THE
AQUATIC
ENVIRONMENT:
Microbial Transformations and
Water Management
Symposium Sponsor*?! by Environmental Protection Agency Offica at Water Program Operation*
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SYMPOSIUM SPEAKERS
Left to Right:
J. LcCall, R. K. Ballentine, F. Verhoff, R. H. Hardy, D. C. Hood, R. R. Colwell, F. Gordeiro, J. W. Payne, R. L. Sansom,
K. M. Mackenthun, W. Fuhs, L. Keup, W. R. Finnerty, L. J. Guarraia.
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THE
AQUATIC
ENVIRONMENT:
Microbial Transformations and
Water Management
Implications
Symposium Sponsored by Environmental Protection Agency s
Office of Water Program Operations - Held in October 1972 £
\
UJ
Edited by Leonard
J. Guarraia and
Richard K. Ballentine
EPA 430/G-73-008
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For sale by the Superintendent of Documents, U.S. Government Printing Office
Washington, B.C. 20402 - Price $2.05
Stock Number 5501-00615
IV
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ACKNOWLEDGEMENTS
The editors wish to express their appreciation to the following
people who helped make the workings of the symposium run
smoothly:
Mrs. T. W. Musser
Mrs. V. M. Fearson
Miss R. Penick
Mrs. K. Shorr
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KEYNOTE SPEAKER
Robert L. Sansom
Assistant Administrator
for Air and Water Programs
Environmental Protection Agency
CHAIRMEN OF THE MEETINGS
Richard K. Ballentine*
Water Quality Protection Branch, Water Quality and
Non-Point Source Control Division, Office of
Water Program Operations
Leonard J. Guarraia
Water Quality Protection Branch, Water Quality and
Non-Point Source Control Division, Office of
Water Program Operations
Kenneth M. Mackenthun
Director, Technical Support Staff, Office of the
Assistant Administrator for Air and Water Programs
Lowell E. Keup
Deputy Director, Water Quality and Non-Point Source
Control Division, Office of Water Programs Operations
Donald Lear
Annapolis Field Office, Region III
* Currently with the Non-Point Source Control Branch, Water Quality and Non-
Point Source Control Division
VI
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THE AUTHORS
F. H. Verhoff
C. F. Cordeiro
W. F- Echelberger, Jr.
M. W. Tenney
W. J. Payne
J. LeGall
W. R. Finnerty
R. Hardy
R. D. Holsten
M. Alexander
C. W. Fuhs
R. R. Colwell
D. Hood
T. C. Loder
R. K, Ballentine
L. J. Guarraia
R. S. Kennedy
D. J. Van Donsel
E. E. Geldreich
University of Notre Dame
University of Notre Dame
University of Notre Dame
University of Notre Dame
University of Georgia
University of Georgia
University of Georgia
E. I. DuPont de Nemours and Co.
E. I. DuPont de Nemours and Co.
Cornell University
New York State Department of Health
Laboratories
University of Maryland
University of Alaska
University of Alaska
Environmental Protection Agency
Environmental Protection Agency
Department of Health, Education and
Welfare
Aquatic Health Center, Alaska, EPA
Water Supply Laboratory, National En-
vironmental Research Center, Cincinnati,
Office of Research and Monitoring, EPA
vii
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Contents
page
Welcoming Address 1
Robert L. Sansom
Interrelationship of the Major Nutrients and Micro-
organisms 3
L. /. Guarraia
Engineering and Modeling Implications of the Microbial
Influences on Nutrient Cycles ....... 9
R. K. Ballentine
Modeling of Nutrient Cycling in Microbial Aquatic Eco-
systems: Theoretical Considerations 13
F. H. Verhoff, C. F. Cordeiro, W. F. Echelberger, Jr.
and M. W. Tenney
Bacterial Growth Yields 57
W. J. Payne
The Sulfur Cycle 75
/. LeGall —--•'
Microbial Influences on Phosphorus Cycling .... 149
G. W. Fuhs
Microbial Recycling of Naturally Occurring Refractory
Material 171
R. R. Celwell
ix
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page
Microbial Interactions with Hydrocarbons: Physiological
and Structural Correlates 187
W. R. Finnerty and R. S. Kennedy
Microbial Conversions of Dissolved Organic Carbon
Compounds in Sea Water 211
D. W. Hood and T. C. Loder
Bacterial Bottom Sampler for Water Sediments ... 237
D. J. Van Donsel and E. E. Geldreich
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KEYNOTE ADDRESS
ROBERT L. SANSOM
Assistant Administrator for
Air and Water Programs
Environmental Protection Agency
It is a pleasure to welcome you to this symposium on aquatic
microbial transformations and their implications in water man-
agement decisions. The projected results of this symposium will
be a state-of-the-art assessment of the scientific knowledge on
the actions of bacteria oil the cycling of carbon, nitrogen,
phosphorus and sulfur in the aquatic ecosystem and the relative
importance of bacterial cycling of these major elements to
water quality. The proposed dialogue on the application of
mathematical models to natural processes is an area that will
promote beneficial discussions between the economist and the
microbiologist.
We of the Environmental Protection Agency are charged
with cleaning up the environment, and it's our opportunity and
our fate to make far-reaching decisions, often with imperfect
knowledge. Any conclusion which can be offered by this type of
symposium will help us to make better water management
decisions.
Subjects that you will discuss affect decisions made by EPA
in at least two areas. I know that recent developments in
aeration, which affect the rate of bacterial action on organic
matter, have influenced the design of sewage treatment plants.
These developments have reduced holding time from 6 to 8-hours
to 4 hours. Obviously this has a key impact on the capacity of
major components of treatment plants and thereby an impact
on their cost. Cleaning up our environment will require the
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building of many additional municipal treatment plants. No
matter how much money we have to construct such plants, it
will never be enough. The more we learn about the role of
bacteria in this process, the more we're going to be able to
accomplish with the money allocated to us.
The second major area is eutrophication. Understanding how
quickly nutrients are recycled is one of the key determinants
of the rate of eutrophication. Discussions such as you are having
here today will help us to understand and possibly exert some
control over the process.
I would like also to reflect briefly on a broader theme. At-
tendance here today is composed of members of the academic
community, EPA, and other Federal agencies. We of EPA want
to remain aware of developments outside the Agency in order
that we may make our decisions in the light of such awareness.
Keep us informed. With assistance from you of the scientific
community, we will be able to make our decisions enlightened
by the latest scientific knowledge and expertise available.
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Interrelationship of the Major Nutrients
and Micro-organism
L. J. GUARRAIA
Development of successful water management programs and
program planning depends upon as complete a knowledge as
possible of both the physical and biological processes working
within a particular system. The turnover rates and exchange of
nutrients with the sediments are in part governed by biological
communities. However, before proceeding, the term "nutrients"
must be defined. Definition of "nutrient" depends upon the
individual involved. "Nutrients" refer to not only organic ma-
terial, simple and complex, but to trace elements, vitamins, and
also the major inorganic elements: carbon, nitrogen, phosphorus
and sulfur.
Although widely recognized as basic agents in the transfor-
mation of all classes of nutrients, the exact role of the microbial
populations in terms of their contribution to the cycling of
nutrients is in part obscure. The reasons for this are many.
Techniques for the definition of microbial populations, size of
these populations, metabolic rates or activities of these popu-
lations in situ, relationship of specific genera with each other
and with higher life forms are for the most part unknown. These
questions have been, and are the subject of many investigations.
The principle of microorganisms in nutrient cycling and bio-
logical transformations is the subject of this symposium.
Whether one recognizes the fact or not man depends basically
on two independent, but not mutually exclusive, processes to
cleanse his immediate environment: oxidation, both chemical
and photo, and biological oxidation or degradation. The first
category with respect to the environment is beyond the im-
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mediate control of society. The second category, that is, bio-
logical treatment or biological processes, has been taken ad-
vantage of and is apparent in the form of oxidation ponds.
Microbial processes in the aquatic environment involve the
cycling, transformation and synthesis of innumerable simple
and complex compounds. The attention of this symposium is
focused on the four basic nutrients—carbon, nitrogen, phos-
phorus and sulphur.
One nutrient which has received widespread attention is
phosphorus. It is known that phosphorus can be limiting to
phytoplankton and other organisms. However, most of the
phosphorus in the aquatic environment is bound in the sedi-
ments as an insoluble phosphate salt with availability of in-
soluble salts being influenced by both the physical-chemical
factors and bacterial metabolism. As seen in Figure 1 loss or
precipitation of phosphates to the sediments and solubili-
zation of insoluble phosphates from the sediments and ex-
change among the various biologic communities, is mediated
in part by the bacterial community. Three general processes
involved in phosphate solubility are: the direct metabolic proc-
PHOSPHORUS CYCLE
Higher Aquatic
Plants
Wastes
Introduction
Water
Mud
Inqirganic
Phosphate
Zooplankton
Soluble Organic
Phosphate
Phytopjanktoi
Bacteria Inorganic Reaction
Loss to Permanent Sfdimaits
FIGURE 1.
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esses involving enzymes, carbon dioxide production leading to
a lower pH, and organic acid production. Inorganic phosphate
is, in turn, used by higher aquatic plants—by zooplankton and
phytoplankton. The microbial involvement in the phosphorus
cycle will be discussed in detail later in the symposium.
As with the cycling of phosphorus, sulfur is cycled by the
microbial populations in the aquatic environment. Although not
widely recognized, the availability of sulfur can limit the pro-
ductivity of the aquatic environment and has been linked to
decreased productivity of fish.
Sulfate can be stoichiometrically reduced to hydrogen sulfide
which, in turn, can be oxidized chemically, in the presence of
oxygen, to elemental sulfur. Elemental sulfur, in turn, can be
oxidized to sulfate.
Also the production of hydrogen sulfide by a specific
class of bacteria, the anaerobic dissimilatory sulfate reducers,
leads to the stoichiometric production of hydrogen sulfide and
consequently an anaerobic environment. On the other side the
oxidation of elemental sulfur by Thiobacilli leads to the pro-
duction of sulfuric acid and their metabolic activity is evident
in the acid mine drainage in certain areas of the country.
Biological nitrogen cycling involves, as does the cycling of
sulfur and phosphorus, the transition of an elemental nutrient
through various chemical states. Figure 2 is a schematic repre-
sentation of the cycling of nitrogen. It is convenient to initiate
the consideration of the nitrogen cycle at a point where fixation
of gaseous nitrogen occurs. Relatively few species of micro-
organisms populating the earth are capable of metabolizing
nitrogen from the air. Once fixed from the atmosphere nitrogen
is converted by a relatively few species of bacteria and blue
green algae to organic nitrogenous compounds.
The biochemical mechanisms involved in denitrification have
only recently been elucidated in significant detail. These re-
actions result in the conversion of nitrate ultimately, to nitrogen
gas and are apparently unique to a limited group of micro-
organisms.
The carbon cycle is composed of an integrated network of
physically and biologically mediated pathways encompassing
the synthesis, degradation, and transformation of innumerable
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MICROBIAL CYCLING of INORGANIC NUTRIENTS
Nitrogen
Bacterial
Denitrification
Bacterial
Fixation
Organic
•Nitrogenous
/, Compounds
Animals
FIGURE 2.
Plants
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 the organic carbon cycle in the aquatic environment
have been examined with the emergent principle that an overall
balance between the production, or synthesis, and decompo-
sition of naturally occurring substances exists in nature. A
simplified carbon cycle is seen in Figure 3. Photosynthetic
carbon dioxide fixation by green plants is a major route by
which carbon enters the organic carbon cycle. However, fixation
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by autotrophic bacteria adds to the total carbon budget in the
ecosystem. 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. Some of the ecological questions
relating to carbon that arise when considering the microbe's
direct relationship to carbon cycling are: what effect does
microbial synthesis of complex molecules such as vitamins,
amino acids, carbohydrates, and Upids have on the aquatic biota;
what is the contribution pf the bacterial biomass as a food source
for zooplankton; and, what is the significance of microbial degra-
dation of suspended soluble or sedimented organic compounds?
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
MICROBIAL CYCLING of INORGANIC NUTRIENTS
Carbon
Bacterial / Fixation
Decomposition n .GrSenAPI?n,tsc .
Certain Bacterial Species
Complex
Molecules _ „ . ,
Bacterial
Synthesis
Vitamins
FIGURE 3.
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MICROBIAL INFLUENCE ON INORGANIC NUTRIENT CYCKS
P04
S04
C02
N2
Solubilization
Organic X
Nitrogen
Organic Carbon
Sulfide
Sediments
Precipitation
FIGURE 4.
various algae and bacteria in the marine and fresh water en-
vironments. Also the degradation of complex naturally occurring
organic compounds such as chitin are affected by the microbial
species.
As seen in Figure 4 microbial metabolic activity affects
the cycling of the four major inorganic nutrients under con-
sideration. The cycling of each of these nutrients—phosphorus,
sulfur, carbon, and nitrogen—are interrelated in that any pertur-
bation in one cycle has far reaching effects in the other cycles.
For example, it has been shown that the sulfate reducing
bacteria are capable, not only of nitrogen fixation, but degra-
dation of carbon compounds to carbon dioxide and also to effect
a solubilization of phosphate as a consequence of precipitation
of insoluble iron sulfides. This is but one example. There are
many examples of these interrelationships in microbial com-
munities and it is these relationships, these rates, and these
transformations which require elucidation for proper water
management.
8
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Engineering and Modeling the
Microbial Influences on Nutrient Cycles
R. K. BALLENTINE
The engineering aspects section was included as part of
this symposium for several reasons. The principal reason,
however, was to demonstrate one type of analytical method
used by engineers and others for the analysis of biological
systems, that being the mathematical model, and how infor-
mation developed by microbiologists is fundamental for the use
of this analytical tool. Additionally, it is hoped that by becoming
more aware of the scientific requirements of these analytical
methods, microbiologists can produce many of the theoretical
and laboratory developed coefficients necessary to allow the
formulation of more realistic and complex mathematical models.
Such developments ultimately will lead to a better under-
standing of ecological relationships in the aquatic environment.
As a brief introduction to the engineering section a few
remarks about mathematical models are in order. I realize that
to many in the audience mathematical models are used and
developed routinely. Others, however, are not intimately fa-
miliar with these analytical tools.
Figure 1 is a schematic of a lake with various sources of
nutrients which can be considered either organic or inorganic
for purposes of the example. These sources include a waste
treatment plant, direct discharges from residences close to the
lake, drainage from agricultural lands and tributary inflow.
Additionally, the outlet stream is shown which actually removes
some nutrients.
Because of the input of these nutrients, a biological response
will occur in the lake. Such response will generally include
9
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DIRECT DOMESTIC
DISCHARGE
INPUT
AGRICULTURAL
RUNOFF ...
SEWAGE
and
INDUSTRIAL
MISCELLANEOUS
Recreational Boating
Rain Out
Fish Harvesting
FIGURE 1.
increased microbial activity on organic nutrients and phyto-
plankton growth or primary production because of increased
inorganic nutrients. In less than nuisance proportions these
processes can result in desirable increases in productivity such
as increased fish production and perhaps increases in recreational
opportunity. When overloaded by either organic or inorganic
nutrients the system becomes unbalanced and nuisance con-
ditions develop (e.g. zero dissolved oxygen or algal blooms).
In an effort to avoid the development of nuisance conditions,
the capacity of the lake to absorb nutrients must be described.
Qualitative descriptions are insufficient for control—only a
quantative description is sufficient and the concept of a mathe-
matical description of the processes must be developed. This
mathematical description is called the mathematical model.
10
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INPUT
SYSTEM MODEL
OUTPUT
1. NUTRIENTS 1. DETERMINISTIC 1. WATER QUALITY
2. GEOMORPHOLQGY 2. STATISTICAL 2. CHLOROPHYLL
3. TEMPERATURE 3. OTHERS
4. OTHERS
FIGURE 2.
The various biological processes themselves or the results of
these processes are represented by mathematical expressions.
These expressions are linked together to represent, or model,
the system. These expressions and coefficients are then evaluated
by field surveys and laboratory studies. This process is called
calibration. The model is then tested against the prototype to
see how well it describes known changes in the prototype. This
process is called "verification". Once "verified" the model is
used to gauge the lake or prototype response to various-changes
in the input variables. Figure 2 schematically represents the
input-output model described.
This figure shows as inputs the nutrients and the geomorpho-
logical characteristic of a lake. The lake reaction mechanism is
shown as a box in which the mathematical expressions are
contained. The inputs are mathematically manipulated and the
predicted output is generated. This output is water quality
described by phenomena such as biological populations or chlo-
rophyll concentrations.
The verified model can now be used to test abatement
schemes including waste treatment or waste diversion in an
attempt to control the adverse effects of excess nutrient addition.
Following the construction and operation of engineering
structures, it is informative to follow the biological changes
that actually occur and compare these to the predicted changes.
In this way mathematical models can be truly verified and
internal improvements incorporated. Such monitoring and vali-
dation requires as much microbiological input as does the initial
formulation.
II
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Modeling of Nutrient Cycling
in Microbidl Aquatic Ecosystems
Part I. Theoretical Considerations
F. H. VERHOFF, C. F. CORDEIRO,
W. F. ECHELBERGER, JR.*
and M. W. TENNEY*
University of Notre Dame
Department of Chemical Engineering
Notre Dame, Ind. 46556
ABSTRACT
This paper summarizes work being done on the construction
of ecosystem models of nutrient cycling through the microbial
populations in an aquatic environment. The principle factors
to be considered are the rate of microbial reactions, i.e., the
microbial growth rate, the stoichiometry of the microbial re-
actions, and the conservation of mass for the major microbial
nutrients.
A new mechanistic model of microbial cell growth is reviewed.
This model assumes that microbial cell growth occurs in two
separate processes, the transport of nutrients into the cell and
the conversion of these nutrients into protoplasm. The rate of
transport is dependent upon the external nutrient concentrations
and the conversion rate is dependent upon light and temper-
ature. This model is compared with known experimental facts
of cell growth.
A model of an aerobic aquatic environment including the
cycling of carbon, oxygen, and phosphorus has been con-
* Department of Civil Engineering
13
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structed. This model has been simulated on a yearly and a
diurnal basis; qualitative comparisons between the predictions
of this model and known experimental facts are made. The
effect of the chemical element composition of the bacteria and
the algae upon the response of the ecosystem is investigated.
A discussion of the model alterations necessary to include the
nitrogen cycling and the bottom sediments is given. The stoichi-
ometry of the bacterial and algal reactions is altered signifi-
cantly with the addition of the nitrogen in its different forms.
The stoichiometry and the rate of the reactions occurring in
the bottom muds of deep water bodies appears to be nebulous
and hence these reactions are difficult to include in the model.
INTRODUCTION
The term modeling usually implies an attempt to quantify
relationships between components of a system; in this particular
paper it means the quantification of the dynamic relationships
among the microbial populations existing in a given aquatic
environment and the nutrients that are associated with their
growth. To prevent the model from becoming unwieldy, the
microbial populations are grouped into large categories based
upon the type of nutrient transformations accomplished by the
microbe. A realistic dynamic description of the growth of these
categories is a necessity, and the stoichiometry of the microbial
reaction, as well as the chemical composition of the microbe,
must be known. To complete the model of nutrient cycling in
aquatic microbial ecosystem, the purely chemical reactions of
the nutrients and the mass transfer of nutrients from the
atmosphere to the water and among different aquatic environ-
ments should be included. This following discourse presents
the approaches of the authors to this modeling task.
This paper summarizes a more realistic quantitative dynamic
description of microbial cell growth. The cell growth process is
assumed to occur via two distinct processes; (1) the transport
of the nutrient from the exterior of the cell to the interior and
(2) the conversion of nutrients to cell protoplasm and the subse-
quent cell division. The transport process is separated from
other cell processes because the rate of this transport process is
dependent upon external nutrient concentrations.
14
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An aerobic water model which includes the cycling of carbon,
oxygen, and phosphorus through the algal and bacterial popu-
lations is included. That the composition of the algae and
bacteria (both assumed to be the same) effects the dynamics of
the ecosystem is shown. The ecosystem model was used to
simulate seasonal and diurnal variations of population and
nutrient population.
Nitrogen in its various chemical forms is included in the last
microbial ecosystem model to be presented. The division of the
microbes into population categories for particular chemical
transformation of nitrogen and the corresponding stoichiometry
of the transformation is presented.
General ecological models have been studied since the original
work of Volterra and Lotka (3) on the interaction of predator
and prey. Since then, various types of investigations into eco-
logical systems have been made; some of these investigations
would have to be considered theoretical and others practical.
The theoretical investigators (see for example Garfinkel (9),
Smith (25), or Verhoff (33) were usually interested in mathe-
matical properties of the system of equations which describe
the ecological system. Stability, effects of density dependent
factors, and number of trophic levels are among the factors
studied by these authors. The practical studies were usually
motivated by some ecological phenomena observed in many
ecological systems (see for example Bloom (1) and Chen (4))
or by the specific ecology of a particular location (see for
example, Parker (20)}. The commonality of these papers is the
attempt to simulate and predict actual concentrations of nutri-
ents and biological organisms as a function of time and possibly
position for the ecological system studied.
Chen (4), Parker (20), DiToro, O'Connor and Thoman (7),
O'Brien and Wroblewski (19), and Patten (21) all have de-
veloped ecological models which consider the movement of
nutrients through the microbial populations. However, none of
these models complete the nutrient cycles, e.g., they do not
consider the exchange of CO2 on N2 with the atmosphere, nor
do any consider the nitrogen fixation capability of certain
aquatic microorganisms. Parker's model follows the passage of
a nutrient through the trophic levels up to the fish and O'Brien
15
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and Wroblewski follow the cycling of a single conserved nutrient
through the lower trophic levels. The O'Brien analysis is similar
to that of Verhoff (33) except that the hydrology of the conti-
nental shelf is included.
DiToro, et al. consider the effects of phosphate and nitrate
nutrients upon the dissolved oxygen of natural waters as caused
by the growth of algae; the cycles of these nutrients are not
completed. Patten considers the effect of these nutrients upon
the growth of algae.
It is believed by the authors that the cycling of nutrients
through the microbial populations plays a significant role in
dynamics of water quality. Hence to determine the effects of
the nutrient cycling, the complete cycle of the nutrients must
be mimicked by the model. The first attempt at the construction
of such a model, including the cycling of carbon, phosphorus,
and oxygen, was reported recently and is summarized herein.
Chen partially completed the cycling of nitrogen, phosphorus,
and oxygen in a stream model.
Also, the quantitative description of the rate of microbial cell
growth plays a key role in the dynamics of nutrient cycling
(and hence water quality) in aquatic ecosystems. All of the
above investigators use a Monod-type expression to relate the
growth rate of microbial organisms with the nutrient concen-
trations in the natural water. To account for two limiting
nutrients, all investigations use a multiplication of Monod
factors. This Monod equation is used despite the fact that it
does not represent the dynamics of microbial cell growth (30).
Although DiToro et al. justify the multiplication of Monod
factors by using data taken for one nutrient limiting at a time.
It is known that this multiplication does not express growth
rate when two nutrients are limiting growth at the same time
(6). This paper then includes a synopsis of the work done by
the authors on microbial cell growth (35).
Microbial Cell Growth
The principal mathematical expression used to relate rate of
microbial cell growth to limiting substrate concentration is the
Monod equation (18). It is recognized that there are many
16
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cases in which observed values of cell concentration do not
correspond to those predicted by this equation in batch culti-
vation. In particular, this expression does not predict the lag
phase, nor does it predict the endogenous phase of batch culti-
vation. In continuous cultivation, this equation rarely predicts
the experimental results partly because the yield of cell mass
from limiting substrate is not constant (see Herbert (12, 13)).
Still other phenomena such as that involved in the biosorption
process of sewage and waste treatment (31) are totally un-
explained by this equation of microbial cell growth.
Other authors have developed different empirical expressions
relating cell growth rate to limiting substrate concentration,
e.g., Teissier's (28) exponential expression for growth rate, and
the two phase proposal of Garret and Sawyer (JO). Kono (16)
developed a model for the case of batch cultivation taking into
account four phases of growth. He bases his model on a similarity
to chemical kinetics and introduces the new concepts of critical
concentration and the coefficient of consumption activity. He
applies the model to various available data and finds the fit to
be good with proper choice of parameters. Other authors (e.g.,
lerusalimskii (15) and Contois (5)) worked on cell concentration
effect on growth rate. Still others (e.g., Powell (23)) modified
Monod's equation. In particular, Powell included the effects of
mass transfer in the Monod growth model. He compared his
growth model with that of Monod and with that of Teissier
and found that his model fitted the experimental data better
than the others. Several of the above authors, as well as others,
also worked on the problems of yield and endogenous respi-
ration; some have corrected their models for this latter effect.
An excellent review of the application of these models is given
by Gaudy (11).
The important point to note about the above models is that
they are primarily empirical, i.e., the experiment data were fit
with an equational form. None of the authors propose a mecha-
nism for growth because it is always assumed that the metabolic
processes are quite complex, involving a variety of rate laws,
and that these processes are among the rate determining steps
for growth. Although, there is extensive knowledge about the
mode of growth (the sequence of processes involved in cell
17
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growth), none of the authors use this information in developing
a model.
The fact that such diverse organisms as bacteria, yeasts, and
even molds (see Pirt and Callow (22) for mold growth) exhibit
the same growth dependency seems to indicate that the meta-
bolic processes do not play a major role in the growth rate
dependency on limiting substrate. In the authors' opinion, only
a few facts about the metabolic activity of the cell are required
along with a knowledge of the mode of growth in order to
explain the growth rate—limiting substrate dependency.
In this paper, a mechanism based upon the mode of growth
is proposed by the authors. We assume that every cell is either
growing (however slowly) or it is lysed. The mode of growth
assumed in this model supposes that the cell growth occurs in
a two-step manner; the first step involves the accumulation
and possible loss of the limiting substrate by the organism and
the second step involves the ingestion of the limiting substrate
by the organism and possibly a subsequent cell division. This
mechanism suggests that the microbial population should be
divided into two categories as well as it suggests certain mathe-
matical forms for the rate of each step. These rates are then
combined to predict the microbial growth rate as a function of
limiting concentration. This mechanism along with a simple
mechanism for microbial lysis are applied to the continuous
stirred tank reactor (chemostat) and to the batch reactor. The
relationship of organism and substrate concentration to such
variables as flow rate and input substrate concentration for
the continuous culture case are developed. The variation of
yield with flow rate is also shown. In batch culture, this mecha-
nism predicts the lag phase through to the endogenous phase.
Mechanism of Microbial Ceil Growth
For a simplified version of the cell growth process, we might
consider a viable cell in a growing culture just after a cell
division. This cell selectively assimilates or adsorbs the nutri-
ents from the culture medium. The cell reaction systems then
convert the nutrients which have been assimulated into proto-
plasmic material characteristic of the particular organism. This
process of assimilation followed by conversion may occur once
18
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or' several times between cell divisions; the theory to be de-
veloped will be the same for all cases with the change of one
parameter. After some chemical rearrangement of the proto-
plasmic material and the production of an increased amount of
nuclear substance, the cell ultimately divides producing two
new viable cells which will repeat the process.
This process can be put into symbolic form to represent the
stoichiometry (conservation of mass) as shown below.
+e)A +p,fo -«)S.. (1)
7-2
This symbolism in words says that a viable cell just after cell
division, A9 combines with rj amount of limiting substrate, Ss,
to form an ingesting cell, B. Since we will be primarily dealing
with mass units, we will define the mechanism as one mass
unit of A combines with a fraction 77 of mass units of Ss to form
1 +97 mass units of B. One mass unit of B then can undergo
one of two processes; it could release the assimilated nutrients
and revert back to its original form of A and Ss, or it could
ingest the substrate and produce 1+e units mass of A. This
ingestion could include cell division; if cell division always
accompanies ingestion as is probably the case with some bac-
teria, then e is approximately equal to one. During the ingestion
and possible cell division, some of the substrate may be returned
to the medium for use again by other cells; thus the weight p2
(17 — e) represents the weight of substrate returned to the
medium. Since p2 is normally considered to be near zero without
changing the mechanism, it could be assumed that part or most
of the ingestion occurs during what has been described as the
assimilation step.
From the symbolic form (equation 1) it can be seen that the
rate of growth will depend upon the three rate processes n, r2,
and r3 indicated. For the development of the rate functions, we
will consider A9 Ss and B to be mass concentration, i.e., mass
per unit volume of solution. The rate of absorption of nutrient
per unit volume of solution n should be linearly proportional to
A with all other quantities held constant, i.e., a larger mass of
cells per unit volume of solution will give a proportionally
larger rate of absorption per unit volume of solution. Also, we
19
-------
would expect the absorption process to be mass transfer limited,
either in the cell membrane or in a liquid film around the cell.
With all other quantities held constant, the rate of absorption
per unit volume is proportional to the difference between the
substrate concentration in solution, Ss, and some low substrate
concentration, Sc, maintained by the cell. Combining these two
facts, the rate of absorption per unit volume can be written as
the following equation.
where
O == t3a i3c*
Because Sc is very small, S is approximately equal to Ss. For
all of the analysis to follow in this paper, we will assume that
ki is independent of all substances whose concentrations change
during growth.
The rate loss of limiting substrate per unit volume by in-
gesting cells B (i.e., rate r2) will be proportional to the concen-
tration of B in the solution assuming all other quantities are
constant. Again we will assume in the subsequent analysis that
k2 in the following law expressing this relationship is inde-
pendent of all other changing substances in the solution.
r2 = k2B.
The rate at which B ingests substrate or ingests substrate
and divides is assumed proportional to B with all the other
quantities held constant. It is also assumed that all other quan-
tities that change during an experiment do not affect the in-
gestion rate; in particular, it is assumed that the ingestion rate
is independent of the concentration of limiting substrate in the
solution. The rate of ingestion and cell division per unit volume
is then
r3 = k3B.
Growth Rate Predicted by Mechanism
To develop mathematical expressions for microbial cell growth
rate dependencies, we will assume that a certain mass of cells
are placed in a media with a constant concentration of limiting
substrate. No cells are assumed to lyse. The equations that
20
-------
express the rate of change of concentration of the absorbing
cells, A, and of the ingesting cells, B, are given below:
3. (2)
= (1 +r,)k,AS -ksB ~k2B. (3)
Associated with these equations are the initial conditions:
A=At
@t=0.
B=B0
Since S is constant, equations (2) and (3) are a linear set of
differential equations.
Specific growth rate, the quantity of interest, is given by the
following formula.
1 d(A +B)
~A+B dt '
Solving for the largest eigenvalue gives for the specific growth
rate
_ +7c3)
M-
This equation can be reduced such that there are three groups
of numbers to be plotted; two on the axes of the graph and one
as a parameter. The form suitable for such plotting is
_
kse \2Ai3€ 2k3e \2k,e 2k3e kse
Figure 1 shows a plot of equation (4) with (&2 +&3) /€^3 as the
parameter. The shapes of these curves closely resemble those of
the Monod equation.
For larger values of substrate concentration, the specific mass
growth rate approaches the rate of ingestion and cell division
times the weight fraction of substrate converted. From a bio-
logical point of view, this would be the expected limiting growth
rate.
In order to see the effect of various parameters, we will
21
-------
0 ID EC 30
to so
FIGURE 1.—Dimensionless growth rate vs dimensionless limiting nutri-
ent concentration.
investigate various limiting cases of them. Taking the limit as
ki gets extremely large, the specific growth rate is a constant
value for all but the very lowest values of substrate concen-
tration S. This would be expected because in this case, the rate
limiting step is the ingestion and cell division.
For the case of ks approaching zero (equivalent to ki and kz
getting large), it can be shown that
JL
eka
+s
22
-------
which is the Monod equation for limiting substrate. This equa-
tion would be expected for this case because it closely corre-
sponds to the equilibrium assumptions involved in the develop-
ment of the Michaelis-Menton equation for enzymatic reactions.
In this case, the reaction rates TI and r2 are approximately in
equilibrium with r3 being much slower. Figure 2 shows the
relationship between steady state growth from this mechanism
and that of Monod. It can be seen that there is very little
difference in the steady state characteristics for one substrate.
To modify the above description of microbial cell growth for
two limiting nutrients is very simple since the only modification
is in the transport process. A multiplication of the concentration
of the two limiting nutrients was used in the ecological models
discussed in this paper, although this is not the true dependency
as can be obtained from the microbiological literature. Work is
progressing on an improvement of transport quantification
along the lines of a recent publication (34).
Also, the dynamics of this model as operating in a chemostat
were investigated (36). This model suggested that under most
circumstances the response of this system should be non-
i.o
0.95
MODEL
0.9
MONOD
0.85
0.80
0.05
0.10
0.15
0.20
0.25
FIGURE 2.—Comparison of model growth rate with rate from Monod's
equation as a function of the dimensionless parameter.
23
-------
oscillatory. However, under certain conditions, oscillation was
predicted by the model; under similar experimental conditions,
these oscillations also were observed.
Application of Model to Aerobic Waters
SEASONAL VARIATIONS
Since the use of system modeling for prediction of water
quality is a recent development, much is unknown about the
effects of various system parameters on the dynamics of such
systems and hence many areas arc available for research. A
cursory search of the literature indicates that the empirical
composition of algae and bacteria is not well known and thus
the stoichiometric constants which describe the composition of
these organisms are among the system parameters whose effect
upon the lake dynamics needs to be investigated. This study
presents the formulation of an aquatic microbial model and the
results of a sensitivity analysis of these stoichiometric constants
by the simulation of a simplified model of the surface waters
of a lake.
The simplified model including only the surface waters (i.e.,
the algae and bacteria in the aerobic zone) rather than the
model which also includes the anaerobic bottom muds was
chosen for this study for two reasons: (1) the stoichiometry of
the reactions in the bottom muds is almost totally unknown,
and (2) the inclusion of the bottom muds only complicates the
investigation of the composition of algae and bacteria in the
upper waters. The model to be simulated in this paper also
applies directly to any lake which never develops an anaerobic
lower water layer.
Stumm (27), Redfield (24), and Eckenfelder (8) suggest that
most mixed algal cultures common in surface waters and mixed
bacterial cultures as found in waste treatment processes, have
an average stoichiometric relation between carbon, nitrogen,
and phosphorus of about 106 to 16 to 1 atoms. They propose
the chemical formula for such algae and bacteria to be
CioeHuoCXeNieP. This is one of the chemical formulas we will
use for the composition of bacteria and algae. McCarty (-Z7),
24
-------
Speece (26), Hoover (14), and Burkhead (2) conclude that a
typical empirical formulation for bacterial cells is C5H7O2N.
Extrapolating this to the same nitrogen to phosphorus ratio
(16 to 1) as that proposed by Stumm (27) and others cited
above gives the formula CsoHmC^NieP for the bacterial and
algal cells. Extrapolating this formulation to the carbon to
phosphorus ratio (approximately 105 to 1) gives yet another
empirical formula CiosHusC^^iP for the composition of these
living cells. Various other formulations as suggested by these
other authors were used in the model of this paper.
The purpose of this modeling venture is to simulate the inter-
actions of microorganisms and nutrient cycling which affect
water quality. Water quality can be defined in different ways,
and in this instance, we are primarily focusing on the algae.
Included in this model must be all quantities thought important
in the growth of algae and all factors affecting these quantities.
The final model will then be essentially closed with only external
influences, such as weather and stream flows (set equal to zero)
serving as inputs to the system.
Essentially a model of the water quality of aerobic waters
involves the nutrient cycling between components in these
waters and the influence of weather on these components. Each
cell of bacteria and algae cannot be considered as a particular
component (nor can each species of bacteria or algae for that
matter) of the system because the size of the system to be
simulated would be prohibitive. Hence the components of the
system must be a lumped group of organisms. In the model
used in this paper, all algae are included as one component
(see Chen (4) for a division of the algae) and all bacteria as
another. The other chemical components of the system include
the HCO3~, H2PO4~, O2, carbonaceous material with phosphorus
included and the carbonaceous material without phosphorus.
These components and the flow between components are shown
in Figure 3.
The bacteria are assumed to have the same composition as
the carbonaceous material with phosphorus included. Thus
when bacteria or algae lyse the products enter this component.
If a different composition had been chosen for the algae and
bacteria, two components of carbonaceous material with phos-
25
-------
CARBONACEOUS
-I MATERIAL
H2P04
CARBON
PHOSPHORUS
MATERIAL
i
AEROBIC
BACTERIA
FIGURE 3.—Flow diagram of components in the surface water model.
phorus included would be necessary. The carbonaceous ma-
terial without phosphorus has the same composition as the
bacteria and algae with a H2PO4~ removed. The requirements
of algae for carbon and phosphorus were assumed to be supplied
by HCO3~ and H2PO4~ respectively; these choices were then
used in the stoichiometric equations. This division of com-
ponents might appear rather simplistic, however, it is required
to make the problem tractable.
Kinetics Used in Model
There are essentially four types of transfers of nutrients in
a lake ecological system: (1) convective, i.e., hydrodynamic,
(2) diffusive, i.e,, simple diffusion of substances, (3) chemical
reaction, i.e., spontaneous reactions occurring in the lake pos-
sibly mediated by radiation, and (4) complex biological reac-
tions, i.e., those involved in bacteria and algae. The kinetics of
the first three types of transfer are understood to some extent
and reasonable transfer rate functions can be formulated. Since
hydrology has been neglected, no transfer rates are required
for this process (see Tenney (29) for hydrological consider-
ations). There are two diffusive transfer mechanisms necessary
for this model; the oxygen transfer between the water and air
and the similar transfer of CO2 or HCO3~. The rate of transfer
per unit volume of shallow lake is then given by the following
26
-------
formulae assuming that the main resistance to mass transfer is
in the water.
7*9 =
where
r8=mass accumulation of HCOs" per unit volume of
lake per unit time.
r9=mass accumulation of O2 per unit volume of lake
per unit time.
fc8=mass transfer coefficient for HCO3~. This constant
varies with time due to lake overturn.
k9 =mass transfer coefficient for 02. This constant varies
with time due to lake overturn.
YeqHcos" — water mass concentration of HCO3~ in equilibrium
with the air.
Yeq.o2 = water mass concentration of O2 in equilibrium with
the air.
YHcos~~ = water mass concentration of HCO3-.
Yo2 = water mass concentration of O2.
There is assumed to be one homogeneous chemical reaction
occurring in the water; the carbon-phosphorus material decom-
poses into carbonaceous material and phosphate H2PO4~. This
reaction is assumed to be first order, i.e., the rate of the reaction
is given by the following formula.
r-i =&7YCp
where
r7=mass of carbon-phosphorus material reacted per unit
time per unit volume of lake.
&7 = first order reaction constant.
YCp=mass concentration of carbon-phosphorus material in
the lake.
The algae and bacteria kinetics are somewhat more compli-
cated since a dynamic description of microbial cell growth as
discussed previously was used. This mechanism essentially di-
vides the algal and bacterial masses into two parts, the ab-
sorbing part and the ingesting part.
The rate functions describing the absorption of nutrients from
27
-------
the waters are then described by the following formulae written
in terms of mass changes in A2 and H2 respectively.
TI ==K.ii AilH^POiJl-HCOs"
r4=/b4YHlY02(Yc+NYcp)
where
TI =mass of A2 formed per unit time per unit volume of
lake.
r4 =mass of H2 formed per unit time per unit volume of
lake.
YAI = mass concentration of algal cells AI.
YHi = mass concentration of bacterial cells HI.
YH2po4~ =mass concentration of H2PO4~.
Yc =mass concentration of carbonaceous material.
N = preference factor for carbon-phosphorus material
over carbonaceous material.
ki = rate constant.
ki =rate constant.
This formulation of the absorption step is very empirical; how-
ever, work is progressing on a more realistic approach using
facilitated and active transport. The disappearance of A2 and
H2 by ingestion is assumed to be first order in mass concen-
trations of A2 and H2. Note that this is equivalent to zero order
reaction inside the algae and bacteria; such zero order reactions
are known to occur, e.g., the replication of DNA.
TZ = »2 A A2
r5=fc5YH2
where
r2 =mass of A2 disappearing per unit time per unit volume
of lake.
r5 =mass of H2 disappearing per unit time per unit volume
of lake.
YAZ =mass concentration of algae A2.
YH2 =mass concentration of bacteria H2.
A;5= first order rate constant; this constant is a function of
time due to light and temperature change.
Also the lysing rates of the algae and bacteria have to be
28
-------
specified; for this the usual first order law is used as shown
below.
re =
In these equations r3, r3', r6, and r6' represent the disappearance
of mass per unit time per unit volume of lake for AI, A2, Hi,
and H2 respectively. The first order rate constants for these
processes are &3, &3, &6, and k& respectively. The lysing constant
for AI and A2 (similarly HI and H2) could have been made
different, but for the purposes of this presentation, the as-
sumption that they are the same will suffice.
The rates of transfer of nutrients throughout the lake eco-
logical system is then described by nine rate constants. How-
ever, before the model is complete, the stoichiometry of these
interactions must be specified.
Stoichiometry Used in Model
Stoichiometric relationships are required for the absorption,
ingestion, and lysing processes of the bacteria and the decompo-
sition of the carbon-phosphorus material. If the composition
of the algae, the bacteria, and the carbon-phosphorus material
is assumed to be given by CaHbOcNdP, then stoichiometry
associated with the decomposition of carbon-phosphorus ma-
terial is given by the following formula
CaHbO0NdP-»CaHb_2Oe_4Nd +H2PO4~
The absorption stoichiometry for the algae and bacteria re-
spectively is as follows.
(CaHb00NdP) A1 +17! [aHCO3- +dNO3- +H2PO4~
+ ((b-a-2)/2)H20]->(CaHbOcNdP
])A2(CaHb00Nd)Hl
(CaHbOcNdP)CP
_(CaHb_2Oc-4Nd)cc +H2P04-_
-c)/202]
29
-------
These stoichiometric relations are essentially mass balances
showing the mass of nutrients which combine with a unit mass
of Ai and Hi respectively to form A2 and H2. Note that the
stoichiometry for the carbonaceous form of bacterial substrate
is quite similar to that of the carbon-phosphorus form.
The ingestion stoichiometry for algae and bacteria respec-
tively is then given by the following expressions.
])A2-^(l+€1)(CaHbOcNdP)Al (5)
(CaHbOeNdP+)H2^(H-e2)(CaHbOeNdP)Hl ' (6)
- «0 [aHCOs- +dN03- +H2P04- + ((b - a -2) /2)H2Q ]
The ingestion reactions given for the algae and bacteria are not
reactions, i.e., the separate production and energy consuming
reactions are not presented for the algae. For the bacteria part
of the substrate is used for synthesis and part is oxidized.
The overall reaction for both the algae and bacteria is given
in the next chemical equation.
aHCO3- +dNO3- +H2PO4- + [(b -a -2)/2 ]H2CMCJEbOcNdP
+ [3a+3d+4 + (b-a-2)/2-c]O2
The algae uses, of course, the forward reaction and the bacteria
the reverse.
The lysing of AI and HI is assumed to produce pure carbon-
phosphorus material and the lysing of A2 and H2 is assumed
to follow the same stoichiometry as equations (5) and (6)
except the produced CaHbOcNdP enters the carbon-phosphorus
pool instead of the Ai and HI pools respectively.
Differential Equations of Model
Mass balances are performed for a volume of the surface
waters resulting in nine differential equations to be simulated.
These equations were simulated with the following rate
constants.
=0.3(0.525 +0.475
= 0.015
30
-------
&4=0.03
&6 =0.5(0.55 +0.45 si
fee =0.1
&7=0.05
&8 = 1.0(0.55 -0.45 si
k<> =0.2(0.55 -0.45 sin0)
771 =1.5
N=2.0
Yeq.Hco8-=0.44
ieq-Oa = y.J-
where
= 6.283 /2t/360 t = days
From experimentation estimates can be obtained for constants
A;2, &3, &5, k 6, A; 8, and &9. The physical meaning of k2 and &5 are
explained by Verhoff (35) and the values given above are
reasonable values. Values for the other four rate constants are
in the same range as used by Chen (4). Constants &i, &4, and fa
were obtained by intuitive guesses along with the four constants
771, 172, €2, and N. The stoichiometric coefficients in the compo-
sition of bacteria, algae, and carbon -phosphorus material were
varied in the simulation.
Results
The differential equations were programmed for the computer
using the Runge-Kutta subroutine in the IBM Scientific Sub-
routine Package. The simulation was performed until a steady
curve was reached; the criteria used to determine if transients
had damped out was that the integral over the year of the
deviation of the oxygen and bicarbonate from the equilibrium
value was zero. This says that at a steady state for the model
assumed the net transfer of oxygen and carbonate over a year
interval should be zero. Various integration step sizes were
31
-------
tested and the longest possible step size that could be used
without significant error was one day. The three values for the
organic composition of algae, bacteria, and carbon-phosphorus
material used in the simulation are shown on the plots of the
concentrations as functions of time. All three substances have
the same composition in a simulation.
Figure 4 is a plot of the algae concentration in mg/1 through-
out the year. The algae peak occurs around October 1 which
might be somewhat late, but other variations of k2 as a function
of time probably would be more realistic and give a peak nearer
the measured value. The choice of the algae composition causes
up to 50 % change during certain times of the year and also
causes changes in the shapes of the curves.
The effect of organic composition on the bacterial curves is
very small as can be seen in Figure 5. The bacterial concen-
tration are much higher during the summer season as would be
expected.
3.50
3.00
2.50
o>
2.00
1.50
Cinrk HicrtOertN
IOO I5050I5
045^16
\
0 (April 1)
1 Yr.
TIME
FIGURE 4.—Modeled seasonal variation of algal concentration.
32
-------
8.00-
6.00-
4.00 -
D>
2.00
0.00
CIOO HI50°50NI5 P
CI06 HI80 °45 NI6 P
I 1
0 (April 1)
1 Yr.
TIME
FIGURE 5.—Modeled seasonal variation of bacterial concentration.
The bicarbonate concentration varies significantly throughout
the year. During the spring, C02 is transferred out of the water,
and it is absorbed by the water during the rest of the year. The
effect of organic composition on the time variation of bicarbonate
is small except at the beginning of summer when the effect is
about ten percent. Figure 6 shows the bicarbonate functionality.
The phosphate variation, shown in Figure 7, exhibits the low
value during the summer months as characteristic of surface
waters. The effect of organic composition on phosphate concen-
tration is as large as 15 % during winter.
The carbon-phosphorus material shows two peaks, one in
early spring and the second much larger in late fall. Since it is
rather difficult to estimate the spontaneous decomposition rate
of this material, these peaks could be much smaller if the rate
were higher. As seen in Figure 8, the organic composition in the
ecological system has a small effect on the carbon-phosphorus
material.
33
-------
0.700
0.600
0.500
0.400
0.300
Hii5 °38 Ni6 p
C,00H,50°50N,5P
I
0 (April 1) lYr.
TIME
FIGURE 6.—Modeled seasonal variation of HCOs"" concentration.
Figure 9 shows that the carbonaceous material is very low
during the summer and relatively high during the winter. The
effect of organic composition is negligible on the carbonaceous
material.
The oxygen concentration in the surface waters drops down
in spring indicating a transfer of oxygen into the water at this
time of the year and transfer out during the rest of the year.
The organic composition can cause as much as a five percent
change in composition as can be seen in Figure 10.
The overall dynamics of the nutrient cycling are indicated by
Figures 4 through 10. During the year, the algal growth rate
gradually becomes more restricted by the HCO3~ concentration.
The phosphates drop rapidly to a low value and remain there.
The HCO3~ needed for rapid algal growth in early spring is
supplied by the bacterial action on the carbonaceous material
left from the winter. Other interactions of this type can be noted.
34
-------
0.400 ~
0.300
0.200
o>
0.100
0.000
HH5
— C,00H.50°50N,5P
I
I
0 (April 1) 1 Yr.
TIME
FIGURE 7.—Modeled seasonal variation of H^PO^r concentration.
Diurnal Model for the Upper Waters
The diurnal model follows essentially the same stoichiometry
and kinetics as that used in the annual model. Changes were
made in the model to follow the light and temperature variations
over a twenty-four hour period. An equinoxial day was chosen
for study giving twelve hour periods of light and darkness.
Temperature was assumed to reach a maximum around 4 p.m.
and a minimum at 4 a.m. Thus it could be approximated with
a sinusoidal function of time.
The algae production rates are mainly light dependent and
this is reflected by setting the ingestion rate constant
&2 =&2°(0.525 +0.475
where
-------
If we assume an 8 % variation in temperature over the day, we
would have
T = Tmax(0.96 +0.04 sin(0 -0
T = temperature at time t
where
= max temperature reached
150 X27T
360
radians
and 0 has the same form as before.
Taking a linear variation of bacterial reproduction rate with
temperature, its rate can be given by
k5=k5°T/T0
Similarly, the transport rates of O2 and HCO3~ into the svstem
can be given by
ks=k8°T/T0
2.50
2.00
1,50
0)
1.00
0.50
C80 HII5 °38 NI6 P
CIOO HI50°50NJ5 P
"™ vine •»
!80
I
I
I
0 (April i)
TIME
FIGURE 8.—Modeled seasonal variation of CP concentration.
lYr.
36
-------
8.00-
6.00
4.00 ~
o>
2.00 -
0.00 -
0 (April 1)
TIME
FIGURE 9.—Modeled seasonal variation of C0 concentration.
Saturation values of O2 and HCO3~ are also temperature de-
pendent in an approximately linear fashion.
Thus
Yeq.HCOs" = Yeq.HCO8~"°(2TV — T) TO
Yeq.Oz = Yeq.Qz X(2TV — T)/T0
TM = T at 0 =cf>T
In order to bring the model to an intransient mode changes
had to be made in some of the parameters used in the annual
model. However, these changes were kept within experimentally
observed bounds. The composition of the bacteria, algae, and
carbon-phosphorus material was not varied.
The constants used in the simulation were:
k =25.0
fa =3.0(0.525 +0.475 sin(0 -0
&3=0.05
37
-------
9.50
9.00
j?8.50
8.00
7.50
0( April 1)
C80 H.I5 °38 NI6 P
CIOO HI50°50NI5 P
H
I80
TIME
FIGURE 10.—Modeled seasonal variation of O? concentration.
k5 =6.00(0.96+0.4 si
1 Yr,
ks = 1.00(0.96 +0.04 si
^9 =0.2(0.96 +0.04 si
771 =1.5
c2=1.0
772=0.7
r =0.44(0.96 -0.04 si
Yeq.02 =9,1(0.96 -0.04 si
0r))
38
-------
Results
The system was run with intervals of half-an-hour using a
Runge-Kutta subroutine. In order to obtain better stability, it
would be necessary to cut down on the interval size, but this
leads to larger computing time. The system was found to be
sensitive to various parameters predominant among them being
the ingestion and lysing rates of the bacteria and algae, and
decomposition rates of the organics. The transport rates into the
organism had less of an effect while change in the mass transport
rates of O2 and HCO3~ showed very little disturbance in the
system.
Oscillatory behavior was also noticed in the system with a
period ranging from 10-30 days depending on the step size,
the machine accuracy and the parameters used.
In the model simulated, the algae (Fig. 11) reached a peak
early in the day with a subsequent die-off. The concentrations
are mass concentrations and this reflects the high ingestion rate
into the system.
FIGURE 11.—Modeled diurnal variation of algal concentration.
39
-------
12
Night
FIGURE 12.—Modeled diurnal variation of bacterial concentration.
Bacteria (Fig. 12) on the other hand, reach a peak at night
due to the higher availability of oxygen produced by the algae
during the day. Then HCO3- given off by the bacteria is again
taken in by the algae. The dissolved oxygen (Fig. 14) peaks at
the end of the daylight period showing the accumulation that
has been occurring through the day. It drops down during the
night due to the consumption by the bacteria.
Both the nutrients H2PO4- and HCOr (Fig. 13) peak at
around the same time and follow essentially the same pattern
in the system. These peaks which occur during the day show
the sum total of their components produced by the bacteria
less that taken up by the algae.
Expansion of the Model
There are two basic directions in which the model is to be
expanded. The cycling of nitrogeneous nutrients is to be in-
(hided into the basic model. The water model can also be
ampliiied to include the bottom waters.
40
-------
INCLUSION OF NITROGEN
The nitrogen compounds thought to be most important in
the growth of algae and bacteria are NH4+, NO3~ and N2. The
nitrite form of nitrogen was not included because of the rela-
tively rapid conversion of NO2~ to NO3~. In order to model
different stages of decomposition of organics in the system,
four types of organic components are included. The first, desig-
nated as CPN, is formed on death of bacteria or algae and has
the same composition as the organisms. CPN may decompose
giving off H2PO4~ to yield CN or may give off NH4+ to yield
CP. CN and CP then decompose with removal of NH4+ and
H2PO4~ respectively yielding a carbonaceous compound C.
Three different forms of algae are postulated to correspond to
each of the nitrogeneous nutrients. They are AI which uses
~" 0.0833
P*
£
0.0832
8
'o*
**• 0.434
&
£
U
0.433
0.432
12
NIGHT
8
12
NOON
8
12
NIGHT
TIME »•
FIGURE 13.—Modeled diurnal variation of H2PO4~ and HCO3- con-
centrations,
41
-------
8.90
o>
8.60
O
I
O
d
8.30
12
NIGHT
8
12
NOON
TIME -
8
12
NIGHT
FIGURE 14. — Modeled diurnal variation of O2 concentration.
NH4+ as a nutrient, A2 which utilizes N2, and A3 which takes
NO3~ as its nutrient. Each of them has its corresponding ingestion
state A2i, A22, and A23.
The bacteria too are of three kinds: the first, BI, oxidizes
NH4+ to NO3~, the second, B2, utilizes both NO8~ and O2 to de-
compose the carbonaceous materials into the nutrients while the
third, B3, uses solely NO3~ as an oxygen source and evolves N2
as a product. They too have their corresponding secondary
stages, B2i, B22, and B23.
The various forms of algae and bacteria will be predominant
according to the availability of the nitrogeneous nutrients and
the growth rates will also be dependent on their concentrations.
Kinetics
The kinetics used are the same as in the surface model.
The diffusive transfer of N2 into the system is given by
N = mass accumulation of N2 per unit volume of lake per
unit time
N =mass transfer coefficient for N2. It is time variant
Ng^water mass concentration of N2 in equilibrium with
the atmosphere.
42
-------
The decomposition of organics is by homogeneous chemical
reactions.
rPl =mass of CPN per unit volume of lake decomposing to CP
rNl =mass of CPN per unit volume of lake decomposing to CN
rN2 =mass of CN per unit volume of lake decomposing to C
rp2 =mass of CP per unit volume of lake decomposing to C
kfw &NI, &N2, k?z = first order reaction constants
YCPN, Yep, YCN =mass concentrations of CPN, CP, CN in
lake.
The rate functions describing the absorption of nutrients from
water have essentially the same form as in the simpler model.
The nitrogeneous components have been included as a factor
in the rate expressions.
For the algae
7*Ala — &AllYAi
JL NO~
For the bacteria.
7"B2a =
rB3a =fcB13YB3YN03-(YcPN 4-NiYcp +N2YcN +N3YC)
rA2a? rA3a = masses of AI, A2, A3 formed per unit time per
unit volume of lake.
rB2a, rB3a= masses of BI, B2, B3 formed per unit time per
unit volume of lake.
The Y's are the mass concentrations of the various components
in the rates. Ni, N2, N3 are preference factors for the other
organic components over CPN. The fc's are rate constants.
The disappearance by ingestion of the components A2i, A22,
A23 and B21, B22, B23 are assumed to be first -order in their mass
43
-------
concentrations. The first-order law is also used for the lysing
rates of the algae and bacteria.
e.g.,
and TBH = k 321 Y 321
would be the ingestion rates of components A2i and B2i. The
rate constants can be set according to the relative activities of
the different components in the system.
Stoichiometry
In order to conserve the nutrients, the Stoichiometry used is
important and is required for the absorption, ingestion, and
lysing processes of the organisms and the decomposition of the
organic components.
The Stoichiometry associated with the decomposition of the
organic components which are assumed to have the composition
CaHb00NdP are given by the following formulae
(CaHb00NdP)PN->(CaHb_4dOcP)cP+dNH4+
(CaHbOcNdP)cPN-^(CaHb_2Oc_4Nd)cN+H2P04-
Lib— 4d— 2^-'c—4JC ~|~-tL2lr O4~~
H b-4d-2O 0-4)0+dNH4+
For algae the absorption Stoichiometry is given by
(C aH bO CN dP) Al +ni [aHCO3- + dNH4+ +H2PO4-
(CaHbOcNdP)A2+n1[|N2+H2P04-+aHC03-
+ ((b-a-2)/2)H2o]-+A22
(CaHbOcNdP) A3 +H! [dN03- +aHCO3-
+ ((b-a-2)/2)H20]^A23
44
-------
For the bacteria the absorption stoichiometry is given by
'CP+dNH4+
(CaHbOcNdP)B1+(62 + (772- €2)SBl)
_C+dNH4++H2PO4-_
21
(eaHbOcNdP)B2
(CaHbOcNdP)Bs
5
3
roi
i
0
»23
'CPN
CP +d(NO3- +2H20 -|O2)
CN+H2P04-
_C +d(NO3- +2H2O -fO2 +H2PO4-)_
'22
CP+d(NO,-+2H,0)
CN +H2PO4-
LC +d(N03- +2H2O) +H2PO4-_
SB3) C(3a +4 + (b -a -2)/2 -c)/3NOr]
45
-------
where
q 62d c _(I.W)No«-_62 5SN
^N~AM; N0 (A.W)0 16; SB8SNo
The ingestion stoichiometry for the algae is given by the fol-
lowing expressions.
A21^(l+€l)(CaHbOcNdP)Al
+ (1 -SAl>?i(3a+4 + (b -a -2 -4d)/2 -c)O2
AM-»(l+ei)(C.HbOoNdP)A,
(l +c:)(CaHbO0NdP)A3
For bacteria they are:
B21->(l+e2)(CaHbOcNdP)Bl
+ fo, -«,) [aHC03-+dNO3-+H2PO4- + (b -a -2)/2H2O]
B22->(l+e2)(CaHbOeNdP)B2
+ 0?« - e.) [aHC03- +dNO3~ +H2PO4- + (b - a -2) /2H2O ]
B23-»(l+€2)(CaHb00NdP)B3
-€2)faHCO3-+H2PO4- + (b -a -2)/2H2O+^ N2)
\ * /
In the above equations several stoichiometric constants are
used which are defined as follows:
„ mol. wt. of organism AI _ AM
Al wt. of corresponding nutrients ANH
„ _ mol. wt. of organism A2 _ AM
A2 wt. of corresponding nutrients AN2
„ mol. wt. of organism A3 AM
As wt. of corresponding nutrients AN
^ mol. wt. of organism BI AM
Bl wt. of corresponding nutrients AN
2 _ mol. wt. of organism B2 AM
Bz wt. of corresponding nutrients AN
<-, _ mol. wt. of organism B3 AM
33 wt. of corresponding nutrients AN2
46
-------
where
AM=12a+b+16c+14d+31
AN2=61a+14d+97+9(b-a-2)
ANH = 61a+18d+97+9(b -a -2 -4d)
AN=61a+62d+97+9(b-a-2)
Extension to the Bottom Waters
The next logical stage in the modelling is the inclusion of the
bottom waters in the system. This would be most essential in
the cases of lakes which show stratification and varying char-
acteristics through their depth.
The lake would be modelled as consisting of two parts, the
upper waters and the lower waters. Both would be considered
as well-mixed bodies not affecting each other physically. The
physical parameters of each of the systems would be different,
e.g., they would have different temperatures and light densities.
Both anaerobic and aerobic bacteria have to be included in
the bottom waters as also other reaction products like CH4 and
organic acids. Transfer of components may occur across the
interface. This may occur in either of two ways: (1) Convective
transport can be modelled with mass transfer coefficients multi-
plied by the difference in concentrations. This would be the
mode in which the nutrients like O2, HCO3~, H2PO4~, the nitroge-
nous compounds and the dissolved organic compounds may be
transferred. (2) Gravitational transport occurs in the cases of
settling of the dead organisms and the rising of gaseous bottom
products. There may also be settling included into the bottom
muds and hence out of the system.
The same kinetics and stoichiometry would hold for the upper
waters and the aerobic components of the bottom waters. How-
ever, they are not very well known for the other components
and this would present an obstacle.
SUMMARY
Nutrient cycling through the microbial populations is an im-
portant factor in the dynamics of aquatic ecosystems and at-
tempts to develop mathematical models of this part of the
ecosystem should lead to a better understanding of the eco-
47
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system as a whole. In the construction of such ecosystems three
major fundamental relationships are used: The conservation of
each chemical element that cycles through the ecosystem must
be preserved, the kinetics of the microbial and chemical re-
actions which occur in the ecosystem should be realistic, and
the stoichiometry of these reactions must be handled in detail.
This paper presents a summary of various research activities
designed to accomplish this goal.
The kinetics of microbial cell growth are developed from a
mechanism which postulates that this growth is accomplished
by two consecutive processes, the transport of the metabolite
from the exterior of the cell to the interior and the chemical
conversion of these chemicals into cellular protoplasm. Only
the rate of the transport process is assumed to be dependent
upon the external nutrient concentrations, while the chemical
conversion rate is assumed to be dependent upen the light and
temperature of the media.
Since the exact elemental composition of the microbial popu-
lations is not known, these parameters were investigated by
simulating a particular ecosystem. This ecosystem was composed
of the algae and the bacteria microbial components along with
the associated nutrients of carbon, phosphorus, and oxygen.
The seasonal simulation indicated that the composition of the
microbes significantly altered the dynamic response of the eco-
system, although it did not change some expected system prop-
erties such as the increase of algae in the summer. Qualitative
comparisons of the mathematically predicted response of the
ecosystem with known occurrences in real aquatic ecosystems
are presented.
This same ecosystem containing the cycling of carbon, phos-
phorus, and oxygen was simulated for diurnal variations of
the water quality. The diurnal response obtained from the
mathematical model resembled that found in a nearly stagnant
lake since this more closely approaches the assumption that
there were no nutrient inputs. As would be expected, all the
nutrients followed the same diurnal cycle indicating that nutri-
ent cycling was a contributing factor to the dynamics of the
ecosystem.
Finally, a discussion of the necessary model modification to
48
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include the cycling of the nitrogen in its various forms and the
inclusion of microbial organisms on the bottom of the water
body. The existence of several nitrogen forms in natural waters
complicates the stoichiometry of microbial and chemical re-
actions involving this nutrient. The relatively unknown stoichi-
ometry of anaerobic microorganisms and the stationarity of
attached algae in flowing streams are two of the major problems
to be solved when the bottom organisms are included in the
microbial nutrient cycling model.
REFERENCES
(Z) Bloom, S. G., Levin, A. A., and Rains, G. E., "Mathematical
Simulation of Ecosystems—A Preliminary Model Applied to a Lotic
Freshwater Environment," Batelle Memorial Institute, Columbus,
Ohio (1968).
(2) Burkhead, C. E., and McKinney, R. E., Proced. Amer. Soc. Civil
Engrs. 95 SA2 253 (1969).
(3) Chapman, R. N. in "Animal Ecology," McGraw-Hill, N. Y., p. 409
(1931).
(4) Chen, C. W., Proceed. Ainer. Soc. of Civil Engr., 96. SA5, 1083
(1970).
(5) Contois, D. E., J. Gen. Microbiol. 21, 40 (1959).
(6) Cooney, C. L., and Wang, D. I. C., "Influence of Environmental
Conditions on Microbial Cell Growth: Experimental and Mathe-
matical Analysis," presented at 63rd Annual Meeting A.I.Ch.E.,
Chicago (1970).
(7) DiToro, D. M., O'Connor, D. J., and Thoman, R. V., "A Dynamic
Model of Phytoplankton in Natural Waters, Enviro. Engr. Sci.
Program. Manhattan College, Bronx, N. Y. (1970).
(8) Eckenfelder, W. W. in "Biol. Treatment of Sewage and Industrial
Wastes," Vol. I, Reinhold, New York (1956).
(9) Garfmkel, D., J. of Theor. Biol., 14, 46 (1967).
(10) Garrett, M. T., Jr. and Sawyer, C. N., Proced. 7th Ind. Waste
Conf. (1952).
(IT) Gaudy, A. F., Jr. and Gaudy, E. T., Annual Review of Microbiology,
20, 319 (1966).
(12} Herbert, D., Continuous Cultivation of Microorganisms, Publ. House
of Czech. Acad. Sci., Prague, 1958, p. 45.
(13) Herbert, D., Elsworth, R., and Telling, R. C., J. Gen. Microbiol.,
14, 601 (1956).
(14) Hoover, S. R., and Porges, N., Sewage and Ind. Wastes 24, 306
(1952).
49
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(15) lerusalimskii, N. D., Continuous Cultivation of Microorganisms, Publ.
House of Czech, Acad. Sci. Prague, 1962, p. 101.
(16) Kono, T., Biotechol. Bioeng., 10, 105 (1968).
(17) McCarty, P. L., "Sec. Int. Conf. on Water Pollution Research,"
Pergamon Press, New York, p. 169 (1965).
(18) Monod, J., Ann. Res. Microbiol., 3, 371 (1949).
(19) O'Brien, J. J., and Wroblewski, J. S., "An Ecological Model of the
Lower Marine Trophic Levels on the Continental Shelf off West
Florida Coast," Masters Thesis, Florida State University (1972).
(20) Parker, R. A., Biometrics 24, 803 (1968).
(21) Patten, B. C., "Mathematical Models of Plankton Production,"
Int. Rev. ges. Hydrobiol. 53, 570 (1968).
(22) Pirt, S. J., and Callow, D. S., J. Appl. Bact. 23, 87 (1960).
(23) Powell, E. O., Evans, C. G. T., Strange, R. E., and Tempest, D. W.,
Eds., Microbiol Physiol. and Continuous Cultures, H. M. Stationary
Office, London, 1967, p. 34.
(24) Redfield, A. C., Amer. Sci. 46, 205 (1958).
(25) Smith, F. E. in "Eutrophication, Causes, Consequences, Cor-
rections," Nat. Acad. Sci., Washington, D. C. p. 631 (1969).
(26) Speece, R. E., and P. L. McCarty, "First Int. Conf. on Water
Pollution Research," Pergamon Press, London, p. 305 (1964).
(27) Stumm, W., and Tenney, M. W., "Waste Treatment for Control
of Heterotrophic and Autotrophic Activity in Receiving Waters,"
Twelfth Southern Municipal and Industrial Waste Conference,
Raleigh, N. C. (1963).
(28) Teissier, G., Ann. Physiol. Physiochim, Biol. 12, 527 (1936).
(29) Tenney, M. W., Echelberger, W. T., Singer, P. C., Verhoff, F. H.,
and Garvey, W. A., Fourth Int. Conf. on Water Pollution Research,
Pergamon Press (to be published).
(30) Tsuchiya, H. M., Fredrickson, A. G., and Aris, R., "Models of
Microbial Cell Growth," Adv. Chem. Eng. 6, 125 (1966).
(31) Ullrich, A. H., and Smith, M. W., Sewage and Ind. Wastes, 23, 1248
(1951).
(32) Verhoff, F. H., Echelberger, W. F. Jr., Tenney, M. W., Singer,
P. C., and Cordeiro, C. F., "Lake Water Quality Prediction Through
System Modeling," Proced. 1971, Summer Computer Simulation
Conference, Boston, Mass., p. 1014-1023 (1971).
(33) Verhoff, F. H., and F. E. Smith, "Theoretical Analysis of a Conserved
Nutrient Ecosystem," J. Theor. Biol. 33, 131-147 (1971).
(34) Verhoff, F. H., and Sundaresan, K. R., "A Theory of Coupled
Transport in Cells," Biochim, et Biophys. Acta 255, 425 (1972).
(35) Verhoff, F. H., Sundaresan, K. R., and Tenney, M. W., "A Mecha-
nism of Microbial Cell Growth," Biotech, and Bioeng. 14,411 (1972).
(36) Vender Haar, J. J., and Verhoff, F. H., "Dynamics of a Microbial
Growth Model in a Chemostat," unpublished results.
50
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Questions and Comments Following
Mr. Verhoff's Talk
QUESTIONS FROM THE FLOOR: I notice you talk a fair amount
about aerobic cycles. Have you purposely neglected the anaer-
obic cycle or did you just dismiss them for the sake of simplicity?
DR. VERHOFF: We are at the present developing or have
included in the model the nitrogen as ammonia, nitrate, and
nitrogen gas. We've ignored the nitrite since it can be assumed
that it goes to nitrate. Ignoring it doesn't cause problems
because every time we add something we have to add bacteria
to that particular operation and that just adds two more com-
ponents to the model. But with regard to anaerobic conditions,
I couldn't find any data which specified the stoichiometry with
regard to nitrogen, phosphorus, carbon, hydrogen, or oxygen,
and what really happens under anaerobic conditions. That is the
problem. We've ignored them at present to get some feel for
problems we will have with modeling but we certainly do intend
to include them. I should say that we've done some modeling
with anaerobes but at more gross level. We do intend to include
them for sediment conditions with exchange of nutrients between
the overlying aerobic water and the anaerobic bottom sediments,
QUESTION FROM THE FLOOR: Where empirical data have
differed from what your model predicted, are there any par-
ticular areas that you've gone back and reevaluated? For
example, your model predicted a late summer or early fall peak
-------
in algae. The two examples that you showed, Lake Constance
and some other lake, one had two peaks which were much
earlier than fall and one had a broad peak which also occurred
much earlier. Did you go back and reevaluate the model?
DR. VERHOFF: We can make that kind of check. But there
was something else going on in those lakes. We never before
saw those types of instabilities that Lake Lenore showed in the
particular model dealt with. I can't tell you exactly why that
occurred. It might have something to do with the nitrogen
cycle. We do not have the nitrogen model developed yet to the
point where can see those kinds of instabilities.
QUESTION FROM THE FLOOR: Where are the grazing zoo-
plankton included in your model?
DR. VERHOFF: The grazing zooplankton operate like bacteria
contributing to the recycle of the nutrients back to the water.
However, I think there should be a separate consideration of
zooplankton in certain ecological conditions because they defi-
nitely graze the algae down to nothing.
QUESTION FROM THE FLOOR: You indicated that the transport
of nutrients was governed by intercellular nutrient concen-
trations rather than extracellular concentrations. Do you have
any evidence of this?
DR. VERHOFF: No. I think it's extracellular nutrient concen-
trations that control transport. We separate the transport
process from the reaction process because we think the external
extracellular nutrient concentrations governs the rate at which
transport occurrs and we think temperature and sunlight
governs the rate which the reactions occur.
QUESTION FROM THE FLOOR: Did you intimate that more than
one nutrient can be growth limiting at one time?
DR. VERHOFF: I think that's the case.
QUESTION FROM THE FLOOR: What evidence do you have of
this? In any of the work we've done we've never seen any more
than one nutrient growth limiting at one time.
DR. VERHOFF: It depends on what you mean by growth
limiting. Some are normally more limiting than another, if you
think you're phosphate limiting you add some phosphate.
Without changing the growth rate too much. There are few
data on mixed systems, which are the kind of data that I need.
52
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I think O'Connor and DiToro developed a model for two
limiting nutrients, or how to handle two limiting nutrients.
QUESTION FROM THE FLOOR: I'm not referring to modelling.
I mean actual laboratory or field data.
DR. VERHOFF: There are a great deal of data available on
limitation of phosphorus and of nitrogen. Very little, I should
say, where both are nutrients low with very slow growth rates.
O'Connor and DiToro summarized the data that they could
find available when they were trying to develop phytoplankton
growth models for the San Joaquin Valley. They summarized
the data that they were able to use. They didn't separate the
transport process from the reaction scheme and used two Monod
expressions multiplied together. There was also a paper delivered
at a American Institute of Chemical Engineers meeting at
Chicago in 1970 which I've never been able to find in print.
Some investigators looked at bacteria and two limiting nutrients
however I do not remember which two limiting nutrients they
used. They tried two Monod expressions multiplied together
in an effort ,to model the two limiting nutrient system and
found that it didn't work. I don't contend that we have the
answer on how to handle two limiting nutrients because the
transport schemes that are going on which I think play the key
role are extremely complex.
QUESTION FROM THE FLOOR: When you speak of two limiting
nutrients, are you speaking of two limiting nutrients for the
same group of organisms or are you splitting them up? Say one
for algae versus one for bacteria?
DR. VERHOFF: I would have to say that we're talking about
a group of organisms because we don't model individual orga-
nisms per-se. If we were to look at bacteria as a group and
talk about two limiting nutrients, we would have to be talking
about groups of organisms. For example, the carbonaceous
material is limiting the one group and phosphorus limiting to
the other. This situation should be modified.
QUESTION FROM THE FLOOR: Well is this a possible situation?
DR. VERHOFF: Yes, it certainly is.
QUESTION FROM THE FLOOR: What I'm saying is that for a
particular alga or for a alga population there cannot be more
than one limiting nutrient at one time, nor for bacteria.
53
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DR. VERHOFF: In that case I think there is one general
limiting nutrient. The normal way of determining it is to add a
certain amount of that particular nutrient to the batch growth
or other culture system and whichever addition shows the best
response is what is called the limiting nutrient.
QUESTION FROM THE FLOOR: I don't agree with some of the
assumptions you have made before you closed the "black box".
I think this tends to run counter to some of our understanding
of the nutrient changes occurring in a heterogenous community
of organisms. One of the things I'm interested in is the basis
for your conclusions that you can separate out contributions
for the turnover of nutrients of algae and bacteria. I wonder if
you can open up the "black box" and indicate the basis for
your decision that you can distinguish between various heteroge-
nous communities of both algae and bacteria, forgetting of
course not only the herbivores but the protozoa and the fungi
in the same communities.
DR. VERHOFF: The problem every modeller faces is how to
separate a system to make it, at least half-way mimic reality.
In chemical engineering we run into exactly the same problem.
Whenever a chemical reaction occurs, there are various kinds
of intermediates that occur and we have to separate out those
intermediates whether we know for sure they're there or not,
and whether we know they're independent of what's going on.
It's just a process of modelling. You have to be able to separate
all components and look at individual transport processes.
QUESTION FROM THE FLOOR: I'm talking of end products not
the intermediates: the algal biomass and the bacterial biomass.
DR. VERHOFF: You mean separate them experimentally?
QUESTION FROM THE FLOOR: Well, in your conclusions you
apparently have been able to separate them.
DR. VERHOFF: In the experimental data that I presented on
algae and bacteria that is what the experimenters contended.
I realize that there is tremendous difficulties in this area and
that instrumentation could be very helpful that could provide
precise separation. One example of separation that we would
like to see and I think is along the line of your question is how
do you distinguish reactions even if you do separate all the
bacteria.
54
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QUESTION FROM THE FLOOR: I question to what extent one
ought to accept the validity of the model in giving these bio-
logical solutions?
DR. VERHOFF: We modellers realize that our models can be
off and any suggestion as to how to handle or how to do this
is important and is going to be a great contribution to describing
what is actually happening. Models are only so good.
QUESTION FROM THE FLOOR: Presumably from your model
you are treating these various groups of organisms as a single
population that exists throughout the year. So one would
think from your model that this is the way the system would
behave if you have a single population of algae, a single popu-
lation of bacteria, and so forth. But this is not in fact what
happens. There are definite qualitative changes. These quali-
tative changes not only change the composition of the com-
munity but also the stoichiometry. They also change the kinetics
of the community. And so, with these qualitative consider-
ations put on top of your model how can we see any sort of
pattern whatsoever? The band of variation around your model
could be 400%.
DR. VERHOFF: That's very true but at the present time all
we're looking for from these models are general phenomena.
With regard to including everything in a model, and one would
like to include everything right down to the species, including
all kinds of interactions. I think an example of the difficulty
one encounters in trying to include everything, is exemplified
by what occurred at an IBP modelling seminar in 1969 con-
nected with deserts. One of the persons there insisted that any
of the models that was developed for the desert must include
the effects of the circling hawk on the motion of the ground
squirrel. Now this is carrying it to an extreme but we just can't
include it and we can't include very easily species changes. We
just have to look at what effect general nutrient cycling between
organisms that tend to degrade and organisms that tend to
synthesize substrate. That's about the only conditions we can
help elucidate with a model.
QUESTION FROM THE FLOOR: Can you give us an order of
magnitude of goodness of fit or of your model as applied to
situations that you have tested against?
55
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DR. VERHOFF: In the case discussed today we really didn't
intend to achieve 50 % fits or anything like that. This discussion
is of general phenomena that are observed from models com-
pared with the general phenomena that are observed in nature
I didn't mean to imply that there was any kind of fit sta-
tistically. All of the data that we have were scaled from some
phenomena to show a peak here, a valley there. We've done
some comparison independent of this particular model and de-
pending on how general or how crass we want to be with the
model we get predictions of nutrients not algae. We are not
going to attempt to predict cover algal blooms populations.
QUESTION FROM THE FLOOR: There are three elements that
you have to consider: generality, precision, or you can be
realistic. I think it's possible to have all three of these in one
model. So if you're a non-modeller, and if you're opposed to the
modelling concept, you can always find something to argue
about in a model. The modeller has to decide what he is opting
for, is he going for a general model, a precise model, or what?
And of course the non-modeller can say, "Well your model
isn't realistic." That's the nature of the beast.
DR. VERHOFF: We can find out if these various instabilities
can be attributed to the nutrient cycling and the kinetics that
are actually occurring in nature. That's the hope. If these
instabilities are due to other things, and they have been pro-
posed as have being caused by other things, such as feed-back
organisms, production of poison by algae affecting bacteria, etc.
then a modeller that doesn't include this kind of phenomena
surely won't explain it.
56
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Bacterial Growth Yields
BY W. J. PAYNE
Department of Microbiology
University of Georgia
Athens, Georgia 30601
ABSTRACT
For prototrophic growth of microorganisms in minimal media
(with carbon, but no mineral nutrients limiting), yields in terms
of available electrons per mole of substrate utilized (i.e., Yave~
values) were found experimentally to approximate 3.14 grams
dry weight of cells per available electron. It was therefore
expected that this value is characteristic of prototrophic growth,
in general, when growth occurs at the expense of ordinary
organic substrates. However, growth on methane and linear
hydrocarbons appears not to be governed similarly. A mean
Yave- of 2.08 was calculated from 32 values for growth on
hydrocarbons. But, extending from the Yave~ values for ordinary
substrates to predict cell yields for either auxotrophic or proto-
trophic growth, it was predicted that a Ykcai value of approxi-
mately 0.118 grams dry weight of cells may be expected for
each kilocalorie of energy taken in any way from a culture
medium by the growing cells. Thus, consistent with expec-
tations, the mean for 94 Yave~ values calculated from growth
yield data found in the literature is 3.07. This represents expendi-
ture on the average of 39 percent of the substrate energy during
growth and incorporation of 61 percent into cell structure. The
mean for 63 YkCai values is 0.121. A figure used in determining
Ykcai values is the mean energy content of bacterial cells. This
figure was predicted from elemental and macromolecular struc-
57
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tural analysis data to lie between 5.3 and 5.46 kilocalories per
gram, ash-free dry weight and was then estimated by bomb
calorimetry to average 5.38. Finally, it is suggested that state-
ment of Bail's M concentration in terms of Yave- or Ykcai may
be preferred over expression in terms of numbers of cells per
unit volume.
INTRODUCTION
In the past, alliances between engineering and microbiology
were likely to be uneasy. The engineer who uses microorganisms
is likely to strive for constancy upon which he can base a con-
tinuous operation that can be modeled—then set in motion and
left to run with minimal upkeep; and in many cases the micro-
organisms appear to behave quixotically. I say "appear" be-
cause, the closer we examine their seeming vagaries, the more
obvious it becomes that microorganisms respond precisely and
reliably to environmental influences. In the domain of growth
yields particularly, it is clear that microorganisms will respond
predictably to easily determined properties of growth substrates.
One outstanding value of this observation lies in the prospect
it offers of meeting the producing engineer's need for reliable
yield data to base cost feasibility projections upon—when he
decides that certain reduced organic material may be converted
to edible or otherwise useful microbial products. Moreover, this
is a "classical thermodynamics" approach that considers prop-
erties only at the beginning and the end of a system's func-
tioning and thus seems more promising for engineering purposes
than "statistical thermodynamics" derived from following one
reaction to the next through biochemical pathways.
Significant interest among microbiological engineers today
lies with production of single cell protein (SCP) by microbial
utilization of waste hydrocarbons; but in the introductory
portion of their report on the growth of Candida utilis on waste
acetate left over from synthesis of cellulose acetate, Cama and
Edwards (10) pointed out that a significant tonnage of SCP
is produced annually from sulfite liquor wastes from the wood
pulp industry. Some of the SCP is used in foods prepared for
human consumption. They further suggest that whey, starchy
58
-------
and cellulose-containing wastes, and citrus and other plant
wastes are additional substrates available for microbial conver-
sion to SCP. Edwards and Finn noted, moreover, that, in
general, chemical process wastes have been neglected as trans-
formable substrates, despite several obvious advantages of their
use (12).
It seems clear that, if microbial transformation of reduced
organic wastes from agriculture and industry can be shown to
be more advantageous than just disposal, both economic and
environmental gains will be achieved. The more so-called wastes
that are converted microbiologically to useful products, the less
there are left to pollute the water ways of the world. Toward
that end, the purpose of this paper is to show that potentially
useful predictive factors have been derived experimentally and
tested for consistency with published figures on growth yields
from various sorts of cultures.
METHODS
Interest in our laboratory in the predictability of growth
yields stemmed from demonstration by Prochazka and Payne
(27) that bacterial growth could be correlated directly with
physico-chemical methods of analysis of the biodegradability
of detergents and their intermediate degradation products—and
growth is easily assayed. We were aware of the hypothesis put
forward by Bauchop and Elsden (4) based on their studies of
the growth of fermentative bacteria that an average of 10.5
grams dry weight of bacteria were produced for every mole of
adenosine triphosphate (ATP) synthesized during growth. This
is the well known YATP value currently under close consideration
and question in several laboratories as to its constancy or
variance (25). But, leaving that question aside, we noted that
Battley proposed, after lengthy but little known investigations
of growth yields of Saccharomyces cerevisiae (3), (i) that energy
obtained by either respiratory or fermentative pathways should
serve equally well for biosynthesis and (ii) that aerobic, proto-
trophic bacteria whose only products of growth in minimal
culture medium are cells and CO2 should be used in model
studies of efficiency of energy conversion by growing cultures.
59
-------
These were attractive arguments, and we set out with them
in mind. As a beginning, yields from growth of our soil pseudo-
monads based on molar consumption of substrate (YSUb values),
molar consumption of carbon (Ycarb values), and molar con-
sumption of oxygen (Y02 values) were determined for the
growth of the pseudomonads on limiting quantities of a variety
of simple substrates that served simultaneously as sole sources
of carbon and energy in minimal, mineral media. Mineral
nutrients were not limiting, and NH4+ served as nitrogen source.
Cultures were incubated at 30 C with aeration. Acting on
advice from Elsden (private communication), the cells were
harvested at the moment that the slope of the oxygen uptake
curve turned sharply over to horizontal. At this time, the spent
culture fluid was analyzed for unused substrate or organic
products. Ysub values were thus based on substrate actually
utilized rather than the quantity provided at the beginning of
the experiments. Maintenance energy for such cultures is con-
sidered to be zero.
RESULTS AND DISCUSSION
It was soon obvious from our results that Ysub, Ycarb and Yo2
values were widely divergent for growth of the pseudomonads
on various substrates. A search for normalizing factors in our
data was thus begun, following at first in much the same vein
as other workers concerned with ATP-related yields, despite
the realization that actual measurements of ATP production
would be operationally impossible. But, basing our calculations
on Hernandez and Johnson's (18) suggestion, the Y02 value for
growth on acetate was assumed to correspond to synthesis of
2 moles of ATP for every mole of oxygen consumed (i.e., an
assumed P/O ratio of 1 for acetate). With this assumption, an
apparent YATP value of 10.7 was obtained for acetate—very
near Bauchop and Elsden's YATP of 10.5 for anaerobic growth.
Moreover, when the Y02 values for growth on the other sub-
strates that we used (21) and that Hajipetrou et al. (15) used
were all then made proportional to the Y0z value for growth on
acetate, and fractional molar ATP-to-oxygen ratios were as-
sumed, then apparent YATP values very near 10.7 were obtained
for the 20 different substrates utilized by our soil pseudomonads
60
-------
and by Aerobacter aerogenes. This set of data could be significant
in that the constancy of the values may reflect screening out,
by our treatment of the data, of energy lost by mixed function
oxidase activity or other nonphosphorylation-linked oxygen
consumption. But, two currently untestable assumptions are
included in the calculations. The potential worth of this ap-
proach thus could not be estimated, and it was abandoned.
Rather than continue to be concerned with quantity of ATP
synthesized, we examined instead the oxidation-reduction state
of the carbon atoms of the growth substrates utilized for pos-
sibly normalizing influences; and at this point an obvious but
useful datum came to light (i.e., the number of available elec-
trons per molecule of substrate). Every organic compound can
be characterized by the number of these electrons, for they are
the ones that are transferred to oxygen when a mole of the
compound is completely burned to CO2 and H20. Kharasch
recorded figures for a great variety of compounds in 1925 (19).
For example, glucose has 24 available electrons per mole, acetic
acid has eight and dodecanol has 72. The value of this property
became apparent when we divided the Ysub values for growth
on each of the 10 substrates we employed by the number of
available electrons per mole of each substrate. This provided
Yave~ values in units of grams dry weight of cells per equivalent
of available electron (21). These Yave- values clustered around
a mean of 3.14. Figure 1 illustrates the simple calculation of
Genera/ consideration:
9 cells/mole substrate g cells
~~ " ~
Tave~ =r == ~^~, " ~ _
ave ave /mole substrate ave
For succJn/c acid: Ysub = 42 . 3
ave~ = 14
Yave- = 3 .02 for Pseudomonas CizB
For tetraethylene glycoh Ysub = 129.9
ave~ = 40
Yave- = 3 .25 for Bacterium TEG-5
FIGURE 1.—Calculation of Yave~ values for aerobic, prototrophic hetero-
trophs.
61
-------
Yave" values for growth of pseudomonads Ci2B and TEG-5 on
succinic acid and tetraethylene glycol, respectively. It can be
seen that the yield per mole of succinic acid consumed was 42.3
grams dry weight. A mole of succinic acid contains 14 available
electrons. Yave~ is thus 3.02, and the calculation of Yave~ for
the ether glycol is similarly uncomplicated.
18
16
14
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£ 10
FREQUENCY
00
w
6<
4-
2
0
MEAN 3. 07
VALUES BASED ON
YIELDS FROM
AEROBIC
CULTURES
••B^MH
X
•••^•M
•••••B
— 1
-.
<2. 0
YAVE-
FIGTJRE 2.—Yave- values for growth on various substrates calculated
from data published in the microbiological and engineering literature
(25; also see Table 1).
62
-------
With these results in hand, it seemed reasonable to hypothe-
size that, irrespective of species or substrate utilized, the growth
of prototrophic heterotrophic bacteria or yeasts will yield ap-
proximately 3.14 grams dry weight of cells per available electron
in the substrate used as sole source of carbon and energy—if
the conditions of our experiments are met or approximated. To
test the hypothesis, an extensive search of the literature was
initiated, and as the frequency diagram in Fig. 2 reveals, the
hypothesis need not be rejected. This graph represents a compi-
lation of data from a review of this topic presented in 1970 (25)
plus Yave~ values from Table 1 that have come to our notice
since that time.
Thus, both the mean Yave- value of 3.07 from the earlier
review (25) and 3.10 for data obtained from these 15 additional
sources over the past two years are close to the predicted mean
of 3.14, as is the overall mean of 3.07 for all the values we have
been able to find. Three quarters of the values lie within 0.5 g
of the mean even though none of the experiments were per-
formed in any of the various laboratories with this type of
analysis in mind. Apparently, prototrophic microorganisms
have the capacity to budget the electrons of an organic substrate
in minimal medium in predictable manner between biosynthesis
and oxidation, and control of this distribution by an "invest-
ment-expenditure" gene has been hypothesized (25).
TABLE 1.—YaVe— Values Not Previously Recorded in (25)
Rieche et al. (29) growing Torulopsis utilis on acetic acid 2 .84
Same studies (29) growing T. utilis on glucose 3 .72
Vernerova and Belonsheva (34) growing Candida tropicalis on acetic acid 2 .25
Tsuchiya and Drake (personal communication) growing Escherichia coli on glucose. ... 2 .93
Cama and Edwards (10) growing Candida utilis on acetate in batch culture 2 .55
Same studies (10) growing C. utiiis on acetate in continuous culture 3 .36
Perry and Edwards (26) growing Pseudomonas fluorescens on maleic acid at 25 C.... 3 .29
Edwards, Kinsella and Sholiton (13) growing P. fluorescens on maleic acid at 30 C.... 3 .46
Same studies (13) growing P. fluorescens on malic acid at 35 C 2.86
Bongers (7) growing Hydrogenomonas eutropha on succinic acid 3 .64
Same studies (7) growing H. eutropha on fumaric acid 3.24
Ahern, Turner and Mohan (1) growing Candida tropicalis on glucose 2 .89
Gaudy, Obayashi and Gaudy (14) growing a Flavobacterium species on glucose.... 3 .35
Harrison and Loveless (16) growing Klebsiella aerogenes on glucose 3 .20
Same studies (1 6) growing Escherichia coli on glucose 2 .90
Mean 3.10
63
-------
One reservation and a significant deviation should be noted
here. For this prediction to hold, the organic substrates utilized
must have at least one oxidized function in the molecule. Growth
on methane or linear hydrocarbons provides smaller Yave~
values with a mean of 2.08 (Fig. 3). Represented here are
values derived from growth of Candida and Torlua species of
yeasts, mixed bacterial cultures, Micrococcus cerificans, and
unidentified species of Mycobacterium., Nocardia and Pseudo*
monas. None are derived from our own studies, but they are
reported here because of the significantly different mean ob-
10
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P
a
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VALUES FROM
CULTURES ON
HYDROCARBONS
MEAN 2. 08
d
1. 25 1. 75 2. 25 2. 75
YAVE-
FIGURE 3.—Yave- values for growth on hydrocarbons calculated from
data published in the microbiological and engineering literature
(1, 6, 25, 32).
64
-------
We predict that:
Y (g dry wt of cells) Y (in g)
Ykcal = - , - = ~
Ea + Ed
Numerically{ therefore:
Yave" 3.14 g/ave
==0.118 g/kcal
c 26.5 kcal/ave- '
Thus, the yield in grams dry weight per kilocalorie of the combined energy assimilated and
dissimilated in a bacterial culture will approximate 0.1 18.
FIGURE 4.—Derivational basis for the value,
served. Only four values have been added to the list (1, 6, 32}
since the earlier review (25). No physiological explanation for
the difference is currently at hand, but an interesting suggestion
for dealing with it in the treatment of data was recently put
forward by Bell (5). He derived an empirical mathematical
factor to be subtracted from the number of hydrogen atoms in
the hydrocarbon substrate molecules prior to analysis of yield
data. This provided less variant Ycarb values. Bell considers
Ycarb figures preferable to Yave- values, for he holds carbon
content rather than energy content of substrate to be the
dominant factor regulating prototrophic growth.
Whether the influences of carbon and energy contents of
organic substrates on growth are indeed separable as Bell sug-
gests is yet to be determined. Meanwhile, we have asked if
Yave- values might not be used to gain insights into growth in
more complex systems. The answer appears to be, yes. As indi-
cated in Fig. 4, the ratio of yield of cells per available electron
to kilocalories of energy that each available electron represents
is proposed as an approximation of the yield of cells that may
be expected per kilocalorie of energy taken in any way from a
culture medium by microorganisms growing either aerobically
or anaerobically. This value is represented as the Ykcai, and its
calculation is based on the average energy potential of the avail-
able electron in organic material considered generally. This
latter figure was approximated as 26.5 kcal/ave- by dividing
the molar heats of combustion of a wide variety of organic
compounds, as specified by Kharasch (19), by the number of
available electrons per mole of each—and then averaging.
65
-------
SAMPLE CALCULATION
Pseudomonas C12B
w
Ykcal = ——— for aerobic growth on benzoate
Ea + Ed
86.8 g/mole
[86.8 g/mole X 5.3 kcal/g]+ [106 kcal/mole X 3 .46 mole/mole]
86.8 g/mole
460 kcal/mo!e + 366 kcal/mole
0.105 g/kcal
FIGURE 5.—Procedure for determining a Ykcai value from experimental
data from an aerobic growth yield.
Actual calculation of Ykcai approximations were undertaken
as indicated in Fig. 5. The molar growth yields from a number
of studies were related directly to the sums (represented as Ec)
of the quantities of energy taken both by assimilation and dis-
similation from the culture media employed. To calculate Ea
(the energy assimilated) Ysub values were multiplied by the
average calorific content of bacterial cells. This value is taken
as 5.3 kilocalories per gram ash-free dry weight (28). To calcu-
late Ed (the energy dissimilated), the number of moles of oxygen
consumed per mole of substrate utilized during growth was
multiplied by the energy equivalent of four available electrons,
for one mole of oxygen requires four electrons for reduction.
SAMPLE CALCULATION
Streptococcus fecalis
Y
Ykcal = for fermentative growth on glucose
Ea+ Ed
22 g/mole
[22 g/mole X 5 .3 keol/g] + [673 kcal/mole - 2(326 kcal/mole)]
22 g/mole
~ 117 kcal/mole + 21 kcal/mole
= 0 .159 g/kcal
FIGURE 6.—Procedure for determining a Ykcai value from experimental
data from a fermentative growth yield.
66
-------
Since each available electron in an organic compound represents
approximately 26.5 kilocalories, four available electrons repre-
sent 106 kilocalories. It can thus be seen that aerobic growth on
benzoate provided a Ykcai of 0.105.
Calculation of Ykcai values for fermentative growth (Fig. 6)
required only a different method for estimating Ed values.
These may be derived from fermentation balance data and are
22
20
18
3 16
P
iH
CO
* 12
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| 10
a
w
£ s
6
4
0
MEAN . 121
LOW
. 075
COMBINED
VALUES FOR
AEROBIC AND
ANAEROBIC
CULTURES
HIGH
. 164
. 075 . 090 . 105 . 120 . 135 . 150 . 165
YKCAL
FIGURE 7.—Ykcai values calculated from figures published in the micro-
biological and engineering literature (24, 25).
67
-------
simply the difference in molar terms between the heat of com-
bustion of the original substrate and the heat of combustion of
all the fermentation products—between a mole of glucose and
two moles of lactic acid in this example. Values for fermentative
growth calculated out slightly higher than those for aerobic
growth. Means were 0.130 and 0.116, respectively. An overall
mean (Fig. 7), when both were considered together, was then
0.121, which is remarkably near the predicted value of 0.118.
Only one value has been added since an earlier review (25)—
0.110 for growth of Saccharomyces varlsbergensis on glucose in a
complex medium (24).
By algebraic rearrangement, the expression Ykcai =YSub/Ec
can be stated as Ee =8.47 xYsub (i.e., the energy in kilocalories
per mole removed in any way from a culture medium is related
to the yield in grams ash-free dry weight per mole by the
constant 8.47). It seems likely that, in one of these forms,
this expression may be usefully applied as a base for prediction
of yield and cost feasibility for projected production runs.
An additional word should be said about the value we em-
ployed to express the calorific content of cells. The experimental
estimate obtained by bomb calorimetry was 5.38 kilocalories
per gram ash-free dry weight (28) as the average for a number
of types of bacteria and yeasts. This agrees closely with the
value projected from the elemental composition of bacteria
(Fig. 8). Neglecting ash content, bacterial cells were determined
by elemental analysis to comprise a hypothetical polymer of
C4H8O2N with a formula weight of 102 (22). This predicts,
the number of available electrons in this formulation, a heat
Energy Content Predicted from
Elemental Analysis of Pseudomonas C12B
C4H8O2N (neglecting ash); F. W. 102
Thus,
21 ave~/F. W. at approximately 26.5 kcal each = 556.5 kcal/102g
= 5.46 kcal/g;
(experimental value, 5.38 kcal/g)
FIGURE 8.—Estimation of probable calorific content of bacteria cells
from elemental analysis and average energy equivalence of each
available electron.
68
-------
AH = AF + TAS
Molar heats of combustion 7 number of available electrons per mole = Average energy
equivalent of each ave~ in organic material generally = 26 .5 kcal/mole~ AH
Average yield Average cellular energy Energy in average yield of cells
3 .07 g/ave- X 5.3 kcal/g = AF 16 .3 kcal/ave~
Thus, energy lost to the system by dissimilation
TAS 10.2 kcal/ave- ~
In sum, aerobic growth represents approximately 61 % conservation at the expense of
39% loss of substrate energy.
FIGURE 9.—Rationale for estimating energy budgeting of aerobic proto-
trophic growth of microorganisms.
content of 5.46' kilocalories per gram. Another close approxi-
mation was obtained by Mennett and Nakayama (23} who de-
termined the macromolecular composition of Pseudomonas fluo-
resce/is, then based their calculations on the average heats of
combustion recorded in the literature for proteins, nucleic acids,
polysaccharides and lipids. Their estimate was 5.3 kilocalories
per gram. Use of this value for calculating Ykcai thus seems
justified. "*"
From the foregoing studies, a strictly controlled energy budget
for aerobic, prototrophic microbial growth has been postulated
(Fig. 9). Reasoning that microbial cultures proceed in accord
with the second law of thermodynamics, we've taken the average
energy content of the available electron in nature, 26.5 kilo-
calories, to represent the culture's enthalpy or AH. Growth
then yields an average of 3.07 grams of cells per available
electron at approximately 5.3 kilocalories per gram. Thus, 16.3
kilocalories of energy are conserved (representing free energy or
AF). By difference, approximately 10.2 kilocalories of energy
are randomized by the system (representing the entropy term,
TAS). When the distribution of the energy represented by
available electrons in the original growth substrate is thus ac-
counted for, conservation into cellular structure averages 61
percent and loss 39 percent.
Such figures are consistent with Terroine and Wurmser's (33)
estimate (as far back as 1922) of 60 percent energy conservation
by Aspergillus niger. Moreover, in 1950 Siegel and Clifton (30)
69
-------
found that Escherichia coli growing in glucose minimal medium
oxidized 40 percent of the glucose carbon and assimilated 60
percent at any time from lag to log growth phase. During the
1960's Herbert and his colleagues (17} obtained similar values
for growth of Aerobacter on glycerol. MacKechnie and I}awes
(20) did likewise for Pseudomonas aeruginosa growing on glucose,
gluconate and 2-oxo-gluconate at 30 and 37 C. Busch and
Myrick (9) found a similar, reliable distribution of 59 percent
of the carbon into cells and 41 percent into CO2 when mixed
sewage and soil cultures were grown on glucose. And, Danforth
(11} observed an identical distribution of the carbon of acetate
by heterotrophically growing, bleached Euglena.
One other persuasive bit of evidence exists that is consistent
with the notion that the "investment-expenditure" control
mechanism for optimal microbial growth couples the amount
of energy that we can measure as biomass accumulated spe-
cifically and predictably with the quantity of energy expended
in the effort. We can accept Bail's (2} original suggestion of an
M concentration for a bacterial culture, measured in numbers
of viable cells per unit volume, as an indication of the constancy
of biomass accumulation for any bacterial species grown under
standardized conditions. But, a significant study was carried
out in 1954 by Smith and Johnson (31) with a pigmented and a
non-pigmented strain of Serratia marcescens. They observed
that, under identical culture conditions in identical volumes of
minimal medium, a larger population of the pigmented than the
colorless bacteria were obtained at M concentration for each.
This was not in keeping with Bail's hypothesis, for the bacteria
were monospecific. Smith and Johnson (31) noted on micro-
scopic examination, however, that the colorless cells were larger
than the pigmented. When we analyzed their yield data 16
years later in terms of the concept of yield per available electron
per mole of substrate utilized (25), there was no difference in
the Yave- values for the pigmented and non-pigmented cells.
M concentration thus appears to be more appropriately ex-
pressed in grams dry weight than in numbers of cells.
Finally, what appears to be the simplest possible growth
system has been examined, and has revealed data in good
accord with our predictions. It is described separately here
70
-------
because all the values presented to this point were derived
from growth yields of heterotrophic microorganisms. But,
Bongers (8) found that for the autotrophic growth of Hydrogeno-
monas in a mineral salts medium gassed with an optimal mixture
of hydrogen, carbon dioxide and oxygen, Ysub (i.e., YH2) values
of 6.2 to 6.6 were obtained. Hydrogen contains 2 ave~ per mole.
Thus, by division, Yave- values for H. eutropha growing auto-
trophically on hydrogen range from 3.1 to 3.3, bracketing the
predicted value of 3.14.
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(I) Ahern, D. G., W. E. Turner, and R. R. Mohan. 1970. Physiology
and ultrastructure of hydrocarbon-utilizing yeasts. Bacteriol. Proc.
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(3) Battley, E. H. 1960. A theoretical approach to the study of the
thermodynamics of growth of Saccharomyces cerevisiae. Physiol.
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(4) Bauchop, T., and S. R. Elsden. 1960. The growth of microorganisms
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(6) Bewersdorff, M., and M. Dostalek. 1971. The use of methane for
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chemical wastes: The growth of Candida utilis on sodium acetate.
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(19) Kharasch, M. S. 1925. Heats of combustion of organic compounds.
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(21) Mayberry, W. R., G. J. Prochazka, and W. J. Payne. 1967. Growth
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(30} Siegel, B. V., and C. E. Clifton. 1950. Oxidative assimilation of
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from hexadecane at high temperature. Appl. Microbiol. 23: 543-546.
(33} Terroine, E., and R. Wurmser. 1922. L'energie de croissance. I. Le
development de YAspergillus niger. Bull. Soc. Chim. Biol. 4: 519-567.
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on synthetic substrates. Kvasny Prumysl 9: 251-253.
ACKNOWLEDGMENT
I am grateful for the many contributions made by Drs. W. R. Mayberry
and G. J. Prochazka to this work.
73
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Questions and Comments following
Dr. Payne's Talk
QUESTION FROM THE FLOOR: Do you know how far you depart
from the growth yield values as you depart from the optimum
conditions?
DR. PAYNE: I haven't made such calculations; however,
when we examined a paper reporting the influence of temper-
ature on yield, we took the optimum values. In many papers
one has to convert from one set of units to another and the
data is usually given in grams of organisms per gram of sub-
strate or grams of organisms per gram mole of substrate. I
simply took the optimum value that the experimenter reported
and did not analyze the various numbers given.
QUESTION FROM THE FLOOR: If you got into a freshwater
situation with substrate concentrations of 10~6 or 10~8 molar,
would you expect the same degree of efficiency and energy
utilization?
DR. PAYNE: I can't answer that because referring back to
my own data, we took non-limiting concentrations of carbon
substrate. We don't know what the situation would be when
there is micro-micro quantities of substrate. The only thing I
can bring to bear on that is the work by Siegel and Clifton,
back in 1950. They showed that at any time during the growth
of E. coli substrate glucose taken up by these organisms would
be divided with 40% of it oxidized and 60% assimilated.
Whether that would pertain to micro molar quantities, I don't
know.
74
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The Sulfur Cycle
J. LE GALL
CJV.B.S., Marseilles, France
INTRODUCTION
Although the general title of this series of communications is
restricted to the influence of microbial populations upon the
aquatic environment, it is necessary to discuss first what we
shall refer to later as the "Global Sulfur Cycle". By global
sulfur cycle, we mean a model for the transfer of sulfur on a
planetarial scale from the seas to the atmosphere to the lands
and so on (see Kellog et a/., (15)). The definition of the Sulfur
Cycle given in most textbooks is generally confined to biological
aspects and particularly to the description of the bacterial
groups which are involved in the reduction or oxidation steps
of elemental sulfur. Such a representation gives but a partial
picture of the different processes by which sulfur is being cycled
on our planet. If we really want to understand and eventually
control the flux of the natural elements which are necessary for
our survival, we need a clear picture of all natural cycles and
their interconnections, including all sources and forms of a
given element, together with the amounts involved, on a
planetary scale. As far as sulfur is concerned it is now possible
to find such data in the literature. In the present paper we shall
discuss the models that have been proposed and point out the
uncertainties that still exist. Then we shall turn to the biological
sulfur cycle and give a few examples of the interactions among
the different groups of microorganisms which are involved in
the cycle with the environment. Examples are so numerous that
it is not possible to give an exhaustive list, but by pointing out
75
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some characteristic examples, we shall try to make clear to the
reader how delicate the balance between the environment and
the sulfur bacteria is and what can be the consequences of
man's bad management of our natural resources.
The Global Sulfur Cycle
Models representing the global sulfur cycle have been pub-
lished. Figure 1 shows a simplification of the model presented
by Kellog, et al. (15). The authors propose that in order to
compensate the sulfur intake living organisms must produce
282 XlO6 tons of "reduced" sulfur per year, that form of sulfur
so2, so4
soz, so4
_ " _. Man Living Volcanoes
Coast Rivers , 6.
organisms
Total s : 282 X10«tons/year
FIGURE 1.—The global sulfur cycle (from Kellog et al, (15)).
Figures are given in million of tons per year. In order to balance
the cycle, 282 X106 tons per year of "reduced" sulfur must be produced
by living organisms. For a discussion on the nature of the "reduced"
form of sulfur, see text.
76
-------
being sent into the atmosphere. It is their conclusion that man
is contributing more and more to this figure and is presently
sending 150 XlO6 tons per year of sulfur, mainly in the form of
sulfur dioxide (SO2) into the atmosphere and within a few years
he will be overmatching nature. Two questions are to be posed
then:
(1) What is the form of sulfur which is sent by living orga-
nisms into the atmosphere and what are the organisms which
are responsible for this process?
(2) What is the effect of man's contribution upon the natural
balance of sulfur?
The answer to the first question is not yet known with cer-
tainty. It was generally assumed that hydrogen sulfide was
playing that role. But since this compound was not detectable,
even in trace'amounts, Lovelock et al (20} concluded that other
compounds could be the actual form under which sulfur is
present in the atmosphere. Indeed, they found that the concen-
tration of dimethyl sulfide (CH3-S-CH3) was sufficient so that
this organic sulfur compound could be the actual form of sulfur
which is balancing the cycle. The question was then to de-
termine the origin of this compound. Challenger (9) reported
that many living systems are able to produce dimethyl sulfide
and, in particular, marine algae. Lovelock et al (20} found that
samples of Laminaria produced dimethyl sulfide at a rate of
50-400 XlO~12 g Xg"1 Xh"1 of fresh wet tissue. Leaves from cot-
ton, spruce, oak and pine trees also were capable of producing
the same compound. The possible role of bacteria in the pro-
duction of organic sulfur was not examined by the authors,
however, it should not be overlooked since Baas-Becking and
Ferguson-Wood (5) demonstrated in 1955 that sulfate reducing
bacteria and thiobacilli were able to produce such compounds.
In conclusion, one has to point out that, surprisingly enough,
we still don't know with certainty which are the compounds
balancing the sulfur cycle and the contribution of the different
groups of living organisms in this balance.
We now must try to answer the second question: Kellog et al
(15) conclude that man's contribution to the atmospheric sulfur
is already too great and will still increase in the future. But it is
important to note that this does not mean that the sulfur thus
77
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sent into the atmosphere has the time to be evenly diluted.
Increasing air pollution above industrialized areas has a tendency
to increase the total concentration of sulfur in rainfalls, thus
creating an artificial "sulfur cycle" limited to these areas. Data
exist supporting the idea: for example in Virginia (3) the sulfur
deposited at Norfolk, a very heavily industrialized area wats of
33.5 pounds/acre/year, when at Halifax, a rural area, it was
only 12:7 pounds/acre/year. In fact, sulfur deficiencies appear
more and more in very wide areas of the earth in addition to
previously well-known areas where sulfur deficiencies were al-
ways present such as Africa, Australia, New Zealand, etc . . .
severe sulfur deficiencies are developing. The reasons for this
have been developed by Tisdale (26):
—continued cropping and increasing yields have decreased
the sulfur content of the soils;
—the change of patterns of fuel consumption (from coal to
oil) has decreased the sulfur deposited in rainfalls;
—and finally the use of modern fertilizers and pesticides
which contain little or no sulfur also has decreased the
sulfur content of soils.
As a consequence sulfur deficiencies appear in the United
States. Examples can be given from Georgia (2) and Virginia
(3). In France a similar situation has been reported in the
Yonne County (25). Man's role in the imbalance of the global
sulfur cycle is then clear, we are sending more and more sulfur
into the atmosphere, but at the same time we are decreasing
the sulfur content of soils without rendering "our" sulfur avail-
able for crop production. In addition, since from Kellog's model
(15) it is evident that coastal areas are important in the balance,
our tendency to over-urbanize and, at the same time, to destroy
the ecological balances of these regions (in particular marshes)
also contributes to the decrease of organic sulfur in the
atmosphere.
The Biological Sulfur Cycle
A simple way of representing the sulfur cycle as it occurs
in soils and waters is given on Figure 2. Although the lower
part of the cycle (namely the synthesis and decomposition of
78
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bacterial
oxidation
bacterial
oxidation
Sulfate Reducing bacteria
SO.
bacterial
degradation
organic
sulfur
absorptioiVby plants
Proteinsyand other
s in vegetals
animals.
FIGURE 2.—The biological sulfur cycle.
organic sulfur-containing substances) is a vast and interesting
field, it is not the purpose of this paper to discuss its various
aspects. Our interest will be mainly centered on what may be
called the "dissimilatory" sulfur cycle which is carried out by
bacteria.
Two groups of sulfur oxidizing bacteria are aerobic and
chemoantotrophic organisms, namely the Thiobacilli and the
Beggiatoaceae. Thiobacilli oxidizes sulfur according to the fol-
lowing reaction:
2H2O+2S°+O2-^2H2SO4
so, the result of their proliferation is a sharp drop of pH which
can have important consequences upon the environment, as
will be discussed later.
The other groups of sulfur oxidizing bacteria, the purple and
green photosynthetic bacteria, are almost exclusively anaerobes
and derive their carbon from CO2 with reduced sulfur as electron
source:
CO2+2H2S-»(CH2O)+2S°+H2O
79
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Since the reduction of sulfur is a strictly anaerobic process,
one can see that the sulfur cycle can turn with little or no
atmospheric oxygen. In fact, such an environment, where low
oxygen tensions are predominant, have been called a "sul-
furetum" by Baas Backing (4) with the following definition
"The natural ecological community of these bacteria (the sulfur
bacteria) is a miniature cycle in itself and will be called
Sulfuretum."
The ecological system thus created is completed by the aerobic
sulfur oxidizers, some algae and even higher organisms such as
protozoa and nematodes able to tolerate low oxygen tensions.
A recent work by Fenchel and Rield (13) has shown that even
more organisms are implicated in sulfureta and that they cover
large areas under the oceans, ranging from high tide level to
the sea bottom.
Such a notion of an anaerobic or semi-anaerobic world living
close to our aerobic world has a very basic importance if one
takes Oparin's view that oxygen was absent from our atmosphere
when life first appeared, then the organisms present in the
sulfureta can represent living relics of the very first stages of
evolution.
The agents responsible for the reduction of sulfates and other
£w»xidized sulfur compounds are called sulfate reducing bacteria.
V\They are all strict anaerobes and have been classified by
Postgate and Campbell (24) and Campbell and Postgate (8)
into two genera: Desulfovibrio and Desulfatomaculum.
They utilize oxidized sulfur on a dissimilatory level leading,
in contrast to the assimilatory pathway (which is used by
organisms for the biosynthesis of organic sulfur compounds
such as amino acids), to the accumulation of large amounts of
hydrogen sulfide (23).
The typical metabolism of the different species belonging to
the genus Desulfovibrio is as follows:
Carbon Substrate + SO 4= —>S"+CO 2= + acetate
They are not true autotrophs since they require organic
substrates for growth. A review of the various aspects of their
metabolism is in press (19).
It is mainly the sulfate reducing bacteria which are responsible
for the accumulation of hydrogen sulfide in nature. It has been
80
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calculated by Deuser (12) that in the Black Sea, where concen-
trations of H2S up to 1 g per liter can be found, only 2.5 percent
Jof this compound conies from organic matter.
During their metabolic activity, the sulfur bacteria can pro-
duce sulfur compounds which are potentially active reagents,
such as sulfuric acid or hydrogen sulfide, and thus able to have
severe consequences upon the environment. Under normal con-
ditions, the sulfur cycle will turn smoothly and will be un-
noticed, although ubiquitous. But if for some reason the cycle
becomes unbalanced, then one of the terminal products may
accumulate, sometimes in a useful way, sometimes with cata-
strophic consequences. The following section will deal with some
of these economic consequences of the proliferation of sulfur
bacteria.
Economic Activities
Table 1 gives a list of the possible interactions of sulfur
bacteria with man's activities. As impressive as it is, this list
is far from being complete. We have chosen a few examples
that will mainly serve to illustrate their actual importance.
OIL TECHNOLOGY
Sulfate reducing bacteria are almost always associated with
oil deposits although their actual role, either in the oxidation
of oil or in oil synthesis, is not yet known with certainty.
As it has been pointed out by Floodgate (14), in contrast to
TABLE 1.—Some Economic Activities of
the Sulfur Bacteria.
—weathering of rocks
—attack of concrete (dam)
—attack of Ankor-Vat temple
—formation, oxidation of metal sulfide ores (acid mine waters)
—formation of sulfur deposits
—pollution of artificial ports (marinas)
—oil technology: sulfur, corrosion in oil wells
—sulfur in town and natural gas
—lethality of various gases
—mortality of fish
—mortality of man
—etc ...
81
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the aerobic mechanism, the evidence for a possible anaerobic
pathway of hydrocarbon degradation remains meager. Further-
more, as has been pointed out by Alexander (I), as a consequence
of their dissimilatory use of sulfate, in order to be of importance
in the degradation of hydrocarbons, they would have to reduce
huge amounts of sulfate, which is not usually present in that
high a concentration. However, Kusnetov (18) states that, when
marine water is pumped into the stratum to maintain stratal
pressure, the oil becomes the source of energy to sulfate reducing
bacteria, and H2S is sometimes formed at the rate of 0.2 mg/
liter/24 hours. The explanation for this apparently contra-
dictory statement probably resides in the fact that another
organism attacks the oil and provides some organic material
for the growth of the sulfate reducers. The presence of such a
syntrophy has been demonstrated by Kuznetsov and Gorlenko
(16) who found that a Pseudomonas was able to provide sub-
strates for sulfate reduction in the Romashkino oil field. Kus-
netzov's observations provide at least an excellent example in
which an unwanted and unexpected imbalance has been created,
leading to the attack of a useful compound.
Whether or not sulfate reducing bacteria play an important
role in oil formation is still an open question. Oppenheimer
(22) demonstrated the presence of "hydrocarbon-like" com-
pounds in mixed cultures of sulfate reducing bacteria. When a
pure culture of sulfate reducing bacteria was used, the organisms
contained 0.8 percent of hydrocarbon-like material and, after
heat treatment, 4 percent of the same material.
The formation of oil is most probably a very intricate process
involving several types of organisms. In an interesting paper,
Ciereszko and Youngblood (10) showed that sediments of
decomposed gorgonia yielded an ri-alkyl disulfide fraction con-
taining a mixture of:
Ri6-S-S-Ri6; Ri6-S-S-Ri8; Ri8-S-S-R18.
Desulfurization of these compounds would give the corre-
sponding hydrocarbons. The disulfides were probably derived
from gorgosterol (C3oH5oO). It is obvious that a close examination
of the ecosystem responsible for the decomposition of the
gorgonia, and in which sulfate reducers are most probably in-
volved, would be of the greatest interest.
82
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Formation of Sulfur Deposits
Several examples of the formation of sulfur deposits have
been given. Kusnetzov (17) has described the mechanism of
this formation in Russia. In that particular example, hydrogen
sulfide originates from an oil field, in the water, and produces
0.2 mg H2S/liter/24 hours. Ground water and H2S containing
oil and water are transported by gas pressure near the surface.
There the water contains oxygen and Thiobacillus thioparus in
large quantities. It is estimated that about 50 percent of H2S
is oxidized to sulfur and that T. thioparus contributes up to
20 percent, the rest being chemical oxidation.
The observations by Butlin and Postgate (7) that certain
lakes in Lybia produce elemental sulfur (in that case the
oxidizing agents are the green photosynthetic bacteria Chro-
matium or Chlordirum) up to 100 to 200 tons of crude sulfur
per annum, has led them to propose that sulfur could be pre-
cipitated out of sewage water. This works on a pilot scale in
London, India and also in Czechoslovakia where sulfur is pro-
duced from industrial wastes (6). In Russia, Murzaev (21)
calculated that the sewage water of Moscow could make a
useful contribution to the sulfur supplies of USSR.
Acid Mine Water
The role of sulfur oxidizing bacteria in the formation of acid
mine water is now well established (11). It is due to the oxi-
dation of pyrites by members of the genus Thiobacillus which
results in the formation of sulfuric acid producing a drop of
pH (with values in the order of 2.8).
Tuttle et al (27, 28) have proposed the utilization of wood
dust and possible sewage water to correct the pH of acid mine
water. In their reported experiments, they were able to raise
the pH from 2.84 to 3.38. The decomposition of wood provides
substrates for the sulfate reducing bacteria and the production
of sulfides raises the pH. Unfortunately, this procedure does not
solve the problem entirely since the sulfides should be removed
before they have a chance to be reoxidized.
CONCLUSION
* \
We have seen that many questions are still unanswered con-
cerning the sulfur cycle. More research is needed in order to
determine what is the actual role of plants and microorganisms
in the balance of the global sulfur cycle and in particular in
the biosynthesis of sulfur compounds. More information is also
83
Environmental Protection Agency
Library Systems Branch, Room 2903
Ml M Street S.W
-------
needed concerning the relations between the different groups of
microorganisms. This has been clearly illustrated by the example
of hydrocarbon degradation and in acid mine water treatment.
When all this information is available we may be able to
understand and eventually control one of the natural cycles
which are necessary for our survival on this planet.
LITERATURE CITED
(1) Alexander, M. 1965. Biodegradations: Problems of molecular re-
calcitrance and microbial fallibility. Adv. Appl. Microbiol. 7, 35-80.
(2) Anderson, O. E. and Futral, J. G. 1966. Sulfur and crop production
in Georgia. Bulletin N.S. 167. Georgia Agricultural Experimental
Stations. Univ. of Georgia College of Agriculture, Athens, Georgia,
Sam Burgess, Ed.
(3) Anonymous. 1966. Virginia Farmers advised to watch for sulfur de-
ficiencies. The sulfur Institute Journal, 2, 7.
(4) Baas Becking, L. G. M. 1925. Studies on the sulfur bacteria. Ann.
Bot. 39, 613.
(5) Baas Becking, L. G. M. and Ferguson Wood, E. J. 1955. Biological
Processes in the Esthuarine environment. I. Ecology of the sulfur
cycle. Proc. Koninkl. Nederl. Akademie van Wetenschappen.
Amsterdam. Series B, 58, 160-181.
(6) Barta, J. 1964. Proc. 2nd Intern. Symp. Continuous Culture,
Prague. Ed: Malek, L, Beran, K. and Hospodka, J. Acad. Press.
325-27.
(7) Butlin, K. R. and Postgate, J. R. 1954. The microbial formation of
sulfur in cyrenaican lakes. In: Biology of deserts Symposium. Inst.
Biol. London, 112-22.
(8) Campbell, L. L. and Postgate, J. R. 1965. Classification of the spore-
forming sulfate-reducing bacteria. Bact. Rev. 29, 359-363.
(9) Challenger, F. 1951. Biological methylations. Advances in enzymol-
ogy, 12, 429.
(10) Ciereszko, L. S. and Youngblood, W. W. 1971. n-hexadecyl and
n-octadecyl disulfides in "Sediment" derived from the Gorgonian
Pseudoplexaura porosa (Houttuyn). Geochim. and Cosmochim.
Acta, 35, 851-3.
(11) Cornier, A. R. and Hinkle, M. E. 1947. The role of microorganisms
in acid mine drainage: A preliminary report. Science 106, 253-6.
(12) Deuser, W. G. 1967. Black sea: microbial mineralization of organic
sulfur. Science 168, 1575-7.
(13) Fenchel, J. M. and Rield, R. J. 1970. The sulfide system: a new
biotic community underneath the oxidized layer of sand bottoms.
Mar. Biol. 7, 255-268.
(14) Floodgate, G. D. 1972. Biodegradation of hydrocarbons in the sea.
84
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Water pollution microbiology, Ralph Mitchell, Ed. Wiley Inter-
science, 153—71.
(15) Kellog, W. W., Cadle, R. D., Allen, E. R., Lazrus, A. L. and Martell,
E. A. 1972. The sulfur cycle. Science, 175, 587-96.
(16) Kuznetsova, V. A. and Gorlenko, V. M. 1965. Mikrobiologiya, 34,
324.
(17) Kusnetzov, S. I. 1963. The role of microbes in the genesis and
weathering of sulfur deposits. Symp. on Marine Microbiol. C. H.
Oppenheimer, Ed. C. G. Thomas Publisher, Springfield, 111. USA,
172-8.
(18) Kusnetzov, S. I. 1967. The role of microorganisms in the trans-
formation and destruction of oil deposits. Proc. World Petroleum
Congress. Elsevier Ed. 8, 171-81.
(19) Le Gall, J. and Postgate, J. R. 1973. The physiology of sulfate
bacteria. Adv. in Microb. Physiology, in the press.
(20) Lovelock, J. E., Maggs, R. J. and Rasmussen, R. A. 1972. Atmos-
pheric dimethyl sulfide and the natural sulfur cycle. Nature 237,
452-3.
(21) Murzaev, P. M. 1965. Izvest. Akad. Nauk, U.S.S.R., Biol. ser. 692.
(22) Oppenheimer, G. H. 1965. Z. Allg. Mikr. 5, 284.
(23) Peck, H. D. Jr. 1962. Symposium on metabolism of inorganic com-
pounds. V. Comparative metabolism of inorganic sulfur Bact. Rev.
26, 67.
(24) Postgate, J. R. and Campbell, L. L. 1966. Classification of Desulfo-
vibrio species, the non-sporulating sulfate-reducing bacteria. Bact.
Rev. 30, 732-38.
(25) Rameau, G. and Concaret, J. 1967. Sulfur deficient soils in the
Yonne County. Comptes Rendus d'Agric. de France, 1388-1394.
(26) Tisdale, S. L. 1966. Research on sulfur in agriculture: Current status
and some further requirements. The sulfur Institute Journal, 2,
15-18.
(27) Tuttle, J. H., Dungan, P. R., MacMillan, C. B. and Randies, C. I.
1969. Microbial Dissimilatory sulfur cycle in acide mine water.
J. of Bacteriol. 97, 594-602.
(28) Tuttle, J. H., Dungan, P. R. and Randies, C. I. 1969. Microbial
sulfate reduction and its potential utility as an acid mine water
pollution abatement procedure, Applied Microbiol. 17, 297-302.
85
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Questions and Comments Following
Dr. leGall's Talk
QUESTION FROM THE FLOOR: Would you predict that with the
massive use of aluminum sulfate and tertiary treatment which
has been proposed for some plants in the United States that
some problems will arise with regard to the sulfate cycle?
DR. LEGALL: It all depends on whether or not there is a
tendency for the water containing the sulfate becoming anaer-
obic. As long as you have dissolved oxygen plus sulfate no re-
duction of sulfate occurs but problems develop when you have
anaerobic conditions. These anaerobic conditions can be created
by organisms besides Desulfovibro:, for example by algae growing,
as they remove oxygen during part of the daily cycle. And I
would wonder about any disturbance of the sediments in doing
the same sort of thing.
QUESTION FROM THE FLOOR: What do you feel would be the
environmental consequence of nitrification inhibitions. In other
words, to the other cycles?
DR. LEGALL: Nothing at all. There were a number of state-
ments, about the disasterous affect of nitrification inhibitors.
Dr. Hardy said that the nitrogen reserve is about 10 million
years so if we inhibit nitrification with a compound which has a
life of 10 million years and cover the earth with 1% of this
inhibitor then we have 1 billion years of reserve.
86
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Global Nitrogen Cycling:
Pools, Evolution, Transformations,
Transfers, Quantitation and Research Needs
R. W. F. HARDY and R. D. HOLSTEN
Central Research Department
E. I. du Pont de Nemours & Co.
Wilmington, Delaware 19898
INTRODUCTION
One of the earliest designations for nitrogen was azote,
meaning without life; however, it is a combination of biological
as well as abiological chemical transformations and purely
physical transfers that constitute the global nitrogen cycle.
Our presentation considers the nitrogen cycle with emphasis on:
nitrogen compounds, nitrogen pools, nitrogen transformations,
nitrogen transfers, global nitrogen cycle and limitations and
research needs in this sequence. Each nitrogen transformation
will be defined and examined for biological and abiological re-
actions, organisms, biochemistry and physiology relevant to
ecological studies especially of aqueous environments, method-
ology, occurrence and rate. An understanding of the biochemis-
try and physiology of each transformation is an essential pre-
requisite for effective investigations of these reactions in the
"gemische" of natural environments. In one striking case funda-
mental biochemical research led to a significant new analytical
application. Difficulties in measurement undoubtedly impose
the greatest limitation on environmental work. New analytical
approaches are essential in order to provide adequate measure-
ments for the development, testing and application of realistic
model systems. For example, 15N mass spectrometric analyses
87
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are widely applied in nitrogen cycle studies, but the method
involves assumptions which are not always valid (13). We can
think of no greater contribution the present paper can make
than to stimulate research to develop novel and facile assays
to enable extensive quantitation of the various nitrogen trans-
formations in the biosphere.
The breadth of the nitrogen cycle makes it difficult, if not
impossible, for any two scientists to be intimately familiar with
all phases of it. Therefore, we will emphasize the input reaction,
dinitrogen fixation, which is our particular research specialty,
and denitrification, the major output reaction on which we can
speak only as reviewers of the literature; brief outlines of the
other transformations will be presented here but will be con-
sidered in detail by the following paper.
Several of the topics—nitrogen pools, possible evolution of
biological nitrogen transformations and quantitative estimates
of global nitrogen cycling are based on a recent monograph
"Nitrogen Fixation in Bacteria and Higher Plants" by Burns
and Hardy (24). Other recent general reviews of the nitrogen
cycle (9, 28, 33, 40, 45, 83, 104, 156, 195) are also available as
are several that specifically emphasize the aquatic environment
(4, 17, 56, 101, 102, 120, 122).
TABLE 1.—Oxidation numbers of nitrogen
Nitrogen Compounds
Environmental Significance
Oxidation
number
6+
5+
4+
3+
2+
1 +
0
1-
2-
3-
Main
HN03
NO2
HNO2
NO
N2O
N2
NH3; RNH2
Possible
intermediate
(HON)2
NH2OH2; N2H2
N2H4
Other
K03
N205
N203
H2N203
NO2NH2
HN3
88
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NITROGEN COMPOUNDS
Compounds containing nitrogen in oxidation states from —3
to +6 inclusive are known (Table 1). The main nitrogen com-
pounds in the environment are ammonia and organic amines
(-3), molecular nitrogen or dinitrogen, the latter designation
introduced by chemists and increasingly used because it dis-
tinguishes N2 from N (0), nitrous oxide (+1), nitric oxide (+2),
nitrous acid or nitrite (+3), nitrogen dioxide (+4) and nitric
acid or nitrate (+5). In addition other oxidation states are
represented by proposed intermediates in biological transfor-
mations e.g., hydrazine ( — 2), hydroxylamine and diazene or
diimide (—1). Other nitrogen compounds are known but their
global abundance is negligible and their role in nitrogen cycling
insignificant.
NITROGEN POOLS
The five major pools of global nitrogen (93, 120, 168) are
diagramed in Figure 1 (24). The largest pool, containing 97.8
percent of all nitrogen, is nitrogen occluded in primary rocks,
mainly as dinitrogen. The second largest pool, making up 2.0
percent of the total, is in the atmosphere. Quantitatively, N2,
with an average turnover time of about 17 million years is by
far the dominant species; however, the traces of other gases—
N2O, NO, NO2, and NH3—and aerosols—NH4+ and NO,-/
N02~—with turnover times as short as several minutes are
highly significant in nitrogen cycling within the atmosphere and
between the atmosphere and the land-sea pool. The third largest
pool occurs in sedimentary rock and the fourth in deep sea
sediments. Finally, the smallest pool is the land-sea pool. The
major components of the land-sea pool listed in order of de-
scending quantitative significance are N2 dissolved in the sea,
soluble inorganic nitrogen mainly as nitrates in the sea, dead
organic matter in the land and sea, insoluble inorganic matter
in the land, biomass in land and sea and soluble inorganic
nitrogen in land. It is obvious that this pool contains only an
.infinitesimal quantity of global N, but with respect to cycling
of nitrogen and environmental concern, the land-sea and the
atmospheric pools are of primary concern.
89
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INVENTORY OF THE DISTRIBUTION OF GLOBAL NITROGEN'
r— ^— ^ —
LAND
10
1
550
1
SEA
PLANTS 3
ANIMALS 1
DEAD ORGANIC 650
SOLUBLE
INORGANIC
NOj 650
N(
i '
3; 5
DISSOLVED
GASES
N2 22,000
N^ 0.25
NH} 5
INSOLUBL
E INORGANIC
DEEP SEA SEDIMENTS
DEAD ORGANIC
540
SEDIMENTARY ROCK
80,000 -400,000
(0.2%)
PRIMARY ROCKS
N2
GAS 40,000,000-190,000,000
(97.8%)
* EXPRESSED IN 109 OF METRIC TONS
FIGURE 1.—Inventory of the pools of global nitrogen from (24).
NITROGEN CYCLES
Chemical Transformations and Physical Transfers
What systems contribute to the cycling of nitrogen? There
are basically two types—chemical transformations of either a
biological or an abiological nature and physical transfers (Figure
2). Biological chemical transformations include fixation of di-
nitrogen to ammonia, assimilation of ammonia into organo-
90
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CHEMICAL TRANSFORMATIONS
Assimilation
•*•
Ammonification
Fixation t \
Nitrification
Denitrification *
PHYSICAL TRANSFERS
Volatilization — rf Precipitation
Runoff
Volcanic Release
Sedimentation
FIGURE 2. — Chemical transformations and physical transfers con-
tributing to nitrogen cycle.
nitrogen compounds, ammonification of organonitrogen to am-
monia, nitrification or oxidation of ammonia to nitrate and
denitrification of nitrate mainly to dinitrogen. Abiological chem-
ical transformations (Table 2) are well known in the atmosphere
(24, 156) and abiological denitrification occurs in soils. The
physical transfers include two counter-directional movements:
(1) volatilization-precipitation and (2) sedimentation-solubili-
zation, and two unindirectional movements: (1) leaching and
runoff which contributes to the transfer of nitrogen from land
to sea and (2) volcanic release which introduces N2 from pri-
mary rocks to the atmosphere.
Evolution of Biological Transformations
A logical but hypothetical sequence (24) for the evolution of
the various types of biological transformations of nitrogen is
shown in Fig. 3. Abiological transformations are not considered.
91
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TABLE 2 (24).—Abiological chemical transformations of nitrogen in the
atmosphere
Ozonization of N2
N2 + O3 -» N2O + O2
Lightning
e.g., 0+ + N2 -» NO+ + N
NO+ + H2O -» 2H+ + NOr
Photolysis of N2O
N2O -> N2 + O
N2O -» NO + N
Ozonization of NO
NO + O3 -> NO2 + O2
Oxidation of NO
2NO + O2 -> 2NO2
Hydration of NO2
3NO2 + H2O -* 2HNO3 + NO
Oxidation of NH3
NH3+ [O]-»->-> NO$~
Hydration of NH3
NH3 + H2O -» NH4+ + OH-
Acid aerosol neutralization of NH3
NH3 + HNO3 -» NH4NO3
2NH3 + H2SO4 -> (NH4)2SO4
The sequence is based upon a series of challenges and responses.
A challenge by the primordial or most recently accumulated
from of nitrogen compound leads to the evolution of a biological
reaction to meet the challenge and at the same time provide an
advantage through satisfaction of a biological need. Ammonia,
generally accepted as the major nitrogen compound in the
reduced primordial environment presented a challenge that was
first met by development of assimilation, the conversion of
ammonia to organonitrogen compounds such as amino acids.
This anabolic biological reaction is essential for all living things,
and therefore is proposed as the initial biological transformation
of nitrogen. Subsequent biological transformations are less
ubiquitous. Ammonification, which reverses assimilation by
release of organonitrogen as ammonia, arose in response to the
challenge of the accumulated organonitrogen compounds. The
next challenge is suggested to have developed from the concur-
rent accumulation of ammonia and O2, the former from pri-
mordial sources and the ammonification reaction and the latter
initially from abiological reactions and subsequently from photo-
92
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synthesis. Nitrification evolved to oxidize ammonia to nitrate,
and the reaction possessed the added advantage of making
energy available to the organism able to do this. The accumu-
lation of nitrate initiated two responses. Possibly the first to
evolve was denitrification, the conversion of nitrate to dinitrogen
since it provided a new nitrogen compound as well as some
energy. The other, nitrate assimilation or reduction of nitrate
to ammonia, provided living organisms with an alternate source
of nitrogen for organonitrogen compounds. Finally, the challenge
of dinitrogen accumulation in the atmosphere as a product of
denitrification was met by the response of N2 fixation which, of
course, provided a third source of nitrogen for organonitrogen
compounds. This hypothetical evolutionary sequence adequately
accounts for the known biological nitrogen utilizing capacities
of organisms and their ability to interconvert N2, NO3~, NH3
and RNH2.
Challenge
Nitrogen utilization
Response capacity of organisms
Ammonia assimilation, NH3
Ammonification, RNH2
Nitrification, NH3, O2
Denitrification, NO3~
Nitrate assimilation, NOs
Nitrogen fixation, N2
NH3->RNH2
RNH2->NH3
[O]
NH3 —> NO3~ + energy
NO3 —> N2 + energy
[H]
r -»NH3
[H]
N2->NH3
NH3-»RNH2
NH3 <=* RNH2
NH3 <=> RNH2
I
NOS~
NH3<=±RNH2
I
NOr -> N2
T
N03~ ->• N2
NO3~ -> N2
FIGURE 3.—A possible evolutionary sequence of biologically-catalyzed
nitrogen transformations from (24).
93
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NITROGEN TRANSFORMATIONS
Ammonia and Nitrate Assimilation
Ammonia or nitrate assimilation is the conversion o£ NH4+
or NO 3- to biomass such as amino acids. Typical biological
reactions are:
NO,--»NH4+ I
O NH2
II - |
NH4+ +RCCOOH->RCCOOH +H2O II
The ability to assimilate ammonia and/or nitrate is common to
all non-animal organisms. Preference for ammonia vs. nitrate
varies with organisms. Assimilation of these compounds in
temperate lakes varies with the season showing a spring and
late summer maxima (48, 102). Sea algae have been shown to
have a low K? for NH3 which enables them to take up ammonia
effectively even at the low concentrations found in the sea (46).
Studies of coastal marine environments indicate that nitrogen,
not phosphorus, is the limiting component for algal growth
(162). The ability to assimilate NO3~ or NH3 is ubiquitous,
occurring in soil, lakes, sea and sediment.
Ammonification (8)
Amnlonification is the release or mineralization of organic
nitrogen to ammonia. A biological reaction is:
NH2
H2CCOOH +^O2-*2CO2 +H2O +NH3
AF'=-176Kcal III
Bacteria and fungi are the primary agents of ammonification
in soil while bacteria or possibly zooplankton are the major
decomposers in aquatic environments (94, 102). Considerable
and rapid re-cycling of nitrogen by assimilation and ammonifi-
cation probably occurs (46, 199). The balance of assimilation
vs. ammonification is influenced by the C: N ratio with minerali-
zation dominant at a low ratio and assimilation at a high ratio.
94
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Ammonification usually exceeds assimilation at temperatures
near freezing and under anaerobic environments (8, 102).
Organonitrogen in sediments is more stable than plant and
animal remains, possibly due to complex formation. Ammonifi-
cation also occurs in all environments—soil, lakes, sea and
sediment.
Nitrification (2, 3, 28, 31, 41, 102, 115, 132, 187, 193, 194}
Oxidation of nitrogen from a reduced state such as NH4+ to
a more oxidized state such as NO3~ is referred to as nitrification.
Biological reactions include:
NH4+ +%6,-»NO,- +2H+ +H2O
AF/=-65Kcal IV
AF'=-18Kcal V
Autotrophs are the most important organisms with members
of the genus Nitrosomonas catalyzing reaction IV and those of
Nitrobacter catalyzing reaction V. Heterotrophs such as fungi
may be responsible for nitrification at acidic pH's. The ratio
of moles of oxygen consumed to ammonia oxidized is 2, and it
is well recognized that nitrification of ammonia can create a
major biological oxygen demand in aquatic systems (36, 183).
Nitrification enzymes have a high affinity for O2 with activity
occurring down to a pO2 of about 1 percent so that diffusion of
02 into the system may be the major limitation. High levels of
ammonia inhibit Nitrobacter and may make the reaction proceed
in stepwise manner. Nitrification occurs at temperatures as
low as 0° with optimum activity at about 30°-40° and in the
case of Nitrosomonas and Nitrobacter at pH's of 6 to 8. Hydroxyl-
amine and urea are alternate substrates. A number of inhibitors
of nitrification are recorded in the patent literature. One,
2-chloro-6-(trichloromethyl) pyridine has been extensively stu-
died and is reported to selectively inhibit Nitrosomonas (66).
It may be a useful experimental tool. Nitrification occurs in
soil, lakes, sea and sediment.
95
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Denitrification (15, 16, 20, 39, 102, 131, 139)
DEFINITION AND REACTIONS
Denitrification is the reduction of NO3-/NO2- to N2 and
nitrogen oxides. Examples of biological reactions are:
C6H12O6 +6NO3-^6CO2 +3H2O +6OH- +3N2O
AF' = —545 Kcal/mole glucose VI
5C6H12O6 +24NOr-*30CO3 +18H2O +24OH-+12N2
AF' = -570 Kcal/mole glucose VII
5S +6KN03 +2CaC03->3K2S04 +2CaSO4 +2CO2 +3N2
.AF' = -132 Kcal/mole S, VIII
while a not yet understood abiological denitrification reaction,
unfortunately named chemodenitrification (14, 20, 53), (all
reactions whether biological or abiological are chemical), occurs:
NO-AN2, NO, NO2 IX
ORGANISMS
A variety of facultative anaerobic bacteria catalyze biological
denitrification. Some, such as Pseudomonas, use sugar or organic
acids as reductant, while others such as Thiobacillus use S and
others such as Micrococcus use H2. Examples of denitrifiers
include:
Pseudomonas denitrificans
P. perfectomarinus
P. aeruginosa
Thiobacillus denitrificans
Micrococcus denitrificans
Corynebacterium nephridii
BIOCHEMISTRY
The biochemistry of denitrification is becoming increasingly
well defined. Payne and colleagues (7, 37, 139, 140, 141) have
used P. perfectomarinus to provide an in vitro system for eluci-
dation of the pathway of dentrification. A four-step sequence
96
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of NO3--»NO2--^NO-^N2O-^N2 has been clearly demonstrated
using separated fractions to catalyze each step (141). Nitrite
accumulates until NO3~ is completely reduced. This control is
mediated by inhibition of the conversion of NO—»N2O (140} by
NO3-. The product mixture of N2O to N2 will vary with con-
ditions, but in at least one case, C. nephridii, N2O is the terminal
product (150). Synthesis of the denitrifying enzymes and their
activity is highly sensitive to O2 but insensitive to NH3 or
N03~ (55). Denitrification couples NO3~ in place of O2 to the
oxidation of a variety of substrates, e.g., sugars, organic acids,
S, H2, with energy production. In P. perfectomarinus the sub-
strate required is asparagine (151) which is converted to malate
and oxidized to NADP, to NAD and FAD.
Both assimilatory and dissimilatory nitrate reductases are
known (139). Oxygen sensitivity of the dissimilatory reductases
differentiates them from the assimilatory reductases that func-
tion in nitrate assimilation. There are two types of dissimilatory
nitrate reductases. Reductase A catalyzes the first step of
denitrification while reductase B catalyzes the respiratory re-
ductase of coliforms, Achromobacter, Rhizobium japonicum, ba-
cilli, Neurospora, Proteus mirabilis, Hemophilus parainfluenzae,
and Spirillum itersonii. Both are Oil-sensitive,1 but reductase A
reduces chlorate and transfers electrons from NADH while B
does not (142, 143).
PHYSIOLOGY
Denitrification is restricted to anaerobic environments con-
taining less than 0.2 ml of O2/l (60). The rate in soil is sensitive
to moisture and increases with degree of saturation. Organic
matter stimulates denitrification by provision of reductant and
consumption of O2. Added electron donors e.g., S or CH3OH,
increase dentrification rate (5, 119). The rate is very rapid at
neutral to alkaline pH's and very slow at pH's below about 5;
the rate increases rapidly from 2 to 25°C and has a high optimum
of 60-65°C (15, 41, 134). The Km for NO3- is low and the rate
in soil is relatively independent of concentration. Denitrification
and assimilation of NO3~ may occur in the same environment
(30, 103, 118). In aquatic and soil systems nitrification in a more
aerobic surface layer may provide NO3~ for denitrification in a
97
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less aerobic subsurface layer (137}. In the equatorial Pacific
Ocean, nitrite produced from nitrate accumulates in regions
with very low populations of denitrifying bacteria and is pre-
sumably a product of other bacteria (57, 81). Denitrification
rates in sediments are reported to be inversely related to C/P
ratio (29).
METHODOLOGY
Development and application of more effective techniques
for measurement of denitrification are urgently needed since
the greatest data limitation in nitrogen cycle studies is the
lack of quantitative measurements of gaseous nitrogen losses
(124). Most global assessments of denitrification are simply the
unaccounted for difference between measured gains and losses
due to other transformations and transfers. (See Figure 8, for
example.)
Denitrification is measured by loss of NO3~ or formation of
gaseous nitrogen products. Indirect measurements based on
NO3~ disappearance are the least reliable since assimilatory
nitrate reduction and denitrification occur simultaneously in
some environments (30, 103, 108). A more direct measurement
is based on formation of the volatile products of denitrification,
N2 and N2O. However, fixation and denitrification also both
occur under anaerobic conditions (98) and measurements of N2
will give only net N2 changes.
Special open and closed chambers (124, 157, 158, 167) are
being used for in situ measurements in soil, e.g., modifications
of the Van Bavel air-sampling well, air lysimeters, aeromatic
apparatus and volatilization chambers. In situ measurements
that cause minimal disturbance of the eco-system are preferred
for denitrification as well as other nitrogen measurements since
disturbances often lead to increased or altered activity.
Methods of analysis are listed in Table 3. Mass spectrometry
is used to measure 15N abundance in volatile or non-volatile
compounds after an appropriate incubation with 15NO3~. It is
also used to measure N2/Ar and/or 15N/14N ratios to determine
excess N2 in aquatic samples (10, 82, 85, 86, 155). Recently a
gas chromatographic separation method utilizing Porapak Q to
separate N2O, NO and N2 has been used to measure denitrifi-
98
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NITROGEN INTER-POOL TRANSFERS
I
C9
CO
CO
<£
0
»—
0
"
I
z
o
•—
«t
o
u_
n*
LU
3
O
m
_J
CO
z
a:
' \ v'C" \'^"\ '" \
\ i l •. l | l
! LJ §
zl z
Ql o
f <
ocl »-
l^t E
3i ^
LLJI ct:
a:! a.
! H
t—
S
u-
1
o
8
_J
o
^^^^ OS
z
2
x
[^
.
3
iE
140 60 170 30
LAND SEA
m
5
1 t
I 15 ' DEEP SEA SEDIMENTS
SEDIMENTARY ROCK
PRIMARY ROCKS
* EXPRESSED IN 106 OF METRIC TONS PER YEAR.
FIGURE 8.—Global Nitrogen Inter-Pool Transfers from (24).
cation in laboratory experiments (7). Other detection systems
e.g., a He detector in place of a thermal conductivity detector,
would increase sensitivity by about 103. Gas chromatography
could be used in series with mass spectrometry to eliminate
errors in measurement of N2 and N2O mixtures introduced by
breakdown of N2O to N2 occurring in the mass spectrometer.
99
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TABLE 3.—Denitrification assays
Manometric
Kjeldahl (75)
Mass Spectrometric
Gaseous loss of 16N using 16NO3~ (30, 63)
Excess N2 in sea based on N2/Ar (70, 82, 85, 86, 154, 755)
15N-Enrichment in sea (82, 755, 70, 85, 86, 754)
Gas Chromatographic
Separation of N2O, NO and N2 on Porapak Q and detection by thermal conductivity (7)
More sensitive gas chromatographic detection system, e.g., He detector
Combination of Gas Chromatography and Mass Spectrometry
Infra-Red Spectrometric (730) N2O
The application of gas chromatography to measurements of
denitrification and N2 fixation was first proposed at about the
same time. In a recent conversation, Dr. Payne noted that only
seven papers have been published on denitrification using this
technique (e.g., 7, 32, 123, 140, 189) in contrast to the over
200X on gas chromatographic assays of N2 fixation (74). Ad-
ditional use of gas chromatography in place of mass spectrometry
in denitrification assays deserves more serious consideration.
OCCURRENCE AND RATE
Denitrification occurs in soil, lakes, sediment and sea. A
tabulation of representative measurements in aquatic eco-
systems is presented in Table 4. Only a few measurements are
recorded and there is a need for more intensive and extensive
analyses. Recorded activity is about 15-20 ^ug N denitrified/1-
day in lakes. Sediments are several times as active with a rate
of 12.4 Kg/ha -yr recently reported for Lake Mendota sediment.
Denitrification occurs in a variety of sea locations; the two
reported rates for the tropical Pacific Ocean are similar to those
for lakes.
N2 FIXATION (24, 25, 38, 71, 72, 73, 75, 76, 79, 102, 116,
129, 145, 148)
Definition and Reactions
N2 fixation is any reaction of N2 that yields nitrogen co-
valently bonded to any other atom. Biological and abiological
fixation reactions are summarized in Table 5. There is only one
100
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TABLE 4.—Denitrification measurements
Occurrence Rate
Soil
Many reports e.g., Broadbent and Clark, 1965
Lakes and sediments
Subarctic lake 15 jug N/L«day (63)
Subarctic lake mud 90 ng N/L-day (63)
Brackish Japanese lake A.D.* (82)
Lake Kizaki A.D. (109)
LakeMendota 8-26 jug N/L-day (19)
Lake Mendota sediment 12.4 Kg/ha-yr (30,103)
Reservoir mud, USSR A.D. (112)
Sea
Oxygen-minimum layer of eastern tropical
Pacific 7.2 pg N/L'day (60)
Island bay in equatorial Pacific 12-18 ng N/L-day (62)
Sea water A.D. (61)
Cariaco Trench—Caribbean A.D. (155)
—Norwegian fjord A.D. (155)
Northeastern tropical Pacific A.D. (1 84)
Coast of Peru A.D. (57)
Central Pacific A.D. (80)
* A.D. = activity detected.
biological reaction, but several abiological reactions of which
lightning and ozonization occur naturally while the remainder
are synthetic reactions. The Haber-Bosch process, developed
in 1914, has become increasingly significant. This reaction is the
basis of the relatively cheap fertilizer nitrogen essential to
support the increased crop yields of developed countries and
will be of equal importance for expanding the green revolution
in undeveloped countries. The Haber-Bosch fixation process
requires extreme conditions of pressure and temperature for
effective conversion of N2 to NH3. Moreover, the low production
cost possible in large plants generating 1000 or more tons per
day are accompanied by almost equivalent costs for transpor-
tation incurred in distributing from such widely separated pro-
duction centers. Novel synthetic fixation reactions have recently
been described in which N2 is converted to NH3 under ambient
conditions. In one process the conversion occurs in an aqueous
environment (763, 165) under conditions similar to those of
the biological reaction. It may lead to an improved system for
providing additional nitrogen. For example, utilization of mem-
101
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TABLE 5. — Biological and abiological fixation reactions of molecular
nitrogen (72, 76)
Biological reaction
Nitrogenase
X ATP + 6e + 6H+ + N2 2NH3 + x ADP + x Pi .............. X
25°,<1 atm
Abiological reactions
Lightning*
O+-f N2->NO+ + N ........................................... Xla
NO+ + H2O -» 2H+ + NO2~ ..................................... Xlb
Ozonization*
N2 + O3 -» N2O + O2 ........................................... XII
Haber-Bosch*
Fe, Mo
N2 + 3H2 2NH3 AF. = - 7.95 kcal/mole .............. XIII
450°, 200 atm.
Dinitrogen Transition Metal Complexes**
?
N2+M-»M'N2 N2H4, NH3 .............................. XIV
26°, 1 atm.
Homogeneous Aprotonic**
Ti
N2+RMgX NH3 ............. . ......................... XV (192)
25e 1 atm.
Homogeneous Protonic**
Mo • Thiol
N2+BH4- NH3 ......................................... XVI (163)
25°, 1 atm.
* Significant natural or industrial inputs
** Exploratory research systems — possible future inputs
branes for N2-enrichment of air and for retention of the catalyst
effective in aqueous systems may provide a system for the
fixation of N2 in irrigation water at the time of plant need, and
thereby more closely couple nitrogen fertilizer production to
crop location and need (136}.
Organisms
Which organisms are capable of fixing N2? We have suggested
that this process is the most recently evolved major biological
nitrogen transformation. N2-fixing organisms possess a covenant
analogous to that of the chosen. They have been given the
unique ability to utilize the sea of atmospheric N2 surrounding
them to fill their fixed nitrogen needs. Possibly only one percent
of living organisms are diazotrophs. We recently coined the
102
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word diazotrophs to designate N2-fixing organisms. They are
distributed somewhat randomly throughout the bacterial and
blue-green algal orders, families and genera (Table 6) (24).
Examples of bacterial diazotrophs found in aquatic ecosystems
include the broadly distributed anaerobe, Clostridium, photo-
synthetic bacteria, Rhodospirillum and Chlorobium, and a facul-
tative anaerobe, Klebsiella:, algal diazotrophs include Anabaena,
Aphanizomenon and Nostoc in lakes and Calothrix and Tricho-
desmium in seas (e.g., 21, 89, 169., 170., 175).
The breadth of N2-fixing relationships extend from asymbiotic
to obligatory symbiotic with various associative relationships
in between (Figure 4). Asymbiotic diazotrophs fix N2 independ-
ent of other organisms and include bacteria and blue-green
algae. Both natural and synthetic asymbiotic diazotrophs are
now known with the recent production of N2-fixing Escherichia
coli (42). The obligatory symbiotic diazotrophs are exemplified
by the legume root nodules based on genetic involvement of the
legume and Rhizobium component, each of which is ineffective
in N2 fixation by itself. The associative symbioses (113) also
involve two partners, but one is an asymbiotic diazotroph. In
the associative relationships morphological changes by the non-
diazotroph may provide an environment more favorable to the
diazotroph e.g., fungal and algal relationship in lichens (127).
The importance of these associative symbioses are only cur-
rently being recognized and they may well contribute a major
amount of fixed nitrogen. For example, a marine associative
TABLE 6.—Molecular nitrogen-fixing organisms (24)
Bacterial Diazotrophs
Orders 3/10*
Families 11 /47
Genera 26/200
Examples: Clostridium, Rhodospirillum, K/efas/e'/a, CWorobium
Blue-Green Algal Diazotrophs
Orders 2/3
Families 6/8
Genera 23/165
Examples: Anabaena, Aphanizomenon, Nostoc, Calothrix, Trichodesmivm
* Ratio of orders, families or genera with diazotrophs to total orders, families or
genera.
103
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BIOLOGICAL N2-FIXING RELATIONSHIPS
OBLIGATORY SYMBIOSES
RHIZOBIA
ACTINOMYCETES (?)
GENETIC
INVOLVEMENT
LEGUME ROOT NODULES
NON-LEGUME ANGIOSPERM
ROOT NODULES
LEGUMES
NON-LEGUME
ANGIOSPERMS
BLUE-GREEN ALGAL
AND BACTERIAL -
DIAZOTROPHS
ASSOCIATIVE (SEMI) SYMBIOSES
MORPHOLOGICAL
INVOLVEMENT
LEAF NODULES
ROOT NODULES
LICHENS
INTRA / INTE
NO MORPHOLOGICAL
INVOLVEMENT
PHYLLOSPHERE
ASSOCIATIONS
RHIZOSPHERE
ASSOCIATIONS
VARIOUS HIGHER
PLANTS AND
•MICROORGANISMS
ASYMBIOSES
NATURAL DIAZOTROPHS
BLUE-GREEN ALGAL
AND BACTERIAL
DIAZOTROPHS
SYNTHETIC DIAZOTROPHS
ESCHERICHIA COU
FIGURE 4.—Biological N2-fixing relationships from (24).
104
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symbiosis has recently been found to fix up to 500 Kg/ha-yr
(138).
Biochemistry
During the 1960's the major aspects of biological N2 fixation
were elucidated. At the beginning of the decade essentially
nothing was known at the molecular level; while at the end the
nature of the enzyme and its reactions had been defined. A new
assay of great application for ecological measurements was de-
veloped from this new biochemical knowledge. Spectacular ad-
vances in biochemistry are not anticipated during the present
decade. On the other hand progress in unravelling the biology of
N2 fixation is expected to dominate this decade.
What aspects of the biochemistry of N2 fixation are relevant
to aquatic studies? Selected highlights are listed in Table 7
and discussed below. Nitrogenase, the enzyme that catalyzes
the biological fixation of dinitrogen is composed of a Mo-Fe
protein and an Fe protein, each named on the basis of their
metal content (Fig. 5). They are essentially similar in all
organisms and are both extremely sensitive to O2, and ad-
ditionally the Fe protein is cold labile. Oxygen-sensitivity in-
fluences the distribution and activity of nitrogenase. It requires
energy in the specific form of ATP and a reductant provided by
TABLE 7.—Biochemistry of nitrogen fixation
(24, 25, 72, 73, 76, 79, 116, 129, US)
Enzyme—Nitrogenase <=^ Mo-Fe protein + Fe protein
—Similar in all organisms
—Cold labile
—CVSensitive
Requirements—Energy—ATP(op to 15-20 ATP/N2 -> 2NH3)
—Reductant—Ferredoxin or Flavodoxin
Reaction —Electron activation
—Substrate reduction
Alternate Substrates—N = N, N = O, C=N and C=C bonded small molecules such as N2, N2O,
N3~, RCN, RNC and RCCH as well as H3O+
Intermediates—None, enzyme-bound diazene and hydrazine proposed
Product—NH3, no feedback inhibition
Activation Energy—>20° 14 kcal/mole
— <20° 35-50 kcal/mole
pH optima—~7
105
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the strongly reducing electron carrier proteins, ferredoxin or
flavodoxin. The isolated enzyme has a large energy requirement
of 15-20 ATP/N2 fixed. The nitrogenase reaction (Figure 6) is
composed of two steps: (1) electron activation and (2) substrate
reduction. The former is the limiting reaction of nitrogenase and
its activity is independent of the latter. The "sensuous" ni-
trogenase by analogy with "The Sensuous Woman" is an un-
usually versatile enzyme coupling with and reducing a heretofore
unequalled variety of small N, O or C, triple-bonded or po-
tentially triple-bonded substrate partners. All reductions cleave
the NN, NC or NO bonds except in the case of CC where the
triple bond is reduced only to the double bond. There are no
enzyme-free intermediates between N2 and the product, NH3,
although diazene and hydrazine are proposed as enzyme-bound
intermediates. Ammonia does not inhibit nitrogenase activity.
The temperature response of nitrogenase is expressed as acti-
vation energy and above 20° an approximate doubling in rate
occurs for a 10° increase while below 20° the change per 10° is
much greater. The pH optimum of nitrogenase is near neutrality.
NITROGENASE
Mo-Fe PROTEIN Fe PROTEIN
(AZOFERMO) (AZOFER)
FIGURE 5.—Mo-Fe and Fe protein components of nitrogenase with
light micrographs of crystals of Mo-Fe protein and electron micro-
graphs and models of each protein from (75, 72).
106
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A
E C
L T
E I
C V
T A
R T
0 I
N 0
N
THE N2ase REACTION
ELECTRON
ACCEPTOR
e1
C
+ Pj
OXIDIZED REDUCTANT
REDUCTION
PRODUCT
S R
U E
B D
S U
T C
R T
A I
ENDOGENOUS 2HT
EXOGENOUS N2
C2H2
2e'
T
E
0
N
H2 (H2 EVOLUTION)
(N2 FIXATION)
REDUCTION)
FIGURE 6.—The nitrogenase reaction showing electron activation and
substrate reduction with either reduction of H+ or of added N2 or
C2H2 to give H2 evolution, N2 fixation and C2H2 reduction respec-
tively from (74).
Biology
The biology of N2 fixation relevant to environmental studies
is tabulated in Table 8. Oxygen concentration is a key variable
(87). Anaerobic, facultative anaerobic and photosynthetic bac-
teria fix N2 only in the absence of O2 while aerobic bacteria
and blue-green algae are more active at subatmospheric oxygen
concentrations. Since nitrogenase is oxygen-sensitive (-Z96), pro-
tection against O2 by enhanced respiration, conformational
change or membranes in aerobic diazotrophs must occur. Am-
monia is a represser, while N2 is not essential for nitrogenase
synthesis (135). The Km of N2 for nitrogenase is about 0.05 atm
so that the enzyme is essentially saturated by the N2 in air.
Diazotrophs consume about 1 g of carbohydrate per 10-20
mg of N2 fixed. Nitrogenase has only been definitively demon-
strated in lower organisms without organized nuclei (126). Sub-
stantial amounts of fixed nitrogen are not excreted but may be
transferred to associated organisms. The optimum temperature
107
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TABLE 8.—Biology of nitrogen fixation (116, 129, 145)
pO2—Type of organisms
—Activity of aerobic diazotrophs
—Protection against O2
Ammonia repression
Km(N2)—0.05 atm.
High CH2O utilization/N2 fixed.
Transfer vs. excretion of fixed N
Temperature—Optimum 20°-35°
—Limit 5°-55°
Nitrogenase restricted to prokaryotic organisms
Heterocysts
Algal activity and available P elevation.
Activity in eutrophic vs. non-eutrophic aquatic systems.
is 20-35°C although activity may be detected as low as 5°C and
as high as 55°C, in hot springs; the optimum pH varies and is
dependent on the particular organisms.
Aquatic Ecology
Algal fixation is correlated with abundance of heterocystous
forms (142, 172}; however, fixation can occur in non-hetero-
cystous filaments as well as unicellular forms (111, 161, 197}.
The activity maxima of N2-fixing algae in aquatic systems
follows elevation of available phosphorus (188). Fixation is
greater in eutrophic than non-eutrophic environments (177). It
is suggested that fixed nitrogen does not markedly decrease
activity in aquatic environments (102).
Measurement
Methods for measurement of N2 fixation are listed in Table 9.
These have recently been evaluated in a review of the C2H2-^
C2H4 assay (74). This assay, first proposed by Hardy and
Knight in 1966 (78), is based on the nitrogenase-catalyzed
reduction of C2H2 to C2H4 coupled with gas chromatography.
The method has gained wide acceptance with over 300 reports
from at least 15 different countries in 5 continents already
published.
Steps of the C2H2-C2H4 assay are tabulated in Table 10
(70, 73, 74, 77, 106, 146, 149, 152, 174, 175, 176, 180). Sample
preparation for aquatic systems involves transfer of a sample,
108
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TABLE 9. — Methods of measurement of N2 fixation (74)
Growth and Morphology
Biomass or O.D. increase on N2-free medium ................ . . ............ 2*
Heterocysts (algae only) ..... . ....... , ................. . .............. 2, 3
N-Based Methods
Kjeldahl or Dumas
N Increase [[[ 2, 3
Inoculated minus Uninoculated ...................................... 2,3
Legume N minus Cereal N ...................................... ... 3
1BN-Enrichment from «N2
Mass Spectrometry (26) .......................................... 1 , 2, 3
Optical Emission (T28) ............................................ 2, 3
13N from 13N2
Radioactive Counting (27) ......................................... 1,2
Ng:Ar Ratios
Mass Spectrometry .................................... .......... 2
NH3
Titrimetry [[[ 1
Colorimetry ....................................... . ............ 1
N2:H2 Uptake-
Manometry [[[ 1
Alternate Substrates
Gas Chromatography (70, 77, 174) ................................ 1, 2, 3
Colorimetry (114) ............................................... 3
Nitrites and Isonitriles ,
Gas Chromatography (59) ....... . ................................ 1/2
Ha-Evolution
Manometry [[[ 1
Gas Chromatography (59) ......................................... 1
Mass Spectrometry ......... , ............... ..................... 1
* Areas of use: 1, nitrogenase in vitro; 2, diazotrophs in culture; 3, field system
possibly following concentration, into a gas tight chamber made
10 percent in C2H2 and incubated in situ for a defined time at
the temperature, illumination etc. of the population being meas-
ured. Sensitivity of the method is extremely high and therefore
.a short incubation is recommended to avoid altered activity due
to derepression of nitrogenase synthesis during incubation. Al-
though it is preferred on at least a theoretical basis that N2 be
removed so as to eliminate its competition with C2H2, in practice
this is not essential since the error introduced by failure to
remove N2 is insignificant relative to the inherent variability
-------
TABLE 10.—Methodology of C2H2-C2H4 assay (74)
Incubation
Sample Preparation
Incubation chambers—serum vials
—syringes
—plastic bags
—fermentors
—micro canopies
Gas Phase—aerobic vs. anaerobic
—removal of N2
—purification of C2H2
Incubation Conditions—temperature
—time
—illumination
Termination of Reaction—removal of gas
C2H4 Analysis
Fractionation—gas chromatography
—oxidation
Detection System
—flame ionization
—colorimetry
Expression of Results
g N2[C2H2] fixed = moles of C2H2 -» C2H4 X 28
that evacuated blood sampling tubes are ideal for transfer and/or
quantitative storage of gas between incubation and analysis.
Termination of reaction by acid addition has recently been
demonstrated to introduce errors due to release of C2H4 from
acid-treated rubber stopalls (182) and results reported by the
several laboratories using this termination technique may be
invalid and require re-examination. Analysis of C2H2 and C2H4
is usually performed by gas chromatography using a flame
ionization detector. Less than one picamole of C2H4 can be de-
tected. Recently a colorimetric assay (114) has been proposed
but its relatively low sensitivity limits its application to high
activity samples such as legumes and eliminates its use for
aquatic systems.
The measurement of C2H2 reduction is converted to an N2
fixation equivalent by the formula shown in Table 10. Moles
of N2 fixed is obtained by division of moles of C2H2 reduced to
C2H4 by 31 This factor is based on the ratio of the requirement
of 6 electrons for reduction of N2 and 2 for C2H2. Twenty-six
reports in which the C2H2 reduced :N2 fixed conversion factor
110
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was experimentally measured for nitrogenase and cultured
diazotrophs validated the ratio of 3 (74). Only long term
anaerobic incubations of soil varied substantially from the
conversion factor of 3. We have recently introduced terminology
to clearly indicate the source of N2 fixation measurements ob-
tained by C2H2-C2H4 assay (74). This terminology utilizes an
italicized [C2H2] following N e.g.,
mgN[C2U2] fixed.
Although the sensitivity, simplicity and facility of the C2H2-
C2H4 assay recommend it for most field measurements, it must
always be remembered that this is an indirect assay and new
systems must be confirmed utilizing 15N2 and measurements of
15N -enrichment.
Occurrence and Rate
N2 fixation occurs in soil, lakes, sediment and sea. The
global rate of biological fixation was recently reestimated by
Burns and Hardy (24). Table 11 indicates the rates and total
TABLE 11.—Estimate of Global biological N2 fixation (24)
Land use
Metric tons
kg N2 fixed per
perhaXyr yr(XK>-6)
Permanent meadows
Total
4400
1400
250
.... 63
34
18
135
1 1 50
135
1015
3000
4100
4900
1500
14 900
36 100
51 000
140
30
5
15
10
2
0
1
35
4
5
45
40
10
0
139
36
175
111
-------
TABLE 12.—Basis for increase in estimates of biological N2 fixation (24}
Increased reports of measurement of N2 fixation in the biosphere doe to improved assays e.g.,
C2H2-C2H4 assay (74).
Recognition of more effective N2 fixation by aerobic bacterial and algal diazotrophs at pO2
<0.2 atm. e.g., lichens (127, 178).
Documentation of associative symbioses in the phylioplane and rhizosphere e.g., Paspalum-Azoto-
bacfer association (43, 44, 1 59, 198).
Recognition of algae as highly significant factors in global N2 fixation e.g., activity in marine en-
vironments and by nonheterocystous forms (23, 49, 111, 161, 197).
Demonstration of N2 fixation in additional environments e.g., digestive tracts of ruminants
Evidence for new non-legume symbiotic N2 fixation e.g., sagebrush (34, 54, 88).
amounts estimated for various environments which collectively
yield a global rate of 175 XlO6 metric tons of N2 fixed/annum.
This total is approximately equally distributed between crop-
land, grassland, forest and woodland and sea with the value
for sea being the most uncertain. We feel this global esimate
may err on the conservative side even though it is 75 percent
greater than a previous widely quoted estimate made about a
decade earlier (45). Some factors that may contribute to further
increases in the size of the current estimate are listed in Table 12.
There have been a number of recent reports of measurements
of N2 fixation in lakes and sediments (Table 13) many of which
have been made with the C2H2-C2H4 assay. With two excep-
tions, activity has been detected in all lakes and sediments
tested including those located in arctic, temperate and tropical
latitudes. One exception involved a newly established reservoir.
Because of the high temporal variability of aquatic N2 fixation
rates it is essential that continuous sampling be made over
periods of up to one year or more in order to obtain meaningful
quantitation. This has been done for Lake Mendota in the
U.S., Lakes Windermere and Esthwaite in England and Lake
Erken in Sweden. Activities were 0.61 to 30.0 Kg N2 fixed/ha «yr
with a two-fold variation recorded for the same lake in successive
years.
Some measurements of N2 fixation in the sea have been made
with 15N2 and a few recent ones with the C2H2-C2H4 assay
(Table 14). No data are yet available for intensive measure-
ments over a long period. It is anticipated that projected testing
will substantially add to the data on N2 fixation in both the
112
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TABLE 13.—Nitrogen fixation in lakes, sediments and rivers
Site N2 fixed*
Lake Windermere, Eng. 1965 / 2.87 Kg/ha-yr (58, 90)
Lake Windermere, Eng. 1966 1.07 Kg/ha-yr (58, 90)
Lake Esthwaite, Eng. 1965 1.27 Kg/ha-yr (58, 90)
Lake Esthwaite, Eng. 1966 0.61 Kg/ha-yr (58,90)
Lake Erken, Sweden soKg/ha (5 months) (68)
Lake Mendota, Wise 4.3 (17)
Lake Mendota, Wise. 1.7-2.04 (64)
Lake Mendota, Wise 0.9(51 days) or 2.4 Kg/ha-yr (177)
Lake Mendota, Wise, algal blooms up to 1032 (175)
Lake Mendota, Wise, sediment A.D. (°) (30)
Lake sediments, Wise.
—oligofrophic 0.5-2.5 jug/Kg -day (117)
—moderately eutrophic 5.0-10 jug/Kg-day (117)
Crystal Lake, Wise N.A.D (160)
Green Bay, Wise. 1971 0-49 (188)
Lake Mary, Wise. Om 0.12 (18)
Lake Mary, Wise. 10 m 0.38 (18)
Lake Mary, Wise. 20 m 1.12 or 4.6 Kg/ha-yr (18)
Lake Mize, Fla. 0 m 0-0.40 (18)
Lake Mize, Fla. 10m 0.73-7.4 (18)
Lake Mize, Fla. 20 m 0.6-2.4 (18)
Lake Mize, Fla 1.9-7.9 or 2.8-11.4 Kg/ha-yr.... (105)
Sediments of lakes, Fla A.D. in 7 of 25 (105)
Sediments of Lakes, Guatemala A.D. in 3 (105)
River, Pensacola, Fla A.D (50)
Sanctuary Lake, Pa 24-144 (47)
Smith Lake, Alaska up to 72 (12)
Lake Erie A.D (92)
Lake Erie sediment 285 /jg/Kg-day (92)
Hot Spring streams 1-5 Mg/plarrt N-day (173)
Farm pond, ok A.D (1 85)
Keystone Reservoir, ok N.A.D (69)
Lake Carl Blackwell, ok A.D (186)
Fresh and Sea water and bottom mud 7.2-108 ng N/L-day (181)
Lake George, Uganda 44 Kg/ha-yr (91)
Fish culture ponds, Japan A.D (99)
USSR reservoir A.D (112)
* Expressed as Mg N2 fixed/L-day unless otherwise indicated; A.D. = activity de-
tected; N.A.D. = no activity detected.
Pacific and Atlantic Oceans. The high activity of Trichodesmium
blooms coupled with their extensive size suggests that this alga
may represent a major source of fixed nitrogen (46). High
activity has also been found for the bacteria associated with
Thalassia in marine environments (138).
113
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TABLE 14.—Nitrogen fixation in sea
Site No fixed
Tropical ocean—Trichodesmium sp up to 0.32 Kg/ha • day (49)
Pacific ocean—bacteria associated with
phytoplankton A.D.* (122)
British rocky shore—Ca/ofhr/x 25 Kg/ha -yr (97, 171)
Florida current—planktonic Trichodesmium sp.... 7.7 pg/mg protein «day (23)
Waccasassa Estuary, Fla.
—water 035-0.11 (ig/L-day (21)
—sediment 0-2 cm 0 /tg/kg -day (21)
2-5 cm 15-144 jig/Kg*day (21)
5-20 cm 0.3-4 fig/Kg-day (21)
Caribbean marine angiosperms
Tha/ass/a 100-500 Kg N/ha-yr (65, 138J
Syringodium A.D (138)
Dip/anfhera A.D (65, 138)
Ruppia A.D (65)
Cymoefocea A.D (65)
Temperate marine angiosperms
Zoostera A.D. (138)
* A.D. = activity detected
Rate of Abiological Fixation
The Haber-Bosch process is the only significant contributor
of synthetically or industrially fixed nitrogen. This process cur-
rently provides 30-31 XlO6 tons of fertilizer nitrogen and it is
estimated that this need will increase to about 50 X106 tons
within a decade (6). Other non-physiological reactions including
lightning, combustion and ozonization are estimated to fix about
10, 20, and 15 XlO6 tons of nitrogen each year (24).
PHYSICAL TRANSFERS OF NITROGEN
Leaching
The transfer of nitrogen from land to sea via runoff and
leaching is of increasing concern since the transferred nitrate
may be a major contributor to eutrophication in lakes and
rivers (2, 35, 200, 220, 290, 292). It is estimated that about
15 XlO6 tons per annum are transferred from the land to the
sea (24).
114
-------
9
8
7
6
5
\
JAN-JULY NO, \
O
o
O
1958
'60 '62 '64 '66 '68 '70
FIGURE 7.—Trends in annual use of fertilizer nitrogen in Illinois and
concentration of NO3~-N in the Kaskaskia River at Shelbysville
Illinois from (I). Data for January-July are plotted since 1967 to
include 1971 data for which only 7 months were available.
The source of leached nitrogen—biological fixation, abiological
fixation by industrial, lightning or other processes or minerali-
zation—has not been established. Some attribute much of the
nitrogen leached from agricultural soils to fertilizer nitrogen.
The parallel between the increasing nitrate concentration of
rivers and the accelerating rate of fertilizer nitrogen used during
the last decade initially supported this hypothesis. However,
the correlation may have broken down in recent years, as shown
in Figure 7 with nitrate concentration decreasing but fertilizer
nitrogen use remaining at least constant (1). One group has
attempted to determine the source through measurements of
abundance of 15N in streams (107). They suggest that the
15N/14N ratio of streams more closely parallels that of fertilizer
nitrogen than other nitrogen sources found in agricultural soils.
This work has been severely criticized (84). In addition to the
sensitivity limitations of this technique, the unfortunate use of
virgin soil in place of an appropriate control such as unfertilized
but tilled agricultural soil appears to invalidate the result. It is
clear that additional work is needed to define the source of
115
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leached nitrogen which is fundamental to the control of nitro-
gen-based eutrophication.
Sedimentation and Solubilization
Nitrogen is transferred from the sea to the deep sea sediment
by sedimentation and returned to the sea by solubilization.
Estimates indicate that 15 XlO6 tons of nitrogen are sedimented
each year and 5 X106 tons are solubilized, providing a net
balance in favor of sedimentation (24).
Volatilization and Precipitation
Ammonia nitrogen at the rate of about 165 X106 tons per
year is volatilized and about 140 XlO6 tons of ammonia nitrogen
and 60X106 tons of NO3~/NO2~ nitrogen are returned to the
land and sea by precipitation (24). The additional nitrogen in
precipitation over volatilization loss is provided by combustion,
lightning and other oxidations. It has recently been demon-
strated that plants may effectively take up and utilize ammonia
from the atmosphere and also that they may give up lesser
amounts of ammonia to the atmosphere (125, 144). Agricultural
areas adjacent to sites of high ammonia volatilization such as
feed lots may obtain a large portion of their nitrogen by this
physical transfer.
GLOBAL NITROGEN CYCLE
The five pools of global nitrogen (Figure 1) and the various
transformations and transfers of nitrogen (Figure 2) are synthe-
sized into a global nitrogen cycle in Figure 8 (24). On the left
hand side of the chart are listed the various transfers and
transformations contributing nitrogen to the atmosphere. These
include outgassing of primary rocks, abiological denitrification,
biological denitrification, combustion and volatilization. These
inputs to the atmosphere are balanced by outputs due to
industrial fixation, biological fixation, precipitation and reab-
sorption. Biological denitrification is a forced value used to
balance the cycle and the value is substantially in excess of
that which present measurements support. The interconversions
of N2, N2O, NO and NO2 in the atmosphere provide a catalytic
116
-------
NITROGEN LAND-SEA TRANSFERS
N2
NHa
ATMOSPHERE
BIOLOGICAL
FIXATION
VOLATILIZATION
LAND
© i
22
o
"n3 INDUSTRIAL tt^
NHi
NOj
NOj
N.
I FIXATION
l~| PRECIPITATION
, , ABIOLOGICAL
- DENITRIFICATION
N2^DENITRIFICATION
N20 *
[*• CO
V.
UJ
fee
L*. ce
5E
-
o
§
.^g
£
2
o
31
h-
LiJ
O
1
O
H~
o
u_
o
«
<
o
fee
•z.
2
-------
TABLE 15.—Comparison of N budgets of two lakes
Lake Lake
Mendota (19) George (91)
Kg/ha. yr
Nitrogen fixation 9.3 44
Precipitation on lake surface 11.3 9.5
Ground water
Streams 9.2 11.1
Seepage 19.7 or 7.3 (103) ?
Urban runoff 3.5
Rural runoff 60 ?
Municipal and industrial waste 5.4
Hippopotamus excreta 3.5
Denitriflcation 19.5 or 7.1 {103) ?
Outlet loss 10.5 109 (Plankton)
Weed removal 0.8 —
Fish catch 2.9 2.5
Ground water recharge ? ?
Other Losses—sediments 42.7 22.5
A comparison of the nitrogen transformation and transfer
budgets of a temperate and a tropical lake are tabulated in
Table 15. One comes from Lake Mendota adjacent to the Uni-
versity of Wisconsin campus and possibly the most intensively
investigated lake in the world. The other is Lake George in
Uganda. Even the data on intensively investigated Mendota is
undergoing substantial modification. For example, note changes
in denitrification rate and contrast the fixation rate recorded
in this table vs. that for the same lake in Table 13.
ADDITIONAL NITROGEN MOVEMENTS
Additional natural movements of nitrogen are only now being
recognized and additional synthetic or man-made movements
are being introduced which will contribute to or modify the
nitrogen cycle. Selected examples in Table 16 include N2 fixation
in rhizosphere associations, phylloplane associations, mam-
malian digestive tracts, white fir decay and Escherichia coli.
Quantitatively, the digestive tracts are not significant con-
tributors of fixed nitrogen. A recent report suggests that
118
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TABLE 16.—Newly recognized or developed sites of nitrogen transfor-
mation
N2 fixation
Rhizosphere associations—
Azofobacfer—Paspalum (43)
Rhizomes of marine angiosperms (65, 138)
Phyl'oplane associations
Leaf algae, lichens, liverworts, bacteria in for example tropical rain forests (51, 96, 166)
Mammalian digestive tracts (11, 52, 67, 77, 147)
White fir tree decay (164)
Escherichia co/i (42, 179)
Denitrification
Methanol to remove nitrogen from sewage (5)
NH3 Assimilation
Foliar uptake and release of NH3 (125, 144)
nitrogen-fixing bacteria may provide nitrogen for the fungi that
decay white fir, a disease that destroys about a million board
feet of lumber per year in the U.S. Northwest. The transfer of
information for N2-fixing activity to E. coli, a bacteria heretofore
unable to fix nitrogen (42), has recently been accomplished by
genetic manipulation. What other man-made diazotrophs may
be developed and what will be their contribution to the nitrogen
cycle? In the denitrification area, methanol additions are being
planned to stimulate denitrification in sewage treatment plants
(5). Foliar uptake of ammonia by plants is a recently recognized
route for direct assimilation of nitrogen from the atmosphere
by plants (125, 144).
LIMITATIONS AND NEEDS FOR NITROGEN CYCLE RESEARCH
The scarcity of reliable data is the major limitation of nitrogen
cycle research and this limitation has been clearly recognized
by many investigators. For example Robinson and Robbins
(156) wrote that "The weak point in the nitrogen circulation
model is primarily the small amount of available data that is
applicable to the task. Differential data for land and ocean
areas would be especially valuable in making more realistic
estimates of the masses of material that enter into various
portions of the cycle."
In conclusion, we would like to reemphasize the need to
develop novel methods for measurement of nitrogen transfor-
119
-------
mations and transfers. Alternate detection systems are needed.
We propose that an example of such an approach may be the
utilization of proton-reaction analysis to determine 15N/14N
ratios in place of mass spectrometry (153). Advantages for
proton-reaction analysis include high sensitivity for 15N vs. 14N
possibly permitting more definitive measurement using samples
with natural abundance. Furthermore, chemical treatments of
a sample to isolate nitrogen and convert it to N2 may not be
needed. Systems capable of continuous monitoring are desirable.
Seasonal or annual profiles of ecosystems are needed and ex-
trapolation from limited measurements of an ecosystem should
be avoided. Sufficient data and a suitable experimental design
should be used to allow statistical evaluation.
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Acetylene reduction in relation to levels of phosphate and fixed
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(193) Watson, S. W. 1963. Autotrophic bacterial nitrification in the
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(194) Watson, S. W. 1965. Characteristics of a marine nitrifying bac-
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(196) Wong, P. P., and R. H. Burris. 1972. Nitrogenase: Oxygen inhi-
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(197) Wyatt, J. T., and J. K. G. Silvey. 1969. Nitrogen fixation by
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(198) Yoshida, T., and R. R. Ancajas. 1971. Nitrogen fixation by bacteria
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(199) Yoshida, Y., M. Kimata, A. Kurata, and T. Maizuru. 1970.
Studies on the marine microorganisms utilizing inorganic nitrogen
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Questions and Comments Following
Dr. Hardy's Talk
QUESTION FROM THE FLOOR: You showed on your charts that
in ocean sediments there appeared to be more nitrogen coming
out than going in. Do you have any idea of the natural processes
in the sediments?
DR. HARDY: The figures presented were obtained from
numbers that were available. Any of the numbers on that sheet
except industrial input are to be considered as current best
estimates. Little, if any information on the specific natural
processes in ocean sediment are available.
QUESTION FROM THE FLOOR: I wonder why in your evolu-
tionary scheme of nitrogen transformations that you put ni-
trogen fixation last or most recent. It's an aerobic system and
it occurs so frequently in so-called primitive bacteria.
DR. HARDY: There are plausible arguments that you can make
for either an early or late appearances for N2-fixers. Our proposal
is a logical sequence based on the stresses on the organisms.
The random distribution of nitrogenase, is not explained in our
proposal of late development. Our way of looking at the nitrogen
metabolism evolutionary scheme makes it easy to understand.
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Environmental Impact of Nitrate,
Nitrite, and Nitrosamines
M. ALEXANDER
Laboratory of Soil Microbiology,
Department of Agronomy,
Cornell University,
Ithaca, New York 14850
Environmental microbiologists have been concerned with
transformations of nitrogen for decades, and both soil and
aquatic microbiologists have actively investigated the behavior
of this element in the ecosystems of interest to them because
of a variety of practical problems in agriculture and water
pollution control. Moreover, a great deal of basic microbio-
logical research has been done on the organisms and the bio-
chemical steps involved in the decomposition, oxidation, re-
duction, volatilization, and fixation of nitrogen compounds.
Recent developments have again focused attention on the
microbiology of nitrogen. A few of the issues of current interest
are not really new. High nitrate levels in wells have been known
for many years, and the danger of methemoglobinemia to in-
fants and livestock consuming nitrate-rich waters has been a
long-time concern of public health and agricultural specialists.
Fertilizers have frequently been cited as poisons of biological
cycles in the past also, and the recent claims that they are
hazardous to critical biogeochemical processes taking place in
natural ecosystems are, to a great degree, a repetition of state-
ments made one or two generations ago.
On the other hand, certain of the concerns with environmental
hazards arising from nitrogen compounds are quite new. Farmers
in some regions of the United States are applying quantities of
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fertilizer nitrogen heretofore unknown. Groundwaters which
previously were reasonably free of nitrate are now occasionally
showing levels in excess of the U. S. Public Health Service
guidelines for potable water supplies. Feedlots with large num-
bers of animals are appearing in many areas, and the vast
concentrations of manure would not likely have showed up
even in the worst nightmares of farmers of 20 years ago.
Eutrophic lakes are being recognized more frequently, and their
increasing fertility may sometimes be attributed to an increased
rate of release of nitrogen from agricultural, urban, or industrial
sources. Furthermore, the possible significance to man's health
of nitrosamines, a new class of toxicants, was not appreciated
until the last few years.
Unfortunately, few microbiologists have been participating
in investigations of these newer aspects of nitrogen pollution,
and most of the research is being conducted by scientists in
other disciplines. Owing to the absence of a sizable body of
microbiological literature relevant to current problems, my
comments will be based upon a few recent investigations, an
assessment of the older literature, and a recent report of a
committee of the National Academy of Sciences, which I was
privileged to chair (4).
Microorganisms are the chief agents responsible for the cycling
of nitrogen in the biosphere. For example, the organic nitrogen
in soils, sediments, and terrestrial and aquatic plants is con-
verted to ammonium by a large assortment of ubiquitous
heterotrophic bacteria, fungi, and actinomycetes. Members of
these microfloras are capable of tolerating essentially all natural
conditions which allow for growth of living organisms, so that
ammonium is produced in a wide array of circumstances. Am-
monium in soil is retained by the clay particles, and thus it
moves very slowly through the profile and into the groundwater.
However, if O2 is present and the pH is not too low, the am-
monium is oxidized to nitrate in a process known as nitrification.
Nitrate is not retained by clay, and hence it moves rapidly
through soils to enter the subterranean water. Nitrate, in turn,
serves as an electron acceptor for bacteria normally using O2,
and the nitrate is reduced and escapes in the form of N2 or
occasionally N2O. The fate of the oxide in the biosphere is
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presently unknown, but N2 serves as a nitrogen source for
blue-green algae, nodulated leguminous plants, and several
bacterial genera and is thereby returned to the water and soil
in a form suitable for subsequent plant growth and animal
development.
Microorganisms are thus responsible for synthesizing most of
the nitrate in soils, groundwaters, inland bodies of surface
water, and presumably the oceans. Although any estimate must
still be considered as only a first approximation, probably in
the vicinity of 21.0 million metric tons of nitrogen are returned
annually or are released in the soils of the United States to
become potentially available for plant growth. Of this total,
about 3.6 million tons is acquired by symbiotic nitrogen fixation
resulting from the activities of Rhizobium and its hosts, 1.2
million tons is estimated to come from nonsymbiotic fixation
largely by free-living bacteria, and 3.1 million tons arises from
the microbial decomposition of organic nitrogen in soil and its
release in the form of ammonium, which subsequently is con-
verted to nitrate. In addition, 7.5 million tons is added to the
soil each year in the form of nitrogenous fertilizers, and most of
this too is acted upon by microorganisms, converting the reduced
nitrogen to the nitrate form. Thus, probably about 75% of
the nitrogen which is available for plant use is transformed by
microorganisms in soil, and a large part of this 15.4 million tons
is oxidized ultimately to nitrate (4).
Many other sources of nitrogen are acted on microbiologically
in waters and soils. These sources include urban sewage, effluents
from septic tanks, industrial wastes, and substances released
from refuse dumps, food processing industries, and feedlots.
The quantity of nitrogen provided in these various forms and
the amount acted upon microbiologically to yield nitrate are
still largely unknown at the present time, but available evidence
suggests that a large part of this nitrogen will be nitrified if the
environment is aerobic and not overly acid.
The remarkable rise in the number of cattle feedlots shown in
Table 1 illustrates the growing problem of point and diffuse
sources giving rise to high local concentrations of nitrate. Such
large feedlots are frequently situated in regions having little or
no adjacent cropland on which to apply the manure, and part
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TABLE 1.—Numbers and sizes of feedlots in three representative years (4)
Animals per Number of feedlots
feedlot
1962 1966 1970
1,000-2,000
2,000-4,000
4,000-8,000
8,000-16,000
16,000-32,000
..32,000
752
373
179
105
26
5
938
486
298
136
55
8
991
543
331
210
105
41
of the nitrogen in the fecal matter is volatilized as ammonia and
part is converted to nitrate by soil bacteria. The anion may
leach through the soil and pollute the underlying waters. On
occasion, this pollution is marked.
No one can doubt that fertilizers are also contributing to the
increasing nitrate levels in certain groundwaters and, after
lateral movement of the anion, in surface waters, too. Investi-
gations of soil scientists have clearly shown that high fertili-
zation rates do indeed give subterranean water concentrations
of nitrate occasionally in excess of the Public Health Service
guidelines for potable water supplies (7). The only real points
of contention are the percentages of the fertilizer nitrogen
added in any particular watershed that are leached as nitrate
after it is nitrified and the percentages returned to the atmos-
phere as a result of denitrification. The heated debate that we
are witnessing involves interested parties who have, sad to say,
either inadequate or no data. Inasmuch as the nitrogen level in
soil that has been cultivated for some time does not change
appreciably, although there is a massive initial loss when virgin
land is first tilled, all the fertilizer nitrogen added to soil is lost.
Taking the 1970 figures for fertilizer use, 7.5 million tons thus
disappear from surface soils.
Of course, only part of the nitrogen is lost by denitrification
or leaching, following the nitrification phase. A considerable
part of the nitrogen is assimilated by growing plants and that
which is not allowed to decay in place is removed from the farm
land. If the efficiency of nitrogen uptake by the plant were to
be increased, then the loss through denitrification or by leaching
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would be diminished. Agricultural scientists have long sought
to increase nitrogen efficiency, but the justification has tra-
ditionally been to improve crop yield per unit of fertilizer
applied. Now the interest is prompted by a desire to minimize
environmental pollution. A number of means are being explored
to increase the proportion of added nitrogen that is assimilated
by the crop. Of particular interest to the microbiologist is the
search for selective inhibitors of nitrifying microorganisms, in-
hibitors which would keep the element in the soil in the am-
monium form. Another means of diminishing the nitrogen flow
into waters is to enhance denitrification, that is, to carry out
some operation in the field to increase the magnitude of the
microbial reduction of nitrate before it enters the groundwater.
At the moment, however, essentially no research is being done
on this approach.
Indeed, surprisingly little data are available on the percentage
of nitrogen actually denitrified under field conditions and the
percentage that is leached. It was commonly believed not too
long ago that denitrification was inconsequential and that most
of the added nitrogen or that released from humus entered
groundwaters. However, now that it is considered to be unwise
to have much nitrate in water, the same groups of individuals
who proposed that leaching was the major mechanism of
nitrogen loss from soil maintain that denitrification is extremely
important. Conversely, a second group states vociferously that
the old view is completely correct. In point of fact, neither
school of thought has presented convincing data, and except
for occasional areas or in greenhouse trials, the significance of
microbial denitrification to nitrogen losses from soil is totally
unclear.
The type of watershed or ecosystem study which ought to be
conducted is illustrated by the work of Biggar and Corey (2).
They estimated the contribution of various sources to the input
of nitrogen into Lake Mendota, Wisconsin. Their data suggest
that a significant amount, namely 45 %, of the nitrogen entering
the lake is derived from the groundwater, an input quite likely
resulting from the intensive dairy farming in the vicinity of the
lake. A high proportion of the nitrogen entering the lake appears
to come from manure washed off the land, and microorganisms
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are here once again the culprits as they decompose the animal
wastes washed into the lake during the spring thaw and heavy
rains. In addition, the lake acquires 14% of its new supply by
nitrogen fixation through the activities of microorganisms in
the water, presumably the blue-green algae blooming so fre-
quently in that body of water.
Some individuals have argued that the problem of increasing
nitrate levels in certain water supplies is simply overcome by
reducing the use of chemical fertilizers. Admittedly, in many
areas this can and should be done because of injudicious use
by farmers. However, high crop yields require much nitrogen,
and it makes no difference to microorganisms whether the
nitrogen is supplied as ammonium, sewage sludge, plant residues,
or manure. The nitrogen is converted to ammonium, if it is
not already in that form, and the ammonium is subsequently
oxidized to nitrate. After analyzing the changes in fertilizer use
and dietary patterns in the United States during the last several
decades, the National Academy of Science committee came to
the surprising conclusion that the recent marked increase in
consumer demand for animal protein, at the expense of plant
protein, has contributed materially to the greater use of ferti-
lizer. An inordinate amount of nitrogen must be used to grow
the plants which are then fed to livestock and poultry so that
we can eat animal rather than plant protein. This is illustrated
by the figures in Table 2.
With reference to making animal protein from fertilizers, the
efficiency of nitrogen utilization has two components: (a) the
efficiency of plants in taking up the nitrogen that is provided
in the soil; and (b) the remarkable inefficiency of animals in
converting plant protein to animal protein. In fact, of the 29.4
kg of nitrogen in plant protein used or consumed per person
per year, only 1.7 kg comes directly from plants that man
consumes. The remaining 27.7 kg, or 94%, is used to feed the
animals which provide us with the meat, poultry, dairy products,
and eggs we consume. Consequently, if Americans are to have an
adequate amount of high quality food and continue to consume
enormous quantities of animal proteins, agriculture is obliged to
use vast tonnages of synthetic fertilizers, much of which will
be lost to waterways.
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TABLE 2.—Protein and nitrogen consumption in U.S.: 1968 (4)
Protein Protein N in plant
source consumed protein
(g/person/day) (kg/person/yr)
Meats
Fish
All plants
36.6
8.8
2.5
6.7
15.8
28 7
19.4a
3.0a
1 .6a
3.7a
1 .7
a N in plants required to feed the animals.
A number of scientists and even a few technically trained
individuals have again raised the alarm that fertilizers are
poisons of microorganisms and inhibit microbial processes. This
restatement of an old and largely forgotten issue is rather
surprising in view of the enormous literature that has developed
since the time that the question was first posed. Certain ferti-
lizers do indeed cause a change in the abundance and activity
of soil microorganisms, but this inhibition is both temporary
and reversible. However, plants are obtaining the nutrients they
need from the fertilizers, nutrients they would have gotten in
only small quantities from natural microbial activities, so that
the temporary suppression is of no practical consequence.
Should fertilizer use terminate, the activities of microorganisms
in releasing nitrogen, phosphorus, and other elements would
resume at probably the same rate as before.
Why is there the concern with nitrate as a health hazard?
If the human infant or the ruminant animal consumes reason-
ably high concentrations of nitrate, the baby or the animal may
develop methemoglobinemia, a disease in which O2 transport
through the blood is impaired by the decreased supply of
hemoglobin. The 10 ppm nitrate-nitrogen recommended stand-
ard was established on the basis of a number of studies in which
it was demonstrated that methemoglobinemia is very infrequent
in infants drinking water containing less than this level and
frequent in infants consuming water with higher concentrations.
141
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Despite the fact that many surface wells contain nitrate in
excess of these recommended limits, the number of reported
cases of methemoglobinemia in infants in the United States is
decreasing (4). The reason for this decline in disease incidence,
despite the apparent increase in some areas of high nitrate
waters, probably results from the greater vigilance of public
health officials, the awareness by pediatricians of the problem,
and the information provided to mothers of infants. However,
society cannot be complacent about its present good fortune
because it is clear that high levels of fertilizers will continue to
be used and that present practices will continue to lead to
nitrification of the added nutrients with the consequent appear-
ance of nitrate in water supplies. Inasmuch as the nitrate prob-
lem will get worse and not better in the absence of new tech-
nologies, a real need exists for imaginative microbiological
research.
Nitrate may also be hazardous to man and animals because
of its presence in food or feed. Certain plants accumulate
enormous quantities of nitrate, and consumption of such vege-
tables or feeds may induce symptoms of methemoglobinemia in
infants and are known to have caused deaths in animals. It
must be emphasized, however, that commercially prepared baby
foods have been implicated in only one instance of methemo-
globinemia in the United States in recent years. This remarkable
safety record attests to the lack of acute toxicity resulting from
the nitrate per se. This point must be emphasized because
irresponsible individuals have blown out of proportion the
problem of this disease in infants due to baby food. However,
high nitrate levels in vegetables fed to infants are of no value,
and it should not be too difficult for plant breeders to develop
varieties and for farmers to employ practices to minimize the
nitrate levels in food and feed.
To a microbiologist, it is astounding that essentially no at-
tention has been given to what happens to the nitrate in foods
stored in the home, either at room temperature or in the re-
frigerator. Recent evidence suggests that nitrate in certain
foods may be only partially reduced during storage, and po-
tentially toxic levels of nitrite may appear in these stored
commodities. No data actually demonstrate that a hazard exists,
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but complacency is again foolhardy, particularly in view of the
indications that pathological effects other than those of methe-
moglobinemia may be associated with the nitrite formed from
nitrate. These include effects on thyroid function and vitamin
A utilization.
Much has been said and written about eutrophication, and
inasmuch as the problem species in many eutrophic lakes are
algae, the issue is one belonging to microbiology as well as other
sciences. In addition to algal growth being a problem of rele-
vancy to microbiologists, one recognized by few of my colleagues,
however, means for removing nitrogen from domestic wastes so
it does not enter bodies of water should be of interest to micro-
biologists. Unfortunately, few of them have been involved in
studies of denitrification and algal harvesting, both of which
appear to be particularly promising ways of removing nitrogen
from such wastes (3). Although these studies require micro-
biological techniques and knowledge, environmental engineers
investigating these methods of nutrient removal have had es-
sentially no assistance from professional biologists.
It would also seem worthwhile for a microbiologist to seek
an organism, or to develop one by suitable genetic techniques,
which is able to oxidize ammonium autotrophically to molecular
nitrogen. This process is exergonic and probably thermo-
dynamically feasible. Such an organism would bring about the
destruction of a widespread cation and nitrate precursor to
yield a volatile product of no concern to man. The organism
should grow by using the energy released in the oxidation.
After nitrification was first described in the 19th century and
shown to be important in natural environments, autotrophic
bacteria able to catalyze the process were obtained in axenic
culture. In the subsequent ninety years, essentially all environ-
mental researchers have assumed that nitrification is always an
autotrophic process and that nitrite, the only product known to
appear outside of the cell, is released only in trace quantities.
However, nitrite may occasionally accumulate in enormous
amounts (5), and it could then constitute a significant albeit
local problem. Investigations of models of poultry waste dis-
posals systems with high organic matter loading rates suggest
that nitrite accumulates here too. The accumulation probably
143
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results from an inhibition of Nitrobacter by ammonia at the high
pH resulting from the decomposition of nitrogen compounds in
the waste (!}.
Nevertheless, it is premature to assume that all products of
nitrification in natural environments are known. In a recent
study, for example, Verstraete and Alexander (unpublished
observations) demonstrated that hydroxylamine and 1-nitroso-
ethanol are formed in sewage treated with simple organic ma-
terials. Hydroxylamine is a potent mutagen, and its presence
in at least laboratory samples of treated sewage suggests the
need for careful testing under natural conditions. A process
identical to that observed in samples from nature has also been
noted in cultures of an Arthrobacter (6). Of particular interest
is the finding of the nitroso compound, the first report of such
a microbial metabolite in natural waters.
C-Nitroso compounds are not known to be health hazards,
but N-nitroso compounds, which are more commonly termed
nitrosamines, are carcinogenic, mutagenic, and teratogenic. The
nitrosation to yield such potent products can occur chemically,
but it usually requires the presence of nitrite and a suitable
secondary amine. On the other hand, nitrite is formed micro-
biologically from both nitrate and ammonium. Secondary amines
are present in foods, plant materials, and other natural products,
and they may be generated microbiologically in the decompo-
sition of a variety of tertiary amines. Thus, one can postulate
that microorganisms may contribute to nitrosamine formation
in four ways: (a) by synthesizing secondary amines; (b) by
forming nitrite; (c) in the nitrosation reaction itself; and (d) by
modifying the environment, such as by acidification, in order
that chemical nitrosation may take place.
Recent work in the author's laboratory has demonstrated
that microorganisms residing in sewage or lake water can pro-
duce secondary amines from tertiary amines and from one
fungicide. They are then able to nitrosate the secondary amine
with the formation of dimethylnitros amine, an extremely toxic
compound. Analogous processes have been shown to occur in
soil. These preliminary studies have been performed with con-
centrations of nitrate, nitrite, secondary and tertiary amines,
or fungicide higher than those which would be formed or added
144
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in nature. However, if one extrapolates from the concentrations
used in these studies to the low levels that are present in nature
and also assumes that the yield of the nitrosamine is linear
with precursor concentration, then the quantities may indeed
be of public health concern*
To verify that the processes observed in lake water, sewage,
and soil actually result from microbial activities rather than
being nonbiological reactions, axenic cultures of representative
organisms have been isolated and their activity in nitrosamine
formation demonstrated. Furthermore, enzymes obtained from
these microorganisms were found to catalyze nitrosation of
representative compounds (Ayanaba and Alexander, unpub-
lished observations).
Microorganisms are thus extremely important in the for-
mation of nitrate, nitrite, and possibly nitrosamines in natural
habitats. The activities of particular applied interest are the
mineralization of organic nitrogen, the formation of nitrate by
autotrophic bacteria, the possible heterotrophic oxidation of
nitrogen compounds to yield products containing oxidized
nitrogen, the reduction of nitrate to nitrogen gases, the for-
mation of nitrite by either oxidative or reductive pathways,
and the possible biogenesis of nitrosamines or their precursors
in water or soil. Despite the critical importance of these micro-
bial activities for man's health and for environmental quality,
the body of information remains sparse. Hopefully, microbiolo-
gists will soon be attracted to such environmental studies to
help bridge the information gap.
LITERATURE CITED
(1} Aleem, M. I. H., and M. Alexander. 1960. Nutrition and physiology
of Nitrobacter agilis. Appl. Microbiol. 8: 80-84.
(2) Biggar, J. W., and R. B. Corey. 1969. Agricultural drainage and
eutrophication, p. 404-445. In Eutrophication: Causes, consequences,
correctives. National Academy of Sciences, Washington, D. C.
(3) Eliassen, R., and G. Tchobanoglous. 1969. Removal of nitrogen and
phosphorus from waste water. Environ. Sci. Technol. 3: 536-541.
(4) National Academy of Sciences. 1972. Accumulation of nitrate.
National Academy of Sciences, Washington, D. C.
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(5) Stojanovic, B. J., and M. Alexander. 1958. Effect of inorganic nitrogen
on nitrification. Soil Sci. 86: 208-215.
(6) Verstraete, W., and M. Alexander. 1972. Heterotrophic nitrification
by Arthrobacter sp. J. Bacteriol. 110: 955-961.
(7) Zwerman, P. J., T. Greweling, S. D. Klausner, and D, J. Lathwell.
1972. Nitrogen and phosphorus content of water from tile drains at
two levels of management and fertilization. Soil Sci. Soc. Amer.
Proc. 36: 134-137.
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Questions and Comments Following
Dr. Alexander's Talk
QUESTION FROM THE FLOOR: It is my understanding that
applications of fertilizers on crops have leveled off the past few
years, and I was wondering if you'd like to predict what the
next crop would be that they're going to hit with nitrogen
fertilizers. Do you think it will be wheat?
DR. ALEXANDER: Do you mean where our agriculturalists
will be putting the nitrogen? I really wouldn't want to predict
this. My own personal concern in this area, and I'm counting
on some values which were calculated by members of our
committee, is in the developing areas where many crops are
going to get nitrogen. But I wouldn't try to guess what crops are
going to be getting more nitrogen.
QUESTION FROM THE FLOOR: Would you say that we're going
to level off the input?
DR. ALEXANDER: You're asking a microbiologist to give you
an answer. If I were to give you an answer, it would be a micro-
biologists answer, and I wouldn't trust that.
QUESTION FROM THE FLOOR: Is there hope of utilizing the
nitrogen build up in water by stripping the algae from waste
treatment ponds and feeding it back to the cattle?
DR. ALEXANDER: There is very little hope if the nitrogen
comes from diffuse sources, such as agricultural nitrogen where
is released into waterways over the country-side. This differs
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from an urban effluent, where one has a discrete point source
which can possibly be used. But the problem here, too, is that
you're not changing the nitrogen. You're recycling the nitrogen
and the question is where it goes; down into the groundwater
or up into the atmosphere. As far as I know, there are no data
indicating that such a recycling will lead to less nitrate pollution
of the groundwaters.
QUESTION FROM THE FLOOR: I'd like you to comment on a
recent finding that indicates, a good deal of nitrogen as nitrate
entering a lake was being denitrified. The nitrate in groundwater
was being denitrified before it reached the lake by traveling
through the sediments. I'd like to know what your comments
would be on that kind of mechanism.
DR. ALEXANDER: I would be surprised that there would be
significant denitrification in the groundwater because of the
absence of appreciable amounts of electron donors for denitrifi-
cation. I would not be surprised in the sediments themselves.
It really is a matter of having an available energy source for the
denitrifying bacteria.
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Microbial Influences on
Phosphorus Cycling
G. W. FUHS
Division of Laboratories and Research,
New York State Department of Health.,
Albany
ABSTRACT
Measurements of phosphorus uptake kinetics and rates of
phosphorus-limited growth show that the maximum uptake
rates for orthophosphate exceed the rates of growth-related
assimilation of phosphorus into biomass, even at maximum
rates of growth. Half-saturation concentrations for phosphorus
uptake, on the other hand, exceed those for growth, often by
one or two orders of magnitude. Known and apparent half-
saturation concentrations of growth for microorganisms are
below 5 jug of phosphorus per liter, and uptake of phosphorus
may exceed the need for this element whenever growth is re-
stricted in the presence of an energy source and certain required
ions (K+, Mg++).
In aquatic ecosystems the basic relationships are modified
by the presence of multiple substrates (organically bound phos-
phorus) and physical factors which act differently on different
species. While certain simple generalizations can be made for
the effects of phosphorus on the growth of attached plants and
microorganisms in flowing waters, an adequate description of
lake ecosystems requires the measurement in situ of important
rates (growth, sedimentation, decay, grazing) for both total
biomass and individual species. These measurements provide a
framework for exploratory and predictive modeling in two
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opposite directions: the dependence of the total system's be-
havior on the limiting nutrient input, and the interdependence
of species properties and system behavior.
Heterotrophic mixed bacterial systems, thriving on naturally
grown organic matter in water or on waste from other hetero-
trophic systems, tend to be controlled by the availability of
carbon and energy rather than of phosphorus. Conditions re-
quired for the known mechanisms of metabolic imbalance with
respect to phosphorus ("luxury uptake") rarelv if ever exist.
Indications of aerobic uptake and anaerobic release of phos-
phorus by bacteria are strong, but the underlying mechanism is
insufficiently understood for control and optimization of the
process on a technical scale.
INTRODUCTION
The role of phosphorus in biochemical energy conversion and
growth has received much attention during the past half-century
of research. Its role as a minimum factor for microbial growth
was recognized early in the fermentation and yeast-growing
industry, and yeast for a long time was the object of choice in
studies on its metabolic role. Concern for the effect of phos-
phorus on water quality stems from observations during the
past 25 years that quite often, particularly in fresh-water eco-
systems, phosphorus regulates the size of algal and possibly
also bacterial populations (21, 25, 34). Not until very recently,
however, have there been investigations into the kinetics of
phosphate utilization and the growth rate of microorganisms
during phosphate limitation.
In this paper I shall attempt to review the present state of
knowledge of the phosphorus nutrition of microorganisms in
relation to water-quality management, including the modeling
of aquatic ecosystems. Following Mortimer (24), I shall empha-
size our understanding of mechanisms and not restrict myself
to the communication of numbers. If many mathematical
models of entire ecosystems have little predictive value, this is
due, not to lack of numbers, but rather to lack of the right kind
of numbers and inadequate understanding of mechanisms. Un-
fortunately the traditional set of measurements of water quality
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is almost useless for successful ecosystems modeling. Accurate
determinations of the appropriate kind are just now becoming
possible as a result of new developments in methodology.
Kinetics of Phosphorus Utilization by Microorganisms
Phosphorus occurs in microorganisms in a number of chemical
compounds which can be grouped by function into three major
categories:
Structural components. These are cell constituents which are
indispensable for the viability and integrity of the cell. They
include both the hereditary material, deoxyribonucleic acid
(DNA), and phospholipids to the extent that they are essential
components of a minimum complement of peripheral and in-
ternal membrane systems. During each cell cycle these con-
stituents must be synthesized in the amount already present in
order to produce two viable cells from one. During slow growth
the synthesis of DNA, a major phosphorus-consuming reaction
in phosphorus-starved microorganisms, can be discontinuous,
possibly resulting in a cyclic demand for phosphorus. This may
affect to some extent the diurnal phosphorus cycle in lakes and
the competition among species.
Functional components. This category includes compounds
which determine the rate of growth, i.e., ribonucleic acid (RNA)
and low-molecular, phosphorylated metabolic intermediates,
including nucleotides. To the extent that additional intra-
cellular membrane systems and chloroplasts are developed dur-
ing rapid growth, their phosphorus would also be included in
this category.
Storage components. In microorganisms this category is repre-
sented by two fractions, which are often incorrectly identified.
The first consists of high-molecular, linear polyphosphates in
the potassium form, which aggregate as solid concretions of
high refractive index and extreme basophilia. These are known
as metachromatic or polyphosphate granules (7). In some bac-
teria and algae these granules appear during unrestricted growth
in a rich, balanced medium. In others, the granules do not
appear until cells are no longer growing but are still exposed to
abundant phosphorus, potassium, and a source of energy. As
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can be predicted from their consistency, the granules are built
up slowly (several to 24 hours are needed for growth into a
microscopically visible particle) and are degraded slowly. This
explains the slow turnover of phosphorus in this fraction. The
granules are soluble in cold 10 percent trichloroacetic acid,
and the fraction therefore is identical with the acid-soluble
polyphosphate of the biochemical literature (16, 35).
The other fraction is an acid-insoluble component, found in
bacteria and other microorganisms, which turns over very
rapidly and which resembles polyphosphates biochemically but
not cytochemically. Its exact chemical nature and its chemical
or structural association are not clear. This fraction has been
referred to as acid-insoluble polyphosphate (16, 35) or poly-
phosphate-ribonucleic-acid complex (2) and recently has been
characterized by Correll (1) as a polymer of phosphoric acid
which contains imido groups. The structure proposed by Correll
is interesting, since it combines high packing of PO3 residues
with a low basophilia (only one negative charge for three PO3
residues), which would explain why this fraction cannot be
stained with basic dyes and requires many fewer K+ ions for
synthesis than the granular form (KPO3)n- The K/P ratio
during synthesis of this fraction is closer to 1:4 (15). Other
authors also report a low K+ requirement during the first
stages of rapid uptake of phosphorus (38). In any case, the
acid-insoluble storage fraction tends to appear within minutes
after exposure of the cells to phosphorus, and it rapidly ex-
changes its phosphorus with that of the nucleic acids, so that
this fraction and the nucleic acids often show complementary
behavior during the growth cycle (29, 33). This fraction is
determined by treating trichloroacetic-acid-extracted cells with
hot 1 N HC1 for seven minutes and correcting for the nucleo-
tide-P which is released simultaneously ("7-min phosphate").
Microorganisms starved for phosphorus or any other element
are "programmed" to form as many cells as possible by cell
division. All phosphorus then becomes concentrated in the
structural components, while in the functional components it is
restricted to a bare minimum. In this state the highest cell
yield is obtained (expressed as the number of cells or the dry
weight per unit of phosphorus consumed). Growing cells contain
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phosphorus in the form of functional components as well as
7-min phosphate. A plot of growth rate (jj, in Equation 1) vs.
cell phosphorus has the form of a saturation curve which inter-
cepts the abscissa (// = 0) at the minimum phosphorus content
per cell (a0) (8). This latter parameter appears to be a species
constant. Half-maximum growth is obtained at two times the
minimum phosphorus content; 95 percent of maximum rate of
growth, at five to six times the minimum content. During
unrestricted growth, and even more during growth restriction
by nitrogen or certain other elements, phosphorus content per
cell (a) increases further but without a concomitant increase in
the rate of growth. Maximum rate of growth (jumax) is a function
of several factors, including light and temperature.
M/Mm«=l-2-<-««>'- (1)*
A complicating factor in the evaluation of Equation 1 is that
it holds only on a per-cell, not on a cell-mass basis. During
phosphorus starvation and in the presence of an energy source,
microorganisms accumulate carbonaceous reserve materials,
with a resultant increase in cell mass. Also, as is evident from
the preceding paragraphs, the popular concept of approximately
constant yield per unit of nutrient spent—a concept derived
from Monod's (23) experiments on heterotrophic bacteria limited
in carbon and energy source—does not apply to phosphorus-
limited microorganisms.
Because of shifts in cell composition during different phases
of phosphorus limitation and shifts of lesser extent occurring
with nitrogen limitation (4), it is not possible to predict the
effects of simultaneous limitation of the growth rate by both
elements from experiments where only one element is limiting.
Both phosphorus and nitrogen limitations have certain effects
in common, such as restriction of nucleic acid synthesis and
excessive (luxury) uptake of carbon.
Although cell phosphorus determines the growth rate directly,
external phosphorus determines the rate of replenishment of the
internal phosphorus pool and therefore is the ultimate regulator
of growth rate. Uptake of phosphorus follows Michaelis-Menten
* Growth rate is in doublings per day (base 2 logarithms). Time basis is one day.
153
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kinetics (Equation 3):
(2)*
(3)
Equations 1-3 can then be combined to give a fourth relation-
ship in a form which, for practical purposes, is indistinguishable
from either Monod's (23) hyperbolic model or Teissier's (32)
exponential model linking growth rate and substrate concen-
tration. The hyperbolic equation has the form:
(4)
Kr
Arriving at Equation 4 via Equations 1-3 appears circuitous;
but it is practical, since growth-limiting phosphorus concen-
trations are often too low to be measured directly in chemostat
experiments where the use of the isotope 32P is impractical (8).
The isotope, however, is easily handled in short-term uptake
experiments used in the evaluation of Equation .3.
The set of Equations 1-3 is not complete, however, because
it does not make provision for a limitation of uptake of phos-
phorus from the environment, i.e., control of V by cell phos-
phorus, or by an active fraction thereof. An effect of cell phos-
phorus on Fm alone was postulated by Jeanjean (14), but our
experiments suggest an effect on both Fm and Ks (9). More
recent experimentation in our laboratory suggests that Haldane's
(12) modification of the Michaelis-Menten formula can be used,
provided that the velocity of the back reaction (Fr) is assumed
to be zero. This assumption is in agreement with results by
other authors who describe phosphorus Uptake as a virtually
unidirectional process (11, 17, 31). The modified Equation 3
reads:
F=—
Assuming that Fr = 0 and substituting cell phosphorus in excess
of the starvation level (a— a0) for the intracellular product, Z,
* Equation 2 unfortunately was misprinted in an earlier article (9). Growth rate and
time basis are as in Equation 1.
154
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1234 tig/IP
FIGURE 1.—Growth rate of two diatoms and three bacterial species as
a function of P concentrations with orthophosphate as a source of P.
A = Cyclotella nana, B = Thalassiosirafluviatilis., C = Pseudomonas
aeruginosa, D = Corynebacterium bovis, E = Bacillus subtilis. From
(9).
we obtain:
V m '
-rr
K ~
where KT is a constant which expresses the degree of end-
product inhibition. In our experience, (a— a0) can also be re-
placed by "7-min phosphate," which suggests that this fraction
is involved in the regulation of phosphorus uptake.
Two principal results have been obtained from studies of
growth and uptake kinetics using orthophosphate as a source
for phosphorus: The half -saturation constant for phosphate -
limited growth (Km) for all algae and bacteria examined so far
is in the range below 5 jug P/l, sometimes below 1 /-ig P/l (9),
as shown in Figure 1. Literature data giving a much higher
range of limiting concentrations, particularly with Scenedesmus
(39 1 i), have not been confirmed in chemostat experiments in
our laboratory (Rheev personal communieatioB!)
155
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By contrast, the half-saturation constant for phosphorus up-
take can be much higher; for various bacteria and algae ex-
amined so far, it is in the range of 5 to 400 ^g PA Short-term
uptake experiments alone, therefore, are not sufficient to predict
the growth rate of an organism from a given phosphorus concen-
tration. This is illustrated in Figures 2 and 3, which are based
on earlier experimental data (9) for a planktonic diatom. It is
evident that variations in Vm and Ka in the order of magnitude
observed do not materially change Km, although, all other
factors being equal, the organism with a higher Vm or a lower
Ka would realize a certain competitive advantage.
In nitrogen-limited algae Ka and Km appear to be of similar
magnitude (4). One possible explanation is the smaller storage
1
FIGURE 2.—The effect of Fm (maximum uptake rate) on substrate-
dependent rate of growth. Solid curve based on experimental data
for Thalassiosira fluviatilis, a planktonic diatom (9). With ^m=1.6
doublings-day"1, a0= 12.5 X10"15 g-atoms P-cell"1, and Ks = 50 /*g P-
liter"1, Vm varied as indicated, in units of 10~12 g-atoms P-min"1-
cell"1. Wide variation of Vm causes Km to vary only between 0.1 and
0.4 /xg P-liter"1 (lower left of graph).
156
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'250
1 2 34 jig/P
FIGURE 3.—The effect of Ks (Michaelis constant for uptake) on P-de-
pendent rate of growth. Solid curve based on experiments with
Thalassiosira fluviatilis (9). Constants as above, except for con-
stancy of Vro. (6 X 10~12 g-atoms P • min"1 • cell"1) and variation of K3
as indicated, in units of jug P-liter"1. As a result of variaiton in Ks,
Km varies between 0.1 and 0.8 jug P« liter"1 (lower left of graph).
capacity of microorganisms for nitrogen as compared with that
for phosphorus.
Other Environmental Factors Affecting Phosphorus Uptake
Rapid uptake of phosphorus from very dilute solutions can
result in the rapid depletion of microlayers around the micro-
organisms. Their repletion with nutrient by diffusion or water
movement therefore can become an important regulating factor.
Canelli and Fuhs (ms. in preparation) found that phosphorus
uptake by diatoms was reduced when medium flow along the
cells was slow or absent. At medium flows of 1 mm/min, uptake
at any concentration between 1 and 100 jug/1 was reduced to
one-half of the maximum value (observed at velocities above
157
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10 to 20 mm/min). Reduction of the surface-to-volume ratio
and impediment of flow at the cell surface by gelatinous sheaths
and in colony-forming microorganisms may also slow the uptake
from very dilute solutions and extend the range of limiting
concentrations.
When orthophosphate supplies are near exhaustion—a con-
dition commonly found in freshwater lakes during summer—
organic phosphates become a major source of phosphorus for
microorganisms. These constitute a complex substrate for which
no single half-saturation constant can be given. Uptake may
vary from species to species and with the nature of the substrate
(27), and the involvement of phosphatases excreted by phyto-
plankters is another complicating factor.
Application of Kinetic Data: Qualitative Considerations
From the facts presented in the preceding section we can
safely conclude that in any standing body of water, phosphorus
limitation of the microbial growth rate exists when orthophos-
phate falls to a concentration of 3 ^ig of phosphorus per liter
(0.1 /xg-at/1) or lower, regardless of the presence of organic
phosphates. At higher concentrations^ the rate may still be
limited for some forms, depending on the replenishment of
phosphorus in microlayers close to the cell surface.
For attached algae in flowing waters, or for suspended algae
in turbulent streams, orthophosphate-phosphorus concentra-
tions of 5 Mg/1 and above indicate with some probability an
absence of phosphorus limitation. Concentrations below 5 /ig/1
are found in many lake effluents and in certain mountain
streams (e.g. in the Adirondack Mountains in New York). If
sewage effluents reach these waters, luxurious growths of at-
tached algae may occur as a result of the increase in phosphate
concentration. It is equally obvious that in such environments
a massive increase in growth rate can result from a minuscule
increase in phosphorus concentration—say, from 0.2 to 1.0
Mg/1—on the steep part of the growth curve (Equation 4). A
strong response may occur on periphyton slides immersed in
such waters, although the development of massive weedbeds
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which exhibit a marked phosphorus-stripping activity, or the
development of an algal hloom in a receiving lake, would require
a more plentiful supply.
Whenever massive weed growths occur helow sewage dis-
charges into streams with naturally high levels of phosphate, it
may be suspected that the principal cause for growth stimulation
is not phosphate but free carbon dioxide derived from sewage.
Application of Kinetic Data: Quantitative Considerations
The critical influence on algal growth of concentrations of
orthophosphate too small to be measured with a high degree
of accuracy, together with the effects of the other interfering
factors mentioned above, poses a serious obstacle to the con-
struction of conventional ecosystem models, because a sub-
stantial uncertainty is inevitable in any prediction of growth
rate of a planktonic microorganism, alga, or bacterium, if that
prediction is based on chemical measurement of substrate con-
centration and if the limiting substrate is phosphorus. Since a
management model requires accuracy within ±25 percent, a
two- or threefold uncertainty in the estimate of the most im-
portant parameter, algal growth rate, obviously cannot be
tolerated.
There is no viable substitute for the measurement of microbial
growth rates in situ. Such measurement offers two advantages:
a major difficulty in determining the principal process rates is
solved, and by relating growth rate to chemical concentrations
and kinetic constants, the interfering factors and lesser known
properties of the species can be explored systematically. The
results of these studies provide valuable feedback for the under-
standing of species dominance and are a necessary step toward
more specific predictions of the response of the ecosystem to
variations in phosphorus loading.
Going a step further, we can make this approach part of a
larger plan for the characterization of a lake ecosystem. This
master plan is being used with success in our studies of New
York State lakes, which are designed both to improve water
quality management and to generate data needed for basic
research.
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Our basic plan is illustrated in Figure 4, which shows a
rigorously simplified model of a phosphorus-limited lake eco-
system. Criteria of phosphorus limitation have been described
elsewhere (9). Only two pools are distinguished, both expressed
as phosphorus. All other elements are rate terms: I (input),
0 (output via effluent, or overflow), dB/dt (change in biomass),
and S (sedimentation). The term (dB/dt) +S can also be ap-
proximately written /* —(G+X), that is, primary productivity
less grazing by zooplankton and other effects, as explained
below. In the equation
I+fl=M-(G + X)+0 (5)
then, the nutrient regeneration rate (jR) is the only unknown,
and it can be calculated for each time interval studied. The
comparison of / and R as forces driving /JL reveals an important
characteristic of the lake. The role of R with respect to /
increases with the degree of eutrophy and with decreasing mean
depth and is affected by wind mixing. Although sedimentation
measurements can also provide us with data on M (minerali-
zation), this parameter does not enter our simplified model.
G (grazing) is not measured at present; consequently the
term G + X stands for all interconversion of biomass and organic
soluble phosphorus by grazing, excretion of organic phosphates
by algae (18), and lysis. This complex remains to be resolved
for a better characterization of population growth, and one
might start by measuring its strongest component, which is
probably grazing. Grazing in this context means the excretion
of soluble phosphorus by all zooplankton and the solubilization
of zooplankton fecal pellets by bacteria.
The growth of bacteria on detritus, as well as the feeding of
protozoa and rotifers on bacteria, constitutes cycling of phos-
phorus within the particulate fraction and is not measured.
One may go so far as to express some doubt whether the sepa-
ration of bacterial processes in the epilininetic cycle is essential
except in rare circumstances. Soluble organic phosphorus com-
pounds excreted by zooplankton may be taken up by algae as
well as by bacteria or may be hydrolyzed by enzymes produced
by either group of organisms. The effect of bacteria here is less
readily identified than their role in the cycling of carbon and
160
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-inorg-
(M)
FIGURE 4.—Simplified conceptual model of P cycle in lakes. 7, input;
P, substrate (dissolved P); 5, biomass; p, rate of incorporation of P
into B; 0, output (overflow); S, sedimentation; M, mineralization;
R, recycling of P. Subscripts indicate that rate and concentration
terms can be determined for individual species as well as for total
mass and rates of transformation.
nitrogen in the epilimnion and their role in the mineralization
of organic matter at the sediment surface.
Subscripted variables in Fig. 4 are those that are determined
not only for total standing crop but also for all major species
of phyto- and zooplankton, using a conversion of basis from
total phosphorus to volume. From dB/dt and S for each species,
the "net" growth rate (/Ji—G — X) is determined. This approach
was used with biweekly surveys in our studies of Lake Cana-
darago (Otsego County, New York). A few major conclusions
deserve mention:
1. The maximum net growth rate of any alga observed so far
was 0.6 doublings per day. A rate of 0.2 doublings was about
average for many forms during active growth.
2. The development of blue-green algal populations can be
accompanied by increases in the pool of soluble organic phos-
phorus, possibly by excretion or light-induced lysis. Algal pro-
duction at the expense of dissolved organic phosphorus demon-
strates indirectly the activity of phosphatase-producing species.
If an algal population contributes to the soluble pool when
disappearing from the water column, this is indirect evidence
of grazing by zooplankton or lysis.
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3. Our contention that grazing and sedimentation are by far
the most important removal mechanisms for plankton algae is
based on the relatively low mineralization rates in our sediment
sampling devices (1 percent or less of cell phosphorus released
into supernatant during a biweekly sampling period). This rate
is in line with observations by Force et al. (5).
4. During the growing season, total biomass production in
terms of phosphorus is about 10 times the total phosphorus
input, less overflow. This shows that incoming phosphorus is
utilized 10 times before being precipitated irreversibly or flushed
out, a characteristic of eutrophic lakes of this type. Our measure-
ments in this lake also show the importance of what E. A.
Thomas (34) has called the phosphorus "macrocycle" (involving
the hypolimnion and the sediments), as compared with the
"microcycle" (cycling within the epilimnion).
The important point is that the descriptive scheme of Fig. 4
does not yet represent a mathematical model. It lacks predictive
value because it operates with a set of factors whose values
vary continually (as determined at biweekly intervals, in our
study). The factors are properties of the planktonic species and
of rapidly changing meteorological conditions. As an example,
growth rate (ju) not only depends on nutrient concentrations,
solar radiation and temperature but also varies from species to
species. Sedimentation (S) is affected by buoyancy, which in
turn depends on the nutritional state and other properties of
the species. Grazing (G) depends on the size and other properties
of the several species, those grazing and those serving as food.
Nutrient recirculation (R) varies with sediment exposure and
wind action, which may cause upwelling of bottom waters or
partial overturn. .
By relating these factors to available information on species
and weather, in addition to nutrient input, concentration data,
kinetic constants, and other laboratory data on nutrient re-
quirements, we can effectively explore the lesser known proper-
ties of the different species or groups of physiologically similar
species, guided by observations rather than assumptions. In
this manner we can come to understand the causes of species
dominance and develop models as predictive tools. In the mean-
time, the simplified descriptive model can be used for qualitative
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estimates and semiquantitative predictions, assuming no change
in species succession and dominance.
This approach to the modeling of a lake ecosystem has the
advantage that it relies on quantities and rate terms which
can be determined with some degree of accuracy in phosphate-
limited, confined systems. By contrast, in nitrogen-limited ma-
rine systems the current approach is one of reliance on nutrient
concentrations and kinetic data for the estimation of growth
rates (4). This appears justified because kinetic data on nitrogen
uptake by microorganisms can be applied more easily to practi-
cal situations than kinetic data on phosphorus utilization and
because sedimentation rates are much more difficult to measure
in vast, open and complex oceanic systems.
Phosphorus and Microorganisms in Sewage Treatment
All organisms, when given ample nutrients and a supply of
energy, are programmed to synthesize preferentially the es-
sential components of their cell such as nucleic acids, protein,
and membrane and cell wall material. Intracellular carbohy-
drates are formed for temporary storage of energy only. This
principle is a useful one, since it permits the production of a
maximum number of progeny containing a minimum comple-
ment of essential constituents.
If a heterotrophic organism utilizes such cell material, part
of it is consumed for energy, which is derived from the degra-
dation and oxidation of the carbonaceous skeletons of the various
cell constituents. Unless the material happens to be almost
pure carbohydrate, nitrogen and phosphorus are released from
the part of the substrate utilized for energy and are excreted
as waste materials.. An excess of nitrogen and phosphorus
relative to energy content is therefore found in most organic
food for heterotrophic organisms—and even more so in waste
from heterotrophic organisms (humans, animals), relative to
the need of other heterotrophic organisms (bacteria) which feed
on it. This is the situation in biological sewage treatment,
where bacteria are degrading carbonaceous material in the
presence of an excess of both nitrogen and phosphorus.
The ability of microorganisms to take up phosphorus in
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excess of immediate need has received much attention and is
summarily referred to as "luxury uptake." In reality this term
subsumes three or possibly four very distinct mechanisms, which
are activated under very specific sets of environmental con-
ditions. The first, which was mentioned in the beginning of this
paper, is uptake to sustain the growth rate. During unrestricted
growth, a microorganism contains five or six times as much
phosphorus as during phosphorus starvation. This additional
amount is indispensable for rapid growth. Although it exists
partly in the form of storage materials (e.g. 7-min phosphate),
reduction of this amount would reduce the rate of growth. If
phosphorus supply were interrupted, the organism would grow
and divide at a steadily decreasing rate until all progeny con-
tained only the minimum amount of phosphorus required for
survival.
Luxury uptake in the stricter sense of the microbiological
literature is dead storage of phosphorus, typically in the form
of solid polyphosphate granules. This phenomenon normally
occurs when bacterial growth is limited by lack of some nutrient
which is not required for phosphorus uptake and storage.
Phosphorus uptake requires, besides phosphorus, a source of
energy, magnesium as an enzyme activator, and potassium as
a neutralizing cation. Growth limitation by lack of any of these
factors will not normally induce luxury uptake of phosphorus.
Nitrogen limitation, however, is a good condition for phosphorus
storage, and so are some less likely conditions, such as sulfur
starvation.
The third condition, known as the "phosphate overplus"
phenomenon, was first demonstrated in yeast and was subse-
quently observed in algae and certain bacteria (13, 19). It
occurs when phosphorus-starved organisms are exposed to
phosphor us-rich media or solutions. In this case, phosphorus
accumulation proceeds at a far higher rate than the synthesis
of other cell constituents, but it is still dependent on the avail-
ability of an external or internal energy source.
If we compare these; conditions for luxury uptake in its
different forms with the conditions prevailing in biblogical sew-
age treatment, it is clear that luxury uptake in the strict sense
is unlikely. In domestic and most other wastes, bacterial growth
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is limited by the supply of carbon and energy; the exceptions
are certain carbohydrate-rich industrial wastewaters. Occurrence
of the phosphate-overplus phenomenon is equally unlikely, as
it requires a phase of phosphorus starvation. Such starvation
does not occur at any time during the biological treatment of
domestic sewage.
A new biological mechanism has been described by Levin and
Shapiro (19) and Shapiro (30). It occurs when activated sludge
cycles between aerobic and anaerobic phases, and its kinetics
resembles those of the phosphate-overplus phenomenon and the
storage of granular (acid-soluble) polyphosphates (with maxi-
mum phosphorus content reached in four hours). Phosphorus
accumulation is faster than during "metabolic upshift" (in-
crease in growth rate) and much slower than chemical precipi-
tation. Selection of suitable species may be involved, since the
bacterial mass must apparently be exposed to the aerobiosis-
anaerobiosis cycle over an extended period of time before the
process is operational. More experimentation is also needed
with regard to the exact nature of the storage material and its
localization, since according to Shapiro phosphorus is stored in
an acid-soluble form (30), whereas Yall et al. (36) find increases
mainly in the acid-insoluble fraction. At present, this mechanism
poses more new questions than have been answered so far,
partly because of the general scarcity of data on the release of
phosphorus from viable microbial cells.
Not to be confused with these mechanisms is the chemical
precipitation of sparingly soluble phosphates on microbial cells
and colonies, a process that occasionally has been confused with
uptake and release of phosphorus by the organisms. A case in
point is the alleged diurnal cycle of uptake and release of
phosphorus by algae in phosphorus-rich natural waters or
laboratory media (26). Reinvestigation of the phenomenon has
shown that together with phosphorus, a stoichiometric amount
of calcium was bound; and autoradiography with 45Ca showed
that calcium phosphate had been precipitated on the cell surface
during daylight hours, when the pH was high due to photo-
synthetic removal of CO2 from the medium. At night, release
of respiratory CO2 lowered the pH to such an extent that the
precipitate dissolved (6). The same phenomenon is easily demon-
165
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strated with activated sludge, particularly in hard water. During
periods of aeration, respiratory CO2 is expelled and the pH
rises; in the settling tank, the pH is lowered by CO2 accumu-
lation (22).
CONCLUSIONS
Bacteria and algae, sharing the basic properties of all organ-
isms and a plantlike mechanism of nutrition (uptake of soluble
or solubilized phosphorus), show similar absolute requirements
for phosphorus and similar uptake and growth kinetics, exempli-
fied in the existence of very effective uptake of orthophosphate
from very dilute solutions.
Interspecies variation which already exists with regard to
orthophosphate utilization is so greatly intensified in the pres-
ence of mixed organic phosphates and under the modifying
action of physical factors that serious errors are introduced in
the prediction of growth rates from simple chemical measure-
ments. Predictions of standing crop are even more dangerous,
as they depend on additional variables which are affected by
the characteristic properties of species. Only the recognition of
species differences and the introduction of more meaningful
measuring techniques, particularly the direct measurement of
certain transfer rates in situ, can prevent ecosystems models
from becoming detached from reality and largely worthless as
predictive tools.
The investigation of the role of phosphorus in heterotrophic
systems reveals the need for research into the operation of
known biochemical mechanisms under environmental conditions
and for the development of a basic explanation of phenomena
which cannot be predicted from existing knowledge of cell
physiology. Capabilities for control and optimization of en-
vironmental processes cannot be expected to develop without
such research.
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Science 151: 819-821.
166
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(2) Ebel, J.-P., G. Dirheimer, A. J. C. Stahl, and M. Yacoub. 1963.
Etude des complexes entre acides ribonucleiques et polyphosphates
inorganiques dans la levure. I. Bull. Soc. chim. biol. 45: 865-874.
(3) Eichhorn, M. 1969. Zur Stoffproduktion kontinuierlicher Kulturen
von Scenedesmus obliquus (Turp.) Kiitzing im Dauerlicht bei Phos-
phat- und Nitratlimitation. Flora 159: 494-506.
(4) Eppley, R. W., J. N. Rogers, and J. J. McCarthy. 1969. Halfr,
saturation constants for uptake of nitrate and ammonium by marine
phytoplankton. Limnol. Oceanogr. 14: 912-920.
(5) Force, E. G., J. W- Jewell, and P. L. McCarty. 1970. The extent of
nitrogen and phosphorus regeneration from decomposing algae.
Adv. Wat. Pollut. Res., Proc. 5th Int. Conf. San Francisco (S. H.
Jenkins, ed.), Pergamon Press, Oxford, 2: III-27/1-15.
(6) Frank, E. 1962. Vergleichende Untersuchungen zum Calcium-,
Kalium- und Phosphorhaushalt von Griinalgen. I. Calcium, Phos-
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(7) Fuhs, G. W. 1969. Interferenzmikroskopische Beobachtungen an
den Polyphosphatkorpern und Gasvakuolen von Cyanophyceen.
Osterr. Bot. Z. 116: 411-422.
(8) Fuhs, G. W. 1969. Phosphorus content and rate of growth in the
diatoms Cyclotella nana and Thalassiosira fluviatilis, J. Phycol. 5:
312-321.
(9) Fuhs, G. W., S. D. Demmerle, E. Canelli, and M. Chen. 1972.
Characterization of phosphorus-limited plankton algae. In: Nutrients
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Spec. Symp. I; 113-132.
(10) Golterman, H. L., C. C, Bakels, and J. Jakobs-Mogelin. 1969.
Availability of mud phosphates for the growth of algae. Verb. Int.
Verein. Limnol. 16: 467-479.
(11) Goodman, J., and A. Rothstein. 1957. The active transport of
phosphate into the yeast cell. J, gen. Physiol. 40: 915-923.
(12) Haldane, I. B. S. 1930. Enzymes, Longmans, Green and Co., London.
(13) Harold, F. M. 1963. Accumulation of inorganic polyphosphate in
Aerobacter aerogenes. J. Bacteriol. 86: 216-221.
(14) Jeanjean, R. 1969. Influence de la carence en phosphore sur les
vitesses d'absorption du phosphate par les Chlorelles. Bull. Soc. fr.
Physiol. veg. 15: 159-171.
(15) Jungnickel, F. 1966. Vergleich des Einflusses der Veratmung en-
dogener und exogener Substrate auf Polyphosphatbildung und
Kaliumaufnahme bei phosphatverarmten Zellen von Candida utilis.
Arch, f. Mikrobiol. 55: 175-186.
(16) Juni, E., M. D. Kamen, J. M. Reiner, and S. Spiegelman. 1948.
Turnover and distribution of phosphate compounds in yeast me-
tabolism. Arch. Biochem. Biophys. 18: 287-408.
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(17) Kempner, E. S., and J. H. Miller. 1965. The molecular biology of
Euglena gracilis. II. Utilization of labeled carbon, sulfur and phos-
phorus. Biochim. biophys. Acta 104: 18-24.
(18) Kuenzler, E. J. 1970. Dissolved organic phosphorus excretion by
marine phytoplankton. J. Phycol. 6: 7-13.
(19) Levin, G. V., and J. Shapiro. 1965. Metabolic uptake of phosphorus
by wastewater organisms. J. Wat. Pollut. Contr. Fed. 37: 800-821.
(20) Liss, E. and P. Langen. 1962. Versuche zur Polyphosphat-Uber-
kompensation in Hefezellen nach Phosphatverarmung. Arch. Mikro-
biol. 41: 383-392.
(21) Lund, J. W. G. 1950. Studies on Asterionella formosa Hass. II.
Nutrient depletion and the spring maximum. J. Ecol. 38: 1-35.
(22) Menar, A. B., and D. Jenkins. 1968. The fate of phosphorus in
sewage treatment processes. II. Mechanism of enhanced phosphorus
removal by activated sludge. SERL Report 68-6. Univ. Calif.
Berkeley.
(23) Monod, J. 1942. Recherches sur la croissance des cultures bac-
teriennes. Hermann, Paris, 211 p.
(24) Mortimer, C. H. 1972. Opening remarks. In: Nutrients and Eutrophi-
cation (G. Likens, ed.) Amer Soc. Limnol. Oceanogr. Spec. Symp.
1: vii—viii.
(25) Ohle, W- 1953. Phosphor als Initialfaktor der Gewassereutro-
phierung. Vom Wasser 20: 11-23.
(26) Overbeck, J. 1962. Untersuchungen zum Phosphathaushalt von
Griinalgen. I. Phosphathaushalt und Fortpflanzungsrhythmus von
Scenedesmus quadricauda (Turp.) Breb. am natiirlichen Standort.
Arch. Hydrobiol. 58: 162-209.
(27) Reichardt, W., and J. Overbeck. 1969. Zur enzymatischen Regu-
lation der Phosphomonoesterhydrolyse durch Cyanophyceenplank-
ton. Ber. deutsch. hot. Ges. 81: 391-396.
(28) Rosenberg, H., N. Medveczky, and J. M. La Nauze. 1969. Phos-
phate transport in Bacillus cereus. Biochim. biophys. Acta 193:
159-167.
(29) Schlegel, H. G., and H. Kaltwasser. 1961. Veranderungen des Poly-
phosphatgehaltes wahrend des Wachstums von Knallgasbakterien
unter Phosphatmangel. Flora 150: 259-273.
(30) Shapiro, J. 1967. Induced rapid release and uptake of phosphate by
microorganisms. Science 155: 1269-1271.
(31) Tanzer, J. M., M. I. Krichevski, and P. H. Keyes. 1969. The
coupling of phosphate accumulation to acid production by a non-
growing Streptococcus. J. gen, Microbiol. 55: 351-360.
(32) Teissier, G. 1942. Croissance des populations bacteriennes et quantite
d'aliment disponible. Rev. sci. 80: 209-214.
(33) Terry, K. R., and A. B. Hooper. 1970. Polyphosphate and ortho-
phosphate content of Nitrosomonas europaea as i function of growth.
J. Bacteriol. 103: 199-206.
168
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(34) Thomas, E. A. 1953. Zur Bekampfung der See-Eutrophierung:
Empirische und experimentalle Untersuchungen zur Kenntnis der
Minimumstoffe in 46 Seen der Schweiz und angrenzender Gebiete.
Monatsbull. schweiz. Ver. Gas- u. Wasserfachm. no. 2-3, 15 p.
(35) Wiame, J. M. 1949. The occurrence and physiological behavior of
two metaphosphate fractions in yeast. J. Biol. Chem. 178: 919-929.
(36) Yall, I., W. H. Boughton, R. C. Knudsen, and N. A. Sinclair. 1970.
Biological uptake of phosphorus by activated sludge. Appl. Micro-
biol. 20: 145-150.
16*
-------
Questions and Comments following
Dr. Fuhs's Talk
QUESTION FROM THE FLOOR: I wonder if you could comment
on aquatic macrophytes as a sink for phosphate wastes.
DR. FUHS: I have been looking for data to show that the
source of phosphorus for aquatic weeds is entirely in the sur-
rounding water. Certainly you see things growing on old mud
banks of streams where sewage treatment has newly been insti-
tuted. But I have no data on the kinetic uptake of phosphorus
from the aqueous phase.
STATEMENT FROM THE FLOOR: There have been radioactive
uptake studies which have shown that aquatic plants do
take up phosphorus.
170
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Microbial Recycling of
Naturally Occurring
Refractory Material
DR. R. R. COLWELL
Department of Microbiology
University of Maryland
College Park, Maryland 20742
ABSTRACT
Sampling devices specifically designed for microbiological
work in the field are needed. The methods presently available
for enumeration and characterization of in situ microbial ac-
tivities are also limited in scope and application.
Detailed information on the cycling of matter in nature is
lacking and would be more easily obtained, if knowledge of
indigenous microbial communities, i.e., the natural flora, were
available. The carbon cycle is, at the present time, described in
the literature in simplistic terms, with much of the present
understanding of the breakdown of carbon polymers derived
from studies of pure substrates, such as, cellulose, chitin, lignin
and pectin. Recycling of carbon compounds in nature can be
shown to involve interrelationships with the annual cycles of
organisms, as exemplified by the association of chitin-digesting
vibrios with zooplankton. These relationships, as well as the
biochemistry of breakdown and assimilation of carbon com-
pounds, should be incorporated into carbon cycle models.
The assigned task of this presentation was to discuss the role
of microorganisms in the carbon cycle of the aquatic habitat,
insofar as the available knowledge of the biochemistry and
ecology of carbon compounds permits. This is at once extra-
171
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ordinarily difficult and yet not burdensome, i.e., difficult because
of the enormous complexities involved in the mobilization of
carbon by microorganisms in the aquatic habitat, yet deceptively
simple because of the paucity of data. A simplistic over-view
thus can be presented without undue embarrassment or
apologies.
Before moving into the main theme of the discussion, some
comment on the problems of obtaining samples for aquatic
microbial ecology is appropriate. Aquatic microbiology, herein
defined as encompassing fresh water, estuarine and marine
microbiology, inevitably involves field work at some point in
any serious investigation. The methods used in the field, in the
main, are primitive, despite the fact that we are in the computer
age. A fair statement of the art would be, I believe, to say that
no new method has been developed by aquatic microbiologists
specifically for use in aquatic microbiology field studies since
the turn of the century. A variety of modifications of the sterile
glass bottle sampler and the adaptation of some potentially
sophisticated practices, such as radiorespirometry for in situ
work, have been done, but expenditure of intellectual creativity
in the design and fabrication of equipment to overcome the
multitude of problems besetting any microbiologist working in
the field has not been made to any significant degree. The only
possible exception to this sweeping statement may be the ATP
assay system devised by Holm-Hansen and his colleagues (1966).
The methods for studying aquatic microorganisms are not
unique and are, in general, taken directly from soil, food and
medical microbiology. In our own work we have employed data
processing by computer to identify bacteria isolated by use of
selective, elective and enrichment culture, grouping bacteria by
enzymatic capabilities, nutritional requirements and level of
complexity of nutritional requirements, with relative incidence
of these groups used to characterize water masses, as was sug-
gested originally for comparison of rhizosphere versus non-
rhizosphere soils (Lochhead and Burton, 1957).
The usual method of quantitating the microflora in a sample
of water or sediment is by dilution plate, membrane filter and
most probable number count methods. These are all indirect
means of enumerating microorganisms in such samples and
172
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possess inherent sources of error due to limitations imposed by
the nutrient medium, temperature, pH, pressure, salinity, etc.
employed. Direct counts, via direct examination of sample
aliquots suspended on slides, the use of fluorescent antibody
microscopy, submerged slide methodology or the more recently
developed infrared microscopy method for visualization of un-
stained microorganisms in natural habitats (Casida, 1968) pro-
vide somewhat greater detail concerning distribution of par-
ticular organisms in water and sediment and in the Aufwuchs
community (Seligo, 1905; Cooke, 1956; Potter, 1964), i.e., on
the surfaces of animals, plants and submerged inanimate objects.
Transmission electron microscopy has also been used by Bae,
Cota-Robles and Casida (1972) to separate and concentrate
indigenous microorganisms from soil and ocean sediment with-
out the occurrence of growth. "Dwarf" cells, less than 0.3 /im
in diameter were thus discovered in untreated soil and sediment
samples. As high as 72 percent of the cells observed in a natural
sample were dwarf cells. Scanning electron microscopy has been
used by Sieburth (1972) to view the alignment of bacteria,
yeasts and fungi on the surfaces of seaweeds, wood and ^and
grains. Fahraeus (1947) observed that the cellulases of certain
Cytophaga spp. were not extracellular and cellulose digestion
required physical contact between the cells and the substrate,
whereas in several fungi the cellulases are freely diffusible exo-
enzymes. Scanning electron microscopy thus may assist in
elucidation of cellulytic activity by Cytophaga spp.
In summary, a major problem faced by the aquatic micro-
biologist is that he must attempt to ferret out details of the
carbon, or any other cycle, while lacking an appropriate sam-
pling technology. Only two, perhaps three, samplers that can
be considered to be reasonably reliable are available for taking
water samples at desired depths. These include the J-Z sampler
(ZoBell, 1946), employing sterile rubber bulbs and restricted to
neritic ocean or coastal region sample-taking, the Niskin sampler
(Niskin, 1962), which consists of a sterile plastic bag, messenger-
triggered for opening at coastal or deep-ocean depths, and the
sterile glass bottle for streams and estuaries (ZoBell, 1946).
Sediment samplers designed strictly for microbiological work
are not yet available. Coring devices with autoclavable plastic
173
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liners are the best compromise of corers available on the market
(Colwell and Zambruski, 1972). However, these must be sent
down open so that contamination with surface water cannot be
avoided. A variety of samplers have been constructed for
chemical and physical oceanography applications. Some cannot
be adapted; for example, the Nansen bottle, and others have
been used because nothing else was available, but the oligo-
dynamic effect of the metal sampler and its non-sterile state
must be contended with. The gravity corer, bucket sampler for
sandy sediments, dredge, oyster dredge and plankton net are
all apparatus not suitable for microbiological work in the
strictest sense. Thus, instrumentation is an immediate need,
and it is in this area that research and development would
provide substantial benefit, since the necessary technology is
sufficiently advanced to meet the requirements of instrument
development. Unquestionably, without the proper tools, aquatic
microbiology is seriously impeded.
Naturally occurring refractory compounds are generally con-
sidered to include the less readily degraded components of plant
and animal residues, such as, cellulose, chitin, pectin, lignin and
humus. In most textbooks, the carbon cycle is very simplistically
presented showing carbon moving from the atmosphere, as
carbon dioxide, to organic carbon via green plant photosynthesis,
also via algae and some bacterial species. The plant and animal
tissues deposited in soil and streams thus become part of the
organic complex of the sediments. Organic carbon degradation
is viewed chiefly as microbial attack on cellulose, lignin, pectin
and humus materials to complete oxidation, producing carbon
dioxide and water, thus completing the cycle. See Figure 1.
Higher plant tissues are essentially boxes or tubes made up
of cellulose, pectin and minor polysaccharide components, such
as, galactans, mannans, xylans and arabans. These cellular
building blocks are cemented together by an interstitial mortar
of pectic substances, which in ageing tissues may contain an
increasing proportion of lignin (Codner, 1971). The pectin,
cellulose and other components of the plant cell are complex
and variable, as are the microbial enzymes degrading these
systems. Pectic substances are a group of colloidal polymeric
materials formed mainly from anhydrogalacturonic acids in
174
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Particulate matter and.
i
organic residues
HUMUS (water column
and sediment)
animals
and zooplankton
Plants, algae and
microorganisms
FIGURE 1.—The Carbon Cycle in the Aquatic Environment.
linear al-4 glycosidic linkage. Compounds referred to as pectic
acids or low methoxyl polygalacturonic acids are compounds
which contain unesterified carboxyl groups of the polymer or
only esterified to a very limited extent by methoxyl groups.
Where greater than 5 percent of the carboxyl groups is esterified
with methoxyl, the term pectinic acids is applied. A wide range
of microorganisms, both terrestrial and aquatic, produce pectin-
methyl esterase capable of hydrolyzing the ester groups with
the production of methyl alcohol. A variety of microorganisms
can also effect hydrolytic cleavage of pectic acid.
Cellulose, one of the world's most abundant polysaccharides,
is composed of repeating 1-4/3-linked glucose units. Microbial
breakdown of cellulose is brought about by hydrolytic glyco-
sidases specific for the /31-4 D linkage, i.e., cellobiase activity.
Most of the cellulose decomposition in nature is catalyzed by
fungal and bacterial enzymes derived from gram negative
saprophytes, anaerobic rumen bacteria of several genera and
anaerobic thermophilic sporeformers.
Cellulolytic, pectinolytic and ligninolytic microorganisms, in-
cluding many species of bacteria and fungi, are common in
soil and aquatic sediments and have been isolated from the
175
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aquatic habitat without great difficulty. Generally speaking, the
breakdown product of cellulose is glucose and other hexoses,
which is readily metabolized by a great variety of microorga-
nisms. Again speaking generally, the aerobic bacteria produce
CO2 and cell material, whereas anaerobes produce gases, fatty
acids and alcohols and other neutral products, i.e., an "incom-
plete" utilization. Plant and animal residues in soil and sediment
and comprising a portion of the particulate matter which in
water, when in the last stages of decomposition, are referred
to as humus: dark-colored, amorphous material containing com-
pounds not readily decomposed by microorganisms. Humus,
then, is used to denote the organic fraction of soil that is gener-
ally resistant to decomposition, distinguishing it from the or-
ganic matter of leaf litter, for example. Initial plant litter con-
tains some water-soluble materials, such as sugars and ammo
acids, which are leached out and readily decomposed. The
humus in soil contributes to the bulky nature of soil, acting as a
buffer and water-holding agent. Krey (1967) reported on studies
of detritus in water samples of the North Atlantic and suggested
that detritus acts as an adsorber of dissolved organic matter
and plays a role in the ocean similar to that of humus in arable
soils.
Interest in organic particles in seawater was focussed by
Nishizawa, Fukuda and Inoue (1954) who published pictures
of "sea snow." Organic matter in the aquatic environment was
the subject of a symposium, where much of the work done on
the subject has been summarized (Hood, 1970). For the pur-
poses of this discussion, the nomenclature proposed by Riley
(1963) is used, i.e., the term organic aggregates refers to the
flat, plate-like forms or flocculent, heterogeneous amorphous
aggregations and detritus to the recognizable particles, which
include zooplankton carapaces, diatom tests, faecal pellets and
plant fibers. The assumption has been made by many investi-
gators that large numbers of bacteria are attached to these
particles, both organic aggregates and detritus. Documentation
by direct observation has been made by only a few investigators
(viz., Jannasch, 1958). Wiebe and Pomeroy (1972) undertook
the examination of the phenomenon of microbial attachment to
particles in seawater and concluded that in the open ocean,
176
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regardless of the location, two types of aggregates were domi-
nant: flat, plate-like flakes and flocculent forms. The chemical
composition of both types was undertaken mainly by staining
procedures. They observed that "mucus-like" material was
more associated with floes than with flakes. Carbohydrate was
a more substantial part of the aggregates and aggregates were
more chemically heterogeneous. Seldom were bacteria or other
recognizable microorganisms found associated with flakes. Wiebe
and Pomeroy (1972) concluded that relatively few bacteria are
directly associated with particulate matter, although bacteria
became extremely abundant and overgrew large floes during
incubation. This finding was true of estuarine as well as open
ocean waters. Thus, it may well be that the flakes comprise the
humus of the aquatic environment, either settling to concentrate
in the sediments or remaining suspended in the water column.
Size distribution of particles in sea water has been studied by
Sheldon et al. (1972).
Thus, organic matter is found abundantly in water and bot-
tom sediments, in solution in the water or as fractions of
planktonic and organic debris, i.e., the seston, and suspended
in the water. Organic material is clearly abundant in sediments.
Organic matter in the aquatic environment derives from two
main sources: (1) incoming waters and wave action at the shore,
which introduces allochthonous matter into lakes, streams and
estuaries. This accounts for part of the total organic matter,
with the nature of the substances brought in being varied,
ranging from leaf litter of shorelines to upland soil leachings
and organic pollution; (2) autochthonous matter produced by
living organisms and by decomposition of plant and animal
bodies (Reid, 1961). The relative contribution of these sources
is difficult to measure, since appropriate techniques are not
perfected.
The nature of the soil that forms in given areas, hence the
allochthonous material contributed to the aquatic regions, is
greatly dependent on climatic conditions affecting microbial
action, as well as the kinds of plants that develop.
Organically poor lakes contain 1-2 mg C/l, with the total
organic content, ca 73 percent carbohydrate and 24 percent
protein. Richer lakes contain up to 26 mg C/l, with about 90
177
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percent of the organic matter carbohydrate and 10 percent
protein. The total organic content of most "average" lakes is as
much as three or four times that of the open seas. Seawater
generally contains about 2 mg C/l and about 0.2 mg organic
N/liter. Most estuaries receive considerable quantities of al-
lochthonous materials from stream inflow and drainage from
surrounding marshes or other land features.
Cycling of refractory materials in the aquatic habit can be
considered to take place chiefly at the water-sediment interface.
Aquatic ecosystem surface sediments act as a boundary between
the circulating dynamic medium primarily dominated by the
properties of water and its solutes and the structurally more
stable medium, the sediment, with properties much like soil.
This boundary area is the site of intense microbial activity in
most natural waters. A variety of bacteria can be isolated from
surface sediments in high numbers relative to the overlying
water column. Harrison, Wright and Morita (1971) examined
A M
MONTH
J A S
FIGURE 2.—Bacterial counts/100 cc water showing relationship of the
incidence of Vibrio spp. (VLO), organisms closely related to Vibrio
parahaemolyticus (VPLO) and V. parahaemolyticus strains with tem-
perature. VPLO appeared in the water column at 14C and V. para-
haemolyticus at 19C.
T78
-------
C c
CD O
04
0>
-o
CD
d
TVC
35
30
25
0°
20:
g
-------
V. parohaemolyticus
A M j
MONTH
FIGURE 4.—Bacterial counts/gram plankton, showing seasonal changes
in the bacterial flora of plankton. Organisms closely related to V.
parahaemolyticus (VPLO) and V. parahaemolytiCUS strains appeared
simultaneously in and on plankton when the water temperature
reached 14C.
Thus, organic materials not mineralized while floating in the
sea, deposits and forms part of the sediments where the residual
organic matter would be further utilized by the bacteria, fungi,
protozoa and benthos. Residual refractory material would
thereby be permanently trapped in the sediment. In most of
the open ocean, the rate of deposition is slow, in the order of
<1 cm/100 years for chitin (Seki, 1965(a)).
Chitin is a naturally-occurring refractory material that has
been best studied in ocean and estuarine waters. Decomposition
of chitin has been shown to occur mainly in the sediments,
180
-------
ANNUAL LIFE CYCLE OF V porohoemolyticus
°c
35
30
25
20
15
10
5
I9-20°C./.. ' £y'4- .PL^ *
.PHNKTPN. V4. V ,5'i
• _ V v A
1:
*\ I4-I5*C
\ ||
-
.
-------
cent of the total bacterial number in the neritic zone. Many
chitinoclasts were found on copepods.
These results are interesting in view of our own studies on a
chitinoclastic bacterium, V. parahaemolyticus, in Chesapeake
Bay. The distribution of this organism in water (Fig. 2), sedi-
ment (Fig. 3) and on plankton (Fig. 4) led us to hypothesize a
relationship between this organism and the copepod, Calanus
sp., in Chesapeake Bay (Fig. 5), (Kaneko and Colwell, 1973).
In conclusion, the bits and pieces of information available on
refractory materials and their synthesis and catalysis in the
aquatic environment suggest that the cycling of these materials
does occur in lakes, estuaries and the oceans. However, the data
are fragmentary, and the patterns of true "cycles" of these
materials remain to be painted in full detail.
ACKNOWLEDGEMENT
The support of the National Science Foundation (Grant No. GB-
35261X) and of the National Oceanic and Atmospheric Administration
(Sea Grant Project Grant No. GH-91) for studies of Vibrio parahaemo-
lyticus quoted in this paper is gratefully acknowledged.
LITERATURE CITED
Aleschina, W. J. 1938. Decomposition of chitin by sulfate-reducing
bacteria and changes in oxidation-reductions during the process of
reduction of sulfates. Microbiologia 7: 850-859 (In Russian). Ab-
stract in Chem. Abstr. 34: 4098, 1940.
Bae, H. C., E. H. Cota-Robles and L. E. Casida, Jr. 1972. Microflora
of soil as viewed by transmission electron microscopy. Applied
Microbiol. 23: 637-648.
Barber, R. T. 1968. Dissolved organic carbon from deep waters resists
microbial oxidation. Nature 220: 274-275.
Casida, L. E., Jr. 1968. Infrared color photography: selective demon-
stration of bacteria. Science 159: 199-300.
Codner, R. C. 1971. Pectinolytic and cellulolytic enzymes in the microbial
modification of plant tissues. J. Appl. Bacteriol. 34: 147-160.
Colwell, R. R. and M. S. Zambruski. 1972. Rodina-Methods in Aquatic
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Cooke, W. B. 1956. Colonization of artificial bare areas by microorganisms.
Bot. Rev. 22: 613-638.
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Fahraeus, G. 1947. Studies in the cellulose decomposition by Cytophaga.
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184
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Questions and Comments following
Dr. Colwell's Talk
QUESTION FROM THE FLOOR: Does Vibrio parahaemolyticus
occur in fresh water?
DR. COLWELL: This organism in general demonstrates a
preference for salinites above 12 parts per thousand, so it is
restricted to coastal and estuarine areas.
QUESTION FROM THE FLOOR: Have you looked at the con-
tamination in a Zobell sampler due to the messenger going
down the slide wire?
DR. COLWELL: I have looked at this problem with a Niskin
sampler but not with the Zobell sampler.
STATEMENT FROM THE FLOOR: I would bring an endorsement
for the record on Dr. Colwell's request for modern equipment
and the development of equipment as a research priority for
deep ocean and ocean sampling and work. Some of the problems
are the need to miniaturize ocean sampling equipment for inland
water, for continuous monitoring of the ocean environment,
and in particular for engaging in rate measurements.
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Microbial Interactions with Hydrocarbons:
Physiological and Structural Correlates
W. R. FINNERTY
and R. S. KENNEDY
Department of Microbiology
University of Georgia
Athens, Georgia 30601
INTRODUCTION
At present, little detailed knowledge exists at the molecular
level or the organismic level as to the means by which diverse
microorganisms grow at the expense of various hydrocarbons.
Advances relevant to hydrocarbon microbiology have appeared
at frequent intervals (1, 6, 11, 21, 28}. These reviews serve to
emphasize our paucity of knowledge concerning detailed mecha-
nisms in hydrocarbon oxidation processes by microorganisms.
A large number of microorganisms have been shown to utilize
hydrocarbons quite efficiently as sole carbon sources. Also,
general classes of hydrocarbons (paraffins, olefins, aromatic,
and non-aromatic polynuclears) have supported growth of bac-
teria, yeasts, and fungi. Summation of these studies have
revealed that products of hydrocarbon utilization are predomi-
nantly acids, alcohols, ketones, and esters. Oxidative mecha-
nisms have been proposed following a consideration for struc-
tural formulae involved with comparisons being made between
starting compounds and products obtained following growth.
Such chemical analogy has allowed meaningful analysis of
microbial hydrocarbon degradation patterns.
While a great number of microbiological studies have been
directed towards a compilation of hydrocarbon utilizing micro-
187
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organisms, very few workers have undertaken serious studies
concerning the detailed mechanisms of hydrocarbon assimilation
and utilization. The exact nature of the biochemical mechanisms
which regulate hydrocarbon oxidations by microorganisms are
generally poorly understood at best.
Micrococcus cerificans represents a microorganism capable of
prolific growth on a large variety of paraifinic and olefinic
hydrocarbons greater than ten carbons in length. Published
work has delineated the nature of compounds arising from
growth of M. cerificans on many of these hydrocarbons (4, 9,
25, 26, 27}. Further studies have provided additional insight
into the biosynthesis of characteristic esters resulting from
growth of M. cerificans on specific hydrocarbons (5, 9). Detailed
biochemical analyses have appeared with this microorganism
defining the comparative aspects of hydrocarbon assimilation
(14, 15, 16, 17, 18, 19).
This report extends these studies with observations concerning
the effect and fate of hydrocarbon on a microorganism in pure
culture. The results of these studies emphasize our lack of
detailed knowledge concerning the impact of petroleum on
biological entities in the environment and the consequences
which arise from this relationship.
MATERIALS AND METHODS
Organism and Growth Conditions. All studies were carried out
with the HO1-N strain of Micrococcus cerificans as described by
Finnerty et al. (3). The organism was grown on a mineral
medium consisting of (in grams per liter): (NH4)2SO4, 2;
KH2PO4, 4; Na2HPO4, 6; MgSO4-7H2O, 0.02; CaCl2.2H2O,
0.001; FeSO4.7H2O, 0.001; pH 7.5.
n-Alkanes and n-1-alkenes (Humphrey Chemical Co., New
Haven, Conn.) were added to a final concentration of 1 percent.
Organisms were also grown on nutrient broth (0.8%)-yeast
extract (0.5%), acetate (2%), and ribose (2%) for comparative
studies. All hydrocarbon and non-hydrocarbon growth sub-
strates were added to the defined mineral medium.
Cultures were grown on a gyratory shaker at room temper-
ature (25 C) to the late exponential growth phase and harvested
188
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by centrifugation at room temperature. Cell pellets were washed
two times with the mineral medium to remove the external
non-metabolized hydrocarbon.
Fixation: Two fixation procedures were used. Method 1 fol-
lowed the techniques described by Kellenberger et al. (10). In
method 2, the procedure of Glauert and Thornley was used (8).
A cell suspension was mixed with equal parts of 5 percent
glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3) containing
2 mg of CaCl2 per ml.
Dehydration and Embedding. The fixed cells were dehydrated
by one of two procedures. Method 1 involved processing the
samples through a graded series of water-ethanol mixtures fol-
lowed by propylene oxide. These samples were infiltrated with
Epon according to Luft (13) or with Maraglas as described by
Freeman et al. (7). In method 2, the samples were dehydrated
by processing through a graded series of Durcopan as described
by Staubli (24) and embedded in Araldite. Ultrathin sections
were cut on a Reichart OMU-2 ultramicrotome and mounted
on uncoated 300 mesh copper grids. The sections were stained
with lead citrate (23) followed by uranyl acetate and examined
in a Philips-200 electron microscope operating at 80 Kv.
Extraction of Cells. Cell pellets obtained from the growth of
M. cerificans on specified substrates were washed 5 times with
distilled water by repeated centrifugation. Each cell pellet was
processed through a graded series of water-ethanol mixtures as
used in the dehydration for EM (50:50, 30:70, 5:95, 5:95,
0:100, 0:100, v/v). The alcohol extracts were pooled and re-
duced to dryness. All alcohol extracts reduced to dryness except
those obtained from hexadecane and hexadec-1-ene grown cul-
tures. The alcohol extracts were dissolved in chloroform, dried
with anhydrous sodium sulfate, and dissolved in hexane for
analysis by gas chromatography.
Gas-Liquid Chromatography (GLC). A Packard gas chromato-
graph, series 7500, consisting of a dual-column oven with coiled-
glass columns (4mm inside diameter, 1.83 m long) was used for
the analysis of alcohol extracts. The detection system was an
argon ionization detector with column support systems consist-
ing of liquid phases of 10 percent Apiezon L and 20 percent
diethylene glycol succinate (DECS) on a support of 70-80
189
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Anakrom A. Operating conditions were: column temperature,
120 C; detector temperature, 190 C; injection temperature,
180 C; outlet temperature, 205 C; argon flow rate, 60 ml min;
chart speed, 2.5 min inch.
X-Ray Diffraction Analysis. Cultures of M. cerificans grown
on specified hydrocarbon and non-hydrocarbon substrates were
washed extensively and lyophilized. These cell preparations were
encapsulated in a Mylar film and cooled to —10 C with a cold
air stream. The cooled specimens were exposed to a 1 mm
diameter beam of Ni-filtered X-rays from a Cu anode and a flat
FIGURE 1.—Photomicrograph of M. cerificans associated with hexa-
decane during the early exponential growth phase. 5000 X.
190
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FIGURE 2.—Scanning electron micrograph of M. cerificans associated
with hexadec-1-ene (HC). The hydrocarbon appears as large ovoid
bodies. 1750 X.
film was set perpendicular to the beam approximately 5.3 cm
behind the specimen (flat-plate forward-reflection technique).
The exposed film was quantitatively analyzed by micropho-
tometry along a diametric line.
Scanning Electron Microscopy. Cell preparations for scanning
electron microscopy were either fixed in glutaraldehyde or dried
unfixed onto specimen stubs, coated with gold-palladium (40:60)
in a vacuum evaporator and viewed in a Cambridge Stereoscan
Scanning Electron Microscope.
191
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RESULTS
A preliminary phase microscopy study of M. cerificans ac-
tively growing at the expense of hexadecane revealed that the
hydrocarbon was dispersed as small microdroplets in the culture.
Bacteria were observed to adhere to the surface of these hydro-
carbon microdroplets as shown in Figure 1. Interestingly, hexa-
decane did not disperse into stable microdroplet emulsions
when shaken with uninoculated growth medium. The presence
of bacteria preinduced to growth on hydrocarbons was essential
and necessary for the formation of stable hydrocarbon-water
emulsions.
This phenomenon was studied in further detail with the aid
of the scanning electron microscope to better determine the
relationship of the bacteria to the hydrocarbon microdroplets.
Figures 2, 3, and 4 are scanning micrographs obtained by
FIGURE 3.—Scanning electron micrograph of M. cerificans cells coating
the surface of the hydrocarbon-eicosene. The surface of the hydro-
carbon is irregular with the bacteria randomly positioned on all
surfaces. 3400 X.
192
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FIGURE 4.—A micrograph of higher magnification showing bacterial
cells on the surface of the hydrocarbon-hexadec-1-ene. The sample
was taken from the mid-exponential growth phase and cells in the
process of division (D) can be seen. 3650 X.
sampling a culture of M. cerificans during exponential growth
on hexadecane. Figure 2 is of low magnification (1750 x) and
includes many hydrocarbon microdroplets. A larger magnifi-
cation (Fig. 3) reveals the surface of the microdroplet to be
uniformly covered with bacteria. Further magnification shows
quite clearly the bacteria densely packed over the surface of
the microdroplet (Fig. 4).
A whole cell preparation negatively stained with phospho-
tungstic acid reveals topographically the presence of intra-
193
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cellular inclusion bodies (Figure 5). This topographical pattern
is characteristic only of bacteria grown at the expense of
hydrocarbons.
A detailed study was initiated into the ultrastructure of
M. cerificans grown on various hydrocarbon and non-hydro-
carbon growth substrates. Cell specimens were prepared for
examination by transmission electron microscopy as specified
in Materials and Methods. Ultra-thin sections obtained from
cells grown on acetate, ribose, or nutrient broth-yeast extract
(NBYE) were identical with respect to their fine-structure detail
(Figure 6). In contrast, cells obtained from growth on hydro-
carbons exhibited unique characteristics that served to dis-
FIGURE 5.—M. cerificans grown on hexadecane and negatively stained
with phosphotungstic acid. Large electron translucent granules of
the hydrocarbon (H) substrate are observed in the whole cell prepa-
ration. 92,000 X.
194
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FIGURE 6.—Electron micrograph of M. cerificans grown on nutrient
broth-yeast extract. Ribosomes (R) and nuclear material (N).
71,500'x.
tinguish them from nonhydrocarbon grown cells (Figure 7).
Multiple lipid inclusion bodies were characteristic features of
only hydrocarbon grown cells. This fine-structure lipid inclusion
was a characteristic feature associated with the growth of M.
cerificans on a homologous set of alkanes varying in chain length
from 12-20 carbon atoms. Alk-1-enes as sole carbon and energy
source exhibited a characteristic fine-structure pattern (Figure
8) in that 1-2 lipid inclusion bodies were observed per cell.
These lipid inclusion bodies have been observed to be present
in yeast and fungal species capable of growth on hydrocarbons.
Under no circumstances have these patterns of inclusion bodies
been observed in bacteria unable to grow at the expense of
hydrocarbons.
The chemical identification of these inclusion bodies was
established by gas-liquid chromatography and X-ray diffraction.
Positive identifications of pentadecane, hexadecane, hexadec-1-
ene, heptadecane, and octadecane were confirmed in alcohol
extracts of cells grown on these respective hydrocarbons by gas
chromatography. Table 1 shows the X-ray diffraction data ob-
tained from analysis of lyophilized cells grown on the specified
hydrocarbons. The cells from each group were compared with
195
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TABLE 1.—X-ray diffraction analyses of M. cerificans
Hexadecane
Hexadec-1-ene
Heptadecane
Control Cells
Standard
Cells
Standard
Cells
Standard
Cells NBYE Acetate
d
20.40
10.40
6.96
5.20
4.63
4.50
4.20
4.03
3.82
3.60
3.45
3.24
2.60
2.31
2.28
2.14
2.04
2.01
1
10
10
5
1
80
70
5
20
100
100
20
5
16
2
30
5
5
5
d
20.41
10.40
4.62
4.50
3.82
3.60
2.60
2.28
2.01
1
6
8
45
30
100
100
7
18
3
d
21 .20
11 .00
7.43
5.59
4.67
4.30
4.22
4.07
3.88
3.82
3.65
2.52
2.29
1
50
50
20
5
20
5
100
30
80
5
5
20
10
d
21 .21
11 .00
4.65
4.22
4.07
3.87
2.53
2.29
1
10
8
5
100
5
30
6
5
d
22.40
11 .90
7.88
5.94
4.76
4.14
4.03
3.93
3.81
3.71
3.54
3.26
2.50
2.32
2.22
2.14
2.07
1
30
50
40
30
5
100
40
5
10
90
5
2
40
30
40
30
20
d
22.4
11 .9
5.94
4.14
4.03
3.81
3.72
2.49
2.30
2.22
2.14
1 d 1 d 1
15
25
20
1 00
6
50
20
15
15 ....
15
-------
FIGURE 7.—Ultrathin sections of M. cerificans grown on hexadecane
medium showing the large translucent hydrocarbon (H) inclusion
granules. Hexadecane grown cells contain numerous granules in each
cell. The cell envelope is similar to that of cells grown on other sub-
strates with an occasional bleb (B) appearing on the cell wall (CW).
117,000 X.
samples of chemically pure hydrocarbon and the D values and
relative intensity (I) determined. Only bacteria obtained from
hydrocarbon cultures exhibited a X-ray diffraction pattern. The
D values obtained from hydrocarbon grown cells enabled posi-
tive identification of the intracellular hydrocarbon pools with
the ability to discriminate the identity of each hydrocarbon.
These results serve to confirm the presence of intracellular
pools of hydrocarbon in M. cerificans.
Infrastructure Modifications Associated with Hydrocarbon
Oxidation
An unexpected ultrastructure feature revealed a new dimen-
sion to the complexities associated with the biochemistry and
enzymology of hydrocarbon oxidations by biological systems.
197
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FIGURE 8.—Electron micrograph of M. cerificans grown on hexadec-1-
ene. Cells normally contain one or two large granules in each cell.
These cells were processed with Durcupan in order to retain the un-
saturated, osmiophilic, hydrocarbon granules as electron dense in-
clusions. 124,000 X.
These ultrastructure differences became apparent in a com-
parative study of the fine-structure of M. cerificans grown with
hydrocarbon and non-hydrocarbon substrates. Hydrocarbon
grown cells exhibited the synthesis of complex intracytoplasmic
membranes. These membranous structures do not appear as a
result of bacterial growth on non-hydrocarbon substrates.
Figures 9 and 10 show the variation and structure of these
membranes in j\I. cerificans when grown on hexadecane. Figure
11 shows the type of membrane structure observed with hexadec-
1-ene grown bacteria. These structures appear as highly ordered
bilayer lamellar membranes extending throughout the cell. An
198
-------
FIGURE 9.—Intracytoplasmic membrane (IM) development in M.
cerificans grown on hexadecane. 89,000 X.
occasional physical relationship between the intracytoplasmic
membrane and a hydrocarbon inclusion body can be observed.
A growth phenomenon currently inexplicable concerns a gross
cellular transformation in hydrocarbon grown bacteria. At ir-
regular intervals cultures of M. cerificans growing on alkane or
alk-1-ene transform into giant cells. Individual bacteria will
become 4-10 times larger, greatly extended and elongated, and
exhibit extensive intracytoplasmic membrane development.
Figure 12 shows a thin-section of one of these giant cells ob-
tained from a culture growing on hexadecane. Figure 13 is a
micrograph of a cell obtained ffom a hexadec-1-ene culture. This
induced transformation occurs only when bacteria are grown
on hydrocarbons. Subculturing these transformed cells to new
hydrocarbon containing media maintains giant cell populations,
with approximately 30 percent of the total cell population
undergoing this transformation. Reversions to normal cell size
and shape occurs by subculturing to non-hydrocarbon nutritive
media, e.g., nutrient broth-yeast extract or acetate. A physical
property associated with these transformed cultures is their
199
-------
FIGURE 10.—A complex intracytoplasmic lamellar membrane system
observed in M. cerificans grown on hexadecane and dehydrated with
Durcopan prior to embedment in Araldite. 94,500 X.
buoyant density. It is impossible to sediment these cells in
centrifugal fields as high as 40,000 Xg. The transformed cells
remain as a floating granular pellicle at the top of the centrifuge
tube.
Quantitative Relationships of Lipids in Relation to Hydrocarbon
Oxidation. A comparative study of lipids in relationship to
200
-------
FIGURE 11.—M. ccrificans grown on hexadec-1-ene and dehydrated in
a graded series of ethanol exhibits a complex intracytoplasmic mem-
brane system (IM). 96,000 X.
FIGURE 12.—Electron micrograph of M. ceriftcans transformed to large
cells by repetitive growth on hexadecane. Intracytoplasmic mem-
brane (IM) and hydrocarbon (H). 50,000 X.
201
-------
FIGURE 13.—A transformed cell of M. cerificans obtained by growth on
hexadec-1-ene. The membrane system (IM) appears to traverse the
length of the cell and is in close association with the electron dense
hydrocarbon inclusions. 49,000 X.
hydrocarbon oxidation was quantitatively determined with M.
cerificans growing on hydrocarbon and non-hydrocarbon sub-
strates. Table 2 shows the quantitative distribution of specific
lipid classes in M, cerificans. Phospholipids doubled as a result
of hydrocarbon metabolism. The induction of intracytoplasmic
membrane in hydrocarbon growTn cells accounts for a doubling
of phospholipid. Free fatty acid pools remain constant under
the two growth conditions while significant differential concen-
trations of free fatty alcohol and wax ester (cetyl palmitate)
were measured only in hydrocarbon grown cells.
The extracellular accumulation of lipids is shown in Table 3.
TABLE 2.—Quantitative distribution of lipids in Micrococcus cerificans
Micromoles/g dry cell weight
Lipid components
Non-hydrocarbon Hydrocarbon1'
Free Fatty Acid
Wax Ester
60
48
75
0
0
120
2 5
8 2
2 7
7 3
" Nutrient broth-yeast extract grown bacteria
' Hexadecane grown bacteria
202
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TABLE 3.—Quantitative distribution of extracellular lipids
Micromoles/liter of culture medium
Lipid components
Non-hydrocarbona Hydrocarbonb
Phospholipids 0 0
Triglyceride 2.4 25.6
Free fatty acid 4 60
Free fatty alcohol 0 0.5
Wax ester 0 280
0 Nutrient broth-yeast extract grown cells
6 Hexadecane grown cells
These results show that triglyceride, free fatty acid, free fatty
alcohol, and wax ester are present in the culture broth of hydro-
carbon grown cells. Triglyceride, free fatty acid, and wax ester
appear as major extracellular lipids in hydrocarbon cultures.
The direct effect of these lipids in solubilizing hexadecane is
undetermined.
DISCUSSION
These studies have served to direct attention to problems
associated with petroleum which have been either totally ignored
or unrecognized. The recent impact of petroleum in the environ-
ment tends to emphasize the cogency of providing concrete
answers to these problems. Specific implications which stem
from this study are:
(a) cellular transformations induced by hydrocarbons;
(b) the sequestering of hydrocarbons by microorganisms
with potential impact on food chain interrelationships;
and
(c) the microbial production of specific solubilizing agents
which aid or promote hydrocarbon metabolism.
The fate and effect of petroleum or petrolic byproducts on
the environment has received cursory attention at best. A
summary discussion of the effects of oil pollution on birds,
mammals, fish, molluscs, plankton, plants, as well as marine
communities has defined a few of the overall aspects of pe-
203
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troleum pollution (22). The examination of shell fish and marine
sediments demonstrated the persistence of oil in sediments and
shell fish several months following the pollution incident. Of
significant interest was the retention of aromatic hydrocarbons
by these shell fish (2). Recent studies have shown the rapid uptake
of heptadecane, 1,2,3,4-tetrahydronaphthalene, toluene, napth-
alene, and 3,4-benzopyrene by marine mussels. The data indi-
cated that mussels did not metabolize these hydrocarbons but
significant amounts were retained by the tissues (12).
We have observed that a microorganism undergoes a morpho-
logical transformation as a result of growth on chemically pure
hydrocarbon. Whether this transformation is the result of syner-
gism between hydrocarbon and a metabolic byproduct or a
singular effect of hydrocarbon is unknown. Further evidence
has demonstrated fine-structure modifications in the form of
induced membrane synthesis. Quantitative biochemical analyses
of the simple and complex lipids of M. cerificans have demon-
strated an increase in the cellular and extracellular lipids as a
result of hydrocarbon metabolism. The pharmacological and
toxicological properties of petroleum and petroleum byproducts
on the aquatic environment are undetermined. It is well known
that many polycyclic aromatic hydrocarbons are carcinogenic.
A hydrocarbon oxidizing microorganism has responded physio-
logically and morphologically in a rather drastic manner to a
hydrocarbon molecule which is tacitly assumed to be innocuous.
The inescapable question of "why?" remains.
Sequestering of hydrocarbons by the microbial flora through
active transport becomes of practical concern. Hydrocarbons
which are pooled by the indigenous microbial flora could relate
to the sequential transfer of hydrocarbons through the food
chain. The phenomenon becomes of fundamental concern as a
mechanism for passage of potentially dangerous chemicals
through the food chain webs to higher life forms. Compounds
which can undergo such processes are: (a) paraffinic and olefinic
hydrocarbons; (b) aromatic and polycyclic aromatic hydro-
carbons; and (c) halogenated hydrocarbons. We have investi-
gated hydrocarbon utilizing bacteria, yeasts, and fungi and
found all to accumulate paraffinic and olefinic hydrocarbons in
the cytoplasm. The critical question becomes just how far does
204
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hydrocarbon move through the food chain progression before it
disappears, is converted into a potentially harmful intermediate,
or is metabolized to non-toxic intermediates?
The conclusion appears warranted that M. cerificans under-
goes a loss or alteration of biochemical control mechanisms for
cell wall synthesis, membrane synthesis, and morphological
shape when grown repetitively on hydrocarbons. The analogy
to neoplastic transformations in higher cellular systems is evi-
dent. The "how" and "why" of what triggers a microbial cell
to "pool" a hydrophobic hydrocarbon against a concentration
gradient, to form complex intracellular membranes, and to
undergo gross cellular transformations are undetermined.
It is a well recognized fact that all microorganisms are not
capable of hydrocarbon assimilation presumably lacking the
requisite enzymatic complement to effect such conversions. A
study of hydrocarbon structure in relationship to microbial
oxidizability has established a set of biological guidelines for
determining the biodegradability of specific substituted hydro-
carbons (20). Hydrocarbons as hydrophobic, water-insoluble
compounds have circumstantially been considered as refractory
to active transport processes. These considerations necessitated
an extracellular modification of the hydrocarbon by the micro-
bial agents with assimilation processes being effected on the
byproducts of this activity. Our findings that hydrocarbons are
pooled suggests a mechanism for active transport of hydro-
carbons. The component parts and requirements for active
transport of a water-insoluble substrate are ill-defined. However,
if hydrocarbon oxidizing microorganisms possess the ability to
synthesize a biodetergent which promotes pseudo-solubilization
of hydrocarbon, a mechanism for active transport against a
concentration gradient can be affected. An analysis of cellular
and extracellular lipids revealed that a number of specific lipids
were present in a hydrocarbon utilizing system that could fulfill
this purpose. The explicit role of these lipids as solubilization
agents for hydrocarbons has not been determined within this
system. Their known physical properties would suggest that an
influential role could be served in maintaining a finite level of
hydrocarbon in aqueous solution.
Future directions for analyzing the impact of petroleum on
205
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the aquatic environment can be formulated as a series of experi-
mental questions:
(a) what is the diversity of microorganisms in the aquatic
environment capable of hydrocarbon metabolism?;
(b) do differential kinetic parameters exist with respect to
uptake of specific hydrocarbons?;
(c) what is the magnitude of hydrocarbon pooling in the
microbial flora and what are the rates of hydrocarbon
elimination from such microbial populations?;
(d) can internal pools of more than one hydrocarbon be
maintained or do selective uptake and oxidation rates
prevail depending on the hydrocarbons available?;
(e) do hydrocarbons induce cellular transformations
throughout all microbial species?;
(f) what pharmacological and toxicological properties do
specific hydrocarbons exert on higher cellular.systems?
Answers to these questions would provide basic information
towards formulating specific policies and procedures in water
quality management and control as well as providing needed
insight and knowledge into the biological effects of petroleum
in the environment.
ACKNOWLEDGMENTS
These studies were supported by National Science Foundations grants
GB-8208 and GB-34120 to W. R. Finnerty. The X-ray diffraction analyses
were conducted by Dr. R. A. Young, Engineering Experiment Station,
Georgia Institute of Technology, Atlanta, Ga. The authors express
gratitude for the use of the Electron Microscopy Laboratory, University
of Georgia, Athens, Ga. and the helpful advice of B. O. Spurlock.
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(11} Klug, M. J. and A. J. Markovitz. 1971. Utilization of Aliphatic
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Academic Press, New York.
(12} Lee, R. F., R. Sauerheber, and A. A. Benson. 1972. Petroleum
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(13} Luft, J. H. 1961. Improvements in epoxy resin embedding methods.
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95: 2102-2107.
(15) Makula, R. A. and W. R. Finnerty. 1968. Microbial Assimilation of
hydrocarbons. II. Fatty acids derived from 1-alkenes. J. Bacteriol.
95: 2108-2111.
(16} Makula, R. A. and W. R. Finnerty. 1970. Microbial assimilation of
hydrocarbons. III. Identification of phospholipids. J. Bacteriol. 103:
348-355.
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of hydrocarbons. IV- Phospholipids metabolism. J. Bacteriol. 107:
806-814.
(18} Makula, R. A. and W. R. Finnerty. 1972. Microbial assimilation of
hydrocarbons. V. Cellular distribution of fatty acids. J. Bacteriol.
112- 398-407.
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(19} McCaman, R. E. and W. R. Finnerty. 1968. Biosynthesis of cytidine
diphosphodiglyceride by a paniculate fraction from Micrococcus
cerificans. J. Biol. Chem. 243: 5074-5080.
(20} McKenna, E. J. and R. E. Kalio. 1964. Hydrocarbon structure:
its effects on bacterial utilization of alkanes, p. 1-14. In H. Heu-
kelekian and N. C. Dondero (eds.), Principles and applications in
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(22} Nelson-Smith, A. 1970. The problem of oil pollution of the sea.
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(24} Staubli, W. 1963. A new embedding technique for electron micro-
scopy, combining a water soluble epoxy-resin (Durcopan) with
water-insoluble Araldite. J. Cell. Biol. 16: 197-199.
(25} Stewart, J. E., W. R. Finnerty, R. E. Kallio, and D. P. Stevenson.
1960. Esters from the oxidation of olefins. Science 132: 1254-1255.
(26} Stewart, J. E. and R. E. Kallio. 1959. Bacterial hydrocarbon oxi-
dation. II. Ester formation from alkanes. J. Bacteriol. 78: 726-730.
(27} Stewart, J. E., R. E. Kallio, D. P. Stevenson, A. C. Jones, and
D. O. Schissler. 1959. Bacterial hydrocarbon oxidation. I. Oxidation
of n-hexadecane by a gram-negative coccus. J. Bacteriol. 78: 441-
448.
(28} Van der Linden, A. C. and Thijsse. 1965. Microbial oxidation of
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208
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Questions and Comments following
Dr. Finnerty's Talk
QUESTION FROM THE FLOOR: Are the hydrocarbons true
emulsions in the sense that they are clear suspensions?
DR. FINNERTY: No they are not. They are turbid suspensions.
QUESTION FROM THE FLOOR: Have you tried growing the
bacteria in a medium in which both alkanes and alkenes are
present?
DR. FINNERTY: No, I haven't. They have all been the single
substrate type of experiments. Granules form even when there
is a vapor phase over the liquid medium.
QUESTION FROM THE FLOOR: If there is to be recycling of
hydrocarbons to the sediments, then there must be a lot of
anaerobic activity for hydrocarbons to be degraded. Have you
observed anaerobic degradation of hydrocarbons?
DR. FINNERTY: I don't know that anyone has shown con-
clusively that there is such a thing as anaerobic degradation of
hydrocarbons.
COMMENT FROM THE FLOOR: I might add that there is evidence
for the anaerobic degradation of the benzene ring with nitrate
as a terminal electron acceptor. This is a special case of the
hydrocarbon degradation and not the degradation of a linear
hydrocarbon but there is no explanation as to how the ring is
cleaved.
COMMENT FROM FLOOR: You also mentioned that you had
looked at yeasts and fungi. I wonder what particular species of
yeasts you looked at.
209
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DR. FINNERTY: Mainly Candida species.
QUESTION FROM THE FLOOR: Have you sampled and looked
at any bacteria in natural oil slicks to see if they have the
characteristics of the organisms you grow in culture?
DR. FINNERTY: I have not. All the studies we have done have
been with pure cultures, under carefully defined conditions.
QUESTION FROM THE FLOOR: Have you established any rates
of degradation for the specific hydrocarbons?
DR. FINNERTY: No definitive rates have been defined, but we
have done some preliminary experiments. We cannot furnish
any numbers right now.
QUESTION FROM THE FLOOR: When you remove the cells from
these certain types of media do you find that there is a time lag
before the cells begin to lose the inclusion bodies which you
showed?
DR. FINNERTY: If you put the cells back in fresh medium
without hydrocarbon intracellular hydrocarbon inclusions dis-
appear in approximately 60 minutes.
QUESTION FROM THE FLOOR: Did you find nitrogen to be a
limiting nutrient in the utilization of the hydrocarbon?
DR. FINNERTY: We have never quantitated the exact utili-
zation of nitrogen in the growth of this organism. We always
grow the organisms in an excess of nitrogen, for the organism
will not grow in the absence of nitrogen.
QUESTION FROM THE FLOOR: Do you think any of the inclusion
bodies could have been polyhydroxybuterate?
DR. FINNERTY: No. We specifically looked for PHB, but our
organism does not make PHB. We grew many microorganisms
on crude oil and we found that many of them had PHB as
inclusion bodies.
QUESTION FROM THE FLOOR: What is the generation time of
the organism grown in hydrocarbon when compared to the
generation time of the organism grown on acetate medium?
DR. FINNERTY: The generation time on hydrocarbon is ap-
proximately 30 minutes and 50 minutes on acetate.
QUESTION FROM THE FLOOR: Does the structure shown on
your slides appear to be correlated with growth phase?
DR. FINNERTY: The structures are seen throughout the
growth cycle.
210
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Microbial Conversions of Dissolved
Organic Carbon Compounds
In Sea Water
D. W. HOOD
Institute of Marine Science
University of Alaska
and T. C. LODER
University of New Hampshire
Interest in the organic components of sea water has a long
history generated largely by biologists, whose thinking was first
summarized by Lucas (1955), in emphasizing the significance
of so-called organic metabolites on the distribution and growth
of marine algae, bacteria, protozoa, and larvae. Most parameters
in the early studies indicate little difference between "good"
and "bad" ocean waters, a terminology based on the ability to
support productivity. Good waters support heavy growth; bad
waters are nearly sterile. Recent studies seem to indicate that
coniplexation of trace metals by organic matter is at least one
consideration in distinguishing possible "good" and "bad"
waters (Barsdate et al. 1973; Ryther et al. 1971). Observations
suggest other critical factors such as growth substances, toxins,
and antimetabolites may hold the key to the productivity of the
sea. Recent reviews (Hood 1963, 1970; Johnson 1955; Provasoli
1963; Vallentyne 1957; Saunders 1957) have pointed out the
importance of dissolved organic matter in the biology of the
sea from six viewpoints:
—Organic compounds, even in dilute solution, serve as
energy sources for bacteria, yeast, fungi, larvae, and
possibly algae;
211
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—Vitamins serve as growth stimulators for algae, bacteria,
yeast, fungi, and larvae;
—Organic toxins such as those derived from Gymnodinium
brevis kill large populations of marine organisms.
—Organic compounds are important in migration, school-
ing, predator-prey relationships and sexual behavior of
organisms.
—Certain organic compounds trigger feeding processes of
organisms, even though these compounds may not be
nutrients;
—Nutrient or toxic trace elements may be associated or
complexed with organic compounds, thus controlling the
level of these elements in the aquatic medium.
Organic matter also has importance in geological and geo-
chemical processes in nature. The literature on this topic is
voluminous and will not be covered in this discussion. It is
clear, however, that the diagenic processes leading to fossil fuel
formation involve microbial alteration of deposited organic
matter. Part of this transition must involve a soluble or at
at least colloidal state for the organic matter. Clay-organic
association in this process is not yet fully understood, but
evidence indicates it has a central role in sedimentary processes
involving organic matter.
Physical processes in the sea that are affected by organic
material include sea-foaming, surface tension, air-sea interaction,
light absorbancy, sound transmission, viscosity, and the char-
acter of surface films.
The importance of organic matter to the chemistry of the
sea is well established. There have been many problems in
obtaining quantitative data even of the gross amounts present,
and the presently used techniques are good only to about
±0.1 mg C/liter. Group-type organic analysis has been used
extensively and is useful in some considerations; however, many
reactions, especially those involving organisms, are compound-
specific. Thus, an understanding of the stimulus, depression, or
activity of organisms influenced by organic matter depends
upon a careful evaluation of the constituents present, their
rates of production and degradation, and distribution in the
ocean. At this time, no single water sample has been analyzed
212
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for all the organic components present. In sea water only about
20 to 30 percent of the total present can be accounted for by
the classes of compounds such as proteins, including amino
acids; lipids, including hydrocarbons; and carbohydrates. The
remaining organic constituents, best classified as "fulvinic acids"
or marine humates, are extremely difficult to classify. They
probably do not consist of specific molecular structures, but
rather are conglomerates of different sized, highly complex
polymeric chemicals with varying numbers of functional groups.
Analysis of this fraction for the purpose of understanding re-
activity in the environment might best be done on a functional
group basis rather than by the conventional compound-specific
means.
In this paper, an attempt is made to review what is known
about dissolved organic carbon distribution in nature, par-
ticularly the ocean, as it reflects information on decomposition
rates. Most attention is directed to those compounds that may
affect the sea on a world ocean scale. Since those materials that
are easily oxidized have already been considered by others in
this meeting as they have dealt with nutrient regeneration, I
will discuss the more refractory material.
Distribution of Dissolved Organic Matter in the Ocean and
Evidence for its Role
Dissolved organic matter is defined in this paper as that
fraction of the organic matter which passes through a filter of
an average 0.4 micron pore diameter. This is an operational
definition and has little to do with the physical-chemical state
of the material. The filtered fraction will contain the crystal-
loidal and colloidal portions; cell contents of ruptured phyto-
plankton, especially nannoplankton and dinoflagellates; small
aggregates; and some minute organisms.
In the case of the decomposition of dissolved organic matter,
however, the ambiguity of the above definition is probably not
critical. Organic material as utilized by most organisms must
go through a single-molecule stage before absorption into the
metabolic pool of the organism. Whether the material starts
as an aggregate and is attacked in this form or is in true solution
would probably affect only the rate of utilization. The concept
213
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of dissolved organic carbon has limited scientific utility, since
observed dissolved organic carbon concentration is the result of
a number of diverse processes such as heterotrophic utilization,
adsorption to particulate matter, transformation between dis-
solved and particulate organic carbon, extracellular excretion
by living plants and animals, and decomposition of dead
organisms. In order to study the dynamics of the carbon cycle,
however, some separation is necessary between living and non-
living carbon; large particles which settle quickly can be sepa-
rated from smaller particles or colloids which remain in sus-
pension. The size fractionation method currently in use by most
investigators is a practical way to reach those goals and is
probably the best technique now available.
Distribution. The largest quantity of organic carbon on this
planet, with the exception of that in sediments and soils, is
contained in the oceans. Nearly all of this is non-living and in
the dissolved form. The concentration of organic carbon in sea
water is less than 1 mg C/liter in the major portion of the
ocean and higher at the surface and near land; yet it exceeds
by an order of magnitude the particulate portion, which in
turn exceeds the living matter present by a proportionate
amount (Wangersky 1965).
There have been a number of studies on the distribution of
both dissolved organic carbon and particulate organic carbon in
the world ocean. Investigations in the Atlantic include work by
Duursma (1961), Parsons (1963), Menzel and Ryther (1964),
Riley et al. (1965), Skopintsev et al. (1968), and Gordon
(1970a,b). Among Pacific studies are those conducted by Holm-
Hansen et al. (1966), Hobson (1967), Barber (1967), Holm-
Hansen (1969), and Gordon (1970c). Work in the Indian Ocean
includes studies carried out by Menzel (1964), Starikova (1967),
Szekielda (1967), Newell and Kerr (1968), and Newell (1969).
Organic carbon in the Arctic has been studied recently by
Loder (1971) and Kinney et al. (1971); work in the Bering Sea
has been reported by Loder (1971), Nishizawa and Tsunogai
(1973); and a glacial inlet has been investigated for organic
carbon by Loder and Hood (1972).
There is general agreement among most of the authors cited
as to the concentrations of DOC and POC both near the ocean
214
-------
surface and at depth with two exceptions: (1) The DOC values
reported by Skopintsev and colleagues from dry combustion of
evaporated samples are several times higher than the concen-
trations found by other workers. (2) There has been a trend
toward finding smaller amounts of POC in the deep sea than
was earlier reported; this is probably due to more careful sample
preparation, larger samples, and more precise methods.
Summary of available data shows that from surface to about
300 m the DOC concentration is about 1.0 mg C/liter (range
0.3-2.0) and the POC concentration is about 100 ug C/liter
(range 30-300). In dense plankton blooms, however, DOC values
may temporarily reach 3-5 mg C/liter and POC values 1000-
2000 ug C/liter. Below this surface zone DOC values are rela-
tively constant with an average of 0.5 mg C/liter (range 0.2-0.8
mg C/liter) and POC values averaging about 10 ug C/liter
2-30 ug C/liter). It should be made clear at this point that the
methodology used for acquiring these numbers is generally that
of Menzel and Vaccaro (1964), in which certain components of
the dissolved organic fraction may become lost. The sparging
process used to free the system of inorganic carbon may also
remove low molecular weight volatile compounds. In addition,
some of the high molecular weight hydrocarbon type compounds
may not be oxidized to carbon dioxide by the procedure used.
The composition of the deep water particulate organic matter
appears to be mainly carbohydrates and proteinaceous material
(Parsons and Strickland 1962, Gordon 1970a and Handa 1970).
There have been conflicting reports on the nutritional value of
this material. Although Menzel and Goering (1966) observed
that particulate organic matter is uniformly resistant to bio-
logical decomposition, Gordon (1968) found that 19-26 percent
of the material (mostly protein) could be removed with a
mixture of digestive enzymes. Lorenzen (1968) found that, al-
though Artema nauplii did not survive feeding on concentrated
deep water particulate organic matter, 43 percent of the POC
nevertheless disappeared when the concentrated portion was
allowed to sit for 90 days at 20 C in the dark. Deep water
zooplankton do ingest organic aggregates, but whether or not
they digest them is unknown. These organisms have an abun-
dance of 0.1 to 1 mg/m3 at 4000 m (Vinogradov 1962) and
215
-------
require about 1 to 2 percent of their body weight per day for
respiration. This energy must come from organic carbon.
In the surface layers, the particulate organic matter consists
mainly of detritus from the death of organisms and organic
aggregates described by Riley (1963, 1970). Living matter
contributes only about 10 percent of the total. The ratio of
DOC to POC ranges from about 3-10 in the surface layers to
200-300 for some deep water. The average deep-water ratio is
about 50 (Williams 1969).
Dissolved organic matter appears to contain small portions
(about 20 percent) of the following compounds: amino acids
(free and combined) (Degens 1970, Pocklington 1970); sugars
(free) (Handa 1970); fatty acids and hydrocarbons (Jeffrey
1970, Blumer 1970); and even smaller amounts of aromatics
(substituted phenols) and vitamins (Natarajan 1968). The
amount of these compounds present in the near-surface waters
is quite variable because of changes in biological activity in-
volving both uptake and liberation of extracellular products and
decomposition (Parsons and Seki 1970). Both Williams (1969)
and Degens (1970) feel that these compounds comprise only a
small percent (10-25) of the total amount of DOC. Based on
low C/N ratios reported by several workers, Degens (1970)
proposed that most of the remaining dissolved organic material
is in the form ofheteropolycondensates., a clathr ate-type molecular
structure. Williams (1969), however, suggests that the remaining
material is humus or lignin-tjpe material and is refractory to
microbial and chemical oxidation. Jannasch (1970) suggested
that the low concentration may effect utilization but found
that bacteria would not utilize DOC from deep water even in
five-fold concentration.
Duursma (1961, 1965) and later Menzel and Ryther (1968,
1970) have suggested that DOC in deep waters appears to be a
conservative property and can therefore be used to trace water
masses. Menzel and Ryther (1968) concluded from their study
of the Antarctic Intermediate Water that oxygen concentrations
observed in the water as it moved north were a function of
mixing and were not primarily due to oxidation of entrapped
organic matter. They suggested that dissolved organic matter
tends to be conservative in nature once water masses sink below
216
-------
the surface and that the cycling of organic matter is restricted
almost entirely to relatively shallow surface water.
Carbon-14 dating and stable isotope studies (carbon-13:
carbon-12 ratios) have provided further insight into the status
of organic carbon in sea water and its age. Williams and Gordon
(1970) reported that the range of 13C/12C ratios for both POC
and DOC was between -22.0 and -24.4 percent relative to
the PDBi standard, meaning the carbon is relatively depleted
in 13C. They found that the ratios are constant with depth,
sample location and time in the northeast Pacific. In addition.
they reported that the ratios found for both DOC and POC
most closely approximate the cellulose and lignin-like fractions
of surface plankton and concluded that paniculate organic
matter is similar in composition to dissolved organic matter.
Williams et al. (1969), using carbon-14 dating techniques, have
reported an average age of 3400 years (b.p.) for the total organic
matter in 2 samples taken at 2000 m off southern California.
This value is almost twice that of inorganic carbon and is sug-
gestive of different input and utilization pathways for the two
fractions.
With the concept of the inert nature of sea water organics in
mind, a review of other evidence infers that a portion of organic
material at depth is in dynamic equilibrium rather than totally
inert. There are a number of problems that remain unanswered
hy Menzel, Williams and their co-workers: Some of these include
the seasonal and spatial variation of POC and DOC in the sea
(Hobson 1967; Menzel 1964; Gordon 1970a,c), and the possible
DOC-to-POC transformation and subsequent role as a food
source for deep sea filter feeders.
The role of DOC-to-POC transformation is still not well
understood, although it is generally accepted that under the
proper conditions particulate organic matter does form from
part of the dissolved fraction. Baylor et al. (1962), and Sutcliffe
(1963) found that phosphate adsorbed to bubbles passing
through sea water. The next year Baylor and Sutcliffe (1963)
found that cultures of Anemia could survive on particles pro-
duced by the bubbling of sea water. Sutcliffe et al. (1963)
suggested this process might be associated with Langmuir
circulation and be a source of organic food particles in the sea.
217
-------
Riley et al. (1965) found that particles could be produced by
bubbling both surface and deep water from the Sargasso Sea,
although deep water produced fewer particles.
In further studies Menzel (1966) found by using ignited,
filtered air and trice-filtered sea water that bubbling is not a
likely mechanism for the production of particulate carbon.
Barber (1966) reported he was unable to produce significant
particulate carbon by bubbling sea water "sterilized" by fil-
tration through 0.22-/j, filters and concluded that bacteria played
a role in particle formation. Batoosingh et al. (1969) explained
these apparent contradictions with a set of very carefully con-
trolled experiments. They found that small particles (0.22-1.2 \i
in size) are important as nuclei in formation of larger particles
during the bubbling process and that the concentration of
particles inhibits further formation until particles are removed.
Thus it appears that entrapment of bubbles near the sea surface
may play an important role in production or organic particles
suitable as food for the small grazing organisms. This process
does not provide particles for the deep sea, however.
Riley et al. (1965) proposed that there is a dynamic balance
between utilization of particles and further adsorption from the
filterable fraction. Sheldon et al. (1967) and Parsons and Seki
(1970) described the formation of particles and the possible role
of bacteria in particle formation and enlargement by bacterial
clumping. Khaylov and Finenko (1968) found that dissolved
high-molecular compounds are sorbed into detritus, broken
down by enzymatic hydrolysis and then utilized by bacteria
populating the detritus. This would move dissolved organic
matter into the particulate phase and make it available to
filter-feeding organisms. The role of adsorption of organic mole-
cules by inorganic particles has been shown by several workers
(Bader et al. 1960; Chave 1965, 1970; Degens and Matheja
1967), but the extent to which this process contributes to
particle formation is unknown. These, then, are some of the
problems concerning the relationship of DOC to POC.
Several theories have been suggested concerning the source of
food for deep-living zooplankton: (1) Non-living particulate
organic material may settle slowly out of the euphotic zone and
provide a continuous supply of food material. (2) Migrating
218
-------
zooplankton populations may utilize food materials above them
in the water column and in turn be eaten by organisms below
them in a series of overlapping food chains (Riley 1951; Vino-
gradov 1962); (3) Detrital organic matter, some of which
originates in the euphotic zone, settles in the water column in a
state of dynamic equilibrium with the dissolved organic matter
through a series of interactions perhaps mediated by hetero-
trophic microorganisms and physical adsorption (Riley et al.
1964; Sheldon et al. 1967; Riley 1963, 1970). (4) High concen-
trations of paniculate matter may exist at various depths,
possibly in discontinuous layers for short periods of time, pro-
viding a concentrated food source (Parsons and Seki 1970).
Since data have been found to support all of these theories to
some extent, it is suggested that the actual mechanisms are a
combination of some or all of these factors, with the amount of
food contributed by each mechanism still unknown.
Several recent studies on the role of POC and DOC in sea
water may help our understanding of the very complex role
played by organic matter. Organisms may release natural
organic chelators which are necessary for increased phyto-
plankton growth (Barber and Ryther 1969). Surface-charge
studies on organic particles may give insight into aggregate
formation (Wangersky 1968; Neihof and Loeb 1972). Studies
on the release of dissolved organic matter by phytoplankton
(Anderson and Zeutschel 1970), zooplankton (Johannes and
Webb 1970) and macrophytes (Sieburth and Jensen 1970) sug-
gest additional sources of DOG.
Considerable advance has been made on the differentiation
of living particulate matter from the non-living by the use of
ATP analysis (Holm-Hansen 1970). This technique is now
being widely used to determine the biomass of living organisms
associated with detrital material.
Decomposition of Organic Matter in the Deep Sea
The distribution of organic matter with depth in the Atlantic
Ocean is shown in Table 1 (Menzel 1970) and for the Arctic
Ocean in Table 2 (Loder 1971). The locations of the samples
designated in Table 1 are shown in the original paper of Menzel
(1970). It may be seen from these data and those obtained by
219
-------
many others that the concentration of dissolved organic matter
ranges from 0.8 to 2 mg C/liter in the surface layer and de-
creases gradually with depth (Duursma 1961; Menzel 1964;
Ogura and Hanya 1967; Barber 1968; Menzel and Ryther 1968;
Ogura 1970; Loder 1971; Loder and Hood 1972; Ogura 1972).
The concentration of DOC below 400 m is fairly uniform and
TABLE 1.—The distribution of dissolved organic carbon (mg C/liter) in
selected areas of the Atlantic Ocean. Each value is the average of
concentrations measured at the indicated number of stations
Area
number of stations
Depth (m)
1
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1 1 00
1200
1 300
1500 P
2000 .'
2500 .1
3000 i1-
wO vv/ • • • •••••*•• •••••• • •
4000
4500
5000
A
t
1
0
0
0
0
0
0
0
0
0
0
0
0
0
o'
~d*
tf
tf
&
k
.04
.70
.67
.67
.66
.64
.64
.64
.70
.66
.66
63
.60
.60
.60
.58
.60'-
.60f
.65
B
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.88
.61
.57
.50
.47
.42
.44
.39
.38
.40
.40
.46
.43
.41
.40
.42 .
.42 .
.40 .
.40 .,
.45 -..-,
F
9
0.82
0.74
0.62
0.50
0.50
0.46
0.46
0.46
0.47
0.46
0.46
0.44
0.40
0.39
0.38
0.40
0.37
0.46
0.39
0.42
0.44
0.42
0.40
0.43
0.43
0.49
G
8
1 .03
0.80
0.65
0.59
0.59
0.56
0.51
0.52
0.49
0.50
0.48
0.46
0.47
0.48
0.48
0.44
0.48
0 44
0.48
0.48
0.45
0.50
0.53
0.52
0.49
0.42
!*••*••
E
1
0
0
0
0
0
0
0
• 0
0
0
0
0
0
0
0
0
0
n
0
n
0
o
n
n
0
0
• • • •
0
.99
.98
.86
.88
.78
.72
.66
.61
.58
.57
.55
.56
.53
.50
.53
.52
.50
.54
.53
.49
.51
.49 ,
.51
.53 ,
.50
.51
• • • •
1-
•
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
n
0
n
0
n
0
0
0
0
0
1
7
.81
.82
.65
.56
.52
.48
.46
.48
.42
.43
.43
.44
.45
.45
.43
.44
.44
.44 ,
.40
.44 ,
.44
.46
.42
.45
.50
.44
.44
D
13
0.87
0.87
0.77
0.67
0.58
0.58
0.52
0.45
0.45
0.45
0.45
0.42
0.40
0.38
0.38
0.37
0.34
0.36
0.34
0.34
0.37
0.36
0.36
0.36
0.34
0.38
0.38
Source: Menzel 1970.
220
-------
TABLE 2.—Organic matter in the water of the Amerasian Basin of the
Arctic Ocean at Station T-3-703, 82 N°, 157°W, from March 29 to
April 6, 1968
Depth
(m)
5
30
60
200
250
500
1 500
2500
3500
POC (tig/liter)
wet combustion
no
110
71
140
88
55
64
140
69
DOC
(mg/liter)
i 'ii
1 -IA
1 -J1
1 30
i OA
0 77
O 7A
n on
0 75
DOC/POC
11 Q
\V
i nx
I Do
1 Q C
1 HO
Q A
OO
1 At
140
110
i i y
^7
TOO
Source: Loder 1971,
ranges between 0.3 and 0.80 mg C/liter if the results of Skopint-
sev are eliminated (Table 3). The remarkable regularity of
these data led Menzel (1970) to an analysis of the way in which
DOC could affect non-conservative constituents in the sea and
thereby reflect on its rate of decomposition. The variability in
DOC in 400 m water is about ±0.2 mg C/liter if absolute values
are disregarded and only variations within any set of obser-
vations are considered. This variability is equivalent to a utili-
zation rate of less than 1.0 ml/liter (2.6:1 atomic ratio of
oxygen to carbon, as derived by Redfield et al. 1963) in areas
where the oxygen concentration is between 0.1 ml to 6.4 ml/liter.
TABLE 3.—The concentration of dissolved organic carbon at depths
below 400 m in the Pacific and Atlantic Oceans
Range (mg C/liter) Location Authority
1 .34-1 .74 (500 m) 10°S 51° N: Atlantic Skopintsev et al. (1966)
0.34-0 .70 35°S 35°N: Atlantic Menzel and Ryther (1970}
0.24-0 .82 40°N 66°N: Atlantic Duorsma (1961)
0.34-0 .70 20°N 28°N: Gulf of Mexico Fredricks (1968)
0.40-0.80 10°S 0°: E Pacific Barber (1967)
0.43-0.79 15°S 10°N: E Pacific Fredricks (1968)
0.36-0.77 27°N 3J9N: N.W. Pacific Ogura (1967)
Source: Menzel 1970.
221
-------
These data indicate the maximum influence that the decompo-
sition of DOC can exert on the oxygen present at depth may
amount to 15 percent of the total change. Even this variation
seems unrealistic, because DOC distribution is not consistent
with other criteria used to differentiate deep water mass distri-
bution, nor are gradients evident in the direction of decreasing
O2 values. Further evidence by Barber (1968) and Ogura (1972)
show that DOC from deep water samples does not decrease
during prolonged storage of water samples and that stable iso-
tope ratios of organic matter are constant as a function of depth
(Williams 1968; Williams and Gordon 1970). Also, Menzel
(1967) and Newell and Kerr (1968) found that particle concen-
tration below 200 m was independent of surface production and
that density discontinuities do not lead to the accumulation
and thus accelerated decomposition at depth. These obser-
vations led Menzel (1970) to conclude that the compounds
included in the DOC which found its way into the deep ocean
were biologically and chemically stable.
What may be considered to be a disagreement on the stability
of the DOC fraction based on other data has recently arisen.
Nishizawa and Tsunogai (1973), in extensive studies of particu-
late organic matter distribution in the Bering Sea, found that
the minimum concentration of particulate carbon occurred close
to the dichothermal layer and concentrations below this depth
were 2 to 3 times higher. Most significant, however, they found
that the average POC in deeper layers correlated closely with
the concentration in the upper 50 m layer. These authors con-
cluded that the major fraction of POC in the deeper layers is
directly derived through rapid sinking from the immediate
overlying layer, implying a much closer coupling between the
surface and depth than determined by Menzel (1970). It must
be clearly understood in comparing these data that in the
Bering Sea, vigorous mixing in some areas may place the pro-
ductivity under physical control—thus allowing excess pro-
duction over consumption. The conditions studied by others
that supported the conclusion of conservative-type organic
matter at depth, were under biological control (probably by the
zooplankton community), thus allowing only limited amounts
of organic matter to remain available for sinking.
222
-------
A second consideration concerns the sources of carbon dioxide
in the water column. The major changes in the concentration
of carbon dioxide in the oceans are effected through exchange
with the atmosphere, production during respiration, consump-
tion during photosynthesis, and in the formation and solution
of particulate calcium carbonate. The oceans of the world con-
tain about 2 XlO~3 moles/liter of total carbon dioxide. The
deep waters of the Atlantic and Pacific contain about 10 and
20 percent, respectively, more total carbon dioxide than the
surface water. The addition of CO2 to the water by solution of
CaCO3 is accompanied by a concomitant increase in calcium
and carbonate alkalinity. Each mole of CO32~ which enters
solution will increase the alkalinity by two equivalents. Tsunogai
(1972), computing differences in total-CO2, Ca2+ and alkalinity
between the surface and deep water, concluded that only about
20-30 percent of the increase in carbon dioxide could arise from
CaCO3 dissolution, the remainder being derived from decompo-
sition of organic matter.
Kroopnick et al= (1971) has estimated the source of carbon
dioxide in deep water based on two methods: the consumption
rate of oxygen and the mean isotopic composition of carbon
isotopes added to the water column. Both of these methods
indicate that about 70 percent of the excess CO2 is derived
from oxidation of organic matter.
Some direct measurements of decomposition rates of DOC
have been made by Barber (1967) and Ogura (1972). Ogura,
during extensive cruises on the Hakuho Maru in the north
equatorial Pacific collected samples from the surface layer and
incubated them aboard ship for a period of up to 75 days while
observing dissolved oxygen and DOC changes with time. His
data were found to best fit a three-component decay system.
The first part (Fi), consisting of 10-20 percent of the total,
was utilized in the first 40 days of storage; disintegration of the
second fraction (Fn), consisting of 20-25 percent of the total,
required a much longer period; and the third portion (Fin),
essentially refractory, constituted 50-60 percent of the total
DOC. Table 4 summarizes these conclusions. From these data,
he was also able to compute rate constants (Table 5).
The decomposition of phytoplankton has been studied both
223
-------
TABLE 4.—Proportion and nature of three organic fractions in surface
sea water.
Fraction Percentage Nature of DOC
Fl
FII
Fin
10-20
30-40
50-60
Easily utilized by microorganisms
Utilizable by microorganisms
Refractory, not easily utilized by
microorganisms
Source: Ogura 1972.
in mixed and single specie culture by several workers (Skopintsev
et al. 1966; Khaylov and Finenko 1968; Grill and Richards
1964; Handa 1970; Ogura 1971). Their results show that de-
composition occurs usually at two different rates: between
0.03-0.10 mg day-1 during the first few days and between
0.0004-0.02 mg day-1 for extended periods. The available data
do not appear to permit a precise breakdown of decomposition
rates for different organisms under various conditions; the
general conclusion may be drawn, however, that there are
rapidly decomposing fractions together with more refractory
materials that decompose at slower rates. The decomposition of
the easily decomposed organic matter occurs mainly in the
surface water, leaving the more refractory materials as the bulk
of dissolved organic matter found in the ocean.
There appears to be no real basis for the apparent disagree-
ment on the stability of DOC in the deep ocean. Zooplankton
in the Pacific Ocean have a biomass of 0.1-1 mg/m3 at 4000 m
(Vinogradov 1969). This biomass is about 10 percent carbon
and will require about 1-2 percent of the body weight per day
as food. Thus, the consumption of carbon would be about
0.1-1 Mg C/ms-day or 0.036-0.36 mg C/m3-year. The distri-
bution of particulate carbon alone is between 3-10 mg C/m3
available for zooplankton consumption. If this were the source
of zooplankton food, a turnover time of approximately 10 years
is indicated.
Based on the above assumptions and the integrated abun-
dance of zooplankton from 400-4000 m, the flux of carbon
required is about 2g C/m2-year. This estimate is similar to that
of Packard et al. (1971), based on potential electron transport
224
-------
TABLE 5.—Rate constant for decomposition and half-life of DOC in
surface sea water
Fraction Rate constant Half-life (days)
(mg X day"1)
25 C 12 C 25 C 12 C
FI
FI + FII + Fill
0.033
0.0052
0.016
0.0030
20
130
40
230
Source: Ogura 1972.
measurements, which indicated carbon requirements of 2-3
g/m2-year. This flux may be provided through the sinking of
plants, animal feces, zooplankton migration, paniculate carbon,
or dissolved organic carbon.
The oxygen depletion rate resulting from the consumption of
this much carbon is between 9-13 /^liters/liter-yr, which would
result in a depletion of about 0.1 ml O2 in 25 years. This is
within the presumed accuracy range of the presently used
method for oxygen determination and, therefore, such a de-
pletion would normally be undetectable in the standard devi-
ations of oxygen data.
There appears to be little basis for the differences in opinion
expressed above on utilization of DOC in deep ocean water to
account for oxygen utilization, excess carbon dioxide or isotope
ratio differences. Although it is clear that the deep water biomass
requires approximately 2g C/m2-yr to sustain its metabolic
requirements, this amount may easily be derived from other
sources. Alternately, a plausible model compatible with existing
data may be advanced: The DOC in deep water is in dynamic
equilibrium, which maintains its concentration at a remarkably
constant value throughout the oceans. The particulate organic
carbon, although low in concentration, is not only a source of
usable carbon but might in fact function in a sorption-desorption
equilibrium with DOC components. Through such a process
POC would serve as a source of oxidizable dissolved organic
matter that may escape, surface oxidation. Both the carbon
dioxide required to satisfy the increase in total carbon dioxide
with depth and the metabolic energy required by zooplankton
225
-------
and deep ocean microorganisms is derived by oxidation of
organic matter on surfaces provided by sinking paniculate
matter. Such a mechanism would also justify the very old dates
obtained for dissolved organic carbon and the correlation of
surface productivity with particulate carbon found at depth.
Some recyclable DOC will be added to the pool by metabolizing
organisms; if such a contribution is equivalent to 10 percent of
the metabolizable carbon taken in, about 0.4-4.0 mg C/m3-yr
would be released, an amount which would not cause concen-
tration changes significantly detectable by present methods of
measurement.
Man-Made Additions of Organic Matter to the Marine
Environment
Easily oxidized compounds constitute the major portion of
organic matter that man adds to the marine environment. Many
of these compounds do not pose a serious pollution problem
unless their concentrations are high enough to reduce the dis-
solved oxygen content. Other compounds or their degradation
products may directly inhibit vital biological processes. Al-
though easily oxidized compounds are eliminated relatively
rapidly by oxidation and thus do not ordinarily constitute a
serious pollutant threat, even their very brief residence time in
the case of certain highly toxic compounds is sufficient to cause
considerable harm through their specific individual properties.
The more resistive organic compounds are of greater concern
because of their potential role as major pollutants, depending
upon the manner in which they interact biologically and chemi-
cally with environmental components.
The effect of contaminants on vital life processes can often be
measured quantitatively, such as inhibition to photosynthesis
or respiration, energy conversion, or even decrease in motility.
Other deleterious effects are subtler, more difficult to detect,
and if not monitored carefully can go unnoticed until a particular
species is eliminated. These latter effects may be manifested
as reproductive failure, thermotactic interference or alterations
of predator-prey relationships. Inhibition of particularly sensi-
tive life processes such as photosynthesis or respiration occurs
often at contaminant concentration levels below those affecting
226
-------
TABLE 6.—United States annual production rate of some synthetic
organic chemicals in 1968
Million metric tons Boiling point (C)
Formaldehyde
Acetone
Dichloro-difluoro-methane
(freon 12)
Carbon disulfide
Methylene chloride
Diethyl ether
Ethanol
Methanol
Cyclohexane
DDT
1 .70
0.59
0.59
0.13
0.34
0.12
0.05
0.86
1 .50
0.86
0.74
-21
21
56
-29
45
40-41
35
78.5
65
81
"™""
Source: U.S. Tariff Commission 1968.
other vital functions. This condition enables use of a control
technique based on a maximum tolerable concentration thresh-
old, below which photosynthesis and respiration are unaffected,
thus providing some degree of protection against the less con-
spicuous effects. Because of the rapid increase and diversity in
production of synthetic organic chemicals over the past few
decades and their release to the environment by multiple
methods of transport, it is difficult to predict the future extent
of contamination. The 1968 U.S. production rate of some
commonly used organic chemicals is presented in Table 6;
hundreds of other synthetic organic compounds are produced in
varying quantities. Since the quantity produced of these com-
pounds tends to follow the gross industrial production of the
world, it is apparent that the environment will continue to be
under ever-increasing stress from additions of man-made organic
chemicals.
Biodegradation and geochemical removal are probably the
two most active factors in depleting the oceans of added organic
contaminants. All organic materials are eventually biodegraded.
Although the rate of biodegradation has been measured for
very few compounds, it is clear that this process proceeds very
slowly in the case of chemicals such as DDT, polychlorobi-
phenyls, some plastics, and certain petroleum fractions.
227
-------
A review of the general topic of critical problems in the
coastal zone has recently appeared in a workshop report The
Water's Edge (Ketchum, ed. 1972). One of the recommendations
of this workshop reads in part:
The Coastal Zone Workshop recommends basic biological,
chemical, and physical research directed toward the following
types of problems in the coastal zone:
(a) Transport, dispersion, upwelling and cycling of nutri-
ents and hazardous chemicals as they affect the func-
tioning and stability of coastal zone ecosystems;
(b) Surveillance of input levels of contaminants, especially
chlorinated hydrocarbons, petroleum and heavy metals.
(c) Effects of chronic, long-term, sublethal contaminants
on organisms and ecosystems;
(d) Assimilative capacity of the coastal zone for all kinds
of waste;
(e) Factors affecting stability, diversity, and productivity
of coastal zone ecosystems.
Until a major portion of these problems are resolved, par-
ticularly concerning the natural capacity of the environment to
degrade and assimilate, the question of organic contaminant
additions will remain in question. Particular emphasis is needed
on refractory compounds such as components of petroleum,
polychlorobiphenyls and pesticides; at the same time a constant
surveillance system is needed to detect any new additions that
may be accumulating in the marine ecosystem. Since biodegra-
dation may be the key factor in removing organic wastes from
the environment, a thorough knowledge of specific reaction rates
under varying environmental conditions is essential.
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Questions and Comments Following
Dr. Hood's Talk
QUESTION FROM THE FLOOR: What about sinking of the oil
and high sediment loading that may occur in Port Valdez?
DR. HOOD: There is a problem with sediments—oil interaction
in glacial inlets. In Cook Inlet, however, where oil spills fre-
quently occur, hydrocarbons have not been found in the sedi-
ments. I know this is not true in general. These are the results
obtained on over a hundred samples in this inlet. We are
watching the developments in Port Valdez. If the oils do sink
there will be probably be active degradation in the surface
sediment.
QUESTION FROM THE FLOOR: In your measurement of carbon,
did you use a carbon analyzer?
DR. HOOD: No. The oceanographers are using a method which
involves a 5 ml sample of ocean water taken in a vial= Persulfuric
acid and phosphoric acid is then added and the vial sealed. The
vials are autoclaved and the CO2 evolved is measured by gas
chromatography or infrared analysis.
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Bacterial Bottom Sampler
for Water Sediments
DALE J. VAN DONSEL*
and EDWIN E. GELDREICH**
An important aspect in assessing the water quality of a lake,
river or estuarine area zoned for swimming may be the bac-
teriology of the bottom sediment. The sediment deposits may
provide a stable index of the general quality of the overlying
water, particularly where there is great variability in the bac-
terial quality of the water (1). Bottom sediments may also
prove to be of critical significance in shellfish culturing waters.
Attempts to collect sediment samples for bacteriological analysis
have met with difficulty because of the limitations of available
equipment. Bottom material is variable in composition, ranging
from sand, gravel, marl, clay and hard rocky outcroppings to
bottoms Uttered with refuse and sludge banks. Dredges are of
limited value when attempting to obtain representative samples
from a fixed area in some of the above sediments and are unsatis-
factory for recovery of bottom sludges. Core sampling devices
were found to be difficult to sterilize and the nature of the
sediment limited the sample size that could be recovered. Field
tests with the Emery bacterial bottom sampler were disap-
pointing in that the spring loaded cut off plate frequently
jammed in gritty substrates, preventing complete closure of the
open end of the glass vial during recovery of the bottom sampler.
In addition to this problem, the sample size was too small for
multiple testing procedures. The need for a device that would
* Research Microbiologist, Arctic Health Research Center, College, Alaska.
** Chief Bacteriologist, Water Supply Research Laboratory, National Environ-
mental Research Center, EPA, Cincinnati, Ohio
237
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function in a wide variety of bottom materials, incorporate a
disposable sterile container of adequate size, and permit hand
operation in shallow water, or remote control operation from a
boat, motivated the development of a new sediment sampler.
GENERAL DESCRIPTION
It is desirable that the sampler (Figure 1) be constructed of
stainless steel, but brass material may be used except for the
nose piece which requires stainless steel for durability. Samples
are collected directly in sterile "Whirl-Pak" type plastic bags
(6-ounce size) that are easily closed after use and serve as con-
venient containers during subsequent handling. A nylon cord
COUNTER BALANCE
and SAFETY LINE
ANCHORAGE
WEIGHT
SPRING LOADED
PLATE
(SAMPLE BAG CLAMP)
SLIDE LATCH
SLIDE
BAR
NOSE
PIECE
'ERFORATED
BOTTOM DISK
FIGURE 1.—Bacteriological Bottom Sampler.
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loop arrangement closes the bag from the outside after the
sampler penetrates the bottom sediment. A slide bar keeps the
bag closed during descent and thus prevents air pressure build-
up or entry of water into the bag. This slide bar permits the
bag to open only during penetration of the sediment and then
only as much as necessary to contain the in-flow of sample.
Adding a heavy metal weight jacket to the assembly and using
a counterbalances hook release makes it possible to use the
bottom sampler in deep water.
Major Components
THE NOSEPIECE
This section holds the sampling bag and provides the cutting
surface for penetration of the sediment. The nylon closing loops
are inside the nosepiece but encircle the open end of the sample
HOLE A
NYLON-CORD
FIGURE 2.—Sampler nosepiece and closing loop.
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bag, which is fitted over this nose piece (Figure 2). There are
two holes on each side of the nosepiece; the upper set (B and BI)
serves only to anchor the free ends of the closing loops, which
pass through the nosepiece and are knotted on the outside.
The lower set of holes (C and Ci) allows the cord to pass through
two hollow rods in the sampler to the operator's hand directly
or indirectly by attachment to the counter-balance drop plate.
SLIDE BAR
The slide mechanism consists of a bar that rides on two solid
rods attached to the nose. A plate is attached to one side of the
slide and is held against the slide by slight tension from two
small springs. Except for the open mouth, the sample bag is
held shut by the spring-loaded plate. A short standoff mount
on each side of the slide bar serves as an attachment point for a
perforated bottom plate or disk. Thus, as the sampler nosepiece
penetrates the bottom sediment, the perforated disk presses
against the surface of the sediment, and the weight of the
sampler causes the slide to traverse a distance equal to the
penetration depth of the nosepiece. This procedure allows the
sample bag to open and the bottom sediment core to enter the
space provided. A latch holds the slide bar in the uppermost
position during sample retrieval to prevent its dropping back
to a lower position and cutting the bag.
CLOSING LOOPS
A #18 nylon cord, 160-lb. lest strength, is used to make
the closing loops that constrict the bag opening in a sphincter-
like manner (Figure 2). The closing loops are made by: passing
one end of the cord through the upper hold C of the nosepiece;
knotting this end; and lightly flaming the free nylon cord tips
to prevent fraying. The unknotted end of the cord is passed
through the lower hole B and threaded through the hollow rod A
to the counterbalance drop plate or extended long enough to be
controlled by manual operation. This portion of the cord is then
returned threaded, through the other hollow rod A; passed
through the lower hole B; inter-looped through the companion
closing loop and then through the upper hole C, where this end
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is locked in a knot and the free tip is flamed. Spreading the inter-
looped cord in the nosepiece forms an opening through which
the sample bag is inserted. The hag is opened and its mouth
turned back over the nosepiece. When the cord at the top of
the sampler is pulled, the bag is tightly closed.
COUNTERBALANCED DROPPING PLATE
In shallow water, a counterbalanced dropping plate is not
needed. Instead, a rod is screwed into the top plate for handling
the bottom sampler and the closing loops are operated by
pulling the cord by hand. In deep water, bottom sediment col-
lections will require attachment of a suitable jacketed weight
and an eye bolt to the top plate of the sampler. In operation,
the sampler is then lowered from a boat by a counterbalanced
dropping plate (Figure 3), which also operates the closing loops.
As soon as the sampler touches bottom, tension is removed from
the dropping plate, permitting disengagement of the hook and
eye. This release then permits the sampler to penetrate the
bottom sediment by force of its own weight. Adjustment of the
line length from the screw hook on the dropping plate to the
sampler eye bolt will influence free fall force, depth of core
collected, and speed of sample bag closure. The initial motion
of retrieving the sampler via the line attachment to the eye
bolt will simultaneously pull the closing cords to cut off the
bottom sample and close the plastic bag. When the sampler is
used in this manner, a safety line should be connected to the
sampler eye bolt, passed through a hole in the dropping plate,
and then joined to the lowering rope to ensure against loss of
the sampler in the event the closing cords break.
SAMPLER OPERATION
The sampler is inverted and the closing loops are opened,
keeping these cords within the nosepiece. Place a bag inside the
loop opening, parallel with the slide. Open the bag, and fold it
back over the rim of the nosepiece. The spring-loaded plate on
the slide is opened and held open with the two stops. While
feeding the free end of the bag through this opening, advance the
slide bar until it comes to rest against the nosepiece and then
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TOP VIEW
SIDE VIEW
ERQP PLATE
FIGURE 3.—Sampler Drop Plate.
242
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return the spring-loaded plate to full tension against the bag.
The slide bar should be held in this position until ready to
sample. The sampler should be lowered slowly to prevent water
resistance from lifting the perforated bottom plate, causing it
to slide upward and permitting the bag to fill with water. When
the counter-balanced hook is used for remote operation, the
lowering line should be slackened after the sample has reached
bottom to ensure that the hook has swung free.
When operating the sampler by hand in shallow water, the
sampler is pushed into the bottom sediment with the rod at-
tachment, then the closing cords are pulled to cut off the sample
and secure it for retrieval. Tension on the closure cords, applied
during retrieval by weight of the sampler is sufficient to keep
the bag closed.
SPECIAL PRECAUTIONS
Because the nylon cord frays easily, all burrs and sharp
edges should be removed from parts with which it comes in
contact. The slide will bind on the two solid rods if all four lips
of the slide holes are not rounded and polished. To ensure
smooth operation of the bag-opening mechanism, the spring-
loaded plate and the bar behind it should be polished and their
edges rounded.
The perforated bottom disk shown in Figure 1 may be unsatis-
factory for repeated operation from a boat because it requires
a slow descent. If the bottom disk is unperforated, the water
resistance of the disk may be reduced by using a smaller one
or by using material having larger perforations, such as heavy
hardware cloth. In instances where very soft sediments are en-
countered, a solid sheet metal disk has been used; this requires
an extremely slow descent. In soft sediments, the depth to which
the nose piece penetrates can be limited by attaching a Hoffman
tubing clamp on one of the solid rods as a slide stop.
Although the sampler was designed for use in relatively
shallow waters, it will operate at considerable depths (up to
at least 200 feet); however, at depths greater than 50 feet,
there is a chance that the Whirl-pak bag may rupture as it is
returned to atmospheric pressure. When a sample is retrieved
243
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from depths greater than 50 feet, the closing loops must be
opened cautiously and the mouth of the bag pointed away
from the operator lest internal pressure sprays the material
over the operator.
REFERENCES
(2) Van Donsel, D. J., and E. E. Geldreich. 1971. Relationships of
Salmonellae to Fecal Coliforms in Bottom Sediments. Water Research
5: 1079-1087.
244
* U. S. GOVERNMENT PRINTING OFFICE : 1974 O - 507-521
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