 ml bottles which
were sealed with a one-hole rubber stopper containing a septum.
Anaerobic conditions were attained by flushing the bottles with helium
until all air was removed.
19
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In order to incubate liquid media under anaerobic conditions, 50 ml of
Giltay's medium were added to each bottle and inoculated with one loop
of a bacterial culture.
For aerobic conditions 125 ml Erlenmeyer flasks were used which were
stoppered with foam plugs. Soil specimens were placed in a vacuum
incubation chamber and a slight negative pressure insured a continuous
exchange of air—also within the flasks. Aerobic liquid samples were
kept on a rotary incubation shaker revolving at 200 oscillations per
minute. All samples—aerobic and anaerobic—were incubated at 30°C for
3 days. The experiments were repeated two or three times and several
replicates were evaluated for each specific treatment.
The comparison of the denitrifying characteristics of the selected
microorganisms in a liquid growth solution as well as in soil is
depicted in Figure 3. Nitrate and nitrite determination as well as the
measurement of the evolved gases during incubation served as indicators
of the microbial activity. After incubation for 3 days it was apparent
that the disappearance of nitrate was slower in the soil than in the
liquid Giltay's medium, but the essential features of nitrate transfor-
mation by the microbes were not affected under the two tested condi-
tions. Isolates A and D produced a considerable amount of nitrous
oxide in the culture medium as well as in soil, but the intermediary
formation of nitrite could not be observed. On the other hand,
Serratia marcescens and Isolate H showed similar characteristics con-
cerning the marked formation of nitrite in Giltay's medium and the
same observation was present but not so apparent under soil conditions.
The smaller accumulation of nitrite in soil could be correlated with
the slow transformation of nitrate and a lower nitrite concentration
could facilitate its subsequent volatilization to nitrogen gas.
The growth of Pseudomonas aeruginosa was very rapid in the culture
solution as indicated by increasing turbidity. At the same time it
20
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75
CONTROL
ISOLATE
A
SOIL
ISOLATE
0
ISOLATE
H
PSEUDOMONAS
AERUOINOSA
SERRATIA BACILLUS
MARCESCENS SUBTILIS
Figure 3:
D N03 • N02 ffl N2 H N20
Denitrifying activity of different bacteria under anaerobic conditions in culture media
and in soil.
-------
could be found that practically all nitrate which disappeared was
recovered as nitrogen gas. However, if the Pseudomonas species was
cultivated in soil, it used the nitrate more slowly and, although N2
was the predominant gas, in this case it was also possible to detect
nitrite and some nitrous oxide. Bacillus subtilis was also included
in these experiments although this bacterium is not considered to be
a denitrifier. It was found that both in soil and in the culture some
nitrate was transformed and it could be recovered as nitrite and
nitrogen gas.
Simultaneously with the release of the nitrogenous gases the formation
of C02 was observed and the results of one representative experiment
are shown in Table 3. It is of interest to note that Isolate H and
Serratia marcescens which accumulate in the culture solution a con-
siderable amount of nitrite show a strong production of C02 if compared
with the other active bacteria, but a weaker nitrate transforming
ability in soil is complemented with a relatively smaller formation of
C02- This fact is especially noteworthy if it is compared with the
bacteria which do not accumulate nitrite: their production of C02 is
by far more extensive in soil than in the culture medium although less
nitrate disappeared. For example, Isolate A produced 10.2 yg of C02
per ml culture solution and 69.3 yg of CC^ per g soil.
A small amount of CC^ formation which was found in the sterile control
of the soil samples appears to be of nonbiological origins, e.g., C02
may be produced by decarboxylation of organic compounds or decay of
free carbonates.
22
-------
TABLE 3: C02 FORMATION UNDER ANAEROBIC CONDITIONS IN GILTAY'S MEDIUM
AND IN SOIL.
Control
Isolate A
Isolate D
Isolate H
Pseudomonas
aeruginosa
Serratia
marcescens
Bacillus
sub til is
C0~ - Formation
Gilt ay's medium (yg/ml)
Sample
1
0
9.0
11.0
29.7
16.9
52.8
4.4
Sample
2
0
10.4
10.8
32.9
17.7
52.0
8.2
Sample
3
0
11.3
18.6
16.0
57.9
8.2
Average
0
10.2
13.4
31.3
16.9
54.2
6.9
Sample
1
8.7
137.6
123.2
71.9
115.7
r>
69.8
58.8
in
Soil
Sample
2
7.0
137.0
125.6
75.5
138.8
68.7
61.1
(yg/g)
Sample
3
7.3
137.6
125.5
76.8
114.9
51.4
Average
7.7
137.4
124.8
74.7
123.1
69.3
57.5
23
-------
SECTION VII
ENVIRONMENTAL FACTORS IN THE DENITRIFYING PROCESS
If one is interested in influencing denitrification in nature by stimu-
lation or inhibition, it is primarily necessary to know the responses
of the predominating denitrifiers to the various environmental condi-
tions. Though there exists a considerable literature on these topics,
many observations are contradictory, e.g., the question how the oxygen
concentration is related to denitrification or what is the most suit-
able pH for denitrifiers still evokes controversial answers; therefore,
it appeared desirable to investigate the influence of various environ-
mental factors on the selected microorganisms in this study.
Denitrification at Different Temperatures
All' metabolic processes can be characterized by their temperature
dependency. Therefore, the response of Isolates A, D, G, and H to
temperatures at 10°, 22°, 30° and 37°C was observed under anaerobic
conditions. Since growth was poor at 10°C and little denitrification
occurred, only the data for the higher temperatures are reported
(Table 4). Best growth and nitrate disappearance were established for
all isolates at 30°C which was also the optimal temperature for growth
under aerobic conditions. Isolate A did not show any growth at 37°C
under either aerobic or anaerobic conditions. An interesting point is
that temporary accumulations of nitrite occurred with strain D at 22°C
and 37°C after 3 days. This indicates that the nitrate reducing
enzyme system of this isolate is more temperature dependent than
nitrate reductase. In all cultures there was a clear correlation
between the rate of growth and nitrate reduction under the various
temperatures.
Effect of pH on Denitrification
It is generally assumed that denitrification is favored in a neutral
to alkaline ecosystem and that denitrifying populations in otherwise
25
-------
TABLE 4: EFFECT OF TEMPERATURE ON DENITRIFICATION BY AHAEROBICALLY GROWN ISOLATES.
Temperature
•c
22
30
37
22
30
37
22
30
37
22
30
37
Incubation
period
days
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
1
2
3
7
Growth,
optical density
at 550 my
Isolate A
0.00
0.02
0.22
0.54
0.00
0.34
0.46
0.47
0.00
0.00
0.00
0.00
Isolate D
0.00
0.05
0.21
0.23
0.04
0.27
0.28
0.32
0.02
0.36
0.39
0.41
Isolate G
0.00
0.04
0.24
O.S5
0.00
0.21
0.57
0.82
0.00
0.00
0.04
.0.09
Isolate H
0.17
0.43
0.66
0.67
0.40
0.57
0.74
1.05
0.00
0.15
0.19
0.22
Nitrate
concentration
ppm N
110
110
96
0
110
33
6
0
110
110
110
110
110
110
36
0
110
12
0
Q
110
16
0
0
110
102
66
0
110
24
0
0
110
110
82
70
96
40
21
8
43
31
25
17
110
102
96
92
Nitrite
concentration
pp» N
0.0
0.0
3.4
0.0
0.0
0.0
1.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
9.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
6.4
0.0
0.0
7.5
28.0
0.0
0.0
41.0
16.5
0.0
0.0
0.0
16.3
7.2
2.5
39.0
65.0
76.0
48.0
51.0
58.0
71.0
0.0
5.2
8.9
10.2
26
-------
optimal environmental conditions fail to release gaseous nitrogen at
high hydrogen ion concentration (Nommik, 1956). It is not surprising
that relatively little denitrifying activity was found at pH 5 and
below, since the metabolic activity of most microorganisms is reduced
under acidic conditions. However, many contradictory statements exist
in the literature on the influence of pH on nitrate volatilization.
Broadbent (1951) reported that denitrification is favored below pH 7,
whereas other investigators (Jansson and Clark, 1952; Bremner and Shaw,
1958b; Valera and Alexander, 1961) concluded that nitrogen loss was
considerably suppressed under acidic conditions. In another report
(Khan and Moore, 1968) it is concluded that no correlation between pH
and denitrification parameters could be found.
As in other instances, a general statement on the effect of pH on
denitrification can be misleading, since the various microbial strains
investigated here showed different responses in their denitrifying
behavior at a changing hydrogen ion concentration.
In Figure 4, the data demonstrate the influence of pH on nitrate reduc-
tion with the four investigated isolates after 2 days of growth under
anaerobic conditions. In no instance was there significant growth or
nitrate disappearance below pH 6.0. Best growth occurred at a neutral
pH and nitrate disappearance was closely related to this phenomenon,
but the accumulation of nitrite varied in relation to the pH with the
different organisms. Isolates A and D produced nitrite at pH 8, but
not at pH 7. Isolate G formed the largest amount of nitrite at pH 6,
although optimal growth and nitrate reduction occurred at pH 7.
Influence of Different Nitrate and Nitrite Concentrations
Information is scarce on the response of the denitrifying population to
increasing concentrations of nitrate. This constitutes an important,
practical problem in agriculture and waste disposal. Wijler and
27
-------
140
70
E"
Q.
Q.
IbULAit A
ISOLATE G
PH
NITRATE
ISOLATE D
0.8
0.4
ISOLATE H
0.8
O
b
8!
o
3
0.4
o NITRITE
7 8
PH
x GROWTH
Figure 4: Effects of pH on growth and denitrification (samples taken
from 2-day-old anaerobic cultures).
28
-------
Delwiche (1954) as well as Nommik (1956) concluded from soil experi-
ments that the initial nitrate concentration does not influence the
rate of gas release. Nitrite is toxic to various microorganisms, yet
it is not known how much can be produced when nitrate is reduced, and
to what extent its accumulation may inhibit the denitrifying process.
The four isolates were tested under anaerobic conditions for their
ability to use nitrate as well as nitrite at very low to toxic concen-
trations during 3 days of growth. It appears from the results in
Figure 5 that, if the growth medium contains 0.5% nitrate, only Isolate
G is able to transform all applied nitrate within 3 days. Isolates A
and D use only a small amount, a part of which can be recovered as
nitrite. The appearance of considerably more nitrite with higher
nitrate concentrations seems to be a general phenomenon. There was no
visible inhibitory effect of 2.0% nitrate on the growth of all isolates
tested, but relatively little of the added nitrate disappeared.
From Figure 5 it is obvious that the optimal and acceptable concentra-
tion for growth is considerably higher with nitrate than with nitrite.
One-tenth of 1 percent nitrate proved to be optimal for denitrification
by these four soil isolates. The same concentration of nitrite was
inhibitory for Isolates A and D; 0.2% nitrite suppressed practically
all growth.
Whereas relatively low concentrations of nitrite exerted an inhibitory
effect on growth and denitrification, there was no indication that
increasing amounts of nitrate suppress the development of micro-
organisms. However, if at higher concentrations unused nitrate
remained in the growth medium, an accumulation of nitrite occurred.
This phenomenon requires more attention since it could constitute an
important factor under certain ecological conditions.
29
-------
100
50
100
50
UJ
O
O
100
50
100
50
ISOLATE A ISOLATE 0 ISOLATE G
0.1% NITRATE
fi
ISOLATE H
t
I
n
n
0.5 % NITRATE
1.0 % NITRATE
2.0 % NITRATE
D GROWTH
ISOLATE A ISOLATE 0 ISOLATE G ISOLATE H
0.01% NITRITE
n
NITRATE
0.05% NITRITE
n
O.I % NITRITE
0.2 % NITRITE
NITRITE
0.6
0.4
0.8
0.4
Ol
O1
O
o.e 3
0.4
0.8
0.4
Figure 5: Influence of different nitrate and nitrite concentrations on the growth and denitrify-
ing characteristics of the isolated bacteria (samples taken from 3-day-old anaerobic
cultures).
-------
Interactions of Microorganisms During Denitrification^
Little attention has been given to the interaction of denitrifying
organisms within an ecosystem. Multiple possibilities of combinations
between microbes are possible; therefore, these experiments are diffi-
cult to perform for clear conclusions on interactions.
The experiments reported here appear to be the first attempt to
investigate this problem. Several interesting observations appeared
during the combined growth of the isolated microorganisms under
anaerobic conditions (Figure 6). Nitrate disappeared under all cul-
ture combinations, but the formed nitrite was attacked differently.
For example, Isolate G produced a nitrite-nitrogen concentration of 55
to 75 ppm after 2 days (Figure 6) while very little was produced by
this organism in combination with either Isolates A or D. This indi-
cates that Isolates A and D are able to degrade nitrite as soon as it
is formed. Nitrite concentration increased temporarily to 95 ppm in a
mixed culture of Isolates G and H.
As previously described, Isolate H produced nitrite which accumulated
in the medium. A mixture of Isolates D and G was able to degrade the
formed nitrite. However, when Isolate A was cultured in combination
with Isolate H, the nitrite concentration was not reduced. This indi-
cates that one microbe may inhibit a second denitrifier. The formation
of nitrite by one bacterium may be toxic for another. Consequently,
the possible interactions of microbes have to be carefully considered
if nitrate volatilization of a mixed population is to be evaluated.
31
-------
NJ
LU
o
Cr
h-
140
70
GH
AG
1234
ADG
1234
AH
ADH
1234
DAYS
DG
AGH
234
DH
234
Figure 6: Denitrifying characteristics during combined growth of the investigated bacteria (the
letters indicate the combination of the tested isolates).
-------
SECTION VIII
NITROUS OXIDE RELEASE BY SOIL FUNGI
The following study was made to investigate the possible participation
of fungi in nitrogen volatilization or denitrification processes in
soil.
Although certain bacteria reduce nitrate and nitrite to nitrogenous
gases by the process of denitrification, there is still no definite
evidence that fungi can perform a similar reaction (Nicholas, 1965).
A dissimilatory nitrate reductase can be present if fungi are grown
submerged where oxygen supply is limited (Walker and Nicholas, 1961;
Nicholas and Wilson, 1964), but the reduction process halts in this
case with the formation of nitrite. A nitrite reductase was also
isolated from Neurospora (Nason et al., 1954), but nitrite was reduced
in this case to ammonia which indicates an assimilatory pathway. Most
fungi can utilize nitrate and nitrite as a nitrogen source under
aerobic conditions in an assimilatory process, but little is known
about the anaerobic growth of fungi and the possible formation of
nitrogenous gases.
Experimental Procedure
Two Fusarium species, F_. oxysporum and F_. solani, were isolated from
soil samples collected from 15 to 50 cm below the surface of a silty
loam soil. The isolation was performed at 30°C under anaerobic condi-
tions using Brewer anaerobic Petri dishes with Czapek-Dox agar con-
taining 0.2% (w/v) sodium thioglycollate. To suppress the growth of
bacteria, 0.1% (w/v) streptomycin (Pfizer) and 100 units ml"-'- of peni-
cillin (Pfizer) were added to the growth medium. Subsequently, it was
possible to transfer and culture the fungi under the described
anaerobic conditions. The liquid growth media were inoculated with
spore suspension from agar-grown slants.
33
-------
For the growth experiments two different media were used: Medium A;
a modified medium of Cove (1966) containing dextrose, 10 g; peptone
(Difco), 2 g; yeast extract (Difco), 3 g; casein hydrolysate, 1.5 g;
KC1, 0.52 g; MgSO -7H20, 0.52 g; KH2P04> 13.2 g; and 1 ml trace
elements solution (Na^O -lOJ^O, 400 mg; CuS04'5H20, 500 mg; FeS04'
2H20, 400 mg; MnSO^f^O, 800 mg; ZnSC^-y^O, 9 g; Na2Mo04'2H 0,
800 mg/1. distilled water) in 1 liter distilled water; Medium B con-
tained nutrient broth (Difco) enriched with 1% (w/v) dextrose, 0.3%
(w/v) yeast extract and 0.1% (v/v) of the same trace elements solution
used in Medium A.
Potassium nitrate was added as required before autoclaving, but sodium
nitrite was added through millipore filteres (0.22 y pore size,
Millipore Corp., Bedford, Mass.) after steam sterilization of the
medium.
For aerobic growth, the inoculated media were incubated at 30°C on a
rotary incubation shaker (200-250 osc min~*). 'French Square1 flasks
(120 ml) containing 50 ml medium were used for incubation under
anaerobic conditions. The flasks were closed with a two-hole rubber
stopper in which cylindrical septum plugs for gas chromatographs were
tightly inserted for gas flushing and sampling. Inside the flask a
glass tube was fixed in the inlet plug hole which extended into the
medium. To flush with helium, a syringe needle was inserted into the
inlet plug and excess gas and air were released from the flask through
the outlet plug which was also temporarily provided with a syringe
needle. After flushing for 2 min, both needles were immediately
removed. Sampling with a gas-tight syringe for gas-chromatographic
analysis indicated that all air was removed by this method.
For some experiments, the flasks were not flushed but tightly closed;
in this case it was assumed that the available amount of oxygen would
provide a better beginning growth for the mycelium, and after use of
34
-------
oxygen a nearly anaerobic environment was generated. These conditions
were characterized as 'initially aerobic'.
All flasks were incubated at 30°C. Carbon dioxide was absorbed in
small test tubes containing 1.5 ml 3 N KOH which were placed into the
incubation flask.
For experiments with resting cells, fungi were cultured in 2-liter
Erlenmeyer flasks under 'initially aerobic' conditions for 5-7 days.
The mycelium was harvested by filtration and washed three times with
0.02 M phosphate buffer, pH 7.0. A 10-ml Erlenmeyer flask with a
rubber stopper containing a septum for flushing and gas sampling was
used as the reaction vessel. Nitrite-transforming activity was assayed
at 30°C by incubating 1 g of mycelium in 0.02 M phosphate buffer
(pH 7.0) with the equivalent of 100 parts/106 N of the N-containing
substrate in a final volume of 4 ml. Boiled mycelium, a mycelium
without substrate, and substrate alone were included as controls in
all assays. At least two replicates were included in each treatment
and experiments were repeated two or three times. Anaerobic conditions
were also provided by flushing with helium.
Experiments with Growing Cultures
The growth of Fusarium oxysporum and F_. solani was observed under
aerobic and anaerobic conditions in different liquid media which had
been enriched with 0.1% (w/v) KNO or NaNO . The fungi grew well in
the various media, but oxygen supply affected their appearance. If
the fungi were cultured in a well aerated atmosphere on a rotary shaker
(250 osc min~ ), the mycelium grew fast, and a thick paste-like mixture
of mycelium and medium was obtained within 3 days. If the fungi grew
under conditions of limited oxygen supply but with initially aerobic
conditions, the yield of cells was approximately ten times less after
6 days than under aerobiosis. Flushing of the incubation flask with
35
-------
helium for 5 min provided conditions which were considered anaerobic,
and in this case even less growth was observed.
When nitrite was supplied to IF. oxysporum grown at 30°C in either
aerobic or initially aerobic conditions, nitrite disappeared from the
culture solution within 6 days. In the case of F_. solani grown under
similar conditions, nitrite disappeared from the aerobic cultures,
while a small amount of nitrite remained in the initially aerobic cul-
tures (Table 5). There was no decrease of nitrate in the fungal growth
medium, however, even though both isolates grew well in the presence or
absence of oxygen. The lack of nitrate disappearance indicates that
the investigated fungi do not have a nitrate-reducing enzyme system.
The growth of the fungi in relation to gas production under initially
aerobic conditions was studied in more detail (Table 6). After 3 days
of growth a small amount of NJD production increased continuously
thereafter. It is possible to deduce from the dry weight determina-
tions of the harvested mycelium that the growth of the fungi stopped
after 7 days, but the amount of N^O increased further. After 10 days
of incubation approximately 12.8 percent of the nitrite-nitrogen sup-
plied could be detected as N^O and all nitrate had disappeared. No
attempt was made to determine the amounts of nitrogen present in the
medium or assimilated by the fungi, since we were interested in the
possible volatilization of the nitrite-nitrogen. The presence of
molecular nitrogen could readily be detected by gas chromatography.
However, conclusions were difficult to make because the 'initially
aerobic' conditions were such that N2 was present at the beginning of
the experiment, but an increase in molecular N2 was still indicated.
Under anaerobic conditions N~0 release was much less—which seems to be
related to less growth—and the formation of N2 was not significant.
Attempts were made to trap nitric oxide or nitrogen dioxide in alkaline
permanganate solution, but our results were negative.
36
-------
TABLE 5: EFFECT OF GROWTH OF FUSARIUM ISOLATES UNDER AEROBIC AND
INITIALLY AEROBIC CONDITIONS FOR 6 DAYS AT 30°C ON THE
DISAPPEARANCE OF NITRATE AND NITRITE FROM THE GROWTH MEDIUM.
NO '-N in parts/10
J
Initially
Aerobic Aerobic
N02'-N in parts/10
Initially
Aerobic
Aerobic
F_. oxysporum
F. solani
110
107
109
108
0
26
0
0
Uninoculated
Control
106
119
224
226
37
-------
TABLE 6: NITRITE DISAPPEARANCE AND NITROUS OXIDE FORMATION DURING GROWTH OF FUSARIUM OXYSPORUM
UNDER INITIALLY AEROBIC AND ANAEROBIC CONDITIONS.
00
Conditions Period of
of incubation
incubation (days')
Initially
aerobic
Uninoculated
control
Anaerobic
Uninoculated
control
0
3
5
7
10
10
0
3
6
9
13
13
Dry weight
of
mycelium
(mg/ml)
0.0
7.6
21.8
37.4
37.3
0.0
0.0
0.0
0.0
23.9
28.1
0.0
Oxygen
concentration
(%)
20.0
12.0
3.5
1.6
0.08
20.0
0.0
0.0
0.0
0.0
0.0
0.0
Nitrite
concentration
(yg N/ flask)
7500
7000
3500
125
0
7520
5030
4150
3440
1300
1130
5000
N20*
formed
Cyg N/flask)
0
29
515
742
956
0
0
7
128
280
513
0
Per cent
of nitrite-N
transformed
to N20
0.0
0.4
6.9
9.9
12.8
0.0
0.0
0.9
1.6
3.6
6.5
0.0
*No corrections were made for the solubility of N70 in the medium.
-------
Resting Cell Experiments
When resting cells of F^. oxysporum were incubated anaerobically at 30°C
in the presence of nitrite, results similar to those of the growth
experiments were obtained (Table 7). An increase of nitrous oxide was
accompanied by a proportionate disappearance of nitrate. A greater
mass of mycelium provoked a stronger reaction: 49.7 mg (dry weight)
mycelium produced 35.3 ug N_0-nitrogen from 400 ug nitrite-nitrogen in
5 hours, while it took 20 hours for 22.7 mg (dry weight) mycelium to
form 36.5 yg of N20-nitrogen.
If nitrite was substituted in the reaction by other nitrogen-containing
substrates like KNO , (NH.)2S04 or NH2OH-HC1, at concentrations of 100
parts/lO^N, the formation of N-0 could not be observed after incuba-
tion of resting cells of P_. oxysporum for 5 hours under similar
conditions.
If the fungal mycelium was grown in an initially aerobic medium without
s
nitrite, no conversion of nitrite to N-O could be detected in a subse-
quent resting-cell experiment. It was also evident that cells of F_.
oxysporum cultivated in a medium containing nitrite under well aerated
conditions were not able to produce nearly the same amount of NJD in a
replacement-culture experiment as cells grown in the presence of
nitrite under initially aerobic or anaerobic conditions.
The production of nitrogenous gases by the reduction of nitrate or
nitrite during anaerobic respiration by fungi has not been reported
before. The fact that fungi cannot grow as well as some bacteria do
under anaerobic conditions (Cochrane, 1958) reduces the possibility
that fungi play a major role in denitrification within soil. Neverthe-
less, participation of fungi in nitrogen volatilization—including
possible chemical interactions—cannot be excluded.
39
-------
TABLE 7: NITRITE REDUCTION AND NITROUS OXIDE PRODUCTION BY RESTING CELLS OF F_. OXYSPORUM.
0
Time of incubation (hours)
2 1/2
Nitrite-nitrogen remaining
Cumulative N20- formation
ug N/ flask %
400 100
0 0
Ug N/ flask %
305 76.2
21.8 5.5
ug N/ flask %
0.0 0
35.3 8.8
-------
There are several recent reports which described the anaerobic growth
of certain fungal species: Mucor rouxii (Bartnicki-Garcia and
Nickerson, 1961); Fusarium oxysporum (Gunner and Alexander, 1965);
Neurospora crassa (Nicholas and Wilson, 1965); and Aspergillus nidulans
(Cove, 1966). The two Fusarium species, F_. solani and F_. oxysporum,
which we isolated from sub-soil, were also capable of growing anaero-
bically. The fact that it was possible to demonstrate that N-0 was
formed during the growth of the fungi as well as by resting cells and
that significantly more N~0 was formed under conditions of limited
oxygen supply provided an indication that a nitrite-reducing dis-
similatory reaction may be involved. Since no N~0 was formed in a
resting cell experiment when nitrate, ammonium or hydroxylamine served
as substrates, these substances seem to be excluded as intermediates or
compounds related to the observed activity.
Denitrifying bacteria are the only microorganisms which have been
reported to produce significant amounts of NO. The possibility of NO
production by other organisms or by other physiological reactions has
rarely been reported. Nitrous oxide was observed during the oxidation
of NFLOH to nitrite by cell-free extracts of Nitrosomonas europaea.
(Nicholas and Jones, 1960; Falcone et al., 1963; Anderson, 1964). A
chemical reaction between hydroxylamine and nitrite can cause the for-
mation of N«0, and we suggest that this reaction may explain the
results described in previous reports (NH-OH + NO ' -*- N90 + HJD + OH').
£. £ £ £
In the experiments of Renner and Becker (1970) where nitrite and
hydroxylamine were supplied simultaneously, the above chemical reaction
was no doubt initiated and the addition of resting cells of
Corynebacterium nephridii probably did not initiate NO formation in
the way they concluded. Furthermore, Nicholas (1965) reported that N^O
is also likely to be formed nonenzymatically when some intermediate at
the nitroxyl or hyponitrite level accumulates.
No fungi have been reported to denitrify, and the observed formation of
N_0 in this study does not imply such a mechanism. The possible
41
-------
release of N?0 by a chemical side-reaction—as mentioned above—cannot
be excluded but requires experimental proof. The finding, however,
that more N20 is produced when the fungal cells were cultured under
initially aerobic or anaerobic conditions could indicate that a cer-
tain degree of nitrite respiration may be involved, but it is not
possible for us to make any firm conclusions.
Nevertheless, it was possible to demonstrate that fungi can partici-
pate—at least indirectly—in nitrite volatilization, and this factor
may be of importance if one is interested in influencing the nitrogen
content of the soil.
42
-------
SECTION IX
DENITRIFICATION IN SOIL SAMPLES
The volatilization of nitrogen-containing compounds from soil into
nitrogenous gases is attributed to biological as well as chemical
reactions. Denitrification, chemical nitrite decomposition, and vola-
tilization of ammonia seem to be the predominant processes, but there
is no clear knowledge of which one of the volatilization mechanisms is
of a greater practical importance. Some investigators hold mostly the
biological reaction of denitrification responsible for nitrogen losses
from the soil (Nommik, 1956; Allison, 1966), whereas other studies tend
to emphasize more the chemical volatilization (Nelson and Bremner, 1969
and 1970; Bulla, Gilmour and Bollen, 1970). There is little doubt that
both processes are influenced by factors like pH, organic matter and
others, but only a few and incomplete approaches have been made in
order to establish to what extent these factors are of dominating
influence for the biological or chemical reaction.
It was the purpose of this investigation to clarify if nitrite, which
is an intermediate during nitrogen transformation in soil, is volatil-
ized rather by biological or chemical means, by comparing its dis-
appearance in sterile and non-sterile soil systems under various
conditions.
Experimental Procedure
The soil used in all the experiments had an original pH of 7.0, an
organic-carbon content of 1.60%, and with a texture composed of 7.4%
sand, 67.5% silt and 25.1% clay. Before use, the soil was air-dried
for 48 hours in layers of 1 cm thickness and passed through a 2-mm
sieve. The original air-dried soil contained 50 ug of NO_-N per gram.
One portion of the air-dried soil was adjusted to pH 5.0 by titration
with approximately 40 ml of 0.2 N HC1 per 100 g of soil. After
43
-------
adjustment of the pH, the chloride was removed by leaching and sub-
sequently the soil was oven-dried at 45°C. A recheck of the pH after
drying gave a value of 5.0. In order to obtain soil with a pH of 8.3,
increments of powdered Ca(OH)2 were added to a 1:2 soil-to-water
mixture until the desired pH was obtained.
For some experiments a portion of the soil in the neutral and alkaline
pH-category was leached with distilled water in order to decrease the
content of water-extractable nitrogen, particularly the nitrate which
was present. Since all of the acidified soil had been leached, one
portion received 1 mg of nitrate dissolved in 10 ml H_0 per 20 g soil
to restore its nitrate level to that in the original soil. The same
10 ml of H_0 also contained 5 mg of nitrite-nitrogen. Where only
nitrite was added, 5 mg nitrite dissolved in 10 ml HJ3 was added to
each 20 gram portion of soil.
Sterilization of soil was achieved by autoclaving the samples at 121°C
and 15 Ib steam pressure three times for 30 minutes with intervals of
at least eight hours within a two-day period. Nitrite and nitrate
solutions which were added to autoclaved soil were sterilized by milli-
pore filtration (0.2-u pore size, Millipore Corp., Bedford, Mass.).
Soil samples which were incubated under anaerobic conditions at 30°C in
125 ml flasks were sealed by a rubber stopper in which a septum was
inserted for gas sampling. Anaerobic conditions were prepared by
flushing helium gas through the flasks until all air was exchanged by
helium as determined by gas sampling and subsequent gas-chromatographic
analysis. In all flasks a constant positive pressure of helium gas was
applied before incubation Cl-25 atm) and the resulting change of gas
pressure by chemical or biological activity did not appear to change
the applied gas pressure significantly. The gases used for the cali-
bration curves were also kept under the same conditions. The volume of
the gas phase was measured in each flask and the amount of gas formed
was calculated from the representative analyzed sample. If incubation
44
-------
was performed under aerobic conditions, the samples were put into
125 ml flasks closed with foam tube plugs and kept in an incubation
chamber which was continuously flushed with filtered air; in order to
diminish the drying of the soil, a large pan of water was placed in the
chamber to increase the humidity. Two replicates were included in each
treatment and the same experiment was repeated once; the results
reported represent average values.
The determination of nitrite and nitrate in soil samples was performed
by extraction of 20 g of air-dried soil with 40 ml distilled water
during 30 min on a 'Wrist-Action' shaker; subsequently the soil was
separated from the aqueous phase by cenitrifugation and the supernatant
analyzed.
Biological versus Chemical Nitrite Decomposition
Since it was the major purpose of this investigation to distinguish
between biological and chemical nitrite volatilization, all experiments
described in this section—except one—were carried out with sterile
and non-sterile soil. It is evident from Table 8 that under anaerobic
conditions at a neutral pH there was very little nitrogen-degrading
activity in the autoclaved soil while all nitrate and nitrite dis-
appeared in the non-sterile soil. It is also interesting to note that
during the first 6 days the dominating gas produced in the latter soil
was nitrous oxide, which changed almost entirely to molecular nitrogen
during further incubation.
One of the most important environmental factors influencing biological
as well as chemical nitrite volatilization is the pH of the soil.
Therefore, the pH of the soil was adjusted from that of the natural
soil, pH 7.O., to values of 5.0 and 8.3, and the soil provided with
nitrite. The decrease of nitrite and the formation of nitrogenous
gases were observed during anaerobic incubation (Figure 7). There is
no apparent difference between sterile and non-sterile soil under
45
-------
TABLE 8: PRODUCTION OF NITROGENOUS GASES FROM AUTOCLAVED AND NON-AUTOCLAVED SOIL AT pH 7 UNDER
ANAEROBIC CONDITIONS.
Remaining amount of nitrite and nitrate Cumulative gas production in yg N/g soil
0 days 12 days 3 days 6 days 9 days 12 days
N02"-N N03~-N N02~-N N03"-N N20 N2 N20 N2 N20 NZ N20 N2
Soil* yg/g soil yg/g soil yg/g soil yg/g soil
Autoclaved 250 50 225 50 00 0 10 0 18 0 20
Non-autoclaved 250 50 0 0 84 22 130 79 78 219 59 250
*Each soil initially contained 50 yg N03"-N/g and 250 yg N02~-N dissolved in 0.5 ml of H20 was added
to each gram.
-------
STERILE SOIL
NON- STERILE SOIL
LU
e>
o
cr
200
100
pH 5.0
0
0> 200
100
o fc
pH 7.0
---
2OO
100
pH 8.3
2 4
DAYS
i
8
o -o
o o NITRITE
o o N20 o « (NO+N02)
Figure 7: Formation of nitrogenous gases from nitrite during anaerobic
incubation of leached sterile and non-sterile soils with
different pH values.
47
-------
acidic conditions (pH 5.0) in the rate of nitrite disappearance, but
there are considerable differences in a neutral or alkaline pH. The
nitrite concentration decreased rapidly within the first 2 days of
incubation at pH 5.0 in sterile and non-sterile soils, but then the
rate of decomposition slowed. In an autoclaved soil at pH 7.0 the rate
of nitrite disappearance was small and no decrease occurred at a pH
8.3. The soils which were biologically active lost all nitrite within
4 days at a pH 7.0 or within 2 days at a pH 8.3.
The composition of the nitrogenous gases which were evolved during
incubation also varied considerably according to the pH. Gas release
from sterile soils in a neutral or alkaline environment was small or
nonexistent, respectively. In non-sterile conditions the formation of
N^O or N was dominant. Again it was observed that during the initial
period of incubation nitrous oxide was the major gas generated, but
later N2 was the predominant volatilization product. These findings
indicated that denitrification is very likely responsible for this
transformation, since essentially microorganisms are able to reduce
nitrous oxide to molecular nitrogen.
No production of NO or N02 could be found in non-sterile neutral and
alkaline soils, and only a very small amount of these gases was trapped
by KMn04 in a sterile and neutral soil. At a pH 5, however, the major
gaseous products evolved in sterile and non-sterile samples were NO and
N02, and only very small amounts of N_ and N20 were released. The
reason for this appearance seems to be the suppression of biological
activity in an acid environment, and it indicates that nitrite volatil-
ization at a low pH is caused mainly by chemical reactions.
As was mentioned earlier, the original soil contained naturally a small
amount of nitrate. To evaluate the possible influence of this nitrate
upon nitrite volatilization, an additional experiment was performed.
Portions of the pH 7 and 8.3 soils which were leached to provide soils
48
-------
free of nitrate and a portion of the pH 5 soil which had been leached
in adjusting the pH, all received nitrate-nitrogen equivalent to that
found in the natural soil. Table 9 shows that the acid soil to which
nitrate was not added was more active in chemical nitrite degradation
than the soil which received nitrate. In the neutral sterile soil
there was relatively little difference between the unleached (nitrate-
containing) and leached (nitrate-free) soil with respect to total
nitrite disappearance. In neutral, non-sterile soils nitrite dis-
appeared more completely in the leached soil, but there was no differ-
ence in the quantity of nitrogenous gases recovered. With the sterile,
alkaline soil there was virtually no difference in the behavior of the
leached and unleached soil. With the non-sterile, alkaline soil
nitrite disappearance was similar in leached and unleached soil, but in
the nitrate-containing soil N~ and N~0 was observed, whereas in the
nitrate-free soil only N2 was recovered after 4 days. This phenomenon
indicates that the presence of nitrate influences the composition of
nitrogenous gases and that the conversion of nitrite to N2 is faster in
the absence of nitrate.
The formation of small amounts of nitrate in some samples under
anaerobic conditions was not further investigated but may have been due
to spontaneous decomposition of the nitrite to produce nitrate and
nitric oxide.
Influence of Oxygen on Nitrite Volatilization
Another environmental factor which is of major importance in influ-
encing nitrite volatilization concerns the oxygen supply in soil. The
decrease of nitrite was followed in aerobic and anaerobic conditions
with sterile and non-sterile soil of different pH values (Table 10) .
Only soil samples at a neutral or alkaline level which were not auto-
claved respond to changing oxygen conditions. No disappearance at all
of nitrite was found in aerobic, alkaline conditions and only 10%
nitrite disappearance was observed at a pH of 7.0 in neutral,
49
-------
TABLE 9: NITRATE AND NITRITE TRANSFORMATION AND FORMATION OF NITROGENOUS GASES IN STERILE AND
NON-STERILE SOIL, AT DIFFERENT pH VALUES, IN LEACHED AND UNLEACHED SAMPLES INCUBATED
UNDER ANAEROBIC CONDITIONS.
l/i
O
Nitrate -nitrogen
in yg/g soil
0 days 4 days
pH 5 sterile
non-sterile
pH 7 sterile
non-sterile
pH 8.3 sterile
non-sterile
- leached + NO ~
- leached
- leached + NO ~
- leached
- unleached
- leached
- unleached
- leached
- unleached
- leached
- unleached
- leached
50
0
50
0
50
0
50
0
50
0
50
0
50
0
60
15
56
0
0
0
50
0
0
0
Nitrite-nitrogen Recovery of nitrogenous gases in
in yg/g soil yg N/g soil after 4 days
as as as
0 days 4 days N20 N2 (N02 + NO) TOTAL
250
250
250
250
250
250
250
250
250
250
250
250
140
90
130
70
235
230
115
0
250
250
20
0
5
5
5
5
0
0
119
108
0
0
85
0
0
12
0
18
0
0
18
34
0
0
61
155
22
94
10
77
0
9
0
0
0
0
0
0
27
111
15
100
0
9
137
142
0
0
146
155
-------
TABLE 10: NITRITE DISAPPEARANCE UNDER AEROBIC AND ANAEROBIC CONDITIONS
FROM STERILE AND NON-STERILE SOILS WITH DIFFERENT pH VALUES.
Disappearance of nitrite-nitrogen after 4 days
AEROBIOSIS ANAEROBIOSIS
SOILS* Ug/g soil % yg/g soil %
pH 5
STERILE
NON-STERILE
165.0
200.0
66
80
180.0
195.0
72
78
pH 7
STERILE 10.0 4 25.0 10
NON-STERILE 25.0 10 250.0 100
pH 8.5
STERILE 0.0 0 0.0 0
NON-STERILE 0.0 0 250.0 100
*Each soil initially contained 50 yg NO ~-N and 250 yg NO~-N was added
per gram.
51
-------
non-sterile aerobic soil. Under anaerobic conditions very little
nitrite disappearance occurred in sterile, neutral or alkaline soil;
however, in the same non-sterile soils, nitrite disappeared entirely
which undoubtedly was due to denitrification.
Chemical nitrite disappearance under sterile conditions should not be
affected by oxygen supply, but at pH 5.0 and pH 7.0, a small influence
was detected. A large decrease of nitrite occurred at pH 5.0 under
aerobic and anaerobic conditions in both sterile and non-sterile media.
This represents a further clear proof that at a low pH chemical nitrite
decomposition can be an important factor responsible for nitrite dis-
appearance when substrate conditions are adverse for microbial activity.
However, it is worthwhile to point out that biological nitrite dis-
appearance is more effective than chemical degradation when it occurs
under optimal conditions, since during the observation period all
nitrite disappeared when biological activity was favored in the neutral
and alkaline soils. Only approximately 80% of nitrite removal was
found in sterile, acid soil where only chemical processes were
operating.
Effect of Inoculation of Sterilized Soil on Nitrite^Disappearance
In order to clarify further the participation of denitrifying microbes
in nitrite volatilization, the investigated soil with different pH
values was inoculated with two bacteria, Pseudomonas aeruginosa and a
newly isolated denitrifier (Isolate A), whose denitrifying character-
istics in culture solution were described previously (Bollag et al.,
1970). Both microorganisms were grown in nitrate broth and when
nitrate had disappeared from the medium, 1 ml of the bacterial culture
was applied to the sterilized soil samples. In additional experiments
with the culture solutions it was established that Ps. aeruginosa pro-
duced mainly molecular nitrogen, whereas Isolate A generated nitrous
oxide. The same physiological activity was observed after inoculation
of the soil with the two bacteria (Table H). Ps_. aeruginosa produced
52
-------
TABLE 11: EFFECT OF INOCULATION OF STERILIZED SOIL AT DIFFERENT pH
VALUES WITH DENITRIFYING BACTERIA ON NITRITE DISAPPEARANCE
AND NITROGENOUS GAS EVOLUTION AFTER 4 DAYS UNDER ANAEROBIC
CONDITIONS.
Soil* and Disappearance Recovery of nitrogenous gases
Denitrifiers of nitrite-nitrogen in yg N/g soil
as as as
in yg/g soil in % N20 N2 (N02 + NO) TOTAL
Soil pH 5.0
Isolate A 125.0 50
Ps. aeruginosa 125.0 50
Soil pH 7.0
Isolate A 10.0 4
Ps. aeruginosa 250.0 100
Soil pH 8.3
5.0 12.0 28.0 45.0
5.0 12.0 25.0 42.0
5.0 0.0 0.0 5.0
0.0 133.0 0.0 133.0
Isolate A 250.0 100 169.0 9.0 0.0 178.0
Ps. aeruginosa 250.0 100 0.0 103.0 0.0 103.0
*Each soil initially contained 50 yg NO ~-N and 250 yg NO ~-N was added
per gram.
53
-------
most molecular N_ in a neutral soil, but Isolate A did not reduce
nitrite at pH 7. The predominant gas in an acid environment was again
nitric oxide or nitrogen dioxide indicating chemical nitrite volatil-
ization, and only minute amounts of N^O and N~ were found.
54
-------
SECTION X
INHIBITION OF METHANE FORMATION IN SOIL
BY VARIOUS NITROGEN-CONTAINING COMPOUNDS
The formation of CH. in soil under anaerobic conditions and in the
presence of organic matter is a very common and well-known process,
especially in poorly drained or waterlogged soil. Anaerobiosis appears
to be a major condition for the activity of CH.-producing bacteria, but
other factors like redox potential, pH, temperature, moisture, and
available organic materials have a major influence on the rate of CH.
production.
It has been observed that nitrate has to disappear before CH. formation
begins. This observation was first made by Barker (1941) in studies of
cultures of the then called Methanobacterium omelianskii in which very
low concentrations of nitrate were used. The suppression of methane
production by nitrate in soil was observed by Takai et al. (1956) and
Yamane (1957) and more recently by Laskowski and Moraghan (1956) and
Bell (1969). The various investigators also concluded that the redox
potential has to be reduced considerably before methane production can
start.
During an investigation of the denitrifying process we observed inhibi-
tion of methane evolution by various nitrogen-containing compounds.
This paper describes these findings and correlates the results with the
oxidation state of the various nitrogenous substances. The influence
of different organic substances on methane accumulation was also
investigated.
Soil containing 75 parts/10 of nitrate-nitrogen and an added amount of
250 parts/10 was incubated under anaerobic conditions with glucose
(2.5 mg/g soil) and denitrification was immediately observed as indi-
cated by the evolution of N2 as well as N20 and nitrite as
55
-------
intermediates (Figure 8). When no further denitrification could be
observed and the N? concentration remained constant, the formation of
CH. began (Figure 8).
If the initial nitrate concentration was less than 325 parts/10 in the
soil, denitrification ceased after a shorter time of incubation and the
evolution of CH. started earlier. Since interest was centered on the
formation of CH., the soil samples were usually pre-incubated until C^
evolution started and then the soils were treated for the different
experiments.
Influence of Nitrogen-containing Compounds on Methane Formation
As mentioned previously, CH. evolution began only when all nitrate had
disappeared from the soil. Subsequently, the activity of CH.-producing
bacteria could be immediately interrupted by the addition of nitrate to
the soil. Evolution of CH. would not recommence until denitrification
was completed.
Table 12 shows the results of an experiment in which the influence of
various nitrogen-containing compounds on the formation of methane was
investigated. The compounds were added to the preincubated soil.
Nitrate, nitrite and the gases nitrous oxide and nitric oxide 'inhibited
the production of methane, but ammonium sulfate and hydroxylamine were
ineffective. The oxidation-reduction state of the nitrogenous com-
pounds affected the formation of CH. and the concentrations of these
substances were also an important factor. Nitrous oxide at concentra-
tions of 50, 100, and 500 parts/10 inhibited CH. production for 1, 4,
and 5 days, respectively, and in the presence of 50, 100 and 500
parts/106 n:
accumulate.
parts/10 nitric oxide it took 4, 5, and 8 days until CH. started to
It is obvious from Table 12 that nitrate was most effective in sup-
pressing the formation of CH.; the addition of 500 parts/10
56
-------
tn
o
w
0>
o>
300
200
UJ
o
o
a:
100 -
o
o-
/
8
10 12 14 16
DAYS
18
/
f
200
ISO
o
I 00
-------
TABLE 12: INFLUENCE OF DIFFERENT NITROGEN-CONTAINING COMPOUNDS ON METHANE PRODUCTION IN SOIL.
(yl CH4 per 1 g of soil]
DAYS OF CONTROL
INCUBATION*
1 4
2 7
3 14
4 20
5 38
6 49
8 75
10 98
12 145
16 311
N03'-N parts/106
50 100 500
000
000
000
000
000
0 00
000
3.3 0.6 0
4.1 0.8 0
6.4 1.9 0
N02'-N parts/ 106
50 100 500
000
000
000
000
000
5.1 0 0
7.0 0 0
31 0.8 0
63 2.9 0
191 9.4 1.8
NO-N parts/106
50 100 500
000
000
000
000
0.6 0 0
1.8 0.7 0
7.4 2.7 0
18 6.2 2.8
69 17 9.1
264 55 37
N20-N parts/106
50 100 500
000
1.1 0 0
1.9 0 0
3.3 0 0
4.9 1.0 0
15 3.8 1.7
28 9.7 5.9
45 29 12
76 51 47
280 132 94
(NH2)OH parts/ 106
50 100 500
453
976
15 10 11
18 11 15
32 19 33
N.D. N.D. 46
51 40 62
N.D. N.D. 78
99 92 102
204 188 230
(NH4)2S04 parts/ 106
50 100 500
585
9 14 11
12 19 16
18 23 24
24 30 30
N.D. N.D. 42
61 68 56
N.D. N.D. 73
128 119 97
204 212 170
tn
oo
* The soil was preincubated for 8 days under anaerobic conditions after addition of 2.5 rag of glucose per 1 g of soil.
N.D. Not determined.
-------
completely inhibited CH. production under the selected experimental
conditions.
Influence of Organic Substances on Methane Formation
Three organic compounds (glucose, starch and alfalfa) at concentrations
of 2.5 and 10.0 mg per 1 g soil were added to soil and the formation of
methane and C02 were determined after 8, 12, 16, and 24 days of incuba-
tion (Table 13). Starch and alfalfa produced a stronger CH. evolution
in relation to increasing concentrations, but glucose reacted differ-
ently. 2.5 mg of glucose per g of soil showed a similar stimulating
effect on CH. formation as a corresponding amount of starch and alfalfa,
but 10 mg of glucose, which caused an intensive formation of C0_,
almost completely inhibited CH4 production. If nitrate was added with
the organic compounds to the soil, there was—as expected—a delay in
the formation of CH., but otherwise the results were similar.
59
-------
TABLE 13: EFFECT OF GLUCOSE, STARCH, AND ALFALFA ON CH4 AND C02 EVOLUTION FROM SOIL.
DAYS
OF
INCUBATION
8
12
16
24
CONTROL
CH4 C02
0 232
0.6 299
13 301
101 354
GLUCOSE
2.5 mg/g soil
CH4 C02
4.6 457
11 587
112 631
880 744
10.0 mg/g soil
CH4 C02
0.8 1086
2.2 1147
5.7 1175
4.1 1148
STARCH
2.5 mg/g soil
CH4 C02
yl/g soil
0.6 277
4.4 386
89 416
429 518
10.0 mg/g soil
CH4 C02
10 465
20 546
179 672
1084 850
ALFALFA
2.5 mg/g soil
CH, C02
6.1 333
13 428
123 554
418 520
10.0 mg/g soil
CH4 C02
7.1 506
39 554
414 729
1351 878
-------
SECTION XI
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Cochrane, V. W. 1958. Physiology of the fungi. Wiley, New York.
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Cove, D. J. 1966. The induction and repression of nitrate reductase
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Falcone, A. B., Shug, A. L. and Nicholas, D. J. D. 1963. Some
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Greenwood, D. J. and Lees, H. 1961. Studies on the decomposition of
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Gunner, H. B. and Alexander, M. 1964. Anaerobic growth of Fusarium
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Jansson, S. L. and Clark, F. 1952. Losses of nitrogen during decompo-
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Khan, M. F. A. and Moore, A. W. 1968. Denitrifying capacity of some
Alberta soils. Can. J. Soil Sci. 48:89-91.
Laskowski, D. and Moraghan, J. T. 1967. The effect of nitrate and
nitrous oxide on hydrogen and methane accumulation in anaerobically
incubated soils. PI. Soil. 27:357-368.
Nason, A., Abraham, R. G. and Averbach, B. C. 1954. Enzymatic reduc-
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62
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63
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SECTION XII
PUBLICATIONS
1. Bollag, J.-M., M. L. Orcutt, and B. Bollag. "Denitrification by
isolated soil bacteria under various environmental conditions."
Soil Sci. Soc. Amer. Proc., 34:875-879 (1970).
2. Bollag, J.-M. "Comparative denitrification of newly isolated
microbial strains." Agronomy Abstracts, p. 89 (1969).
3. Bollag, J.-M., and G. Tung. "Nitrous oxide release by soil fungi."
Soil Biol. Biochem., 4:271-276 (1972).
4. Drzymala, S., J.-M. Bollag, and L. T. Kardos. "Nitrite transfor-
mation in soil by biological and chemical reactions." Agronomy
Abstracts, p. 81 (1971).
5. Bollag, J.-M. and S. T. Czlonkowski. "Inhibition of methane for-
mation in soil by various nitrogen-containing compounds." Soil
Biol. Biochem., in press (1973).
6. Bollag, J.-M., S. Drzymala, and L. T. Kardos. "Biological versus
chemical nitrite decomposition in soil." Soil Science, in press
(1973).
7. Bollag, J.-M. and C. L. Nash. "Comparative denitrification of
specific microorganisms in liquid media and in autoclaved soil."
Submitted for publication.
t U. S. GOVERNMENT PRINTING OFFICE : 1973 — 5^6-309/50
65
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
if: ReportX0,
Accessson No.
w
A. Title
NITRATE AND NITRITE VOLATILIZATION BY MICROORGANISMS
IN LABORATORY EXPERIMENTS
~, Authcr(s)
Bollag, J.-M.
r 8," 'Perforating Qi&anix*tian
.jK«pttffNo, ,-,.
9, Organization
Pennsylvania State University, University Park, Pa.
16802. Laboratory of Soil Microbiology, Department of
Agronomy.
16080 BIT
?/. Contract/ Grunt No.
i%%t&K>ll!&~^!f3rZI'''
v \- ~ > ••-
Environmental Protection Agency report number,
EPA-660/2-73-002, August 1973.
9C 3fw«?JB§»rti5i«
Period Covered •
16. Abstract
Microbial nitrate and nitrite volatilization was considered as a means to eliminate
nitrogen from soil and water in order to inhibit the accumulation of nitrogenous sub-
stances as pollutants or health hazardous compounds. Therefore it was attempted to
compare nitrate reducing microorganisms in their reactions to different environmental
conditions in laboratory experiments. Changing oxygen concentration, pH, temperature,
nitrate or nitrite concentration affected differently the denitrification process of
various isolated microorganisms. Unfavorable growth conditions led to the accumulation
of nitrite if nitrate served as substrate. It was found that certain soil fungi are
also capable of volatilizing nitrogen as nitrous oxide.
Biological and chemical factors were evaluated during nitrite transformation in auto-
claved and non-autoclaved soil by determination of the evolvement of nitrogenous gases.
During chemical nitrite volatilization, which occurred essentially at a low pH, the
major gases evolved were nitric oxide and nitrogen dioxide, but if biological activity
was predominant in a neutral and alkaline environment, nitrous oxide and molecular
nitrogen were formed. The validity of laboratory observations in relation to field
studies in the domain of denitrification is discussed and evaluated.
i?a. Descriptors *Denitrification, *Nitrates, *Nitrites, *Nitrogen cycle, Soil
microorganisms, Bacteria, Fungi, W c-obial cultures, Nitrogen, Nitrous oxide,
Environmental effects.
l~b. Identifiers
Optimizing denitrification, Nitrate-volatilizing microorganisms
17c. CO WRR Field & Croup 05C, 05G
18. Availability
Zt, Ptice '"'
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
LJ.S DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. ZO24O
Abstractor
institution
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