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The Pymatuning Symposia in Ecology

THE ECOLOGY OF ALGAE

A Symposium Held at the Pymatuning

Laboratory of Field Biology on

June 18 and 19, 1959

Edited by: C. A. Tryon, Jr. and R. T. Hartman

Special Publication Number 2

Pymatuning Laboratory of Field Biology

University of Pittsburgh

The publication of this symposium was made possible by a grant from
the Wherrit Memorial Fund, The Pittsburgh Foundation.

Printed April, 1960.

Pymatuning Special Publications.

1. Man and the Waters of the Upper Ohio Basin 1955 $2.00

2. Ecology of Algae 1960 $2.00

Prices are postpaid. The publications may be ordered from The
Pymatuning Laboratory of Field Biology, University of Pittsburgh,
Pittsburgh 13, Pa.

Lithoprinted in the U. S. A.

by

Edwards Brothers, Inc.

Ann Arbor, Michigan

Acknowledgments

We would like to acknowledge our Indebtedness
to Mr. Stanton Belfour and the Trustees of
The Pittsburgh Foundation whose grant from
the Wherrit Memorial Fund made the symposium
possible .

ill

CONTENTS

Page

Ecological Distribution of Fresh-Water Algae.

L. A. Whitford, North Carolina State College 2

Algal Populations in Flowing Waters.

John L. Blum, Canisius College 11

Biological Disturbances Resulting From Algal Populations in

Standing Waters .

G. W. Prescott, Michigan State University 22

Algae and Metabolites of Natural Waters.

Richard T. Hartman, University of Pittsburgh 38

Algae in Relation to Oxidation Processes in Natural Waters.

Alfred F. Bartsch, United States Public Health Service 56

Organic Production by Plankton Algae, and Its Environmental Control.

John H. Ryther, Woods Hole Oceanographic Institution 72

Artificial Media for Fresh-Water Algae: Problems and Suggestions.

L. Provasoli and I.J. Pintner, Haskins Laboratories 84

77231

INTRODUCTION

The papers which make up this volume were presented at a conference
held June 18 and 19, 1959 at the Pymatuning Laboratory. The focal point
of the conference and of this publication is encompassed in the title Ecology
of Algae . The emphasis in the papers is on fresh-water algae but several of
them present data from marine situations and implications can be extended
to this related field.

Stimulation for many of the recent developments in our understanding
of algae has come from outside the traditional boundaries of botanical
sciences. Much has been contributed from those areas of ecology concerned
with the hydrosphere, limnology and oceanography. The emphasis on the role
of algae as the primary producers in aquatic ecosystems has resulted in a
great deal of work on the physiology of algae. Certain problems of engineers
concerned with sewage disposal, mass culture, and space travel have also
been a great stimulus to algal studies.

The ecology of algae is here considered from the standpoint of ecological
phycology i .e. , we are interested in algae and their environmental relation-
ships. These algal-environment interactions may be viewed from the traditional
descriptive approach or they may be dealt with experimentally, either in terms
of changes occurring in the environment due to the algae or in terms of changes
that occur in the algae as a response to the environment.

The fact that algae are able to alter decisively the biotic factors of their
environment, is brought out in the papers by Dr. Prescott and Dr. Hartman.
Dr. Blum and Dr. Whitford present a picture of the occurrence and composition
of algal communities in various ecological situations. Dr. Bartsch dwells on
the functional changes or processes that occur in a unique algal community.
Dr. Ryther continues this approach by presenting the results of algal activities
in terms of productivity, which is, to many ecologists, the philosopher's
stone that will provide understanding of the ecosystem. Dr. Provasoli shows
what may be necessary in an environment in order for growth and development
of algal communities to occur .

It is obvious that time did not permit a full exploration of the ecology
of algae, even to the limited extent that the present development of the field
would permit .

ECOLOGICAL DISTRIBUTION OF FRESH-WATER ALGAE

L. A. Whitford
North Carolina State College

Ecology is one of the younger divisions of
biology, and yet the desire and search for eco-
logical data is as old as man's culture of plants
and animals. Needham and Lloyd (1915) say the
science of limnology began with the musings of
the contemplative fisherman. We can likewise
say the science of ecology began when the cave
man first began to wonder why his patches of
half-wild peas and cereals grew better at certain
times and places than others, and that the study
of algal ecology began when the Chinese began
to try to manage fish ponds, probably a thousand
years ago .

Just as the ecology of land plants began
with, and was stimulated by, such works as
Kerner's Plant Life of the Danube Basin, and by
Humbolt's studies of world floras, algal ecology
also had its beginnings in floristic studies.
Many early students of algal floras were good
observers and worked at such a leisurely pace that
they took many notes. Furthermore, page space
for publication was not at such a premium then as
now. Many papers of a few decades ago have
valuable ecological data among the more volumi-
nous floristic and taxonomic material. Among
those who have published material of this type are
the Wests (W. and G. S.), Chodat, Fritsch, and
Pascher. More recently in this country Smith,
Taylor, Transeau, and Tiffany have published
floristic and taxonomic papers from which eco-
logical data can be gleaned. Some like Fritsch,
Taylor, and Tiffany have also later written eco-
logical papers .

As early as 1904 West pointed out that
regions with drainage from pre-cambrian rocks
have an algal flora richer in species than regions
of more recent rocks. Later, a flora few in
species but rich in numbers of individuals was
recognized. These became known as the Cale-
donian and the Baltic floras. By 1920 this first
classification of fresh-water algal floras was
established in the literature. While the original
theory as to the reason for the two types is now
held to be invalid, the names can still be used to
designate the types of flora.

The rapidly developing science of limnology,
of course, has contributed most to algal ecology.
Welch (195 2) has given an excellent summary of
the history of ecology from which an idea of its
influence on algal ecology can be gained . We
have taken over, however, a number of limnologi-
cal terms and concepts without, perhaps, too
critical an evaluation of their use and value.
Thienemann and Naumann's oligotrophic , eu-
trophic , and dystrophic lake types are a case in

point. Welch (195 2, p. 344) says these terms
have "drifted slowly into a certain limited, un-
crystallized acceptance in American limnology,
although no serious attempts have yet been made
to reduce the diagnostic characters to positive
specifications valid for American inland waters . "
The terms suggest a type of lake or type of habitat
and they have also been defined in a number of
different ways . If used in phycology they should
designate a habitat and not a type of flora .

During the past sixty years phytoplankton
studies have been by far the most numerous.
Many limnological papers including phytoplankton
and papers on algae alone have been published
both in Europe and in this country. In the United
States , the Wisconsin lakes , the New York lakes ,
and the Great Lakes have all received attention.
Until recently the south and the west have been
relatively neglected .

The algae of streams have also been inves-
tigated in the course of limnological studies. The
Russians are reported to have worked especially
on the large rivers . The algae of other European
streams have received considerable attention and
series of papers have been published by Budde,
Butcher, and Fritsch. In this country stream algae
have received relatively little attention, especially
non-planktonic species. Published material is
found mostly in limnological surveys . There are
plankton papers by Allen (1920), Chandler (1937),
Eddy (1932), Purdy (1916), Reinhard (1931) and
Roach (1932) . Recently there have been papers on
non-plankton algae by Blum (1954, 1956, 1957)
and Whitford (195 6).

During the nineteen hundred forties a system
using organisms to indicate limnological condi-
tions was developed by Liebmann (1951) . It seems
to have some merit but it has not been widely
tested by investigators. This is the saprobien
system mentioned by Dr. Blum. Symoens also in
1951 outlined a classification of fresh-water algal
communities . An acceptable system of ecological
classification should be based on experimental
data, but before it will be generally adopted, it
must be widely tested under experimental condi-
tions .

Algal ecology will someday have its Warming
or its Forel, who will write a definitive treatise as
Warming did for land plant ecology and Forel did
for limnology. We probably do not yet have a suf-
ficient body of data for such a paper on fresh-
water ecology .

In a consideration of the ecological distribu-
tion of algae it is obvious that the effects of
light, temperature, and water quality should be

considered . A fourth factor , the effect of current ,
seems of considerable importance in streams and
on wave- swept shores .

Since algae are photosynthetic plants , no
one can deny that light is a very important habitat
factor. Algae are thus restricted to the photic
zone. The depth of this zone varies not only
locally with the turbidity or color of the water,
with the cloud cover, and in small streams, as
pointed out by Blum (1956), with the leaf canopy;
but also with season and latitude. Rodhe (1955)
has recently postulated, however, that some
species may grow heterotrophically in arctic winter
darkness. Welch (1952) cites two marine obser-
vations indicating that the effective day length at
some depth (10 to 40 m) is markedly less than near
the surface. This effect is probably of less im-
portance in fresh waters except at high latitudes
or in winter.

As is true of other habitat factors, we do not
have nearly enough data on the effect of light on
algal distribution. It has not been easy to make
under-water light measurements. Until recently
there was no standard or commercially made meter
for under-water measurements . Light and dark
bottle experiments have been most common. Total
diurnal incident light measurements are still diffi-
cult to make. Most fresh-water algae are probably
"shade plants . " Exceptions are algae growing on
soils, in seeps, or epiphytically on land. Certain
species of Zygnemataceae, Mesotaeniaceae, and
Desmidiaceae growing in seeps seem to need full
sunlight. Tilden (1935) proposed a theory on the
relation between algal pigments, especially the
accessory pigments, and algal evolution and dis-
tribution. This theory has never gained general
acceptance except in one part not original with
Tilden. It seems to have been adequately proved
that the accessory pigments in some red algae
render the light penetrating to some depth, more
effective in photosynthesis . This seems to be
true of fresh-water Rhodophyceae as well as
marine species . Batrachospermum . Hildenbrandtia ,
Chantransia , and Thorea have been found growing
at from ten to thirty meters in depth. The fresh-
water red algae are said to be red only when grow-
ing in deep water. I have collected Thorea ra -
mosissima , deep red in color, growing in Vaucheria
mats at ten meters depth in Silver Springs, Florida,
(Whitford 195 6) . We had thought that Batracho -
spermum is a cool water genus, but we have good
evidence that low light is an equally important
factor. Batrachospermum is found at cool seasons
and persists longest in cool shady streams, but it
is probable that low light is a more important
factor than low temperature . Plants are found later
in the spring under shady banks and in the shade
of stones than elsewhere, although the water tem-
perature here is identical with that in sunny
reaches. We were surprised to find Batracho -

spermum much less widespread in the higher North
Carolina mountains in summer than water tempera-
tures would lead one to believe. On the other
hand, at least one species, J., macrosporum , is
abundant in the deep brown waters of Coastal
Plain streams throughout the summer. It is most
abundant in shady places . Water temperature may
exceed 27°C where it is growing.^

We have made another observation which
seems of some interest. In North Carolina spring
and autumn are fairly long . In autumn we notice
a reversal of the spring flora as regards species
and time of abundance . Late spring species come
in earliest in autumn and early spring species
later. We note, however, that abundance of indi-
viduals in autumn is usually less than in spring
although the period of the same water temperatures
are approximately equal. In autumn some species
seem to be caught between a fairly low tempera-
ture requirement and a high light requirement. In
spring light intensity and duration are increasing
as water temperatures become favorable but in
autumn total incident light is decreasing as tem-
peratures become favorable for certain species.
We definitely note that if bright clear weather pre-
vails in autumn we get a more nearly exact rever-
sal of the spring flora than when it is cloudy.
Oedoqonium kurzii , an important stream species
with us, most species of Draparnaldia , as well as
some plankton algae such as Asterionella and
Dinobryon have a low temperature, high light re-
quirement .

It seems apparent that many green algae are
high-light species and red algae are low-light
species. Diatoms and Chrysophyceae seem more
indifferent in light requirement . Perhaps they will
respond to fairly high light intensities if other
conditions are ideal . The high diatom populations
in the cool Florida springs in summer seems to be
an indication that this is true (Whitford 1956) .
Blue-greens probably respond more to high summer
temperatures than to high light intensities . This
would be an interesting laboratory problem as will
be pointed out later .

Oberdorfer (1928) published detailed data on
the distribution of algae in relation to the light
factor in Lake Constance. His work and that of
others has been summarized by Fritsch (1931).

Schiller (1930) showed by means of culture
experiments that cold water (11-12°C) is fre-
quently more productive than warm (23-25°C) . The
productivity of the northern fishing banks is well
known and apparently diatoms are more abundant in
northern lakes than in those at lower latitudes .
The diatoms and Chrysophyceae are in general
microthermal (oligothermal) . Some species of

^Schumacher and Whitford, unpublished data.

Peridinium (P. limbatum, £. willei ) and ap-
parently some blue-greens are also. In some
blue-greens, however, such as Chamaesiphon low
light and current may be more important than low
temperature . We have recently found Chamaesi -
phon abundant in mid-Peruvian streams at summer
temperatures . The Chlorophyceae seem more
eurythermal . Many green algae have good vege-
tative growth at fairly low temperatures , if light
is adequate, and they continue growth at summer
temperatures . The larger species of Spirogyra and
Oedoqonium and many of the Chlorococcales seem
mesothermal (eurythermal) . Among the diatoms
Melosira granulata and Eunotia pectinalis are much
more eurythermal than most species. In brown-
water streams in North Carolina Eunotia pectinalis
replaces the genus Fraqilaria of northern waters in
late winter and spring, and it is abundant after the
water temperature well exceeds 20°. West (1909)
cites Rhizosolenia as a diatom growing at warmer
temperatures than most. We agree with this and
also suggest that Terpsinoe may be another . Many
species of fresh-water algae which are most abun-
dant in spring and autumn may be mesothermal but
data is lacking which will clearly separate light
from temperature effects .

There are probably few megathermal algae in
spite of the fact that certain blue-greens espe-
cially grow in hot springs . Some workers report
that most thermal species grow as well at ordi-
nary room temperatures as at high temperatures.
Other reports differ, however. Synechococcus
eximus is said to make maximum growth at 79°C
and will not survive at a temperature below 70°.
Oscillatora filiformis is said to tolerate a tem-
perature above 85°C and it will survive at a tem-
perature no lower than 59°C .

Recently we (Schumacher and Whitford) have
been collecting algae in winter in North Carolina.
We find that 15° seems to be the critical tempera-
ture for many microthermal species . A species of
Chrysophyceae, Phaeosphaera perforata is perhaps
the most remarkable. It has been under observa-
tion for more than ten years . It grows only at
temperatures between 4 and 10°C, and disappears
rapidly as the temperature approaches 15°. It is
very abundant in Coastal Plain streams in Febru-
ary and March following cold winters , and fairly
common in bogs and brooks somewhat later at
Raleigh, 150 miles inland. We do not have con-
clusive evidence, but apparently it is rare near
the coast southward because water temperatures
do not drop below 10 for a long enough period for
it to become abundant.

Another species in the Xanthophyceae ,
Chlorosaccus fluidus Luther grows in somewhat

Whitford, unpublished data .

similar habitats at about the same temperatures.
The diatom, Meridion circulare seems another
microthermal species. It is found growing with
us only in winter and early spring except in the
mountains . Single living but depauperate cells
can occasionally be collected in the ooze of
stream bottoms at any season but the typical half-
wheel colonies occur only at temperatures below
15°. Actinella punctata Lewis, a supposedly rare
diatom, has been collected in ten counties in the
Coastal Plain. It is an abundant epiphyte on
algae and stems during the cold months but, like
Meridion, is present only as an occasional single
cell in the ooze during warm seasons . Chae-
tophora incrassata is common in brooks in late
winter but disappears rapidly when the water tem-
perature exceeds 15°. Fragillaria is very rare at
lower elevations except in late winter but is com-
mon in streams at high elevations , where tempera-
tures remain low, in summer.

The factor of water quality is the most com-
plex and diverse especially if one includes such
things as water color, turbidity, and pH, in addi-
tion to mineral content, and dissolved gases.
Nevertheless they are all closely related and in-
teracting and have to be considered together.
There is a voluminous literature on water quality
and it is useless to try to summarize it in a paper
of this length. There have been numerous attempts
to generalize by using types of flora, or groups of
organisms as indicators of over all conditions .
Frequently single supposedly important factors
have been considered so important that such terms
as Galciphilic and halophytic have been coined .
Much investigation has been done on phosphorus
and iron as limiting factors. Undoubtedly these
as well as nitrate are often limiting. There is no
doubt that lack of available silicon limits diatom
growth, for instance. In communities of micro-
organisms, however, conditions can change so
rapidly that stenoeclous species (that is those
with a narrow range of tolerance) may become
abundant or disappear with amazing rapidity. As
Hutchinson said (1944) it is the interaction of a
complex of both physical and chemical factors
which produces both seasonal fluctuations and
sporadic blooms .

The terms eutrophic and oligotrophic have
been most used to indicate general productivity,
but we cannot yet reduce them to positive specifi-
cations . We can only say that a eutrophic habitat
is one with a high pH where available organic mat-
ter is rapidly reduced to liberate an abundance of
the vital mineral elements . The oligotrophic habi-
tat has a low pH and the mineral nutrients are low.
Thus pH seems the best single indicator of the type
of algal flora. At least several American phycolo-
gists agree with this statement. The pH results,
of course , from the interaction of a complex of
factors .

As mentioned above, I am not qualified to
judge whether the saproblen system which uses
indicator organisms will prove to be useful and
workable. Dominant species and indicator organ-
isms have been widely used in other branches of
ecology .

Separate studies of individual species might
go a long way toward solving some of the problems
of the effects of water quality.

One habitat factor which is of considerable
importance in stream ecology seems to be mis-
understood by many llmnologists and phycologists .
This is the effect of current. For a long time it has
been known that certain species grow only in a
current of water or grow better there . This fact
was at first explained by assuming that running
water has a higher content of dissolved gases and
a lower temperature than still water. There is now
much evidence that these assumptions are incor-
rect, but there is evidence that some rapids
species have a higher respiratory rate than cor-
responding lenltlc species. Many investigators
recognize this "inherent current demand" of such
species but offer no explanation. Ruttner (1926)
seems to have offered the first partial explanation .
His statement as translated by Frey and Fry
(Ruttner, 195 3) is as follows: "In quiet or in
weakly agitated water the organisms are surrounded
by a closely adhering film of liquid, which speedily
forms around the animal or plant, a cloak Impover-
ished of substances Important for life. In a rapid
current, however, the formation of such exchange-
hindering Investitures Is prevented, and the ab-
sorbing surfaces are continually brought into con-
tact with new portions of water as yet unutilized. "
Moving water, he continues, "is not absolutely
but rather physiologically richer In oxygen and
nutrients . "

The reason for this "physiological richness"
can be explained, I believe, by the laws of co-
hesion and diffusion which are familiar to most
llmnologists and phycologists. It Is a simple mat-
ter of the diffusion gradient being steeper around
plants in rapid water. If all other factors are
equal, diffusion will occur twice as fast at half the
distance. There is formed around a cell In still or
slowly moving water a gradient of concentration of
diffusing materials. For a material diffusing In-
ward the concentration In the medium is least at
the cell wall and the material Increases in concen-
tration outward a certain distance until it reaches
100% of that In the surrounding water . For the
smaller Inorganic molecules this distance seems to
be about 1/4 mm. Even in a current cohesion holds
a film of relatively still water against the surface.
In a rapid current at least part of this film Is swept
away bringing the region of high solute concentra-
tion closer to the surface — In other words making a
steeper diffusion gradient and therefore increasing
the rate of diffusion. Ferrell, Beatty, and Richard-

son, (1955) have shown that the speed of current
necessary to displace this film is of the order of
one-half foot (15 cm.) per second.

In case of oxygen and carbon dioxide, which
are more soluble In cold water, low temperature
may reduce or eliminate the "current demand . "
Cedergren (1938) reports that certain algae which
grow in still water during the cooler seasons are
found only in rapids in summer. I discovered this
to be true for Stigeoclonlum and Draparnaldla in
North Carolina, many years before seeing a ref-
erence to Cedergren's work. One species of Sti-
geoclonlum grows in summer in very swift water at
a temperature averaging close to 25°C.

We^ have data which indicate that some fif-
teen or more species of algae require a rapid cur-
rent at least at temperatures above 15°C. These
include species of Stlgeoclomum , Chaetophora ,
Gongosira , Oedogonlum , and Spirogyra as well as
most of the species of red algae we have collected.
A few crust-forming species of blue-green algae
almost certainly belong In this group also.

One of our most Important rapids species is
Oedogonlum kurzii . It Is a perennial In rocky
rapids especially in the Piedmont of the southeast.
It seems to occupy the same place In soft water
streams as does Cladophora In hard-water regions .
In late spring It forms great masses and skeins
over a meter in length, but by late summer it is re-
duced to short tufts attached to rock in the swiftest
water. During long sunny autumns it may again
become fairly abundant and it never completely
disappears all winter.

The problem of communities has received the
attention of numerous llmnologists and phycolo-
gists. Everyone who does ecological work must
consider It. Since there has been more work done
on the plankton than on other communities there
have been more attempts to classify the phyto-
plankton than other communities . Several hundred
papers deal at least in part with community rela-
tionships . Perhaps the best summaries and lists
of literature are those of Str^m (1924), Fritsch
(1931), Symoens (1951), Tiffany (1951) and more
recently Prescott (1956), and Blum (1956). In his
summary of the ecology of fresh-water algae.
Tiffany says, " . . .the ecological factors are iden-
tical with those affecting the larger land plants,
but the degree of intensity, the availability, and
distribution of such factors are different;. . .atten-
tion must be directed more and more to the micro-
environments of algae. Algal communities, though
quite distinct In many habitats , are more difficult
to define and delimit than associations of many
terrestrial seed plants . Successlonal phenom-
ena in the algae are often matters of seasonal

Schumacher and Whltford , unpublished data.

periodicity, determined by the occurrence and
length of the vegetational span of the species in-
volved . . . .Climax associations are approached
in some instances , but they are scarcely to be con-
sidered as the counterparts of such aggregations
among the higher plants of terrestrial habitats . "

He says also that it is almost impossible to
make a classification of algal communities that
will entirely separate one community from another
or that has ecological significance.

Each ecological investigator should deal with
the problem in his own way. He should use the
classification which seems to fit his data best, or
make his own. It is only in this way that a good ,
basic, and generally acceptable system of com-
munity classification will sometime be achieved.
I cannot refrain from again quoting from Tiffany be-
cause I recently found a statement of his which
clearly and succinctly expresses my own views,
" . . .we are as yet not in a position to formulate
very definite principles regarding algal associa-
tions /and7 successions, or even direct relation-
ships between algal productivity and special
causal environmental factors."

The last part of the above quotation is a good
way to introduce the problem of seasonal distribu-
tion . We have a relatively large amount of data on
the specific organisms abundant at the different
seasons, and on many of the associated habitat
factors . This is especially true of the phytoplank-
ton . Can we, however, even occasionally be sure
just what the chief causal factors are for seasonal
abundance, or more particularly for the sporadic
blooms of phytoplankton? This is even more true
for the littoral and lotic communities where we have
much less data. There is general agreement that
temperature and light are important in seasonal dis-
tribution, but these factors act on a whole spectrum
of autotrophic, heterotrophic, and parasitic organ-
isms. As pointed out above, we cannot sometimes
easily separate the effect of temperature from that
of light. Can we ever be quite sure, on the basis
of present data, that phosphorus or nitrogen is
really a limiting factor in a natural community, ex-
cept perhaps for a very short period of time? Some
of you have read numerous papers on phosphorus as
a limiting factor, but the problem does not seem to
be settled yet. Here, it seems to me, is where
combination laboratory and field studies of species
could give us some exact answers. Laboratory cul-
ture work should always be checked against a study
of the same species in natural communities . Only
such studies give promise of solving the problem of
the sporadic, objectional bloom as well as that of
seasonal fluctuations .

It is amazing to those who have had an in-
terest in fresh-water algae for many years, how
frequently a species first described from one local-
ity will turn up next in a locality very remote from
the first one. William Bailey (1851) wrote an

excellent paper on fresh-water algae collected
along the south Atlantic, coast. In this paper he
described a new desmid genus, Triploceras . His
two species were next collected in Scandinavia, I
believe. Triploceras is now known from all five
continents. Bailey's Ceratium carolinianum was
also next found in Scandinavia. The genus Oedo -
cladium was first collected in Germany, then
Virginia, Puerto Rico, India, and Australia. Oedo-

cladium operculatum described from Puerto Rico, is
common in India and is known also from Missis-
sippi. These few illustrations give the pattern, or
rather the lack of a pattern, in the known world
distribution of fresh-water algae. Sometimes one
thinks there is a pattern of distribution for certain
species. In 1935 Tiffany described a genus (Clon-
iophora) from Puerto Rico which somewhat resem-
bles both Stiqeoclonium and Draparnaldia . Re-
cently when I found the plant common in Peruvian
rivers in the Amazon watershed, I thought, "Here
is a tropical species . " When I came to examine
the literature, however, I found that the species
has been collected widely in the northern hemi-
sphere but reported under the name Draparnaldia
mutabilis .

In a review of the geographic distribution of
one of the best known families of green algae, the
Oedogoniaceae, Tiffany (1957) concludes that some
species (of Oedogonium and Bulbochaete ) are cos-
mopolitan, the largest aggregation of species
occurs in the temperate zone (where most collecting
has been done), a few species are arctic, many
species are both temperate and tropical, and a few
large species are almost exclusively tropical.
These conclusions are anything but concise and
definite but they indicate the state of our present
knowledge. Some of the supposedly rare species
have an interesting history. Cyclonexis annularis
Stokes, Chrysophyceae, was described from New
Jersey in 1886 . It was next found in Germany about
1910, then in Ohio in 1933 and in Massachusetts
in 1939 . A closely related species was described
from Russia a few years ago, and last winter we
found Cyclonexis annularis again, in North
Carolina. We have three second records of species
described in Europe, 30, 40, and 50 years respec-
tively after the original collection. The diatom,
Actinella punctata , until two years ago was known
from only two or three collections in the United
States . Since then we have found it at 12 locali-
ties in 10 counties in the Coastal Plain of North
Carolina . As many as 50 cells were seen in one
clump this past winter. Is it possible that if we
knew when and where to look there would be no
really rare species of fresh-water algae? I believe
this is probably true. Most genera have been in
existence for a very long time and individuals have
become widely distributed into suitable habitats
throughout the world . Just the other day I found
this statement in Str>:fm's 1924 paper, "They all

have In common that they do not possess any sharp
geographical distribution. They occur where their
claims upon the habitat are fulfilled".

Some species are more stenoecious in habi-
tat requirements than others; that is, they will
multiply and become abundant only within a narrow
range of habitat conditions. These are the so-
called rare species which are sometimes very
abundant when found. Sometimes they are thought
to be indigenous to a particular locality because
they have been found a few times in one locality
only. Is it not possible that most of them are
really widespread? Suppose a species is just
barely able to survive during unfavorable seasons
or conditions and becomes abundant only when the
habitat is exactly favorable. It would long be re-
garded as a rare species . If this theory is true how
do species survive during unfavorable periods.
Fritsch (1931) has discussed this problem. He
postulates that they may survive as spores or other
"resting cells, " but also suggests that they sur-
vive in the bottom ooze of the littoral zone, in case
of plankton species . We have considerable evi-
dence that this is true of both lotic and lenitic
species . Synura uvella can always be found dur-
ing the warmest weather in massive net collections
from North Carolina lakes. The colonies are very
rare and contain only a few cells, but they are
there . We have found occasional living but depau-
perate cells of Actinella in the bottom ooze of
streams where the water temperature reaches 27 C .
Batrachospermum seems to survive the summer as
very tiny rhizoidal, microscopic colonies on stones
in streams . I could give many other such in-
stances .

Many species of algae are more euryecious
in habitat requirements . They are abundant enough
under fairly favorable conditions to be collected
widely or at all seasons of the year. These are
the so-called cosmopolitan species. I suppose
Mlcrasteria radiata is likely to turn up in any col-
lection in the world where desmids are found at
all. Perhaps this species is just somewhat more
tolerant of unfavorable conditions than some, but
like them only becomes abundant when conditions
are exactly right .

I propose a theory of micro-habitats in both
time and space for species of fresh-water algae.
It Is suggested that stenoecious species survive
unfavorable periods or in unfavorable places as
resting cells or as vegetative cells in very small,
uncollectable numbers, that more euryecious
species survive in the same way but in numbers
great enough to be more often collected; but that
all species become abundant only under precisely
suitable conditions . This theory would explain
the sudden blooming of plankton species, the
marked seasonal pulses of others, and also the
apparent rarity of certain species and the general
and cosmopolitan distribution of others.

If an ecological approach were made on this
basis, that is on the basis that each species or
perhaps small group of species occupies a micro-
habitat, I believe it would help solve many of our
ecological problems. It would help in the matter
of an ecological classification, because the
smallest group of species occupying a particular
micro-habitat would be our smallest community. A
sufficient body of data regarding these habitats
would enable us to group them logically into larger
units. It would help solve the problem of seasonal
pulses and of sporadic blooms. When a narrow
band of blooming Microcystis occurs across one of
our larger lakes, the habitat factors in this area
must be just slightly different from those on each
side. If we knew the exact habitat of one of the
rare species we could predict where in the world it
could likely be found . We already have a fairly
large body of ecological data some of which might
be interpreted on this basis.

In conclusion, I should like to suggest to
phycologists; first, that students of flora try to re-
cord and publish more than bare date and locality
records, at least on apparently interesting species;
and second that there be more studies of species
ecology. Only from a backlog of accurate and de-
tailed data on species can a really good ecology
of fresh-water algae ever be derived.

The ecology of fresh-water algae will, for
many years, be very productive both of important
data and of new ideas . I strongly recommend it to
students with an interest in the algae .

Summary

The contribution of floristic studies and of
limnology to algal ecology has been pointed out
and also that certain concepts and terms have been
borrowed from these disciplines.

The importance of such habitat factors as
light, temperature, water quality, and speed of
current have been discussed together with some of
the problems encountered in obtaining accurate
data .

The fact that there is no entirely satisfactory
system of community classification was empha-
sized .

The problems of seasonal distribution and of
world distribution of fresh-water algae were briefly
discussed. It was indicated that data are lacking
for good correlation of seasonal distribution with
habitat factors, and likewise that we cannot yet
hypothesize accurately as to the character of world
floras .

A theory was presented that fresh-water algae
occupy micro-habitats and species frequently are
not abundant enough to be collected except when
growing under precisely suitable conditions .

Selected References

Allen, W. E . 1920 . A quantitative and statistical study of the plankton of the San Joaquin River and its
tributaries near Stockton , California, in 1913. Univ. Calif. Publ . Zool . 22:1-292.

Atkins, W. G. R. 1913. The phosphate content of fresh and salt water in its relation to the growth of
the algal plankton. J. Marine Biol. Asso. 13:119-150.

Bailey, J. W. 1851. Microscopical observations made in South Carolina, Georgia, and Florida.
Smithson. Contr. to Knowl . 2:1-48.

Blum, J. L. 1954. Two winder diatom communities of Michigan streams. Pap. Mich. Acad. Sci . , Art.,
Lett. 34:3-7.

Blum, J. L. 1956. The ecology of river algae . Bot . Rev . 22:291-341.

Blum, J. L. 1957. An ecological study of the algae of the Saline River, Michigan. Hydrobiologia
9:361-408.

Budde, H. 1928. Die Algenflora des Sauerlandischen Gebirgsbachen . Arch. Hydrobiol . 19:433-520.

Budde, H. 1932. Die Algenflora Westfallischen Salinen und . Salzgewasser . Arch. Hydrobiol.
23:462-490.

Butcher, R. W. 1933 . Studies in the ecology of rivers. I. On the distribution of macrophytic vegeta-
tion in the rivers of Britain. J. Ecol. 21:58-89.

Butcher, R. W. 1940. Studies in the ecology of rivers . IV. Observations on the growth and distribution
of the sessile algae in the river Hull, Yorkshire. J. Ecol. 28:210-223.

Butcher, R. W. 1942. Studies in the ecology of rivers . II. The microflora of rivers with special
reference to the algae on the river bed. Ann. Bot. 46:813-861

Butcher, R. W. 1946. Studies in the ecology of rivers. VI. The algal growth in certain highly cal-
careous streams. J. Ecol. 33:268-283.

Cedergren, G. R. 19 38. Reofila eller det rinnande vattnets algsamhallen . Svensk . Bot. Tidskr.
32:362-373.

Chandler, D. C. 1937. Fate of typical lake plankton in streams. Ecol. Monogr . 7:445-479.

Eddy, S. 1925. Fresh-water algal succession. Trans. Amer. Mic . Soc . 44:138-147.

Eddy, S. 1932. The plankton of the Sangamon River in the summer of 1929. Bull. 111. State Nat. Hist.
Surv. 19:469-486.

Fritsch, F. E. 1906. Problems in aquatic biology with special reference to the study of algal perio-
dicity. New Phytol. 5:149-169 .

Fritsch, F. E. 1929. The encrusting algal communities of certain fast-flowing streams. New Phytol.
28:165-196.

Fritsch, F.E. 1931. Some aspects of the ecology of fresh-water algae . J. Ecol . 19 :233-272 .

Fritsch, F. E. 1949. The lime-encrusted Phormidium -community of British streams. Verb . Int. Ver.
Theoret. Ang . Llm. 10:141-144.

Ferrell, J. K. , K. O. Beatty , Jr., and F. M. Richardson. 1955. Dye displacement technique for
velocity distribution measurements. Ind . and Engr . Chem. 47:29-33.

Hutchinson, G. E. 1944. limnological studies in Connecticut . VII. Examination of the supposed rela-
tionship between phytoplankton periodicity and chemical changes in lake waters . Ecol . 25:3-26.

Kofoid, C. A. 1903. The plankton of the Illinois River, 1894-1899, with introductory note upon the
hydrography of the Illinois River and its basin. Part I, Quantitative invesUgations and general
results. Bull. 111. State Lab. Nat. Hist. 6:95-629.

Lackey, J. B. 1939. AquaUc life in waters polluted by acid mine waste. (U.S.A.) Public Health Rep.
4:740-746.

Lackey, J. B. 1942. The effects of distillery wastes and waters on the microscopic flora and fauna of a
small creek. (U.S.A.) Public Health Rep. 58:253-260.

Lackey, J. B. 1942. The plankton algae and protozoa of two Tennessee rivers. Amer. Midi. Nat.
27:191-202.

Liebmann, H. 1951. Handbuch der frischwasser und abwasserbiologie , 5 39 p . R. Oldenbourg , Munchen.

Needham, J. G. and J. T. Lloyd. 1916. The life of inland waters. Ithaca. Comstock Pub. Co.

Newcombe, C. L. and J. V. Slater. 1950. Environmental factors of Sodon Lake - a dichothermic lake
in southeastern Michigan. Ecol. Monogr. 20:207-227.

Oberdorfer, E. 1928. Licgtverhaltniss und Algen-besiedlung im Bodensee . Zeitscgr. f. Hot. 20:465-5 68

Pascher, A. 1914-30. Die Susswasserflora Deutschlands , Osterreich u.d. Schweiz. Jena.

Powers, E. B. 1929. Fresh-water studies . I. The relative temperature oxygen content, alkali reserve,
the carbon dioxide tension, and pH of the waters of certain mountain streams at different altitudes
in the Smoky Mountain National Park. Ecol. 10:97-111.

Prescott, G. W. 1939. Some relationships of phytoplankton to limnology and aquatic biology. A.A.A.S.
Publication No. 10:65-78.

Prescott, G. W. 1951. Algae of the Western Great Lakes Region. Cranbrook Press.

Prescott, G. W. 1956. A guide to the literature on ecology and life histories of the algae. Bot . Rev.
22:167-240.

Purdy, W. C. 1916. Potomac plankton and environmental factors . In. H. S. Gumming. Hygien. Lab.
Bull. Publ. Health Serv. 104:130-191.

Relnhard, E.G. 1931 . The plankton ecology of the upper Mississippi, Minneapolis to Winona. Ecol.
Monogr. 1:395-464.

Roach, L. S. 1932. An ecological study of the plankton of the Hocking River. Bull. Ohio Biol. Surv.
5:253-279.

Rodhe, W. 1948. Environmental requirements of fresh-water plankton algae . Symb. Bot. Upsal.
10:1-149.

Rodhe, W. 1955. Can plankton production proceed during winter darkness in sunarctic lakes? Proc .
Inter. Asso. Thero. Appl . Lim. 12:117-122.

Ruttner, F. 1926. Bermerkungen uber den Sauerstoffgehalt der Gewasser und dessen respiratorischen
Wert. Naturwissenschaften. 14:1237-1239.

Ruttner, Franz. 1953. Fundamentals of Limnology. Trans, Frey, D. G. and F. E. J. Fry. Toronto.
The University Press.

Schiller, J. 1930. Kulturversuche uber den Temperature influss auf die Producttivitat des Wassers.
Zeltschr. f. Bot . 23:132-149.

Schumacher, G.J, A qualitaUve and quantitative study of the plankton algae in southwestern Georgia.
Amer. Midi, Nat. 56:88-115.

Smith, G. M. 1920. Phytoplankton of the inland lakes of Wisconsin. Parts I, II. Wis. Geol . and
Nat. Hist. Surv. Bui. 57.

Str;5m, K. M. 1924. Studies in the ecology and geographical distribution of fresh-water algae and
plankton. Rev. Algol . 1:127-155 .

Symoens, J. J. 1951. Esquisse d'un systeme des associations algales d'eau douce. Verh . Int. Ver.
Theoret. Ang , Lim. 11:395-408.

Tiffany, L, H. 1936. Willie's collection of Puerto Rican fresh-water algae. Brittonia 2:165-176,

Tiffany, L. H. 1951. Ecology of fresh-water algae . In Smith, G. M., Ed. 1951. Manual of Phycology .
Chronica Botanica Co., Waltham, Mass.

Tiffany, L. H. 1957. The Oedogoniaceae III. Bot. Rev. 23:47-63 ,

Tllden, J. E. 1935. The algae and their life relations. Univ. Min. Press.

Transeau, E. N. 1916. The periodicity of fresh-water algae. Amer. J, Bot, 3:121-133.

Wade, W. E, 1949, Some notes on the algal ecology of a Michigan lake. Hydrobiologica 2:109-117.

Welch, P. S. 1947. Eutrophication of lakes by domestic drainage. Ecol . 28:383-395.

Welch, P. S. 1952. Limnology. McGraw-Hill.

West, G. S. 1904. A treatise on the British fresh-water algae. Cambridge.

West, G. S. 1909. The algae of the Yan Yean Reservoir Victoria . J. Lin. Soc . of London 39:1-88 .

West, W. and G. S. West. 1912. Periodicity of the phytoplankton of some British lakes. J. Linn. Soc,
Bot, 40:395 ,

Whitford, L. A. 1943. The fresh-water algae of North Carolina. J. Elisha Mitch. Sci . Soc. 59:131-170.

Whitford, L. A. 1956. The communities of algae in the springs and spring streams of Florida. Ecol.
37:433-442,

Wien, Janet D. 1959. The study of the algae of irrigation waters. 2nd. annual progress report,
(mimeographed) Arizona State University, Tempe .

10

ALGAL POPULATIONS IN FLOWING WATERS

John L. Blum
Canisius College

The problems encountered in dealing with or-
ganisms that inhabit a current are different in many
ways from those met in the study of communities of
standing water. The fickle, unstable nature of both
the surrounding medium and the solid substratum;
the insecurity of being unattached in a current; the
enhanced chances of survival bestowed by a pro-
tected nook wherein to live and grow; the physio-
logical and mechanical stress which continued
pulling, twisting, and abrasion provide; the linear
alternation of the strikingly different environments
of riffle and pool; the continual supply of fresh
nutrients, along with silt and other debris from up-
stream area — all these factors so influence river
algae as to render the problems inherent in sam--
pling, counting, and even in determining them, dif-
ferent in certain ways, and often more difficult,
than those involved in working with algae from still
water .

QUANTITATIVE METHODS
OF SAMPUNG STREAM ALGAE

Most of the special methods of sampling river
algae which have been devised are intended for
studies of the bottom vegetation. Of course, this
vegetation can simply be pulled up or scraped off
with a knife. Other methods better suited to give
quantitative data on the vegetation have been de-
vised by many workers . Probably the best known
method is that of Butcher (1932) who used a sub-
merged photographic frame of the type used for
printing postcards. The frame, holding five micro-
scope slides, is fastened horizontally in the river
and held by chains which are stretched tightly on
iron stakes driven into the river bed .

This method was used throughout a long
series of government studies of British streams, in
which Butcher took part. These studies provided
the foundation for the most extensive information
we have about stream vegetation of any given
region of the world . It permits quantitative ap-
praisal of the early stages of algal growth. The
method has not been adapted to long periods of ob-
servation, and no proof has apparently been given
that algae colonize the slides in quite the same
way that they colonize the river bottom. There is
some evidence that numerical results from slide
counts are not, in fact, comparable to those from
the river bottom, but that the species present are
generally similar (Reese, 1935). However, a few
species of algae, which are almost universally
present on such slides, are little known from natu-
ral substrata . Large algae may easily be torn off

smooth surfaces , and some algae may colonize
such a glass surface very slowly or simply show
slow growth (Lund and Tailing 1957). This method
of Butcher is hence somewhat inadequate, and par-
ticularly so, in recording changes in the mass or
volume of the vegetation or its components . This
is a serious drawback, for such changes can be
spectacular, as well as inexplicable.

Among measures designed to measure epi-
phytic growth may be mentioned the method of
Thurman and Kuehne (195 2) . They used this method
for epiphytes of Cladophora glomerata (L.) Kutz.,
but it could be easily applied to those of many
other filamentous algae. A cylinder of algae 1 cm.
thick is prepared, and pieces 1 cm. in length are
cut from this. The individual 1 cm. X 1 cm. cylin-
der pieces are placed in preservative, shaken, al-
lowed to stand for a day; and counts of the epiphy-
tic algae are made from the sediment contained in
the liquid .

Algae, such as Oscillatoria and various other
blue-greens which live unattached on sediment, are
difficult to collect by means which permit quantita-
tive comparison between different collections . Soft
deposits may be sampled with a suction-tube sam-
pler, using a hand- or foot-operated pump and
sucking the surface deposit through a funnel which
is passed over the deposit in the manner of a vac-
uum cleaner. The method does not permit the col-
lection of comparable samples from different types
of substrate, nor give the same ratio of mud to
water from any one area on each sampling (Lund and
Tailing 1957).

Margalef (1949a) has devised a method for
stripping the algae from surfaces of rocks from the
stream bed. After fixing, staining, and dehydrating
the algal layer in situ , a collodion solution is
poured over it and, when dry, is removed from the
surface of the rock. The method, similar to the one
commonly used for fossil material, is claimed by
Margalef to permit accurate counts of the popula-
tions present .

As a means of estimating algal growth on the
bottoms of shallow rocky streams, I employed a
modification of the transect method. A rope was
marked off into decimeter and meter units and
stretched across the stream at a riffle, just above
the water surface. Presence was recorded in alter-
nate decimeters of all visible algal species, an in-
dividual or colony of which, is crossed by the rope.
The importance of the species was thus determined
by the relative number of decimeters within which
that species was present. Frequency was deter-
mined for each visible species by estimating the
relative percentage of total decimeters in which it

11

was found in relation to the total number of deci-
meters whose composition was recorded. All tran-
sects were located with the intention of providing
a typical cross-section of the riffle and were ori-
ented at approximately right angles to the direc-
tion of water movement (Blum, 195 7) .

This method also has many drawbacks . It is
not applicable to pools and is workable only with
difficulty on riffles when the water is 20 cm. or
more in depth and turbidity is high — conditions
which are likely to be synchronous in a small
stream .

Douglas (1958) has devised several useful
instruments for working with epilithic algae . One
of these consists of a short, tuft-like brush of
nylon bristles, provided with a handle of steel rod.
After removing a specimen rock from the water, a
50 ml. polythene bottle with bottom sawn off is in-
verted over the surface to be sampled . The brush
is inserted into the bottle and the rock surface de-
limited by the neck of the bottle is scrubbed clean.
Washing and brushing are carried out over an en-
amel sorting tray so that none of the sample is
lost. From the area of the polythene bottle neck
and the number of stones used in sampling, it is
possible to calculate the total area of stone sur-
face thus cleaned.

Another, more complex tool was devised for
the sampling of algae from stones under water.
Here the brush used is similar but has a hollow,
tubular handle. The upper end of this handle con-
nects by means of a length of rubber tubing to a
specimen tube. The area brushed is delimited by
a steel casing which shields the sampling area
from the current and prevents the algae from wash-
ing away. A sponge rubber ring at the base of the
casing tube provides a tight seal against the rock
surface. During sampling with this equipment,
algal material brushed from the rock surface is
sucked up into the specimen tube through the hole
in the brush. From the area covered by the inner-
most part of the casing and the number of such
areas cleaned, the total area sampled can be cal-
culated .

A third device, employed for the sampling of
epiphytes on aquatic bryophytes, was developed by
Douglas. This consists essentially of a hardened
steel tube or borer sharpened at one end and a wire
plunger which can be pushed through it. The borer
is forced through the moss layer against the under-
lying rock, thus cutting out a small cylinder of
vegetation which is subsequently removed by the
plunger. An elaborate cleaning process follows.
As in the other two methods, counts of the sample
are made microscopically after delivering portions
of the sample to a counting cell (Douglas, 1958) .

Among the algae, which the stream collector
records by these, or other methods, are forms
which are characteristic of standing waters, as
well as others which are indifferent; still others

which are practically limited to running water.
Some of these latter forms have been recorded from
so many streams, particularly of Europe, that it
becomes possible to correlate the data from many
surveys and to a degree, to characterize the usual
habitat of each alga . Much of the following infor-
mation on algal species has been taken from the
recent paper of Hornung on the Echaz (1959).

ECOLOGICAL CHARACTERIZATION
OF RHEOPHILIC ALGAE

Achanthes lanceolata Breb . This is a diatom
common and widespread in alkaline streams, and
particularly common in springs and small brooks.
It exhibits a degree of resistance to certain poi-
sons and to sewage pollution (Schroeder, 1939).
It exhibits a spring growth maximum, according to
Hornung (1959), and a maximum in spring and fall
in the results reported by Schroeder. Schroeder
states further that in polluted water, its growth
maximum is attained in winter.

Cocconeis placentula Ehr . , frequently an
epiphyte, grows best and is most common in run-
ning water, in mildly alkaline streams. It is
somewhat sensitive to pollutants and seems to ex-
hibit a growth maximum in autumn .

Diatoma vulqare Bory is common and wide-
spread, especially in running water, and prefers
somewhat alkaline water. According to Kolkwitz
(1950) and Hustedt (1944), it is characteristic of
the ^-mesosaprobic zone or the region of advanced
oxidation of pollutants in a polluted stream. But,
I have found its best growth in the oligosaprobic
portion of a polluted stream, and various authors
have credited it to every one of the principal zones
of the saprobic system. In the streams of southern
Michigan, which I studied, it was the dominant
form, covering all available rocks in the current
during the fall months, and again, in spring,
(Blum, 195 7). A related species, D_. hiemale
(Lyngb.) Heiberg is characteristic of mountain
brooks .

Gomphonema angustatum (Kutz.) Rabh . is
typical of small brooks and is adapted to alkaline
waters. Kolkwitz (1950) considers it to be an oli-
gosaprobic form, but Hornung reports it from highly
polluted localities .

The similar Gomphonema olivaceum (Lyngb.)
Kutz. is not confined to running water but forms
thick, gelatinous mats in favorable stream situa-
tions. In the Michigan streams investigated, it
replaced Diatoma yulgare in winter and became a
strong competitor for favorable sites with Diatoma
in the following spring. In European streams this
form seems to be associated with polluted waters,
occupying the mesosaprobic zone, according to
Kolkwitz (1950), Fjerdingstad (1950), and Liebmann
(1951). Melosira varians C.A.Ag. is found in both

12

standing and flowing eutrophic waters. Both
Kolkwitz and Liebmann consider it to be character-
istic of the/9-mesosaprobic zone. Growth maxima
of this species have been found both In spring and
In summer. Nltzschia linearis W. Smith is a true
rheobiont, characteristic of springs and flowing
water. The related species, Nltzschia palea
(Ku'tz.) W. Smith is perhaps the most resistant and
most tolerant of all diatoms , a eurytopic, euryhaline
eurythermlc form which grows conspicuously in
polluted water where it forms a rich brown surface
layer on rocks of rapids, as well as in quiet water
on shallow silt banks. Both Kolkwitz and Liebmann
place it In the <l-mesosaprobic zone of incipient
oxidation In a stream, and It is often used as an
Indicator of polluted water although Its ubiquitous
tendencies permit it to grow throughout the entire
gamut of water purity.

Hildenbrandia rivularis (Liebm.) J. Ag. is a
rheobiont, a crust-forming red alga which is found
In many hard waters of Europe. It seems to be ab-
sent, however, from streams with extremely high
calcium content . In a paper reviewing its occur-
rence, Luther (1954) has concluded that it is
limited to shaded situations. Thus, it is often
found in rather deep clear water, being at the ex-
treme outpost of macroscopic vegetation due to
light limitation. In sunny situations and in clear
water it may be found only on the under side of
rocks . Whereas, if the water is turbid, it is more
likely to be found on the upper side. In the waters
of Lough Neagh in Ireland it was not found on
basalt or other dark-colored rocks but was found on
the under side of quartz rocks where it apparently
received most of its light directly through the rock .
It seems to have a wide amplitude of tolerance for
temperature, as well as for organic pollution. Its
absence from otherwise suitable waters has been
Interpreted as brought about, in some cases, by
competition with other forms; in other cases, by
deposition of silt around rocks inhabited by young
plants (Luther, 195 4) .

Ulothrix zonata Kii'tz. is a rheobiont in-
habiting cool, well-oxygenated streams. It shows
a vegetative growth maximum in late spring and
early summer. Fjerdingstad (1950), Kolkwitz
(1950), and Liebmann (1951) characterize it as
ollgosaprobic in habit, but there is some indica-
tion that its growth is enhanced in polluted waters.

Cladophora glomerata L.) Kiltz . is one of the
largest and most widespread of rheoblontic algae.
It is known to be highly sensitive to Iron and rela-
tively tolerant of high pH values . It seems to re-
quire alkaline and calcareous water. It is tolerant
of relatively large amounts of sewage in the water
and of weak salinity. The length of Its summer
growth period varies from a few days to several
months . Usually it is Inconspicuous In winter al-
though Its full development has been recorded in
February and March, as well as in October; and

Jaag (1938) has observed it throughout the winter.
It can apparently produce swarmers throughout the
entire year but is probably sensitive to tempera-
tures higher than about 25° C . After periods of
turbidity, the plants of Cladophora often become
heavily loaded with silt and epiphytes, a factor
which may be significant in the suppression of
growth or in the mechanical detachment of the
thallus .

Stigeoclonium tenue Ku'tz. is a large, com-
mon stream alga which thrives in polluted waters
of the H- and^-mesosaprobic zones and is sel-
dom found growing luxuriantly elsewhere . Budde
(1930) concluded that it favored cool waters, but I
have found it attaining its best growth in early
summer and midsummer. Like Cladophora glom -
erata it is likely to be found in the most rapid
water of a brook, occupying the crest of a shal-
lows. Its preference for polluted water is not
limited to organically enriched water, for it can
frequently be found downstream from industrial out-
falls (which, however, frequently contain sewage
wastes) whereas it is apparently absent upstream
from the outfall .

Hydrurus foetidus (Vill.) Trev . , a filamentous
member of the Chrysophyceae, is a rheobiont
adapted to cold water (approximately 0°-16 C.) .
Like Hildenbrandia it appears to favor shaded situ-
ations . In Europe, where it has been found much
more frequently than in North America, its growth
maximum is apparently attained in spring and in
autumn .

Phormidium autumnale (Ag.) Gom. is common
in hard-water streams and is frequently associated
in polluted water with Sphaerotilus and with Stige -
oclonium tenue . With the sheaths which surround
its filaments, it forms mats of medium thickness
and rubbery consistency which adhere to the sur-
faces of rocks in the stream bed .

The species I have taken up here are only a
few of many common and widespread stream forms ,
and I have tried to give merely general information
on the ecological preferences of most of them.
Much more precise data on the diatom species as
concerns their halobion spectrum, the pH spectrum,
and the current spectrum are available in the recent
work of Foged (1947-48, 1954) .

ECOLOGICAL CLASSIFICATION
OF RHEOPHIUC ALGAE

The Naming of Communities. — In streams it
is the exception, rather than the rule, to find ex-
tensive areas inhabited by recognizable groups of
algal species which maintain a definite order of
dominance or inferiority with respect to each other
throughout all the seasons, although this relation-
ship is more commonly found If higher plants are
considered. Many algal populations appear to be

13

relatively haphazard mingling s of two to many un-
attached species. These groupings may exhibit no
constant composition from place to place within the
stream. Frequently , too , their composition changes
rapidly from week to week. In the composition of
any community of aquatic algae, chance probably
plays a major role throughout the formative period ,
which may last for many years, perhaps centuries.
Thus, it is only in large, old, aquatic situations
that one may hope to find associations so stable
that the absence of one form or another would be of
real biological significance (Symoens , 1951). The
availability of numerous diatom and other species,
any one or any combination of which is able to take
over a site and quickly achieve local dominance,
makes it particularly difficult and unrewarding to
apply formal names to such groupings. Only when
there is (l) a permanent, distinctive community
structure which can be found repeated again and
again in a series of streams with essentially the
same species composition of algal dominants
throughout the series, or (2) a consistent reap-
pearance of a ;pecific algal vegetation in a given
season of the year, does there seem to be suffi-
cient grounds for applying a formal ecological name
to the vegetation. It seems preferable to deny for-
mal status to the less frequently observed combin-
ations . Just as in land vegetation, the application
of formal names to all types of variant transition
combinations found along ecotones is liable to
create confusion without serving any redeeming
practical purpose. It is futile to name, aquatic
communities without evidence that they are of more
than purely local occurrence. Panknin (1945)
points out that phytoplank* on can, in this way, be
divided up into an endless series of associations .
In the benthos, extensive stands of one or more
species of dominant algae are relatively more com-
mon than among land communities . Often these
stands are unialgal, or nearly so, to the exclusion
of other green plants below them. Often they grow
with great rapidity , carpeting the stream floor in
ten days or less when they experience favorable
environmental conditions . Many small species are
epiphytes (or endophytes) on larger algae which
support, and to a certain extent protect, them. The
ephemeral nature of attached algae is, furthermore,
one of the unique features of the stream communi-
ties . With the detachment of a large thallus, it,
and its entire collection of epiphytes, is immedi-
ately taken downstream. All these organisms with
the nutrients they contain are effectively lost to
the local ecocosm.

These are rather deep-seated differences
from the conditions on land and from the conditions
in standing water. It has been suggested that we
may be basically in error in attempting to apply to
the hydrosere terms such as "climate" and "climax"
which were first used for land vegetation and which
may, upon scrutiny, carry with them too many im-

plications of no validity in hydrobiology . For one
thing, a plant community which takes over a site
and reproduces there, maintaining dominance and
successfully resisting invasion so long as en-
vironmental conditions remain substantially un-
modified, has fulfilled the conditions for a climax
community . But , with reference to river algae ,
this community may not have been preceded by any
other green plants whatever — colonization and dom-
inance are thus synchronous . With no previous
community occupying this site, succession has not
occurred and use of the term "climax" seems in-
appropriate (Blum, 1956a) .

Eddy has concluded that permanent fresh-
water communities exist, reach maturity and show
aspects comparable to terrestrial communities, and
he points out that the maintenance of given cli-
matic conditions necessary for the establishment
of a "climatic" climax is not confined to land com-
munities, but can also be found in permanent
streams (Eddy, 1934). The generation and the life
span of dominant algae are so much shorter than
those of most dominant vascular plants that "perm-
anent" climatic conditions can be achieved in a
relatively shorter time and more rapid succession
is to be expected. For extremely short-lived mic-
rophytes, a single growing season in temperate
regions may be sufficiently long to represent a
"permanent" climate (Blum, 1956a).

Panknin (1941) has discussed the classifica-
tion of seasonal communities which assume tem-
porary dominance upon a given site and has con-
cluded that such algae should not be regarded as
constituting seasonal associations but rather as
making up seasonal aspects of the entire associa-
tion. He considers that community names based
on only two diagnostic species are inadequate if
only one season has been studied, and the aspect
of the vegetation in other seasons is unknown.
The "true" algal community recognized by Panknin
includes no higher plants and occurs only on the
seacoast, in deep mountain lakes, in the phyto-
plankton, and in streams. All other algal com-
munities are "dependent, " the algae being merely
an undergrowth in an assemblage of higher plants .
(Panknin, 1945) . This refusal to recognize many
common algal communities separate from those of
higher plants around them led, as Symoens (1951)
has pointed out, to lumping them with plants
mostly larger than themselves and to designating
such heterogeneous communities as the Micraster-
ieto-Sparganietum. Various communities have been
named exclusively on the basis of desmids present
or on diatoms present while, at the other extreme,
Margalef (1947, 1949) understandably introduces
even Protozoa, Gladocera, Copepods, and insect
larvae into associations made up partly of algae
(Symoens, 1951) .

The Classification of Stream Algae According
to Structural Adaptation. — Among schemes of

14

classification which have been devised for the
forms of plant body possessed by rheobiontic algae,
that of Cedergren, sketched in brief form In 1938,
is worth attenUon. Cedergren points out that ben-
thic algae, which inhabit a current, exhibit adap-
taUons of four principal types: (1) Richly branched
filaments. These have strong anchorage and water
may pass freely between adjacent filaments. The
presence of a slippery gelatinous external coat is
noteworthy in many of these such as Batrachosper -
mum, Draparnaldia , and Stigeoclonium . (2) Long
flexible cylinders. These align themselves per-
fectly with the current and thus offer little resis-
tance to the water. (Encyonema spp. , Tetraspora
cylindrica (Wahlenb.) Ag . or Splrogyra fluviatilis
Hilse) The presence of a firm attachment and a
slippery exterior is remarkable in this group.
(3) Spheric, wart-like or cushion-like colonies.
These show numerous contained filaments as in
Chaetophora or Nostoc which, because of the
smooth external surface of the colony, offer little
resistance to water movement. (4) Reduced, sim-
plified, plate-like forms. These grow in a thin,
appressed sheet. Although diverse in internal
structure, externally such sheets are similar in
usually following the contours of the rock or other
substrate and in exposing relatively little surface
as a colony to the water in relation to the enormous
surface of the individual filaments or other units .
Hildenbrandia and Phormidium are examples .

These four types can actually be further re-
duced. Groups 1 and 2 are those with flexible
thalli which permit water to run through them and
thus expose a large surface to the water. Groups
3 and 4 have inflexible bodies and a greatly re-
duced surface . A number of low-growing green and
blue-green algae do not seem to fit into this system
very well. Nevertheless, a great majority of
stream algae would fall under Cedergren 's groups
1 and 4 .

Named Benthic Communities of Algae. — A
number of authors have catalogued stream commun-
ities which have come under their observation, but
relatively few have made a synthesis of observa-
tions from other regions with their own. Among re-
cent contributors to algal sociology, Symoens, who
has done important work on streams in Belgium, has
presented an outline of algal communities based on
(1) floristics, (2) aspect (physionomie ) , (3) syn-
genetic relationships, and (4) synecology. The
system includes eighteen alliances which he lists
"timidly and provisionally. " These include three
alliances of stream algae, as follows: (1) epil-
ithic algae and crustose lichens, (2) benthic dia-
toms and (3) filamentous green and red algae
(Symoens 1951) . Mention was made of certain
associations represented by each heading, but
without extensive discussion or description. These
associations were amplified in a subsequent study
of streams in the Ardennes region (Symoens, 1957) .

In England, Butcher (1946) has recorded what
he considers to be two distinctive communities.
The first of these, Achnanthes microcephala —
Chaetopeltis , with associated Diatoma hiemale and
and Eunotia, is the characteristic oligotrophic com-
munity in the streams studied. With increasing
eutrophy in downstream areas, it may be replaced
there by the Cocconeis - Ulvella - Chamae siphon
community. This is a eutrophic association which
shows no seasonal periodicity and which is com-
posed principally of Cocconeis placentula Ehr . ,
Achnanthes minutissima Kiitz . , Ulvella frequens ,
Chamaesiphon incrustans , Grun . and Chamaesi -
phon irregularis .

Butcher's group observed the dominance of
the first of these communities in the upper reaches
of the Tees. This was replaced by the second
wherever sewage drains flowed into the river in
quantity. The first, or Achnanthes - Chaetopeltis ,
reappeared wherever the dilution was sufficient and
decomposition rapid. Margalef (1948) has referred
under a different name (Asoclation Hydrococcetum
rivularis) to what appears to be a directly com-
parable community living in streams of the
Pyrenees .

Another community characteristic of the upper
reaches of hard water streams is a crustose com-
munity dominated by Phormidium , Schizothrix , and
Audouinella , and which is known also as the Chan-
transieto-Phormidietum incrustans (Symoens, 1957).
It has been identified in Austria, Belgium, England,
and the United States of America. It is probably
widespread in hard water streams, perhaps through-
out the world. The principal blue-green components
of this crust, Schizothrix fa sciculata , S . pulvinata
and S. lacustris grow on submerged rocks, forming
dense tufts of radiating filaments which may be
only .5 mm. in height. As the Schizothrix grows,
two other species frequently invade the tufts: Phor -
midium incrustatum , whose filaments mingle with
the tufts and grow, with either horizontal or verti-
cal orientation, twisted in and out between the
Schizothrix filaments; and Audouinella sp. , which
grows radially like the Schizothrix out to the limits
of the tufts, where it branches while its upward
growth keeps pace with, or slightly surpasses, that
of the Schizothrix and Phormidium species . These
four or five organisms constitute a single layer, and
apparently all of them may secrete calcium carbon-
ate abundantly, thus converting the entire tuft, or
stratum of continuous tufts, into a crust. This
crust becomes especially stony and resistant in the
presence of a dense admixture of Phormidium in-
crustatum . After growth for a year or more, the
tufted layer may eventually attain a thickness of
4-5 mm. Frequently, at seasons when other ben-
thic algae are abundant within the stream, it
becomes an inferior layer, shaded and more or less
enveloped by temporary dominants such as Clado-
phora qlomerata (Blum, 1956).

15

Among the many benthic diatom communities
which have been named, I shall mention only a few.
The Pi a to ma vulgare - Melosira varians community,
characterized by a substantial development of
species of alkaline and eutrophic waters . Under
typical conditions, at least in the Belgian streams
studied by Symoens (1957), few non-diatomaceous
algae are included. With time, Ulothrix , Clado -
phora , Vaucheria and Oscillatoria make their ap-
pearance. The latter is associated especially with
organically enriched waters . When these algae be-
come dominant, the physiognomy of the vegetation
changes completely, and its formal classification
under the alliances listed by Symoens would shift
from group 2 (benthic diatoms) to group 3 (green and
red filamentous algae) (Symoens, 1957).

Another diatom community is the Diameto r
Meridionetum of Margalef (1948) and others . It is
represented in various forms in different streams,
with many species included, but the diagnostic
ones are Diatoma hiemale and Meridion circulare
Ag. In the Ardennes, Symoens (1957) considers
this community to be associated with considerable
elevation (150-2500 m.) and Margalef states that
it Is confined to waters of temperature between 10°
and 15° C.

Under the associations of green and red fila-
mentous algae Symoens has listed, among others a
Cladophora qlomerata association, known from
Belgium, France, Western Germany, Catalonia,
Switzerland, Balearic Isles, the U.S.A., and, with
associated Podostemaceae, from central Africa.
The diatoms associated in the Ardennes region are
those of the Diatoma vulgaris - Melosira associa-
tion . Cocconeis pediculus is a widespread, epi-
phyte on the Cladophora .

Another filamentous association listed is
that of lime-rich waters dominated by Vaucheria
spp. , especially (in the Ardennes) V. debaryana
Woronin, and characterized by much the same dia-
toms that are associated with the Cladophora vege-
tation.

An additional Vaucheria Association is listed.
However, this is Vaucheria of soft waters and is
accompanied by diatoms of the Diatoma hiemale-
Meridion circulare community .

It is clear from these brief examples taken
from the literature that a beginning has been made
in the cataloguing and classification of stream
communities. Work in future years will, no doubt,
increase our knowledge of the validity and floris-
tic affinities of the associations named thus far
and give us more than a pioneer explorer's view of
the vegetation of streams of the world .

DISTRIBUTION OF ALGAL POPULATIONS IN TIME

Colonization and Succession. — From his
work on the Hull (England), Butcher has concluded

that colonization there reaches a maximum in May.
Individual dominant species have periods of most
rapid reproduction in various months from March
through October, and the period of least coloniza-
tion is in winter. In summer, colonization appears
to be complete in about 20 days, but in cold months
when growth is slower, 30 to 40 days are required.
It might be supposed that vigorous current would
impede algal colonization on so smooth a surface
as a glass slide, but more algae are produced on
the slides in regions of rapid flow than elsewhere.

In southern Michigan, colonization of rock
surfaces by winter-dominant diatoms is very rapid,
and macro scopically visible colonies can form in as
little as ten days . The period within which Gom-
phonema olivaceum colonized bare rock surfaces
extended from late November to early April, and
colonization appeared to be possible at any time
within this period. No evidence of succession
was found prior to the establishment of this com-
munity, and the same may be said for the commun-
ity of Diatoma vulgare characteristic of late fall —
both these forms were at one and the same time
colonists and seasonal dominants (Blum, 1956).

In the work of Butcher there is likewise little
evidence of succession in the algal communities he
has investigated . In the Cocconeis — Chamaesi -
phon community, Chamaesiphon arrives in the en-
semble later than the other forms, but there is no
true succession. Certain of these communities are
regarded as representing a true climax, but their
development is apparently accomplished with no
steps separating invasion from climax conditions
(Butcher, 1946) .

Periodicity of Algal Populations. — The plank-
ton algae of a great many streams have now been
investigated for sustained periods. Most of these
streams are in Europe, but some of those in the
United States have been extensively studied also,
and a few tropical streams are represented. Many
streams exhibited considerable consistancy, during
the period and to the extent of the observations
made, as regards the time of development of the
greatest densities of their plankton or of their ben-
thic vegetation, whereas other studies have pointed
out sharp differences in the year-to-year develop-
mental pattern. A mere year's work upon a single
stream may prove inadequate in that it reveals con-
ditions essentially unlike the preceding and fol-
lowing years . In nearly all streams investigated ,
the principal phytoplankton pulse, if there is one,
is to be expected at some time during the warm
season, but individual plankters, e.g., Crucigenia
rectangularis , Pediastrum boryanum , Fraqilaria
capucina , Meridion circulare and Synedra ulna ,
frequently exhibit population maxima in winter.
Many streams are characterized by two separate
periods of maximum plankton abundance. Some
species of algae exhibit great variability in pro-
duction, remaining uncommon in one year and

16

becoming a dominant in the next, exhibiting a
pulse at widely variable times throughout the year,
or a single pulse in one year and a bimodal one in
another. Certain diatoms tend to exhibit a pulse
in spring and/or in autumn rather than in midsum-
mer .

Occasionally a river may develop a "bloom",
although this phenomenon is more frequently seen
in ponds. Organisms which have been found re-
sponsible for such blooms include Thalassiosira
fluviatilis (Weser, Germany), Synedra delicatis -
sima (Potomac, U.S.A.), Microcystis flos - aquae
(Bug, U.S.S.R.), Anabaena spiroides (Mayenne,
France), Aphanizomenon flosaquae (Don, U.S.S.R.)
and Pandorina morum (Cumberland, U.S.A., and
Kentucky, U.S.A.) (Blum, 1956). Lackey lists no
fewer than 25 separate blooms for the Clinch River
and adjacent waters in 1956 with a great variety
of organisms represented (Lackey, 1958).

The best correlations which have been ob-
tained by plankton work and chemical analyses
over a period of years point to a time relationship
between abundant nutrients and abundant phyto-
plankton, the plankton pulse usually following the
period of highest concentration of the nutrients, as
nitrates and nitrites, in such a way that the de-
crease in nutrients precedes by a few days to a
few weeks the maximum development of phytoplank-
ton.

A diurnal plankton pulse has been observed
in a polluted stream, apparently dependent upon
and produced by midday sunlight which causes
benthic forms to rise into the current and be car-
ried downstream . Evidence that planktonic forms
reproduce as they are carried downstream has been
presented by various workers, but there remains the
suspicion that much of the actual cell division oc-
curs on the bottom and that the apparent increase
in phytoplankton downstream is largely the result
of more extensive nutrient beds there, and of
denser populations of benthic individuals, many of
which rise every day into the plankton. The vege-
tative dissemination of Spirogyra and Oscillatoria
communities was observed by the author on warm
summer days in 195 2 and '5 3. These communities
were especially characteristic of quiet shoals or
bays of the stream. Here the algae remained on
the bottom in contact with nutrient-rich silt de-
posits, as masses of filaments easily visible from
a distance. The surface water of such shoals and
bays is usually in slow circular movement set up
by the main current of the stream, which by-passes
the shoal or the bay in a tangent to the circular
current which it produces there . At times of rapid
photosynthesis, individual masses of the algal
filaments are detached and buoyed upwards by
trapped oxygen bubbles . Once the algal mass has
quit the floor of such a shoal, it is carried slowly
along in the eddying surface water. After moving
for some time in this circular manner, it may even-

tually be picked up by the tangential current of the
main stream which removes it definitively from the
shoal. As the algal mass travels downstream. It
disseminates live filaments along the way. The
progress of these filaments Is arrested on obstruc-
tions or on new shoal areas or other sediments
downstream, which in this way are themselves
colonized. The elevation of algal masses by en-
trapped bubbles can be observed from about noon
until about 2-3 p.m. on sunny days in summer, and
the movement downstream of these floating masses
can be observed throughout an entire afternoon
(Blum, 1956).

Under benthic algae are included both sea-
sonal and perennial species. While a single alga
may be dominant over relatively long reaches of the
stream's course, it is more common to find a num-
ber of dominants with different parts of the stream
having different dominant communities. In some
streams the algal vegetation remains much the
same throughout the year, whereas in others there
are marked seasonal aspects. As with plankton
algae, the seasonal variation may be summarized
broadly as maximum development in warm months,
followed by minimum development in cold months.
But, many algae behave quite differently. To what
degree these seasonal changes are due to some
environmental factor, has not been established.

DISTRIBUTION OF ALGAL POPULATIONS IN SPACE

Depth zonation .--The relation of depth to
the algae of rivers has received relatively little
attention . On the bank of the polluted Mulde
River, Schroder (1939) has illustrated a series of
algal zones with Spirogyra spp . just below the
water surface, Stigeoclonium tenue and then Oscil-
latoria spp. further down, and Sphaerotilus spp.
and Nitzschia spp. in the deepest water. In cer-
tain lower portions of the Meuse, Symoens has de-
scribed a distinctive zonation consisting of
Rhizoclonium sp . just above the usual water level,
Banqia atropurpurea at the level washed by the
water's edge, in a band 10 to 20 cm. wide, and
finally Cladophora glomerata , below the water
level and mixed with certain mosses (Symoens,
1957). It is probable that nearly all streams which
maintain a given level for several weeks exhibit
some zonation of the attached algae. Figure 1
illustrates a type of zonation, involving two or
three species , which has been noted in southern
Michigan streams. Scheele (1954) has recorded
diatom zonation in drains (SchleusenwSnde) in
Germany . The most outstanding difference with
increasing depth was a decrease in numbers of
Nitzschia palea . N . frustulum was found most
commonly in a transition zone intermediate in
depth and in available light.

17

UiothriXv

water surface

Gomphonema

DiatOTTlQ

other on the stream profile determine the laws of
distribution of the animal population as well.
Succeeding riffles or shallows frequently carry the
greater volume of water on alternating sides of the
stream, so that erosion is greater first against the
right bank and then against the left. This asym-
metrical pattern results in asymmetric distribution
of the benthos biocoenoses of the pools, with an
accompanying break in their continuity at every
riffle . Ulothrix spp . , Stigeoclonium tenue and
Diatoma vulgare are all characteristic of riffles
and regularly drop out as massive components of
the vegetation wherever pool conditions obtain.
Spirogyra spp., Euglena spp . and other mostly un-
attached forms naturally collect where current is
minimal. Within a riffle or shallows itself, cer-
tain areas are apparently much more favorable than
others for the larger algae. Growth of Diatoma
vulgare has been observed to be inhibited in the
portions of the riffle downstream from large rocks,
where water movement is relatively slow. (Fig. 2)

Fig . 1 . Diagram of spring zonation of prin-
cipal algae on rocks of shallows (average depth
ca . 15 cm.) in the Saline River, southeastern
Michigan. Dark area represents Ulothrix tenuis -
sima filaments, present only on rocks in swift
water immediately below the water surface where
the current is broken by protruding emergent rocks
as at A. Irregular oval patches represent Gom-
phonema olivaceum occupying the upstream face of
rocks such as A and B in rapid current but slightly
below the Ulothrix level. Irregular short lines
represent the Diatoma vulgare community which is
partly intermingled with the Gomphonema but is
dominant at a slightly lower level. Rocks such as
C, below a given depth are not colonized by mas-
sive Gomphonema colonies or by Ulothrix. Arrow
shows direction of water movement. Note that this
zonation is not uniquely related to depth, but is
also influenced by current rate .

-2 H.

DISTRIBUTION WITHIN THE BASIN AS A WHOLE

Most small streams consist of a series of
alternating shallow areas ("shallows", "riffles")
and deep areas ("pools") . The shallow areas
naturally receive greater abrasion by the water and
the molar agents it carries. The water flows faster
here and in a thinner sheet, and significant chemi-
cal differences are to be expected between the
shallows and pools, although little effort has been
made to demonstrate them. Many algae are con-
fined to shallow parts of headwaters streams, just
as others are characteristic of the slower and
deeper waters of pools or deeps. Peculiarities of
the deeps and shallows as they alternate with each

Fig . 2 . Diagram of colonization by Diatom a
vulgare in a shallows of the Saline River, Michigan.
Heavy continuous line represents the water's edge.
Heavy broken lines are isobaths at 1 and 2 ft.
depths . R = large, emergent rocks; pattern of
short lines represents the Diatoma vegetation.
Note that this Diatoma is limited to shallow (swift)
water, and that it is absent from shallow areas
near the stream edge and from areas to the lee of
emergent rocks where water movement is much
slower. Arrow represents direction of water move-
ment .

Eddy presented the view that the amount of
plankton in river water is dependent upon the

length of time required for the water to pass down-
stream from headwaters sources, or, as he regarded
it, as the "age" of the water. Thus there is an
initial increase in plankton with time and distance
going downstream. Certain streams (e.g., the
Illinois River and the Rock River, both in U.S.A.)
exhibit a headwaters area low in plankton and a
middle region rich in plankton, followed by a con-
sistent decline in plankton in the lower course.
This decline is, however, not universal, and the
phenomenon can not yet be correlated with a given
length of course or degree of eutrophy. Conditions
in a mature stream may be expected to approach
those of late or middle-age ponds in the neighbor-
hood of the stream whose waters have a similar
chemical composition (Blum, 1956).

An ambitious attempt to compare the flora of
different streams has been made by Budde (1930)
who reviewed several published works on European
and other rivers. Of the British streams studied by
Butcher and his co-workers, the Itchen, Test,
Bristol Avon, Hampshire Avon, and Lark seem to be
somewhat similar physico-chemically and to have

many parallels in their algal vegetation (Butcher,
193 2) . The work of Scheele on diatoms of the
Fulda (Germany) has shown that the tolerant, ubiq-
uitous species increase from source to mouth and
that the species characteristic of springs show a
corresponding decrease (Scheele, 1952).

The changes in benthic algae over the course
of a stream from source to mouth have been inves-
tigated by many workers. One of the more exhaus-
tive treatises of this nature is the work of Lauter-
born (1910, 1916, 1918) on the Rhine vegetaUon.
The great variability in size, profile, geology, de-
gree of pollution, and other atrributes of the
streams studied does not admit of many compari-
sons of their respective floras, and little informa-
tion of general application can be drawn from these
studies until an extensive analysis of the data con-
tained in them has been assembled.

As an example of some of the changes which
may be expected in the algal flora on the profile of
a stream, Table 1, condensed from Butcher (1947)
shows some of the changes as determined quanti-
tatively in his work. The table shows the response

TABLE 1

(Condensed from Butcher, 1947)

ALGAL COLONIZATION ON BLANK GLASS SLIDES SUBMERSED IN A POLLUTED STREAM

Totals under the different algal species identified show average growth in numbers per square millimeter of
sUde surface on slides submersed in the River Trent (England) above and below outfalls from tar disUUing
industries. Collections were taken from slides at intervals of 20 days from April to October, 1938. The
river water showed a pH of about 7.5. The station above the source of pollution, and that at 35 miles
below this source of pollution may be considered oligosaprobic . Stations from 2-8 mi. below may be con-
sidered polysaprobic, 10-17 mi. below, mesosaprobic .

Miles from

pollution

source

Stigeo-
clonium
tenue

Nitzschia
palea

Gompho-

nema

parvulum

Stiqeo-
clonium
farctum

Ulvella
frequens

Chamae-

siphon

spp.

Cocco-
neis pla-
centula

above

130

+

820

below
2

30

130

20

30

+

3

190

680

130

20

20

5

1620

2380

600

20

8

15,300

5250

3390

20

40

10

50

620

690

1880

13 1/2

45

+

660

270

60

20

17

180

250

3000

300

260

330

35

220

150

1280

210

480

200

1480

19

of certain algae to pollution due to gas liquor and
organic chemical wastes from tar distilling . The
effluent from this industry included tar acids, cy-
anides, and ammonium. The totals, shown under
the different algal species identified, represent
average growth in numbers of plants per square
millimeter of slide, surface on slides submerged in
the River Trent (England) . Noteworthy here is the
striking build-up in numbers of Nitzschia palea
and Stiqeoclonium tenue throughout the polluted

(polysaprobic) portion from 2 to 8 miles below the
source of pollution, and the great abundance at the
lower end of this region. Also significant is the
striking elimination of Cocconeis placentula from
the flora with the accession of the chemical wastes,
and the reappearance only after many miles of the
stream's course had permitted sufficient reduction
and oxidation of the wastes, dilution, and mixing
adequate to return the water to a condition once
more suitable for growth of the Cocconeis.

References

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Blum, J. L. 195 6a . The application of the climax concept to algal communities of streams. Ecology
37: 603-604.

Blum, J. L. 1957. An ecological study of the algae of the Saline River, Michigan. Hydrobiologia
9: 361-408.

Budde, H. 1930. Die Algenflora der Ruhr . Arch. Hydrobiol . 21:559-648.

Butcher, R. W. 1932. Studies in the ecology of rivers . II. The microflora of rivers with special
reference to the algae on the river-bed. Ann. Bot. 45: 813-861.

Butcher, R. W. 1946 . Studies in the ecology of rivers . VI. Algal growth in certain highly calcareous
streams. J.Ecol. 33:268-283.

Butcher, R. W. 1947. Studies in the ecology of rivers. VII. The algae of organically enriched water.
J. Ecol. 35: 186-191 .

II

Cedergren, G. R. 1938. Reofila eller det rinnande vattnets algsamhallen . Svensk Bot . Tidskr .
32: 362-373.

Douglas, B. 1958. The ecology of the attached diatoms and other algae in a small stony stream.
J. Ecol. 46: 295-322.

Eddy, S. 1934. A study of fresh-water plankton communities . 111. Biol. Monogr . 12: 1-93.

Fjerdingstad, E. 1950. The microflora of the River Mfz^lleaa with special reference to the relation of the
benthal algae to pollution. Fol . Limnol. Scandinav. No . 5 . 123 pp.

Foged, N. 1947-48. Diatoms in water-courses in Funen. Dansk Bot. Ark. 12 (5): 1-40; (6): 1-64;
(9): 1-53; (12): 1-110.

Foged, N. 1954. On the diatom flora of some Funen lakes . Fol . Limnol . Scandinav. No . 6 . 75.

Hornung, H. 1959 . Floristisch-6kologische Untersuchungen an der Echaz unter besonderer
Berucksichtigung der Verunreinigung durch Abwasser . Arch . Hydrobiol . 55: 52-126.

Hustedt, F. 1944. Diafomeen aus der Umgebung von Abisko in Schwedisch-Lappland . Arch. Hydrobiol.
39: 82-174.

Jaag, O. 1938. Die Kryptogamenflora des Rheinfalls und des Hochrheins von Stein bis Eglisau. Mitt.
Naturf. Ges. Schaffhausen 14:1-158. PI. 1-18.

20

Kolkwitz, R. 1950 . Okologie der Saprobien. Uber die Beziehungen der Wasserorganismen zur Umwelt.
Schriftenr . Ver . Wasser-, Boden- , Lufthyg . No . 4 . 64 pp.

Lackey, J. B. 1958. The suspended microbiota of the Clinch River and adjacent waters in relation to
radioactivity in the summer of 1956. Florida Eng . Indus. Exp. Sta . Tech. Pap. No. 145. 36 pp.

Lauterborn, R. 1910. Die Vegetation des Oberrheins. Verh . Naturhist .-Mediz . Ver. Heidelberg, n.
F.20: 450-502.

Lauterborn, R. 1916-18. Die geographische und biologische Gliederung des Rheinstroms. Sitzber.
Heidelberg. Akad . Wiss. Kiasse 7b (Biol. Wiss.) (6): 1-61. (1916); SB (5): 1-70. (1917); 9B
(1): 1-87. (1918).

Liebmann, H. 1951. Handbuch der Frischwasser- und Abwasserbiologie . Miinchen.

Lund, J. W. G. and J. F. Tailing. 1957. Botanical limnological methods with special reference to the
algae. Bot. Rev. 23: 489-583.

Luther, H. 1954. Uber Krustenbewuchs an Steinen fliessender Gewasser, speziell in Sudfinnland.
Acta Bot. Fenn. 55: 1-61 .

Margalef, R. 1947. Limnosociologia . Jn Monogr. Cienc. Moderna Inst. Espan. Edafol., Ecol . y Fisiol.
Veget. Madrid No. 10. 93 pp. (Not seen).

Margalef, R. 1948. Flora, fauna y comunidades bioticas de las aguas dulces del Pirineo de la
Gerdana . Monogr. Estac. Estud. Pirenaicos No . 1 1 (Biol. No. 2). 226 pp.

Margalef, R. 1949. Las asociaciones de algas en las aguas dulces de pequeno volumen del noreste de
Espana. Vegetatio 1: 258-284. 1948 (1949).

Margalef, R. 1949a. A new limnological method for the investigation of thin-layered epilithic com-
munities. Hydrobiologia 1: 215-216.

Panknin, W. 1941 . Die Vegetation einiger Seen in der Umgebung von Joachimsthal . Bibl . Bot. H,
119. vil, 162 pp.

Panknin, W. 1945 . Zur Entwicklungsgeschichte der Algensoziologie und zum Problem der "echten" und
"zugehorigen" Algengesellschaften. Arch Hydrobiol . 41: 92-111.

Reese, Mary J. 1935 . Report on the microflora of the Rheidol and Melindwr above and below sources of
pollution by lead mines. Min. Agr. Fish. Ser. 123, Rep. 510. 17 pp.

Scheele, M. 1952. Systematisch-okologische Untersuchungen uber die Diatomeenflora der Fulda .
Arch. Hydrobiol. 46: 305-423.

Scheele, M. 1954. Die Diatomeenflora der Schleusenwande in der unteren Fulda und die Lichtabhangig-
keit einiger Diatomeenarten . Arch. Hydrobiol. 49: 581-589.

Schroder, H. 1939. Die Algenflora der Mulde . Ein Beitrag zur Biologie saprober Flusse. _In R . Kolkwitz,
Pflanzenforschung 21 . vi , 88 pp . 1 pi .

Symoens, J.-J. 1951. Esquisse d'un systeme des associations algales d'eau douce . Verh . Int . Ver .
Theoret. Angew. Lim. 11: 395-408.

Symoens, J.-J. 1957. Les eaux douces de I'Ardenne et des re'gions voisines: Les milieux et leur vegeta-
tion algale. Bull. Soc. Roy. Bot. Belg. 89: 111-314.

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(Chlorophyceae) near Dallas. Field and Lab. 20: 26-28. /r^^'^TT^^

21 j^fuBKAf'Y)?

BIOLOGICAL DISTURBANCES RESULTING FROM ALGAL POPULATIONS
IN STANDING WATERS

G. W. Prescott
Michigan State University

Nowhere can there be found more striking ex-
amples of interactions between, and interdepend-
ence of organisms in nature than in the ramifying
effects produced by algal populations . The roles
which these plants play in aquatic biology and in
limnology are clearly recognized but often incom-
pletely understood. Whereas the place of algae in
nature may involve some benefits to man and to
other organisms , some of the effects produced are
radically and offensively disturbing, sometimes
costly and even lethal. The putrefying masses of
algae in recreational lakes and in water supply
reservoirs are aesthetically disturbing and econom-
ically aggravating . But of greater importance are
the indirect and far-reaching effects algae have on
other aquatic organisms, including their ability to
kill.

It is from populations of algae in standing
water, of course, that effects are most obvious
and most disturbing because concentration of num-
bers of individuals is possible. To be sure, some
of the same disturbances occur in slowly flowing
waters which have ecological conditions condu-
cive to bloom- forming algae. We are not primar-
ily concerned with marine waters here, but it is
appropriate to recall that some of the same effects
are produced by algae in the sea (Red Tides; fish
poisoning) .

It is well-known that of the countless
species of fresh-water algae, only a few produce
disturbances which attract our attention. These
belong almost entirely to three phyla, the Cyano-
phyta, Chrysophyta, and Pyrrhophyta . The
species with which the present subject mostly
deals are listed for reference. It will be recog-
nized immediately that these are mostly so-called
"bloom "-producing species.

Ill . Pyrrhophyta

Gymnodinium brevis
G . veneficum

Ceratium

hirundinella

I. Cyanophyta
Microcystis

aeruginosa
M . toxica II .

Coelosphaerium

Kuetzinqianum
Oscillatoria

rubescens
O. lacustris
Anabaena circinalis
A . flos - aguae
A . Lemmermannii
Anabaenopsis

Elenkinii
Nodularia spumigena

Aphanizomenon flos -
aguae

Chrysophyta
Dlnobryon sertularia
D . sociale
Svnura uvella
Fraqilaria spp.
Tabellaria fenestrata
Asterionella gracillima
Coscinodiscus spp .
Melosira aranulata
Stephanodiscus
Niaqarae

When the algae guilty of causing disturb-
ances are examined and their ecology analyzed, a
few generalities appear:

1 . Disturbances occur in waters which are
enriched with phosphates and nitrates; usually the
algae occur in bloom condition, and the disturb-
ances arise in relatively shallow lakes where it is
possible for nitrates and phosphates to be recircu-
lated from bottom decomposition.

2. Excessive and troublesome growths can
occur only in lakes which are amply supplied with
CO2 or with bicarbonates from which carbon di-
oxide necessary for photosynthesis can be ex-
tracted . Hence, hard water lakes support blooms,
whereas soft water or acid lakes are spared.

3 . Most of the adverse effects of algal
blooms occur when one species dominates in a
standing body of water . A population density in-
volving one species is often followed by succes-
sions of other dominants in a repeating sequence.

4. The most drastic and often lethal effects
of algal blooms appear in a curious distribution in
both the Eastern and the Western Hemispheres.

5 . The disturbing effects of algae are both
directly and indirectly produced .

6 . Effects are mostly related to algal physi-
ology, but in some instances the simple morphology
of algae is involved.

ECOLOGICAL CONDITIONS

Some of these generalities can now be con-
sidered in limited detail to illustrate the inter-
actions of those factors which are responsible for
algal disturbances.

First to be considered are the conditions of
the environment which are responsible for super-
abundant algal populations . Of all the nutritive
elements and dissolved substances known to be
used by algae, those which are most often control-
ling or critical are phosphorus and nitrogen. Phos-
phates and nitrates are essential nutrients for all
chlorophyll-bearing organisms; but when these are
present in unusual quantities, a lake can support
tremendous populations of algae which recur year
after year. Those who are familiar with the lakes
in north mid-America know the relationship between
polluted lakes and algal blooms . This relationship.

22

simply stated In the negative Is: no phosphates
and nitrates, no blooms; hence, no disturbances.
We naturally Inquire why, of all the dozens of
species of algae In a lake, do only a few respond
to a rich supply of nitrates and phosphates by ex-
plosive reproductions. From one Montana valley
lake, for example, over one-hundred species of
algae have been listed; yet only one, Alphanizo -
menon flos-aquae developed a bloom. It is diffi-
cult, if not impossible, to explain this "selec-
tivity" partly because of the multiplicity of spe-
cific physiological requirements, the presence or
absence of critical trace elements (so far undeter-
mined) , the inhibiting actions of antibiotics, and
the presence of growth stimulators in the medium.

Most likely the response of some species by
producing bloom populations, and no such response
from others, is related in part to the high reproduc-
tive rate possessed by particular plants. For ex-
ample, when conditions are favorable for both
Dinobryon and Synura , they occur together espe-
cially in the early spring plankton of hard water
lakes. But Dinobryon spp. quickly assume bloom
proportions and vastly outnumber Synura because of
the rapid, zoospore method of reproduction used by
the former. The reproductive rate is usually paral-
leled by the speed with which nutrients are ab-
sorbed , as with Aphanizomenon , Microcystis and
Anabaena among the Cyanophyta which develop
superabundant populations in a short time. Also,
this group of the algae possesses a more rapid
photo synthetic rate than any other.

Among the blue-green algae there is at least
one characteristic which explains why they develop
especially well only in waters well-supplied with
phosphates and nitrates. The protoplasm of most
blue-green species is highly proteinaceous , more
so even, than some animal protoplasms. Crude
protein analyses (dry weight) show that Micro -
cystis aeruginosa is 55.58 per cent protein; Ana -
baena flos - aquae is 60 .5 6 per cent and Aphani -
zomenon flos - aquae is 62.8 per cent. Hence,
their requirement for nitrates for the elaboration
of proteins is much greater than that of green algae
such as Spiroqyra , for example, which is 23.82
per cent protein , or Cladophora 23.56 per cent .
Nitrogen alone in Aphanizomenon amounts to 10 .05
par cent (dry weight) as compared with 3.81 per
cent in Spirogyra . Some other data give inferential
evidence to support the thought that phytoplankton
depends upon nitrogen for bloom production. Juday
(1943) analyzed plankton of Waubesa Lake and
found that it was composed of 49 per cent protein,
5 per cent fat, 6 per cent pentosans, 4 per cent
crude fiber. The total annual plankton yield of
this lake was 2592 pounds per acre, which inci-
dentally supported 295 pounds of fish per acre.

It has been demonstrated in laboratory cul-
tures and inferred from numerous water and plank-
ton analyses that phosphorus is more critical than

nitrogen in determining phytoplankton production.

Phosphorus is also important in that it, in turn,
facilitates the assimilation of nitrogen. In fertili-
zation experiments where attempts are made to in-
crease phytoplankton, it has been found desirable,
or even necessary, to use a sulphate to fix iron
and so prevent this element from taking up the
phosphorus and thus lessening the well-known de-
sirable effect of phosphate on plankton production.
In respect to fertilizers, it is pertinent to mention
here that such an operation frequently leads to
disastrous upsets in a fish pond because super-
abundant growths of algae decay, the oxygen is
depleted, and winter-kill results.

The work of Atkins illustrates the importance
of phosphorus . He found that a pure culture of
Nitzschia closterium developed a concentration of
300,000 organisms per cc . when Miquel's solution
was used. All the P was consumed and it was de-
termined that 1.12 mg. of P2O5 was used to pro-
duce 1 X 109 diatoms during the first phase of
growth. One gram of P-pentoxide suffices for
9 X 10^1 diatoms. Krumholz (1954) found that Os-
cillatoria accumulated P 800 ,000 times that of the
medium. These figures are derived from culture
studies but it is reasonable to infer that they are
applicable in a general way to situations in nature.
Atkins (1923, 1925) found that in the sea one liter
of water can produce 26.8 million diatoms for each
0.03 mg. of P consumed. Here, P2O5 -content at
37 mg . per cubic meter, the plankton removed all
but 7.4 mg. per cubic meter. It is claimed that in
the tropics where phytoplankton development is
more uniform throughout the seasons the sea water
is persistently low in P2O5 -content. Correspond-
ingly, in the Arctic the P2O5 is high in winter and
low in daylight periods, paralleling the action of
plankton populations in taking up P. In the English
Channel P and N are completely exhausted at the
peak of phytoplankton production.

Rice (195 3) and others have demonstrated the
use and uptake of P by radioactive-P in laboratory
cultures. Hasler (1958) has suggested that strati-
fied lakes be given artificial circulation to bring
phosphorus from the bottom layers into the photo-
synthetic zone so as to increase productivity.

The disappearance of nitrates and phosphates
from standing water in summer and the increase
again in winter is taken as evidence that these sub-
stances are taken up by phytoplankton. In fact, it
appears clear that the exhaustion of P2O4 in a body
of water by plankton uptake in late spring or in mid-
summer acts as a brake or increment and explains
the plateau in population numbers. Therefore, it is
only in those lakes which receive a continuous
supply of phosphate that tremendous blooms of
algae can appear and reappear throughout the en-
tire 'growing' season. It has been noted that phy-
toplankton increases sharply after the decay of
larger aquatic plants whereby substances are

23

returned in a soluble form to a lake.

It follows that lakes which have been en-
riched by various kinds of pollution from human
habitats, run-off from agricultural lands, wastes
from farm animals, etc. are the ones in which
blooms appear. Accordingly, blue-green algae fol-
low man about as he colonizes and pioneers, cre-
ating situations favorable for his worst aquatic
pests. It is common knowledge that virgin lakes
and lakes in unsettled areas rarely, if ever suffer
from blooms and have no fish deaths caused by
algae. Also, it is well-known that oligotrophic
lakes are spared algal blooms since they are low in
electrolytes, nitrates, and phosphates. Such lakes
have a high Na2 - K2O ratio (3.2) and support a

CaO - MgO
desmid and planktonic chlorophycean flora together
with such Pyrrhophyta as Peridinium Willei . Un-
contaminated lakes, therefore, are not enriched,
have a flora that is 62 .4 per cent desmids and a
diatom flora of 11 .1 per cent; blooms do not de-
velop. When the Na/Ca ratio is low (1 .1) eu-
trophic conditions and a predominant blue-green
flora exist. Two English lakes, for comparison,
were found to have:

Lake Postherne - Nitrates .012;

Hardness 18.0

Chlorine 3.45;

Flora Myxophyceae

Lake Katrina - Nitrates .001;
Hardness 9 .0
Chlorine .85;
Flora Desmids

Eutrophic lakes, on the other hand, by their
chemical nature may be veritable garden spots for
blue-green algae when phosphates and nitrates are
plentifully supplied. Here at Pymatuning Lake,
Tryon and Jackson (195 2) have shown that blue-
greens predominate in summer months, with 90 per
cent of the plankton consisting of Microcystis , the
remaining 10 per cent being made up of Chloro-
phyta and Pyrrhophyta . In their middle lake station
HCO3 varied from 35 to 20 ppm.; CO3 from 15 to
25 ppm. This is the situation found, in general, in
all basic, eutrophic lakes with a high pH.

It is purely conjectural, but at least worthy
of mention, that trace-elements and/or growth
stimulators are present in water where peak popu-
lations of a single species explode. In our igno-
rance it appears possible that there is an unde-
tectable agent (or agents) present — some ingredient
coupled with animal excretory matter, possibly Bi2-

Numerous other studies and analyses, both
marine and fresh-water, in the field and in the lab-
oratory, have demonstrated the importance of N
and P in plankton production . Reference might be
made to numerous researches on this problem.
Harvey (1940), for example, determined in labora-
tory studies that phytoplankton used 10.5 times

more nitrogen than phosphorus, but of course this
does not mean that a maximum amount of P is criti-
cal. Hasler and Einsele (1948) found that for every
1000 Kg of N in lake water, 20,000 Kg. of plankton
material was possible but that P to the amount of
10 per cent of the available N was necessary before
the N could be used.

As mentioned above, lakes well-supplied with
bicarbonates provide a CO2 reserve that makes
blooms possible. The demand for carbon dioxide by
such a mass of vegetation during photosynthetic
hours is tremendous. Obviously CO2 from respira-
tion provides for some photosynthesis, but addi-
tional amounts from bicarbonates can support a
mass of vegetation when, or if the dissolved CO2
is reduced or depleted. Photosynthesis has been
shown to be proportional to concentrations of bi-
carbonates. In distilled water, free, from car-
bonates, which has been saturated with CO2 photo-
synthesis occurs but very little. Two- thousand
six-hundred and fifty-four units of blue-green algae
per cc . of lake water were found at the upper one-
foot zone of a lake where carbonic acid was 1 ppm.
Whereas, at the 23-foot depth where carbonic acid
was 8 ppm., only 756 units per cc . were found. At
46 feet with carbonic acid at 16 ppm. there were
only 120 units of algae per cc .

As Birge and Juday (1911) have pointed out,
an ample supply of dissolved carbonates benefits
aquaUc plants in two ways: 1) by supplying CO2
from the half-bound carbonates , and 2) in that
mono-carbonates take on a greater amount of CO2
from the atmosphere than would be absorbed other-
wise. Further, they also take up and hold CO2 of
respiration in the water and thus retain the gas for
subsequent use by the plants.

Plankton population in eutrophic lakes where
electrolytes are abundant are characteristically
greater than in oligotrophic where electrolytes and
C02-content are low. For example, Rawson (195 3)
measured 10 to 40 Kg. of plankton per hectare in
Canadian oligotrophic lakes as compared with 100
Kg . per hectare in eutrophic lakes . He refers to
Lake Mendota (Wisconsin) in which Birge and Juday
measured 177 Kg. of plankton per hectare.

An attending phenomenon in lakes which are
well-supplied with calcium carbonate is the pre-
cipitation of lime on stems of submerged plants,
stones, and other objects. When carbon dioxide
is withdrawn by photosynthesis , mono-carbonates
are formed. Lime may also be formed by oxidation
of calcium bicarbonates. Hence, dense blooms of
algae may lead to the formation of a thick layer of
lime which directly or indirectly has a profound
effect upon the limnology and the biology of an
aquatic habitat. This layer becomes inhabited by
lime-precipitating, benthic blue-green algae and
the deposition is accordingly amplified. Marl
weights down vegetation, thus forming bottom
layers of dead and decaying vegetation and so

24

forms another link In

Ca(HC03)2 + O2 = H2O + CaCOa + CO2 + O2

a complex chain of chemical-biological events .

A still unexplained aspect of algal blooms Is
the phenomenon of dominance and succession of
species. (An arbitrary index of 33-1/3 per cent of
the flora can be used to define dominance) . When
a bloom develops, invariably a single species is
involved. Aphanizomenon flos-aquae , for example,
is never in abundance, or scarcely present at all,
when Microcystis aeruginosa is in peak production,
and vice versa. Anabaena Lemmermannii com-
pletely takes over in some lakes early in the sum-
mer and after a week or two, disappears as Gloeo-
trichia echinulata assumes a bloom condition .
Algal species , like other organisms have their own
metabolic rate, time of development and maturation,
and then have a decline. No doubt some of the
sequences commonly observed of algal species in
lakes are related to natural life histories. Also,
dominance is related to specific rates of cell di-
vision. The fission method used by blue-green
algae gives them an 'edge' over more slowly repro-
ducing Chlorophyta .

But at the same time, there are incompati-
bilities between species which must be explained
other than by differences in life histories. For ex-
ample, two lakes within less t.han a hundred feet
of one another may at the same time support in one
a bloom of Microcystis , in the other a bloom of
Anabaena . Or, as in the Yahara River system in
Wisconsin, Lake Waubesa in the chain often has a
pure'e of Aphanizomenon (as many as 16 , 000 ,000
filaments per L.), whereas Lake Kegonsa, about
three miles below in the chain, supports a dense
bloom of Microcystis . The masses of Aphanizo-
menon carried into the lower lake are completely
eliminated, and one might say annihilated as de-
termined by plankton sample counts from Lake
Kegonsa .

Ideas are expanding in respect to the causes
of and explanations for such incompatibilities and
successions of species and dominance. Many
points are difficult of clarification because of the
highly refined chemical analyses that are called
for. The best explanation of these phenomena is
that there are extracellular by-products produced
by one species which inhibit the growth of another,
or of others . These by-products well may be
classed as antibiotics and, indeed, there is ample
evidence to indicate that they are . The sudden
'crash' of a dense population of a single species
is due to its inability to tolerate its own growth-
inhibiting substances . After a growth period when
extracellular products have accumulated, it is
thought that these act as deterrents and that the
plant, in a sense, manufactures its own algacide .
The extracellular substances (possibly of a toxic

nature) have prevented the growth of selected or
certain other species, and thereby the plant which
begins its development first in a body of water
quickly assumes a dominance (depending upon its
inherent rate of cell division) . With autodestruc-
tion comes a reduction in the inhibitor, thus per-
mitting another species or group of species to de-
velop. We say "selected" species because when
one plant, such as Microcystis aeruginosa , for
example, is in dominance, a combination of a few
other phytoplankters, relatively sparse in numbers,
occurs along with the bloom. The accompanying
phytoplankters are mostly species in other phyla of
algae, and the species which apparently are elimi-
nated are members of the phylum to which the in-
hibiting alga belongs. Akehurst (1931) long ago
pointed out the succession of oil-producing and
carbohydrate-producing algae. The brown-pig-
mented algae undergo self-destruction by their own
toxins and/or antibiotics, which In turn serve as
stimulators to Chlorophyta . Although there is some
variability from lake to lake, or region to region
(as ecological factors vary) , it has been noted that
the same combination of associated species occurs
and recurs along with the dominant. Species which
are usually associated with Microcystis aeruginosa
when this plant is in a bloom condition, are:
Anabaena circinalis; Coelosphaerium Kuetzingia -
num; Stephanodiscus Niagarae ; Asterionella gracil -
lima ; Tabellaria fenestrata ; Ceratium hirundinella .
With Aphanizomenon flos-aquae are usually found
Anabaena Lemmermannii ; Go mpho s pha eri a aponina;
Lyngbya Birgei ; Stephanosphaera Niagarae ; Cosci -
nodiscus spp.; Melosira granulata ; Pediastrum
spp.

As mentioned elsewhere, we do not know
wherein antibiotics of algae differ from exotoxins,
if indeed they are different. From the many studies
which have been made , we know that extracellular
substances occur in the dissolved organic matter of
lakes. In Wisconsin, for example, dissolved or-
ganic substances vary from 2.9 to 39.6 ppm . ,
whereas as much as 300 ppm. have been measured.
Secreted nitrogenous and carbohydrate materials
from algae have been detected (Bishop eta^, 1954;
Fogg, 1951). Some are liberated by autolysis
(Aleyev, 1934). Complex proteins , peptides, leu-
cine , aspartic acid, tyrosine, and many other sub-
stances have been found in the medium of the algae
and in bottom deposits (Petersen et al, 1925;
Wangersky, 1952). It is among these organic sub-
stances that we must search for specific antibiotics
and toxins; growth stimulators and growth inhibi-
tors, sometimes both, depending upon the concen-
tration .

Laboratory experiments (Pratt, Akehurst,
Proctor and others) have demonstrated incompati-
bility of two algal species and the inhibition of
growth of one plant in the presence of another.
Chlorellin from Chlorella vulgaris is a well-known

25

antibiotic to some other species of algae, and It
has been found further (Pratt and Fong , 1942; Rice,
1954) that two species may be mutually antagonis-
tic. Rice used varying concentrations of Nitzschia
and Chlorella and by evaluating growth rates he de-
termined that there were varying degrees of mutual
inhibition. His findings, when applied to condi-
tions in nature, could explain seasonal fluctua-
tions. Sampaio (1946) discovered that Closterium
acerosum produced an antibiotic to species of bac-
teria, especially Bacillus subtilis . Pandorine from
Pandorina morum produces marked effects and ab-
normal shapes in Scenedesmus ; whereas scenedes-
mine III inhibits completely the development of
Pediastrum Boryanum . Not exactly pertinent, but
of interest, is the fact that Elodea and Potamoqeton
foliosus in Wisconsin were found to produce anti-
biotics which inhibited populations of phytoplank-
ton and rotifers .

Laboratory experiments by Proctor, and by
Lefevre and his co-workers have clearly demon-
strated antibiotic effects of algae. In Proctor's
work (1957) two closely related species of green
algae were employed — one inhibiting the growth of
the other. His supposition is that antibiotics are
located in unsaturated fatty acids liberated from the
algae. The hypothesis is advanced that antibiotics
interfere with oxidation of nutritive substances,
and that they interfere with chlorophyll behavior .

Both Microcystis and Chlorella secrete sub-
stances active against Staphylococcus and Clostri -
dium , and it is thought highly possible that many
other species of algae have the same capacity.

Antibiotics may even have Inhibitory effects
on fish . In Europe it was found that the fish Gor-
donus rutilus was stunted in habitats dominated by
Aphanizomenon .

If one allows a plankton collection domi-
nated by Aphanizomenon to stand in the laboratory
for a time , Ankistrodesmus , Pediastrum and Oocys -
tis will flourish after the dominant has died . As
long as the Aphanizomenon is alive, it completely
inhibits development of the other plankters .

The idea has been advanced that by-products
of one species, or the substances characteristi-
cally produced by one phylum of the algae, actually
serve as stimulators to another species or to a
group of species belonging to another phylum. Thus,
the death and destruction of one protoplasmic mass
frees substances that are veritable fertilizers for
other algae. In the research on this facet, the sur-
face film over a great sea of ignorance has been
punctured only in a few places . Most of the
studies on stimulators has been done with marine
organisms and their ecology, but some investiga-
tions have been carried out using fresh-water algae.
In any case, results and implications are appli-
cable to fresh-water algae and their ecology, and
to the determination of blooms .

The present state of our knowledge concern-

ing growth stimulators has been derived largely
from culture studies which, because of their arti-
ficiality, introduce an element of questionability
when laboratory-derived concepts are applied to
situations in nature. Possibly Pringsheim (1921)
was the first to recognize and to publish on growth
stimulators in the form of acetate-peptone and
acetate-ammonium present in his soil-extract cul-
tures of Polytoma spp. and other unicellular forms.

Since then Hutner and his co-workers in-
cluding Provasoli, Lwoff and co-workers, Lewin,
Starr and others have determined the existence of
growth stimulators and have used microorganisms
for their assay. These workers have found that
6^2 is an essential requirement, especially for
Dinoflagellata . In fact, the seeming universality
of the demand for B12 by algae has led Provasoli to
postulate that this factor alone might be a deter-
miner of algal blooms . Inferential but strong evi-
dence for this is to be found in the appearance of
marine blooms in coastal waters periodically fol-
lowing rainy seasons and floods which wash large
amounts of B]^2 i^^^ other growth promoters) into
the sea. Thus the death-dealing red tide may be
related to B,, increment because these blooms seem
to follow periods of flooding .

Measurements have shown that B12 occurs in
marine waters such as Vinyard Sound (0.03 to 2.0
iig/L). near Nova Scotia (O.Ol^ag/L), at Millport
Marine Station (0.005 to O-Oljag/L). These
amounts are apparently normally present (and well
they should be if they are as critical as observa-
tions indicate; otherwise there would be no coastal
phytoplankton) . Robbins , Hervey and Stebbins dis-
covered that in one fresh-water habitat the 8^2
varied greatly seasonally from 0.1 to 2.0^g/L.
Hence in lakes receiving pollution or where B12
analogues can be formed by bacteria, it is possible
for blooms to develop. This would be especially
true for species whose requirements for growth pro-
moters are not highly selective. They would have,
therefore, an advantage over competing organisms,
the requirements for which are highly specialized .

It has been shown that there is a specificity
for various analogues of B12 which are produced by
bacteria. Thus the activity of these microorganisms
is caught up in the complex cycle of exchange of
metabolites. Provasoli, for example, reports Am-
phora spp . , Skeletonema sp . (diatoms) , and Phorm-
idium (a blue-green genus) to use all cobalorains,
including factor B (one of the fractions of 8^2) ■ Two
dinoflagellates were found to require factors A, H,

Bj2/ arUficial B^2 ^"^^ ^12"^^^' ^^^ ^^"^^ "°^ '"^~
sponsive to pseudovitamin 822-

What such specificity means in respect to
our present consideration is that the appearance
and disappearance of algal populations and bloom-
producers are related to growth promoters and vita-
mins . Thus, both the regular sequence of peak
populations of different groups of algae, and also

26

the erratic and sudden appearances of blooms might
be determined mainly by B12 -content.

Doubtless very many instances of disturb-
ances by algae have not been reported in the liter-
ature; so, we are ignorant of a true pattern of their
distribution over the earth. But it is interesting
and somewhat enigmatic that those cases which
have been reported do form a pattern . In North
America, especially, disturbances involving the
death of animals are localized in the midpart of the
continent. Almost all instances of cattle deaths
have been reported from Michigan, Wisconsin,
Iowa, Minnesota, the Dakotas, and south-central
Canada. Further, it is from this general region
that reports are recurrent. There is another area in
Australia where lethal blooms of algae develop, and
one in south central Africa . The author is aware of
but one report of lethal blooms in Europe (Finland)
and none from Asia or South America . Algal blooms
occur in many parts of the world, of course, but it
is the distribution of toxic effects which invites
curiosity. Whatever the distribution of bloom-pro-
ducing algae is, we can state, rather tritely to be
sure, that waters in scattered parts of the earth
have critical ecological factors in common: (high
nitrate- and phosphate-content , an abundance of
CO2, a high pH and suitable temperatures) .

It has been mentioned that disturbances at-
tributable to algae are both directly and indirectly
produced. These effects will be discussed later.
By way of explanation, it may be mentioned here
that direct effects are those arising from sub-
stances given off by certain objectionable algae,
or through their physiology wherein nutrients nec-
essary for the growth of less offensive forms are
taken up; clogging of fish gills because of their
concentration in shallow water; producing disa-
greeable tastes and odors in drinking water, etc.
Indirect effects arise as a result of a chain reaction
in modification of ecological conditions leading to
oxygen depletion, formation of ptomaines as a re-
sult of bacterial decomposition, forming poisons in
shellfish eaten by man, birds, etc.

Whereas most of the disturbances of algae
are related directly to their physiology, it may be
noted in the instances of cyanophycean trouble-
makers especially, that their effects are related to
morphology and to their habits. For example, the
sticky sheath possessed by blue-green algae makes
it possible for plants to adhere to one another, thus
forming dense mats . Many blue-green algae, es-
pecially the trouble-makers, possess gas vacuoles
(pseudo-vacuoles) which permit or force the plants
to float high in the water . Because of their tre-
mendous numbers, the mat is actually elevated
above the water level. Thus, in intense illumina-
tion and in warm water layers, where oxygen is
low in any case, a decaying blanket forms in which
oxygen-consuming bacteria thrive. If the species
involved were evenly distributed through the water.

some of their objectionable direct and indirect ef-
fects would not occur. Species of Lyngbya , for
example, are sometimes extremely abundant, as
are some members of the Volvocales. But these
forms do not have the mechanisms for producing
sticky scums and for causing serious disturbances.

One instance of an apparent toxic effect has
been noted in a subalpine lake just outside Glacier
National Park. This is an unusual condition be-
cause all of the lakes in the area are relatively
soft and are relatively unproductive of plankton.
This particular body of water, about five acres in
area, has received during the recent past drainage
from a small farm and barnyard; sufficient drainage
apparently to raise the nitrogen and phosphorus
content so that a bloom of Aphanlzomenon flos-
aquae is supported . Each year that this bloom de-
velops , mud-puppies (Necturus ) die by the scores
and those which are not dead are sluggish and roll
about on the bottom .

Deaths of farm animals have been reported
from Iowa, Minnesota, North and South Dakota,
Wisconsin, Montana, Bermuda, and elsewhere (as
mentioned previously) . The greatest economic loss
of cattle has been in central South Africa . Here in
the shallow pans and reservoirs in the cattle-
grazing country, one species of blue-green algae.
Microcystis toxica dominates the phytoplankton,
producing 'soupy' blooms. Cattle drinking these
waters have died by the thousands . Symptoms in-
clude convulsions (similar to strychnine poisoning),
paralysis, jaundice, constipation, loss of apetite,
falling off in milk production, abortion, and skin
sensitization. Cases can be acute, with death oc-
curing in a few hours after animals have drunk from
Microcystis -infested water, or chronic, wherein
death does not occur until after some weeks or
months . It is thought that the toxin contains two
poisons, one of which is found in phycocyanin .
This poison, after damaging the liver, is carried
by the blood stream to the surface of the body
where, being stimulated by ultra-violet light, it
produces tenderness, dryness of the skin, and
lesions . The other toxin is said to be a liver-
neural type or a fucoin .

Also from Africa, a serious disease of sheep
is reported which has now been referred to a photo-
sensitizing toxin. Stewart et al (1950) found that
cattle, dead from blue-green algal poisoning in
Canada , had experienced lung hemorrhages and
liver lesions. As reported by Olson (1951), serious
outbreaks of animal deaths occurred in 1948 on
Round and Fox Lakes in Minnesota where horses,
cattle, hogs, and dogs were killed by drinking
water infested with Microcystis aeruginosa (flos -
aquae ?) and Anabaena Lemmermannii .

Poisoning is not confined to mammals, how-
ever, for from several localities throughout central
North America come authentic reports of deaths
among water fowl, pigeons, and even song birds.

27

Botulina poisoning has been definitely excluded as
a possible cause of these bird deaths . It seems
highly possible that many instances of water fowl
deaths in the past were not caused by botulina as
claimed. In one year 7000 Franklin gulls and 60
mallard ducks were killed by Anabaena flos-aquae
in Iowa .

In addition to these animal deaths, there are
many instances on record of the killing of fish by
algal toxins. Carl (1937) reported fish deaths in
British Columbia from Anabaena flos - aquae . Prym -
nesium parvum secretes a toxin which kills gill-
breathing organisms. Also, it has been found
(Ryther, 1954; Lucas, 1936) that extracellular sub-
stances have a profound effect on the biology and
feeding habits of microorganisms. AA^ereas these
cannot be construed as highly detrimental, they do
constitute examples of disturbances that may have
far-reaching and ramifying effects on aquatic biota.

Mention should be made here of the critical
studies of Abbott and Ballantine (1957) on the
toxins of the dinoflagellate, Gymnodinium vene-
ficum which plays a role in the wholesale death of
fish. Material obtained from laboratory cultures of
the flagellate was found to be toxic to the nervous
system of animals by depolarizing nerve and mus-
cle membranes .

Although in need of further study and con-
firmation, evidence indicates that fish are killed
in Iowa lakes by toxins and scanty but strong in-
ferential evidence is at hand to support the belief
that epidemics of human intestinal disturbances
are caused by them. (See Spencer, 1930; Tisdale,
1931) . That there are not more cases of this sort
of disturbances is related to the simple fact that
domestic drinking water is seldom allowed to de-
velop concentrated algal blooms . Persons would
naturally refrain from drinking water directly from
ponds which are obnoxiously overgrown with algae,
especially blue-green species which produce dis-
agreeable tastes and odors.

That toxins actually exist in extracellular
excretions has been demonstrated by both labora-
tory and clinical tests. Fitch, et al (19 34) are the
first to have investigated waters after an epidemic
of cattle deaths . The toxins with which they
worked were produced by Aphanizomenon , Ana-
baena and Microcystis in Minnesota . They fed
animals in the laboratory with algal material
taken fresh from the lake and also administered
injections . The animals died within a matter of
minutes in some instances, or after 12 hours in
others .

Mason and Wheeler (1942) also found that
small animals were killed within three hours after
being injected with Microcystis medium. Veteri-
narians in Iowa injected 10-15 cc . of a bacteria-
free filtrate from Anabaena flos - aquae into guinea
pigs which lived but 12 minutes. Only 0.04 cc .
injected into a mouse caused its death in four

minutes . They also found that after one year the
filtrate was ineffective. Olson (1951) points out
that only 0.02 cc . of raw algal material injected
into a 20-gram mouse produced death in one hour.
More time is required for death to occur when lab-
oratory animals are given algal material orally.
An interperltoneal injection of 10 cc . killed a
chicken in 20 minutes, whereas 50 cc . fed to a
chicken produced death one and one-half hours
later according to Olson (1. c).

Shelubsky (195 1-b) obtained toxin from Mi -
crocystis by drying the plants and by precipitating
it with phosaphotungstic acid at a pH of 2.0 .
Also, he demonstrated a toxin to be present in
living cells experimentally and that it was not an
antigen. Living Microcystis injected in several
small animals, including carp, produced death.

The deaths mentioned above are supposedly
caused by exotoxins present in the water in which
algae have been living (in a bloom condition) .
These exotoxins have not been Isolated. This is
understandable since extracellular substances in
quantities sufficient for analyses would have to be
separated from the other substances in solution
within the medium. Can they be filtered, or pre-
cipitated, or distilled? It is known that the exo-
toxins are alkali-labile, which indicates a possi-
bility that a technique can be developed that will
permit chemical analysis. Laboratory tests using
water and/or algal material have determined that
the toxins of Microcystis and Anabaena occur in
the medium of healthy cells; that they are non-
filterable; that like endotoxins , they are non-ionic,
are not alkaloid, nor albuminoid, are non-volatile
and are heat stable . They have a low molecular
weight and may be soluble in alcohol . Exotoxins
are not destroyed by air-drying, by freezing or by
ultra-violet radiation. Louw (1950) claims to have
detected two alkaloids from Microcystis , however.
One of these has a suspected formula of C^oHig
NO2. The other is a picrate and is the one that
acts as a hepatotoxin.

Molecules of chlorellin are smaller than
15 A in diameter. The exotoxin effects of Micro-
cystis have been shown by Ashworth and Mason
(1946) to be similar to the symptoms of poisoning
by Amanita . From a marine alga a toxin has been
identified as a form of polysaccharide.

Some workers have found that decayed blue-
green algae are toxic , a fact which might be ex-
plained by the presence of endotoxins which have
been released. Thus, it is clear that either living
or in a decomposed state algal substances are
toxic. Louw (1950), for example, found that fil-
trations of decayed algae were toxic, whereas
fresh or living plants were non-poisonous to lab-
oratory animals . It has been found also that the
'maturity' of the alga involved is related to varia-
tions in the strength of the toxin. Just what this
age-time factor is, has not been determined, but

28

it Is known that laboratory cultures several days
old are not so potent as fresh material or young
cultures.

Bishop, Anet and Gorham (1959) have suc-
ceeded in extracting an endotoxin from Microcystis
aeruginosa (flos - aquae ?). The toxin was not iso-
lated, but a syrup- like dialyzate was obtained and
many properties were determined. It was found
that the toxin is a non-volatile peptide, that it be-
haves as a non-ionic substance in electrophoresis
in a pH of 2.2 but is negatively ionic at a pH
above neutral . Five fractions were found to be
present and only one (peptide No. 2) was proven
toxic to laboratory animals. This component also
included leucine and one other amino acid residue.

In addition to their lethal effects, some
algae produce other types of pathological condi-
tions, in human beings for example. Mention was
made of intestinal disorders, epidemic in nature,
as reported by Spencer and Tisdale (1 .c .) . Further,
it seems clear that allergies, asthmatic conditions,
and epidermal irritations result from algal toxins .
Many persons experience skin irritations and sting-
ing rashes after bathing in algal-infested waters.
These symptoms are sometimes ascribed to cer-
cariae and accordingly inflamations are regarded
as the well-known swimmer's itch. Phycocyanin
from Anabaena is one particular irritant . Gioeo -
trichia will cause similar irritation when it is in
bloom condition .

Mention was made of the indirect effects
produced by dense algal populations . Examples
are to be found in fish deaths and in human poi-
sonings from eating fish and shellfish. Because
blue-green algae especially are high in proteins,
when they decompose it follows that proteinaceous
by-products are formed, some of which are poison-
ous. It has been demonstrated that when algal
blooms are dense in shallow water, the decomposi-
tion products may be concentrated enough to kill
fish. Large scale deaths of fish have been noted
in East Okoboji Lake, Iowa during periods of dense
Aphanizomenon blooms. Examination of the fish
(by specialists) indicated that they had not died
from suffocation nor from parasitism. Hence, poi-
soning was suspected . A large mass of algae was
collected and allowed to decay in vats. A variety
of fish, freshly seined from a nearby lake, were
placed in two large concrete tanks and the D. O.
adjusted and maintained at a safe level by intro-
ducing oxygen from oxygen tanks . Then the de-
cayed algae were poured into the aquaria , and the
behavior of the fish observed. After behaving er-
ratic for a time the fish all died within a few hours.
Chemical analyses of the decayed algae showed
the presence of hydroxylamine in quantities theo-
retically sufficient to be lethal, and also 8.5 ppm
of H2S . A similar experiment to confirm these ob-
servations, and with similar results, was con-
ducted by constructing lakeside ponds in which

fish and decaying algae were placed . Adequate
D. O. was maintained by allowing fresh lake water
to flow into the ponds periodically .

Mackenthum et al (1945) likewise experi-
mented by placing fish in aquaria with lake water
in which Aphanizomenon had occurred and in which
a decaying plant mass had developed. Perch and
crappies were all dead at the end of a 34-hour
period although the oxygen was maintained at 8.3
ppm.

It cannot pass unnoticed that disastrous ef-
fects of algal blooms are caused by those species
in which pseudovacuoles occur. I suggest that
there is more than a casual relationship and that
research may show that pathological conditions are
caused by substances contained in these vacuoles,
possibly a gas . I have never read a discussion of
this and apparently pseudo-vacuoles have never
been investigated in this connection.

Another type of indirect effect from algal pop-
ulations is experienced when human beings and
other animals eat fish or shellfish that have fed
upon phytoplankton in which the Pyrrhophyta are
abundant ( Gonyaulax , Ceratium , Prorocentrum) .
Shellfish, especially, store toxins in their diges-
tive tract and liver in quantities sufficient to pro-
duce death, or at least to cause serious illness in
animals who partake. The toxin appears to have
no effect however, on the fish or shellfish.

Puffers (Tetraodon ) store tetraodontoxin which
forms a white, water-soluble powder after extrac-
tion. It has a suspected formula of C2gH32N02^g .
Like other exotoxins, referred to above, this one
is non-alkaloid. It has produced death in 60 per
cent of the reported cases of poisoning. Persons
who eat moray eel ( Gymnothorax) are poisoned but
only 10 per cent of the cases are fatal. It is
claimed that more than 400 Japanese soldiers died
from eating fish in Micronesia, attributable to con-
centrations of Pyrrhophyta toxins .

Another indirect effect chargeable to algae is
that of fish-kills by oxygen depletion. In such in-
stances algae supply the basic food for bacteria
which actively oxidize masses of dead plants.
Dissolved O2 is often reduced to zero (or to an
amount too low to be read by usual oxygen tests) .
As the oxygen decreases below the threshold nec-
essary for them, various groups of organisms are
suffocated. In Iowa lakes the sequence of deaths
in a necritic cycle appears to be first green algae
and Protozoa; yellow-green algae and microcrust-
aceans; certain species of fish, including carp and
sheepshead; then other and all species of fish;
finally chironimid larvae and bottom organisms . In
one observed climax in an Iowa lake the oxygen
dropped from 4.5 ppm. at midday to zero the fol-
lowing morning at 1:00 a.m. Later the same day
not a single living organism of any kind could be
found in this lake. The lake shore was bordered by
a 25-foot zone of carcasses, and the water was a

29

puree of microcrustacea , chironimids , and decay-
ing algal masses. A few days later the dead fish
floated to the surface and were washed ashore,
forming a veritable "windrow" along several miles
of lake front. Such events often occur, unfortu-
nately when water temperatures are high (22 to 30
degrees C.) and when the oxygen-content is ac-
cordingly low. Rarely, blooms of algae, diatoms,
and planktonic blue-greens decay under winter ice.
This leads indirectly to fish deaths. Such events
not infrequently follow fertilization experiments
(as previously mentioned) due to oxidation of the
surplus organic matter, as well as to the break-
down of the dense population of algae which has
accrued as a result of the fertilization practice. A
thick blanket of snow over winter ice decreases
illumination so that a growth of winter-time plank-
ton 'crashes' and decays.

Whereas blue-green algae are accountable
for toxic effects, other disturbances are caused
mostly by those organisms in which yellow and
brown pigments predominate and in which oil and
leucosin accumulate as food reserve. These are
representatives of the Chrysophyta and the Pyrrho-
phyta, especially the former. Two classes of the
Chrysophyta , the Chrysophyceae and the Bacillar-
iophyceae contain several species which are in-
famous pests in aquatic habitats. Dinobryon spp.
and Synura uvella become abundant enough to pro-
duce obnoxious odors and fishy tastes. These
genera are often most abundant early in spring
when silicon- and calcium-content are low, these
elements having been exhausted by winter pulses
of diatoms .

In suitable habitats (eutrophic lakes, pH
7.2-8.5) diatoms often develop dense blooms or
form thick encrustments on submerged surfaces
(Asterionella , Melosira , Fragilaria , Tabellaria ) .
The 'fishy' odor of water is caused not by fish but
by diatoms which either alive or dead, but espe-
cially after a peak development, release large
quantities of oil into the water.

Chrysophyceae and the diatoms are physio-
logically suited to development in cold water and
in weak illumination. As classes they often de-
velop peak populations in the winter, or in early
spring, following a fall dominance of blue-green
species. For example, diatoms composed 100 per
cent of the plankton during winter and spring
months in Cleveland harbor of Lake Erie . Accord-
ingly disagreeable tastes in domestic water sup-
plies may appear during winter as well as in sum-
mer .

The Pyrrhophyta that cause trouble are mostly
marine and are not of direct concern here . As men-
tioned previously, Noctiluca , Gymnodinium spp.
and Gonyaulax are responsible for fish deaths (red
tide) along shores. The former is reported to be
especially troublesome in the eastern Pacific (East
Tndies) . In fresh-waters only Ceratium hirundi-

nella appears to form blooms sufficiently dense to
cause objectionable tastes in drinking water. This
species, although it does not form scums, may
color water a distinct gray-brown with as many as
13,000,000 organisms per liter. To my knowledge,
Ceratium hirundinella has never brought about fish
deaths .

Another incidental way in which diatoms
cause disturbances is by clogging sand filters in
city water systems which use impounded water.
This calls for additional work and expense for san-
itary engineers. Diatoms are successful in this
because of their persistent silicious walls and be-
cause of their large numbers which produce a
dense film over a filter bed .

Some chlorophycean algae cause minor dis-
turbances . Unlike the blue-green algae and the
diatoms , they have a slower reproductive rate and
seldom form superabundant growths. The product
of photosynthesis is starch and not ill-smelling
fats and oils. They have no pseudovacuoles and
do not form surface scums . Even the Volvocales
which frequently form conspicuous blooms cause
no particular disturbance. They are excellent oxy-
genators and remain evenly distributed throughout
the medium.

Such coarse filamentous forms as Rhizoclon-
ium and Hydrodictyon , however, develop thick mat
mats which do cause some minor troubles. Boating,
fishing and vacation sites are spoiled by these and
other species. The weight of filamentous blankets
causes larger aquatic plants to sink and to undergo
decay with attendant objectionable effects.

30

Selected References

Abbott, B. C. and Ballantine, D. 1957. The toxin from Gymnodinlum veneficum Ballantine. J. Mar.
Biol. Assoc. U. K. 36: 169-189.

Abbott, W. 1957. Unusual phosphorus source for plankton algae. Ecology 38: 152.

Akehurst, S. C. 1931 . Observations on pond life, with special reference to the possible causation of
swarming of phytoplankton. J. Roy. Microsc. Soc . 51: 237-265.

Aleyev, B. S. 1934. Secretion of organic substances by algae into the surrounding medium. Mikrobiol .
3: 506-508.

Al Kholy, A. A. 1956. On the assimilation of phosphorus in Chlorella pyrenoidosa . Physiol. Plant.
9: 137-143.

Arthur, J. C. 1883. Some algae of Minnesota supposed to be poisonous. Bull. Minn. Acad. Sc . 2
(Appendix): 1-12 .

Arthur, J. C. 1886. Some algae of Minnesota supposed to be poisonous. 4th Bienn. Rep., Bd . of
Regents, University of Minnesota 1 (Suppl.): 97-103.

Ashworth, C. T. and Mason, M. F. 1946. Observations on the pathological changes produced by a

toxic substance present in blue-green algae (Microcystis aeruginosa) . Amer . J. Path. 22: 369-384,

Atkins, W. R. G. 19 23. Phosphate content of waters in relationship to growth of algal plankton. J.
Mar. Biol. Assoc. U. K. 13: 119-150.

Atkins, W. R. G. 19 25 . Seasonal changes in the phosphate content of sea water in relation to the
growth of the algal plankton during 1923 and 1924. Ibid . 13: 700-720.

Barnum, D. A., Henderson, J. A. and Stewart, A. G. 1950. Algae poisoning in Ontario. Ontario Milk
Producer 25: 312.

Birge, E. A. and Juday, G. 1926. Organic content of lake water . U. S. Bur. Fish. Bull. 42: 185-205.

Birge, E. A. and Juday, C. 1934. Particulate and dissolved organic matter in inland lakes. Ecol .
Monogr. 4: 440-474.

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31

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32

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33

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34

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35

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36

Tlsdale, E. S. 1931a. The 1930-1931 drought and its effect upon water supply . Ibid . 21 : 1203-1215 .

Tryon, C. A. and Jackson, D. F. 195 2. Summer plankton and productivity of Pymatuning Lake,
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Wangersky, P. 1952. The isolation of ascorbic acid and rhamnosides from sea water. Science 115: 685.

Watanabe, A. 1951 . Production in culture solution of some amino acids by atmospheric nitrogen fixing
blue-green algae . Arch. Biochem. Biophys . 34: 50-55.

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Kiitz. Bull. Soc. Bot . France 96: 49-50.

37

ALGAE AND METABOLITES OF NATURAL WATERS

Richard T. Hartman
University of Pittsburgh

Increased attention has been given in recent
years by investigators working in various aspects
of algal ecology to the quantity, chemical nature,
and source of the organic constituents of natural
waters. This study of a long-ignored portion of the
aquatic environment has resulted from a growing
awareness of the role of organic substances in het-
erotrophic algal nutrition and from an appreciation
of the possible effect of these substances in influ-
encing the size and species composition of algal
communities by stimulating or inhibiting the growth
of particular species . Two important and compre-
hensive reviews have recently appeared on the sub-
ject of organic matter in natural waters. Vallen-
tyne (1957) has brought together the available in-
formation on the organic compounds Isolated or
identified from water, seston, and sediments of
lakes and oceans. Saunders (1957) has reviewed
studies on the interrelationships between dissolved
organic matter and phytoplankton pointing out four
functions which dissolved organic substances may
have for algae: (1) direct nutritional value,
(2) source of accessory growth factors, (3) toxins
or growth inhibitors , and (4) chelation of trace
minerals.

In the present paper it is my intent to review
primarily the evidence in support of the existence
of organic metabolites produced by algae , to ex-
amine the properties of these substances , and to
consider the possible role of such substances as
inhibitors, stimulators, or regulators of growth in
natural algal populations . The role of organic
metabolites in direct nutrition of the algae and the
production of substances toxic to fish and other
vertebrates will not be discussed since these
phrases have been or will be treated by other pa-
pers in this symposium.

Biological literature is replete with attempts
to relate fluctuations in algal populations to vari-
ations in chemical constituents of the water or to
physical factors of the environment. The problem
is most difficult, and the answers least satisfac-
tory, when attempts are made to explain the rapid
development of bloom-producing species and the
apparent decline of other species under environ-
mental conditions which appear to be equally sat-
isfactory for each organism. Hutchinson (1941,
1944) in discussing these problems some years ago
pointed out that a clear-cut correlation between
chemical conditions and the qualitative composi-
tion of the phytoplankton is not to be expected . He
suggested the possible importance of accessory or-
ganic substances in development of specific popu-
lations .

As early as 1931 it was suggested by Ake-

hurst (1931) that toxins produced by algae were re-
sponsible for fluctuations in phytoplankton popula-
tions . He theorized that changes in the rate of
development of certain algal genera were in re-
sponse to excreted toxins which could serve as
"accessory food" and could inhibit or stimulate
growth . Phytoplankton was divided into two groups,
"starch" and "oil", according to the chemical na-
ture of their food reserves . The autotoxins pro-
duced by the oil group were postulated as an ac-
cessory food of the starch group. It was thus pos-
sible to account by this theory for many succes-
sional patterns shown by algal communities .

This proposal was advanced with essentially
no direct evidence for the production or existence
of such toxic substances and was based almost en-
tirely on observations of the sequential changes in
phytoplankton populations over the seasons. Data
from many investigators covering a variety of geo-
graphical locations were gleaned from the litera-
ture and analyzed in support of his proposal.

Direct evidence for the existence of such ex-
tracellular growth modifiers in natural waters has
been slow to accumulate. Even now, most of the
experimental data confirming the production of ex-
tracellular substances are based on results from
investigations of algae in laboratory cultures.
Physiologically active organic substances from
algae may originate in nature in either of two ways:
(1) by extracellular excretion from living algal cells
or (2) from the decomposition of the remains of dead
cells . While it may be possible to demonstrate the
existence of such active substances in nature, it
is almost impossible to determine the specific
source of the active materials. Furthermore, even
though many intensive studies have been carried
out in the laboratory with certain algal species,
little has been accomplished in determining the
actual chemical composition of the active sub-
stances or in understanding the nature of the physi-
ological response of algae to these substances.

SOLUBLE EXTRACELLULAR SUBSTANCES

The excretion of soluble, extracellular sub-
stances by algae has received the attention of a
number of investigators . Studies have been con-
cerned with algae from various taxonomic groups,
with the chemical nature of the excreted products ,
and with the environmental and internal conditions
influencing excretion. Myers (1951) after review-
ing the work of Krogh , Lange and Smith (19 30) on
Scenedesmus and of Myers and Johnston (1949) and
Spoehr and Milner (1949) on Chlorella concluded

38

that "conservation of carbon and a minimal level of
excretion is probably generally characteristic of the
green algae". Fogg (1953), on the other hand,
notes that the amount of extracellular carbon pro-
duced by species of green algae may be relatively
high, with quantities as high as 12.5 per cent
being given off by old cultures .

Studies of motile green algae, particularly
species of Chlamydomonas , indicate that produc-
tion of soluble organic materials in the media by
these organisms Is quite extensive. Lewin (1956)
using cultures of eighteen species isolated mostly
from soil samples, showed that all species tested
liberated some polysaccharides into the growth
medium. Chlamydomonas mexicana released as
much as .1 gram per liter, equivalent to 25 per
cent of the total organic matter produced by the
cells of this organism. Allen (1956) similarly
demonstrated excretion of oxidizable organic mat-
ter by six species of Chlamydomonas . Organic
acids as well as polysaccharides were identified
in the media . It was apparent from the results of
the studies on Chlamydomonas that the appearance
of organic substances in the media did not result
primarily from decomposition of dead cells. In-
creased quantities of extracellular organic material
in the media has been shown to parallel growth of
the algae .

In mucilaginous species it is obvious that
much, perhaps most, of the soluble polysaccharide
arises from disintegration of the external portion of
the enveloping sheath. Lewin (1956) states "a
clear distinction cannot be readily drawn between
a soluble liberated polysaccharide and diffluent
sheath, between the latter and a firm capsule, or
between a capsule and the cell wall itself" .

The production of extracellular nitrogenous
materials in the growth medium is characteristic of
many blue-green species, for example: Nostoc
spp. (Henrikson, 1951); Tolypothrix tenuis ,
Calothrlx brevissima , Anabaenopsis sp . , and
Nostoc sp . (Watanabe, 1951) Anabaena variablis ,
A. gelatinosa (De , 1939): and A. cylindrica (Fogg,
1942) . Filtrates from healthy cultures of these
algae were shown to contain up to 50 per cent of
the total nitrogen fixed by these organisms. These
soluble nitrogenous compounds appear to be pres-
ent largely as polypeptides, but amides (Fogg,
195 2) and amino acids (Watanabe, 1951) have also
been identified in the filtrates. Phormidium unci-
natum has also been shown by Lefevre and Nisbet
(1948) to produce quantities of soluble organic
material In the medium (as determined by oxidation
with KMn04) , increasing in 40 days from 4.8 mg/1
to 30.4 mg/1 .

It has been suggested (Fogg, 1953; Fogg and
Westlake, 1955) that extracellular nitrogenous
compounds are liberated by most algae. Further
evidence in support of this was supplied by Fogg
and Boalch (195 8) who showed that the brown algae

(Ectocarpus confervoides) growing in a bacteria-
free culture liberated sizeable amounts of nitro-
genous material to the media. The ecological
significance of the presence of extracellular nitro-
genous compounds in natural waters, has been dis-
cussed by Fogg and Westlake (1955) and Fogg
(1956) who have emphasized the possible role of
excreted polypeptides in forming complexes with
sparingly soluble salts and with toxic ions . The
availability of the nitrogenous materials to organ-
isms unable to fix nitrogen may also be of great
ecological importance. It has been pointed out,
however, that not only is Anabaena in bacteria-
free culture unable to make use of its own excre-
tion products, but that Chlorella sp. and Oscilla -
toria sp. are likewise unable to use these materials
as sources of nitrogen.

Of ecological importance, at present not fully
evaluated in the case of algae, is the production of
extracellular substances capable of influencing
growth of the producing organism itself or of assoc-
iated species. Harder (1917) has been credited
(Jpfrgensen, 195 6) with the first report of the pro-
duction of growth-inhibiting substances, in this
case as autoinhibitor produced by Nostoc puncti -
forme . The classical work of Pratt and Fong (1940)
established firm evidence for the production of an
autoinhibitor by Chlorella vulgaris . Subsequent
investigations by Pratt and his co-workers (Pratt
1942, 1943; Pratt etai 1944, 1944a, 1945, 1948)
and by various other investigators over nearly a
score of years has resulted in the accumulation of
a great deal of information about this species. The
inhibiting material, termed "Chlorellin" by Pratt
has not been isolated nor chemically defined al-
though it has been concluded that the active agent
is probably an organic base.

Evidence for the production of extracellular
inhibitory or stimulatory substances by algae has
come largely from laboratory studies carried out in
either of two ways: (1) by growing certain test
species in cell-free media (filtrates) in which an
alga had previously been grown or (2) by growing
two species in mixed culture using a media which
would permit satisfactory growth of either organism
when grown alone .

The use of filtrates permits following the
production of active substances throughout dif-
ferent growth phases of the culture . Mixed-culture
techniques, while providing perhaps a closer ap-
proach to the natural situation than procedures
using culture fiifrates, produce no concrete data on
the rate of production of active metabolites nor do
they permit recognition and evaluation of responses
resulting from interactions of the mixed strains in
culture. Difficulties may arise, for example,
when two organisms have different inherent growth
rates under a given set of conditions . Active
growth and rapid photosynthesis of one species
may bring about the pH changes in the media which

39

can alter subsequent development of the other
species. Such effects are difficult to separate
from those resulting directly from the presence of
metabolites. Various modifications of these basic
procedures have been made, including the tech-
nique used by McVeigh and Brown (1954) in which
two species were grown together in a flask but kept
separate by a dialyzlng membrane .

The effect most frequently observed in cul-
ture studies has been the modification of the rate
of cell division which may result in reduction or,
more rarely, in stimulation of growth of the popula-
tion. Morphological modifications, particularly
changes in cell size, have been noted along with
increased accumulation of storage products within
the cell. There appears to be such variability in
the nature of the physiological action induced, and
in the degree of response shown by various species
belonging to the same taxonomic group that is often
difficult to generalize from reported results . It is
true of course that some of the variability reflects
differences in experimental procedures. Diversity
in culture media used, variations in temperature
and light conditions under which the organisms
were grown, age of the culture at the time of ex-
traction of active substances, methods of extrac-
tion or concentration of the active materials, and
physiological conditions of the test inoculum, all
may influence the results and make comparison of
the findings of several workers very difficult, even
when the same algal species are being considered.

In an attempt to examine the results obtained
with a number of different algal organisms in lab-
oratory culture, observations reported by a number
of different workers have been tabulated in Table I.
(Table at end of paper) The most commonly ob-
served effect of algal metabolites has been their
influence on the rate of multiplication of cells .
Relatively few authors (Pratt, 1942; J;5rgensen,
1956; Lefevre and Jakob, 1949; McVeigh and Brown,
1954; Lefevre and Nisbet, 1948; Lefevre, Jakob,
and Nisbet, 1952) have reported an increase in the
rate of this process, most attention having been
given to those interactions in which a reduction in
the rate of cell division appeared. Reports of in-
duced morphological changes have likewise been
relatively few .

A certain amount of ambiguity exists in the
terms used to describe the reactions of algae to
the physiologically active substances. Lefevre
and co-workers plainly distinguish in most cases
between "toxic" substances which bring about the
death of affected cells and "algostatic" substances
which retard cell division but do not cause death
of the organism. Cells affected by "algostatic"
substances resume multiplication if transferred to
fresh media . Other workers are not always so def-
inite in their descriptive terminology and it is not
always clear whether cells were actually killed or
merely prevented from undergoing cell division.

It is apparent from Table I that the algal
species which have been demonstrated to be pro-
ducers of inhibitory or stimulatory substances rep-
resent a relatively few taxonomic groups . The di-
vision Chlorophyta is represented by several gen-
era in the orders Chlorococcales , Volvocales, and
Zygnematales . Chrysophyta is represented by
diatom genera of the order Pennales and the Cya-
nophyta by various genera in Oscillatoriales and
Chroococcales . The nature of the problem of
studying growth modifying substances has de-
manded eliminating any possible effects of other
organisms from the observed reactions. For this
reason most of the studies have been carried out
with bacterially-free algal cultures. The list of
active organisms, therefore, reflects largely those
species which may be easily cultivated in pure
culture .

A few investigators (Lefevre , et al, 1950,
1951; Rice, 1954; Johnston, 1955; Proctor, 1957)
have used natural waters containing high popula-
tions of particular species in their studies and
have thus extended the laboratory observations to
field conditions . Results from studies of this type
carried out in fresh-water are grouped in Table II.
(Table at end of paper)

To generalize as to the mode of action or the
nature of the physiological processes concerned
in the observed response is difficult from present
data . The effect produced by one filtrate , for ex-
ample, may vary from one test species to another.
It may produce stimulating effects on some species,
inhibitory effects on others, and have no observ-
able effects on still others (Lefevre and Jakob,
1949; J/Jrgensen, 1956). The composition of the
media may affect the interaction between species
in mixed culture as shown by McVeigh and Brown
(1954).

It is the opinion of many investigators
(Denffer, 1948; Lefevre, and Nisbet, 1948; Lefevre,
Jakob, and Nisbet, 1952) that inhibition of growth
induced by algal metabolites results from inter-
ference with the cell division process and not, in
most cases, with nutritional processes. The few
morphological or cytological modifications which
have been reported are in many cases apparently
the result of the accumulation of abnormal amounts
of storage products within cells which failed to
divide .

NATURE OF THE INHIBITING SUBSTANCES

It seems extremely unlikely that the variety
of effects attributed by various investigators to
extracellular algal metabolites are the result of a
single chemical substance. Such widely differing,
specific physiological effects as interference with
the dark reaction in photosynthesis (Pratt, 1943),
reduction of oxygen-consumption of bacteria

40

(Steeman Nielson, 1955, 1955a), blockage of mi-
tosis (Denffer, 1948), interference with the filter-
ing action of E)aphnia (Ryther, 195 4) , as well as
such ambiguous and all inclusive terms as "stimu-
lation" and "inhibition of growth", have all been
laid to algal metabolites .

A growth- modifying substance may, of course,
be stimulating to a specific reaction in low concen-
trations and inhibitory in higher concentrations .
The action of auxins is illustrative of this effect.
Bentley (1958) has demonstrated that auxin-like
substances are produced by a variety of algae and
that these substances are excreted into the media .
While 3-indoleacetic acid was shown to have a
stimulating effect on algal growth, Bentley con-
cludes that it is not the major natural auxin pro-
duced by algae. It should be noted that in the ex-
traction and study of these materials the petroleum
ether soluble fraction was extracted from the algae
or the media and discarded in order to forstall any
inhibitory effects by fatty substances. This latter
group is perhaps most suspect as the agents active
in inhibitory or antibiotic reactions among the
algae. The work of Spoehr, et al (1949) , for ex-
ample, indicated that the antibacterial properties
of chlorellin resulted largely from photooxidizable
unsaturated fatty acids. Proctor (1957a) has shown
that the inhibition of Haematococcus pluvlalis by
Chlamydomonas reinhardi resulted from fat- like
extracellular substances. He suggested that the
inhibitor produced by Chlamydomonas was probably
a long-chain fatty acid or a mixture of such acids .
Eight long-chain fatty acids were tested against
six algae in culture, all of them showing varying
degrees of inhibition. Considerable differences in
sensitivity could be observed with Haematococcus
being most sensitive, among the algae tested, and
with Chlorella vulgaris and Scenedesmus quadri -
cauda the least. Proctor is of the opinion that the
release of the toxic substance is dependent largely
on the death of the cell. Although there has not
been sufficient evidence to establish this with
other species, it is obvious that age of the culture
is important in determining the activity of a fil-
trate . Phormidium uncinatum, for example, was
found by Lefevre and Jakob (1949) to be stimulated
by a filtrate from a young culture of Scenedesmus
quadricauda but to be inhibited by a filtrate from an
old culture. This does not appear to be a consist-
ent reaction, however, since the filtrate of the
young culture of this species brought about com-
plete inhibition of Scenedesmus oahuensis . Proc-
tor (1957a) has observed stimulatory effects of fil-
trates from young cultures of Chlamydomonas rein-
hardi and inhibitory effects from filtrates of older
cultures when tested against Haematococcus
pluvialis . This early stimulation, he feels, re-
sults from a conditioning of the medium, pre-
sumably by removal of heavy metals.

In the greater number of cases studied

(Table I), strongest Inhibition has been obtained in
filtrates from old cultures. In only a few cases,
however, has the production of inhibitor been fol-
lowed through the growth period of a culture by
quantitative procedures. In studies on Chlorella
vulgaris , Pratt, Oneto, and Pratt (1945) have shown
that almost as much of the inhibiting substance,
chlorellin, could be extracted from two day old cul-
tures as could be extracted from cultures that had
attained their full growth. During the period of
rapid growth, however, the chlorellin content de-
creased rapidly, increasing again as the rate of
growth of the colony decreased . Whether similar
conditions prevail in the case of inhibiting sub-
stances from other algae has not been adequately
determined .

The higher concentration of inhibiting sub-
stances observed in old cultures may of course re-
sult from an increased rate of production by these
cells; it may represent merely the accumulation of
large quantities of the material from prolonged
growth of the algae; or it may result from release
of larger quantities of cellular substances through
changes in permeability of the cell membrane or
from actual autolysis of old cells.

EFFECT OF DECOMPOSITION PRODUCTS

While attention has been directed thus far
largely to the action of substances released during
growth and development of algal organisms and pre-
sumably formed and excreted as a part of normal
metabolic processes, another source of soluble or-
ganic substances must be considered. The vast
quantity of material, both plant and animal, re-
leased in natural waters from organisms which
undergo a seasonal decomposition by aquatic bac-
teria and fungi give rise to a multitude of degrada-
tion compounds which makes identification or
quantitative estimation of active substances ex-
tremely difficult. Nevertheless it must be recog-
nized that such substances, particularly when they
result from the decomposition of highly concentrated
bloom communities represent a potentially important
source of soluble organic materials. Critical
studies of the effect of such degradation products
on algae in culture have been few in number.

Lefevre and Farrugia (1958) have recently re-
ported the results of studies of the effect of de-
composition products from the alga Cladophora
glomerata and the gudgeon (Gobio fluviatilis) on the
growth of seven algae in culture. Their experiments
were carried out by adding various concentrations of
the decomposition products from these organisms to
distilled water and inoculating with pure cultures of
algae. Results indicated that concentrations of the
decomposition products as low as 1 mg./l. per-
mitted growth of some algal species without the ad-
dition of mineral salts . Higher concentrations

41

resulted in stimulation of growth of certain species
and in complete inhibition of others. Decomposi-
tion products of algae and of fish gave comparable
results but affected cultures in different ways .
Each had a specific effect and each demonstrated
individual thresholds of activity for particular algae.
The action was essentially unchanged whether the
decomposition products were added in combination
to distilled water or were added separately to min-
eral media. As noted previously in studies of sub-
stances interfering with cell multiplication, dis-
tinct cellular anomalies were observed in inhibited
cells .

Preliminary studies of a somewhat similar
nature are under way in our laboratories (Hartman
and Demoise, unpublished). In these investiga-
tions, extracts have been prepared from large quan-
tities of algal material collected from Pymatuning
Reservoir at the end of the summer bloom. The
species present in the material tested were mostly
Microcystis aeruginosa and Coelosphaerium kuetz -
inganum . When water extracts, prepared by freez-
ing, repeated centrifugation , and millipore filtra-
tion were added to cultures of Scenedesmus obli -
quus and Dictyosphaerum ehrenbergianum the growth
rate of these organisms was approximately double
that of control cultures, as measured by gravimetric
or colorimetric techniques .

Extrapolation of generalizations based on re-
sults from laboratory culture studies to conditions
existing in natural waters must always be made
with an appreciation of the many difficulties in-
volved . Excreted substances which remain in
fairly concentrated form in confined culture media
may become diluted below their limit of physio-
logical activity in the great unconfined volume of
the natural situation. Activities of associated
fresh-water bacteria may result in rapid decompo-
sition of substances which remain active for long
periods in bacterially-free cultures. Active ma-
terials may also be rendered inactive in natural
waters by formations of complexes with other sub-
stances . Finally, some consideration must be
given to the fact that modifications of algal strains
under conditions of the laboratory may have given
rise to alterations in enzyme systems or to changes
in metabolic pathways which differ from those in
naturally occurring algal species .

The significance of these physiological active
substances in the ecology of the algae of natural
waters is difficult to appraise. Many workers since
the time of Akehurst have leaned heavily on exter-
nal metabolites in their attempts to explain such
seasonal succession of species and the often-ob-
served situation where a single species completely
dominates a phytoplankton bloom community. Much
of the efforts of Lefevre and his group have been
attempts to explain these phenomenon by the action
of algal inhibitors (Lefevre and Nisbet, 1948;
Lefevre, Jakob and Nisbet, 1950, 1951; Lefevre and

Farrugia, 1958). Lucas (1947, 1949, 1955) has
brought together much of the findings from other
areas of aquatic biology in support of the "exclu-
sion theory" as applied to plant and animal rela-
tionships. From his interpretation of the available
evidence he has suggested that many organisms
have adapted themselves to tolerate or take advan-
tage of the external metabolites of their neighbors
and has postulated further that many "stimulating"
and "inhibiting" ecological relationships may have
arisen in this way .

The work of Proctor (1957) represents one of
the few attempts to follow the presence of inhib-
iting substances in natural waters over several
seasons and to relate the occurrence of these sub-
stances to community structure. Work of this type
carried the criticism that the measurement of in-
hibition or stimulation is based on the reactions in
the laboratory of a single organism, in this case
Haematococcus pluvialis . The method is also open
to criticism on the grounds that there is not direct
evidence that phytoplankton populations are the
source of the active substances being studied . In
spite of such objections this type of approach com-
bining seasonal studies on natural waters with lab-
oratory culture studies of bloom-producing species,
promises to be the most rewarding in terms of under-
standing the role of algal metabolites in influencing
the structure and occurrence of many phytoplankton
communities .

In summary, it would appear that ample evi-
dence now exists to indicate that many species of
fresh-water algae are capable of producing physio-
logically active metabolites which may function as
toxins, growth inhibitors, or growth stimulators to
themselves or to associated algae. Natural waters
supporting bloom concentrations of algae have been
shown to contain substances inhibitory to various
species although evidence is not absolute that the
active substances are derived directly from algae .
Chemical and physical studies of the active sub-
stances have indicated that in a number of cases
these materials appear to be related to fatty acids .

There remain many unsolved problems and un-
answered questions: One is concerned with the
chemical nature of the inhibiting or stimulating sub-
stance and the mechanism of physiological action
on affected organisms . A second is concerned with
the nature of the production of the active materials
and its release from the source organism. Is it a
metabolite produced and excreted during normal
growth of the organism or is it produced only on
death and subsequent breakdown of cells? Finally
there remains the question most important perhaps to
to the algal ecologist - Are these substances pro-
duced in physiologically effective concentrations in
nature and do they remain at these concentrations
for sufficient time to affect the growth of other
algae?

42

Test Organism

TABLE I
Summary of laboratory studies on the production of physiologically
active metabolites by algae in culture
Interacting Organism Observed Effects

Reference Source

Chlorella vulgaris
C . vulgaris

C. \ailgaris

G. vulgaris
C . vulgaris
C . vulgaris
C . vulgaris
C_. pvrenoidosa
C . pvrenoidosa
C . pyrenoidosa

C . pyrenoidosa

C . pyrenoidosa

Scenedesmus
quadricauda

S^. quadricauda
S. quadricauda
S . quadricauda

Chlorella vulgaris
Chlorella vulgaris

Nitzschia frustulum

Scenedesmus quadricauda

Chlamydomonas reinhardi

Haematococcus pluvialis

Daphnia magna

Euglena deses

Pediastrum duplex

Pediastrum clathratum
var . punctulatum

Nitzschia pa lea
Scenedesmus quadricauda
Phormidium uncinatum

P. uncinatum

Scenedesmus Oahuensis

Pediastrum borvanum

Autoinhibition observed
in culture filtrates

Stimulating effect of
filtrate in low concen-
tration

Mutual inhibition in
mixed culture and in-
hibition in filtrate

Inhibition in mixed
culture

Inhibition in mixed
culture

Inhibition in mixed
culture

Inhibited rate of
filter feeding

Inhibition in mixed
culture

Inhibition in mixed
culture

Inhibition in mixed
culture

Inhibition by filtrate

Acceleration by filtrate

Stimulation by filtrate
of young culture

Inhibition by filtrate
of old culture

Inhibition by filtrate
of old culture

Inhibited by culture
filtrate

Pratt (1940)
Pratt (1942)

Rice (1954)

Proctor (1957)
Proctor (1957)
Proctor (1957)
Ryther (1954)

Lefevre and

Nisbet (1948)

Lefevre and

Nisbet (1948)

Lefevre, Nisbet
and Jakob
(1950)

J^rgensen (1956)

Jpfrgensen (1955)

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre and Nisbet
(1948)

43

Test Organism

Interacting Organism

Observed Effects

Reference Source

^. quadricauda

S . quadricauda
S . quadricauda
^ . quadricauda

S . quadricauda
S . quadricauda

S. quadricauda

S . quadricauda

S . quadricauda

S. quadricauda

S^. quadricauda

S . quadricauda

S. oahuensis
^. obliquus
S . obliquus
^. obliquus
Pediastrum tetras

Dictyosphaerium
ehrenbergianum

Gosmarium botryti s

Nitzschia pa lea
Chlorella pyrenoidosa
Haematococcus pluvialis

S . quadricauda
Daphnia magna

Pediastrum clathratum
var . punctulatum

Gosmarium lundellii

Gosmarium ochthodes

Phormidium uncinatum

Microcystis aeruginosa

Microcystis aeruginosa

Dictyosphaerium
ehrenbergianum

Scenedesmus obliquus

Microcystis aeruginosa

Inhibited by culture
filtrate

Inhibited by filtrate

Inhibited by filtrate

Mutual inhibition in
mixed culture

Inhibited by filtrate

Inhibited rate of filter
feeding

Inhibited by filtrate

Mesotaenium caldoriorum Inhibited by filtrate

Inhibited by filtrate

Inhibited by filtrate

Gomplete inhibition
by filtrate

Achnanthes microcephala No action by filtrate

Achnanthes microcephala Inhibited in mixed

culture

Inhibited in mixed
culture

Inhibited by filtrate

Inhibited by filtrate

No effect of filtrate

Inhibited by filtrate

Lefevre and

Nisbet (1948)

Xffrgensen (1956)

Jj5rgensen (1956)

Proctor (1957,
1957a)

Jprgensen (1956)

Ryther (1954)

Lefevre, Nisbet
and Jakob
(1949)

Lefevre, Nisbet
and Jakob
(1949)

Lefevre, Nisbet
and Jakob
(1949)

Lefevre, Nisbet
and Jakob
(1949)

Lefevre, Jakob
and Nisbet
(1952)

Lefevre , Jakob
and Nisbet
(1952)

Lefevre and Jakob
(1949)

Hart man

(Unpublished)

Hartman

(Unpublished)

Hartman

(Unpublished)

Hartman

(Unpublished)

Hartman

(Unpublished)

44

Test Organism

Interacting Organism

Observed Effects

Reference Source

Pandorina

morum

Pandorina

morum

P. morum

P. morum

P. morum

P. morum

P . morum

P. morum

Cosmarium lundellii

Scenedesmus ovalternus

S . ovalternus

Cosmarium spp .

Micrasterias spp.

Cosmarium obtusatum

Pediastrum boryanum

P . clathratum var .
punctulatum

Inhibited by filtrate

Inhibited by boiled or
frozen filtrate

Stimulated by untreated
filtrate

Inhibited by filtrate

Inhibited by filtrate

Not inhibited by filtrate

Not inhibited by filtrate

Inhibited by filtrate

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre and Jakob
(1949)

Lefevre, Nisbet
and Jakob
(1949)

P. morum

Scenedesmus oahuensis

Inhibited by filtrate

Lefevre, Nisbet
and Jakob
(1949)

P. morum

Achnanthes microcephala

Inhibited by filtrate

Lefevre, Nisbet
and Jakob
(1949)

P. morum

Phormidium uncinatum

Good growth for 1 week
then cells die

Lefevre, Nisbet
and Jakob
(1949)

P. morum

Mesotaenium caldariorum

Not inhibited by filtrate

Lefevre, Nisbet
and Jakob
(1949)

P. morum

Cosmarium lundellii

Cells accumulate reserve
materials, fail to divide,
die.

Lefevre, Nisbet
and Jakob (1949
(1949)

P. morum

Cosmarium ochthodes

Cells accumulate reserve
materials, fail to divide,
die.

Lefevre, Nisbet
and Jakob
(1949)

Chlamydomonas
chlamydoqama

Haematococcus pluvlalis

Stimulation or inhibition
depending on media and
time in culture

McVeigh and
Brown (1954)

Chlamydomonas sp .

Chlorella sp .

Filtrates permit growth in
situation where Chlorella
could not otherwise grow

Allen (1955)

45

Test Organism

Interacting Organism

Observed Effects

Reference Source

C . reinhardi

C . reinhardi

Haematococcus pluvialis

Killed in mixed culture

Proctor (1957)
(1957a)

Scendesmus quadricauda Inhibited in mixed culture Proctor (1957)

Haematococcus pluvialis Chlamydomonas chlamydogma Stimulation or inhibition

depending on media and
time in culture

H . pluvialis

Scenedesmus quadricauda Mutual inhibition in

mixed culture

Selanastrum minutum Cosmarium impressulatum No inhibition in mixed

culture

Cosmarium impressulatum Selanastrum minutum

No inhibition in mixed
culture

McVeigh and
Brown (1954)

Proctor (1957)
(1957a)

Lefevre and

Nisbet (1948)

Lefevre and

Nisbet (1948)

DIATOMS

Nitzschia frustulum

Nitzenhia palea
N. palea
N . palea

N. palea

N. palea

N. palea

N . palea

N . palea

N . palea

N. palea

Chlorella vulgaria

Nitzschia palea
Chlorella pyenoidosa
Asterionella for mo s a

Senedesmus quadricauda
Pediastrum boryanum

Scenedesmus quadricauda

Cosmarium lundelli

C . obtusatum

Nitzachia palea

Phormidium uncinatum

Mutual inhibition in
mixed cultures .
Inhibition by filtrate

Inhibited in filtrate

Accelerated in filtrates

Inhibited in filtrate
and mixed cultures

Some multiplications ,
cells clumped

Very few multiplications

Inhibition by filtrate

Cells killed by filtrate

Good development
in filtrate

Slight development in
filtrate

Rice (1954)

Jprgensen (1956)
J^rgensen (1956)
J;5rgensen (1956)

Accelerated in filtrate J;irgensen (1956)

Lefevre, Jakob
and Nisbet
(1952)

Lefevre , Jakob
and Nisbet
(1952)

Lefevre , Jakob
and Nisbet
(1952)

Lefevre, Jakob
and Nisbet
(1952)

Lefevre , Jakob
and Nisbet
(1952)

Lefevre , Jakob
and Nisbet
(1952)

46

Test Organism

Interacting Organism

Observed Effects

Reference Source

Asterlonella formosa

A. formosa

Nltzschia pa lea

Asterionella formosa

A. formosa

A. formosa

Fragllarla crotonensis

Phormidlum uncinatum

P . uncinatum

_P. pachydermaticum

_P. pachydermaticum

P. pachydermaticum

_P. pachydermaticum

P . pachydermaticum

Ph . uncinatum

Ph . uncinatum

Ph . uncinatum

Ph . uncinatum

Ph . uncinatum

No s toe gelatimosum
N. gelatlnosm

N. gelatinosum
N. gelatlnosm

Nltzschia palea
Fragllarla crotonensis

Asterlonella formosa

Scenedesmus quadrlcauda

Achnanthes microcephala

Phormidlum uncinatum

Nostoc zetterstedti

N . verrucosum

Inhibition by filtrate
and in mixed cultures

Accelerated in filtrate

Accelerate in filtrate

No inhibition In mixed
culture or filtrate

No inhibition in mixed
culture or filtrate

Inhibited by filtrate

Stimulated in mixed
culture and by filtrate

Killed by filtrate

Unaffected by filtrate

Unaffected by filtrate

Cylindrospermum alatosporum Inhibited in mixed

culture

Nostoc gelatinosa
Pediastrum boryanum

P . clathratun var .
punctulatum

Scenedesmus oahuensis

Ankistrodesmus falcatus

Achnanthes microcephala

Oscillatorla acutissima
Anabaena cyllndrica
Nostoc verrucosum

N . carneum

Mutually inhibitory on
solid media

Inhibited by filtrate
of old cultures

Inhibited by filtrate
of old cultures

Inhibited by filtrate
of old cultures

Stimulated by filtrate
of young cultures

Stimulated by filtrate
of young cultures

Inhibited by filtrate
Inhibited by filtrate
Unaffected by filtrate
Unaffected by filtrate

Jdrgensen (1956)

J;<rgensen (1956)

J;irgensen (1956)
Tailing (1957)

Tailing (1957)

Lefevre and

Nisbet (1948)

Lefevre and

Nisbet (1948)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Lefevre, Jakob
and Nisbet
(1952)

Lefevre , Jakob
and Nisbet
(1952)

Lefevre, Jakob
and Nisbet
(1952)

Lefevre , Jakob
and Nisbet
(1952)

Lefevre, Jakob
and Nisbet
(1952)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

47

Test Organism

Interacting Organism

Observed Effects

Reference Source

N.

qelatinosum

N.

gelatinosum

N.

gelatinosum

N.

zellerstedti

N.

zellerstedti

N.

zellerstedti

N.

zellerstedti

N.

zellerstedti

N.

zellerstedti

N.

zellerstedti

N.

verrucosum

N.

verrucosum

IL-

borneti

Anacystis nidulans
A. nidulans
A^. nidulans
A. nidulans

N ■ borneti

Phormidium rubroterricola

Anabaena cylindrica

Nostoc Broneti

N . verrucosum
Phormidium pachydermaticum
P . uncinatum
Oscillatoria acutissima

Nostoc verrucosum

Nostoc borneti

Cvlindospermum alatosporum

Nostoc zellerstedti

N . zellerstedti

Chlorella vulgaris
Scenedesmus quadricauda
Chi a mydo mo na s reinhardi
Haematoccus pluvialis

Microcystis aeruginosa Scenedesmus obliquus

Microcystis aeruginosa Scenedesmus obliquus

M. aeruginosa

Cylindropermum
alatosporum

A. cylindrica

Dictyosphaerium
ehrenbergianum

Nostoc verrucosum

A. cylindrica

Sarcina subflava

Corynebacterium faciens

Unaffected by filtrate

Mutually inhibitory on
solid media

Mutually inhibitory on
solid media

Little affected by filtrate

Little affected by filtrate

Little affected by filtrate

Inhibited by filtrate

Inhibited by filtrate

Mutually inhibitory on
solid media

Mutually inhibitory on
solid media

Mutually inhibitory on
solid media

Mutually inhibitory on
solid media

Mutually inhibitory on
solid media

Inhibited in mixed culture

Inhibited in mixed culture

Killed in mixed culture

Killed in mixed culture

No effect in mixed
culture

Slight inhibition by
filtrate

Inhibition by filtrate
Inhibition in filtrate

No appreciable affect
by concentrated filtrate

No appreciable affect
by concentrated filtrate

Jakob (1954)
Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Jakob (1954)

Proctor (195 7)

Proctor (1957)

Proctor (1957)

Proctor (1957)

Hart man

(Unpublished)

Hartman

(Unpublished)

Hartman

(Unpublished)

Jakob (1954)
Fogg (195 2)
Fogg (195 2)

48

Test Organism

Interacting Organism

Observed Effects

Reference Source

A. cylindrica

A. cylindrica

Blue green spp.
(Later identified
as Fischerella)

Rhodotorula rubra

Torulopsis utitis

No details

Mesotaenium caldariorum Phormidium uncinatum

M. caldariorum

M. caldariorum

M. caldariorum

M. caldariorum

Scenedesmus oahuensis

Cosmarium ochthodes

Pediastrum clathratum

Anklstrodesmus falcatus Euglena gracilis

A. falcatus

A. falcatus

A. falcatus

A. falcatus

A. falcatus

Scenedesmus oahuensis

S . falcatus

A. falcatus

Pediastrum boryanum

P . clathratum var.
punctulata

Cosmarium botrytis

Mesotaenium caldariorum

No appreciable affect by
concentrated filtrate

No appreciable affect by
concentrated filtrate

Production of antibiotic
substances

No effect by filtrate

No effect by filtrate

Scenedismus quadricauda No effect by filtrate

Little effect by filtrate

Complete inhibition by
filtrate

No effect of filtrate

No effect of filtrate

No effect of filtrate

No effect of filtrate

No effect of filtrate

Inhibition by filtrate

Slight inhibition by
filtrate

Fogg (1952)
Fogg (1952)

Flint and Moreland
(1946);Proc.La.
Acad . Sci .
10:30-31.(1957)

Lefevre , Jakob and
Nisbet (1952)

Lefevre, Jakob and
Nisbet (1952)

Lefevre , Jakob and
Nisbet (1952)

Lefevre, Jakob and
Nisbet (1952)

Lefevre , Jakob and
Nisbet (1952)

Lefevre, Jakob and
Nisbet (1952)

Lefevre , Jakob and
Nisbet (1952)

Lefevre , Jakob and
Nisbet (1952)

Lefevre , Jakob and
Nisbet (1952)

Lefevre , Jakob and
Nisbet (1952)

Lefevre, Jakob and
Nisbet (1952)

Lefevre , Jakob and
Nisbet (1952)

r, n, I /;

49

TABLE II
Summary of Studies of the Effect of Natural Waters containing
bloom communities on the growth of algae in culture.

Water Source

Interacting Organism

Observed Effects
in Filtered Water

Reference Source

Canal water containing
bloom of Aphanizome-
non gracile

Canal water containing
bloom of Oscillatoria
planctorica

Canal water containing
bloom of Oscillatoria
planctorica

Pond water containing
bloom of Anabaena
planktonica

Cosmarium lundellii

Inhibition of growth

Micrasteris papillifera Inhibition of growth

Pediastrum boryanum Inhibition of growth

_P . clathratum v . punctulalum Inhibition of growth
Phormidium uncinatum Inhibition of growth

No effect

Nitzschia pa lea

Micrasteris papillifera
Chlorella pyrenoidosa

Cosmarium lundellii
C . obtusatum

Micrasteris pacillifera
Pediastrum boryanum
Phormidium uncinatum
P. autumnale

Scenedesmus quadricauda

Pediastrum clathratum
var . punctulatum

P ■ Boryanum

Cosmarium lundellii

C . obstusatum

No effect
Inhibition of growth

Inhibition of growth
Inhibition of growth
Inhibition of growth
Inhibition of growth
Inhibition of growth
Inhibition of growth
Inhibition of growth

Complete inhibition
with death of cells

Complete inhibition
with death of cells

Complete inhibition
with death of cells

No effect

Lefevre, Jakob,
and Nisbet (1950)

Lefevre, Jakob,
and Nisbet (1952)

Lefevre , Jakob ,
and Nisbet (1950)

Lefevre, Jakob
and Nisbet (1950)

Lefevre , Jakob
and Nisbet (1952)

50

Water Source

Interacting Organism

Observed Effects
in Filtered Water

Reference Source

Pond water containing
bloom of Anabaena
planktonica

Pond water containing
bloom of Anabaena
spiroides

Pond water containing
bloom of Anabaena
spiroides

Pond water containing
bloom of Microcystis
flos aquae

Pond water containing
bloom of Ceratuim
hirundinella

Phormidium uncinatum

Scenedesmus quadricauda

Nitzschia palea

Chlorella pyrenoidosa

Pediastrum clathratum
var . punctulatum

_P. boryanum
Cosmarium lundelll

C . obtusatum

Pediastrum clathratum
var . punctulatum

P . boryanum

Cosmarium lundellii
C . obstusatum

Pediastrum clothratum
var . punctulatum

_P. boryanum

Scanedesmus quadricauds

Cosmarium lundelli

C . obstusatum

Chlorella pyrenoidosa

Phormidium uncinatum

Nitzschia palea

Pediastrum clathratum
var . punctulatum

_P . boryanum

Inhibition of growth

Slight inhibition
Slight inhibition
Inhibition of growth
Inhibition of growth

Slight inhibition

Complete inhibition
with death of cells

Inhibition of growth

Complete inhibition,
with death of cells

Inhibition of growth
Slight inhibition

Increased growth

over control

Complete inhibition
with death of cells

Lefevre, Jakob
and Nisbet (1952)

51

Water Source

Interacting Organism

Observed Effects
in Filtered Water

Reference Source

Pond water containing
bloom of Ceratuim
hirundinella

Pond water containing
bloom of
Spirogyra (sp)

Pond water containing
bloom of Pandorina

Pond water containing
bloom of Anabaena
spiroides and Micro -
cystis aeruginosa ;
also blooms of
Crypto monas sp . , and
Lyngbya aesturaii

Cosmarium lundellii

C . obstusatum

Phormldium uncinatum

Nitzschia palea

Scenedesmus quadricauda

Ghlorella pyrenoidosa

Pediastrum clathratum
var . punctulatum

_P . boryanum
Nitzschia palea
Cosmarium lundellii
Phormidium uncinatum

Ghlorella vulgaris

Nitzchia frustulum
Haematococcus pluvialis

Complete inhibition
with death of cells

Inhibition of growth

Inhibition of growth

Complete inhibition
with death of cells

Inhibition of growth

Inhibition of growth
Variable Inhibition

Lefevre , Jakob
and Nisbet (195 2)

Rice (1954)

Rice (1954)
Proctor (195 7)

52

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Allen, M. B. 1955 . General features of algal growth in sewage oxidation ponds. Calif. State
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Allen, M. B. 1956. Excretion of organic compounds by Chlamydomonas . Arch. Mikrobiol . 24:163-168.

Bentley, Joyce A. 1958. Role of plant hormones in algal metabolism and ecology. Nature 181:1499-1502.

De, P. K. 1939 . The role of blue-green algae in nitrogen fixation in rice fields. Proc . Roy. Ser. B
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Flint, Lewis H. and Charles F. Moreland . 1946. Antibiosis in the blue-green algae. Amer . J. Bot.
33:218 (an abstract) .

Fogg, G. E. 1942. Studies on nitrogen fixation by blue-green algae. I. Nitrogen fixation by
Anabaena cylindrica Lemm. J. Exp. Biol. 19:78-87.

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Fogg, G. E. 1953. The Metabolism of Algae . London: Methuen & Co . , Ltd.

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Fogg, G. E. and D. F. Westlake. 1955 . The importance of extracellular products of algae in fresh-
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Harder, R. 1917. Ernahrungsphysiologishe Untersuchungen an Cyanophyceen , hanptsachlich dem
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Hartman, Richard T. and Charles F. Demoise. Effects of extracts from algal bloom communities on the
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Henrikson, Elisabet. 1951. Nitrogen fixation by a bacterial free, symbiotic Nostoc strain isolated
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Hutchinson, G. E. 1941. Ecological aspects of succession in natural populations. Amer. Nat.
75:406-418.

Hutchinson, G. E. 1944. Limnological studies in Connecticut . VII. A critical examination of the
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Jakob, H. 1954. Compatibilities et antagonismes entre algues du sol. C. R. Acad. Sci . 238:928.

Johnston, R. 1955. Biologically active compounds in the sea. J. Mar. Biol. Assn. U. K. 34:185-195.

53

IfJrgensen, Erik G. 1956. Growth inhibiting substances formed by algae. Physiol. Plantarum.
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Lefevre, Marcel and Gisele Farrugia . 1958. De I'influence sur les algues d'eau douce, des produits
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Hydrobiologia . 10:49-65.

Lefevre, M. and H. Jakob. 1949. Sur quelques proprletes des substances activee tirees des cultures
d'Algues d'eau douce. G. R. Acad. Sci . Paris 229:234-236.

Lefevre, M., H. Jakob, and M. Nisbet. 1950. Sur la secretion, par certaines Gyanophytes, de
substances algostatiques dans les collections d'eau naturelles . G. R. Acad. Sci., Paris
230:2226.

Lefevre, M., H. Jakob, and M. Nisbet. 1951. Gompatibilities et anta-gonismes entre algues
d'eau douce dans les collections d'eau naturelles. Verh . Int. Ver. Limnol . 11:224.

Lefevre, M., H. Jakob, and M. Nisbet. 1952. Auto- et heteroantagonisme chez les algues d'eau
douce. Annales Station Gentr. Hydrobiol . Appl . 4:5.

Lefevre, Marcel and Maud Nisbet. 1948. Sur la secretion, par certaines especes d'algues, de
substances inhibitrices d'autres especes d'algues . G . R. Acad. Sci . , Paris 226:107-109.

Lefevre, M., M. Nisbet, and H. Jakob. 1949. Action des substances excretees, en culture, par

certaines especes d'algues, sur le metabolisme d'autres especes d'algues. Verh. Int. Ver. Limnol,
10:259.

Lewin, Ralph A. 1956. Extracellular polysaccharides of green algae . Gan. J. Microbiol. 2:565-672.

Lucas, S. E. 1947. The ecological effects of external metabolites. Biol. Rev. 22:270.

Lucas, S. E. 1949. External metabolites and ecological adaptation. Symp. Soc . Exp. Biol. III.
Selective toxicity and antibiotics, pp. 336-356.

Lucas, S. E. 1955. External metabolites in the sea. Deep-Sea Research. 3 Suppl:139.

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81:218-233.

Myers, J. 1951. Physiology of the algae . Ann. Rev. Microbiol. 5:157.

Myers, Jack and James A. Johnston. 1949 . Carbon and nitrogen balance of Ghlorella during growth.
Plant Physiol . 24:111-119.

Pratt, Robertson. 1942. Studies on Ghlorella vulgaris . V. Some properties of the growth inhibitor
formed by Ghlorella cells. Amer . J. Bot. 29:142.

Pratt, Robertson. 1943. Studies on Ghlorella vulgaris . VI. Retardation of photosynthesis by a
growth-inhibiting substance from Ghlorella vulgaris . Amer. J. Bot. 30:32-33.

Pratt, Robertson. 1944a. Studies on Ghlorella vulgaris . IX. Influence on growth of Ghlorella of
continuous removal of chlorellin from the culture solution. Amer. J. Bot. 31:418-

Pratt, Robertson. 1948. Studies on Ghlorella vulgaris . XI. Relation between surface tension and
accumulation of chlorellin . Amer. J. Bot. 35:634.

54

Pratt, Robertson and Jane Fong . 1940. Studies on Chlorella vulgaris . II. Further evidence for a growth
inhibiting substance. Amer . J. Bot . 27:431-436.

Pratt, R. , J. F. Oneto, and J. Pratt. 1945 . Studies on Chlorella vulgaris . X. Influence of the age of
of the culture on the accumulation of chlorellin. Amer. J. Bot. 32:405-408.

Pratt, Robertson et . a_l^. 1944. Chlorellin, an antibacterial substance from Chlorella . Science.
99:351-352.

Proctor, Vernon W. 1957. Some controlling factors in the distribution of Haematococcus pluvalis .
Ecology. 38:457-462.

Proctor, Vernon W. 1957a. Studies of algal antibiosis using Hematococcus and Chlamydomonas .
Limnol . and Oceanog. 2:125-139.

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U. S. No. 87, 54:227.

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Spoehr , H. A. and Harold W. Milner. 1949. The chemical composition of Chlorella; effect of
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I^'r'i- VVO^

55

ALGAE IN RELATION TO OXIDATION PROCESSES IN NATURAL WATERS

Alfred F. Bartsch

Water Supply and Water Pollution Research

Robert A. Taft Sanitary Engineering Center

of the

United States Public Health Service

Cincinnati, Ohio

Agencies concerned with management of water
quality are especially interested in the following
oxidation processes that occur in natural waters:
(a) stabilization by microorganisms of natural or in-
troduced organic matter, (b) respiration by the
aquatic biota, and (c) direct oxygen demand of
chemical substances. Each process to some ex-
tent draws upon the oxygen resources of the water
so that replenishment must occur to prevent serious
and lasting oxygen depletion. Replenishment oc-
curs chiefly by reaeration from the atmosphere and
through photosynthesis in algae and other aquatic
plants. As will be shown, algae are involved in
several ways in both the oxidation processes and
the broader oxygen relations in natural waters .

Direct Algal Oxidation of Organic Matter

Algae have direct as well as indirect effects
upon oxidation processes . It has been shown that
a number of algae are able to use organic sub-
stances directly for energy and growth (Samejima
and Meyers, 1958; Eny, 1951; Wilson and Danforth,
1958) . A recent summary by Saunders (1957) lists
32 species of phytoplankton that possess this ca-
pability. Also listed is a wide variety of the or-
ganic substances known to be used, grouped as
carbohydrates, aldehydes, ketones, alcohols, es-
ters, fatty acids, organic acids, amino acids, and
related compounds .

Raw and treated sewages have not generally
been analyzed thoroughly for their content of or-
ganic compounds of the groups cited above. The
origin and history of domestic sewage leave little
doubt, however, that many if not all of these or-
ganics are present, along with others not men-
tioned. Presumably, select members of the phyto-
plankton in natural waters utilize some of these or-
ganics if they are present. When such organics in
polluted waters are utilized by phytoplankton, the
resulting reduction in their concentration would be
beneficial to water quality in the same sense that
bacterial oxidation is beneficial. In this sense the
phytoplankton participate directly in the oxidation
processes that occur in natural waters, although
the extent of such action has not been determined .
From casual observation it would appear that the
over-all effect of this type of algal activity upon
both the dissolved organics content and the
stream's oxygen resources would be of little con-

sequence . The algae would be merely competing
with and perhaps to some extent displacing the ox-
idation activities of the customary assemblage of
heterotrophic organisms that stabilize such pol-
lutants .

Shallow waste stabilization ponds are becom-
ing popular for treating municipal sewage and some
industrial wastes and have recently received con-
siderable study (Gotaas , Oswald andLudwig, 1954;
Oswald and Gotaas, 1955; Hermann and Gloyna, 1955;
Gloyna and Herman, 1956; Anon . , 1957; Bartsch and
Allum, 1957) . Successful operation depends es-
sentially on bacterial decomposition of the waste
organics and algal photosynthesis to provide dis-
solved oxygen for the aerobic processes. Obser-
vations on many sewage stabilization ponds have
shown that ability to reduce organic matter, as
measured by the B.O.D. test, is not seriously im-
paired during winter in spite of cold weather con-
ditions that generally impede biochemical proces-
ses . B.O.D. reduction persists at 70% or more,
in spite of anaerobic conditions under semiopaque
ice cover 9 to 30 inches thick. Undoubtedly, a
combination of processes makes possible this con-
tinued reduction . Much of the phytoplankton
gathers on the bottom, but appreciable quantities
of Scenedesmus dimorphus , S. quadricauda ,
Chlorella sp . , Euqlena sp . , Phacus longicauda ,
and other algae have been observed suspended in
the water, motile if flagellated, and obviously ac-
tive. With extremely low light intensity below the
ice, the presence and active condition of these
algae raise the question of heterotrophic use of or-
ganic substances as a contribution to the stabiliza-
tion performance. Interestingly, these algae are
still active in the prolonged absence of dissolved
oxygen as shown by the Winkler procedure and by
the presence of sulfides up to 117 ppm. Such ac-
tivity of Chlorella , Chlamydomonas , and Euglena
in the presence of hydrogen sulfide has been ob-
served also by Abbott (1951). The full significance
of these observations is not presently apparent,
and further study is needed to establish clearly the
role of algae along with other more obvious proces-
ses in the winter performance of waste stabiliza-
tion ponds .

Along this same line, studies are in progress
at the Sanitary Engineering Center to determine the
ability of specific algae to utilize sugars in spent
sulfite liquor. The study was designed to explore
possibilities that algae may serve in treating this

56

industrial waste .

Provision of Dissolved Oxygen

In relation to water quality management, the
most valuable contribution of phytoplankton is pro-
vision of oxygen for use in oxidation by the total
aerobic biota. Oxygen is vitally important because
it affects the capacity of streams to receive and
oxidize sewage and other organic wastes without
unduly impairing water quality. Multiple use of
water resources is increasingly necessary as
water-hungry population and industries grow while
the total water supply remains constant. Use of
water for waste disposal presently is inescapable.
It is therefore appropriate to point out some of the
fundamental oxygen relationships related to waste
disposal. Aside from the natural cyclic effects of
phytosynthesis by aquatic plants, significant
changes in the dissolved oxygen content of a
stream result primarily from the oxidation of or-
ganic matter entering as waste. A combination of
available dissolved oxygen and suitable biota -
especially aerobic bacteria - results in progres-
sive oxidation and stabilization of the organic mat-
ter. It has been postulated (Streeter and Phelps,
1925) that the rate of biochemical oxidation of or-
ganic matter is proportional to the remaining con-
centration of unoxidized substance measured in
terms of oxidizability. Unpolluted water tends to
hold in solution the maximum amount of oxygen it
is capable of holding at the existing temperature
and partial oxygen pressure of the atmosphere, but,
when organic pollutants are introduced, use of the
dissolved oxygen supply in progressive satisfac-
tion of the demand tends to reduce the oxygen con-
tent below this saturation value. Sources of oxy-
gen available for this use are (l) that initially dis-
solved in the water, (2) that absorbed from the at-
mosphere by partially deoxygenated water, and
(3) oxygen made available through photosynthesis
of aquatic plants . These interrelations are shown
graphically in a recent publication (Bartsch and
Ingram, 1959) . At present, our interest is directed
to the last oxygen source only.

Equations have been developed and used
widely to elucidate the dynamics involved in the
oxygen resources of streams (Streeter and Phelps,
1925) . Although others have taken a modified ap-
proach and added refinements to evaluating these
relations, photo synthetic oxygen production, un-
fortunately, has remained elusive of precise quan-
titative expression as a part of the over-all pic-
ture . That photosynthesis in aquatic plants af-
fects dissolved oxygen levels in surface waters
has been known for a long time . General relation-
ship of photosynthesis to stream sanitation, how-
ever, is a more recent interest. Studies of
Potomac River plankton and rooted plants in 1913

by Purdy (Gumming, 1916) included measurement of
oxygen changes caused by photosynthesis. The
importance of plant-covered flats below the Dis-
trict of Columbia in accelerating recovery from
pollution originating at that point is discussed at
some length. Oxygen production in one area was
shown to be 17.7 pounds per acre per day, mainly
by submerged aquatic plants growing on flats out-
side the river channel .

Calvert (1933) studied diurnal fluctuations of
dissolved oxygen in the White River below the
Indianapolis sewage treatment plant in relation to
varying B.O.D. load and amount of sunshine.
Daytime oxygen concentrations frequently reached
5 or 6 ppm . but usually dropped to zero at night,
showing the importance of algal photosynthesis in
maintaining a daytime supply of dissolved oxygen
and preserving desirable aerobic conditions .

Since the early observations cited , diurnal
variations in dissolved oxygen have been noted
and reported many times . Typical variations are
shown in Figure 1 for two stations on French Creek
near Meadville, Pennsylvania, where a study was
made in August, 1955 , to determine stream condi-
tions and assimilation capacity characteristics.
Obviously, monetary oxygen data, such as might
be obtained by sampling as chance and conven-
ience bring the technician and the stream together,
have little value in showing stream character. If
station A had been sampled only at 5:00 p.m. , the
early morning depression would have been missed;
if sampled only at 8:00 a.m., the period of super-
saturation would not have been detected . The sit-
uation would have been the same at station O but
the differences less pronounced. Sampling defi-
ciencies such as these are discussed thoroughly by
Gameson and Griffith (1959) . It is obvious that
stream technicians cannot properly discern oxygen
conditions in surface waters by restricting them-
selves to field sampling and analyses only during
the usual working hours between 8 and 5 .

Newly developed equipment that automati-
cally analyzes and records dissolved oxygen
(Macklin, Baumgartner, and Ettinger, 1959) now
makes collection of diurnal data both less tedious
and more accurate. Desired oxygen data are now
obtainable with less nocturnal sampling under the
adverse conditions of darkness . Figure 2 shows
dissolved oxygen records for a two-day period ob-
tained with such equipment at an experimental
sewage stabilization pond. The record for both
days shows dissolved oxygen was absent at sun-
rise. Factors affecting photosynthesis and oxygen
retention by the water obviously were more favor-
able on November 19 than November 14 according
to dissolved oxygen levels attained. The periodic
irregularities resulted from variation in illumina-
tion . It is sometimes taken as a "rule of thumb"
in reference to algal photosynthesis that the higher
the oxygen level climbs above saturation in the

57

0800

1200

OUOO

0800

1200

OUOO

0800

1200

AM

NOON

PM

PM

MIDNIGHT

AM

AM

NOON

0^00

PM

DISSOLVED OXYGEN VS. TIME OF DAY IN FRENCH CREEK.

PENNSYLVANIA. AUGUST 17. 18, 1955

Figure I

daytime the lower it is likely to drop below it at
night. The record for the Little Miami and Ohio
Rivers (Figure 3) seems to substantiate this "rule"
when compared with the stabilization pond in Fig-
ure 8. For the period shown, the Ohio deviated
only slightly from air saturation whereas the Little
Miami, with more numerous attached algae and a
habitat more favorable for photosynthesis, exhibits
a little more vigorous photo synthetic oxygen pro-
duction .

Recently, a six- month record of dissolved
oxygen has been obtained for a small polluted
stream in England (Gameson and Griffith, 1959)
using photographic equipment to record the dial
readings from a dropping mercury electrode (Briggs,
Dyke, and Knowles , 1959). Data for the six- month
average diurnal oxygen curve in Figure 4 were ob-
tained in this way. While obviously the daily ir-
regularities and extremes have been eliminated, the
curve shows the times of day when the maxima and
minima might be expected for this stream. About
two-thirds of the maxima were found to occur be-
tween noon and 6:00 p.m.; 85 per cent of the mini-
ma , between 7:45 p.m. and 7:45 a.m. This curve
is not unusual; it agrees generally with the much
shorter term observations on other waters where the
minimum commonly has been noted around 3:00 to

5:00 a.m. and the maximum around 4:00 or 5:00
p.m.

A number of approaches have been made to
detect and evaluate the influence of algal photo-
synthesis on oxygen resources in streams . For a
given reach of a hypothetical stream, Odum (195 6)
has attempted to interrelate existing oxygen con-
centration, provision of oxygen by inflowing water,
the atmosphere, and photosynthesis with losses
through respiration and diffusion (Figure 5) . Three
main daily processes are noted to affect the oxygen
and carbon dioxide concentrations of water flowing
between two established points . These are release
of oxygen into the water during daylight through
photosynthesis (A in Figure 5) , absorption of dis-
solved oxygen by all organisms (B) , and oxygen ex-
change between water and air (G) . Interplay of
these three processes determines the rate of oxygen
change (D) and the resulting oxygen concentration
(E) that would appear in a homogeneous medium.

Toward assessing the effect of large quanti-
ties of blue-green algae on oxygen resources in
Wisconsin's Lower Fox River, Wisniewski (1958)
modified the customary B.O.D. procedure to meas-
ure algal respiration and photosynthesis . Results
for a sample from one station are shown in Figure 6 .
When incubated for five days at a light intensity of

58

X

o

o

O

>-
X

o

o
to

60-
50 -
UO —
30 -

20 —
10 —

6

NOV. 19, 1958

NOV. lU. 1958

8

12

U
AM

6

Dl

URNAL VARIATION IN DISSOLVED OXYGEN IN A SEWAGE STABILIZATION POND

LEBANON. OHIO
Figure 2

120

—

'

1 1

1 1 TTI r IJ 1 AKi 1

RIVER

1

1 1 1 1 1

(OXYGEN VALUES DETERMINED WITH A CCNTIN
RECORDING DISSOLVED OXYGEN ANALYZER)

1

uous

—

1 lU

i^

DEC. 15. 1958

OHIO RIVER

"^-^^_^^

icn

^~

/

NOV. 8, 1958

90,

1 1 1 1 1 1 1 1 1 1 1

3

8

10 12

2

U

6 8 10 12 2

u

6

AM

PM

AM

DIURNAL VARIATION IN DISSOLVED OXYGEN IN TWO OHIO RIVERS

Fiflure 3

300 foot-candles: 2.8 ppm. algae produced 3.4
ppm. oxygen; 11.0 ppm. algae produced 7.9 ppm.
oxygen; and 13.7 ppm. algae produced 18.4 ppm.
oxygen . It was also noted that oxygen production
in relation to algal density, expressed as sus-
pended solids, increased as samples were col-
lected progressively downstream. Concurrently,
the blue-green planktonic algae from Lake Winne-
bago, from which the Fox River flows, were re-
placed by algae more suited to the flowing water
habitat. How the laboratory results are to be ap-
plied to river conditions was not determined.

A third approach used the "light and dark
bottle test" to measure photosynthesis and respira-
tion simultaneously. Although raw sewage stabi-
lization ponds are not natural waters, the intensity
of processes occurring there makes studying them
especially instructive in relation to other waters.
In general essentially all photosynthesis occurs in
the surface water layer that absorbs 99% of the

light. For this reason water transparency must
serve as a guide in determining desirable depths at
which to expose light and dark bottles within the
euphotic zone . Typical relations between oxygen
production and oxygen use in such ponds are shown
in Figure 7 . During mid-morning with intense light
and abundant carbon dioxide accumulated during
nocturnal bacterial decomposition, photosynthetic
oxygen production reaches a high rate of more than
2 grams per square meter per hour. At the depth
where light intensity is 20% of the surface value,
oxygen production proceeds at 70 times the rate
found at only 1% of surface value intensity. Be-
cause the density of phytoplankton is much greater
than that commonly found in other surface waters ,
extinction of light is rapid and the euphotic zone
is only about 120 centimeters thick. In the Ohio
River, on the other hand, turbidity is caused
largely by inorganic suspensoids, but they are suf-
ficient to absorb 99% of the light in about 130

59

-TEMPERATURE

Modified from Gameson
and Griffith, 1959

I I I

20 24

4 8 12 16

TIME - DAY

SIX MONTH AVERAGE DIURNAL CURVES

rOR RIVER H\l, ENGLAND

Figure U

centimeters. Here, however, with a much lower
phytoplankton population, the oxygen production
rate never exceeded 1 gram per square meter per
hour for the period studied .

The quantity of oxygen produced by photo-
plankton can be both impressive and important. In
raw sewage stabilization ponds in the northern part
of the country, production of as much as 26 grams
per square meter per day by a biological system
using 19 grams per square meter per day for total
respiration (a P/R of 1 .4) assures a favorable oxy-
gen balance during illuminated periods. It also
provides a surplus that can be mixed by wind and
convection currents into deeper, less illuminated
strata so that they also can participate in aerobic
stabilization. Some dissolved oxygen persists in-
to the night, but usually it is completely used be-
fore the next period of illumination . Although the
quantities of oxygen in question may seem small,
the customary daily addition to these ponds of
oxygen-demanding material in the form of sewage
is only about 1-1/4 grams per square meter. Even
in rivers, unless their pollution load is unusually
heavy, an oxygen production rate of 7 .4 grams per
square meter per day and respiratory rate of 5 grams
per square meter per day (P/R of 1 .5) , as found in
the Ohio River, leave a surplus sufficient for the
usual total demand .

Both in sewage stabilization ponds and in
streams, surplus oxygen produced by phytoplankton
is not efficiently used and may be unavailable for

oxidizing organic matter within a short time bfter it
is produced. During periods of intense oxygen pro-
duction the water becomes supersaturated and oxy-
gen escapes to the atmosphere. Surface agitation
by wind accelerates such loss as suggested by the
lower oxygen concentrations in stabilization ponds
on windy days than on calm days {Figure 8) . In
quiescent, organically rich waters such as these,
oxygen concentration decreases rapidly with depth,
but in rivers with their constant movement, such
gradients are rare. Nocturnal oxygen depletion
through continued intense respiration may become
serious - more so in ponds than in rivers.

While no one disclaims the importance of
photosynthetic oxygen, the present state of knowl-
edge does not show how such oxygen may be uti-
lized more efficiently. The whole subject of phyto-
plankton in relation to oxygen production and use
has not yet reached a state of development permit-
ting practical application to the analysis of oxygen
resources or use in the oxygen sag equation. Even
when data are available on momentary rates of photo-
synthesis in relation to phytoplankton density, light
intensity, limits of the euphotic zone, and other
factors , it is not clear how such data can be ap-
plied to practical problems . There is need for
wider and more intensified study in this area. Also,
there is need to relate photosynthetic potential of
the waters to some numerical expression of the phy-
toplankton, whether this is given as pigment con-
centration, number of cells per volume, packed cell

60

L IGHT ^iiiiiiiiiiiiiMMMMiMJ

'V""T"*"*''''H''"'''n

6 12 18

TIME OF DAY IN HOURS

DIURNAL OXYGEN RELATI ONS I N A
HYPOTHETICAL STREAM SECTION

F i Qure 5
Modi fled from Odurr, 1956

61

8

6

U

2

E
a.
o

-2

a
o

OQ

-U

-6

-8

.

10

-12 —

lU

BCD OF SUSPENDED MATTER AND ALGAE

BOD OF SOLUBLE MATTER

EXCESS D.O. PRODUCED IN CONTINUOUS LIGHT

1. SAMPLE AS RECEIVED; INCUBATED IN DARK

2. SAMPLE CENTRIFUGED; INCUBATED IN DARK

3. SAMPLE AS RECEIVED; INCUBATED IN LIGHT

6
TIME

8
DAYS

lU

EFFECT OF NATURALLY OCCURRING PHYTOPLANKTON ON RATE OF

OXIDATION IN A FOX RIVER SAMPLE

STATI ON C. JULY 25. 195G

Fiqure 6
Modified from Wisniewski, 1958

volume, or some other expression. Toward this
end the Sanitary Engineering Center is continuing
to study photosynthetic oxygen production in rivers
and sewage stabilization ponds and also is co-
operating with the Tennessee Valley Authority on
similar studies on rivers immediately below large
impoundments .

Costs of Dissolved Oxygen
from Algal Photosynthesis

Oxygen production by algae is not a free ben-
efit to the aquatic habitat. At times, in fact, it is

probable that the benefits do not outweigh the
costs .

Stimulation of algal production by nutrients
derived from sewage, other organic wastes, and
their decomposition products can lead to formation
of a mass of organic matter greater than that of the
original waste (Renn, 1954) . This result is demon-
strable in the laboratory. It was also observed by
Renn at two study stations on the Potomac River
that, during the bright light period, rise and fall of
B.O.D. concentrations were parallel with rise and
fall of dissolved oxygen. This observation was
interpreted to indicate accelerated production of
algae that became a part of the total B.O.D.

62

t^.O

3.0

to

5 2.0

o

CO

CO

I .0

TYPICAL RAW SEWAGE POND
(AUGUST 10, 1955)

OHIO RIVER - BROMLEY
(OCTOBER 3, 1957)

PHOTOSYNTHESIS

RESPIRATION

.■;ovi-'

H 6 8 10 12 m 16 18 20 22 2>4
TIME - DAY

OXYGEN RELATIONS IN SURFACE WATERS
Figure 7

In sewage stabilization ponds algae are im-
portant for the oxygen they produce. California
pilot plant studies of such ponds (Gotaas, Oswald
and Golueke, 1954) showed an average yield of
1 .65 pounds of photosynthetic oxygen for each
pound of algae produced. In spite of this benefit,
the fact remains that an algal residue is left and
must be disposed of. In the pond, or in other re-
ceiving waters, the algae continue to draw upon
available oxygen for their respiration while alive
and are oxidized by bacteria when dead . Experi-
ence to date indicates that algae in such effluents
generally die and decompose at a sufficiently slow
rate that their deoxygenating influences are spread
over a long stream reach and, consequently, do
not produce acute oxygen depressions. In theory,
however, an equitable materials balance must show
not only the oxygen produced in growing the algae,
but also the oxygen required to oxidize them to a
stable state. In such a balance, the benefits of
algal photosynthesis appear less attractive .

The necessity to include in evaluations of the
oxygenating benefits of algae the oxygen required
to decompose them is shown strikingly by recent
developments in Lake Washington (Sylvester,
Edmondson and Bogan, 1956). Domestic eutrophi-
cation has stimulated an increasing abundance of

algae. It has been noted that oxygen consumption
in the hypolimnion has increased greatly during the
past 22 years because of the dropping down of in-
creasing quantities of algae and organic derivatives
from above. In 1933, 825 tons of oxygen were re-
moved per month; in 1950 the rate was 1400 tons,
and in 1955, 2190 tons. During the same period
the minimum dissolved oxygen concentration near
the bottom decreased from 6.4 ppm . to 3.5 ppm.

Elsewhere in lakes, severe odor problems
have been encountered where planktonic blue-green
algae, having abandoned their more uniform dis-
persion in the water, clump together in windrows
and accumulate as a surface scum in protected
areas. As they decay, such concentrated algal
masses soon exhaust the available oxygen in the
immediate surroundings so that gaseous products
of anaerobic processes befoul the air.

In polluted water conducive to intense algal
photosynthesis, super- saturation which so com-
monly occurs is wasteful, inefficient, and appar-
ently even reportedly dangerous. In sewage stabi-
lization ponds, oxygen concentrations exceeding
400% saturation have been observed. Because the
rate of the B.O.D. reaction unfortunately is not ac-
celerated by Increasing the oxygen concentration,
the surplus oxygen cannot be used for more rapid

63

' 7 ' ■ ■ ■^••.•.". V***'.'.'.*.' I

(WINDY) mm

NIGHT

NIGHT

J I iiiii I I

NIGHT

1200 1800 2U00 0600 1200 1800 2M00 0600 1200 1800 2400
AUGUST 10 AUGUST II AUGUST 12

EFFECT OF WIND ON DISSOLVED OXYGEN IN A

TYPICAL RAW SEWAGE POND

Figure 8

64

6 8 10 12 |i| 16
RIVER MILES FROM LAKE

20 22

EFFECT OF LAKE KEGONSA ALGAE ON YAHARA RIVER
OXYGEN RESOURCES

Figure 9

waste stabilization and is readily lost to the at-
mosphere . This type of loss applies in principle to
rivers also but usually occurs there at a much lower
degree of intensity. For waste stabilization it
would be far better that oxygen be available con-
tinuously at a lower but constant level than the
"feast and famine" situation that occurs naturally.

Death of fish has been reported in natural
waters where high concentrations of dissolved oxy-
gen were produced by a bloom of Chlamydomonas
(Woodbury, 1941). At oxygen concentrations of
30-32 ppm. , characteristic lesions of the fish con-
sisted primarily of gas emboli in the gill capillaries
and gas bubbles in the subcutaneous tissues. Al-
though not tested, the gas was believed to be oxy-
gen. Occurrences such as this are not common.

The Lower Fox River mentioned above is an
example of a stream that receives its principal flow
from a lake (L. Winnebago). During late summer
and early fall, tons of algae produced in the lake
enter the river and move downstream. Prominent in
the phytoplankton are large quantities of Anabaena ,
Aphanizomenon , and Microcystis , which are more
characteristic of standing than flowing water.
These algae are reduced in numbers progressively
downstream, apparently by dying off. Field and
laboratory study showed that seasonal decomposi-
tion of such algae is a serious factor in decreasing
the assimilation capacity of the river, which re-
ceives residual wastes from several cities and from
pulp and paper mills.

Another more explosive example of excessive

use of oxygen resources by algae occurred in the
Yahara River in Wisconsin (Mackenthun, Herman
and Bartsch, 1945, Published in 1948) where, in
October of 1946, tremendous quantities of blue-
green algae, almost entirely Aphanizomenon flos
aquae , entered from Lake Kegonsa . Decomposing
as they passed, wave-like, downstream, they de-
pleted the oxygen supply, thus causing the death
of tons of fish. Although algal toxicants were
present, oxygen conditions were sufficiently severe
to account for the fish mortality. The pattern of
progressive oxygen depression and recovery that
accompanied the mass movement of algae is shown
in Figure 9. Fish mortality occurred 3-1/2 miles
from the lake three days after algae entered the
river, at 6 miles after five days, and at 18 miles
after eight days .

Effects of Algae on the B.O.D. Determination

The oxidation processes of algae that con-
tinue to occur in the absence of light in nature per-
sist also in the laboratory in conduct of the widely
used standard B.O.D. test (Anon., 1955). Ordi-
narily, the test result is used with other data to
estimate the oxygen conditions that will occur at
selected stream points in response to waste loading
at others . Sufficient numbers of algae in stream
samples collected as a step in such stream analysis
affect applicability of the B.O.D. result because:
(a) incubation in darkness for the standard five-day

65

n —

e

Cl

d

3 —

2 —

OHIO RIVER WATER-DARK

OHIO RIVER WATER-DAYLIGHT

2 U 6 8 10 12

t, DAYS

INFLUENCE OF ILLUMINATION ON B.O.D. OF SAMPLE CONTAINING ALGAE

AUGUST 26, 1955

Figure 10

m

period may give an unduly high value because of
algal respiration, death, and decay; (b) incubation
with illumination as an attempt at improvement may
give unduly low or negative B.O.D. (Figure 6) be-
cause of the addition of oxygen by photosynthesis;
and (c) incubation with intermittent illumination,
although superficially attractive, does not suffi-
ciently simulate stream conditions in terms of tem-
perature, illumination, availability of nutrients ,
and other factors.

A number of attempts have been made to mod-
ify the B.O.D. procedure to make the result more
meaningful with samples containing algae . Abbott
(1948) incubated duplicate samples in B.O.D. bot-
tles for 48 hours, one bottle of the pair in darkness

and the other exposed to light at a north window.
Test results were expressed as a ratio, L ~ D

Oo- Od

when Oq is initial dissolved oxygen, Ol and Od
are concentrations after light and dark incubation
respectively. Later, the procedure was modified
further (Abbott, 1952) to measure the light energy
during incubation with a hydrogen iodide actino-
meter .

In studying the influence of blue-green algae
on the B.O.D. result, Wisniewski (1958) modified
the test to determine separately the influence of
living, dead, and variable concentrations of algae.
Further studies on this problem are in progress at
theUniversity of Wisconsin, the Sanitary Engineering

66

e
c
a.

o

— 2

HEAN B.O.D.

From Bartsch, 1956

2

AM

8

II

t!oo:i

B

a.

(X

c

m

o
m

8

II
PM

INFLUENCE OF DIFFUSED DAYLIGHT OH B.O.D. SAMPLE
CONTAIN IMG ALGAE - AUGUST 26, 1955

Figure I I

Center, and perhaps elsewhere.

Three areas of relationship between algae
and the B.O.D. test are of particular interest.
They are: (a) the effect of illumination, (b) quan-
tity of algae, and (c) the influence of dead as op-
posed to living algae. Water from the Ohio River
containing limited quantities of phytoplankton was
incubated in an uncovered water bath at 20°C at an
east window. Half the bottles were exposed to
daily fluctuations in natural light; the others were
covered with opaque material. At the same hour
each day, duplicate bottles of each group were
processed for B.O.D. concentration. The 13-day
record is shown in Figure 10. The five-day B.O.D.,
after more closely simulating natural conditions, is
only about half that derived by closer adherence to
the standard procedure. The fluctuation in B.O.D.
that occurred during the first five days resulted
from intermittent sunny and cloudy weather . With
continuous illumination, there was generally an ex-
cess of dissolved oxygen in the sample. In another
test, when replicates were incubated in the dark
for 19 hours, the B.O.D. was 2.77 ppm. , but with
natural light it was only 1 .15 ppm. Furthermore,
as shown in Figure 11, the mean B.O.D. of sam-
ples processed only at noon would have been, for

those illuminated, 0.55 ppm., but for those in the
dark, 2.10 ppm. - a difference of 400%! It is ap-
parent that incubation in neither continuous light
nor continuous darkness can effectively adjust for
the photosynthetic and oxidation reactions related
to algae .

To explore the influence of plankton quantity,
B.O.D. samples were prepared so that one con-
tained the phytoplankton removed from 20 volumes
of Ohio River water, the other the quantity from 40
volumes. Three samples are referenced "X algae"
and "2X algae" in Figure 12, which shows increas-
ing amounts of algae result also in increasing
B.O.D.

In the standard B.O.D. test with its five-day
incubation in the dark , respiratory oxygen demand
of living algae gradually is replaced by an oxygen-
consuming attack by bacteria , protozoa , and other
organisms as the algae die. The extent and rapid-
ity of such transition in bottled samples is not
known although, as pointed out, the same situation
in principle has been observed repeatedly in natu-
ral waters .

Chlorella variegata and sewage were added
to standard dilution water to give a fixed sewage
concentration of 0.5% and an algal density of 1 .22

67

E
a.

c

2 X ALGAE-

X ALGAE

From Bartsch, 1958
L_

INFLUEtJCE OF QUANTITY OF ALGAE ON B.O.D.
AUGUST 26, 1955

FiQure 12

million cells per ml. The algae added to one por-
tion of the dilution water-sewage mixture were
killed by short exposure to heat at 70°C; the others
were living. B.O.D. values with living and dead
algae were compared with values obtained when
the medium contained sewage alone (Figure 13) .
Although oxygen demand with dead cells was a lit-
tle greater, it is believed that the ultimate first-
stage B.O.D. (Lg) in either case would have been
about the same . Under identical laboratory condi-
tions, when dead Chlorella cells were added to
samples in relative quantities of 1 , 10, and 66
(18,330, 183,300, and 1,222,000 cells/ml. re-
spectively), the resulting B.O.D. values were
3.21, 3 .73 , and 7 .74 ppm. respectively . On the
basis of the Chlorella tests , it appeared that the
contribution to the five-day, 20°C B.O.D. ap-
proximated .2 ppm. per billion cells .

Some workers believe that dense populations
of algae in polluted waters are always beneficial
and desirable. This view undoubtedly is supported

by the common observation of high dissolved oxy-
gen concentrations in their presence and failure to
note the nocturnal depressions. Concurrent rapid
production of a more stable mass of organics in the
form of algae from readily putrescible wastes in ef-
fect postpones satisfaction of the oxygen demand to
some later time at a different place. When condi-
tions are favorable, such postponement may be de-
sirable; when unfavorable, as in examples cited,
algae make existing pollution conditions even
worse. The oxygen released to the water in algal
photosynthesis is momentarily beneficial in spite
of the frequent inefficiency in its use . To evaluate
equitably the algal source of oxygen, the processes
by which algae along with the other biota also con-
sume oxygen must be included in the tally.

68

6

a

a
o

CD

SEWAGE + DEAD ALGAE
L5-6.5, La-7.8

SEWAGE + LIVING ALGAE
L5-5.

Lr-2.5, La-3.3

Bartsch, 1958

8

2 U 6

t, DAYS

NFLUENCE OF ALGAE ON BOD

Fi flure 13

10

Acknowledgment

The data used in preparation of Figures 10-13 were obtained jointly with Dr. W. M. Ingram,
Dr. E. C. Tsivoglou, and Mr. D. G. Ballinger, all of the Sanitary Engineering Center. Their willing-
ness to allow the information to be used here is gratefully acknowledged.

69

References

Abbott, W.E. 1948. Oxygen production in water by photosynthesis . Sew . Wks . J . 20; 538-541 .

Abbott, W.E. 1951. Note on tolerance of green flagellate protozoa to hydrogen sulfide . Sew. and
Ind. Wastes 23: 1310.

Abbott, W.E. 1952. Analysis of polluted waters capable of photosynthesis . Sew . and Ind . Wastes
24: 666-659.

Anon. 1955 . Standard methods for the examination of water, sewage, and industrial wastes. Tenth
Edition. Amer. Public Health Assoc. N. Y. 1955.

Anon. 1957. Sewage stabilization ponds in the Dakotas. Volume I. An evaluation of the use of

stabilization ponds as a method of sewage disposal in cold climates. A joint report with North
Dakota Dept . of Health, South Dakota Dept. of Health, and the U . S. Dept . of Health, Education,
and Welfare .

Bartsch, A. F. and M. O. Allum. 1957. Biological factors in treatment of raw sewage in artificial
ponds. Limnol. & Oceanog. 2: 77-84.

Bartsch, A. F. and W. M. Ingram. Stream life and the pollution environment. Public Wks. Magazine.
7 pages . In Press .

Briggs, R., G. V. Dyke and G. Knowles . 1959. Electrical recorder for dissolved oxygen. The Water
& Waste Treatment J. Ipage. Jan. -Feb.

Calvert, C. K. 1933. Effect of sunlight on dissolved oxygen in White River . Sew . Wks . J . 5 : 685-694 .

Gumming, H. S. Investigation of the pollution and sanitary conditions of the Potomac Watershed, with
special reference to self-purification and the sanitary condition of shellfish in the Lower Potomac
River. U. S. Public Health Service, Treas. Dept., Hygienic Lab. Bull. No. 104, February 1916.

Eny, D. M. 1951. The effect of organic acids, inhibitors and enzymes on the respiration of Chlorella.
Biochem. 50: 559-564.

Gameson, A. L. H. and Susan D. Griffith. 1959. Six months' oxygen records for a polluted stream.
The Water & Waste Treatment J. 4 pages. Jan. -Feb.

Gotaas, H. B., W. J. Oswald and C. Golueke. 195 4. Algal-bacterial symbiosis in sewage oxidation
ponds - Fifth Prog. Rept. Univ. Calif. Inst, of Eng . Research Bull. Ser. No. 44, Issue No . 5 :
1-88.

Gotaas, H. B., W. J. Oswald and H. F. Ludwig . 1954. Photosynthetic reclamation of organic wastes.
Paper from San. Eng. Research Lab. of Univ. of Calif.

Gloyna, E. F. and E. R. Hermann. 1956. Some design consideraUons for oxidation ponds . Proc . Amer .
Soc . Civil Eng . 82 , No . SA 4 .

Hermann, E. R. and E. F. Gloyna. 1955. The design of oxidation ponds . Paper presented to U . S . -
Mexico Border Conf. 13 pages. Mimeographed.

Mackenthun, K. M. , E. F. Herman and A. F. Bartsch. 1945 . A heavy mortality of fishes resulting
from the decomposition of algae in the Yahara River, Wisconsin. Trans. Amer. Fish. Soc. 75:
175-180.

Macklin, M. O., D. J. Baumgartner and M. B. Ettinger. 195 9. Performance test of continuous recording
dissolved oxygen analyzer. Sew. and Ind. Wastes. In Press.

70

Odum, H. T. 1955. Primary production in flowing waters . Limnol. and Oceanog . 1; 102-117.

Oswald, W. J. and H. B. Gotaas. Photosynthesis in sewage treatment. ASCE Proc . 81, Separate
No. 686, 27 pages (May 1955) See Discussion 82 (SA 2 , No . 932) 9-12 (Apr. 1956) .

Renn, C. E. 1954. Allowable loading of Potomac River in vicinity of Washington, D. C. A Report on
Water Pollution in the Washington Metropolitan Area. SEC. Ill - Appendixes, Feb. 1954:
AB-1 - AB-17.

Samejima, H. and J. Meyers. 1958. On the heterotrophic growth of Ghlorella pyrenoidosa . Jour.
Gen'l. Microbiol. 18: 107-117.

Saunders, G. W. 1957. Interrelations of dissolved organic matter and phytoplankton . Bot . Rev. 23:
389-410.

Streeter, H. W. and E. B. Phelps. 19 25 . A study of the pollution and natural purification of the Ohio

River. III. Factors concerned in the phenomena of oxidation and reaeration . U.S.P.H.S., Public
Health Bull. No. 146: 1-75.

Sylvester, R. O., W. T. Edmondson and R. H. Bogan. 1956. A new critical phase of the Lake
Washington pollution problem. The Trend in Engin. 8; 8-14.

Wilson, B. W. and W. F. Danforth. 1958. The extent of acetate and ethanol oxidation by Euglena
gracillis . J. Gen'l. Microbiol. 18: 535-542.

Wisniewski, T. F. 1958. Algae and their effects on dissolved oxygen and biochemical oxygen demand.
Oxygen Relationships in Streams. U.S. Public Health Service: 157-176.

Woodbury, L. A. 1941 . A sudden mortality of fishes accompanying a super- saturation of oxygen in
Lake Waubesa, Wisconsin. Trans. Amer . Fish. Soc . 71: 112-117.

71

ORGANIC PRODUCTION BY PLANKTON ALGAE, AND ITS ENVIRONMENTAL CONTROL

John H . Ryther
Woods Hole Oceanographic Institution

Roughly three quarters of the earth's surface
is inhabited by an algal flora. Only on land and in
a few shallow water areas do the higher plants pre-
vail . Does it follow, then, that the non-vascular
plants dominate the earth in terms of the biomass
and productive capacity? In some coastal waters
there are vast beds of giant seaweeds representing
several thousands of grams of organic matter per
square meter (i.e. Blinks, 195 4), some of the
densest stands of vegetation known. However,
over 99% of the oceans are too deep to permit the
growth of attached plants and the flora is repre-
sented almost entirely by the microscopic, unicell-
ular algae, the so-called phytoplankton . The sole
exception to this is the sporadic occurrence of
floating seaweeds which may accumulate locally,
as in the Sargasso Sea, but are quantitatively in-
significant in the oceans as a whole.

There have been but few attempts to measure
directly the standing crop, in terms of biomass, of
the phytoplankton . But there have been a large
number of measurements of their photosynthetic
pigments, particularly during 1957 - 1958, as part
of the oceanographic program of the International
Geophysical Year . Chlorophyll a values for the
upper, illuminated layers of the Atlantic Ocean
were found to range usually between .1 and .5
mg/m , averaging perhaps .25 . If we may assume
a chlorophyll a^:dry weight relationship of 1:100 in
phytoplankton (Harris and Riley, 195 6) the dry
weight/m of phytoplankton to a depth of 100
meters averages no more than 2.5 grams. Adding
a generous .5 grams to allow for the richer coastal
waters and to include the benthic algae, the oceans
support an average standing crop of about 3 .0 g/m
or, for the 361 x 10 kmof the hydrosphere, a
total weight of some 1 .1 x 10 ■'■^kg .

From various sources (Schroeder, 1919;
Fawcett, 1930; Show, 1949; Brown, 1956) we may
divide the land surface into the following relative
proportions:

1. Wasteland (desert, artic regions ,

mountains) 50%

2. Cultivated land, grasses, sedges,

brush, etc. 20%

3. Forests 30%

Let us assume that the first category, half
of the land area, supports a negligible fraction

of its vegetation. From various agricultural statis-
tics, crop yields were found to average about 1000
g/m /year. According to Pearsall and Gorham
(1956) natural stands of grasses, sedges, bracken,
etc. produce from 400 to 1400 g/m /year, about
the same as cultivated land. Since these crops are
seasonal in most of the world, the average standing
crop on an annual basis would be less, perhaps
5 0% of the annual production. The mean standing
crop of this second category may be taken, then,
as 5 00 g/m^ over an area of roughly 30 x 10 km
for a total biomass of 1.5 x lO-'-'^kg.

Ovington and Pearsall (1956) have estimated
the annual production of forest trees in Great
Britain from the weight of selected samples and the
age of the trees . Working backwards from their
data , the standing crop of their forest trees was
found to range from 10,000 to 40,000 g/m^ , an
average of 25 ,000 g/m . Extropolating this to the
45 X 10 km of the world's forests, we may esti-
mate a crop of trees of 1.1 x lO-'-^kg.

Summarizing these standing crop estimates,
we have:

Oceans
Land

Wasteland

Crops, grasses, etc.

Forest

1.1 X 10-^ ^kg.

1.5 X lO^^kg,
1.1 X lO^^kg,

Thus it appears that the higher plants , oc-
cupying no more than 1/8 of the area inhabited by
the algae, maintain a biomass more than 1,000
times greater. Crude though these figures may be,
they probably minimize the contrast between the
two plant groups, since the values for the aquatic
plants, if anything, have been exaggerated, while
the terrestrial stands were probably underestimated.

What of the productive capacity of the land
and sea ? Does it follow that the algae are equally
insignificant in the annual production of the earth's
organic matter? There have been several recent
studies of organic production in restricted marine
areas on an annual basis (Steemann Nielsen, 1937,
1951; Riley, 1956, 1958; Ryther and Yentsch, 1958;
Menzel and Ryther, in press), and many more scat-
tered single observations in different parts of the
world's oceans (i.e. Riley etal, 1949; Steeman
Nielsen and Jensen, 1957) . From these sources,
we may place the mean value for net oceanic

Contribution No. 1049 from the Woods Hole Oceanographic Institution and Contribution No.
from the Bermuda Biological Station. Supported in part by contracts AT(30-1)-1918 and AT(30-l)-2078
with the U.S. Atomic Energy Commission and by NSF Research Grant G-3234.

72

production (that part of the plant's production in
excess of its respiratory requirements) at approxi-
mately 100 g/m /year or 3.6 x 10l3kg/year for the
oceans as a whole.

Returning to our earlier consideration of land
plants, it was estimated that the category of agri-
cultural crops, grasses, etc. produce about 1000
g/m /year or 3.0 x lO^^y^g/year over its alloted
30 X lO^km^. Making another bold assumption that
the average forest tree is 50 years old (actually,
this could be halved or doubled without greatly af-
fecting the end result) the crop of 1 . 1 x lO^^kg of
trees would yield an annual production of 2 .2 x
lO^^kg/year. This is equivalent to about 500
g/m /year, roughly the lower limit of the values
obtained by Ovington and Pearsall (1956) for 20 -
40 year-old forests .

Summarizing the production estimates, we
have:

Ocean
Land

Wasteland

Crops, grasses, etc.

Forests

3 .6 X lO^^i-g/year

3.0 X ^_ _

2.2 X 10^ -^kg/year

10^ ^kg/year

Calculations of this second type have been
made before, but it is of some interest and value
to repeat them using independent methods and data.
Schroeder's (1919) value for the land (3.8 x 10^3
kg/year) and Steemann Nielsen and Jensen's (1957)
value for the oceans (2.4 - 3.0 x 10-^3)^g/year) are
both somewhat lower than those reported above, but
are nevertheless in surprisingly good agreement
with them .

The interesting fact which emerges from all
this is that the annual rate of organic production
on land and in the sea is about the same despite
the fact that the latter is accomplished by a flora
less than one thousandth the biomass of the ter-
restrial vegetation. The explanation for this is
that most of the bulk of land plants is in the form
of slowly growing, non-photosynthetic structural
tissue. If we were to consider the relative quan-
tities of photosynthetic pigments in the two en-
vironments, the results would be quite different.
In fact, it has been proposed by Gessner (1949)
that the highest concentration of chlorophyll that
can be attained in nature (1-2 g/m ) is actually
the same for both water and land. What does this
mean in terms of the photosynthetic potential of
the land and sea? This depends not upon the
amount of chlorophyll per se , but upon the amount
of sunlight absorbed by this pigment. Steemann
Nielsen (1957) correctly points out that the chloro-
phyll which lies below the illuminated or "eu-
photic" zone of lakes or oceans has no bearing on
the productive potential of the area. In this con-
nection, however, it would be difficult to estimate
the chlorophyll in a comparable "euphotic zone" of

a forest, where all of the pigment is probably never
Illuminated at any one time.

This difficulty can be circumvented if we look
at the problem from another viewpoint and consider
a situation in which all of the sunlight falling on a
unit of the earth's surface is effectively absorbed
by photosynthetic pigments. Stipulating these con-
ditions in both a terrestrial and aquatic environ-
ment , how much of the solar energy will be con-
verted to organic matter in each case? This, of
course, depends upon the efficiency of the photo-
chemical process, the quantum yield of photosyn-
thesis .

The quantum yield of photosynthesis has been
one of the most thoroughly studied aspects of plant
physiology. Reviews by Rabinowitch (1951) and
others reveal that, under similar conditions, ap-
proximately the same number of quanta of light are
required to reduce one mole of CO2 to carbohy-
drate by a wide variety of plant types; the process,
in other words, seems to be largely species in-
dependent .

We have recently attempted to calculate the
quantum yield of photosynthesis under completely
natural conditions , considering the efficiency of
utilization of light falling on a square meter of the
earth's surface (Ryther, in press). The assumption
was made that all of the light, except that lost by
reflection and back scattering , was absorbed by
photosynthetic pigments and that all other condi-
tions for photosynthesis were optimal. In this
treatment it was necessary to take into account the
spectral composition of daylight and the photosyn-
thetic utilization of light of this mean spectral
composition. Particularly important was a consid-
eration of the effects of light intensity. Photo-
synthesis is proportional to intensity up to about
1,000 foot candles, approximately 10% of full sun-
light. Above this, the process becomes light satu-
rated, and at still higher intensities may be actu-
ally depressed. Obviously, photosynthetic effi-
ciencies decrease rapidly as plants are exposed to
increasing intensities above the saturation point.
At the same time, however, the higher intensities
are effective in illuminating organisms deeper in
the water of a planktonic community or the leaves
farther down in a thick forest. Thus, while effi-
ciencies are decreasing, production continues to
increase with higher intensities of solar radiation.
Individual algal cells or leaves become light satu-
rated but entire plankton communities or forests do
not. Figure 1 shows the striking similarity between
plankton algae (Ryther, 195 6a) and pine trees
(Kramer and Clark, 1947) in this respect.

The resulting efficiencies which were ob-
tained after corrections for respiratory loss, were
equivalent to a yield ranging from 8-19 grams of
dry orgaruc matter/m^/day for radiation values of
200 - 400 langleys/day (the range normally encoun-
tered over most of the earth) . These theoretical

73

100
90
80
70
60
50
40
^ 30
[2 20
^ .0

I 90
O 80

a. 70

60
50
40
30
20
10

- / \ ^^^PULATiCN

- / y

-/ / Vell

f/

>

1 1 1 1 1 1 1 1 1 .

/ ^y^ ^NEEDLE
/ ^y

/

] / /

./seedling

1 1 1 1 1 1 1 1 1 1

DD

Ill

I 23456789 10

0^ FOOT CANDLES

Figure Legends

Figure 1 . Photosynthesis-light intensity curves for (A) individual phytoplankton organisms and entire
phytoplankton populations (from Ryther, 1956) and (B) individual pine needles and entire seedlings (from
Kramer and Clark, 1947) .

74

Table I

Net organic production of various natural and cultivated systems in grams dry weight pro-
duced per square meter per day (from Ryther, in Press) .

A. Theoretical potential

average radiation (200 - 400 langleys/day)
maximum radiation (750 langleys/day)

B. Mass outdoor Chlorella culture (Tamiya , 1957)

mean
maximum

C. Land (maxima for entire growing seasons) (Odum, 1959)

sugar cane

rice

wheat

spartina marsh

pine forest (best growing years)

tall prairie

D. Marine (maxima for single days)

coral reef (Odum and Odum, 1955)

turtle grass flat (Odum, 1954)

polluted estuary (Ryther, et al, 1958)

Grand Banks (April) (Ryther and Yentsch, unpublished)

continental shelf (May) (Ryther and Yentsch, 195 8)

Sargasso Sea (April) (Ryther and Menzel, in press)

8-19
27

12.4
28.0

18.4
9.1
4.6
9.0
6.0
3.0

9.6
11.3
8.0
6.5
3.7
2.8

values were compared with some of the highest
yields of organic matter which have been observed
in nature. The data are reproduced here as Table I
and they reveal that in cultivated crops and natural
communities, on land or in the sea, in algae or in
the higher plants, the maximum rate of production
is very nearly the same and may closely approach
the theoretical potential. As in laboratory quantum
yield experiments, organic production in nature ap-
pears to be species independent and determined
primarily by a photosynthetic potential common to
all plants .

But the ideal conditions necessary for the
attairiment of this potential are seldonvmet in
nature. Rather than producing 5 kg/m /year, the
land averages no more than 10 - 20% of this, the
sea perhaps 2 - 3% . The low mean production of
the land is not hard to understand. Soils are fre-
quently poor in quality or essential nutrients . Ad-
equate moisture is often lacking. Carbon dioxide
is thought to be usually, if not always, limiting to
land plants, their yields being increased 2-3 fold
when this gas is artificially introduced in green-
house experiments (Maximov, 1930). Finally tem-

perature, or climate, accounts for much of the dis-
crepancy between the potential and observed pro-
duction of the land . A large portion of the cultur-
able land on earth is found in temperate or semi-
tropical climates where the plants have a limited
growing season. An agricultural yield of 500 -
1000 g/m^ may be achieved in such regions in 4 to
6 months .

But what of the ocean ? Here there are no
substrate problems; no lack of moisture. Carbon
dioxide is probably never limiting due to the great
reservoirs of dissolved carbonates and bicarbonates.
Oceanic temperatures are always favorable for the
growth of some species of phytoplankton . In the
words of Henderson (1913) "No philosopher's or
poet's fancy, no myth of a primitive people has
ever exaggerated the importance, the usefulness,
and above all the marvelous beneficence of the
ocean for the community of living things . " This
ideal environment yields an average crop of organic
matter which is almost two orders of magnitude less
than the biotic potential of its producers . Only be-
cause of its vast area does the ocean equal the
land in its over-all organic production.

75

The reasons for the apparent paradox lie in
the restriction and limitations which the planktonic e
existence impose upon plants with respect to two
basic requirements, light and nutrients. We shall
consider these separately.

Though the oceans are some three miles deep,
over 95% of their waters are in virtual darkness,
uninhabitable by photosynthetic organisms. The
light intensity at which photosynthesis balances
respiration, the "compensation intensity " , varies
somewhat with the species and physiology of the
plant, but is of the general order of 100 foot can-
dles. This is equivalent to about 1% of full noon
sunlight incident to the surface . Below this com-
pensation intensity, no growth of plants is possi-
ble. Thus the oversimplified but useful concept
has come into use of a "euphotic zone", the depth
through which plant growth can occur, which has as
its lower limit the depth of penetration of 1% of the
incident surface radiation . In the clearest ocean
water the euphotic zone extends to about 120
meters .

But this clearest ocean water contains no
plants, so a euphotic zone of 120 meters is hypo-
thetical. Let us introduce an algal population into
this clear solution which we will further stipulate
to be adequately enriched with all the vital plant
nutrients. Immediately, the plants themselves
will absorb and scatter the light, and the euphotic
zone will become progressively shallower as the
population grows . Thus, the phytoplankton , shut-
ting out its own light, becomes self-limiting. But
as the euphotic zone becomes shallower, a pro-
gressively larger fraction of the light is absorbed
by the plants , a proportionately smaller fraction by
the water itself . Consequently, organic produc-
tion increases with a decreasing euphotic zone.
However, this relationship holds only as long as
the decreasing euphotic zone results from the in-
crease of living plants. Not all turbid waters are
highly productive, for the turbidity is frequently
caused by non-living particulate or dissolved mat-
ter. In Long Island Sound, for example, Riley
(1956) estimated that two thirds of the incident
radiation was absorbed by such material.

We may conclude, then, that photosynthesis
in the ocean is not only restricted to a shallow
surface layer, no more than 100 meters deep, but
that for the process to proceed at its maximum po-
tential rate, the plants must be concentrated in the
upper five meters or less of otherwise clear ocean
water. Let us now turn to a consideration of the
nutrients available to support this production. For
a convenient example we shall take nitrogen.

The highest concentrations of inorganic ni-
trogen in the oceans are present as nitrate at in-
termediate depths of approximately 1000 meters,
and range from about 40 to 60 tig A NO3 - N/l or
.56 - .84 gN/m~^ . Water from this depth rarely
reaches the surface. The highest known concen-

trations of nitrogen within the euphotic zone are
found in restricted areas where upwelling or diver-
gences of water masses bring water from several
hundred meters of depth to the surface . Rudd
(1930) for example, reports values of about 40 fig
A NO3 - N/l in the surface layers of the Antarctic,
one of the most fertile oceanic regions.

In most temperate and northern waters, the
surface layers are enriched by winter cooling and
mixing to depths of some 300 - 500 meters. The
highest concentrations of nitrogen brought to the
surface in this way range from 10 to 20 }ig A NO3 -
N/l (R . F . Vaccaro, unpublished data for the
North Atlantic) . Let us assume that winter mixing
has resulted in a surface enrichment of 15 pg AN/1
or 210 mg N/m^ . In clear ocean waters with a 100
meter euphotic zone, the phytoplankton will have
a reservoir of 100 m x 210 mg N/m^ or 21,000 mg
of nitrogen per square meter to draw upon . How-
ever, as the population grows, it not only depletes
this supply, but creates in the process a progres-
sively shallower euphotic zone with a correspond-
ingly smaller reservoir of nitrogen. By the time the
phytoplankton have increased to a population con-
taining 10 mg of chlorophyll/m , production is
limited to a euphotic zone of about five meters
(Riley, 1956) . This water contained an initial
amount of only 1050 mg N and, of that, 500 mg
were utilized in producing the population (assum-
ing the plants to contain about 1% chlorophyll and
10% nitrogen) . We may calculate the daily rate of
production from the chlorophyll and depth of the
euphotic zone for a day of average incident radia-
tion , (300 langleys) according to the equations of
Ryther and Yentsch (1957) . From these calcula-
tions it is estirrBted that 1 .8 grams of organic mat-
ter will be produced and about 180 mg nitrogen con-
currently consumed within the five meter euphotic
zone each day. Thus, after a population equiva-
lent to 10 mg chlorophyll/m3 has developed, there
is sufficient nitrogen remaining in the resulting
shallow euphotic zone to sustain production for
less than three days. Clearly in a static situation
such as we have pictured, high levels of organic
production approaching the potential discussed
above can scarcely be attained, much less main-
tained .

The oceans as a whole are not, of course, a
static environment , but their surface waters are
highly variable in this respect. In certain re-
stricted areas hydrographic or meteorological forces
bring water from intermediate depths to the surface.
This happens when two water masses diverge, as
in the equatorial Pacific (Sette , 1955), and most
notably along the West Coasts of continents, where
prevailing offshore winds produce a surface current
which moves seaward, this water being replaced
with upwelling, rich, deep water. It is for this
reason that the coastal waters off Peru and parts of
West Africa are among the most fertile regions of

76

the sea. However, if one were to follow a given
parcel of water as it is brought to the surface and
subsequently is transported horizontally, one
would probably observe the same sequence of
events which we discussed above. Steemann
Nielsen and Jensen (1957) have described this for
the coast of Africa , pointing out that the freshly
upwelled water, though rich in nutrients, is poor in
phytoplankton . It is "new" water, in which the
plants have not had time to grow. As one moves
seaward, following the path of the surface current,
the plankton becomes more and more dense, passes
through a maximum, and then decreases ultimately
to a very sparse population by the time the water
has reached mid-ocean. The time course of this
sequence is probably not very different from that of
a perfectly stable water mass which is enriched by
winter mixing. The difference is that high produc-
tion in an upwelling area is maintained at a given
geographical location. Sette (1955) has described
a similar geographical sequence "downstream" from
the mid-Pacific equatorial divergence.

In contrast to these dynamic situations,
which are comparatively rare in the oceans as a
whole, we described above a static system in
which the surface waters are enriched by winter
mixing. The term "static" refers here to the ab-
sence or minor effects of horizontal advection, not
to the absence of vertical water movements . Let
us now return to this situation and consider it in
more detail .

During the winter in temperate and northern
regions, surface waters cool sufficiently to destroy
the summer thermocline, and the waters become
mixed to 300 - 500 meters, several times the depth
of the euphotic zone . Not only are the nutrients
from below the euphotic zone brought up and mixed
with the impoverished surface layers, but the
plankton algae are transported downward and spend
a considerable fraction of their time in darkness.
As a result, though nutrients are plentiful, pro-
duction is severely curtailed due to the limitation
of light.

With the return of spring , the surface waters
begin to warm up, a seasonal thermocline develops,
and the euphotic zone becomes stabilized against
vertical mixing. At the same time, radiation in-
creases. Those phytoplankton which find them-
selves in the euphotic zone are held there and
suddenly have access to both light and nutrients .
The stage is set for the "spring bloom", a feature
characteristic of the temperate oceans. We have
shown on the previous pages the succeeding
events, terminating in the exhaustion of the nu-
trient supply . During a period of fine , calm
weather in March or April, the whole process may
run its course within a week or two. More typi-
cally, the formation of the summer thermocline is
interrupted by storms, periods of cold weather,
etc . and the spring flowering may then be pro-

longed, at a lower level, for a period of one or two
months . But its days are numbered by the supplies
of nitrogen, phosphorus, and the other essential
elements which are limiting to plant growth in the
sea. As we have seen, the amounts of these sub-
stances brought to the surface by mixing are small
to begin with, and they are quickly consumed.

What happens next is a matter of some con-
troversy. Some believe that most of the nutrient-
deficient plants sink, their density increasing with
old age. Evidence for this is the accumulation in
summer of relatively high concentrations of chloro-
phyll at or near the lower limit of the euphotic zone.
Others hold that the ultimate fate of the plants is
to be eaten by the animal members of the plank-
tonic community (i.e. Harvey e^t_al,, 1935; Gushing,
1958). Whatever happens , the spring maximum
soon gives way to a summer minimum during which
time production proceeds at a very low level which
is probably maintained by the complete recycling
(assimilation, death or consumption, and regenera-
tion) of a small fraction of the winter nutrient bud-
get within the surface layers.

In the fall, when cooling again destroys the
seasonal thermocline, there may be minor and ir-
regular outbursts of plant growth as the surface
layers are alternatively cooled and mixed to a
slight degree and then restabilized . These small
blooms terminate with the final disappearance of
thermal stratification and the return of winter con-
ditions .

At the semi-tropical latitude of Bermuda, in
the Sargasso Sea, the annual cycle of organic pro-
duction is much the same as that pictured above,
with the major difference that production persists
at a relatively high level throughout most of the
winter. This is due to the fact that winter cooling
and mixing is less pronounced than in more north-
ern waters, and never, in fact, extends below the
depth of the permanent tliermocline at 300 - 400
meters. In addition, incident radiation is higher
at these latitudes, and the water is exceptionally
clear. As a result of this combination of factors,
the plants are never carried down out of the light
for sufficiently long periods to prevent their growth.
Figure 2 shows the seasonal cycle of organic pro-
duction in the Sargasso Sea off Bermuda. The ac-
companying seasonal profile of temperature in the
upper 700 meters will illustrate how hydrographic
conditions influence plant growth . Note that high
production is correlated with a well-mixed, largely
isothermal layer in the upper 400 meters; low
production, with the thermally-stratified summer
conditions .

The nutrient supply available to the plants
in this 400 meter deep reservoir in the Northern
Sargasso Sea is extremely low--an order of magni-
tude less than that present in the temperate seas.
Nitrate, for example, seldom exceeds 1.0 ug AN/1
in the euphotic zone. Consequently, plant

77

Q|.o

Figure 2. The seasonal profile of temperature to 700 meters and net organic production in the Sargasso
Sea off Bermuda for the period January - July, 1958 (from Menzel and Ryther, in press) .

production per unit volume is never high — the
process cannot build up towards its biological po-
tential . In contrast to a dense flowering in the up-
per five meters of temperate seas, the Sargasso
Sea plankton grow over a euphotic zone which is
never less than 50 - 100 meters deep. What main-
tains this organic production in the face of such
poverty of nutrients? Low as they are, the con-
centrations of nitrate, phosphate, and silicate (to
name a few essential elements which have been
studied) never change appreciably in these surface
waters. We don't yet understand this, but it ap-
pears that in these relatively warm waters the
whole cycle of assimilation, consumption, death or
excretion and remineralization occurs very rapidly.
The amounts of plants and animals and minerals
are small, but the metabolic wheels turn fast.

No study of the seasonal cycle of organic
production has been made in tropical waters, but
we can imagine what it must be like. In the true
tropics there is no winter cooling of the surface
waters, no mixing, no replenishment of the eu-

photic layer with rich aphotic waters . The sea -
sonal thermocline of temperate and semi-tropical
latitudes becomes a permanent, thermal barrier to
vertical mixing .

On March 1, 1959, a short oceanographic
section was made by Research Vessel Crawford
between 24° and 35° North latitude at the longitude
of Bermuda. At that time of year, the surface water
temperatures in the North Atlantic Ocean are at
their seasonal minimum; vertical mixing is most
pronounced. Actually the 1959 season was some-
what early. Our seasonal study at Bermuda re-
vealed the beginnings of warming and stratification
by March 1 , and the spring phytoplankton pulse
was in full bloom on that date. Figure 3 shows the
temperature profile and values of organic produc-
tion for this section running from North to South.
It is remarkably similar to the seasonal cycle at
Bermuda. The well-mixed, almost isothermal ,
highly productive stations at the northern end of
the section merge into stratified, low productive
stations to the South much as the spring flowering

78

528 527

NORTH

BERMUDA

525 517 524
STATION No.

522 520

SOUTH

Figure 3 . The profile of temperature to 700 meters and values for net organic production
as measured on a section between 35° and 24° N. latitude at the longitude of Bermuda
in March, 195 9.

79

declines to the summer minimum at Bermuda. One
can assume from this alone that organic production
in the tropics is maintained at a low, rather steady
rate throughout the year, probably maintained by
the recycling of nutrients entirely within the eu-
photic zone, perhaps occasionally stimulated into
brief minor outbursts of growth by the limited mix-
ing action of storms .

It is clear, then, that organic production in
the oceans is limited most of the time by either
light or nutrients . Both are available in plentiful
supply in the oceans as a whole, but both seldom
occur together. At the few times and places where
neither of these factors is limiting, production may
proceed at rates comparable to the highest levels
of production observed on land .

As mentioned earlier, the concentrations of
nutrients in the tropics and semi-tropics are far
less than those present in the high latitude seas.
As a result much smaller populations of plants can
develop. Yet, due to the rapid turnover of these
materials , a low to moderate rate of production can
be maintained throughout a deep euphotic zone . If
one integrates production over the entire water col-
umn, the annual rate beneath a square meter of sea
surface is as high or higher than that of presumably
far richer waters . As an extreme example of this ,
let us compare the vertical profile of photosynthe-
sis in the Sargasso Sea during a period of peak pro-
duction (April 19, 1958) with that of a shallow,
highly enriched sewage oxidation pond in South
Dakota (from Bartsch and Allum, 195 7) . The chlor-
ophyll concentration of the former averaged less
than 1 mg/m'^, the latter some 450 mg/m^ . The
oxygen production values of Bartsch and Allum have
been converted to carbon fixation assuming an
assimilatory quotient of 1.25 (Ryther , 1956b), and
the depth curve of photosynthesis has been rather
subjectively extrapolated to the surface. Exami-
nation of the depth profiles of daily production from
the sewage oxidation pond and the Sargasso Sea
(Figure 4) reveals an interesting fact . In the oxi-
dation pond, the euphotic zone is two orders of
magnitude smaller while production per unit volume
is two orders of magnitude greater than in the Sar-
gasso Sea . If one integrates the two curves, or-
ganic production beneath a square meter for the two
areas is found to differ by less than 20% . Actually,
the value for the oxidation pond is probably some-
what low, for the measurements were made from
10:00 a.m. to 3:00 p.m. rather than for an entire
day. But even if this figure is increased by 25% -
50%, the similarity between the two situations is
striking .

This may appear to contradict the earlier
statement that production per unit area may be ex-
pected to increase with a decreasing euphotic zone.
It should be reiterated here that such is true only
in cases where living plants alone contribute to
the turbidity of the water . In a pond receiving raw

sewage wastes, this would hardly be the case.

But the point which I wish to make in com-
paring these two situations is this: that the daily
rate of production of organic matter, as it is cur-
rently defined by ecologists, is very nearly the
same in two bodies of water in which the amounts
of living plant material are respectively of the or-
der of 100 grams and 0.1 grams per cubic meter.

AAHiat, then, does this rate of primary produc-
tion actually mean? One looks in vain for evidence
of it in the clear, blue waters of the Sargasso Sea .
The major fisheries of the world are located in the
temperate or high latitudes, or in the few regions of
divergences and upwellings which we discussed
above, not in places like the Sargasso Sea. Is it
realistic to compare fertility of northern and tropical
seas, of the ocean and the land, of a plankton
bloom and a cornfield, all on the basis of their rel-
ative rates of natural photosynthesis ?

In modern, dynamic ecology, it has become
unfashionable to speak of the "standing crop" of
organisms . The important question is not "how
much is there?" but "how fast is it being produced?"
There is no doubt that this concept has opened up
new and extremely interesting avenues of ecologi-
cal research. But the population ecologist or fish-
eries biologist should beware of these values. The
sociologist who compares the productive capacity
of the land and sea may be sadly deluding himself.
For animals eat food, not photosynthesis. What is
the significance of organic matter which is produced,
consumed, decomposed and remineralized almost
simultaneously? Why add up a daily production
which is daily expended into a non-existent annual
total. Is this comparable to a barn full of corn?
The study of the rate of organic production has al-
ready and will continue to reveal fundamental phys-
iological and ecological principles . But the person
who examines these data with the hope of feeding
an overpopulated earth on marine resources would
do well to remember, when he picks a pound of
beans from his kitchen garden, that to get the same
weight of rather undigestable and unappetizing
plankton algae from the open sea, he would need to
filter some five million gallons of water.

80

mg Carbon assim./m^/day

cr

LU
UJ

1000 2000 3000 4000 5000

T

X

A -

1.90 gC/mVday

B -

1.56 gC/mVday

10

20

30

40

50

Figure 4. The vertical profile of dally photosynthesis in (A) a sewage oxidation pond in
Lemmon, South Dakota (from Bartsch and Allum, 1957) and in the Northeastern Sargasso
Sea (from Menzel and Ryther, in press) .

References

Bartsch, A. F. and M. O. Allum. 1957. Biological factors in treatment of raw sewage in artificial ponds.
Limnol . & Oceanogr., 2: 77-84.

Blinks, L. R. 1955. Photosynthesis and productivity of littoral marine algae. J. Mar. Res., 14:
363-373.

Brown, H. 1956. The challenge of man's future . The Viking Press , New York . 290 pp.

Gushing, D. H. 1958. The effect of grazing in reducing the primary production: a review. Rapp.et
Proc. Verb. Cons. Internal. Explor . Mer., 144: 150-154.

Fawcett, C. B. 1930. The extent of the cultivable land . Geogr . Rev ., 76: 504-509 .

Gessner, F. 1949. Der Chlorophyllgehalt im See und seine photosynthetische Valenz als geophysik-
alisches Problem. Schw . Zeitschr. f. Hydrol , 11: 378-410.

Harris, E. and G. A. Riley. 1956. Oceanography of Long Island Sound, 195 2 - 1954. VIII Chemical
composition of plankton. Bull. Bing. Oceanogr. Coll., 15: 315-322.

Harvey, H. W. , L. H. N. Cooper, M. V. Lebour and F S. Russell. 1953. Plankton production and its
control. S. Mar. Biol. Assoc. U.K., 20: 407-442.

Henderson, L. J. 1913. The fitness of the environment. The Macmillan Co. , New York.

Kramer, P. J. and W. S. Clark. 1947. A comparison of photosynthesis in individual pine needles and
entire seedlings at various light intensities. Plant . Physiol., 22: 51-57.

Maximov, N. A. 1938. Plant physiology. 2nd. Eng . Ed. McGraw-Hill Book Co., Inc., New York,

Menzel, D. W. and J. H. Ryther. In press. The annual cycle of primary production in the Sargasso Sea
off Bermuda . Deep-Sea Res .

Odum, E. P. 1959. Fundamentals of ecology. 2nd Ed. W. B. Saunders Co., Philadelphia.

Odum, H. T. 1957. Primary production measurements in eleven Florida springs and a marine turtle-
grass community. Limnol. & Oceanogr., 2: 85-97.

Odum, H. T. , and E. P. Odum. 1955. Trophic structure and productivity of a windward coral reef
community on Enlwetok Atoll . Ecol . Monogr . , 25: 291-320.

Ovington, J. D. and W. H. Pearsall . 1956. Production ecology II. Estimates of average production by
trees. Oikos, 7: 202-205 .

Pearsall, W . H . and E. Gorham. 1956. Production ecology I. Standing crops of natural vegetation.
Oikos, 7: 193-201.

Rabinowitch, E. I. 1951. Photosynthesis and related processes . Vol.1. Interscience Publishers , Inc . ,
New York .

Riley, G. A. 1956. Oceanography of Long Island Sound , 1952 - 1954 . II Physical oceanography . Bull.
Bing. Oceanogr. Coll., 15: 15-46.

Riley, G. A. 1958. Phytoplankton of the North Central Sargasso Sea , 1950 - 1952. limnol. &
Oceanogr. , 2: 252-270.

82

Riley, G. A., H. Stommel and D. F. Bumpus. 1949. Quantitative ecology of the plankton of the
western North Atlantic . Bull. Bing . Oceanogr . Coll., 12: 10-69.

Rudd, J. T. 1930. Nitrates and phosphates in the southern seas. J. Cons. Internat. Explor. Mer . ,
5: 347-360.

Ryther, J. H. 1956a. Photosynthesis in the ocean as a function of light intensity. Limnol. &
Oceanogr., 1:61-70.

Ryther, J. H. 1956b. The measurement of primary production. Limnol. & Oceanogr. , 1: 72-84.

Ryther, J. H. In press. The potential productivity of the sea. Science.

Ryther, J. H. , and C. S. Yentsch . 1957 . The estimation of plankton production in the ocean from
chlorophyll and light data. limnol. & Oceanogr. , 2: 281-286.

Ryther, J. H., and C. S. Yentsch. 1958. Primary production of continental shelf waters off New York .
Limnol. & Oceanogr., 3: 327-335.

Ryther, J. H., andC. S. Yentsch, E. M. HulburtandR. F. Vaccaro . 1958. The dynamics of a diatom
bloom. Biol. Bull., 115: 257-268.

Schroeder,H. 1919. Die jahrliche Gesamptproduktion der griinen Pflanzendecke der Erde . Naturwiss.,
7: 23-29.

Sette, O. E. 1955 . Consideration of mid-ocean fish production as related to oceanic circulatory
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Show, S. B. 1949. The world forest situation. U.S. Dept. Agricult . Yearbook of Agriculture, Trees.
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Steemann Nielsen, E. 1937. The annual amount of organic matter produced by the phytoplankton in the
Sound off Helsingdr. Medd . Komm. Danmarks Fiskeri-og Havndenis Ser: Plankton., 3(3): 1-37.

Steemann Nielsen, E. The marine vegetation of the Isefjord — a study on ecology and production. Ibid. ,
5: 1-11.

Steemann Nielsen, E. 1957. The chlorophyll content and the light utilization in communities of plankton
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Steemann Nielsen, E. , and E. A. Jensen. 1957. Primary oceanic production, the autotrophic production
of organic matter in the oceans . Galathea Repts., 1: 49-136.

Tamiya,H. 1957. Mass culture of algae . Ann . Nev. Plant Physiol ., 8: 309-334 .

83

ARTIFICIAL MEDIA FOR FRESH-WATER ALGAE:
PROBLEMS AND SUGGESTIONS^

L. Provasoli and I.J. Pintner
Haskins Laboratories

Fresh-water algae seemingly are ubiquitous:
as knowledge Increases the same species are re-
corded from all continents. Because most fresh-
water habitats are ephemeral, most species survive
only if they can form stages resistant to desslca-
tlon (Evans, 1958). Overcoming this ecological
disadvantage permits species to be transported by
high winds and birds all over the globe as constant
inocula In search of opportune environments .

It is also well known that fresh-waters, un-
like the almost homogeneous marine environment,
display a wealth of environments and algal flora.
The distribution of algal species in fresh-waters
depends on one hand on the selective action of the
chemlco-physical environment and, on the other,
on the organism's ability to colonize a particular
environment and to compete with the other species
living in the same ecological niche . The ecolo-
gist and the sanitation scientist seeking a rapid
way to distinguish various environments rely more
and more on biological markers, the indicator spe -
cies which typify each environment more subtly and
precisely than can laborious chemical analysis.

This knowledge is being extended and refined
continuously. Meanwhile the ecologist would like
to know more precisely the physico-chemical char-
acters and boundaries of the ecological niches . A
frontal chemical attack is difficult at present be-
cause many organic and Inorganic substances are
biologically effective and present in waters in ex-
treme dilution. Another way to define the chemical
environment Is to study the nutritional needs of the
indicator species . These, because of their re-
stricted occurrence and narrow fit to their environ-
ments, are, presumably, outstandingly exacting for
at least some Important parameters (stenobionts) .
Few, if any, of these species have been cultured
aseptlcally, the only way to determine their nu-
tritional requirements . Ridding the algae of the
unwanted microbial flora, though often tedious and
delicate, is only occasionally the main obstacle.
Agar-plating, washing and dilution techniques com-
bined with the use of antibiotics are well-estab-
lished methods [Pringshelm (1946); Provasoli
etal., (1951), Lewln (1959), Provasoli and Holz
(In press)] . The main difficulty is to design media
which will support the aseptic growth of algae
whose nutritional requirements are still unknown.
A practical approach, when the indicator species of
a specialized environment do not grow In present

media, is to study the nutrition of a species of the
same environment which can also live in closely re-
lated environments . An analysis of their nutritional
requirements may show how to construct media ade-
quate for the indicator species . Selection and con-
struction of media would perhaps be facilitated if
one had: a) data on the relative Importance of the
chemical components of the waters , b) data on the
nutritional requirements of the different groups of
algae, c) some guideposts in solving some of the
problems of designing artificial media.

Relative Importance of the Chemical Factors
of the Environment

In dealing with organisms of unknown nutri-
tional requirements one can obviously benefit
greatly in mimicking as closely as possible the
chemical environment .

Due to the diverse composition of fresh-
waters one should know the following parameters in
order of Importance: a) total solids, b) the prevail-
ing major ions, c) pH, d) main trace metals, e) ra-
tios of monovalent/divalent cations and of Ca/Mg,
and_f) content of organic matter (which may act as
trace metal solubllizers or as growth factors) .

The pertinence of these considerations is em-
phasized by Rodhe's success (1948) in studying in
detail the mineral requirements of Anklstrodesmus
falcatus . At the end of his studies he compounded
a medium in which all the major minerals were
added at optimal concentration. This medium is
very close to the composition of the waters of Lake
Erken, where Anklstrodesmus blooms, and the aver-
age composition of the waters of many Swedish
lakes. This medium proved to be satisfactori' for
most of the algal species Inhabiting Lake Ekren and
would probably sustain some growth of all the spe-
cies when enriched with vitamins (at the time it
was not known that many algal species require vit-
amins) .

The medium of Rodhe might have been quite
different had he chosen to study an eurybiont and
not a stenoblont. The eurybiont, depending upon
the species selected, would have led to construct
several media specific for the species in question,
and therefore very different from the natural waters .

The most important parameter, especially for
oligotrophic algae, seems to be the total-solids

■^ Aided in part by research grant G-3216 from National Institutes of Health.

84

concentrations. Chu (1942) succeeded ingrowing
several oligotrophic fresh-water algae by simply
diluting the well known old medium of Benecke .
Later, by studying the mineral nutrition of several
oligotrophic diatoms and chlorophytes , he com-
pounded a medium (Chu 10) of wide use for oligo-
trophic organisms. The media experimentally de-
signed by Chu, Rodhe and us (Provasoli and
Pintner, 1953) are very similar. Further studies
(Provasoli, McLaughlin and Pintner, 1954) con-
firmed that oligotrophic species in general require
very low total-solids concentrations and are steno-
bionts toward this parameter. In the same analysis
it was found that algae may prefer a mono - or di-
valent ion, but that they are in general far less
exacting toward the Ca/Mg or Na/K ratio, and that,
within large limits, the two monovalent and two
divalent ions are interchangeable when the lower
limits of the preferred ion have been satisfied ,
(Provasoli, McLaughlin, Pintner 1954, Droop 1958).
However, it is possible that organisms living in
dystrophic lakes may show the need for special
ratios of major elements besides total solids con-
centrations .

Trace-metal content and pH are related fac-
tors because the solubility and availability of
trace-metals varies with pH . Iron and Mn seem
to be the two trace-metals ions which are quanti-
tatively important . Zn, Co, Cu , Mo, and V are
also important because required for growth, even
though they are generally present in waters in far
lower concentrations . They are generally present
as impurities of other chemically pure salts and,
depending upon the concentrations of major ele-
ments employed (especially in sea water media) ,
one of them may be introduced in media at concen-
trations approaching inhibition if not toxicity. The
use of metal chelators may be of advantage in che-
lating these metals and in giving the limited steady
supply allowed by the dissociation constant typi-
cal for each divalent ion. Hence in mimicking nat-
ural conditions one should try to estimate the kind
and content of organic substances in the water .
"Humates" in alkaline or peaty waters are trace-
metal chelators less strong in their chelating power
than the chelator most used in artificial media
(EDTA) , but apparently more versatile in that they
can be employed at different ph"s without causing
toxicity (i.e. soil extract can be employed at pH's
form 5-8.5). "Pollution" is generally thought ben-
eficial to algae as a source of N and P. However,
in the degradation of organic matter by microorgan-
isms organic acids may be produced, especially
amino acids, many of which are good trace-metal
chelators . This is especially important in alka-
line waters when the solubility of heavy divalent
metals is almost nil. Iron was found by Uspenski
and Uspenskaja (1925) to be the important trace-
metal for several Volvox species . They were the
first to introduce chelators (citrate) in fresh-water

media to keep the iron available to the algae in
neutral and alkaline media, because they sus-
pected that this was the mechanism operating in
nature. Volvox generally blooms in waters rich in
organic matter. Further investigations on the nu-
trition of Volvox (Pintner and Provasoli, 1959) show
that V. qlobator and V. tertius have scant hetero-
trophic abilities and that they do not need any pre-
formed organic compound as sources of energy; the
only organic compound needed is vitamin Bi2-
Their colonizing of water rich in organic matter is
therefore due to the need of finding vitamins and,
perhaps even more, to the need of having Fe solu-
bilized by the "organic acids".

Generalities on the Nutritional Requirements
of Different Algae

The known requirements of the algae have
been recently reviewed (Krauss, 1958; Provasoli,
195 8) . We will therefore consider only a few
points .

a) Some nutritional requirements seem pre-
dominant or even unique in some algal groups .
Silica is important and often a limiting factor for
diatoms and perhaps also for the chrysomonads
bearing silica plates . High Fe and trace metals
are important for many euglenids and cryptomonads
colonizing acid waters. Sodium and/or potassium
are essential elements for blue-green algae. Dia-
toms have calciophilic and calciophobic species .
Most algae utilize nitrates preferentially but the
euglenids, so far cultured, can only utilize am-
monia, and, some, amino acids as N sources.
Many blue-green algae utilize atmospheric nitro-
gen .

b) Organisms in the same ecological niche
often have common nutritional features. Most oli-
gotrophic algae cannot withstand total solid con-
centrations above 100-200 ppm.; many are steno-
haline . Algae in environments high in some sub-
stances (as the high concentrations of Fe and trace
metals in bogs and ditches and the high S in heav-
ily polluted waters) may merely be withstanding
these conditions; some actually require them. The
organisms of barnyards and sewage oxidation ponds
in general withstand ammonia even at alkaline pHs .

c) Algae lacking photo synthetic pigments ob-
viously need non photosynthetic sources of energy.
Acetate, glutamate, aspartate, glycine, and glu-
cose seem to be the preferred carbon sources for
many. So far we know no chemotrophic algae.

However the possession of photosynthetic
pigments does not exclude the possibility of heter-
otrophy, indeed, they may prefer it. While per-
haps most pigmented algae are phototrophic , many
are heterotrophic .

The taxonomic position of a species to be
cultured offers indications of the probabilities.

85

Elsewhere (Provasoli, 1956) we have enumerated
some of the nutritional hints derivable from the
phyletic tendencies of algae so well described by
Fritsch (1934) . The Chlorophyceae and the Dia-
toms have strong vegetal tendencies; most of them
can be expected to be phototrophic . Heterotrophy
of the osmotrophic type is to be expected in genera
which contain colorless species. The Chrysophy-
ceae have a strong rhizopodial tendency even
among the pigmented species and the Eugleninae
and Dinophyceae have a strong tendency toward
loss of pigments; many species can be expected to
be heterotrophic or even phagotrophic . Two ex-
amples of the nutritional versatility of pigmented
algae are Ochromonas and Euglena . Ochromonas
malhamensis (Hutner et aL- / 1953; Aaronson and
Baker, 1959) , though pigmented and able under op-
portune conditions (high CO2) to live photoauto-
trophically, is preferentially an heterotroph . In
rich organic media, Ochromonas through many di-
visions, and until the organic nutrients are re-
duced to a low level, synthesizes only subopti-
mally its photosynthetic pigments: the cultures
appear white and the large chloroplast is reduced
to an anterior faint brown spot. However, when
the organic solutes are low and nutrient particles
(bacteria) are offered , it becomes a phagotroph
(Aaronson and Baker, 1959) . While Ochromonas
can utilize three methods of nutrition the Euglena
gracilis group is less versatile, but its photoauto-
trophy is as efficient as its osmotrophic hetero-
trophy. Therefore deductions based solely on the

presence of photosynthetic pigments and type of
environment may be misleading. Species living in,
or restricted to polluted waters, may colonize
these waters for entirely different needs and are
not necessarily heterotrophs: Vol vox grows there
because it needs soluble iron and vitamins, while
Ochromonas prefers heterotrophy and Euglena , be-
sides its heterotrophic abilities , needs and toler-
ates NH4 in alkaline waters .

c) Many algae require vitamins; a detailed
tabulation is given in a recent review (Provasoli,
1958) . No correlation has been detected between
need in vitamins and source of energy employed or
any particular environment; photosynthetic or color-
less species, species living in oligotrophic or
polysaprobic environments may need or not need
vitamins . However, the incidence of auxotrophic
species differs in various algae groups. (Table I) .
The Chlorophyceae and Bacillariophyceae have the
lowest number of species requiring vitamins. The
great majority of the Dinophyceae and Chrysophy-
ceae needs vitamins and all the species so far
studied of the Eugleninae and Gryptophyceae re-
quire vitamins. The need for vitamins seemingly
predominates in algal groups having strong animal
tendencies; most species of the algal groups having
strong vegetal tendencies (Chlorophyceae, Bacil-
lariophyceae, and probably Cyanophyceae) do not
need vitamins. Although the sampling is very
small, the data should be valid because the re-
cently studied species (all except the Chlorophy-
ceae) were not preselected by the choice of

Table 1 . Vitamin requirements of algae

Algal group

Number
of

No

Require

Species Vitamins Vitamins Q12

Chlorophyceae 40 25

Eugleninae 9

Cryptophyceae 9

Dinophyceae 18 2

Chrysophyceae 13 1

Bacillariophyceae 37 20

Totals 124 46

Thia-

15

6

8

9

2

1

9

2

2

16

11

12

3

1

17

10

3

78

34

15

B12 +

Thia-

mine Biotin mine

Biotin

+
Thia-
mine

B12 +
Biotin +
Thiamine

1
6
5
1
5
4-
22

Total requirement
for single Vitamins

61

44

86

isolation media (i.e. they were isolated from nature
in media containing a mixture of known water-solu-
ble vitamins) .

It is remarkable that all the species which
have photosynthetic pigments and the related color-
less forms require only three vitamins; in order of
decreasing incidence, vitamin Bj2, thiamine, and
biotin (Table I). Only the phagotrophic flagellates
(e.g. Peranema ) seem to need other vitamins and
"building blocks".

Problems in Designing Artificial Media

It is not generally realized that the classic
media for algae (Knop, Beijerinck, Detmer) have
been designed before 1900. No pH is mentioned,
nor the type of phosphate (mono-, di-, tri-basic) ,
and, often, if the salts are anhydrous or not. Un-
fortunately even the more recent media (up to 19 20
and in some cases later) have the same defect. It
is not surprising therefore that there exist different
interpretations and modifications of these formulas.
These solutions were apparently meant to be em-
ployed at their natural pH (i.e. pH 5 for Knop, 6.2
for the Detmer and 7.2 for the Beijerinck); they pre-
cipitate in the more alkaline pHs, yet they have
been used successfully even at different pHs.
These solutions are in general too concentrated and
it was soon found that dilutions of 1:2 to 1:20 per-
mitted growth of many more species . Most Chloro-
phyceae and many diatoms grow in these media .

In most algal media, especially the old ones,
the only trace metal added is iron. This does not
mean that the other trace metals are not needed: it
is well known that algae need Mn , Zn , Co, Cu, V,
Mo. It only reflects the degree of impurity of the
major mineral constituents of the medium. It is
worth noting that the industrial methods of purifica-
tion of the "chemically pure" salts have undergone
many changes since Knop's time resulting in an
ever-changing sets of impurities . Besides, with
the advent of plastic containers in industry we may
expect increasingly pure salts and therefore possi-
ble nutritional deficiencies. New deficiencies
(and perhaps toxicities) will develop when the nor-
mal glassware of the laboratory will be substituted
by plastics with consequent absence of the impuri-
ties leaching from glass.

The addition of trace metal-chelate mixtures
of media offers the possibility of minimizing the
impurities (toxic or favorable) as well as furnishing
a metal pool of available trace metals. Therefore
chelated media should be more reproducible and
withstand fluctuations in impurities of the chemi-
cally pure components of media . A further advan-
tage is that chelators help in preventing precipita-
tion - one of the main goals for reproducibility.
A number of chelated media have been developed
for a few species of fresh-water algae: Euglena

(Hutner et al . , 195 6); Ochromonas malhamensis
(Hutner et al . , 195 7); O. danlca (Aaronson and
Baker, 1959). These media were designed for
maximal growth and rapid cell division. To obtain
this goal, the media have been enriched, often up
to the limits of osmotic tolerance, with as many
preformed key metabolites as possible, to spare the
organism most of the work of synthesis. Since the
success of these media depends upon their exploit-
ing all the externally accessible synthetic path-
ways (i.e. permeability) and any useful potential
and tolerance of the organism in question, they be-
come so highly tailored that seldom are they suit-
able for other organisms. Only a few chelated
fresh-water media are of more general, though still
restricted, use, as the Kratz-Myers (1955) medium
for Anacystis nidulans , Anabaena variabilis , and
Nostoc muscorum , which is also good for other
blue-green algae (Phormidium autumnale , Synecho -
coccus cedorum , Anabaena cylindrica) , and the
medium for Chlamydomonas (Hutner and Provasoli ,
1951) which serves for its colorless counterpart
Polytoma (Cirillo, 1957) and a few other colorless
flagellates (Chilomonas , etc.). Perhaps the flexi-
bility of these media is due to their being designed
for photoautotrophic nutrition, while the others are
for heterotrophic nutrition and for eurybionts . Good
heterotrophic media contain several amino acids
and often other building blocks, some with strong
chelating abilities, therefore the trace-metal mix-
tures developed for these media are too high in
metals for the photoautotrophs .

The prerequisites for reproducible and ver-
satile media for photoautotrophic algae can now be
specified:

a) total-solids concentrations.

b) concentrations of major elements to suit
the prevalent ions required .

c^) adequate sources of N, and growth fac-
tors .

d) sources of P and avoidance of precipi-
tates in alkaline pHs .

e) pH buffering

f) trace-metal buffering

As stated in section I, the best chances to fulfill
a) and b) , at least for devising media for isolation
and moderate growth, are to mimic as closely as
possible the conditions of the natural waters in
which the organisms normally bloom. Natural con-
ditions in respect to carbonates are hard to repro-
duce because carbonates, during sterilization dis-
integrate releasing CO2 with resultant alkaliniza-
tion and precipitation of the medium. Filter-steri-
lized carbonates or CO2 can be added aseptically.
Fortunately, carbonates can be substitutes by other
anions such as Gl, SO4, PO4 , and NO3 . It is op-
portune to introduce as little as possible of chlo-
rides and sulphates by employing as much nitrate
as is compatible with the organism. Therefore the
prevalent ions, often Ga , can be introduced as

87

nitrate as Knop did .

Most algae utilize nitrates except the eu-
glenids which, so far as we know, prefer ammonia
or amino acids. Ten to 20 mg.% nitrates are in
general well tolerated . Ammonia tends to become
toxic above 3-5 mg.% in alkaline media except for
eurybionts living in polluted waters. Since it is
difficult to know priori if an alga needs vitamins ,
it is advisable to include in the isolation media the
three vitamins required by most auxotrophic algae:
vitamin B12 .01 jug.%, thiamine 10 )dg .% , and bio-
tin 0.05>ag.%. Mineral phosphates should be
avoided because they cause precipitates , especial-
ly in alkaline media . In a recent survey (Provasoli ,
McLaughlin, Pintner, (in Provasoli , 1958, p. 294)
we have found that glycerophosphate was utilized
by all the algae tested. Glycerophosphates have
the advantage of forming far more soluble salts with
the divalent anions than the phosphates, thus pre-
cipitation is often avoided even in slightly alka-
line media or in the absence of chelators . Con-
centrations of .5-3 mg.% are generally adequate.

The problem of avoiding precipitates while
supplementing the necessary trace-metals was
partly solved by Uspenski and Uspenskaja (19 25)
by using opportune ratios of citric acid and Fe .
But chelation of Fe with citric acid does not prevent
precipitates in alkaline media. Hutner (1948) and
Hutner et al. (1950) explored the possibility of em-
ploying stronger chelating agents to study the es-
sentiality and role of trace metals. The criteria for
selection of a "good" chelator for studying essen-
tiality were that: a) it should form very stable
complexes; b) it should be a bulky non-penetrable
molecule so that chelation will take place in the
external medium and not within the cells; c) it
should be photostable and thermostable; d) it should
be non- metabolizable and non-toxic for reasons
unconnected with its metal-binding properties .
Ethylenediamine tetracetic acid (EDTA) met all
these specifications. EDTA, or other chelators
tried, could not, as hoped, serve to determine the
essentiality of single trace metals, but were very
effective in providing a non-precipitable metal pool
of trace metals, thus approaching the goal of
metal-buffering (pM) , and became of general use.
No difficulties were found for fresh-water eurybi-
onts (Euglena , Ochromonas , etc.) and for some
marine fungi (Vishniac, 1955). Enormous quanti-
ties of EDTA (50-100 mg.%) were employed at
first and the trace metals had to be raised to cor-
respondingly high and unphysiological levels prob-
ably to prevent the excess EDTA from binding other
necessary ions such as Ca and Mg . It was soon
found that most organisms (even the marine algae)
could not tolerate the high content of free trace
metals and free chelator resulting from the equa-
tion: K = Civrd . Provasoli et al. (1957) found

[M] [Y]
that artificial synthetic marine media are suitable

for the greatest variety of organisms when the trace
metals pool is low and the trace metals are offered
as a mixture slightly overchelated (ratio of chela-
tor/trace metals = 1:1 to 3:1) . Since these mix-
tures (PI, PII, and TMII are the most widely used
for marine organisms) are over-chelated , Droop
(1959) has rightly raised the point that when the
salinity of the marine media is varied and lowered
(by lowering the Ca, Mg , Na , K concentrations) it
might be better to employ citrate as a metal buffer
because citrate has less affinity for Ca and Mg
than EDTA . The free EDTA (of the over-chelated
mixture) binds these cations more strongly and will
reduce drastically their availability, especially at
the lowest salinities. In fact it is possible to em-
ploy these over-chelated mixtures even for fresh-
water organisms . One can substitute with 1-2
ml/100 of PII mixture the metal mixtures which had
been experimentally found to compensate for the
various chelations employed in the media for Pha -
cus pyrum , Volvox globator , V . tertius , and Wolo-
szynskia limnetica . But this does not hold for
Synura media which are low in Ca and Mg (respec-
tively .4 and .05 mg.%) while the other media
contain from 2-4 mg.% of Ca and from .4 to 2
mg.% of Mg. This seems to confirm Droop's
assertion . Experiments with Synura are in progress
to see whether increased Ca and Mg will allow the
use of over-chelated mixes; this might not be feas-
ible because of the very poor tolerance of Synura
toward total solids .

The other important difficulty in fresh-water
media is pH buffering. Inorganic phosphate can not
be employed because: a) most fresh-water algae,
especially those of oligotrophic waters, cannot
withstand the concentrations of phosphates needed
for buffering (phosphates often become toxic above
5-20 mg.% except for organisms living in polluted
waters and often for blue-green algae); b) heavy
precipitates result because the fresh-water media
are generally rich in calcium. Several amines have
been employed successfully for buffering in the al-
kaline range. Of these the most useful are tris
(hydroxy- methyl) aminomethane (TRIS) and triethan-
olamine (TEA); ethanolamine is in general more in-
hibitory. TRIS is a very good buffer for marine
media: most marine algae are not inhibited by 100
mg .% and several withstand much higher concentra-
tions. However, TRIS and other amines are toxic
to certain pathogenic bacteria. MacLeod and
Onofrey (1954) found that the toxicity of amines
could be counteracted by the addition of K, Ca ,
Mg , and Na , alone or in combinations. This is
probably why Tris is not toxic at useful pH buffer-
ing concentrations in media rich in those ions, like
the marine media . TEA and TRIS can be employed
for fresh-water algae fairly resistant to high total-
solids concentrations so that it is possible to raise
the cations and counteract the inhibition of the
amines without approaching osmotically inhibitory

concentrations of salts. Different species of algae
react differently toward the various amines. The
toxicity of TEA is counteracted by increasing con-
centrations of Ca for Vol vox globator and by Ca and
Mg for y. tertius and Woloszynskia . The toxicity
of TRIS for Cyanophora paradoxa can be removed by
additions of Mg . and K; Ca and Na are of no use
and even in presence of TRIS, they can be dis-
pensed with in media allowing optimal growth.
Since the amines heighten requirements for the
major cations, they may be used to reveal the pre-
dominant necessary ions. But the amines can be
toxic through other, unknown mechanisms: the
toxicity of TRIS (20 mg.%) for Vol vox globator can-
not be counteracted by Ca , Mg , Na , K or trace
metals .

Another pitfall of TEA, at least for Volvox , is
that minor variations in pH (from 7.0-7.6) are im-
mediately reflected in a change in availability of

trace metals, particularly iron: the same concen-
trations of iron act at one pH as if Fe were avail-
able in excess (toxic) and at a slightly different
pH as if Fe were lacking. A similar inflexibility
toward the trace metal mixtures was noted for other
algae. The first success was the buffering of the
extremely dilute media for Synura at pH 5.0 (Table
2) . Buffering these media was particularly diffi-
cult yet Imperative because solutions are so dilute,
owing to the sensitivity of the organisms to con-
centarions of any solute. Histidine adequately
solved the problem: it is well tolerated at 15-30
mg.%, just enough to buffer the medium effectively
at pH 5.0-65 . Much later, following the sugges-
tion of Droop (personal communication) , who had
found glycylglycine less toxic than TRIS for Oxyr -
rhls marina , we tried this buffer for Volvox , Wolo-
szynskia , and Phacus . Surprisingly, minor changes
in pH no longer affect metal availability in Volvox

Table 2 . Culture medium for Synura media

Metal -buffered

pH-buffered

EDTA
NaN03
KH2PO4
Mg (as 304=)
Ca (as CI")
K (as Cl~)
Fe (as Cl")
Zn (as Cl")
Mn (as Cl")
Mo (as Na salt)
Co (as Cl")
Cu (as Cl")
Na H glutamate
Na acetate. 3H2O
pH 5.5

mg.%

mg.%

5

Na3 citrate. HgO

2

2

(NH4)2S04

6

1.4

Na2 glycerophos

phate.5H20*

5

0.2

Mg (as Cl")

0.05

1.3

Ca (as Cl")

0.4

0.5

K (as Cl")

0.2

0.07

Fe (as Cl")

0.05

1.0

Na2Si03.9H20

3

0.2

Mn (as Cl")

0.001

0.001

L-histidine (free

base)

20

0.003

B12

0.04ng

0,0005

pH 6.0

10

4

* a mixture of oC - and p- glycerophosphates,

89

Table 3. Culture Medium for Volvox globator and V_- tertius

Ca (as NO3 )

2 mg.%

^12

.01

pg.%

MgS04 .

7H2O

4 mg.%

Biotin

.01

pg .%

Na2 glycerophosphate. 5H2O

5 mg.%

P. IV Metals*

.3 ml./lOO - 4 ml./lOO

KCl

5 mg.%

V . globator.

V. tertius,

average

average

optical density

optical density

pH 6.0

histidine

20 mg .%

0.13

0.16

pH 6.4

histidine

20 mg .%

0.23

0.24

pH 6.6

histidine

20 mg.%

0.26

0.23

pH 7.2

glycylglycine

50 mg.%

0.22

0.28

pH 7.5

glycylglycine

50 mg.%

0.29

0.24

pH 8.0

glycylglycine

50 mg.%

0.23

0.24

1 ml . of PIV metals contains:
HOEDTA ^^^ 1 mg.

Fe (as Cl~) 0.04 mg .

Mn (as CI") 0.01 mg .

Zn (as CI') 0.005 mg .

Co (as C1-)
Mo (as Na+)

0.001 mg,
0.005 mg.

(1) see note Table 5

media (Table 3) when glycylglycine is used as a pH
buffer in place of TEA. Not only could good growth
be obtained with the same trace metal mixture be-
tween pH 7 and pH 8, but we could also employ a
wide range of concentrations (1-4 ml ./1 00) .

During our work with Phacus pyrum and Wolo -
szynskia limnetica , we designed suitable media for
the alkaline and acid region, with the hope of eli-

citing more growth by changing the penetrability of
the substrates . With TEA and succinic or malic
acids as buffers, we had to vary the media and the
chelation to suit the pH and the buffer system. The
resultant acid and basic media gave less or no
growth when the pH was changed. After suspecting
that the choice of the pH buffer affected chelation,
we tried both the acid and alkaline media at

90

Table 4. Culture Medium for Phacus pyrum

pH

5 .5 Base
mg.%

pH 7.6 Base

mg.%

pH 5.5 Base
mg.%

pH 7 .6 Base

mg . %

Ca (as CI )

2

4

Zn (as CI")

1

ug.

5 ^ig.

MgS04-7H20

30

4

Mn (as CI")

9

>ug.

0.04

Na2 glycerophos-
phate. 5H2O

7

4

Co (as CI")

0.3

jug.

1/jg.

KCl

3

IS

Cu (as CI")

0.03 jjg.

—

(NH4)2S04

50

-

Mo (as Na+)

0.2

yg.

—

{NH4) acetate

-

50

Boron

-•

-

0.2

Na2 EDTA

-

1

Fe (as NH4 citr.)

0.1

0.07

pH 5 .5 Base
Optical density

pH 7 .6 Base
Optical density

pH 5 .8 malic acid

30 mg.%

0.37

0.64

pH 5 .8 succinic acid

30 mg.%

0.28

0.52

pH 6 .5 L-histidine

30 mg.%

0.23

0.52

pH 8.0 triethanolamine

30 mg.%

0.24

0.40

pH 8 . 1 TRIS

30 mg.%

0.25

0.09

pH 8.2 glycylglycine

80 mg.%

0.30

0.50

NB. The low pH medium was buffered with amalic acid; the high pH medium with TEA.
* a mixture of Of- and S - glycerophosphates .

different pH but with histidine and glycylglycine as
buffers (Table 4 and 5) . The pH 7 .6 base for Pha -
cus gives, with suitable buffers, good growth from
pH 5.2 - 8.2; the toxicity of TRIS in the pH 7.8
medium is puzzling, since TRIS is not toxic for the
pH 5 .5 base . The pH 6 .0 base for Woloszynskia
llmnetica is more versatile than the pH 8.0 base.

In any attempt to understand the reason for the
successful use of histidine and glycylglycine as
pH buffers, we have to consider:

a) that histidine is a metal chelator almost as
strong as EDTA and that glycylglycine is much
weaker; b) that in the zone pH 5 .0-8 .5 , an in-

crease in pH results in more chelated metal ions
and less free metal ion. Therefore, if a chelated
metal mixture is kept constant and the pH is raised,
this rise will result in a metal deficiency (i.e.
more metals should be added or the chelator should
be reduced); decreasing the pH results in an excess
of free metals (i.e. one should add more chelator
or reduce the metals); c) how much the chelator is
in excess of the quantity needed for the 1:1 chela-
tion of the trace metals (and therefore how much Ca
and Mg will be chelated); d) whether the chelator
employed is a bulky molecule unable, to or slowly
penetrating, the cell, or whether it is small enough

91

Table 5 . Woloszynskia limnetica

pH

8.0 Base

pH 6.0 Base

pH

8.0 Base

pH 6.0 Base

mg .%

mg.9i

D

mg.%

mg.%

Ca CI2

21.6

10.8

Na2 glycerophos-
phate. 5 H2 0+

4

2

MgS04.7H20

15

20

Mo (as Na+)

4;ag,

0.02

KCl

1

1

Co (as Cl~)

4^9

0.03

NaNOa

20

20

Cu (as CI")

OAjdQ

3^ig.

B12

0.1 pg.

0.1

pg.

Zn (as CI")

0.32

0.1

HOEDTA (as Nas)*

7

■"

""

Mn (as CI")

0.32

0.8

Na2 EDTA

6

Fe (as NH4 citr.)

0.1

0.2

pH 8.0 Base

pH 6.0 Base

Optical density

Optical density

pH 5 .6 succinic acid

30 mg.%

0.02

0.14

pH 6.3 succinic acid

30 mg.%

0.015

0.13

pH 6.3 L-histidine

30 mg.%

0.07

0.85

pH 7.8 TRIS

80 mg.%

0.54

0.52

pH 7.8 glycylglycine

80 mg.%

0.13

0.54

NB . The pH 8 medium was buffered with TRIS and the pH 6 medium with succinic acid ,
* hydroxyethyl ethylenediamine triacetic acid

t a mixture ofOt'- andy^ - glycerophosphates .

to penetrate the cell .

In our case we have kept constant the trace
metal/chelator mixture and added pH buffers, some
of which are metal chelators . Therefore according
to b) when one decreases the pH (e.g . when the
pH 8 .0 medium is lowered to pH 6 .0 and 5.0), one
should add more chelator and when one brings the
acid medium to alkaline pH, one should decrease
the amount of Chelator. TRIS does not control the
availability of trace metals at pH 7 to 8 (Cheno-
weth, 1956) nor probably does , TEA. Therefore
when TEA is substituted and the alkaline media of
Volvox and Phacus are brought to pH 6 .0-6 .5 ,

more chelator should be introduced. This is what
we did with the addition of histidine as the buffer
for the pH 6 .0-6 .5 zone . However we obtain good
growth of Phacus at pH 5 .8 with addition of suc-
cinic or malic acids which are poor chelators . The
pH 6.0 base of Woloszynskia gives better growth
when the pH is raised and succinic acid is re-
placed by histidine, TRIS, or glycylglycine. Since
the succinic medium is under-chelated, it may give
poor growth at pH 5 .6 because it has too much
metals; addition of a chelator like histidine could
have adjusted the balance, but more growth is also
obtained with TRIS, a non-chelator and by glycy-

92

glycine, a weak chelator. The behavior of these
pH buffers in our media cannot therefore be ex-
plained as a pure chelating effect, nor can the two
Synura media (Table 2) . If the miUiequivalents of
all the metals, including Ca and Mg, are com-
pared with the miUiequivalents of the chelators,
we find that the EDTA-glutamate medium is over-
chelated 1.3:1 and the histidine medium 5:1;
yet they give similar growth . The media for Oxyr-
rhis marina of Droop (1959a) present a similar puz-
zle: at the same pH and with the same amount of
trace metals and major elements similar growth can
be obtained by chelating with 0.6 mg .% EDTA or by
the joint chelation of 20 mg.% histidine, 4 mg .%
citric acid and 50 mg.% glycylglycine; 2-6 mg.%
EDTA on the contrary allows far less growth. Again
the level of chelation does not explain in the data:
hisUdine alone chelates > 60:1; 0.6 mg.% EDTA,
2:1 and 6 mg.% EDTA, 20:1.

These discrepancies can be explained satis-
factorily if one considers the consequences of the
different molecular size of the chelators used. As
mentioned, EDTA was chosen because it was sup-
posed that the bulkiness of its molecule would pre-
vent penetration into the cells and that it would be
photo-stable. It was later found that Fe-EDTA
chelates decompose in light (Jones and Long, 195 3)
and that some EDTA or its breakdown products pen-
etrate in the algal cell (Krauss and Specht, 1958) .
However, the majority of the iron apparently does
not enter as intact iron chelate because, on a mol-
ecular basis, 15 to 50 times more iron was ab-
sorbed by the cells than EDTA. Tiffin and Brown
(1959) employing the iron chelate of ethylenedia-
mine di (o-hydroxyphenylacetic acid) (EDDHA)
found that roots of decapitated sunflower plants ab-
sorbed only about .3% of the total EDDHA and
large amounts of iron, leaving most of the EDDHA
in the nutrient solution.

Therefore for practical purposes EDTA is a
non-absorbable chelator and the cells depend al-
most exclusively: a) on the available free metal
ions which are very low because of the high sta-
bility constants of EDTA chelates, though in the
case of iron more free ions may be made available
by the partial disintegration of EDTA in light and
b) on the ability of the organisms to compete for
the metals in the EDTA chelates . This transfer is
uphill since EDDHA and EDTA accumulate in the
medium. The data in fact show that the algae be-
have as if they depend mostly on free ions present
in the medium because any conditions, like varia-
tions in pH, over- and under-chelation, which up-
set the ratio metal chelates: free metal ions favor-
able for an organism and a given pH, result in tox-
icities or deficiencies which inhibit or suppress
growth .

Histidine and other chelating small molecules
are readily absorbed. Since in this case the free
chelator, the metal chelates, and the free metals

presumably all can be absorbed, the effect of over-
and under-chelation and pH changes should, and
do, affect far less the availability of the trace
metals . The transfer and the competition for trace
metals by the different biological internal chela-
tors now can proceed freely in the interior of the
cells. Furthermore the absorbable chelators when
employed in large quantities as pH buffers have
the power to smooth out, perhaps by mass action,
the inflexibilities caused by the presence in the
media of weak , slightly over-chelated EDTA-trace
metal mixtures .

This way to supply metals by employing pen-
etrable chelators parallels what may happen fre-
quently in nature. Lichens and other plants grow-
ing on rocks must be able to secrete organic com-
pounds which dissolve and perhaps chelate the
mineral elements. Various fungi and bacteria pro-
duce and release in their media extremely strong
chelating substances which are specific for iron
such as: coprogen, produced by bacteria, actino-
mycetes and fungi, (Hesseltine et al . , 195 3),
"terregens factor", produced by Arthrobacter
pascens (Lochhead and Burton, 195 3) and ferri-
chrome, produced by Ustilago sphaerogena (Nei-
lands, 1952). Ferrichrome, amazingly, has a sta-
bility constant ten times higher than EDTA (Nei-
lands, 1957) for ferric iron yet is an effective way
to supply iron to tomato plants grown hydroponi-
cally . Arthrobacter terregens and other soil bac-
teria (Burton, 1957), Microbacterlum sp . (Demain
and Hendlin, 1959), and the fungus Pilobolus
kleinii (Hesseltine et al . , 1953) have a growth
factor requirement which is satisfied equally well
by terregens factor, coprogen and ferrichrome.
These substances, though apparently different
chemically, provide an extremely effective way of
supplying iron. Because of their special biological
activities at very low concentrations (ug./ml.)
they are considered by Demain and Hendlin (1959)
as "iron-transport factors" . Nielands (1957), in
a thoughtful review, postulated that they may act
as coenzymes for the intracellular transfer of iron.
Only a few of the great variety of molecules
tried, many of which are known chelators, can re-
place them. Outstanding are the compounds formed
upon heating sugars with amino acids , the ketose-
amino acids. Glucosyl-glycine is active for
Microbacterlum sp . , fructose-phenylalanine and
the products derived from autoclaving glucose and
glutamic acid are active for Micrococcus lysodeik -
ticus ■ Other bacteria , like Lacto-bacillus gayoni
and Proprionibacterium freundenreichii require glu-
cosyl-glycine , suggesting that they may also need
"iron transport factors". It is interesting to note
that the fructose- amino acids stimulate haem syn-
thesis and amino acid incorporation into globin.
(Kruh and Borsook, 1955) . Other compounds like
citric acid and 8-hydroxyquinoline are active for
M. Ivsodeikticus and aspergillic acid for Micro - -^

93

/

*

bacterium sp . , while EDTA is unable to satisfy the
requirement for both organisms . So far the active
substances are all strong chelators able to pene-
trate through the cell membrane . But many other
penetrable metal chelators are inactive indicating
that penetrability and chelating properties are not
the only prerequisites for activity . A strong speci-
ficity for binding iron and other properties seem es-
sential . For instance, the effectiveness of the
transport function may require an easy release of
iron to the apoproteins of the iron containing en-
zymes and this may be achieved in a number of
ways. The activity of the ketose- amino acids as
iron transport substances may be related to their
role in stimulating, and perhaps participating in,
haem production and amino acid incorporation in
proteins . These new developments suggest means
of making better, more flexible media for algae.
Hutner et al^. (1951) had briefly mentioned
that it is preferable , for the sake of obtaining heavy
growth, to have the complexing agent serve as an
auxiliary substrate. There is no evidence that his-
tidine nor glycylglycine are utilized to any extent
as substrates by Synura , Woloszynskia ,and Volvox
because these organisms are unable to utilize exo-
genous carbon sources under our experimental con-
ditions . Perhaps this is an advantage. If the chel-
ator employed is utilized as the sole substrate or is
a needed building block, metal toxicities may re-
sult because the chelator may be utilized more

rapidly than the metals. This obstacle may how-
ever be circumvented by offering several substrates
or other building blocks along the same pathway of
synthesis, so as to balance the rate of uptake of
the metabolizable chelator. Another procedure
would be to employ several chelators; some marine
algae seem to prefer media chelated jointly by
EDTA and nitrilotriacetic acid (Provasoli et al^. ,
1957).

Though far more experiments are needed to
find more versatile fresh-water media, the use of
penetrating pH buffers endowed with chelating
properties like histidine and glycylglycine seem to
offer great advantages for the most important pH
range (pH 6.0-8.5). Tracer studies are required
again to tell the extent to which these buffer-
chelators penetrate .

The supply of trace metals in the acid range
offers a different set of problems . At pH below 5 the
heavy metals are quite soluble. A chelator is not
needed to prevent precipitates , but may still be very
useful as a metal buffer to prevent toxicities and to
stabilize the metal pool. EDTA and other chelators
having acidic coordinating groups offer little prom-
ise: their chelating power decreases with increas-
ing acidity because the hydrogen ions compete
more and more favorably with the heavy metal for
the coordinating group. Very little has been done
with highly soluble, penetrating but non-toxic
chelators having a predominance of basic groups.

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