Special Report 81-33 December 1981
EFFECT OF SOIL TEMPERATURE AND pH ON NITRIFICATION KINETICS IN SOILS RECEIVING A LOW LEVEL OF AMMONIUM ENRICHMENT
L.V. Parker, I.K. Iskandar and D.C. Leggett
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Special Report 81-33 5. TYPE OF REPORT & PERIOD COVERED
EFFECT OF SOIL TEMPERATURE AND pH ON NITRIFICATION KINETICS IN SOILS RECEIVING A 6.
LOW LEVEL OF AMMONIUM ENRICHMENT
PERFORMING ORG. REPORT NUMBER
8. CONTRACT OR GRANT NUMBERfs)
7. AUTHORfs;
L.V. Parker, I.K. Iskandar and D.C. Leggett 9.
10. PROGRAM ELEMENT, PROJECT, TASK AREA & WORK UNIT NUMBERS
PERFORMING ORGANIZATION NAME AND ADDRESS
U.S. Army Cold Regions Research and
CWIS 31633 CWIS 31297
Engineering Laboratory
Hanover, New Hampshire 11.
03755
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December 1981
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Washington, D.C.
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20314
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SUPPLEMENTARY NOTES
19. KEY WORDS (Continue on reverse side If necessary and Identify by block number)
Ammonium
Nitrites
Microorganisms
pH factor
Land Treatment
Removal
Nitrates
Waste water
2GL ABSTRACT (Coxrt&me art rmveram afflto tf rt0x»avaey and Identity by block number)
Two soil samples from an on-going field study of land application of municipal wastewater were spiked with low levels of ammonium to determine the effect of temperature on nitrification kinetics. The concentrations of ammonium and.
nitrite-plus-nitrate, and the number of autotrophic ammonium and nitrite oxidizers' were monitored periodically during the study.
There was a lag period prior to
nitrite-plus-nitrate production at all temperatures, and the length of this lag period was temperature-dependent, with the longest period occurring at the lowest temperature.
DD.ETnUn
The maximum rate of nitrification increased with temperature
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20. Abstract (cont'd).
as expected.
While nitrite-plus-nitrate production appeared logarithmic, sug
gesting a growing nitrifier population, the MPN counts of the nitrifiers did
not exhibit logarithmic growth. To study the effect of soil pH on nitrifica tion kinetics, soil samples from field plots having the same soil type but dif ferent pHs (4.5, 5.5, and 7.0) were spiked with low levels of ammonium and the rate of nitrite-plus-nitrate production was measured.
The maximum rate of ni
trification was greater at pH 5.5 than at 4.5. Unexpectedly rapid disappear ance of ammonium, nitrite and nitrate, caused by immobilization, obscured the expected effects of pH on the nitrification rate at the highest pH.
11
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PREFACE
This report was prepared by L.V. Parker, Microbiologist, Dr. I.K. Iskandar, Research Chemist and D.C. Leggett, Research Chemist, all of the Earth Sciences Branch, Research Division, U.S. Army Cold Regions Research and Engineering Laboratory.
Financial support for the study was provided from Civil Works project CWIS 31633, Optimization of Automated Procedures for Design and Management
of Land Treatment Systems, and CWIS 31297, Nitrogen Transformations in Land Treatment.
The authors acknowledge the technical assistance of Dr. E.L. Schmidt
of the University of Minnesota, St. Paul; Dr. D.R. Keeney/, of the Univer sity of Wisconsin, Madison; Col. John Atkinson, U.S. Army Reserve; B. Blake, former Physical Science Technician, CRREL; P. Coderre, former
Biological Lab Technician, CRREL; and Dr. A.P. Edwards, Visiting Scientist, CRREL.
Col. Atkinson provided statistical support for this project as part
of his annual active duty.
Dr. Keeney also provided soil samples of
different pH.
This report was technically reviewed by Dr. D.R. Keeney and Dr. E. Schmidt.
•
-
.
The contents of this report are not to be used for advertising or pro
motional purposes.
Citation of brand names does not constitute an official
endorsement or approval of the use of such commercial products.
111
CONTENTS
Page Abstract
i
Preface
.^ .
Introduction
iii 1
Materials and methods.
.
4
Effect of temperature on nitrification kinetics
4
Effect of pH on nitrification kinetics .
5
Results and discussion
6
Effect of temperature on nitrification kinetics
Effect of pH on nitrification kinetics
6
13
Summary and conclusions
16
Literature cited
17
Appendix A:
Data collected for the temperature experiment . .
21
Appendix B:
Statistical analysis of data collected. .....
25
FIGURES
Figure
1. The effect of temperature on NHt utilization and (NO2+NO3) production in Windsor soil
2.
9
The effect of temperature on NH^ utilization and (NO2+NO3) production in Charlton soil
9
3. Ammonium oxidizer population at 5°, 15° and 23°C ....
10
4. Nitrate oxidizer population at 5°, 15° and 23°C
12
5. Production of (NO^+NOp-N and loss of NHj-N in Piano silt loam at different soil pHs
14
1. Changes in pH in the Piano soils with time
13
2. Equations for In (NO2+NO0) concentration with time ...
15
TABLES
Table
IV
EFFECT OF SOIL TEMPERATURE AND pH ON
NITRIFICATION KINETICS IN SOILS RECEIVING A LOW LEVEL OF AMMONIUM ENRICHMENT
L.V. Parker, I.K. Iskandar and D.C. Leggett INTRODUCTION
All land treatment systems for wastewater must strive towards the removal of N (nitrogen) in the percolate and minimization of the N03~
(nitrate) leached into groundwater. The concentration of N in domestic wastewater applied to land rarely exceeds 50 ug N/mL, of which
approximately 85-90% is in the mh+ (ammonium) form (Iskandar et al. 1976). Once in contact with the soil, NH^"1" is sorbed on the soil exchange sites and taken up by plants or microorganisms (immobilization) or oxidized by soil microorganisms (nitrification). In contrast N03" is only slightly sorbed by most soils and thus moves down the soil profile with the water unless it is first taken up by the plants or reduced to N2 or N20
(nitrogen, nitrous oxide) gases by soil microorganisms (denitrification). It was first demonstrated a century ago that nitrification is mediated
by microorganisms when Schloesing and Muntz (1877) discovered that the pro
duction of N02" (nitrite) and N03" from NH^"1" in sewage percolating through soil could be terminated by the addition of chloroform.
Nitrification is
generally believed to be mediated by autotrophic nitrifiers. These organisms derive the energy necessary for growth from the oxidation of either NH1++ or N02" and do not require organic growth factors. Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio are the
terrestrial NH^* oxidizers. However, only Nitrosomonas, Nitrosospira, and Nitrosolobus are widely distributed in soils (Belser 1979).
The other two
genera have been only occasionally isolated from the soils.
Nitrobacter is
believed to be the only terrestrial N02~ oxidizer.
Nitrifying bacteria are in the highest numbers at the soil surface (Ardakani et al. 1974), consistent with the highest levels of organic
matter, total N, 02, and cation exchange capacity. Ardakani et al. (1974) also found that the number of NH^* oxidizers in soils declined more drastically with depth than did the number of N02~ oxidizers.
explained by the soils' ability to retain NH^+ at the surface.
This can be
The rate of nitrification depends on several environmental conditions
including water content, dissolved 02, temperature, and pH.
The effect of
water content on nitrifier activity involves at least two factors: the
effect of pore space in which enough water is retained to support life and
activity, and the rate limiting factor of 02 diffusion (Seifert 1962).
The
optimum water content for nitrification is a soil water tension of 0.15 to
5.0 kPa (Miller and Johnson 1964, Sabey 1969).
In the sandy and silty loam
soils used for wastewater treatment, moisture tensions are invariably within this range.
Skinner and Walker (1961) and Laudelout et al. (1976) present evidence to suggest that 02 can also become limiting in the presence of relatively
high concentrations of NH^"*". However, the NHI++ concentration is normally low in land-treated soils.
Nitrification occurs at a wide range of temperatures, from 2° to 35°C
(Frederick 1956).
(Chandra 1962).
The optimum is reported to vary between 27° and 30°C
Focht and Chang (1975), in a review of the literature,
indicated a higher optimum, between 30° and 36°C.
These differences in
optimum temperature might exist because of differences in nitrifier
adaptation to the temperature regimes of the areas from which they originate (Mahendrappa et al. 1966, Anderson et al. 1971).
The optimum pH for NH^"*" and N02~ oxidizers is neutral to slightly alkaline (Focht and Chang 1975).
Nitrosomonas is quite sensitive to acid
conditions, while Nitrobacter is more susceptible to alkaline conditions. The susceptibility of Nitrobacter to alkaline pHs results in an accumula
tion of N02~ in alkaline soils (Morrill and Dawson 1967, Dancer et al. 1973).
Most studies in the past were conducted using a relatively high NIL + concentration.
Since Michaelis-Menten kinetics are expected (Leggett and
Iskandar 1980, 1981) low concentrations are needed to reveal the inter
actions with pH and temperature and to avoid oxygen limitations.
There
fore, one objective of this study was to investigate the effect of soil
temperature and pH on nitrification kinetics in soils receiving a level of
NH^
enrichment which would ensure N limitation and simulate most closely
the slow addition of wastewater N.
Several methods are available for studying nitrification in soils.
Column techniques usually involve a soil-sand mixture to allow proper
drainage. The nitrification rate is then determined by measuring the concentrations of NH^"1", N02", and N03" in the effluent with time. Static incubation methods involve treating soil samples with a specific amount of
oxidizable N, and then analyzing the soil sample for NH4+, N02", and N03". This method has two advantages over column techniques: it reflects more
closely the true ecological conditions, and the soil may be sampled for microbial counts without disturbing the system.
For these reasons the
static incubation method was selected for this study.
Enumeration of nitri.fiers has been hindered by the lack of a quick and accurate method.
Several methods exist; the most commonly used one is the
most probable number (MPN) technique (Alexander and Clark 1965). This method has two disadvantages: it requires a long incubation time (3 weeks to 4 months), and there is a large statistical uncertainty inherent with the method. The degree of uncertainty depends on the number of tubes used
per dilution and the dilution factor used. The number of nitrifiers may be underestimated if the growth conditions and media do not allow all of the
nitrifiers present to grow, or if the cells are not separated from the soil particles so that each cell is individually dispersed. Two other techniques for enumerating nitrifiers, which have been
developed recently and may yield better precision and require less time to
execute, are the microtechnique MPN (Curtis et al. 1975, Rowe et al. 1977) and the fluorescent antibody (FA) technique (Bohlool and Schmidt 1973, Rennie and Schmidt 1977, Belser and Schmidt 1978a). The microtechnique MPN needs further testing. The major drawback of the FA technique is the existence of multiple serotypes requiring many FAs for each genus (Belser 1979). The problem is compounded by the difficulty in isolating nitrifiers and preparing antibodies to them (Belser 1979). Since both of these techniques are still being developed, the tube MPN technique was used for this experiment.
MATERIALS AND METHODS
Effect of temperature on nitrification kinetics
Windsor sandy loam and Charlton silty loam soils were collected from
an experimental slow infiltration land treatment facility at CRREL.
For
information on the test facilities and the soil characteristics the reader
should consult Iskandar et al. (1976) and Iskandar et al. (1979).
Fresh
soil samples were collected from the top 7.5-cm layer, air dried overnight, and sieved through a 2-mm mesh sieve.
Forty-gram subsamples were incubated with 5 mL of a 120-ppm NH. CI solution in 125-mL Erlenmeyer flasks, which were fitted with one-hole
rubber stoppers to allow air exchange while keeping moisture loss to a
minimum.
The soil moisture content was maintained at approximately
two-thirds of field capacity to prevent N losses due to denitrification.
Denitrification ceases when soils become drier than field capacity (Bremner and Shaw 1958, Mahendrappa and Smith 1967, Pilot and Patrick 1972, Abd-el-Malek et al. 1975).
The flasks were incubated at 5°, 15° and 23°C to mimic field conditions at the CRREL slow infiltration test sites.
Iskandar et al.
(1979) reported a maximum soil surface temperature of 21°C for the test site during the period from September 1976 to April 1978.
Samples were
sacrificed on days 0, 3, 6, 9, 15, 21, and 30 for analyses. Inorganic forms of N were determined by the steam distillation method of Bremner and Keeney (1966).
The relative rate of nitrification was
determined from the rate of (N02~ + N03~) production.
Soil pH was deter
mined on soil slurries made with deionized water on days 0 and 30.
Mois
ture content was determined by oven drying at 105°C for 24 hr.
The method selected for enumerating nitrifiers in this study is a
modified MPN method as outlined by Belser and Schmidt (1978b and pers. coram.).
Briefly, 10 g of soil was mixed with 90 mL of basic Walker medium
and 5 drops of Tween 80.
The mixture was then shaken 1 hr.
Serial 1:10
dilutions in 1-mM potassium phosphate buffer (pH 7.4 - 7.6) were performed on the suspended soil solution.
Ammonium-oxidizing microorganisms were
enumerated in Walker medium with 0.04% bromthymol blue solution (0.5 g
[NH^ ^SO^/L).
Nitrite oxidizers were enumerated in Watson's Nitrosomonas
medium by replacing the (NHl+)2SOt+ with KN02 (0.00085 g/L).* Five tubes
were used for each dilution. The NH^+ oxidizers were incubated 6 weeks at 28°C, and the N02~ oxidizers were incubated 8 weeks at 23°C. After incubation, growth was determined in the MPN tubes for the NH4+ oxidi zers by testing for production of N02~ by a modification of the spot test of Strickland and Parsons (1972).
The concentration of the reagents was
changed slightly so that Greiss reagent A was 0.5% sulfanilamide in 2.4 M HC1 and Greiss reagent B was 0.3% N-1-naphthylethylenediamine dihydrochlo-
ride in 0.12 M HC1.
Growth was determined for the N02~ oxidizers by test
ing for the disappearance of N02~ in the MPN tubes using the same spot test.
Effect of pH on nitrification kinetics
To study the effect of pH on the nitrification rate at low NH^"1" con centrations, it is essential that other soil physical and chemical charac teristics remain constant.
Piano silt loam, classified as a Typic Argiu-
doll, from the University Experimental Farm at Arlington, Wisconsin,was selected for this study.
The soil had an initial pH of 4.8, organic matter
content of 3.5%, and a clay content of 22% (Dancer et al. 1973).
In 1972
the plots were limed with different amounts of dolomitic limestone. treatment resulted in soils of different pH.
7.0, were selected for this study.
This
Three soils, pH 4.5, 5.5, and
Over the years these soils may have
developed populations of nitrifiers adapted to each of the different pHs.
Field moist samples were collected and stored for approximately 6 months at 8°C, preincubated at 23°C for 1 week, air dried for 24 hr at 23°C, and then sieved through a 2-mm sieve.
Samples were incubated with NH4+ (as NH^Cl) in Erlenmeyer flasks at 23°C as described previously.
Subsamples were randomly selected and sacri
ficed on days 0, 3, 7, 10, and 14.
The soil was analyzed for NH4+ and
* Later correspondence revealed that this concentration should have been 0.085 g/L. The concentration used in this medium was detectable using
the spot test for N02".
However, this low concentration of N02" may have
been subject to chemical breakdown leading to falsely positive growth of
N02~ oxidizers in a random manner.
For this reason the tubes were read
after 8 weeks incubation instead of the recommended 4 months incubation.
(N02~ + N03~) using the steam distillation method of Bremner and Keeney (1966).
Soil pH was determined in deionized water slurries on days 0, 7,
10, and 14. at
Moisture content was determined by oven drying of subsamples
105°C for 24 hr.
RESULTS
AND DISCUSSION
Effect of temperature on nitrification kinetics
Figures 1 and 2 show the changes in NH^"*" and (N02~ + N03~) concentra tions in relation to temperature and time of incubation for Windsor and
Charlton soils respectively.
At all temperatures there was a lag phase,
after which there was an increase in the (N02~ + N03~) concentrations with
time.
The rate of (N02~ + N03~) production was slowest at 5°C and greatest
at 23°C. The concentration of NHtt+ decreased at 23°C, remained steady at 15°C, and increased at 5°C in both soils.
Statistical analyses were performed on the actual values of the (N02~
+ N03 ~) and NHlf+ concentrations, and the numbers of N02~ and NH^"*" oxidi zers, and on the natural logarithms of those values.
Natural logarithms
were used because of the exponential nature of microbial growth.
Here
after, instead of saying, for example, "the natural logarithm of the number
of NH^
oxidizers," the shorthand "In NH^
oxidizers" will be used instead.
Least significant difference (LSD) calculations on the In (N02~ + N03~) concentrations showed that the length of the lag or delay phase was dependent on the temperature of incubation.
After 9 days there was a
significant increase in (N02~ + N03~) concentration at 23°C. and 5°C, the lag phase was 15 and 21 days respectively.
While at 15°
The dependence of
the length of the lag phase on soil temperature has been previously
reported by Sabey et al. (1969). In both soils, the rate of nitrification increased with increasing
temperature.
The maximum rates of nitrification for Windsor soil at 5°,
15°, and 23°C were 0.0049, 0.0056 and 0.0089 meq N/100 g soil per day, re spectively, or 1.6, 1.8 and 2.8 kg N/ha per day.
The corresponding maximum
nitrification rates for the Charlton soil were 0.0009, 0.0034, 0.0069 meq
N/100 g soil per day, or 0.3, 1.1, and 2.2 kg N/ha per day.
In a separate
experiment, Schmidt and co-workers (pers. comm.) obtained values for nitri fication rates of 0.0104 and 0.0073 meq N/100 g soil per day for similar
Windsor and Charlton soils, respectively, or 3.3 and 2.3 kg N/ha per day. When one considers that those nitrification rates were obtained at 28°C,
they compare well with the values"obtained in the present study at 23°C. The higher nitrification rates in the Windsor soil as compared with the
Charlton soil may have been due to the higher pH or the higher initial NHl++ concentration in the Windsor soil.
Analysis of variance was performed to test the effect of temperature
and soil on In (N02~ + N03~) production (Table Bl). It showed that soil type and the interaction between time and temperature were highly signifi cant, and that time and temperature were both highly significant within
that interaction.
The (N02~ + N03~) values for the Charlton soil (Fig. 1
and 2) were significantly lower than those for the Windsor soil.
Since
none of the interactions of other variables (time, temperature) with soil
were significant, the soils do not have to be considered independently. The effect of temperature was similar for both soils.
Regression analysis
showed that there was significant statistical evidence for a linear rela tionship at all three temperatures and that the slope of the line, repre
senting the increase in the production of (N02~ + N03~) with time, increased with temperature.
The linear equations for both soils and the
coefficients of determination (r2) for these equations at each temperature are:
at 5°C
In (N02~ + N03~) = (0.02132)x - 3.1516 r2 =
at 15°C
In (N02" + N03") = (0.02694)x - 3.0145 r2 =
at 23°C
0.74
In (N02~ + N03-) = (0.03932)x - 2.9482 r2 =
where x = time (days).
0.83
0.94
Although the relationship between concentration and
time could also be expressed as linear, statistically it was better repre
sented by the logarithm of the concentration with time.
A logarithmic
increase in concentration is indicative of a corresponding logarithmic increase in nitrifiers.
'
•
.
Since in nitrification, production of N02~ and N03~ is accompanied by
a corresponding decrease in NH^ concentration, it was of interest to look at the change in concentration of NH(++ with time at the three tempera tures.
Analysis of variance to test the effect of temperature, time and
soil type (Table B2) on the In NH^
concentration indicated that the inter
action of time with temperature was significant.
Since the interactions of
soil with time and soil with temperature were not significant, the effect
of temperature on NHlf+ concentration was the same for both soils. lowest temperature (5°C) there was a net increase in NH^
At the
while at the
intermediate temperature (15°C) there was no significant change in the NH^"*" concentration.
At 23°C the loss in NH^
was nearly equal to the (N02~ +
N03~) produced, although there was a slight net decrease in total mineral N at 23°C.
Belser (1979) reported that low temperature affects nitrification
more adversely than mineralization.
At 15°C mineralization equaled NH^"*"
losses, which were due to nitrification and immobilization (microbial
assimilation).
At 5°C mineralization was greater than nitrification and
immobilization, which resulted in an accumulation of NH^ . Regression
analysis of the In NH^ values by day at each temperature gave significant statistical evidence for a linear relationship between the In NH^ tration and time (in days) at 5° and 23°C.
concen
Statistical evidence was
stronger for a linear relationship between the In NH^
concentration and
time than for the concentration and time. This indicates that NHl++ was being used by a growing population, either nitrifiers or microorganisms
which assimilated NH^"1". The pHs of the soils were determined at the beginning and conclusion
of the experiment.
The pH of the Windsor soil was 5.55 on day 0.
30 the pH of the Windsor soil at 5°C was 5.80.
By day
This increase was probably
due to a depletion of the organic matter (acids).
The pH of this soil at
15°C on day 30 was nearly the same as on day 0, 5.53.
At this temperature
the rate of nitrification was slightly greater and neutralized any increase
in pH that resulted from depletion of the organic matter.
At 23°C the rate
of nitrification was much greater and the pH had dropped by day 30 to 5.31.
The Charlton soil showed very similar trends. , The initial pH of
this soil was 5.50.
By day 30 at 5°C the pH of the soil had risen to
A0.26 0.24
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Incubation Time (days)
Incubation Time (days)
b. Production of (N02" + N03~)
a. Utilization of NH1+ +
Figure 1. the effect of temperature on NH1++ utilization and (N02~ + N03~) production in Windsor soil. 0.24
i
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30
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9
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Incubation Time (days)
b. Production of (N02~ + N03")
Figure 2. The effect of temperature on NH^"*" utilization and (N02 + N03 ) production in Charlton soil.
30
5.80.
At 15°C the pH had dropped slightly to 5.36 by day 30, and at 23°C
the pH had dropped to 5.23 by day 30.
Many modelers assume that the rate of nitrification is determined by
the number of nitrifiers (Ardakani et al. 1973, Beek and Frissel 1973, Day et al. 1978, Leggett and Iskandar 1980, 1981) and that growth and nitrifi cation are coupled, provided growth is not limited by other factors.
In
this study oxygen and moisture content should not have been limiting.
The
MPN data for the NH^* oxidizers in the Windsor and Charlton soils at 5°, 15°, and 23°C can be found in Table A3. The mean initial number of NH^"*"
i/\>
oxidizers was 7.5x10 /g soil for the Windsor soil and 6.3xl05/g for the Charlton soil.
These numbers agree well with MPN determinations made by
Schmidt and co-workers on similar samples where they found 105 to 106 organisms/g soil (pers. comm.).
Analysis of variance was performed on the In
number of NH4+ oxidizers (Table B3). There was no significant difference in the number of NH^* oxidizers in the two soils. The interactions of soil with temperature and soil with time were also not significant.
Therefore
the data for the two soils were pooled and plotted for each temperature
(Fig. 3).
There may not have been a large enough difference between the
nitrification rates of the two soils to result in a statistically signifi cant difference in the number of nitrifiers, or the MPN technique may not
1
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i
1
in6 IO
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Figure 3.
i
30
Ammonium oxidizer
population at 5P, 15° and 23°C.
Time (days)
10
be sensitive enough to reflect these differences.
Analysis of variance
indicated that temperature was highly significant within the interaction of time with temperature, which also was significant.
Examination of the dif
ference between the In number of NH^+ oxidizers at the different tempera tures (by LSD calculations) demonstrated that there was no significant difference between those values at 5° and 15°C, while the values at 23°C,
surprisingly, were significantly lower than those at 5° and 15°C. Least significant difference determinations performed for each day at
each temperature showed that the In NH^* oxidizers did not change signifi cantly with time at 5° and 15°C.
However, by day 6 at 23°C the number of
In NH^4" oxidizers was significantly lower than it was at day 0. Linear regression and analysis of variance of the regression demonstrated that the
relationship between In NH^+ oxidizers and time was not linear at 5° and 15°C.
At 23°C the probability of obtaining the estimated slope, given that
the true slope was zero, was significant. did exist at 23°C.
Therefore, a linear relationship
The slope for this line was negative, indicating that
death of the NH^"1" oxidizers was logarithmic. The substrate was rapidly depleted at 23°C, but not at 5° and 15°C (Fig. 1 and 2), which may explain
why the NH^"*" oxidizers died at 23°C. The low level of substrate at 5° and 15°C was apparently enough to allow maintenance of the population but not growth.
Regression analyses were performed to determine if a correlation
existed between (N02~ + N03") production and growth of the NH4+ oxidizers. The data from each population were treated separately to allow for the sig nificant effect of temperature.
Both the actual values and the natural
logarithms of the values were tested for correlation.
There was no signi
ficant correlation between the production of (N02~ 4- N03~) and the number
of NH^4" oxidizers at either 5° or 15°C. There was significant negative correlation at 23°C.
Since the original soil for this experiment was taken
from an on-going wastewater treatment site, it is likely that the number of nitrifiers was close to the maximum population density for the given NH4
concentration.
Therefore, growth would not be expected at this relatively
low NH4+ concentration. The number of N02~ oxidizers for each day and temperature are given in Table A3.
The mean number of N02" oxidizers on day 0 for the Windsor soil
11
was 1.7xl07/g soil and 2.4xl07/g soil for the Charlton soil.
These numbers
agree well with MPN counts made by Schmidt and co-workers (pers. comm.) on
similar samples where they estimated the number of N02" oxidizers to be 106 to 107/g soil. Analysis of variance of the In N02~ oxidizers indicated that time was the only significant factor (Table B4).
Since temperature, soil, and their
interactions were not significant, the data for the two soils were pooled and plotted for each temperature (Fig. 4). Linear regression and analysis of variance of the regression demon
strated that the relationship between the In N02~ oxidizers and time was not linear at any temperature or for all temperatures combined.
Least
significant difference calculations were performed for each day at each
temperature.
At 5°C the number of N02~ oxidizers was significantly lower
(at the 0.05 significance level) for days 9 and 15 than the number of N02" oxidizers on day 0.
Day 21 was only significantly lower at the 0.10 level,
not at the 0.05 level.
level.
At 15°C day 9 was significantly lower at the 0.10
At 23°C there was no significant change in the number of N02~
oxidizers at either the 0.05 or 0.10 levels.
The significant decrease in
the bacterial number by day 9 at 5° and 15°C may be correlated with the lag
period that was observed prior to (N02~ + N03~) production.
During this
lag period N02~ was not produced and was thus unavailable for nitrite oxidizer growth and maintenance.
Although the decrease in the number on
day 9 was not significant at 23°C, the observed decrease may also reflect
the lag in (N02~ + N03~) production.
10
The decrease in the bacterial number
Figure 4.
20
Nitrite oxidizer
population at 5°, 15° and 23°C,
Time (days)
12,
was largest and of the greatest duration at 5°C where ,the lag in (N02~ +
N03~) production was most pronounced. Regression was used to analyze the relationship between the In (N02~ + N03~) values and In number N02~ oxidizers for days 9 through 30. considered the starting day for growth of the N02~ oxidizers. statistically significant correlation at 5°, 15° or 23°C.
Day 9 was
There was no
However, when
the mean concentration of (N02~ + N03~) and number of N02~ oxidizers for each day at 5°C were used, a highly significant correlation was found.
Therefore, there was a real positive correlation between (N02~ + N03~) pro duction and N02~ oxidizer growth at 5°C, although there was a lot of noise in the data.
Use of the means in similar analyses at 15° and 23°C did not
indicate any significant relationships.
This was partially due to the
decrease in N02~ oxidizers on day 30, and no doubt the statistical uncer tainty inherent in the MPN technique was also a factor.
In general, while there appears to be no correlation between nitrifi
cation and growth of the NH^"*" oxidizers, which is most likely due to. their initially high numbers, there appears to be a correlation between growth of
the N02~ oxidizers and nitrification, at least at the lower temperatures. This correlation occurs after the N02~ oxidizers have died off, their death
being due to the lag in N02~ production. Effect of pH on nitrification kinetics
Figure 5 shows the reduction of NH^4" concentration and the production of (N02~ + N03~) with time at pH 4.5, 5.5 and 7.0 for the Piano soils, which are from the experimental field plots at the University of Wisconsin.
Production of (N02~ + N03") equals the loss of NH4+ during the first week for all three soil pHs. Therefore nitrification of the added NHl++ was complete within the first 7 days. Table 1.
Changes in pH in the Piano soils with time. Incubation time (days)
Initial
j>H
0
7
10
14
4.50
4.63
4.55
4.52
4.33
5.52
5.56
5.35
5.37
5.31
7.00
7.00
6.80
6.74
6.79
13
2
6
4
8
10
12
Time (days)
Figure 5.
Production of (N02~ + N03")-N
and loss of NH^+-N in Piano silt loam at different soil pHs.
After the first 7 days there was some loss of (N02~ + N03~) from the soil at pH 7.0.
At pH 5.5 there was an increase in the (N02~ + N03~)
levels, and at pH 4.5 there may have been a very slight increase in (N02~ + N03~) concentration.
Least significant difference calculations revealed
that the increase in the In (N02~ + N03~) concentration from day 7 to day 14 was significant at pH 5.5, and that the decrease in In (N02~ + N03~) concentration from day 7 to day 14 at pH 7.0 was also significant.
How
ever, there was no significant difference between the In (N02~ + N03~)
values on days 7 and 14 at pH 4.5.
The loss of (N02~ + N03~) from the soil
at pH 7.0 was most likely due to immobilization (microbial assimilation). Microbial growth, and therefore assimilation, appears to be most rapid at the neutral pH.
The continued production of (N02~ + N03~) after the first week at pH 5.5 indicates that in this soil organic N was mineralized and subsequently
nitrified.
The continued production of (N02~ + N03~) was not significant
at pH 4.5, indicating that mineralization and nitrification were much slower at this pH, typical of an acid soil.
The pHs of the three soils were monitored during the course of the ex
periment (Table 1). ceeded.
All three soil pHs dropped as nitrification pro
The decline in pH was more rapid at pHs 5.5 and 7.0. 14
Analysis of variance of the In NH4+ and (N02~ + N03~) concentrations indicated that the interaction of pH and time was significant, or highly significant, and that the effects of pH and time were significant or highly
significant within this interaction (Table B5).
Therefore, pH had a signi
ficant effect on the rates of NHt++ loss and (N02~ + N03~) production. Linear regression analyses were performed on the In (N02~ + N03~) concentration data from the first 7 days (Table 2). proved to be significantly linear.
inadequate number of data points.
None of the equations
This lack of linearity may be due to an
Nitrification was more spontaneous than
expected, and it is clear that more samples should have been taken earlier in the experiment.
Table 2.
Equations for In (N02~ + N03~) concentration with time. Significance
pH
r2
Equation
level
4.5
In (N02" + N03") = (0.0175)x - 0.2871
0.9805
NS
5.5
In (N02" + N03~) = (0.0232)x - 0.3199
0.7744
NS
7.0
In (N02~ + N03") = (0.0215)x - 0.3389
0.8921
NS
x = Time (days) NS = Not significant
Maximum rates of NH^"4" loss and (N02 + N03 ) production were deter mined from values on days 0 and 3, and these maximum rates were then used
for comparisons. The maximum rate of NH1++ loss was greatest at pH 7.0 (0.0250 meq N/100 g soil per day) and least at pH 4.5 (0.0133 meq N/100 g
soil per day). The maximum rate of NH^ loss at pH 5.5 was 0.0212 meq N/100 g soil per day.
The maximum rate of (NQ2~ + N03~) production was
greatest at pH 5.5 (0.0378 meq N/100 g soil per day or 12.0 kg N/ha per day) and least at pH 4.5 (0.0175 meq N/100 g soil per day or 5.6 kg N/ha per day).
The apparent rate at pH 7.0 was less (0.0277 meq N/100 g soil
per day or 8.8 kg N/ha per day) than at pH 5.5 due to the loss of (N02~ +
N03~) and NH^"1" by immobilization. These maximum rates were tentative since more data points were required. The high rate of immobilization was most likely a result of the hand
ling the samples received prior to the start of the experiment.
These
samples had been refrigerated for 6 months and were then brought to room
15
temperature for 1 week prior to being used.
The microbial population could
then have used the newly added NH^4" and newly formed N03~. This growth was most rapid at the neutral pH. SUMMARY AND CONCLUSIONS
Windsor sandy loam and Charlton silty loam soils from a slow infiltra tion land treatment test facility were used to study the effect of tempera
ture on nitrification kinetics at low NH^4" concentrations. The net rate of nitrification was determined by measuring the rate of (N02~ + N03~) produc tion at various temperatures.
There was a lag period prior to (N02~ +
N03~) production at all temperatures that was longest at the lowest temperature.
The maximum rate of nitrification increased with increasing
temperature ranging from 1.6 kg N/ha per day at 5°C to 2.8 kg N/ha per day at 23°C in the Windsor soil, and from 0.3 kg N/ha per day at 5°C to 2.2 kg N/ha per day at 23°C in the Charlton soil. the Windsor soil at all temperatures.
Nitrification was more rapid in
This may have been due to its higher
pH, or higher initial NH^4" values. The concentration of NH^4" was also monitored. At 23°C nitrification proceeded rapidly, and the added NHl++ had been used by day 21 in the Windsor and by day 30 in the Charlton soil.
Since at the lower tempera
tures (5° and 15°C) the rate of nitrification was slower, the relative rate of mineralization became greater at these temperatures.
Therefore there
was no net loss of NH^4" at these temperatures, and at 5°C NH^4" accumu lated.
The number of NH^4" and N02~ oxidizers was monitored throughout the
experiment. The number of NH^4" oxidizers remained relatively constant at 5° and 15°C, while at 23°C it decreased significantly.
This pattern
appeared to be most closely related to the available NH^ . At 5° and
15°C, Nl^4" remained available while at 23°C it was depleted. The lack of correlation between nitrification and growth of NH^4" oxidizers may be because the number of NH^4" oxidizers in these soils had reached the maximum
population density allowed by this level of NH^4" enrichment. This may have been the result of the periodic wastewater treatment these soils received prior to this experiment.
The number of N02" oxidizers decreased significantly after 9 days at 5° and 15°C because of the lag in production of N02". At 23°C this decrease was not significant, but the lag period was not as long either. 16
The decrease in N02
oxidizers was most pronounced at 5°C where the lag in
N02~ production was also longest.
After this reduction in number, the N02"
oxidizers grew as nitrification proceeded and there was a statistically
significant correlation between nitrification and growth of the N02~ oxidizers at 5°C.
The Piano soil was selected for the experiment testing the effect of pH on nitrification kinetics because this would keep soil factors constant
while changing only the pH.
The experiment was performed at 23°C so that
temperature would not be a limiting factor.
Lowering the pH from 5.5 (the
approximate pH of the Windsor and Charlton soils) to 4.5 resulted in a
lowered rate of NH4+ loss and (N02~ + N03~) production. The maximum rate of (N02~ + N03~) production went from 12.0 kg N/ha per day at pH 5.5, to 5.6 kg N/ha per day at pH 4.5.
Raising the pH from 5.5 to 7.0 resulted in
a greater rate of NH^4" loss. However, raising the pH from 5.5 to 7.0 did not increase the rate of accumulation of (N02~ + N03~) (8.8 kg N/ha per day).
Instead, there was a loss of mineral N from the system at pH 7.0.
This loss was most likely accounted for by losses to immobilization, a
result of the rapid microbial growth following cold storage. LITERATURE
CITED
Abd-el-Malek, Y., I. Hasny and N.F. Eman (1975) Studies on some environmental factors affecting denitrification in soil. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2, vol. 130, p. 644-653 (Cited in Focht and Verstraete 1977.)
Alexander, M. and F.E. Clark (1965) Nitrifying bacteria. soil analysis. Part 2.
Black, Ed.).
In Methods of
Chemical and microbiological properties (C.A.
Madison, Wisconsin: American Society of Agronomy Inc.,
p. 1477-1483. Anderson, O.E., F.C. Boswell and R.M. Harrison (1971) Variations in low temperature adaptability of nitrifiers in acid soils. Soil Science Society of America Proceedings, vol. 35, p. 68-71. Ardakani, M.S., J.T. Rehbock and A.D. McLaren (1974) Oxidation of am monium to nitrate in a soil column. Soil Science Society of America Proceedings, vol. 37, p. 53-56.
Beek, J. and M.J. Frissel (1973) Simulation of nitrogen behavior in soils. Wageningen, Netherlands: Centre for Agricultural Publishing and Documentation.
Belser, L.W. (1979) Population ecology of nitrifying bacteria. view of Microbiology, vol. 33, p. 309-333.
17
Annual Re
Belser, L.W. and E.L. Schmidt (1978a) Serological diversity within a ter restrial ammonia-oxidizing population. Microbiology, vol. 36, p. 589-593.
Applied and Environmental
Belser, L.W. and E.L. Schmidt (1978b) Diversity in the ammonia-oxidizing nitrifier population of a soil. Applied and Environmental Micro biology, vol. 36, p. 584-588. Bohlool, B.B. and E.L. Schmidt (1973) A fluorescent antibody technique for determination of growth rates of bacteria in soil. Bull. Ecol. Res. Commun., vol. 17, p. 336-338.
Bremner, J.M. and K. Shaw (1978) Denitrification in soil, II. Factors affecting denitrification. Journal of Agricultural Science, vol. 51, p. 40. (Cited in Focht and Verstraete 1977.) Bremner, J.M. and D.R. Keeney (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils: 3. Exchangable am monium, nitrate, and nitrite by extraction-distillation methods. Soil Science Society of America Proceedings, vol. 30, p. 577-582. Chandra, P. (1962) Note on the effect of shifting temperatures on nitrification in a loam soil.
42, p. 314-315.
Canadian Journal of Soil Science, vol.
(Cited in Chopp 1979.)
Chopp, K.M. (1979) Microbial populations and activity in soil irrigated with municipal wastewater effluent. University of Minnesota: Master's Thesis.
Curtis, E.J.C., K. Durrant, and M.M.I. Harman (1975) Nitrification in rivers in the Trent Basin.
Water Research, vol. 9, p. 255-268.
Dancer, W.S., L.A. Peterson, and G. Chesters (1973) Ammonification and nitrification of N as influenced by soil pH and previous N treat
ments.
Soil Science Society of America Proceedings, vol. 37, p.
67-69.
Day, P.R., H.E.^ Doner, and A.D. McLaren (1978) Relationships among microbial populations and rates of nitrification and denitrification in a Hanford soil.
In Nitrogen in the Environment.
Vol. 2. Oil-
Plant-Nitrogen Relationships (D.R. Nielson and J.G. MacDonald, Eds.). New York: Academic Press, p. 305-363.
Delwiche, C.C. and B.A. Bryan (1976) Denitrification.
Annual Review of
Microbiology, vol. 30, p. 241-262.
Focht, D.D. and A.C. Chang (1975) Nitrification and denitrification processes related to wastewater treatment. Advances in Applied Microbiology, vol. 19, p. 153-186.
Focht, D.D. and W. Verstraete (1977) Biochemical ecology of nitrification and denitrification. In Advances in Microbial Ecology, vol. 1 (M. Alexander, Ed.). New York: Plenum Press, p. 135-214.
18
Frederick, L.R. (1956) The formation of nitrate from ammonium nitrogen in soils: I. Effect of temperature. Proceedings, vol. 20, p. 496-500.
Soil Science Society of America
Iskandar, I.K., S. Quarry, R. Bates, and J. Ingersoll (1979) Changes in soil characteristics and climatology during five years of wastewater application to CRREL test cells. U.S. Army Cold Regions Research and Engineering Laboratory Special Report 79-23.
Iskandar, I.K., R.S. Sletten, D.C. Leggett, and T.F. Jenkins (1976) Waste water renovation by a prototype slow infiltration land treatment system. CRREL Report 76-19.
Laudelout, H., R. Lambert, and M.L. Pham (1976) Influence du pH et de la pression partielle d'oxygene sur la nitrification. Ann. Microbiolo. (Institute Pasteur), vol. 127(A), p. 367-382.
Leggett, D.C. and I.K. Iskandar (1980) Improved enzyme kinetic model for nitrification in soils amended with ammonium.
ture.
1.
Review of
litera
CRREL Report 80-1.
Leggett, D.C. and I.K. Iskandar (1981) Evaluation of a nitrification model.
Chapter 12 in Modeling wastewater renovation: land treatment (I.K. Iskandar, Ed.). New York: Wiley-Interscience. Mahendrappa, M.K. and R.L. Smith (1967) Some effects of moisture on denit rification in acid and alkaline soils.
ca Proceedings, vol. 31, p. 212.
Soil Science Society of Ameri
(Cited in Focht and Verstraete
1977.)
Mahendrappa, M.K., R.L. Smith, and A.T. Christiansen (1966) Nitrifying organisms affected by climatic region in western United States. Science Society of America Proceedings, vol. 30, p. 60-62.
Soil
Miller, R.D. and D.D. Johnson (1964) The effect of soil moisture tension on carbon dioxide evolution, nitrification, and nitrogen mineraliza tion. Soil Science Society of America Proceedings, vol. 28, p. 644-647.
Morrill, L.G. and J.E. Dawson (1967) Patterns observed for the oxidation of ammonium to nitrite by soil organisms. Soil Science Society of America Proceedings, vol. 31, p. 757-760.
Pilot, L. and W.H. Patrick, Jr. (1972) Nitrate reduction in soils: Effect of soil moisture tension. Soil Science, vol. 118, p. 78. (Cited in Focht and Verstraete 1977.)
Rennie, R.J. and E.L. Schmidt (1977) Immunofluorescence studies of Nitrobacter populations in soils. Canadian Journal of Microbiology, vol. 23, no. 8, p. 1011-1017.
Rowe, R., R. Todd, and J. Waide (1977) Microtechnique for most-probable number analysis.
Applied and Environmental Microbiology, vol. 33, p.
675-680.
19
Sabey, B.R. (1969) Influence of soil moisture tension on nitrate accumu lation in soils.
Soil Science Society of America Proceedings, vol.
33, p. 263-266.
Sabey, B.R., L.R. Frederick and W.V. Bartholomew (1969) The formation of nitrate from ammonium nitrogen in soils. IV. Use of the delay and maximum rate phases for making quantitative predictions. Soil Science Society of America Proceedings, vol. 33, p. 276-278.
Schloesing, J.J.T. and A. Muntz (1877) Sur la nitrification par les fer ments organises. Comptes Rendus, vol. 84, p. 301-303. Paris: Academy of Sciences.
Seifert, J. (1962) Influence of soil structure and moisture content on number of bacteria and degree of nitrification. Folia Microbiologia, vol. 7, p. 234-238.
Skinner, R.A. and N. Walker (1961) Growth of Nitrosomonas europea in batch and continuous culture.
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20
APPENDIX A:
DATA COLLECTED FOR THE TEMPERATURE EXPERIMENT
Table Al. The NH^-N meq/100 g soil for short term nitrification experiment testing the effect of temperature.
Incubation
Soil and incubation
temp.
time
(days)
Before
NH^Cl
15
21
30
addition
Charlton
5°C A
0.1688
0.1661
0.1794
0.2151
0.1572
0.1987
0.2382
B
0.1661
0.1672
0.1807
0.2044
0.2135
0.1926
0.2432
15°C A
0.1887 0.1781
0.1692
0.1886 0.1864
0.1734
0.1332
0.1663
0.1886 0.1913
0.1557
B
0.1811
0.1759-
0.1396
23°C A
0.1763
0.1636
0.1422
0.1467
0.0697
0.0255
0.0302
B
0.2044
0.1712
0.1502
0.1511
0.0917
0.0311
0.0328
Windsor
5°CA
0.0616
0.1696
0.1920
0.1772
0.1925
0.2003
0.2151
B
0.0608
0.1663
0.1854
0.1786
0.1937
0.2052
0.2120
0.2695 0.2664
15°C A
0.1647
0.1674
0.1651
0.1836
0.1694
B
0.1746
0.1558
0.1725
0.1762
0.1690
0.1797 0.1720
0.1616
23°C A
0.1737 0.1786
0.2022 0.1918
0.1161 0.1842
0.1774 0.1802
0.1435 0.1414
0.1129 0.0916
0.0265
B
21
0.1646
0.0271
The (N02~ + N03") -N meq/100 g soil for short term nitrification experiment testing the effect of temperature.
Table A2.
Incubation time Soil and incubation
temp.
(days)
Before
NH^Cl
0
3
9
6
15
21
30
addition
Charlton
0.0497 0.0422
0.0385 0.0398
0.0463 0.0498
0.0617 0.0512
0.0565 0.0600
0.0625
0.0729
0.0754 0.0761
0.0542 0.0462*
0.0508 0.0497
0.0561 0.0607
0.0668 0.0802
0.0497 0.0322
0.0851 0.0738
0.0965 0.1244
23°C A
0.0525
0.0508
0.0566 0.0581
0.0687 0.1003
0.0660
B
0.0523 0.0697
0.0970
0.0981 0.1156
0.1455 0.1930
0.0607 0.0495
0.0390 0.0429
0.0537 0.0379
0.0488 0.0568
0.0476 0.0560
0.0483 0.0677
0.1031 0.1018
15°C A
0.0622
,0.0932
0.0661 0.0794
0.0810
0.0428
0.0578 0.0588
0.0799
B
0.0459* 0.0566
0.1324 0.1449
23°C A
0.0746 0.0614
0.0498 0.0591
0.0510 0.0610
0.0787 0.0948
0.0825
0.1202
0.0947
0.1639
5°C A
0,.0381
B
0 .0393
15°C A B
Windsor
5°C A
0 .0329
B
0 .0393
B
22
0.0945
0.1815 0.1960
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APPENDIX B:
STATISTICAL ANALYSIS OF DATA COLLECTED
Table Bl. Analysis of variance of In (NO +N0,) concentration for the shortterm nitrification experiment testing the effect of temperature. Term
DF
Mean square
T
2
1.25815
S
1
0.175031
D
6
1.31364
TS
2
3.75524xl0"2
TD
12
SD
Num./den.
F
Level of significance
T/TD
HS 0.001
S/WCE
HS 0.010
D/TD
HS 0.001
1.77
TS/WCE
NS
6.76592xl0-2
3.19
TD/WCE
HS 0.005
6
2.98343xl0-2
1.38
SD/WCE
NS
TSD
12
3.15924xl0-2
1.42
TSD/WCE
NS
WCE
42
2.11963xl0~2
S
=
Temperature (°C) Soil type
D
=
Time (days)
T
=
Table B2.
18.60
8.26
19.42
WCE
= Within--cell error
-
= Not significant = Highly significant
NS HS
Analysis of variance of In NH£ concentration values for
the short-term nitrification experiment testing the effect of tem perature.
Num./den Num./den.
Level of significance
Term
DF
Mean square
T
2
-3.07924
3.94
T/TD
NS
S
1
0.237304
2.81
S/TSD
NS
D
6
0.544662
D/TD
NS
TS
2
0.168097
1.99
TS/TSD
NS
TD
12
0.780581
9.26
TD/TSD
HS 0.001
SD
6
9.89457/xl0" 1 1.17
SD/TSD
NS
TSD
12
8.43071xl0"2 12.74
TSD/WCE
HS 0.001
WCE
42
6.6198xl0-3
T = Temperature (°C) S = Soil type
D = Time (days)
F
<1
WCE = Within-cell error
NS = Not significant HS = Highly significant
25
T+ oxidizers for the short-term Table B3. Analysis of variance of In number NH7" nitrification experiment testing the effect of temperature.
Ten1
1^lean
DF
square
HS 0.001
S/TSD
NS
1.36
D/TD
NS
0.471907
1.30
TS/TSD
NS
12
1.16444
3.20
TD/TSD
S
6
0.24623
SD/TSD
NS
2
6.18455
S
1
0.04734
D
6
1.58879
TS
2
TD
SD
16.99 <1
<1
0.050
0.364002
12
T
=
S
=
Temperature (°C) Soil type
D
=
Time (days)
Table B4.
Level of significance
T/TD
T
TSD
Num./den.
F
NS HS
= Not significant = Highly significant
Analysis of variance of In number N02 oxidizers for the short-term
experiment testing the effect of temperature. Term
DF
Mean square
T
2
0.374663
S
1
5.08408
D
6
TS
2
2.27132
TD
12
1.19563
SD
6
0.416234
TSD
12
11.8639
Level of significance
T/TSD
NS
S/TSD
NS
D/TSD
S 0.050
TS/TSD
NS
1.46
TD/TSD
NS
1.97
SD/TSD
NS
2.93 <1
3.48 <1
1.35107
T = Temperature (°C) S = Soil type
Num./den.
F
NS = Not significant
S = Significant
D = Time (days)
26
Table B5. Analysis of variance of mineral N levels for the short-term nitrification experiment testing the effect of pH. Term
DF
Num./den.
Mean square
Level of significance
In NHk+ concentration p
2
2.5017
14.59
P/PD
HS 0.001
D
4
4.73449
27.61
D/PD
HS 0.001
PD
8
0.171497
WCE
30
2.70
S 0.025
0.06356
In (N02 -2
+ N03~) concentration 5.27
P/PD
S.
4.014914xl0"2
11.11
D/PD
HS 0.001
8
3.63872xl0"3
4.26
PD/WCE
HS 0.005
30
8.53266xl0_i+
p
2
1.91657x10
D
4
PD
WCE
PD/WCE
0.050
P = pH
D = time (days) WCE = Within-cell error
S = Significant HS = Highly significant
27
* U.S. GOVERNMENT PRINTING OFFICE: 1982 500-590/276
