OCLC15691319
FACTORS AFFECTING
THE ALGAL ASSAY PROCEDURE
by
George P. Fitzgerald
University of Wisconsin
Madison, Wisconsin
Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
1975
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ABSTRACT
Evaluations of the Algal Assay Procedure (AAP) have demon-
strated its value for determining the level of nutrients
in water samples which is available for the growth of algae
as contrasted to chemical analyses of total nutrient con-
tents. The maximum specific growth rate, v max, has been
shown not to be affected by the supply of N, P, or carbon
when tests are carried out using the suggested AAP. The
maximum yield of algae is affected by the initial concen-
tration of N and P (and Fe, Ca, Mg, K, and S in other nutri-
tion studies). The maximum yield is not affected by the
supply of carbon in normal AAP cultures (foam or cotton
plugs), but increased incubation times might be required
for the maximum yield to be attained if comparatively con-
centrated nutrient solutions are used. The inoculum levels
of algae suggested in the AAP are sufficiently low to make
use of fluorometry as a measurement of the growth of algae
in relatively dilute culture media. If cell counts or
absorbance measurements are to be used to follow the growth
of cultures, higher initial cell densities may be employed.
The u max of algae cultures i^s influenced by the light
intensity, but maximum yields "merely require longer incuba-
tion periods if less than suggested light intensities are
used. The applications of the AAP with the suggested algal
species or in vivo algae have demonstrated its value in
determining~~which algal nutrient will limit the growth of
algae in water samples.
11
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CONTENTS
Page
Abstract ii
Figures iv
Tables v
Acknowledgments vi
Sections
I Introduction 1
II Comparison of Results and Interpretations
Using Different Methods of Measurements
of Algal Growth 5
Measurements 5
When and What to Measure 6
III Effects of Some Physical Factors 17
Size of Inoculum 17
Flask Size and Shaking 20
Effect of Light Intensity 24
IV The Application of AAP 26
Comparisons Using the Three AAP Algae 26
Use of in situ Algae in the AAP 26
V References 30
111
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FIGURES
No. Paqe
1 Rate of Growth of Selenastrum; Comparison of
Fluorometry, Absorbance and Cell Counts 7
2 Rate of Growth of Selenastrum in AAM: Effect
of Added Nutrient Spikes' 9
3 Rate of Growth of Selenastrum; Effect of
Concentration of PO.«-P 11
4 Rate of Growth of Selenastrum: Effect of
Concentration of NO^-N 12
S'
5 Rate of Growth of Selenastrum: Gorham's ys
AAM 14
6 Rate of Growth of Selenastrum: Effect of
Nutrients Added to Lake Waters 16
7 Rate of Growth of Selenastrum: Effect of
Size of Inoculum 18
8 Rate of Growth of Selenastrum; Effect of
Size of Inoculum 19
9 Rate of Growth of Selenastrum: Effect of
Culture Volume 21
10 Rate of Growth of Selenastrum in AAM:
Effect of Limiting Carbon 23
11 Rate of Growth of Selenastrum in AAM:
Effect of Light Intensity25
12 Rate of Growth of Lake Kegonsa Diatoms
in Lake Waters 29
IV
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TABLES
\;o. Page
1 Comparisons of Yields and u Max of
Selenas trum Grown in Algal Assay Medium
with Different Concentrations of
Phosphorus and Nitrogen 13
2 Comparisons of Soluble POi,-P and
Calculated Available Phosphorus from AAP
Growth Tests of Water Samples from the
Outlets of Madison, Wi.s Lakes 27
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ACKNOWLEDGMENTS
The technical assistance of Mrs. S. L.
Faust and Mrs. C. R. Nadler is gratefully
acknowledged. This work was supported
by a grant from the Water Quality Office
of the Environmental Protection Agency.
VI
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SECTION I
INTRODUCTION
The general purpose of the Algal Assay Procedure (AAP) is
to measure the biologically available fertility of a water
sample, as contrasted with chemical analyses of the com-
ponents of the sample. By the algal response to the addi-
tion of nutrients, alone or in combinations, one can also
determine the nutrient or nutrients limiting algal growth
and the potential changes in algal growth with changes in
nutrient concentrations. In other words, this is a prac-
tical test to compare the fertilities of water samples and
predict the algal responses to changes in the water. The
value of the test lies in the fact that one can differen-
tiate between available nutrients and total chemical com-
position of water samples. Many sources of nutrients, such
as the nitrogen and phosphorus of aerobic lake muds, are
relatively unavailable for the growth of algae although
chemically present (3,4,5,6,7,9).
Of critical importance is the fact that the relative fer-
tility of water samples is what is being measured with the
AAP. Thus, the selection of the sampling site, depth, season,
and other possible factors will have an influence on the
value of the results. As much care and consideration must
be given to the sample collecting as would be expected to
be given to carrying out the AAP. By careful selection
of sampling sites and times of sampling, very worthwhile
information of ecological importance can be obtained from
results with the AAP; the comparative fertility of surface
lake waters during the different seasons will indicate which
lakes become deficient in one or more algal nutrients in
midsummer and the effects of storms, lake turnover, or other
natural versus man-caused changes in the available nutrient
content. Potential sources of algal nutrients can be eval-
uated and their quantitative effect on the fertility of
the receiving waters predicted by proper sampling. Sources
of nutrients that are relatively unimportant during winter
and spring due to the relatively high nutrient levels
in the receiving waters can become important sources of
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limiting algal nutrients, such as phosphorus, during the
summer period when algal nutrients in the receiving waters
are at minimal levels. Thus, it must be emphasized that
samples of water are being analyzed in the AAP, and the
interpretation of the results will depend upon a logical
approach to selection of the samples to be analyzed. If
one wanted to obtain more direct information on the week-
to-week level of available algal nutrients in a body of
water, it would be proper to analyze the nutrients con-
tained in in situ algae. The algae growing in the environ-
ment can be used as in situ continuous monitors of the
available algal nutrients in the water (8,10,11). Thus,
predictions of changes in levels of algal nutrients based
upon results with the AAP and manipulations in the environ-
ment can be evaluated by nutritional changes in in_ situ
algae with minimal work effort.
Water samples for algal assays sometimes must be preserved
for more convenient analysis times. The treatment to be
given water samples before evaluating their nutrient con-
tent will depend upon the test to be used. Usually nutrient
sorption tests using nutrient-limited algae require so
little time and space to carry out that tests can readily
be carried out on samples as fast as they are received or
even in the process of bringing them to the laboratory,
if necessary (6). Since the usual handling procedures in
harvesting sorption tests consists of removing the test
algae with a forceps or coarse plankton net funnel, most
planktonic in, situ algae in the water sample would not be
harvested and analyzed. However, the presence of in situ
algae in the sample during the sorption incubation may have
an effect on the results because of competition with the
test algae (5,8) . The majority of in_ situ algae can readily
be removed from water samples by plankton nets or centri-
fugation, and samples can be stored for short periods in the
dark or under refrigeration. Since growth tests require a
relatively large amount of laboratory space for the number
of flasks and long incubations involved, it is frequently
necessary to preserve water samples for more appropriate
times of analyses. In situ algae may grow in some samples
and compete with the test algae of growth tests or cause
confusing results, such as when nitrogen-fixing algae might
grow in tests of the available N content of water samples.
Therefore, it is convenient to remove most organisms by
membrane filtration (0.45 u) or kill all organisms by auto-
claving. Both of these preservation techniques will affect
the nutrient content of the water samples. Membrane filtra-
tion removes particles that are insoluble at the time of
filtration. It has been shown (3,4,6) that algae can
utilize some forms of P, N, and Fe that are relatively
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insoluble (shark teeth, hair, and pyrite crystals). AAP
tests of water samples from four lakes in the Madison,
Wisconsin area (January, 1973) after membrane filtration
of the samples, indicated all four of the waters were
deficient in Fe (stimulation of growth when Fe-EDTA was
added), whereas there was no response to added Fe when
autoclaved samples were tested. Thus, these water samples
must have originally contained adequate available Fe which
was removed by filtration.
There are several effects that autoclaving can have on the
nutrient status of water samples. The method used to pre-
serve water samples for AAP tests by autoclaving has been
to autoclave the samples, cool, gas with COz for 1/4 to
1/2 hour to resolubilize some of the precipitated materials,
and aerate for 8 to 12 hours to remove excess C02• As has
been mentioned, available P, N, and Fe can be released from
in_ s itu algae in autoclaved samples. During July, 1972,
2~4" lake water samples were tested for soluble PCK-P, total P,
and available P (sorption tests with Cladophora). Only two
of the 24 raw or filtered (Whatman #2) samples had more than
0.02 mg P/l as soluble PCu-P or available P. Six autoclaved
samples had significantly higher soluble PCH-P than the raw
samples, and in all six autoclaved samples the available P
was 2 to 4 times higher than the concentration of soluble
POi»-P. Thus, both soluble POi»-P and other forms of avail-
able P were released from the iri situ algae by autoclaving.
In contrast to the increase of soluble and available P caused
by autoclaving lake water samples containing in situ algae,
autoclaving muddy river water samples caused a decrease of
soluble POL.-P in 11 of 12 samples. This loss in soluble
POi»-P in muddy samples is believed to be due to sorption
of P by the mud (3,9).
The general conclusions that can be drawn about water sample
treatments are:
Raw samples - The nutrient content of untreated stored
samples may change because of the growth of algae. Also,
in situ algae can compete with test organisms in either
sorption or growth tests. For sorption tests the effects
of in situ algae can be removed by reducing their numbers
to below competitive status by crude filtration through
plankton nets or coarse filter papers. Minimal changes
appear to take place in samples stored refrigerated in the
dark.
Preserved samples - Membrane filtration will remove competi-
tive organisms to nutrition tests as well as insoluble
nutrients which might or might not be available nutrients.
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Such treatments allow the measurement of nutrients soluble
at the time of treatment. The filtration of samples of
more than a few hundred ml, however, requires very special
equipment not normal to routine laboratories. Autoclaving
cauces a release of nutrients from ijri situ algae and an
increase in the sorption of nutrients by muds. However,
the total available nutrient content of water samples (the
availability of nutrients in the water and contributed by
the death of in, situ algae) can be measured after auto-
clavinq. Samples to be stored for long periods could be
crudely filtered to remove the majority of iri situ algae
or muds and then autoclaved as a compromise of the effort
required and the integrity of results.
This report will demonstrate some useful techniques, for
carrying out the AAP, evaluations of the measurements that
have been suggested for use, and some of the factors which
might affect the results of the AAP as well as suggestions
for short-cut modifications and expansions using in situ
algae as the test organisms for the AAP.
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SECTION II
COMPARISON OF RESULTS AND INTERPRETATIONS USING
DIFFERENT METHODS OF MEASUREMENTS OF ALGAL GROWTH
MEASUREMENTS
Dry weight - The amount of dry weight (suspended solids)
"in a culture of algae is probably the most reproducible
measurement of growth when comparing different nutritional
levels or species of algae. However, dry weight measure-
ments require the greatest waste of cultures and are not
suitable for low density cultures. Consequently, other
techniques are usually used and the results calculated to
dry weights.
Absorbance - The absorbance of algal cultures is preferably
measured at 750 mp in order to correlate results with sus-
pended solids and not be affected by chlorophyll changes
in the cultures. Absorbance measurements are well corre-
lated to dry weights, and they can be followed without
waste in cultures grown in tubes or in flasks with side
arms fitting a colorimeter. However, the sensitivity of
this method using 1 cm cells is less than 1/10 that of
fluorescence measurements when green algae are used.
Cell counts - The use of haemocytometer slides and a micro-
scope has advantages over other measurements in that one
sees what is being measured, and contaminating algae can
be detected, as well as changes in cell size or shape under
different environments. The cells of algae do vary in size
in young versus old cultures, and there are variations in
shapes in SeJenastrum capricornutum (AAP) ; N-limited cells
are truly "capricorn"-shaped (long, curled ends) whereas
P-limited cells are very stubby. In order to have reason-
able accuracy in cell counts one should count at least
100 cells (95% confidence equals ± 5 cells). When using
a 430 magnification with the microscope and a haemocytometer
25 fields would be required to be scanned for 100 cells if
the culture contained 1,000,000 cells/ml. If the culture
contained only 100,000 cells/ml, 250 fields would have to
be searched to count 100 cells. Thus, unless one has the
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use of a properly calibrated electronic particle counter,
considerable effort would be required to attain 95% con-
fidence in cell counts of cultures containing less than
1,000,000 cells/ml.
Fluorometry - The fluorescence of the chlorophyll a of
algae is easily measured without extractions or much waste
of culture volume. Direct measurements with live algae
can use as little as 2 ml, and some fluorometers can be
adapted to very simple flow-through systems with minimal
waste. In our measurements we used an Aminco fluoromicro-
photometer with the standard photomultiplier tube (4-6250)
and filters of 360 nm and 415 nm. The filters used in a
fluorometer must be selected carefully since different
algal species will require different filters for best re-
sults. With this instrument one Aminco fluorescent unit
was equivalent to 0.045 mg chlorophyll a/1. The in vivo
fluorescence measurement of Selenastrum was the most sensi-
tive and reproducible technique: as few as 500 cells/ml
could be readily detected. However, the fluorescence of
Selenastrum cells in different media does not necessarily
correlate with the absorbance of the culture. Selenastrum
growing in the Algal Assay Medium (AAM) with a culture
absorbance of 0.10 would have about 20 fluorometry units,
whereas Selenastrum growing in Gorham's Medium (12) with
an absorbance of 0.10 would have 40 fluorometry units. In
a later section it will be pointed out that the fluorometry
of cultures decreases after they pass their peak growth
and become senescent (less chlorophyll a per mg dry weight)
Thus, fluorometry is well suited to follow the growth of
cultures up to their maximal level and compare different
nutritional and environmental factors on these stages of
growth, but is suitable for only gross comparisons with
cultures that have passed their peak.
Correlation of measurements - Using Selenastrum growing
In AAM, a culture with 20 fluorometry units would have an
absorbance (1 cm, 750 my) of 0.10, a dry weight of 40 mg/1,
and have 2,000,000 cells/ml.
WHEN AND WHAT TO MEASURE
The results of a typical experiment following the growth
of Selenastrum in AAM and AAM supplemented with N and P
(final concentration 2 x normal) are presented in Figure 1
as the fluorometry (Aminco units), cell counts, or ab-
sorbance (1 cm, 750 mu) of the cultures at different incu-
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r:,
rig. l. Rate of growth of Selenostrum:
Comparison of Fluorometry, Absorbance and Cell Counts
+AI+P
FLUOROMETRY (Units)
CELL COUNT (Millions/ml)
ABSORBANCE (XIO)
15
TIME (Days)
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bation times. The 95% confidence limits of the fluorometry
data are presented for Day 3.
It can be seen that the maximum rate of growth in AAM is
the same as that in AAM supplemented with more N and P.
It must be emphasized that the period of maximal rate of
growth in Selenastrum cultures takes place at very low
cell densities. In actual fact, once Selenastrum cultures
take on a definite green color, they have passed their
period of maximal rates of growth used to calculate jj max.
At about 7 days the AAM cultures stop growing, but the
supplemented AAM cultures continue to grow, at reduced
rates, until about 15 days. All three measurements --indi-
cate the maximum yield of these cultures, but only fluo-
rometry measurements could be used for calculating the
maximum specific growth rate, y max. However, the fluo-
rescence of the chlorophyll a of these cultures decreased
after the maximum yield had been attained and a definite
yellowing of the cultures took place. Thus, the importance
of selecting the proper time for yield measurements if one
is using fluorometry cannot be understated. Wfeb:n cultures
with different nutritional characteristics are to be com-
pared it is sometimes necessary to harvest them at different
times in order to detect the maximum yield by fluorometric
measurements. Cell counts and absorbance measurements do
not decrease with the age of cultures under these condi-
tions, but it must be remembered that they are not very
sensitive methods at low cell densities.
Spikes of AAM level of P, P -t- N, and N + Fe were added
to AAM and the rate of growth and final yield of Selenastrum
measured using fluorometry (Figure 2) in order to demonstrate
which nutrient in AAM first becomes limiting to Selenastrum
and what effect spikes of nutrients have on the rates of
./ growth as compared to final yield of algae. (All cultures
had the same growth through Day 4 (see 95% confidence
limits), indicating that the maximum specific growth rate
was not affected by the different levels of nutrients
being tested.) The maximum yield in AAM was reached at
Day 7, but cultures with added P or P + N continued to
grow for 2 or 3 more days. Thus, P is the first nutrient
to limit the growth of Selenastrum in AAM, but the cultures
with added P soon run out of N also. The effect of added
N -i- Fe was to stimulate the depletion of P in the cultures,
and these cultures turned yellow before those in normal
AAM. Therefore, without added P, the addition of extra
N + Fe does not help the growth of Selenastrum in AAM and
might be slightly inhibitory.
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I-
'•i1
ct:
i—
O
1.0
O.It-
Fig. 2
. The rote of growth of Selenastrum
in AAM: Effect of added nutrient spikes.
10
12
AGE (Days)
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A series of studies have been made to demonstrate how dif-
ferent levels of nutrients in a culture medium will affect
the yield of algae, bu_ will not affect the maximum specific
growth rate, u max. This is in contradiction to the sup-
position (1,2) that u max can be correlated with limiting
nutrients when one is dealing with P or N. In the first
test different concentrations of P in P-free AAM were in-
cubated with 1,000 cells/ml of Selenastrum, and the growth
of the alga was followed for 9 days (Figure 3). The
second test was with different concentrations of N
(Figure 4).
The results of these tests indicate that whereas the maxi-
mum yield of Selenastrum depends upon the concentration
of P or N in the media, the rate of growth of the cultures
is the same up to the point when the cultures become
deficient in P or N. This latter point takes place con-
siderably past the time of the maximum growth rate. A
summary of the final yields and maximum specific growth
rates for the different concentrations of P and N is pre-
sented in Table 1. Thus, it can be seen that the same rate
of growth was attained in 0.025 mg P/l as in 0.3 mg P/l,
and a lower y max was attained in 8 mg N/l than in the
medium with 0.5 mg N/l. The relationship between the
fluorometry, absorbance, and cell counts of these cultures
is also presented.
Further evidence that the maximum rate of growth in bottle
tests of the AAP is not related to the composition of the
culture medium is presented in Figure 5, which follows the
growth of Selenastrum in AAM and Gorham's medium, a con-
siderably more concentrated algal culture medium (737 versus
66 mg/1 dissolved solids). The growth of Selenastrum Tn
the two media is identical until Day 5 when growth in AAM
slows, while growth continues, but at a slower rate, in
Gorham's medium. The average u max for all cultures in
AAM was 1.80 ± .18, and for cultures in Gorham's medium it
was 1.66 ± .14. Thus, media with such different composi-
tions support the growth of Selenastrum at comparable rates
of growth up to the stage when the maximum rate no longer
is supported. There is further growth in more concen-
trated media, but the rate of growth is not at maximal
levels.
As a test of whether natural waters would produce similar
results, samples of water from Lakes Wingra and Kegonsa
(Madison, Wis), collected 4/11/73, were used in AAP tests
10
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10. *-
O
CxL
O
0. 025/L
Fig. 3, Rate of growth of Selenastrum:
Effect of concentration of PCty-P.
-P
4 6
TIME (Days)
10
11
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100.
o
(V
i- Fig. A, Rate of growth of Selenastrum:
Effect of concentration of
10
0
4 6
TIME (Days)
4and8/L
2/L
I/L
•-» r 'I
u. yi
I'J
12
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COMPARISONS OF YIELDS AND y MAX OF SELENASTRUM GROWN
IN ALGAL ASSAY MEDIUM WITH DIFFERENT CONCENTRATIONS OF PHOSPHORUS
AND NITROGEN. FINAL YIELDS AFTER 9 DAYS OF INCUBATION.
Nutr L'-nt
concentration
(infi/1)
-F
0.025
0.05
0.1
0.2
0 . ';
-N
0.5
1.0
2.0
4.0
8.0
Maximum yield
Fluorometry
(Aminco units)
0.05
2.2
6.0
14.
30.
54.
0.65
5.0
9.0
22.
36.
36.
Absorbance Cell count
(1 cm, 750 my) (cells/ml)
0.0
.03
.07
.15
.17
.21
0.01
.06
.10
.16
.16
.16
-
230,000
1,100,000
2,100,000
2,800,000
6,700,000
_
1,600,000
2,800,000
4,000,000
4,700,000
5,300,000
y max
-
1.22 ±
1.23 ±
1.29 ±
1.32 ±
1.24 ±
_
1.43 ±
1.37 ±
1.35 ±
1.32 ±
1.29 r.
.18
.08
.02
.10
.023
.08
.03
.11
.053
.087
13
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5,
ot growth of Selenostrum: GorhcmVvs. AAM.
e
ID
246
TIME (Days)
14
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with and without various spikes. The data summarized in
Figure 6 indicate that so little growth took place in Lake
Wingra water that no definite M max could be calculated
(0.48 t .44), the growth going from 0.04 to 0.2 Aminco
units. When the Lake Wingra water was supplemented with
P (AAM level) there was increased growth (u max-1.03 ±
.62) to 0.7 units, thus indicating that Lake Wingra at
this time was relatively low in P. The rate of growth
of Selenastrum in Lake Kegonsa was similar to that in AAM.
When P was added to Lake Kegonsa waters there was no stimu-
lation of growth of the algae, but increased yields did
result with the addition of N + Fe. The rate of growth
(,j max 1.48 ± .75), however, was not increased with this
latter spike even though it contained the nutrient which
limited growth in Lake Kegonsa water. Other tests indi-
cated that spikes of only N would result in the same in-
crease in yield of algae in Lake Kegonsa waters, collected
at this time of year, as spikes of N + Fe or N + p + Fe.
-)(- The general conclusions from the results presented thus
far are that the yield of algal cultures in the AAP-bottle
test is dependent upon the nutrient content of the media
being tested, whereas the maximum specific growth rate is
independent of the media. The maximal yield of algae can
be readily measured by fluorometry, cell counts, or ab-
sorbance, and the final yield can be either calculated as
dry weight (suspended solids) from these data or measured
directly in those cultures containing at least 5 mg of
algae.
15
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Fig. 6, Rote of growth of Selenastrum: Effect of nutrients
added to lake waters.
f
4 6
TIME (Days)
16
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SECTION III
EFFECTS OF SOME PHYSICAL FACTORS
SIZE OF INOCULUM
The results in the previous section when Selenastrum was
grown in Lake Wingra water (Figure 6) point out the neces-
sity of using relatively low inoculum densities when deal-
ing with oligotrophic waters. In that test, an initial
concentration of 1,000 cells/ml was used and there was
about a 5-fold increase in growth of the algae. It can be
seen that if 10,000 cells/ml had been the initial cell
concentration there would probably not have been enough
nutrients in the lake water sample to bring about even a
doubling of the cell density. Thus, there is need for very
sensitive measuring methods even though they may not be
perfect for all uses. If one was interested in the nutri-
tion of oxidation ponds fed with different sewages,, the use
of absorbance measurements and relatively high initial
cell densities would be appropriate.
The sensitivity of the available means for algal measure-
ment will determine how low the nutrient levels can be
which will be detected and differentiated in lake waters.
By using in. vivo chlorophyll a_ fluorescence measurements
one can detect as low as 500 Selenastrum cells/ml. Thus
measurements of growth of Selenastrum can be followed from
this level upwards. Without modification of the method
to make it more sensitive the use of less dense initial
cell concentrations would be of little value. However,
except for measurements of the growth of algae in extremely
oligotrophic waters there is no real necessity to start
with such low cell densities. The fact that the initial
density of cells does not affect either the M max of algae
cultures nor the final yield is demonstrated in Figures 7
and 8. Cell densities of from 500/ml to 8,000/ml were
tested in AAM in the first test, and the results indicate
that all the cultures reached the relatively same concen-
tration in the AAM after 8 days of incubation. The u max
for the different inocula ranged from 1.44 ± .25 with
17
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FLUOROMETRY (Units)
-------
FLUOROMETRY (Units)
CO
a,
o
m
o
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O
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13
O
CO
o
CD
O
-------
500 cells/ml to 1.71 ± .20 for 1,000 cells/ml; the y max
with 4,000 cells/ml was intermediate at 1.56 ± .04. When
1,000 and 50,000 cells/ml were compared in AAM, the same
yield was attained at 6 days and the y max were 1.92 ± .091
and 1.41 ± .11, respectively. Thus, the initial cell
density affects only the sensitivity of differentiation
that can be made between algal cultures, and the sensi-
tivity of the available means of measuring the algae will
determine how low an initial cell density is practical.
Fluorometry allows us to start with 1,000 Selenastrum
cells/ml, but at least 10 times this concentration would
be appropriate if absorbance measurements were to be used.
FLASK SIZE AND SHAKING
Under the assumption that available carbon (C02 or
would limit the growth of algae in the AAP, various rela-
tively low volumes of liquid per flask are suggested for
use. This is to allow the atmosphere to replenish the
carbon used by the algae. At relatively low nutrient levels,
and consequently low concentrations of algae, the rate of
supply of available carbon from the atmosphere is sufficient
to keep up with the rate of growth of the algae. Also,
lower volumes require relatively lower amounts of carbon.
If the algal mass increases sufficiently to become'carbon-
limited there will be an increase in the pH of the algal
culture. This usually takes place after the cultures have
taken on a definite green color and consequently occurs
after the period of maximal rate of growth. Thus, the rate
of maximal growth of algae will not be affected if the sug-
gested culture volumes per flask size are used. The fact
that later growth periods may be carbon-limited has no
effect on the final yield of the cultures, merely more
time is required to reach the maximum yield. This is
pointed out by data on the growth of Selenastrum cultures
in AAM in which volumes of 150, 300, and 450 ml per 500 ml
Erlenmeyer flask were compared (Figure 9). The 95% con-
fidence limits on all cultures at Days 3 and 4 indicate
that all the cultures were growing at the same rate up to
Day 5. After six days of incubation the effect of the
different volumes on the growth of Selenastrum became
apparent. The 95% confidence limits at Day 9 indicate
there were significant differences in the yields of the
cultures at that time. However, at Day 13 there was no
difference between the growths in 150 and 300 ml, so the
final yield of cultures can be assumed to be equal regard-
less of the culture volume if one waits long enough, in
contrast to results with different initial concentrations
20
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Fi(]. g. Rate of growth of Selenostrum: Effect oi culture volume.
10.
I 1.0
>-
O
C£
O
ID
—1
u_
0.
0.0!
150 ml/500 ErI.
450 ml/500 ErI.
6 8 * 10
TIME (Days)
12
21
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of N, P, or Fe, since the atmosphere is a constant supply
of COj to looroly plugged cultures.
In order to demonstrate that the atmosphere is the source
of CC>2 for these cultures, a series of flasks were com-
pared which had been plugged with the usually-used plastic
foam plugs (or plugging cotton in some tests) or which •
were plugged with solid rubber stoppers (Figure 10). The
rate of growth with either type of flask closure was the
same up to Day 4. Apparently, after this time the cultures
plugged with the rubber stoppers ran out of available carbon
since growth ceased at Day 5 while the growth in foam-
plugged cultures continued to Day 7 and resulted in a final
yield of nearly 10 times that in the rubber-stoppered flasks.
As further evidence that the exclusion of atmospheric COi
caused the cessation of growth in the rubber-stoppered
cultures, the rubber stoppers of two cultures were replaced
with foam plugs. Within one day these cultures had started
growing again, and by Day 11 their growth had nearly equaled
that in the original foam-plugged cultures.
When cultures become carbon-limited, the pH of the cultures
increases. This was shown in the previous cultures using
different volumes; the pH of the 150, 300 and 450 ml cul-
tures at Day 13 were 8.3, 8.9, and 9.4, respectively. How-
ever, if similar cultures were aerated (200 ml per minute)
similar yields were obtained with all volumes at Day 9
(20-26 fluorometry units), and the pH of the cultures were
only 8.1-8.2. Therefore, aeration of cultures containing
larger liquid volumes than the recommended levels would
serve to prevent the pH of the cultures from rising sig-
nificantly as long as relatively dilute culture media were
used. In more concentrated media, such as Gorham's medium
or sewage effluents, the mass of algae grown is so great
that aeration cannot supply the carbon-demand of the cul-
tures and the air must be supplemented with CO2. A concen-
tration of 0.5% CO2 in air is sufficient to maintain the
pH of cultures of algae in Gorham's medium at pH 7.0-7.5
up to culture densities of at least 1,000 mg/1.
Shaking of cultures has been suggested as an alternative
to merely leaving the .fcultures quiescent in a culture
room. In order to test' if shaking improved the rate of
growth of SelenastruroVln AAM, tests were carried out with
different volumes and with or without shaking (100 oscil-
lations per minute). Quiescent cultures with 25 ml/50 ml
Erlenmeyer flasks had u max of 1.53; 50 ml/125 ml Erlen-
meyer flasks had u max of 1.76 ± .099; and 150 ml/500 ml
Erlenmeyer flasks had u max of 1.82 ± .17. Cultures of
22
-------
t
SJ. I-
Cl i
O
01
NORMAL (Foam Plugs) —
Rubber
Stopper
Fig. JO, The rate ol growth of Selnnostrum
in AAM: Effect of limiting carbon.
4 6
TIME (Days)
10
23
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150 ml/500 ml Erlenmeyer flasks which were shaken had y max
of 1.83 -t .091. Thus, shaking of cultures results in no
improved maximum rates of growth as long as relatively low
volumes of liquid per flask are used.
EFFECT OF LIGHT INTENSITY
Thus far, different concentrations of sources of nutrients
have been shown to not affect the maximum rates of growth
of the green alga Selenastrum. One factor not evaluated
thus far has been the source of energy to the cultures,
light. When cultures of Selenastrum in AAM were incubated
at different light intensities we found that within .3 days
there was an effect of light intensity on the rates of
growth (Figure 11). By Day 7, cultures in 50 ft C of light
had yields of approximately 15% of those in the 400 ft C
suggested for AAP tests. Cultures at 100 ft C were inter-
mediate with about 25% growth. Higher yields were attained
in cultures with 1,000 ft C up until Day 9 when cultures
from 200, 400 and 1,000 ft C had similar cell densities.
By Day 11 the algal growth of cultures in 100 ft C had also
reached those with the higher light intensities and cul-
tures in 50 ft C were within 50%. . Thus, light intensity
will affect the rate of growth of algae in nutrition studies,
but with increased incubation the algal growth in cultures
with less than the recommended 400 ft C will catch up.
24
-------
Fig. 11, The rote of growth of Selenastrum
n AAM: Effect of light intensity.
TIME (Days)
25
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SECTION IV
THE APPLICATION OF AAP
COMPARISONS USING THE THREE AAP ALGAE
As a demonstration of the utility of the AAP for measuring
the available nutrients in different lake waters, results
of tests using Selenastrum (1,000 cells/ml), Microcystis
aeruginosa (50,000 cells/ml), and Anabaena flos aquae
(50,000 cells/ml) are summarized in Table 2 as the calcu-
lated concentrations of available P from growth tests as
compared to the soluble POu-P of the lake waters. There
is some variability in the results using the different
algae, but it is evident that reasonably good results were
obtained with any of the three algae. More detailed
studies of this nature have been published elsewhere (7,8).
USE OF IN SITU ALGAE IN THE AAP•
The suggested algae for use in the AAP were selected to
represent the green algae, non-nitrogen-fixing blue-green
algae, and nitrogen-fixing blue-green algae. These repre-
sentative algae can be maintained in laboratories as stock
cultures and are thus available for use at any time. How-
ever, other algae can also be used in AAP tests, and they
do not necessarily have to be laboratory cultures. Mix-
tures of algae of ecological importance can be readily used
to compare results obtained with the AAP algae. For
instance, a series of tests of the growth of algae from
Lake Kegonsa (May, 1973) (mostly Centrales Diatoms) have
been carried out. The first test compared the rates of
growth of this mixture of diatoms versus Selenastrum in
Gorham's medium. Ten ml of Lake Kegonsa water were added
to 150 ml of medium whereas an initial Selenastrum concen-
tration of 1,000 cells/ml was used. Either type of algae
grew very well in this medium. At Day 4 the Lake Kegonsa
algae consisted mostly of diatoms (Centrales and Pennales)
with some green algae. The u max of this mixture of algae
26
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Table 2. COMPARISONS OF SOLUBLE POi»-P AND CALCULATED
AVAILABLE PHOSPHORUS FROM AAP GROWTH TESTS OF WATER SAMPLES
FROM THE OUTLETS OF MADISON, WIS AREA LAKES
Chemical
analyses
Lake Soluble PO^-P -
Date sampled (mg P/l)
l/30/73d Mendota
Monona
Wingra
Waubesa
Kegonsa
l/18/73e Mendota
Monona
Wingra
Waubesa
Kegonsa
0.12
.076
.010
.060
.020
.14
.10
.020
.066
.042
aSelenastrum capricornutum (AAP) ,
^Microcystis aeruginosa (AAP), 50,
cAnabaena flos aquae
^Low flow period
eHigh flow period
(AAP), 50,000
Available phosphorus by growth tests
Selenastruraa
0.14
.080
0
.062
.010
.16
.11
.010
.062
.020
1,000 cells/ml
000 cells/ml
cells /ml
(mg P/l)
Micr,ocystisb
0.082
.050
.005
.035
.025
.12
.080
.010
.05
.025
Anabaenac
0.12
.12
.005
.050
.025
.13
.070
.005
.035
.010
27
-------
was 1.92 + .35 whereas the y max of the Selenastrum was
1.72 ± .21. Similar results were also obtained when tests
were carried out in AAM.
Mixtures of algae from Lake Kegonsa were used in AAP tests
with different lake waters with and without spikes of
nutrients. When raw lake waters (150 ml) were inoculated
with 10 ml of Lake Kegonsa water the initial fluorescence
of the cultures varied from 0.03 to 0.084 Aminco units
depending upon the source of water (Figure 12). The maxi-
mum yield in Lakes Kegonsa and Mendota waters was reached
by Day 5 (1.6 and 1.8 fluorometry units/ respectively).
Lake Mendota or Kegonsa waters supplemented with N or N + Fe
(AAM levels) continued to grow until Day 7. There was little
growth of algae in Lake Wingra waters unless P04-P was added.
Since the waters of Lakes Kegonsa and Mendota at the time
of sampling (May, 1973) contained 0.06 and 0.13 mg PO«»-P/1,
respectively, there was no stimulation of growth over that
in unspiked cultures when more POi,-P was added. However,
increased growth occurred in either lake water when N was
added. Thus, tests with these mixtures of iii situ algae
indicated that algal growth in these lake waters would be
limited by available N and available P in Lake Wingra water.
These conclusions are the same as arrived at when Selenastrum
was used (Figure 6). Therefore, one can obtain logical
results using in situ algae in AAP tests, but the convenience
and reproduciblTity of tests with the selected three species
of laboratory algae make them the preferred organisms of use
for most applications. Special tests, if warranted, using
iri situ algae could be used as checks on the results with
Selenastrum.
28
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01
n
O
-^o
O
ui SIUOJDIP osuo6a>| a>
jo y$v& '
-------
SECTION V
REFERENCES
Environmental Protection Agency. "Algal Assay
Procedure: Bottle Test." U.S. Gov't. Printing Office,
Washinton, D.C. Report No. 1972-795-1461. Region
No. 10. August 1971. 82 p.
Environmental Protection Agency. "Marine Algal Assay
Procedure: Bottle Test." U.S. Gov't. Printing Office,
Washington, D.C. Report No. 1975-697-829. December
1974. 43 p.
Fitzgerald, G. P. "Aerobic Lake Muds for the Removal
of Phosphorus from Lake Waters." Limnol. and
Oceanogr. 15^550-555, 1970.
Fitzgerald, G. P. "Evaluations of the Availability of
Sources of Nitrogen and Phosphorus for Algae."
J. Phycology 6:239-247, 1970.
Fitzgerald, G. P. "Nutrient Sources for Algae and
Their Control." U.S. Gov't. Printing Office, Washington,
D.C. Report No. EPA 1.16:16010 EHR 8/71; NTIS No.
S/N 5501-0214. U.S. Environmental Protection Agency.
1971.
Fitzgerald, G. P. "Bioassay Analysis of Nutrient
Availability." p. 147-169, In: Nutrients in Natural
Waters. Allen, H. E., and J. R. Kramer (eds). John
Wiley & Sons, New York. 1972.
Fitzgerald, G. P., S. L. Faust, and C. R. Nadler.
"Correlations to Evaluate Effects of Wastewater
Phosphorus on Receiving Waters." Water and Sewage
Works 120(1) :48-55, 1973.
Fitzgerald, G. P., and P. D. Uttormark. "Applications
of Growth and Sorption Algal Assays." U.S. Gov't.
Printing Office, Washington, D.C. Report No. EPA-
660/3-73-023. U.S. Environmental Protection Agency.
February 1974. 176 p.
Fitzgerald, G. P., and S. L. Faust. "The Release,
Sorption and Availability to Algae of Phosphorus from
Lake Muds." Submitted for publication, 1975.
30
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.. r., V^''. .„ ;,,; • -
I .J..J • '••"•".' ,. i-""' '
I.' .' _ '(': t-'/ " " '- ' ,. ;..'>' ''
10 Fitzgerald, G. P./ M. S. Torrey, and G. C. Gerloff.
"The Self-Purification of Green Bay of Contributed
Algal Nutrients." Submitted for publication, 1975.
11 Fitzgerald, G. P., and G. C. Gerloff. "The Nutrition
of Great Lakes Cladophora. " Comp±e'LlJon—Report.
U.S. Environmental Protection Agency. 1975':,.^
12 Hughes, E. 0., P. R. Gorham, and A. Zehnder.
"Toxicity of a Unialgal Culture of Microcystis
aeruginosa." Can. J. Microbiol. 4:225-236, 1958.
31
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