EPA 660/3-73-002
. . iq,- Ecological Research Series
NITROGEN SOURCES AND CYCLING
IN NATURAL WATERS
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
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been grouped into five series. These five bread
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EPA 660/3-73-002
July 1973
NITROGEN SOURCES AND CYCLING
IN NATURAL WATERS
By
Patrick L. Brezonik
University of Florida
Department of Environmental Engineering Sciences
Gainesville, Florida 32601
Research Grant No. 16010-DCK
Project Officer
Dr. Charles F. Powers
National Eutrophication Research Laboratory
Pacific Northwest Environmental Research Laboratory
Corvallis, Oregon 97330
Prepared for
OFFICE OF RESEARCH AND MONITORING
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 $2.35
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EPA Review Notice
This report has been reviewed by the Environ-
mental Protection Agency, and approved for
publication. Approval does not signify the
views and policies of the Environmental Pro-
tection Agency, nor does mention of trade names
or commercial products constitute endorsement
or recommendation for use.
ii
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Abstract
Sources of nitrogen were reviewed to determine their significance
in lacustrine budgets. Nutrients in rainfall were found significant
although their variability obviates precise conclusions. Using litera-
ture values for nutrient export from various land uses, nutrient bud-
gets were calculated for 55 Florida lakes. Critical N and P load-
ing rates (above which eutrophication is likely) were estimated from
the calculated budgets and lake trophic conditions.
Algal fixation in two eutrophic Florida lakes was studied in detail;
the total annual N fixed and factors affecting the occurrence of
fixation were evaluated. A survey of fixation in 55 Florida lakes
showed significant fixation only in eutrophic lakes. Bacterial fixa-
tion in the anoxic hypolimnion of a small lake contributed substantial
nitrogen to the lake, and N fixing activity was found in both estuarine
and lacustrine sediments. The acetylene reduction assay for N fixa-
tion was evaluated; short incubations were found essential. Reduction
was light dependent and N2 acted as a competitive inhibitor.
A preliminary experiment suggested that lacustrine sediments act as
ammonia buffers; estuarine sediment sorbed ammonia strongly with little
tendency to release ammonia to the water. Interferences from high
organic color were evaluated for automated inorganic N and P analyti-
cal methods. Various amino acids were also shown to interfere with
the indophenol ammonia procedure.
This report was submitted in fulfillment of Project Number 16010 DCK,
under the sponsorship of the Office of Research and Monitoring,
Environmental Protection Agency.
ill
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CONTENTS
Section Page
I Conclusions 1
II Recommendations 3
III Introduction 5
IV Review of the Nitrogen Cycle in Natural Waters 7
V Transport of Nitrogen Into Lakes 21
VI Nitrogen Fixation as an ln_ Situ Nitrogen Source
for Natural Waters. I. Algal Fixation in Lakes 39
VII Nitrogen Fixation as an I_n Situ Nitrogen Source
for Natural Waters. II. Bacterial Fixation in
Lakes and Sediments 63
VIII Other In Situ Nitrogen Sources and Sinks 93
IX Analytical Investigations 109
X Acknowledgements 139
XI References 141
XII Publications and Patents 157
XIII Appendix 159
A. Sampling and Analytical Methods
B. Routine Data Collected on Bivin's Arm and
N Newnan's Lake during Nitrogen Fixation Study
C. Enrichment and Isolation Procedures for Nitrogen
Fixing Agents in Lake Water and Sediments
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FIGURES
PAGE
NO.
1 Simplified nitrogen cycle showing main molecular
transformations °
2 Nitrogen cycle reactions in an idealized stratified
lake 9
3 Bathymetric map of Newnan's Lake 44
4 Bathymetric map of Bivin's Arm 45
5 Nitrogen levels, nitrogen fixation rates and primary
production in Bivin's Arm 47
6 Nitrogen levels, nitrogen fixation rates and primary
production in Newnan's Lake 49
7 Temperature, primary production and nitrogen fixation
during Aphanizomenon bloom in Newnan's Lake 50
8 Areal variations in primary production and nitrogen
fixation in Newnan's Lake, 8 December 1969 53
9 Diel variations in nitrogen fixation, primary production
and light intensity in Newnan's Lake, 16 December 1969 55
10 Diel variations in primary production and related para-
meters in Newnan's Lake, 21 April 1971 56
11 Bathymetric map of Lake Mize, Florida 64
12 Liinnological characteristics of Lake Mary, Wisconsin 65
13 Limnological characteristics of Lake Mize, Florida 65
14 Temperature profiles in Lake Mize during 1969 and 1970 69
15 Dissolved oxygen profiles in Lake Mize during 1969 and 1970 70
16 Depth profiles of nitrogen fixation in Lake Mize during
1969 and 1970 72
17 Total hourly fixation in Lake Mize during 1969 and 1970 74
18 Depth distribution of acetylene reduction rates in
Waccasassa Estuary sediments
77
vi
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19 Areal variations of acetylene reduction in Waccasassa
Estuary sediments 78
20 Lineweaver-Burk plot showing effect of N on acetylene
reduction by estuary sediments 81
21 Lineweaver-Burk plot showing effect of N~ on acetylene
reduction by Lake Kanapaka sediments 89
22 Sediment C/N ratio vs. trophic state ranking of lakes 98
23 Total organic nitrogen vs. total phosphorus in Florida
lake sediments 99
24 Decline in aqueous ammonia levels in aquaria containing
undisturbed estuary sediments 100
25 Ammonia uptake by fresh and sterilized estuary sediments 102
26 Ammonia uptake by Bivin's Arm sediments 105
27 Ammonia leached from Bivin's Arm sediments 105
28 Typical chromatogram showing elution order of acetylene,
ethylene and other gases 110
29 Effect of incubation time on acetylene reduction by
estuary sediments 114
30 Effect of N2 and light on time course of acetylene re-
duction by natural population of Anabaena 115
31 Theoretical Lineweaver-Burk plot for competitive
inhibition 117
32 Competitive inhibition of acetylene reduction in Anabaena
population by N~ 117
33 Inhibition of acetylene reduction in Anabaena population
by carbon monoxide 119
S
34 Lineweaver-Burk plot of CO inhibition data from Figure 33 119
35 Ethylene production by natural Anabaena population vs.
acetylene concentration at various levels of NoO addition 120
36 Immediate effect of ammonia on ethylene production by
Anabaena 123
37 Effect of ammonia on ethylene production by Anab aena
24 hours after ammonia addition 124
VI1
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38 Effect of organic color on apparent nitrate concentration
corrected by method of blank substraction 128
39 Effect of organic color on apparent nitrite concentration
corrected by method of blank subtraction 129
40 Effect of organic color on apparent ammonia concentration
cannot be corrected by method of subtraction 130
41 Effect of increasing organic color concentration on calibration
curves for alkaline-phenol ammonia procedure 132
42 Absorption spectra for products of alkaline phenol-ammonia
procedure at various levels of organic color 134
Vlll
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TABLES
No. Page
1 Sources and sinks for the nitrogen budget of a lake 22
2 Nitrogen and phosphorus contents of various nutrient 24
sources
3 Variations in nutrient content of rainfall in Gainesville
area 28
4 Nutrient content of rainfall in. coastal and rural areas 30
5 Temporal variations in nutrient contents of rainshowers 31
6 Contribution of rainfall to nutrient budget of Anderson-
Cue Lake 33
7 Regression analyses of trophic state index (TSI) vs.
nitrogen and phosphorus loading rates 35
8 Critical loading rates for nitrogen and phosphorus 36
9 Summary of the occurrence of nitrogen fixation in Florida
lakes 40
10 Nitrogen fixation rates in selected Florida lakes ^2
11 Chemical characteristics of Newnan's Lake and Bivin's Arm 46
12 Areal variations in physical and biogenic parameters in
Newnan's Lake, 14 April, 1969 51
13 Summary of areal and vertical variations in nitrogen
fixation and related parameters in Newnan's Lake,
8 December, 1969 54
14 Multiple regression analysis of primary production and
limiting factors in Newnan's Lake during Aphanizomenon
bloom 59
15 Contributions of nitrogen fixation to nitrogen budgets
of Newnan's Lake and Bivin's Arm
61
ix
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16 Nitrogen fixation rates in Lakes Mary, Wisconsin, and Mize, 67
Florida, in summer, 1968
17
18
19
20
21
22
23
24
25
26
27
28
29
Chemical characteristics of Lake Mize
Changes in nutrient levels of Lake Mize from 1968 to 1970
Effect of added organic substrates on rates of acetylene
reduction by estuary sediments
Acetylene reduction in Bivin's Arm sediments, August, 1968
Lake surveyed for sediment nitrogen fixation
Acetylene reduction rates in Florida lake sediments
Stratification of acetylene reduction in Florida lake sediments
Effect of organic substrates on rates of acetylene reduction
by Lake Kanapaha sediments
Sediment characteristics of north central Florida lakes
Ammonia uptake by estuary sediments in short-term shaker-
flask experiment
Replicate data on algal acetylene reduction
Replicate data on acetylene reduction by estuary sediments
Ammonia effects on nitrogenase in natural population of
Anabaena
68
71
81
84
85
86
87
90
95
103
112
113
122
30 Application of external compensation method of nitrite and
nitrate samples containing varying color concentrations
31 Response of two automated ammonia methods to free amino
. j 136
acids
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SECTION I
CONCLUSIONS
1. Rainfall contributes substantial amounts of nitrogen to lakes. The
great variability in concentrations obviates simple assessment of the sig-
nificance of this source, and experimental evaluation for each lake seems
necessary for accurate results. Nutrient concentrations decline rapidly
with length of rainfall, indicating the role of precipitation as an atmos-
pheric cleansing agent.
2. Using literature values for nutrient export from various land uses
and easily evaluated watershed characteristics, one can compute nutrient
budgets for lakes that, if not totally accurate,are nevertheless meaningful
and useful in assessing the eutrophication potential of a lake. This
approach, with refined export values would seem especially useful for small
but still recreationally important lakes, where the time and expense of
experimental nutrient budget evaluation cannot be justified.
3. Nitrogen fixation by blue green algae in eutrophic surface waters is
of doubtless ecological significance, but in terms of contributions to
the total nitrogen budgets of lakes fixation probably represents a rela-
tively minor source. Fixation is found only in moderately or highly
eutrophic waters where it likely exascerbates the problem by supplying
more nitrogen during times of relative depletion. However, if external
nutrient supplies are decreased below critical (eutrophying) levels, fix-
ation itself is unlikely to cause continued problems.
4. Bacterial fixation in anoxic lake waters is now an established fact,
and in some cases it can supply significant amounts of nitrogen to a lake
system. Fixation in sediments is also established, and heterotrophic
bacteria have been identified as the responsible agents in the sediments
studied here. The ecological significance of this phenomenon remains
puzzling, but the occurrence of fixation suggests that nitrogen is rela-
tively unavailable at least in some sediments.
5. Non-enzymatic chemical equilibria, in particular sorption reactions,
play an important, perhaps dominant role in controlling ammonia inter-
change betVeen sediments and water. Sorption of ammonia onto sediments
is, at least in some cases, rapid and largely irreversible.
6. The acetylene reduction method is a useful, precise and simple method
for assaying nitrogen fixation in environmental samples. Short incuba-
tions (approximately one hour) should be used for accurate rate determi-
nations. Acetylene reduction by algae is strongly light dependent.
Molecular nitrogen inhibits acetylene reduction, and a pattern of compe-
titive inhibition is obtained from Lineweaver-Burk plots. This fact is
useful in establishing the enzymatic nature of acetylene reduction in
environmental samples. Atmospheric levels of N9 decrease acetylene
reduction rates bv about 25-30 percent, and if only approximate results
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are desired, removal of Nn may not be necessary.
7. The interference of organic color in automated nitrite, nitrate and
ortho phosphate analyses can be simply compensated for by the method of
blank subtraction, but this cannot be done with the indophenol ammonia
method. Organic color or some constituent associated with color (e.g.
iron) causes positive interferences with this method which cannot be corrected
by eliminating a key reagent and measuring the color "blank." Further, a
variety of amino acids also react as ammonia in the indophenol method,
in some cases giving greater responses than equivalent ammonia-N levels.
This method should thus be used with caution, especially when free amino
acids are suspected in a water sample. When high color is present, a
alternate ammonia procedure would be preferable.
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SECTION II
RECOMMENDATIONS
1. Various diffuse sources of nitrogen, such as agricultural runoff,
sanitary landfill sites, and urban runoff, need much further study to
assess their quantitative significance in lake nutrient budgets.
2. The extent of denitrification in wetlands and sediments needs to be
evaluated to properly assess the importance of this reaction as a nitro-
gen sink for lakes.
3. The relationships between nitrogen-fixing blue-green algae and other
bloom forming algae need further investigation in terms of the transfer
of nitrogen from fixers to non-fixers.
4. The role and ecological significance of nitrogen fixing activities
in lacustrine and estuarine sediments need further exploration.
5. The role of sediments as nitrogen reservoirs or sinks needs further
clarification. Experiments involving laboratory model systems and
in situ methods should be undertaken.
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SECTION III
INTRODUCTION
Of the major nutrient cycles in natural waters, the nitrogen cycle is
perhaps the most interesting, most complex and least understood from a
quantitative point of view. The geocycle of nitrogen is largely a biochemi-
cal phenomenon; in natural waters it is nearly wholly so. As such, the
nitrogen cycle, like the carbon and phosphorus cycles, is inextricably
related to aquatic organic productivity. While many elements and com-
pounds are required for biosynthesis, nitrogen and phosphorus have long
been considered the prime limiting nutrients for primary production;
recent evidence suggests carbon may also limit production in some situa-
tions. The great recent concern over cultural eutrophication has stimu-
lated much new research into the chemistry and biochemistry of these
nutrients in aquatic systems, into quantifying the sources and sinks of
nutrients and into the dynamics of nutrient uptake and release. This
report discusses these subjects with emphasis on the cycle of nitrogen in
natural waters.
The nitrogen cycle in natural waters has been studied over a long period
of time. Qualitative aspects of the cycle are well known, but quantitative
studies are much less advanced. For example, in situ rates of nitrogen
cycle reactions have only recently been determined in a few lakes using
1^N. Rates of ammonia release from lacustrine sediments are virtually
unknown, and determination of the nitrogen contributions from various
sources is still in a crude and elementary state. The significance of
natural and uncontrollable cultural sources like urban and agricultural
runoff must be placed in proper perspective with the one nutrient source
most often cited in the popular press, i.e. domestic waste effluent. If
uncontrollable sources are alone sufficient to effect eutrophy in a lake,
then a priori decisions to limit waste effluent as a nutrient source may
be highly costly to the taxpaper without the anticipated benefits in
improved water quality. On the other hand, it may be found that reduction
of nutrieYits in controllable sources may significantly improve or completely
restore water quality. In any event, decisions to limit nutrient flux by
constraining certain sources must arise from knowledge of the entire nitro-
gen budget rather than from ignorance or speculation. To understand the
factors controlling the concentrations and temporal variations of nitro-
gen forms in natural waters requires two types cf research efforts. The
first involves quantitative studies of the sources and sinks comprising
the nitrogen budget of a water body; the second involves determination
of the internal dynamics or in situ turnover rates of the different nitro-
gen forms in the water body.
The overall objective of the project was to study the factors controlling
the concentration and forms of nitrogen in natural waters. To this end
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the project consisted of two principal phases:
1) delineation of the relative importance of certain source and
sink components in nitrogen budgets and;
2) investigation of the internal cycling of nitrogen compounds
within a body of water. The study was conducted on lakes of varying
trophic states (from oligotrophic to hypereutrophic) in the region around
Gainesville, Florida, and thus, has special relevance to lakes in humid,
subtropical regions like the southeastern United States where information
of this type is especially sparse. However, the results should also have
a general significance in other geographical areas, for many aspects of
the nitrogen cycle are similar in widely varying climates.
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SECTION IV
REVIEW OF THE NITROGEN CYCLE IN NATURAL WATERS
Nitrogen occurs in the biosphere in a variety of forms ranging in oxida-
tion state from +5 to -3. Inorganic nitrogen is present primarily as
highly oxidized nitrite and nitrate, as reduced ammonia and as molecular
nitrogen. A variety of intermediate gaseous oxides of nitrogen are im-
portant in atmospheric chemistry but not in natural waters. Naturally
occurring organic nitrogen consists primarily of amino and amide (pro-
teinaceous) nitrogen along with some heterocyclic compounds present
as cellular constituents, as non-living particulate matter, as soluble
organic compounds, and as inorganic ions in solution. All these forms
are interrelated by a series of reactions known collectively as the "nitro-
gen cycle," which portrays the flow of nitrogen from inorganic forms in
soil, air and water into living systems and then back again into inor-
ganic forms.
This cyclic phenomenon can be illustrated diagramatically in various
ways; Figure 1 presents a simplified reaction sequence of the interconver-
sions between organic nitrogen and the raain inorganic forms, and it
labels the principal reactions. This figure indicates the central role
of ammonia in the cycle as the link between the organic and inorganic
phases. The major reactions of the nitrogen cycle are (1) ammonia and
(2) nitrate assimilation, (3) ammonification, (4) nitrification, (5)
denitrification, and (6) nitrogen fixation. Reactions (1) and (2)
represent and major pathways for conversion of inorganic nitrogen into
organic (cellular) forms. Reaction (3) represents a series of complex
reactions breaking down organic forms (proteins, amino acids, nucleic
acids, etc.) into ammonia. Reaction (4), nitrification, is the aerobic
oxidation of ammonia to nitrite and nitrate. Reactions (1) - (4) can
be considered to comprise a potentially closed system of reactions
(the internal cycling of nitrogen among its various forms within a body
of water). Reactions (5) and (6) are respectively in situ sinks and
sources for nitrogen within given ecosystem. Nitrogen fixation (reac-
tion 6) is\he reduction of molecular (atmospheric) nitrogen to ammonia
and then to cellular (organic) nitrogen. Molecular nitrogen is not nor-
mally considered part of the nitrogen reserve in an ecosystem since it
(N9) is not utilizable by most organisms; thus fixation is a source of
nitrogen to the exosystem. Denitrification (reaction 5) is a nitrogen
sink since it converts utilizable nitrogen in the forms of nitrate and
nitrite to molecular nitrogen under anoxic conditions.
Figure 2 illustrates the nitrogen cycle as it may occur in an idealized
stratified lake. With the exception of ammonia exchange with sediments
it is apparent that all reactions are biologically mediated. By far the
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oo
COMPLEX
HIGH WEIGtHV
ORG/tf
2 xPURINES
PYRIMIDINES
PEPTI
AMINO ACIDS
AMINES
UREA
1NO.
FIGURE 1, SIMPLIFIED NITROGEN CYCLE SHOVING MAIN MOLECULAR TRANSFORMATIONS:
1, NITRATE ASSIMILATION, 2, AMMONIA ASSIMILATION, 3, AMMONIFICATION/
^i, NITRIFICATION/ 5, DENITRIFICATION, 6, i\!ITROGEN FIXATIO?]
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EPILIMNION
INORGANIC N
ASSIMILATION
THERMXLINE
lYPOLIfttlON
SEDIMENT
FI"URE 2, NITROGEN CYCLE REACTIONS IN AN IDEALIZED STRATIFIED LAKE,
"OTE T-iAT 30TJ AER03IC AND A^AEROT;iIC TRAiXS
ARE WO'v': I\| T:t L'V^OLI^;iOr;, II! A REAL LAKE
HEY ^OULD OF COURSE :JOT OCCUR SIMULTA'IEOUSLY,
REDRAWN AND ADAPTED FRO" \UZ;!3T30V (1Q59) ,
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greatest influx of inorganic nitrogen into organisms results from ammonia
and nitrate assimilation. These reactions predominate in surface waters
and are mediated primarily by phytoplankton and macrophytes. While nitrate
tends to be the predominant inorganic nitrogen form in surface waters,
there is considerable evidence that ammonia is the preferred form for plank-
tonic assimilation since it is already at the reduction level of organic
nitrogen. Nitrate can be used by most plants (Strickland, 1965, and
others cite some flagellates as exceptions and a few algae have been
reported to prefer nitrate over ammonia (Proctor, 1957)). Organisms using
nitrate as their nitrogen source must first reduce it to the level of
ammonia before incorporating it into organic forms, which process re-
quires a reduction system including the enzyme nitrate reductase. This
inducible enzyme is present in algal cells only when nitrate is being
used as the nitrogen source (Eppley, Coatsworth et_ a.l_. , 1969), which
suggests a mechanism for determining the form of nitrogen an algal pop-
ulation is using.
Reduction of nitrate also requires the oxidation of organic matter to
C02> and this oxidation-reduction sequence is apparently not linked to
the respiratory chain and to ATP synthesis. Thus utilization of nitrate
is in a sense energetically wasteful.
Ammonia is a weak base:
NH,+ £ NH,, + H+ , pKa = 9.3.
4 J aq.
Thus in poorly buffered waters where photosynthesis may raise the pH
as high as 10-11, the predominant form is NH-j while in well buffered
and in low productivity systems the cationic form (NH, ) dominates.
The fact that ammonia assimilation can continue at acidic pH values
(where [NHo ] is negligible) implies that the cation itself is trans-
ported through the cell membrane. At high concentrations NHo is toxic
to organisms and this fact may explain the reports of earlier workers
that ammonia is a less suitable source of nitrogen for algal cultures
than is nitrate. Growth media frequently contain 5 or more mg N/l (as
ammonia or nitrate) . If CC>2 uptake by photosynthesis is sufficient to
raise the pH to 10 or so, then the NH, could act as a toxicant. On the
other hand assimilation of ammonia and its incorporation into organic
matter tends to lower the pH (Fogg, 1965) since NH is a base and its
removal tends to increase acidity. Whichever phenomenon is in fact res-
ponsible for the inhibitory effects noted with high ammonia concentra-
tions, it is clear that a well buffered system is essential to maintain
proper growth conditions in ammonia containing media.
Rates of ammonia and nitrate assimilation in aquatic environments have
recently been determined by several workers using 15N tracer techniques.
Such studies are relevant to an increased understanding of the metabolism
of the aquatic community since the availability and utilization of
nitrogen profoundly influences and in some cases controls primary pro-
duction. Of interest in this regard is such information as the rates
of inorganic nitrogen turnover, the relative nutritional importance of
the various nitrogen forms, the effect of concentration on rates of
utilization, and the relationship between nitrogen assimilation and
10
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primary production. Althougn the N tracer approach to studying aquatic
nitrogen cycle dynamics is only about 10 years old, considerable gains
have already been made through its use. The nitrogen cycle can no
longer be viewed as a slow, primarily seasonal cycle. Turnover times
for inorganic nitrogen are on the order of days or even hours in many
waters, which implies the essentiality of rapid organic nitrogen decom-
position or a constant influx of new nitrogen into the system.
Dugdale and Dugdale (1965) were the first to report In situ ammonia
and nitrate assimilation rates in surface waters. Using ' bN techniques
they found two main pulses of assimilation in Sanctuary Lake, Pennsyl-
vania. During the first pulse, in June, ammonia was assimilated most
rapidly with a maximum rate of about 110 yg N/l.-day. The maximum
nitrate assimilation rate during the same period was about 40 yg N/l-day.
A second pulse of assimilation in early September utilized primarily N~.
Some of the high assimilation rates reported may be an artifact of
methodology. For example, the nitrate concentration at the time of
the June assimilation maximum was only about 10 yg N/l. The amounts of
NO-j" and NH~ added to samples were not specified, but a large amount
of tracer relative to the initial unlabeled nitrogen would stimulate
assimilation beyond the normal rate.
Rates of ammonia and nitrate assimilation in Lake Mendota, Wisconsin,
during 1966 were reported by Brezonik (1968, 1971a). Ammonia assimi-
lation rates were greater than nitrate assimilation rates in all cases,
and highest assimilation rates in the surface water occurred in late
spring and late summer. No correlation was found between ammonia and
nitrate concentrations and assimilation rates. Depth profiles of
ammonia assimilation during holomixis and early stratification were
fairly uniform and no trends were indicated. However, by early June
a pronounced stratification of assimilation was found with high rates
in the epilimnion and much lower rates in the hypolimnion.
In nearly every assimilation study published thus far ammonia was by far
more important than nitrate as a nitrogen source. In view of the ease
with which ammonia can be assimilated this is not surprising, but prior
to these tracer studies most marine biologists considered nitrate to
be the only significant nitrogen source in the sea. It now seems cer-
tain that both marine and fresh water algae derive most of their nitro-
gen from ammonia often in spite of higher nitrate concentrations. There
may be a certain amount of "wheel-spinning" involved in ammonia assi-
milation; rather than representing new production, at least part of
the uptake may derive from the necessity to recapture nitrogen from
compounds which apparently continually leak through cell walls (Whitta-
ker and Feeney, 1971; Hellebust, 1965; Stewart, 1963). Dugdale and
Goering (1967) derived a simple model for the nitrogen cycle in marine
surface waters in which they considered primary production associated
with ammonia assimilation to be "regenerated" production,and primary
production associated with nitrate assimilation was regarded as "new"
production. Only the latter would be available for export to higher
trophic levels since nitrate input from the deep water is the principal
source of nitrogen to the surface. Ammonia in sea water is the product
of short term regeneration, and primary production associated with its
11
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assimilation is essentially that needed to maintain the standing crop.
Extension of this approach to the more complicated nitrogen cycles in
shallow lakes would be of perhaps questionable validity.
An exception to the general rule of more rapid ammonia than nitrate
assimilation was recently reported by Goering &t_ al. (1970) in the eastern
subtropical Pacific. Phytoplankton in a nutrient-rich discontinuity
layer at the bottom of the euphotic zone were found to use nitrate pri-
marily while algae in the impoverished surface water used ammonia.
Dugdale (1967) has developed a model for nutrient limitation in the sea
in which assimilation of ammonia and nitrate were assumed to follow
Michaelis-Menten kinetics:
VS
v =
Kt+S ,
where v is the rate of nutrient assimilation; V a maximum rate, constant
under a given set of conditions; S is nutrient concentration; and Kt a
transport or half-saturation constant with units of concentration. The
numerical value of Kfc is equal to the nutrient concentration at which
v is one-half the maximum for the system ( V/2). Subsequent l5N measure-
ments on natural populations by Maclsaac and Dugdale (1969) and direct
uptake measurements on algal cultures by Eppley, Rogers _et^ a^Li (1969)
have verified this model and estimated Kt values for various marine
surface waters. Kt values calculated by the former authors ranged from
less than 0.2 to 4.2 ug.-atom/1, for nitrate and from 0.1 to 1.3 yg.-atom/1,
for ammonia. Lower transport constants correlated with oligotrophic
(nutrient-depleted) areas while higher K values were found in nutrient
rich waters. It was thus suggested that phytoplankton in oligotrophic
waters are adapted to low ambient concentrations and can assimilate
nutrients more rapidly under these conditions than can phytoplankton
from nutrient-enriched regions.
The reverse of assimilation is ammonification, whereby organic nitrogen
is returned to the inorganic nitrogen pool as ammonia. This is a compli-
cated process involving several mechanisms. Early workers considered
only bacterial decomposition of soluble organic nitrogen and organic
detritus as important (vonBrand et. al. , 1937), but more recent studies
have shown significant excretion of ammonia and amino acids by zooplank-
ton feeding on phytoplankton and detritus. Johannes (1969) suggested this
as the dominant mechanism of ammonification in surface waters and
reviewed previous work indicating that net zooplankton release amounts
of dissolved nitrogen and phosphorus equal to their total body content
of these nutrients in 20 to 200 hours. A third mechanism for ammoni-
fication is direct autolysis after cell death, which may account for 30
to 50 percent of the nutrients released from plant and animal material
(Johannes, 1969; Krause, 1964; Golterman, 1960). The excretion of amino
acids and other small nitrogenous substances from photosynthesizing
cells (Hellebust, 1965) and their subsequent direct uptake by other algae
or bacteria represents a short-circuiting complication of ammonification
pathways, the significance of which is not yet understood.
12
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Current information and theories suggest that bacterial decomposition
of organic matter accounts for a minor portion of nutrient regeneration
in marine surface waters and in shallow lakes (although this is probably
not not true in systems receiving large organic inputs from sewage out-
falls) . In soils and bottom sediments and in anoxic hypolimnia of
lakes, nutrient regeneration by bacteria and fungi is the dominant pro-
cess. Ammonification is obviously important in reviewing a limited
supply of nitrogen for assimilation and growth of primary producers.
In situ ammonification rates can be measured by an isotope dilution
technique (Zilversmit e_t_ _al. , 1943; Dugdale, 1965; Brezonik, 1968).
Assuming steady state conditions for assimilation and ammonification,
15N label added to the ammonia pool will gradually become diluted as
both labeled and unlabeled ammonia is assimilated but comparatively
unlabeled N is returned to the ammonia pool by ammonification of organic
N. The decrease in l5N enrichment in the ammonia pool can be quantified
and related to the rate of ammonification. Because of the difficulties
encountered in l5N measurements, only a few in situ ammonification
rates in lakes have been reported. Alexander (1970) reported that
maximum ammonification rates in Smith Lake, Alaska, were associated
with a spring bloom of Anabaena and with the period of maximum ammonia
assimilation. Relatively high rates were measured in Lake Mendota,
Wisconsin (Brezonik, 1968, 1971a) with a tendency for higher rates in
the bottom waters. From steady-state kinetics, ammonia turnover times
were calculated to range from 7 to 62 hours in Lake Mendota surface
waters during the summer. Because steady state conditions do not
exactly apply and because of the errors inherent in the methodology,
these calculations are only approximate (Brezonik, 1971a); nonetheless
they suggest a much more rapid cycling of nitrogen than measurement of
concentration changes alone would suggest.
Ammonia is oxidized to nitrite and nitrate in the process of nitrifica-
tion by a select group of aerobic autotrophic bacteria which obtain
their energy by nitrogen oxidation and their cellular carbon by reduc-
tion of C0?. A variety of heterotrophic bacteria, actinornycetes and
fungi (Schmidt, 1954; Hatcher and Schmidt, 1971) have also been reported
capable of nitrification, generally at much slower rates, but the signi-
ficance of these organisms in aquatic nitrification is not well known.
The biochemistry and metabolism of nitrifying organisms has been reviewed
by Painter (1970) particularly with reference to its occurrence in sewage
treatment. The significance of nitrification in the nitrogen cycle lies
in the conversion of labile ammonia (which tends to be lost from solu-
tion by sorption onto sediments and by volatilization at high pH) to a
more stable form (nitrate). On the other hand, nitrate can be reduced
to molecular nitrogen by the process of denitrification, and thus nitri-
fication has a second and opposite role that of producing the reac-
tants for this nitrogen sink. The oxidation of ammonia to nitrate re-
quires almost 4.5 mg 02 per mg of ammonia - N oxidized. Thus nitrifi-
cation can act as an important oxygen sink in streams which receive high
ammonia concentrations from unnitrified sewage effluents. Attempts to
quantify and model these effects have recently been described by a
number of workers (Stratton and llcCarty, 1967; Wezernak and Gannon,
1969: Thomann et al., 1971).
13
-------
In situ nitrification rates can be estimated by adding 15NE^ to a water
sample and determining the amount of label in the nitrate and nitrite
fractions after incubation (Brezonik, 1968). The latter is accomplished
by reducing the oxidized nitrogen to ammonia with Devarda's alloy
(Bremner and Keeney, 1966), and subsequent conversion of ammonia to N?
by hypobromite oxidation (Rittenberg «!t_ al., 1939; Neess et_ al_. , 1962)
for isotope ratio analysis. Dugdale and Goering (1967) were unable to
detect nitrification in the surface waters of the Sargasso Sea using
incubations as long as a week, and it appears that regeneration of
nitrate in the sea occurs primarily below the euphotic zone. A few
nitrification rates were reported for Lake Mendota by Brezonik (1968,
1971a). During 1966 rates increased with depth in spring but were very
low in the surface water during summer. The fact that nitrate levels
invariably decline in lake surface waters during spring and summer
suggests that nitrifying bacteria are unable to compete with algae for
ammonia, and nitrification is thus felt to be of minor significance
in surface waters during the growing season (Brezonik, 1968). However,
Brezonik and Lee (1968) also found that substantial nitrification evi-
dently occurred at mid-depths (7-17 m.) in Lake Mendota, Wisconsin,
during late spring and early summer. Largest increases occurred in the
12-15 meter zone where nitrate increased from less than 0.2 mg. N/l. in
June to 0,5-0.7 mg. N/l. in late July. Nitrification in the hypolimnion
is especially significant since the water eventually becomes anoxic
during late summer, and denitrification takes place. Thus nitrifica-
tion increases the importance of denitrification as a nitrogen sink in
Lake Mendota. Vollenweider (1963) has also reported nitrification at
mid-depths during summer stratification in Lake Orta, Italy.
In the process of denitrification nitrate is used as a terminal electron
acceptor by facultative and anaerobic bacteria in the absence of oxygen.
Nitrite is formed as the first intermediate in the process, and nitrous
oxide can sometimes be formed along with molecular nitrogen although it
is not an essential intermediate. Since the principal end product (N2)
is a nitrogen form not utilizable by most organisms, this reaction acts
as a nitrogen sink and can assume importance in the nitrogen balances
of lakes and other aquatic systems subject to intermittent anoxia,
(e.g. soils, Delwiche, 1965). The biochemistry of denitrification has
been reviewed extensively (Delwiche, 1956, 1965; Kessler, 1965; Painter,
1970), who describe the long and considerable controversy over the re-
quirement of completely anoxic conditions for this reaction to occur.
The consensus now is that denitrification occurs only when oxygen is
absent from the system or at least sufficiently low enough to allow
anoxic microzones to develop.
Denitrification may account in part for the difficulty encountered in
obtaining nitrogen balances in waste treatment plants (Wuhrmann, 1954;
Symons et al., 1965) . The reaction may occur in anoxic microzones of
activated sludge floe or in anoxic settling basins. Nitrification
followed by intentional denitrification has been proposed as a means
of nitrogen removal from biological waste treatment plants (Johnson and
Schroepfer, 1964; Wuhrmann, 1964) . Denitrification has been shown to
be an important nitrogen sink in lakes (Brezonik and Lee, 1968; Goering
and V. Dugdale, 1966). Its role in the nitrogen cycle of the oceans is
14
-------
less defined but there is no doubt of its occurrence in some oceanic
environments (Goering and R. Dugdale, 1966; Goering, 1968).
Not all the nitrate that is reduced is lost to the system as N_. A
significant but variable amount of assimilatory reduction to ammonia and
organic nitrogen occurs simultaneously (but different organisms may be
involved in the two processes). This was first noted by this author
in denitrification experiments with sewage sludge digesters; only 50
percent of added nitrate was recovered as N2 (Brezonik and Lee, 1966).
An early study of denitrification in lakes (Goering and V. Dugdale,
1966) suggested that assimilatory nitrate reduction was unimportant
based on low measured rates of ammonia production from labeled nitrate into
organic nitrogen. Brezonik and Lee (1968) found that reduction to ammonia
and organic nitrogen occurred at comparable but somewhat lower rates
than denitrification in Lake Mendota, Wisconsin. Of the 4.43 x 107g.,
nitrate-N present in the lake hypolimnion below 14 m. in mid summer of
1966, an estimated 2.81 x 107g. were lost by denitrification while the
balance (1.62 x 107g.) was reduced to ammonia and organic nitrogen.
Goering (1968) also reported ratios of N2-N formed/N03~-N lost ranging
from 1/10 to 8/10 in the eastern Pacific, indicating that assimilatory
reduction is of great but variable importance.
Denitrification in lake sediments may be an important nitrate sink in
ground water seeping into lakes as Keeney et al. (1971) have recently
described for Lake Mendota, Wisconsin. Finally, denitrification may be
be the mechanism whereby the high N/P ratios of fresh waters are trans-
formed to the lower ratios encountered in estuarine waters (Stumm, 1971).
No In situ experiments of denitrification have been conducted in such
environments, but if density stratification is sufficient to produce
anoxic or near anoxic conditions in the bottom layers of an estuary, this
reaction could occur.
Probably more attention has been devoted to nitrogen fixation than to all
the other nitrogen cycle reactions combined. This process is important
at several levels. Geochemically nitrogen fixation is essential in main-
taining a nitrogen balance in the biosphere, which would otherwise become
depleted as a consequence of denitrification over a time scale of
millions of years. In agriculture the reaction is important in maintaining
or increasing soil fertility. In natural waters nitrogen fixation acts as
a source of nitrogen and permits continued organic production when the
supply of fixed nitrogen becomes depleted. A variety of organisms are
capable of nitrogen fixation including a number of blue-green algae,
apparently all photosynthetic bacteria, various aerobic bacteria (e.g.
Azotobacter), anaerobic bacteria (e.g. Clostridium) , many facultative
bacteria (but only under anoxic conditions (Wilson, 1969)),legume root
nodules and nonleguminous root nodulated plants such as Podocarpus anr*
the alder tree (Alnus sp.). Most studies of nitrogen fixation in natural
waters to date have emphasized the role of filamentous, heterocystous
blue-green algae. The occurrence of Azotobacter and Clostridium in the
Black Sea (Pshenin, 1959, 1963) and in other aquatic habitats has been
established, and photosynthetic bacteria have been found in dense popu-
lations in a variety of lakes (e.g. Triiper and Genovese, 1968). However
the presence of bacteria capable of fixation does not necessarily imply
15
-------
the actual occurrence of fixation in any given environment. On the other
hand these organisms have the potential for fixation presumably because
it is useful to them at least in some habitats and it would be surprising
if fixation bv these organisms were not found in the environment. Most
biologists have de-emphasized the role of these organisms (especially
the heterotrophic bacteria) in nitrogen fixation because they have felt
that insufficient oxidizable carbohydrate (or other substrate) would
be available in the environmental Stewart (1966, 1968, 1970) has reviewed
the agents and occurrence of nitrogen fixation in the biosphere in greater
detail.
Nitrogen fixation is generally considered to be an adaptive process used
by organisms only when the supply of fixed nitrogen is depleted. Nitrogen
fixing alyae usually bloom in lakes only after nutrients have been de-
pleted by blooms of other algae (i.e. late summer in temperate lakes).
However, contrary to earlier opinions, small to moderate concentrations
cf ammonia do not necessarily inhibit fixation although synthesis of
the enzyme nitrogenase is repressed at high levels. Stewart (1969)
suggests that the levels of combined nitrogen nitrogen in most natural
ecosystems are insufficient to inhibit fixation immediately or even to
persist long enough for existing nitrogenase to be diluted out. Low
levels of combined nitrogen may actually be advantageous to nitrogen-
fixing plants by enabling more efficient and healthy growth than could
be achieved on N2 alone.
Reduction of N2 to the level of ammonia requires energy and a source of
reduced hydrogen, both of which may be obtained either from chotesynthetic
production or from oxidation of organic carbon. Nitrate reduction to
ammonia similarly requires energy obtainable by oxidizing organic sub-
strates. While the net reactions in each case are exergonic, they are
nevertheless energy sinks since the cells apparently have no means of
trapping and storing the energy (as ATP) given off by the reactions. To
this extent use of nitrate and of molecular nitrogen rather than ammonia
for assimilation is wasteful of energy.
Molybdenum has been shown to be a specific requirement for organisms
using nitrate or fixing nitrogen, and it apparently is a constituent of
the enzymes directly involved in the processes (nitrate reductase and
nitrogenase, respectively). This element has been shown to be a limiting
nutrient in various soils and at least one lake has demonstrated a
deficiency. Goldman (1960) found a planktonic growth response when
molybdenum was added to Castle Lake, California; this Mo limitation
was presumably linked to nitrogen metabolism. Nitrogen fixing organisms
also require iron in greater amounts than is required by organisms
growing on fixed nitrogen, and cobalt (or vitamin B^) has also been
implicated as an essential nutrient for fixation.
The biochemical pathway of nitrogen fixation is poorly understood.
A long history of efforts to isolate intermediate reduction products is
recorded in the literature, but thus far only ammonia the final in-
organic intermediate before conversion to organic nitrogen compounds
has been definitely shown to be a step in the process. Because of this
distinct lack of success in finding intermediates, most biochemists
16
-------
currently favor a pathway in which the intermediates are tightly bound
to the enzyme active site. Burris (1966, 1969) has reviewed the bio-
chemistry of fixation in several informative articles.
Suggestions that nitrogen fixation occurs in lakes date back at least
as far as far as Hutchinson (1941), who thought that Anabaena was res-
ponsible for an increase in fixed nitrogen in Linsley Pond (Connecticut)
during the summer. The first in situ measurements of lacustrine fixa-
tion were reported by Dugdale et_ ad. (1959). These workers developed the
15N technique (Neess _et_ al_. 1962) and demonstrated the occurrence of
low rates of fixation in several lakes. More thorough studies were
reported by these workers for several Alaskan lakes (Dugdale and Dug-
dale, 1961) where only very low rates were detected; in Sanctuary Lake,
Pensylvania, (Dugdale and Dugdale, 1962) where a maximum rate of 130 yg
N fixed per liter per day was associated with a late summer bloom of
Anabaena; in Lakes Mendota and Wingra, Wisconsin, (Goering and Neess,
1964); in Smith Lake, Alaska (Dugdale, 1965); and in the Sargasso Sea
(Dugdale e_t £l. , 1964). High rates of fixation have been consistently
correlated with blooms of blue-green algae, primarily Anabaena,
Gloeotrichia, and Aphani z omen on in lakes, Trichodesnium in subtropical
marine waters and Calothrlx in shallow temperate marine environments
(Stewart, 1965).
The expense and difficulties associated with !5N tracer techniques pre-
cluded routine and detailed investigations of nitrogen fixation in
aquatic environments until recently when a simple, indirect method of
assessing nitrogen fixation rates was developed (Stewart _e_t al. , 1967) .
This method utilizes the fact that the nitrogen fixing enzyme complex
reduces acetylene to ethylene, the production of which can be sensitively
and easily determined by gas chromatography. Acetylene (H-C=C-H) is
isoelectronic with molecular nitrogen (N=N), and the nitrogenase system
is incapable of distinguishing between the two molecules. In fact a
number of other similarly shaped molecules such as cyanide (C~N)~ and
carbon monoxide (C=0) also react at or attach to the nitro-
genase active site and thus act as enzymatic inhibitors of fixation
(Burris, 1969). Based on the fact that nitrogenase reduces acetylene
to ethylene (H2C=CH2) , Dilworth (1966) proposed that di-imide (HN=NH)
is the first intermediate in the reduction of nitrogen. Of the various
compounds that react at the nitrogenase active site, acetylene is best
suited for indirect assay of nitrogenase activity. Neither acetylene
nor its redaction product is toxic to the organisms. Furthermore
acetylene reduction yields a single, easily measured product; (ethylene
is not further reduced to ethane by the enzyme). Finally the affinity
of nitrogenase for acetylene is large, in fact larger than for its
normal substrate. Thus even in the presence of molecular nitrogen,
acetylene is preferentially reduced. The advantages of the acetylene
reduction assay over 15N techniques are both a greatly simplified metho-
dology and a greater sensitivity. These properties have facilitated
such fundamental studies on aquatic nitrogen fixation as the effects of
sunlight intensity, detailed depth profiles, and diel variations (Stewart
et_ jil. , 1967; Rusness and Burris, 1970). In addition the fact that
acetylene reduction yields a gaseous product has enabled the study of
17
-------
low fixation rates in lacustrine and estuarine sediments (Brooks et al.,
1971; Keirn and Brezonik, 1971); see Section VI.
In work supported by this project Brezonik and Harper (1969) reported
evidence for nitrogen fixation in anoxic lake hypolimnia, and Stewart
(1969) has similarly reported on fixation in anoxic waters of a Nor-
wegian fjord. Brooks (1969), Brooks ejt _aJL. , (1971) and Keirn and Brezonik
(1971) in work described later in this report have also found low but
measurable rates of nitrogen fixation in estuarine and lacustrine sedi-
ments, and Howard et^ jil_. (1970) have reported low fixation rates in Lake
Erie sediments. Thus the known distribution of nitrogen fixation in
aquatic habitats has been extended considerably by recent work.
The rate at which newly fixed nitrogen is transferred from agents of
fixation to other aquatic organisms is of great interest. Jones and
Stewart (1969) have reported that extracellular nitrogen liberated by
the marine nitrogen fixer, Calothrix scopuloruci, is assimilated by
marine algae, fungi, and bacteria and could serve as the sole nitrogen
source for a species of Chlorella. Based on limiting nutrient bio-
assays, Fitzgerald (1969) concluded that transfer of nitrogen newly fixed
by Aphanizomenon is of little significance to colonies of Microcystis
grox-jing in the same water, but this is perhaps an overextension of the
data. It seems clear from Fitzgerald's results that Aphanizomenon is
unable to supply Microcystis with sufficient nitrogen to satisfy the
organisms, but the actual magnitude of supply is still unclear.
With the exception of sediment-water interactions (to be discussed in a
later section), the major nitrogen cycle reactions of natural waters
have been discussed above. As presently understood the nitrogen cycle
is almost exclusively a biological phenomenon. However, the possible
importance of chemical reactions and of heretofore undetected (but
potential) biological transformations should not be overlooked. A
variety of other reactions are thermodynamically feasible, and some of
these could have a substantial impact on the overall cycle. The mechanisms
of No formation from fixed nitrogen have been the subject of a long con-
troversy and many pathways have been proposed and rejected. Formation
of N? directly from ammonia has been hypothesized to occur in sludge
digestion (Malina, 1961; Crane, 1962) and anoxic lake waters (Koyama,
1964) , but others have found no evidence for such a reaction (Brezonik
and Lee, 1966; Wijler and Delwiche, 1954). The latter authors used l5N
methods to show that all N in denitrification derives from oxidized
nitrogen (NO^" ,NC>2~) sources. The reaction of nitrous acid with amino
groups under acid conditions:
RCHCOOH + HN02 > RCHOHCOOH + N? + H,,!)
(commonly known as the Van Slyke reaction) has been proposed by some
workers to be significant in acid lakes (Hutchinson, 1957) and soils
(Reuss and Smith, 1965) but has been rejected by others (Bremner and
Nelson, 1968; Nelson and Bremner, 1969). However the latter authors have
provided evidence for significant chemical decomposition of nitrite to
a variety oC gaseous products in soils. Bremner and Nelson (1969) demon-
strated that nitrite decomposition in sterilized soils is due to reaction
-------
of nitrite with soil organic matter and to self decomposition of nitrous
acid (2HNO- = NO + NOo + H~0) under acid conditions. The nitrite-or-
ganic matter reaction was found to involve phenols and polyphenols such
as lignins and tannins. The mechanism is thought to involve reaction
of nitrous acid under slightly acid conditions (pH 5) with phenolic
compounds to form nitrosophenols which tautomerize to quinone oxiir.es.
The latter are decomposed by nitrous acid to form ^ and ^0 (Austin,
1961). This reaction could be of importance in the nitrogen cycle of
natural waters. Colored lakes are high in polyphenolic substances
(tannins, lignins, "humic acid") and generally have acid pH values.
While nitrite levels in natural waters are normally low, such a reaction
could act to decompose nitrite as rapidly as it if formed.
While photochemical reactions are highly important in the atmospheric
chemistry of nitrogen, photochemistry is presently thought to play an
insignificant role in aquatic nitrogen transformations. Since most photo-
chemical reactions are induced by UV light, such processes should be
limited to a narrow surface layer. Photochemical nitrification in the
sea was proposed by various workers in the 1930's (ZoBell, 1935;
Rakestraw and Hollaender, 1936; Cooper, 1937) partly as a result of
difficulties in culturing marine nitrifiers (Waksman et al., 1933), but
Hamilton (1964) concluded that such a reaction is of no significance
in the marine nitrogen cycle. Hamilton found slight photoreduction of
nitrite but concluded that this too was insignificant.
Several thermodynamically possible nitrogen reactions have never been
shown to occur as biological phenomenon. For example, N? could be used
as a terminal electron acceptor in organic carbon oxidation with the
release of sufficient energy for organism growth:
glucose + 4N2 + 8H+ + 6H20 = 6CO + 8NH* , AG° = -84.5 kcal/mole,
The above reaction of course occurs when heterotrophic organisms fix
nitrogen, but it is not thought that any of this energy is trapped and
retained by nitrogen fixing cells for other uses. However, it is con-
ceivable that this reaction could be a source of energy for some anaerobic
bacteria. Oxidation of molecular nitrogen to nitrate by molecular oxygen
is also exergonic at pH 7:
N2 + ~°2 + H2° = 2N03~ + 2H+ (w)' AGw = ~15-2 kcal/mole.
With the large reservoir of available reactants for this process, it is
surprising that no organisms have evolved to take advantage of the situa-
tion. However, this is probably fortunate considering the nature of the
product (i.e. nitric acid).
19
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SECTION V
TRANSPORT OF NITROGEN INTO LAKES
The concentration and forms of nitrogen in a lake at a given time are a
product of input rates, of the interconversion reactions occurring within
the lake and the rates of loss via outflow, denitrification and sediment
deposition. Lakes act in general as nutrient traps, implying an accumu-
lation of nitrogen in bottom sediments as a lake gradually fills in. Both
natural and cultural nitrogen sources can be significant in lacustrine
nitrogen budgets. The natural nutrient input to a lake is a function of
its drainage basin geochemistry (i.e. the potential for nutrient leach-
ing from soils and substratum), drainage basin size, hydrology, precipi-
tation patterns, etc. (Brezonik et_ jil_. , 1969). Superimposed on these
factors are a multitude of human or cultural factors, which can be ex-
pressed in terms of drainage basin land use patterns and population
characteristics. Quantitative information on lacustrine nitrogen budgets
and the significance of individual sources is sparse, but concern over
cultural eutrophication has stimulated much needed measurements of the
nitrogen (and phosphorus) contributions from various sources along with
development of elementary nutrient models.
Table 1 lists the possible nitrogen sources and sinks which must be con-
sidered in calculating the nitrogen budget for a lake. The classical
approach to evaluation of a lake's nutrient budget is actual measurement
of nutrient concentrations and flows for each source over a reasonable
time span (e.g. one year). Obviously this requires a large time and man-
power expenditure, especially for large lakes, and some diffuse sources
(e.g. ground water seepage) may not be amenable to direct or accurate
evaluation. Because of these difficulties it is probable that complete
measurements of all sources and sinks have not been accomplished for any
single lake although approximate nutrient budgets have been established
for a few American and European lakes (see Brezonik ejt^ jil_. , 1969, and
Vollenweider, 1968, for reviews). An alternative to actual measurement
is development of a simple simulation model of nutrient transport based
on knowledge of the lake's drainage basin size, population, hydrology and
land use patterns and on literature values for nutrient contributions
from the various sources. This approach was taken by Lee e_t_ _al_. (1966)
in their well-known budget for Lake Mendota, Wisconsin. Actual measure-
ments of nutrient contributions are undoubtedly more accurate than litera-
ture estimates, but as the literature on nutrient export rates from various
land uses and other sources becomes more complete, the accuracy of
literature-based nutrient budgets will increase. The accuracy of such
nutrient budgets depends not only on the accuracy of the original measure-
ments but also on their applicability to the particular system under
consideration. However considering the expense and difficulties of direct
21
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Table 1. Sources and Sinks for the Nitrogen Budget of a Lake
Sources
1. Surface
Agricultural (cropland) runoff
and drainage
Aniraal waste runoff
Marsh drainage
Runoff from uncultivated and
forest land
Urban storm water runoff
Domestic waste effluents
Industrial waste effluent
Wastes from boating activities
2. In. situ
Nitrogen fixation
Sediment leaching
3. Airborne
Rainwater
Aerosols and dust
Leaves and miscellaneous
debris
4. Underground
Natural ground
Subsurface agricultural and
urban drainage
Subsurface drainage from
septic tanks near lake
shore
Sinks
Effluent loss
Groundwater recharge
Fish harvest
Weed harvest
Insect emergence
Volatilization (of N
Evaporation (aerosol) forma-
tion from surface foam)
Denitrification
Sediment deposition of detritus
Sorption of ammonia onto
sediments
22
-------
measurement, literature-based models seem the only reasonable approach
for many lakes receiving cultural stress. Thus refinement of nutrient
contribution values from the various types of sources is of great impor-
tance.
Clearly experimental evaluation of the quantitative importance of the
myriad sources of nitrogen was beyond the scope of the present project.
Rather it was concerned primarily with the importance of in situ sources
and sinks, especially nitrogen fixation and sediments (see Sections VI-
VIII). However, in conjunction with a concurrent study of eutrophication
in Florida lakes a significant effort has been made to gather information
from the literature regarding the significance of natural and cultural
sources and to apply this information to the development of nitrogen
budgets and simple nutrient-eutrophication models for Florida lakes. In
addition the significance of rainfall as a nutrient source received some
experimental attention during this project.
The significance of particular nitrogen sources has received considerable
attention in various recent reviews, and an exhaustive literature review
is not in order herein. For details the reader should consult the ori-
ginal literature or the specific reviews cited below. This section will
present a summary of the nitrogen (and to a lesser extent phosphorus)
contributions from various sources and describe the use of such data in
developing nutrient budgets and enrichment models. Feth (1966) reviewed
the sources and concentration range of nitrogen in natural waters empha-
sizing natural sources and a geochemical viewpoint. Agricultural sources,
especially fertilizer and soil losses have been treated by Biggar and
Corey (1969), and C. F. Cooper (1969) has reviewed nutrient export from
forest land. Weibel (1969) reviewed his and other studies on urban nutrient
sources. Schraufnagel j2t_ al. (1967) and Lee et_ jil_. (1966) reviewed the
natural and cultural sources of nitrogen especially as applied to lakes
and streams in Wisconsin, and Vollenweider (1968) has provided a broad
ranging review on both nitrogen and phosphorus sources for lakes.
Table 2 summarizes the ranges of nitrogen and phosphorus contributions from
various natural and cultural sources and provides further references which
have evaluated or reviewed their significance. Nutrient contributions
from undisturbed forest land are generally considered to be small, but
fertilization and clear-cutting tend to increase nutrient export (Cole
and Gessel, 1965; Bormann e_t^ aJL 1968). For example, the former authors
found that^nitrogen output via percolotion water from a Douglas fir
forest increased from 0.54 kjj./ha. to 0.69 and 1.04 kg./ha. when plots
were fertilized with urea and ammonium sulfate, respectively. Clear-cutting
increased nitrogen export to 0.96 kg,/ha., and this practice also tends
to increase runoff and sediment transport. Bormann ^t a_l_. (1968) have
found large alterations in the nitrogen cycle of a watershed after its
hardwood forest was leveled and regrowth prevented by herbicide applications.
Greatly increased nitrate levels were noted in stream water (greater than
lOmg. N/l.), and nitrogen export rose from less than 2 kg./ha.-yr. to more
than 6.0. Thus properly managed and undistrubed forests retain their
accumulated nutrients efficiently and not surprisingly should be of little
concern in eutrophication control. However management and harvest prac-
tices can alter this state of affairs and produce considerable nutrient export,
23
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Source
Natural
Table 2. Nitrogen and Phosphorus Contents
of Various Nutrient Sources 1
References
Forest runoff
Forest Percolation
water
Swamp and marsh
runoff
Meadow land and
runoff
Precipitation on
lake surface
Aquatic birds
Leaves and pollen
Cultural
Domestic sewage
Agricultural areas
Citrus
Pasture
Cropland
1.3-5.0
1.5-3.4
0.54
?
?
.58
.18-. 98
.48-. 95
?
39.4
22.4
8.5
1-5
5-120
.084-. 18
.83-. 86
.034
9
?
.044
.015-. 060
.09-. 18
?
0.80
0.18
0.18
0.15-0.75
0.22-1.0
Cooper, 1969;
Sylvester, 1961
Cole & Gessel, 1965
Vollenweider,1968
Lee et al. 1971
Vollenweider,1968
Brezonik e_t al . , 1969
Vollenweider, 1968
Hutchinson, 1957, Feth
1966; Veibel et al. ,1966b
Sanderson, 1954
Paloumpis and Starrett,
1960; Gates, 1963
Vollenweider, 1968
Goldman, 1961, Hynes
Kaushik, 1969, Richard-
son, et al. , 1970
Vollenweider, 1968 Saw-
yer, 1947; Mackenthun et al.
1964;Englebrecht&Morgan, 1959
Montelaro,1970
Miller, 1955
Vollenweider , 1968
Johnson ,et .al , 1965 Moe
1967; Biggar&Corey, 1969
Farm animals,
feedlots
Vollenweider, 1968; B. A.
Stewart, 1970; Loehr,
1969; Miner et_ al 1966;
Hutchinson & Viets, 1969
Urban runoff
8.8
1.1
Weibel ej: al, 1964;1966ab
Weibel, 1969; Palmer,
1950; Sawyer, ^t al, 1945
Septic tanks
Polta, 1969; Patterson
et al, 1971
Marsh and landfill
drainage
Quasim, 1965
units in kg/ha-yr. except rainfall (g/m^-lake area-yr.), birds
(kg/duck-yr.), and domestic sewage (kg/capita-yr.) .
24
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Undisturbed meadowland is probably camparable to forestland in retention
of nutrients, but intensively used pastureland appears to be a somewhat
greater source. In many areas pasture land is fertilized to increase the
yield of grass, and this fertilizer coupled with animal wastes undoubtedly
increases nutrient export rates. Swamp and marshland have frequently been
cited as important both in trapping nutrients before they reach a lake
and in contributing to the nutrient budget of a lake (Benson, 1965; Lee
1970a), but quantitative information is sparse. Recently Lee et. al. (1971)
described the annual nutrient balance in several Wisconsin marshes as
in approximate steady state with the nitrogen and phosphorus income equal
to the outflow. During growing season in these temperate marshes nutrient
inflow is retained by the marsh but these nutrients appear to be exported
during a short period of high flow in spring. On the other hand, draining
a marsh and converting it to dry land tends to release large amounts of
nitrogen and phosphorus as a result of soil oxidation and mineralization.
Pollen was recently shown to be an insignificant source of nitrogen for
Lake Tahoe (Richerson et. al., 1970), but it may be of greater importance
(a few percent or so) in small alpine or subalpine lakes with limited
nutrient inflow and greater shoreline to volume ratio. Leaves are known
to exert a significant effect on water quality in streams (Slack and Felz,
1968), but quantitative assessment of their role in lacustrine nutrient
budgets is a difficult matter.
Nutrient contributions of water fowl are not well known but may be signi-
ficant in some cases, especially where large rookeries and migratory bird
preserves occur on small water bodies. A community of about 50 mallard
ducks maintained for research purposes on the shore of Lake Mize, a small,
deep lake near Gainesville, Florida, was recently estimated to have increased
the nitrogen and phosphorus loading rates beyond those suggested as cri-
tical for eutrophication (Brezonik, 1971b). Duck contributions were cal-
culated to represent 50 and 75 percent of the nitrogen and phosphorus loading
rates of 8.1 and 0.73 g./m?-yr., respectively. Waste production of small
and medium sized birds (ducks, chickens) have been assessed in relation
to pollution from commercial breeding, production, and processing (San-
derson, 1954; Gates, 1963). The role of wild aquatic birds in the nutrient
regime of a water body may be more that of cycling agents than of direct
sources (i.e. much of their food may be taken from the lake itself - fish-
or from tt^e immediate watershed). However, such activities can still stim-
ulate increased production and apparent eutrophication by accelerating
nutrient cycling and degrading a normally more stable (organic) nutrient
reservoir.
Agricultural sources of nitrogen are difficult to summarize because of
large local variations in soil retention capacities, in irrigation and.
fertilization practices, and in differences associated with climate and
type of crops grown. However, some data are available to indicate the sig-
nificance of agricultural runoff as a nutrient source. Johnston et. al.
(1965) found large percentages of nitrogen applied to crop lands in the
San Joaquin Valley of California appeared in the tile drainage effluents.
From 9 to 70 percent and phosphate concentrations ranged from 0.053 to
25
-------
0.23 ppm., but even these comparatively low concentrations are sufficient
to stimulate algal growths. Moe et^ aL_. (1967) found considerably smaller
losses of nitrogen from soils in southern Indiana. A maximum of 15 per
cent of applied fertilizer (NH^NCs) was found in surface runoff after
simulated heavy rains.
Waste output for the common farm animals (cattle, hogs, sheep, horses)
are reasonably well known (Vollenweider, 1968; B. A. Stewart, 1970),
and total animal populations are readily available in areal units at
least as small as counties. However, the amount of animal waste which
enters and is transported by the source waters to a lake depends largely
on local circumstances, and few quantitative figures are available. Ammonia
volatilized from cattle feed lots has been shown to contribute signifi-
cantly to the nitrogen budgets of down wind lakes (Hutchinson and Viets,
1969). For example, this source was estimated to add about 0.6 mg.
NH3~N/l.-yr. to Seely Lake, Colorado, a small lake 2 km. down wind from
a 90,000 unit cattle feed lot. Ground water in areas of cattle feed lots
is frequently contaminated with high nitrate levels, but present data are
insufficient to allow prediction of nitrogen fluxes for individual situa-
tions .
Nitrogen contributions from sewered populations are well known on a per
capita basis, and the total nitrogen concentration in raw and treated
domestic sexvage is probably sufficiently constant to permit evaluation
of this source for nutrient budget purposes from readily available sewage
flow and population records. Non-sewered human waste contributions, as
from septic tank drainage, are far more difficult to evaluate. Septic
tanks are undoubtedly important factors in the cultural eutrophication of
many recreational lakes. However there are almost no data available to
quantify this source (see Patterson et al. 1971 for a detailed discussion).
Urban runoff composition has been studied in detail in a few places
(Weibel et_ al. , 1964, 1966a; Palmer, 1950; Sawyer e_t al. 1945), and storm
drainage flows are more widely known, but the general applicability of
existing information outside the original study areas is unknown.
Rainfall has recently received attention as a source of nutrients, especially
nitrogen. Menzel and Spaeth (1962) correlated the ammonia content of
Sargasso Sea surface water with the rainfall of the previous five days,
and Gambell (1963) found rainfall to be a major source of nitrate and sul-
fate in certain Virginia and North Carolina streams. Chalupa (1960) found
significant rainfall contributions of phosphate to a Czech reservoir.
Parker (1968) has found bloom stimulating concentrations of vitamin Bio
in rainfall of the St. Louis area. Weibel ej^ al. (1964, 1966a,b) reported
extensive studies on rainfall and urban and rural runoff as nutrient sources
in the Cincinnati, Ohio, region. Cincinnati rainfall averaged more than
one mg/1 in total nitrogen and 0.24 mgP/1 in hydrolyzable phosphate.
Reviews on rainfall as a nutrient source have been compiled by Feth (1966) ,
Hutchinson (1957) and Vollenweider (1968), and if previous studies have
confirmed one point, it is that nitrogen concentrations in rainfall are
(unfortunately) highly variable. Both nitrate and ammonia occur in signi-
ficant amounts but the latter is usually higher. In the tropics nitrate
is said to be somewhat more important than in temperate rains but even there
26
-------
ammonia is usually higher (Hutchinson, 1957). Total nitrogen concen-
trations frequently approach 1/mg. l./l., and both natural and cultural
sources are responsible. The correlation of high rainfall ammonia levels
with alkaline soils and of low rainfall ammonia content in regions with
acid soils suggests that sorption of ammonia onto soil clays may be an
important factor in the hydrospheric nitrogen cycle (Feth, 1966). Al-
though a number of cultural sources (ammoniated fertilizers, nitrogen
oxides in auto exhaust) would seem to make important contributions to
atmospheric fixed nitrogen, there is conflicting evidence regarding the
correlation of high rainfall nitrogen levels with areas of industrial
or cultural activity (see Vollenweider, 1968, and Feth, 1966, for details).
Careful analysis of historical records to discern possible long term
trends toward increased rainfall nitrogen resulting from intensified
inorganic fertilizer use, from increases in vehicular traffic and internal
combustion engines (sources of nitrogen oxides), or from other cultural
sources has not been reported.
While relatively large amounts of data have been collected on nitrogen
in rainfall in several areas of the country, little information is avail-
able for the Florida peninsula. Because of the variabilities in concen-
trations and amounts, it is not yet possible to make reliable estimates
of rainfall contributions to nutrient budgets without a thorough field
study. To further knowledge concerning these variabilities several
studies on nutrients in rain were conducted during the project. The
great variations in amount of rainfall within a small geographical area
are indicated in Table 3. r.ight stations scattered 8 miles long were set
up in August, 1953, and 9 stations were similarly established during the
rainy season in August, 1%9. Rainfall was collected for 7 days in 1968
and during 3 days of intermittent showers in 1963, in pyrex bottles
through 4 inch dianeter glass 'funnels (see Appendix A for analytical
details). The results from 1968 show an inverse correlation between rain-
fall amounts and nutrient content, in agreement with the role of rainfall
as a cleansing agent for the atmosphere. The marked variations in rain-
fall amounts even in this small geographic araa were surprising. Whether
the sampling stations would have such large differences over longer periods
is not known, but presumably the differences would become small over the
period of a year or so. Ammonia and nitrate were much higher in the 1963
collections\than in I960, which may be related to differences in amounts
of rainfall during the days preceding sampling in the two years. Since
rainfall seems to cleanse the atmosphere a large amount of rain prior
to a collection period would result in lower concentrations in collected
samples. The ratio of nitrate to annaonia varied markedly among samples in
both years- this would seem to reflect the importance of local terrestrial
sources such as dust and airborne fertilizer since the larger scale atmos-
pheric movements and patterns of circulation would tend to homogenize
rail!fall composition over as small an area as sampled here. It is interest--
ing to note that orthophosphate concentrations in the samples were with
few exceptions higher than the critical concentration said to stimulate
blooms in lakes. Thus, rainfall might stimulate blooms rather than providing
a dilution effect; this seems especially likely during summer when surface
waters are nutrient depleted.
-------
Table 3. Variations in Nutrient Content of
Rainfall in Gainesville Area
Sampling
Station
Volume
Collected5
Ortho PO-P
4
t-P04-P
NH -N
NO -N
1
2
3
4
5
700
370
150
120
115
35
27
August, 1968
0.03
0.23
0.40
0.13
0.07
0.06
0.29
0.08
0.79
0.28
0.81
0.27
0.79
1.26
0.004
0.024
0.006
0.007
0.007
0.018
0.008
August, 1969
0.25
0.41
0.40
0.52
0.52
0.94
1.11
A
B3
C
D
E
F
G
H3
I
250
135
290
20
130
180
130
290
170
0.006
0.043
0.009
0.018
0.011
0.018
0.004
0.021
0.002
.033
0.65
0.125
0.076
0.061
0.045
0.32
0.022
0.35
0.10
0.20
0.05
0.14
0.02
0.05
0.10
0.15
0.02
0.09
0.11
0.07
0.07
0.07
0.05
0.05
0.06
Concentrations in mg/1.
2Volume in ml collected in 1 liter pyrex bottle with 4 inch diameter glass
funnel over 7 day period in 1968 and 3 day period in 1969.
3Sample contained large amounts of particulate matter evidently from
dry fallout
28
-------
While Gainesville is not heavily industrialized, the high nutrient levels
in Table 3 may be partly the result of man's activities. To determine the
nutrient concentrations in rural rainfall, a series of sampling stations
were set up near Cedar Key, Florida, a small town on the Gulf of Mexico
about 65 miles from Gainesville. Eight stations were set up within the
town and on a line from the Gulf up to 20 miles from the coart. Results
for a rainfall in late August, 1968, are shown in Table 4. Ammonia and
nitrate were considerably lower in these rain samples indicating possible
cultural effects in the Gainesville samples. However, the Gainesville
rains were brief intense showers, while the Cedar Kay rain was steady fine
rain lasting for 24 hours. Also the former samples were collected over
a week's time while the latter samples represent 24 hours of rain. The
effect of saline Gulf water on the ionic content (especially Na ) of rain-
fall is clearly seen in the first two stations on Cedar Key. These were
the only stations with detectable ammonia, but nitrate and phosphate show
no trends with distance from the coast.
Previous studies (Putnam and Olson, 1960; Weibel jBt_ _a_l. , 1964) have des-
cribed rainfall as a cleansing agent for the atmosphere. Nutrient and
particulate concentrations in precipitation were generally found to decrease
during the course of an extended rainfall and to increase in rain with the
length of antecedent drought. Several experiments were conducted in this
project to study the washing out of airborne nutrients over short periods
of time during rainfalls. Rainwater was collected on the roof of the
Environmental Engineering Building at various time intervals (five minutes
to one hour) after the beginning of showers on several occasions during
August and September, 1969. Samples were collected in pyrex bottles with
a large funnel and preserved with mercuric chloride for analysis of nitro-
gen and phosphorus forms.
Results from three rain showers (Table 5) indicate a rapid cleansing of
nutrients from the atmosphere. A fourth rainfall (Table 5) did not show
such marked decreases; this was a light steady rain rather than an intense
shower, and it was preceded by a heavy rain on the previous day. Most or
the phosphorus in rain was present as ortho-phosphate. Initial concentra-
tions were considerably higher than the threshold levels for algal bloom
problems, but after ten minutes or so, the concentrations dropped to
0-lOyg P/l. Organic nitrogen and ammonia were the predominant forms of
nitrogen in the rain, and initial levels were comparable to those in eu-
trophic waters. Both forms generally decreased to O.lmg N/l or less
within an hour. Nitrate levels were lower tlo more than 0.08 ing N/l
initially -- and declined to trace levels rapidly. A trace of nitrite
was found on only one occasion. The rapid decline of nutrient forms in
rain showers suggest that the nutrients may originate as low altitude
aerosols. If this is the case, the high initial concentrations probably
reflect local, cultural activity (e.g. auto exhaust, airborne fertilizer
from plowed fields, etc.), whereas the low concentrations afterward may
reflect natural or general levels of nutrients in the atmosphere.
It should be apparent from the above results that estimation of total
nutrient contributions to a lake via rainfall is not so simple a matter
as one might have first thought. In particular, the use of literature
values for nitrogen concentrations is likely to produce inaccuracies
29
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Table 4. Nutrient Content of Coastal and Rural Rainfall1
OJ
o
Location2
Cedar Key a
Cedar Key b
1
3
5
10
15
20
Amount 3
580
530
490
490
640
770
1070
710
KH3-N
0.08
0.03
0.00
0.00
0.00
0.00
0.00
0.00
NO-3-N
0.05
0.05
0.04
0.06
0.03
0.05
0.05
0.06
0-PO -P
4
0.012
0.036
0.010
0.007
0.027
0.028
0.024
0.010
Ca+2
1.34
0.61
0.30
0.31
0.22
0.31
W +2 »T +
Mg Na
3.05 11.0
0.24 2.0
0.05 0.26
0.04 0.38
0.05 0.45
0.03 0.31
K+
1.7
0.3
0.08
0.15
0.18
0.12
Concentrations in mg/1.
2Stations were located at the waters' edge on Cedar Key (a), on the island interior (b),
and on a line approximately due east from Cedar Key with the distances (in miles) from
the coast as noted.
3Amount in ml collected in 1 liter pyrex bottles with 4" diameter glass funnels during
24 hour period.
-------
Table 5. Temporal Variations of Nutrients in Rain Showers
in Gainesville, August and September, 1969:
Date
Time2
o-PO ,-P
4
t-PO,-P
4
TON
NH -N
NO^-N
4 Aug. Type of rain: thunder shower following weekend of dry weather
0-5
5-10
10-20
20-30
30-60
0.056
0.032
0.010
0.005
0.005
0.058
0.032
0.015
0.005
0.005
1.85
1.00
0.83
0.10
0.92
0.84
0.09
0.09
0.08
0.08
0.00
0.00
0.15
0.10
0.00
10 Sept. Type of rain: shower following day with trace of rain
0-5
5-10
10-20
20-30
30-60
0.080
0.031
0.010
0.00
0.00
0.150
0.031
0.015
0.00
0.00
0.85
0.75
0.10
0.15
0.09
0.85
0.75
0.10
0.10
0.10
0.08
0.05
0.01
0.01
0.01
16 Sept. Type of rain: thunder shower following day with light shower
0-5
5-10
10-20
20-30
30-60
0.038
0.010
0.010
0.00
0.00
0.045
0.010
0.010
0.00
0.00
1.60
1.30
0.0
0.0
0.0
0.50
0.40
0.18
0.0
0.13
0.05
0.01
0.00
0.00
0.00
3 Sept. Type of rain: light and steady following day with 2 inches of rain
> 0-5
5-10
10-20
20-30
30-60
0.008
0.005
0.00
0.00
0.00
0.008
0.005
0.00
0.00
0.00
0.15
0.10
0.00
0.00
0.10
0.10
0.10
0.08
0.00
0.00
0.08
0.005
0.00
0.00
0.00
concentrations in mg/1
JTime in minutes after start of rainfall
31
-------
because of the large local and temporal variations which characterize
the nitrogen content of rain. For phosphorus in rainfall there are almost
no data available in the literature, so this flux must be evaluated
experimentally. In conjunction with a concurrent project on eutrophica-
tion factors in Florida lakes, the total nitrogen and phosphorus contri-
butions of rainfall to the nutrient budget of a small lake were determined
in 1968. A recording rain gauge was established at the lake site 30 miles
east of Gainesville, and samples were collected at weekly intervals for
ammonia, nitrite, nitrate and phosphate analyses. Rain was collected in
a pyrex bottle with a 4-inch diameter glass funnel. In order to prevent
contamination of collected rainfall by dust, leaves, dead insects and
other particulate matter a wad of glass wool was placed over the funnel
drain. Results of this study are summarized in Table 6. Because of the
inverse correlation between nitrogen content and amount of rainfall,
use of a simple mean value of total N for all measurements times the volume
of rainfall on the lake surface would not be very accurate. Consequently
nitrogen contributions were calculated for each sampling increment and
these results then summed. Rainfall is a significant although not
dominant nutrient supplier to Anderson-Cue Lake. If the artificial nutrient
input to this lake is not considered, rainfall then is seen as an even more
important source, especially for oligotrophic lakes in nutrient impoverished
watersheds (as is Anderson-Cue Lake).
In summary, a large volume of raw data on nutrient sources is available,
but generalizations on the importance of various sources can in only few
cases be estimated. A few point sources (e.g. domestic sewage) are
reasonably well characterized, and some diffuse sources (e.g. urban runoff)
can probably be estimated with fair accuracy using literature values.
However the quantitative aspects of nutrient budgeting are still relatively
undeveloped.
From a management point of view it is more important to determine the contri-
butions from man-made sources since they are more readily controlled than
natural sources. However the importance of natural sources must be known
in order to determine whether control of man-made sources will result in
significant improvements in water quality. The most significant cultural
sources of nitrogen are domestic sewage, agricultural (cropland) runoff,
animal wastes from farming operations, and urban runoff. Nitrogen fixation,
rainfall, ground water, and natural stream flow are probsbly the most sig-
nificant natural sources. Undoubtedly sediment deposition, flow through
the outlet (in lakes) and denitrification are the largest sinks for nitro-
gen in natural waters. Outlet losses would seem to be directly propor-
tional to the nitrogen concentration within the body of water. Denitri-
fication is limited in lakes to those that stratify thermally and lose their
oxygen in the bottom water.
The collected information on nutrient export from various sources has been
used in conjunction with land use and population data to compute nitrogen
and phosphorus budgets for 55 lakes in north and central Florida (Shannon,
1970; Shannon and Brezonik, 1971a). Because this work was performed pri-
marily in relation to a concurrent project (Brezonik, 1971c), detailed
results are not presented here. However, the findings are of interest
in relation to the preceding discussion of nitrogen sources, and a summary
32
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Table 6. Contribution of Rainfall to Nutrient
Budget of Anderson-Cue Lake
Total rainfall in 1968: 136.7 cm (53.8 in)
Lake area:
Lake volume:
7.7 x 10* m2
1.85 x 105 m3
Nutrient
Rainfall nutrients in g/ra:
Mean
Range
TON
NH3-N
NOo~'N
Ortho PO^-P
Total PO^-P
0.31
0.21
0.21
0.009
0.033
0.07-0.67
0.01-0.86
0.04-0.94
0.002-0.033
0.020-0.070
Budget
N
Source kg g/m3
Rainfall1 49.7 0.27
Artificial nutrient
mixture2 124 0.67
Other sources
(surface runoff)3 134 0.72
Total 308 1.66
Percent of total
contributed by rain 16
P
kg g/m3
3.4 0.019
10.6 0.057
4.4 0.024
18.4 0.100
18
1Rainfall nitrogen levels were inversely proportional to amount
of rain collected; budget value was calculated from total N
measured at each collection times the amount of rain collected
since last analysis and summing these amounts over the entire
lakV surface. Since relatively few total phosphate analyses
were run and no inverse correlation with rainfall amount could
be discerned, rainfall phosphorus in the budget was calculated
from the mean for all samples (0.033 g/m3) times the total 1968
rainfall volume over the lake surface.
2Ammonium chloride and sodium phosphate dissolved in sewage as
a controlled enrichment experiment (Brezonik et al., 1969).
Calculated from watershed (land use) patterns from Shannon (1970)
and Shannon and Brezonik (1971a).
33
-------
is presented below. Watershed land use patterns were obtained from recent
aerial photographs, lake and watershed areas were measured from U.S.G.S.
topographic maps, and population estimates for each watershed were obtained
by counting the dwellings visible on aerial photographs. Nitrogen budgets
were then calculated from appropriate export rates (i.e. kg N exported
per hectare of a particular land use per year, kg N per capita per year etc.)
and the measured watershed parameters. Budgets were calculated per unit
area of lake surface and also per volume of lake water. On an areal basis
nitrogen loading rates ranged from 1.2 g./m. -yr., for a small oligotrophic
lake (Swan Lake, Putnam County) to over 90 g./m.2-yr., for Lake Alice, a
shallow, hypereutrophic lake which receives 1.5-2.0 mgd of treated sewage
from the University of Florida waste treatment plant. Expressed on a
volumetric loading basis, nitrogen extremes were 0.18 to 106 g./m.J-yr.
Loading rates generally correlated with lake trophic conditions. In order
to study relationships between lake conditions and watershed characteris-
tics (e.g. nutrient loading rates), a numerical index (TSI) was developed
from values for 7 quantitative trophic indicators (e.g. primary pro-
duction, total N and N levels, Secchi disc transparency). Annual mean
values for the parameters from the 55 Florida lakes and the multi-variate
statistical Technique of principal component analysis were used in this
derivation (Shannon and Brezonik, 1971b). Increasing values of the index
corresponded to increasingly eutrophic conditions, and various analyses
of the results suggest the index does in fact quantify the concept of
trophic state.
Regression analyses of the trophic state index (TSI) vs. N and P
loading rates (expressed on both areal and volumetric bases) were per-
formed using an additive model (TSI = f(N + P) and a multiplicative model
(TSI = f(N + P) . Some representative results are given in Table 7.
In that phosphorus was incorporated into most of the stepwlse regression
equations first and was hence the most important variable in a statisti-
cal sense, it might be inferred that phosphorus rather than nitrogen is
the controlling input for eutrophication in Florida lakes. However,
regression analyses are inherently eianirieal, and such inferences must
be approached with ~" ''.-.:..
Of great interest in control of cultural eutrophication is the develop-
ment of critical loading rates for nitrogen and phosphorus, above which
one can expect eutrophic conditions to ensue. Sawyer (1947) was the
first to propose quantitative guidelines of this sort. Based on data
from Wisconsin lakes he suggested that 0.015 mg/1 of inorganic phosphorus
and 0.3 mg/1 of inorganic nitrogen at the spring maximum are critical
levels, above which algal blooms can normally be expected. In the ab-
sence of any other studies, these values have been widely quoted and
applied to many tvpes of lakes in diverse geologic and climatic situa-
tions. Recently Vollenweider (1968) analyzed the available data on
nutrient loading rates and corresponding trophic conditions and proposed
permissible and critical loading rates for nitrogen and phosphorus as
a function lake mean depth. Using the regression equations shown in
Table 7, similar critical loading rates were estimated for Florida
lakes (see Shannon and Brezonik, 1971a, or Brezonik, 1971c for details
of this derivation). Results are shown in Table 8, and VollenweiderTs
34
-------
Table 7. Regression Analyses of Trophic State Index (TSI)
vs Nitrogen and Phosphorus Loading Rates for 55 Florida Lakes (from Brezonik, 1971a)
Multiple Percent Variance
Loading Rate , Correlation Explained by
Model Units Equation F Ratio Coefficient Equation
Additive
(1) per unit lake TSI = 0.62(N ) + 10.06(P ) 46.44 0.804 64.5
,- o L» o L»
surface area
(2) per unit lake TSI = 26.1(P ) + 0.90(N ) 43.20 0.793 62.9
1 VL V LJ
volume
Ln
Multiplicative
/ o o r\
(3) Per unit lake TSI = 0.84(P_T ) ' ' (NST ) * 14.08 0.600 36.0
SL 3L,
surface area
(4) per unit lake TSI = 1.08(Ptrr ) '42(N ) ^ 15.64 0.620 38.5
, VL V L
vo lume
'Abbreviations: TSI = trophic state index (ditnensionless) ; Ng-^ and Pg-^ = nitrogen and phosphorus
surface loading rates in g/m2-yr; Ny^ and Py^ = nitrogen and phosphorus volumetric
loading rates in g/m3-yr.
All significant at the 99% confidence level.
-------
Table 8. Critical Loading Rates for Nitrogen and Phosphorus
Loading Permissible Loading Dangerous Loading
Reference Rate Units (up to) (in excess of)
N P N - P
Shannon and
Brezonik 1971a Volumetric (g/m3-yr) .86 .12 1.51 .22
Ibid. Areal (g/m2-yr) 2.0 .28 3.4 .49
Vollenweider
(1968)a Areal (g/m2-yr) 1.0 .07 2.0 .13
aFor lakes with mean depths of 5 m or less.
36
-------
(1968) values for shallow lakes (z < 5m.) are given for comparison.
While differences are evident between the two analyses, the estimates
are of the same magnitude. Florida lakes appear capable of assimilat-
ing somewhat greater quantities of nutrients before becoming mesotro-
phic or eutrophic than is indicated by Vollenweider's values.
The above values should not be regarded as final. In a sense they are
quite unrefined because of uncertainties and even complete ignorance
regarding nutrient export rates from various types of terrain and land
uses. However, as these values are experimentally determined the em-
pirical analysis described here should become more precise, and it
offers the potential for quickly and economically predicting water
quality conditions from watershed characteristics.
37
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SECTION VI
NITROGEN FIXATION AS AN _IN SITU NITROGEN SOURCE FOR NATURAL WATERS
I. ALGAL FIXATION IN LAKES
Aside from the external nitrogen sources discussed previously, several
mechanisms within a lake can effect an increase or decrease in the total
N content of the lake. Nitrogen fixation is an in situ source, while
denitrification is a sink. Sediments must act as nitrogen sinks over
the geological life span of a lake, but over shorter periods of time they
may act as sources to the overlying water. The role of sediments as
a source or sink and of denitrification is discussed in Section VIII.
A wide variety of studies were undertaken to assess the significance
of nitrogen fixation as a nitrogen source in lacustrine systems. For
the purpose of this report the studies can be grouped into four phases:
1.) an extensive survey of nitrogen fixation in the 55 Florida lakes
described in the previous section, 2.) an in depth study of fixation
in two eutrophic lakes, Bivin's Arm and Newnan's Lake, 3) investiga-
tions on bacterial fixation in lakes, and 4.) investigation of the nitro-
gen fixing properties of sediments. The first two phases are described
in this section, the latter two in Section VII. Analytical procedures
used throughout these studies are described in Appendix A.
In order to estimate the overall significance of nitrogen fixation as
a nutrient source, fixation measurements were included as part of a broad
ranging study of the limnology and trophic conditions in Florida lakes
(Brezonik, 1971c). The 55 lakes included in the study were of widely
varying trophic and chemical conditions (Shannon and Brezonik, 1971c) ,
and most are located within a 40 mile radius of Gainesville. All 55
lakes were sampled 4 times, and 19 of them were sampled 7 times over
one year. For ease in sample handling, all incubations were carried
out under ^Laboratory conditions (22°C in the light) in a shaker-bath;
samples were assayed on the same day as they were collected. This pro-
cedure undoubtedly yielded results somewhat different from in situ
data, but laboratory incubation permitted many more samples to be pro-
cessed. For assay and comparative purposes, the method is well suited.
Assuming the lakes represent a cross-section of Florida lakes as a whole,
it is pertinent to consider the frequency of nitrogen fixation in them.
Out of a total of 272 lake samplings (not including separate samples
taken in depth profiles on some of the lakes), 42 samples from 15
different lakes gave positive fixation rates using the acetylene reduc-
tion method (Table 9). L?ke Mize, in x^hich fixation was found only in
the anoxic hypolimnetic waters is not included here, but the 7 samplings
39
-------
Table 9. Summary of the Occurrence of
Nitrogen Fixation in Florida Lakes
Number of lakes sampled: 55
Number of lakes in wbich fixation was detected: ^5
Total lake samplings (not including separate
depth profiles on some lakes) : 272
Number of positive rates detected: 42
Range of measured rates (nM C2H^/l-hr) : 1-262
(ng N/l-hr): 93-2450
Frequency distribution of fixation: //positive samples #lakes
1 5
2 5
3 0
4 2
5 0
6 2
7 1
Nitrogen fixing algae detected: Anabaena sp. , Aphanizomenon
40
-------
of Newnan's Lake and Bivin's Arm conducted as part of the 55 lake study
are included. The infrequent sampling routine probably missed fixation
in 4 or 5 more lakes which seem likely environments for sporadic
nitrogen fixation (based on their trophic conditions and the nature of
their plankton). Low (undetectable) fixation may be more widespread or
more frequent in lakes giving occasional high rates, but no attempt was
made to concentrate the algae by filtration or centrifugation before
incubation. It is doubtful whether such low rates (as requiring organism
concentration for detection) would be ecologically significant. Fixa-
tion was detected on only one occasion in 5 of the 15 lakes and in 5
other lakes it was found twice. Overall fixation is thus not a typical
feature of the lakes. However, in five lakes it apparently occurs
commonly; rates in these lakes are summarized in Table 10. Rates ex-
pressed as equivalent N fixed in ng. NH-j-N/l.-hr. ranged from just
detectable (about 0.5) to a high of 175, but most rates were low (less
than 10). However even rather low rates can contribute to a lake's
nitrogen budget if the rates continue for a long period. For example,
fixation was found in Lake Dora on all 7 sampling dates ar.d in Lake
Hawthorne on 6 out of 7 sampling dates. Mean fixation rates in the two
lakes are 116 and 127 np,. N/l.-hr. respectively. Assuming this mean
rate occurs over a year and that fixation occurs on the average for 8
hours a day, fixation would then contribute about 0.34 and 0.37 mg. N/l.
to Lakes Dora and Hawthorne, respectively, on an annual basis.
Detectable fixation was found in the lake surface waters only in the
presence of blue-green algae, and the phenomenon seems limited to eutro-
phic or mesotrophic lakes. None of the oligotrophic Trail Ridge lakes
gave detectable fixation, and species of algae capable of fixation were
not found in their plankton. Anabaena and Aphanizomenon were the only
two nitrogen fixing algae aetected in the lakes, and the former was by
far the more common.
The seasonal pattern of nitrogen fixation in temperate lakes has been
well established (Dugdale and Dugdale, 1962; Goering and Neess, 1964),
and fixation is primarily a late summer phenomenon. In subarctic lakes
significant fixation is apparently limited to parts of the short ice-
free season (Billaud, 1968). In tropical and subtropical lakes seasonal
cycles are less pronounced and fixation is possible year-round, but
no data are available to substantiate this point. Consequently, a
detailed ye^r study of nitrogen fixation was conducted on two highly
eutrophic lakes near Gainesville in order to (1) determine seasonal
variations in nitrogen fixation, (2) estimate the total contribution
of fixation to the nitrogen budgets of these lakes, (3) determine the
environmental factors controlling fixation, and (4) relate fixation
activities to other biological cycles and phenomena in the lakes, viz.
nutrient cycles, primary production, algal blooms.
The two lakes chosen for the detailed study are Newnan's Lake and Bivin's
Arm, both of which are located near Gainesville, Florida, and are within
10 miles of each other. Both lakes are highly eutrophic, but the chemical
characteristics and causes of these conditions in each lake are quite
different. Newnan's Lake is the catchment for a large pine forest and
41
-------
Table 10. Nitrogen Fixation Rates in Selected Florida Lakes1
Date
1969
June
August
October
December
Bivin'
Arm
67.2
1020
0
0
s Lake
Dora
19.6
280
26.6
133
Lake Newnan '
Hawthorne Lake
134
392
9.8
49
0
0
19.6
384
' s Unnamed
#20
0
0
95
47
.2
.6
1970
February
April
June
165
16.8
440
57.4
88.2
210
151
506
0
133
30.8
0
0
39.2
26.6
ZA11 rates in ng N/l-hr, derived from nmoles ethylene produced/1-hr,
using theoretical factor of 1.5 moles ethylene produced per mole
ammonia fixed.
42
-------
and partially swampy area. The drainage basin north of the lake is
rich in phosphatic minerals (Clark et al., 1964), and while some
cultural enrichment may enter the lake as runoff from fertilized pas-
ture and leachate from City of Gainesville sanitary landfill operations
northeast of the lake, Newnan's Lake appears to be largely naturally
eutrophic. The bathymetry of Newnan's Lake is shown in Figure 3; its
large surface area (2433 ha.) and shallow depth (z = 2m) coupled with
its loose, unconsolidated organic sediments insure frequent wind
generated resuspension of the surficial sediments and their recircula-
tion to the surface waters. On the other hand, Bivin's Arm receives
much of its nutrient budget from cultural sources, including a stream
which drains a large urbanized area and into which poorly treated
sewage has in the past been dumped, septic tank drainage, and cattle
wastes from University of Florida experimental farms on its western edge.
A large rookery on the northern shore of the lake may supply substantial
nutrients and effect a more rapid cycling of nutrients in the lake.
This lake is also shallow (z = 1.5m; see Figure 4 for a bathymetric map),
and even in the absence of cultural inputs the lake would probably be
highly productive. In their presence the lake can only be described
as grossly enriched. Algal blooms are present continuously through
the year. Secchi disc transparencies greater than 0.6m (2 ft.) are
uncommon, and the water frequently has a rather sickly green color.
Table 11 summarizes the chemical characteristics of these two lakes.
Newnan's Lake is highly colored from the swamp and pine forest drainage,
and its water is both low in conductance and in hardness. The pH of the
lake is neutral to slightly alkaline, but the low alkalinity implies
a poor buffer capacity. A rather high iron content (average .23 mg/1)
is associated with the high color, but manganese is very low as are other
heavy metals. Total phosphate in Newnan's Lake, while high and re-
flecting eutrophy, is less than one-fourth the level in Bivin's Arm.
Bivin's Arm has considerably more dissolved solids and hardness which
probably arise from cultural sources. Its pH is usually greater than
that of Newnan's Lake and its alkalinity is much greater. Organic color
is comparatively low and that which is present is at least partially
autochthonous.
The biota of these two lakes is somewhat different although blue-green
algae are dominant in both. Aphanizomenon is the predominant bloom former
during winter in Newnan's Lake whereas Anabaena nay bloom in summer;
Microcystis is commonly present in abundance in both lakes. Anabaena
is the common nitrogen fixing bloom former in Bivin's Arm, but the flora
of this lake are much more diverse than in Newnan's Lake, and several pulses
of diatom blooms also occur during the year (Harper, 1971). Water hya-
cinths (Eichornia crassipes) have been a serious problem in both lakes,
and their surfaces have been nearly covered by this species within the
recent past. Chemical spraying now keeps the growth of hyacinths in
check, but the effect of the herbicides employed on other parts of the
ecosystems is a matter of question and concern.
Figure 5 shows the concentrations of major nitrogen forms, rates of
nitrogen fixation and primary production in Bivin's Arm from May, 1969,
-------
1
N
FIGURE 3, SATiYfTETRIC MA" OF :Tr'Av/c: LAKE,
ALACHUA COUNTY, FLORIDA, DE^TH Ca^TOURS IK FEET,
44
-------
1
"
FIGURE L\, BATi-iYMETRIC MAP OF 3WIN'S ARM/
ALACHUA COUNTY, FLORIDA, DEPTH CONTOURS IN FEET,
-------
Table 11. Chemical Characteristics of Newnan's Lake and Bivin's Arm1
Parameter2 Newnan's Lake Bivin's Arm
Dissolved oxygen
PH
Alkalinity (as CaCO )
Acidity (as CaC03)
Conductivity (y mho cm"1)
Turbidity (JTU)
Color (as Pt)
Total organic N
Total P
Cl~
S04=
Na +
K+
Mg+2
Ca+2
Fe
Mn
COD
8.6
7.8
7.3
1.0
65.7
4.8
235
1.55
0.10
11
3
8.4
0.6
1.5
5.3
0.23
0.002
68
10.0
9.0
105
0
267
11
48
1.94
0.45
16
7
11.5
1.7
4.7
30.3
0.04
0.002
60
values for six samplings (every 2 months) over the period
June, 1969 to April, 1970.
Concentrations in mg/1 except as noted.
46
-------
1.0 _
M
FIGURE 5. INORGANIC NITROGEN LEVELS, NITROGEN FIXATION AND
PRIWRY PRODUCTION IN BIVIN's ARM/ FLORIDA, FROM MAY, 1969 TO MAY, 1970,
-------
tc May, 1970. Routine data for each sampling data are listed in
Appendix B. Fixation occurred at moderate to high rates throughout
the summer of 1969; a maximum of 330 nmoles ethylene/l.-hr. (equivalent
to about 3.1]jg. N/l.-hr.) was found in late August. Lower rates were
noted during winter, and fixation was detected on about 50 percent of
the sampling dates. Fixation rates in this lake do not seem to be signi-
ficantly correlated with either primary production or nitrogen concen-
trations. A rather definite seasonal trend in primary production is
apparent, with peak rates in summer (June to October) and lower rates
from November to May, but the pattern of algal activity is really much
more complex, being characterized by a pronounced short-term variation
in both production and nutrient levels.
Seasonal patterns of nitrogen forms, fixation rates and primary pro-
duction in Newnan's Lake are displayed in Figure 6, and routine data
collected on each sampling date are listed in Appendix B. In spite of
the geographical proximity and similar enriched conditions in the two
lakes, their patterns of fixation are quite dissimilar. Except for
one high rate of nitrogen fixation in July, 1969, during a bloom of
Anabaena and Microcystis and for very low rates on several occasions
in spring and fall, fixation in Newnan's Lake is associated with a
dense winter and early spring bloom of Aphanizomenon. In winter of
1969-1970 the bloom occurred in two pulses. The first pulse began in
mid-December and died out in early January, apparently because of ad-
verse weather (an abrupt cold wave and an extended period of heavy rain)
The population stayed low in January and gradually increased during
February to high levels through most of March. Maximum fixation rates
during the Aphanizomenon bloom were about 40 nmoles ethylene/l.-hr,
roughly equivalent to 0.37yg. N/l.-hr. Fixation was highly correlated
with primary production during the winter bloom period (Figure 7) in
Newnan's Lake, but no obvious correlation exists between fixation and
concentrations of nitrogen forms.
It should be noted that algal fixation occurred in both lakes in the
presence of moderate to high (i.e. for lake waters) ammonia levels. In
fact the maximum rate measured in Bivin's Arm occurred when the ammonia
concentration was 0.5 mgN/1. High rates in Newnan's Lake occurred at
ammonia levels as high as 0.6 mgN/1. Thus while fixation occurs fre-
quently on nutrient depleted waters, it is not limited to them. Pre-
vious studies have demonstrated simultaneous assimilation of ammonia,
nitrate and molecular nitrogen (Dugdale and Dugdale, 1965; Billaud,
1968); no doubt this also occurs in the lakes studied here.
Several measurements of areal variability in nitrogen fixation and
related parameters were made on Newnan's Lake during the project in
order to evaluate the homogeneity (or lack thereof) of biogenic para-
meters in the lake and hence determine the number of sampling stations
required for adequate representation of the lake. Table 12 presents
the results of an areal study conducted on April 19, 1969. Top, mid
and bottom samples were composited at 10 stations scattered around the
entire lake. All samples were collected within one hour and brought to
shore for processing. Primary production and nitrogen fixation samples
48
-------
MJJASONDJF
MONTH
FIGURE 6. INORGANIC NITROGEN CONCENTRATIONS, NITROGEN FIXATION, AND
PRiriARY PRODUCTION IN NEWMAN'S LAKE/ FLORIDA FROM MAY, 1369 TO fW, 1970
49
M
-------
Ul
o
f
c
o
>,
i
280
240
200
160
120
80
40
0
I
I
I
28
24
20
16
12
11 21
DEC,
31 10
20
JAN,
9 19
FE3,
DATE
1 11
MAR,
21 31
APR,
FIGURE 7, TEMPERATURE, PRIMARY PRODUCTION AND NITROGEN FIXATION
IN NEMAN'S LAKE DURING '-/INTER (1953-1970) APHANIZQMENON BLOOM,
c
o
PH
C
-------
Table 12. Areal Variations in Physical and Biogenic Parameters in Newnan's Lake, 19 April, 1969
Station
Mean and 95%
Parameter1
Sfc. Temp.
Secchi
Disc
Tot. depth
D. 0.
pH
Alk.
TON
NH3-N
NO^-N
o-PO^-P
t-PO^-P
Prim. Prod.
1
22.0
0.6
1.35
6.40
7.05
10.4
1.51
0.61
0.07
0.023
0.167
20.8
2
22.4
0.6
1.5
6.00
7.05
10.7
1.64
0.71
0.03
0.010
0.144
32.4
3
22.4
0.6
2.0
5.80
6.90
9.95
1.55
0.78
0.03
0.010
0.156
43.5
4
22.5
0.6
2.0
5.80
7.05
10.0
1.68
0.82
0.02
0.010
0.184
71.0
5
22.3
0.6
2.6
5.90
7.00
10.0
1.40
0.84
0.02
0.012
0.152
41.7
6
22.3
0.6
1.8
5.70
7.00
10.1
1.77
0.76
0.02
0.010
0.108
92.0
7
22.5
0.6
1.8
4.50
6.95
9.71
1.88
1.06
0.02
0.012
0.188
51.1
8
22.0
0.6
1.8
5.90
7.00
10.1
1.48
0.77
0.03
0.019
0.160
38.8
9
22.1
0.6
1.8
5.50
7.00
10.3
1.46
0.94
0.04
0.025
0.140
16.5
10
22.4
0.6
1.8
6.50
7.05
10.4
1.44
0.92
0.00
0.020
0.160
51.1
Confidence Coefficient o
Interval Variation (7<,
22.3
0.6
5.8 ± 1.0
7.00 (median)
10.2 ± 0.5
1.58 ± 0.29
0.82 ± 0.24
0.028 ± 0.003
0.015 ± 0.011
0.162 ± 0.029
46.0
28.3
8.3
30.4
47.6
17.9
118
28.7
Temp, in °C; Secchi disc visibility and total depth in meters; pH = -log (H ); alkalinity in mg/1 as CaCO ;
primary production in mg C/m3-hr; all other values in mg/1.
-------
shore for processing. Primary production and nitrogen fixation samples
were then returned to the lake for incubation at a near shore station.
Although nitrogen fixation was not occurring in the lake on this day,
the data are nevertheless useful in describing the variability in bio-
logical conditions. The variations in biogenic parameters was much
larger than in the physical parameters and in pH and alkalinity. Pri-
mary production had by far the greatest variability - from 21 to 92 mg
C/m-hr. Newnan's Lake obviously cannot be considered homogeneous, and
any one sampling station may not be representative of the entire lake
at a given time. In order to obtain representative data and still keep
the sampling and analyses within reasonable limits, it was decided to
composite 6-8 stations scattered at random around the lake into one
sample for the routine bi-weekly analyses. Thus the data in Figure 6
represent determination on a composite lake water sample and are felt
to reflect closely the average conditions in the lake at a given time.
Because Bivin's Arm is a much smaller and presumably better mixed lake,
it was not felt necessary to composite so many stations. Consequently
the data in Figure 5 represent composite samples fros three stations
and 3 depths.
A second areal variation study was conducted on Newnan's Lake December 8,
1969, at the onset of the Aphanizomenon bloom. Again 10 stations were
sampled and nitrogen fixation was found at each station. Figure 8 shows
the distribution of primary production and nitrogen fixation rates at
the 10 stations; the former varied in a much smaller range on this date
than in the April study. Nitrogen fixation was much more variable than
primary production and the two rates do not seem correlated. Table 13
presents depth variations found at two stations and summarizes the
variations found in nitrogen fixation, primary production and chlorophyll.
It is interesting to note that areal variations with the latter
biomass measure were somewhat smaller than those of primary produc-
tion. The large vertical differences in these parameters in so shallow
a water column are probably the result of self-shading. Note that
chlorophyll decreased less than did the activity parameters - primary
production and nitrogen fixation which are light dependent phenomena.
Diel variations in Newnan's Lake were measured on thr?e occasions.
\pril 21, 196?, December 16, 1969 and April 20, 1971. Nitrogen fixa-
tion was found in the lake only in the December study. The April, 1971,
study apparently just missed the annual Apnanizomenon bloom, which was
quite evident in the lake a few days earlier. Figure 9 shows the time
course of primary production, nitrogen fixation and light intensity
during the December 16, 1969, study. Sampling began at 7:00 A. M. and
continued at about one and a half hour intervals until dark. Primary
production reached a maximum value at mid-morning and decreased markedly
during the afternoon, at least partly because an increased cloud cover
reduced light intensity. Peak nitrogen fixation lagged behind photo-
synthesis and occurred in early afternoon. Although sampling was not
continued after dark the data suggest that nitrogen fixation is limited
to daylight hours or at least occurs at much lower rates during the night,
Figure 10 presents some results of a more detailed diel study conducted
in April, 1971. Unfortunately the lake was not fixing nitrogen at this
52
-------
FIGURE 8, AREAL VARIATIONS IN NB^IAN'S LAKE/ DECEfBER 8, 1969,
STATION NUMBERS IN PARENTHESES; UPPER VALUE: PRIORY
PRODUCTION IN m§ C/m3-hr; LOWER VALUE1. NITROGEN
FIXATION IN W N/m3-hr.
53
-------
Table 13. Vertical and Areal Variations in
Nitrogen Fixation and Related Parameters in Newnan's Lake, 8 December 1969
Depth Primary Nitrogen
m. Production Fixation Chlor. a_. Temp,
mg C/m3-hr nM N/l-hr mg/m3 °C
Station 4
Sfc 38 20 70 13.8
1 19 14 69 13.3
2 20 10 64 13.3
Station 8
Sfc 38 19 70 14.8
2 19 18 71 13.6
4 17 6 64 13.3
Summary of Areal Variations1
Mean 36.3 18.1 67.4
Range 31.0-44.1 9.8-33.6 60.7-75.1
1Means of ten stations, eight of which were samples
composited with depth and two of which (stations 4 and
8 above) were samples taken at discrete intervals.
Mean values over depth for these two stations were used
for calculation of the areal means.
54
-------
0.7
PRIMARY PRODUCTION
0700
1730
FIGURE 9, DIEL VARIATIONS IN NEWNAN'S LAKE/
DECE^ER 16, 1969,
55
-------
26
~ 24
G
3
« 22
-------
time but the data are interesting in illustrating the large changes
in biogenic parameters that can occur on a daily basis in eutrophic
lakes. The lake was very calm during this study, and a slight, tem-
porary temperature stratification occurred during the day. Dissolved
oxygen values rose during daylight hours but fell much below saturation
after nightfall. The small decline in oxygen after several hours of
sunlight seems to be a case of photorespiration, as recently described
by Odum (1971). The total change in oxygen content during the day was
dramatic; considering the calm conditions most of the increase would
appear to arise from photosynthesis rather than gas transfer from the
atmosphere. Nutrients declined somewhat irregularly during the day-
light hours, but the ratios of carbon, nitrogen and phosphorus net assi-
milation during the period of peak photosynthesis can be approximated
from the data in Figure 10. During the period 9:30 A. M. to 4:15 P. M.
photosynthesis resulted in a net increase of 2.1 mgO~/l in the surface
waters. Before and after this period photosynthesis was less than or
about equal to respiration as the dissolved oxygen values indicate.
During the period of maximum photo synthesis a net decrease of 0.16 mg
NHy-N/l and 0.022 mg ortho PO^-P/1 occurred in the surface water. Con-
verting the oxygen increase to equivalent carbon fixed indicates a net
of about 0.75 mgC/1 assimilated. On a molecular basis then these
values indicate net C:N:P uptake ratios of 100:18:1*1 on a molar basis,
xtfhich ratios are quite similar to the average C:N:P composition ratios
reported for algal cells (i.e. 106:16:1).
The rather large diel changes in biogenic parameters reflect the dynamic
nature of nutrient cycling especially in eutrophic lakes. These changes
also imply that routine sampling for seasonal or other long term studies
should be conducted at approximately the same time of day. Otherwise
changes resulting from diel cycles may erroneously be thought of as
reflecting longer term variations.
The severity and duration of the annual Aphanizomenon bloom in Newnan's
Lake is apparently quite variable. In winter 1968-1969 the bloom occurred
from late December to mid February, and at its height was more intense
than during the following year. Two samples taken near shore on January 20,
1969, were thick with clumps of Aphanizomenon, and yielded fixation rates
of 70 and 163 nM C2H,/l.-hr., which values are considerably in excess
of the maximum values for the winter 1969-1970 bloom. On the other hand
massive concentrations of the algae near shore frequently result from
sustained winds, so the above values are not necessarily representative
of fixation rates in the entire lake during the January, 1969, bloom.
The bloom of Aphanizomenon in 1971 appeared much later (late March) and
was of much shorter duration than in the previous year. These differences
are no doubt associated with differences in weather conditions (rainfall,
temperature) from year to year. However this qualitatively simple
explanation would be very difficult to verify in a quantitative manner,
and attempts to correlate the bloom occurrence and duration with climate
data collected during this study were not fruitful. Perhaps a detailed
analysis of past weather records and bloom conditions (if they were
available) could yield an empirical explanation, but that is beyond the
scope of this project. While the details and absolute amounts of fixa-
tion in the lakes may vary yearly, the pattern seems qualitatively similar
57
-------
from year to year. Thus the data presented here are regarded as typical,
and in this sense can be utilized for more general conclusions on the
importance of fixation in Florida lakes.
An obvious but unanswered question about the patterns of fixation in these
two lakes concerns its occurrence primarily as a winter phenomenon
in one lake but as a summer phenomenon in the other lake in spite their
proximity and essentially identical climate. The large volume of data
(Appendix B) collected during the Aphanizomenon bloom in Newnan's Lake
during winter, 1969-1970 has been scrutinized and subjected to statis-
tical analysis in order to elicit trends and potential causal factors
for the appearance and eventual demise of the bloom. However there does
not appear to be any clear answer, and bloom formation and disappearance
(hence also the occurrence of nitrogen fixation) may be controlled by
a subtle interaction of physical and chemical factors, or by biological
causes that were not measured. Several possibilities are suggested:
initiation of the bloom by temperature changes (e.g. an increase in
temperature following a cold period during which Microcystis and the
other algae present in abundance during the bulk of the year are elimi-
nated) ; changes in light intensity or in photoperiod, or perhaps a com-
bination of temperature and light conditions are necessary. There do
not appear to be any substantial chemical changes in the lake prior to
bloom onset, but the possibility of a biologically induced change in an
organic growth factor or in a required trace metal such as iron or moly-
bdenum should not be dismissed. Possible reasons for bloom disappearance
are even more numerous. This strain of algae may have rather narrow
temperature requirements, and the pulse shown in Figure 7 can be ex-
plained on this basis. When the lake temperature declined because of
a severe cold spell in early January, 1970 (Figure 7), the bloom rapidly
died out. Only after the temperature rose above 14°C in late February
were the algae able to reach dense bloom conditions again, but once the
temperature rose above about 20° in late March the algae again died out
or alternatively were no longer able to compete successfully against
other forms. In support of this hypothesis a multiple regression ana-
lysis (Table 14) of primary production vs. temperature, light, ammonia
and ortho phosphate in the lake during this bloom found temperature
to be the most important variable (statistically). In fact using a
multiplicative model (logarithmic transformation) temperature alone
explained nearly as much of the variance as die! all four variables com-
bined. However temperature alone is probably too simple an explana-
tion and there are discrepancies in the argument. There is no informa-
tion in the literature suggesting that Aphanizomenon has such a narrow
temperature tolerance (Hammer, 1964). In fact blooms of this algae in
temperate lakes (e.g. in Wisconsin) are common in mid and late summer
when water temperatures are between 20 and 25°C. Further, the drop in
Newnan's Lake temperature in January 1970 was also associated with large
quantities of rain which raised the lake level as much as three feet.
Consequently the Aphanizomenon bloom was both diluted and transported
out of the lake in the unusually high out-flows generated by the rain.
Alternative explanations for the blooms demise also exist, including
the possibility of nutrient exhaustion (probably a trace nutrient since
inorganic phosphate remained low but not depleted during the entire bloom
58
-------
Table 14. Limiting Factors for Primary
Production in Newnan's Lake, Florida During Aphanizomenon Bloom
Multiple Regression Analysis
Dependent Variable: Primary Production
Independent Variables: Light, Temperature, NH-j-N, Ortho-PO^-P
1. Additive Model:
Y = a1X1 + a2X2 + a^ + a^
PP = 4.76(Temp) + 4.32(o-P04> - 6.05(Light) - 69.4(NH3~N)
Var. Increase
Entered R In R2
Temp.0.65790.6579
o-PO, 0.6812 0.0233
Light 0.6953 0.0141
NH? 0.6995 0.0042
* 9
Rz - measure of portion of variance accounted
for by regression
2. Multiplicative Model:
Y = X blX b2X b3X b4
J- 2. j 4
InY = b InX + b2lnX2 + b3lnX3 +
PP = 1.65 (Temp) - 0.26(Light) - 0.14(o-P04> + 0.07(NH3-N)
Var.
Entered
Temp.
Light
o-P04
NH -N
R2
0.9416
0.9425
0.9432
0.9433
Increase
In R2
0.9416
0.0008
0.0007
0.0001
59
-------
and nitrogen would not seem a limiting element to a nitrogen fixing
bloom). Viral attack is currently a popular explanation among aquatic
scientists for otherwise unexplainable bloom disappearances, as is
autointoxication (i.e. synthesis of a substance by the algae which
causes their own death), but neither possibility has ever been proved
to cause bloom death in the environment. In brief, the complexities
of algal bloom cycles are still beyond simple explanation and compre-
hension. While many causes can be conjectured, unfortunately they
remain only that.
Seasonal patterns of nitrogen fixation, nutrient concentrations, pri-
mary production and other biogenic parameters are poorly defined in
these Florida lakes in comparison with classic cycles reported for
temperate lakes. Rather, production, nutrients and algal populations
are characterized by a bloom-crash cycle throughout the year. Pro-
nounced differences were noted between sampling dates even when sampling
was done as frequently as twice a week. The large measured changes in
chemical and biological parameters from one sampling to the next were
matched by obvious visual changes in the lakes, and many of the rapid
fluctuations can be ascribed to rapidly changing vjeather conditions.
The diel variations coupled with the large oscillations in fixation
from one sampling date to the next obviate accurate assessment of the
total quantities of nitrogen fixed in the lakes during a year. How-
ever, the data in Figure 5 and & should permit at least a rough estimate.
Assuming nitrogen fixation is primarily a daylight phenomenon, and during
periods of fixation it occurs in the lakes for about 8 hours a day at
the rates shown in Figures 5 and 6, the nitrogen fixed in the 2 lakes
over the year study was calculated, and the results are summarized in
Table 15. While the total N fixed is large in both lakes, fixation
represents a relatively small contribution to the total nitrogen bud-
gets previously calculated for these lakes by Shannon (1970) (see Sec-
tion V). Based on the survey of Florida lakes described earlier, it is
unlikely that nitrogen fixation would play a substantially greater
role in the nutrient budgets of other Florida lakes. Kence, nitrogen
fixation in lakes seems to be of greater significance in an ecological
sense than as a nutrient source.
60
-------
Table 15. Contributions of Fixation to the
Nitrogen Budgets of Newnan's Lake and Bivin's Arm
Newnan's Lake
Bivin's Arm
N Fixed
0.15
0.52
External
N Supply
g/m3yr
2.60
6.86
Total Percent of
N Supply Total Supplied
by Fixation
2.75 5.5
7.38 7.0
'Calculated from watershed characteristics (land use and population
patterns) by Shannon and Brezonik (1971a) - see Section V.
61
-------
SECTION VII
NITROGEN FIXATION AS AN IN_ SITU NITROGEN SOURCE FOR NATURAL WATERS
II. BACTERIAL FIXATION IN LAKES AND SEDIMENTS
While numerous studies have identified and isolated nitrogen fixing
bacteria from lakes and marine waters (see Section IV), most previous
investigators have considered heterotrophic (bacterial) fixation to
be of little consequence. A lack of oxidizable carbohydrate has usually
been cited as the limit-rrrgfactor for heterotrophic fixation in both
soils and natural waters ( Stewart 1969). Various reports (see Stewart,
1966, for a review) indicate that heterotrophic fixation under labora-
tory (pure culture) conditions is highly inefficient in terms of nitro-
gen fixed per unit of carbon oxidized. However it is always dangerous
to extrapolate highly artificial laboratory results to in sjLjtja con-
ditions. Studies conducted as a part of this project indicate that
heterotrophic (bacterial) fixation can be important in certain anoxic
lacustrine environments as well as in sediments. In addition the ability
of photosynthetic bacteria to fix nitrogen is well known (Stewart, 1966)
and while these organisms occupy a rather special niche (i.e. they
require anoxic conditions and low light intensity) and are not generally
abundant in the environment, they may fix substantial amounts of nitro-
gen in favorable habitats. For example, Stewart (1969) correlated
significant uptake of :5N2 at a depth of 7m. in a Norwegian fjord with
the presence of the photosynthetic bacterium Pelodictyon. Triiper and
Genovese (1968) have reported high concentrations of photosynthetic
bacteria occurring at intermediate depths in certain Italian lakes.
The first evidence for bacterial nitrogen fixation in lakes was re-
ported by Brezonik and Harper (1969) in work supported by this project.
They found fixation in two dystrophic lakes chosen for their extensive
anoxic environments. Lake Mary, Wisconsin, is a colored meromictic
lake, permanently anoxic below 5 meters (maximum depth = 21 meters).
Lake Mize, Florida, is a dystrophic, highly colored lake with a maxi-
mum depth around 25 meters and a surface area of 0.9 ha. The morpho-
metry of Lake Mize (Figure 13) does not promote good vertical circula-
tion, but the lake is monomictic with a circulation period from October
or November to the end of February or early March depending on weather
conditions. Oxygen is lost during the long period of stratification,
and the lake Is anoxic below about 3 to 5 meters from June to November.
Lake Mary was sampled June 28, 1968, and Lake Mize on July 11, and
August 21, 1968. Profiles of some pertinent lake conditions are shown
in Figures 12 and 13 for Lakes Mary and Mize, respectively. Rates
of acetylene reduction at various depths in the two lakes appear in
63
-------
.
FIGURE 11
BATHYMETRIC WP OF LAKE MIZE, ALACHUA COUNTY, FLORIDA. CONTOURS IN PETERS, MAP COURTESY OF F, G, NORDLIE,
-------
Temperature (°C) ; Dissolved 0~ (mg/1)
6 12 18 24
Dissolved Oo (mg/1)
4 " 6
Temperature (°C)
16 24
32
10
p.
cu
Q
15 _
20
20
I
80
320
160 240
Color (ppm as ft)
FIGURE 1?, DEPTH PROFILES FOR TEMPERATURE (o),
DISSOLVED OXYGEN () AMD COLOR (A) IN
28
60
L^.!<- MARY, !'/ISCOMSr-L JUNE /
ARRO1-' INDICATES DEPTH OF SECCHI DISC VISIBILITY,
120 180
Color (nr>^ as Pt)
FIGURE 15, DEPTH PROFILE? FOR TEMPERATURE (o)/
DISSOLVED OXYGEN () AMD COLOR (A) IN
LAKE MIZE, AUGUST 1, 1%8, ARRC"'' IMDJ-
CATES DEPTH OF3ECCHI DISC VISIBILITY,
40
-------
Table 16. Rates measured in this initial study were generally low
compared with fixation rates found in some eutrophic surface waters
containing blue-green algae. The highest rate in Lake Mary occurred
near the bottom, but in Lake Mize maximum activity was noted at a
depth of 15 m.
A detailed study of nitrogen fixation in Lake Mize was conducted
during 1969 and 1970 to determine the extent and significance of
bacterial fixation in the lake (Keirn and Brezonik, 1971). Table 17
presents a summary of the general chemical characteristics of Lake
Mize. The lake has a high but variable color, which is evidently
leached from pine needle litter in the drainage basin, and has typi-
cal characteristics of dystrophy, i.e. low values of pH, calcium,
and primary productivity. Figures 14 and 15 present temperature and
dissolved oxygen profiles of the lake for 1969 and 1970. In 1969 the
lake was only slightly thermally stratified in February but a marked
depletion of dissolved oxygen was found in the hypolimnion. By April
anoxic conditions had developed below 7 meters and by July no oxygen
was present below 3 meters. Stratification began somewhat later in
1970, but dissolved oxygen was partially depleted in the bottom waters
during February. Anoxic conditions occurred below 5 meters by early
April and below 3 meters by early June; it is also interesting to note
that dissolved oxvgen values were low even in surface waters during
the summer of 1970.
Conditions in Lake Mize have changed during the last several years as
a result of an increased nutrient loading. In fall of 1968 an enclosure
housing about 50 ducks was placed at the north shore of the lake. In
response to enrichment, a lush growth of emergent grass occurred
along the previously nearly bare shoreline and the rate of primary
production increased markedly. Rates ranging from 1.9 to 10.4 mg.
C/m.3-hr. were found prior to 1970, but samples taken in April and June,
1970, yielded rates of 42.3 and 36.2 mg. C/m.3-hr., respectively. Con-
centrations of nitrogen and phosphorus also increased substantially
from 1968 to 1970 (Table 18), presumably reflecting duck enrichment.
Low concentrations of ammonia occurred throughout the water column in
spring of 1969 and an inverse clinograde distribution occurred in summer.
By September 1969 epilimnetic ammonia concentrations surpassed those
in the bottom water. Concentrations were higher throughout 1970 and
generally showed more complex profiles. Total organic nitrogen was
rather uniform throughout the water column during the two years, and
nitrate levels remained low (typically < 0.05 mg. N/l.) but somewhat
variable during the study.
Nitrogen fixation as measured by the acetylene reduction method was
found during summer stratification in 1969 and 1970. Figure 16
illustrates the relationship of fixation rate to depth during the
periods of fixation; it is noted that fixation occurred only in the
hypolimnion during both years. Rates were highest during the summer
of 1969, and the depth at which peak fixation occurred became greater
over the course of this summer. This tendency was not found during
1970 when fixation started later than 1969 and ended sooner. Why a
66
-------
Table 16. Acetylene Reduction in Lakes Mary and Mize
Lake Mary- June 28
Depth l
meters
0 a
2 b
5 b
7 b
10 b
15 b
20 b
TKN2
mg/1
0.93
3.1
1.8
2.9
2.5
3.3
4.5
nM C2H4/
1-hr.
0.54
0.36
0.89
0.98
1.70
1.16
5.00
, 19£8
ng N/ 3
1-hr.
5.0
3.3
8.3
9.2
15.8
10.8
46.6
Lake Mize-July 11, 1968
Depth1
meters
0 a
1.5 a
3.0 a
6.1 a
6.1 a
9.1 b
18.3 b
TON2
mg/1
0.56
0.56
0.63
0.65
0.65
0.70
0.69
nM G2H4/
1-hr.
17.8
14.3
12.5
11.6
8.9
33.0
10.7
ng N/ 3
1-hr.
166
133
117
108
83
308
100
Lake Mize-A.ugust 21
Depth a
meters
0 a
2 a
5 a
1 b
10 b
15 b
20 b
TKN 2
mg/1
0.38
0.60
0.69
0.73
0.77
1.29
0.66
nM C2H4/
1-hr
0
0
0
2.98
3.28
8.93
2.68
, 1968
ng N/ 3
1-hr
0
0
0
27.8
30.6
83.3
25.0
1 Samples were purged with oxygen-argon mixture (a) or helium (b) before incubation.
2TKN = total Kjeldahl nitrogen; TON = total organic nitrogen, both in mg N/l.
q
Calculated from nanomoles of ethylene produced per liter-hour assuming theoretical ratio of
1.5 moles ethylene produced per mole of ammonia fixed.
-------
Table 17. Chemical Characteristics of Lake Mize, Florida1
pH 5.03
Acidity (mg CaCO,/!) 29.4
Alkalinity (mg CaCOj/1) _ 1.7
Specific conductance (ymho cm ) 52.9
Color (mg Pt/1 at pH 8.3) 434
Turbidity (JTU) 1.4
Chloride (mg/1) 10.3
Sulfate (mg/1) 5.0
Na (mg/1) 6.7
K (mg/1) 0.32
Mg (mg/1) 0.94
Ca (mg/1) 3.4
Fe (mg/1) 2.4
Mn (yg/1) 19
F (mg/1) 0.02
SiO (mg/1) 3.19
COD (mg/1) 69
Total solids (mg/1) 77
Suspended solids (mg/1) 2
1Average of Data collected from June 1968 to June 1970
(Shannon 1970).
68
-------
(X
Q
3
6
9
L2
.15
18
FIGURE 14, TEMPERATURE PROFILES IN LAKE MIZE DURING 1969 AND 1970
-------
FIGURE 15. DISSOLVED OXYGEN PROFILES IN LAKE MIZE DURING 1369 AND 1970,
-------
Table 18. Changes in Nutrient Levels in Lake Mize, 1968-19701
August, 1968 June, 1969 June, 1970
Parameter2
Total organic N
Ammonia-N
Ortho phosphate-P
Total phosphate-P
Top
0
0
0
0
.25
.20
.000
.005
Bottom
0
0
0
0
.63
.40
.063
.063
Top
0
0
0
0
.82
.03
.018
.087
Bottom
0
0
0
0
.66
.07
.030
.059
Top Bottoi
1
0
0
0
.35
.50
.058
.15
1.02
0.28
0.10
0.19
Values represent means of top 3 meters and bottom 15 meters,
or approximately the mean epilimnetic and hypolimnetic
concentrations.
2Values in mg/1 as N or P.
71
-------
0
9 .
g-12
Q
15
18
1.0 2.0 3.0
Nitrogen Fixation (yg N/l-hr)
6
>w'
4J
o.
o
12 .
15
18
I
6-12-70
6-18-70
6-30-70
7-9-70 A
0.5 1.0
Nitrogen Fixation (pg N/l-hr)
1.5
FIGURE 16, DEPTH PROFILES OF NITROGEN FIXATION IN LAKE MIZE DURING 1969 AND 1979,
-------
a shorter period of fixation occurred in 1970 is not completely clear;
however the noted nutrient enrichment may have been a factor. Maximum
nitrogen fixation rates ranged from 0.08 to 3.26 Ug. N/l.-hr.; the
upper value is within the range of rates reported for blue-green algae
in lakes (Dugdale and Dugdale, 1962; Goering and Neess, 1964; Billaud,
1968) .
Nitrogen fixation in Lake Mize is apparently restricted to mid-summer
stratification. No fixation was detected in samples assayed in April,
September, October, and December 1969 as well as February, April and
May 1970. The annual cycle of nitrogen fixation for 1969 and 1970 is
illustrated in Figure 17, which shows total hourly nitrogen fixation
in the lake over the year. It is apparent that fixation was much
greater during 1969 both in duration and rate. Values for total nitro-
gen fixation per hour were estimated by summing the product of the
volumetric fixation rate at each depth times the calculated lake volume
for each depth. The annual input of nitrogen to Lake Mize by fixation
can be extrapolated from the data in Figure 17. Calculation of yearly
rates from a few one-hour incubations is undoubtedly risky, but even
an approximate value would be instructive. Assuming that during the
period of fixation the reaction occurred 24 hours a day at the rates
shown in Figure 16, the total nitrogen contribution in 1969 was 39.2 kg.
and in 1970 was 9.6 kg., or 1.14 and 0.28 g./m.3 of lake water per year,
for 1969 and 1970, respectively. Shannon and Brezonik (197la) com-
puted a nitrogen budget for Lake Mize (excluding fixation) of 2.05
g./m.3-yr, (see Section V). Thus fixation represents about 56 and 14
percent of the total nitrogen income from other sources in 1969 and
1970, respectively, and at least for this admittedly unusual lake, bac-
terial nitrogen fixation is a highly significant nutrient source.
Because of the unusual nature of the above results an intensive effort
was undertaken to determine the agents of fixation in Lake Mize. Bac-
teriological samples were collected aseptically at the same depths
assayed for nitrogen fixation on several occasions, and samples from
those depths where fixation occurred were subjected to enrichment culture
for groups of nitrogen-fixing organisms. Three enrichment schemes
were utilized covering the spectrum of likely microbial agents: 1) an-
aerobic or facultative heterotrophic bacteria, 2) photosynthetic bacteria,
3) yeasts and fungi. Details of the enrichment and isolation procedures
are given in Aopendix C.
Enrichment techniques indicate that at least two nitrogen fixing groups
of bacteria, one heterotrophic, the other autotrophic, exist in the
depths of Lake Mize. Six isolates of gram positive spore forming rods
which grew anaerobically but not aerobically on nitrogen free media
were isolated from water samples taken at 3, 5 and 7 meter depths on
June 18 and at 5 and 7 meters on July 9. A total of seven transfers
of the anaerobic isolates were made, each time to nitrogen free media,
and the tests for acetylene reduction were positive at each step. Cul-
ture of the six isolates grown in liquid media for 24 hours gave acety-
lene reduction rates in the range 1.5 to 10 nmole C2H? reduced/mg
organism N-hr.
73
-------
a
00
w
0)
4-1
tfl
o
X
H
PM
0)
00
O
t-i
4-1
H
SB
01
n)
cfl
4-J
O
30
20
10
Jan Feb Mar Apr May June July Aug Sept Oct Nov Dec
MONTH
FIGURE 17, HOURLY RATE OF NITROGEN FIXED IN ENTIRE VOLUME
OF LAKE MIZE DURING 1969 AND 1970,
-------
Colonies of purple sulfur bacteria began to appear after 4 weeks
incubation in water from seven meters depth. Growth began at the
glass cellulose interface, which hampered observation of morpholo-
gical types. Transfers of pigmented colonies were made into liquid
media containing only Na7S and other inorganic salts at pH 8.5 and incu-
bated in the light. Phase contrast microscopy showed two morphologi-
cal forms, a long motile, spiral shaped organism which we identified
as a species of Thiospirilium and a small motile rod, apparently a
species of Chromatium. The latter form predominated in mixed cultures
and grew rapidly under the culture conditions.
No growth on nitrogen free media occurred within 7 weeks in any of
the 65 yeast and fungal isolates picked from the June 18 and July 9
samples. However, growth was observed within 2 weeks for all isolates
incubated in the test medium plus nitrogen.
The agents responsible for fixation in Lake Mize are probably hetero-
trophic bacteria. Fixation occurs only in a region of low or no light
and no dissolved oxygen. The fact that fixation is maximum at inter-
mediate depths suggests photosynthetic bacteria may be the agents,
and they are in fact present in the subsurface layers of this lake.
However, the high rates of acetylene reduction found in Lake Mize sam-
ples incubated in the dark would tend to rule out photoautotrophic
forms as dominant agents of fixation since photosynthesis apparently
provides the source of energy for fixation by these forms. Rhodospi-
rillum rubrum, for example, has been shown to fix nitrogen anaerobi-
cally in the light, but intact cells of this bacterium cease fixing
immediately when placed in the dark (Pratt and Frenkel 1959). Further,
photosynthetic bacteria were isolated from only one of the sampled
depths at which fixation occurred (7 meters). The long lag before
significant growths of these organisms were noted in enrichment media
and the fact that microscopic examination of raw lake water and seston
retained on Millipore filters failed to reveal the presence of photo-
synthetic bacteria imply very low populations in the lake.
On the other hand, heterotrophic growth on a nitrogen-free medium was
rapid for samples from the various depths at which fixation occurred.
The taxonomic characteristics of the isolates (i.e. gram-positive, spore-
forming, obligate anaerobes, capable of utilizing N2 as their sole N
source are those of the genus Clostridium. Whether this is the only
heterotrophic bacterium fixing nitrogen in Lake Mize cannot be answered
by the present study. Other enrichment procedures may isolate differ-
ent forms; Arthrobacter has been suggested as another likely agent
(J. Sieburth, personal communication). No aerobic or facultative bac-
teria and no fungi or yeasts were found which could grow on nitrogen
deficient media. Cyanophyceans are not likely to be agents of fixa-
tion in Lake Mize since algal nitrogen fixation is related to photo-
synthesis and conditions at the depths of fixation indicate no oxygen
production. No blue-green algae were found in samples examined micro-
scopically. The observed stratification of fixation and its annual
periodicity may be influenced by such factors as a requirement for low
light and narrow tolerance to high or two sulfide concentrations or
Eh values, which form gradients in the anoxic zone of this lake.
75
-------
Sediments are generally considered to be relatively enriched in nutrients
and as such are unlikely environments for nitrogen fixation. However,
studies supported by this project have detected fixation in both estua-
rine and lacustrine sediments from subtropical and tropical environments.
In addition the report of Howard £t al. (1970) on nitrogen fixation in
Lake Erie sediments indicates this phenomenon is not unique to warm
climates.
Sediments were collected at various locations in the Waccasassa Estuary
and in various lakes with a Viomemade coring device. Cores were returned
to the laboratory and sectioned for profile analyses of nitrogen fixation.
Since the sediments were anoxic, purging of N~ was accomplished with
helium (see Apendix A for procedured details).
The optimum incubation time for the estuarine sediments was determined to
be one hour. The amount of ethylene production was linear for incubation
up to that time, but the rate decreased markedly after that (see Section IX)
Rates of ethylene production in embayment sediments ranged from 0.00 to
0.54 nM C~Hx/g. dry wt.-hr. for all the determinations (over 60) made
in this study. A distinct layering was found within a typical sediment
core (Figure 18). In the flocculent, unconsolidated 1 to 2 cm at the
core surface, no Cft^ reduction was found at any time. Significant re-
duction rates were consistently noted in the next 2 to 5 cm of consoli-
dated, grey-black ooze. From 5 to 20 cm the typical core consists of
coarse organic material, which overlies the limerock substrate. Acety-
lene reduction was consistently found in the bottom zones of sediment
cores, but rates were low compared with those in the upper portions.
An areal survey of the Waccasassa embayment sediments showed acetylene
reduction to be a consistent phenomenon in the upper sediments. Dupli-
cate cores were taken at eight stations in the embayment, and the upper
2 to 5 cm portion of each was blended to obtain a homogenous sample.
Acetylene reduction was then run on a portion of the blended sediment.
The distribution of ethylene production rates in the estuary sediments
is shown in Figure 19. A range of 0.175 to 0.54 nM C^L/g- dry wt.-hr.
was found, but most of the values were near the mean rate of 0.33
nM C~H,/g. dry wt.-hr. Expressed in terms of the equivalent amount of
nitrogen that would have been fixed (using the theoretical ratio of 1.5
moles C2H, produced per mole NH- fixed) the results indicate a range of
1.63 to 5.0 ng N/g. dry wt.-hr.
Several experiments were performed to evaluate the environmental condi-
tions for fixation in sediments and to define the agents of fixation.
The effect of a nitrogen-free oxygen atmosphere vs. an anoxic (helium)
environment was determined in one experiment on five sediment samples.
In all but one case, the rate of ethylene product was slightly higher
under a helium atmosphere, but the differences were not as great as
expected if strict anaerobes were the fixing agents. Probably the or-
ganic sediments exerted a sufficient oxygen demand to maintain anoxic
microzones in the samples even in an oxygen atmosphere. It was also
found that exposure of incubating samples to light decreased the acety-
lene reduction activity. The mean rate of ethylene production in five
replicates incubated in the light was only one third of the mean value
76
-------
FLOC
ETHYLENE PRODUCTION
(ng C2H^/g dry wt.-hr)
1.62 - 11.4
COARSE
ORGAN ICS 5~20 cm
0.03 - 0.40
FIGURE 13, SECTION OF CORE ILLUSTRATING
VARIATION OF ACETYLENE REDUCTION WITH
DEPTH IT! VACCASASSA ESTUARY SEDIMENTS,
RANGE OF ETHYLENE PRODUCED REPRESENTS
AT LEAST SIX DETERMINATIONS IN EACH LAYER,
-------
FIGURE 19, DISTRIBUTION OF ETHYLENE PRODUCTION RATES
WITHIN WACCASASSA ESTUARY SEDIMENTS (in nB G2H4/§ dr
DUPLICATE CORES AT EACH OF EIGHT STATIONS, STATION 11 WAS IN MUD FLAT,
78
-------
for replicates of the same sediment incubated in the dark. The reasons
for this cannot be fully stated; however, it is well known that many
non-photosynthetic bacteria have pigments and that these organisms can
be inhibited by visible radiation.
Since the agents of ethylene production (hence nitrogen fixation) in
the sediments are presumably anaerobic or facultative bacteria, addi-
tion of easily assimilable carbonaceous substrate to sediment samples
should enhance their activity. Several experiments have given somewhat
conflicting results in this regard. In an early experiment acetate
gave apparent stimulation of acetylene reduction at concentrations of
0.02 and 0.2 M but glucose gave no response. However no acetate stimu-
lation was found in three later experiments in the range 10"1* to 1C"1 M,
and in fact 10"1 M acetate and butyrate actually inhibited fixation by
about 30 percent. No evidence for glucose stimulation or inhibition
was found in any of the four enrichment experiments in the concentration
range lQ~k - 10~1 M, but sucrose gave a definite stimulation (50-100
percent increases over controls) in each of the three experiments in
which it was added (Table 19) . These results are similar to responses
of lacustrine sediments to organic additions (described later in this
section).
Several experiments were performed to evaluate the impact of inhibitors
on acetylene reduction. In 30-40 ml. sediment slurries fixation was
inhibited by 1 ml. of fifty percent trichloroacetic acid or 3 ml. of
saturated mercuric chloride solution. If acetylene reduction is re-
lated to nitrogen fixing activity, it would be expected that molecular
nitrogen would inhibit the rate of acetylene reduction. Since acetylene
is a competitive inhibitor of N~ (Schb'llhorn and Burris, 1967), the con-
verse should be true, i.e. added molecular nitrogen should reduct the
rate of acetylene reduction according to the competitive enzyme inhi-
bition pattern (see Cleland (1963) for a complete discussion of enzyme
kinetics). Thus a reciprocal (Lineweaver-Burk) plot of reaction velo-
city (ethylene production rate) versus substrate (acetylene) concentra-
tion should be linear, and added inhibitor (molecular nitrogen) should
give the classical competitive inhibition plot (see Section IX). To
verify this for Waccasassa sediments, two sets of samples were set up;
one series was exposed to various concentrations of acetylene in a
helium (nitrogen-free atmosphere, and in another series using the same
sediment, samples were exposed to various acetylene concentrations in
a 70% N2 - 30% C02 atmosphere. A Lineweaver-Burk (reciprocal) plot of
the ethlene production rate vs. acetylene concentration (Figure 20)
shows the classical competitive inhibition pattern. The maximum velo-
city (V ) as obtained from the y-intercept of the Lineweaver-Burk plot
is approximately the same for both curves implying that high concentra-
tions of acetylene negate the inhibitory effect of N2- Competitive
inhibition patterns are given by enzyme inhibitors that react at the
same active site on the enzyme as the substrate.
The organisms most likely to be responsible for the above phenomena
would seem to be .anaerobic bacteria such as Clostridium sp. An
enrichment and isolation procedure was conducted (see Appendix C for
details on methodology) to determine whether such organisms could be
79
-------
Table 19. Effect of Organic Substrate Addition
on Ethylene Production by Estuarine Sediment
Relative Response1
Concentration Added (Molar)
Substrate
Acetate
Butyrate
Glucose
Sucrose
Hf1
-
-
0
+
1CT2
0
0
0
+
1C' 3
0
0
0
0
i
-------
00
0.12
0.10
M
rC
'
n
oo
u
W)
c
0.08
0.06
> 0.04
0.02
I
0.2
0.4
I/Substrate (cc
atm O
He atn
0.6
0.8
-1
FIGURE 20, LINECAVER-BURK PLOT OF ETHYLENE PRODUCTION VS ACETYLENE CONCENTRATION
INDICATES COMPETITIVE INHIBITION BY N2 IN WACCASASSA ESTUARY SEDIMENT,
-------
detected in Waccasassa sediments. Three isolates were obtained x^hich
could grow in a nitrogen-free medium and reduce acetylene to ethylene
(hence presumably fix molecular nitrogen). The characteristics of the
final isolates vere highly indicative of a Clostridium (or clostridium-
like) culture; i.e. the isolates were gram-positive, strict anaerobic,
large rods, which ferment sugars (and fix molecular nitrogen). Further
physiological tests would of course be necessary to establish specific
taxonomy of the cultures, especially down to the species level. How-
ever the taxonomy of marine and estuarine anaerobes is not highly
developed, and to place the cultures in a particular genus would be
questionable at this time. Nonetheless, this work has demonstrated
that "clostridia-like" nitrogen-fixing bacteria are present in Wacca-
sassa Estuary sediments. The results do not exclude the possibility
of other nitrogen-fixing forms being present in the sediment since the
isolation method used was somewhat selective for clostridia-like bac-
teria.
Sufficient evidence has been obtained to conclude x^ith reasonable
assurance that the acetylene reduction activity is directly related
to nitrogen fixing organisms in the sediments. All lines of evidence
point to ethylene production as a biological phenomenon; both trich-
loioacetic acid and mercuric chloride completely inhibit acetylene
reduction. Organic substrate (in some cases) affects the rate of ethy-
lene production. The fact that N» acts as a competitive inhibitor of
acetylene reduction strongly suggests that ethylene is produced by
nitrogenase. Nitrogen-free media produced growths of gram positive
spore-forming rods from sediments under an atmosphere of pure nitrogen,
and a pure culture similar to Clostridium sp. was isolated from the
sediment and was shown capable of nitrogen fixation by acetylene re-
duction
A rough estimate of the total amount of nitrogen fixed in the 2-5 cm
layer of sediments in the Waccasassa can be obtained from the data pre-
sented in Figure 19. Assuming that the mean value of 3.1 ng N fixed/g
sediment-hr. is a reasonable estimate of the nitrogen fixation rate in
this stratum of sediment throughout the estuary, the amount of nitrogen
fixed on an annual basis is 37 yg N cm2-yr. or for the entire 7 km2
estuary, 2.6 x 103 kg N/yr. These values are based on an average sedi-
ment dry weight of 0.455 g/cm3 in the 2 to 5 cm zone. These extrapo-
lated values are obviously rather crude, but they serve to illustrate
the point that the seemingly low rates found in the sediments in fact
represent significant amounts of nitrogen fixed on an annual basis.
Because sediments are normally thought to be enriched in nutrients, we
were somewhat surprised at our initial results indicating nitrogen fixa-
tion in Waccasassa estuary sediments. Free ammonia was determined on
sediments from 14 widely scattered stations in the estuary (Brooks,
1969); a range of 0.01 to 0.37 and mean of 0.06 mg NH^-N/g sediment was
found. Not all this ammonia would necessarily be available to micro-
organisms; much or perhaps all of it could be loosely sorbed to solid
material in the sediment. Some evidence was found to support this hypo-
thesis (see Section VIII); Waccasassa Estuary sediment was shown to
contain 3 to 5 percent clay mineral and to sorb ammonia rapidly from
solution.
82
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The first indication that lacustrine sediments supports nitrogen fixing
activities was uncovered in 1968 when a short core from Bivin's Arm
yielded positive rates which decreased rapidly with depth (Table 20).
Sediment cores were obtained from 16 lakes in north central Florida in
September, 1969, and from these and 9 additional lakes in June and July
of 1970 to determine whether lacustrine sediment fixation is a more
widespread phenomenon. These lakes were all sampled periodically in
conjunction with other studies (Shannon and Brezonik, 1971c; Brezonik,
1971c), and their water and sediment characteristics are thus known.
A list of the 25 lakes and their trophic states is presented in Table 21
along with pertinent sediment characteristics. Three general types
of sediment occur in the lakes: A) brown flocculent or unconsolidated
material often with undecomposed plant remains, B) black, jelly-like
sediment, and C) sandy sediment.
Short cores (30-50 cm) from the lakes either blended in their en-
tirety and an aliquot taken for measurement of nitrogen fixation or
segmented into several zones which were then tested for fixation indi-
vidually. Another aliquot of sediment was dried and weighed, and results
were reported as rate of fixation per gram dry weight of sediment. Seven
of the twenty-five lake sediments showed apparent nitrogen fixation in
one or both surveys (Table 22) . Four of the seven sediments were peat-
like (Type A), and except for Lake Apopka, these also gave the highest
rates of ethylen production. Peat sediments might be expected to have
a lower available nitrogen content than muck sediments. Sediment from
lake #20 appeared tobbe a mixture of sand and sludge. This shallow lake
(maximum depth is 3.5 m) receives some domestic waste effluent and is
at times anoxic below 1 meter. Sediments from Lakes Apopka, Bivin's Arm,
Kanapaha and Orange emitted a musty or moldy odor when blended, while
the other sediments were either odorless or gave off a hydrogen sulfide
odor. Lake Alice was the only sediment with a pronounced H~S odor that
showed significant fixation. This lake receives a large proportion of
its inflow as treated sewage effluent. Five cores taken from this lake
in 1969 showed fixation, but no fixation was found in a core taken during
the 1970 survey.
In order to determine the layers of sediment most active in nitrogen fixa-
tion, profiles of acetylene reduction rates were measured on sediment
cores from Orange Lake in the 1969 survey and on all lakes sampled in the
1970 survey. The loose, unconsolidated nature of most of the sediments
exhibiting nitrogen fixation precluded detailed segmenting of the cores,
and only the core from unnamed lake 20 was easily divisible into segments.
Table 23 presents the 1969 profiles for Orange Lake and profiles from
the 4 lakes exhibiting fixation in the 1970 survey. Highest rates were
found in the upper layers; this trend was most marked when the rates
are considered per gram dry weight of sediment rather than per milligram
of sediment nitrogen. Sediment from lake #20, while not as active as
the other three sediments when compared on a sediment dry weight basis,
showed comparable activity on per sediment nitrogen basis.
Sediment samples from lakes in the Peten region of Guatemala surveyed
in conjunction with another project during the summers of 1969 and 1970
also showed nitrogen fixation by the acetylene reduction method. Loca-
tions and descriptions of these lakes are given by Fox ejt. al. (1970).
83
-------
Table 20. Acetylene Reduction in
Bivin's Arm Sediment Collected in August, 1968
Ethylene Production
Sample Treatment1 nM C^/g. dry wt-hr.
Ekman dredge -
Surface to Control (TCA) He2 0.043
3" mixture He 2.09
OojAr 0.54
500 mg NH3~N/1 added, He 0.27
Core
Surface
Surface
3" deep
5" deep
7" deep
9" deep
0 Ar
He
He
He
He
He
0.31
2.15
0.25
0.21
0.10
0.09
1Control treated with 1 ml 50% trichloroacetic acid before adding
acetylene. Samples purged either with helium (He) or with gas
mixture (20% 02, 0.03% CO-, balance Ar) to remove dissolved N .
2Equal to "blank" reading, i.e. background ethylene present in
acetylene.
-------
Table 21. Lakes Surveyed for Sediment Nitrogen Fixation1
Hypereutrophic Eutrophic Mesotrophic
Apopka (B) Hawthorne (A) Cooter Pond (B)
Unnamed 20 (D) Clear (B) Lochloosa (B)
Bivin's Arm (A) Wauberg (A) Calf Pond (A)
Griffin (B) Newnan's (A) Orange (A)
Alice (A)
Eustis (B)
Kanapaha (A)
Oligotrophic Ultraoligotrophic
Watermelon Pond (B) Anderson-Cue (A)
Unnamed 10 (A) Gallilee (B)
Jeggord (3) Cowpen (B)
Moss Lee (B) Sand Hill (C)
Altho (B) Swan (C)
Classification into trophic class after Shannon and Brezonik (1971b).
2Sediment types found:
A-Brown flocculant unconsolidated material with or without
undecomposed plant remains.
B-Black Jelly like sediment.
C-Sandv bottom.
D-This sediment was a mixture of sand and brown, foul-smelling sludge.
85
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Table 22. Nitrogen Fixation Rates in Florida Lake Sediments
Lake
Sediment
Characteristics
Nitrogen Fixation Rate1
1969 19702
ng.N/g-hr. ng.N/g-hr. Ug.N/g N-hr.
Bivin's Arm
Kanapaha
Orange
Moss Lee
Apopka3
Alice
Unnamed 20
A
A
A
B
B
A
D
28
36
9.5
1.8
17
1.2"
0
22
59
28
0
-
0
16
2.0
1.4
1.3
0
-
0
23
Calculated assumed a C^H^/NH molar production ratio of 3/2.
2Maximum rate found in profile (Table 23).
30ne dredge sample Nov. 3, 1969.
"* Average of 5 cores.
86
-------
Table 23. Stratification of Nitrogen
Fixation in Florida Lake Sediments
Stratum
(cm)
Orange Lake
1969
°~37 Shallow Water Core
37-50
0-36 Deep Water Core
36-50
1970
0-33
33-42
Lake Kan ap ah a
1970
0-5
5-15
15-25
25-38
Lake Bivin's Arm
1970
0-2
2-5
5-10
10-15
20-25
Lake Unnamed 20
1970
0-2
2-4
4-6
6-8
8-10
10-12
12-15
15-20
20-25
Nitrogen Fixation
ng N/g - hr. yg N/;
1.09
0.33
0.98
0.29
28
1.1
59
10
3
0
14
22
18
5
0
3
12
16
3
4
6
0
0
Rate
g N-hr
1.3
0.78
1.4
1.4
0.37
0
1.4
2.0
1.9
0.8
0
0.51
2.1
2.3
0.71
0.25
0.24
0
o
0
87
-------
In 1969, Laguna Petenxil sediment fixed at a rate of 3.1 yg. N/g.
sediment N-hr., while green flocculant surface sediment from Laguna
Echixil fixed at a rate of 5 yg. N/g. sediment-hr. Sediments from
two Peten lakes (Eckixil and Sal Peten) were sampled in detailed pro-
files in 1970, and high rates of fixation were found in both lakes (up
to 2.4 yg. N/g. sed. N-hr.). Sal Peten sediment exhibited three distinct
bands of fixation. The near surface sediments of these lakes are un-
usual in being composed of flocculent, pigniented organic particles. The
green to pink color of the sediment apparently derives from undecomposed
algae and perhaps partially from photosynthetic bacteria.
Because the acetylene reduction technique is an indirect assay for nitro-
gen fixation and because fixation had never before been measured in a
sedimentary environment, it was felt essential to demonstrate that the
measured ethylene production indeed reflected nitrogen fixing activities.
This was accomplished by a variety of experiments. First, biological
poisons were shown to inhibit acetylene reduction. Addition of 1 ml of
50% trichloroacetic acid or 3 ml of saturated mercuric chloride to sedi-
ment samples (25 ml slurries) immediately and completely stopped ethy-
lene production. Second, if acetylene reduction is related to nitrogen
fixation, addition of N2 should inhibit the rate of acetylene reduction
as was shown for Waccasassa Estuary sediments and for nitrogen fixing
algae (see Section IX). A similar experiment was performed with sediment
from Lake Kanapaha, a shallow, highly eutrophic lake near Gainesville,
Florida. Varying amounts of acetylene were added to serum bottles con-
taining 25 ml of sediment slurry which had either been purged with an
02~Ar mixture to eliminate N or left unpurged, and the samples were
incubated for 1.5 hours at 22°C in the dark. A Lineweaver-Burk (recipro-
cal) plot of the resulting rates vs. substrate'(acetylene) concentrations
(Figure 21) fit a pattern of competitive inhibition of ethylene produc-
tion by N£. This corroborates the experiment on Waccasassa Estuary sedi-
ment discussed previously and is in agreement with Schollhorn and Burris'
(1967) finding that acetylene is a competitive inhibitor of nitrogen fix-
ation.
Heterotrophic nitrogen fixation is known to depend on the availability
of organic carbon. Table 24 shows the results of an experiment designed
to show the effect of various organic substrates on acetylene reduction
in lake sediment. Twenty-five ml. samples of sediment slurry were purged
and injected with acetylene in the usual manner except that prior to
injection of acetylene sufficient carbon source was added to give the
concentrations shown. Sucrose definitely stimulated acetylene reduction
while the 0.10 M concentrations of sodium acetate and butyrate caused
inhibition. The above experiments strongly support a biological media-
tion of ethylene production from acetylene which is directly related to
nitrogen fixing activities. Fixation in both estuarine and lacustrine
sediments is most likely mediated by heterotrophic bacteria, and sucrose
additions stimulated fixation in both sediments, Photosynthetic bacteria
are obviously unlikely agents for the observed activities since fixation
extends into fairly deep layers of sediment and blue-green algae are
rare or absent in the Waccasassa Estuary and in some of the lakes whose
sediments show fixation. Blue-green algae cannot be completely ruled
out in all cases, for Hoare at al. (1971) have demonstrated that Nostoc
88
-------
5.0
J-i
,e
4.0
£ 3.0
G
o
o
3
TD
O
S 2.0
w
1.0
O
0.68 atm N2
0.00 atm N0 O
0.2
0.4
0.6
0.8
1/S = I/(Acetylene Added) (cc C2H2)
-1
i.o
FIGURE 21, COMPETITIVE INHIBITION OF ETHYLEME PRODUCTION
(BY NITROGENASF.) CAUSE BY !io IN U\KE KANAPAHA SEDIMENT,
89
-------
Table 24. Effect of Organic Substrates on Rates of
Acetylene Reduction by Lake Kanapaha Sediment
Relative Rate of Acetylene Reduction1
Substrate
10
10
10 3M
10~"M
Glucose
Sucrose
Pyruvate
Acetate
Butyrate
1.2
1.9
1.1
0.8
0.8
1.1
1.7
1.0
1.1
1.0
1.1
1.2
1.0
1.2
1.1
1.0
1.0
1.0
1.0
1.1
Relative to acetylene reduction rate of 0.50 nmoles
C2H2 reduced/mg N-hr. (average of 4 controls) . Each
value is the mean of duplicate samples.
90
-------
and other species can metabolize heterotrophically even under anoxic
conditions. However, bacteria are at least as likely an explanation
for fixation under these circumstances.
While nitrogen fixation does not occur universally in lake sediments,
its occurrence at least sometimes in 7 of the 25 Florida lake sediments
suggests that the phenomenon is more than just an environmental curiosity.
The occurrence of fixation in the three Guatemala sediments tested in
Lake Erie and Waccasassa Estuary sediments indicates a fairly widespread
distribution of low nitrogen fixation rates in the sediments underlying
natural waters. The range of fixation rates measured in Florida sedi-
ments was 0.33 - 59 ng. N/g. sediment-hr. in surface layers and 0.02 -
1.1 ng. N/g. sediment-hr. in the bottom strata of 30-50 cm. cores.
Comparable rates were found in Guatemalan lake sediments and in sediments
from the Waccasassa Estuary. The rates for Lake Erie sediments (Howard
et al., 1970) are also in the same range.
It would be of great interest to determine why fixation occurs in some
sediments but not in others. Examination of sediment characteristics
of the Florida lakes shows no obvious correlation between the occurrence
of sediment fixation and sediment ammonia, total organic nitrogen, phos-
phorus, or percent volatile solids, carbon or nitrogen. Fixation acti-
vities should be inversely related to ammonia concentration. On the
other hand, even though sediments may be apparently nitrogen-rich, much
of the ammonia may be sorbed onto clays and other particles and may
unavailable to microorganisms (see Section VIII).
Increased activity in the upper layers of sediment probably reflects
higher concentrations of oxidizable substrate in these layers. Depth
profiles of fixation were not nearly so narrow in the lake sediment cores
as those found in Waccasassa Estuary sediments, where fixation was con-
fined largely to the 2-5 cm. stratum. The broader depth distribution is
undoubtedly related to wind-induced mixing of relatively unconsolidated
sediments in the shallow lakes. This hypothesis is supported by the
results for lake #20, which had a sharper stratification and which also
has a fairly compact sediment. Stratification of fixation is much more
pronounced when the rates are expressed per gram dry weight sediment than
when expressed per unit of organic nitrogen in the sediment.
Nitrogen fixed in the sediments will be only partially released to the
overlying water, depending on the degree of mixing effected by wind
action. In the loose unconsolidated sediments such as found in Lakes
Kanapaha, Orange and Bivin's Arm, this may be considerable; in compacted
sediments release to the overlying water may be controlled by much slower
diffusion processes. Extrapolation of the sediment fixation rates in
Tables 22 and 23 to annual amounts indicates that the process may contri-
bute substantial quantities of nitrogen to the lake basin as a whole and
may in this sense be geochemically significant.
91
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SECTION VIII
OTHER IN SITU NITROGEN SOURCES AND SINKS
Although sediments are generally described as nutrient traps, this is
only true as a net reaction. Transfer of nutrients between water and
sediment is at least partially reversible and to an extent cyclic rather
than one-way. Over geological time sediments build-up acts as a net
sink, but on a seasonal or shorter time basis the possibility of sedi-
ments acting as nutrient sources must also be considered. Nitrogen is
lost to sediments by deposition of particulate matter (detritus, silt)
and by sorption of ammonia onto clays in or being deposited onto the
sediments. Nitrogen can be released from sediments by activities of
burrowing animals, decomposition of organic nitrogen to ammonia and
its diffusion into the overlying water, and desorption of ammonia from
clays and other sorbents in the sediment.
Nitrogen exchange at the sediment-water interface has received scant
attention compared to phosphate exchange. Several classic studies
have discussed the role of oxygen and redox potential in controlling
diffusion of ammonia from sediments. Mortimer (1941, 1942) reported
that little ammonia is released from sediments as long as an oxidized
microzone exists but that large quantities of this (and other nutrients)
are liberated when this surface layer is reduced. He felt that this
general increase could be explained by sorption of ammonia onto a com-
plex organic floe containing iron (III). The apparently low rate of
nitrogen release from oxidized sediments could also be partially ex-
plained by nitrification at the sediment surface causing nitrate rather
than ammonia release, and by the fact that ammonia is unlikely to
accumulate in oxygenated water as it will either be oxidized or assi-
milated. The role of the oxidized microzone in influencing sediment-
water nutrient cycling would be a fruitful area for future research.
For purposes of nutrient exchange, lake sediments can be divided into
at least two main types based on the presence or absence of a defined
water-sediment interface. In the former case nutrient exchange may
be limited by rates of diffusion, the presence of an oxidized surface
layer, and the activities of burrowing animals (Gahler, 1969). Many
shallow, eutrophic Florida lakes have no defined sediment-water inter-
face. A flocculant suspension covers the bottom in these lakes with
gradual compaction from thin "soup" to consolidated sediment occurring
over a depth of perhaps several feet. In this case considerable ex-
change may be effected by wind generated currents and turbulence,
causing the sediment suspension to mix with overlying water. Rates
of anaerobic decomposition of organic sediment are probably a primary
factor limiting nutrient exchange in such sediments.
93
-------
Thus,sediment-water nitrogen interactions involve both biological and
chemical processes, and rates of exchange may be further controlled by
physical (e.g. diffusion) phenomenon. Qualitative aspects of biologi-
cal decomposition in sediments are fairly well understood (e.g. Foree
et_ al. , 1970; Lee, 1970b; Rittenberg et aJL. , 1955) and need no further
elaboration here. The importance of burrowing animals (e.g. worms,
larvae, crustaceans) in aerating sediments and in physically trans-
porting nutrients from the sediment and excreting them into the over-
lying water was suggested (Brooks, 1969) in work supported by this
project. When surface sediment from the Waccasassa Estuary (Florida)
was covered with estuary water in laboratory aquaria, a defined sedi-
ment-water interface and oxidized surface layer quickly arose, and with-
in several days extensive burrowing and tube building by amphipods and
annelids were evident through the aquarium glass wall. However quan-
titative data on regeneration rates induced by this mechanism are
lacking.
Ammonia may be sorbed onto clays and organic colloids in sediments, in
which case an equilibrium between aqueous ammonia and ammonia in sedi-
ments would be set up. Principles of soil chemistry and cation adsorp-
tion processes in soils should be applicable to the study of this phe-
nomenon in sediments (Toetz, 1970; Toth and Ott, 1970). Carritt and
Goodgal (1954) listed clays, gels of ferric hydroxide and silicic acid,
humus colloids, polymorphic inorganic and organic complexes and sur-
faces of living and dead organisms as possible adsorbents active in
sediments. If sorption plays an important role in ammonia equilibrium
between sediment and water, it should be possible to describe the
equilibriun between ammonia adsorbed and ammonia concentration in the
interstitial water by one of the well-known sorption isotherms such as
the Langmuir or Freundlich. Since other cations compete with ammonia
for sorbent binding sites, the extent of ammonia adsorption should be
affected by changes in concentrations of other ions in the bulk solu-
tion. On the other hand a considerable amount of "trapping" may occur
such that the sorbed ions are not freely in contact with bulk or insti-
tial water but have migrated by intraparticle diffusion. Such a situa-
tion would render the sorbed ammonia relatively immune to changes in
composition of the bulk solution, in effect making aamonia sorption
irreversible.
A problem in defining the role of sorption in sediment-nutrient inter-
actions is to separate the effects of biological activities. Use of
poisoned or irradiated sediments is possible, but there is always a
danger in changing the chemical nature of sediment by such treatment.
The importance of chemical processes can be inferred if rapid uptake
or release occurs during short (several hour) incubations since bio-
logical decomposition and assimilation are inherently slow processes.
A further problem also arises in relating the results of laboratory
shaker-flask experiments on sorption and leaching to in situ conditions.
The high concentrations of ammonia which typically arise in anoxic lake
hypolimnia during summer stratification have long been known (Domo-
galla et^ cd. , 1926; Hutchinson, 1957). Release of ammonia from sedi-
ments has been a widely accepted mechanism for these increases, but
the decomposition of sinking organisms and detritus offers an alternate
94
-------
explanation for at least some of the increase. Attempts to determine
the relative importance of these two mechanisms have not yet been
successful. Rates of detrital deposition in sediment traps and changes
in the elemental composition of detritus as it sinks have been reported
(Kleerekoper, 1953), and sediment leaching experiments involving labo-
ratory incubations have been described by Mortimer (1941, 1942), Sawyer,
et al. (1945) and others. But the quantitative significance of sediments
HLS 1m ammonia source remains elusive. Recently Fitzgerald (1970) des-
cribed experiments in which algae grown in contact with sediment from
Lake Mendota, Wisconsin, maintained a phosphorus deficiency in spite
of the high phosphate level of the sediments. The results qualitatively
suggest that phosphate incorporation into sediments is largely irre-
versible. Whether this also applies to nitrogen in sediments and if
so, under what conditions, remains unknown.
In summary the processes of nitrogen cycling and interchange in sedi-
ments are'complicated and poorly understood. The mechanisms whereby
nitrogen is exchanged between water and sediments are probably known,
and in some cases qualitative rankings can be given to their importance.
However almost no information is available to establish the in_ situ
rates and controlling factors for these processes.
The chemistry of nitrogen in Florida sediments has been studied in two
phases. The chemical characteristics of sediments from the 55 lakes
described in previous sections have been evaluated to provide a basis
for later studies on the dynamics of nutrient exchange. In conjunction
with a separately funded project the sediments were analyzed for total
organic nitrogen, free ammonia, ortho and total phosphate, iron, man-
ganese, volatile solids, C, H, and N contents by elemental analysis,
chlorophyll and carotenoid degradation products, benthos, and gross
physical description. Of primary concern here are the first two analy-
ses, but the other parameters are important in describing the sediments
and'may later be useful in relating exchange rates and capacities to
sediment type.
Results for surface sediments collected by Ekman dredge from the lakes
are presented in Table 25. For comparative purposes the type of sedi-
ment and organic content are listed along with nitrogen content. Methods
used in the analyses are described in Appendix A. Highest concentra-
tions of sediment ammonia were generally associated with enriched or
polluted lakes (e.g. Hawthorne, Dora, Griffin) or with undisturbed lakes
having a high sediment organic content. Ammonia concentrations ranged
from less than 10 ppm to over 1000 ppm on a dry weight basis. Total
nitrogen values ranged from 0 to 4.0 percent on a dry weight basis, but
low levels (less than one percent) were generally associated with low
carbon and low volatile solid samples. For example, no nitrogen was
detected in the Swan Lake (Putnam County) sample, but this sediment
was composed largely of sand and had only 1.2 percent carbon and 2.3
percent volatile solids. This lake is also very clear and can be con-
sidered ultra-oligotrophic. Highest values of total nitrogen were
associated with high volatile solids and carbon and usually with eutro-
phic or polluted conditions. Lake Hawthorne had the highest sediment
nitrogen; this eutrophic lake receives sewage effluent from the town of
95
-------
Table 25. Sediment Characteristics of North Central Florida Lakes
Lake1
Sante Fe
Little Santa Fe
Hickory Pond
Altho
Cooter
Elizabeth
Clearwater
Hawthorne
Little Orange
Unnamed (#10)
Moss Lee
Jeggord
Still Pond
Lochloosa
Orange
Palatka
Newnan's
Calf
Unnamed (#20)
Meta
Alice
Bivin's Arm
Clear
Unnamed (#25)
Beville's Pond
Unnamed (#27)
Kanapaha
Watermelon
Long Pond
Burnt
Wauburg
Tuscawilla
Apopka
Dora
Harris
Eustis
Griffin
Weir
Kingsley
Sumter Lovry
Magnolia
Brooklyn
Geneva
Sediment Volatile
Type2 Solids%
DBr,Bl-N-Si
DBr,Bl-N-Si
DBr-O-S, Si
DBr-0-S,Si
DBr-O-S, Si, Or
DBr-T-S,Or
Br-0-S,Or
LBr-N-S,Or
DBr-T-S,Or
LBr-N-S,Or
DBr-T-S
DBr-T-S
LBr-0-S,D
Br-O-S, Or
Br-0-S,Or,P
Br-N-D
Br-O-P
Br-N-P,D
G-O-S
G-0-S,D
Br-O-P, D
Br-O-P, D
Br-O-S
Br-O-S ,D
Br-O-P ,D
Br-O-P, D
Br-O-P, D
B1-N-M,S
B1-0-M,D
DBr-0-S,M,D
Bl-0-S,Or
G, Br-O-S, D
DBr-O-S ,M
DBr-O-S ,M
DBr-O-S ,M
DBr-O-S ,M
DBr-O-S ,M
G, Br-O-S, Si
Bl, Br-O-S, Si
Bl-0-S,Si,D
Bl-0-S,Si
Bl-0-S,Si
Bl-O-S
18.7
58.8
6.8
61.5
51.5
13.2
8.1
82.4
15.0
88.3
87.3
62.2
24.7
15.3
47.9
95.1
52.8
84.3
25.7
29.6
43.0
65.7
4.9
2.1
84.4
41.7
76.5
64.6
49.6
34.5
39.2
52.7
52.2
59.7
32.5
56.4
63.4
54.7
34.4
24.4
32.2
7.4
16.6
Ammonia-N
Pg/g
30
30
10
40
70
10
10
1010
80
180
180
20
140
10
50
520
320
10
310
30
40
570
10
10
270
50
240
60
60
60
460
80
250
480
250
150
820
150
30
10
30
10
< 10
Carbon3 Nitrogen3
% %
11.5
45.2
4.6
36.6
26.6
8.1
6.1
44.9
8.2
49.1
49.6
42.3
1.6
8.7
26.0
48.2
28.1
50.1
12.8
18.3
14.8
35.5
2.3
1.2
46.2
23.6
40.3
43.4
27.3
18.6
23.6
27.3
29.7
27.4
16.9
26.9
33.7
31.8
18.3
14.2
14.9
3.3
8.9
0.7
2.4
0.3
1.8
2.1
0.4
0.4
4.0
0.6
3.5
3.5
1.4
0.1
0.9
2.4
3.1
2.4
3.5
0.9
1.3
1.2
3.2
0.2
0.1
3.9
1.9
3.6
2.9
2.1
1.3
1.7
2.1
2.4
2.2
1.6
2.3
2.7
2.4
1.1
0.7
0.7
0.2
0.6
C/N
wt . /wt .
16.6
18.7
18.4
20.6
12.7
19.6
15.3
11.1
14.8
14.1
14.3
29.6
12.4
9.8
11.0
15.5
11.9
14.2
14.4
14.4
12.7
11.2
11.7
13.7
11.9
12.7
11.2
14.8
12.8
14.2
13.6
13.1
12.5
12.5
10.6
11.7
12.3
13.3
17.4
20.0
22.6
15.0
15.8
96
-------
Lake
Table 25. (Contd.)
Sediment Volatile Ammonia-N3 Carbon3 Nitrogen3 C/N
Type2 Solids% yg/g % % wt./wt,
Swan
Wall
Santa Rosa
Adaho
McCloud "
Anderson-Cue
Suggs
Long
Cowpen
Gallilee
Bl-O-S
Br-0-S,P
LBr-N-S
DBr-0-S,Si
DBr-0-S,Si
DBr-0-S,Si,D
DBr-0-S,Si
DBr-N-S
Bl-O-S, Si
Bl-O-S, Si
2.3
66.8
14.9
58.9
67.4
85.3
40.5
31.6
31.4
49.2
<10
30
410
30
510
80
50
150
10
20
1.2
41.5
0.4
35.9
38.4
50.2
6.4
0.7
18.9
29.4
0.0
2.6
0.02
1.8
2.4
2.7
0.4
0.03
1.2
1.8
__
16.3
21.0
19.5
16.5
18.5
17.3
24.3
16.0
16.5
'Lakes Santa Fe to Tuscawilla are in Alachua County; Lakes Apopka to Weir
are in the Oklawaha River Basin; remaining lakes are in the Trail Ridge
(sandhill) region of the central highlands. See Brezonik et_ al. (1969)
for lake locations.
2Sediments typed according to color, odor, and texture: Br=brown, DB=dark
brown, LBr=light brown, Bl=black, G=gray, 0=H S odor, N=No odor, S=sand,
Si=silt, Or=fine organic matter, P=peat, D=debris (roots, leaves, etc.),
M=muck.
3Expressed on a dry weight basis.
97
-------
Hawthorne (Alachua County).
Only one lake had a C/N ratio less than ten (on a weight-weight basis) .
The total range in C/N ratios was 9.8 to 29.6, but most of the ratios
were between 11 and 18. Thus all the lake sediments are relatively
enriched in carbon compared to nitrogen. There is a slight trend toward
lower C/N ratios in the more eutrophic lakes, but the data are highly
scattered. Figure 22 presents a plot of sediment C/N ratio vs. a rela-
tive ranking of trophic state (Shannon and Brezonik, 1971b) for the 55
lakes. Similarly a plot of total organic nitrogen vs. total phosphorus
in the lake sediments (Figure 23) shows no trend toward a constant
ratio; N/P ratios vary from 0.7 to over 40 in these sediments. The
"ideal" N/P ratio (by weight) in organisms is about 7 (assuming the fre-
quently cited elemental ratios C:N:P of 105:16:1 by atoms). Most of
the sediments have N/P ratios much larger than 7, indicating relative
nitrogen enrichment. Thus on this basis alone one would conclude that
phosphorus is the more likely limiting nutrient in most Florida lakes.
However too much emphasis should not be placed on these figures. The
ecological significance of nutrient ratios in sediments remains obscure;
certainly the activity or mobility of the species (i.e. their relative
abilities for recycling) are more important in determining what is limi-
ting than are static concentration ratios.
The second phase of sediment studies involved evaluation of the rates
of nitrogen exchange between sediments and the overlying water. Most
of the work on this aspect has been conducted on estuarine sediments
(from the Waccasassa Estuary on the Florida Gulf Coast), (Brooks,
1969). Results from a series of ammonia exchange experiments demon-
strate the significance of sediments as nutrient sinks and indicate
that at least some sediments have a greater tendency to remove nutrients
from solution rather than to release them to the overlying water.
An initial experiment with estuarine sediment incubated in aquaria
indicated that ammonia levels tended to decline in the overlying water
with time whether the aquatic microorganisms were eliminated by various
sterilization techniques or not. These preliminary results suggested
that these sediments act as a nutrient trap probably by simple sorption
onto clays or organic colloids and that from a net viewpoint release of
ammonia to the overlying water is unimportant. Several further experi-
ments were conducted to verify this supposition.
Three sediment-water aquaria were set up as closed systems in which
any free ammonia evolved as a gas could be trapped and measured.
Ammonia free air was bubbled through the water to keep it in slow cir-
culation but the sediments were left undisturbed and developed a sharp
sediment-water interface and oxidized microzone in a few days. After
the aquaria were equilibrated ammonia was added to two aquaria to raise
aqueous concentrations by about 0.1 and 0.5 mg N/l respectively, and
concentrations in the water were monitored over a six day period (Fig-
ure 24). In the aquarium with no addition, ammonia remained relatively
constant, but in both aquaria with added ammonia there was a rapid loss
from the water with final values in the vicinity of 0.10 mg N/l. In
no case was a significant amount of ammonia stripped from the water;
98
-------
25
O
I-
fc
CO
ILJ
UJ
a
20
15
10
o o
O
o o
o
00
o
oo°o°
o
o
00
o
o
o
o
o o
o
o
o
o
o
o
o
o
o
o
o
o o
o
10
20
30
40
Trophic State Rank
50
60
FIGURE 22, CARBON TO NITROGEN RATIO IF; 55 LAKE SEDIMENTS AS A FUNCTION OF A
RELATIVE TROPHIC STATE RANKING (FROM BREZONIK l?71c), MOST EIFTROPHIC
LAKE AT LOWEST END OF RANK; MOST OLIGOTROPHIC LAKE AT HIGMEST E^SD,
-------
0.40
4-1
| °'32
(G
3
O
X
o, 024
en
o
iH
to
0
H
0.16
0.08
0.0
O '
/ 0
o/
o/
/
/
/ o
o f
' ° /
' 0 0 0
,00 0
/ 0 0 O
0 'Q£ 0 ° ° 0 g o $
° /* 0 °
0.8 1.6 2.4 3.2
Total Nitrogen (% dry wt.)
4.0
FIGURE 23, TOTAL PHOSPHORUS VS TOTAL NITROGEN IN 55 FLORIDA LAKE
SEDIMENTS, DASHED LINE REPRESENTS IDEAL N:P RATIO OF 7:1
(VIT.AfT,) IN MICROORGANISMS, VALUES BELOW THE LINE INDICATE
RELATIVE ENRICHMENT OF N IN SEDIMENT; SEDIMENTS OCCURRING
ABOVE THE LINE ARE RELATIVELY ENRICHED IN P,
100
-------
o
0.6
0.5
0.2
0.1
O Control
C 0.1 mg N/l added
0.5 mg N/l added
Day
FIGURE 2L\, UPTAKE OF AMMONIA BY F.STUARINE SEDIMENTS IN AQUARIA,
V,'AS COMTINUALLY ilIXED BY BUBBLING AIR 3UT SEDIMENTS WERE UNDISTURBED,
-------
the maximum ammonia caught in the acid trap was 5vig as N, and the pH
levels in all three aquaria remained in the range 8.0-8.3 through-
out the experiment. Aside from sediment uptake, the only likely sink
would be assimilation by algae and bacteria. The water was filtered
before being added to the aquaria, so initial organism levels were low,
and there was no visual evidence for growths either in the water or
on the aquaria walls. While not perhaps unequivocal, the results
strongly suggest th&t sediments from the Waccasassa Estuary act to sorb
ammonia from solution.
One question raised by the above experiments was whether ammonia uptake
by sediments was biologically mediated or a strictly chemical pheo-
memon. A sediment-water system which was sterilized by cobalt irra-
diation gave similar rates of ammonia uptake as an unsterilized system
did (Figure 25). The results fit neither a first nor second order
rate expression but could be approximated by two zero order expressions:
a rapid initial uptake (k=1.3mg/1-day) followed by a slower rate (k=0.03
mg/l-day) occurring after several days. This result is reminiscent of the
kinetics of phosphate sorption by estuarine sediments reported by Carritt
and Goodgal (1954), uptake of pesticides by clays (Haque et. al., 1968),
and sorption of alkyl benzene sulfonates by activated carbon (Weber and
Rumer, 1965). In all these cases the rates can be explained by a mechanism
involving rapid sorption onto the particle (sediment) surface followed by
a slower (rate limiting) intra particle diffusion controlled process.
Several short term ammonia sorption-leaching experiments also indicated
the non-biological nature of the phenomenon with rapid uptake of ammonia
from the aqueous phase to the sediments. Table 26 summarizes the results
from a three hour experiment in which 300 ml. of estuary water was added
to four flasks, and 22 g. sediment was added to two of these. An identical
amount of ammonium chloride solution was added to one of the pure water
and one of the sediment water flasks, and samples were immediately taken
from all four flasks for ammonia determinations. The flasks were kept
in a constant temperature shaker-bath and replicate analyses were also
made after one and three hours. No change was noted in the water only
flasks and in the sediment-water flask without added ammonia during the
three hours, but a rapid uptake of ammonia occurred in the sediment
flask with added ammonia. About ninety-four percent was lost from the
water within the three hour period and over sixty percent was sorbed onto
the sediment immediately as indicated by the 0 hour concentration of 2.7mg
N/l in this flask compared 6.8mg N/l in the flask with added ammonia but
without sediment.
The above experiment was also repeated using estuary water from which the
background ammonia was removed by boiling and with sediment samples from
various depths in a core (interface (top cm), middle (1-10 cm) and the
bottom of the core (below 10 cm). In no instance was free ammonia detected
in the water phase at the termination of the experiment. Uptake of various
ammonia concentrations (1.5 to 4.5 mg N/l) by 10 to 30 g. fresh sediment
was also determined using shaken flasks as above with a contact tine of 6 h
hours. Free ammonia levels in the water after incubation ranged from O.-O
to 1.8 mg N/l, but results did not conform to the Freundlich isotherm.
Finally, several experiments were conducted
102
-------
O Fresh Sediment
Sterilized Sediment
Time (days)
FIGURE 25, DECRFASE IN A^FiOUS AMMOTJIA CONCENTRATION '/ITH TF'E IM
AQUARIA V'lTH FRESH AMD STERILIZED ESTUARY SEDU^NT
-------
Table 26.
Uptake of Ammonia by Waccasassa Estuary Sediment
in Short-Term Shaker-Flask Experiment1
Estuary
Water"2
Control (no sediment)
5 min
1 hr
3 hr
0.
0.
0.
0.
11
11
11
10
Estuary Wat
Added NHj
4
6.80
2.70
1.06
0.35
1From Brooks (1969). Four flasks had 300 ml estuary water added.
Ammonium chloride solution was added to 2 flasks in identical
amounts and 22 g fresh sediment was added to one of these. Of
the 2 remaining flasks, one was a control and the other had 22 g
sediment added to it.
2Ammonia concentrations in mg N/l; controls (no sediment) remained
constant throughout the experiment.
104
-------
with blended sediments. In these cases as much as 0.34 mg NH^-N could
be obtained per gram (dry weight) of freshly homogenized sediment.
Ammonia within soil (and presumably) sediment exists both in a form
exchangeable by other cations such as sodium, calcium or potassium and
in the fixed or non-exchangeable state (Bremner, 1965; Jackson, 1958).
In an estuarine environment with high concentrations of sodium, magne-
sium, potassium and calcium present, it might be predicted that exchange-
able ammonia on the sediments would be small, and the absorbed ammonia
might be present in the non-exchangeable form. This agrees with the
above results in which little tendency for ammonia leaching was ob-
served. However it should be noted that the concentrations of sodium
and other cations in a marine or estuarine environment do not approach
the levels used in standard soil chemistry to displace exchangeable
ammonia (Jackson, 1958). Bremner (1965) has indicated that grinding
of sediments would release a portion of the fixed ammonia. This was
found to be the case with Vaccasassa Estuary sediments homogenized in
a Waring blender. Probably some of this ammonia resulted from dis-
ruption of cellular material.
The above experiments indicate that ammonia uptake and release by
Waccasassa Estuary sediments is largely controlled by strictly chemical
(sorption) phenomena. X-ray diffraction analysis of the sediment showed
the presence of small amounts of clays, primarily of a kaolinitic nature,
but further experiments are necessary to describe this sorption mathe-
metically.
Rapid losses of ammonia from solution have also been found with lake
sediments in some preliminary experiments. Figure 26 shows the ammonia
remaining in solution vs. amount of ammonia added to sediment (dry weight)
from Bivin's Arm, a highly eutrophic lake near Gainesville, Florida.
In this experiment 10 ml of fresh sediment, equivalent to 0.4 g. dry
weight, was suspended in 100 ml demineralized water in 125 ml Erlenmeyer
flasks, which were continuously shaken on a shaker table for 2 hours.
At low concentrations of added ammonia the levels after incubation were
higher than the added levels; for example at 0.1 mg N added 0.45 mg N
(both per 100 ml sample) was measured in solution after incubation.
But at higher levels of added ammonia the amounts left were relatively
small (e.g. with 5.0 mg N added 0.92 mg N remained in the 100 ml sample).
These results can be interpreted more readily in light of Figure 27
which plots the amount of ammonia leached from sediment vs. the amount
of sediment added to 100 ml of deionized water. Thus, with 0.4 g. (dry
weight) sediment suspended in 100 ml of water, 0.37 mg annonia N is
released, but the amount of leaching is highly dependent on the sediment
/water ratio. At 2.0 g. (dry weight) per 100 ml of water (a 5-fold
increase) only 0.65 mg ammonia N is released (a less than 2-fold in-
crease). These results seem to suggest that sediments from Bivin's Arm
act as a buffer to maintain a certain level of ammonia in their overlying
105
-------
9.0 .-
10 20 30 40 50
Ammonia Added (mg N/l)
DO
a
-a
0)
.o
ij
o
tfi
-a
c
o
6.0 n
4.0
2.0
-2.0
-4.0
0
1.0 2.0 3.0 4.0
Ammonia Added (mg N)
5.0
7.0
25
sc
FIGURE 26, AMMONIA UPTAKE BY LAKE SEDIMENT: (A) AMMONIA CONCENTRATION
REMAINING AFTER 2 HOURS INCUBATION AS FUNCTION OF INITIAL AQUEOUS
CONCENTRATION; (B) NET SORPTION OF AMMONIA (AMDUNT ADDED MINUS
AMOUNT REMAINING) vs INITIAL AMOUNT OF AMMONIA ADDED TO WATER
o
M
01
5.0
O
o
E
3.0
1.0
0.4 0.8 1.2 1.6 2.0
Sediment, g. (dry weight)
FIGURE 27. AMMONIA LEACHED AS A FUNCTION OF AMOUNT
OF LAKE SEDIMENT ADDED TO 100 ML DEIONIZED WATER
106
-------
(or interstitial) waters. When water low or entirely depleted in ammonia
is placed in contact with these sediments, they tend to release ammonia
to the water, but when more concentrated ammonia solutions are contacted
with the sediments they sorb the ammonia decreasing solution concentra-
tion toward some equilibrium value.
Obviously much remains to be done to characterize the equilibria and
kinetics of ammonia exchange between sediments and water. However, the
above experiments indicate that rapid and large exchanges apparently
mediated by simple sorption phenomena are possible for nitrogen as well
as for phosphorus. While the nitrogen cycle within the water column
is nearly exclusively a biochemical phenomenon, non-enzymatic processes
may be more important within sediments and at their interface with water.
While sediments can act as either in situ sources or sinks, denitrifi-
cation acts solely as a sink. As described in Section IV, this reaction
occurs in water when oxygen is depleted and is mediated by facultative
bacteria who use nitrate as a terminal electron acceptor (in the place
of 02) with the major reduction product being N2. However, varying
amounts of ammonia and organic nitrogen are apparently always produced
in situ simultaneous with denitrif ication, as has been shown by a num-
ber of investigations (see Section IV) including one supported by this
project (Tang. 1968). In the latter study from 70 to 80 percent of
added nitrate nitrogen was recovered as N~ in laboratory sludge digesters,
and the remainder was reduced to ammonia and organic nitrogen. This
phenomenon occurred even at high ammonia levels where nitrate assimi-
lation would seen a superfluous nitrogen source. It is not possible to
ascertain from such mixed culture experiments as these and others
(B-ezonik and Lee, 1968; Goering, 1968; and Keeney £t al., 1971) whe-
ther the biological agents of denitrification are the same as those
reducing nitrate to the level of ammonia, but from an ecological view-
point this is perhaps unimportant. What is important however is that
both reactions occur whenever oxygen is depleted in a nitrate containing
environment. Thus denitrification (i.e. nitrogen lost to the system)
is not equivalent to nitrate lost and any attempts to assess the sig-
nificance of denitrification as a nitrogen sink must take this into
account.
Denitrification in Florida lakes would seem to be of minor importance
although there may be unique situations where this statement is not
valid. In the study of 55 lakes in north central Florida (Brezonik,
197Ic) only about 8-9 of them appear to develop stable summer strati-
fication, and of these only 4 develop anoxic conditions in a signifi-
cant volume of their hypolimnia. In addition several other lakes
develop temporary stratification and lose their oxygen in the bottom
half to one meter, probably because of a high sediment oxygen demand.
Denitrification would be possible in these regions; however from a
quantitative point of view they represent only a minor fraction of the
water volume in the lakes. Further nitrate concentrations in Florida
lakes are typically low. Most of the waters are soft and acidic, and
nitrification is not favored by such environments. Hence most of the
inorganic nitrogen in Florida lake waters occurs as ammonia. In those
lakes which stratify nitrate levels greater than 0.10 mg N/l are rare.
107
-------
Thus, denitrification does not appear to be a significant nitrogen sink
in Florida lakes and it vas felt that a detailed evaluation of its
occurrence was not worth pursuing. On the other hand, denitrification
may be important in areas of Florida where nitrate rich ground water
seeps through organic and anaerobic sediments or where surface drainage
enriched in nitrate from agricultural fertilization passes through
anoxic soils or sediments. However, this aspect was beyond the scope
of the present project.
108
-------
SECTION IX
ANALYTICAL INVESTIGATIONS
While research on the analytical aspects of nitrogen in natural waters
was not a primary function of this project, some work was undertaken to
evaluate procedures used in the main phases of this research. Because
the acetylene reduction method was used extensively in the nitrogen fix-
ation studies and since this is a relatively new method (first reported
by Stewart et_. at. , 1967), an evaluation of the procedure was considered
essential. Also, a large number of inorganic nitrogen (ammonia and
nitrate) analyses of natural waters were performed during this project
and also in conjunction with other investigations on lake eutrophication
conducted in this laboratory. A number of difficulties were encountered
..-;ith the usual automated procedures for these constituents, especially
interferences arising from the high color content of many Florida lakes.
Consequently a study of these procedures was undertaken to devise methods
of overcoming thesa interferences. The acetylene reduction assay ralies
on the fact that acetylene acts as a competitive inhibitor of nitrogen
with the enzyme nitrogenase, which reduces it to ethylene. Production
of the latter gas can be quantified by flame ionization gas chromato-
graphy. and the method, though an indirect assay, is rapid, highly
sensitive, and inexpensive. Laboratory experiments were conducted to
determine optimum incubation conditions, to evaluate the method as an
indirect assay for nitrogen fixation, and to study environmental con-
ditions affecting nitrogen fixation by blue-green algae.
The basic procedures used for acetylene reduction assays of sediments
and waters are described in Appendix A. Figure 28 shows a typical
chromatogram obtained from one cc. of gas phase in an incubated sample.
The column of Poropak R cleanly separated ethylene from any traces of
ethane (sometimes present in the acetylene) and from the large peak
of acetylene. h'arly in the development of this technique for assays
of lo*T fixation rates in sadiments it was recognized that controls were
necessary with each set- of samples. These consisted of samples carried
through the basic sequence (Appendix A) except that 1 nil of 50 percent
trichloroacetic acid was added before inserting the acetylene. Con-
trols were necessary because rather wide variations of background
ethylene occurred in the purified" acetylene used. Ethylene levels
were especially high in full tanks, but contaminant levels declined
with use. In order to avoid complications arising from very high
backgrounds it was ^our.d expedient to waste about the first third of
a t ank.
Sediments for these evaluation studies wsra obtained from the Wacea-
sassa Estuary (Gulf Coast of Florida) and from Lake Kanapaha, near
Gainesville/Florida. Water samples for this phase were obtained from
109
-------
CMROMATOGRAPH
RESPONSE ^
APPROXIMATE TIME OF TRAVEL (MIM)
o
0.5
n
"*
cr«
2.0
2.5
4.0
INTEGRATOR
TRACING
FIGURE 28, TYPICAL CHTOMATOGRAM ILLUSTRATING IDEMTIFICATION
OF PEAKS AND MEASUREMENT OF PEAK AREA FOR ACETYLENE REDUCTION ASSAY,
-------
a small pond on the University of Florida golf course, which conveniently
has a dense bloom of Anabaena (2 species) throughout the summer. Table
27 presents replicate data for 3 pond samples incubated for one hour
and 8 samples incubated for 2 hours under laboratory conditions. Mean
C2H^ production from the two hour incubations was 51.0 ± 1.9 nmoles
c2H4/m£ N-hr. at the 95% confidence level. The mean one hour incuba-
tion rate was 84.2 nmoles/mg N-hr. The replication is obviously within
acceptable limits for biological phenomena. However, the rates decreased
rather significantly with longer incubation times, probably because of
bottle effects. Possibly, removal of N2 from the organisms causes nitro-
gen starvation over this period of time. Table 28 presents results of
a replication using Waccasassa Estuary sediments. A mean of 11.5 ng,
ethylene per g. dry wt. sediment-hr. with a relative standard devia-
tion of 3.6 percent was found for six replicates, again indicating the
methodogy has adequate precision. Figure 29 shows the effect of incu-
bation tine on the amount of ethylene produced by estuary sediments.
Up to about one hour the rate appears linear, but after that it drops
markedly. Thus the efficacy of short incubations is apparent.
The effect of light and the presence of N2 on the time course of acety-
lene reduction was also studied on water from Golf Course Pond. One
set of samples was purged with the 02-C02-Ar mixture to remove N2;
another set was left unpurged. A set of purged and unpurged samples
were each incubated in the light and in the dark for times ranging from
0.5 to 3 hours (Figure 30). Correlation of nitrogen fixation rates
with primary production is evident from the much higher rates of ethy-
lene production in the light than in the dark. However, fixation is
evidently not completely dependent on photosynthesis to supply reduced
hydrogen (NADPH2) for fixation; otherwise fixation would have ceased
nearly immediately after light was removed. Reduced hydrogen apparently
is also supplied by respiration (oxidation of organic compounds). In
all cases, the rate of ethylene production decreased with longer incu-
bation times but the effect was most dramatic in the light incubated,
unpurged set. Rates were linear for about an hour in the light and
for about the same time in the dark although variability in the dark
incubation data do not permit great accuracy in this conclusion. The
decline in production with time could result from bottle effects, nitro-
gen starvation (in purged samples), organism shock from manipulation
or from incubation conditions (since temperature and light conditions
in the lab differed from the natural conditions). Removal of N2 did
not significantly affect the rates measured in dark incubations, but
these rates were low and not as precise. In the light, the presence
of N2 has a depressant effect, especially over longer incubation times.
However the magnitude of the depression is not too large to preclude
the direct addition of acetylene to unpurged samples if field conditions
prevent purging. Based on Figure 30 results obtained in this manner
would be 25 to 30 percent too low (assuming incubations of 0.5 to 1
hour) but this error may be acceptable in surveys or in difficult field
circumstances where transport of purging tanks would be cumbersome or
otherwise undesirable.
The effect of N2 on acetylene reduction was studied in greater detail
in a further experiment on Golf Course Pond water. Since C2H2 is
111
-------
Table 27. Replicate Data on Nitrogen Fixation by
Natural Population of Anabaena sp. from Golf Course Pond
Sample No.
1
2
3
4
5
6
7
8
9
10
11
Incubation
Time
(Hours)
1
I
I
2
2
2
2
2
2
2
2
C2H, Produced
Nanomoles
12.0
11.2
9.7
11.6
13.7
13.6
16.6
16.9
16.9
12.8
15.6
Fixation Rate
nrooles 02*1 //mg N-hr
85.7
86.2
80.8
48.3
48.9
52.3
55.3
52.8
52.8
53.3
52.0
1See text for details of incubation and statistical analysis
of results.
112
-------
Table 28. Replication of Acetylene
Reduction by Estuary Sediments
Sample
Dry Weight
g.
4.827
4.631
4.971
4.961
4.582
4.760
Ethylene
Nanomoles
2.00
1.83
2.16
2.08
1.83
1.91
Production
nM/g. dry wt
0.414
0.396
0.436
0.418
0.400
0.403
x = 0.410
s2 = 0.0062
113
-------
M
JZ
u 20
-o
00
CM
u
00
c
o
^
o
u
PL,
0)
0>
W
10
0
t
O
i.o
2.0
Incubation Time (hr)
3.0
4.0
FIGURE 29, EWLENE PRODUCTION VS, TIME FOR WACCASASSA ESTUARY SEDIMENTS
-------
100
0)
4J
td
P
o
t-i
03
80
oo
0^60
'O
Q)
0
3
T3
O
J-i
0)
c
4J
40
20
1.0
2.0
Tine (hr)
3.0
FIGURE 3H, TIME COURSE OF ACETYLENE REDUCTION BY NATURAL ANA?AEf'A "OPULATIO'
UNDER CONDITIONS OF LIGHT AND DAR!< IM T-E PRESENCE OF 1,78 ATM :^
(opE^! CIRCLES) AND r^ T-F. A~SE;CE OF ',q (CLOSED CIRCLE),
-------
apparently competitive inhibitor of N^ the converse should be true,
and a reciprocal (Lineweaver-Burk) plot of reaction velocity vs. sub-
strate concentration should give a competitive inhibition pattern
(Figure 31) . This pattern was found for the natural Anabaena popu-
lation in the pond (Figure 32). In this experiment varying amounts
(05 1.0, 3.0, 5.0 ml) of acetylene (the substrate) were added to
serum bottles with 25 ml of pond water both in the presence of inhi-
bitor N? (0.78 atm) and its absence (by purging air from the bottles
with the 02-CO?-Ar gas mixture). Samples were incubated for one hour
in the light at 22°C. Competitive inhibition patterns (Cleland, 1963)
are given by inhibitors that combine with or react at the same active
site on the enzyme as the normal substrate. The molecular similarity
of N9 and C7H9 suggests they would react at the same site on nitroge-
nase and that each should thus competitively inhibit reaction of the
other. Schollho'rn and Burris (1966, 1967) found competitive inhibi-
tion of N2 reduction by acetylene, but Burris (1969) reported that
Hwang and Burris (1968) did not.
Differentiating between competitive inhibition and other patterns
(e.g. non-competitive inhibition) is not always a simple or unequi-
vocal matter. The distinguishing feature of the former pattern is
that the reciprocal plots for different inhibitor levels intersect on
the ordinate; in the latter pattern the plots intersect to the left
of the ordinate. The physiological significance of this is that in
competitive inhibition the maximum reaction velocity (V_ ) is un-
affected by the presence of inhibitor. Vmax is obtained from the
y-intercept of the 1/v vs. 1/S curve on a Lineweaver-Burk plot; at
1/S = 0, S = °° or in physical terms S is at a saturating concen-
tration'and v is at a maximum level ( = V ). Since the y-intercept
is unaffected by inhibitor in competitive inhibition (see Figure 31),
V is unaffected, meaning that very high (saturating) levels of
substrate can completely overcome inhibitor effects. If inhibitors
change the Lineweaver-Burk plot y-intercept values, Vmax is changed,
and even saturating levels of substrate cannot completely overcome
inhibitor effects. Note however, that placement of lines through 1/v
vs. 1/S data is subject to a certain amount of experimental error.
Whether or not curves for varying inhibitor levels actually intersect
exactly on the y-intercept is always subject to some uncertainty,
the magnitude of which increases with scatter of the data.
What is perhaps more significant about Figure 32 than whether the data
yield an exact pattern of competitive inhibition is that acetylene
reduction rates can be described by Michaelis-Menten kinetics since
the reciprocal (Lineweaver-Burk) plots are linear (within experimental
error). This fact supports the enzymatic basis for acetylene reduction,
and the fact that N2 inhibits (in some way) the rate of acetylene re-
duction is further evidence that ethylene production from acetylene
is mediated by nitrogenase and is therefore an indirect assay of nitro-
gen fixation. There is at present no evidence in the literature for
the production of ethylene from acetylene by any non-nitrogen fixing
organisms; however the possibility that such organisms might exist
should be kept in mind. These techniques (i.e. determination of N2
inhibition and of fit to enzyme (Lineweaver-Burk or Michaelis Menten)
116
-------
1 = 0
I/(Substrate)
FIGURE 31. THEORETICAL LINEWEAVER-BURK PLOT FOR COMPETITIVE INHIBITION
OF ENZYME REACTION RATE, WHERE h/ I2/ 13 REPRESENT INCREASING INHIBITOR LEVELS,
0.08 P-
i
z.
0.06
sc
E
0.04
0.02
0.5 1-0
l/(Substrate) (cc C2H2)"
FIGURE 32, COMPETITIVE INHIBITION OF ACETYLENE REDUCTION
IN NATURAL ANABAENA SP, POPULATION BY ^2'
117
-------
kinetics) should be useful in relating ethylene production in an
environmental sample to nitrogen fixation, and they were used for
this purpose in the studies of fixation in sediments (Section VII).
As a further evaluation of the acetylene reduction method, the effects
of two other nitrogenase inhibitors on C2H, production were studied
with Golf Course Pond water containing Anabaena sp. Both carbon mono-
xide and nitrous oxide are reported to inhibit nitrogen fixation.
Presumably, they should also inhibit nitrogenase reduction of C^^2
to C^i,- In one experiment varying amounts of 02^ and CO were added
to pond samples and incubated in the light for one hour. The results
(Figure 33) indicate that CO is a very potent inhibitor of acetylene
reduction, and the data seem to fit a Lineweaver-Burk plot (Figure 34).
However, for some inexplicable reason the curves for various CO levels
intersect to the right of the y-axis. The author is unaware of any
enzyme kinetic expressions yielding this pattern, and this aspect of
the result would seem an artifact. Schbllhorn and Burris (1966, 1967)
found competitive inhibition of C2H2 reduction by CO, but Hwang and
Burris (1968) did not. Perhaps the intact organism systems are too
complex to consistently yield the simple, pure enzyme inhibition
patterns. The inhibition of C2H2 reduction by CO is good evidence for
the biochemical nature of the reduction, but the data are inconclusive
regarding the nature of the enzyme inhibition pattern and hence the
mechanism of inhibition.
In another experiment, samples of Bivin's Arm lake water were purged
to remove ^j and varying amounts of C2H2 and N20 were added. The re-
sults indicated a typical Michaelis-Menten response to added C2H2» but
no significant inhibition by N^O was found in the range 0.04 to 0.20
atm (Figure 35). There is no information in the literature on the
range and nature of ^0 inhibition of nitrogenase, but Hardy et al.
(1968) reviewed other studies showing that nitrogenase reduces N^O to
N2- Possibly ^0 is much more loosely bound by nitrogenase than is
C2H2, so that even at the lowest C£H2 concentration and highest ^0
concentration, C2H2 may essentially saturate the enzyme active site,
at least in comparison to ^0. The lack of inhibition by ^0 cannot
be explained by solubility differences, for N20 is somewhat more solu-
ble_than C2H2, respective Henry's Law constants at 20°C being 1.98 x
10"* and 1.21 x 10~3. Regardless of the explanation for these results,
^0 would not appear useful in inhibition studies with nitrogenase.
It is well known that nitrogenase is an adaptive enzyme and that cells
fix nitrogen only when other available sources (^3, NOp are depleted.
In this sense, ammonia can be considered to inhibit fixation. The
question arises whether ammonia inhibits the activity of existing
nitrogenase or represses synthesis of new enzyme. Another pertinent
question concerns the level of ammonia which produces enzyme inhibition
or repression of enzyme synthesis. Recent data by Hardy ej^ al. (1968)
indicates that ammonia does not directly inhibit fixation but merely
represses synthesis of nitrogenase enzyme. According to Stewart (1968)
levels of ammonia in the environment are unlikely to be high enough
to inhibit fixation immediately, but the quantitative aspects of this
question are poorly understood. The immediate and longer term effect
118
-------
60
~ ^5
p*
i
30
c
c
| 15
cu
c
01
1 cc C?H9
3 cc
5 cc C H
2
0
0 1.0 2.0 3.0 4.0 5.0
Carbon Monoxide (cc. added)
FIGURE 33, INHIBITION OF ETHYLENE PRODUCTION IN NATURAL ANABAENA
POPULATION BY CARSON MONOXIDE AT SEVERAL LEVELS OF SUBSTRATE (ACETYLENE)
s:
c
0.4
0.3
P. 0.2
c-
No Co 0
1 cc CO 9
0.2 0.4 0.6 0.8
1/S = I/(Acetvlene) (cc C^)"1
FIRIRE 34, REpLOT OF DATA FROM FIGURE 33 ACCORDING TO
Llf-JE'/EAVER-BURK EXPRESS ION,
119
1.0
-------
100
4-J
C3
u
80
CO
a.
,60
o
c
o
o
P.-;
0)
IH
£ 20
1.0
4.0
2.0 3.0
Acetylene (cc added)
FIGURE 35, ETHYLENE PRODUCTION RATES FOR NATURAL AWBAENA
5.0
DOPULATION VS ACETi^E AT FOUR LEVELS OF NITROUS OXIDE
SHO's' ^ SIGNIFICANT INHIBITION, SYhBOLS: CLOSED CIRCLES, 0 ATM
OPFN CIRCLES/ 0.04 ATM N; DOTS, 0.12 ATM r; TRIANGLES, 0,20 A
120
-------
of ammonia on nitrogen fixation by a natural Anabaena population
(from Golf Course Pond) was studied in an attempt to clarify the
matter. Two liter samples from the pond were inoculated with
0.10 and 0.50 ing NH-^-N/l and one control (no ammonia added) was also
set up. Samples were taken from each bottle for acetylene reduction
assay within 20 minutes of ammonia addition, and a second set of sam-
ples were assayed after the bottles had been incubated in situ for
24 hours. The pond had an especially heavy bloom of Anabaena and
Microcystis species at this time. Samples for ammonia analysis were
taken initially, immediately after ammonia addition and at the end of
24 hours. Results from this experiment are presented in Table 29 and
Figures 36 and 37. Ammonia (up to 0.5 mg N/l) has no direct inhibitory
effect on C2H2 reduction; i.e. the initial samples showed no decrease
in C2H^ formation with increasing ammonia concentration. There was a
depression in C2H^ production possibly related to ammonia concentration
after 24 hours incubation; however, the control bottle also showed a
lower rate after the twenty-four hour incubation. This was probably
caused by bottle "wall effects," e.g. death of some N fixing organisms.
Furthermore the differences in acetylene reduction between the control
and ammonia enriched samples after 24 hours were much larger (on a rel-
tive basis) for the lowest acetylene concentrations (Ice/bottle) than
for the highest acetylene levels. Experimental errors tend to be lower
at higher activities and higher substrate levels, so more weight should
be put on these values. Lineweaver-Burk plots tend to emphasise the
differences found at lowest rates and substrate levels, where precision
is poorest, and in this sense such plots can be misleading. Thus the
effect of ammonia on nitrogen fixation after 24 hours is somewhat am-
biguous. A slight depression seems evident, even considering the above
cautionary comments, but the difference in rates between the control and
the sample with 0.5 mg N/l added was only about 8 percent (for the 5cc
acetylene samples). These results therefore suggest that ambient
ammonia levels (which in lakes are infrequently higher than 0.5 mg
NHo-N/1) should not directly influence nitrogen fixation rates at least
over short periods of time.
The second analytical study performed in relation to this project con-
cerned the evaluation of automated inorganic nitrogen methodologies,
particularly in reference to interferences from organic color and
dissolved free amino acids. As part of our investigations of trophic
conditions in Florida lakes (Brezonik, 1971c) and in relation to the
studies of nitrogen in these lakes conducted as part of this project,
a large number of ammonia, nitrite and nitrate analyses have been per-
formed in this laboratory over the past several years. Because of the
volume of work involved, analyses were performed by automated (Technicon
Auto Analyzer) techniques. In the nitrite and nitrate procedures
chemistries employed in Standard Methods (A.P.H.A., 1965)(i.e., the
diazotization of sulfanilic acid and a-naphthylamine by nitrous acid
to form a pink dye and the well-known brucine method for nitrate) were
automated directly.
The Standard Methods procedures for ammonia are either inappropriate
for the waters being analyzed (viz. direct Nesslerization) or are un-
suitable for automation (viz. distillation and Nesslerization or titra-
121
-------
Table 29. Ammonia Inhibition of Nitrogenase in a
Natural Population of Anabaena sp.
Initial Effect
Effect After 24 Hours
Ammonia
Added1
None
(Control)
0.1
0.5
Measured C2H2
Ammonia Added
0.00 0.5
1.0
3.0
5.0
0.09 1.0
3.0
5.0
0,48 1.0
3.0
5.0
Production
Rate of C2H^3
19.2
34.2
102
111
33.9
127
117
31.7
104
123
Measured ^2^?
Ammonia Added
0.03 1.0
3.0
3.0
5.0
0.06 1.0
3.0
5.0
0.09 1.0
3.0
5.0
Production
Rate of C2H^3
2.40
5.28
58.0
67.8
21.3
42.7
60.0
18.5
38.1
62.3
mg N/l
cc. acetylene to 70 ml serum bottle with 25 ml water sample.
3nM C-H, /1-hr . ; also equal to nM
water had 1 mg particulate N/l.
particulate N-hr. since the
122
-------
O Control
0.1 mg N/l added
A 0.5 mg N/l added
0.5 1.0
I/(Substrate)
(cc
1.5
^-l
2.0
FIGURE 35, ADDITION OF AfWNIA TO NATURAL AMA3AENA SP,
POPULATION HAS NO IMMEDIATE EFFECT ON RATE OF NITROGEN FIXATION,
123
-------
0.25
0.50
l/(Substrate)
(cc
0.75
T N-l
1.00
FIGURE 37, EFFECT OF AMMONIA ON NITROGEN FIXATION BY NATURAL ANABAENA
SP, POPULATION AFTER INCUBATION WITH ADDED NH3 FOR 24 HOURS,
OPEN CIRCLES/ NO AMMONIA ADDED; TRIANGLE/ 0,10 MG N/L
ADDED; CHOSED CIRCLES/ 0,50 MG fVL ADDED, DASHED LINE
REPRESENTS INITIAL LINEVJEAVER-BURK LINE FOR ALL SAMPLES (SEE FIGURE
124
-------
N ==0 + HC1 (fast)
tion) . Rather the most common automated method for ammonia employs
the Berthelot or indophenol reaction in which ammonia, hypochlorite
and alkaline phenol react to form a product believed related to indo-
phenol. The product is intensely blue with an absorption maximum
around 630 nm. Methods employing this reaction are highly sensitive,
but because of its complex chemistry, irreproducible results often are
obtained by manual procedures. Bolleter et_ jil_. (1961) have proposed
the following reaction sequence;
1) NH3 + HOC1 t NH2C1 + H20 (fast)
2) C1H2N ~\ ^ } QH + 2HOC1 -> Cl-N =( VO + 2H20 + 2HC1 (slow)
3) HO-=+ Cl-N =
(fast)
indophenol blue
But the overall reaction is doubtless more complex than this. Weich-
selbaum et_ al. (1969) noted that the reaction has a "very odd behavior"
with respect to temperature, order and timing of reagents additions,
reagent concentrations, and so forth. They felt that many side and
competing reactions (some irreversible) result in a situation where the
same equilibrium condition is never reached twice. Many of the pro-
blems associated with this method are overcome by use of the Auto Ana-
lyzer since all samples and standards are treated identically with res-
pect to order and timing of reagent addition, temperature, reaction
time before measurement, etc. With the Auto Analyzer this method
essentially becomes a kinetic analysis, i.e. the concentration of
ammonia in a sample is determined by the amount reacted after a cer-
tain time, not by the amount of product that would be formed at equili-
brium. However, because of this fact, the method is particularly sus-
ceptible to errors caused by catalysts or inhibitors, which nay occur
in samples but not in standards, causing variable acceleration or dec-
celeration of reaction rate. Iron, chromium and manganese are known to
catalyze the reaction, while oxidants such as persulfate inhibit color
development. It has also been reported (Russell, 1944) that certain
amino acids respond similarly to ammonia in this procedure.
A less common automated ammonia procedure is the Grasshof method. This
sensitive method was developed for sea water analysis (Grasshof, 1966)
and is listed as Technicon Auto Analyzer "Industrial Method 42-69W." The
chemistry of this procedure is based on the formation of a bromamine
intermediate from the reaction of ammonia and bromine. The intermediate
reacts with iodide to form iodine, which combines with starch to form
the familiar starch-iodine blue complex with an absorption maximum at
570 nm. The method is attractive in certain respects, but it has a
highly critical and narrow pH range. This is not an overwhelming pro-
blem in seawater analysis since seawater is well buffered within a
narrow range, but it is a problem in fresh waters. For example, typical
125
-------
pH values of the 55 Florida lakes ranged from about 4.5 to 9.5 or more
(Shannon and Brezonik, 1971c). The Grasshof method was tried in this
laboratory for some time, but satisfactory results could not be obtained
with Florida waters, and the procedure was abandoned.
Organic color is a common but highly variable constituent of Florida
lakes. Many of the oligotrophic lakes in the sand hills are essentially
colorless, but in lakes receiving swamp or pine forest runoff color
concentrations may reach several hundred units (on the Pt. scale). In
fact color levels up to 700 units have been found in Lake Mize. Since
the analytical procedures for inorganic nitrogen analysis all involve
measurement of color produced by a chemical reaction, it was felt essen-
tial to evaluate the influence of naturally occurring organic color on
these tests. Empirical evidence indicated at an early date that color
was affecting the results of inorganic nitrogen analyses. In particular
more problems were encountered with the ammonia analyses than with nitrite
and nitrate. Assuming no interaction between organic color and the
reagents in the various nitrogen analyses, one would expect the degree
of interference from organic color to be greatest for nitrate and least
for ammonia since organic color absorbs most strong at the yellow end
of the visible spectrum (around 400 nm) . The absorption maxima for the
nitrate (brucine), nitrite (diazotization) and ammonia (indophenol)
procedures are 410,520 and 630 nm., respectively.
To apply compensation methods to these analyses requires careful consi-
deration of the reactions involved. In external compensation, whereby
a "key reagent" is removed from the analytical stream to provide a color
blank, the "key reagent" must fulfill several criteria. Removing it
from the system must not affect the baseline, and since organic color
intensity is pH dependent, reagent removal must not alter the reaction
mixture pH. Finally, the key reagent must not react with organic color,
and its removal obviously must completely eliminate color formation in
the reactions involving the analyte. For the most part, selecting rea-
gents which met these criteria was accomplished by trial and error.
It was found that sodium hypochlorite was the most suitable reagent to
remove in the indophenol procedure for ammonia. Removal of brucine
from the mixed reagent of the nitrate procedure was found satisfactory,
and in the case of nitrite analysis, removal of the N (1-naphthyl) -
ethylenediamine reagent met the established criteria. In order to keep
the concentration of the remaining reagents unaltered, deionized water
was used to replace the key reagent when color blanks were run.
To determine whether the selected key reagents allowed satisfactory
correction of analyses by external compensation (i.e. color blank sub-
traction), a series of experiments were undertaken with the various
analytical procedures. For each analytical procedure a standard or
calibration curve was prepared in the usual manner using the range of
concentrations normally selected for the particular constituent. A
second, similar curve was prepared by adding an identical amount of
organic color concentrate to each standard, thus obtaining a series of
artificial colored waters having various concentrations of one of the
inorganic nitrogen constituents. The color concentrate was obtained
by vacuum evaporation at a temperature of about 40°C of colored lake
126
-------
water from Lake Mize in a manner similar to that of Black and Christ-
man (1963). The original sample was concentrated about 50-fold and
then filtered through a 0.45 ym Millipore filter and passed through
a mixed bed ion exchange resin to remove concentrated inorganic ions.
The "purified11 concentrate was then stored at 4°C in the dark in filled
glass containers to minimize oxidation and photodecomposition. The
inorganic nitrogen analysis was repeated on each of the color ' spiked'1
samples resulting higher absorbance values for each sample compared to
the absorbances of corresponding concentrations in the standard solu-
tions without color. Kext, the key reagent was removed from the analy-
tical stream and color blanks were run on each sample. The results for
each constituent were plotted on one graph and a curve representing
the difference between the absorbance values of color "spiked
standards and their corresponding color blanks was computed and also
drawn on each graph.
The results for nitrate, nitrite and ammonia are shown respectively
on Figures 38, 29, and 40. In the case of nitrite and nitrate it is
apparent that subtraction of the color blank values from the absor-
bances of the color "spiked" samples results in curves which coincide
exactly with the calibration curve using pure standards. This is good
evidence that no interaction takes place between the key reagents and
organic color, and the results indicate that the external mathod of
compensating for effects of organic color can be successfully applied
to nitrate and nitrite analyses by these procedures. The validity
of this correction technique for nitrate and nitrite assays was veri-
fied by attempting recovery of a known amount of standard from solutions
of varying color. Solutions of 0.10 mg NOj" - N/l were spiked with
color concentrate in levels ranging from 17 to 405 units, and nitrite
standards were spiked with color to give final concentrations ranging
from 12 to 287 units. The analyses and color blanks were determined,
and the latter values subtracted from the former gave the corrected
value. Table 30 summarizes the results and shows that application of
external compensation yields good agreement with known concentrations.
Results from the ammonia experiment (Figure 40) were at considerable
variance with the above data. Subtraction of the color blank curve
froa the absorbance curve for the spiked standards produced a curve
(D) that was still higher than the calibration curve. Further the
slopes of the color "spiked" curves were greater than that of the cali-
bration curve, yielding an increasing divergence between them at in-
creasing ammonia levels. Thus, the differences between the calibration
curve and the curves for the colored samples cannot be explained
solely by assuming that some ammonia was added to the test solutions
via the color concentrate. Since all samples received the same amount
of color concentrate, the diverging nature of the curves remains un-
explained. Interaction of the organic color or of some constituent
associated with organic color with the reagents seemed likely. This
interaction is further shown by the data shown in Figure 41. These
results were obrained by preparing six series of standards solutions.
Each series was given a different color, ranging from 0 units for
series A to 244 units for series F; within a given series of ammonia
standards (ranging from 0 to 1.0 mg N/l) each solution had an equal amount
of color. The increasing divergence of the curves as color increased
127
-------
0.28
0.24
0.20
O 0.16
o
C/3
0.12
0.08
0.04 -
0.0
O "Spiked" Colored Water (A)
* "Color Blank" (B)
* Calibration Curve and (A)-(B)
0.2 0.4 0.6
Nitrate (np N/l)
0.8
FIGURE 38, EFFECT OF ORGANIC COLOR ON APPARENT NITRATE IN
BRUCINE TEST CAN BE ACCURATELY CORRECTED FOR BY .METHOD
OF BLANK SUBTRACTION, COLOR LEVEL IN TEST SOLUTIONS
WAS 63 UNITS (PT SCALE) AT p,H 8,3,
128
-------
0.16
0.14 ,
0.12
0.10
0.08
0.06
0.04
0.02 _
O "Sulked" Colored Water (A)
3 "Color Blank" (B)
Calibration Curve and (A)-(B)
Nitrite
6 8
(yg N/l)
FIGURC 7\ EFFECT OF ORGANIC COLOR ON APPARENT NITRITE CONCENTRATION
CAN BE ACCURATELY CORRECTED FOR BY METHOD OF BLANK SUBTRACTION,
ORGANIC COLOR IN TEST SOLUTIONS WAS W UNITS (FT SCALE) AT PH t.*>,
129
-------
,36
,32
,28
.24
.20
LU
CJ
.16
.12
.08
.04
A = Calibration Curve
B = "Spiked" Colored water
C = "Color Blank"
D = B-C
0.0
0.2 0.4
Ammonia
1.0
0.6 0.8
(mg N/l)
P 40, FFRCT OF ORGANIC COLOR ON APPARENT AWONIA CONCENTRATION
CANNOT BE ELIMINATED BY BLANK SUBTRACTION, ORGANIC COLOR LEVEL
IN TEST SOLUTIONS WAS 1P5 UNITS (PT SCALE) AT pH 8,3,
130
-------
Table 30. Application of External Compensation
Method to Nitrate and Nitrite Samples
Containing Varying Color Concentrations
Synthetic
Water
A
B
C
D
E
F
G
H
A
B
C
D
E
F
G
Color
Units
0
17
25
50
84
119
208
405
0
12
21
50
130
173
287
Known
N03-N,
mg N/l
0.10
0.10
0.10
0.10
0.10
0.10
0.10
0.10
N02 -N ,
yg N/i
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Apparent
N03-N,
mg N/l
0.10
0.10
0.12
0.15
0.16
0.18
0.25
0.38
N02 -N ,
yg N/I
5.0
5.8
6.0
6.9
9.7
11.2
17.2
Color
Blank
as N03-N,
mg N/l
0.00
0.02
0.03
0,05
0.07
0.10
0.17
0.30
as N02-N,
yg N/I
0.0
0.8
0.9
2.0
4.8
6.1
12.2
Corrected
N03-N,
mg N/l
0.10
0,08
0.09
0.10
0.09
0.08
0.08
0.08
N02-N
yg N/I
5.0
5.0
5.1
4.9
4.9
5.1
5.0
All analytical values are the average of three replicates
131
-------
0.36
0.32
0.28
0.24
0.20
o
t/5
0.16
0.12
0.08 -
0.04 -
0.0
B
A
Color Units
0,0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 9.9 1.0
Ammonia (mg N/l)
FIGURE 41, EFFECT OF INCREASING COLOR CONCENTRATION ON
CALIBRATION CURVES FOR ALKALINE-PHENOL AMMONIA PROCEDURE
132
-------
supports the suoposition that organic color (or some associated con-
stituent) interacts with the reagents in a manner which precludes
correction by external compensation. In effect addition of organic
color concentrate caused the analytical reaction to proceed toward com-
pletion at a rate increasing in proportion to the amount ef color plus
the concentration of ammonia present.
A. possible explanation for these effects derives from consideration of
the nature of the analytical reaction (see p. 1?5) for the indophenol
technique and the nature of organic color. The slow or rate determin-
ing step of the indophenol reaction involves the reaction of monochlora-
nine with phenol (step 2) . Organic color is known to be aromatic in
character with many phenolic groups present (Christman and Ghasserai, 1966).
Thus, some of these phenolic groups could possibly react with
monochloramine under the reaction conditions. In effect, the presence
of organic color would increase the concentration of phenol, shift the
equilibrium of step ?. to the right, and thus, accelerate the rate deter-
mining step. As mentioned earlier, the Auto Analyzer measures the
reaction products in this test before the reaction reaches completion.
Thus, varying amounts of color would cause various responses to the same
ammonia concentration.
If organic color reacted in the manner just described it might be
supposed that the formation of extraneous color products (i.e. con-
taining substituted phenols from organic color) would cause a shift in
the absorption spectrum of the reaction products. This possibility was
studied by measuring the spectra for several reaction mixtures, each
containing 0.5 me y^-N/l but with color concentrations from 0 to 378
units (?t. scale). However, the abosrption maximum for all samples was
the same (63<~t nm.), and the shapes of the spectra were identical
(^igure 42). Thus, the possibility of side reactions with phenolic
groups from organic color is not substantiated, but it cannot be ruled
out solely on this experiment.
An alternate, perhaps simpler explanation for the results with ammonia
and organic color is the presence of the catalyst in the color concentrate.
It is well known that organic color is frequently associated with high
iron levels, and in fact hake v±z& water has a mean iron concentration
of 2.4 me/1 (Table 17). Since this iron is associated with the organic
color, perhaps as P chalate, or even as an integral part of the color
molecule, nassine the concentrate through an ion exchange resin would
not separate all the iron from the concentrate. As mentioned previously,
the indophenol procedure is catalyzed by iron, and in fact some refer-
ences on the indophenol procedure call for the use of sodium nitro-
prusside (i.e. sodium pentacyanonitrosyloferrate) to catalyze the reaction,
(e.g. see Weichnelbaum zt_. .<_., 1%°) . This reagent was not generally
used in cur analyses since sensitivity ^s sufficient without it. Fur-
ther, solutions of this species are unstable and have a relatively short
life tin-p, and the appropriate concentration for proper catalysis is
fairlv critical. That iron associated with organic color is the cause
Of t-n^ r>roblem with the indophenol procedure was indicated by some
recent experiments in our laboratory. Addition of the sodium nitro-
prusside catalvst seems to eliminate th= interference due to color
(o^c/^nt for ti'is interference due to absorption ^y color itself, which
133
-------
AMMONIA LEVEL: 0,5 mg N/l
1.10
1.00
0.90
0.80
0.70
;0.60 -
c
C/5
0.50 -
0.40
0.30 .
0.20
0.10 -
0.00
83
249
378
0 Color Units
it n
it it
II U
480 510 540 570 600 630 660 690 720 750 780 810
Wavelength (nm)
FIGURE 42, ABSORPTION SPECTRA FOR PRODUCTS OF ALKALINE
PHENOL-AMMONIA PROCEDURE AT VARIOUS LEVELS OF ORGANIC COLOR
134
-------
can be corrected by the external compensation method). However, further
work will be required to verify this and to determine the optimum level
of catalyst to add.
Free amino acids have been known, to exist in lake water since the work of
Peterson et. al. (1925) on Lake Mendota, Wisconsin. Since then low levels
have been reported in English lakes (Fogg and Westlake, 1955) and in marine
surface waters (e.g. Degens et. al., 1964). The excretion of free amino
acids by zooplankton is a well known phenomenon (Webb and Johannes, 1967),
and blue-green algae have also been shown to excrete nitrogenous materials
including peptides, amides and amino acids especially in enriched lakes
should not be unexpected. Limited analyses of amino acids in Florida lakes
(Yorton, 1971) indicated the presence of low levels (1.5-12 yg/1) of total
amino acids, but no definitive work has been undertaken. Because of the
chemically similar behavior of amino acids to aamonia in various reactions,
a brief study was undertaken to determine whether any amino acids would
react in the ammonia procedures employed in our laboratory.
Solutions of 15 common amino acids and urea were prepared so that equiva-
lent N concentrations were approximately 0,5 mg/1 in each. Aliquots of
each solution were analyzed for ammonia nitrogen by microdistillation
followed by Nesslerization, and all were found to be free of ammonia.
Aliquots were also digested by snicro-Kjeldahl methods to determine the
total nitrogen content of each. Each solution was then analyzed by both
the indophenol and the Grasshof methods for ammonia nitrogen. The results
shown in Table 31 indicate that both methods are susceptible to considerable
errors from various amino acids. The Grasshof procedure is much more prone
to error than the indophenol method, but all the amino acids tested gave
at least some response in both methods. Interestingly urea was not found
to react in either method.
In the Grasshof method leucine and methionine give approximately the
same response as ammonia. Considering only amino groups as reactive,
histidine and argine also fall in this category since only one-third
and one-half, respectively, of their nitrogen is in amino groups. The
other amino acids gave responses ranging from 23 to 290 percent of
equivalent ammonia nitrogen in the Grasshof method. In the indophenol
method glycine gave a response equivalent to that of asimonia, and
threonine gave a somewhat greater response; the other amino acids gave
responses ranging from 4 to 70 percent of the equivalent ammonia nitro-
gen.
The responses greater than 100 percent can be explained by the fact
that the procedures do not measure equilibrium concentrations, but only
the extent of reaction after a certain time. The amino acids giving
responses greater than 100 percent evidently react more rapidly than
ammonia; hence relatively more product has been formed at the time of
measurement than is the case for an equivalent ammonia solution. The
extent of reaction of non-predictable from the amino acid structure
or isoelectric point. The reasons for the relative responses of the
various amino acids in these procedures would be an interesting study,
but this was beyond the scope of the present project.
135
-------
Table 3.1. Response of Automated Ammonia
Procedures to Free Amino Acids
Grasshoff Method
Alkaline-Phenol Method
Amino Acid
Alanine
Glycine
Arginine
Asparagine
Aspartic Acid
Cysteine
Histidine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Valine
Urea
Free NH
Present
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
<.001
Total N
Present
.495
.476
.405
.48
.48
.33
.35
.495
.374
.47
.462
.445
.478
.495
.47
.445
Apparent
NH -N
0.60
1.38
.18
.11
.28
.10
.11
.53
.74
.49
.52
1.03
.59
.25
.60
.08
% of Avail-
able N
121
290
44
23
58
31
31
107
198
104
113
231
122
51
128
18
Apparent
NH -N
.23
.47
.02
.02
.06
.05
.03
.07
.05
.31
.15
.17
.34
.60
.04
00
% of Avail-
able N
46
99
5
4
13
15
9
14
13
66
32
38
70
121
9
00
All values in mg N/liter.
-------
The need for better automated procedures for ammonia should be
apparent from the studies reported above. The interference resulting
from color or color correlated species is significant and not correct-
able by usual compensation methods. The source of the interference is
still not known with certainty, but iron is the most attractive candi-
date. If this is substantiated, at least this error can be eliminated
by swamping out the natural iron in the sample with added catalyst
(sodium nitroprusside). However, the interferences resulting from
amino acids may be more serious. The normal levels of free amino
acids in natural waters , compared to the levels of ammonia are not
well known, but it should be noted that ammonia concentrations in the
ug N/l range are of great interest in limiting nutrient studies. Limi-
ted information suggests that free amino acids occur in waters in these
concentrations, and the introduction of serious errors in low level
ammonia analyses is thus a distinct possibility.
137
-------
SECTION X
ACKNOWLEDGEMENTS
The able assistance of Michael A. Keirn and Roger A. Yorton in con-
ducting the research culminating in this report is gratefully acknow-
ledged. Mr. Keirn conducted or directed most of the nitrogen fixation
studies and was responsible for the bacterial isolations. Mr. Yorton
performed or directed the various chemical analyses throughout the
project.
Carol C. Harper worked on initial phases of the nitrogen fixation
studies, and Roger King assisted in various aspects of sampling and
analysis. Glenn T. Brasington was most cooperative in performing
sampling and other duties especially during diurnal and other extended
studies. The assistance of W. II. Morgan in fiscal matters greatly
simplified administration of this grant. The principal investigator
was Patrick L. Brezonik, who directed the project and wrote this report,
The support of the project by the Office of Research and Monitoring,
Environmental Protection Agency, and the cooperation and assistance
of C. Powers, grant project officer, are acknowledged with sincere
thanks.
139
-------
SECTION XI
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154
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155
-------
SECTION XII
PUBLICATIONS AND PATENTS
The following publications have resulted from work supported wholly or
in part by this project:
Brezonik, P. L. 1971. Nitrogen: Sources and transformations in natural
waters. Presented at 164th National American Chemical Society Meeting,
Symposium on Nutrients in Natural Waters, Los Angeles, Calif., April,
1971, proceedings to be published by J. Wiley, Inc.
Brezonik, P. L. and C. L. Harper. 1969. Nitrogen fixation in some
anoxic lacustrine environments. Science 164, 1277-1279.
Brooks, ?,. ".., P. L. Brezonik, H. D. Putnam, and M. A. Keirn. 1971.
Nitrogen fixation in an estuarine environment: The Uaccasassa on the
Florida Gulf coast. Limnol. Oceanogr. I6_ (5), 701-710.
Keirn, M. A. and P. L. Brezonik. 1971. Nitrogen fixation by bacteris
in Lake Mize, Florida, and in some lacustrine sediments. Limnol.
Oceanography 1_6_ (5), 720--731.
Keirn, M. A. and P. L. Brezonik. Significance of nitrogen fixation in
the surface waters of eutrophic lakes. In preparation for submission
to Ecology.
Shannon, II. L. and P. L. Brezonik. 1972. Relationships between lake trophic
state and nitrogen and phosphorus loading rates. Environ. Sci. Technol.
6_, 719-725.
Tang, Tsye-Lang. 1968. Methane formation and associated nitrogen cycle
reactions in sediments and sludges. M.S. thesis, University of Florida,
Gainesville.
Yorton, R. A. 1°71. Effects of color and araino acids on automated
nutrient analyses. M. 3. thesis, University of Florida, Gainesville.
Yorton, R. A. and P. L. Brezonik. Interferences in the analyses of
inorganic nitrogen and phosphate in natural waters and procedures for
correction. "'anuscript in preparation for submission to J. Am. Wat.
Works Assoc.
157
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SECTION XIII
APPENDIX A
SAMPLING AND ANALYTICAL METHODS
1. Sampling Procedures.
Water samples for routine chemistry were taken by Van Dorn sampler;
bacteriological samples from the depths were taken aseptically in BOD
bottles attached to a special apparatus which opens the bottle from
the surface at the pull of a string. Sediment cores 30 to 50 cm. in length
were collected in plexiglass tubes. Surface sediments were collected
with an Ekman dredge.
2. Routine Chemical Analyses.
Acidity, alkalinity, and pH were run potentiometrically in the labora-
taory within a few hours after collection of samples in BOD bottles
to avoid contact with air. Dissolved oxygen was determined by the
Winkler-azide method (A.P.M.A. 1965). Cations were measured by atomic
absorption spectrophotometry. Other determinations were done according
to Standard Methods (A.P.H.A. 1965).
3. Nutrient Analyses.
Inorganic nitrogen and phosphorus forms were determined with a Tech-
nicon Auto Analyzer on samples preserved with one ml saturated mercuric
chloride per liter of sample. Ammonia was determined using modifica-
tions (Yorton, 1971) of the phenol-hypochlorite method (Technicon Corp.,
1969). Nitrate was determined by the automated brucine technique
(Kahn and Brezenski, 1967), and nitrite by adaption of the Standard
Methods diazotization procedure to the Auto Analyzer. Ortho phosphate
was measured by the Murphy and Riley (1962) single reagent method. Flow
diagrams and details of the automated version of these methods are pre-
sented by Yorton (1971). Total organic nitrogen was determined by the
macro-Kjeldahl method according to Standard Methods; recovered ammonia
was measured by titration or by the Auto Analyzer. Particulate organic
nitrogen was measured by micro or macro-Kjeldahl on solids scraped from
an MgCO~ mat placed on a 0.45 ym. Millipore filter. Total phosphate
analyses were performed by persulfate-sulfuric acid autoclaving for one
hour at 15 psi followed by manual determination of ortho phosphate
by the Murphy and Riley (1962) method.
4. Biological Methods.
The acetylene reduction technique (Stewart et al., 1967) was used to
measure rates of nitrogen fixation. The technique used on water
159
-------
samples was described by Brezonik and Harper (1969) and on sediments
by Brooks e_t_ _al. (1971). Sediment and water samples (20 to 25 ml) were
placed in 70 nl serum bottles, capped and purged with helium or, in
the case of waters containing dissolved oxygen, with a gas mixture
(20% 0?, 0.03% C02, balance Ar) . Following this 5 cc. of gas phase was
remove? with a gas tight syringe and replaced with 5 cc purified acety-
lene (Matheson Co.)- After incubation (usually for 1-2 hours) activity
was stopped by adding 1 cc. 50% trichloroacetic acid with a needle and
syringe. Gas separation and ethylene analysis was accomplished with a
Varian-Aerograph 600 D gas chromatograph with a hydrogen flame ioniza-
tion detector and a 1/8" x 6' Poropak R column at room temperature.
Newnan's Lake and Bivin's Arm samples were incubated in situ. Other
lake and sediment samples were brought to the laboratory for processing
and incubation at 22°C in a water bath-shaker. Sediments and anoxic
water samples were incubated in the dark; aerobic samples were incubated
under daylight-type fluorescent lighting. Controls run with each set
of samples were carried through the identical procedure except that
1 ml 50% TCA was added before acetylene addition.
Primary production was assayed by 11+C techniques with in situ incubation
for 1 to 3 hours and Geiger counting of the 1!*C incorporated into par-
ticulate matter (filtered onto 0.45 vim. Millipore filters). Chloro-
phyll a was measured by standard acetone extraction and spectrophoto-
metry TCreitz and Richards, 1955) using Parsons and Strickland's (1963)
equations to calculate chlorophyll concentration.
5. Sediment Analyses.
Volatile solids were determined by Standard Methods (A.P.H,A., 1965).
Total organic nitrogen and free ammonia determinations were made on
fresh-sediments diluted with demineralized water into a thin slurry.
Ammonia was distilled from the slurry buffered at pH 8.3 with sodium
bicarbonate and the recovered ammonia measured by titration. Total
organic nitrogen was determined similarly by micro-Kjeldahl digestion.
Results were converted to a dry weight basis by drying and weighing a
known volume of the sediment slurry. Elemental analyses were accom-
plished with a Perkin-Elmer C-H-N analyzer in oven (60°C) dried sedi-
ment samples which were pulverized to a homogenous mixture in a mortar
and pestle. Total phosphate was run on fresh sediment slurries using
persulfate oxidation and the Murphy and Riley (1962) manual method for
ortho phosphate.
References cited in this appendix are listed in Section X.
160
-------
Appendix B
Routine Data Collected on Newnan's Lake and Bivin's Arm During Nitrogen Fixation Study
Bivin's Arm
Parameter
Air Temp.
Surf, water
temp.
Cloud cover
Wind
Secchi Disc.
Dis. 07
L
surface
bottom
PH
Alkalinity
as CaC03
Tot. org. N
NH3-N
NC>2-N
N03-N
Ortho P04-P
Total PO,-P
Prim prod.
Nofixed (a)
Z.
(b)
21
May
29.0
27.3
20
0-2
0.6
10.5
8.4
8.9
97
2.7
0.70
0.008
0.03
0.5
1.0
256
0
0
6
June
25.7
28.2
100
5
0.3
13.0
12.5
9.5
88
3.3
0.11
0.003
0.00
0.042
0.37
1070
7.2
0.067
25
June
32.0
31.1
0
0-2
0.45
12. 1
12.0
9.2
95
4.0
0.80
0.001
0.00
0.063
0.56
790
114
1.08
9
July
__
90
0
0.3
10.5
10.0
9.5
113
5.4
0.0
0.001
0.00
0.091
0.58
2760
25
0.24
29
July
36.0
31.5
80
5
0.3
12.3
10.6
9.9
115
2.9
1480
46
0.43
7
Aug
38.4
31.3
80
0
0.6
13.3
9.4
9.5
145
2.30
0.21
0.01
0.3
0.61
117
109
1.02
21
Aug
27.6
27.6
5
10
0.6
12.6
12.1
9.5
100
3.0
0.48
0.000
0.00
0.25
0.49
1960
330
3.06
3
Sept
28.6
27.5
90
5
0.75
3.8
3.7
8.7
100
1.7
0.76
0.003
0.04
0.27
0.49
100
46
0.43
18
Sept
33.9
27.2
30
5
0.45
9.9
9.8
9.8
125
3.25
0.62
0.03
0.23
0.72
1580
0
0
2
Oct
25.3
25.9
100
5
0.9
4.6
4.1
8.8
96
1.1
0.38
0.006
0.21
0.19
0.54
166
0
0
16
Oct
32.0
29.0
5
0
0.45
15.0
14.4
9.6
105
2.4
0.35
0.000
0,00
0.13
0.54
1220
42
0.39
30
Oct
20.5
20.5
100
10-15
0.45
10.0
9.4
9.2
155
2.6
0.73
0.004
0.06
0.30
0.58
420
0
0
13
Nov
16.4
17.8
5
5
0.45
10.8
11.0
8.9
160
2.4
0.42
0.006
0.00
0.02
0.51
81
0
0
-------
Bivin's Arm
Parameter*
Air temp.
Surf, water
temp.
Cloud cover
Wind
Secchi disc.
Dis . O^ surf.
bottom
pH
Alkalinity
Tot. org. N
NH3-N
N02-N
N03-N
Ortho PO^-P
Total P04-P
Prim. prod.
N2 fixed (a)
(b)
24
Nov
23.3
15.8
10
0-3
0.45
11.4
11.4
8.8
1.8
0.00
0.000
0.00
0.15
0.41
47
0
0
11
Dec
16.3
15.3
15
5-10
0.3
8.8
8.6
8.4
110
1.8
0.07
0.001
0.00
0.16
0.51
324
12.9
.12
2
Jan
10.5
12.8
100
10
0.45
8.4
8.1
9.0
100
2.0
0.23
0.000
0.03
0.055
0.21
236
15.7
.15
12
Jan
10
9.3
100
5
0.85
10.2
10.8
8.0
110
1.0
0.16
0.036
0.14
0.30
0.54
84
0
0
21
Jan
8.5
12.9
0
5
0.75
9.5
9.4
7.9
120
1.8
1.0
0.045
0.67
0.33
0.50
650
13.4
.12
4
Feb
10
13.0
0
5
0.45
8.8
8.7
8.3
80
1.2
0.08
0.000
0.14
0.031
0.63
168
0
0
18
Feb
15.5
16.1
95
0-5
0.6
11.8
10.9
8.9
100
1.7
0.00
0.005
0.03
0.43
0.62
25
17.7
.17
9
Mar
21
17.8
15
5
0.45
6.9
6.7
8.4
115
1.8
0.20
0.006
0.02
0.48
0.70
68
0
0
31
Mar
24.5
24.5
30
10
0.75
8.5
6.5
8.4
98
1.0
0.22
0.002
0.03
0.40
0.62
263
0
0
7
Apr
22.7
21.9
5
5
0.75
7.9
8.6
8.0
105
1.1
0.34
0.006
0.03
0.23
0.73
81
1.8
.016
28
Apr
27.5
26.8
75
0
0.55
12.2
8.7
8.9
120
1.7
0.13
0.000
0.02
0.43
0.59
345
3.6
.034
12
May
26.8
25.3
50
0-5
0.45
9.9
8.2
9.0
185
2.1
0.55
0.000
0.01
0.21
0.65
400
0
0
26
May
28.8
24.8
10
5
0.55
7.5
7.5
8.8
110
1.6
0.56
0.000
0.00
0.13
0.52
585
0
0
iTemperature °C; cloud cover in percent; wind in mph; Secchi disc in m.; all concentrations in mg/1 of
species noted; primary production in mg C/m -hr. ; nitrogen fixation (a) in nM ethylene/m^-hr; and nitrogen
fixation (b) in ng N/l-hr. assuming a theoreticl molar ratio of 1.5 moles ethylene produced per mole of
ammonia.
-------
Newnan's Lake
1969
01
OJ
Parameter
Air temp .
Water temp.
surf.
bottom
Cloud cover
Wind
Secchi Disc.
Dis Oo surf.
bottom
PH
Alkalinity
as CaC03
Acidity
as CaC03
Tot. org. N
NH3-N
NO^-N
NO^-N
Ortho PO,-P
Total P04-P
Prim. prod.
N2 fixed (a)
(b)
20
May
26.6
28.0
25.1
,
0.6
8.6
8.1
8.8
8.4
0
1.9
0.69
.006
0.16
0.012
0. 16
223
0
0
3
June
26.0
27.0
27.0
0
0.4
8.3
8.0
7.1
2.9
1.4
1.8
0.80
.006
0.22
0.028
0.15
15
0
0
24
June
32.7
31.7
28.9
40
0
0.45
8.7
8.0
8.6
6.8
0
1.5
0.07
.001
0.00
0.015
0.15
169
0
0
8
July
31.2
31.5
31.0
20
5-10
0.3
7.7
7.6
7.7
10
0
1.7
0.07
.002
0.00
0.09
0.25
229
0
0
24
July
32.5
30.0
29.7
80
10
0.45
8.0
8.3
8.0
12.5
0
2.1
331
76.5
0.71
6
Aug
36.4
29.1
27.9
20
5
0.6
9.1
6.5
9.1
8.5
0
1.2
0.03
0.02
0.009
0.10
247
0
0
22
Aug
31.1
30.8
30.5
30
0.45
7.9
7.9
7.5
7.5
1.8
0.18
.000
0.03
0.010
0.14
67
0
0
9
Sept
24.1
28.1
28.0
90-100
5-10
0.45
8.0
7.9
7.4
7.2
1.5
0.04
.000
0.11
0.004
0.09
23
0
0
17
Sept
37.1
32.3
26.5
50
0
1.05
7.8
7.2
7.1
7.2
1.9
1.3
0.02
.000
0.00
0.000
0.15
54
0
0
25
Sept
26.8
27.5
27.0
20
0.6
6.2
6.2
6.6
7.2
3.0
1.3
0.10
.000
0.01
0.000
0.14
86
0
0
14
Oct
32.0
28.5
26.0
40
0.6
8.0
6.3
7.2
7.7
2.1
1.3
0.29
.001
0.02
0.003
0.08
63
21
0.02
23
Oct
23.1
24.6
23.8
45
10
0.6
8.0
7.9
7.1
8.6
1.7
2.3
0.32
.003
0.06
0.010
0. 12
48
0
0
7
Nov
20.9
16.9
16.4
60
5
0.45
9.4
8.6
6.9
7.2
2.5
1.4
0.48
.003
0.16
0.012
0.10
39
0
0
See footnote at end of table for parameter units.
-------
Newnan's Lake
1969
Parameter
Air temp.
Water temp. surf.
" " bottom
Cloud cover
Light intensity
Wind
Secchi disc.
Dis. Oa surf.
" " bottom
pH
Alkalinity as CaCOs
Acidity as CaCOa
Tot. org. N
NH3-N
NOj-N
NO-3-N
Ortho P04-P
Total PO^-P
Chlorophyll a
Prim, production
N fixation (a)
(b)
20 4
Nov Dec
26.3 15.7
14.3 13.4
14.0 13.1
100 30
10 10
0.55 0.45
9.9 9.3
10.0 10.5
7.1 7.8
8.2 8.7
1.6 0.6
1.7 2.4
0.33 0.60
0.002
0.05 0.09
0.007 0.010
0.15 0.12
36 70
0 41
0 0.38
8
Dec
19.3
14.5
13.4
5
0-5
0.4
11.1
8.4
8.9
0
2.0
0.31
0.02
0.004
0.17
66.6
34
24.5
0.23
11
Dec
14.4
15.0
15.0
25
6080
5
0.25
9.7
7.0
8.7
2.3
2.1
0.06
0.000
0.06
0.007
0.14
67.9
119
20.3
0.19
16
Dec
15.7
13.7
13.5
40
5000
5
0.45
11.9
8.5
8.6
0
3.9
0.23
0.000
0.02
0.009
0.14
78
41
28.6
0.27
18
Dec
18.5
13.9
12.5
20
6400
0-5
0.5
1.6
0.09
0.001
0.05
0.007
0.05
10.4
160
31.2
0.29
23
Dec
13.2
13.0
12.6
10
6400
0
0.45
11.4
7.8
2.0
0.19
0.14
0.013
0.15
77
11.4
0.11
26
Dec
13.0
13.8
13.8
0
7040
15
0.4
10.4
7.0
12
2
2.1
0.19
0.14
0.005
0.19
10.4
11.2
15.2
0.14
30
Dec
22.0
14.6
14.4
60
4160
10
0.25
2.2
2.2
0.18
0.05
0.012
0.19
10.9
35
22.1
0.21
1970
2
Jan
10.6
13.6
13.6
100
510
10
0.25
10.1
6.8
1.2
2.8
0.7
0.20
0.07
0.005
0.15
10.0
4.8
5.7
0.053
7
Jan
9.0
12.5
12.5
75
2720
20
0.3
10.1
_i_
6.7
1.5
4.6
0.7
0.02
0.000
0.05
0.005
0.21
16.5
15.5
6.5
0.06
13
Jan
7.0
8.2
8.1
100
640
10
0.4
11.4
6.6
6.1
3.2
1.8
0.10
0.003
0.05
0.007
0.14
73
43
2.7
0.025
20
Jan
11.9
12.8
12.5
75
720
0
0.4
12.4
6.9
0
2.2
2.2
0.000
0.04
0.013
0.050
60
32
0.5
0.004
footnote at end of table for parameter units.
Cont'd.
-------
Newnan's Lake
1970
Ui
Parameter
Air Temp.
Water temp. surf.
bottom
Cloud cover
Light intensity
Wind
Secchi Disc.
Ms Oy surf.
hot turn
PH
AlkaliniLy
Acidity as CaGO.,
Tot. org. N
NH3-N
NC>2~N
NO--N
Orf-ho P04-P
Total PO^-P
Chlorophyll a
Prim, production
N2 fixation (a)
(b)
30
Jan
11.0
13.7
13.4
70
2880
15
0.45
12.6
6.8
0
2.7
1.7
0.23
. 000
0.07
0.007
0.060
57
2.8
0.025
3
Feb
12.5
14.2
14.2
100
1030
15-20
0.45
8.5
7.0
1.6
2. 1
1.1
0.10
.000
0.05
0.090
0.11
67
29
""
16
Feb
18.9
13.9
12.1
100
1020
5
0.45
10.5
7.1
5.8
1.2
0.7
0.06
.000
0.02
0.008
0.09
49
14.3
0.13
27
Feb
16.4
16.4
13.0
0
3860
0
0.7
8.9
6.7
8.0
2.9
1.0
0.21
.001
0.05
0.011
0.09
78
85
17.9
0.17
4
Mar
19.2
17.5
16.3
60
4970
5
0.75
9.9
7.3
12.7
3.2
1.4
0.15
.000
0.05
0.006
58
117
23
0.21
11
Mar
22.4
19.0
18.5
40
8000
10
0.8
8.0
7.4
16.0
4.0
1.1
0.33
.006
0.02
0.012
0.08
62
281
39
0.36
20
Mar
28.5
18.3
17.2
50
800
10
0.8
10.2
6.7
0.4
2.9
1.2
0.06
.002
0.11
0.007
0.12
51
281
27.6
0.26
26
Mar
20.1
18.5
18.2
100
1280
0
0.75
10.2
7.2
6.3
0.9
1.5
0.08
.000
0.05
0.001
0.12
68
270
11.8
0.11
2
Apr
23.0
22.5
22.5
100
25
.35
7.5
7.5
6.9
4.2
1.8
1.3
0.28
.003
0.03
0.010
0.15
55
22
0
0
7
\pr
15.2
19.1
19.0
0
5
0.6
7.5
6.7
6.8
6.5
2.2
1.2
0.40
.003
0.04
0.017
0.13
87
197
3.2
0.03
24
Apr
30.4
27.0
25.4
95
6400
0.5
7.0
6.8
6.8
3.5
1.9
1.2
0.79
.008
0.03
0.042
0.064
68
29
1.5
0.014
6
May
27.1
26.5
25.5
50
7000
5
1.0
8.6
8.0
7.2
10
2.5
1.1
0.0
.000
0.03
0.008
0.12
43
129
1.6
0.015
22
May
31.8
27.0
26.0
40
6400
15
0.5
8.8
8.4
7.5
6.4
0.6
1.9
0.25
.001
0.08
0.011
0.056
80
185
0
0
2
June
52.1
28.2
28.0
100
5000
5
0.8
8.2
8.0
7.0
6.3
2.1
2.0
0.36
.003
0.02
0.011
0.063
101
65
0
0
^Temperature in °C; cloud cover in percent; wind in mph: Secchi disc in meters; chemical concentrations
in mg/1 of species noted; chlorophyll a in mg/m primary production in mg C/m -hr.; nitrogen fixation
(a) in nM &2 H^/m -hr.; and nitrogen fixation (b) in ng N/l-hr. assuming a theoretical producation
ratio of 1.5 moles ethylene produced per mole of ammonia fixed.
-------
APPENDIX C
ENRICHMENT AND ISOLATION PROCEDURES FOR NITROGEN FIXING
AGENTS IN LAKE WATER AND SEDIMENTS
Water samples were collected from various depths in Lake Mize asepti-
cally by lowering a closed, autoclaved BOD bottle to the appropriate
depth and opening the stopper with a string. Lake water samples were
subjected to enrichment schemes for heterotrophic anaerobic, faculta-
tive, or aerobic bacteria, photosynthetic bacteria, and yeasts and
fungi. In addition, samples were examined microscopically for blue-
green algae and photosynthetic bacteria but visual examination of all
samples yielded negative results.
Heterotrophic nitrogen fixing bacteria, both aerobic and anaerobic,
were enriched intially using a nitrogen free, modified Winogradsky's
medium (Grau and Wilson, 1962) with sucrose as the carbon source.
Aliquots from each depth were mixed with equal amounts of double strength
medium and incubated at 20°C both aercbically and anaerobically under
one atm No. Control aliquots were carried through the same procedure
with the exception that 1.0 g/1 NH.C1 was present in this medium. After
growth was noted by increased turbidity, transfers were made to fresh
nitrogen free media. After three transfers streak plates were made on
to the same medium plus 1.5% agar.
Enrichment for photosynthetic bacteria was done with three media.
Athiorhodaceae (non-sulfur purple bacteria) were enriched by placing
100 ml of lake water from each depth into A BOD bottle and filling with
a medium consisting of sodium acetate (2 g/1), NH,CL (1 g/1), l^HPO^
(0.5 g/1), MgCl2 (0.1 g/1), yeast extract (0.05 g/1), and adjusted to
pH 7.0. The bottles were stoppered and incubated at 25°C under 250
foot candles of continuous fluorescent light. Enrichment for Thiorho-
daceae (green and purple sulfur bacteria) was accomplished by inocu-
lating 200 ml of lake water into each of two BOD bottles containing
100 ml of a sterile slurry of cellulose (3 g.), CaSO^ (3 g.) , NH^,C1
(0.139 g.), KH2P04 (0.33 g.), and Ha2S7H20 (0.07 g.), one adjusted to
pH 7.3 (for green sulfur bacteria) and the other adjusted to pH 8.5
(for purple sulfur bacteria). The bottles were capped and incubated as
described above for the Athiorhodaceae. Controls, consisting of sedi-
ment samples from a lake known to contain these groups (Lake Alice,
Gainesville, Florida (Lackey et_ al. , 1964), were carried through the
procedure to assure that the scheme was valid.
Enrichment for nitrogen fixing yeasts of fungi was made by filtering
aliquots of water through 0.45 urn. membrane filters which were incu-
bated at 25°C and 35°C on Sabouraud dextrose agar. All morphological
types (about 30% of the total colonies or 65 isolates) were picked
166
-------
after 4 days growth, gram stained and inoculated into a nitrogen de-
ficient medium modified from Metcalfe and Brown (1957), containing
0.3% sodium benzoate; glucose, sucrose, and mannitol glucose as car-
bon sources at 5.0 g/1; phosphate, and trace metals and buffered to
pH 7.2. The isolates were also inoculated into a control medium (i.e.
the above medium plus nitrogen as NH/C1). All cultures were incubated
in the dark at 25°C.
Anaerobic bacteria such as occur in the genus _Clp_st_ri_dium were consi-
dered the most likely organisms responsible for fixation in the anoxic
estuarine sediments, and the following enrichment and isolation scheme
for clostridia-like nitrogen-fixing organisms was used to determine
whether such organisms occur in Waccasassa Estuary sediments. A sedi-
ment sample from the 2-5 cm. depth was heated at 80°C for 10 minutes
to destroy vegetative forms and then inoculated into a nitrogen-free
salt medium containing 4 g/1 of one of the following carbon sources:
acetate, maltose, mannitol, sorbitol or sucrose. All of these except
acetate have been reported as fermentable carbon sources for Clostridium
pasteurianum. After incubation at room temperature (22-25°C) under
a pure nitrogen atmosphere for three days, growth was apparent (by in-
creased turbidity and gas production) in all samples except the medium
containing acetate as carbon source. Most rapid growth occurred ini-
tially with mannitol; however in subsequent transfers sorbitol gave
quickest response. A test for acetylene reduction activity in the
mannitol culture gave positive results. Gram studies showed an abun-
dance of gram-positive rods accompanied by a few gram-variable coccoid
forms. Many of the rods showed sub-terminal swellings characteristic
of developing spores.
The enrichment cultures were incubated five more days to promote
sporulation, then heated to 80°C for 10 minutes and transferred to
basal salts media containing sorbitol. Transfers were incubated under
an Np atmosphere at room temperature for 48 hours, and another transfer
was made to similar media. Following incubation for 5 days, a third
transfer was made. Micro-Kjeldahl analysis of each batch of fresh
media showed no detectable ammonia or organic M. A test for acetylene
reduction on the 8 bottles (duplicates of the 4 original carbon sources
showing growth) from the third transfer gave positive results for 3
bottles.
Streak plates were made onto the nitrogen-free basal salts medium plus
sorbitol in 2 percent agar from the incubated material of the third
transfer, and the plates were incubated for 72 hours in an anerobic
jar (N^ atmosphere). Colonies on the plates were all similar in
appearance and did not display chromogenesis. Ten colonies which were
gram-positive rods were picked from plates streaked with media from
bottles which exhibited acetylene reduction and were transferred to
basal salts plus sorbitol. Each isolate was grown under both aerobic
and anaerobic (^ atmosphere) conditions. Four colonies grew only
anaerobically; the remainder grew under both conditions. The latter
were tested for acetylene reduction and gave negative results. The
four obligate anaerobic isolates were also tested for acetylene reduc-
tion, and three of the isolates showed positive rates.
fiU.S. GOVERNMENT PRINTING OFFICE: 1973 546-303/35 1-3
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
1. Report No.
2.
3. Accession No.
w
4. Title
NITROGEN SOURCES AND CYCLING IN NATURAL WATERS
7. AuthoT(s)
Patrick L. Brezonik
9. Organization
University of Florida
Department of Environmental Engineering
Gainesville, Florida
12. Sponsoring Organization
35. Supplementary Notes
Environmental Protection Agency report number,
EPA-660/3-73-002, July 1973.
5. Repoit Date
6.
8, Performing Organization
Report No.
JO. Project No.
16010 DCK
11. Contract/Grant No.
13. Type of Report and
Period Covered
16. Abstract Sources of nitrogen were reviewed to determine their significance in lake
nitrogen budgets. Nutrients in rainfall were evaluated and found to be significant.
Nitrogen and phosphorus budgets were calculated for 55 Florida lakes and critical load-
ing rates established by comparing calculated budgets with data on trophic state.
Nitrogen fixation by Cyanophyceae was studied in detail in two eutrophic
Florida lakes for one year. Also a survey of fixation in Florida lakes was conducted
and fixation found only in eutrophic lakes. Bacterial fixation was found to contribute
significant nitrogen to the anoxic hypolimnon of a small stratified lake. Nitrogen
fixation was found in both lacustrine and estuarine sediments.
Sediments of 55 lakes were characterized chemically and results suggest that
such sediment may act as an ammonia buffer, sorbing ammonia at high concentrations and
releasing it to ammonia depleted water. Estuarine sediment sorbed ammonia strongly
but failed to release it to overlying water.
The acetylene reduction assay for nitrogen fixation was evaluated. Inter-
ferences in automated nutrient determinations due to organic color were studied and
simple color correction found for nitrite, nitrate and orthophosphate but not ammonia
as determined by the indophenol method. Amino acids also interfered with the ammonia
analysis. (Keirn-Florida)
17a. Descriptors
17b. Identifiers
*Eutrophication, *Limnology, *Nutrients, *Nitrogen Fixation
Water Quality, Trophic Level, Blue-Green Algae, Bacteria
Florida Lakes, Waccassa Estuary
17c. COWRR Field & Croup
18. Availability
19. Security Class.
(Report)
20. Security Class.
(Page)
21. No. of
Pages
22. Price
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CFNTFR
US DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C 20210
Abstractor Michael Keirn
\Institution University of Florida
WRSIC 102 (REV. JUNE 1971)
------- |