EPA-GOO/3-84-016
January 1984
THE NUTRITIONAL ECOLOGY
OF GREAT LAKES
CLADOPHORA GLOMERATA
by
Gerald C. Gerloff
and
J. Vic Muth
Department of Botany
University of Wisconsin-Madison
Madison, Wisconsin 53706
Grant No. R804402010
Project Officer
Nelson A. Thomas
Large Lakes Research Station
Environmental Research Laboratory-Duluth
Grossc Ilse, Michigan 48138
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
DULUTll, MINNESOTA 55804
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2.
EPA-600/3-84-016
3. f^CiPlENT'S ACCESSION NO.
fmk 116571
4. TITLE AND SUBTITLE
The Nutritional Ecology of Great Lakes Cladophora
glomerata
5. REPORT DATE
January 1984
6. PERFORMING ORGANIZATION CODE
7. AUTMOR1S)
G. C. Gerloff and J. V. Muth
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NA>4E AND ADDRESS
Department of Botany
University of Wisconsin-Madison
Madison, Wisconsin 53706
10. PROGRAM ELEMENT NO.
It. CONTRACT/GRANT NO.
R804402
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/03
15. SUPPLEMENTARY NOTES
.tract Various bloassays, primarily plant analysis, were utilized to evaluate relative nutrient
supplies and orlnary growth limiting nutrients for Cladophora glomerate growth In parts of Green Bay,
Lake Michigan, known "ho differ marked I y In degree of pollution"! Pre 11 ml nary studies indicated emphasis
should be on evaluations of five nutrients: phosphorus, nitrogen, boron, sulfur, and vitamin Bj• The
bIoassays Indicated that phosphorus very likely Is the critical nutrient In nuisance C. glomerate
growths and that at times phosphorus supply actually Is reduced to growth-Ilmlting concentrations.
However, the possibility that vitamin B, "Vay at times be critical to C. glomerate cannot as yat be
eliminated. '
. The bloassoys employed (total P, hot-watei—extractable P, and alkaline phosphatase
activity) were In agreement and coos I stent In Indicating phosphorus availability limited C. glomerata
growth.
In further development of plant analysis as a bloassay, nitrogen and phosphorus critical
concentrations were demonstrated to be relatively constant In C. glomerata of different ages and grown
under various environmental conditions which would affect the rate and snount of growth.
A requIrement for vitamin Bjj aid very high requirements for sulfur and boron were
confirmed as unusual nutritional features of C. glomerata.
Data obtained from solution replacement cultures indicated that, even when adequate total
phosphorus Is available, the growth of C. glomerata becomes less than optimum at phosphorus solution
concentrations of 0.014 ppm and less, and at potassium and calcium concentrations below 0.18 and 0.24
own, respectively.
Various algae, primarily from Lake Michigan, failed to produce Inhibitors of C. glomerata
growth to a degree that would suggest they are Important In the ecology of C. glomerata In the Great
Lakes.
17.
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
ii
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FOREWORD
Pollution from municipalities and industries has affected the Great
Lakes adversely with increased growths of nuisance algae, particularly
Cladophora glomerata, as one pollution response. Great masses of these
algae, attached to rocks, floating on the surface, or blown onto swimming
beaches, have become all too common in the Great Lakes.
While the correlation between nuisance Cladophora growths and pollution
is obvious, the development of specific and effective control measures
will be most efficiently carried out if the nutritional requirements of
C1adophora glomerata arc understood and if the key nutritional factors in
pollutants can be identified. For a number of years, the laboratory of
Dr. G. C. Gerloff in the Department of Botany at the University of
Wisconsin-Madison has been involved in (1) laboratory studies on the
nutrition of nuisance aquatic plants, (2) application of the nutritional
data in the control of nuisance growths, and (3) development of plant
analysis as a reliable procedure for evaluating nutrient supplies in aquatic
ecosystems.
This is a report on recent studies of the laboratory on C1adophora
glomerata, with essentially the objectives mentioned above. The work
has progressed to the point that practical applications are possible, for
example in the conclusion that phosphorus is the critical factor in the
development of nuisance Cladophora growths. This makes possible the develop-
ment of control measures, based on reduction of phosphorus input, with
confidence that the measures will be effective.
Michael S, Adams
Professor of Botany
Laboratory of Aquatic
Plant Ecology
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ABSTRACT
Various bioassays, primarily plant analysis, were utilized to evaluate
relative nutrient supplies and primary growth limiting nutrients for
Cladophora glomerata growth in parts of Green Bay, Lake Michigan, known to
differ markedly in degree of pollution. Preliminary studies indicated
emphasis should be on evaluations of five nutrients: phosphorus, nitrogen,
boron, sulfur, and vitamin The bioassays indicated that phosphorus
very likely is the critical nutrient in nuisance C. glomerata growths and
that at times phosphorus supply actually is reduced to growth-limiting
concentrations. However, the possibility that vitamin Bj may at times be
critical for Cladophora glomerata cannot as yet be eliminated.
The bioassays employed (total P, hot-water-extractable P, and alkaline
phosphatase activity) were in agreement and consistent in indicating
phosphorus availability limited C. glomerata growth.
In further development of plant analysis as a bioassay, nitrogen and
phosphorus critical concentrations were demonstrated to be relatively
constant in Cladonhora glomerata of different ages and grown under various
enviornniental conditions which would affect the rate and amount of growth,
A requirement for vitamin B12 an^ very high requirements for sulfur
and boron were confirmed as unusual nutritional features of Cladophora
glomrrata. Comparisons with the requirements for other green and blue green
algae established the uniqueness of the high sulfur requirement for
iv
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C. glomerata. Tlicrc was no indication of sulfur deficiency in Cladophora
sp. from the Great Lakes.
Hate obtained from solution replacement cultures indicated that, even
when adequate total phosphorus is available, the growth of Cladophora
glomerata becomes less than optimum at phosphorus solution concentrations
of 0.014 ppm and less, and at potassium and calcium concentrations below
0.18 and 0.24 ppm, respectively. This supports the concept of critical
concentrations in the water for optimum C. glomerata growth.
Trace clement studies indicated Cladophora glomerata requires an in-
organic nutrient not generally recognized as essential for plant growth.
The alga responded positively and significantly to a mixture of elements
of suspected essentiality. However, systematic elimination of elements
from the mixture failed to identify one element as responsible for the
yield increases. The situation is more complex than anticipated.
The culture and nutrition of Nitzschia palea, a very common diatom
in the Great Lakes, was investigated. Silicon concentrations well above
those in available media were essential for successful laboratory growth
of Nitzschia palea.
Various algae, primarily from Lake Michigan, failed to produce
inhibitors of C. glomerata growth to a degree that would suggest they are
important in the ecology of Cladophora glomerata in the Great Lakes.
Laboratory studies were initiated on the toxicity of heavy metals
to Cladophora glomerata.
This report was submitted in fulfillment of Contract No. R80440210 by
Hnvironmental Research Laboratory under the sponsorship of the U. S.
Environmental Protection Agency. This report covers the period May 1, 1976,
v
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September 30, 1978. nnd work was completed as of September 29, 1978
vi
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CONTENTS
Foreword iii
Abstract iv
Figures viii
Tables ix
Acknowledgement xii
1. Introduction 1
2. Conclusions and Recommendations 4
3. Controlling Nutritional Factors in Nuisance Cladophora
glomerata Growths in Lake Michigan 7
4. Further Refinement of the Plant Analysis Bioassay for
Nutrient Supplies in Natural Waters . 28
5. Confirmation of Unique Nutritional Features of
Cladophora glomernta 36
6. Growth of Cladophora gloroerata at Element Concentrations
Approximating Lake Concentrations 43
7. An Additional Essential Element for Cladophora glomerata 51
8. Heavy Metal Toxicity to Cladophora glomerata 58
9. The Culture and Nutrition of the Diatom N'tzschia palea 65
10. Tests on the Production by Algae of Cladophora glomerata
Growth Inhibitors 78
References 86
90
Appendices
A. Statistical Analyses ¦ 9b
B, Additional lixpurimcntni Data 130
vii
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FIGURES
Number Page
1 Total nitrogen (%) in Cladoohora sp. 10
2 Boron (ppm) in Cladophora sp. 10
3 Sulfur (%) in Clndophora sp. 11
4 Total phosphorus (%) in Cladophora sp. 11
5 The effect of culture period length on N critical
concentration in Cladophora gloroerata 29
6 The effect of culture period length on P critical
concentration in Cladophora glomerata 30
7 The effect of low K concentration on P critical
concentration in Cladophora glomerata 34
8 The effect on Nitzschia pa 1ea yield of increasing
the media Si concentration and substituting
Ca(N0a)2 for NaNCb. 68
j
viii
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TABLES
Number Page
1 Response of Cladophora glomerata to Differential Nutrient 15
Enrichment of Lake Michigan Water Collected at Green Bay
Campus
2 Response of Cladophora glomerata to Differential Nutrient 17
Enrichment of Lake Michigan Water Collected at Bayshore Park
3 Response of Cladophora glomerata to Differential Nutrient 19
Enrichment of Lake Michigan Water Collccted at Egg Harbor
4 Response of Cladophora glomerata to Differential Nutrient 21
Enrichment of Lake Michigan Water Collected at Gill's Rock
5 Response of Cladophora glomerata to Differential Nutrient 23
Enrichment of Lake Michigan Water Collected at Bailey's
Harbor
6 Biological Assays for Adequacy of P Supply for Cladophora 25
glomerata Growth at Various Green Bay and Lake Michigan Sites
7 Assays for Adequacy of P and N Supply for Cladophora glomerata 26
Growth at Various Green Bay and Lake Michigan Sites Sampled
September 18, 1977
8 The Effect of Different Patterns of Addition of a Growth- 31
limiting Amount of N to Cladophlora glomerata Cultures
9 The Effect of Different Patterns of Addition of a Growth- 32
limiting Amount of P to Cladophora glomerata Cultures
10 The Effect of Low Temperature (15C) on P Critical Concentration 33
in Cladonhora glomerata
11 Confirmation of Quantitative Responses of Cladophora glomerata 37
to Vitamin Bj2 in Culture Medium
12 Critical S Concentrations and the Range of Concentrations in 38
Various Algae
13 Sulfur Analysis of Cladophora sp. Collected from the Great 40
Lnkrs
ix
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14 Yield and B Concentration of Cladophora glomerata Isolated
from l.nko P.ru' jiikICrown in Culture Medium DilTerinu in B
Concent rat ion
15 Response of Cladonhora ulomcrata to Low Concentrations of
Essential F.lemcnts Made Available in Nutrient Solution
Replacement Cultures
16 Response of Cladonhora glomerata to the Addition of Potentially
Essential Micronutrients
17 Response of Cladonhora glomerata to the Addition of B7
Micronutrients
18 Response of Cladonhora glomerata to the Addition of Ci3
Micronutrients
19 Response of Cladophora glomerata to the Addition of Ci3
Micronutrients Using Zoospores as Inoculum
20 Response of Cladophora glomerata to Various Concentrations
of Copper
21 . Response of Cladonhora glomerata to Various Concentrations
of Manganese
22 Response of Cladonhora glomerata to Various Concentrations
of Cadmium
23 Response of Cladophora glomerata to Various Concentrations
of Lead
24 Response of Cladophora glomerata to Various Concentrations
of Zinc
25 Recommended Culture Solution for Nitzschia palea
26 Comparison of Silicon Sources of Nitzschia palea
27 Response of Ni tzschia pa 1ea to Various Levels of the Essential
Trace Elements
28 Response of Nitzschia pa lea to Changes in Various Physical
r.nvironmcnrn I Factors
29 Critical Concentrations and Range in Concentrations of Four
lissential Nutrient Elements in Nitzschia palea
50 I:ft'eet of Lake Michigan Seoncdesmus quadricauda on Growth of
Cladophora glomerata in a Combined Culture
41
48
52
53
54
56
59
60
6 I
62
63
70
72
73
74
75
80
x
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31 liffcct of Lake Michigan Chlorrlla sp. on Growth of Cladophora 81
glome rat a in a Combined Culture
32 Effect of Lake Mendota Sccnedesmus qtiadri cauda on Growth of 82
Cladophora ulumcrata in a Combined Culture
33 Tests on the Production by Various Algae of. a Cladophora glomerata 83
Growth Inhibitor
xi
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ACKNOWLEDGMENTS
The assistance of John Devereux, Hope Lester, Richard Norby, Corinne
Scofield and Lynn Zimmerman in performing the experiments reported is
gratefully acknowledged. The laboratory help of James Dietrich and Kathy
Gallagan also is recognized.
We thank Beverley Helms for her patience and endurance when typing
the manuscript.
Appreciation also is expressed for the assistance and interest of
Mr. Nelson Thomas, the Grant Project Officer.
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SECTION 1
INTRODUCTION
The pollution of the Great Lakes with municipal and industrial wastes
has resulted in increased growths of nuisance algae. Filamentous green
algae in the genus Cladophora are among the most troublesome of the nuisance
organisms. The development of measures for the reduction of the Cladophora
growths, based on nutrient limitation, will be facilitated by an under-
standing of the importance of specific nutrients in the growth of Cladophora
in the field, in other words, in the nutritional ecology of the Cladophora.
A primary goal of this project has been to identify the nutrients, both
inorganic and organic, most likely to limit and control the growth of
Cladophora glomerata in Lake Michigan.
The knowledge of the qualitative and quantitative nutritional require-
ments necessary to evaluate the field nutrition of Cladophora glomerata
usually can be obtained most effectively in the laboratory. An earlier
report (Gerloff and Fitzgerald, 1976) described successful efforts to bring
Cladophora sp. into laboratory culture in a synthetic medium and to study
its nutrition in detail. Requirements for vitamins Bi and Bi 2 arid very high
requirements for boron (B) and sulfur (S) were among the unusual nutritional
features of C1adophora glomerata that were identified. Additional nutrition-
al studies will be presented in this report, particularly studies on nutri-
tional features which might be significant in nuisance Cladophora growths in
1
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Lake Michigan.
The bioassay known as plant or tissue analysis has been the primary
procedure used on this project to evaluate the nutrition of Cladophora sp.
in the Grout Lakes and to identify tlie nutrients most likely to control
excessive growths. Hie initial results of plant analysis, described in an
earlier report (Gcrloff and Fitzgerald, 1976), suggested phosphorus (P)
to be the primary limiting element. However, this conclusion was based on
relatively few samples. It seemed worthwhile therefore to extend the survey
by more thoroughly sampling a specific area in Lake Michigan and by
verifying plant analysis results with results from other bioassays. One
such bioassay involved a lake water enrichment technique similar to the Algal
Assay Procedure (National Eutrophication Research Program, 1971). Specific
bioassays for P included total tissue P, hot-water extractable P (Fitzgerald,
1969), and a procedure based on the activity of the enzyme alkaline phos-
phatase (Fitzgerald and Nelson, 1966). Total N, uptake of NHi»-N in the dark
(Fitzgerald, 1969) and accumulation of NO3-N in the algae (Ulrich, 1961;
Cataldo, et al, 1975) were tested as bioassays for available N. Bioassays
based on algae analyses and growth avoid many problems of sampling and
data interpretation associated with chemical analyses of water samples
(Gcrloff, 1969: Gcrloff, 1973; Gerloff and Fishbeck, 1973).
Studies on the primary growth limiting nutrients for Cladophora
glomcrata, and other nuisance aquatic organisms, seem justified even though
it is generally agreed that reduction in P input usually is the most
practical procedure for a nutritional control program. However, it cannot
be assumed that P always is the key limiting nutrient in nuisance algae
growths (Ryther and Dunstan, 1971; Gerloff, 1975). This is particularly
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true for an organism such as Cladophora which requires external supplies of
organic factors, vitamins Bi and Bi2, as well as inorganic. The presence
of the vitamins in organic pollutants or their production by bacteria
could be critical in Cladophora growth. Also, reliable and simple assays
arc needed to predict the degree of success of control measures based on
reduced P input. It cannot be assumed that every reduction in the level of
P in a body of water will reduce nuisance aquatic growths, because the
element nay not be reduced to a 1imiting level. Assays which would
determine whether P, or some other nutrient, 1imits Cladophora growth in a
specific area at a specific time would be useful.
3
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The conclusions from this project can most readily be evaluated when
related to the primary objectives. Those goals were (1) to identify the nu-
trient which is the key or limiting factor in the development of nuisance
Cladophora glomerata growths in Lake Michigan and (2) to continue laboratory
experiments on aspects of the nutrition of Cladophora glomerata which will
be useful in understanding the ecology of that organism.
The primary conclusions are:
1. Cladophora glomerata becomes phosphorous deficient in the least polluted
parts of Green Bay during late summer. Phosphorus seems to be the key
nutrient in the development of nuisance C. glomerata growths.
2. There is a close correlation between pollution of Green Bay and the
development of phosphorus deficiency in Cladophora glomerata.
3. Cladophora glomerata has extremely high and unique requirements for
sulfur and boron, but these elements are not critical in the nutritional
ecology of the organism.
4. While phosphorus very likely is the key factor in nuisance Cladophora
glomerata growths, the possibility that vitamin Bi is a critical
nutrient cannot be eliminated at this time.
5. Relatively simple bioassays are available for evaluating nutrient
supplies for Cladophora glomerata.
4
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6. Critical nitrogen and phosphorus concentrations, which are the basis
of nutrient assays through plant analysis, are relatively constant for
Cladophora gloinerata under a variety ot' environmental conditions and
at various stages of growth. This is a critical point of support for
plant analysis as a reliable nutrient bioassay.
7. Cladophora glomerata growth seems to be less than optimal when the
phosphorus solution concentration decreases to 0.014 ppm and below.
8. Inhibitor production by common algae probably is not a major factor in
the ecology of Cladophora glomerata.
9. An adequate supply of soluble silicon is a critical factor in the
successful laboratory culture of the diatom Nitzschia palea. Media
commonly used for culturing blue-green and green algae contain in-
adequate silicon for optimum growth of the diatom.
The primary recommendations which seem justified from the information
obtained are:
1. Reduction of the phosphorus input probably is an effective means of
reducing nuisance Cladophora glomerata growths in the Great Lakes and
should be implemented.
2. Further studies should be carried out on the vitamin Bt field nutrition
of Cladophora glomerata in the Great Lakes and on the possible key role
of vitamin Bj in nuisance growths under some conditions.
3. Plant analysis and other bioassays should be more widely used in
evaluating nutrient supplies and critical nutrients for nuisance growths
of Cladophora glomerata and other species.
4. Further laboratory studies on the trace element nutrition, particularly
heavy metal toxicity, of Cladophora glomerata, and the other primary
5
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algae of rhe Great Lakes, should be carried out and the results should
be related to the ecology of the algae in the Great Lakes.
6
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SECTION 3
CONTROLLING NUTRITIONAL FACTORS IN NUISANCE
Cladophora glomerata GROWTHS IN LAKE MICHIGAN
The results of various bioassays to identify the critical or control-
ling nutrients for nuisance growths of Cladophora glooerata are presented
in this section. Primary emphasis is on "plant analysis" as a diagnostic
technique, partly because of the long-term interests of the Principal
Investigator in this procedure and also because it indicated a close
correlation between pollution and the algae concentrations of N and P on
samples of Cladophora sp. randomly collected from the Great Lakes (Gerloff
and Fitzgerald, 1976). This was particularly apparent in samples from
Green Bay (Fitzgerald, Torrey, and Gerloff, 1975) in which the P concentra-
tion in Cladophora sp. progressively decreased with increasing distance
between the collection site and the point of entry of the nutrient-rich
Fox River into lower Grcon Bay, Green Bay seemed a particularly desirable
7
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area to sample in further studies.
The initial studies on nutrient supplies in Green Bay, involved samples
collected only on one date. It seemed worthwhile to sample algae at specific
sites several times during the growing season. Also, because Cladophora
",1 omernta was shown to have very high requirements for S and 3, these elements,
in addition to N and P, were considered possible key nutrients, and were
included in the plant analysis assay.
GENERAL EXPERIMENTAL PROCEDURES
For the bioassays, samples of £. glomerata and of lake water were
obtained primarily at five Lake Michigan sites (Figure 1). Four of the
sites were on the Green Bay side of Door County; the fifth site was on
the Lake Michigan side. The sites were selected to represent a range of
pollution and fertility, resulting primarily from the entry of the Fox
Uivcr into lower Green Hay.
Statistical analyses were run on the dry-weight algae yields from the
experiments included in this and other sections of the report. Homogenicity
of variance within an experiment was tested using Bartlett's and F -tests
max
(Sokal and Rohlf, 1969). If the variances of the treatments were not
homogeneous, these tests were used to create homosccdastic groups within an
experiment. Homosccdastic groups were then tested individually to determine
significant difference at the 59« level using the Student-Newman-Keuls (SNK)
multiple range test (Sokal and Rohlf, 1969). An approximate test, the
equality of means with heterogeneous variances (EMHV), was used to indicate
significant difference at the 5% level between non-homogeneous or
heteroscedastic groups (Sokal and Rohlf, 1969).
8
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C. glorcerata samples were obtained by clipping filaments, indicated as
healthy by green color, from heavy growths attached to rocks close to the
shore or from rock-breakwaters which extended out from the shore. The
algae samples were repeatedly rinsed in distilled water to remove attached
algae and other obvious, contaminating debris. After rinsing, as much
water as possible was hand-squeezed from the algae. The algae then were
placed in nylon bags and transported to the laboratory under refrigeration.
The algae were oven-dried at 65C and ground in a Wiley Mill. Analyses
for N, P, S, and B were by generally accepted quantitative techniques.
Unfortunately, all of the samples from a June sampling were lost because
of the failure of a laboratory deep-freeze unit.
PLANT ANALYSIS
In the plant analysis bioassay, reduction in the supply of an element
to a growth-controlling level for a particular species at a particular
site is indicated by a concentration of the element in a sample of the
species at or below the critical concentration, the minimum plant concentra-
rion which permits optimum growth. A concentration above the critical level
indicates the algae from the site sampled were adequately supplied with the
element. The dates of Cladophora glomcrata sampling and the results of
algae analyses for N, B, S, and P are presented in Figures 1-4, respectively.
The critical N concentration for C. glomerata is 1.10% (Gerloff and
Fitzgerald, 1976). It is apparent from Figure 1 that no N concentration
was close to that value. The lowest concentration was 2.00% in the August
15 sample from Bayshore Park; all other values were more than double the
critical concentration. There was no indication that C. glomerata became N
9
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GREEN
HARBOR
r». 3.S3
b. 2,00
«, 2.30/BITSHORE
'-'PARK
LAKE
MICHIGAN
-6RCEN BAY
'.'tlCAMPUS
FOX C 4 4»
RIVER
Collect Ion dates -
a. July 2S, 1976
b. August IS, 1IH
c. September 12, 1976
Figure 1. Total Nitrogen (%)
in Cladophora
c
LAKE
MICHIGAN
Collection dates -
». July 25. 1976
b. August IS, 1976
c. September 12. 1976
Figure 2. Boron (ppm) in
Cladophora
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2.4
2. 1
.bay shore
PA*K '
LAKE
MICHIGAN
foixr?:
H1VEB.
r_ CKCtX BAY
^CAMPUS
¦ 20
b I b U:.v
1.4 «:•:•
Collection ditci -
¦. July JS, 1974
b. August IS, 1976
e. September 12. 1976
Figure 3. Sulfur (%) in
Cladophora
•mm
' vKvji
• . .13
b. .11
«• .26^4®*TSHORE
'CHCCN ¦**
CAMPUS - '
.44
b. ,*s
'o* C. .»
RIVCK
LAKE
MICHIGAN
Collection dates -
t. July 2S, 19T6
b. Aufusi IS, 1976
c. September 12, 1926
Figure 4. Total Phosphorus (4)
in Cladophora
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deficient at any of the five sample areas during July, August, and Septembe
The high N concentrations in the samples from the University of Wisconsin
Campus at Green Bay correlate with the entrance of pollution at the lower
end of Green Bay via the Fox River.
The critical B concentration in C. glomerata is 110 ppm. As with N,
there was no indication that B supply became growth-limiting at the sample
sites (Figure 2). The lowest B concentration was 173 ppm in the July 25,
Bayshore Park sample; most values were at least double the critical
concentration.
Relatively, S concentrations in the samples were closer to the critical
value of l.S% than were the N and B concentrations (Figure 3). In fact,
the 1.6% S concentration in the August 15 sample was almost at the critical
concentration. The plant analysis results, in general, did not indicate S
supply was critical for C. glomerata growth in Green Bay. It seems inter-
esting that the two lowest algae S concentrations were in samples from the
most eutrophic part of Green Bay.
The analyses for total P in the C. glome rata samples are presented in
Figure 4. One interesting aspect of the P data is the correlation between
the degree of pollution of Green Bay and the P concentration in the algae.
The average P concentration in the three Green Bay Campus samples was 0.42%;
in the Bayshore Park samples, 0.26%; and in the samples from the other three
sites, 0.13%.
No C. glorncvat.i P concentration was as low as the 0,08% critical
concent rat ion. However, in seven of the samples the P concentration was
below 0.15% and in two of the samples was only 0.09%. In an earlier phase
of this project, the plant analysis technique indicated that P did become
-------
a growth-1imiting nutrient in the upper portions of Green Bay (Fitzgerald,
Torrcy, and Gerloff, 1975; Gerloff and Fitzgerald, 1976).
DIFFERENTIAL NUTRIENT ENRICHMENT OF LAKE WATER SAMPLES
Lake water samples were collected at the same sites as Cladophora
glomerata and immediately were packed in ice for transport to the laboratory.
After filtering through Reeve-Angel 934AH glass fibre filters, the samples
were frozen and kept in that condition until utilized in experiments.
Aliquots of the lake water from specific sites were enriched with
nutrients as indicated in Tables 1 to 5. Concentrations of added nutrients
were those in the synthetic medium developed for Cladophora sp. culture
on this project (Gerloff and Fitzgerald, 1976). All the essential inorganic
and organic nutrients were added to lake water in Treatment B; Treatment A
was the synthetic Cladophora medium prepared with distilled water. In the
remaining treatments, one or several essential nutrients were omitted in
each treatment. This approach made possible evaluations of the relative
amounts of specific nutrients present in lake water samples.
The C. glomerata to be used as inoculum was transferred to a solution
containing only NaaCOj and Na2SiC>3 for 48-72 hours prior to inoculation of
an experiment. All cultures were incubated for 21 days under continuous
fluorescent light of approximately 500 t't-c and at a temperature of 23 1C.
Details of procedures used for culturing and harvesting Cladophora can be
found in Gerloff and Fitzgerald (1976).
In most tests, the cultures were autoclaved prior to inoculation.
Because of concern over possible destruction of vitamins Bi and Bx2 and
precipation of iron (Fe) in non-available forms, several duplicate treat-
ments wore included in which the lake water was not autoclaved but passed
13
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through a Whatman GF/F filter. A control with all nutrients added to the
filtered water also was prepared.
The results of the enrichment tests on water collected at the five
Lake Michigan sites are presented in Tables 1 through 5. July and September
water samples from all the sites were used in the enrichment tests. Because
of insufficient time to prepare the experiments, the August water samples
were used from only several sites. Due to its location on lower Green Bay,
the sample site designated as Green Bay Campus - University of Wisconsin
provided water relatively high in nutrients. This was supported by the
appearance of heavy blue-green algae blooms, as well as Cladophora glomerata,
at this location. The Bayshore Park site also was at a rather heavily
polluted section of Green Bay. Egg Harbor, Gill's Rock, and Bailey's Harbor
arc in relatively unpolluted areas of Green Bay and Lake Michigan.
As anticipated, some differences in the results related to variations
in water fertility, for example, the greater growth in the minus P treat-
ment prepared with Green Bay Campus and Bayshore Park water. Nevertheless,
for evaluating primary limiting nutritional factors, variations between
sample sites were not significant.
Any element present in lake water in amounts adequate to support 50
per cent of the yield in the complete medium in lake water (Treatment B)
was considered unlikely to limit algae growth under field conditions. Of
the nutrients tested, this eliminated calcium (Ca), magnesium (Mg),
manganese (Mn), molybdenum (Mo), zinc (Zn), copper (Cu), boron (B),
sulfur (S) and vitamin B12 as critical in £. glomerata growth. The average
yields of 12% of Treatment B in the minus S and 60% in the minus B cultures
prepared in low B glassware are of particular interest because of the
-------
TABLE 1. RESPONSE 01= CtaJophora glomcratri TO DIFFERENTIAL
NUTRI liNT L;NRlc:lIMIiNT OP l.AKli MICHIGAN WATER COL-
LECTLD AT GREEN BAY CAMPUS
Date sampled'
July 26, 1976 September 12, 1976
Ave, nig % Ave, mg %
Designation Treatment liter of B liter of B
A
Cladophi
ora medium*
562
71
584
94
B
C lad.
medium in lake water
793
100
620
100
C
As
B,
-
vit. Bi
82
10
132
21
D
As
B,
-
vit. Bi2
435
55
556
90
E
As
B'
-
N
87
11
141
23
F
As
B,
-
P
140
18
287
• 46
G
As
B,
-
l:e
116
15
249
40
H
As
B.
-
S
704
89
686
111
I
As
B.
-
B, pyrex
SIS
103
-
-
.]
As
B.
-
R, low 1) glass
396
50
356
57
K
As
B.
-
K
319
40
271
44
L
As
B,
In,
- Ca, Mg, Mn, Mo,
Cu
749
94
538
87
M
As U, but filtered 1. w.
.. + vit.
-
-
773
125
N
As
M,
-
vit. Bi
-
82
13
0
As
M.
-
vit. Bi2
-
-
638
103
P
As
M,
-
I'c
-
-
461
74
15
-------
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K. 7.5 ppm Mg, .7 ppm S, 1.12 ppin Fo. 1/5 Johnson's trace elements, B?
and Ci3 solutions, 4 ppm vitamin Eh, 0.5 pg/1 vitamin BJ2 and Na2C03 and
Na2Si03•
t Average of five replicates in each treatment except J, July, which has
three.
very high requirements for those two elements. The enrichment tests gave
no indication of a growth-limiting role for S and B.
Potassium (K) seemed appropriately designated as an element "unlikely
to be limiting but should not be disregarded." Yield in the minus K
treatment varied from 23% in the July 25 water from Bailey's Harbor to
44% in the September 12 Green Bay Campus sample.
Every enrichment test indicated that four nutrients (N, P, Fe, and
vitamin Bi) were most likely to limit growth. The average yield in the -N
treatment in the samples from the three least fertile sites (Egg Harbor,
Gill's Rock, and Bailey's Harbor) was 7%; in the -P treatment, 15%;
in the -Fe, 16%; and with vitamin Bi omitted, 12%.
Some yield differences among the four most limiting nutrients were
significantly different; others were not. For example Table A-8 in Appendix
A shows that in the July 25 sample from Gill's Rock yield was significantly
different in the minus P cultures compared to the minus N (heteroscedastic
group VII) but minus P was not significantly different from minus vitamin
Bi or minus Fc. All evaluations and summaries of the enrichment data
indicated N to be the most likely growth-limiting nutrient in the lake
water samples.
16
-------
TABLE 2, RESPONSf: OF Cladophora glomerata TO DIFFERENTIAL NUTRIENT
FNR ICIIMIiNT OF I.AKF MICH THAN IVATP.R COLLECTED AT RAYSIIORE PARK
+
Oate sampled
July 26, August 15, September 12,
1976 1976 1976
Desig- Ave, mg % Ave, mg % Ave, mg %
n:i t V on Tre.i tmoii t liter of B liter of B liter of B
A
Cladophora medium*
649
83
. 494
84
564
74
B
Clad. medium in lake
water
778
100
586
100
761
100
C
As
B, - vit. B!
71
22
134
23
'80
11
D
As
B, - vit. B]2
324
42
706
120
525
69
E
As
B, - N
52
7
73
12
94
12
F
As
B, - P .
164
21
177
30
190
25
G
As
B, - Fe
155
16
124
21
102
13
11
As
B, - S
501
64
-
-
476
63
1
As
B, - B, pyrcx
869
112
-
-
-
-
J
As
B, - B, low li glass
420
54
338
58
430
56
K
As
B, - K
280
36
-
-
240
32
L
As B, - Ca, Mg, Mn, Mo,
Zn, Cu
709
91
-
-
254
33
¦ M
As B, but filtered l.w.
~ vit.
• -
-
718
123
543 '
• 71
N
As
M, -vit. Bt
-
-
169
29
75
10
0
As
M, - vit. Bl2
-
-
-
-
458 •
60
l»
As
M, - Fe
-
-
-
-
70 .
9
17
-------
* The nutrient medium is the same as
t Average of five replicates in each
has three and D, August, which has
that described in Table 1.
treatment except for J, July, which
two.
ADDITIONAL BIOASSAYS
Because no single test seems sufficiently reliable to permit firm
conclusions on primary growth-limiting nutrients, several additional bio-
assays which have been proposed and tested for estimating N and P avail-
ability for aquatic plant growth (Fitzgerald and Nelson, 1966; Fitzgerald,
1969) were used to evaluate Lake Michigan nutrients. This included hot
water extractable P and alkaline phosphatase activity as P assays and
ammonium-nitrogen uptake in the dark and nitrate accumulation as N assays.
Borderline or critical concentrations had to be established which
would distinguish between £. glomcrata adequately supplied with N or P and
plants deficient in these elements. It could not be assumed that the values
established for other aquatic plants would apply to C. glomerata.
As indicated in Figure B-l, and Table B-l in Appendix B, the critical
concentration for the Fitzgerald hot-water extractable P test was 0.013%.
The alkaline phosphatase assay for P is based on the observation that
increased alkaline phosphatase enzyme activity is induced in plant tissues
under P deficiency. As a result, the more P deficient the environment
from which plants are collected, the higher the alkaline phosphatase
activity. The calibration test reported in Table B-2, and other unreported
tests, indicated the threshold alkaline phosphatase level for C, glomerata,
to be 1 SO units per mg of dried algae.
18
-------
TABLE 3. RESPONSE OF Cladophora glomerata TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COL-
LECTED AT EGG HARBOR
Date sampled
July 25, 1976 September 12, 1976
Ave, mg % Ave, mg %
Resignation Treatment liter of B liter of B
A
Cladophora medium*
597
90
690
90
B
Clad, medium in lake water
660
100
764
100
C
As B, - vit. Bi
107
16
76
10
D
As B, - vit. B)2
-
-
606
79
E
As B, - N
21
3
86
11
F
As B, - P
64
10
149
19
G
As B, - Fe
72
,11
288
38
li
As B, - S
543
82
493
65
]
As B, - U, pyrex
713
108
-
J
As B, - 15, low B glass
545
83
342
45
K
As B, - K
200
30
220
29
L
As B, - Ca, Mg, Mn, Mo, Zn,
Cu
580
88
565
74
M
As B, hut filtered 1. w. +
vit.
-
539
71
N
As M, - vit. Bj
-
-
44
6
0
As M, - vit. Bi2
-
-
389
51
P
As M, - l'c
-
-
142
19
19
-------
* The nutrient medium is the same as that described in Table 1•
+ Average of five replicates in each treatment except for J, July, which
has three.
The uptake of ammonium-nitrogen in the dark bioassay for N availability
recognizes that as plants become N deficient, their capacity for NHi,-N
uptake during a specific time interval increases (Fitzgerald, 1969).
Nitrate also is known to accumulate in many species under conditions of
high N supply. This has frequently been used as an assay for environmental
N supply in agricultural and horticultural crops, for example by Ulrich
(1961). Efforts to calibrate these two tests for Cladophora glomerata are
reported in Tables B-3 and B-4 in Appondix B. Neither test was satisfactory.
When cultured over a wide range of N supply in the nutrient medium, there
was neither a marked increase in the rate of NHi»-N uptake nor an accumula-
tion of NO3-N in the critical zone in which Cladophora yields first
readied a maximum with increasing external N supply. In other words,
NHh-N uptake and NO3 .accumulation were not sufficiently sensitive indicators
of the changing N status of the Cladophora in the critical zone of
yield change.
. Results of three bioassays for P availability to £. glomerata collected
from five Green Bay sites during July and August are presented in Table 6.
No analysis of the July 11 samples indicated P was limiting algae growth.
This is not surprising because July 11 is relatively early in the growing
season, and nutrient supplies had not come under stress.
There was excellent correlation between.the various assays on the
A
samples taken August 24. All three tests indicated that Green Bay Campus
-------
TABI.H 4. RESrONSP OP Clmtophora glomcrata TO DIFFERENTIAL
NIJTU11-NT I.NI\ I CIIMIiNT OI~ IAKK MlCIHfiAN WATIIR
COLLECTED AT HILL'S ROCK
Date sampled
July 25, August 15, September 12,
1976 1976 1976
Dosiij- Ave, mg % Ave, rag % Ave, mg %
nation Treatment liter of B liter of B liter of B
A
Cladophora medium*
840
81
635
76
742
100
B
Clad, medium in lake
water
1038
100
829
100
741
100
C
As
B,
- vit. Bi
239
23
81
10
103
14
P
As
B,
Vit. B i 2
566
55
381
46
.665
90
E
As
B,
- N .
43 •
4 .
130
16
66 •
¦ 9
F
As
B,
- P
181
17
43
5
185
25
G
As
B.
- Fe
152
15
85
10
81
11
11
A?
B,
- S
458
44
473
57
665
90
I
As
B,
- B, pyrex
920
89
-
-
-
-
J
As
B,
- B, low B glass
578
56
415
50
742
100
K
As
B,
- K
350
34
228
27
231
31
L
As
B,
Zn,
- Ca. M;;, Mil, Mo,
Cu
712
69
866
105
582
79
M
As
B,
- Mn
-
-
784
95
-
N
As
B, but filtered l.w.
*¦ vit.
-
-
680
82
806
109
0
As
N,
- vit.. Bi
-
-
57
7
329
44
1'
As
N,
- vit. B,2
-
-
-
-
631' •
85
Q
As
N.
- Fo
-
• -
-
-
59
8
21
-------
* The nutrient medium is the same as that described in Table 1.
t Average of five replicates in each treatment except D and H, September,
which have 4, .) and 0, August, and J, July, which have three and N,
August which has two.
and Bayshore Park algae were well supplied with P. In addition, all tests
indicated the Egg Harbor C. glomerata was P deficient. Total P in the
C_. glomerata was at the critical total P concentration of 0.08%; the hot
water extractable P was just below the 0.013% critical level; the alkaline
phosphatase activity was at 191 which is considerably above the 150 threshold
value. All three tests also indicated the Gill's Rock and Bailey's Harbor
algae, to be close to P deficiency. . .
Several of the bioassays were run on Cladophora glomerata, collected
from six sites on September 18, 1977 (Table 7). This is near the end
of the period of peak algae growth. The total N concentration in every
sample was far above the 1.1% critical concentration. In contrast, the
total P concentration in the Egg Harbor and Newport State Park samples
was below the 0.08% critical level. The hot water extractable values for
those two samples were very close to the 0.013% critical concentration.
The algae were P-deficient. Both the total and hot water extractable
values were much higher on the samples from the other four sites.
DISCUSSION
The primary result from the enrichment tests was the evidence that
only four nutrients, N, P, Fe, and vitamin Bj, need be seriously considered
as growth-limiting factors for nuisance Cladophora glomerata growths in
Green Bay. Although the data are somehwat inconsistent, the results
-------
TABLE 5. RESPONSE OF Ciadophora glomerata TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COL-
LECTED AT BAILEY'S HARBOR
-j*
Date sampled
July 25,
1976
September 12,
1976
Designation
Treatment
Ave. mg
1 iter
%
of B
Ave. mg
liter
%
of B
A
Ciadophora medium*
821
90
653
86
3
Clad, medium in lake water
912
100
763
100
C
As
B, - vit. Bi
58
6
58
8
D
As
B, - vit. B12
720
79
433
57
" E • .
As
B, - N
42
5
58
8
'• • P
As
B» : P
84
9
156
20
G
As
B, - Fc
146
16
84
11
H
As
H, - S
593
65
505
66
1
As
B, - B, pyvcx
907
99
-
-
J
As
13, - li, low B glass
584
64
346
45
K
As
B, - K
213
23
201
26
L
M
As 8, - Ca, Mg, Mn, Mo, Zn,
Cu
As B, but filtered 1. w. +
vit.
543
60
465
388
61
51
• N
As
M, - vit. Bi
-
-
41
5
0
As
M, -vit. Bi2
-
-
123 '
16
P
As
M, - Fe
_
-
8
1
23
-------
* The nutrient medium is the same as that described - .in Tabic 1.
1" Average of five replicates in each treatment except D, G, and L, July,
and B, September, which have four and J, July, and P, September which
nave three.
further indicated that of those nutrients N was the most critical. In
evaluating this result, it should be recognized that the enrichment
procedure is subject to a source of error which may be responsible for
errors in all assay procedures involving water samples withdrawn from a
lake at a specific site and time. The nutrients no longer are replenished
as they are utilized, for example, from organic matter decay and through
release from bottom sediments. The failure of plant analysis to indicate
even borderline N deficiency in any sample confirms the above source of
error and emphasizes the need to translate the enrichment test data to
field conditions with considerable caution.
Two general points relating to the culture and nutrition of £. glomerata
were apparent from the results. First, the data from all sites showed that
yields consistantly were slightly better when nutrients, in the concentra-
tions normally found in the synthetic culture medium, were added to lake
water rather than distilled water (Treatment A vs. B). This suggests that
lake water contains an unrecognized essential trace nutrient or that the
concentration ratios of nutrients in the synthetic medium are not optimal.
Second, autoclaving lake water does not inactivate Fe or destroy vitamins
R! and to a degree that C. glomerata growth is affected. If in-
activation or precipitation did occur, responses to the omission of the vita-
mins and Fe would have been greater in autoclaved than in filtered lake water.
24
-------
TABLE 6. BIOLOGICAL ASSAYS FOR ADEQUACY OF P SUPPLY
FOR CI;u1opltor;i nlomorata CROWTII AT VARIOUS
GRLLN BAY AND LAKE MICHIGAN SITES
July 11, 1977 August 24, 1977
Alkaline Alkaline
Hot H20 phosphatase,+ Total Hot 1^0 phosphatase,
Sampling Total extract. Enzyme units* P, extract. Enzyme units*
site
P,%+
P 9- +
r t
mg algae
O,
•o
P %
r , o
mg algae
Green Bay Canpus
.25
.040
25.4
.32
.027
14.6
Bayshore Park
.23
.019
30.0
.24
.023
8.1
Egg Harbor
.17
.023
134.9
.08
.012
190.8
Gill's Rock
.29
.017
52.3
.12
.014
161.7
Bailey's Harbor
.49
.052
660.9
.12
.015
137.1
* One unit of alkaline phosphatase activity equals the amount of enzyme
• which liberates one mi 1limicromole of nitrophenol/hr, under the
prescribed conditions.
1* ' The critical concentration for total P was 0.08%, for hot-water
extractable P, 0.013%, and for alkaline phosphatase, 150.
They were not consistently different (Treatments B, C, D, G and M, N, 0, P).
Overall the results indicate P is the key nutrient in nuisance
glomerata growths in Green Bay of Lake Michigan. Further sanpling probably
would result in a similar conclusion for other parts of Lake Michigan.
It seems.signifiennt that at certain times and at certain sites, £. glomerata
were collected that were actually P deficient. This indicates not only
that P is the key nutrient but also that reduction in P input into Green
*
Bay would reduce nuisance algae growths.
25
-------
TABLE 7. ASSAYS FOR ADEQUACY OF 1' AND N SUPPLY FOR
t;iaik>l>l)orn glomerata GROWTH AT VARIOUS GKEliN
BAY AND LAKE MICHIGAN SITES SAMPLED SEPTEMBER
18, 1977
Sampling
Total N,
Total P,
Hot H20
s ite
%
%
extract.
P * %
*
Green Bay Campus
2.98
.26
.031
Bayshore Park
2.16
.13
.023
Egg Harbor
2.25
.06
.014
Newport State Park
2.07
.05
.015
Gill's Rock
4.28
.24
.073
Bailey's Harbor
3.29
.14
.035
* Analysis run on dried, ground material rather than fresh material.
While the evidence suggests P is the key Factor in £. glomerata growths,
the possibility that vitamin B, at times might be in that role cannot be
eliminated. The evidence for this statement was obtained from the enrich-
ment tests in which yield was reduced as much by omitting vitamin Bj as
by omroitting 1'. Unfortunately, at this time other assays are not available
to confirm this observation. Further studies on the vitamin nutrition
of C. glomerata under field conditions are needed and should be initiated.
Whenever possible, evaluations of relative nutrients in natural waters
should be based on several techniques. Several of the bioassays tested,
particularly plant analysis, showed promise as simple and reliable tests
for nutrient supplies and growth-1imiting nutrients for aquatic plant growth.
26
-------
The general agreement in the three P hionssnys w.'is pnrticulnrly encouraging.
The importance of obtaining relatively clean and healthy Cladophora sp.
in order to obtain reliable data from plant analysis and other bioassays
should be recognized. In this study, sampling was limited to £. glomerata
plants that seemed healthy as indicated by a green color. Cladophora glomerata
is recognized to have heavy growths of epiphytes which could affect in-
organic analyses of the algae. As a result, careful attention must be given
to removal of as many of the contaminating organisms as possible.
27
-------
SECTION 4
FURTHER REFINEMENT OF THE PLANT ANALYSIS
BIOASSAY FOR NUTRIENT SUPPLIES IN NATURAL WATERS
Plant analysis has been the primary bioassay used on this project to
evaluate nutrient supplies and limiting factors for nuisance Cladophora
glomerata growths. Because plant analysis only recently has been applied to
aquatic plants and aquatic nutrient problems (Gerloff, 1973; Gerloff and
i
Fishbeck, 1973), further studies are needed on the reliability of the tech-
nique and of factors which might affect the critical concentration values.
The consistency of a specific critical concentration when an organism
is grown under widely different environmental conditions is one item of
concern. Questions :ilso arise regarding changes in a critical concentration
as an organism ages and matures. These changes could be associated with
differences in the ratio of protoplasmic to non-protoplasmic constituents
arising, for example, from continued deposition of cell wall components or
increases in carbohydrate or fat reserves. The results in this section are
from several experiments designed to determine the consistency of critical
concentrations in C. glomerata under varying environmental conditions.
EXPERIMENTAL PROCEDURES
Cladophora glomcrata was grown, harvested, and analyzed by procedures
described or referred to in Section 3. Deviations from these standard
28
-------
800 f-
o»
e
600
>-
oc
a
i
400
u
O 200
0
\\.
M
¦•14 DAY CULTURE PERIOD
~ ~ 21 DAY CULTURE PERIOD
0.5 1.0 1.5 2.0
TISSUE CONTENT OF N, %
2.5
Figure S. The effect of culture period length on N critical concentration
in Cladophora Rlomerata.
conditions will be mentioned in the presentation of individual experiments,
RCSULTS . . •
To determine whether N and P critical concentrations decreased as C.
g]oiiicraui aged and matured, these values were compared after 14-day and
21-day culture periods. The N data are plotted in Figure 5; the P data, in
29
-------
800
E 600
£
v 400
(C
Q
I
z
W 200
o
0
/
»
I
~
~
i
i
»
t
0
I
• 14 DAY CULTURE PERIOD
~ 21 OAY CULTURE PERIOD
j i i :
.1 .2 .3 .4 .5
TISSUE CONTENT OF P, %
.7
Figure 6. The effect of culture period length on P critical concentration
in Cladophora glonierata.
Figure 6. . Detailed data and statistical evaluations are in the Appendices
(Tables B-5, B-6, A-14, and A-15).
In the N experiment, the maximum yield was approximately 70% greater
at 21 days than at 14. Nevertheless, the critical concentration changed
only slightly, from approximately 0.9 to 0.8%. The lower value was for
30
-------
TABLE 8. THE EFFECT OF DIFFERENT PATTERNS OF ADDITION OF A
ClUOWlll-LlMli INlj AMOUNT OF N TO Cladophora glomerata
CULTURES
Treatment*
Ave, algae
dry-wt. yy;ld,
mg/1
Ave. N conc.
in algae.
%
16.4 ppm N, initially
311
3.75
1.6 ppm N, initially
219
.94
0.8 ppm N, initially;
at 9 days, .16 ppm N
each of 5 days
172
1.10
10 additions of .16 ppm
N during 14-day period
160
1.13
.* The nutrient medium contained 2.8 ppm P, 9.7 ppm Ca, 18 ppm K, 7.5 ppra Mg,
9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace, NajCOs and NajSiOj, B7 and
C1 3 solutions, and vitamins Bj and Bi2.
~ Average of five replicates in each treatment; 14-day culture period.
the longer culture period. In the P experiment, yield at 21 days was
about 60% greater than at 14 days and the critical concentration was lower
by approximately 0.01%, that is, it decreased from 0.08 to 0.07%,
The procedure for establishing critical element concentrations in £.
alomcrata involves "batch culture" of the alga. The total amounts of an
element to he made available to the algae in all treatments, from optimal
ro suhont inwI, arc added at the 51art of an experiment. Because of concern
tli.it this net hod o L" culture might influence critical concentrations, these
values were established tinilvr several patterns of element addition, as
indicated in Table" 8 and 9. There were no significant differences in yields
31
-------
TABLE 9. T1IK EFFECT OF DIFFERENT PATTERNS OF ADDITION OF
A GROWTH-LIMITING AMOUNT OF I' TO Cladophora
glomerata CULTURES
Treatment*
Ave. algae
dry-wt.
yield, mg/1
Ave. P conc.
in algae,
%
SNK§
2.8 ppm P, initially
311
.77
a
.07 ppm P, initially
164
.06
b
.035 ppm P, initially:
at 9 days, .007 ppm
P each of 5 days
152
.06
b
10 additions of .007 ppm
P during a 14-day period
179
.06
b
* The nutrient mcdium contained 16.4 ppm N, 9.7 ppm Ca, 18 ppm K, 7.5 ppra
Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace, NajCOj and NajSiOj,
B7 and C13 solutions, and vitamins Bi and B12.
+ Average of fiye replicates in each treatment; 14 day culture period.
§ Yields with a common letter arc not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
and no variations in the critical concentrations when a growth-limiting
amount of N or P was m;jde available in one addition or in as many as ten.
additions during a 14-day culture period.
The experiment reported in Tabic 10 was designed to determine if
growth at a much reduced rate might influence the critical P concentration
f°»' Cladophora glomerata. A culture temperature of 15C, rather than the
-------
TABU' 10. THH F.FFFCT OP LOW TF.MPF.RATURF. (IS C, ON P
(.'UITICAL L'ONCPNTUATION IN C t.idophora glomeratn
P added to
culture soln.,*
ppm
Ave. algae
dry wt. yield,
mg/1
Ave. P conc.
in algae,
%
§
SNK
.07
116
.08
a
. 14
173
.08
ab
.28
241
.11
b
.56
270
.15
b
1.12
257
.22
b
2.80
216
.51
ab
* The nutrient medium contained 16.4 ppm N, 18 ppm K, 7,5 ppm Mg, 9.7
- • ppm S. 23.4 ppm Ca, 1.12 ppm Fe, 1/5 Johnson's trace, B7 and C13
solutions, vitamin B| and B12," and NaaCOj and Na2Si0j.
i Average of five replicates in each treatment.
§ Yields with a common letter are not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
usual 23C, was used to reduce the growth rate, As indicated, yields were
considerably less than in most C. glome rata experiments. Nevertheless,
the critical P concentration was in the range of 0.08 to 0,10% and not
significantly different than the value established at 23C.
In establishing critical concentrations, all elements are present in
33
-------
300
250
200
150
100
50
0
1
1
1
0 .05 .10 .15 .20 .25 .30 .35 .40
TISSUE CONTENT OF P, %
Figure 7. The effect of low K concentration on P critical concentration
in Cladophora glomerata.
tin* culture medium in optimal concentrations except the clement for which
the critical concentration is being determined. In contrast, under field
conditions the supply of more than one element might be growth limiting, or
close to growth limiting, at the same time. In the experiment reported in
Figure 7. and Appendix Table ?>-7; the P critical concentration was established
34
-------
when flu* K concentrat ion in :ill cultures was constant hilt less than adequate
for optimal growth. Maximum growth was below the average for a 17-day
culture period indicating that at the higher P concentrations K supply
limited growth. Nevertheless, the P critical concentration was approximately
0.08%, the same as in other tests.
DISCUSSION
The critical N and P concentrations for Cladophora glomerata were
found to be relatively unaffected by the age or maturity of the algae and
by several environmental factors which affected the rate and amount of
algae growth. While additional experiments with C. glomerata, and other
organisms as well, are needed, the results obtained contribute significantly
to the concept of the critical concentration of an element as a specific
value and to the validity of plant analysis as a bioassay for environmental
nutrient supplies. With additional confirming data and with careful
standardization of sampling procedures, it is anticipated that plant
analysis will find the same widespread use for nutrient evaluation in aquatic
environments that it currently enjoys in measuring nutrient availability for
economic crops in terrestrial environments.
35
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SECTION 5
CONFIRMATION OF UN I QUI"; NUTRITIONAL MATURES OF Cladophora glomerata
Establishing the exact qualitative and quantitative nutritional require-
ments of Cladophora glomerata in laboratory studies has been a primary
aspect of this project. In the early stages, there were indications that
C. glomerata from Lake Michigan had very high and unusual requirements for
S and B. The alga also was shown to require external supplies of vitamin
Bt and very likely vitamin B,2. unique requirements for an autotrophic
freshwater alga. Because any unusual nutrient requirement could be the
basis of measures to control nuisance Cladophora sp. growths and because of
conflicting reports on the vitamin B12 essentiality, further studies were
carried out to more closely define requirements for S, B, and vitamin Bj2.
EXPERIMENTAL PROCEDURES
Cladophora glomerata was cultured, harvested, and chemically analyzed
for S and B by procedures detailed or referred to in Section 3.
RESULTS
In the experiment reported in Table 11, Cladophora glomerata responded
to vitamin Hi? with approximately a 100% yield increase. There also were
statistically significant increases between the first three treatments
representing progrcssivcly higher Bia concentrations. It should be noted
that the use of an inoculum which has been through several transfers in
36
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TABLE 11. CONFIRMATION OF QUANTITATIVE RESPONSE OF
Cladophora glomerat a TO VITAMIN Bi2 IN
CULTURE MEDIUM*
Concentration of
vitamin Biz"'", Mg/1
Ave. algae dry-wt.
yield, mg/1
0.00
300 a
0.25
459 b
0.50
622 c
1.0
SIS be
2.0
440 b
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements, B7
and Ci3 solutions, vitamin Bj, and Na2C03 and NajSiOj.
t Inoculum grown through three transfers in rainus-vitamin Bi2 medium during
a seven week period.
5 Average of five replicates in each treatment; yields with a common letter
are not significantly different at the 5% level using Duncan's multiple
range test.
a medium lacking vitamin B is essential in demonstrating the requirement.
Preparation of vitamin B 17 was described in an earlier report (Gerloff and
Fitzgerald, 1976).
Sulfur
On this project, inorganic nutrient requirements of Cladophora glomerata
have been quantitatively expressed primarily as cell critical concentra-
tions, that is the lowest concentrations of elements in algae cells wh ich
permit maximum yield. In earlier studies, the critical S concentration in
a Cladophora glomerata isolate from Lake Michigan was shown to be 1,50%.
37
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TARl.r. 12. CRITICAL R CONCENTRATIONS AND TilP. RANCH OF
CONCENTRATIONS IN VARIOUS ALCAK
Algae
Critical S
concentration, %
Range in
concentration
Lake Huron
Cladophora sp.
Lake liric
Cladophora sp.
Lake Ontario
Cladophora sp.
Drnparnaldia plumosa
Nostoc muscorum
Microcystic aeruginosa
1.70
1.20
1.30
0.15
0. 13
0.13
.90 - 3,46
1.06 - 3.06
.44 - 2.00
.02 - .69
.11 - .34
.07 - .21
The critical S concentrations for other algae had not been established;
however, for agricultural and horticultural species the range is usually
0.08%-0.25?o (Chapman, 1966). To verify the uniqueness of the high C.
glomernta S requirement, the critical concentration of this element was
established for isolates from three additional Great Lakes, for another
filamentous'green alga (l)raparnaldia plumosa). and for two species of
blue-green algae (Nostoc muscorum and Microcystis aeruginosa). Data from
the individual experiments are reported in Appendix B, Tables B-8 through
B-13; the critical concentration established for each organism and the range
of S concentration in the algae in each experiment are presented in Table 12.
38
-------
The critical S concentrations for the three Cladophora sp. isolates
were within the range of 1.2 to 1.7%. The correctness of the 1.5% value
for the Lake Michigan isolate was verified. The uniqueness of the very high
value for £. gioiucrata was supported by the much lower S critical concent ra-
tions for the other algae, 0. 13"» for the blue-green algae and 0.15% for
Draparnaldia plumosa. Both Cladophora glome rata and Draparnaldia plumosa
are filamentous green algae, yet the critical S concentration is 7x greater
for C. glomerata.
In an early phase of this project, samples of Cladophora sp, were
collected from a number of sites on four of the Great Lakes. Where sufficient
plant material remained these samples were analyzed for S, As shown in
Table 13, in spite of the very high requirement, there was little indication
that S supply became a limiting factor in the growth of Cladophora sp.
In one sample, from inside the Milwaukee breakwater on 7-8-74, the S concen-
tration was only 1.5*; in most samples, the concentration was 2.0% or more.
Boron
In the initial phase of this project, the critical B concentration for
Cladophora glomernta from Lake Michigan was established as 110 ppm which
is far above the 5-25 ppui critical concentrations for most plant species
and for other algae (Chapman. 196fi; Gcrloff, 1968). The data in Table
14 are from an effort to establish the critical B concentration in a C.
glomerata isolate from Lake F.rie. There were stat i stical ly
significant yield decreases at the lowest levels of B in the culture
medium. However, even in the B deficient algae the B concentration was in
excess ol 150 ppm which not only confirms the earlier result but suggests
the 110 ppm critical concentration was conservatively low.
-------
TABLE 13- SULFUR ANALYSIS OF Cladophora sp. COLLECTED FROM
Till: fiREAT LAKES
Lake
s amp 1 ed
Sampling site
Date
sampled
S cone
o.
Michigan
Hoi land
10-18-73
2.3
Michigan
Manistique
7-29-74
>3.0
Mich igan
Milwaukee, N. Pt. Park
9- 6-73
3.0
Michi gan
Milwaukee, N. Pt. Park
10-14-73
1.8
Michigan
Milwaukee, N. Pt. Park
7- 8-74
2.2
Michigan
Milwaukee breakwater, shore, inside
6-17-74
2.5
Michigan
Milwaukee breakwater, shore, outside
6-17-74
>1.9
Michigan
Milwaukee breakwater, shore, inside
7- 8-74
1.5
Michigan '
Milwaukee breakwater, shore, outside
7- 8-74
2.5
Michigan
Milwaukee breakwater, 0.5 mile, inside
7- 8-74
2.0
Mich i gan
Milwaukee breakwater, 0.5 mile, outside
7- 8-74
2.3
Mich igan
Milwaukee, Pier 2, inside
7-16-74
1.9
Michigan
Milwaukee, Pier 2, outside
7-16-74
>3.0
Michigan
Milwaukee, Pier 4, inside
7-16-74
2.0
Michigan
Door County, Egg Harbor
9-11-74
1.9
Michigan
Door County, Gill's Rock
9-11-74
3.4
Michigan
Menominee
9-12-74
>2.9
Erie
Western, Port Clinton, Ohio
6-20-74
1.9
Erie .
Eastern, Geneva, Ohio
6-20-74
2.5
Erie
Eastern, l:t. Erie, Ontario
8-11-74
2.9
Erie
Eastern, Pt. Colburnc, Ontario
8-12-74
2.6
Ontario
Olcott, New York
6-20-74
1.9
Huron
Pt. Sanilac, Michigan
10-19-73
2.3
40
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TABLE 14. YIELD AND B CONCENTRATION OF Cladophora glomernta
ISOI.ATHl) 1'PvOM l,AKI:. I1RII! CROWN IN ClJI.TIIftl: MilDl"
D11-1'liRINfG IN B CONCENTRATION
B added to
culture so In.,* ppm
Ave. algae dry-wt.
yield,!" mg/1
Ave, B conc.
in algae, ppm
snk!
0.00
287
178
a
0.00054
343
198
ab
0.0011
423
161
be
0.0054
458
238
c
0.054
520
249
c
0.27
525
264
c
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.5 ppm Mg, 9.7 ppm S. I.12 ppm l'c, .1/5 Johnson's trace elements (~B),
Na^COa, Na2Si03, B7 and C13 solutions, and vitamins Bi and 812. Culture
flasks were low boron, alkali-resistant glassware.
i Average of six replicates in each treatment except for the two highest
treatments which had five replicates each.
!i Yields with a common letter are not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
, DISCUSSION
The unique requt rencnt of Cladophora glome rata for an external supply of
vitamin B12 and its unusually high requirements for S and B have been
verified. Nevertheless, there is no indication from the results in this
section, and from Section 3, that those nutrients play a critical role in
the field nutrition of Cladophora glomerata. The key nutrient instead is
the element I> for which the critical concentration is relatively low (0.08%)
compared to values for other algae and macrophytes (Gerloff, 1969). Natural
41
-------
nml jtollntion sources of S, R, arul vitamin Hi;. apparently arc providing
adequate amounts of these nutrients to tlic parts of the Great Lakes sampled.
Even though the very high S and B requirements seem to be of little
practical importance, they should he of considerable basic physiological
interest. The distribution in the cells and the biochemical utilization
of S which accounts for the 7x greater S requirement of C. glomerata
compared to Draparnaldia plumosa deserves further study. The high B
requirement of C. glomerata is particularly of interest in view of the
complete lack of a requirement in some algae and fungi (Gerloff, 1968).
The physio logical basis for these variations undoubtedly will remain un-
settled until the functions of B in plant structure and/or metabolism are
established. Because of its unique requirement, Cladophora glomerata
should be a useful organism in such studies.
42
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TABLE B-13. Y1 lit,I) AND S CONCENTRATION IN THE BLUE-GREEN ALGA
Microcystis ;i<-rug i nosn CROWN IN CULTtlRf; MF.DIUM
"dTfFERING IN' S CONCENTRATION
S added to
culture solit.,* ppm
Ave. algae dry-wt.
yield,t mg/1
Ave. S cone,
in algae, %
0.12
54
.07
0,24
61
.08
0.49
166
.10
0.97
203
.15
1.94
207
.16
4.85
192
. 16
9.70
207
.19
19.4
177
.21
* The nutrient medium contained 82 ppm N, 18 ppm K, 7 ppm P, 7.5 ppm Mg,
9,7 ppm Ca, 1.12 ppm 1-e, i/5 Johnson's trace elements and Na2C03
and NajSiOj, .
+ Average of four replicates in each treatment.
43
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SECTION 6
GROWTH OF Cladophora glomerata AT ELEMENT CONCENTRATIONS
APPROXIMATING LAKE CONCENTRATIONS
Aquatic plants usually are cultured in the laboratory in nutrient media
containing much higher concentrations of the essential elements than are
present in lakes and streams. This is necessary to provide the total amounts
of the elements necessary for high yields in the small volumes of nutrient
solution provided. As a result, the responses of aquatic organisms to
element concentrations determined in the laboratory usually cannot be
directly related to field conditions.
Responses of Cladophora glomerata to the very low concentrations of N",
P, and other elements characteristic of natural waters can be measured in
various ways. This includes the use of very large volumes of culture medium,
continuous-flow cultures, the frequent addition of an element to a culture
to maintain a very low concentration, and the uptake of a radioactive tracer
at low concentrations over relatively short time periods. Our approach was
to progressively lower the concentration of one element, while making an
adequate total amount available, until a concentration was attained at
which the algae could not absorb the element rapidly enough to produce
maximum growth. This point was evaluated by comparisons with growth in cul-
tures provided with a normal supply of the element under study. The relative
aggressiveness of CIadophora in the uptake of specific elements could be
44
-------
evaluated by comparisons of minimum solution concentrations for maximum
growth of that species with comparable concentrations for other nuisance
algae and macrophytes.
EXPERIMENTAL PROCEDURES
glomerata
Cladophora was confined to a culture vessel of relatively small volume
(2 1) and large volumes of solution were made available to the alga by
draining away and replacing the culture medium at frequent intervals.
Because of the large volumes of nutrient solution required, only two treat-
ments, which were replicated, could be included in each experiment. In
every experiment, one treatment always was the control solution consisting
of Cladophora medium (Gerloff and Fitzgerald, 1976) at one-quarter strength;
the other treatment was similar except the clement under study was provided
at a very low concentration. Experiments were repeated, with a progressively
lower concentration of the limiting element made available in each successive
experiment, until a concentration was established at which rate of absorption
was inadequate to provide yields equal to those in the control solution.
For significant differences in dry weight to be detected in the solution
replacement cultures, growth periods of 10 to 14 days were necessary,
yielding tissue dry weights of 75 to 100 mg per replicate. The lowest
solution concentration that could be tested was a function of the critical
concentration (%) for thr controlled nutrient multiplied by the increment of
totnl growth (mg] produced in the last day of the culture period divided by
the largest culture medium volume (liters) it was practical to make avail-
able to the algae in 24 hours. At least twice this "minimum daily
requirement" of tho controlled element was uukle available to the C i ..dophora .
even at the lowest concentration tested.
-------
Two additional procedures wore tested for establishing minimum nutrient
concentrations for optimum Cladophora glomerata growth. One approach was to
monitor the depletion of I1 using 3?P as a radioactive tracer. Whenever
tlie activity of a culture fell below 50% of the activity of a sterile
uninoculatcd standard, sufficient I1 was added to the culture to equal the
activity in the standard. In this way, the P concentration was controlled
between 0.5 and 1.5x the concentration of the standard.
Radioactivity counts were made both on unfiltered and millipore-filtered
aliquots from the cultures. This was to determine if any separated particles
or microorganisms were sequestering a substantial pool of the P. This was a
critical point, because P tied up in particulate debris might not be available
to the £. glomerata yet would be indicated as available P by radioactivity
counts. No substantial pool of organic P developed in the medium. However,
because of problems from algae contaminants introduced by the repeated
opening of the culture vessels, this technique was abandoned in favor of
frequent replaceinnt of the culture solution.
Another approach attempted was to reduce the algae tissue cultured to
a microscopic quantity and thus avoid the need for nutrient solution replace-
ment altogether. Quarter-strength medium was placed in petri dishes into
which C. glomerata filaments of 5 to 8 cells each were placed. Growth of the
filaments was to he followed by microscopic examination to determine increase
in cell members and filament volume.
The problems of maintaining sterility and concentration were obviated
by this approach. However, two problems with the system could not be
solved. First, the variation in the vigor of randomly selected filaments
was very large, and second a practical and objective procedure to determine
-------
the growth of individual filaments could not be established. Counting cells,
counting branch points on the filaments, and measuring the length of the
filaments were attempted, but the considerable variation in the size of
C. glomerata cells, the degree of branching, and the width of filaments
made all of these indices unsatisfactory.
RESULTS
The data in Table 15 show the results from three experiments in which
Cladophora glomerata was grown at P concentrations approximating those in
lakes and streams. In Experiment 1, the lower P concentration was 0.028
ppm. Yield was not significantly different than in the control culture
containing 1.4 ppm P. In experiments 2 and 3, the P concentration was
reduced to 0.014 ppm, and in both cases glomerata growth was reduced
significantly. In Experiment 2 the dry weight yield was 51% of the control;
in Experiment 3, 80%.
It was anticipated that P concentrations in £. glome rata grown at 0.014
ppm P would be near or below the critical concentration of 0.08%. This was
true in Experiment 3 in which the P concentration was 0.09%. However, in
Experiment 2 the £. glomerata P content was surprisingly high at 0.16%.
Only 26 and 36" of the P added in the 0,014 ppm solution was absorbed
in Treatments 2 and 3 respectively. This supports the view that, as desired,
the pattern of solution replacement employed made much more P available to
the algae than could be absorbed,
Cladophora glomerata yield was significantly reduced when the K con-
centration in the culture medium was reduced from 9.0 to 0,18 ppm. Of
perhaps greater interest was the nearly complete failure of C_. glomerata
47
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TABLE 15. RESPONSE OF Cladophora glomerata TO LOW CONCENTRATIONS OF ESSENTIAL
ELEMENTS MADE AVAILABLE IN NUTRIENT SOLUTION REPLACEMENT CULTURES
Element
varied
Clement
conc.,
ppm
So In.
volume,
1
Element
added,
mg
Cladophora
Dry weight,
mg
yield
Relative,
%
Element in
Conc.,
%
Cladophora
Weight,
mg
Added
element
absorbed,*
%
t-test
1.4
14.2
19.90
73.9
100
0.69
0.51
3
P
N. S.
0.028
14.2
0.40
71.7
97
0.13
0.09
23
1.4
22.2
31.08
99.5
100
0.75
0.75
2
P
<0.05
0.014
22.2
0.31
50.6
51
0.16
0.08
26
1.4
14.2
19.88
100.1
100
0.87
0.87
4
P
< 0.02
0.014
14.2
0.20
79.8
80
0,09
0.07
35
9.0
17.2
154.80
147.7
100
4.92
7.27
5
K
c 0.10
0.18
17.2
3.10
101.3
69
2.47
2.51
81
4.77
5.2
24.69
30.2
100
Ca
< 0.001
0.24
5.2
1.24
4.3
14
--
—
--
* Includes amount of element added in inoculum.
-------
to grow when the Cn concentration was lowered to 0.24 ppm. The amount of
plant material produced was insufficient for Ca analyses.
Two tests were made on £. glonerata response to low N concentrations.
The data were not sufficiently conclusive to include in this report.
DISCUSSION
As suggested in an earlier report (Gerloff, 1975), the data presented
in this section are of considerable interest in relation to the interpreta-
tion of chemical analyses of water samples. A water concentration considered
limiting for nuisance plant growths may be limiting not only because the
amount of an element required for a specific amount of growth is inadequate,
but also because the concentration is so low that the rate of absorption
cannot keep pace with needs for luxuriant growths. In other words, it
cannot be assumed that each unit of a nutrient is equally available to
organisms and equally effective in promoting growth over the range of
concentrations present in natural waters.
The minimal solution concentrations at which maximum growth can be
maintained undoubtedly varies for different aquatic species, but so far only
limited data arc available for such comparisons. One example is the C.
glomerata results and data on the macrophyte Elodea occidental is (Gerloff,
1975). At 0.03 ppm P in solution replacement cultures, Elodea growth was
significantly below the control; C_. glome rata yield was not reduced at 0,028
ppm but was at 0.014 ppm. Cladophora glome rata, therefore, seems more
aggressive than EJ^odon_ in P uptake and utilization.
Cladophora glomornta is most abundant in hard water lakes in which Ca
and Mg concentrations are relatively high. This correlates with the
complete inability of glomerata to grow with only 0.24 ppm Ca in the
-------
nutrient solution, Again comparisons with other organisms become of
interest. In the study mentioned above (Gerloff, 1975), the filamentous
green alga Prapanuil J ia pluniosa made optimal growth in a solution containing
(1,80 ppm whi le r.lodca occidental Is made almost no growth in the same
solution. Information obtained with solution replacement cultures, or
another similar technique, might contribute significantly to an interpreta-
tion of aquatic plant distribution in hard water and soft water lakes.
Solution replacement cultures as described require much time and effort.
However, they have the advantage of indicating responses to element concentra-
tions that are reflected in actual plant growth. This may not always be
true when potential for clement utilization is measured with radioactive
isotope uptake over short intervals. Techniques based on plant growth also
minimize another complication of isotope uptake studies, namely that uptake
of an element is greatly influenced by the degree of deficiency of that
element in the plants used as experimental material.
50
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SECTION 7
AN ADDITIONAL ESSENTIAL ELEMENT
FOR Cladophora glomerata
In the early stages of this project, evidence was obtained that
Cladophora glomerata requires at least one inorganic nutrient currently
unrecognized as essential for plant growth. This was demonstrated through
C. glomerata yield response when B7 and Cu solutions (mixtures of elements
of suspected essentiality) were added to £. glomerata cultures. It was •
anticipated that systematic omission of elements from the B7 and C13 solu-
tions would quickly establish which element was responsible for the
beneficial effects of the supplemental mixtures. This has been the approach
in extending the list of essential elements for other species. As indicated
in the following paragraphs, in spite of a number of experiments, the
results have not been as anticipated and the issue has not been settled.
EXPERIMENTAL PROCEDURES
Establishing the essentiality of additional inorganic nutrients usually
requires special purification procedures to remove element contamination
from the reagent salts from which the synthetic culture medium is pre-
pared. The definite response when B7 and C13 were omitted from the culture
medium made these lengthy purification procedures seem unnecessary on
this project. Careful attention was given to contamination from other
sources, for example in thorough cleansing of all glassware in acid, in the
-------
TAHI.I; 16. [ll-Si'ONSI; ()!•• Cladophora I'lomenita IX) TUP. ADHITIUN 0I:
IHVn.NTtAI.I.Y "liSSiiNTIAl, ~M I CUONI/tIu I .NTS
Ave. algae dry-wt.
yield,* mg/1
Treatment
Exp. I
Exp. II
CIadophora medium4
59a
113a
Cladophora medium, plus
®7> 3
536b
291b
Cladophora medium, plus
B7
--
lUa
Cladophora medium, plus
3
--
246b
* Average of ten replicates in each treatment in Experiment I, and' four
replicates in each treatment in Experiment II; yields with a common
letter are not significantly different using Student-Newman-Keuls
multiple range test.
+ The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P,
18 ppin K, 7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements,
Na?CO3, Na^SiO,, and vitamins Bt and B]2.
§ The B7 trace element mixture contained 0.001 ppm of each of the following:
V, Cr, Ni, Co, IV, Ti, and Sn. The C]3 trace element mixture contained
0.0005 ppm of each of the following: A1, As, Cd, Sr, Hg, Pb, Li, Rb,
Br, 1, I7, Se, and Be,
use of water double-distilled from g.1 ass in preparing culture media, and
in carrying C. glomeratn to be used as innoculum through several transfers
in a minus B7 C,3 medium.
HIiSULTS
The data in Tabic 16 show the response of C, glomerata to the elements
in P? and Ci3 solutions in two experiments. Yield increase was statistically
significant in both experiments. In Experiment II, omitting the C13 solution
i
decreased yield from 291 to 111 m.c/liter; omitting the By solution was
52
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TABLE 17. RESPONSE OF CJadojphora glomerata TO THE ADDITION OF B?
MICUONUTRliiNTS"
Ave. algae dry-wt.
Treatment yield,* mg/1
CIadi-nhora medium,
phis B 7 ^
529
Cladophora medium,
minus B7
582
Cladophora medium,
minus V
588
Cladophora medium,
minus Cr
529
Cladophora medium,
minus Ni
633
Cladophora medium,
minus W
525
Cladophora medium,
minus Ti
581
Cladophora medium,
minus Sn
515
Cladophora medium,
minus Co
498
* Average of five replicates in each treatment; yields are not significantly
different at the S% level using Student-Newman-Keu1s multiple range test.
t The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm F, 18 ppra
k, 7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements,
C13 solution, Na2C03, Na2Si03, and vitamins B, and B12. The B7 tTace
element mixture contained 0.001 ppm of each of the following: V, Cr,
Ni, Co, W, Ti, and Sn.
without significant effect. The clement, or elements, responsible for
the yield increase seem to he among the Cj •components.
Tables 17 and 18 present data from experiments designed to establish
the specific B? , C components that stimulated growth. As anticipated,
there was no significant yield reduction when the B? solution or each element
of the r7 solution was omitted in individual treatments [Table 17),
53
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TABLE 18. RESPONSE OF Cladophora glomerat.a TO TIE AUDITION OF Cn
M1CRONUTRTENTS
Ave. algae dry-wt. yield,* mg/1
Treatment
Exp. I
Exp. II
Cladophora medium,
plus C 13i"
427
408
Cladophora medium,
minus
C 1 3
327
289
Cladophora medium,
minus
C 13 "7U§
238
304
Cladophora medium,
minus
As
407
334
Cladophora medium,
minus
Cd
447
360
Cladophora medium,
minus
Hfi
416
384
Cladophora medium,
minus
Pb
454
366
Cladophora medium,
minus
F ¦ "
520
346
Cladophora medium,
minus
Se
434
341
Cladophora medium,
minus
Be
427
375
* Average of five replicates in each treatment; yields are not significantly
different within each experiment at the 5% level using Student-Newman-
Kculs multiple range test.
+ The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements, B7
solution, Na2C03, Na2Si0s, and vitamins Bi and B12. The Ci3 solution
contained 0.0005 ppm of each of the following: A1, As, Cd, Sr, Hg, Pb,
Li, Rb, Br, I, F, Se, and Be.
§ The Ci3 "7" solution contained the elements individually tested in the
experiment: As, Cd, Mr, Pb, F. Se, and Be. The remaining six elements
(Al, Sr, Li, Rb, Br, and I) were added.
• There was reason ro'belicvu that the Cj-j response in Table 16 was due
to one of the following Cj 1 elements: arsenic (As), cadmium (Cd), mercury (Hg),
54
-------
lc:id (Pl>), fluorine (1). selenium (Se), or beryllium (lie). However, omitting
each of those elements from the culture medium was without effect on yields
in both experiments reported in Table 18.
Poor agreement among replicates within treatments seemed to be a major
reason for the inconsistencies in the B7, C,3 experiments. The poor
replication in turn was due to uneven initiation of growth from the vegeta-
tive filaments used as inoculum. To provide more uniform yields, an
effort was made to induce C. glomerata to produce zoospores which in turn
would serve as inoculum. It was established that zoospore release could be
accomplished when a two-week old C. glomerata culture which had been under
conditions of high continuous light (800 ft-c) and high temperature (25C)
for three to four days was transferred to fresh culture medium and placed
under short days (8 hours), low light (200 ft-c or less), and low temperature
(21C). The cultures were watched closely for zoospore release at approxi-
mately three days. When this occurred, the zoospores were concentrated
by mil 1 ipore fi1trat ion and small volumes were used as ir.noculum. The
zoospores immediately began to attach to the sides of the flasks.
Because of termination of the project, only one experiment with zoo-
spore innoculum has been possible (Table 19). Overall the results confirm
the data from Table 16. Omitting the Ci3 solution, or the Cj 3 "7" components
of the C13 solution, decreased yield from 741 to only 12 to 14 mg/liter.
However, completely unexpected results were obtained when the C, , ,,7" .
components were individually omitted from the culture solution. No element
had a significant effect on £. glomerata yield. Although detailed data
nve not given, replication was improved when zoospore? rather than
vegetative filaments were used as innoculum.
-------
TABLE 19. RESPONSE OF Cladophora glomerata TO THE ADDITION OF
C (3 MIf.RONUTRIi*NTS USING ZOOSrOltHS AS TNOCtll.UM
Treatment Ave, algae dry-wt. yield,*mg/l
Cladophora
medium,
plus Cl3
741
Cladophora
medium,
minus C, 13
12
Cladophora
medium,
§
minus C13
14
Cladophora
medium,
minus As
563
Cladophora
medium,
minus Cd
564
Cladophora
medium,
minus Hg
776
Cladophora
medium.
minus Pb
783
Cladophora
medium,
minus F
778
Cladophora
medium,
minus Se
626
Cladoi>hora
medium,
minus Be
736
* Average of five replicates in each treatment; 28-day culture period,
J.
1 The nutrient medium contained 23,4 ppm C;i, 16.4 ppm N, 2.8 ppm I5, 18 ppm K,
7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson1s trace elements, B7
solution, NaaCOj, NazSiOs, and vitamins B» and B12. The Cij solution
contained 0.0005 ppm of each of the following: Al. As, Cd, Sr» Hg,
Pb, Li, Rb, Br, 1, F, Se, and Be.
§ The Cj 3 "7" solution contained the elements individually tested in the
, experiment: As, Cd, Hg, Pb, F, Se, and Be. The remaining six elements
(AI. Sr, Li, Rb, Br, and I) were added.
DISCUSSION
Tht; inability to translate the strong response to the Ci j solution
into a requirement for an additional essential element has been frustrating
and unlike experience in the studies that.led to the essentiality of other
56
-------
elements, for example Mo (Arnon and Stout, 1939) and CI (Broyer et al,
1954) for all plants and Co for some plants (Ahmed and Evans, 1961; Delwiche,
et al, 1961). One possible explanation for failure in this study is that
the unrecognized essential element is an impurity in several of the C13 "7"
salts. The presence of any one of those salts provides enough of the un-
recognized essential element to meet the needs of Cladophora glomerata,
This seems doubtful bccau.se of the extremely low concentration in which
the C]3 components were added (0.0005 ppm of each element). Another
possibility is that an additional element is essential for C glomerata
but another clement can substitute for it. Such substitutions have been
established among other elements, for example between K and Na and Sr and
Ca (Walker, 1956; Brian, 1967).
Considerable additional work probably will be necessary to clarify
the situation discussed. The improved replication within treatments in
the last experiment suggests that for future experiments zoospores should
be used as innoculum. Because of the very small quantities that would be
required, it seems unlikely that the unknown essential element would be
of practical importance in the nutrition and ecology of Cladophora in the
Great Lakes. Nevertheless, the discovery of a new essential element for
even a single species is an exciting scientific event. For that reason,
furl her srud'ies to clarify tin; situation discussed seem worthwhile.
57
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SUCTION 8
HEAVY METAL TOXICITY TO Cladophorn glomerata
Because of increased interest in the effects of heavy metals likely
to be introduced into aquatic environments on plants and animals, tests
were made on the responses of Cladophora glomerata to several common heavy
metal pollutants often toxic at relatively low concentrations. This was
not a part of the original project proposal. However, the experience in
culturing Cladophora glomerata on this project made expansion into toxicity
studies relatively simple and worthwhile.
Ovganisra response to toxic agents can be expressed in various ways.
A common expression is the environmental concentration which results in a
f»0*. depression in organism numbers or growth in a standardized laboratory test.
A disadvantage of this approach is that the volume of a solution, as well
as the concentration of a toxic agent, determines the total amount of a
toxic agent to which an organism is exposed. For this reason, the Principal
Investigator prefers expressing toxicity of metal.c as minimum concentrations
in organisms, tissues, and cells which are responsible for significant
decreases in yield.
EXPERIMENTAL PROCEDURES
Cladophora glomerata was grown in the medium and under the conditions
developed as standard culture technique on this project and described in
earlier sections. Each culture involved 200 in 1 of medium in a 500 ml
58
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TABLE 20. RLSPONSE 01; Cladophora gjomerata TO VARIOUS
CONCHNTRATIONS OP COPPER
Cu added to Ave. algae dry-wt.
culture soln.,* ppm yield,^ mg/1 SNK§
0.03 726 a
0.15 800 a
0.18 773 a
0.21 G5S ab
0.24 530 b
0.30 252 c
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements (-Cu),
NazCOa, NajSiOj, B7. and C13 solutions, and vitamins Bi and B1Z."
4*
Average of five replicates in each treatment.
9 Yields with a common letter are not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
Grlenmeyer flask. All heavy metals were aseptically added to the cultures
after autoclaving.. The inoculum was a 1-2 mg oven-dry weight segment of
vegetative filaments from a vigorous culture. ¦
unsui/rs
The yield responses to various concentrations of copper (Cu), manganese
(Mn), cadmium (Cd). lead (Pb), and zinc fZn) are presented in Tables 20
through 24, Copper significant1y reduced yields at solution concentrations
of 0.24 ppm and higher. Cladophora glome rata tolerated 1.3S ppm Mn but
wa^ at footed adversely by 2.16 ppm of that elemetlt. ¦ Cadmium was not harmful
59 .
-------
TABLE 21. RESPONSE OF Cladophora glomerata TO VARIOUS
(".ONCr.NTRATIONS* OF MANGANP.SP.
Mn added to
culture soln.,* ppm
Ave. algae dry-wt.
yield,'"" mg/1
0.00
356
0.27
698
1,35
606
2.16
342
2.43
296
2.70
188
* The nutrient medium contained 23,4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.5 ppm Mg» 9.7 ppm S, 1,12 ppm Fe, 1/5 Johnson's trace Elements
(-Mn), Na2C03, NaaSiOa, B7 and C13 solutions, and vitamins Bj and B^.
. ^ Average of five replicates in each treatment; a statistical analysis
of the data can be found in Appendix A (Table A-24).
at I .0 ppm; 2.5 ppm of the element, however, completely killed C_.
glomerata. Lead was relatively non-toxic with no reduction in yield at 3.0
ppm, the highest concentration tested. Zinc was surprisingly toxic with
growth nearly stopped at a solution concentration of 0,65 ppm,
DISCUSSION -
Time was not available in the final stages of this project for analyses "
of Cladophora glomerata and expressions of toxicity as cell concentrations
of the heavy metals tested. The samples have been stored for possible
analyses in the future. At this time, only general observations on
toxicities to C. glomerata in relation" to solution concentrations and by
60
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TABLE 22. RESPONSE OF Cladophora glomerata TO VARIOUS
CONCLNTIIATIONS OI: CADMIUM
C(J added to
culture so In.,* ppm
Ave. algae dry-wt.
yield,"'' mg/1
0,0
396
0.1
399
0.4
290
0.6
303
1.0
286
2.5
0
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18
ppm K, 7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace ele-
ments, B7 and C1j solutions, NajCOj > NazSi03, and vitamins Bi and Bj2.
Average of five replicates in each treatment; yields of treatments
0.0 through 1.0 ppm Cd are not significantly different at the 5% level
using Student-Newman-Keuls multiple range test.
comparison with toxicities to other organisms arc possible.
Copper salts are among the most widely used algicides at copper ion
concentrations of 0.2 to 0.5 ppm. This correlates with the yield reduction
to about ono-third of maximum in a solution containing only 0.3 ppm Cu,
Zinc and manganese are both essential elements for plants and elements
for which the range between toxicity and essentiality often is relatively
narrow, Probably because Mn toxicity occurs in the field in very acid
soils, much information is available on the response of various plant
species to Mn. Some legumes are affected by only 1.0 ppm Mn in the culture
soltuion; some cereals will tolerate much higher concentrations of the
61
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TABLE 23. RESPONSE OF Cladophora glomerata TO VARIOUS
CONCENTRATIONS OF I.F.AP
i'b added to Ave. algae dry-wt.
culture so In. , * ppm yield,"'" mg/1 SNK§
0.0 213 ab
0.1 99 a
0.6 287 ab
1.0 386 b
2.0 250 ab
3.0 252 ab
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K. 7.5 ppm Mr, 9.7 ppm S., 1.12 ppm Fe, 1/5 Johnson's trace elements,
N-:C03, NazSiOj, 1*7 and Cn solutions, and vitamins Bi and B12.
1 Average of five replicates in each treatment.
§ Yields with a common letter are not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
element [Hewitt, 1966). On the basis of higher plant data, Cladophora
glomerata seems moderately susceptible to Mn toxicity. In contrast,
glomerata appears quite susceptible to Zn toxicity. The Zn concentrations
employed in higher plant cultures generally are in the range of 0,1 to
0,5 ppm. CIadophora glnmvrata growth was almost completely stopped by 0,65
ppm of Zn.
Cladophora glomerata seemed quite tolerant of Cd, one of the two non-
essential heavy metals tested, Some testing of the response of economic
plants to Cd has been carried out because of concern over possible problems
resulting from land disposal of sewage sludge. Growth of beets, beans, and
¦ 62
-------
TABLE 24. RESPONSE OF Cladophora glomerata TO
VARIOUS CONCENTRATIONS Of ZINC
Zn added to
culture soln,,* ppm
Ave. algae dry-wt.
yield, mg/1
0.00
637
0. 13
486
0.65
23
0.91
20
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18
ppm K, 7.5 ppm Mg, 9.7 ppm Fe, 1/5 Johnson's trace elements (-Zn),
Na2C03, Na?Si03, B7 and Cjq solutions, and vitamins Ri and Bi2.
t Five replicates in each treatment.
turnips was reduced by 50% "at a Cd solution concentration of 2 ppm; similar
growth reduction in corn resulted at 1 ppm Cd and in tomatoes and barley
at 5 ppm. Cabbage was the most tolerant plant tested with 9 ppm Cd requi red
for a 50'1. yield reduction (Page, ct al, 1972). Cladophora glomerata
tolerance of Pb cannot be evaluated until additional experiments are carried
out to establish concentrations which decrease growth,
On the basis of experience with higher plants, and particularly crop
plants, it can be anticipated that aquatic species will be highly selective
in their accumulation of heavy metals and in their responses to high
concentrations of the metals. It seems important to begin accumulating
b.isic information of this nature for the most prevalent aquatic plants in
the Great Lakes. With the input of large amounts of specific heavy metals in
local areas, 'those metals may become critical in the ecology of the polluted
63
-------
water. Knowledge of ihc responses of aquatic species to the metals could
be useful in predicting changes in the dominant species as a result of
heavy metal pollution.
64
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SECTION 9
THE CULTURE AND NUTRITION OF THE DIATOM Nit2schia pa lea
Explanations of the abundance of Cladophora glomerata in certain
areas and under specific conditions probably would be facilitated if detailed
information were available not only on the nutritional requirements and
responses of C. glomerata but also on the primary organisms with which it
occurs and competes. Diatoms were selected for additional nutritional
studies because they arc the dominant organisms in all the Great Lakes
(Schelske and Stoerraer, 1971).
This section reports the isolation from Lake Michigan C. glomerata
filaments of the diatom Ni12schia palca, the laboratory culture of the
organism in a synthetic medium, and its general nutrient requirements.
EXPERIMENTAL PROCEDURES
Diatoms were collerted from Green Bay near the entrance to Sturgeon
Bay by wiping filaments of Cladophora glomerata across solidified medium
which contained 2S". lake water and 1.5* agar in petri dishes. The medium
was a slightly modified Hughes, Gorham, Zehnder (1958) so]ution. The petri
dishes were transferred to the lab the same day and placed in a culture
mom.
Within a week, algal colonies appeared on the agar. Diatoms were trans-
fer red aseptically with a flamed wire loop to liquid culture medium to
which 2 by volume lake water had been added. The organisms were al lowed
65
-------
to grow for approximately two weeks before restreaking onto agar. This
not only facilitated isolation but also selected organisms which grow well
in liquid media. This procedure was repeated until unialgal cultures were
obtained as determined by microscopic examination. One isolate which grew
faster and more abundantly than the others was selected for detailed study.
Efforts were made to obtain bacteria-free cultures by several procedures.
Although bacterial numbers could be reduced by treatment with an antibiotic
mixture, axenic cultures were not obtained.
Cleaned diatom frustules were obtained by the standard procedure given
by Patrick and Reimcr (1966). Permanent slides were obtained by using
pleurax, a synthetic mounting medium developed by Manna (1949) which is
commonly used in diatom identification because of its high refractive index.
Permanent slides and micrographs were sent to Dr. Reimer at The Academy
of Natural Sciences in Philadelphia who identi ficd the organism as Nitzschia
palea var. tropica Hust. This assumes that no changes in wall structure had
taken place during the time the organism was in culture.
Apparatus and Culture Conditions
Unless otherwise stated, cultures were maintained at 23 * 1C under
continuous fluorescent light of 350-500 ft-c derived from T-12 cool-white
bu llis. • .
The diatoms were grown in 5U0 ml tirlenmcyer flasks containing 200 ml
of liquid culture .medium, .Humidified and filtered air was provided by
incorporating a double-distilled water rrap and a polyester fiberfill filter,
into the aeration system. The treated air was then gently bubbled through
the culture medium.
66
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Rout ine Culture Procedure
All glassware coming into contact with the culture media was washed,
rinsed in distilled water, soaked in 4N UNO 3, and thoroughly rinsed in
distilled and double-distilled water (second distillation in a glass still).
Media were prepared in large batches, poured into the 500 ml Erlenmeyer
culture flasks, and sterilized by autoclaving at 15 psi for 15 min. Only
water which had been double-distilled was used in media preparation.
The diatoms generally were harvested between the period of most rapid
growth and senescence, the exact timing depending upon the type of experiment.
Harvest was by centrifugation at 2,000 RPM for one minute at OC in tared,
plastic 50 ml centrifuge tubes. The tubes were then oven-dried, cooled,
reweighed, and the dry weight was recorded for cells from each flask.
Statistical analyses were run on the results of the experiments using Duncan's
multiple range test.
RESULTS
Media Modification Experiments
Figure 8 presents the combined results of the initial experiments which
wore most effective in i »cve;is ing N i t zschi a palcu yield. This was accom-
plished by increasing the Na2SiO}•91120 concentration in the medium (5.7 to
ppm Si) and substituting CafNOjl^ f"r NaNOj. The combined modifications
resulted in a yield of approximately 800 mj>/1 in contrast to 80 mg/1 in the
original medium.
Although these modifications produced an adequate yield, the resulting
medium was unacceptable for N. pa 1ea nutritional studies. The high con-
centrations of N^SiO, yH,0 and Ca(N03)3 produced a heavy precipitate which
67
-------
o»
e
«k
h-
£
>
OC
Q
I
Z
LU
>
o
800
700
600
500
400
300
200
100
I
i
I
EXPERIMENT NITROGEN NUMBER OF
SOURCE REPLICATES
1
2
3
4
~
N0NO3
N0NO3
NaNOj
Ca {NO3)2
Ca (NO3)z
T
30
6
6
2
4
T
40
50
0 10 20
Si IN MEDIA, ppm
Figure 8. The effect on Nitzschia palea yield of increasing the media
Si concentration and substituting Ca{N0j)2 for NaNOa•
formed immediately upon addition of the salts, It was difficult to deter-
mine the quantity of precipitate remaining at the end of the culrure period,
thus making jt impossible to obtain accurate diatom yields.
The precipitate problem finally was resolved by using silicic acid as
a silicon source. This chemical, although sparingly soluble in water at a
68
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pH near neutral, dissolves upon heating in a strong basic solution, Experi-
ments showed that at low silicon concentrations, the silicic acid remained
in solution upon readjustment of the pH to neutral with IICl. Because a
gel formed at high silicon concentrations, it was most convenient to pre-
pare a silicon solution of neutral pH in large quantities instead of smaller
amounts of a highly concentrated stock solution. Specific details on
preparation of the recommended silicon solution (containing approximately SS
ppm Si) are given in Table 25.
Sodium silicate and silicic acid solutions arc compared as a silicon
source for N_, pa lea in Table 26. The recommended silicic acid solution not
only gave a higher yield but also produced very little precipitate (less
than 5 mg/1). When a base was not added to the solution, yield was much
lower.' . •
To determine if micronutrient concentrations in the recommended "CS"
medium arc near optimum, the trace element mixture was doubled and halved
in an experiment summarized in Table 27. There was no significant difference
between the treatments, although observations during the culture period
indicated a slightly lower growth rate for the highest micronutrient
concent rat ions.
Physical environmental factors
l'actoi's other than nutrient media composition must be considered when
specifying optimum conditions for the culture of an organism, for example,
lij'ht intensity temperature, and availability of CO2. Results from experi-
ments to determine the sensitivity of N_. nalea to changes in these factors
are summarized in Table 28.
00
-------
TABLE 25. RECOMMENDED CULTURE SOLUTION FOR Nitzschia pa lea.
fCS" MEDIUM)
Silicic acid solution*
0.132S g/1 silicic acid (88.8% S1O2)
15 drops/I brointhymo 1 blue
5 ml/1 2N KOH or NaOHt
Approx. 5 ml/1 2N HC1
Mai or nutrients
Stock
Concentration in
Salt
solution,
Final solution,
final
solution,
g/1
ml stock/1
ppm
§
Ca(N03 )2»4H20
69.16
10.0
N
=
82
KH2 POi,
3.08
10.0
Ca
s
117
MgSOi, • 7H2 0
7.50
10.0
P
r
7
Na2C03
2.00
10.0
K
9#
Fe Citrate^
0.60
10.0
Mg
7.5
Citric acid^
0.60
10.0
S
=
9.7
EDTA 2Na
0.10
10.0
Fe
—
1.12
Micronutrients^
KC1
3728
0.2
CI
S2
0.35
li2 BO3
1546
0.2
B
s:
0.054
MnSOi, • 1I20
845
0.2
Mn
=
0.054
ZnS0„ • 71i2 0
575
0.2
Zn
=
0.026
CuSOu • 5H20
125
0.2
Cu
0.006
(NHlt)6Mo70zlt.4H20
18.4
0.2
Mo
=
0.002
B£ and CJ3 solutions
1.0 each
* 'This solution is prepared in large quantities with a low silicon concentra-
tion and used in place of distilled water during media preparations. Solu-
tion containing silicic acid, indicator, and base is heated to 80°C,' or
allowed to stand for 1-2 days to dissolve the silicic acid. pH adjusted
to 7.2 with the aid of bromthymol blue by addition of HC1.
+ NuOH gives slightly higher N_. pa lea yields. Experience proved this ratio
of silicic acid to base works the best, although proportionally higher
quantities of silicic acid can be dissolved in higher concentrations of
base, The diatoms were able to tolerate up to 10 ml of 2N KOH, higher •
concentrations were not investigated.
5 If a completely.precipitate-free medium is desired, this stock solution
should be autoclaved separately and added aseptically.
70
-------
TABI,E 25 continued
# Does not include KOH in silicic acid solution.
f Prepared as one stock solution and heated to 80°C to hasten solution,
cj) Modified from Johnson, et al (1957).
Of the two temperatures investigated (24 and 19C), N. palea produced
slightly more growth at 24C. This experiment was conducted in two control led
environment cabinets adjusted to provide a light intensity between 390
and 420 ft-c.
Nitzschia palea yields were not influenced by differences in the light
intensities used in nutrition experiments (250-550 ft-c). However, higher
intensities (650-700 ft-c) were slightly inhibitory.
Non-aerated cultures depend mainly on diffusion from the surrounding
air for a supply of CO2. The first aeration experiment showed that dif-
fusion was not rapid enough to supply CO2 for N. palea cultures. Yields
from flasks with cotton plugs were significantly less than yields from
flasks with aerators, even with a medium low in silicon. Enriching the
air supply with C02 in the second experiment further increased yield.
Critical concentrations •
The results from experiments to establish the critical concentrations
for N,. K, P, and Ca are summarized in Table 29.
In all experiments, the effects of a deficiency of an element were
readily observable before harvest. This was especially true for the
phosphorus experiment in which the diatoms not only- showed an obvious
71 "
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TABI.E 26. COMPARISON OF SILICON SOUIO.S 1-Olt Ni tzschin palco,
t
I) in torn yield, mg/I
Si source*
I'rcci pitate
mg/1
Replicates
Mean
5
Sodi um
si licate
89
348,377,365,380
373 a
Silicic
acid
< S
221,224,229,227
225 b
Silicic
acid dissolved
x S
414,394,450,398
416 c
in K0H#
* 25 pprn Si.
t Weight of precipitate subtracted from total dry weight values.
S Means with a common letter are not significantly different at the 5%
level using Duncan's multiple range test.
" 737 ppm K in final medium; pH adjusted with UC1.
difference in biomass but also in color, which ranged from yellow in
deficient cultures to brown in cultures with adequate phosphorous. Because
there has not been time for confirmatory experiments, approximate critical
concentrations rather than specific values are presented,
DISCUSSION AND CONCLUSIONS .
Because diatoms require relatively large amounts of silicon, establishing
the critical concentrations Cor the essential nutrients in Nitzschia palea
presented problems, High concentrations of Na?SiO *9H 0 produced pre-
cipitates i:i culture media containing calcium and phosphorous salts.
72
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TABLE 27. RESPONSE OF Nitzschia palea TO VARIOUS
LEVELS OF THE ESSENTIAL TRACE ELEMENTS.
Trace element Mean yield,*
Basal medium level mg/1
CS (Table 25) 0.5 x 676
1.0 x 670
2.0 x 677
* Mean of four replicates; means with a common letter are not significantly
different at the 5% level using Duncan's multiple range test.
t Concentration in modified Johnson's inicronutrients.
This caused errors in dry weight determinations and precluded the synthesis
of a defined medium. The high silicon requirement also meant that palea
might exhaust the supply of that element before the nutrient under investiga-
tion became limiting. The problem was compounded when Ca(NO3)2 produced
higher yields than NaN03 or KN03.
These problems were solved in the development of the recommended
medium. The primary difference between this medium and others reported in
The literature for the culture of diatoms is that silicon is added as a
solution of silicic ac id instead of the conventional Na2Si03*9M20.
A vitamin requirement could not be demonstrated for N.palea, at
least in the bacterized state. This is not surprising in view of the fact
that species of N. palea have been isolated and cultivated since the late
IROti's in purely inorganic media.
Nitzschia pa len showed no obvious toxicity to the high concentrations of
73
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TARIP. 28. KliSI'ONSr Of NitzscMa pa lea TO CHANGES IN
VARIOUS PHYS1 CAL~l;NVi KONMliNTAL [••ACTORS.
Physical
cnvi ronmcntal
factor Medium
Temperature
Low S i
6
19C
67
a
24C
75
b
Light intensity
Low S i
6
250-300 ft-c
84
a
500-550 ft-c
88
a
650-700 ft-c
71
b
Aeration
Low Si
6
Aeration
98
a
Experiment I
No aeration
81
b
Aerat ion
25 ppm Si, pre-
4
- C02
242
a
Experiment II
cipitate dissolved
~ C02
302
b
by IIC]and pH ad-
justed by NaOH
* Means with a common letter are not significantly different at the 5% •
level using Duncan's multiple range test.
sodium, potassium, or chlorine in the silicon solution. This conflicts
somewhat with early work which showed a preference by diatoms for a
monovalent/divalent ratio below 1.5 (Provasoli, 1958).
Because substitution of Ca(N03)2 for NaN03 stimulated growth, the
calcium salt is recommended as a N source even though the exact nature of
the effect has not been determined. Early workers (Chu, 1942; Vollenweider
1950; and Provasol), et al, 1954) reported a preference for a high calcium
concentration in media for some freshwater diatoms and gave evidence for
interaction between Ca, K, anit Mg ions. In the present study, the stimulatory
effect of C;i (NO ^ ), on N. palea was not due to a high calcium requirement,
No. of Mean yield,
replicates Treatment mg/1
*
74
-------
TABLE 29. CRITICAL CONCENTRATIONS AND RANCH 01-" CONCENTRATIONS
01-' IUUK liSSliNTIAL NUTIUHNT i-UiMHNTS IN Nitzschia pa lea.
Element Critical concentration Range in concentration
N 2.3-2.5% 1.49 - 4.56%
P 0.20 - 0.25% 0.07 - 0.61%
K 0.30 - 0.45% 0.28 - 0.89%
Ca <0.4%
nor was it the result ot" a toxicity due to high concentrations of sodium
or potassium.
Caution must be exercised in comparing the critical concentrations
presented in Table 29 to critical concentrations reported for other organisms.
Because of a "dilution factor" caused by differences in the relative amounts
of structural material in various organisms, comparisons of critical con-
centrations between species sometimes do not accurately reflect variations
in protoplasmic requirements for an element. This would especially seem
a j)rob 1 em with diatoms, wlio.se cell walls not only have a unique structure
and composition, but also exhibit large intraspucific differences in thick-
ness. However, if the amount of cell wall material in the t.ixon remains
relatively constant, then critical concentrations can provide information on
nutritional requirements that are of ecological interest.
Of some concern is the observation by Lund (1950).and Lewin (19S7J
that cell wall rhiefcness in diatoms varies with the division rate. Be-
cause of these reports, the ash content of samples of N. palea from various
75
-------
experiments was determined and was found to he a consistent 31%. This
reflects a fairly constant SiO ? percentage.
I a comparison with values reported lor other freshwater algae and
aquatic angiosperms (Ccrloff, 197S; and Gorloff and Fitzgerald, 1976),
N. pa tea has an unusually high phosphorous critical concentration (0,20-0.25%).
This range is typical of critical concentrations in index segments on ter-
restrial plants. An obvious question is the extent of the high phosphorous
requirement among other diatoms. Although there have been many reports on
the phosphorous concentration in diatoms which limited growth (Ketchum,
1959; Goldberg, et al, 1951; Mackereth, 1953; Quenzler and Ketchum, 1962;
and Carpenter, 1970), these values were all expressed on a per cell basis.
Values generally were between 5 x 10 and 2 x 10 -IS"grams P/cel1. On
the basis of data obtained in the present study, the--limiting'phosphorous"
concentration for N. p.-»1 pa was estimated to lie between 9.6 and 12.0 x
10" J1* grams P/cel 1. Because N_. pa lea cells are much smaller than most
diatoms, the actual phosphorous requirement may be greater than this estimate.
It is difficult to draw conclusions from comparisons on a per cell basis
unless reliable information on cell size is available.
Although the critical concentration range for nitrof.cn in N_. palea is
higher than most values reported by GerSoff (1975) for freshwater algae
and aquatic angiosperms, it.is groatly exceeded by the critical concentra-
tion reported for Microcyst is at' rug inosa {4,0%), The critical concentration
range for potassium in _N_. jviloa is slightly lower than the values given for
most other aquatii* organisms.
It would be interesting to determine if rhe high requirement for
nitrogen and phosphorous shown by Nitzschia paipa in these experiments, along
-------
with the rapid growth rate observed i n the cu] tiurcs, hinder the organism'
ability to compete for these nutrients at the low concentrations present
the natural envi ronmeitt. It has long been observed that diatom blooms
tend to form at the time of spring and fall turnover in lakes, when the
level of nutrients is high, and then to fall off sharply after lake
strati fication.
77
-------
SECTION 10
TESTS ON THE PRODUCTION BY ALGAE OF
Cladophora glomerata GROWTH INIIIBITORS
The production of inhibitors either toxic to the producing algae or
to other algae has been demonstrated to be a rather common phenomenon under
laboratory conditions (Harris, 1971; Fogg,1975). It is, however, difficult
to establish that these inhibitors are present in high enough concentrations
to influence the presence and growth of algae under field conditions.
Because a very potent inhibitor could be more critical than nutrient supply
in controlling Cla^nnhora glomerata occurrence, it seemed worthwhile to assay
for the production of C. glomerata inhibitors by several algae species widely
distributed in niidwestern lakes.
EXPERIMENTAL PROCEDURES
Two procedures were used in the inhibitor tests. In one, two species
were inoculated into a culture flask simultaneously and after a specific
growth period the yield of each organism was determined. An inhibitory
effect of one organism on another was indicated by comparisons with yields •
in cultures containing the individual species. Success in this approach
required a nurrient medium in which both organisms would, grow satisfactorily,
It also was essential that one organism did not grow so much more rapidly '
than the other thar. reduced growth of the latter was due to restricted
light supply or nutrients rather than inhibitor production. To avoid this
-------
possibility, C. glomerata, which grows rather slowly, was inoculated into
cultures f> to 7 days before the inhibitor organism.
The second procedure involved testing for the release into the culture
medium during the growth of a specific alga of a substance inhibitory to
C. j'Jomerata. The organism tested was cultured in 200 ml of medium in 500
ml flasks using standard procedures. When heavy growth was observed, the
cultures were filtered through an 8 p millipore fi 1 ter. Efforts then were
made to grow C. glomerata in 40 ml volumes of the filtrate, with and without
nutrient enrichment and autoclaving, in 21 x 150 mm test tubes. The solu-
tions were vigorously aerated.
RESULTS
The data in Tables 30 through 32 show the capacity of Cladophora
glomerata to grow in the presence of two common unicellular green algae,
Scenedesnus quadricauda and a species of Chlorella. The first two treat-
ments show yields of a pair of organisms when grown separately; the third
indicates the yield of each organism after the two were inoculated into
a culture at the same time and grew together.
A decreased C. glomerata yield could result from extremely rapid
growth of a competing organism which overwhelmed C. glomerata in competition
for light and nutrients,. To recogniie this possibility, in the fourth
treatment the competing organism was introduced into the culture 7 days
after the C. glomerata..
As shown in Tables 3D and 31, the Ch1 ore 11 a species and Scenedesmus
quadricauda from l.akc Michigan were without major effect on Cladophora
glomerata growth. . The re was a marked reduction in yield of C, glomerata
when both organisms were inoculated together. However, when C. glomerata
79 •
-------
TAHI.I' 30. CI'I'liCT OI; I.AKI: MICHIGAN Sci'iu'ilesmus quadricauda ON
(JUUWTll 01-' Cladophora r!omerata IN A COMBINED CULTURE
Ave. dry-wt. yield, mg/1
Treatment*
Scenedesmus Cladophora
Cladophora only
295
Scenedesmus only
462
Clad. 5 Seen.
both inoc. at start
469
44
Scenedesmus
inoc. 7 days after Clad.
122
223
Clad, after 7 day
42
culture period
* The culture solution was the Cladophora medium with the N increased to
32.4 ppm and P to 7 ppm; 14 day culture period; 450-500 ft-c.
was inoculated 7 days prior to the green algae, the C. glomerata continued
to grow throughout the culture period,
The results with a species of Scenedesmus quadricauda isolated from
Lake Mendota were somewhat different (Table 32), Cladophora glomerata
growth was completely inhibited when the two organisms wore inoculated at
the same time; growth also was severely inhibited when the S. quadricauda
was- inoculated a week after the C. glomerata. In fact, there was almost
iio growth following the introduction of the S. quadricauda. The production
of a C.. glomerata inhibitor is strongly suggested,
The data in Table 33 are from experiments in which efforts were made
to grow C. glomerata in the filtrate from cultures of several common Great
80
-------
TABU; 31. EFFECT OF LAKT; MICHIGAN Chlorella S]>. ON GROWTH OF
Cladophora glome rata IN A CQMBI NI:.D CULTURE
Ave. dry-wt. yield, mg/1
Treatment*
Chlorella Cladophora
Cladophora only
421
ChlorelIn only
592
Clad. 5 Chlorella
both inoc. at start
623
165
ChlorelIn
inoc . T days after Cl_ad.
133
383
Clad, after 7 day
67
culture period
* The culture solution was the Cladophora medium with the N increased to
32.4 ppm and P to 7 ppm; 14 day culture period; 450-500 ft-c.
Lakes algae. The critical data are in the columns indicating growth in the
inhibitor organism culture filtrate after various treatments. For comparison
purposes, growth in the standard Cladophora medium also is indicated. Fail-
ure of the C. glomerata to grow in the filtrate after passage through an 8
millipore filter is of little significance, because the nutrients in the
medium could have been depleted by the inhibitor organism. Growth in filtrate
on nutrient-enriched filtrate approximating that in autoclaved Cladophora
medium indicates an inhibitor was not produced. This was true for Scenedesmus
dimorphus, in two tests with Selenastrum gracile, with the Chlamydomonas sp.,
and with the diatom Nitzschia palea.
Initially the rcsforat ion of the potential for C. glomerata growth
when the nutrient-enriched medium was autoclaved was interpreted as due
81
-------
TABLE 32. EFFECT OF LAKH MHNDOTA Scenedesmus quadricauda ON
CROWNI OI: Cladophora wlomcrata IN A" COMBINED CiJL/IURG
Ave. dry-wt. yield, mg/1
T reatinent*
Scenedesmus
Cladophora
Scenedesmus only
620
CIadophora only
435
Clad. f, Seen. , both
~ Fnoc. at start
717
6
Scenedesmus
inoc. 7 days after
527
69
Clad.
* The culture solution was the Cladophora medium with the N increased to
.32.4 ppn and P to 7 ppm: 21 day cultore period; 500-600 ft-c.
to heat destruction of an inhibitor produced by an organism from the left
column. This would be consistent with the results obtained in other
investigations. However, another explanation is possible and cannot be
eliminated. The autoclaving could inactivate some of the nutrients
which reached toxic levels as a result of enrichment of the filtrates.
The results suggest production of a heat-labile inhibitor by Scenedesmus
quaiiricauda but until further experiments are carried out the alternative
explanation is a possibility. The differences in the results in the two experi-
ments with the same Scenedesmus species perhaps was associated with the use of
different culture media.
DISCUSSION
The preliminary results on alfi-'ie production of compounds inhibitory
to Cladophora glomerata were comparable to results from studies with other
82
-------
TABLE 33. TESTS ON THE PRODUCTION BY VARIOUS ALGAE ON Cladophora glomerata GROWTH INHIBITOR
A1 gae
Ave. yield of Cladophora, nig/1
Inhibitor organism culture filtrate
Yield of
inhibitor Ave. dry wt. Autoclaved
inoculum Cladophora CIadophora
culture, mg/1 inoculum, mg medium* MPF
8M MPF
8p MPF ~nutrients
+ nutrients ~autoclave
Scenc-desraus
quadricaudf
368
85
376
Scenedesmus
30
f
quadricauda'
cenedesnus
bijuga
Scenedesmus
dimorphus
Selenastrura
gracile
Selenastrun
gracile
Chlamydomonas sp.
Chlorella sp.
Chlorel la sp.^
Nitzschia^
palea
240
443
300
200
250
282
735
443
1.3
2.2
2.5
2.6
2.6
2.3
1.7
1.3
2.0
398
178
269
214
184
209
233
270
285
148
89
249
215
168
177
124
159
517
189
191
240
162
126
60
101
97
362
192
232
205
258
131
198
208
383
-------
TABLE 33. (continued)
* Culture period varied from 7 14 days.
+ The culture solution for both Cladophora and the inhibitor organism was Cladophora medium except in
the second S. quadricauda and Chlorella sp, experiments in which Gorham's Medium was used, and in
Nitzschia pa lea in which Gorham's Medium with increased silicon was used.
-------
algae. 1t has been relatively simple to obtain laboratory data which
surest inhibitor production occurs. However, laboratory studies for
the most part have involved inhibitor assays in small-volume cultures in
which organisms were present in far greater numbers than even in major
blooms. The possibilities for dilution of any natural inhibitor under
lake conditions make it doubtful that inhibitors are a significant ecological
factor.
From the present study, the common unicellar green alga Scenedesmus
quadricnudn seems the most promising organism for any further inhibitor
investigations. However, the evidence fcrproduction of a highly toxic
inhibitor was limited and, as with other algae, the possibility it is
important under field conditions seems slight.
-------
SUCTION 11
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Plants Grown under Symbiotic Conditions. Soil Sci. 90i205-210.
Arnon, D. I., and P. R. Stout. 1939. Molybdenum as an Essential Element
for Higher Plants. Plant Physiol. 14:599-602.
Brian, R. C. 1967. Action of Plant Growth Regulators. IV. Adsorption of
Unsubstituted and 2,6-Dichloro-aromatic Acids to Oat Monolayers.
Plant Physiol. 42:1209-1214.
Uroycr, T. C. , A. B. Carlton, C. M. .Johnson, and P. R. Stout. 1954.
Chlorine - A Micronutrient Element for Higher Plants. Plant Physiol.
29:526-532.
Carpenter, E. J. 1970. Phosphorus Requirements of Two Planktonic Diatoms
in Steady State Culture. J. Phycol. 6:28-30.
Cataldo, D. A., M. Maroon, L. E.Schrader, and V. I. Youngs. 1975. Rapid
Colorimetric Determination of Nitrate in Plant Tissue by Nitration
of Salycilic Acid. Commun. Soil Science and Plant Analysis. 6(l):71-80.
Chapman, U. D. 1966. Diagnostic Criteria for Crops and Soils. University
of California Div. of Agricultural Sciences, Riverside, California.
793 pp.
Chu, S. P. 1942. The Influence of the Mineral Composition of the Medium on
the Growth of Planktonic Algae. I. Methods and Culture Media. J, Ecol.
. 30:2S4 325,
Dclwiche, C. C., C. M. Johnson, and H. M. Reisenaur, 1961, Influence of
Cobalt on Nitrogen Fixation by Medicago. Plant Physiol. 36;73-78.
l-'Lt ngor;i Id, l», P, 1969. Field and Laboratory Evaluations of Bioassays for
Nitrogen and Phosphorus with Algae and Aquatic Weeds. Limnol, Oceanogr.
14:206-212,
Fitzgerald, (1. P.. and T. C. Nelson. 1966. Extraction and Enzymatic Analyses
for Limiting or Surplus Phosphorus in Algae. J. Phycol. 2;32-37.
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Fitzgerald, G. P., M. S. Torrey, and C. C. Gerloff. 1975. Green Bay Self-
puritics Algal Nutrients. Water and Sewage Works. I22iS8-6U. (
Fogg, G. E. 1975. Algal Cultures and Phytoplankton Ecology, University of
Wisconsin Press, Madison, Wisconsin. 175 pp.
Gcrloff, G. C, 1968. The Comparative Boron Nutrition of Several Green and
Blue-Green Algae. Physiol. Plant. 21:369-377.
Gerloff, G. C. 1969. Evaluating Nutrient Supplies for the Growth of Aquatic
Plants in Natural Waters. In: Eutrophication: Causes, Consequences,
Correctives, Proc. Symp. National Academy of Sciences, Washington,
D. C. 661 pp.
Gerloff, G. C. 1973. Plant Analysis for Nutrient Assay of Natural Waters.
EPA-R1-73-001, U. S. Environmental Protection Agency, Washington, D.C.
66 p.
Gerloff, G. C. 1975. Nutritional Ecology of Nuisance Aquatic Plants.
EPA-660/3-75-027, U. S. Environmental Protection Agency, Corvallis,
Oregon. 78 pp.
Gerloff, G. C., and K. A. Fishbeck. 1973. Plant Content of Elements as a
Bio.issay of Nutrient Availability in Lakes and Streams. In: Bioassay
Techniques and Environmental Chemistry, G. E. Glass, ed. Ann Arbor
Science Publishers, Inc., Ann Arbor, Michigan, p 159-176.
Gerloff, G. C., and G. P. Fitzgerald. 1976. The Nutrition of Great Lakes
Cladophora. EPA-600/3-76-044, U. S. Environmental Protection Agency,
Duluth, Minnesota. 111 pp.
Goldberg. I:. D. , T. .J. Walker, and A. Whisenand. 1951, Phosphorus Utiliza-
tion by Diatoms. Biol. Bull. 101:274-284.
Hanna, G. D. 1949. A Synthetic Resin which has Unusual Properties. J. R.
Mic rose. Soc. 50:424-426.
.Harris, D. 0, 1971, Growth Inhibitors Produced by the Green Algae
(Volvocaceael. Arch, Mikrobiol, 76;47-50,
llcwirr, li. J, 1966. S,-ind and Water Culture Methods Used in the Study of
Plant Nutrition, Tech. Commun. No. 22 of the Commonwealth Bureau of
the Hurt iculture and Plantation Crops, Second Edition, East Mailing,
Maidstone, Kent, 547 pp.
Hughes, I:. 0,. P. R. Gorham, and V, A. Zehnder. 1958, Toxicity of a Unialgal
Culture of Microcystis aeruginosa. Canadian .Jour. Microbiol, 4:225.
Johnson, C. M. , P. II. Stout, T. C. Broyer, and A. B, Carlton. 1957,
Comparative Chlorine Requirements of Different. Plant Species. Plant
Soil. 8:337-353.
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Kctchum, B. II. 1939. The Absorption of Phosphate and Nitrate by Illuminated
Cultures of Nit~sclna closterium. Am. .1. Hut. 26:31)9-407,
kuennler, li. J., and K. II. Kctchum. 1962. Unto of Phosphorus Uptake by
Phacodactylum tricornutum. Biol. Bull. 123(134- 145,
Lew inf., .1. C. 1957. Silicon Metabolism in Diatoms. IV, Growth and Frustule
Formation in Navicula pelliculosa. Can, J. Microbiol. 3:427-433.
Lund, J. W. G. 1950. Studies on Asterionella formosa Hass. II. Nutrient
Depletion and the Spring Maximum. J. Ecol. 38:15-35,
Mackereth, F. J, 1953. Phosphorus Utilization by Asterionella formosa.
J, Exp. Bot. 4:296-313.
National Eutrophication Research Program. 1971. Algal Assay Procedure:
Bottle Test. Environmental Protection Agency, Corvallis, Oregon. 82 pp.
Page, A. L., F. T. Bingham, and C. Nelson. 1972. Cadmium Absorption and
Growth of Various Plant Species as Influenced by Solution Cadmium
Concentrations. J. Environ. Quality 1:288-291.
Patrick, R., and C. W. Rcimer. 1966. The Diatoms of the United States
Exclusive of Alaska and Hawaii. Vol. I. Fragilariaceae, Eunotiaceae,
Achanthaceae, Naviculaceae. Acad. Nat. Sci. Philadelphia, Monogr.
13:1-688.
Provasoli, L. 195S. Nutrition and Ecology of Protozoa and Algae. Annu.
Rev. Microbiol. 12:279-308.
Provasoli, L., J. J. A. McLaughlin, and I. J. Pinter. 1954. Relative and
Limiting Concentrations of Major Mineral Constituents for the Growth of
Algal Flagellates. Trans. N. Y. Acad. Sci. 16:412-417.
Ryther, J. H,, and W. M. Dunstan, 1971. Nitrogen, Phosphorus and Eutrophica-
tion in the Coastal Marine Environment. Science 171:1008-1013.
Schelke, C. L., and K. F. Stoermer, 1971. Eutrophication, Silica Depletion
and Predicted Change? in Algal Quality in Lake Michigan. Science 173:
423-424,
Sokal. R. R., and F, J. Rohlf, 1969. Biometry, W, H, Freeman and Company,
Snn Francisco, California, 776 pp,
Ulrich, A. 1961, Plant Analysis in Sugar Beet Nutrition, In: Plant Analysis
and Fertilizer Problems (Pub. 8), W, lleuther, ed. Amer. Inst Biol,
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Vollenweider, R. A. 1950. Okologische Untersuchungen von Planktischen
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88
-------
kcr. H. 1956. Strontium Inhibition of Calcium Utilization by a Green
Alga, Arch. Bioch. Biophys. 60:264-265.
89
-------
SECTION 12
APPENDICES
Page
A. Statistical Analyses 30
B. Additional Experimental Data 129
90
-------
APPENDIX A
STATISTICAL ANALYSES
91
-------
TABLE A-l. STATISTICAL ANALYSES OF YIELD OF Cladophora Rlomerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT GREEN BAY CAMPUS
JULY 26, 1976
Momoscedastic
group
Treatment wi thin
homoscedastic group
Ave. algae
dry-wt. yield,
mg/1
SNK*
A
Cladophora medium
562
c
B
CI ad. medium in l.w.
793
d
D
As
B, - vit. B 1 2
43S
be
I
G
As
B, - Fe
140
a
J
As
B, - B, low B glass
396
be
. K
As
B, - K
319
b
L
As
B, - Ca, Mg, Mn, Mo, Zn, Cu
749
d
C
As
B, - vit. Bi
82
a
E
As
B, - N
87
a
II
F
As
B, - P
140
b
H
As
B, - S
704
b
I
As
B, - B, pyrex
818
b
(continued)
-------
TABLE A-1.(continued)
Hcteroscedastic
group
Treatment within
heteroscedastic group
Ave. algae
dry-wt. yield,
mg/1
EMMY5-
I
B
Clad.
. med. in l.w./C As B, -vit. Bj
793/82
SD
11
B
Clad.
med. in l.w./E As B, - N
793/87
SD
III •
B
Clad.
med. in l.w./F As B, - P
793/140
SD
IV
B
Clad.
med. in l.w./H As B, - S
79 3/704
SD
V
B
Clad.
med. in l.w./I As B. - B, pyrex
793/818
NS
VI
G
As B,
- Fe/C As B, - vit. Bi
140/82
NS
. VII
G
As B,
- Fe/E As B, - N
140/87
NS
VIII
G
As B,
- Fe/F As B, - P
140/140
NS
IX
J
As B,
B, low B glass/I As B, - B, pyrex
396/818
SD
* Yields with a common letter are not significantly different at the 5% level using Student
Newman-Keuls multiple range test.
t NS = not significant at the 5% level; SD = significantly different at the 5% level using the
equality of means with heterogeneous variances test.
-------
TABLE A-2. STATISTICAL WALYSES OF YIELD OF Cladophora glomerata RESPONSE TO DIFFERENT1AL
• NUTRI F.NT ENRICHMENT OF LAKE MICHIGAN WATER "COLLECTED AT GREEN BAY CAMPUS
SEPTEMBER 12, 1976
Homoscedastii.
group
Treatment within
homoscedastic group
Ave. algae
dry-wt. yield,
mg/1
SNK*
A Cladophora medium
584
a
B Clad. medium in l.w.
620
r.
I
D As
3, - vit. B, 2
556
a
L As
B, - Ca, Mg, Mn, Mo, Zn, Cu
538
a
0 As
M, - vit. Bl2
638
a
C As
B, - vit. Bj
132
b
II
E As
K As
B, - N
B, - K
141
271
b
c
N As
M, - vit. Bj
82
a
F As
B, - P
287
a
G As
B, - Fe
249
a
III
H As
B, - S
686
c
J As
B, low B glass
356
a
M As
B, filtered l.w. ~ vit.
773
c
•
P As
M, - Fe
461
(continued)
b
-------
TABLE A-2 (continued)
Heteroscedastic
group
Treatment within
heteroscedastic group
Ave. algae
dry-wt. yield,
mg/1
EMH\-
I
Clad. med.
in
l.w./C As
B, - vit.
V
620/132
SD
II
B
Clad. med.
i n
1.w./E As
B, - N
620/141
SO
III
B
Clad. med.
in
l.w./F As
B, - P
620/287
SD
¦ IV
B
Clad. med.
in
1.w./G As
B, - Fe
620/249
SD
. V
B
Clad. med.
in
l.w./H As
B, - S
620/686
NS
VI
B
Clad. med.
in
l.w./J As
B, low B
glass
620/356
SD
VII
8
Clad. med.
in
1.w./K As
B, - K
620/271
SD
VIII
B
Clad. med.
in
l.w./fj As
B, filt.
l.w. + vit.
620/775
NS
IX
B
Clad. med.
in
1.w./N As
M, vit
Bi
620/82
SD
X
B
Clad. med.
in
l.w./F As
M, - Fe
620/461
NS
XI
E
As B, - N/
F As B, - P
141/287
SD
XII
E
As B, - N/G As
; B, - Fe
141/249
SD
XIII
C
As B, - vit
. B
l/F As B,
- P
132/287
(cont
SD
inued)
-------
TABLE A-2.(continued)
Heteroscedast ic
group
Treatment within
heteroscedastic group
Ave. algae
dry-wt. yield,
mg/1
EMHV'-
XIV
C As B, - vit. Bj/G As B, - Fe
132/249
SD
* Yields with a common letter are riot significantly different at the 5% level using Student-Newman-
Keuis multiple range test.
!" NS = not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
table A-3. STATISTICAL ANALYSES OF YIELD OF Cladophora glomerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT BAYSHORE PARK JULY 26, 1976
Ave. algae
Honoscedastic Treatment within dry-wt. yield,
group homoscedastic group mg/1 SNK*
A Cladophora medium 649 be
B Clad, medium in l.w. 778 c
D As B, - vit. Bi2 324 a
I F As B, - P 164 a
I As B,'- B, pyrex 869 c
J As B, - B, low B glass 420 ab
L As B, - Ca, Mg, Mn, Mo, Zn, Cu 709 c
C As B, - vit. Bi 71 a
E As B, - N 52 a
II G As B, - Fe 155 b
H As B, - S 501 b
K As B. - K 280 b
(continued)
-------
TABLE A-3.(continued)
Heteroscedastic
group
Treatment within
heteroscedastic group
Ave. algae
dry-wt. yield,
rag/1
EMHV"
I
B
Clad.
med.
in 1.w./C As B, -
vit. Bi
778/71
SD
II
B
Clad.
med.
in 1.w./E As B, -
N
778/5.2
SD
III
• B
Clad.
med.
in l.w./G As B, -
Fe
778/155
SD
IV
B
Clad.
med.
in l.w./H As B, -
S
778/501
SD
• V
B
Clad.
med.
in l.w./K As B, -
K
778/280
SD
VI
F
AS B,
- P/C
As B, - vit. B,
164/71
SD
VII
F
As B,
- P/E
As B, - N
164/52
SD
VIII
F As fi,
- P/
G As B, - Fe
164/155
NS
* Yields with a common letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
i NS = not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
TABLE A.-4.STATISTICAL ANALYSES OF YIELD OF Cladophora glomerata RESPONSE TO DIFFERENTIAL NUTRIENT
ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT BAYSHORE PARK AUGUST IS, 1976
Homogeneous
group
Treatment within
homogeneous group
Ave. algae
dry-wt. yield,
mg/1
SNK"
A Cladophora medium
494
a
B
Clad.
medium in l.w.
586
ab
I
D
As
B,
- vit. B12
706
b
J
As
B,
- B, low B glass
338
c
M
As
B,
filtered l.w. * vit.
718 •
b
C
As
B,
- vit. B2
134
a
E
As
B,
" 'N
73
b
II •
F
As
B,
- P
177
c
G As
B.
- Fe
124
a
N
As
M,
- vit. Bj
169
c
* Yields with a common letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
-------
table A-5. STATISTICAL a.-.ALYSES OF YIELD or Cladophora gloraerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT BAYSfJORE PARK
SEPTEMBER 12, 1976 . .
Homoscedastic
group
Treatment within
homoscedastic group
Ave. algae
dry-wt. yield,
mg/1
SNK*
A Cladophora medium
564
b
B Clad, medium in l.w.
761
c
D As B, • vit. B12
525
b
F As B, - P
190
a
H As B, - S
476
b
J As B, - B, low B glass
430
b
M As B, .filtered l.w. + vit.
543
b
0 As M, - vit. Bj2
458
b
C As B, - vit. Bj
80
a
E As B, - N
94
a
G As B, - Fe
102
a
K As B, - K
240
b
L As B, - Ca, Mg, Mn, Mo, Zn, Cu
254
b
N As M, - vit. Bi
75
a
P As M, - Fe
^ (continued)
a
-------
TABLE A - 53. (continued)
Ave. algae
Heteroscedast ic
Treatment within
dry-wt. yield,
group
heteroscedastic group
mg/1
EMHY-
1
B
Clad.
med.
in l.w./C As
B, -
vit. Bj
761/80
SD
II
B
Clad.
med.
in l.w./E As
B, -
N
761/94
SD
MI
B
Clad.
med.
in l,w./G As
B, -
Fe
761/102
SD
IV
B
Clad.
med.
in 1.w./K As
B, -
K
761/240
SD
' V
B
Clad.
med.
in 1.w./L As
B, -
Ca, Mg, Mn, Mo, Zn, Cu
761/254
SD
VI
B
Clad.
med.
in 1.w./N As
M, -
vit. Bi
761/75
SD
VII
B
Clad.
med.
in l.w./P As M, -
Fe
761/70
SD
VIII
F
As B,
- P/C
As B, - vit
Bi
190/80
SD
IX
F
As 3,
- P/E As B, - N
190/94
SD
X
F
As B,
- P/G As Bf - Fe
190/102
NS
* Yields with a conation letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
+ NS * not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
TABLE A-6. STATISTICAL ANALYSES OF YIELD OF Cladophora glomerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT EGG HARBOR JULY 25, 1976
Ave. algae
Homoscedastic •
Treatment within
dry-wt. yield,
group
homoscedastic group
mg/1
SNK*
A Cladophora medium
597
b
B
Clad, medium in l.w.
660
b
T
• C
As
B, - vit. Bi
107
a
H
As
B, - S
543
b
I
As
B, - B, pyrex
713
b
J
As
B, - B, low B glass
545
b
L
As
B, - Ca, Mg, Mn, Mo, Zn, Cu
580
b
E
As
B, - N
21
a
F
As
B, - P
64
b
ii
G
As
B, - Fe
72
b
K
As
B, - K
200
c
(continued)
-------
TABLE A-6. (continued)
Ave. algae
Heteroscedastic Treatment within dry-wt. yield,
group heteroscedastic group mg/1 EMHY"
I B Clad, med. in l.w./E As B, - N" 660/21 SD
II 3 Clad, mcd. in l.w./F As B, - P 660/64 SD
III B Clad, med. in l.w./GAs B, - Fe 660/72 SD
IV B Clad, med. in l.w./K As B, - K 660/200 SD
V C As B, - vit. Bl/E As B, - N 107/21 SD
VI C As B, - vit. Bj/F As B, - P 107/64 NS
VII C As B, - vit. Bt/G As B, - Fe 107/72 NS
* Yields with a common letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
t NS « not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
TABLE A-7. STATISTICAL ANALYSES OF YIELD OF Cladophora glomerata RESPONSE TO
DIFFERENTIAL NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED
AT EGG HARBOR SEPTEMBER 12, 1976
Ave, algae
Homoscedastic Treatment within dry-wt. yield,
• group homoscedastic group mg/l SNK*
A Cladophora medium
690
ef
B Clad, medium in l.w.
764
f
D As B, - vit. B12
606
de
G As B, - Fe
288
b
H As B, - S
493
cd
J As B, - B, low B glass
342
b
L As B, - Ca, Mg, Mn, Mo, Zn, Cu
565
de
M As B, filtered l.w. + vit.
539
de
0 As M, - vit. B1Z
389
be
P As M, - Fe
142
a
C As B, - vit. Bj
76
a
E As B, - N
86
a
F As B, - P .
149
b
K As B, - K
220
b
N As M, - vit Bj
44
c
(continued)
-------
TABLE A-7.(continued)
Ave. algae
Iieteroscedast ic
Treatment within
dry-wt. yield,
group
heteroscedastic group
mg/1
EMHVt
I .
G
Clad.
med.
in l.w./C As B, - vit. Bj
764/76
SD
II
B
Clad.
med.
in l.w./E As B, - N
764/86
SD
III
• B
Clad.
med.
in l.w./F As B, - P
764/149
SD
IV
B
Clad.
med.
in l.w./K As B, - K
764/220
SD
V
B
Clad.
med.
in l.w./N As M, - vit, Bj
764/44
SD
VI
G
As B,
- Fe/C As B, - vit. B]
288/76
SD
VII
G
As B,
- Fe/E As B, - N
288/86
SD
VIII
G
As B,
- Fe/F As B, - P
288/149
SD
* Yields with a common letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
t NS = not significant at the 5% level; SD ¦ significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
' TABLE A-8.STATISTICAL ANALYSES OF YIELD OF Cladophora gloroerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT GILL'S ROCK JULY 25, 1976
Homoscedastic
group
Treatment within
homoscedastic group
Ave. algae
dry-wt. yield,
mg/1
SNK*
' A
Cladophora medium
840
ab
D
As
B, - vit. B12
566
c
I
F
As
B, - P -
181
d
I
As
B, - B, pyrex
920
a
L
As
B, - Ca, Mg, Mn, Mo, Zn, Cu
712
be
B
Clad, medium in l.w.
1038
a
C
As
B, - vit. Bi
239
b
E
As
B, - N
43
c
II
G
As
B, - Fe
152
d
H
As
B, - S
458
e
•
J
As
B, - B, low B glass
578
f
K
As
B, - K
350
g
(continued)
-------
TABLE A-8.(continued)
Ave. algae
Heteroscedast ic
Treatment within
dry-wt. yield,
group
heteroscedastic group
mg/1
EMHYt
I
B Clad.
med. in l.w./A Clad, medium
1038/840
SD
II
B Clad.
med. in l.w./D As B, - vit. B12
1038/566
SD
III
B Clad.
med. in l.w./F As B, - P
1038/181
SD
IV
B Clad.
med. in l.w./l As B, - B, pyrex
1038/920
NS
V
B Clad.
med. in l.w./L As B, - Ca, Mg, Mn, Mo, Zn, Cu
1038/712
SD
' VI
F As B,
- P/C As B, - vit. Bj
181/239
NS
VII
F As B,
- P/E As B, - N
181/43
SD
VIII
F As B,
- P/G As B, - Fe
181/152
NS
1 As B,
- B, pyrex/J As B, - B, low B glass
920/578
SD
* Yields with a common letter are not significantly different at the S% level using Student-Newman-
Keuls multiple range test..
t NS = not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
TABLE A-9.STATISTICAL ANALYSES OF YIELD OF Cladophora glomerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT GILL'S ROCK AUGUST 15,
1976
Homoscedastic
group
Treatment within
homoscedastic group
Ave. algae
dry-wt. yield,
mg/1
SNK*
A
Cladophora medium
635
c
C
As
B, - vit. B!
81
ab
E
As
B, - N
130
b
I
F
As
P
43
a
G
As
B, - Fe
85
ab
H
As
B, - S
473
c
K
As
B, - K
228
c
0
As
N, - vit. Bi
57
a
B
Clad, medium in l.w.
. 829
b
D
As
B, - vit. Bl2
i
381
a
II
M
J
As
As
B, - Mn
B, - B, low B glass
784
415
b
a
L
As
B, - Ca, Mg, Mn, Mo, Zn, Cu
866
b
N
As
B, filtered l.w. + vit.
680
(continued)
b
-------
TABLE A-9.(continued)
Heteroscedastic
group
Treatment within
heteroscedastic group
Ave. algae
dry-wt. yield,
mg/1
EMHV~
I
B
Clad. med. in
1.w./A Clad.
medium
829/635
SD
II
. B
Clad. med. in
1.w./C As B,
- vit. Bi
829/81
SD
III
B
Clad, med. in
l.w./E As B,
- N
829/130
SD
IV
B
Clad. med. in
1.w./F As B,
- P •'
829/43
SD
V
8
Clad. med. in
l.w./G As B,
- Fe
829/8S
SD
VI
B
Clad. med. in
1.w./H As B,
- S
829/473
SD
VII
B
Clad. med. in
l.w./K As B,
- K
829/228
SD
VIII
B
Clad. med. in
1.w./0 As N,
- vit. B i
829/57
SD
* Yield with a common letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
+ US » not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
TABLE A-10. STATISTICAL ANALYSES OF YIELD OF Cladophora glomerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRIC1C-ENT OF LAKE MICHIGAN WTO COLLECTED AT GILL'S ROCK SEPTEMBER 12,
1976
• Ave. algae
Homogeneous Treatment within dry-wt. yield,
group homogeneous group mg/1 SNK*
A. Cladophora medium 742 c
B Clad, medium in l.w. 741 c
C As B, - vit. Bi - 103 a
D As B, - vit. 8i2 665 c
F As B, - P 185 ab
I HAs B, S 665 c
J As B, - B, low B glass 742 c
L As B, - Ca, Mg, Mn, Mo, Zn, Cu 582 c
N As B, filtered l.w. + vit. 806 c
0 As N, - vit. Bj 329 b
P As N, - vit. B12 631 c
E As B, - N 66 a
G As B, - Fe 81 a
II . K As B, - K 231 b
Q As N, - Fe 59 a
(continued)
-------
TABLE A-10. (continued)
Heterogeneous
group
Treatment within
heterogeneous group
Ave. algae
dry-wt. yield,
rag/1
E.MHY"
I
B Clad.
med. in l.w./E As B, -
N ' . ••
741/66
SD
11
B Clad.
med. in l.w./G As B, -
Fe
741/81
SD
III
B Clad.
med. in 1.w./K As B, -
K
741/231
SD
fv
B Clad.
med. in l.w./Q As N, -
Fe
741/59
SD
V
C As B,
- vit. Bi/£ As B, - N
103/66
NS
VI
C As B,
- vit. Bj/G As B, - Fe
103/81
NS
VII
F As B,
- P/E As B, - N
185/66
MS
VIII
F As B,
- P/G As B, - Fe
185/81
NS
* Yields with a common letter are not significantly different at the 5% level using Student-Newcan-
Keuls multiple range test.
t . N'S = not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test. .
-------
TABLE A-ll. STATISTICAL ANALYSES OF YIELD OF Cladophora glomerata RESPONSE TO DIFFERENTIAL
NUTRIENT ENRICHMENT OF LAKE MICHIGAN WATER COLLECTED AT BAILEY'S HARBOR JULY 25,
1976
Homogeneous
group
Treatment within
homogeneous group
Ave. algae
dry-wt. yield,
mg/1
SNK"
A
Cladophora medium
821
cd
B
Clad, medium in l.vi.
912
d
D
As
B, - vit. Bj 2
720
bed
I
G
As
B, - Fe
146
a
I
As
B, - B, pyrex
907
d
J
As
B, - B, low B glass
584
be
L
As
B, - Ca, Mg, Nln, Mo, Zn, Cu
543
b
C
As
B, - vit. Bj
58
a
E
As
B, - N
42
a
II
F
As
B, - P
84
a
H
As
B, - S
593
b
K
As
B, - K
213
b
(continued)
-------
TABLE A-ll. (continued)
Heterogeneous
group
Treatment within
heterogeneous group
Ave algae
dry-wt. yield,
mg/1
EMHV-
I
B Clad. med.
in l.w./C As B, - vit. B,
912/58
SD
II
B Clad. med.
in l.w./E As B, - N
912/42
SD
III
B Clad, med.
in l.w./F As B, - P
912/84
SD
IV
B Clad. med.
in l.w./H As B, - S
912/593
SD
V
B Clad. med.
in l.w./K As B, - K
912/213
SD
VI
G As B, - Fe/C As B, - vit. B,
146/58
NS
VII
G As B, - Fe/E As B, - N
146/42
SD
VIII
G As B, - Fe/F As B, - P
146/84
NS
* Yields with a common letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
t NS - not significant at the 5% level; SD = significantly different at the 5% level using the equality
of means with heterogeneous variances test.
-------
TABLE A-12:' STATISTICAL ANALYSES OF YIELDS OP Cladophora glomerata RESPONSE TO
DIFFERENTIAL NUTRIENT ENRICHMENT OF"LAKE MICHIGAN WATER COLLECTED
AT BAILEY'S HARBOR SEPTEMBER 12, 1976
Ave. algae
Homogeneous
Treatment
within
dry-wt. yield,
group
homogeneous
group
mg/i
SNK*
A Cladophora medium
653
c
*
B
Clad, medium
in l.w.
763
c
D
As
B, - vit.
B I ?
433
b
I
J
As
B, - B, low B glass
346
b
L
As
B, - Ca, Mg, Mn, Mo, Zn, Cu
465
b
M
As
B, filtered l.w. * vit.
388
b
0
As
M, - vit.
Bl 2
123
a
c
As
B, - vit.
B i
58
ab
E
As
B, - N
58
ab
F
As
B, - P
156
c
G
As
B, - Fe
84
b
II
H
As
B, - S
505
c
K
As
B, - K
201
c
N
As
M, - vit.
Bi • '
41
ab
P
As
M, - Fe
8
a
(continued)
-------
TABLE A-12. (continued)
Heterogeneous
group
Treatment within
heterogeneous group
Ave. algae
dry-wt. yield,
mg/1
EMHV
I
B
Clad.
med.
in
1. w./C As
B, -
vit. B i
763/58
sd
II
B
Clad.
med.
in
l.w./E As
B, -
N
763/5S
SD
III
B
Clad.
med.
in
1.w./F As
B, "
P •
763/156
SD
' IV
B
Clad.
med.
in
1. w. /G As
B. -
Fo .•
763/84
SD
V
B
Clad.
med.
in
l.w./HAs
B, -
S . ,
763/505
SD
VI
B
Clad.
med.
in
l.w./K As
B, -
K
763/201
SD
VII
B
Clad.
med.
in
l.w./N As
M, -
vit. Bj
763/41
SD
VIII
B
Clad.
med.
in
1. w./P As M, -
Fe
763/8
SD
* Yields with a common letter are not significantly different at the 5% level using Student-Newman-
Keuls multiple range test.
t NS * not significant at the 5% level; SD = significantly different at the 5% level using the equality
. of means with heterogeneous variances test.
-------
TABLE A-13. STATISTICAL ANALYSES ON Till; YIELD IN THE FITZGERALD
TEST l-Oil HOT WATER EXTRAd'AIU.E l» IN Cladophorn glomerata
Treatment in group,
Ave, algae
Momoscedast ic.
P in culture
dry-wt. yield,
group
so In., ppin
mg/1
SNK*
I
0.07
221
a
0.14
342
b
0.28
414
c
11
0.56
546
a
1.12
625
a
2.80
589
a
Treatment in group,
. Ave. algae
Heteroscedastic
P in culture
dry-wt. yield,
group
.soIn., ppm
mg/1
EMHV'
I
0.28/0.56
414/546
SD
* Yields with a common letter arc not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
t NS * not significant at the 5% level; SD = significantly different at the
5% level using the equality of means with heterogeneous variances test.
116
-------
TABLE A-14. STATISTICAL ANALYSES ON THE YIELD OF Cladophora glomerata
GROWN IN CUI.TliRi: MILIUM 111 l;l;l;KINU IN N CONCENT HAT I ON AND
CULTURL I'lilUOD
Treatment in group,
Homoscedastic
N added to culture
Ave. algae dry-wt.
H roup
soln., ppm
yield, mg/1
SNK*
I
14 day
1.0
96
a
culture
2.0
209
b
period
II
14 day
4.0
394
a
culture
6.0
423
a
period
III
14 day
8.2
386
a
culture
16.4
344
a
pe ri od
I
1.0
120
. a
21 day
2.0
264
b
culture
4.0
429
c
period
II
6.0
S6S
a
21 day
8.2
660
b
culture
16.4
708
b
period
(continued)
117
-------
TABl.Li A-14(cont tnucd )
llctcroseedast ic
group
Treatment in group,
N added to culture
soln., ppm
Ave, algae dry-wt.
yield, mg/1
EMHV+
14 day
culture period
I
2.0/4.0
209/394
SD
11
6.0/8.2
423/386
NS
21 day
culture period
I
4.0/6.0
429/56S
SD
* Yields with a common letter are not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
t NS = not significant at the 5% level; SD = significantly different at the
5% level using the equality of means with heterogeneous variances test.
118
-------
TABLE A-15. STATISTICAL ANALYSES ON TUP. YIELD OH Cladophora
ulomerata li UOWN IN CUl.TIIRi! MEDIUM niFR-IUNG IN
l> CONCENTRATION AND CULTURE PERIOD
llomosccdast ic
group
Treatment in group,
1' added to culture
soIn., ppm
Ave. algae
dry-wt. yield,
mg/1
SNK*
I
.07
88
a
14 day
. 14
230
b
culture
.28
266
b
period
.56
307
c
1.12
353
c
2.80
343
c
I
21 day
.07
117
a
culture
.28
377
b
pcrjod
.56
435
b
1.12
632
c
2.80
509
b
Ucteroscedastic
group
Treatment in group,
P added to culture
s o1n., ppm
Ave. algae
dry-wt. yield,
mg/1
EMHV+
21 day culture
period
I
.07/.14
117/282
SD
i ]
.14/.28
282/377
SD .
119
-------
Yields with a common letter arc not sipnifj cantly different at the 5%
lcvol using Student-Ncwman-Kiml s multiple rnngo test.
NS = not significant at the 5* level; SD = significantly different at the
5°j level using the equality of means with heterogeneous variances test.
120
-------
TABlli A-16. STATISTICAL ANALYSIS ON IHIi YlliLL) OF Cladophora
glomerata AND THE EFFECT OF DIFFERENT PATTERNS
01- ADDITION OF N
Homo-
scedastic
group
Treatment
in
group
Ave. algae
dry-wt.
yield,
mg/1
SNK*
I
16 .4 ppm N, initially
311
a
1.6 ppm N, initially
219
b
II
0.8 ppm N, initially;
at 9 days, .16 ppm N
each of 5 days
172
a
10 additions of .16 ppm N
during 14 day period
160
a
Hetero- .
scedastic•
group
Treatment
• in'
group
Aye. algae
dry-wt.
yield,
mg/1
EMHV1"
I
16.4 ppm N, initially/0.8 ppm N, etc.
311/172
SD
II
16.4 ppm N, initially/10 additions, etc.
311/160
SD
III
1.6 ppm N, initially/0.8 ppm N, etc.
219/172
SD
IV
1.6 ppm N, initially/ 10 additions, etc.
219/160
SD
* Yields with a common letter arc not significantly di fferent at the 5%
level using Student-Newman-Keuls multiple range test.
i NS = not significant at the 51'. level; SD = significantly different at
the So level using the equality of means with heterogeneous variances tost
121
-------
TAUI.n A-17. STATISTICAL ANALYSIS ON Till! YJI!|,|) OF Clndophora rivularis
ISOLATI-D l;UOM 1.AK1: HURON MOWN IN UULTURli MliUIUM UIFl-'LRING
IN S CONCENTRATION
Homoscedast ic
group
Treatment in group,
S added to culture
solution, ppm
Ave. algae dry-wt.
yield, rag/1
SNK*
1
1.21
149
a
1.90
131
a
2.90
169
a
4.85
270
b
11
2.43
184
a
9.7
554
b
14.6
542
b
19.4
494
• b
ileterosccdast ic
group
Treatment in group,
S added to culture
solution, ppm
Ave. algae dry-wt.
yield, mg/1
EMHV+
I
1.90/2.43
131/184
NS
II
2.43/2.90
184/169
NS
III
4.-S5/9.7
• 270/554
SD
* Yields with a common letter are not significantly different at the 5%
level using Studcnt-Ncwman-Kculs multiple range test.
. NS = not significant ;it the 5o level; SD = significantly different at
the 5% level using the equality of means with heterogeneous, variances test.
122
-------
TABLE A-18
. STATISTICAL ANALYSES ON
THE YIELD OF Cladophora
glomerata
r.SOI.AIT.n FROM LAKH HUT
GROWN IN CULTURE MEDIUM
DIFFERING
IN S CONCENTRATION
Treatment in group,
llomoscedastic
S added to culture
Ave. algae dry-wt.
*
group
solution, ppm
yield, mg/1
SNK
I
1.21
96
a
1.90
168
b
2.43
212
c
2.90
238
d
II
4.85
362
a
9.7
468
a
14.6
475 •
a
19-4 • •
445
• a _.
Treatment in group,
. -
Heteroscedastic
S added to culture
Ave. algae dry-wt.
,
group
solution, ppm
yield, Mg/1
EMHV*
I 2.90/4.85 238/362 SD
* Yields with a common letter are not significantly different at the 5%
level using Student-Newman-Kculs multiple range test.
t NS = n0t significant at the 5% level; SD = significantly different at the
5* level using the equality of means with heterogeneous variances test.
1 23
-------
TAHLE A- 19. STATISTICAL ANALYSES ON "ITII- YIELD OF Cladophora glomerata
I SOI ATI U) l-'UOM LAKE ONTARIO MOWN IN CULTURE MEDIUM D!PEERING
IN S CONCENTRATION
Homoscedast ic
group
Treatment in group,
S added to culture
solution, ppm
Ave. algae dry-wt.
yield, mg/1
SNK*
I
1.21
183
a
1.90
206
a
2.43
196
a
2.90
221
a
4.85
431
b
14.60
641
b
11
9.?0
659
a
19.4
653
a
Heteroscedast ic
group
•Treatment in group,
S added to culture
solution, ppm
Ave. algae dry-wt.
yield, mg/1
EMHV+
I
4.85/9.7
431/659
SD
11
9.7/14.6
659/641
NS
1 11
14.6/19.4
641/6S3
NS
* Y i o 1 ds with ;i common letter arc not significantly different at the 5%
level using Studeiit-Newnian-Kculs multiple range test.
t NS = not significant at the S% level; SD = significantly different at the
5"« level using the equality of means with heterogeneous variances test.
124
-------
TABLE A-20.
STATISTICAL ANALYSES
Pr.ijirirn.'i 1 il i ;i jOumosn
Mlfl) HIM nlH-TiRlNG IN t
ON TIE YIELD OF
(MOWN in cni.Timi;
i CONCENTRATION
Treatment in group,
llomosccdastic S added to culture
"roup solution, ppm
Ave. algae dry-wt.
yield, mg/1
SNK*
I
0. 12
173
C
0.24
196
c
0.49
306
c
0.97
466
b
1.94
702
a
4.85
598
a
II
9.7
827
a
19.4
778
a
Treatment in group,
Heteroscedastic S added to culture
group solution, ppm
Ave. algae dry-wt.
yield, mg/1
emhvt
I
4.85/9.7
598/827
SD
* Yields with a common letter are not significantly different at the 5%
level using Student-Newman-KcuIs multiple range test.
t NS = not significant at the 5% level; SD = significantly different at the
^ 5% level using the equality of means for heterogeneous variances test.
-------
TABLE A-21. STATISTICAL ANALYSES ON THE YIELD OF Nostoc muscoruci
GROWN IN CULTURE MLPIUM I) I ITER INC IN S CONCENTRATION
llomosccdastic
i; roup
Treatment in group,
S added to culture
.solution, ppm
Ave. algae dry-wt.
yield, rag/1
SNK*
!
0.12
115
d
0.24
192
c
0.49
284
b
4.85
- 258
a
II
0.97
249
a
1.94
243
a
9.70
245
a
19.40
230
a
Heteroscedastic
group
Treatment in group,
S added to culture
solution, ppm
Ave. algae dry-wt.
yield, rag/1
EMHV+
1
4.85/9.7
258/245
NS
II
1.94/4.85
243/258
NS
III
0.49/0.97
284/249
NS
* Yields with a common letter are not significantly different at the 5% level
using Studcnt-Ncwinun-ICculs multiple range test,
f NS = not s.i gni f i emit ;tt. the 5% level; SD = significantly different.at the .
5".. level using the equality of means with heterogeneous variances test.
126
-------
TAI*LI- A-22. STATISTICAL ANALYSIS ON Till- YIELD 01- Microc^stis aeruginosa
ciujkn in cuLTtmi; mhhum ini-ii*;kin«; in s concun'I'ration*
Homoscedastic group,
S added to culture
solution, ppm
Ave. algae dry-wt.
yield, mg/1
SNK*
0. 12
54
c
0.24
61
c
0.49
166
b
0.97
203
a
1.94
207
a
4.8S
192
ab
9.70
207
a
19.40
177
ab
* Yields with a common letter are not significantly different at the 5%
level using "Student-Newiiian-Keuls multiple range test.
-------
TAB Li: A-23. STATISTICAL ANALYSES ON Till; YIELD OF Cladophora
clonic rata IN RESPONSE TO Till- ADDITION OP C13 MICRONUTRIENTS
USING ZOOSPORES AS INOCULUM
Treatment
Ave. algae
Ilomnsccdj.st ic
in
dry-wt. yield,
group
group
mg/1
SNK*
Clad, medium,
plus C13
741
a
Clad, medium,
minus As
563
a
Clad, medium,
minus Cd
674
a
Clad, medium,
minus Hg
776
a
I
Clad, medium,
minus Pb
783
a
Clad, medium,
minus F
778
a
Clad, medium,
minus Se
626
a
•
Clad, medium,
minus Be
736
a
Clad, medium,
minus Ci3
12
a
II
Clad, medium,
minus Ci3 "7"
14
a
Treatment
Ave. algae
Meteroscedastic
in
dry-wt. yield,
group
group
mg/1
EMHV'
I
Plus Ci3/minus Ci3
741/12
SD
11
• Plus Ci3/minus C13 "7"
741/14
SD
* Yields with a common letter are not significantly different at the 5%
level using Student-Newman-k'eu Is multiple range test,
^ NS = not significant at the 5% level; SD = significantly different at
the 5% level using the equality of means with heterogeneous variances
test. • .
128
-------
TABLE A-9 4 STATISTICAL ANALYSES ON THE YIELD RESPONSE OF
Clmlophorn pJomernt£ TO VARIOUS CONCENTRATIONS OP
MANlIANliSli"
llomosccdast ic
i:rou|>
Treatment in group,
Mn added to culture
soIn., ppm
Ave. algae dry-wt.
yield, mg/1
SNK*
0.00
356
a
I
2.16
342
a
2.43
296
a
2.70
188
b
11
0.27
698
a
1.3S
606
a
Hoteroscodastic
group
Treatment in group,
Mn added to culture
soln:, ppm
Ave. algae dry-wt".
yield, mg/1
EMHV+
I
0.00/0.27
356/698
SD
11
1.35/2.16
606/342
SD
* Yields with a common letter are not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
^ NS = not significant at the 5% level; SD = significantly different at the
S" level using the equality of means with heterogeneous variances test.
129
-------
APPENDIX 6
ADDITIONAL EXPERIMENTAL DATA
ISO
-------
TABLE B-1. STANDARDIZATION OF FITZGERALD TEST FOR HOT WATER
UXTUACTAHLH PHOSPHORUS IN Cladophora glomerata
P in culture
scln.,* ppm
Ave. algae dry-wt
yield,' mg/1
Extractable
P %
>. » » 0
0.07
221
0.005
0.14
342
0.007
0.28
414
0.008
0.56
546
0.011
1.12
625
0.013
2.80
589
0.042
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 18 ppm K, 7.5 ppm
Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements, B7 and Cj 3
• solutions, vitamins Bi and B12 and Na2C03 and NajSiOj.
t Average of three replicates per treatment; 21-day culture period.
131
-------
600
o»
E
^ 400
>
(Z
Q
2
UJ
> 200
o
0
J L
1
1
J L
o
.01 .02 .03
EXTRACTABLE P, %
.04
FIGURE B-1. Relationship between yield and hot-water extractable P in
Cladophora r lomcr.it a cultured under various levels of P.
132
-------
TABLE H-2. STANDARDIZATION OF Al.KAl.INF. PHOSPHATASE TEST
I OK I'llOSI'ilUIUJS AVAI LA HI LITY TO ClaJophora
glomerata
1' in culture
soln., * ppm
Ave. algae
dry-wt,'
rag/1
Phosphatase
activity,
§
.035
27S
304
.07
238
309
. 14
291
353
.28
433
246
.56
515
136
1.12
558
62
2.80
706
12
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 18 ppra K, 7.5 ppm
Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements, B7 and Cj3
solutions, vitamins Hi and Bi2, and NazCOj and NajSiOa.
+ Average of three replicates per treatment.
§ One unit of alkaline phosphatase activity equals the amount of enzyme
which liberates one ini11imicromole of nitrophenol/hr, under the prescribed
conditions.
133
-------
TABU; B-3. STANDARD I ZATION 01- Nllu-N Ul'TAKU TEST TO INDICATE
NITKlHlliN NUTRITION 01' L' Indophora omerata
N in culture
so In.,* j >pm
1.5
313
22.5
a
2.5
366
32.1
ab
4.0
411
34.9
abc
6.0
472
31.7
abc
10.0
569
29.4
c
16.4
510
24.6
be
32.4
491
15.0
abc
* The nutrient medium contained 2.8 ppm P, 23.4 ppin Ca, 18 ppm K, 7.S ppm
Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements, B7 and C13
solutions, vitamins B, and Bi2i and Na2C03 and Na2Si03.
t Average of two replicates per treatment; 22-day culture period.
§ Yields with a common letter are not significantly different at the 5%
level using Student-Ncwmnn-Kouls multiple ranye test.
Ave. algae dry-wt.
yield,^ NIU-N uptake,
ing/1 pg N/10 mg algae/hr SNK®
134
-------
TABI.n B-4. STANDARD IZAT ION OF NITRATE ACCUMUI.ATION TEST TO
INDICATIl N1 *1 liOt.l N NUTRITION ()1: C ladophora glomorata
N in culture
so In.,* ppm
Ave. algae dry-wt.
yield,'!" mg/1
NO 3 in
algae, ppm
1.5
280
14
2.5
387
8S
4.0
455
140
8.0
700
96
16.4
747
97
24.6
742
126
32.8
606
692
* The nutrient medium contained 2.8 ppm P, 23.4 ppm Ca, 18 ppni K, 7.5 ppm
Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements, B7 and Ci3
solutions, vitamins Bj and B12, and Na2C0a and NajSiOj.
"I Average of two replicates per treatment.
135
-------
TAHLI- H-S. Till: lU-T'liCT 01- CULTIJKU I'P.RIOI) U-NCTH ON N CIUTICAL
CONCENTRATION IN Cladophora glomerata
14-Uay culture period 21-day culture period
?1 added Ave. algae Ave. P cone, Ave, algae Ave. P cone.
to culture dry-wt. yield,"f" in algae, dry-wt. yield,t in algae,
soln.,* ppm
mg/1
Q,
'O
mg/1
%
1.0
96
.89
120
.77
2.0
209
.79
264
.67
4.0
394
O
CO
429
.75
6.0
423
1.09
565
.64
8.2
386
1.27
660
.97
16.4
344
2.85
708
1.6S
* The nutrient medium contained 23.4 ppm Ca, 2.8 ppm P, 18 ppm K, 7.5 ppm
'•1r. 9.7 ppm s, 1.12 ppm Pe, 1/5 Johnson's trace elements, and Na2C03 and
Nn?Si03, 1^7 and C)3 solutions, and vitamins Bi and B12.
Average of five replicates in each treatment.
136
-------
TABl.F. B-fi. Till: EFFECT OF CULTURE PF.RIOI) l.PNCTII ON P CRITICAL
l.'ONCliN'THAT! ON IN C l;uli»plnn a glume rata
(
14-day culture
period
21-day culture period
P added
to culture
so In. , * ppm
Ave. algae
dry-wt. yield,1
mg/1
Ave. P conc.
in algae,
%
Ave. algae
dry-wt. yield,^
mg/1
Ave. P conc.
in algae,
%
0.07
88
.07
117
.05
0.14
230
.05
282
.04
0.28
266
.09
377 ;
.05
0.56
307
.07
435
.18
1.12
353
.26
632
.14
2.80
343
.64
509
.43
* The nutrient medium contained 23.4 ppra Ca, 16.4 ppm N, 18 ppm K, 7.5 ppm
Mr, 9.7 ppm S, 1.12 ppm Fe, 1/5 Johnson's trace elements, Na2C03 and
Nn ,Si03, and CJ3 solutions, and vitamin BA and B12.
t Average of five replicates in each treatment.
137
-------
TABl.lv B-7. THE UFFliCT OF LOW K CONCENTRATION ON P
C1UTICAL CONCENTRATION IN Cladophora
glomerata
P added to
culture soln.,*
ppm
Ave. algae
dry wt. yield,"!"
mg/1
Ave. P conc.
in algae,
%
SNK5
.028
102
.05
a
.042
121
.03
a
.07
122
a
.14
164
.06
b
.28
259
.07
c
.56
292
.13
c
1.12
295
.29
c
2.80
255
.39
c
* K level in all treatments was 1.8 ppm; the nutrient medium contained
16.4 ppm N, 23.4 ppm Ca, 7.5 ppm Mg, 9.7 ppm S, 1.12 ppm Fe, 1/5
Johnson's trace, NazCOj and Na2Si03, B7 and Cj 3 solutions, and vitamins
Bi and Bj 2.
'!" Average of five replicates in each treatment; 17-day culture period.
§ Yields with a common letter arc not significantly different at the 5%
level using Student-Newman-Keuls multiple range test.
138
-------
TABLE B-8.YIELD AN!> S CONCENTRATION IN Cladophorn rivularis ISOLATED
i'KOM I.AKi; HURON C.KOWN IN CUI.TUKL MlflHUM UI1" 1-liKINCi IN S
CONCENTRATION
S added to
culture sola.,* ppm
Ave. algae dry-wt.
yield,+ mg/1
Ave. S Qonc
in algae, %
1.21
149
0.90
1.90
131
1.26
2.43
184
1.54
2.90
169
1.62
4.85
270
1.41
9.7
554
1.80
14.6
542
2.28
19.4
494
3.46
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.S ppm Mg, 1.12 ppm Fe, 1/5 Johnson's trace elements, Na2C03, NajSiOj
B? and C13 solutions, and vitamins Bj and Bi2.
t Average of five replicates in each treatment; 22-day culture period.
139
-------
TAR LP B-9.YIKLH AND S CONCENTRATION IN Clndopliora glomerata ISOLATED
l-'UOM I.AKI-; lilt IC (MOWN IN CUl.TllKlT MliDllIM DIFFF.RING IN S
CONCIiN'iliATlON
S added to
culture soln.,* ppm
Ave. algae dry-wt
yield,'!" mj:/ 1
Ave. S conc.
in algae, °i
1.21
!>G
1.24
1.90
168
1.25
2.45
212
1.11
2.90
238
1.14
4.85
362
1.06
9.7
468
1.24
14.6
475
1.77
19.4
445
3.06
* The nutrient medium contained 23.4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.5 ppm Mg, 1.12 ppm Fe, 1/5 Johnson's trace elements, NazCCh,
Na2Si03, By and C13 solutions, and vitamins Bj and B j 2 -
!' Average of five replicates in each treatment; 18-day culture period.
140
-------
TABLE B-10. YIELD AND S CONCENTRATION IN Cladophora glomerata ISOLATED
I'UOM lAKi: ONIAUIt) I'.UOWN IN CULTURE MEIMUM UlIFERJNG IN S
CONCENTRATION
S added to
culture solo.,* ppm
Ave. algae dry-wt.
yield,' mjj/1
Ave. S conc.
in algae, %
1.21
183
0.44
1.90
206
0.98
2.43
196
0.55
2.90
221
1.09
4.85
431
0.85
9.7
659
1.32
14.6
641
2.00
19.4
653
>1.97
* The nutrient medium contained 23,4 ppm Ca, 16.4 ppm N, 2.8 ppm P, 18 ppm
K, 7.5 ppm Mr, 1.12 ppm Fe. 1/5 Johnson's trace elements, Na2C03,
Na2Si03, b7 and Cn solutions, and vitamins and B,2.
t Average of five replicates in each treatment; 22-day culture period.
141
-------
TAB 1.0 B- 1 1. YUiLD AND S CONCENTRATION IN Tim GREEN ALGAE
Draparnaldia plumosa GROWN IN CULTURE MHDIUM
DIFFERING IN S CONCENTRATION
S added to
culture so In.,* ppm
Ave. algae dry-wt.
yield.t tag/1
Ave. S conc.
in algae, %
0.12
173
.02
0. 24
196
.06
0.49
306
.06
0.97
466
.12
1.94
702
. 16
4.85
598
.25
9.70
827
.29
19.4
778
.69
* The nutrient medium contained 82 ppm N,18 ppm K, 7 ppm P, 7.5 ppm Mg,
9.7 ppm Ca, 1.12 ppm Fe, 1/5 Johnson's trace elements, and Na2CO3 and
Na?Si03.
'/ Average of four replicates in each treatment.
-------
TABLl-i R-12. YIT.LD AND S CONCENTRATION IN TI1C Bl-Ui:-GREHN ALGA Nostoc
muscurum GROWN IN CUL'lURIi MliDIUM DI 11-tHlNC IN S CONCENTRATION
S added to
culture so In,* ppm
Ave. algae
yield,!-
dry-wt.
mg/1
Ave. S conc.
in algae, %
0.12
115
0.11
0.24
192
0.12
0.49
284
0.13
0.97
249
0.06
1.94
243
0.13
4.85
258
0.26
9.70
245
0.28
19.4
230
0.34
* The nutrient medium contained 82 ppm N, 18 ppm K, 7 ppm P, 7.5 ppm Mg,
9.7 ppm Ca, 1.12 ppm Fe, 1/5 Johnson's trace elements and NazC03 and
NaaSiOj.
•'.* Average of four replicates in each treatment.
143
------- |