INFLUENCES OF MICROBIAL
POPULATIONS ON AQUATIC
NUTRIENT CYCLES AND SO
ENGINEERING ASPECTS
PRO^°
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Influences of Microbial Populations on
Aquatic Nutrient Cycles and Some Engineering Aspects
By:
Leonard J. Guarraia, Ph.D.
Richard K. Ballentine, P.E.
Technical Studies Report
TS - 00 - 72 - 06
U.S. Environmental Protection Agency
Office of Water Programs
Water Quality and Non-Point Source Control Division
Water Quality Protection Branch
Fresh Water Pollution Control Section
Washington, D.C. 20460
May 1972
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Acknowledgements
The authors wish to thank our secretary Mrs. Nyla
Linthicum who has labored tirelessly and with patience and
good humor in the preparation of this report and who
suffered excessive eye strain in the translation of the
written word to the final typed word.
We appreciate the reviews and helpful comments of K.M.
Mackenthun and L.E. Keup.
Cover Photograph
Courtesy Mr* Curtis Ross - U.S. Environmental Protection
Agency - From Project Hypo, an intensive study of the Lake
Erie Central Basin Hypolimnion and Related Surface Water
Phenomena.
Front Cover
Picture of a "black patch" where the sulfide layer
has penetrated up to the surface of the sediment.
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TABLE OF CONTENTS
SUMMARY 1
CONCLUSIONS 3
INTRODUCTION 8
PHOSPHORUS 10
SULFUR 13
NITROGEN 16
CARBON 21
ENGINEERING ASPECTS 26
ALGAL MODELING 35
EUTROPHICATION CONTROL 36
OPTIMUM OR COST EFFECTIVE CONTROL 38
POST PROJECT EVALUATION 38
RECOMMENDATIONS 40
NUTRIENT CYCLES 40
ANAEROBIC MICROORGANISMS 42
MICROBIAL PHOTOSYNTHESIS 42
INTERSPECIES INTERACTION 43
ENGINEERING ASPECTS 4 3
METHODS 4 3
BIBLIOGRAPHY
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LIST OF FIGURES
Follows Page No.
FIGURE 1
Phosphorus Cycle 11
FIGURE 2
Sulfur Cycle 13
FIGURE 3
Nitrogen Cycle 18
FIGURE 4
Phytoplankton, Zooplankton and 37
Total Inorganic Nitrogen
FIGURE 5
Mean Trophic State Index Values for 37
Five Trophic Groups vs. Annual
Phosphorus Loading
FIGURE 6
Trophic State Indes Values for 37
Five Trophic Groups vs. Annual
Nitrogen Loading
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LIST OF TABLES
Follows Page
TABLE 1
Biodegradable Compounds 25
TABLE 2
First Principal Components and
Trophic State Indices 36
TABLE 3
Stepwise Regression Analysis of 38
Trophic State Index vs. Eutrophication
Factors Expressed Per Unit Lake Volume
TABLE 4
Critical Loading Rates for Nitrogen 39
and Phosphorus
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SUMMARY
Domestic and industrial wastes, chemical fertilizer
runoff from farms, suburban lawns and golf courses,
heavy metals, and silt from land development all
contribute to the eutrophication process. The addition
of such agents to the aquatic ecosystem can result in
an algal bloom. However, the cause and effect
relationships between many changes in water quality and
the causative flora and fauna may not be clearly
recognizable as due to bacterial metabolism.
Proliferation of endogenous microbial populations
caused by eutrophication leads to the establishment of
anaerobic environments through increased respiration or
biochemical oxygen demand (BOD). One of the causes of
excessive eutrophication in water is the release of
carbon dioxide, organic carbon compounds and the
mineralization of nutrient elements which are the end-
products of microbial catabolism. As a consequence of
an increased rate of eutrophication, lakes age
prematurely, rivers may become choked with algae or
aquatic plants and benthic fauna may become smothered.
Further distortion of biological productivity is
brought about by the ability of the bacteria to
concentrate heavy metals such as mercury. It is the
bacterial population that serves as a base for the food
chain. Zooplankton further enhance metal concentration
by feeding on bacteria. The plankton are in turn
utilized by such organisms as shrimp, mollusks, and
small fish as a food source. The consequence of this
contamination in the food chain is that commercially
important organisms are either killed cr are rendered
unfit for human consumption.
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Establishment of species of bacteria new to an area can
be important. Alterations in microbial flora can be
caused by the discharge of human or industrial sewage
into waterways. The introduction of pathogenic flora
renders an area unsuitable for human use- An
alteration of the nutrient cycles can also cause a
change in the fauna.
Engineering control of natural systems in various
stages of eutrophy must consider microbial responses to
system changes. Limitation of one nutrient without
limiting others may cause unexpected environmental
responses. Development for predicting system response
thus far includes deterministic ecological models which
consider bacteria, phytoplankton and zooplankton
populations based on nutrient concentrations and
statistical models which predict eutrophic conditions
based on watershed development or waste inputs. Using
these tools various control schemes can be tested.
Clearly, all bacterial metabolic activities are not
detrimental to the environment. Bacteria are involved
in the cycling of phosphorus, nitrogen, carbon, sulfur,
hydrogen, and other elements thus providing a
nutritional base for the food chain. Degradation of
complex organic molecules such as the hydrocarbons,
detergents, phenolics, and alcohols is brought about by
bacterial metabolism. Natural organic compounds such
as cellulose, starch, and chitin are also degraded in
the normal catabolic process of bacteria. All of these
organic molecules ultimately are degraded to carbon
dioxide and water for use by green plants. These
catabolic activities are part of the normal life
process in the environment and allow for the cycling of
nutrients for reuse in the food chain.
2.
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CONCLUSIONS
Microbial influence on water eutrophication is
generally accepted as significant. Aquatic microbial
populations have been shown to be either the basic
agents or among the primary effectors in the cycling of
phosphorus, sulfur, nitrogen and carbon. There is a
general qualitative understanding in nutrient cycling;
however, many quantative cycling rates, as governed by
microbial populations, are unknown. With respect to
microbially mediated nutrient cycling, the following
conclusions can be made:
1. In the phosphorus cycle the involvement of
microbes has been demonstrated. However, the
extent and mechanisms of the involvement/the
effects of physical and chemical perturbations
caused by man's activities, and the
implications to the total biological system
are not well defined.
2. The transition of inorganic sulfur in the
aquatic environment is regulated by two
metabolically distinct groups of bacteria:
the sulfate reducers and sulfate oxidizers.
Hydrogen sulfide of biological origin is
exclusively the result of the sulfate
reducers, conversely, sulfuric acid of
biological origin is the result of sulfur
oxidizing bacteria. These two metabolic
groups also are implicated in the nitrogen,
phosphorus and carbon cycles. Excessive
organic pollution which depletes available
oxygen supplies apparently will enrich for the
sulfate reducing bacteria resulting in high
sulfide concentrations. However; the direct
relationships between various types of
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pollution to sulfur availability, sulfide or
sulfate production and environmental dystophy
is not well defined.
3. Fixation of elemental nitrogen is the result
of a select group of free living microbes;
however, the transition of nitrogen through
the various valence states is mediated by a
large number of microbes. The influence of
various pollutants on the nitrogen fixers and
the possible consequences are not understood.
4. Microbial involvement in the carbon cycle is
complex and involves not only carbon dioxide
fixation but the degradation and synthesis of
innumberable complex organic compounds.
Subsequently, microbial biomass serves as a
food source for higher organisms. When
microbial activity is incapable of degrading
organic material a dystrophy results. Peat
bogs and accumulation of non-biodegradable
synthetic organic compounds are examples of
results from an imbalance in bacterial
degradation. As added stresses are placed on
the aquatic ecosystem, the consequences to the
natural biological synthetic and degradative
processes are often not predictable.
5. The effects of many types of pollutants on the
microorganisms involved in the nitrogen,
sulfur, phosphorus and carbon cycles are not
well known. An understanding of the
relationships of these cycles with each other
and their function within the total ecosystem
is needed. Such an understanding would permit
more more efficient and effective waste
management practices and the ability to
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anticipate and perhaps prevent pollution
disasters.
6. Whether certain pollutants selectively
stimulate or inhibit the growth of specific
microbial species is unknown. The ecological
consequences of species selection processess
and how to deal with these possibilities are
not known. Species selection may be
manifested in a variety of ways; for example,
death of fish may be caused by growth of fish
pathogens, excessive sulfide pollution, or
establishment of an anaerobic environment.
7. The consequences to the aquatic environment
when the indigenous microbial flora is altered
are unknown. For example, shellfish feed on
certain microflora; alteration of the foodbase
can cause shellfish to die or become unfit for
human consumption. The specific microbial
flora can be changed by introduction of
agricultural, domestic and industrial
pollution into the water.
8. Since there are a variety of pollutants,
organisms other than fecal coliform and fecal
streptocci bacteria may be introduced and need
to be recognized as indicators of pollution.
This is important both from a health as well
as economic point of view.
9. Engineering control of lake eutrophication
requires the development of mathematical
models which includes phytoplankton
"production function" which accurately predict
the ecosystem response to watershed changes.
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Ultimately, the inclusion of political and
economic factors must be incorporated in such
models to produce the most publicly acceptable
cost-effective solutions to culturally
accelerated eutrophication. Continued
surveillance must be incorporated in projects
to evaluate actual changes in eutrophy versus
predicted changes.
10. To model the microbial effects on
eutrophication, a review of existing
formulation of microbial activities is
required, changes in bacterial densities in
streams have been mathematically described at
least since the publication of Public Health
Bullentine 143 in July, 1924. The
formulations adopted reflected the earlier
work of Chick and consisted of composite
exponential equations to describe the
bacterial die-away.
11. Bacterial growth and accumulation in waste
treatment systems have been correlated with
substrate energy. The kinetics of this growth
has been described by Monod using the enzyme-
substrate model of Michaelis. For simplicity
linear models are frequently adequate for
waste treatment system design because only a
small portion of the growth curve is
represented.
12. Historically phytoplankton modeling in the
engineering literature considered primarily
dissolved oxygen relationships. Modeling by
limnologists considered nutrient relationships
but did not consider symbiotic relationships
among bacteria, phytoplankton and zooplankton
populations.
6
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More recent models evaluate eutrophication by
consideration of the bacterial and plankton
interaction with organic and inorganic
nutrients or other models use statistical
relationships among drainage basin use, waste
inputs and plankton response.
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INTRODUCTION
Pollution and eutrophication originate basically from
introduction of organic and inorganic products into the
aqueous environment. Man has depended primarily on the
abilities of microorganisms to remove these products
from the environment through metabolic proclivities
inherent only in this biological category. As one
advances phylogenetically towards more complex
organisms, the inherent ability to react metabolically
to a vast array of products is rapidly diminished. The
principle of microorganisms in nutrient cycling and
biological transformations is presented.
Man, whether he recognizes the fact or not, depends
basically on two independent, but not mutually
exclusive, processes to cleanse his immediate
environment: (1) Oxidation (chemical or photo)and
(2) biological processes. The first category with
respect to the environment is beyond the immediate
control of society. Examples of the second category
are apparent in the form of biological waste treatment
systems.
Pollution problems have also developed as a result of
technology wherein molecules of diverse chemical
structures have been synthesized and result in
compounds which are refractory to microbial
degradation. The refractory nature of many of these
chemical structures to biodegradation is well
documented in scientific literature. Outstanding
examples can be found with detergents, pesticides, and
plastics. The inescapable conclusion is that basic
ground rules in product development and
biodegradability by microorganisms must evolve as
direct relationship to molecules entering the
environment. A principle of absolute nondegradability
for certain structures must be recognized and
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established. Historical reference to oxidation ponds,
activated sludges, polluted lakes, rivers, and bays
attests to the inability of the indigenous microbial
flora to effect biodegradation of these compounds.
Toxic levels progressively limit the productivity of
all higher biological systems creating a polluted
aquatic environment.
The terms "eutrophication" and "pollution" need
defining since they are often interchangably and
incorrectly used. Pollution from a biological point of
view has been defined as an alteration of the
environment so as to alter the diversity of aquatic
life, eventually destroying the balances in that
environment (1). In general, however, pollution means
an alteration of the aquatic environment so as to
change the beneficial uses. Eutrophication, as
originally defined, refers instead to the fertilization
of streams (2). Eutrophication is a natural phenomenon
which may be aggravated or accelerated by man's
activities. Pollution is an unnatural process
resulting from the introduction of compounds such as
pesticides, heavy metals, mine tailings, and industrial
wastes into the aquatic environment. The two terms are
not always synonymous.
Excellent bibliographies are available presenting
references detailing the effects of nitrogen and
phosphorus (3) as well as other nutrients (4) on the
aquatic ecosystem. General references detail the
effects of excessive fertilization on algae and
taxonomic orders of flora and fauna higher than
microrganisms (3-6). The specific role of the
microorganism in the cycling of elements, sequestering
of metals and the effect on solubility of nutrients
will be considered here.
9.
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PHOSPHORUS
The primary productivity of the aquatic environment may
be limited by inorganic nutrients. Phosphorus is one
nutrient which can be limiting to the phytoplankton and
other organisms. However, most of the phosphorus in
the aquatic environment is bound in the sediment as
insoluble phosphate salts with availability of the
insoluble salts being influenced by both physico-
chemical factors (7) and bacterial metabolism (8). The
fact that microbial flora influences the availability
of phosphate directly through the release of their
metabolic end products was recognized by Sackett, et
al. in 1908 (9). Soil bacteria utilizing glucose as a
carbon source were found to be capable of affecting the
solubilization of various insoluble phosphate salts.
Later workers demonstrated that the rate of solubili-
zation of phosphate salts was dependent upon the type
of carbon and nitrogen sources available to the
bacteria (10). The cause-effect relationship between
bacterial metabolism and phosphate solubility results
from a combination of factors and has been investigated
by various workers (9 - 15) . Three general processes
involved in phosphate solubility are the direct
metabolic processes involving enzymes, carbon dioxide
production leading to a lowered pH, and organic acid
production. A variety of organic acids, which are the
end-products of bacterial metabolism, have been shown
to increase the solubility of phosphate salts
(17,18,19). The mechanism involved may be a chelation
like effect.
Another important factor influencing phosphate
solubility is the concentration of sulfide. Sperber
(20) demonstrated that hydrogen sulfide released
soluble phosphate in soils. Hayes and coffin (21) cite
data suggesting that sulfate reduction to sulfide by
selected microorganisms could lead to release of
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soluble phosphate as a consequence of the conversion of
ferric phosphates to the less soluble ferric sulfides.
Mackenthun and Ingram (82) also noted that bacteria are
capable of utilizing particulate phosphorus, thus
making this element available to the rest of the food
chains. Biological production of sulfide is limited to
two genera, the anaerobic sulfate reducers
Desulfovibrio and Desulfotomaculum (29, 30). These
organisms also require organic carbon with the release
of carbon dioxide and fix elemental nitrogen (31, 32).
These findings offer a link between the basic nutrient
cycles. Phosphate availability is partially regulated
by the same bacteria which affect the sulfur, nitrogen
and carbon states in a particular ecological niche.
The direct relationship between bacterial metabolic
activity and phosphate availability is reinforced by
other lines of evidence. For example, changes in
dissolved oxygen (DO) have been shown to affect release
of inorganic phosphate (P04 ). Anaerobic conditions
will certain bacteria to release P04 , (22) while at
high DO concentrations the bacteria concentrate Pa in
excess of their needs (23).
A cycle is suggested consisting of a algal bloom,
subsequent creation of anaerobic conditions, and
finally, release of phosphate, since blooms of algae
cause decreased levels of oxygen in water as a result
of the decomposition of the biomass, the resulting
anaerobic conditions lead to the release of phosphate.
As the blooms decline, natural mixing will result in
increased DO and the phosphates released from the dead
zooplankton and phytoplankton are reabsorbed by the
bacteria (22). However, as the nutrients are utilized
by the microorganisms, the associated increase in
bacterial metabolic activity causes a lower DO and
release of phosphates which become available for algal
growth resulting in a bloom. This provides an example
11.
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Figure 1 .
PHOSPHORUS CYCLE
Wastes
Introduction
Water
Mud
Higher aquatic
Zoo plankton
Inorganic
phosphate
Bacteria
Soluble organic
phosphate
Bacteria
Inorganic
reaction
Loss to Permanent Sediments
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where the eutrophication cycle is intimately associated
with and regulated by bacterial metabolism (22,24).
The suggestion that zooplankton are primarily
responsible for phosphate regulation in the sea (25) is
inconsistent with the data since it has been
demonstrated that axenic, or bacteria-free, cultures of
zooplankton are unable to concentrate phosphate or to
use organic phosphate (26, 33, 34). Further, the
zooplankton were found to supply only one-third to one-
tenth of the phosphate necessary for phytoplankton
(27) . Thus the bacterial systems appear to be a major
agent in the regeneration and solubilization of
phosphate in aquatic environments. Conclusive
demonstration of direct biological exchange of
phosphate is difficult, as most of the activity occurs
below or at the sediment surface. However, it has been
shown that the bacteria are at least partially
responsible for the exchange of phosphates, both
organic and inorganic in the water (28). Nevertheless,
as seen in Fig. 1, a generalized scheme for phosphate
cycling in an aquatic environment has been suggested.
Phosphate turnover or regeneration in aquatic systems
at the sediment-water interface is greatly influenced
by bacterial metabolic activity. The bacteria are
capable of both sequestering phosphate in excess of
their needs, and at the proper stimulus, release the
phosphate (8). Aquatic bacterial populations can
degrade organic phosphates to inorganic phosphates and
influence phosphate solubility as a consequence of
their metabolic end-products. As Hayes and Phillips
(8) have shown, addition of antibiotics to the aquatic
system will greatly reduce phosphate availability. The
data all point to the need for a more complete
understanding of the microbial process as related to
phosphate regeneration and the influence other
nutrients exert on phosphate availability. However,
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the actual extent of these reactions and the effects of
various pollutants in the form of pesticides, heavy
metals, industrial acid and alkali wastes on the
bacterial flora as related to the solubility of
phosphate is unknown.
SULFUR
Biological cycling of carbon, nitrogen and phosphorus
and the importance of these elements to the fertility
of soil and water is widely recognized. Nevertheless,
other elemental nutrients undergo similar cyclic
processes and are of equal importance to the fertility
of the ecosystem. One of the most easily definable
cycles in nature is the sulfur cycle. Although not
widely recognized, the availability of sulfur can limit
productivity of the aquatic environment and has been
linked to decreased productivity of fish (67).
The biochemical and physiological events resulting in
the transition of sulfur through the various valence
states have been the subject of many reviews (35-38)
and will not be considered here. Rather, the
availability of sulfur in the aquatic environment as
regulated by microorganisms will be considered. The
dynamics of sulfur cycling through the gaseous, liquid,
solid, organic and inorganic compounds, oxidized or
reduced states, can be represented by a cycle as shown
in fig- 2(39-43).
There are a variety of reactions involved in the sulfur
cycle and these can be divided into two principal
pathways: 1) the reduction of sulfate to sulfide and 2)
the oxidation of the reduced compounds to sulfate (Fig.
2). Biological oxidation and reduction of inorganic
sulfur compounds in nature is the result of a
relatively select group of ubiquitous microorganisms
(44). Chemical reactions may also occur because of the
13.
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Figure 2.
THE SULFUR CYCLE
REDUCED ORGANIC SULFUR
IN LIVING MATTER
Plants ^ Animals Bacteria
T
Utilization of sulfate
(plants, aicreer|aaisuis)
\
Bacterial decomposition
of organic natter
+>
Desulfovibrio
Desulfotomaculum
©
I
Sulfur oxidation
(colorless and photosynthetic
snlfnr bacteria)
Oxidation of H^S
(colorless and photosynthetic
sulfur bacteria, or
spontaneously)
o
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relative instability of certain sulfur compounds (45),
however, these chemical reactions occur exclusively
with the highly reduced states of sulfur such as
sulfide resulting in deposition of elemental sulfur.
The extent of microbial involvement in the sulfur cycle
is evident to even the most casual observer by the wide
spread production of hydrogen sulfide resulting in
black muds and soils. Also, the extremely acid
conditions found near acid mine drainage is a result of
the metabolism of sulfur oxidizing bacteria. Both of
these phenomena are a direct consequence of the
metabolic processes of sulfur bacteria.
Microbial oxidation of reduced inorganic sulfur
compounds in nature is mediated by one of three
different ways: 1) oxidation by "colorless sulfur
bacteria" i.e. Thiobacilli 2) by the photosynthetic
sulfur bacteria, and 3) incidental oxidation by various
heterotrophic or autotrophic microorganisms. The
relative importance of the chemical oxidation and of
the three microbial processes is discussed by various
workers (41,46,47). There is presently no means for in
situ measurement of the actual contribution of each
process in sulfur oxidation (48). However, it is
generally agreed that the "colorless sulfur bacteria"
are of greatest importance for oxidation of reduced
sulfur compounds in nature (49,50,51).
Examples of the colorless sulfur bacteria are the
Beqqiatoa and Thiothrix which are filamentous and
associated with polluted waters and found in polluted
waters. Both of these organisms can be found easily in
ponds which have become polluted by either sewage,
fallen leaves or other organic wastes. Presence of
these organisms is evidenced by the white scum which
often is sulfur which forms on the top of the water.
Specialized sulfur oxidizers are also found in sea
14.
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waters and are grouped into the genera Thiovulum and
Thioploca (68). These specialized aerobic sulfur
oxidizers are responsible for the biological oxidation
of sulfide to sulfur.
The anaerobic oxidation of hydrogen sulfide to sulfur
is also mediated by certain photosynthetic bacteria
which can be divided into the red Chromatium and
Thiopedia and green or Chlorbrium and
chloropseudomonas. The red and green sulfur bacteria
are capable of an anaerobic photosynthesis coupled to
the oxidation of sulfide to sulfur. The red sulfur
bacteria may form large zoogleal masses. Carbon
dioxide and nitrogen are fixed by various members of
these two groups (68). Other sulfur oxidizing bacteria
are capable of oxidizing elemental sulfur to sulfate.
The colorless sulfur oxidizers, the Thiobacilli. are
able to oxidize a variety of reduced sulfur compounds
both in aerobic and anaerobic conditions, and to use
either organic carbon (heterotrophic growth) or carbon
dioxide (autotrophic growth) (49,52). Representative
species of Thiobacilli have been isolated from a
variety of aquatic environments such as sea water (53),
fresh water (54), marine sediments (55), and soils
(56). Thiobacilli are especially abundant in estuarine
waters and sediments (57,58) and in volcanic sulfur
springs (59,60,61).
Biological reduction of sulfate can proceed by one of
two separate pathways, assimilatory and dissimilatory
(35). The assimilatory pathway involves the
incorporation of sulfate into organic compounds and is
carried out by the majority cf bacteria, fungi, yeast,
algae and plants (see 44). The dissimilatory pathway
involves the direct reduction of sulfate via sulfite to
sulfide. In this pathway, the function of sulfate is
analogous to that of oxygen in aerobic systems in that
sulfate is "the -terminal electron acceptor.
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Dissimilatory sulfate reduction is a property exclusive
of two genera of ubiquitous microorganisms the
Desulfovibrio (30) and pesulfotomaculum (29). The
dissimilatory sulfate reducers have been found in
marine environments (69,70), Antarctic waters (71,72),
fresh water (73), inland lakes (74), and soils (75,76).
In a balanced ecosystem, the relationship between the
sulfur oxidizers and sulfate reducers is an intimate
one. The sulfur oxidizers yield sulfate and are
capable of autotrophic growth fixing carbon dioxide to
yield organic carbon. The autotrophic sulfur oxidizer
Thiobacillus denitrificans also reduces nitrate to
nitrogen (77). The sulfate reducers have been found to
fix elemental nitrogen (78,79,80,81), reduce sulfate to
sulfide and require organic carbon for growth (35)„
Hence, the microbial activity which is associated with
the sulfur cycle influences the cycling of other
essential nutrients.
Although animal systems can oxidize reduced sulfur
compounds such as sulfide (62), thiosulfate (63), and
sulfite (64), and can incorporate sulfate into various
organic molecules (65), they are unable to reduce
sulfate to sulfide (66). To accomplish this they must
depend upon plants and bacteria to provide them with
reduced sulfur compounds (33, 35). Thus it becomes
apparent that the sulfur cycle, as regulated by
microbial life, influences aquatic life not only by
making sulfur available in various oxidation states,
but also affects nitrogen, carbon, and phosphorous
concentrations.
NITROGEN
Biological nitrogen cycling involves, as does cycling
of sulfur and phosphorus, the transition of an
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elemental nutrient through various chemical states. It
is convenient to initiate consideration of the nitrogen
cycle at the point where the fixation of gaseous
nitrogen occurs, i.e.. where atmospheric nitrogen is
converted to ammonia. Relatively few species of
microorganisms populating the earth are capable of
metabolizing nitrogen from the air. Among the first
investigators contributing to an understanding of the
role of symbiotic microorganism in leguminous plants
was Beijerinck (87). His work has been continued and
expanded by many, and the history of our understanding
of the biology of bacterial nitrogen fixation in
leguminous plants has been thoroughly reviewed (89)•
We now know that the only organisms capable of nitrogen
fixation are certain free-living and symbiotic bacteria
and blue-green algae (85,86,88,90). The biological
transformation of molecular nitrogen through its
inorganic chemical states by this select group of
microbes makes nitrogen available to all the world*s
flora and fauna which are dependent, either directly or
indirectly, upon these microorganisms.
The fact that nitrogen fixation is unigue to a few
species of bacteria and blue-green algae, gives rise to
important ecological questions. Among the most
challenging are those relating to discovery and
elucidation of the mechanisms of biologic nitrogen
fixation. It was not until 1960 that cell-free
fixation was demonstrated using extracts prepared from
the anaerobic bacterium Clostridium pasteuranium (91,
92)• This fundamental break through has permitted a
rapid expansion of our knowledge concerning the
biochemical mechanisms involved in nitrogen fixation,
and many reviews are available detailing the
physiological processes carried out (85, 86, 88, 93,
94). In several respects, it is now clear how nitrogen
gas can be utilized as a source of nitrogen for the
synthesis of amino acids, nucleic acids and other
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nitrogen-containing cellular components by these
microorganisms, and how the nitrogenous compounds are
transferred to higher plants and animals along the food
webs in nature and in agriculture.
Another striking aspect of nitrogen cycling is the
variety of the effects that alterations in the quality
and quantity of organic carbon and concentrations of
phosphorus and sulfate have on the activities of the
microbes involved in nitrogen transformations.
It has been demonstrated that increased availability of
an easily ulitized carbon source like glucose will
generally depress the amount of atmospheric nitrogen
fixed in the aquatic environment. In contrast,
increasing availability of soluble phosphorus will tend
to increase nitrogen fixation (95). The biochemical
bases for these phenomena apparently are found in the
complex nutritional relationship of the various
heterotropic organisms responsible for nitrogen
fixation and their requirement for various carbon
substrates, and the interactions among the enzymatic
processes involved.
The activity of the organisms responsible for sulfate
reduction and sulfide oxidation will affect both
nitrogen fixation and denitrification (32, 75, 77, 80,
81). increased sulfate concentrations in an ecosystem
will enrich the capacity for outgrowth of sulfate
reducing bacteria which fix nitrogen. Furthermore, as
a consequence of the hydrogen sulfide evolved by these
bacteria, a drop in eH will occur The lower eH favors
the development of other anaerobes capable of nitrogen
fixation and in the denitrification reactions as well.
Neither phenomenon can be viewed independently of the
other.
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Figure 3.
REVIEW OF THE NITROGEN CYCLE
Reduced Nitrafen
in organic Matter
(H2N-NH2)
(HNsNH)
(death ft
¦-NH "
lM|n
Fixation
Atmospheric
Nitrogen
Oj Mwitive)
'Diaaiailatonr redaction"
"Aninulatery redaction"
(aerebic.
(neatly aerobic)
(aerebic and alkaline)
"Nitrification
-------
The availability of oxygen in aquatic systems affects
the operation of the processes of denitrification,
nitrification. These reactions are illustrated in Fig.
3 and are the major components of inorganic nitrogen
metabolism. Depending upon the various chemical and
physical parameters impinging on a system, there may be
increased or decreased quantities of nitrate, ammonia
and in rare instances, nitrite in the aquatic
environment.
It has been suggested that the overall contribution of
fixed nitrogen by bacterial and blue-green algae in
certain fresh water lakes amounts to 15 mg per square
meter in a day (95). While this appears to be
inconsequential when compared to the contribution that
is due to agricultural runoff, the total in situ
microbial fixation of nitrogen in a Wisconsin lake has
conservatively been estimated to be in excess of 80,000
lbs per year (178). This amount of fixed nitrogen is
thought to be introduced into the aquatic environment
during certain seasons and is exclusive cf any excess
which may be introduced due to agricultural or sewage
runoff.
Subsequent to fixation the relative concentrations of
the inorganic nitrogen compounds in water, i.e.,
nitrate, nitrite, and ammonia, depend, in part, on the
amount of oxygen available and the oxygen
concentrations are dependent upon the organic carbon
load and seasonal variations in solubility of oxygen in
winter. Attempts to develop a nitrogen balance in
lakes and other aquatic environments are hampered by
the fact that there are several possible sources for
loss of nitrogen. For example, fixed nitrogen can be
lost via: (1) lake effluents; (2) loss cf volatile
nitrogen such as ammonia and nitrogen gas (3) ;
denitrification by certain microbes; (4) precipitation
of nitrogenous compounds into either permanent or
19.
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semipermanent sediments; and (5) removal of organisms
by fishing, weed harvesting or other methods of flora
or fauna depletion (95) .
In general, well oxygenated waters will contain higher
concentrations of nitrate than ammonia and very little,
if any, nitrite- One possible reason for this is the
action of the nitrifying bacteria which will
aerobically convert ammonia to nitrate autotrophically.
However, in waters containing lower oxygen
concentrations there will be higher concentrations of
nitrite and ammonia (96, 97, 98). During summer
months, inorganic nitrogenuous compounds may completely
disappear from the epiliminon in some lakes even though
abundant in spring (98).
The biochemical mechanisms involved in denitrification
have only recently been elucidated in significant
detail (99-102). Those reactions that result in the
conversion of nitrate to nitrogen gas are unique to a
limited group of microorganisms found in the family
Pseudomonadales.
A generalized interrelationship between the
concentrations of ammonia, nitrate, nitrite, and oxygen
in various systems can be developed based on the data
from both laboratory and field studies. It has been
shown that oxygen will inhibit denitrification (nitrate
to nitrogen) in Pseudomonos perfectomari um. an aerobic
marine bacterium. However, when the oxygen level
becomes limiting for aerobic growth these aerobic
organisms can use either nitrate or nitrite as an
electron accepter, that is, as a substitute for oxygen
(99-102), in their respiration. It has been found that
in lakes and in the ocean, nitrate concentrations
follow temperature or seasonal variations and vary with
depth (95, 106-108 ). The oxygen levels also parallel
20.
-------
these variations so that as the oxygen concentration
falls, the organisms begin to utilize nitrate as a
terminal electron accepter. The result is nitrate
concentration reduction or depletion. The field
results are in agreement with laboratory studies of
Payne, et al, (99-102). By the process of
nitrification (ammonia to nitrate), respirable oxygen
in the form of nitrates becomes available as a stored
oxygen resource to be liberated by the denitrification
process (109).
Although findings from both laboratory and field
studies are consistent and tend to support the above
conclusions (95* 99-109), it must be noted that there
have been no definitive studies which actually link all
of the above detailed conditions at the same location
and time. Nevertheless, the above relationships serve
further to emphasize that all the nutrients are co-
egual in importance and any alteration in components of
one cycle can affect other nutrient availability. For
natural populations in aquatic systems, the theory of
nutritional control by a "master nutrient" is not
consistent with data obtained from laboratory
experiments and from field observations.
CARBON
The carbon cycle is composed of an integrated network
of physical and biologically mediated pathways
encompassing the synthesis, degradation, and
transformation of innumerable simple and complex
organic molecules. Superimposed on the carbon cycle
are the controls exerted by nutrient availability, and
the fixation and evolution of carbon dioxide. Various
aspects of organic carbon cycling in the aquatic
environment have been examined (103, 104) with the
emergent principle that an overall balance between
production or synthesis and decomposition of naturally
21
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occurring substances exists in nature. Fhotosynthetic
carbon dioxide fixation by green plants is the major
route by which carbon enters the organic carbon cycle.
However, fixation by autotrophic bacteria add to the
total carbon budget in the ecosystem (120,121) . Once
organic material has been introduced into the aquatic
environment the endogenous flora and fauna can either
utilize or contribute to, depending upon conditions, an
existing reservoir of organic material (105). Some
ecological questions relating to carbon arise when
considering the microbes direct relationship to carbon
cycling. For example, what affect does microbial
synthesis of complex molecules such as vitamins, amino
acids, carbohydrates and lipids have on the aquatic
biota; what is the contribution of the bacterial
biomass as a food source for zooplankton; and, what is
the significance of microbial degradation of suspended,
soluble and sedimented organic compounds? The health
related aspects of bacterial growth are of importance.
Growth of known pathogenic microorganisms can result
from the introduction of raw sewage into water.
Further, recent studies demonstrated that
microorganisms normally considered of no medical
significance are capable of causing human illness
(122). The introduction of wastes from citrus
processing plants and waste paper pulp mills into
rivers and lakes has been demonstrated as providing an
environment suitable for the growth of bacteria capable
of causing human disease. These complex
interrelationships of bacteria to the aquatic habitat
can affect water quality from a public health point.
Another dimension is added with respect to carbon
cycling by the addition of synthetic organic compounds.
Examples of such synthetic compounds are certain
pesticides, detergents, and various other industrial
materials. The majority of the biodegradation process
22.
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involving these compounds are achieved exclusively by
the bacterial population.
Direct and complex relationships between diverse
organisms have evolved based on the needs for various
growth factors. Examples of these relationships are
seen in the association of various algae and bacteria
in the marine and fresh water environment (110-112,
119). Apparently, the microbial flora is one of the
major producers of surplus vitamins which stimulate the
growth of other algae (113-118). In practical terms,
the reason that various eutrophic agents, such as
excess phosphates, sulfate, nitrate and certain carbon
compounds, cause characteristic nuisance blooms can be
traced to the abundant growth of certain bacteria which
produce the necessary growth factors for algae (110).
In addition to providing essential growth factors, the
bacteria further serve as a source of food for higher
aguatic organisms. Specific marine invertebrates were
shown to consume bacteria, as well as require a select
microbial flora to aid in their digestion (123).
Bacteria and protozoa adsorbed onto detritus also
provide nutrition to certain fish (124). Marine
benthic organisms such as bivalves and bottom feeding
fish have been shown to consume bacterial cells as a
food material (125). A generalized scheme becomes
apparent. Bacteria and fungi initiate the primary
stages of oxidation, hydrolysis and assimilation of the
carbon structure of plant debris. The bacteria are
consumed by protozoa creating a bacteria-protozoa-
detritus system which serves the role of a food source
for a larger organisms such as snails, bivalves and
fish. Such cycles have been suggested as providing
food sources under controlled conditions (121).
Credence is given to the above suggested cycle
since the bacteria are responsible for the
23.
-------
decomposition of nutrient organic matter and the
bacterial biomass in certain marine environments is
equivalent to that of the benthic organisms (126).
Bacteria have also been shown to act as concentrators
of nutrients in dilute solution. Protoza, for example,
grow poorly in a dilute nutrient medium but flourish in
the presence of bacteria (127). Brine shrimp have been
shown to grow only over sediments which support a
bacterial-protozan population and not over a
particulate sediment (128, 129), and are apparently
dependent upon the bacterial populations.
An interesting relationship between nutrient
availability and bacterial growth has been observed in
the presence of excess phosphate ions. Eacteria are
incapable of utilizing organic carbon material adsorbed
onto clay particles. However, once the clay was
exposed to phosphate ions (0.3M concentrations) the
organic material became desorbed and the bacteria were
able to multiply (130). This fact suggests a second
role of phosphate in water in addition to constituting
an essential mineral element for growth. Physical
factors involving phosphate ions and the solubility of
organic compounds are of importance in determining the
primary and secondary role of phosphate as a nutrient.
Soluble organic material in waters is composed of free
sugars, (see 131, 135), and amino acids (132-135) where
a relationship appears to exist between these organic
nutrients and heterotophic bacteria (135-137). Both
bacteria (139) and fungi (140) are responsible for the
decomposition of the soluble or suspended organic
material and the higher aquatic plants and benthic
organisms are responsible for the primary production
(141,142). Of all the soluble or suspended organic
material in the water column, it has been suggested
that only 10-20 percent reaches the bottom (145) with
80 to 90% being consumed by bacteria in the upper
24.
-------
layers. Physical factors, including the height of the
water column and turbulence, will affect the
sedimentation rates making it impossible to extrapolate
results between bodies of water due to variations in
the physical environment (144).
Soluble organic material does adsorb onto clay
particles at the sediments by mud water interface
(130). However, the adsorption may not be permanent
and an equilibrium is reached between the adsorbed and
soluble material (145). Thus, the clay acts as a
potential nutrient sink and the associated bacterial
activity creates an appreciable oxygen demand (144).
Consumption of oxygen at the bottom creates an
anaerobic benthic environment which favors the growth
of the methane producing and other anaerobic bacteria.
A vigorous anaerobic bacterial activity is evidenced by
the fact that methane hydrogen sulfide, and carbon
dioxide has been found coming up from lake bottoms
(146,147). The bacteria, fungi, and heterotrophic
fauna in the sediments utilize the biological debris
and act as major recyclers of dead fauna and flora.
The importance of microbial activity in decomposition
of biological debris is best exemplified in certain
bogs. When the water quality is such that pH and
dissolved oxygen become limiting factors to bacterial
growth resulting in decreased biological activity, a
corresponding accumulation of peat occurs (plant and
animal debris). However, this build up can be reversed
if the physical conditions are controlled allowing a
resumption of bacterial metabolic activity (148-150).
Thus the role of the microbes (bacteria and fungi) is
concerned directly with the breakdown and cycling of
the relatively resistant skeletal material of plants
and animals. It has been suggested that bacteria
maintain the organic material at low concentrations
25
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TABLE 1
Biodegradable Compounds3!1
ORGANIC COMPOUND DEGRADING MICROBIAL ORGANISMS
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Phenol
Chlorophenoxyl
ethyl sulfate
Long-chained
alcohols C12-C20
Phenoxyalkyl
carboxylates
Primary alcohol
sulfates
Secondary alco-
hol sulfates
Ether glycols
Chloroacetate;
dichloracetate
4-Chloro-2-methyl
phenoxyacetate
Bacillus, Micrococcus
Pseudomonas species
gac&Uus ceyeyq
Pseudomonas C12B
species
Pseydppipqfls C12B
Aerobacter cloacae.
Bacterium TEG-5
Pseudomonas species
Stra ight-ch ai ned
isomers of alkyl
benzene sulfonate
Polyvinyl alcohol
Trifluralin
Aresin
ZlavsbaStefjjgn
BSESariimffl# &£tb£2*
bacter species
C12B
species
wesentericus
Pseudomonas species
£aSiU3iS# Psey^pn^onas
species
Pseudomonas K62
15.
16.
17.
18.
Phenyl mercuric
acetate; ethyl
mecuric phosphate;
methyl mercuric
chloride
Prometryne Aspergillus species
Acetonitrile activated sludge
Nitrobenzoates activated sludge
Chlorinated activated sludge
alkyl and aryl
compounds
REFERENCE
152
156
157
158
159,160
161
153,162
163
164
154,155
165
166
167
168
169
170
171
172
~Adopted from Payne, al. (151).
-------
(138) and are exclusively responsible for the uptake of
nutrients even in high concentrations (136).
Assimilation and biodegradation of naturally occurring
organic compounds as a dynamic biological continuum of
activity is tacitly assumed and accepted. When natural
bacterial processes become inhibited, a build-up of
organic debris leading to the creation of bogs. As man
has progressed culturally and technologically, new
stresses have been placed on the environment in the
form of man-made, synthetic organic material which may
or may not be biodegradable. Build-up of the non-
degradable agents, such as certain non-linear alkyl
sulfonates (151) and certain pesticides, are of general
concern since they accumulate in the environment.
Fortunately, most synthetic compounds are subject to
bacterial degradative processes (see table 1).
Microbes, however, are not infallible and are incapable
of degrading certain halogenated organic molecules.
Examples of presistent compounds are the pesticides
aldrin, dieldrin, endrin, toxyphene, DDT, benzene
hexachloride, chloradene, and others (173). These
compounds are not a part of the nutrient cycle since
they do not provide an energy source. However,
molecules can be concentrated by microbes (174* 175,
176) providing one entry point into the food cycle for
further degradation or deposition in sediments.
Bacteria, as noted above, do provide a food base for
many benthic animals, and as has been suggested are a
major source of food for aquatic fauna (177). of
naturally occurring substances exists in nature.
ENGINEERING ASPECTS
The engineering phase of any study, stated simply, is
the evaluation of all available scientific information
and subsequent determination of the optimum control
26
-------
plan within political, economical and scientific
constraints to obtain an objective. By this definition
most papers discussing the control of eutrophication by
limiting some essential nutrient are engineering
oriented.
The basic steps in controlling the eutrophication
of a lake for example are:
1. Determination of the source of nutrients and
the biological response of the system to those
nutrients by means of field surveys and
laboratory studies.
2. Analysis of the information by mathematical
models or other methods which include
consideration of alternative control schemes
and which incorporate economic factors in
addition to scientific factors.
3. Implementation of the control scheme by
facility construction and/or system
modification and operation.
4. Evaluation of the effectiveness of the
solution by surveillance of water quality
changes.
Items 1 and 2 in the above listing actually are
interrelated and occur simultaneously in well conceived
projects. Mathematical models must be adapted to or
developed for a particular waterway to take into
account geomorphological characteristics. Data
collection likewise must be tailored to supply proper
input to such models.
The implementation phase is the classic engineering
design and construction of structures such as sewage
27.
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treatment facilities with nutrient removal or the
installation of sewers to collect direct discharges for
treatment or diversion out of a drainage basin-
Operation of these facilities to maximize nutrient
removals is becoming more important.
Surveillance is an often overlooked phase but which is
extremely important, especially currently when large
sums of money are to be spent for nutrient removal. It
is important to study and compare actual lake water
quality changes versus those predicted and to establish
true cost per improvement amount.
Engineering Analysis
The primary interest of this report is the microbial
cycling which has been described for the various
inorganic nutrient cycles. To tie these cycles
together and predict biological production at various
levels in the ecological system is one approach to
determining the critical nutrient for a given system.
Another approach is statistical and consists of
correlating land use patterns and nutrient runoff loads
versus various measures of eutrophy. By calculating
various reductions in loading corresponding reduced
levels in the eutrophy indicators occur.
Modeling Bacterial Systems
The mathematical modeling of bacterial systems
described in the sanitary engineering literature
generally concern either descriptions of bacterial
densities downstream from waste discharges or the
increases in the mass of organisms in treatment
systems.
Public Health Bulletin No. 143 (179) which reports on
an extensive water quality study of the Ohio River
28.
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contains one of the first attempts of quantitatively
describing bacterial behavior downstream from waste
imputs in a natural system. Two types of formulations
were fitted to the data collected from the Ohio River.
The first curve was the autocatalytic or logistic
function: b
y - 1 + 16« (1)
where y = bacteria as a percentage of the maximum
density
t = time of flow from sewer outfalls
a#b#c,d= constants requied to fit data points
The second formulation was a composite exponential type
formulations
y - a(10"bt) + c(10~dt) (2)
where the symbols are the same as above. Equation (1)
was fitted to crested shape curves which were
indicative of a growth phase before a die-way phase.
Equation (2) was simpler to evaluate and was used to
fit only the declining portion of the curve.
Equation (2) is actually an extension of results
attributed by Streeter (180) to Chick for describing
bacterial mortality caused by disinfectants. The
equation attributed to her work was:
1 log y1/y2 8 K or yx « y2 exp(-K )
t2-ti
yi#y2 ® numbers of live bacteria
tlrt2 88 time
K * proportionality constant
29.
-------
However, this simple formulation was inadequate to
explain the Ohio River die-away curves and led to the
formulation of the composite exponential mathematical
form to explain the die-away portion of the curve.
Subsequently, streeter (180) developed a composite
exponential to explain the crested shaped curve as well
as the die-away portion during the Ohio River Studies.
Thp Pnnat--i on nno^ uas nf tho f/irm*
where: y=denotes numbers of bacteria
t=time
y =initial bacterial numbers
a#b#c=constants
n=constant which determines the sharpness of
the crest
k=reaction constant
The values of all constants were determined
graphically.
Hoskins (181) analyzed bacterial data collected from
the Ohio River and the Illinois River. For these data,
two sets of die-away curves were developed to
distinguish between summer (T 15 C) and winter (T 15 C)
conditions. Each set contained curves which were
proportional to the initial bacterial density.
It is interesting to note that these early workers
expended as much effort describing the bacterial
densities associated with pollution as with the oxygen
sag formulations. However, only the latter
formulations came into general use by sanitary
engineers.
(4)
30.
-------
Fair and Geyer (182) suggested another formulation
which is of Chick*s law and includes a non-uniformity
coefficient. Their equation is in the form of a
retardant and is written :
N/N0 « (1+nkt) "1/n (5)
where: N = the model number of bacteria in a
stream at time zero
N « the modal number of bacteria at
subsequent times downstream
K = is the initial die-away rate constant
n = coefficient of non-uniformity
It can be shown that equation (5) becomes equation (3)
when n=0 by evaluating the indeterminate form that
results using the mean value theorems of the calculus
(183) .
Much of the early work concerned bacterial groups other
than the coliform bacteria. Streeter's work considered
additionally the enumeration of standard plate counts
on nutrient agar andoon gelatin (179). The agar counts
were incubated at 37 C and were considered indicative
of heterotrophic bacteria acclimated to the bodies of
warm blooded animals. Gelatin plates were incubated at
20"C and were considered indicative of heterotrophic
bacteria favoring locations in nature outside of warm
blooded animals such as soils. Overlap between these
two groups were recognized but the extent was
undetermined (179).
None of these formulations considered bacterial numbers
as part of the overall food chain, streeter (180)
presented simplified calculations which alluded to the
relationship between a given number of bacteria and the
31.
¦V.c"t,(nroh *•»-- -
a. W. 33th Streak —*«««» UMr
1m
-------
BOD observed„ His analysis was limited to the
heterotrophic oxidative assimilation of organics.
Although streeter (184) recognized the phenomena of
nitrification, he did not include the effect in the
oxygen sag formulation because of the belief that it
occurred after the critical point (maximum dissolved
oxygen deficit) of the sag curve was reached.
Others in the U.S. Public Health service Cincinnati
group (185, 186# 187) in addition to Streeter
recognized and described the bacterial-zocplankton
relationships in natural systems. Butterfield, al.
(186) presented both bacterial density and BOD data as
a function of the presence or absence of zooplankton.
In fact, bacterial regrowth in streams was postulated
as resulting from an interruption of the bacteria-
predator relationship (187). This phenomenon was not
explicitly formulated in any of the descriptive
equations for bacterial survival presented although the
effect was implicitly included in the various die-away
coefficients. The increase in biological mass (sludge)
for treatment systems of the activated sludge type is a
significant design parameter. To accomplish prescribed
treatment in terms of organic constituent removal,
treatment systems must provide a suitable environment
in terms of waste-sludge contact time, sedimentation
time and sludge wasting facilities. Pilot plant
empirical determinations of these parameters are as old
as the activated sludge process itself. An excellent
literature review of the systematic study of sludge
production has been prepared by servizi and Bogan
(188) .
Servizi and Bogan (188) suggest that the amoant of
synthesis of activated sludge is proportional to the
free energy of the substrate. Laboratory evaluations
of the proportionality constants yielded the following
equations:
32
-------
carbohydrates y = 0.38 COD (6)
aromatics & aliphatic acids: y = 0.34 COD (7)
where: y = gross synthetic yield in grams of
cells per mole of substrate
COD = chemical oxygen demand by dichrornate
oxidation in grams per mole of substrate
The concept of bacterial growth yields as a function of
substrate energy was further validated by the work of
Mayberry, et al (189). They calculated yields by four
different methods: grams of cells per mcle of
substrate, gram atom of carbon used, mole of oxygen
consumed, and equivalent of "available electrons" in
the substrates. The "available electrons" calculation
or energy available system proved the most consistent.
Although the amount of sludge produced could be
determined, the rate of production still needed
quantification. For simple pure culture systems using
a single substrate, Monod (190) used the enzymatic
kinetics of Michaelis as a model to predict rates of
growth. Such kinetics are the generally accepted model
today for purposes of modeling biological systems. For
mixed cultures such as activated sludge which oxidizes
mixed substrates of varying complexity, simpler kinetic
models have been adopted.
33
-------
For example, Eckenfelder and 0*Connor (191) adopted a
linear model:
a s = ai'r - b^, (8)
Where: as = increase in sludge concentration
vr = the BOD (or COD) removed per day
S« - the initial sludge mass per unit volume
a = fraction of BOD removed synthesized to new
sludge per day (see constants on equations
(6) & (7)
b = rate of endogenous respiration expressed
as a fraction per day
This model proved adequate for design when the system
operated over only a small portion of the growth curve
(e.g. declining growth - endogenous respiration).
All of the foregoing bacterial models assumed that
sufficient growth factors and trace elements are
present. Very little work has been done to determine
what critical levels of trace elements are required.
Eckenfelder and O'Connor (191) reported that other than
nitrogen and phosphorus, trace elements are generally
available in sufficient quantities in natural waters.
Helmers, et al. showed that for optimum activated
sludge process efficiency a minimum nitrogen and
phosphorus content of the sludge expressed as a percent
of the total volatile solids is 7 percent and 1.2
percent respectively. This same study indicated that
the critical amounts of nitrogen and phosphorus per 100
lbs. of BOD removed were 3-4 lb and 0.6 lb.
respectively (cited in 191).
34
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Algal Modeling
Mathematical modeling of algal systems in the
engineering literature has been concerned
predominantly with net oxygen production. Oxygen sag
formulations of the Streeter-Phelps type freguently
include the terms photosynthetic oxygen production (P)
and algal oxygen respiration (R). The difference
between production and respiration is the net oxygen
production which may be positive or negative. Several
authors (192# 193, 194, 195) have presented models
which include such phenomena. Such models are solved
for steady state conditions after the algal mass has
developed. No consideration was made of the growth
phase or the die-away phase of these algae. Inorganic
nutrients were therefore not incorporated into the
formulations. Additionally, such formulations describe
the effects caused by the algae rather than the
kinetics of the basic algal processess involved.
Limnologists have produced sophisticated mathematical
productivity models of two basic types. The first type
considers the dynamics of growth in relation to growth
factors. The second type considers productivity in
relation to depth of the water column. These
distinctions and descriptions of the various
developments are presented in an excellent review of
the subject prepared by Patten (196). Patten presented
a qualitative model which has terms considering the
factors affecting plankton populations:
income - loss
£fl (influx of producer biomass) + f2(photosynthesis) +
f3 (influx of higher trophic level biomass) +f^(auxo-
trophic and heterotrophic growth]] - (f5(respiration)+ fg
(grazing+f7 (efflux biomass from all trophic levelsj
B = plankton biomass in a unit volume of water
at some depth Z
t = time
f(i)t i-1, 2...7 are processes expressing
major
JLS =
>jt
where
35
-------
Each of the subsequent models presented by Patten
considered various combinations of terms of equation 9,
as well as different functional relationships for these
terms. Many of these relationships can be used in the
development of control strategies. These models,
however, do not consider symbiotic relationships
between bacteria, phytoplankton and zooplankton
populations.
Eutrophication Control
Recently, several researchers have developed
mathematical models which attempt to describe
phytoplankton population dynamics by consideration of
nutrient inputs and bacterial metabolism specifically
for the purpose of providing a basis for developing
eutrophication control programs through nutrient input
limitations.
Two types of models are available. The first type
describes algal dynamics using kinetics such as those
described by Patten (196) in his review. The second
type uses statistical procedures to analyze nutrient
inputs and lake responses from many lakes by applying
multiple regression statistical techniques. The
resulting equations are used to predict eutrophic
conditions in homologous lakes by incorporating the
input of the requisite nutrient data with other
physical and chemical information.
Examples of the first type mathematical model have been
developed by Echelberger, et al. (197), Di Toro, et al.
(198), and Water Resource Engineers, Inc. (199).
Examples of the second type include the work of
Vollenweider (200) and that of Brezonik and shannon
(201) .
36.
-------
Table 2
First Principal Components (yco and yc]_)
and Trophic State Indices (TSIco and TSIci )
(a) Colored Lakes:
Ycon" •848(1/SD) + .809(COND) + .887(TON) + .768(TP)
+ .930 (PP) + .780 (CHA) + .893(1/CR)
Cumulative Percent of Total Variance Explained by y « 72%
TSI^q = ye© + 9.33
(b) Clear Lakess
y cl= .936(1/SD) + .827(COND) + .907(TON) + .748(TP)
+ .938(PP) + .892(CHA) + .579(1/CR)
Cumulative Percent of Total Variance Explained by yc^ « 71%
TSId - yci + 4.76
-------
Figure 4 illustrates the degree of real data
simulation afforded by the predictive model of DiToro,
et. al. (198) for the San Joaquin River near Mossdale,
California. Phytoplankton, zooplankton and total
inorganic nitrogen simulations are shown. Inputs to
the model included inorganic nitrogen (limiting
nutrient), temperature, solar radiation and the
advective flow.
Brezonik and Shannon (201) developed a Trophic State
Indicator (TSI) which is computed from seven
indicators: 1. primary production (PP), 2. Chlorophyll
a (CHA), 3. total organic nitrogen (TON), 4. total
phosphorus (TP), 5. Secchi Disc tansparency (SO), 6.
conductivity (COND), and 7. cation ration (CR= Na + K
Table 2 taken from their publication presents the Mg + Ca
relationships derived for colored and clear water
Florida Lakes.
By calculating regression equations between the TSI and
land use factors, the equations on Table 3 were
developed. The first equation considers TSI as linear
function of land use patterns within the watershed plus
immediate and remote cultural units. The second
equation considers TSI as a function of land use
patterns and total cultural units (Sum of remote,
immediate and sewage treatment cultural units). Table
4 and figures 5 and 6 present the calculated critical
phosphorus and nitrogen loading rates for Florida
Lakes.
Although the results obtained by Brezonik and Shannon
cannot be directly applied to lakes in other sections
of the country without extensive verification, similar
relationships may exist for other sets of lakes
perhaps, regional location or morphological similarity.
Such relationships could then be established by using
similar methodology.
37
-------
Figure 4
100,000
80,000
h , 60,000
* ^
3 ^ 40.000
CL -J
0 w
> ° 20,000
1
a.
z
o
h
< S
3 5
a. z
o
o
N
16,000
12,000
8.000
4,000
360
•
-
• r
• • I
%
•
•#
m
• A#
M •
\
\
I / 1
_J
. 1.
1. 1
1
120
240
360
120
240
360
<£
(9 S
a.
o z
Z u
©
J O
< a:
H t-
120 240 360 120 240
1966 TIME - DAYS '*67
360
PHYTOPLANKTON, ZOOPLANKTON, AND TOTAL INORGANIC NITROGEN.
COMPARISON Of THEORETICAL CALCULAT IONS AND OBSERVED DATA.
SAN JOAQUIN RIVER, MOSSDALE CALIFORNIA, 1966-1967 (198)
-------
Figure 5
PHOSPHORUS SUPPLY (g/m2/yr)
0 .10 .20 .30 .40 .50
1 1 1 1 1 1
I2.CXXP)
VOLUMETRIC LOADING CURVE
SURFACE LOADING CURVE
I
O .05 .10 .15 20 .25 30
PHOSPHORUS SUPPLY (o/««Vvr)
MEAN TSi VALUES FOR FIVE TROPHIC GROUPS v*. ANNUAL PHOSPHORUS
LOADING IN g/m'-yr Ana g/m®-yr.
BRACKETS INDICATE RANGE FOR ONE STANDARD ERROR. SYMBOLS OF
TROPHIC GROUPS ARC: ULTRAOLIGOTROPHIC (U). OLIGOTROPHY (O).
MESOTROPHIC (M). EUTROPHIC IE). HYPEREUTROPH1C (H) (201).
Figure 6
NITROGEN SUPPLY (fl/m2/yr)
0 2.0 4.0 6.0 8.0
) 1 1 1 1
20
M
rrve. •«?«)
-(TSI". Sit
x 12
u
r- .996
4 -
VOLUMETRIC L0A0IN6 CURVE
SURFACE LOADING CURVE
J.
40
3.0
50
1.0
NITROGEN SUPPLY (g/m3/yr)
MEAN TSI VALUES FOR FIVE TROPHIC ©ROUPS vs. ANNUAL
NITROGEN LOADING IN g/m*-yr and g/m* -yr.
SEE FIGURE s FOR EXPLANATION OF SYMBOLS (201^
-------
Optimum or Cost-effective Solution
The models developed in previous sections in essence
are "production functions." That is, for a given level
of nutrient input, a certain level of phytoplankton
biomass or trophic state level will be predicted. By
testing the reduction of nutrient inputs in the model,
a corresponding reduction in biomass for the prototype
system is predicted.
Evaluation of all available and feasible alternatives
by using technical, economic and political criteria,
the various possible solutions can be formulated in
monetary terms. Testing these available solutions with
the corresponding natural system responses can
ultimately lead to the most cost-effective solution of
a problem.
As an example, assume that the phytoplankton biomass
production function for a specific lake has been
determined. The various wastewater treatment
alternatives available to a city located on the lake
might be advanced waste water treatment, disposal on
nearby farmland by spray irrigation, or diversion
around the lake of all waste discharges. Each of these
alternatives have costs which can be evaluated. A
model can be used to calculate which individual or
combination of alternatives can meet the lake
requirements in the most cost-effective manner. A
solution would then be recommended. Suitable nutrient
removal techniques and estimated costs have been
described by Rohlich and uttormark (202) and Swanson
(203) .
Post Project Evaluation
Projects involving nutrient removals as part of the
waste treatment system are relatively new. The actual
38
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Table 3
Stepwise Regression Analysis of Trophic State Index vs.
Eutrophication Factors Expressed Per Unit Lake Volume
(1) Regression Equation:
TSI = 14.9 5(HFA) + .64(FOR) + 2.72(ICU) + 1.59(URB)
59.6 73.9 80.0 81.2
- .35(UCA) + .06(RCU) - .02(PA)
81.5 81.5 81.5
F Ration = 28.98***
Multiple Correlation Coefficient (r) «= .903
Percent of total variation explained by the
regression equation - 81.5%
(2) Regression Equations
TSI - 14.49(HFA) + .61(FOR) + 2.23(URB) + .53(TCU)
59.6 73.9 79.4 80.0
+ .31(UCA) - .01(PA)
80.3 80.3
F Ration = 31.91***
Multiple correlation coefficient (r) * .696
Percent of total variation explained by the
regression equation = 80,3%
Key to Eutrophication Factor Symbols:
HFA = Heavily fertilized cropland (n?/m3)
FOR ¦ Forested area (m?/m3)
ICU » Immediate cultural units (#/m xlO )
URB * Urban area (nr/m3) 2 _
UCA ¦= Unproductive waste cleared area (nr/m )
RCU - Remote cultural units (#/m^xl0^)
PA «= Pastured area (m?/m3)
TCU «= Total cultural units (#/m3xl04)
***Denotes significant F value at the 99% confidence level.
Values below sympols in regression equation indicate
cumulative percent of total varieance explained by
independent variables up to that point.
-------
changes which occur in bodies of water following
institution of such treatment should be thoroughly
documented on a year by year basis. Such accumulated
information will provide knowledge on actual system
responses versus those predicted. The validity of
assumptions, bio-kinetic formulations, species
dominance changes and bottom mud-water interactions are
types of data needing verification. Such information
is required before nutrient removal waste treatment
programs reach maximum expenditure levels so as to
avoid many treatment mistakes and still provide for
corrective action.
In addition to receiving system responses, actual
operating experience with new treatment systems is
necessary to obtain actual removals, consistency of
operation and costs.
Another problem which must be answered is model
extrapolation especially with mathematical models that
must be extended far outside of the data ranges used in
their development. Such situations can occur with
models developed for grossly over-fertilized lakes and
are used to extrapolate conditions for achieving
oligotrophy. Another facet of this problem is the
calculation of lake response following the removal of
only one nutrient to extremely low levels. Current
models might not identify actual effects even
approximately and severely unbalanced biological
systems might develop in the prototype. It is
conceivable that extremely effective removal of
phosphorus without complementary carbon removal might
lead to the development of detritus accumulations
because some link in the food chain may be limiting.
Such facets of mathematical modeling of the
eutrophication problem have not received adequate
attention.
39
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Table 4
Critical Loading Rates for Nitrogen and Phosphorus
Loading Permissible Loading Dangerous Loading
Reference Rate Units (up to) (in excess of)
N P N P
201 Volumetric .86 .12 1.51 .22
(g/m -yr)
201 Areal 2.0 .28 3.4 .49
(g/m2-yr)
20Q Areal 1.0 .07 2.0 .13
aFor lakes with mean depths of 5 i or less.
From? Brezonik and Shannon (201)
-------
RECOMMENDATIONS
An approach to understanding the role of microorganisms
in the environment can be developed as separate
categories. All of these areas are interrelated, but
each can be studied independently. Results from each
of these studies can be collated and used to evaluate
problems, develop better waste disposal methods and
water management, allow for the establishment of
meaningful criteria, and enforce existing regulations.
I.
NUTRIENT CYCLING
The following areas are in need of consideration and
investigation with respect to nutrient cycles:
(a) Kinetics and dynamics of organic and inorganic
nutrient turnover in microbial populations:
The approach necessitates dealing not only
with the effects of single pollutants, but
also with the possible synergistic effects of
combinations of pollutants in relation to
physical and biological environmental
conditions.
(b) Identification of microorganisms which
contribute characteristic tastes and odors in
waters: For example, Actinomyces species are
highly suspect as causative agents for many
noxious odors and tastes. Other microbes can
deposit metallic iron and other elements such
as sulfur, certain bacteria produce slimes or
taste producing substances (83)• The
relationship between selective growth of these
40_
-------
odor producing organisms and eutrophication
requires investigation. Identification of
indicator organisms of specific classes of
pollutants is a possibility. Clostridium
perfringens, a causative agent for gas
gangrene, is an anaerobic spore former found
as a normal inhabitant in the digestive tract
of man and other animals. However, little
information is available on the role this
organism might play as an indicator of fecal
pollution (85). This is especially important
since, as a spore former, samples can be
stored for long periods of time without loss
of viability. The medical significance of
this organism in relation to gastroenteritis
is not clearly understood and requires
clarification.
(c) Defining food chain relationships beginning
with the microorganisms is particularly
important, especially when considering
possible modification of toxic pollutants and
sequestering of metals that in high
concentrations, become toxic for humans and
other animals. Cadmium, lead and mercury, as
well as certain pesticides, of which DDT is an
example, are concentrated by microorganisms
and are further magnified in the food chain.
(d) The effect of environmental, physical, and
chemical limitations on microbial growth; e.g.
carbon dioxide, oxygen* pH, light and
temperature, is not known with respect to the
overall aquatic ecosystem. Alterations of any
of these parameters through excessive
eutrophication or pollution needs additional
research.
41
-------
(e) Nitrogen fixation by microorganisms in various
fresh water, and estuarine and ocean
environments should continue to be considered.
Investigations into the quantity of nitrogen
fixed as related to the total productivity are
required.
(f) The sulfur cycle is an area that has not been
fully explored and a delineation of the cycle
as a primary energy cycle in certain estuaries
and lakes requires investigation.
III.
ANAEROBIC MICBOORGANISMS
A consideration of the metabolic activities as related
to eutrophication processes, self-purification capacity
of water, and potential problems related to septic
conditions of strict anaerobic bacteria and other
microorganisms and benthic organisms needs to be
initiated. Little is known about the effect on either
the viability or selection various nutrients or
pollutants have on the anaerobic flora.
IV.
MICROBIAL PHOTOSYNTHESIS
An evaluation of the role of photosynthetic
microorganisms to eutrophication and to oxygen
requirements is required. This includes not only the
blue-green algae, but also the purple sulfur bacteria
as well as other species capable of photosynthesis.
42
-------
V.
INTERSPECIES INTERACTION
An examination of the interplay between microorganisms
as related to and determined by the water quality is
required. For example, if bacteria of one species is
given a growth advantage over the rest of the bacterial
flora can the whole microbial relationship be altered
with a consequent impact on the food chain.
VI.
Engineering Aspects
a. Further study to determine the limits of model
extrapolation and to obtain cost-effective
solutions of accelerated eutrophication problems is
required before large sums of money are spent on
remedial measures based on such extrapolations.
b. Surveillance of systems where nutrient removal
is instituted to verify the validity of
assumptions, bio-kinetic formulations and predicted
water quality changes.
VII.
METHODS
The methods would involve:
(a) Separation and enumeration of microbial
species in natural populations.
(b) Estimation of activity rates in situ with
respect to synthesis, degradation and cycling
of organic and inorganic materials, this would
include a study of the following general
areas:
1. anaerobic environments
2. aerobic environments
43
-------
3. biological vs. abiological oxidation
(c) Identifying microbial populations in specific
ecological niches. For example, do specific
microorganisms associate with specific higher
life forms and# if so, how does the
elimination of these bacterial species affect
these higher life forms, i.e., protozoa,
fungi, rotifers, and plants?
(d) Development of techniques required for the
identification on higher microorganism, i.e.,
rotifers, algae, protozoa
44.
-------
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