REGION
*iwest Water Labor
OF WASHINGTON
ington
I
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OOOR66103
ENVIRONMENTAL REQUIREMENTS
OF BLUE-GREEN ALGAE
Proceedings of a Symposium
jointly sponsored by
University of Washington
and
Federal Water Pollution Control Administration
Pacific Northwest Water Laboratory
September 23-24, 1966
U. S. DEPARTMENT OF THE INTERIOR
Federal Water Pollution Control Administration
Northwest Region
Pacific Northwest Water Laboratory
Corvallis, Oregon
October 1967
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FOREWORD
The problem of accelerated eutrophication is of growing public
concern. It is scrutinized increasingly not only by the scientist but
by the public and its representatives in Government. There are many
facets to the problem, and some are being examined through research to
find the keys for solution. One of the most challenging areas is the
need to understand better the environmental requirements of blue-green
algae. Without this understanding it is doubtful the tools of ecolog-
ical control now available can give relief from the ravages of such
algal blooms. This subject area, therefore, cries out for research
attention. It is for this reason that this symposium was arranged so
that the knowledge now available can be evaluated, the gaps identified,
and the researcher stimulated to pursue his inquiry in the light of
this knowledge.
A. F. BARTSCH, Chief
National Eutrophication
Research Program
111
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CONTENTS
Why Study Blue-Green Algae - W. T. Edmondson
Problems in the Laboratory Culture of Planktonic
Blue-Green Algae - W. R. Eberly
Aspects of the Nitrogen Nutrition of Some
Naturally Occurring Populations of Blue-
Green Algae - V. A. (Dugdale) Billaud 35
Environmental Requirements of Thermophilic
Blue-Green Algae - R. W. Castenholz 55
Growth Requirements of Blue-Green Algae
as Deduced From Their Natural Distribution -
W. C. Vinyard 81
Recent Advances in the Physiology of
Blue-Green Algae - 0. Holm-Hansen 87
Discussion - G. P. Fitzgerald 97
Summary of Floor Discussions - J. C. Tash 103
General Discussion of Nutrient Measurement
and Nuisance Control - A. M. Dollar,
M. Parker, and W. A. Dawson 107
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WHY STUDY BLUE-GREEN ALGAE?
W. T. Edmondson
Department of Zoology
University of Washington
Seattle, Washington
The purpose of this introductory note is to give a brief review of
some of the problems associated with blue-green algae that make it
possible and useful to organize a symposium such as this.
One of the problems of current public interest is that many of the
species of algae that cause the most difficult nuisance problems in
lakes are blue-green algae. This is not to say that most species of
blue-green algae are nuisances; many are inconspicuous and one never
hears about them except in specialized literature. Nor does it mean
that other kinds of algae do not cause problems; diatoms and Synura
are well known to water filtering plant operators, but I do mean that
any of a certain group of species can become very prominent in lakes,
given the right environmental conditions, and there create the familiar
nuisance conditions of scum and odor so well known in many parts of the
world.
This feature is especially impressive to people who have seen an
unproductive lake with an inconspicuous blue-green alga component in
the phytoplankton turn into a productive lake with a relatively dense
blue-green population. Lake Washington in 1950 was in relatively good
condition in regard to algal population. It had at the maximum
1.5xlC)6 (j.3/ml of total phytoplankton volume of which 15% consisted of
species of blue-green algae. In 1962 the corresponding figures were
10.5 and 95%. By this time the lake had given rise to such public con-
cern that construction of an elaborate and expensive sewage diversion
program was underway (Edmondson, 1968; Clark, 1967).
But even beyond the special feature of creating public nuisance
conditions, the blue-green algae would attract investigation by scien-
tists because of their special properties. Anyone interested in work-
ing out the mechanisms of control of productivity and population density
in aquatic communities would have to give special attention to these
algae because they have special nutritional and other properties. Also,
the blue-greens are conspicuous in a number of special or extreme kinds
of habitats, such as hot springs. They are very important in forming
and stabilizing soil. And, taking a broader view beyond the scope of
this symposium, the specialized morphology and life history provide
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
much additional motivation for study. Thus, there are scientific
reasons as well as practical ones for working on these organisms and
for holding a review symposium about progress.
Which special features of the blue-greens are involved? One is
that the relation to gravity of some planktonic species is upside
down. In calm weather, most of the nonflagellated algae settle toward
the bottom of the lake as the diatoms do. In contrast, many of the
blue-green algae float under these conditions and accumulate as dense
scums on the surface which can be thrown into thick windrows and piled
up on the shore. It seems probable that much of the material produced
by the blue-green algae decomposes in the epilimnion rather than being
deposited into the hypolimnion. This can have very interesting reper-
cussions in the way the consumers in the different parts of the lake
are supported.
We are concerned here with environmental conditions. Nutritional
conditions are of considerable importance in the control of algal pro-
ductivity and will be taken up first, but there is much more to the
environment than the dissolved nutrients, and to interpret the effect
of the supply of nutrients in nature, one must pay considerable atten-
tion to other processes. A real evaluation of the control of phyto-
plankton populations must recognize the quantitative importance of
grazing, parasitism, antibiosis, and the difference in the light and
temperature climate at different depths in the lake.
The blue-green algae for a long time have had a reputation for
needing an organic source of nitrogen or at least for doing best
under these conditons. This reputation apparently developed because
the lakes in which the most prominent populations of blue-green algae
occurred were lakes that had the highest concentration of organically
combined nitrogen (e.g. Pearsall, 1934). On the other hand, it has
subsequently been shown that the major nuisance species can be grown
in pure mineral media without organic additives (e.g. Gerloff, Fitz-
gerald, and Skoog, 1952).
Is there an alternative explanation to this apparent paradox?
A possible hypothesis is that while organic molecules are not obliga-
tory, some organic compounds might be acting as a nutrient-sparing
factor. Another reasonable hypothesis can be established on the
basis that the blue-green algae on a relative basis need higher con-
centrations of inorganic nutrients for a given amount of growth than
their normal competitors, such as diatoms and green algae. Now it
happens that the lakes in Pearsall's group that had the highest con-
centrations of nutrients were the lakes that were enriched with sew-
age. Naturally, these lakes also had the highest concentrations of
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Why Study Blue-Green Algae?
organically combined nitrogen. This coincidence was interpreted as
a causal sequence by Pearsall.
It is still a good question which, as far as I know, has not yet
been fully investigated, to what extent blue-green algae may be
affected by organic substances even though they do not require them.
The lesson to be learned from Rodhe's phosphate sparing factor should
not be forgotten. Rather, this phenomenon should be actively looked
for with other kinds of algae (Rodhe, 1948).
If it were true that the bloom species of blue-green algae need
higher concentrations of nutrients to support a given rate of growth
than do diatoms, one must ask why. It seems reasonable that this
should be so because the bloom species form colonies. The cells in
the middle of a colony of Microcystis are separated from the medium in
which nutrients are dissolved. To support these cells with a given rate
of nutrient absorption and growth equivalent to the same cells free in
the medium, nutrients would have to be supplied to the colony at a
higher concentration. The same argument could be applied to filamen-
tous forms like Oscillatoria in which significant fraction of a cell
surface is out of contact with the medium. If the colonial blue-green
algae are put at a disadvantage by the colonial habit so widespread in
these forms, one wonders how coloniality could have evolved. A possible
answer in this seems to be that the colonial species may be less readily
eaten by some zooplankton, particularly Daphnia. Diaphanosoma, and
Diaptomus. Experimental data support this idea (Sorokin, et al.,
1965). Thus, the colonial habit represents an antipredator device of
some effectiveness. Of course, it must be emphasized that it is a
relative protection. When a Diaphanosoma is seen to wad up a bunch of
Oscillatoria and discard it, it seems obvious that the Oscillatoria
has not been eaten. Nevertheless, small fragments may be broken off
in this process and contribute to the nutrition of the animal.
To make a complete accounting we must consider in some detail
what the predatory loss to the zooplankton is, and generalizations at
the moment do not seem obvious. For example, Hrbacek has found fish-
ponds near Prague which support a dense population of Aphanizomenon
and also Daphnia in good conditions as judged by appearance and presence
of eggs (personal communication). It is not at all clear what is going
on but it has been supposed that a large proportion of the material
photosynthesized by blue-green algae is leached out into the environ-
ment and supports a production of bacteria, which in turn supports
Daphnia. One can also suggest that the feeding mechanism and behavior
of the various species of Daphnia need comparative examination.
One may question the proposition that blue-green algae need high
concentrations of nutrients because large populations are found with
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
low concentrations of nutrients. For instance, in 1962 a dense popula-
tion, dominated by Oscillatoria agardhii, was maintained at a high con-
centration in Lake Washington for several months with concentrations of
P.P04 less than 10 jj.gm/1 and N.N03 less than 50. But during the period
of rapid growth in April and May, the concentrations were more than 30
and 300, respectively.
Thus, the proper evaluation of field data is critical here. It
seems incorrect to describe as optimal the nutrient conditions at the
time a population is not able to grow or even about to crash.
The matter is probably not quite so simple as implied in the fore-
going discussion which put emphasis on the response of growth to nutri-
ent concentration. Provasoli (1958) has suggested that the proportions
of the major cations may be important in determining which particular
species shall predominate in enriched lakes. Lakes which receive con-
siderable sewage are relatively enriched in sodium and chloride. Since
some species of Myxophyceae have a requirement for sodium or potassium,
there is opportunity for a selection of species to be made on a basis
other than competition for nitrate, phosphate, or other exhaustible
nutrients. Lakes enriched primarily with agricultural fertilizers
should have ionic ratios somewhat different from those enriched primar-
ily with sewage, and a close examination of the situation in such lakes
should be instructive.
At present it seems not possible to clarify this question with
existing data. We need to have more specific quantitative information
about how the rate of cell growth and division varies with nutrient
concentration. A considerable body of experimental literature has
accumulated that shows the maximum amount of algal material develops in
media of different nutrient concentration (e.g. Gerloff and Skoog,
1957), but few reports show how the growth rate varied during the period
of growth during which the accumulation took place.
The effect of selection or differential filtration by planktonic
and benthic animals needs further study to establish or disprove the
mechanism hypothesized here. While it has been shown qualitatively in
a general way that the larger colonial species of blue-green algae are
not consumed as readily as the unicellular greens, we seem not to be
in a position to make meaningful calculations of rates of production
and consumption of the different algal types. While this degree of
detail may not be required to settle this problem, it would be desirable
to have that much knowledge.
What other mechanisms are there of population control other than
nutrient limitation and grazing? There has been a great deal of dis-
cussion of antibiotic substances liberated by algae. While literature
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Why Study Blue-Green Algae?
is to some extent ambiguous and contradictory, it appears from many
works that algal antibiosis is a real phenomenon in nature (e.g.
Vance, 1965). Again, we have a qualitative evaluation and need to
know much more quantitatively about such matters as the degree to
which cell division is slowed by under specified conditions. Para-
sitism is another process for which quantitative evaluation is desir-
able.
To conclude I should like to mention the Oscillatoria rubescens
problem.
It has been said that in 1955 Lake Washington was obviously on
its way to deterioration. What was really meant was that suddenly a
large population of Oscillatoria rubescens appeared. This species has
frequently shown up early in the deterioration of lakes brought about
by sewage enrichment. Of course, it is not restricted in its occurrence
to sewage-enriched lakes, but there is something about these conditions
that makes it do well and form dense populations. This species has been
present in Lake Washington since 1955, but not uniformly. It has come
and gone, being succeeded or accompanied at times by other species,
including Oscillatoria agardhii.
It seems a worthwhile task to define the conditions in which
Oscillatoria rubescens does well. Staub (1961) published a very thick
paper on the problem which he described as a first small step forward.
And, in fact, it is difficult to state on the basis of this why
Oscillatoria rubescens occurs where and when it does.
So there are still bits and pieces that need to be fitted together.
I hope no one will take offense at the description of the very large
literature and hard work that has been done on these organisms as bits
and pieces. All this means is that there are some very interesting
ecological problems, centered on environmental requirements, that
still need to be investigated.
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REFERENCES
Clark, E. 1967. "How Seattle is beating water pollution." Harper's
Magazine. June. 234:91-95.
Edmondson, W. T. 1966. "Changes in the oxygen deficit of Lake Wash-
ington." Proc. Internat. Soc. theor. and appl. Limnol. 16:153-158.
Edmondson, W. T. 1968. "Water quality management and lake eutrophica-
tion: The Lake Washington case." In: Water Resources Management
and Public Policy. Univ. of Washington Press, Seattle (in press).
Gerloff, G. C., G. P. Fitzgerald and F. Skoog. 1952. "The mineral
nutrition of Microcystis aeruginosa." Amer. Jour. Bot. 39:26-31.
Gerloff, G. C. and F. Skoog. 1957. "Nitrogen as a limiting factor for
the growth of Microcystis aeruginosa." Ecology 38:556-561.
Pearsall, W. H. 1932. "Phytoplankton in the English Lakes. II. The
composition of the phytoplankton in relation to dissolved sub-
stances." Jour. Ecol. 20:241-262.
Provasoli, L. 1958. "Nutrition and ecology of protozoa and algae."
Ann. Rev. Microbiol. 12:279-308.
Rodhe, W. 1958. "Environmental requirements of freshwater plankton
algae." Sympolae Botanicae Upsaliensis 10:1-149.
Sorokin, U. E., A. V. Monakov, E. D. Mordukai-Boltovskaya, E. A.
Tsikon-Lukanina, and R. A. Rodova. 1965. "Experiments with
radiocarbon for the study of the trophic role of blue-green
algae." In Russian. Ecol. and physiol. of bluegreen algae.
235-240. Publ. Freshwater Biol. Inst. Acad. Sci. USSR, Moscow.
Staub, R. 1961. "Ernahrungsphysiologisch-autokologische Untersuchungen
an der planktischen Blaualge Oscillatoria rubescens D.C." Schweitz.
Hydriobiol. Zeits. 23:82-198.
Vance, B. D. 1965. "Composition and succession of cyanophycean water
blooms." J. Phycol. 1:81-86.
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PROBLEMS IN THE LABORATORY CULTURE
OF PLANKTONIC BLUE-GREEN ALGAE
William R. Eberly
Manchester College
North Manchester, Indiana
Both major orders of blue-green algae, the Chroococcales and the
Hormogonales, are represented in the common plankton communities of the
fresh water. Furthermore, forms may be either occasional or rare
members of the community, or they may be dominant, i.e., comprising the
vast majority of a given sampling, often reaching bloom proportions.
Still another approach to plankton algae divides species into epilim-
netic forms (warm temperature, bright light) and hypolimnetic or meta-
limnetic forms (cool temperature, dim light). Generally speaking,
epilimnetic blooms may be dominated by either members of the Chroococ-
cales (Microcystis) or by some filamentous forms (Anabaena or Aphani-
zomenon), while deep-water blooms are nearly always composed almost
exclusively of one of several species of Oscillatoria.
With one exception, the writer has been concentrating on the fila-
mentous planktonic forms, with special reference to the Oscillatorias
associated with deep-water oxygen maxima. In the United States, meta-
limnetic oxygen maxima may be produced by several types of phytoplankton
communities, one of which is designated the "Oscillatoria type" (Eberly,
1964b). In two lakes in Indiana which have been extensively investigated,
the alga has been identified as Oscillatoria agardhii (Eberly, 1959,
1964a). This alga produces intense mid-summer blooms at depths where the
temperature is 10-12°C and the light intensity usually less than 1% of
the surface intensity. Under these conditions, and with a stability
imposed by the thermal gradient and various morphometric characteristics,
oxygen saturations exceeding 30 ppm have developed on occasion.
High oxygen concentrations in lakes of central Europe are associated
with Oscillatoria rubescens (Eberly, 1964b). Most of these lakes also
reflect an increasing rate of eutrophication due to nutrient additives
present in treated sewage effluent. In fact, 0. rubescens is often
taken as an indicator of pollution or eutrophication. In LaKe Washington,
Q. rubescens has succeeded Q. agardhii as eutrophication has advanced
over the years (Edmondson, 1961; Anderson, 1961). Preliminary evidence
seems to suggest that a similar transition is taking place in McLish
Lake (Indiana), though this has not been determined with any certainty
yet. This, then, provides a second reason for studying these and
related species.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Still a third reason for studying this group of organisms is
related to their taxonomy. There are a number of planktonic Oscilla-
torias with non-constricted cell walls and prominent pseudovacuoles
which are very nearly indistinguishable. Preserved specimens differ
chiefly in diameter of the cells. Living cultures show delicate dif-
ferences in shading, ranging from olive green to deep red, with all
shades in between. Various species grown in different media and under
different light and temperature conditions will exhibit different
colors and shades. Hopefully, some progress might be forthcoming in
the area of the systematics of this very similar cluster of species.
Obviously, the objectives or purposes of an investigation affect
the design and conduct of the experiment. Various studies of blue-
green algae purport to study processes or functions of the organism
(photosynthesis, mineral nutrition, etc.), while other studies intend
to relate the organism to the environmental conditions under which it
lives in nature. Most experiments will contribute some degree of
understanding to both aspects of the problem, and, indeed, a fine line
between the two types of experiments is difficult to draw. Neverthe-
less, the difference does exist. My own work has been largely ecolog-
ically and taxonomically oriented. Consequently, the pure physiologist
or biochemist may find much to be desired in the data presented here.
It should indicate, however, areas where further work may prove to be
of value. Cultures of most of the species and strains described in
this paper may be obtained from the author.
Some of the work reported here was done while the writer was
participating as a visiting scientist at the Institute of Limnology of
the University of Uppsala in Sweden during the academic year 1963-64
(Eberly, 1965). Seven species were studied while at Uppsala, of
which three were described as new species or new varieties (Eberly,
1966). A number of these cultures have been maintained at the Insti-
tute of Limnology by Dr. Wilhelm Rodhe and his co-workers and were
forwarded to North Manchester after facilities were developed there.
Five cultures were sent the author by Dr. Olav Skulberg of the Norsk
Institutt for Vannforskning at Blindern, Norway. A word of thanks is
due both Dr. Rodhe and Dr. Skulberg and their respective institutions
for their assistance in this study. The writer is also deeply appre-
ciative of the continued and generous support of the National Science
Foundation. Thanks, also, to Misses Bonnie Kirby and Jane Henney,
who served as research assistants during much of this study, and to
Miss Cec Widup, who typed the manuscript.
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Laboratory Culture of Planktonic Blue-Green Algae
METHODS
All of the work reported here has been done with unialgal cultures,
none of which is bacteria-free. Single filaments were easily isolated
by plating several drops of culture on a 1.5% agar mixture of standard
medium. Most species of Oscillatoria soon crawled away from the site
of the original inoculum and could be picked up with a fine needle and
placed in test tubes. Single filaments of blue-green algae grow very
slowly in this kind of isolation. Many do not survive. This may be
related to a dependency upon some diffusable metabolic factor, as Fogg
(1965) suggests in connection with the development of the lag phase of
the growth curve, or it may be a purely physical factor related to the
buoyancy of the filament, which causes it to creep up the water film
along the side of the test tube and, subsequently, to dry out with the
evaporation of the medium. Needless to say, regardless of the number
of isolates prepared, successful growth of only one is necessary to
provide the desired culture. Experimental cultures were maintained in
125 ml Erlenmeyer flasks stoppered with a plug of medical quality
cotton. Inoculations were made into about 50 ml culture medium. The
flasks were shaken continuously at about 100 cycles per minute. Illumi-
nation was provided by 40-watt Sylvania F-40 natural tubes, operating
on a 12-hour, on-off cycle. Intensity at the level of the flasks was
about 70-85 ft.-c. Temperature in the two growth chambers was main-
tained at 15° and 20°C. Growth of the cultures was measured turbido-
metrically, using a Fisher Model 7-101 electrophotometer with a color
filter with a peak transmission at 650 mjj,. The optical density of the
measurements is multiplied by 100 to eliminate the decimal. No
attempt was made to relate optical density to absolute quantity of
algae present, though this has been done by other authors,, For example,
Staub (1961) measured growth of cultures of Oscillatoria rubescens with
a filter of 610 mp. and prepared a graph giving the dry weight of algae
as mg/1 plotted against the extinction value. This gave a straight
line, so it was felt that a similar condition would exist with the
algae used in this investigation (though with likely different absolute
values).
Cultures were grown both in the ASM and ASM-1 media of Gorham et
al. (1964). Table 1 gives the composition of these media in micromoles
of ingredients. In almost every case, growth was faster and reached
higher population densities in ASM-1 than in ASM. Specific results
will be presented later in connection with the discussion of each
species. No further attempt was made to define a specific formula for
each culture used. No doubt, some differences might be discovered,
but the filamentous forms do show quite successful growth in the one
medium.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Cultures Currently Under Investigation
At the present time, 19 species and strains are under investiga-
tion in the writer's laboratory. Nine of these were isolated from the
8-meter-deep sample from McLish Lake (Indiana) obtained on August 3,
1965. These comprise two species, Oscillatoria agardhii and Oscillatoria
limnetica. Their culture numbers bear the prefix E (Eberly). Five
cultures are forms isolated from Swedish waters and bear the prefix UP
(Uppsala). A fuller description of these forms is found in Eberly (1966),
Five cultures were sent to the writer by Dr. Olav Skulberg from Norway
and are designated by the prefix Sk. Four of these were isolated from
Norwegian lakes, and the fifth is from the clonal culture of C).
rubescens isolated by Richard Staub from Lake Zurich in Switzerland
(Staub, 1961). The nomenclature is presented here in the traditional
sense. The systematics of these forms is under study and will be dis-
cussed elsewhere.
E7.
E8.
E10.
E12.
E18.
E19.
E16.
E20.
E40.
Upl.
UP2.
UP3.
UP4.
Up6.
SkA.
SkB.
SkC.
Oscillatoria agardhii.
Oscillatoria agardhii.
4.5-5.0 p. thick.
Oscillatoria agardhii.
4.5-4.7 )i thick.
.Oscillatoria agardhii.
thick.
Oscillatoria agardhii.
ca. 6.0 p. thick.
Oscillatoria agardhii.
ca. 6.0 p. thick.
Oscillatoria limnetica.
McLish Lake.
McLish Lake.
Brown.
Reddish brown.
McLish Lake. Gray brown.
McLish Lake. Brown, ca. 6.0 |j.-
McLish Lake. Olive brown.
McLish Lake. Olive brown.
McLish Lake.
McLish Lake.
McLish Lake.
Deep blue-green.
Green blue-green.
Bright(iblue-green.
Lake Hangstaorn, Sweden.
Baltic sea off Stockholm, Sweden.
Ojscillatoria limnetica.
Qscillatoria limnetica.
Oscillatoria agardhii isothrix.
Green blue-green.
Oscillatoria baltica.
Green blue-green.
gynecocystis parvula. Baltic waters near Stockholm.
Oscillatoria limnetica. Lake Norrviken, Sweden. Blue-
green.
Oscillatoria baltica. Lake Norrviken.
Oscillatoria agardhii. Lake Gjersjo, Norway. Green blue-
green.
^scillatoria agardhii isothrix. Lake Froilandsvatn,
Norway. Green blue-green.
Oscillatoria rubescens. Lake Zurich, Switzerland.
(Staub). Red brown.
10
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Laboratory Culture of Planktonic Blue-Green Algae
SkD. Oscillatoria rubescens. Lake Steins fjord, Norway. Light
red-brown.
SkE. Oscillatoria agardhii. Lake Akersvatn, Norway. Deep
blue-green.
EXPERIMENTAL RESULTS
Oscillatoria agardhii Com.
Most of the work on 0. agardhii was done with strain E10 from
McLish Lake in northern Indiana. In ASM medium, a rather characteristic
growth curve is exhibited. The stationary phase was generally reached
at an optical density of about 45 and lasted about 15 days before declin-
ing in the death phase (the terminology is that of Fogg 1965). The
so-called "lag phase" did not seem to be a static period, but, rather,
indicated a steady but small amount of growth from the beginning. The
duration of the lag phase varied rather directly with the concentration
of the culture at the beginning of the experimental period, i.e., the
higher the concentration of algae at the beginning, the shorter the lag
phase (Figure 1). With an initial optical density of 5 or greater, a
lag phase was scarcely apparent at all. No attempt was made to ascertain
the physiological state of cells of the inoculum, but, in general, the
stock cultures were maintained within the exponential phase of growth.
Brief work with the pH of the culture solutions indicated that,
regardless of the initial pH of the medium, within a matter of a few
days a rather uniform pH is attained. Adjustment of pH was accomplished
by adding HC1 or NaOH. Cultures started at pH 7 to 10, dropped to 6.6
to 7.4 by the second day, while cultures at pH 6 or below rose to about
6.5 to 6.8. No attempt was made to adjust the pH at daily intervals as
has been done by other workers. In cultures with initially varying pH
values, the exponential phase of growth was reached quickest in the
cultures with the higher pH, but the level at which the stationary phase
developed was generally similar in all cases and persisted for similar
periods of time (Figure 2). The curve for an initial pH of 9 is close
to that of pH 8 and 10, while the curve for pH 5 is similar to that of
pH 6. The pH in each culture at the time of the beginning of the expo-
nential phase was about 7.1 to 7.3. At the height of the exponential
period, the pH rose to 9 to 9.2 for each culture, with the highest
maximum pH values observed being in those cultures with initial pH values
both higher and lower than pH 7. During the stationary period, all pH
values declined to 7.9 to 8.1. As a standard procedure for subsequent
experimental work, the pH of the medium was adjusted to 7.5-8.0 before
inoculating with algae.
11
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
In a simple experiment to see if any unknown substance had any
effect on growth, a culture of 0. agardhii was augmented by the addi-
tion of 1% and 107o soil extract prepared from ordinary loam silt soil
according to the directions of McLachlan et al. (1963). The results
(Figure 3) indicate that the soil extract stimulated growth earlier
than the plain ASM and produced maximum growth at a somewhat higher
concentration, but the duration of the stationary phase was roughly
the same for all three cultures. A comparison of the three curves
indicates that the differences are quantitative rather than qualita-
tive. This suggests that there is no mysterious growth substance
involved, but simply an increased concentration of a vital inorganic
nutrient in the soil extract. It is, of course, impossible to stan-
dardize any kind of soil extract so that comparisons can be made with
different solutions.
It was noted early that somewhat more rapid growth was achieved
in ASM-1 medium, as well as greater concentrations during the station-
ary period nearly twice that of the ASM cultures (Figure 5). In ASM-1,
strain E10 leveled off at an average optical density of about 80. In
order to determine which ingredient of the ASM-1 medium was responsible
for the increased growth, replicate cultures of strain E10 were started
in ASM medium and cultured under conditions described earlier until
about the middle of the stationary phase. Then each nutrient that was
present in ASM-1 in a different concentration than in ASM was added to
separate cultures in the amount representing the actual difference
between the amount of that nutrient in 50 ml of ASM-1 medium and 50 ml
of ASM. These additions were made on the 18th day. Only the addition
of NaNOg produced increased growth which approximated the level achieved
with ASM-1 medium (Figure 4). It is clear from this that nitrate is
the limiting factor in the ASM medium under these growth conditions.
Whether 0. agardhii can obtain nitrogen from any other substances is
not known. This experiment may also cast some doubt on the ability of
this alga to utilize free nitrogen.
The growth curves representing growth after the addition of ali-
quot amounts of FeCl2, H3BC>3, and CaCl2 are almost identical with the
growth in untreated ASM medium. The addition of Na2HPO^ depressed
growth. This appears not to be due to the addition of sodium, since
additional sodium was added in the culture with the NaN03 supplement.
Perhaps the increased amount of phosphate could not be tolerated in
the presence of a depleted supply of nitrate. The most striking effect
came with the addition of the cobalt-copper-zinc salts (which were
added in a common solution). The algae were killed and began to lyse
very quickly. Since the three ions were added together, it is not
known whether this is a result of one or several. This provides an
12
-------
Laboratory Culture of Planktonic Blue-Green Algae
excellent illustration of the fact that the toxicity of many heavy
metals is determined in part by the concentration of other substances
present in the water.
The two Norwegian strains of 0. agardhii (SkA and SkE) also responded
best in the ASM-1 medium (see Figures 6 and 7). Unfortunately, the maxi-
mum growth exceeded the capacity of the instrument to measure it. It
should be remembered, that an O.D. of 100 represents a maximum absorbance
of 90%, so the actual maximum yield of any alga giving an O.D. over 100
could not be over 10 to 157» greater than the maximum yield of (). agardhii
(strain E10) in ASM-1.
Figures 5-13 present the results of an experiment in which nine
different cultures were grown in both ASM and ASM-1 media at both 20°
and 15°C. Initial densities were similar in all cases. The particular
pattern of the four growth curves for each culture gives something of a
systematic characterization of that form in comparison with the others.
Suffice it to say at this point that SkA and SkE appear very similar to
each other and that they both give a somewhat different picture than
strain E10. How this relates to the taxonomy of the species remains to
be seen.
The general effect of the lower temperature is merely to retard
the rate of growth. In an earlier experiment with strain E12, cultures
at 15°C reached nearly the same maximum density as cultures at 20°C,
but about two weeks later. In Figures 6 and 7, the retardation of the
colder cultures is apparent, but it is not possible to determine the
maximum yield of these cultures yet. It is clear that these two strains
grow as fast and produce higher yields in ASM-1 at 15°C as they do in
ASM at 20°C. The growth of strain E10 was more rapid in ASM-1 at 15°C
than in ASM at 20°C. This points up the fact that often the growth
response of an alga to a particular combination of light intensity and
temperature is determined in part by the ionic concentration of the
environment.
Oscillatoria agardhii var. isothrix Skuja
Of the two strains of this variety, only one (SkB) has been subject
to experimentation so far in our laboratory. Some work was done on
strain Upl at Uppsala and will be referred to later. The growth response
pattern of SkB (Figure 8) is similar to that of SkA and SkE. If isothrix
is a valid subspecies of 0. agardhii, one would not expect to find
drastic differences in physiological adaptation. The slight difference
in growth of SkB in ASM at 20°C and ASM-1 at 15°C may or may not be
significant. The extremely slow growth in ASM at 15°C might also be
13
-------
ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
pointed out as a minor deviation of this form. This particular growth
curve nearly parallels that of strain E10, though the two cultures
differ in other respects.
Oscillatoria baltica Eberly
One strain of this species (Up_2) was used in experimental work.
The growth response pattern (Figure 9) bears a striking resemblance
to that of strain E10. The maximum concentration was not so great as
in E10, and the stationary phase was very short preceding the rather
abrupt decline of the culture. There is a consistent difference
between growth in ASM-1 at 15°C and ASM at 20°C, with the former being
decidedly greater. The relatively minor effect of temperature in the
case of the two ASM cultures might also be pointed out.
Oscillatoria rubescens D.C.
The difference in the response pattern of the two cultures of ().
rubescens SkC and SkD may be noted in Figures 10 and 11. Though both
strains grew best in ASM-1, SkC reached a peak density of about 65 and
maintained a prolonged stationary phase. On the other hand, SkD
peaked at an optical density of about 86 and began to decline rather
quickly. Strain SkC produced a pair of growth curves in ASM similar
to those of ASM-1, showing rather simple quantitative differences pro-
duced by the differing temperature and ionic concentrations of the two
media. Strain SkD, however, did not grow at all in medium ASM. In the
light of the profuse growth in ASM-1 at 20°C, the results in ASM are
difficult to explain. It should be recalled that SkC is originally
from Lake Zurich in Switzerland, while SkD is from Norway. The iden-
tifications are by the original isolators of the cultures.
Because of its association with polluted and eutrophic waters, an
experiment was set up to determine the effect of the addition of
treated sewage effluent to cultures of 0. rubescens in ASM medium at
20°C. The results are shown in Figure 13. The sewage supplement pro-
duced a more rapid growth, but the maximum yield was lower than in plain
ASM medium. These results were hardly what had been expected and did
not illuminate the problem very much. The particular sample of sewage
effluent, obtained from the outlet of the disposal plant at Wabash,
Indiana, was not analyzed chemically.
14
-------
Laboratory Culture of Planktonic Blue-Green Algae
Oscillatoria limnetica Lemm.
The growth pattern of this form (E40) is shown in Figure 12. The
rate of growth of cultures at 15°C compared with the growth in the same
medium at 20°C was more nearly the same than in any of the other cultures.
The quantitative relationship of maximum growth indicates rather clearly
that the final yield (during the stationary phase) in ASM-1 is about
double that in ASM. This is perhaps true of some of the others but
shows most clearly in this experiment with 0. limnetica.
Synechocystis parvula Perfiljev
The origin and taxonomy of this culture were discussed earlier by
the writer (Eberly, 1966). The role of this organism in natural plank-
tonic communities is unknown. In culture it grows very rapidly in a
variety of media. In the present experiment the growth rate during the
early part of the exponential phase was identical in ASM and ASM-1, at
both temperatures, though the density of the culture during the stationary
period was considerably less in ASM (Figure 14). The final density in
ASM-1 considerably exceeded the 907° absorbency which the instrument was
capable of measuring. Though it may not contribute too much to our
knowledge of planktonic forms, a study of this organism may reveal a
number of interesting variations in blue-green algae physiology.
TAXONOMIC NOTES
Although a fuller discussion of the taxonomy of the forms included
in this study is under preparation, a few comments might be made here.
The role of biotic characteristics in systematics, as contrasted with or
superimposed on morphologic characters, is very uncertain at the present
time, with the notable exception of the bacteria. In the blue-green
algae, two biotic characteristics have contributed to the present state
of confusion in the taxonomy of the group. One is the way in which the
actual morphology of the organisms (size and dimension, cell shapes,
presence or absence of sheaths, gross form of colonies or mat formation,
etc.) vary with differences in environmental conditions. By growing
pure cultures of a single species, Schizothrix calcicola, in a great
variety of culture media and under a variety of environmental conditions,
Drouet (1963) was able to classify the final growth forms as 54 species
in nine different genera now included in the commonly used manuals.
Other workers are investigating the same phenomenon in various groups of
blue-green algae.
15
-------
ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
The second factor is the variability of physiological response
observed under different conditions. This may, in part, be merely the
physiological counterpart of the morphological variability noted above,
or it may actually represent genetically determined responses, i.e.,
differences valid at the species level. The latter kind of differ-
ences might be expected to show up when a series of cultures are grown
under identical conditions and in identical nutrient solutions. The
growth response graphs representing the growth of each culture in two
different media and at two different temperatures present this kind of
data (see Figures 5-11). Other biotic characters include color of
cultures (preferably during the exponential phase of growth) and cer-
tain other characters, such as length of filaments, degree of bending
of filaments, presence or absence of clumping, sheath, etc. Observa-
tions of this latter sort have been made, for example, on Aphanizomenon
(McLachlan et al., 1963) and Anabaena (Gorham et al., 1964). How much
(and even whether) such observations should enter the taxonomic picture
is a very current topic. Certainly such information otight not to be
disregarded. In the present study, color of cultures and growth
response patterns are considered along with typical morphological param-
eters.
Strains SkA and SkE identified as Oscillatoria agardhii from two
lakes in Norway are almost certainly identical. The color and general
morphology are very much alike and the growth patterns are very simi-
lar (Figures 6, 7). Strain SkB, 0. agardhii isothrix, also from
Norway, is similar enough to the previous two cultures (Figure 8) that
it very well could be the same species but with minor differences
(chiefly morphological) at the subspecies level. The color is a some-
what lighter green. The strain from Sweden (Upl) has almost the same
color as SkB, though the former has not yet been studied experimentally
in our laboratory at North Manchester. Oscillatoria baltica, Up2, has
the same green blue-green color as the above forms but has a different
growth response pattern (Figure 9). Its continuation as a discreet
species so far seems justified on the basis of its morphology and
physiological response.
On the other hand, the form from northern Indiana identified as
jjscillatoria agardhii (E10 and the others) does not seem at all like
the 0. agardhii from Norway. The color is brownish, ranging in the
various strains from reddish-brown to gray-brown. The growth response
graph of E10 (Figure 5) is more like that of (). rubescens (Figures 10
and 11), especially SkD. Assuming that SkC is the valid 0. rubescens,
some question could be raised about SkD. The filaments of SkD are
about 9-10 \i thick compared with 5-6 u in SkC. However, the fila-
ments of E10 are only 4.5-5 u thick.
16
-------
Laboratory Culture of Planktonic Blue-Green Algae
We are certainly dealing with two groups of algae: the green ones,
that might be called the agardhii group, and the brownish ones, the
rubescens group. How _0. prolifica fits into the picture is not known.
In (). rubescens the degree of reddish or brownish tint seems to be
greatly affected by the growth conditions, particularly nutrients
available. This is also true of E10. In the experiment on E10 shown
in Figure 4, the culture with the added nitrate became almost as red
as 0. rubescens, strain SkC. The confusion has been that when dried
or preserved specimens of these alga are examined they sometimes appear
alike, while in the living condition they are very much different. It
appears likely that what we have been calling 0. agardhii in our lakes
in Indiana is not that. Perhaps, also, what has been reported to be a
transition from "0. agardhii" to "0. rubescens" is not actually a change
from one species to another, but a transformation of a dull variety
(such as E10) to a bright red appearance of the same form, with chang-
ing nutrient conditions in the lake. These relationships remain so far
largely as speculations, but they form the basis of some experimental
work for the future.
SOME PROBLEMS REMAINING
Besides the problems in the area of taxonomy alluded to above, some
other problems remain yet unsolved. One of these relates to the diffi-
culty of maintaining the algae in a truly planktonic or suspended state.
At the present time, this can only be accomplished by artificial means,
such as shaking, rotation, or some other mechanical means of inducing
turbulence, or by bubbling air through the culture vessel. The degree
of turbulence thus required to maintain the alga in a suspended condi-
tion is certainly greater than the actual amount of turbulence existing
in a natural situation. In fact, one might say that usual culture
methods produce such drastic conditions that it is a wonder the algae
survive at all. A means of assessing the effect of this artificially
induced turbulence on the growth data thus obtained is needed. Fogg
(1965) has discussed this problem very briefly. He cites work that
indicates that "growth of Anabaena cylindrica was doubled when culture
flasks were shaken at 90 instead of 65 oscillations per minute and
entirely prevented at 140 oscillations per minute." Further, we ought
to try to maintain cultures in suspension without any artificially
induced turbulence in the culture.
A second problem concerns the role of bacteria in the nutrition
of blue-green algae. This, of course, is not a problem unique to
planktonic forms. Only a few workers have attempted axenic cultures of
blue-green algae which possess slime sheaths in which (or on which)
bacteria may feed. In some cases (again see Fogg, 1965) various algal
17
-------
ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
cultures (not necessarily blue-green) grew poorly or not at all in
axenic bacteria-free cultures. Whether any symbiotic dependency on
bacteria exists in planktonic blue-green algae is not known yet. A
related problem is the possible production of antibiotic substances
by the alga. These might be anti-bacterial or anti-algal. In nature,
the massive deep-water blooms of this type of Oscillatoria are remark-
ably homogeneous, containing very few forms other than the dominant
one. Whether this is a purely phyaical result of shading and tempera-
ture or the result of heteroantagonism (Lefevre, 1964) remains to be
seen.
Another problem concerns the duplication of the spectral quality
of light as it exists in nature. The attenuation of certain wave lengths
of light in water is well known. It is, however, difficult to deter-
mine the actual spectral distribution of subsurface light and equally
difficult to duplicate that quality in the laboratory. If chromatic
adaptation is a reality in blue-green algae (as the writer is inclined
to feel), then the actual color of an alga in nature is a response to
the spectral composition of the light. This color could obviously not
be attained exactly in laboratory culture unless the light quality was
the same as in nature. To what extent a different quality of light
gives deviant results in growth experiments is not known. It is fairly
easy to use bulbs of differing and known spectral emissions, but this
still does not quite present a true picture of growth under natural
conditions.
Related to this is the growth response of the alga to light inten-
sity and temperature. Rodhe (1948) was one of the first to point out
that the response of an alga to light intensity depends on the tempera-
ture and vice versa. That is, a particular light intensity may be sub-
optimal at a certain temperature, optimal at another, and supraoptimal
(induce light saturation) at still another temperature. In theory, it
is reasonably simple to expose replicate cultures to various combina-
tions of light and temperature, but in practice it is somewhat more
difficult. In addition to providing cross-gradients of light intensity
and temperature for a number of samples, some method of keeping the
algae suspended must be devised and the entire apparatus must be con-
structed with a particular method of measuring growth in mind.
Recent attempts to deal with this problem with algae other than
blue-greens include that of Maddux and Jones (1964) and Jitts et al.
(1964). While at the Institute of Limnology in Uppsala, this matter
was investigated briefly with some of the cultures of blue-greens
available at that time. Experiments were carried out in the photo-
thermostats in Dr. Rodhe's laboratory. Two units were used at tempera-
tures of 12° and 20°C. Growth was measured by the relative uptake
18
-------
Laboratory Culture of Planktonic Blue-Green Algae
of carbon-14, with the greatest amount of uptake in any one experiment
arbitrarily taken as 100%. Results are in cpm of a filtered aliquot of
the sample. Cultures were acclimatized in the photothermostat in the
dark for 12 hours before the light cycle came on. Light exposure was
six hours. Cultures were placed in 125 ml pyrex bottles which were
placed in a circular holder which rotated at right angles to the light
source in the top of the chamber. This meant that each bottle was
exposed to a varying light intensity at different points during the
rotation of the culture holder, but that during a complete rotation each
bottle received a similar amount of illumination. The actual amount of
illumination received (or the average intensity) was not measured, but
maximum intensity immediately under the lamps was perhaps close to 1,000
ft.-c. This is considerably higher than that used to cultivate blue-
greens under normal laboratory conditions. In order to obtain varying
levels of intensity, bottles were wrapped in one, two, or three thick-
nesses of copper screen which reduced the intensity to 52%, 26%, and 13%
of the unshielded bottles.
Even though this was a somewhat crude way of measuring this partic-
ular response pattern, the data obtained did indicate some remarkable
things. Data for four species are presented in Figure 15. At 20°C,
Oscillatoria agardhii isothrix (Upl) had not yet reached a level of
light saturation in the full unshielded bottles, while at 12°C some
degree of light saturation was reached at 26% of the full illumination.
Oscillatoria limnetica (Up4) likewise did not show any inhibition at
100% intensity but did show saturation at 12°C under 52% illumination.
Synechocystis parvula (Up3) is apparently a real stenothermal alga. At
20°C, optimum light intensity is reached at 26% illumination, while
growth at 12°C in the higher level of illumination exceeds that at 20°C.
A similar light saturation of NRC-48 (an unidentified algae provided by
Dr. Paul Gorham of the National Research Council at Ottawa) occurred at
20°C and 52% illumination, after which the amount of growth at full
illumination fell below that at 12°C. A graph for Aphanizomenon f los-
aquae was nearly identical with that of 0. limnetica. These data are
only preliminary, but they do indicate the kind of results that would
be worth obtaining under more precise conditions.
The work of Maddux and Jones (1964) demonstrated that the growth
response of a diatom Nitzschia indicated variable light and temperature
optima depending on the ionic concentration of phosphate and nitrate in
the medium. At high concentrations of these two nutrients, both the
light optimum and temperature optimum were higher than at a lower con-
centration of nutrients. If this is true of the planktonic blue-greens,
this might shed some light on the occurrence of various forms at certain
limited depths in lakes. Under low concentrations of nutrients (i.e.,
oligotrophic or mesotrophic waters) optimum growth conditions would be
19
-------
ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
reached at depths with reasonably low temperatures and limited light
penetration. With an increase of nutrients (eutrophication) light and
temperature optima might be raised, with the result that the maximum
concentration of algae (the bloom) would move closer to the surface.
This might also explain the downward movement of a bloom during the
summer with the gradual depletion of nutrients which lowers the light
and temperature optima. Such a seasonal downward movement of the
bloom of Oscillatoria has been observed in Myers and McLish Lakes.
The greatest difference between ASM and ASM-1 is concentration of
certain nutrients. Some of the results presented in Figures 5-12
might be interpreted in the light of the above principle. It would
not be possible to determine exactly the light and temperature optima
without experiments such as have been referred to before. It would
also seem that this phenomenon would be operative only within a lower
range of concentration of nutrients; that is to say, above a certain
level of concentration, increasing the nutrient level would not have
any further effect on light and temperature optima. This hypothesis
also needs to be tested. All in all, this area of investigation
promises to be a fruitful field of inquiry into the ecological physi-
ology of planktonic blue-green algae.
20
-------
Table 1
Composition, in micromoles, of ASM and ASM-1 media
SALT
ASM
ASM-1
NaN03 .
MgS04 .
MgCl2 .
GaCl2 .
K2HP04 .
Na2HP04
FeCl3 .
HoBOo .
—* J
MnCl2 .
ZnCl2 .
CoCl2 .
CuCl2 .
Na2EDTA
1000
200
200
100
100
2
10
7
0.8
0.02
0.0002
20
2000
200
200
200
100
100
4
40
7
3.2
0.08
0.0008
20
21
-------
0)
CO
co
o
(VI
CO
Q
i— 1 -r-l
•H
0) .fi
j3 -o
4J H
CO
C W
O CO
C CO
O -r^
•i-l M
4J o
CO 4-1
M CO
4J i-l
C 1-1
V -i-l
CJ 0
C co
0 O
o
M i
HH
1-1 O
CO
•1-1 (1)
4-1 >
•H )-i
C 3
•.-1 0
4-1
O
M
4-1 CU
O J3
d) 4-1
14-1
4-1 M-l
W O
tot)
(\r
099.
a -o
-------
o
(O
o
in
<
Q
O
iO
O
CM
W CO
OJ
a 3
•H i-l
(0 CS
M >
4-1
ra ffl
-^ a.
T3
bt
(0
60
K
O
en
o -H
•a
1s
o S
M W
O <
O)
S-l
099.
Q'O
-------
60
50
40
8
* 30
Q
6
20
+10
20 30
DAYS
40
50
Figure 3.
Enhancement of growth of Oscillatoria
agardhii (strain E10) in ASM medium
with the addition of soil extract.
-------
80
60
o
•n
-------
o
CN|
01 i-(
.-t 4-1
,n cd
co cd
3 -H
O T3
•H 01
S 6
> <-*
^> S
O CO
m <
CQ T3
01 C
> cd
O CO
<
0)
ro C
C -rH
o
o. a
CO g
0) O
4-J Q)
^ cd
o so
-------
00
60
0)
O
\
\
*
\ «
\
«k
\
\
*
\ .
\ '*
>N l
>. «
^>
O
CO
O
cv
0)
S-i
60
•H
0)
LJ
«:
CO
—
I
Q
cr
<
o
<
-^ o
-------
f \
\
\
\
1 \
\
X
K 4\
t N-
< x>
rvi U
CD
D
CL
\\
W--
0
o
o
00
o
(0
o
-------
o
CM
O
0
0
00
0
(O
" 0
o
CVJ
o
sr
UJ
<
O
H
QJ
T3
O
M-l
u~l
0)
3
bO
••-I
V
0)
CO
-------
50
40
30
o
W
Q
*
O
20
10
+10
10
Figure 13.
20 30
DAYS
40
50
Enhancement of growth of Osc11la toria
rubescens (SkC) in ASM medium with the
addition of 1% and 10% treated sewage
effluent.
-------
100
10 20 30 40
14. S. PARVULA UP 3
See Figure 5 for legend.
-------
DC
<
Q_
4-1 a
•H -r-l
CO
a bO
Cl
01
bO
0)
O
Ol
M
C 0)
6 E -o
0) 0) 0)
i— I 4-1 g
Cu
e -a a
o c w
o to <
3
60
•r-l
o
(O
-------
REFERENCES
Anderson, G. C. 1961. "Recent changes in the trophic nature of Lake
Washington - A review." In: Algae and Metropolitan Wastes.
Robert A. Taft Sanit. Eng. Center Tech. Report W61-3, pp. 27-33.
Drouet, Francis. 1963. "Ecophenes of Schizothrix calcicola (Oscilla-
toriaceae)." Proc. Acad. Natural Sciences of Philadelphia 115
(9):261-281.
Eberly, William R. 1959. "The metalimnetic oxygen maximum in Myers
Lake." Invest. Indiana Lakes and Streams 5(1):1-46.
Eberly, William R. 1964a. "Primary production in the metalimnion of
McLish Lake (Northern Indiana), an extreme plus-heterograde
lake." Verh. Internat. Verein. Limnol. 15:394-401.
Eberly, William R. 1964b. "Further studies on the metalimnetic oxygen
maximum with special reference to its occurrence throughout the
world." Invest. Indiana Lakes and Streams 6(3):103-139.
Eberly, William R. 1965. "Preliminary results in the laboratory
culture of planktonic blue-green algae." Proc. Indiana Acad.
Science 74:165-168.
Eberly, William R. 1966. "Notes on some new and rare Myxophyceae in
laboratory culture." Trans. Amer. Micros. Soc. 85(1):130-138.
Edmondson, W. T. 1961. "Changes in Lake Washington following an
increase in the nutrient income." Verh. Internat. Verein. Limnol.
14:167-175.
Fogg, G. E. 1965. "Algal Cultures and Phytoplankton Ecology." The
University of Wisconsin Press. 126 pp.
Gorham, P. R., J. McLachlan, U. T. Hammer, and W. K. Kim. 1964.
"Isolation and culture of toxic strains of Anabaena flos-aquae
(Lyngb. de Breb.)." Verh. Internat. Verein. Limnol. 15:796-804.
Jitts, H. R., C. D. McAllister, K. Stephens, and J. D. H. Strickland.
1964. "The cell division rates of some marine phytoplankters as
a function of light and temperature." J. Fish. Res. Bd. Canada
21(1):139-157.
Lefevre, Marcel. 1964. "Extracellular products of algae." In:
D. F. Jackson, ed. Algae and Man. Plenum Press.
33
-------
Maddux, W. S. and R. F. Jones. 1964. "Some interactions of temperature,
light intensity, and nutrient concentration during the continuous
culture of Nitzschia closterium and Tetraselmis sp." Limnol. and
Oceanog. 9(l):79-86.
McLachlan, J., U. T. Hammer, and P. R. Gorham. 1963. "Observations on
the growth and colony habits of ten strains of Aphanizomenon flos-
aquae." Phycologia 2(4):157-168.
Rodhe, Wilhelm. 1948. "Environmental requirements of fresh-water
plankton algae." Symbol. Bot. Upsalienses 10(1):1-149.
Staub, Robert. 1961. "ErnHhrungsphysiologisch-autb'kologische Unter-
suchungen an der planktischen Blaualgae Oscillatoria rubescens
DC." Schweiz. Zeit. f. Hydrol. 23(1):82-198.
34
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ASPECTS OF THE NITROGEN NUTRITION OF SOME
NATURALLY OCCURRING POPULATIONS OF BLUE-GREEN ALGAE
Vera A. (Dugdale) Billaud
Institute of Marine Science
University of Alaska
College, Alaska
INTRODUCTION
Although nitrogen is a key element required by living organisms
and its abundance in natural systems is vitally important in problems
of aquatic productivity, little is actually known about its role in
lacustrine systems. The study of the ecological role of nutrients is
still in its infancy, with a great need for the development and evalua-
tion of techniques. A perusal of Hutchinson's (1957) chapter on the
nitrogen cycle in lakes shows that, although a fair amount of informa-
tion exists concerning levels of nitrogen compounds and gross nitrogen
balance in lakes, there is much less on the actual assimilation of
nitrogen sources by algae; and that, of the information available, a
large part is derived from laboratory studies.
The cycle of a nutrient in nature is always a complex process,
with many organisms contributing to the transformation. The bacterial
population is especially important, since the "feed rate" of a nutrient
in lakes is at least in part the result of the heterotrophic breakdown
of organic material by these organisms. This in situ regeneration is
one of the aspects of the nitrogen cycle which has been largely ignored,
especially in considering the nutrient sources available to the photo-
synthetic population in the euphotic zones of lakes. Nutrient cycling
in its most direct pathways of uptake, release, and regeneration has
thus represented a "black box" situation and yet Riley (1951) has sug-
gested that regeneration in the upper 200 meters accounts for 90% of
the organic matter produced annually in the oceans. Grill and Richards
(1964) have measured the release of nitrogen, phosphorus, and silica
from a diatom population in a darkened polyethylene drum. Although the
release of ammonia was observed, there appeared to be no nitrification
judging by the nitrate accumulation.
This work was supported in part by National Institutes of Health Grant
WP-0042-03 and WP-0-42-04 and is contribution No. 25 from the Institute
of Marine Science. I am grateful to Dr. K. V. Natarajan for his able
assistance in the identification of the Anabaena.
35
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Although this discussion has had no direct relationship to blue-
green algae, it has been included to show the problems which must be
faced when considering the nutrition of naturally-occurring populations.
For this reason, we have chosen a direct approach and have attempted to
measure the actual utilization rates of nitrogen compounds in the water;
simultaneously, we have tried to assess the magnitude and importance of
in situ regeneration of these compounds. Blue-green algae play a very
important role in the nitrogen cycle of lakes, and the behavior of these
parameters during blue-green algal blooms has been interesting. My
discussion will center around such populations; but more specifically,
I will emphasize nitrogen-fixing blooms of planktonic, blue-green algae.
NITROGEN FIXATION BY BLUE-GREEN ALGAL BLOOMS
Fogg and Stewart (1965) have recently reviewed the current status
of ecological information on the role of nitrogen fixation by blue-green
algae. The role of planktonic blue-green algae in the fresh water nitro-
gen cycle has attracted interest especially because of the unique ability
among algae of members of this group to fix atmospheric nitrogen. This
is believed to account for the observation that although Myxophyceae are
known to have a high nitrogen content on a unit weight basis, they are
often found in waters containing little combined nitrogen. Certainly,
a major role for Anabaena has been well documented. Hutchinson (1957)
attributed the addition of 490 mg rrr^ of nitrogen to a bloom of Anabaena
in Linsley Pond, and the work of Prowse and Tailing (1958) on a reser-
voir in the White Nile has indicated that there Anabaena is important in
determining the seasonal succession of algae. A bloom of Anabaena dur-
ing the low nitrogen period following impoundment was followed by a popu-
lation of algae which were apparently using nitrate regenerated from the
nitrogen fixed by the initial population of blue-green algae. This not
only confirms the possible importance of nitrogen-fixing blooms of algae,
but suggests that nitrogen assimilated into an algal population can
become available rather quickly to a subsequent population, presumably
as a result of the release of ammonia upon the death of the cells. The
implications for the control of population succession are obvious.
Although the evidence for nitrogen fixation was not direct in any of
these cases, the indirect evidence supports the conclusion that major
nitrogen fixation occurred in connection with these populations. In
each case, large numbers of organisms with high nitrogen content grew
without any apparent adequate nitrogen source; and in each instance,
the population consisted predominantly of members of a genus known to
fix nitrogen in pure culture. Indeed, many of the constituent species
have been critically tested—for instance, Anabaena flos-aquae (Gorham,
et al., 1964) and Anabaena cylindrica (Fogg, 1942). Both these species
are frequent components of planktonic freshwater blooms. There are
other examples in which nitrogen fixation is strongly suggested for
36
-------
Aspects of Nitrogen Nutrition of Some Blue-Green Algae
algal blooms, and it is sufficient to say here that the circumstantial
evidence has been convincing.
We have made measurements of nitrogen fixation rates using -N in
Sanctuary Lake, a temperate reservoir, and found high rates of fixa-
tion, having fortuitously selected a lake in which nitrogen fixation
is an important feature during much of the summer. A positive corre-
lation between fixation rates and light was found, such that after a
period of darkness the fixation rates became very low. Clearly the
algae were involved directly or indirectly. The population in this lake
was diverse, but dominated by Anabaena sp. during the periods of high
nitrogen fixation. Goering and Neess (1964) found light-dependent fixa-
tion at fairly high rates in one of the Madison, Wisconsin, lakes. The
work of Stewart (1962, 1964) has provided information on nitrogen fixa-
tion, both in the laboratory and in situ, by marine blue-green algae
from the water and from a sand dune slack; and Goering, Dugdale, and
Menzel (1966) have measured significant nitrogen fixation in connection
with populations of Trichodesmium in the open ocean. The close inter-
relationship between nitrogen fixation and photosynthesis (Fogg and
Than-Tun, 1960) results in a readily available source of energy for
nitrogen fixation by blooms in the euphotic zone of a lake. This fact,
coupled with the potential independence of such populations from the
nitrogen nutrient levels in the water, may allow nuisance blooms to
develop under conditions unsuitable for non-nitrogen-fixing algae. Con-
sequently, the environmental requirements for nitrogen-fixing blooms of
algae pose an important problem. Many bloom- forming blue-greens are
not among the nitrogen-fixers and, of course, these also play an impor-
tant role in the phytoplankton communities of lakes.
METHODS
Although well-established limnological techniques were used to
obtain most of the information reported here, some of the experimental
techniques need a brief explanation.
The N methods have been described in the literature (Neess et al.,
1962; Dugdale and Dugdale, 1965). In the method for nitrogen fixation,
dissolved nitrogen is removed from the sample by sparging at reduced
pressure with a helium-oxygen or helium-oxygen-carbon dioxide mixture,
and is then replaced by nitrogen containing a known enrichment of 1%2-
For uptake measurements of nitrate and ammonia salts, ^N enriched com-
pounds are simply added to the water sample enclosed in a bottle. Incu-
bation may either be carried out in situ or under standard conditions,
and afterwards the •'•^N enrichment of the particulate fraction, which
contains largely algae, is determined.
37
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
The isotope dilution method for measuring the rate of supply of
ammonia into the dissolved ammonia fraction of the water uses -"N ammonia,
which is added to a sample of lake water. The system is equilibrated and
immediately an aliquot is filtered through a glass filter; the isotope
ratio of the ammonia fraction in the filtrate is determined following
vacuum distillation and conversion to N£ using alkaline hypobromite. This
initial aliquot gives a value for the ammonia concentration in the water
and also a zero time value for the isotope ratio. Subsequently, aliquots
filtered at regular intervals and treated similarly give a rate of dilu-
tion of the added labeled ammonia by unlabeled ammonia from another nitro-
gen fraction in the water.
Quantitative estimates of the algal population have not been made,
although Lugol's acetic acid preserved samples are available for each
experimental day. Usually, chlorophyll a or total chlorophyll estimates,
along with determinations of the particulate nitrogen content of the
water, have been used as an estimate of algal population, with only quali-
tative information on the composition of the populations. Particulate
nitrogen is a particularly useful parameter to measure for this type of
study, and can be determined with high precision using a Coleman Nitrogen
Analyzer on material collected on a glass filter.
NITROGEN METABOLISM DURING THE ANNUAL SMITH LAKE ANABAENA BLOOM
We have studied Smith Lake, a small lake in the Tanana River Valley
of Central Alaska, near Fairbanks. Four years of work are now completed
on this lake. The major annual bloom in this lake is dominated by
Anabaena flos-aquae, and it typically lasts only two weeks.
The conditions preceding the bloom are highly reproducible from
year to year, probably because of the climatic conditions peculiar to
the subarctic. The most remarkable feature, apart from the long severe
winter, is the absence of a normal spring. This is because break-up,
(which refers to the melting of the snow and ice on the lakes and sur-
rounding terrain), corresponds to a period of long daylight hours and
intense insolation. The temperature rise of the water upon break-up is
spectacular, and it is during this period of rapid temperature increase
that the Anabaena population builds up. Thus, temperature may be of
direct significance in this precipitous population increase. Under these
conditions, a lake only three meters deep can become stably stratified
and resist complete mixing for most of the summer in spite of consider-
able winds. Dilute ice-melt water may then float over the water which
has remained unfrozen during the winter. This happens in Smith Lake,
which has a maximum depth of three meters and an estimated mean depth
of two meters. Examples of the depth distribution of temperature during
the bloom period are shown in Figure 1. The relationship between bloom
38
-------
Aspects of Nitrogen Nutrition of Some Blue-Green Algae
formation and temperature can be seen in Figure 2, in which surface tem-
perature and particulate nitrogen are plotted simultaneously. Table 1
shows some physical and chemical properties of the lake during the bloom.
The nutrient content of the water at this time is strongly influ-
enced by the biological events preceding the ice breakup. February,
March, and April are characteristically months of complete anoxia in the
water, and no algae are present. In May, a population of |i-flagellates
and small green cells appear and are apparently influential in terminat-
ing the anoxic status of the water. There is, as yet, no contact of the
water with the atmosphere, but considerable light penetrates the ice
allowing quite high rates of photosynthesis. This population is influen-
tial in reducing the very high nutrient content of the water and, indeed,
the population decline corresponds to the time when the 40 u.g-atoms of
ammonia present have been completely removed into the particulate nitro-
gen fraction. Nitrate is not present, having been lost by denitrifica-
tion during the anoxic period (Goering and Dugdale, 1966). The Anabaena
population which follows develops at a time when the nutrient content of
the water is low but beginning to build up.
The Anabaena population is virtually unialgal and builds up at a
very rapid rate exhibiting high rates of photosynthesis in the surface
water. In Figure 3, we can see the chlorophyll 21 content of the water
during a typical summer season. The first peak represents the under-ice
population, and the second, the Anabaena. Figure 4 shows the seasonal
distribution of photosynthesis measured by the l^C method. The two major
periods of blue-green algal dominance are the high peak (Anabaena), and
the lower, but steady, mid-summer production during which time Aphani-
zomenon is the most abundant form. The extremely high rate of photosyn-
thesis at the June peak, when sustained for virtually 24 hours, allows
the tremendously rapid growth in population. I am deliberately including
data from several years in the figures in order to emphasize the annual
precision in timing.
Measurements of the nitrogen uptake by this bloom show that in the
early phases, ammonia is the predominant form used, with nitrate second.
Nitrogen fixation rates are low during this period, and initially the
bloom seems to build up on ammonia. The source of this ammonia was
unclear and isotope dilution experiments were carried out to see whether
the rate of ammonia supply within the water was adequate to allow the
uptake rates measured without assuming advection. Table 2 shows the
results of these determinations. Although the precision of the experi-
mental procedure is not very good yet, these results are reliable in
approximate magnitude. The value for June 3 indicates that the supply
rate is more than adequate for the uptake rate measured, which agrees
with the analytical observation that the ammonia concentration is increas-
ing in the water. The extremely high rates of supply measured following
39
-------
ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Table 1
Some physical and chemical properties -
Smith Lake Anabaena bloom
1964
Alkalinity Specific
Depth mg liter"-"- 02 T cond.
(m) pH CaCO-} mg liter"1 °C |j,Hmos/cm2
30 May
0 7.10
1 7.10
2 6.90
16 June
0 9.35
1 9.00
2 6.91
18 June
0 7.97
1 7.65
2 6.87
22 June
0 7.34
1 7.26
2 6.93
13 July
0 7.45
1 7.27
2 6.99
18.0
23.0
40.0
-
-
27.0
27.7
34.3
30.0
29.0
40.0
28.0
28.1
28.0
-
-
11.5
10.2
1.7
8.7
6.4
1.2
6.7
6.4
6.2
7.6
7.2
1.9
13.5
5.6
5.2
20.4
18.6
10.2
22.2
18.7
10.7
18.8
18.8
9.6
19.7
18.0
15.1
60.2
52.8
154.0
75.8
77.2
103.0
83.9
85.1
113.4
76.4
80.3
110.5
95.9
97.5
115.5
nutrients
NO^-L-N
0.1
0.0
0.5
0.2
0.2
0.6
0.1
0.1
0.4
0.3
0.3
0.4
-
—
(ug-at liter"1
NH^-N PO/, -P
0.4 2.2
1.7
3.5
2.8 0.6
1.5 0.2
7.7 2.1
_
-
16.5 0.7
0.9
2.6
5.8
-
— ~
the bloom are surprising; much of this ammonia must be taken up by the
macrophytes which show tremendous development at about this time. This
does seem to confirm the immediate availability of nitrogen incorporated
40
-------
Aspects of Nitrogen Nutrition of Some Blue-Green Algae
in a bloom. The rate of supply of ammonia from the previous population
is also apparently adequate to initiate the rapid growth of Anabaena in
spring.
Table 2
Results of ammonia uptake and ammonia
supply experiments. Smith Lake, Alaska
Date
3 June 1965
23 June 1965
21 July 1965
13 Aug. 1965
31 Aug. 1965
30 Sept. 1965
20 Jan. 1966
3 Feb. 1966
15 Feb. 1966
15 March 1966
22 April 1966
*Particulate
NnJ-N ug
liter"1
28.3
361.2
29.4
20.3
47.4
31.5
227.5
338.8
319.9
515.8
676.1
nitrogen
*PN ug
liter"1
371.6
102.3
88.9
81.2
64.5
77.8
62.7
110.6
104.2
105.9
Uptake
rate ug
liter'*
hr-1
7.0
16.4
1.5
2.1
0.5
0.7
0.6
0.0
8.2
Supply rate
ug liter~lhr~
13-3K v
o/o i^Anabaena b
2.9
2.8
12.8
7.6
100.1
67.8
16.0)
107.3) anoxic
123.7)
1
loom
Table 3 shows the uptake rates of ammonia, nitrate, and molecular
nitrogen during the bloom. A progressive shift is evident from a very
high ammonia utilization rate to a relatively greater importance for
nitrogen fixation which, at one point, accounts for half the nitrogen
assimilated. The highest absolute rates of nitrogen uptake, taking into
account all three forms measured, are found early in the bloom, during
the time when the population is increasing at its maximum rate. This
rate approaches 24 ug liter~lhr~l. During the nitrogen-fixing phase,
the algae become noticably concentrated near the surface, resulting in
very high photosynthetic rates on a unit volume basis of surface water.
This concentration can be seen from the chlorophyll data shown in Table
4. As a result of this intense activity at the surface, there is an
abrupt rise in pH near the surface, which lasts perhaps one or two days.
This is a unique and rather closely-timed annual event. The annual pH
curves for Smith Lake surface water during three years are shown in
Figure 5. Shortly after this, the population of Anabaena declines.
41
-------
ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Table 3
Uptake of NH^-N, NO^-N, and N2-N
during the spring Anabaena bloom, Smith Lake
Particulate
Nitrogen
tig liter"1
Date
NH^-N
M.g
liter"1 hr"1
N03L-N
N2-N
4
6
8
10
12
15
23
25
June
June
June
June
June
June
June
June
1964
1964
1964
1964
1964
1964
1964
1964
125.3
263.6
358.8
405.4
640.1
753.1
71.3
0.82
17.23
5.99
6.41
8.21
1.70
0.94
0.36
4.88
4.12
0.75
0.37
0.15
0
.02
1.76
2.45
2.88
2.28
0.19
0.02
1.20
23.87
12.56
10.04
10.86
5.10
0.96
Table 4
Depth distribution of chlorophyll £ during Anabaena bloom, 1964
Depth
(m)
8 June
10 June
12 June
liter-1
18 June
30 June
0.0
1.0
2.0
22.6
10.7
10.7
17.1
34.7
6.8
132.3
19.4
22.2
18.7
10.7
7.7
8.2
3.2
The filaments composing the bloom undergo changes in appearance
as the population progresses through its growth and decline. At first,
the filaments are long, somewhat coiled, with very even, healthy green
cells. Initially there are no heterocysts, but these develop as the
nitrogen fixing activity of the algae increases. Akinetes appear at
about the time of the pH rise, and following this there is clumping
and disintegration of the filaments, so that free akinetes and hetero-
cysts are visible in the water. Following this, there are no Anabaena
42
-------
Aspects of Nitrogen Nutrition of Some Blue-Green Algae
in the water. The cause of the decline is an interesting problem. Per-
haps the pH has a role, either directly or indirectly by causing C02
limitation, or perhaps by increasing the strength with which a metal ion
is bound to an organic chelating agent. The water is strongly colored
by a brown organic material, which behaves in a manner similar to that
described by Shapiro (1964). The influence of pH in increasing the
strength with which cations are bound could be important where high
growth rates are causing rapid removal of a metal.
OTHER BLUE-GREEN ALGAL POPULATIONS
Following the Anabaena bloom decline in Smith Lake, Aphanizomenon
becomes the dominant form. The nutrient levels never become very low,
but rather remain at a constant level, and the growth of the population
appears to be controlled by grazing, with a very dense population of
zooplankton present now. The diversity in algal forms present is much
greater, and nothing approaching a unialgal population is found.
Anabaena appears again in this mixed population, with very long fila-
ments composed of healthy green cells. The macrophyte population is
building up fast at this time, and must continue to drain considerable
quantities of nutrients from the water. Ammonia is predominantly used
by the phytoplankton, and although nitrogen fixation was detected at
one time during later summer, significant rates were never found except
in connection with the spring Anabaena bloom.
An Aphanizomenon-dominated population is found in a second small
lake only two miles from Smith Lake. This lake is similar in surface
area and in the strong brown coloration of the water, but differs in
depth, being 6 meters deep at the maximum depth found. Nitrate accu-
mulates in this lake in spite of much of the water remaining anoxic
during the entire summer--! do not know why this nitrate is not denitri-
fied under these conditions. Water below 2.5 meters remains anoxic
during the summer. The Aphanizomenon population, initially concentrated
in the surface layers, spreads down into the anoxic waters through the
thermocline later in the summer. Ceratium becomes quantitatively domi-
nant later inthe summer, with large numbers of Aphanizomenon concurrently
present. No nitrogen fixation has been detected here, and ammonia is
the chief nitrogen source used.
NITROGEN UTILIZATION IN CIRCLE HOT SPRINGS
Blue-green algae are characteristic components of the populations
in hot springs. Only 50 miles south of the Arctic Circle, Circle Hot
Springs represents a unique environment. The dominant algae here are
43
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Mastigocladus laminosus and Phormidium sp. The springs emerge from a
small opening in the rock and descend rapidly, with one major pool near
the source. The rest of the water drains down, and is used to supply
a swimming pool and also to heat the resort buildings. Nitrogen uptake
measurements show that nitrogen fixation is the quantitatively most
important nitrogen uptake, although low rates of uptake of nitrate and
ammonia were detected. These data are shown in Table 5. The water
temperature at the source is about 55°, and the optimal growth of the
Mastigocladus in the laboratory was at 45°C, with no growth below 35°C.
Phormidium grows at room temperature and seems to tolerate a broader
range of temperatures.
Table 5
Uptake rates of nitrogen sources by particulate fraction
in Arctic Circle Hot Springs water, Alaska - June 1963
Nitrate - 15N
Ammonia - N
Nitrogen gas -
(90%)
(98%)
15N2 (95%)
Atom %
Excess 1
.096
.280
2.552
ug N
.04
.10
.99
-1
Total nitrogen
concentration
in particulate
fraction, 885
|j.g/liter
Algae present: Mastigocladus laminosus
Phormidium sp.
Incubated "in situ" in hot spring pool at ~'45°C.
DISCUSSION
The significant questions which have been asked in this work are:
a. What is the nitrogen nutrition of naturally occurring blooms
of blue-green algae, and especially, how important is nitrogen fixation?
b. Under what conditions do major nitrogen-fixing blooms develop?
c. How do these populations fit into the seasonal patterns of
algae production?
44
-------
Aspects of Nitrogen Nutrition of Some Blue-Green Algae
Nitrogen fixation is apparently highly significant under some con-
ditions. In general, shallow depth, high organic content, high tempera-
tures, and high insolation appear to be prerequisites. The availability
and presence of combined nitrogen sources in the water does not appear
to prevent nitrogen-fixing blooms from developing. Shallow warm waters
in the Arctic and Subarctic apparently provide suitable conditions for
such blooms in the summer. In Table 6, I have summarized some rates of
nitrogen fixation measured in lakes ranging from Pennsylvania to the most
northern tip of the United States, at Point Barrow, Alaska. In Smith
Lake, the total nitrogen fixed during a season is approximately 500 |ag
liter"-*-, which represents a significant proportion of the budget.
The role of bacteria in algal cycle is unfortunately little known,
although a start is evident in the paper on microbiotic cycles by Silvey
and Roach (1964). Whereas in Sanctuary Lake there is important inflow
of sewage-laden water with varying nutrient concentrations, which seem
to influence the algal periodicity, in Smith Lake internal factors appear
to be more important. The rapid release rate of ammonia from algal popu-
lations suggests that the development of bacterial populations in response
to algal cycles is a common feature.
Ammonia, the energetically economical form of nitrogen, is assimi-
lated readily by blue-green algae, and Anabaena in particular can grow
rapidly on ammonia at a low concentration but with a high supply rate,
and under these conditions supplement its nitrogen income by nitrogen
fixation.
45
-------
ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Table 6
A summary of some nitrogen fixation rates
Sanctuary Lake,
Pennsylvania
Smith Lake, Alaska, 1963
Smith Lake, Alaska, 1964
Circle Hot Springs
Paul's Pond, Pt. Barrow
(from Dugdale and
Toetz, 1961)
Little Kitoi Lake (from
Dugdale and Guillard,
1966)
Anabaena circinalis
Anabaena spiroides
Anabaena flos-aquae
Anabaena flos-aquae
47 |j.g liter" day"
1 |ig liter"1hr"1
3 ,ig liter^hr"1
Mastigocladus laminosus 1 u.g liter hr
Nostoc sp.
Anabaena sp.
3.34
liter~1day"1
.04 |ag liter" day"
46
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8
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Figure 5.
10 20
JUN
10 20
JUL
Some representative pH curves for
Smith Lake surface water.
-------
REFERENCES
Dugdale, R. C. and D. Toetz. 1961. "Sources of nitrogen for arctic
Alaska lakes." In: Dugdale, R. C., ed. Final Report of Investi-
gations of the Nitrogen Cycle in Alaska Lakes. A.I.N.A., Subcontr.
ONR-253.
Dugdale, R. C. and R. R. L. Guillard. 1966. "Nitrogen fixation in
lakes on Afognak Island." In: Final Report to the Arctic Institute
of North America, Nutrition of Algae in Subarctic Lakes. Subcontr.
ONR-276.
Dugdale, V. A. and R. C. Dugdale. 1965. "Nitrogen metabolism in lakes.
III. Tracer studies of the assimilation of inorganic nitrogen
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Fogg, G. E. 1942. "Studies on nitrogen fixation by blue-green algae.
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Fogg, G. E. and W. D. Stewart. 1965. "Nitrogen fixation in blue-green
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Fogg, G. E. and Than-Tun. 1960. "Interrelations of photosynthesis and
assimilation of elementary nitrogen in a blue-green alga." Proc.
Roy. Soc. (London), B. 153:111-127.
Goering, J. J. and J. C. Neess. 1964. "Nitrogen fixation in two
Wisconsin lakes." Limnol. and Oceanog. 9(4):530-539.
Goering, J. J., R. C. Dugdale and D. W. Menzel. 1966. "Estimates of
in situ rates of nitrogen uptake by Trichodesmium in the tropical
Atlantic." Limnol. and Oceanog. (In press).
Goering, J. J. and V. A. Dugdale. 1966. "Estimates of the rates of
denitrification in a subarctic lake." Limnol. and Oceanog. 11(1):
113-117.
Gorham, P. R., J. McLachlan, U. T. Hammer and W. K. Kim. 1964.
"Isolation and culture of toxic strains of Anabaena flos-aquae
(Lyngb. de Breb.)" Verh. Internat. Verein. Limnol. 15:796-804.
Grill, E. V. and F. A. Richards. 1964. "Nutrient regeneration from
phytoplankton decomposing in seawater." Journ. Mar. Res. 22(1):
51-69.
Hutchinson, G. E. 1957. "A treatise on limnology. I. Geography,
physics, and chemistry." John Riley & Sons, New York. 1015 pp.
52
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Neess, J. C., R. C. Dugdale, V. A. Dugdale and J. Goering. 1962.
"Nitrogen metabolism in lakes. I. Measurement of nitrogen
fixation with ^N." Limnol. and Oceanog. 7:163-169.
Prowse, G. A. and J. F. Tailing. 1958. "The seasonal growth and suc-
cession of plankton algae in the White Nile." Limnol. and Oceanog.
3(2):222-238.
Riley, G. A. 1951. "Oxygen, phosphate, and nitrate in the Atlantic
Ocean." Bull. Bingham Oceanog. Coll. 13:126 pp.
Shapiro, Joseph. 1964. "Effect of yellow organic acids on iron and
other metals in water." J. Amer. Water Works Assoc. 56(8):1062-
1082.
Silvey, J. K. G. and A. W. Roach. 1964. "Studies on microbiotic cycles
in surface water." J. Amer. Water Works Assoc. 56(l):60-72.
Stewart, W. D. P. 1962. "Fixation of elemental nitrogen by marine blue-
green algae." Annals of Botany 26(103):439-445.
Stewart, W. D. P. 1964. "Nitrogen fixation by Myxophyceae from marine
environments." J. Gen. Microbiol. 36:415-422.
53
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ENVIRONMENTAL REQUIREMENTS
OF THERMOPHILIC BLUE-GREEN ALGAE
Richard W. Castenholz
Department of Biology
University of Oregon
Eugene, Oregon
INTRODUCTION
This discussion will relegate itself primarily to that habitat and
those organisms above 45°C. This is not a completely arbitrary breaking
point between the mesophilic and the obligate thermophilic algae. Most
of the latter find optimal conditions above 45°C and will generally not
grow at temperatures much below 30°C. With one confirmed exception,
the eucaryotic algae reach their upper limit at about 45-47°C. Only
the enigmatic Cyanidium caldarium breaks beyond this barrier well into
the thermal range. Anacystis nidulans, so widely used as an experi-
mental organism, has an upper growth limit of about 45°C and an optimum
of 41°C (Kratz and Myers, 1955), falling somewhat short of a true
thermal range. Probably, a majority of the blue-green algae have tend-
encies in the direction of thermophily. R. Y. Stanier (personal comm.)
has informed me that holding the temperature at 35°C is a simple enrich-
ment method for obtaining a wide variety of blue-green algae from crude
collections containing many other types of algae. Subsequently, I have
had similar success with a number of non-thermal collections at 37°C.
Naturally, a considerable number of blue-green species will not fall
into this category. Nor would this method be well suited for collec-
tions from the non-thermal portion of a hot spring drainway, since in
this environment there are a number of green algae and diatoms adapted
to temperatures of up to 45°C. It is not unusual for micro-algae to
grow at maximal rates at temperatures significantly higher than they
would normally encounter in nature (e.g. Braarud, 1961), but blue-green
algae seem to lie 5 to 15 degrees higher up the scale than the others.
The non-planktonic blue-green algae demonstrate their greatest
development in hot springs, particularly in the formation of macroscopic
mats of a wide variety of textures, colors, and thicknesses (e.g.
Schwabe, 1962a, 1964). Above the 45°C border the blue-green algae are
essentially alone in their nutritional mode with only an unknown quan-
tity of heterotrophs, chemolithotrophs, and a few photosynthetic bac-
teria as unlikely competitors for the same niches. Under the upper
photosynthetic layer of the gelatinous or leathery mats one usually
finds several multicolored layers which either lack photosynthetic
55
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
capacity or consist primarily of dead material. The accumulation of
this material is a strange phenomenon. In a temperate environment
this material would normally be mineralized by heterotrophs more in
pace with primary production. Heterotrophic, thermophilic eubacteria
can be isolated from any temperature at which blue-green algal mats
occur, but they do not seem to be abundant. When such mats reach an
encumbering thickness or age, peeling or washouts by catastrophic
rains result in transport to a non-thermal environment where break-
down may occur rapidly.
Perhaps one of the most important reasons for the great mat
development in thermal waters is the absence of grazers above about
45 to 47°C. For example, in Yellowstone Park, Ephydrid larvae erode
the algal mat significantly in some alkaline springs below 43°C. In
Hunter's Hot Springs, Oregon, a very abundant ostraced of the genus
Potamocypris grazes on the surface of the Phormidium and Pleurocapsa
mat between 47° and 30°C.
The most obvious environmental requirement of a thermophilic
blue-green alga is high temperature, but many have probably adapted
to some extraordinary water chemistries as well. Certainly, not all
hot spring waters are potable, nor are they balneal "cure-alls". Some
of the thermal waters are certainly highly eutrophic, containing an
ample or excessive supply of macro- and micro-nutrients. They exhibit
relatively high salinities and may contain various metals in concen-
trations so high that they should be labelled poisonous. It is pos-
sible, however, that higher concentrations of certain metals may
impart greater thermal stability to some organisms (e.g. Ljunger, 1963;
Militzer and Burns, 1954).
A general adaptation to high light intensity should also be
expected in most thermophilic algae. The usual shallow outflow from
a hot spring results in exposures to extremely high light intensities,
with little or no refuge.
Much of the information relating to thermophily has come from
the thermophilic bacteria. In addition, so much must be inferred from
work with mesophilic Cyanophyta. True thermophilic blue-greens have
not been generally available from stock collections, and few research
institutions are located within commuting distance of hot springs.
Also, the inconvenience of maintaining high temperature equipment and
the general belief that thermophilic algae would be difficult to
culture have kept their use in the laboratory to a minimum.
The gross chemistry of quite a number of hot springs is known,
and there are some published data on the responses of thermophilic
blue-greens under laboratory conditions. However, little synthetic
56
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Thermophilic Blue-Green Algae
ecology has emerged from these two sets of information. Only now has
some experimental work begun on the organisms within their native
environment.
GEOGRAPHIC AND HISTORICAL FEATURES
Although isolated warm or hot spring groups exist in many parts of
the world, there are only a few areas of extensive thermal activity.
The concentrated areas are in the western United States (Yellowstone
Park), Iceland, New Zealand (Rotorua), and Japan. Hot springs are scat-
tered through central and southern Europe, Asia, the East Indies,
Melanesia, Africa, and South America, but they are missing entirely from
several large land areas. Most are found in areas of Quaternary or cur-
rent vulcanism, although many groups of springs are associated entirely
with diastrophism and lie near major faults. Of the four main areas,
only Iceland lies above 60° of latitude.
Although aquatic thermal habitats of similar types are widely iso-
lated geographically, there is a great similarity of the flora through-
out the world. However, lacking physiological, biochemical, or genetical
evidence of similarity, it becomes difficult to state that two similar
blue-green algae are similar in anything besides morphology. Schwabe
(1962b) brings up the difficult problem of species characterization and
meaning in the Cyanophyta. It is obvious that there is a considerable
amount of- endemism among the thermal blue-green algae, even with respect
to strictly morphologically defined species.
The major thermal areas of the earth have apparently been active
continuously since some portion of the Pleistocene. Although little is
known about dispersal mechanisms in the thermophilic Cyanophyta and
survival capacities during transport, one might suppose that close to a
million years or more would be sufficient time for all species to have
been inoculated several times into all of their potential habitats.
This is fallacious. Many hot spring areas have developed in quite recent
times, and it is probably safer to assume that many of the blue-green
algae disseminate in manners similar to those of the eucaryotic micro-
algae which they resemble in cell size and general morphologic pattern.
In some of the freshwater Chlorophyta, time and distance are demonstrable
factors in distribution patterns. The absence of some species in many
thermal waters may be due to the lack of a suitable habitat of lasting
duration rather than the lack of inoculation opportunity, but the latter
is still a real possibility.
Barghoorn and Tyler (1965) and others have recently found good
micro-fossils of blue-green algal-like filaments and cells in Precambrian
57
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
strata. There is evidence indicating that some of this material was
in an aquatic paleoenvironment with a high silica content, similar to
that of present hot springs. It becomes attractive and reasonable to
believe that some, at least, of the present thermophilic Cyanophyta are
directly descended from Precambrian thermophiles and are not more recent
invaders of the thermal environment. Such a hypothesis assumes that
there was no discontinuity in the aquatic thermal activity during the
interim.
TEMPERATURE CHARACTERISTICS AND LIMITATIONS
Constancy is one of the most characteristic features of thermal
waters. Many spring sources, ranging from 40°C to boiling, vary
little in temperature or dissolved mineral composition even over
periods of many years. Yet, it is not safe to assume that this is the
case for every spring encountered. Pools of geyser craters and super-
heated springs often vary considerably in temperature in the course of
a few hours, and, in the case of geysers, the changes in overflow
volume naturally result in temperature changes along the drainway. Some
streams from geyser pools may flow only intermittently during and pre-
ceding an eruption. Many thermophilic or "semi-thermophilic" algae
form aerial mats and depend entirely on frequent wetting by geyser spray
(Fig. 1).
Little is known about the resilience of thermophilic blue-green
algae subjected to temperature changes. Peary and Castenholz (1964)
showed that several strains of Synechococcus lividus (one growing as
high as 70°C) are able to tolerate abrupt changes in temperature within
their growth range or from room temperature to any point in that range
without apparent damage. Temperature shocks to above the long-term
lethal limit are also tolerated provided that these temperatures are
not held for long (Peary, 1964). Resilience of this type appears to
be characteristic of obligate thermophilic bacteria as well, but rapid
shifts up the thermal scale are usually fatal to facultative thermo-
philic bacteria (those that grow well at 37° and 55°C--Bausum et al.,
1961).
A lack of resilience was noted by Marre and Servettaz (cited in
MarreN, 1962) in Aphanocapsa thermal is. This species was very sensitive
to changes from its temperature of acclimation, whatever that happened
to be. Lowering the temperature several degrees depressed both respir-
ation and photosynthesis, but particularly the latter. Raising the
temperature only a few degrees caused a decline in photosynthesis which
was irreversible, followed by a similar decline in respiration. The
gap between the acclimation temperature and the inactivating high
58
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Thermophilic Blue-Green Algae
temperature became less the higher the temperature of acclimation. In
Synechococcus lividus (collected at 53°C), Peary (1964) found no such
indications of acclimation or deadaptation. The clone culture was
grown for over one year at 50°C before a sub-culture was established
and maintained simultaneously for eight additional months at 30°C. The
normal growth rate at 50°C was 8 doublings per day, and at 30°C it was
between 1 and 2 doublings per day. The optimum was at 45°C at over 9
doublings. At the end of the entire period the growth rates for both
30°- and 50°-maintained cultures were both normal throughout the total
range from 30° to 55°C. Exponential growth commenced at 45° and 50°C,
with essentially no lag with inoculum from 30° or 50°C stock. Allen
(1959) and Lb'wenstein (1903), on the other hand, found that the main-
tenance of a thermophilic alga below its optimal growth temperature for
a 15ng period resulted in a deadaptive lowering of the optimal growth
temperature and the upper tolerance limit. The organisms were Cyanidium
caldarium (non-Cyanophyta) and Mastigocladus laminosus, respectively.
Little is known of genetic adaptation in the blue-green algae. Muta-
bility has been demonstrated only a few times (Singh and Singh, 1964a,b;
Van Baalen, 1965).
The upper temperature limit for the growth of blue-green algae is
still unresolved. Copeland (1936) made observations of blue-green algae
in hot springs of Yellowstone Park at temperatures above 75° and up to
85°C. Others (cited in Peary, 1964) have indicated the presence of
blue-green algae above even 90°C. Recently, however, there has been
no incontestable evidence of the growth of chlorophyll-containing
organisms at constant temperatures above 74-75°C. Kempner (1963), look-
ing at upper temperature limits of life in a few adjacent hot springs
in Yellowstone Park, concluded, on the basis of P" incorporation into
nucleic acids, that 73°C was about the upper temperature limit for
growth. This corresponds well with observed demarcations of Synechococ-
cus mats in hot springs of the same area in Yellowstone Park. In a
recent two-week visit to Yellowstone I explored a number of the hot
spring and geyser basins. Some of this was with Thomas D. Brock, of
Indiana University, who is currently working on the productivity and
general ecology of some thermal springs there. In all of the alkaline
and neutral springs investigated, 73-74°C seemed to be the absolute
upper limit for a visible cover of Synechococcus (Fig. 2). Some of
the areas explored were those mentioned by Copeland as containing one
or more of the five blue-green species whose upper limits were above
80°C. It is indeed true that there are non-photosynthetic organisms
that live in springs of the Yellowstone at temperatures of over 80°C.
Brock (personal comm.) found no chlorophyll in a pink-colored filamen-
tous organism growing up to about 88°C and forming luxuriant trailing
tufts at this temperature. It is possible that three of Copeland's
high temperature species of Phormidium and Oscillatoria should be
59
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ENVIRONMENT REQUIREMENTS OF BLUE-GREEN ALGAE
referred to the non-photosynthetic "flexibacteria" or apochlorotic
blue-green algae. Neither Setchell (1903) nor Nash (1938) reported
blue-green algae above temperatures in the mid-70s in Yellowstone
springs, which agrees with our findings. The most intriguing recent
report of a higher limit is that by Anagnostidis (1961) of Syne-
chococcus at 81°C in a salt spring of Greece. A demonstration of
growth is, of course, required to confirm any of the above field
observations.
The 74-75°C upper limit for blue-green algae does not extend to
thermal waters of all chemical types or to all geographical locations.
In Iceland Schwabe (1936) found blue-green algae at no higher than
60.2°C. Brock and Brock (1966) and Jon Sperling (personal comm.) have
indicated that the highest blue-green habitat was about 60° and 63°C,
respectively. These temperatures reflect the approximate upper
growth limit of Mastigocladus laminosus in Iceland; this is also the
upper limit for this cosmopolitan species elsewhere (Schwabe, 1960).
Iceland definitely has an impoverished thermal flora. Petersen
(1923), who studied all the freshwater Cyanophyta, identified about
39 thermal species. This is less than one-half the usual number
found in hot spring groups of lower latitudes. Schwabe (1936)
reduces this number to six definite thermal species typical of the
alkaline springs. Species of Synechococcus, which generally contri-
bute the upper temperature forms in North America, Japan, and Greece,
appear to be entirely missing from Iceland. Either this group has
not been inoculated into Iceland yet, or the environmental conditions
are not suitable for it. Although the volcanic activity has appar-
ently been continuous for somewhat less than a million years, an ice
cap covered the island until Recent times. The thermal springs
present during the ice period may have been severely handicapped and
variable. At present, the chemical characteristics of many North
American springs with Synechococcus are essentially duplicated in
many Icelandic springs. The winter light condition may be the most
detrimental facet of the environment.
In the New Zealand hot springs, an upper limit for blue-green
algae of 61°C has been cited by Nash (1938). Again, a wide variety of
thermal and chemical conditions exist, but again Mastigocladus lamino-
sus forms the upper boundary (along with Phormidium sp.). Some
species of Synechococcus are present in the springs. Since North
Island lies at about 40° latitude, it shares with Iceland only the
properties of being insular and at a great distance from other
thermal areas.
Besides the differences in upper temperature limits between
Yellowstone and Iceland, Brock and Brock (1966) report that standing
60
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Thermophilic Blue-Green Algae
crops of blue-green algae also reach maxima at different temperatures in
the two locations. In an alkaline spring in Yellowstone, maxima of
chlorophyll, protein, and RNA occurred at about 54°C, whereas, in an
Icelandic spring, the same components of standing crop peaked at about
48°C. The difference could easily be attributed to the species composi-
tion, which might in turn reflect chemical differences or geography, or
both. Brock (personal comm.) has reported that primary productivity
in the Yellowstone spring, as measured by C^ assimilation per unit area,
peaks in the area of maximum standing crop to somewhat lower tempera-
tures. A standing crop and productivity maximum in the mid-50s was
apparent in the Drakesbad Hot Springs of Mt. Lassen National Park,
California (Lenn, 1966).
From the temperature of maximum abundance to the upper limit of
photosynthesizing blue-greens, standing crop and the number of species
declines steeply (e.g. Brock and Brock, 1966; Vouk, 1950). This is
quite apparent visually in North American hot springs, at least above
60-65°C. In Hunter's Hot Springs, Oregon, the one or two strains of
Synechococcus of the highest temperature type form only a thin mat and
grow at relatively poor rates in culture (Peary and Castenholz, 1964).
Without aeration and additional C02 the medium may have been a limiting
factor at 65° and 70°C, however. In the case of the Oregon spring,
Svnechococcus formed a continuum from about 74° to 54°C, at which point
Oscillatoria terebriformis entered the spectrum and became predominant.
A minimum of four temperature strains of the Synechococcus may form an
overlapping series in the continuum.
Marrd (1962), in a recent review of temperature and algae, alludes
to the lower growth rates of thermophilic algae. In fact, little to
nothing is known of their growth rates in nature. In culture, however,
the growth rates of many are considerably higher than the norm for well-
known mesophilic and psychrophilic algae. Synechococcus lividus (Clone
53), in my laboratory, can reach 10 doublings per day at 45°C, and
Oscillatoria terebriformis can approach 5 doublings at its optimum of
50°C. Dyer and Gafford (1961) have also reported 9 doublings per day
for Synechococcus lividus from Yellowstone.
A property generally ascribed to thermophilic algae, in particular,
is the ability to grow or photosynthesize at maximal rates up to a
temperature close to the lethal upper limit (e.g. Marre4, 1962; Prat,
1956). In nature this may result in the formation of a prominent uni-
algal mat which is neatly delimited at its upper end from the neighbor-
ing species up the thermal gradient. Sudden increases in temparature
down the range should result in easily observed changes in the visible
pattern of such mats. The sharp banding and fine lines of demarcation
are some of the most striking features of hot springs the world over
61
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
(Fig. 3). The observed temperature range of a particular species in a
hot spring usually corresponds to the growth temperature range in the
laboratory only with respect to the upper limit. The sub-optimal
growth temperature range in the laboratory is masked in the field by
the competitive predominance of another species which finds its opti-
mum within that range. In Hunter's Hot Springs Oscillatoria terebri-
formis successfully competes with at least one of the Synechococcus
strains in the optimum temperature range of both (Peary and Castenholz,
1964).
A maximal growth rate just below the upper temperature limit does
not appear for all strains of Synechococcus. The optimum was often
10-12 degrees below the limit. Holton (1962) found a similar case in
Mastigocladus laminosus when the culture was aerated with line air.
The broad growth optimum was narrowed considerably and moved towards
the upper temperature limit with 170 CC>2 in air. However, the upper
limit was depressed about 5 degrees to 55°C under the latter conditions.
Aeration might also change the pattern in the temperature response
curves of Synechococcus (Peary and Castenholz, 1964). Oscillatoria
terebriformis, however, maintains its growth peak to only 1 or 2 degrees
below its upper limit of 54°C, with or without aeration.
The lower temperature limit of growth in obligate thermophilic
blue-green algae differs greatly with species; it is often well
within the temperate range. The loss of growth potential at very
moderate temperatures may also be accompanied by lethal lesions
(Forrest, et al., 1957). A very high temperature strain of Synecho-
coccus (Clone 75) finds its lower growth limit somewhere between 50°
and 55°C. Other strains ceased growth between 45° and 50° (Clones 66
and 71), between 35° and 40° (Clones 55 and 60), and at some point
below 30°C (Clones 45, 48, and 53) (Peary and Castenholz, 1964).
Oscillatoria terebriformis, from the same Oregon hot spring, will not
grow under normal circumstances below about 30°C (Castenholz, unpubl.).
I have recently investigated the effect of low temperatures on this
species. It is one of the most abundant organisms in Hunter's Hot
Springs, forming dense mats of entangled and tufted trichomes from
about 54° to 47-45°C in the thermal gradient (Fig. 3 and 4). Although
peaking at about 50°C, the general growth optimum for this species in
culture fits the distribution of the mat in nature, even at the lower
end. Below approximately 43°C in culture growth declines to the mini-
mum at 30°C. This species exhibits rapid oscillating movements which
result in a spectacular aggregation phenomenon (Castenholz, 1967).
The rate of this form of motility is saturated only above 37°C, but
it occurs even as low as 12°C in the light and at a respectable rate
between 20° and 30°C. Below about 27°C, however, the motility can be
accounted for almost entirely by energy supplied through photo-
62
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Thermophilic Blue-Green Algae
phosphorylation. Presumably, the enzymes associated with respiration or
oxidative phosphorylation are most inactivated at this temperature and
below. Below 27°C, the Oscillatoria will hardly aggregate in the dark,
while at optimal temperatures dark aggregation takes place with almost
the same rapidity as in the light. Prat (1954, 1956) demonstrated that
photosynthesis continued at least 20 degrees below the lower growth
limit of Mastigocladus laminosus (28-30°C) and that respiration occurred
for about 15 degrees below. However, growth, photosynthesis, and
respiration all reached maximal rates at about 40° C in this strain.
Tentatively, I would consider the aggregative behavior of 0.
terebriformis as an adaptive feature which enables the trichomes to
hold together as a "mat" within the optimal temperature range in flow-
ing water (Fig. 4). As may be seen in the photograph, trichomes are
not embedded in a gelatinous mat but are free or roped together. Vigor-
ous agitation causes almost complete dispersal of the natural mat. The
great majority of blue-green algae in thermal spring drainways hold
their position in the flow by the formation of a gelatinous matrix or
else by embedding or multiplying within the matrix formed by other
organisms.
Being washed downstream in a hot spring to moderate or cold temper-
atures may be lethal to most of the obligate thermophiles, although the
fate of thermophilic algae at low temperatures is largely unknown even
in the laboratory. Oscillatoria terebriformis dies when exposed to
lower than growth temperatures for extended periods (Castenholz, unpubl.).
The deterioration preceding death is more rapid the lower the temperature
and the higher the light intensity. For example, at the end of 6 hours
at 14°C and a light intensity of about 2,000 ft.-c. or 12-24 hours at
450 ft.-c. complete loss of viability occurred, as evidenced first by
the loss of motility when raised to normal temperatures and then by the
leakage of the phycobilin pigments into the medium. Increasing the
temperature or lowering the light intensity prolonged the period of
viability, so that at 23°C and 450 ft.-c. death occurred only after 30
days. In darkness, at 45°C, (). terebriformis dies in from 24-48 hours,
but at 25°C it will remain in good condition for over a week. In the
same period there was poor survival in darkness at 11°C and even less
at 3°C. Synechococcus lividus (Clone 53), on the other hand, is rela-
tively insensitive to low temperature storage in light or darkness. A
high temperature strain (Clone 75), which grew from 55°C to over 70°C,
evidenced some survival after 10 days at 30°C in the light (Peary, 1964).
The survival of thermophilic algae outside of their milieu is a
problem continuously encountered but about which little is known. Few
of the thermophiles have morphological resting spore stages, but many
can probably tolerate desiccation, nevertheless. This may be true in
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
particular for those cells drying within the "gel" of a mat. Synecho-
coccus lividus (Clone 53) will not survive drying much above room
temperature, and the survival rate is apparently very low even at 25°C
after drying 24 hours over anhydrous calcium sulfate. Oscillatoria
terebriformis, however, will not tolerate drying under any conditions,
even momentarily (Castenholz, unpubl.).
Tolerance to freezing in blue-green and green algae was recently
investigated by Holm-Hansen (1963). He found evidence of adaptation
in that the various algae from the Antarctic were able to survive
freezing while all but one green alga from Wisconsin was killed by
either slow or rapid freezing. For most of those algae that survived
freezing, slow freezing was less damaging than rapid. On the other
hand, all the strains of thermophilic Synechococcus in my laboratory
were able to tolerate slow freezing (5-10 minutes) and retain a high
survival percentage after storage at -20°C for a period of at least
10 months. Oscillatoria terebriformis will not survive freezing,
either rapid or slow.
In the middle or temperate latitudes the winter survival of
thermophilic algae should require no special mechanism, since there is
adequate light for the maintenance of growth. The thermal gradient
contracts, and productivity may be reduced considerably, but no diffi-
culties in species survival should arise. In Hunter's Hot Springs in
winter there is little change in appearance from summer except for a
contraction of the species bands toward the source. Also, there is
little change in the relative species abundance (Peary, 1964). In
the extensive springs of Iceland, where the sun may appear for only
three hours or less at a very low elevation in mid-winter, the algal
cover apparently breaks down and does not recover fully until late
spring or summer (Schwabe, 1936; Brock, personal comm.). Some forms
may survive without growth in tepid or cold niches in the drainways.
Others may survive dried or frozen along the edges of the springs.
Heterotrophy, rare in blue-green algae, is an unlikely winter survival
mechanism. Forms such as Oscillatoria terebriformis theoretically
should not be able to survive an Icelandic winter. It is a wonder
how this species has been disseminated any distance anywhere. It is
reported from thermal habitats of western North America, southern
Europe, and Japan, but not from Iceland. The species name is also
used for a non-thermal Oscillatoria from a much greater geographical
range. Certainly several taxa are represented. They may all fit
within the dimensions of the original description, but temperature
ranges, color, and growth habits do not agree.
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Thermophilic Blue-Green Algae
RESPONSES TO LIGHT INTENSITY
As mentioned earlier, many or most of the hot spring environments
are subjected seasonally to very high light intensities because of
locations at high elevations, in treeless areas, or in areas where the
surrounding trees have been destroyed by the springs and fumaroles.
The Yellowstone hot spring basins lie between 6,000 and 8,000 ft.
elevation. Hunter's Hot Springs of south central Oregon occur in the
prairie at about 4800 ft. elevation. In Yellowstone a maximum of about
0.6 g cal/cmr per min. of radiation in the visible range is received
at the surface in summer; a maximum of about 0.2 g cal/cm^ per min. is
received on a clear winter day (see Gates, 1961, 1962). Since most of
the thermal pools are very clear and the stream outflows very shallow,
little light extinction would occur. One would expect a large number
of sun-adapted forms little affected by the highest summer intensities
and exhibiting rather high growth saturation and compensation inten-
sities. Few laboratory studies have been made.
One of the most striking visual aspects of hot springs is the
multicolored algal pattern in which oranges and yellows are conspicu-
ous, particularly in summer. Sargent (1934) was one of the first to
investigate the color change in blue-green algae, using fairly modern
methods. He found that a high intensity of white light or of any broad
color band caused a change from dark blue-green to yellow in the non-
thermal Gloeocapsa montana, and that lowering the intensity reversed
this process. The color change could be explained solely by the reduc-
tion in cell chlorophyll content which was inversely proportional to
light intensity. Brock (personal comm.) has controlled the greening
and yellowing of high-temperature Synechococcus populations in situ
through the placement and removal of shades. With 0. terebriformis,
using light intensities from 200 to 4,000 ft.-c., I found a drop in
chlorophyll content per unit dry weight to less than 20% of the highest
value at 200 ft.-c. (coolwhite fluorescent). Total carotenoid pigments
dropped less than 507o in the same range of intensities. Thus, the
carotenoids become relatively more important with increasing light
intensity. The typical low-light or aggregated Oscillatoria is a dark
reddish-brown; the dominant color imparted by phycoerythrin. The tri-
chomes change to a pale ochre color with high light; the phycoerythrin
is reduced in parallel with chlorophyll (Castenholz, unpubl.). Similar
results were obtained with Anacystis nidulans (Myers and Kratz, 1955).
Thus, pigment adjustments occur in response to light intensity. Pre-
liminary evidence suggests that in _0. t:erebriformis the adjustment of
pigment ratios is a necessary prerequisite to the unrestrained maximal
mass increase. The photo-protective role of carotenoids has already
been well established, particularly in photosynthetic bacteria (e.g.
65
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Sistrom et al., 1956). Complementary chromatic adaptation and the
inverse type are also confirmed in the recent literature of the blue-
green algae (Fujita and Hattori, I960; Jones and Myers, 1965).
It is difficult to discuss the effect of light intensity inde-
pendently from temperature, since the two factors are so completely
entangled (e.g. Sorokin and Krauss, 1962). For example, at 45°C,
Oscillatoria terebriformis showed growth saturation at about 1,200 ft.-c.
while at 31°C saturation occurred at about 350 ft.-c. and the growth
rate was cut to about 20% of the 45°C value (Castenholz, unpubl.).
High light (3,000 to over 4,000 ft.-c.) is not inhibitory to the growth
of this organism at 45°C, but at 31°C, 1,500 ft.-c. depressed the rate
significantly. The aggregated mat in nature at high summer intensities
showed an average carotenoid to chlorophyll ratio equivalent to that
which would be expected with a constant exposure to about 1,000-1,500
ft.-c. in the laboratory (dispersed trichomes). The nature of the mat,
with possibly moving and interweaving trichomes, may prevent individual
trichomes from being exposed to a constant daytime high light intensity
or to the low intensity of continual submergence in the mat. The mat
as a unit might be operating at less than a saturating light intensity,
particularly in a light-dark regime.
For Synechococcus lividus (Yellowstone--50°C), Sheridan (1966)
also found a high light intensity for photosynthetic saturation at
45°C (600-900 ft.-c.). Dyer and Gafford (1961) showed lack of satura-
tion at 400 ft.-c. in Synechococcus lividus from 45° to 55°C; 1,500
ft.-c. was the next highest intensity used. Fukuda (1958) showed that
photosynthetic saturation in the acidophilic thermophile, Cyanidium
caldarium, fell between about 1,000 and 10,000 ft.-c. when CC>2 was not
limiting. The highest intensity (10,000 ft.-c.) was not inhibitory.
In acid thermal creeks in Yellowstone Park (e.g. Roaring Mt. creeks--
pH 2-3) it is obvious that above 50-55°C, high natural light intensity
becomes quite limiting to growth near the upper temperature limit
(ca. 60°C). In this thermal range visible covers of Cyanidium appeared
only in shaded niches.
In thermal springs there is some evidence to indicate that photo-
synthetic incorporation of Cl^ into the algal mat fluctuates in paral-
lel with natural sun-plus-sky radiation even during the summer period
of generally high energies (Lenn, 1966). Stockner (1967) has shown
that the growth of Schizothrix calcicola (=Synechococcus sp.) in
Ohanapecosh Hot Springs (Mt. Rainier National Park) paralleled very
closely the average sun-plus-sky radiation flux over the course of a
year. A difficulty encountered in measuring the native rates of photo-
synthesis or growth relative to light intensity is the thickness of
the algal mat. The active chlorophyll-bearing cells occur only in the
66
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Thermophilic Blue-Green Algae
upper few millimeters, but the density of this top layer may be great
enough to cause significant reductions in the light intensity received
by the lower cells. Thus, light intensity reaching the surface cells
may seldom be limiting while a large portion of the buried cells may
be receiving light well below saturation levels. C^ incorporation by
sections of the mat may represent the average responses to a complete
gradient of light intensities.
DISSOLVED SOLIDS OF THERMAL WATERS AND NUTRITIONAL ASPECTS
The total dissolved solid content of hot springs varies considerably
from area to area, but there are few with less than 500 mg/1 TDS and
more than 2,500 mg/1. An average value might lie somewhere above
1,000 mg/1. These values are well above those of the normal stream
and lake systems which receive most of their water from relatively
recent precipitation. Practically all of the 3,000 or more hot springs
in Yellowstone Park have salinities between 1,000 and 3,000 mg/1 (Allen
and Day, 1935). Extremes are of interest. Medicine Hot Springs,
Montana, has less than 250 mg/1 TDS and a hot spring in Iceland issues
seawater enriched to over 47,000 mg/1 but with a pH of 5. The latter
appears to be devoid of algal growth (Brock, personal comm.). Other
very saline hot springs are known in the salt dome regions of northern
Utah, and springs with salt concentrations of over 15,000 mg/1 in
Greece have an abundant blue-green algal flora (Anagnostidis, 1961).
Halophilic blue-green algae are well known as some of the predominant
organisms in inland saline lakes.
The mineral content of many or most hot springs probably remains
very constant. However, there are exceptions in which the concentration
at the source may vary erratically or seasonally, often under the
influence of rainfall in the watershed. The ratios of certain elements
may also change considerably within a short period (Uzumasa, 1965).
Most hot spring waters represent a mixture of heated ground water and
magmatic water arising from superheated steam. One source may be
enriched at the expense of the other.
Hot springs may be classified in several ways. A simple method is
to recognize three principal types: alkaline, acid-sulfur, and
calcareous springs. The alkaline type comprises over three-fourths of
the earth's hot springs. They are generally rich in sodium, bicarbonate,
and chloride. Silica is usually quite high (>200 mg/1) and sulfate
varies with locale. The pH may range from about 6.0 to 9.0 and siliceous
sinter is the main deposit. Acid-sulfur springs are almost invariably
associated with recent or current volcanic activity. They commonly
have pH values of less than 3.0 and are initially high in hydrogen
sulfide. Sulfur compounds of various oxidation states, including
sulfuric acid, abound. They, of course, lack bicarbonate, have little
67
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
chloride, but may also be very rich in silica. Calcareous hot springs
are near neutrality and are enriched in calcium, magnesium, and
bicarbonate. They have far less silica than the other types. A rich
supply of CC>2 results in CaC03 (travertine) deposition instead of sinter.
The very acid springs, although often presenting a full thermal
gradient, seem to lack all photosynthetic organisms below pH 4 or 5
with the exception of Cyanidium caldarium which inhabits the thermal
range from about 60° to 35°C. Cyanidium is certainly not a blue-
green alga but has a pigment system resembling that of the Cyanophyta
(see Mercer et al., 1962).
Most hot springs, including the abundant alkaline types, are greatly
enriched in certain elements which occur in temperate lakes and streams
in traces only. Some of these are required micro-nutrients. Many are
known to be poisonous to microorganisms at concentrations far lower
than those occurring in many hot springs. High temperature, anaerobic
conditions, and a source of super-heated magmatic steam contribute to
the enrichment of several rarer elements in thermal spring issuances.
The continued solubility of most, after the water has reached the surface,
is greatly dependent on pH among other factors. Uzumasa (1965) reports
the following extreme values of some of these elements in hot springs
of Japan (in mg/1): aluminum--1,000, arsenic--5.1, cobalt--2.19 ,
copper--68.0, fluorine--16.0, iron--l,000, lead-^2.60, nickel--9.38,
manganese--278.0, and zinc--2.00. The greatest enrichment of the metal-
lic elements occurs in the more acid waters. Arsenic concentrations may
be high in either alkaline or acid waters. Fluorine is more prevalent
in mildly alkaline waters. The median value for fluorine was between
1.0 and 2.0 mg/1 in Japan. In Yellowstone Park, values from 15.0 to
20.0 mg/1 were common (Allen and Day, 1935). In Japan the mean value
for arsenic in 174 neutral or alkaline springs containing the element was
0.28 mg/1. It was about twice this value in 16 acid springs (Uzumasa,
1965). Values of over 2.0 mg/1 were common in Yellowstone. In the acid
springs of Hokkaido the mean concentration of copper was about 98 u.g/1 and
in the neutral to alkaline springs between 20 and 30 [j.g/1. Manganese
levels are higher in the acid to neutral springs but may be significant
at least up to a pH of 8.0. The average value for manganese-containing
Japanese springs is 2.3 mg/1. Manganese is also one of the enrichments
of some neutral to alkaline hot spring groups in Yellowstone. Black
Mn02 precipitates are formed for great distances in the drainways, often
well below the thermal range.
There are some reports of manganese toxicity to algal growth at
normal limnetic and at higher (hot spring level) concentrations (cited
by Wiessner, 1962). The toxic effect of copper on algae is well known
even at levels below 10 ug/1. The toxicity of arsenic and fluorine
68
-------
Thermophilic Blue-Green Algae
compounds is also well documented. However, the tolerances of most non-
thermal and thermal algae to the above elements have not been established.
In the few biological investigations of hot springs, analyses of the
minor elements present were not made. I am implying, however, that the
chemical composition of some hot springs may have a severe excluding
effect. Toxic factors may account in part for floristic or productive
differences among various hot springs which may appear alike superficially,
There is at this time no evidence to suggest that any of the known
micronutrients of blue-green algae occur in suboptimal limiting amounts
in thermal waters. Thermal waters, having high salanities in general,
might be expected to be eutrophic in a complete sense. Unlike saline
lakes, however, which are usually rich in all macronutrients, including
combined nitrogen and phosphorus, thermal springs can be quite deficient
in ammonium, nitrate, and phosphate. Very minute concentrations,
nevertheless, may be adequate in a constantly flowing environment.
Unfortunately, few springs have been analyzed for these important
nutrients.
In Japan ammonium was detected in less than one-half of the 860
hot springs analyzed, but in those it averaged about 1.6 mg/1 (Uzumasa,
1965). Nitrate, also derived directly from percolating ground water
or from the oxidation of ammonium, was found in only 1570 of the Japanese
springs and in those averaged less than 0.3 mg/1. Although detectable
in only 37% of the Japanese springs, phosphate averaged about 6.5 mg/1
in those, which is a very high biological value. In Hunter's Hot
Springs, Oregon, nitrate-N was undetectable at a main source, but
increased substantially about 20 meters downstream after passing over
algal mats and receiving some drainage water from the shore. Values
from 42 to 142 |j,g/l were recorded at different seasons at the downstream
station where temperature had decreased to 45° C. Phosphate-P also
increased greatly downstream (to over 100 ug/1), possibly from shore
drainage, although values as high as 40 ug/1 were found at the source.
Brock (personal comm.) found more than 2.0 mg/1 phosphate in an
alkaline Yellowstone spring in which he is studying productivity. He
indicated that there was little change downstream. Values of phosphate
as high as those mentioned for Yellowstone and Japan would be considered
inhibitory levels for some "oligotrophic" algae. On the other hand,
Brock found little bound nitrogen in his spring. ^ is one of the
common gases in alkaline hot springs unassociated with volcanic activity,
but may be extremely rare in others. The volcanic (magmatic) gases are
generally C02, ^S, and H2. Vera Dugdale (in this symposium) reported
active nitrogen fixation in mats of Mastigocladus in an Alaskan hot
spring. Sheridan (1966) found no fixation in an axenic culture of
Synechococcus in my laboratory (Y50BII). Probably no species of this
unicellular genus are capable of utilizing N2. Some spring waters
69
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
initially low in bound nitrogen may greatly restrict the development of
high temperature Synechococcus strata, while N-fixing algae, characteristic
of the middle and lower parts of the thermal range, would not be impaired.
The eutrophic nature of some hot springs is probably reflected by
results seen after a number of hot spring drainways have emptied into
a small river which drains a normal type of watershed in its upper
reaches. The standing crop of algae and macrophytes in the Firehole
River, Yellowstone Park, increases noticeably below the tributaries of
the Upper Geyser Basin. Greatly increased photosynthetic rates were
also measured below these additions (Harrington & Wright, 1966).
Presumably any toxic factors would be sufficiently diluted when enter-
ing the river.
In general, the thermophilic blue-green algae seem to act as eutrophic
organisms. A large variety of species from Oregon, Yellowstone, and
Iceland have been cultured in my laboratory in a heavily fertilized
defined medium (Table 1). The medium was formulated by R. P. Sheridan
in my laboratory, particularly for S_. lividus (Sheridan, 1966) . It is
also used for the experimental work with Oscillatoria terebriformis.
The addition of vitamins B]^, thiamine, and biotin does not appear to
stimulate growth in Synechococcus or 0_. terebrif ormis . Sheridan (1966)
failed to obtain heterotrophic growth of S_. lividus in the dark. However,
the lack of heterotrophic ability is the general rule for the Cyanophyta.
Among photosynthetic thermophiles it is well documented only in Cyanidium
caldarium, in which growth in the light is also stimulated by galactose
and malt extract (Ascione et al., 1966).
It would be interesting to have information on the concentration
of various dissolved organic compounds at different stations in thermal
streams and on the nature and abundance of heterotrophs. The major
portion of the gel-like mats of most hot springs, under the thin photo-
synthetic upper layer, is probably composed of non-chlorophyllous,
carotenoid-containing filaments of 1 u or less in diameter. These
should probably be referred to the "flexibacteria" instead of the
blue-green algae (see Lewin, 1962). The detailed composition of
thermal mats and the reasons for the slow mineralization are of
continuing interest.
I would like to acknowledge with thanks the aid of the National
Science Foundation grants G-19972 and GB 2951 for work done in my
laboratory. I would also like to thank Mr. John Good, Chief Naturalist,
Yellowstone National Park, for permission to collect algae in the Park.
70
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JChermophilic Blue-Green Algae
Table 1
Culture medium for thermophilic blue-green algae
(after Sheridan, 1966)
The components are added in the order listed. For normal use the pH
is adjusted to 8.2 with NaOH. The turbidity resulting from steam
sterilization clears completely in less than 24 hours. The final pH
is 7.5. A ten-fold concentrated stock solution is usually used; it
is stored at 4°C unsterilized. NaOH is added after dilution and before
autoclaving.
Distilled water 1,000 ml
Nitrilotriacetic acid (NTA) 0.1 g
Nitsch's Trace Element Solution 0.5 ml *
FeCl3 solution 1.0 ml **
CaS04.2H20 0.060 g
MgS(V7H20 0.100 g
NaCl 0.008 g
KN03 0.103 g
NaN03 - 0.689 g
Na2HP04 0.111 g
* Nitsch's Trace Element Solution
Distilled water 1,000 ml
H2S04 (cone.) 0.5 ml
MnS04.H20 2.280 g
ZnS04-7H20 0.500 g
H3B03 -- - 0.500 g
CuS04-5H20 0.025 g
Na2Mo04.2H20 0.025 g
CoCl2'6H20 0.045 g
**FeCl3 Solution
0.2905 g FeCl3 in 1 liter water (1 ml = 0.1 mg Fe)
71
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Lbwenstein, A. 1903. "Uber die Temperaturgrenzen des Lebens bei der
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Marre, E. 1962. "Temperature." In: R. A. Lewin (ed.), Physiol.
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Militzer, W. and L. Burns. 1954. "Thermal enzymes. VI. Heat
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Sargent, M. C. 1934. "Causes of color change in blue-green algae."
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GROWTH REQUIREMENTS OF BLUE-GREEN ALGAE
AS DEDUCED FROM THEIR NATURAL DISTRIBUTION
William C. Vinyard
Humboldt State College
Arcata, California
Blue-green algae constitute an assemblage of organisms ranging
over a vast array of ecological situations. Their physiological capa-
bilities are more varied than any other algal group. Their ancient
origin has permitted much in the way of evolutionary advance, but this
has resulted in physiological variants far in excess of morphological
types. It would be hopeless, then, to attempt a discussion of environ-
mental requirements of all blue-green algae. This discussion will be
limited to those primarily of concern to man in his water supplies.
Relatively few genera of blue-green algae have become important
economically, but studies of these have resulted in sufficient informa-
tion to permit certain generalizations as to environmental requirements.
Growth appears to be favored where conditions of low mineral concentra-
tion and high organic material content exist. Organic pollution almost
always insures an overabundance of blue-green algae. Additionally,
excessive blue-green algal growth occurs during periods of high water
temperature and light intensity, and these are, of course, related to
climate and weather, and also to latitude. These generalities suggest
that some areas or bodies of water may become susceptible to troublesome
growths whereas others may remain safe indefinitely, or at least until
the above conditions are met.
Numbers of organisms are of concern to this discussion. The mere
presence of a given species does not cause water quality problems even
though a given organism is well known as a troublemaker. It is only in
excessive amounts that any organism becomes ecologically or economically
important. The environmental conditions that permit mere presence, or
existence, of individuals differ from those which stimulate a bloom;
but it is possible that the conditions differ only as a degree of con-
centration of certain environmental factors. It appears to be usual,
however, that when the growth curve of a population of blue-green algae
starts up, it continues to a maximum.
The identity of an organism, or organisms, becomes important
because it is well established that some—but by no means all--species
of blue-green algae become involved in blooms or otherwise are of
nuisance value. Thus, taxonomy is of importance.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
It has been said that the greatest hindrance to the ecologist is
taxonomy. However, this statement may be interpreted as: Even the
taxonomist is hindered by the taxonomy of the blue-greens. The problems
here are met at the generic as well as at the specific level, and much
has yet to be accomplished in providing a system acceptable to most
taxonomists. There are now in use basically two approaches to taxonomy
of the genera of coccoid blue-green algae (Drouet and Daily, 1956;
Geitler, 1932), and each has its followers among the taxonomists. As
yet, there is no more recent critical study of the filamentous forms
than that of Geitler (loc. cit.). Hopefully, a meeting such as this
will provide the encouragement for a concerted effort to update our
taxonomy, perhaps by incorporating information from physiological studies
and by stimulating cultural studies which contribute to the understanding
of range of morphological variability in at least the more important
members of the blue-green algae.
Any consideration of distribution of organisms should include the
geographical aspects, or patterns of distribution. Apart from the
purely descriptive results, clues to environmental requirements might
then be forthcoming. It is useful to describe species distribution by
the terms endemic, disjunct, or cosmopolitan, depending in turn on
whether they are limited to a very small geographic area, are found
in a few scattered localities but are absent between, or whether they
are universally found. Each of these suggests genetic adaptation to
an environment of a very restricted sort, or to most environments. But
here we lack information on distribution—except for troublesome occur-
rences which get the publicity—and almost nothing is known about blue-
green algal genetics, even of the troublesome species. It is probably
true at this time that any summary of our knowledge of geographical dis-
tribution would only expose the distribution of the blue-green algal
taxonomists.
Another aspect of distribution is that of succession, or distri-
bution in time. Some species are found in the same body of water when-
ever sampled, but others may be seasonal or of sporadic occurrence.
This, of course, complicates the mapping of patterns of distribution.
The possibility of migration must also be considered, and the environ-
mental changes as wrought by man will continue to have their influence.
Some specific cases may be useful in illustrating some of the
problems just outlined, and, hopefully, they may suggest new means of
approach.
First is a problem of taxonomy at the generic level. During the
summer of 1964 a group of us from the University of Montana Biological
Station observed a bloom of Gloeotrichia echinulata in a small lake
(Rainbow Lake, formerly Dog Lake, 50 miles southwest of Flathead Lake).
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Growth Requirements of Blue-Green Algae
Except for its planktonic habit, this species here differs from other
Gloeotrichia in lacking akinetes (gonidia), and this characterizes
Rivularia when present. Some authors, incidentally, include the two
genera under the name Rivularia. Blooms of Gloeotrichia in this lake
have been observed in successive summers as well.
I have observed a case of a more or less continuous bloom of blue-
green algae (with Botryococcus) for a period of five years in Freshwater
Lagoon just south of Orick, California, on the coast. This lagoon is
separated from the ocean by a sand bar, along which U. S. Highway 101
passes. Apparently, marine water does not get carried into this lagoon.
The blue-green algal components of the bloom include the genera Micro-
cystis, Coelosphaerium, and Anabaena. Limnological data are not avail-
able, but it is notable that this condition occurs in a relatively cool
coastal environment.
A situation with many ramifications is illustrated by the algae in
oil field sump ponds, on which, apparently, nothing has been published.
I have discovered a rather startling array of algal types associated
with crude oil and tars in investigations over a period of years in the
States of Oklahoma, Kansas, and California. A few observations seem
pertinent here, but the details will be published elsewhere.
Blue-green algae predominate in these habitats, often to the
exclusion of other algae (diatoms, green algae, Trachelomonas spp.).
In one Kansas sample which appeared to be a semi-solid tar from the
bottom of a sump pond, Schizothrix Friesii Com. made up the bulk of
the "tar," with but few water droplets apparent. This species,
incidentally, is a common soil alga in Kansas. At the opposite extreme
of substrate "texture," more or less clear samples contained minute oil
droplets in suspension with diatoms entrapped within, but these samples
lacked blue-green algae.
Floating "tarry scums" from California— contained predominantly
Oscillatoria spp. and clearer samples included coccoid blue-greens.
The algal growths in oil field sump ponds may be conspicuous, or
they may not, as compared to the substrate. When apparent as floating
scums, the color ranges from pale blue-green (pea soup color) to
yellowish or brownish. When not apparent, as in the benthic tar
Schizothrix mentioned above, the algae may be present in abundance.
i/The California collections were made with the help of, and studied under
contract with,the California Department of Water Resources, San Joaquin
Valley Drainage Investigation.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
There were significant differences in algal floras depending on
whether the oil field contained brine water or non-brine ("freshwater"
might not here be equivalent). The blue-greens predominated in the
non-brine oil fields.
Though quantitative measurements were not made (methods have yet
to be devised), certain deductions appear to be worthy of consideration.
It can be presumed that a minimal amount of light, or none, can pene-
trate the medium, though this will vary with the relative concentra-
tions of water and oil or tar. It appears that certain blue-green
algae can thus thrive in crude oils in the absence of light. Further-
more, there appears to be no possibility of diffusion of gases into,
or out of, the medium.
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REFERENCES
Drouet, F. and W. A. Daily. 1956. "Revision of the coccoid
Myxophyceae." Butler Univ. Bot. Stud. 12:1-218.
Geitler, L. 1932. "Cyanophyceae." In: L. Rabenhorst, Krypto-
gamen-Flora von Europa 14:1-1196.
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RECENT ADVANCES IN THE
PHYSIOLOGY OF BLUE-GREEN ALGAE
Osmund Holm-Hansen
Scripps Institution of Oceanography
La Jolla, California
I would like to reschedule the topic of this talk from "Recent
Advances in the Physiology of Blue-Green Algae" to just "Odds and
Ends," because that is just about what this is going to be. Having
been cloistered for eight years in a temporary building at the Uni-
versity of Wisconsin with George P. Fitzgerald, I had no idea that so
many blue-greenologists could be assembled at one time in one place.
It is indeed a most pleasant surprise to find this. Another surprise
not so pleasant to me, however, is finding myself on this platform
right now. I had withdrawn my contribution for this occasion several
months ago, but due to a bad confusion in the U. S. mail and to my
being away in Tokyo, I had not planned to be here. But then at the
last minute, I decided to come and listen to the speakers and absorb
a lot of good information and meet old friends. I arrived last night
and was rapidly persuaded by Drs. Dollar and Oglesby to present a few
remarks which might be slightly provocative to a few people. I hope
they are. I also hope that you will excuse any serious omissions I
might make due to this completely off-the-cuff presentation and correct
an erroneous statement or concept which I might give forth.
In addition to the reasons for studying blue-green algae which were
stated this morning, I would like to add a few of my own. First, due
to their cellular organization, their chemical composition, and many of
their physiological attributes, the blue-greens, as you all know, occupy
a position of great importance and interest in regard to questions of
phylogeny and evolution of higher plant forms. In this connection, I
would like to call your attention to the numerous statements which you
hear and read in the literature and textbooks and reviews concerning
the blue-greens--the relationships between blue-greens and the colorless
blue-green algae, and to such organisms as Cyanidium and Glaucocytis,
and to the rather peculiar and ubiquitous group of organisms which Ralph
Lewin and Soriano have written quite a bit about, the flexibacteria.
The relationships within this great assemblage of microorganisms demand
some very good analytical chemical work in the future to either rein-
force or to refute the proposed closeness of all this vast assemblage
of microorganisms, both photosynthetic and colorless forms. In view of
the obligate autotrophic nature of many blue-green algae, a subject
which I will discuss later on today, I personally find many of these
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
assumptions and statements in this connection about the closeness of
all these microorganisms to be rather questionable. It remains for
further analytical work to show which way the phylogenetic pathways
will go.
The second major area for studying blue-green algae, in my esti-
mation, is to obtain information on basic problems in molecular biology.
Now, for problems in molecular biology, you might as well use E. coli
or Chlorella or a spinach leaf obtained from the local A&P supermarket
as long as that does the job. However, if a blue-green algal cell can
supply some special characteristic or information to your problem, then
the blue-green is advantageous to use, of course. In this connection,
I would like to run through about ten various problems in microbiology
where I think blue-greens have contributed, and will continue to con-
tribute, much information. The first one is involved with the basic
morphology and function of the blue-green cell. As you all know, the
basic structure of the blue-green is very similar to that of bacteria.
It is a procaryotic cell and does not have the organized organelles of
all higher plants and animals. In all eucaryotic cells, you have the
packets of enzymes organized in such organelles as the mitochondria,
the chloroplasts, the nuclei, etc. Evidence is now accumulating that
the enzymes are oriented very specifically so that substrates can be
passed down along the whole chain of enzymes much in the fashion of a
zipper. Now what happens in the case of blue-green algae? We don't
have any organelles there. If you look at the microscopic picture of
blue-greens, what do you see? You see the various layers surrounding
the cell, you see an ill-defined peripheral portion containing many
lamellae, and a nebulous area of the centroplasm, which is very often
traversed by the lamellae which are found predominantly around the
peripheral portion. But they do transverse the central portion as well.
Where do you get the packets of enzymes and how are they oriented in
the blue-greens compared with all higher plant and animal cells? Now,
this is a question which is of the utmost importance in cell growth and
differentiation. With the blue-greens, unfortunately, it is very hard
to study this problem because, for one thing, you break the cell and
you cannot isolate nice, clean entities such as chloroplasts, mito-
chondria, or a nucleus. So the nature of the problem is exceedingly
difficult, and although many people are interested in it, I know of no
good data which have been accumulated, or do I see any real hope for
achieving any breakthroughs in this area.
The second area involves gliding movements of many of the blue-
greens, most fascinating movements to watch under the microscope, as
many of you know, but also it is a phenomenon which is not explained
at all. There is little evidence as to means of locomotion as there
are no flagella or cilia, and no muscular threads of any kind. It is
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Physiology of Blue-Green Algae
apparently associated, as we heard this morning, with phosphorylation
and ATP, but the basic mechanism of how a filament can glide through
agar or wave back and forth is entirely unknown. It is an extremely
interesting problem.
Another area is nitrogen fixation. Here the blue-greens have con-
tributed quite a bit of information. Blue-green cells have been broken,
and cell-free systems have been isolated by the Wisconsin group of
Burris, Wilson, Singh, and Nicholas in which they have actually gotten
nitrogen fixation. One interesting point which has come out is that
cobalt apparently is intricately involved in one of the early steps of
nitrogen fixation, although it is not pinpointed at all, as yet. It
is extremely difficult to get at because of the lack of any suitable
radioactive isotope of nitrogen. They use •'•-'N, which is the heavy,
stable isotope.
Another area, the fourth one, is the function of accessory pig-
ments in photosynthesis, an area in which the blue-greens and some of
the reds have contributed immensely to our understanding of the physical
chemistry and biochemistry associated with photosynthesis.
A fifth area is the question of the seemingly obligate autotrophic
nature of all blue-green algae. Many of you will disagree with this
statement, but please wait until the end of the talk to question me on
this point. But for now, I'll call them obligate photoautotrophs.
Another area involves the microelements and nutrition of blue-
green algae, problems which have taken a lot of time for many investi-
gators. It looks rather routine to me for the essential macro- and
microelements, with one exception. That is the situation with cobalt,
where cobalt has been demonstrated to be an essential microelement for
all blue-green algae which have been investigated. It is implicated
in nitrogen fixation by Burris1 and Wilson's results, and also by my
earlier results at Wisconsin. It might well play--in fact, I'm quite
sure it does—some non-nitrogen-fixing role in the blue-greens. It is
required by Goccochloris, by Microcystis, and many of these cells that
don't fix nitrogen, so it is involved in a number of metabolic sequences,
such as in methylation, nucleotide reduction, vitamin B-*- , etc.
Another area, the seventh one, is the area of the excretory
mechanism and the excretory product of blue-green algae. Here we get
into a factor which is very important ecologically in that the blue-
greens, apparently from laboratory studies, at least, do excrete lots
of organic metabolites from the cell back into the extra-cellular solu-
tion. How they do this is not known, and in addition to the oxidizable
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
substrates they kick out, there is also the very important factor of
the toxins they produce which, as you know, can be economically very
important.
The eighth area is the effects of light in a non-photosynthetic
mechanism. There is evidence from Lazaroff that light also plays a role
in the differentiation of the cell and in the completion of what he
calls the sexual life cycle of blue-green algae. To my knowledge, his
work has not been duplicated or repeated successfully by anybody. It
looks like nice work to me, but a lot more work has to be done to know
exactly what is happening here.
In line with that, there is the question of how the genetic infor-
mation (the DNA) is passed along from generation to generation with
such consistency in the blue-green algae. There is no mitosis, as we
know it in higher plants and animals, and the exact mechanism of this
division of DNA between two daughter cells is not well understood. The
question of sexuality is also one which is not at all understood; most
textbooks claim that they are asexual, putting the poor blue-greens in
a rather dismal pile on the bottom. People like Lazaroff claim that
they do fuse and engage in sexual transformations. Again, this has
not been duplicated, to my knowledge, by anybody else. That was mostly
a microscopic examination, but I'm still waiting for more work before
I decide for myself whether that is really so or not.
Another area is the area of cellular differentiation where I think
blue-greens are very promising organisms for experimental work. As you
know, there are several types of vegetative cells, akinetes, hetero-
cysts, various spores, and the formation of these can be controlled to
some extent. There has not been much experimental work along this line,
but I think it is a very fruitful one for future investigators.
Well, these are some of the areas I wanted to talk on, but then
last night after saying I'd chat for a few minutes I read this brochure,
and I read that we are supposed to limit our comments to the environ-
mental requirements of blue-green algae, so I realized that I would have
to dispense with all those former topics and talk about something else.
So I decided to say a few words in the remaining minutes on six subjects-
it's going to be fast, though. One is temperature, the second is des-
sication, the third is light, the fourth is nutrients, the fifth is pH,
and the sixth is heterotrophy, on which I'll spend about five minutes.
In regard to temperature, we heard quite a bit from Dr. Castenholtz
this morning on the effects of high temperature, so I will say nothing
more about high temperatures. As the temperature goes down and passes
through the zero centigrade point, there are two major areas of concern
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Physiology of Blue-Green Algae
to the cell. One is with decreased temperature; there is a decrease in
the enzymatic rate of all the chemical reactions and the various meta-
bolic patterns and routes can be upset, which can lead to damage or
death of cells. The second major area of concern to the cell would be
the fact that when the freezing point is reached there is a phase
change where liquid water freezes to give the solid state. This is the
crucial point, the most damaging point of freezing damage, it appears.
First, there is the possibility of deformation or physical damage from
rupture of the cell due to ice formation. Secondly, freezing out of
solutes occurs so that there are osmotic changes within the cell which
can lead to denaturation of the proteins, etc. So for the initial
phase change, it is a period of dramatic concern and potential damage
to all cells. As you heard this morning from Dr. Castenholz, I collected
quite a few blue-greens, greens, and diatoms in the Antarctic about four
or five years ago; during the past few years, I have used these isolates
plus about 20 species isolated from the Wisconsin lakes, in low-tempera-
ture studies. I studied them in regard to survival after rapid freezing
versus slow freezing, retention of viability after freezing, thawing,
freezing, thawing, up to 15 or 20 times. All the Antarctic forms I
isolated were, as you would predict, very capable of surviving the
freezing and thawing process. Of the ones isolated from the Wisconsin
and Midwest area, a good many showed fairly good survival. Some of
them, such as Nostoc, were very good; they were as hardy as anything I
got in the Antarctic. Many of the ones, such as Phormidium and Oscil-
latoria, were horribly sensitive; they would not survive even one freez-
ing. Many from the Antarctic isolates, such as Schizothrix and Nostoc
and some of the Anabaenas, could be frozen and refrozen 19 or 20 times,
with no difference in the apparent viability. Many of them could be
frozen with liquid nitrogen, or slow frozen by gradual decline from
zero to minus 15 or 20 degrees. Most of the Antarctic ones survived
slow freezing better than rapid freezing. This is true for nearly all
the temperate as well as all the polar algae I worked with. With slow
freezing there is less damage than with rapid freezing, which is rather
contradictory to what you would expect and predict by reading the ele-
mentary textbooks. Slow freezing leads to bigger ice crystals and
potentially more damage, you would think, but it apparently does not
work that way. One other interesting thing about the blue-greens is
that the ability to survive freezing is not aided in any way by the
addition of organic adjuvants to the suspension of cells before freez-
ing. This is in sharp contrast to all the greens, where the addition
of horse serum or glycerol, glutamate, milk powder, etc. will increase
very greatly the number of cells that survive. But with the blue-greens,
it has absolutely no effect. You can do it in distilled water or horse
serum or blood, or anything else that you like, and the results are
always the same. In regard to freezing damage, Levitt has theorized on
why some cells are so capable relative to other forms of life in regard
to surviving freezing. It involves the sulfide-disulfide linkages and
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
the fact that you apparently have fewer new intermolecular disulfide
bonds formed by the proteins of low temperature resistant cells. There
is some experimental evidence for this, and it is a rather interesting
and fruitful area for further investigation. In this regard, I was
going to ask Dr. Castenholz this morning about speculations on the
characterization of the proteins which enable them to survive 90°, but
I got too hungry, so I'll have to see him tomorrow. So much for topic
one.
Dessication from the frozen state is of immediate concern in both
Polar regions. Freeze-drying involves first freezing the cells and
then sublimation of all the water molecules directly from the solid
state to the gaseous state. In this way, drying can be effected with
minimal shrinkage and deformation of the tissue. Unless the initial
freezing has resulted in damage to the cells, the cells should be
undamaged. Theoretically, it should be possible to freeze-dry almost
any cell if you can get rapid enough temperature gradations to inner
parts of the cell. I have worked with both Antarctic and Wisconsin
algae a great deal in connection with freeze-drying. In the Antarctic
this is a natural phenomenon and a danger which cells have to face
every day of the year the whole year round. The microclimate is
extremely important, depending on whether the sun is shining, whether
the cell is in the lee of a rock, etc. The low air temperatures
coupled with the very low humidity in the atmosphere result in a freeze-
drying of any cells exposed to the atmosphere. My laboratory studies
with the Antarctic blue-green algae indicated that most of them were
capable of surviving freeze-drying very well. All these tests, by
the way, are of necessity semi-quantitative because I have not been
able to plate out any unicellular blue-green alga and get a 100%
recovery of the number of cells. In other words, if you plate out
100 cells of Anacystis, you cannot get 100 colonies as you can if
you plate out 100 cells of Chlorella, plus or minus 2 or 3% for the
statistical error. But with blue-greens I have not found any method
for doing this, so I place the sample into a liquid culture in an
Erlenmeyer flask and examine it once or twice a day and count the
days until the growth becomes visible. This is roughly proportional
to the number of viable cells, and this is a very rough, semi-
quantitative procedure, but it is the best we have available until
somebody can tell us more about how to culture these tenacious blue-
greens. For any filamentous blue-green algae there is the additional
problem of how to separate the whole chain of cells into a unicellular
suspension. One interesting thing about a freeze-dried cell is that
the ability to survive high temperature is greatly increased. I can
take lyophilized samples of my blue-green algae and heat them at 100°C
for over an hour and rehydrate them, and they will have undergone no
damage during that heating process. In the hydrated state they cannot
survive 35 or 40° C for a few minutes.
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Physiology of Blue-Green Algae
I would like to add just a few comments on pH because we had quite
a bit of discussion this morning and I hope we have more tomorrow.
Blue-green algae require very different pH conditions than do most
other types of algae. Some blue-greens will grow at pH values down to
about 7.0, but the optimum pH range seems to be about 8.0 - 10.0. In
some healthy laboratory cultures the pH has been measured very close
to 11.0. Such a high pH requirement is interesting in regard to the
question of the form of inorganic carbon utilized in photosynthesis.
In other algal groups it has been shown that C02 or bicarbonate is
preferentially assimilated during photosynthesis. By reference to a
curve showing distribution of CC^, HC(>j, and C0=j as a function of pH
(e.g., see Photosynthesis by Rabinowitch, Vol. I, p. 177), it is clear
that at pH 11 there is extremely little free CC>2, a few percent bicar-
bonate, and a high concentration of carbonate ions. Does this mean
that the blue-green algae are assimilating carbonate ions preferentially,
in contrast to most of the other algal groups? To my knowledge there
has been no work on this problem with the Cyanophyta, so I cannot give
you any answer to this question.
Concerning light, one thing that has always bothered me in the
laboratory with blue-green algae is that you have to use low light
intensity. With all the blue-greens I have grown I have used a light
intensity less than 1000 ft.-c., and usually about 100 or 200 ft.-c.
If you have a very dense suspension of Nostoc, which will attenuate
the light markedly, a much higher light intensity can be used. But
with a dilute suspension relatively low light intensities must be used.
Now this disturbs me very much because in their natural environment
they are exposed to very high light intensity of 8,000-10,000 ft.-c.
This contradiction between laboratory growth and field occurrence is
a troublesome one, and to me it indicates that our culture methods are
probably pretty lousy--that we are not obtaining healthy, normal cells,
but are obtaining pretty debilitated, semi-sick cells. The great
change in morphological shape of many blue-green algae from a lake to
a laboratory would also lend evidence to this conclusion. I hope I
don't step on anyone's toes when I describe your cultures as sick cells.
But there is--I think most of you will agree — the fact that morpho-
logical appearance of cultured cells, cells which have been cultured
for a long time in the lab, really do deviate quite a bit from a nice,
healthy cell which you pick out of the lake. So much for the light.
The next section is nutrients. Again, to my knowledge, there is
no organic nutrient requirement for any blue-green alga. In regard to
inorganic nutrients the essential major elements (C, H, 0, N, Ca, Mg,
S, K, P) are the same for all organisms. Some people might question
the calcium requirement, but I would be prepared to put up a good argu-
ment for that one. The essential microelements are iron, boron, manga-
nese, molybdenum, copper, zinc, and cobalt; to this list I think chloride
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
and sodium should also be added. There are a number of other elements
which may be essential, but no convincing case has as yet been made
for any of them with blue-green algae. Manganese is a rather interest-
ing nutrient in regard to pigmentation. In a Mn deficient culture of
Nostoc muscorum the chlorophyll content is extremely low while the
phycocyanin content is more or less like the normal cells; the cells
thus have a distinct bluish color. Boron, molybdenum, and cobalt also
cause changes in pigment complement, but the changes are not as strik-
ing as with Mn deficiency.
Concerning the quantitative requirement of all the inorganic
nutrients, George Fitzgerald is following me on the platform; from my
knowledge of George, I am sure he will do a thorough job there, and
I'll dismiss the subject with that.
We then get to heterotrophy, which is the last thing I'll be talk-
ing about. Many years ago, back in the twenties and thirties, people
like Harder, Allison, Hoover, and a handful of other people, published
some rather nice work claiming that various blue-greens, particularly
Nostoc and Anabaena, could grow in the dark. This information, coupled
with the occurrence of blue-greens in Nature, where they are very com-
monly found in water which is very rich in organic nutrients, such as
we have heard several times today, led to the general belief by laymen
and many phycologists and biologists in general that nearly all, if
not all, the blue-green algae are capable of both autotrophic or
heterotrophic growth. Now this statement has crept into all sorts of
reviews and long essays on blue-green algae. I fooled around with it
for many years, because I got slightly perturbed when investigators
would write that they grew Nostoc in the dark without giving any data
and no good description of exactly how it was done. This was done by
many well-known people in this country and in other countries without
any published data; I would like to see if anyone can give me any
data, either today or tomorrow, in favor of heterotrphic growth in
complete darkness of any blue-green alga. For quite a few years now
I have used quite a few different species of blue-greens and a great
variety of organic substrates, and the net result is that I have
found absolutely no heterotrophic growth as long as you have complete
darkness.
Now, in the last few minutes I would like to give you some
information which I think might well explain the many discrepancies
in the literature regarding heterotrophic growth. To a number of
flasks containing inorganic nutrient solution add 1% (by weight)
glucose or sucrose to half of them; inoculate them all then with a
small inoculum of a blue-green alga such as Nostoc muscorum and
place them in complete dark or in light (200 ft.-c,) for one month.
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Physiology of Blue-Green Algae
There is no measurable cellular material in any of the dark flasks; the
inorganic flasks in the light will have about 200 mg dry weight (per
400 ml suspension), while the glucose-fortified flasks in the light will
have about 400 mg dry weight. This is a very dramatic increase caused
by the addition of glucose (or sucrose), but I noticed in harvesting
these cells that they were very gelatinous. The usual procedure in
obtaining such dry weight was to centrifuge, decant the supernatant, and
then wash the cells once with distilled water before centrifuging again.
As the light-grown cells with glucose were so gelatinous, I wondered if
the increase in dry weight might be accounted for by the presence of
large amounts of extracellular material which could be washed off by
thorough rinsing. In another experiment similar to the above one, some
of the light-grown cultures with glucose were washed eight times with
distilled water. This caused the dry weight to drop from 400 mg to
about 250 mg, which, however, was still significantly higher than the
200 mg in the inorganic flasks in the light. There was no fixed nitro-
gen source in any of the above experiments, so that all cellular nitrogen
was derived via fixation of molecular nitrogen. In addition to dry
weight of the cellular material noted above, I also ran Kjeldahl nitrogen
determinations on all samples. Results were as follows: all samples in
the dark, 0; inorganic cultures in the light, 10 mg N; cultures with
glucose in the light and rinsed once, 12 mg N; cultures with glucose in
the light and rinsed eight times, 12 mg N. I concluded from these
experiments that glucose was not being assimilated in complete darkness,
and that in the light it was penetrating the cell and affecting the
metabolism of the cell such that more molecular nitrogen was being
fixed, which led to a greater dry weight yield per unit volume of sus-
pension. I did a little work with long dark periods (23 hours) and
short light periods (1 hour) which indicated that continuous light was
not necessary for assimilation of the glucose; periods as short as 30
minutes per day gave substantial amounts of growth.
I also examined the rate of assimilation of organic substrates by
blue-green algae by the use of radiocarbon-labelled compounds. After
incubating Nostoc muscorum with C -labelled glucose or sucrose for one
month in the dark, there was no measurable amount of radiocarbon incor-
porated into the cellular material. When light was given to these cul-
tures (either continuously or for a short period of each day), the cellu-
lar material incorporated much of the radiocarbon from the organic sub-
strate. When the cells were hydrolyzed and the products chromatographed,
it was seen that the radiocarbon was distributed in a wide variety of
amino acids, carbohydrates, and organic acids.
All this leads me to believe that all blue-greens that I have
looked at are obligate autotrophs, but that they can assimilate organic
compounds at a very slow rate by a photoactivation process, which we do
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
not understand at the present time. There is some direct evidence of
this by the students of Watanabe at the Institute of Applied Micro-
biology in Tokyo. In view of this rather harsh conclusion, harsh to
some of you, at least, it is advisable that studies be done with blue-
green algae which have been isolated from lichens and also with a
variety of blue-greens isolated from the roots of cycads and other
rather peculiar places where you would almost certainly expect hetero-
trophic forms of life to exist. At the present time, however, no such
data are available as far as I know. Thank You.
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DISCUSSION
George P. Fitzgerald
Water Chemistry Laboratory
University of Wisconsin
Madison, Wisconsin
It seems kind of silly to get up here and tell all these people
about blue-green algae after Dr. Holm-Hansen talked. He knows every-
thing that goes on at Wisconsin. Luckily, we've done a few things
since he left that he didn't touch on. I suppose we have to do a few
things besides freeze-dry mice around there. In order to prepare for
a discussion session, I listed some of the problems to be resolved, and
I think everyone of these problems has already been brought up. But,
I'll bring them up again.
One of the problems is the availability of nutrients, how do you
know if nutrients are available, where are they coming from, and what
is their importance? One of the first ways of measuring this is by
nutritional bioassay, and one of the methods is that which has been
mentioned by Skulberg in Norway. The bioassay technique, which came
down through a long series of workers, consists of adding different
nutrients to a sample of lake water or river and finding out which ones
stimulate the growth of algae, and saying, "Ah, ha, phosphorus in this
area of the river was limiting to the growth of algae because when you
added phosphorus the algae grewl Down here below a source of pollution
where phosphorus is present, there was no stimulation due to the addi-
tion of the phosphorus." Whether you believe in this technique or do
not believe in it, at least you can compare different waters and find
out what is the effect of adding nutrients to this water. This nutri-
tional bioassay type of thing is something that I think is just in the
very exciting phase and could be expanded considerably. We can get some
very valuable information from this type of an assay, but one of the
problems involved here which some people forget is the fact that you are
studying the nutrition of only the algae in the Erlenmeyer flasks that
you're working with. Unless you work on the microscale of increase in
growth, you would lose the effect of the addition of nutrients. A heavy
bloom in Wisconsin lakes means you can't see your hand in six or eight
inches of water. This amounts to 5-10 mg/1, while 5-10 mg/1 in the
laboratory--! mean, you'd think you'd sneezed in the bottle, that's all
the growth there is. You can just barely detect this amount of growth.
So, if you add one milligram of phosphorus or a tenth of a milligram of
phosphorus and wait to find out how much growth you get in dry weight or
optical density measurements, you are expanding your data. What you're
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
actually finding out is the amount of growth you'll get with the addi-
tion of one milligram of phosphorus, whereas in the lake or river that
you're dealing with, you would get that same amount of growth increase
with 1/50 or 1/100 that amount of phosphorus. In other words, you've
got a concentration factor in the laboratory of 50 to 100 times, and,
actually, results of these bioassays may not be related whatsoever to
what may take place out in the field, except in a very rough, qualita-
tive situation. In the field you're dealing with perhaps 2 or 3 mg/1
concentrations of algae and adding minute quantities of nutrients. In
the lab you're adding wholesale quantities, you might say, of nutrients,
and you're dealing with many milligrams of algae per liter.
There has been some very nice work in England by MacKereth and
co-workers where they've actually done all their work with cell counts.
They were concerned with finding out how much growth would take place
when you take algae from the bottom water versus the surface water. It
wasn't a matter of milligrams growth, I mean, they were measuring the
counts — the actual algal count—increases. This type of a bioassay
where you're actually measuring algal counts of from maybe 10,000-
15,000 per liter is a more realistic approach than the type of work we
do with our milligrams per liter type of measurement. So there are some
problems with nutritional bioassays, but it is a technique that I think
will be very good in the future.
One of the things that has been going on recently is to study the
nutrition of algae by their chemical composition or by enzymatic analyses.
Bar-Akiva, in Israel, has been using enzymatic analyses to determine
whether certain types of citrus trees are limited by the available nitro-
gen, manganese, molybdenum, and iron. We at Wisconsin have been doing
this same thing in phosphorus nutrition. We found that the alkaline phos-
phatase activity of algae is extremely high in algae where phosphorus
is limiting the amount of growth of algae. The alkaline phosphatase of
algae with surplus phosphorus is relatively low: a difference of 20- to
40-fold activity. My interpretation of why enzyme induction takes place
is the fact that when algae are phosphorus-starved, the algae get hyster-
ical, like students before an exam, and seek other methods of getting
phosphorus. They increase the alkaline phosphatases in order to break
down other sources of phosphorus: the polyphosphates, the metaphosphates,
and the organic phosphates. Alkaline phosphatase analysis is a matter
of just taking a pill from a pharmaceutical company and dropping it into
a suspension of algae. The supernatant will turn yellow or pink after
incubation if there's any alkaline phosphatase activity. Theoretically,
you can go to a lake and can find out if the algae in that environment
are limited in their growth by the amount of phosphorus present. If
there is no alkaline phosphatase activity, supposedly they had a surplus
98
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Discussion
amount of phosphorus in their environment for the amount of algae that
was produced.
Before we found out about this cute little alkaline phosphatase
analysis, we were extracting algae and fractionating the phosphorus
of starved versus algae with surplus phosphorus. We tried to find out
how excess phosphorus had been stored in algae and to see if we could
detect it. We don't know what the storage phosphorus is, but we know
if we take algae that have a surplus amount of phosphorus available to
them and kill them--we boiled them—we will find orthophosphates in the
supernate or the extract. In algae that have a limited amount of
phosphorus, little or no orthophosphates are extracted. We do not say
that there is orthophosphate in these algae (in the literature they
talk about polyphosphates, metaphosphates, and metachromatic granules),
but we know if we just kill the algae (and it doesrn't make any difference
whether you boil them, kill them with Algimycin /an algicide_/ or freeze
them) as soon as the algae are dead, orthophosphate comes out of the
algae if phosphorus has been available to the algae in surplus quantities.
So we have a system by which we can extract algae and find if there is
a surplus amount of phosphorus present. At the same time, we can take
another sample of algae and add either a commercial alkaline phosphatase
pill or paranitrophenylphosphate and if alkaline phosphatase is present,
phosphate will be split off and you'll have paranitrophenol, which under
alkaline conditions, gives a bright yellow color. You put some algae
and a pill in a bottle, wait 30 minutes at 37 degrees, and it turns
yellow. It's very simple test. This works on the diatoms, blue-green
algae, and green alga that we tested in the lab. All the algae in the
Madison area last summer had surplus amounts of phosphorus. This is
reasonable, as the most fertile water in the country is probably in this
good dairy land of ours in Wisconsin. What we have often wondered was,
in Nature, is there such a thing as phosphorus-limited algae cultures?
Luckily, this last summer when we were checking several lakes in Wisconsin
to see if there is an algal community that is actually limited in the
amount of phosphorus available to it, every once in awhile algal samples
would give tremendous activities of alkaline phosphatase in the same
sample as high, extractable phosphorus. We found a nice example of
this in a sample from Lake Monona, one of our Madison Lakes, where there
was a mixture of what I would call Anabaena and Microcystis. So, I went
back and sat on a pier with a long glass tube and sucked up an Anabaena
colony and put it in one bottle, and a Microcystis colony into another
bottle, until I had a nice, green soup in each bottle to take back to
the lab. We actually had fractionated the bloom: about 987= Microcystis
and about 98% Anabaena. The Microcystis had extremely/ high extractable
phosphorus, but no alkaline phosphatase, and the, supposedly, nitrogen-
fixing organism (Anabaena) had low extractable phosphorus and very high
alkaline phosphatase activity. Thus, peculiar results obtained with
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGA.E
mixed blooms could be separated out because there were two different
types of species there. Now, the question I raise is a most natural
question to ask this group: Where do algae absorb their nutrients?
I don't believe that algae absorb their nutrients in the surface waters
where they're growing or we say they're growing. I think they absorb
them down in the bottom waters. I don't know why they go up, but I
think when the algae get to the surface, they no longer are concerned
with nutrition, and they're up there for some other reason. Now, this
work with nitrogen-fixers and non-nitrogen-fixers, I think is logically
open to question. If Microcystis and some of the other algae in our
area absorb their nutrients in bottom water and they come up to the
surface, they'll run out of some nutrients, obviously. I always assume
it's going to be nitrogen before it's phosphorus. If they run out of
nitrogen, all the surplus, stored phosphorus they pick up down in the
bottom waters will still be surplus phosphorus in the surface water if
they do not continue to grow. If the nitrogen-fixer comes up from the
bottom, it absorbs the same amount of surplus phosphorus (and we know
a nitrogen-fixer can have a surplus amount of phosphorus because we've
tested it in the lab). If they were to absorb the same amount of phos-
phorus in the bottom, then come up to the top, and use up all the
nitrogen they have absorbed, they would be forced into a nitrogen-
fixation situation. They could then utilize their surplus stored
phosphorus up to the point where they were actually limited and they
would start seeking other forms of phosphorus. So this is one of the
theories I've had: that algae do not rely on the nutrients in surface
waters of the lake—there probably aren't any as far as I'm concerned.
They have absorbed the nutrients from bottom waters that they're using
for growth. But it was very fortunate that only the nitrogen-fixers in
our very eutrophic lake in Madison were the ones that were actually
limited, by my measurements, by their phosphorus content. Well, this
is the question: Where do algae absorb their nutrients? And until we
really know, we really don't know too much about what are the environ-
mental factors taking place in the lake where we usually find the algae.
Whether the nutritional absorption is important in the epilimnion or
the hypolimnion, this is one of the major questions for answering in
the algae field. It's touched on every once in awhile by people like
Lund and MacKereth, where they've taken algae from the bottom and found
that such algae would produce more cells than the algae from surface
waters. Maybe this is where they get their nutrients, and when you take
them from the environment, they might be more viable or have more nutri-
ents. We know if we want to culture algae in the laboratory, it's much
easier to take a bottom water samples, bring it into the laboratory, and
wait for the little tiny green spots to show up than it is to try and
get algae from the epilimnion to grow. I guess we've readily cultured
every bloom-forming organism that appears in the Madison Lakes by using
bottom water, whereas only one out of a hundred cultures would grow from
100
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Discussion
the surface or upper part of the lake. The algae in the bottom are
entirely different from the surface algae, but I don't know about the
quantity involved. Are there really enough algae at the bottom with
nutrients in them to create these dense blooms in the surface? I don't
know. But this is one of the questions that we could talk about further.
One of the questions brought up recently, and touched on today, was
the question of nitrogen fixation. I've often wondered if the nitrogen
that is fixed is more available for rapid degradation or availability
to algae than the nitrogen from green algae or diatoms. If you put
algae in the bottom of a tube, the release of nutrients can be measured.
The phosphorus comes off very rapidly if the algae are killed and if
you do various things to these algae to try to leach them they die.
But the nitrogen usually doesn't come out, or very little nitrogen would
come out, of these algae. If this is the case with the nitrogen-fixing
organism, then its nitrogen really isn't very available to other algae
without a long period of time for degradation. The question I would
like answered eventually is: If you took a nitrogen-fixing organism and
Chlorella or a desmid and did this same type of an experiment, would
you find that the nitrogen would be released? We know phosphorus would
come out very rapidly, but would the nitrogen come out at a different
rate from the nitrogen-fixers than from the green alga? This is some-
thing I hope you will be able to tell me.
I was so happy to see Dr. Prowsa here because I thought that he
could solve all the problems about temperature. Here's a man that
doesn't have to worry about temperature. It doesn't vary, and yet they
have nice, beautiful blooms of algae. (I am a little prejudiced. To
me the nice, pea-soupy, bluish-green color of a lake is beautiful.)
They have continuous blooms in the tropics and so can eliminate many of
the things that we worry about: temperature and weather changes. The
algae grow there despite these things and it kind of knocks in the head
many of the theories on effects of weather. One of the questions I
would like Dr. Prowse to talk about, and maybe Dr. Hammer from Canada,
is: From farm ponds and eutrophic lakes we get vague references to the
fact that people, who are using these farm fish ponds for the culture of
fish and actual production of a utilizable harvest of fish, can control
the growth of algae to the species that they want. I've never had any
details and I never knew what species they want in the way of blue-green
algae in these fish ponds, but I've heard vague references to the fact
that by the proper fertilization of these ponds they can eliminate the
Aphanizomenon or increase the Aphanizomenon, whichever it is, and get a
specific type of algae that they want. This is the question for
Dr. Prowse. I hope this is going to be a thing of the future. If a
fish farmer can tell us how to control the growth of Aphanizomenon and
lead it into Anabaenopsis, this might be a very good ecological clue for
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
us to go on. Quite a few people have mentioned the fact that these
beautiful blue-green algae blooms that we get in reservoirs and lakes
are usually associated with sewage effluent or most frequently associated
with sewage effluent. Perhaps we could control the obnoxious forms
(other people call them obnoxious forms), the floating blue-greens, as
opposed to the algae causing green water. Pea soup green water is not
objectionable, except to water treatment people, but the floating blue-
green algae do cause some objections. If you could control them or
know what it is that causes their ecological control, we might be able
to shift the balance from one species to another.
Well, I think this has brought up questions that we can put back
to the speakers that have already spoken or some of the people who
haven't. Well, Dr. Prowse, why don't you start and tell us something
about fish ponds and lack of temperature changes.
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SUMMARY OF FLOOR DISCUSSIONS
summarized by
Jerry C. Tash
Eutrophication Research
Pacific Northwest Water Laboratory
Corvallis, Oregon
Discussion following paper
"Why Study Blue-Green Algae?"
by W. T. Edmondson
At the Tropical Fish Culture Research Institute, Malacca, Malaysia,
Tilapia and Silver Cod are well-known fish that feed on phytoplankton.
These fish can eat and digest Anabaenopsis but most of the cells of
Anabaena pass through the digestive tract intact. In ponds where fish
feed predominantly on Anabaenopsis, algal blooms never develop, but
when Anabaena takes over (usually when high phosphate conditions occur),
pea soup blooms occur. Little is known about the mechanisms involved
in the resistance of digestion by algae but the ability obviously has
evolutionary advantages.
Increases in blue-green algae often occur in lakes after sewage is
introduced, but blue-greens seldom are a problem in oxidation ponds.
The causes for this difference are unknown.
It is important to be aware of the immediate past history of algae
when trying to evaluate the standing crop, because the standing crop is
partially a result of the rate of production minus the rate of consump-
tion (other factors include algal nutrition, light conditions and tem-
perature). For example, the species of algae and their numbers in oxi-
dation ponds are changed by the consumption of smaller algal species by
zooplankton herbivores. Along the California coast, in salt evaporation
ponds, Artemia consumes some species of algae, but through some form of
differential digestion, allows other species to pass through the diges-
tive tract intact.
Discussion following paper "Problems
in the Laboratory Culture of Plank-
tonic Blue-Green Algae" by W. R. Eberly
Short incubation periods, such as six hours, probably do not provide
information on long-term growth rates (days or weeks) and data from such
studies should not be used to extrapolate growth curves for long periods.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
It is difficult to interpret what happens to growth rates after
the addition of microelements; this difficulty is compounded if the pH
of the culture medium is not controlled. If the pH goes above approxi-
mately 9.0, all of the carbon in the water will be in the form of
carbonate. If maximum growth occurs in cultures above a pH of 9.0, the
algae may be showing a preference for carbon in the carbonate form; if
true, after maximum density is reached and after the pH drops, the
carbonate form will no longer be available and carbon may become a
limiting factor.
Oscillatoria rubescens shows different pigmentation under differ-
ent growth conditions; although little is known about the chemistry of
the pigments, such knowledge may be useful in dating the past cycles
of Oscillatoria in various lakes by analyzing pigments trapped in the
sediments.
In major lakes in northern Minnesota, Oscillatoria agardhii and
Merismopedia marssonii are found in one stratified layer, whereas
Oscillatoria redecii and Merismopedia trolleri are found in another
layer of the same lake. Perhaps the presence of hydrogen sulfide
explains the difference by providing sulfur as a form of nutrition for
0. redecii and also for M. trolleri, both of which are found in the
lower stratifications where H2S is most prevalent.
Discussion following paper "Aspects of the
Nitrogen Nutrition of Naturally Occurring
Populations of Phytoplankton Dominated by
Blue-Green Algae" by V. A. Dugdale
In Saskatchewan, Canada, Anabaena in lakes blooms about a month
after ice break-up. Following the relatively short Anabaena bloom,
Aphanizomenon takes over. Nitrogen usually never shows an increase
after the disappearance of the Anabaena bloom.
Under several feet of ice and snow during the winter in both
Alaska and Canada, much light still penetrates into the water. Chloro-
phyll measurements under the ice indicate some organic production.
Discussion following paper "Environmental
Requirements of Thermophilic Blue-Green
Algae" by R. W. Castenholz
A hot spring at Mount Rainier has a very conspicuous mass of
algae (Schizothrix calcecola) that affords refuge for herbivores. One
of the herbivores, an oligochaete unique to this type of algal mass,
has its own network of channels wherein it grazes. Temperature on the
mat surface can be up to 55°C with temperatures under the mat at 40°C,
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Summary of Floor Discussions
even though water the mat is growing in is at the higher temperature.
The herbivores bore upward through the mat surface at night to graze
but return to subsurface channels during the day; they probably use the
surface at night because of the absence of predators.
Discussion following paper "Growth
Requirements of Blue-Green Algae as
Deduced from their Natural Distribu-
tion" by W. C. Vinyard
Some organisms have been observed growing in diesel and high octane
fuels in which water content was less than 2%. It has been established
that certain species of the fungi and bacteria are able to live in vari-
ous petroleum products. Species of some algae have also been found in
petroleum by-product wastes. Some blue-green algae were observed in the
tar of pools in oil fields where they were existing under such darkened
conditions as to make it plausible to assume that they were deriving
nourishment from the hydrocarbons in the wastes. Interestingly, these
algae had retained their bright blue-green color. In some waste ponds,
the algae formed heavy mats intermixed with the tar and oil; the algal
forms varied in color from brown to reddish, indicating that they con-
sisted mainly of diatoms.
Water pumped up with the oil is separated and pooled in many oil
fields; both brine and non-brine waters can be pumped. In non-brine
water, diatoms dominate.
Discussion following paper "Recent
Advances in the Physiology of Blue-Green
Algae" by 0. Holm-Hansen
Diamino phenolic acid is present in all blue-green algae; phenolic
derivatives are also present in lichens, some bacteria, and some fungi.
Diamino phenolic acid has not been found in Chlorella. Since some
organisms produce special pigments and other chemical derivatives that
are specific to their groups, it would enhance the field of taxonomy to
utilize these characteristics as well as morphological characters. In
some instances, chemical or biochemical evidence can be utilized better
than morphological evidence. However, it is quite difficult at this
stage of science to isolate and identify most organic compounds of
various organisms, and this is probably the major reason more bio-
chemical evidence is not used in defining taxa.
In studying productivity in laboratory cultures, pH is probably not
an important factor per se, but rather, indirectly affects productivity
by changing chelation of such elements as iron and manganese, permitting
them to stay in solution.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
In nature, pH usually rises as algal populations increase; in
areas where bloom conditions occur, the pH can be as high as 10.0 to
10.5. In the tropics, the pH fluctuates diurnally from 7 to 10.5
during 24 hours, most likely due to photosynethesis in the algae. If
pH is considered an important factor in primary productivity, it
should be measured continuously during the period of work, whether in
the laboratory or in the field. Since there is little known about the
influence of pH on growth, research should be carried out to assess
the role of pH as it directly and indirectly affects growth.
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GENERAL DISCUSSION OF
NUTRIENT MEASUREMENT AND NUISANCE CONTROL
September 24, 1966
Alexander M. Dollar, Michael Parker, William A. Dawsoni/
INTRODUCTION
Successful multiple use of lake and stream water systems entails a
thorough understanding of the productivity of these systems. Lack of
knowledge and understanding of the causes of algal blooms now prevents
effective control. Principal offenders are blue-green algae, whose
intense blooms can form floating masses of algae, which eventually die
and decompose.
To initiate discussion in the morning session, participants were
invited to present ideas regarding a watershed's contribution of nutri-
ents to its lakes. A study of Upper Klamath Lake, Oregon, was chosen
as a starting point for the discussion. It was not possible to record
the session verbatum nor even to note the specific contributions of
each person. The following is a summary of the session as a whole,
often with information rearranged to make a more logical presentation
than could be maintained or even anticipated in a free-flowing discussion.
MEASURING THE INFLOW OF NUTRIENTS
The morning's discussion began with a consideration of methods and
measurements to evaluate a watershed's contribution of nutrients to its
lakes. It was generally concluded that given adequate manpower and
facilities, contributions of nutrients from the watershed can be evalu-
ated fairly completely. Upper Klamath Lake, Oregon, (130 square miles,
with a 3810 square mile watershed, and an average depth of 8 feet) was
used as an example for discussion. A. F. Bartsch and other staff of
the Federal Water Pollution Control Administration, in Corvallis, out-
lined the work they have done on this lake to measure the input from
several sources:
— Respectively, State of Hawaii, Department of Agriculture, Honolulu;
Department of Zoology, University of Washington, Seattle; and College
of Fisheries, University of Washington, Seattle.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
Inflow from tributary streams. Nitrate, nitrite, ammonia,
orthophosphate, iron, cobalt, molybdenum, manganese, boron,
vanadium, and other elements were all analyzed in both the
particulate and soluble (passing through a 0.45 u membrane
filter) fractions of samples.
Farm land irrigation. Some areas of lake bottom have been
reclaimed from the lake by erecting low earthen dams.
Return water from irrigation is collected in sumps before
being returned by pumpage to the lake. Nutrients have been
measured in the returning water (as for the tributaries).
Springs and seepage. The U. S. Geological Survey made a
detailed determination of the surface water budget of the
lake and by difference the volume of water contributed from
springs and seepage was calculated at 17%. Analyses of the
nutrients in visible springs flowing into the lake and arte-
sian wells in the area were made, weighted, and used to
estimate the amount of nutrients contributed from seepage
and springs within the lake.
Waterfowl. The lake is on one of the major flyways, and
some attempt has been made to evaluate the nitrogen and
phosphorus contributed to the bottom sediments by water-
fowl. It appears that less than 2% of the phosphorus enter-
ing the lake comes from this source.
METHODS
During the course of the morning several points were raised which
have general pertinence in relation to specific methodology. The need
for pretreatment of Millipore filters to avoid contaminating filtrates
was emphasized. This is important in relation to organic matter,
nitrogen, phosphorus, and some micronutrients. In nutrient-poor waters
the amount of micronutrient (as Al, Si) contributed by an unwashed
filter may exceed that present in the sample. The problem may not be
as critical in richer waters, but should still be considered. Methods
for decontaminating filters included rinsing with several hundred ml
of distilled water or dilute HC1 or soaking the filters in distilled
water overnight.
Empirical testing should be used to determine whether the ruptur-
ing of cells during filtration or storage of samples prior to treat-
ment introduces significant changes in nutrient concentration. As a
general rule, filtration should be as gentle as possible and the time
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General Discussion
between taking a sample and its treatment should be minimum. A filtra-
tion vacuum of not more than one-half atmosphere has often been used.
Obtaining adequate samples to represent the whole lake both tem-
porally and spatially is another area where preliminary investigations
are needed. In most cases there will have to be some compromise between
number of samples taken and time available for analysis and/or cost.
In Upper Klamth Lake it was determined that three transects were ade-
quate to describe the lake as far as the objectives of the study were
concerned. Samples were taken by lowering a tube to the bottom at vari-
ous points on each transect and pooling these samples for each transect.
It is important to remember that the objectives of each study will
probably be different and that each sampling program should be considered
in relation to the problem to be solved.
Algal standing crop is often measured on the basis of cell numbers,
cell volume, wet or dry weight, or chlorophyll content. Dr. Dollar sug-
gested the use of Swinney glass filters for sampling small volumes of
water containing relatively large amounts of algae. Material retained
by the filters can be analyzed directly for total nitrogen and can be
used for chlorophyll determinations.
The differences between much oceanographic work and that in pro-
ductive fresh waters were mentioned by several discussants. Productivity
in the ocean is usually less than in productive lakes, and there are
also often lower concentrations of nutrients. Consequently, the methods
for analysis must be more sensitive in many cases. Fluorometry is an
example of a sensitive technique which has been used for the determina-
tion of chlorophyll. As used in this method, the fluorometer measures
light emitted by chlorophyll molecules which have been excited by an
activating wavelength of light. The method, used successfully with
chlorophyll extracts, measures only chlorophyll a. or total chlorophyll
and cannot resolve other pigments. It has also been used to continually
monitor the content of chlorophyll in a stream of untreated sea water
passing through the instrument. Since the amounts of chlorophyll were
near the limit of the machine's sensitivity in the latter case, special
instrumentation was needed to accurately detect these responses. In
fresh waters the standing crop is often much higher and this difficulty
might not be such a problem. Calibration by the use of standard cul-
tures of organisms could also aid in the method's application to rapid
in situ sampling and measurement. It would seem the method has good
possibility of being useful to provide at least relatively accurate in
situ estimates of chlorophyll in lakes, although many technical diffi-
culties remain to be solved.
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ENVIRONMENTAL REQUIREMENTS OF BLUE-GREEN ALGAE
IMPORTANCE OF SEDIMENTS AS A SOURCE OF NUTRIENTS
The study of Upper Klamath Lake also raised another question.
Although it is possible to evaluate the input of nutrients to a lake,
the question arises as to the relative importance of allochthonous
nutrients as opposed to those regenerated from sediments. This question
is of acute importance for those charged with the responsibility of pre-
dicting changes in lakes resulting from changes in the composition of
the nutrients in the incoming waters. In Upper Klamath Lake it was cal-
culated that the upper one inch of sediment has an amount of nutrients
equivalent to that brought into the lake by approximately 60 years of
allochthonous supply. However, there seems to be a lack of data which
can be used to quantitatively evaluate the contribution of nutrients
which are recycled from the sediments.
If, in any given case, it is decided that the contribution of
nutrients from the sediments has a decisive influence on the algal
population, can anything be done to eliminate this contribution? One
possibility which has already been investigated involved placing about
six inches of nutrient-poor soil over rich marsh land. This effectively
sealed off the nutrients in the march from regeneration and release into
the water after the basin was filled.
Similar approaches might also be used in lakes which are already
formed. However, the intended purpose of any such treatment would need
to be carefully evaluated. Any procedure which completely eliminated
the contribution of nutrients from the sediments would also of necessity
eliminate much or all of the benthic fauna living there. This, in turn,
would have an effect on other organisms, as fish, which are in some way
dependent on benthos. Thus, if such methods are to be utilized as a
management tool, careful consideration of the management's objectives
will need to be made. Complete elimination of nutrient regeneration
from sediments might lead to more aesthetic conditions as far as algal
nuisance blooms are concerned, but at the same time might severely
hamper recreational activities such as fishing.
CONTROL OF ALGAL BLOOMS
The problem of controlling algal blooms was raised several times,
and there was some discussion of what can be done to alleviate their
development. One proposal, already discussed, was to seal off the
bottom of the lake, or a significant proportion of it, to reduce the
availability of recycled nutrients. This approach would seem to be
most practical in the case of smaller impoundments which have yet to
be filled with water and much less so in larger lakes already existing.
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General Discussion
Secondly, although the precise importance to algal blooms of the
release of nutrients from sediments is uncertain, it is known from
experience that decreasing or changing the nutrient composition of the
incoming water may lead to changes in the algal flora. Thus, in the
Madison, Wisconsin, area, after sewage was diverted from several lakes,
surface blooms of algae were greatly reduced. In Lake Waubesa the
amount of algae, in mg/liter, remained the same as before diversion,
but instead of a community composed largely of one or two species which
formed blooms, there are now several scores of species, none of which
predominates to form the previous nuisance.
Several similar situations are under study in the State of Wash-
ington. The city of Seattle has almost finished completely diverting
sewage from Lake Washington, and studies are in progress to follow the
changes which will occur as the lake is flushed. The main source of
incoming water is quite low in nutrients.
In Green Lake an experiment is under way in which the lake is
being flushed by large volumes of the city of Seattle's water. The
total phosphate concentration has been reduced to one-half its normal
value at the beginning of the summer but returns to "normal" levels
later in the summer. Although data available are inconclusive, there
is some indication that blooms early in the year have been reduced
even though conditions toward the end of summer appear unchanged.
It was felt that in many cases sealing off a lake's bottom or
reducing nutrient concentrations by flushing would lead to changes in
the relative proportions and/or types of algae present in the system.
Dr. Prowse, Dr. Edmondson, and others felt that a possible factor con-
tributing to this change, or reinforcing it, would be changes in the
zooplankton and in their grazing patterns. This effect of selective
feeding by zooplankton necessitates further studies on their distribu-
tion and abundance and their role in controlling algal populations.
Ill
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